Highlights of 2004 (http://www.nig.ac.jp/hot/index.html)

1. The origin of the parathyroid gland; persistence of gills in tetrapods
Okabe M, Graham A.
PNAS, Vol. WI No.51, 17716-17719,2004

2. A DNA-binding protein can slide along a helical groove of DNA
Kumiko Sakata-Sogawa, Nobuo Shimamoto.

Proc. Not. Acad. Sci. U.S.A, 101,14731-35,2004

3. Autophagy defends cells against invading group A Streptococcus
Nakagawa I, Amano A, Mizushima N, Yamamoto A, Yamaguchi H, Kamimoto T, NaraA, Funao J, Nakata M, Tsuda K, Hamada S, Yoshimori T

Science, 306, 1037-1040, 2004

4. A transposon-mediated gene trap approach identifies developmentally regulated
genes in zebrafish.
Kawakami, K., Takeda, H., Kawakami, N., Kobayashi, M., Matsuda, N., Mishina, M.

Developmental Cell, 7, 133-144, 2004.

5. MBF 1 role in the oxidative stress defense
Jindara M, Gaziova I, Uhlirova M, Okabe M, Hiromi Y, Hirose S.

EMBO Journal, 23, 3538-3547, 2004

6. Chromosome segregation and RNAi in vertebrate cells
Fukagawa T, Nogami M, Yoshikawa M, Ikeno M, Okazaki T, Takami Y, Nakayama T,
Oshimura M.

Nature Cell Biology, 6,784-791, 2004

7. Essential role for de novo DNA methyltransferase Dnmt3a in paternal andmaternal imprinting.
Masahiro Kaneda, Masaki Okano, Kenichiro Hata, Takashi Sado, Naomi Tsujimoto, En li& Hiroyuki Sasaki

Nature, 429, 900-903, 2004

8. DNA sequence and comparative analysis of chimpanzee chromosome 22
H. Watanabe, A. FUjiyama, M. Hattori, T D. Taylor, A. Toyoda, Y Kuroki, H. Nuchi,
A. BenKahla, H. lehrach, R. Sudbrak, M. Kube, S. Taenzer, P. Galgoczy, M. Platzer,
M. Scharfe, G. Nordsiek, H. BloANcker, I. Hellmann, P. Khaitovich, S PaANaANbo,
R. Reinhardt, H.J Zheng9, X.-l. Zhang9, G.-F. Zhu9, B.-F. Wang9, G. Fu9, S.-X. Ren9,
G.-P. Zhao, Z. Chen, Y-S. lee, J-E Cheong, S.-H. Choi, K.-M. Wu, T-T liu, K.J Hsiao,
S.-F. Tsai, c.-G. Kim, S. OOta, T Kitano, Y Kohara, N. Saitou, H.-S. Park, S.-Y. Wang,
M.-l. Yaspo, and Y Sakak
Nature, 429, 382-388, 2004

9. Diverse sensory organs arose via modification of a proto-sensory organ.
Niwa N, Hiromi Y, Okabe M.

Nature Genetics, 36 (3), 293-297, 2004

10. Imprinting of plants over generations
Kinoshita T, Miura A, Choi Y, Kinoshita Y, Coo X, Jacobsen SE, Fischer Rl, and Kakutoni T

Science, 303,521-523,2004

11. migS, a cis acting site for bipolar positioning of oriC on the Escherichia coli
chromosome
Yamaichi, Yond Niki, H.

EMBO Journal, 23, 221-233, 2004

CONTENTS
Introduction
STAFF

1
2

ADVISORY COMMITTEE·················································································· 6
ADVISORY BOARD·························································································· 7
A. DEPARTMENT OF MOLECULAR GENETICS

A-a. Division of Molecular Genetics
Tatsuo Fukagawa Group
Identification of new centromere proteins in higher vertebrate cells
Masahiro Okada, Tetsuya Hori, Mi-Sun Kwon, Mayumi Takahashi and
Tatsuo Fukagawa

8

Molecular analysis of the Nuf2-Heel complex that transiently localizes to centromere
during mitosis
Tetsuya Hori, Yoshikazu Mikami, Kazuko Suzuki and Tatsuo Fukagawa
Functional roles of the RNAi machinery in verterbrate centromeres
Tomoko Motohashi, Masahiro Nogami, Atsushi Fukushima andTatsuo Fukagawa

89A-b. Division of Mutagenesis
Fumiaki Yamao GroupMultifunctional role of Mcll/Slr3 in DNA replication, repair and chromatin structure
Yasuhiro Tsutsui, Toyoaki Natsume and Fumiaki Yamao
10
A-c. Molecular Mechanism Laboratory
Hiroshi Mitsuzawa Group
Identification of glyceraldehyde-3-phosphate dehydrogenase and actin as proteins thatinteract with the Rpb7 subunit of RNA polymerase II
10
Hiroshi Mitsuzawa
Identification of the general transcription factor TFIID in S. pombe
Hiroshi Mitsuzawa

11

A-c. Molecular Mechanism Laboratory
Hiroaki Seino GroupAn in vitro ubiquitination assay of mitotic cyclin
Hiroaki Seino

11

Ubiquitin-conjugating enzyme(s) responsible for degradation of SCF substrates
Hiroaki Seino

12

A-d. Division of Nucleic Acid Chemistry
Saburo Aimoto Group
Development of a method for chemical synthesis of protein by using two different ligation
methods
Saburo Aimoto
12
Preparation of peptide thioesters by the Fmoc-solid phase method via an on-resin N-S acyl
shift
Saburo Aimoto

12

Synthesis of post-translationally modified histone H3
Saburo Aimoto

13

Synthetic studies of G protein-coupled receptor, opioid receptor like-1 (ORL-1)
Saburo Aimoto

13

CONTENTS

Design of the inhibitors to human T-cell leukemia virus type-l protease
Saburo Aimoto

13

A-d. Division of Nucleic Acid Chemistry
Tsutomu Katayama Group
Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in
Escherichia coli: Interaction of DnaA with the sliding clamp-loaded DNA and the sliding
clamp-Hda complex
Masayuki Su'etsugu, Makoto Takata, Toshio Kubota, Yusaku Matsuda and Tsutomu
Katayama
14
DiaA, a novel DnaA-binding protein, ensures the timely initiation of E.coli chromosome
replication
Takuma Ishida, Nobuyoshi Akimitsu, Tamami Kashioka, Masakazu Hatano, Toshio
Kubota, Yasuyuki Ogata, Kazuhisa Sekimizu and Tsutomu Katayama················ 15
Reactivation of DnaA by DNA sequence-specific nucleotide exchange in vitro
Kazuyuki Fujimitsu and Tsutomu Katayama················································ 15
Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in
Escherichia coli
Toh-ru Shimuta, Kiyotaka Nakano, Yoko Yamaguchi, Shogo Ozaki, Kazuyuki Fujimitsu,
Chika Matsunaga, Kenji Noguchi, Akiko Emoto and Tsutomu Katayama············· 15
B. DEPARTMENT OF CELL GENETICS

B-a. Division of Cytogenetics
Tamotsu Yoshimori Group
Three mammalian homologues of yeast Atg8 localize to autophagosomal membrane
depending on post-translational processing
Yukiko Kabeya, Noboru Mizushima, Akitsugu Yamamoto, Satsuki Oshitani-Okamoto,17
Yoshinori Ohsumi and Tamotsu Yoshimori
Autophagy plays a pivotal role in survival during the early neonatal starvation period
Akiko Kuma, Masahiko Hatano, Makoto Matsui, Akitsugu Yamamoto, Haruaki Nakaya,
Tamotsu Yoshimori, Yoshinori Ohsumi, Takeshi Tokuhisa and Noboru Mizushima...................................................... ···················································17Autophagic machinery provides a cellular defense system against invasion by group A
Streptococcus
Ichiro Nakagawa, Takahiro Kamimoto, Akitsugu Yamamoto, Noboru Mizushima,
Kayoko Tsuda, Atsuki Nara, Junko Funao, Masanobu Nakata, Atsuo Amano, Shigeyuki
Hamada and Tamotsu Yoshimori
18
Intracellular Shigella escapes from Autophagy
Michinaga Ogawa, Tamotsu Yoshimori, Toshihiko Suzuki, Hiroshi Sagara, Noboru
Mizushima and Chihiro Sasakawa
18
The intracellular inclusions containing mutant a I-antitrypsin Z are propagated in the absenceof autophagic activity
Takahiro Kamimoto, Shisako Shoji, Noboru Mizushima, Kyohei Umebayashi, Tunda
Hidvegi, David H. Perlmutter and Tamotsu Yoshimori
18
Analysis of invasion of Porphyromonas gingivalis into cells by using beads coated with thebacterial vesicles
Kayoko Tsuda, Ichiro Nakagawa, Kyohei Umebayashi, Atsuo Amano and Tamotsu
Yoshimori
19
Reconstitution of initiation of autophagosome formation by using semi-intact cells
Shunsuke Kimura, Atsuki Nara, Yoshitaka Nagai and Tamotsu Yoshimori ··········19

CONTENTS

Ubiquitin-dependent sorting of the epidermal growth factor receptor in the endocytic pathway
19
Kyohei Umebayashi and Tamotsu Yoshimori
B-b. Division of Microbial Genetics
Hiroyuki Araki GroupThe interaction between replication proteins for the initiation of DNA replication
Sachiko Sakamoto, Kazuyuki Hirai, Yoichiro Kamimura and Hiroyuki Araki········· 21
Regulatory mechanism of the complex formation between Sld2 and Dpbll by cyclin-dependent
kinase (CDK)
Yon-Soo Tak, Yoshimi Tanaka, Yoichiro Kamimura and Hiroyuki Araki············· 22
CDK targets in the initiation of DNA replication·
22
Seiji Tanaka and Hiroyuki Araki
Mechanisms for generating genomic instability
Seiji Tanaka and Hiroyuki Araki···· · .. · .. ·23
B-b. Division of Microbial Genetics
Seiichi Yasuda Group
Mechanism of DnaA interaction with phospholipids
Seiichi yasuda······················································································ 24
B-c. Division of Cytoplasmic Genetics
Kazuto Kobayashi Group
Mechanisms of Neural Circuitry Underlying Control of Behaviors
Kazuto Kobayashi·················································································· 24
B-c. Division of Cytoplasmic Genetics
Masayuki Yamamoto Group
Fission yeast Mes1p ensures the onset of meiosis II by blocking degradation of cyclin Cdc13p
Masayuki yamamoto·············································································· 25
The p150-Glued Ssm4p regulates microtubular dynamics and nuclear movement in fission
yeast
Masayuki Yamamoto
26
C. elegans DAZ-1 directs proper nuclear organization and cytoplasmic core formation during

oogenesis
Masayuki Yamamoto

26

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

C-a. Division of Developmental Genetics
Yasushi Hiromi GroupMulti-dimensional axon network formation
Masaki Hiramoto and Yasushi Hiromi
Intrinsic sub-axonal patterning in Drosophila neurons
Takeo Katsuki, Masaki Hiramoto and Yasushi Hiromi:
Identification of BP102 antigen which shows specific sub-axonal localization
Tohru Umemura, Takeo Katsuki and Yasushi Hiromi
Lamina-specific connection in mouse hippocampus
Fumikazu Suto, Hajime Fujisawa and Yasushi Hiromi

27
27
27
28

seven-up controls switching of transcription factors that specify temporal identities ofDrosophila neuroblasts
28
Makoto Kanai, Masataka Okabe and Yasushi Hiromi

A search for Seven-up-interacting molecules
Takayuki Hondoh and Yasushi Hiromi

28

Glial-expression of Prospero integrates inputs from multiple signaling

CONTENTSYoshihiro Yuasa and Yasushi Hiromi

29

A screening for genes involving germline stem cell establishment and niche formation in
Drosophila
Miho Asaoka, Shuji Shigenobu, Satoru Kobayashi and Yasushi Hiromi
29
The origin of the parathyroid gland
Masataka Okabe and Anthony Graham
29
C-a. Division of Developmental Genetics
Toshitaka Fujisawa Group
Systematic identification of peptide signaling molecules in Hydra
Toshitaka Fujisawa, Chiemi Fujisawa, Eisuke Hayakawa, Yukihiko Noro, Akemi
Hayashiuchi, Hiroshi Shimizu and Toshio Takahashi .. ··30
Cob. Division of Gene Expression
Susumu Hirose Group
Role of histone modifications and chromatin remodeling in epigenetic gene expression
Takahiro Nakayama, Tsukasa Shimojima, Kazuma Hanai, Kenichi Nishioka, Koji
Akasaka and Susumu Hirose
31
Chromatin transcription
Mikage Nakajima, Kenichi Nishioka, Tadashi Wada, Hiroshi Handa and Susumu Hirose...................................................... ···················································32
Role of DNA topology in the formation of active chromatin
Kuniharu Matsumoto, Hirofumi Furuhashi, Youhei Ogasawara and Susumu Hirose........................................................................................................ 32
Nucleosomal histone kinase-1
Hitoshi Aihara, Takeya Nakagawa, Kiyoshi Yasui, Tsutomu Ohta, Susumu Hirose,
Naoshi Dhomae, Koji Takio, Mayumi Kaneko, Yukio Takeshima, Masami Muramatsu
and Takashi Ito32
Tips in analyzing antibodies directed against specific histone tail modifications
Kavitha Sarma, Kenichi Nishioka and Danny Reinberg

32

Functional analysis of transcriptional coactivator MBF1
Marek Jindra, Ivana Gaziova, Mirka Uhlirova, Masataka Okabe, Yasushi Hiromi, Kenichi
Tsuda, Toshiro Tsuji, Ken-ichi Yamazaki and Susumu Hirose
33
Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast
asymmetric division
Yasushi Izumi, Nao Ohta, Asako Itoh-Furuya, Naoyuki Fuse and Fumio Matsuzaki...................................................... ···················································34
Introduction of p161NK4a inhibits telomerase activity through transcriptional suppression ofhuman telomerase reverse transcriptase expression in human gliomas
Masahiro Saito, Kou Nakagawa, Katsuyuki Hamada, Susumu Hirose, Hironobu Harada,
34
Shohei Kohno, Shigeyuki Nagato and Takanori Ohnishi
Coco Division of Molecular and Developmental Biology
Koichi Kawakami Group
Gene trap and enhancer trap approaches in zebrafish
Koichi Kawakami, Yasuyuki Kishimoto, Kazuhide Asakawa, Tomoya Kotani, Saori
35
Nagayoshi and Akihiro Urasaki
Genetic analysis of zebrafish maternal-effect mutations affecting early embryogenesis
Yasuyuki Kishimoto, Sumito Koshida, Makoto Furutani-Seiki, Atsushi Kawakami, Hisato
Kondoh and Koichi Kawakami
36Transposition of the Tol2 element in mouse embryonic stem cells
Koichi Kawakami and Tetsuo Noda

CONTENTS·····36

A transposon-mediated gene trap approach identifies developmentally regulated genes inzebrafish
Koichi Kawakami, Hisashi Takeda, Noriko Kawakami, Makoto Kobayashi, Naoto
Matsuda and Masayoshi Mishina
37
Excision of the Tol2transposable element of the medaka fish Oryzias latipes in Xenopus laevis
and Xenopus tropicalis
Koichi Kawakami, Kosuke Imanaka, Mari Hoh and Masanori Taira37
The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene
Collection (MGC)
Gerhard D. S. and 113 others
37
Cod. Division of Physiological
Fumitoshi Ishino Group
Epigenetic view of mammalian development
Fumitoshi Ishino

38

Cod. Division of Physiological Genetics
Yukiko Gotoh Group
The Wnt- S -catenin pathway directs neuronal differentiation of cortical neural precursor
cells
Yusuke Hirabayashi, Yasuhiro Itoh, Norihisa Masuyama and Yukiko Gotoh ......... 39
Hes binding to STAT3 mediates crosstalk between Notch and JAK-STAT signaling
Sachiko Kamakura, Koji Oishi, Takeshi Yoshimatsu, Norihisa Masuyama and Yukiko···········································39
Gotoh
Notch promotes survival of neural precursor cells via mechanisms distinct from thoseregulating neurogenesis
Koji Oishi, Sachiko Kamakura, Yuko Isazawa, Takeshi Yoshimatsu, Keisuke Kuida,
39
Norihisa Masuyama and Yukiko Gotoh
JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins
Fuminori Tsuruta, Jun Sunayama, Yasunori Mori, Norihisa Masuyama and Yukiko···········································40
Gotoh
D. DEPARTMENT OF POPULATION GENETICS

D-a. Division of Population Genetics
Naruya Saitou GroupDNA sequence and comparative analysis of chimpanzee chromosome 22
The International Chimpanzee Chromosome 22 Consortium [Hidemi Watanabe,
Choong-Gon Kim, Satoshi Oota, Takashi Kitano, Yuji Kohara, Naruya Saitou andYoshiyuki SakakiJ
41
Human specific amino acid changes found in 103 protein coding genes. Molecular Biology
and Evolution
Takashi Kitano, Yuhua Liu, Shintaro Veda and Naruya Saitou
41
Genetic variation versus recombination rate in a structured population of mice41
Aya Takahashi, Yuhua Liu and Naruya Saitou
Phylogenetic analysis of proteins associated in four major energy metabolism systems:
photosynthesis, oxidative phosphorylation, nitrogen metabolism and sulfur metabolism
Takeshi Tomiki and Naruya Saitou
42
Mitochondrial DNA Genealogy of Chimpanzees in Nimba Mountains and Bossou, West Africa
Makoto Shimada, Sachiko Hayakawa, Tatyana Humle, Shiho Fujita, Satoshi Hirata,
Yukimaru Sugiyama and Naruya Saitou···42
Evolution of 0 alleles of the human ABO blood group gene

CONTENTSFrancis Roubinet, Stephanie Despiau, Francese Calafell, Fen Jin, Jaume Bertanpetit,
Naruya Saitou and Antoine Blancher
43
Comparative genetics of functional trinucleotide tandem repeats in humans and apes
Aida Andres, Marta Soldevila, Oscar Lao, Victor Volpini, Nayuya Saitou, Howard Jacobs,···········43
Ikuo Hayasaka, Francese Calafell and Jaume Bertranpetit
Polymorphisms in the Trace Amine Receptor 4 (TRAR4) Gene on Chromosome 6q23.2 Are
Associated with Susceptibility to Schizophrenia
Jubao Duan, Alan Sanders, Cuiping Hou, Naruya Saitou, Takashi Kitano and Pablo········································44
GejmanEvolutionary functional genomic analysis of cis-regulation in mammalian Dlx gene system
Kenta Sumiyama
44
D-a. Division of Population Genetics
Toshiyuki Takano GroupInter-locus nonrandom association of polymorphisms in Drosophila chemoreceptor genes
Toshiyuki Takano-Shimizu, Yuriko Ishii, Akira Kawabe, Masanobu Itoh, Nobuyuki
46
Inomata, Rumi Kondo and Yutaka Inoue
A high frequency null mutant of an odorant-binding protein, ObpS7e, in Drosophilamelanogaster
Aya Takahashi and Toshiyuki Takano-Shimizu
46
Analyses of the mechanism and genetics of mate choice by Drosophila melanogastermales
Aya Takahashi and Toshiyuki Takano-Shimizu
47
Null alleles at Drosophila chemoreceptor genes
Akira Kawabe, Yuriko Ishii and Toshiyuki Takano-Shimizu···············47
Genetic and molecular basis of sex-comb-tooth-number variation in Drosophila
Haruki Tatsuta, Yuriko Ishii and Toshiyuki Takano-Shimizu··········47
D-b. Division of Evolutionary Genetics (Until May)
Toshimichi Ikemura Group
Phylogenetic classification of environmental DNA sequences including those from theSargasso Sea and of rDNA sequences with Self-Organization Map (SaM)
Takashi Abe, Jian-Ping Song and Toshimichi Ikemura
48
SaM classification of mammalian genomic and eDNA sequences according to function
Takashi Abe, Yoko Kosaka and Toshimichi Ikemura
49
Compilation of "GenomeWordDictionary"Takashi Abe, Yoko Kosaka, Kiyomi Kita and Toshimichi Ikemura
49
D-c. Division of Theoretical Genetics
Sumio Sugano Group
Functional Genomics Based on the full-length eDNA collection
50
Sumio Sugano
E. DEPARTMENT OF INTEGRATED GENETICS

E-a. Division of Human Genetics
Hiroyuki Sasaki Group
Establishment and maintenance of genomic imprinting in the germline and in early embryos
Hiroyuki Sasaki, Masahiro Kaneda, Ryutaro Hirasawa, Kenichiro Hata, Maki Kusumi,
Takashi Sado, Kenji Kumaki, Hiroyasu Furuumi, Masaki Okano, Naomi Tsujimoto, En
Li, Tomohiro Suzuki, Shigeharu Wakana and Toshihiko Shiroishi
53
Comparative analyses of the distal imprinted domain on mouse chromnosome 7 and theorthologous domain on chicken chromosome 5
Hiroyuki Sasaki, Takaaki Yokomine, Wahyu Purbowasito, Hisao Shirohzu, Chikako

CONTENTS

Suda, Takashi Sado, Hisakazu Iwama, Kazuho Ikeo, Tetsuya Hori, Masaoki Tsuzuki,
Shigeki Mizuno, Yo-ichi Matsuda, Chiyoko Sato, Katsuzumi Okumura, Tsunehiro Mukai,
Mohamad Zubair, Ken Tsutsui, Reiko Kato, Atsushi Toyoda, Masahira Hattori and
Yoshiyuki Sakaki
53
Imprinting mechanisms of the mouse Igf2/H19 sub-domain
Hiroyuki Sasaki, Yuzuru Kato, Ko Ishihara, Melanie Ehrlich, Walter Reith and Mitsuyoshi···········································54
NakaoComputer-assisted search for sequence features common to imprinted DMRs
Hiroyuki Sasaki, Hisato Kobayashi, Takashi Abe and Toshimichi Ikemura ..... ·····54
Molecular pathology of ICF syndrome
Hiroyuki Sasaki, Hiroyasu Furuumi, Tadashi Kajii, Tomohiro Kamoda, Nobuaki Iwasaki,
Naomi Tobita, Nobuko Fujiwara, Akira matsui, Yu-ichi Goto and Takeo Kubota...................................................... ···················································54Development of a universal DNA chip system applicable to any organism
Hiroyuki Sasaki, Shin-ichi Mizuno, Tadafumi lino, Hidetoshi Ozawa, Kosuke Tashiro
and Takashi Gojohbori
54
Antisense regulation at the Xist locus
Takashi Sado, Tatsuya Ohhata, Yuko Hoki and Hiroyuki Sasaki ·······················54
X-inactivation in mouse embryos deficient for histone methyltransferase G9a
Tatsuya Ohhata, Makoto Tachibana, Hiroyuki Sasaki, Yoichi Shinkai and
Takashi Sado······55
Role of Dnmt3L in spermatogenesis and genomic imprinting during oogenesis
Kenichiro Hata, Maki Kusumi, En Li and Hiroyuki Sasaki

55

E-b. Division of Agricultural Genetics
Tetsuji Kakutani Group
Developmental abnormalities induced by DNA methylation mutation of Arabidopsis
Tetsuji Kakutani, Tetsu Kinoshita, Asuka Miura, Yuki Kinoshita, Masaomi Kato and
Hodetoshi Saze
57
Epigenetic behavior of CACTA transposon
Asuka Miura, Masaomi Kato, Kazuya Takashima, Yuki Kinoshita and Tetsuji Kakutani...................................................... ···················································57
Inheritance of epigenetic developmental abnormality
Yuki Kinoshita, Asuka Miura, Tetsu Kinoshita and Tetsuji Kakutani
58
Epigenetic control of FWA gene expression in endosperm
Tetsu Kinoshita, Asuka Miura, Yuki Kinoshita and Tetsuji Kakutani
58E-b. Division of Agricultural Genetics
Keiichi Shibahara Group
Mechanism of nucleosome assembly during DNA replication
Yasunari Takami, Tatsuya Ono, Tatsuo Nakayama and Kei-ichi Shibahara ......... 59
Physiological implications of CAF-l and CAF-l- dependent nucleosome assembly in higher
eukaryotes
Tatsuya Ono, Hidetaka Kaya, Shin Takeda, Jerzty Paszkowski, Takashi Araki and
Kei-ichi Shibahara
59
Role of histon macroH2A in gene repression
Yuya Ogawa, Tatsuya Ono, and Kei-ichi Shibahara
Differential methylation of Xite and CTCF sites in Tsix in mouse X-inactivation
Rebecca Maxfield Boumil, Yuya Ogawa, Bryan K. Sun and Jeannie T. Lee

59
60

CONTENTSE-c. Division of Brain Function
Tatsumi Hirata Group
Theoretical Consideration of Olfactory Axon Targeting with an Activity-Dependent Neural
Network Model
Hirokazu Tozaki and Tatsumi Hirata .... ··············· .. ········ .. ············· .... ·· .. ······ 60
Systematic Screening and Identification of the Antigens Recognized by Monoclonal Antibodies
Raised against the Developing Lateral Olfactory Tract
Takahiko Kawasaki, Yoshiko Takagi, Hitoshi Yamatani and Tatsumi Hirata········ 61
Chronotopic Organization of Olfactory Bulb Axons in the Lateral Olfactory Tract
Hitoshi Yamatani, Yasufumi Sato and Tatsumi Hirata···· .. 61
E-d. Division of Applied Genetics
Takashi Araki Group
Physiological roles of chromatin assembly factor-l in epigenetic control of meristem activity
during post-embryonic development in plants
Takashi Araki, Mitsutomo Abe, Hidetaka Kaya and Kei-ichi Shibahara
61
Genes involved in the integration of various genetic pathways for regulation of the floraltransition
Takashi Araki, Mitsutomo Abe, Sumiko Yamamoto, Yasufumi Daimon, Ayako
Yamaguchi, Yoko Ikeda, Michitaka Notaguchi and Masaki Kobayashi ··62
E-d. Division of Applied Genetics
Kunio Shiota GroupThe Sa1l3 locus is an epigenetic hotspot of aberrant DNA methylation associated withplacentomegaly of cloned mice]un Ohagane, Teruhiko Wakayama" Sho Senda, Yukiko Yamazaki, Kimiko Inoue, Atsuo
Ogura, Joel Marh, Satoshi Tanaka, Ryuzo Yanagimashi and Kunio Shiota · .. ·...... ·63
Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblaststem cells
Naoko Hattori, Koichiro Nishino, Yeoung-Gyu Ko, Naka Hattori, ]un Ohgane, Satoshi
Tanaka and Kunio Shiota
63
DNA methylation-mediated control of Sry gene expression in mouse gonadal development
64
Koichiro Nishino, Naoko Hattori, Satoshi Tanaka and Kunio Shiota
Skewed X-inactivation in cloned mice
Sho Senda, Teruhiko Wakayama, Yukiko Yamazaki,]un Ohgane, NakaHattori, Satoshi
Tanaka, Ryuzo Yanqgimachi and Kunio Shiota64
Non-coding RNA directed DNA demethylation of Sphkl CpG island
Takuya Imamura, Soshi Yamamoto, ]un Ohgane, Naka Hattori, Satoshi Tanaka andKunio Shiota64
Preference of DNA methyltransferases for CpG islands in normal cells
Naka Hattori, Tetsuya Abe, Masako Suzuki, Tomooki Matsuyama, Shigeo Yoshida, En
Li and Kunio Shiota
65Stage-by-stage change in DNA methylation status of DNA methyltransferase 1 (Dnmtl) locus
during mouse early development
Yeoung-Gyu Ko, Koichiro Nishino, Naoko Hattori, Yoshikazu Arai, Satoshi Tanaka andKunio Shiota
65
F. GENETIC STRAINS RESEARCH CENTER

F-a. Mammalian Genetics Laboratory
Toshihiko Shiroishi GroupElimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh

CONTENTS

expression and truncation of the mouse limb
Tomoko Sagai, Masaki Hosoya, Youichi Mizushina, Masaru Tamura and Toshihiko
Shiroishi
67
Genetic incompatibility between X-linked loci and two autosomal regions causes hybrid
breakdown between two mouse subspecies
Ayako Oka, Nobuo Takagi, Kiyotaka Toshimori, Toshiyuki Takano, Akihiko Mita, Yoichi
Mizushina, Noriko Yamatani, Hiromi Yamamoto, Kazuo Moriwaki and Toshihiko
Shiroishi
67
Comparison of BAC-end genome sequences of Japanese wild mice-derived MSMIMs strain
with the whole genome sequence of standard laboratory strain C57BLl6J detected vast
amount of SNPs
Kuniya Abe, Hideki Noguchi, Keiko Tagawa, Misako Yuzuriha, Atsushi Toyoda, Toshio
Kojima, Kiyoshi Ezawa, Naruya Saitou, Masahira Hattori, Yoshiyuki Sakaki, Kazuo
Moriwaki and Toshihiko Shiroishi
68
Characterization of M00745 mutants resembling Rim3 mutants
Shigekazu Tanaka, Hiroshi Masuya, Shigeru Wakana, Masaru Tamura and Toshihiko
Shiroishi
68
F-b. Mammalian Development Laboratory
Yumiko Saga Group
Molecular mechanism of somite segmentation
Mitsuru Morimoto, Yu Takahashi and Yumiko Saga
Regulation of Mespi and Mesp2 expression·· .. ·
Masayuki Oginuma, Yukuto Yasuhiko and Yumiko Saga
Search for target genes of Mesp2 transcription factor
Kaoru Mitsui, Yoshiro Nakajima and Yumiko Saga
Cardiovascular development and Notch signaling
Hiroki Kokubo, Yusuke Watanabe, Yoshiaki Okamura, Wataru Saito andYumiko Saga

69
·70
70

71

Functional analysis of mouse nanos genes
Masayuki Tsuda, Atushi Suzuki, Hitomi Suzuki, Makoto Kiso and··································72
Yumiko Saga
F-c. Mouse Genomics Resource Laboratory
Tsuyoshi Koide Group
Identification of genetic factors responsible for difference in spontaneous home-cage activity
between KJR and C57BLl6
73
Juzoh Umemori and Tsuyoshi Koide
A systematic analysis of genetic factors associated with the diversity of mouse behavior
Akinori Nishi, Toshihiko Shiroishi and Tsuyoshi Koide
73
Descriptive and Temporal Analysis of the Open-field Behavior in Wild-derived Mouse Strain
74
Aki Takahashi, Toshihiko Shiroishi and Tsuyoshi Koide
F-d. Model Fish Genomics Resource
Noriyoshi Sakai Group
Gene targeting system with RNA interference (RNAi) in zebrafish
74
Minori Shinya, Kimiko Saka and Noriyoshi Sakai
Analysis of functionally distinctive testicular cell lines of zebrafish to support male germ
cell development
Kayoko Kurita, Kataaki Okubo, Masaru Matsuda, Yoshitaka Nagahama and Noriyoshi···········································75
SakaiCulture condition for zebrafish spermatogonial stem cells

CONTENTSKenji Saito and Noriyoshi Sakai·································75
Analysis of the ability of cultured embryonic cells derived from different developmental stages
to induce the anterior-posterior axis
Megumi Hashiguchi and Noriyoshi Sakai····················································· 76
F-e. Plant Genetics Laboratory
Nori Kurata Group
Molecular cytogenetics of plant meiosis analyzed with rice mutants
Ken-ichi Nonomura, Mutsuko Nakano, Mitsugu Eiguchi, Akio Miyao, Hirohiko Hirochika
and Nori Kurata
77
Structural and functional analysis of rice OsHAP3 genes
Yukihiro Ito, Thiruvengadam Thirumurugan, Kazumaru Miyoshi and Nori Kurata...................................................... ···················································77
Regulation of expression of KNOX family class 1 homeobox genes of rice
Yukihiro Ito and Nori Kurata
Generation and screening of enhancer trap lines of rice
Yukihiro Ito and Nori Kurata··········78······78

Positional cloning of a segregation distortion gene detected in a progeny of a cross between
Japonica and Indica rice
78
Yoshiaki Harushima and Nori Kurata
Identification of nuclear proteins showing various sub-nuclear distribution in riceTadzunu Suzuki, Kazuki Moriguchi and Nori Kurata······79
Comparative genomics among cultivated and wild rice species
Yukie Sano, Hiroyuki Kanamori, Nobukazu Namiki, Yukiko Yamazaki andNori Kurata
79
Rice genetic resource project in NBRP and Oryzabase
Nori Kurata, Ken-ichi Nonomura, Yukihiro Ito and Yukiko Yamazaki ·················79
F-f. Microbial Genetics Laboratory
Akiko Nishimura Group
Dynamics of bacterial tubulin: coupling model between DNA replication and cell division
Ippei Inoue, Kenji Yasuda and Akiko Nishimura············································ 80
A complete set of Escherichia coli open reading frames in mobile plasmids and their successful
application to the systematic identification of cell division mutant (fts) genes
Kimiko Saka, Maki Tadenuma, Shinsuke Nakade, Noriko Tanaka, Hideaki Sugawara,
Ken Nishikawa, Nobuyuki Ichiyoshi, Masanari Kitagawa, Hirotada Mori, Naotake
Ogasawara and Akiko Nishimura······························································· 81
Global regulation of cell division: Isolation of a whole set of cell division mutants
Kimiko Saka, Noriko Matsumoto and Akiko Nishimura··································· 81
F-g. Invertebrate Genetics Laboratory
Ryu Veda GroupRNAi mutant fly bank for comprehensive analyses of gene function in Drosophila
Ryu Veda, Misako Taniguchi, Yukiko Sado, Kaoru Saigo and Kuniaki Takahashi...................................................... ···················································82
F-h. Laboratory for Frontier Research
Takako Isshiki Group
Analysis of temporal specification within late phases of Drosophila neuroblast lineage
Takako Isshiki and Ayupli Kusano

84

Investigation of molecular functions of Kriippel type zinc finger proteins in Developmentof the CNS
Ayumi Kusano
85

CONTENTSIn situ Detection of microRNA
Katsutomo Okamura·············································································· 85
G. CENTER FOR GENETIC RESOURCE INFORMATION

G-a. Genetic Informatics Laboratory
Yukiko Yamazaki GroupSHIGEN project
E coli Databases-E. coli strain DB and PEC (Profiling of Escherichia coli Chromosome)database.
86
Takehiro Yamakawa, Junichi Kato, Akiko Nishimura and Yukiko Yamazaki
CARD R-DB and IMSR (International Mouse Strain Resources)
Takehiro Yamakawa, Hideki Kato, Naomi Nakagata, Kenichi Yamamura and Yukiko
Yamazaki······································86
Mouse polymorphism database
Takehiro Yamakawa, Eri Kibukawa, Toshihiko Shiroishi and Yukiko Yamazaki ····87
Oryzabase and Plant Ontology
Takehiro Yamakawa, Nori Kurata and Yukiko Yamazaki ································87
KOMUGI
Takehiro Yamakawa, Takashi Endo, Yasunari Ogihara, Hitoshi Tsujimoto, Taihachi
Kawahara, Tetsuro Sasakuma and Yukiko Yamazaki ·····································87
NBRP-databases...................................................... ···················································88
NBRP-C. elegans
Miharu Ikizawa, Shohei Mitani, Hiroshi Kagoshima, and Yukiko Yamazaki
88
NBRP-Silkworm
Miharu Ikizawa, Yutaka Banno, Hiroshi Fujii, and Yukiko Yamazaki ··················88
NBRP-Legume (Lotus and Glycine) database
Takehiro Yamakawa, Shoko Isobe, Masatsugu Hashiguchi, Ryo Akashi, Satoshi Tabata,and Yukiko yamazaki·························88
NBRP-Medaka database
Takehiro Yamakawa, Yuko Wakamatsu, and Yukiko Yamazaki
88
NBRP-Drosophila database
Takehiro Yamakawa, Masatoshi Tomaru, Masatoshi Yamamoto, Ryu Ueda, and Yukiko
Yamazaki······································89
G-b. Publicity and Intellectual Property Unit
Munehiro Tomikawa Group
2004 Annual Publicity and Intellectual Property Unit Report··································· 90
Acquirement and utilization of Intellectual Property Rights···································· 90
Acceleration of public relations activities··························································· 91
Collaboration Research with academia, industry and government, andSocial Action Work················································································ 91
H. STRUCTURAL BIOLOGY CENTER

H-a. Biological Macromolecules Laboratory
Makio Tokunaga Group
Single Molecule Imaging and Quantitative Analysis of Nuclear Transport in Cells using Highly
Inclined and Laminated Optical sheet microscopy
Makio Tokunaga and Naoko Imamoto
92
RNG105: A Novel Regulatory Protein in Neuronal RNA Granules Responsible for

CONTENTS

Stimulation·Dependent Local Translation
Nobuyuki Shiina, Kazumi Shinkura and Makio Tokunaga

92

Single Hydrogen Bonds of DNA Base Pairs Detected in Unzipping Force by Intermolecular
Force Microscopy
92
Michio Hiroshima and Makio Tokunaga
Mechanism for passive force generation of invertebrate connectin revealed by single molecule
measurement
Michio Hiroshima, Atushi Fukuzawa, Koscak Maruyama, Sumiko Kimura and Makio
Tokunaga·····································93
Asymmetric nucleocytoplasmic transport revealed by a novel assay system using plannerreconstituted nuclear envelope
Atsuhito Okonogi, Michio Hiroshima, Nobuyuki Shiina, Shingo Kose, Naoko Imamoto
and Makio Tokunaga
93
Molecular Imaging of translation initiation factors in neuronal dendrites
Hiraku Miyagi, Nobuyuki Shiina and Makio Tokunaga·
H-b. Molecular Biomechanism Laboratory
Nobuo Shimamoto GroupThe branched mechanism of transcription initiation in E. coli
Motoki Susa, Shouji Yagi and Nobuo Shimamoto·· .. · 94

94

Applicability of thermodynamics to equilibria in biology
Nobuo Shimamoto and Jyun-ichi Tomizawa ·················································95
Systematic search for promoters encoded in the genomic DNA sequences of E. coli
Nobuo Shimamoto, Hideki Nakayama and Hironori Aromaki
96
H-c. Multicellular Organization Laboratory
Isao Katsura Group
Analysis of synthetic dauer-constitutive mutants in the nematode Caenorhabditis elegans
Tomoko Yabe, Kotaro Kimura, Takeshi Ishihara and Isao Katsura
97
C. elegans mutants in the "associative" learning with odorants and foodIchiro Torayama, Hiroshi Ichijo, Kotaro Kimura, Takeshi Ishihara and Isao Katsura...................................................... ···················································98
Molecular genetic studies on sensory integration and behavioral plasticity in C. elegans
Takeshi Ishihara, Yuichi lino, Akiko Mohri, Ikue Mori, Keiko Gengyo·Ando, Shohei
99
Mitani and Isao Katsura
Genetic analysis of plasticity of avoidance behaviors in C. elegans
Kotaro Kimura and Isao Katsura
Class 1 flr mutants of the nematode Caenorhabditis elegans
Yuri Kobayashi, Kotaro Kimura, Takeshi Ishihara and Isao Katsura
Class 2 flr mutants of the nematode Caenorhabditis elegans
Akane Oishi, Kotaro Kimura, Takeshi Ishihara and Isao Katsura
H·d. Biomolecular Structure Laboratory
Yasuo Shirakihara Group
Crystallographic Study of ATP synthaseYasuo Shirakihara and Aya Shiratori

100
100
101

102

Comprehensive Crystallographic Study of Transcription factors and Genome-partitioning
Factors from E.coli
Yasuo Shirakihara and Aya Shiratori
103
Comprehensive structure-based functional studies on transcription factors
Hiroshi Hou and Yasuo Shirakihara
Crystallographic Study of Transcription factors from Pseudomonas aeruginosa

CONTENTS103

Yasuo Shirakihara and Aya Shiratori

103

Crystallization of Kid, a chromosome mover
Yasuo Shirakihara and Aya Shiratori

104

H-e. Gene Network Laboratory
Emiko Suzuki Group
Molecular and fine morphological studies on the phototransduction in Drosophila
Emiko Suzuki

104

Fine morphological studies of the synaptic target recognition
Emiko Suzuki······················································································ 105
Fine morphological analysis of the function of a Ser/Thr kinase, UNC51 in Drosophila nervous
systemHirofumi Toda, Hiroaki Mochizuki, Emiko Suzuki and Katsuo Furukubo-Tokunaga...................................................... ··················································105
I. CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPANI-a. Laboratory for DNA Data Analysis
Takashi Gojobori Group
Comparative analysis of gene expression for convergent evolution of camera eye between
octopus and humanAtsushi Ogura, Kazuho Ikeo and Takashi Gojobori
106.
Molecular hierarchy in neurons differentiated from mouse ES cells containing a single humanchromosome 21
Chi Chiu Wang, Mitsutaka Kadota, Ryuichi Nishigaki, Yasuhiro Kazuki, Yasuaki
Shirayoshi, Michael Scott Rogers, Takashi Gojobori, Kazuho Ikeo and Mitsuo Oshimura...................................................... ··················································106
Strong positive selection and recombination drive the antigenic variation of the PilE proteinof the human pathogen Neisseria meningitidis
Daniel T. Andrews and Takashi Gojobori
106
Difference of organelles involved in membrane traffic
Hajime Ohyanagi and Takashi Gojobori

107

Highly conserved upstream sequences for transcription factor genes and implications forthe regulatory network
107
Hisakazu Iwama and Takashi Gojobori
Computational prediction of microRNA (miRNA) target gene in human and its experimental
varidation
Hitomi Sakurai, Roberto Barrero, Shiho Hayakawa, Takuro Tamura, Yoshio Tateno,
Ikeo Kazuho, Imanishi Tadashi and Takashi Gojobori
107
Detection of apoptosis during planarian regeneration by the expression of apoptosis-related
genes and TUNEL assay
lung Shan Hwang, Chiyoko Kobayashi, Kiyokazu Agata, Kazuho Ikeo and Takashi
Gojobori
108
Analysis of sexual dimorphism of gene expression in mouse brain
Kazuya Yuge, Kazuho Ikeo and Takashi Gojobori
108
Seeking for signes of aging in Hydra, a primitive metazoan
KengoYoshida, Hwang lung Shang, Chimi Fujisawa, Toshitaka Fujisawa, Kazuho Ikeo
and Takashi Gojobori
108
A large variation in the rates of synonymous substitution for RNA viruses and its relationshipto a diversity of viral infection and transmission modes,
108
Kousuke Hanada, Yoshiyuki Suzuki and Takashi Gojobori

CONTENTSEvolutionary processes of gene splicing and gene silencing
109
Lihua Jin, Yoshiyuki Suzuki, Kazuho Ikeo and Takashi Gojobori
The bioinformatics challenges in comparative analysis of cereal genomes-an overview
110
Matthew Bellgard, Jia Ye, Takashi Gojobori and Rudi Appels
Various adaptations for the perpetual darkness in the diversification process from an eyedsurface-dwelling form to an eyeless cave-dwelling form of Mexican tetra, Astyanax
mexicanus, by eDNA microarrays
Nobuhiko Tanaka, Shozo Yokoyama, Kazuho Ikeo and Takashi Gojobori
110
Evolutionary analysis of transcriptional coactivator MBF1
Qing-Xin Liu, Kazuho Ikeo, Susumu Hirose and Takashi Gojobori
110
Pigment cell lineage-specific expression activity of the ascidian tyrosinase-related gene
Reiko Toyoda, Akiko Kasai, Shigeru Sato, Shuichi Wada, Hidetoshi Saiga, Kazuho Ikeo,
Takashi Gojobori, Takaharu Numakunai and Hiroaki yamamoto····················· 111
DDBJ in the stream of various biological data
Satoru Miyazaki, Hideaki Sugawara, Kazuho Ikeo, Takashi Gojobori and Yoshio Tateno........................................................................................................ 111
Note on the maximum likelihood estimation of haplotype frequencies
Shuhei Mano, Norikazu Yasuda, Toru Katoh, Kenichi Tounai, Hidetoshi Inoko, Tadashi
Imanishi, Gen Tamiya and Takashi Gojobori
111
The evolutionary rate of a protein influenced by features of the interacting partners
Takashi Makino and Takashi Gojobori

112

Integrative annotation of 21,037 human genes validated by full-length eDNA clones
Tadashi Imanishi, (154 authors), Takashi Gojobori and Sumio Sugano
112
Japanese domesticated chickens derived from Shamo traditional fighting cocks
Tomoyoshi Komiyama, Kazuho Ikeo, Yoshio Tateno and Takashi Gojobori ......... 113
The evolutionary origin of long-crowing chicken: its evolutionary relationship with fighting
cocks disclosed by the mtDNA sequence analysis
Tomoyoshi Komiyama, Kazuho Ikeo, Yoshio Tateno and Takashi Gojobori
113
Novel algorithm for automated genotyping of microsatellites
Toshiko Matsumoto, Wataru Yukawa, Yasuyuki Nozaki, Ryo Nakashige, Minori Shinya,
Satoshi Makino, Masaru Yagura, Tomoki Ikuta, Tadashi Imanishi, Hidetoshi Inoko,
Gen Tamiya and Takashi Gojobori
113
Evolution of vitamin b6 (pyridoxine) metabolism by gain and loss of genes
Tsuyoshi Tanaka, Yoshio Tateno and Takashi Gojobori
114
Biased biological fu~ctions of horizontally transferred genes in prokaryotic genomes
Yoji Nakamura, Takeshi Itoh, Hideo Matsuda and Takashi Gojobori
114
False positive selection identified by ML-based methods: examples from the Sigl gene ofthe diatom Thalassiosira weissflogii and the tax gene of a human T-celllymphotropic
virus
Yoshiyuki Suzuki and Masatoshi Nei
114
Negative selection on neutralization epitopes of poliovirus surface proteins: implications forprediction of candidate epitopes for immunization
Yoshiyuki Suzuki················································································· 115
New Methods for Detecting Positive Selection at Single Amino Acid Sites
Yoshiyuki Suzuki················································································· 115
Three-dimensional window analysis for detecting positive selection at structural regions ofproteins. Yoshiyuki Suzuki················································································· 115
Evolutionary process of amino Acid biosynthesis in corynebacterium at the whole genome

CONTENTS

levelYousuke Nishio, Yoji Nakamura, Yoshihiro Usuda, Shinichi Sugimoto, Kazuhiko Matsui,
Yutaka Kawarabayasi, Hisashi Kikuchi, Takashi Gojobori and Kazuho Ikeo
116
I-b. Laboratory for Gene-Product Informatics
Ken Nishikawa Group
Eigenvalue analysis of amino acid substitution matrices reveals a sharp transition of themode of sequence conservation in proteins
Akira R. Kinjo and Ken Nishikawa··································· .. ······················· 118
Estimation of the number of authentic orphan genes in bacterial genomes
Satoshi Fukuchi and Ken Nishikawa
118
Alternative splice variants encoding unstable protein domains exist in the human brain
Keiichi Homma, Reiko F. Kikuno, Takahiro Nagase, Osamu Ohara and Ken Nishikawa........................................................................................................ 118
Construction and characterization of chimeric proteins composed of type-1 and type-2
periplasmic binding proteins MglB and ArgT
Kenji Kashiwagi, Kaoru Fukami-Kobayashi, Kiyotaka Shiba and Ken Nishikawa'" 119I-c. Laboratory for Gene Function Research
Yoshio Tateno Group
Submission of microarray data to public repositories
Catherine A. Ball, Alvis Brazma, Helen Causton, Steve Chervitz, Ron Edgar, Pascal
Hingamp, John C. Matese, Helen Parkinson, John Quackenbush, Martin Ringwald,
Susanna-Assunta Sansone, Gavin Sherlock, Paul Spellman, Chris Stoeckert, Yoshio
Tateno, Ronald Taylor, Joseph White and Neil Winegarden .. ·120
The origin of eukaryotes is suggested as the symbiosis of pyrococcus into proteobacteria
by phylogenetic tree based on gene content
Tokumasa Horiike, Kazuo Hamada, Daisuke Miyata and Takao Shinozawa
120
Integrative annotation of 21,037 human genes validated by full-length cDNA clones
Tadashi Imanishi, Takeshi Itoh, Yutaka Suzuki, Claire O'Donovan, Satoshi Fukuchi,
Kanako O. Koyanagi, Roberto A. Barrero, Yoshio Tateno, Zhu Chen, Michio Oishi, Peter
Tonellato, Rolf Apweiler, Kousaku Okubo, Lukas Wagner, Stefan Wiemann, Robert L.
Strausberg, Takao Isogai, Charles Auffray, Nobuo Nomura, Takashi Gojobori and Sumio120
Sugano. et al
Structural and functional differences in two cyclic bacteriocins with the same sequences
produced by lactobacilli
Yasushi Kawai, Yasuyuki Ishii, Kensuke Arakawa, Koichiro Uemura, Boku Saitoh, Junko
Nishimura, Haruki Kitazawa, Yukiko Yamazaki, Yoshio Tateno, Takatoshi Itoh andTadao Saito
121
Japanese domesticated chickens have been derived from Shamo traditional fighting cocks
Tomoyoshi Komiyama, Kazuho Ikeo, Yoshio Tateno and Takashi Gojobori ......... 121
DDBJ in the stream of various biological data
Satoru Miyazaki, Hideaki Sugawara, Kazuho Ikeo, Takashi Gojobori and Yoshio Tateno...................................................... ··············· .. ·································122
Molecular chaperones: proposal of a systematic computer-oriented nomenclature andconstruction of a centralized database
Haitham Sghaier, Thuy Le Huyen Ai, Tokumasa Horiike and Takao Shinozawa ... 122
Extensive analysis of ORF sequences from two different cichlid species in Lake Victoria
provides molecular evidence for a recent radiation event of the Victoria species flock
Identity of EST sequences between Haplochromis chilotes and Haplochromis sp.
"Redtailsheller"

CONTENTSMasakatsu Watanabe, Naoki Kobayashi, Tadasu Shin-I, Tokumasa Horiike, Yoshio
122
Tateno, Yuji Kohara and Norihiro Okada'International public gene expression database (CIBEX) and data submission
Yoshio Tateno and Kazuho Ikeo

123

Analysis of biological networks in eukaryotes using the whole genome sequences
Tsuyoshi Tanaka and Takashi Gojobori

123

Microarray gene expression database
Kazuho Ikeo and Yoshio Tateno

123

Discovery and annotation of forty seven non-protein coding human RNAs
Roberto A. Barrero, Inna Dubchak, Charles Auffray, Laurens Wilming, Jun-ichi Takeda,
Yutaka Suzuki, Erimi Harada, Marie-Anne Debily, Esther Graudens, John Quackenbush,
Takuro Tamura, Dmityiy V. Ryaboy, Sandrine Imbeaud, Kazuho Ikeo, Peter Tonellato,
Nobuo Nomura, Sumio Sugano, Tadashi Imanishi, Takashi Gojobori and Libin Jia...................................................... ··················································123
Evolutionary rate of enzymes in the metabolic network
Tsuyoshi Tanaka, Kazuho Ikeo and Takashi Gojobori

..

124

Development of a method for constructing a phylogenetic tree using a comprehensive
orthologous gene cluster, and phylogenetic analysis of cyanobacteria
Tokumasa Horiike and Yoshio Tateno
124
We are in collaboration with Prof. Tadao Saito of Tohoku University and his laboratoryon the function and evolution of glucosidase and galactosidase genes in Lactobcillus
Tadao Saito, Yukiko Yamazaki and Yoshio Tateno
124
We are in collaboration with Prof. Shintou Eguchi of the Institute of Mathematical Statistics
and his laboratory on the statistical analyses of SNP and gene expression data
Shinto Eguchi, Kazuho Ikeo and Yoshio Tateno
124
I-d. Laboratory for Research and Development of Biological Databases
Hideaki Sugawara Group
Information systems for molecular biology and its related disciplines
From Web services to a Bioportal
Yasumasa Shigemoto, Haruka Sakai, Takashi Abe, Satoru Miyazaki and Hideaki
Sugawara
125
Expansion of Genome Information Broker (GIB)
Masaki Hirahata, Naoto Tanaka, Takashi Abe, Satoru Miyazaki and Hideaki Sugawa...................................................... ··················································126
Information systems on microbes
WFCC-MIRCEN World Data Centre for Microorganisms (WDCM)
Yasumasa Shigemoto, Junko Nagaya and Hideaki Sugawara
126
Development of an e-Workbench for Biological Classification and Identification (InforBIO)
Naoto Tanaka, Kouji Koorikawa, Takashi Abe, Satoru Miyazaki and Hideaki Sugawara........................................................................................................ 126
An information system for pathogenic microorganisms
Masaki Hirahata, Naoto Tanaka, Yasumasa Shigemoto and Hideaki Sugawa ....... 126
Applications of IT to the International Nucleotide Sequence Database
Development of Open Annotation System
Satoru Miyazaki, Takashi Abe and Hideaki Sugawara
127
Exhaustive evaluation of microbial genome information by use of GRID
Takehide Kosuge, Toshihisa Okido, Yasumasa Shigemoto, Masaki Hirahata, Naoto
Tanaka, Yuzuru Maruyama, Takashi Abe, Satoru Miyazaki and Hideaki Sugawara...................................................... ··················································127

CONTENTSGenomics
Development of the H-Invitational Database
Yasumasa Shigemoto, Satoru Miyazaki and Hideaki Sugawara
127
Splicing Profile Based Protein Categorization between Human and Mouse Genomes
A ke Vastermark, Yasumasa Shigemoto, Takashi Abe and Hideaki Sugawara ..... 127
Phylogenetics Analyses of Environmental Samples on the Basis of Self-Organizing Map (SOM)
Takashi Abe, Toshimichi Ikemura and Hideaki Sugawara
128
I-e. Laboratory for Gene-Expression Analysis
Kousaku Okubo Group
Expression profiling of human genes
Data integration (BodyMapII): "Do you know how much of our genes have reliable expression
patterns on the net?"
Osamu Ogasawara and Kousaku Okubo····················································· 129
Data generation and analysis; "Are you satisfied by the present resolution of anatomical
expression data?"
Makiko Otsuji and Koichi Itoh130
Knowledge encoding and computation with gene functions (BOB): "Are you confident in yourmassive data interpretation?"
Kousaku Okubo, Koichi Itoh and Osamu Ogasawara
130

J. RADIOISOTOPE CENTER
Chromosome partitioning mechanism of Escherichia coli; Involvement of a cisacting site on
the chromosome
Yoshiharu Yamaichi and Hironori Niki
132
Partitioning mechanism of F plasmid; elucidation of motive force for bipolar migration of
the plasmid DNA
Toshiyuki Hatano and Hironori Niki
132
Subcellular localization of Escherichia coli proteins in living cells
Yasuyuki Ogata, Toshiyuki Hatano and Hironori Niki

132

Stationary phase-induced illegitimate recombination in Escherichia coli
Yasuyuki Ogata and Hideo Ikeda······························································ 133
K. EXPERIMENTAL FARM

Examination of genetic and phenotypic nature of newly prepared core collections for wild
rice comparative genomics
Toshie Miyabayashi, Mitsugu Eiguchi, Ken-Ichi Nonomura and Nori Kurata
134
L. TECHNICAL SECTION

BIOLOGICAL SYMPOSIUM 2004
FOREIGN VISITORS IN 2004

136
138

CONTENTS

Introduction
The National Institute of Genetics (NIG) was established in 1949 as the central instituteto study various aspects of genetics. It was reorganized in 1984 as an interuniversity research
institute to promote collaborations with researchers at universities. Since 1988, NIG has been
participating in graduate education as the Department of Genetics of the Graduate University
for Advanced Studies (SOKENDAl). NIG also serves as a center for various genetic resources
such as mutant strains, clones and vectors, and houses DDB], the DNA Data Bank of Japan,
and a DNA sequencing center.
The history of NIG overlaps the period of a revolution in the field of life science. Genetics
is no longer a discipline to study the rules and mechanisms of heredity, but has become thebasis for all fields of life science. Molecular techniques now allow us not only to decipher theentire genome sequence of organisms including humans, but also to understand the detailsof higher biological phenomena: cell differentiation, morphogenesis, brain function, andevolution - the history of life itself. Currently, 36 research groups are actively performing
pioneering and cutting-edge researches in these fields at NIG.
Recent generation of massive information on biological systems and their environment
calls for new directions in life sciences, such as bioinformatics, system-level analysis, andtheoretical approaches to extract knowledge from databases. To this end NIG and three othernational institutes, the National Institute of Informatics, The Institute of Statistical Mathematics
and the National Institute of Polar Research have formed a new organization, the Research
Organization of Information and Systems (ROIS) in April 2004, as a part of the reform of national
universities and research institutes in Japan. Interinstitutional collaborations within the neworganization are in progress.
We welcome your comments and suggestions on our research activities and endeavors.
Yuji Kohara, Director-General

Introduction

1

STAFF (as of December 31,2004)
Director-General

KOHARA, Yuji, D. Sc.
Vice- Director

HIROSE, Susumu, D. Sc.
Member

1. Department of Molecular Genetics
YAMAO, Fumiaki, D. Sc., Head of the Department
Division of Molecular Genetics
FUKAGAWA, Tatsuo, D. Sc., Associate Professor
OKADA, Masahiro, D. Sc.
Division of Mutagenesis
YAMAO, Fumiaki, D. Sc., Professor
TSUTSUI, Yasuhiro, D.Med.
Molecular Mechanism Laboratory
MITSUZAWA, Hiroshi, D. Sc.
SEINO, Hiroaki, D. Sc.
Division of Nucleic Acid Chemistry
AIMOTO, Saburo, D. Sc., Adjunct Professor
KATAYAMA, Tsutomu, D. Sc., Adjunct Professor
2. Department of Cell Genetics
ARAKI, Hiroyuki, D. Sc., Head of the Department
Division of Cytogenetics
YOSHIMORI, Tamotsu, D. Med., Professor
UMEBAYASHI, Kyohei, D. Ag.
Division of Microbial Genetics
ARAKI, Hiroyuki, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate Professor
KAMIMURA, Yoichiro, Dc. Med.
TANAKA, Seiji, D. Sc.
Division of Cytoplasmic Genetics
KOBAYASHI, Kazuto, Ph. D., D. Ag., Adjunct Professor
YAMAMOTO, Masayuki, D. Sc., Adjunct Professor
3. Department of Developmental Genetics
HIROMI, Yasushi, D. Sc., Head of the Department
Division of Developmental Genetics
HIROMI, Yasushi, D. Sc., Professor
FUJISAWA, Toshitaka, Ph. D., Associate Professor
SHIMIZU, Hiroshi, D. Eng.
OKABE, Masataka, M. D., Ph. D.
ASAOKA, Miho, D. Sc.
Division of Gene Expression
HIROSE, Susumu, D. Sc., Professor

2

STAFF

NISHIOKA, Kenichi, Do Medo
FUSE, Naoyuki, Do Sc.
Division of Molecular and Developmental Biology
KAWAKAMI, Koichi, Do Sc., Associate Professor
KISHIMOTO, Yasuyuki, Do SCo
Division of Physiological Genetics
ISHINO, Fumitoshi, D. Sc., Adjunct Professor
GOTOH, Yukiko, Do SCo, Adjunct Associate Professor
4. Department of Population Genetics

SAITOU, Naruya, Pho Do, Head of the Department
Division of Population Genetics
SAITOU, Naruya, Ph. Do, Professor
T AKANO, Toshiyuki, Do SCo, Associate Professor
SUMIYAMA, Kenta, Do Sc.
TAKAHASHI, Aya, Do Ago
Division of Evolutionary Genetics
IKEMURA, Toshimichi, D. SCo, Professor
Division of Theoretical Genetics
FUJIYAMA, Asao, Do SCo, Adjunct Professor
SUGANO, Sumio, Do Med., Adjunct Professor
5. Department of Integrated Genetics

SASAKI, Hiroyuki, D. Med., Head of the Department
Division of Human Genetics
SASAKI, Hiroyuki, Do Med., Professor
SADO, Takashi, Do Sc.
HATA, Kenichiro, D. Medo
Division of Agricultural Genetics
KAKUTANI, Tetsuji, D. Sc., Associate Professor
SHIBAHARA, Kei-ichi, M. Do, Pho Do, Associate Professor
KINOSHITA, Tetsu, Do SCo
OGAWA, Yuya, Do Sc.
Division of Brain Fanction
HIRATA, Tatsumi, D. Medo, Associate Professor
KAWASAKI, Takahiko, Do SCo
Division of Applied Genetics
SlOTA, Kunio, D. Ago, Adjunct Professor
ARAKI, Takashi, Do Sc., Adjunct Associate Professor
6. Genetics Strains Research Center

SHIROISHI, Toshihiko, D. Sc., Head of the Center
Mammalian Genetics Laboratory
SHIROISHI, Toshihiko, D. SCo, Professor
TAMURA, Masaru, D. Sc.
Mammalian Development Laboratory
SAGA, Yumiko, Do SCo, Professor
KOKUBO, Hiroki, Do SCo
MITSUI, Kaoru, Do Med.

STAFF

3

Mouse Genomics Resource Laboratory
KOIDE, Tsuyoshi, D. Med., Associate Professor
Model Fish Genomics Resource Laboratory
SAKAI, Noriyoshi, Ph. D., Associate Professor
SHINYA, Minori, D. Sc.
Plant Genetics Laboratory
KURATA, Nori, D. Ag., Professor
ITO, Yukihiro, D. Ag.
Microbial Genetics Laboratory
NISHIMURA, Akiko, D. Ag., Professor
Invertebrate Genetics Laboratory
UEDA, Ryu, D. Sc., Professor
TAKAHASHI, Kuniaki, D. Sc.
Labolatory for Frontier Research
ISSHIKI, Takako, D. Sc., Associate Professor
KUSANO, Ayumi, D. Sc.
7. Center for Genetic Resource Information

NISHIMURA, Akiko, D. Ag., Head of the Center
Genetic Informatics Laboratory
YAMAZAKI, Yukiko, D. Sc., Associate Professor
Genomu Biology Laboratory
KOHARA, Yuji, D. Sc., Professor
ANDACHI, Yoshiki, D. Sc.
Comparative Genomics Laboratory
Publicity and Intellectual Property UnitTOMIKAWA, Munehiro, D. Pharm, Professor
8. Structral Biology Center

KATURA, Isao, D. Sc., Head of the Center
Biological Macromolecules Laboratory
TOKUNAGA, Makio, D. Sc., Professor
SHIINA, Nobuyuki, D. Sc.
Molecular Biomechanism Laboratory
SHIMAMOTO, Nobuo, D. Sc., Professor
NAKAYAMA, Hideki, D. Eng.
SUSA, Motoki, D. Sc.
Multicelluar Organization Laboratory
KATURA, Isao, D. Sc., Professor
KIMURA, Koutarou, D. Ag.
Biomolecular Structure Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate Professor
ITO, Hiroshi, D. Sc.
Gene Network Laboratory
SUZUKI, Emiko, D. Med., Associate Professor
9. Center for Information Biology and DNA Data Bank of JapanGOJOBORI, Takashi, D. Sc., Head of the Center
Laboratory for DNA Data Analysis

4

STAFF

GOJOBORI, Takashi, D. Sc., Professor
IKEO, Kazuho, D. Sc., Associate Professor
SUZUKI, Yoshiyuki, M. D., Ph. D.
Laboratory for Gene-Product Informatics
NISHIKAWA, Ken, D. Sc., Professor
FUKUCHI, Satoshi, D. Sc.
KINJO, Akira, D. Sc.
Laboratory for Gene Function Research
T ATENO, Yoshio, Ph. D., D. Sc., Professor
BARRERO, Robert A., D. Sc.
Laboratory for the Research and Development of Biological Databases
SUGAWARA, Hideaki, D. Eng., Professor
ABE, Takashi, D. Sc.
Gene-Expression Analysis
OKUBO, Kousaku, M. D., Ph. D., Professor
ITO, Koichi, M. D., Ph. D.
10. Radioisotope Center
NIKI, Hironori, D. Med., Associate Professor Head of the Center
OGATA, Yasuyuki, D. Pharm. Sci.
11. Experimental Farm
KURATA, Nori, D. Ag., Head of the FarmNONOMURA, Ken-ichi, D. Ag.
12. Technical Section
ISHII, Yuriko, Chief of the Section
13. Department of Administration
IKINAGA, Tadatoshi, Head of the Department
ISHIDA, Yuzo, Chief of the General Affairs Section
KAWAGUCHI, Kenji, Chief of the Finance Section

STAFF

5

ADVISORY COMMITTEE (as of December 31, 2004)
Chairman
HIROSE, Susumu; Professor, Department of Developmental Genetics, National Institute of Genetics
Vice-chairman
SEKIGUCHI, Mutsuo; Adjunct Professor, Fukuoka Dental College
Outside Members (Alphabetical order)

NAKAMURA, Haruki; Professor, Institute for Protein Research, Osaka University
NISHIDA, Eisuke; Professor, Graduate school of Biostudies, Kyoto University
OGAWA, Tomoko; Vice-Director, Iwate College of Nursing
OKADA, Norihiro; Professor, Department of Bioscience and Biotechnology, Tokyo Institute of Technology
OSUMI, Noriko; Professor, Graduate School of Medicine, Tohoku University
SHINOZAKI, Kazuo; Chief-Scientist, RIKEN
T ACHIDA, Hidenori; Professor, Faculty of Sciences, Kyusyu University
TAKAGI, Toshihisa; Professor, Graduate School of Frontier Sciences, The University of Tokyo
TSUJI, Shoji; Professor, The University of Tokyo Hospital

Inside Members (Alphabetical order)

ARAKI, Hiroyuki; Professor, Department of Cell Genetics
GOJOBORI, Takashi; Professor, Center for Information Biology and DNA Data Bank of Japan
HIROMI, Yasushi; Professor, Department of Developmental Genetics
KATSURA, Isao; Professor, Structural Biology Center
NISHIMURA, Akiko; Professor, Genetic Strains Research Center
SAITOU, Naruya; Professor, Department of Population Genetics
SASAKI, Hiroyuki; Professor, Department of Integrated Genetics
SHIMAMOTO, Nobuo; Professor, Structural Biology Center
SHIROISHI, Toshihiko; Professor, Genetic Strains Research Center
YAMAO, Fumiaki; Professor, Department of Molecular Genetics

6

ADVISORY COMMITTEEADVISORY BOARD (as of December 31, 2004)
Members (Alphabetical order)

GEHRING, Walter J.; Professor, Biozentrum, University of BaselGO, Michiko; Dean & Professor, Nagahama Institute of Bio-Science and Technology
HONJO, Tasuku; Dean & Professor, Graduate School of Medicine, Kyoto University
HUNT, Tim; Principal Scientist, Cancer Research UK London Research Institute
IWATSUKI, Kunio; Professor, University of the Air
OKAZAKI, Tsuneko; Director, JSPS Stockholm Office
SAKAKI, Yoshiyuki; Director, Genomic Sciences Center, RIKEN
SUGIMURA, Takashi; President Emeritus, National Cancer Center
SULSTON, John; Former Director-General, Wellcome Trust Sanger Institute
T AKEICHI, Masatoshi; Director, Center for Developmental Biology, RIKENADVISORY BOARD

7

A. DEPARTMENT OF
MOLECULAR GENETICS

A-a. Division of Molecular Genetics
Tatsuo Fukagawa GroupRESEARCH ACTIVITIES(1) Identification of new centromere proteins in higher
vertebrate cells

Masahiro Okada, Tetsuya Hori, Mi-Sun Kwon,
Mayumi Takahashi and Tatsuo Fukagawa
The centromere plays a fundamental role inaccurate chromosome segregation during mitosis andmeiosis in eukaryotes. Its functions include sister
chromatid adhesion and separation, microtubule
attachment, chromosome movement and mitotic
checkpoint control. Although chromosome segregation
errors cause genetic diseases including some cancers,
the mechanism by which centromeres interact withmicrotubules of the spindle apparatus during cell
division is not fully understood.
To understand the function of the centromere, wewere led to develop a genetic analysis method thatutilizes the hyper-recombinogenic chicken B
lymphocyte cell line DT40. The high level ofhomologous recombination in DT40 cells permit
efficient targeted disruption of genes of interest. We
have improved this system and have created several
cell lines with conditional knockouts of several
centromere proteins to investigate the molecularmechanism of centromere assembly and function. We
have proposed a model for kinetochore assembly invertebrate cells (Fukagawa et al., EMBO ]., 2001;
Nishihashi et al., Dev. Cell, 2002). In this model we
explained that many unidentified proteins could be
involved in kinetochore assembly. Therefore, we tried
to identify new kinetogore proteins using theproteomics approach. We prepared cell lines in which
expression of CENP-H was replaced by expression of

8

DEPARTMENT OF MOLECULAR GENETICS

CENP-H-Flag or CENP-H-GFP. We also prepared celllines in which expression of CENP-I was replaced byexpression of CENP-I-Flag or CENP-I-GFP. We
extracted chromosome fraction from these cell lines
and performed immunoprecipitation with anti-Flag or
anti-GFP antibodies. Immunoprecipitates were thenseparated by SDS-PAGE and we identified several
common bands from all cell lines. We analyzed thesebands by LC-MS/MS and identified amino acids
sequence of all bands by a peptide-mass-fingerprinting
method. We newly identified five proteins and cloned
eDNA of these proteins. We then made expression
constructs for these cDNAs fused by GFP and investigated localization of these proteins. These proteins
perfectly co-localized with CENP-H throughout the cell
cycle. We conclude that these five proteins areconstitutive centromere proteins (manuscript in
preparation). Weare creating conditional knockout
cells for these proteins to understand mechanism of
kinetochore assembly in vertebrate cells.
(2) Molecular analysis of the Nuf2-Hecl complex thattransiently localizes to centromere during mitosis

Tetsuya Hori, Yoshikazu Mikami, Kazuko Suzuki and
Tatsuo Fukagawa
Nuf2 and Heel are evolutionarily conserved
centromere proteins. To clarify the functions of these
proteins in vertebrate cells, we characterized them inchicken DT40 cells (Hori et al.,]. Cell Sci., 2003). We
generated GFP fusion constructs of Nuf2 and Heel toexamine in detail localization of these proteins during
the cell cycle. We found that Nuf2 is associated with
Heel throughout the cell cycle and that this complex
is localized to the centrosomes during Gl and S phases
and then moves through the nuclear membrane to thecentromere in G2 phase. During mitosis, this complex
is localized to the centromere. We also createdconditionalloss-of-function mutants of Nuf2 and Hecl.
In both mutants, the cell cycle arrested atprometaphase, suggesting that the Nuf2-Hecl complex
is essential for mitotic progression. The innercentromere proteins CENP-A, -C, and -H andcheckpoint protein BubRl were localized tochromosomes in the mutant cells arrested atprometaphase, but Mad2 localization was abolished.
Furthermore, photobleaching experiments revealed
that the Nuf2-Hecl complex is associated stably withthe centromere and that interaction of this complex
with the centrosome is dynamic.
We also observed that CENP-H, which is aconstitutive centromere component that localizes to thecentromere throughout the cell cycle, interacts with theNuf2 complex by yeast two-hybrid analysis.
Co-immunoprecipitation experiments revealed that
CENP-H interacts with the Nuf2 complex duringmitosis in chicken DT40 cells. Photo bleachingexperiments showed that both Heel and CENP-H form
stable associations with the centromeres duringmitosis, suggesting that Heel acts as a structuralcomponent of centromeres during mitosis. On the basisof these results and published data, we propose that
the Nuf2 complex functions as a connector between the
inner and outer kinetochores.
We started the proteomics approach to identify
other proteins that interact with the Nuf2-Heelcomplex. We identified chicken homolog of Spc24 andSpc25. We also identified several components that
localizes centrosome and centromere and are now
characterizing these components.

to cell death with accumulation of abnormal mitotic
cells that show premature sister chromatid separation.
Aberrant accumulation of transcripts from a-satellite
sequences, which consist of human centromeric repeat
DNAs, was detected in Dicer-deficient cells. Immunocytochemical analysis revealed abnormalities inlocalization of heterochromatin proteins, Rad21 cohesin
protein, and BubRI checkpoint protein, but core
kinetochore proteins such as CENP-A and -C werenormal. We conclude that Dicer-related RNAi
machinery is involved in formation of theheterochromatin structure in higher vertebrate cells.
We also examined expression profile of non-coding
region using high-density DNA-microarray, when
expression of Dicer was lost. We could identify several
non-coding RNAs from this method. We arecharacterizing biological function these RNAs. We also
started to create conditional knockout cells for othercomponents involved in RNAi machinery such as
Ago-family proteins. We would like to comprehensive
understand relationship of RNAi machinery withcentromere function.

Functional roles of the RNAi machinery inverterbrate centromeres

PUBLICATIONS(3)PapersTomoko Motohashi, Masahiro Nogami, Atsushi
Fukushima and Tatsuo Fukagawa
RNAi-mediated silencing of gene expression occurs
when double-stranded RNAs (dsRNAs) are cleaved by
Dicer into 21- to 23-nt small interfering RNAs (siRNAs).
These siRNAs guide a multicomponent nuclease,
RNA-induced silencing complex (RISC), to degrade
specific mRNAs. Although the Dicer-mediatedgene-silencing is evolutionarily conserved system, thebiological functions of the RNAi machinery are notfully understood. Genetic strategies have been used to
examine the biological functions of the RNAi
machinery in C. elegans, Arabidopsis, Drosophila andfungi. There are reports that the RNAi machinery is
related to chromosome segregation in fission yeast.
However, it is unclear whether the RNAi machinery
is associated with chromosome segregation invertebrate cells. To examine the biological function of
the RNAi-related pathway in vertebrate cells, we
generated a conditionalloss-of-function mutant ofDicer in a chicken-human hybrid DT40 cell line that
contains human chromosome 21. Loss of Dicer leads

1. Fukagawa, T., Nogami, M., Yoshikawa, M., Ikeno,
M., Okazaki, T., Takami, Y, Nakayama, T. andOshimura M. (2004). Dicer is essential for formetion of
the heterochromatin structure in vertebrate cells.
Nature Cell BioI. 6, 784-791.
2. Fukagawa, T. (2004). Centromere DNA, proteinsand kinetochore assembly in vertebrate cells.
Chromosome Res. 12,557-567.
3. Fukagawa, T. (2004). Assembly of kinetochores invertebrate cells. Exp. Cell Res. 296,21-27.
EDUCATION

1. Dr. Fukagawa gave several lectures at The
Graduate University for Advanced Studies, Hayama(in Japanese).
2. Dr. Fukagawa was invited a seminar on"Kinetochore Assembly" at Kyoto University, Kyoto,
August, 2004 (in Japanese).
3. Dr. Fukagawa gave a public lecture of National
Institute of Genetics at National Museum of Emerging
Science and Innovation (MIRAIKAN), Tokyo, October,
2004.

Division of Molecular Genetics

94. Dr. Fukagawa gave a special lecture at Tokyo
Institute of Technology, Yokohama, December, 2004
(in Japanese).
5. Dr. Fukagawa was invited a seminar on"Kinetochore Assembly" at Tokyo Institute of Technology, Yokohama, December, 2004 (in Japanese).

A-b. Division of Mutagenesis
Fumiaki Yamao GroupRESEARCH ACTIVITIES(1) Multifunctional role of Mcll/Slr3 in DNA replication,
repair and chromatin structure

Yasuhiro Tsutsui, Toyoaki Natsume and Fumiaki
Yamao
Slr3 in fission yeast is a mem ber of eukaryotic
family of WD repeat proteins and important for
genome stability. slr3 gene was originally identified as
mutants showing synthetic lethality with Rad2 defect.
Fission yeast Rad2 is a FEN-I family nucleaseprocessing Okazaki fragment during lagging strand
DNA synthesis. Since recombination activity isessential for survival of cells defective in Rad2 function,
genes that show, when mutated, synthetic lethality with
Rad2-defect was expected to be involved inrecombination or recombination repair of damaged
DNA. Actually slr3 mutants are sensitive to DNA
damage reagents such as MMS, UV, bleomycin andhydroxyurea. Null mutant cell is temperature sensitivefor growth, and shows severe growth retardation evenat the permissive temperature. The mutant cells arrest
at G2 phase with 2C DNA content at the restricted
temperature, showing that the bulk DNA synthesis
occurred. However, DNA double-strand breaks were
found to accumulate in the arrested cells, which wasa similar phenotype caused by defect of Okazaki
fragment processing in such as dna20r cdc17mutants.
It was further shown that Slr3 interacts not only
genetically but also physically with DNA polymerase
a. These results strongly suggest that Slr3 functions
in lagging strand synthesis and Okazaki fragmentprocessing in addition to DNA repair. On the other
hand, slr3 gene was found allelic to mcll that isrequired for proper chromosomal segregation.
Concomitantly slr3mutant cells were sensitive to TBZ.

10

Division of Molecular Genetics

Consequently, highly frequent chromosome loss
observed in mcll mutant cells was attributed to
defective cohesion of sister chromatids in centromere
region. Recently, we also found that Slr3 affects
heterochromatin structure, which is now under an
extensive investigation (Natsume et. al). Thus, Slr3/
Mcll protein plays multifunctional role to maintainchromosomal stability by connecting DNA replication,
recombination, damage repair, cohesion andheterochromatin structure. This work was collaborated
with H. Shinagawa's and H. Iwasaki's group in Osaka
University and Yokohama-City University,
respectively.
EDUCATION
1. Dr. Yamao was invited to give a seminar on a titleof "Ubiquitin as a Multi-Player in Cell" at KeihannaInteraction Plaza, Kyoto, March (in Japanese).
2. Dr. Yamao gave a lecture at Department of
Veterinary Medicine, Yamaguchi University, December(in Japanese).

A-c. Molecular Mechanism Laboratory
Hiroshi Mitsuzawa GroupRESEARCH ACTIVITIES

Identification of glyceraldehyde-3-phosphate
dehydrogenase and actin as proteins that interact with
the Rpb7 subunit of RNA polymerase II(1)

Hiroshi Mitsuzawa
Eukaryotic RNA polymerase II (pol II), the enzymethat synthesizes all mRNAs and some non-coding
RNAs, plays a central role in transcription. pol II
consists of evolutionarily conserved 12 subunits,
designated Rpb1 to Rpb12. The catalytic activity
resides in a lO-subunit core, whereas the initiation oftranscription requires two additional subunits, Rpb4
and Rpb7, which form a heterodimer to associate withthe core.
To gain insight into the role of the Rpb7 subunit,
we performed a two-hybrid screen by using the fission
yeast Schizosaccharomyces pombe Rpb7 protein as bait.
The screen yielded glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and actin. GAPDH and actinwere also found to be associated with S. pombe pol II
complexes. Moreover, GAPDH and actin wereaffinity-purified from S. pombeextract with an Rpb4/
Rpb7-coupled column. Although GAPDH is awell-characterized glycolytic enzyme abundant in thecytoplasm, nuclear GAPDH in human cells has been
identified as a key component of a coactivator complex
for histone H2B transcription. In addition, it has
recently been reported that actin is required forformation of the preinitiation complex. Our results are
consistent with these observations and further suggestthat GAPDH and actin play roles in pol II transcription
through the interaction with Rpb7.

RNA polymerases and accessory factors. In The
Molecular Biology of Schizosaccharomyces pombe. R.
Egel, ed. (Heidelberg, Germany: Springer-Verlag),pp.329-342.

A-c. Molecular Mechanism Laboratory
Hiroaki Seino GroupRESEARCH ACTIVITIES(1) An in vitro ubiquitination assay of mitotic cyclin

Hiroaki Seino(2) Identification of the general transcription factor
TFIID in S. pombe

Hiroshi Mitsuzawa
TFIID, one of the general transcription factors
required for transcription initiation by pol II, comprises
the T ATA-binding protein (TBP) and a set of
TBP-associated factors (TAFs). Unlike those in human,
Drosophila, and Saccharomyces cerevisiae, T AFs in S.
pombe have not been characterized until recently. We
have identified five S. pom be T AFs biochemically:
spTAFlll/130 (spT AFl), spTAF72 (spTAF5),
spTAF73 (spTAF5b), spTAF50 (spTAF6), and Ptr6
(spTAF7).I. 2) (Given in parentheses are new unified
names proposed recently.) Of particular interest are
spTAF72 and spTAF73, which are likely to regulate
genes involved in progression through M phase of thecell cycle. spTAF50 was shown to interact with the
WD40-repeat domain of spTAF72. These interacting
T AFs were found to be shared by the TFIID and
SAGA complexes. Furthermore, we carried out BLAST
searches for putative T AFs, revealing that the S. pombe
genome contains nearly all T AF genes identified inother organisms. I. 2)
PUBLICATIONS
Reviews

Mitsuzawa, H. and Ishihama, A. (2004). RNApolymerase II transcription apparatus in Schizosaccharomyces pombe. Curro Genet. 44,287-294.
1.

Books

2. Kimura, M., Mitsuzawa, H. and Ishihama, A. (2004).

Cell cycle events are regulated by sequential
activation and inactivation of Cdk kinases. Mitotic exit
is accomplished by the inactivation of mitotic Cdk
kinase, which is mainly achieved by degradation of
cyclins by a ubiquitin-proteasome system.
Previously we reported that two ubiquitin-conjugating enzymes, UbcPllUbc4 and UbcP4!Ubcll, were
responsible for degradation of mitotic cyclin Cdc13 infission yeast. Each of these two ubiquitin-conjugating
enzymes is essential for cell viability and responsiblefor degradation of Cdc13. These results suggest thatthe functions of these two ubiquitin-conjugating
enzymes are not redundant and they have distinct
functions for ubiquitination of Cdc13. Furthermore, we
found that ubiquitin chains of Cdc13 were totally
reduced in ubcP4/ubc11 mutant cells, whereas
ubiquitin chains were short and not reduced in ubcPl/
ubc4 mutant cells. Thus, we proposed a hypothesisthat UbcP4/Ubcll might be involved in initiation ofubiquitination, and UbcPllUbc4 might be involved inelongation of ubiquitin chains of Cdc13. However, thishypothesis has not been elucidated yet.
To clarify the functional differences between
UbcPl/Ubc4 and UbcP4/Ubc11 for degradation of
Cdc13, development of an in vitro assay system for
ubiquitination for Cdc13 by using fission yeast
components is required. Currently, I am attempting
to develop this assay system for Cdc13. A
ubiquitin-activating enzyme, these two ubiquitinconjugating enzymes and substrate Cdc13 wereexpressed as recombinant proteins in bacterial cells
and purified. One component of Cdc13-specificubiquitin ligase anaphase promoting complex/
cyclosome (APC/C) was tagged and expressed in

Molecular Mechanism Laboratory11

fission yeast cells, and APC/C was purified fromfission yeast cells. Now I am examining the conditions
for reconstitution of a ubiquitination reaction of Cdcl3.
(2) Ubiquitin-conjugating enzyme(s) responsible fordegradation of SCF substrates

Hiroaki Seino
One of the important ubiquitin pathways
responsible for cell cycle regulation is the pathway
involving APC ubiquitin ligase. Another important
pathway for cell cycle regulation is the pathway
involving SCF (Skpl-Cullin-F-box) ubiquitin ligase. The
SCF-pathway mainly regulates the progression of Gland S-phases of the cell cycle. Furthermore, several
putative SCF components have been reported to be
involved in many cell regulations, e.g., a DNA damage
checkpoint mechanism, maintenance of chromosomes
and other regulations in fission yeast and otherorganisms.
However, ubiquitin-conjugating enzyme(s)responsible for degradation of substrates of SCF
ubiquitin ligase in fission yeast have not been
elucidated yet. We found that polyubiquitinatedproteins were significantly reduced in whole cell extractfrom ubcPl/ubc4 mutant. It is possible that thesubstrates of SCF are also ubiquitinated by the UbcPl/
Ubc4-pathway. Thus, I am examining the stability ofthe substrates of SCF ubiquitin ligase Cdcl8in ubcPlI
ubc4 mutant cells, and the results are now being
obtained.

A-d. Division of Nucleic Acid Chemistry
Saburo Aimoto GroupRESEARCH ACTIVITIES(1) Development of a method for chemical synthesis ofprotein by using two different ligation methods

Saburo Aimoto (Institute for Protein Research, Osaka
University)
A thioester method and the native chemical
ligation method are useful for protein synthesis.
Though both methods use peptide thioesters asbuilding blocks, no route was known to use the12

Molecular Mechanism Laboratory

thioester method after the peptide bond formation
reaction by the native chemical ligation method. In thethioester method, the SH groups of the cysteine
residues have to be protected by a group that is stablein the presence of silver ions as an activator of thioester
groups. On the other hand, the native chemical ligation
method produces an SH group at the condensation site
for chemoselective ligation is carried out at -X-Cys- siteusing the cysteine residue at the N-terminal of the
C-terminal building block. In order to overcome thisproblem, we searched protecting groups for the thiolgroups that are easily introduced to the free SH groupin polypeptide under mild conditions and stable in thepresence of silver ions. We examined several thiol
protecting groups. Among them, thiosulfonate group
was the most promising protecting group. This group,
however, was revealed to partially decompose underalkaline conditions. Thus new protecting groups arestill under investigation.
(2) Preparation of peptide thioesters by the Fmoc-solid
phase method via an on-resin N-S acyl shift

Saburo Aimoto (Institute for Protein Research, Osaka
University)
Peptide thioesters are common intermediates incontemporary methods for protein synthesis. The
peptide thioesters can be directly prepared either by
Boc solid-phase peptide synthesis (SPPS) or by Fmoc
SPPS using Fmoc(2-F)-amino acid derivatives. Peptide
thioesters can be prepared by Fmoc SPPS indirectly
via a safety catch resin, too. However each preparation
method based on the Fmoc solid phase method has
intrinsic difficulties such as low yields of products andrecemization of the C-terminal amino acid residue.
Therefore an innovative method was urgently
requested. A new method under development is basedon an on-resin N-S acyl rearrangement. Previously, we
introduced the 2-mercapto-4, 5-dimethoxybenzyl(Dmmb) group as an auxiliary for extended chemical
ligation. This auxiliary group can be removed by acidtreatment following the ligation reaction. Unexpectedly,
it was observed that N-S acyl rearrangement occurred,in part, during the acid treatment. Base on this finding,we searched for conditions to use this mechanism for
preparation of peptide thioesters. For polypeptide
synthesis, the prevention of a reverse acyl rearrangement is of prime importance in this context. A

protected peptide was assembled on a resin using theDmmb-group as a linker followed by Fmoc-based
SPPS. After peptide assembly, the resin was treatedwith a reagent containing trifluoroacetic acid to resultin the removal of the protecting groups. At the sametime, the N-S acyl rearrangement occurred, resulting
in resin-bound peptide thioester. Finally, free peptide
thioester was obtained following treatment with 2-mercaptoethanesulfonic acid in the presence of a base andsubsequent resin wash. This method permits us toprepare peptide thioesters by a Fmoc solid-phasemethod without racemization.
(3) Synthesis of post-translationally modified histone
H3

Saburo Aimoto (Institute for Protein Research, Osaka
University)
Histones can undergo posttranslational
modifications such as methylation, acetylation or/and
phosphorylation. These modifications can playa rolein gene regulation in an epigenetic manner. Focusedon the N-terminal region of histone H3 we searched astrategy that would provide a synthetic procedure forpost-translationally modified histones. First, synthesisof the N-terminal region of histone H3 peptides that
contained Lys(MeJ) was examined by Boc and Fmoc
SPPS. A desired product was obtained by the bothmethods though the Boc method gave better results
than the Fmoc SPPS. Lys(Me3)-containing peptide
thioesters that were building blocks for histone H3synthesis were prepared by Boc SPPS. Furthermore,
two chemically synthesized building blocks covering[Lys(MeJ)4] -histone H3(l-l2) and [Lys(MeJ)36] -histone
H3(l3-43) were condensed by the thioester method togive [Lys(MeJ)4.36]-histone H3(l-43). Along with thistotally chemical synthetic method, we are developinga method that uses a biologically expressed histone H3segment for the total synthesis of modified histone H3.
In the near future, a variety of modified histone H3 witha full sequence will be synthesized by condensing
chemically synthesized modified-amino-acid-containing
peptide thioesters and biologically expressed histone
H3 segments.

(4) Synthetic studies of G protein-coupled receptor,
opioid receptor like-l (ORL-l)

Saburo Aimoto (Institute for Protein Research, Osaka
University)
About 30% of the human genome encodes
membrane proteins. Among them G protein-coupled
receptor family is the largest family of membrane
receptor protein and is the target of mostpharmaceuticals. Much of the information concerning
the structure and function of these membrane proteins,
however, remains to be uncovered because of the
difficulties associated with biochemical sample
preparation. As an alternative approach to obtaining
membrane proteins, chemical synthesis represents a
viable candidate. ORL-l is a receptor of an opioid
peptide, nociceptin. The strategy that is employed forthe synthesis of the C-terminal region of ORL-l is the
combination of the native chemical ligation method
and the thioester method. The first coupling between
ORL-l(288-328) and ORL-l(329-370) was carried out in
SDS solution by the native chemical ligation method.
The coupling reaction conditions were thoroughly
inspected in terms of pH of the solution, chemicalcomposition of the reaction media. According to thedefined conditions, ORL-l(288-370) that contained one
transmembrane region and the C-terminal cytosolic tailregion was obtained in the yield of 47%.4) We arecontinuously searching the route to accomplish thetotal synthesis of ORL-l.
(5) Design of the inhibitors to human T-cell leukemia
virus type-l protease

Saburo Aimoto (Institute for Protein Research, Osaka
University)
Human T-cell leukemia virus type 1 (HTLV-l), a
retrovirus associated with a number of humandiseases, was the first human retrovirus isolated frompatients with adult T-cell leukemia/ lymphoma by
Gallo et al. An HTLV-l gene codes an asparatic
protease (PR), which processes its own polyproteins,which are transcripted owing to three reading frames.
As the result of a series of cis processing, a set ofproteins is produced, which are necessary for viral
replication. Thus, HTLV-l PR plays a key role in the
duplication of HTLV-l in a manner analogous to theDivision of Nucleic Acid Chemistry13human immunodeficiency virus type-l protease inacquired immunodeficiency syndrome. In the designof potent protease inhibitors for this virus, theknowledge of the characteristics of HTLV-l PR itself
and its substrate specificities is critical. Then, we
synthesized HTLV·l PR and examined its substratespecificities. Based on the obtained data we designed
HTLV-l protease inhibitors of an olefin-containing
cyclic peptide. l )ing", between Institute for Protein Research, Osaka
University and Seoul National University, Osaka, June,
2004.

2. ;fll*=:~~, B*A:7'7'" F¥~JW~!'t

A-d. Division of Nucleic Acid Chemistry
Tsutomu Katayama GroupRESEARCH ACTIVITIESPUBLICATIONSPapers

1. Bang, J.K, Hasegawa, K, Kawakami, T., Aimoto,S. and Akaji, K (2004). Synthesis of an olefin-containing
cyclic peptide using the solid-phase Honer-Emmons
reaction. Tetrahedron Lett., 45,99-102.
2. Sekine, S., Kataoka, K, Tanaka, M., Nagata, H.,
Kawakami, T, Akaji, K, Aimoto, S. and Shizukuishi,
S. (2004). Active domains of salivary statherin on
apatitic surfaces for binding to Fusobacterium
nucleatum cells. Microbiology. 150, 2373-9.
3. Yamada, H., Sasaki, T, Niwa, S., Oishi, T, Murata,
M., Kawakami, T. and Aimoto, S. (2004). Intact
Glycation End Products Containing CarboxylmethylLysine and Glyoxal Lysine Dimer Obtained from
Synthetic Collagen Model Peptide, Bioorg. Med. Chern.
Lett., 14, 5677-80.
4. Sato, T., Saito, Y and Aimoto, S. (2004). Synthesisof the C-terminal Region of Opioid Receptor Like 1 in
an SDS Micelle by the Native Chemical Ligation: Effectof Thiol Additive and SDS Concentration on Ligation
Efficiency. J. Peptide Sci., Epub. Dec. 20.
5. Onoda, A., Yamamoto, H., Yamada, Y, Lee, K,
Adachi, S., Okamura, T. A., Yoshizawa-Kumagaye, K,
Nakajima, K, Kawakami, T., Aimoto, S. and Ueyama,N. (2005). Switching of turn conformation in an
aspartate anion peptide fragment by NH ... 0(-)
hydrogen bonds. Biopolymers, Epub., Jan 4.
EDUCATION
1. Dr. S. Aimoto was invited to give a lecture at Kyoto

University, January, 2005.
SOCIAL CONTRIBUTIONS AND OTHERS
1. Dr. S. Aimoto organized a joint symposium entitled"Protein Researches in Bioscience and Bioengineer-

14

Division of Nucleic Acid Chemistry(1) Molecular mechanism of DNA replication-coupled
inactivation of the initiator protein in Escherichia coli:
Interaction of DnaA with the sliding clamp-loaded DNA
and the sliding clamp-Hda complex

Masayuki Su'etsugu, Makoto Takata\ Toshio
Kubota2, Yusaku Matsuda and Tsutomu Katayama(Present addresses: ISumitomo Pharmaceuticals Co.,
Osaka, Japan; 2Department of Pharmacy, Kyushu
University Hospital, Fukuoka, Japan)
In Escherichia coli, the ATP-DnaA protein initiates
chromosomal replication. After the DNA polymerase
III holoenzyme is loaded onto DNA, DnaA-bound ATPis hydrolyzed in a manner depending on Hda protein
and the DNA-loaded form of the DNA polymerase III
sliding clamp subunit, which yields ADP-DnaA, aninactivated form for initiation. This regulatory
DnaA-inactivation represses extra initiation events. In
this study, in vitro replication intermediates andstructured DNA mimicking replicational intermediates'were first used to identify structural prerequisites in
the process of DnaA-ATP hydrolysis. Unlike duplex
DNA loaded with sliding clamps, primer RNA-DNA
heteroduplexes loaded with clamps were not associatedwith DnaA-ATP hydrolysis, and duplex DNA provided
in trans did not rescue this defect. At least 4O-bp duplex
DNA is competent for the DnaA-ATP hydrolysis whena single clamp was loaded. The DnaA-ATP hydrolysis
was inhibited when ATP-DnaA was tightly bound toa DnaA box-bearing oligonucleotide. These results
imply that the DnaA-ATP hydrolysis involves the
direct interaction of ATP-DnaA with duplex DNA
flanking the sliding clamp. Furthermore, Hda protein
formed a stable complex with the sliding clamp. Basedon these, we suggest a mechanical basis in the
DnaA-inactivation that ATP-DnaA interacts with the
Hda-clamp complex with the aid of DNA binding. 4)

(2) DiaA, a novel DnaA-binding protein, ensures thetimely initiation of E.coli chromosome replication
Takuma Ishida, Nobuyoshi Akimitsu\ Tamami
Kashioka2, Masakazu Hatanoa, Toshio Kubota4,
Yasuyuki Ogata5, Kazuhisa Sekimizu 1 and Tsutomu
Katayama (Present addresses: lGraduate School of
Pharmaceutical Sciences, University of Tokyo, Japan;
2Institute for 'Chinese Medicine, Nakamura City
Hospital, Kochi, Japan; 3Santen Pharmaceutical Co.,
Nara, Japan; 4Department of Pharmacy, Kyushu
University Hospital, Fukuoka, Japan; 5Radioisotope
Center, National Institute of Genetics, Mishima,
Shizuoka, Japan)
The DnaA protein is the initiator of E. coli
chromosomal replication. In this study, we identify anovel DnaA-associating protein, DiaA, which is
required for the timely initiation of replication during
the cell cycle. DiaA promotes the growth of specifictemperature-sensitive dnaA mutants and ensures
stable minichromosome maintenance, while DiaA does
not decrease the cellular DnaA content. A diaA::Tn5
mutation suppresses the cold-sensitive growth of an
overinitiation-type dnaA mutant independently of
SeqA, a negative modulator of initiation. Flowcytometry analyses reveal that the timing of replication
initiation is disrupted in the diaA mutant cells as wellas wild-type cells with pBR322 expressing the diaA
gene. Gel-filtration and chemical cross-linking
experiments show that purified DiaA forms a stablehomodimer. Immunoblotting analysis indicates thata single cell contains about 280 DiaA dimers. DiaA
stimulates minichromosome replication in an in vitro
system especially when the level of DnaA included islimited. Moreover, specific and direct binding between
DnaA and DiaA is observed, which requires a DnaA
N-terminal region. DiaA binds to both ATP- and
ADP-bound forms of DnaA with a similar affinity.
Thus, we conclude that DiaA is a novel DnaAassociating factor that is crucial to ensure the timely
initiation of chromosomal replication. 21(3) Reactivation of DnaA by DNA sequence-specificnucleotide exchange in vitro
Kazuyuki Fujimitsu and Tsutomu Katayama
In Escherichia coli, ATP-bound DnaA protein caninitiate chromosomal replication. After initiation,
DnaA-ATP is hydrolyzed by interactions with acomplex containing a replicase subunit to yield theinactive ADP-DnaA. However, the mechanisms which
regenerate ATP-DnaA from ADP-DnaA are not wellunderstood. We report here that a 70-bp DNA segment
promotes exchange of the DnaA-bound nucleotide ina sequence specific manner, thus reactivating theinitiation function of DnaA in vitro. This segment
contains a typical DnaA-binding 9-mer motif, the DnaAbox, and two DnaA box-like sequences. The presence
and precise composition of these three motifs are
required for the DnaA-reactivating activity, whichsuggests that a highly ordered complex which includes
multimeric DnaA molecules is formed for isomerizationof DnaA. We named this DNA segment DARS, for
DnaA-reactivating sequence. The role of DARS in
regulation of DnaA function in vivo is discussed. l )(4) Novel heat shock protein HspQ stimulates thedegradation of mutant DnaA protein in Escherichia coli
Toh-ru Shimuta, Kiyotaka Nakano\ Yoko Yamaguchi,
Shogo Ozaki, Kazuyuki Fujimitsu, Chika Matsunaga,
Kenji Noguchi 2, Akiko Emotd and Tsutomu
Katayama (Present addresses: IChugai PharmaceuticalCo., Tokyo, Japan; 2Kaketsuken Institute, Kumamoto,Japan; 3Department of Pharmacy, Saga University
Hospital, Saga, Japan)

Escherichia coli DnaA protein initiates
chromosomal replication and is an important
regulatory target during the replication cycle. In thisstudy, a suppressor mutation isolated by transposon
mutagenesis was found to allow growth of thetemperature-sensitive dnaA508 and dnaA167mutantsoat 40 e. The suppressor consists of a transposon
insertion in a previously annotated ORF, here termed
hsPQ, a novel heat shock gene whose promoter isrecognized by the major heat shock sigma factor 0 32•
Expression of hsPQ on a pBR322 derivative inhibitsogrowth of the dnaA508 and dnaA167mutants at 30 e,
whereas growth of dnaA46 and other dnaA mutants
is insensitive to changes in the level of hsPQ. Cellular
DnaA508 protein is degraded rapidly at elevatedtemperature, but hspQ disruption impedes this process.
In contrast, DnaA46 protein is rapidly degraded in an
hspQ-independent manner. Gel-filtration and chemical
cross-linking experiments suggest that HspQ forms a

Division of Nucleic Acid Chemistry15stable homodimer in solution and can form
homomultimers consisting of about four monomers.
Heat shock-induced proteases such as Clp containhomomultimers of subunit. We propose that HspQ is
a new factor involved in the quality control of proteins
and that it functions by excluding denatured proteins. 3)
PUBLICATIONSPapersK. and Katayama, T. (2004).
Reactivation of DnaA by DNA sequence-specificnucleotide exchange in vitro. Biochem. Biophys. Res.
Commun., 322, 411-419.
2. Ishida, T., Akimitsu, N., Kashioka, T., Hatano, M.,
Kubota, T., Ogata, Y, Sekimizu, K. and Katayama, T.
(2004). DiaA, a novel DnaA-binding protein, ensures theinitiation timing of Escherichia coli chromosome
replication. J. BioI. Chern., 279, 45546-45555.
3. Shimuta, T., Nakano, K., Yamaguchi, Y, Ozaki,
S., Fujimitsu, K., Matsunaga, C., Noguchi, K., Emoto,
A. and Katayama, T. (2004). Novel heat-shock protein
HspQ stimulates the degradation of mutant DnaA
protein in Escherichia coli. Genes Cells, 9, 1151-1166.
4. Su' etsugu, M., Takata, M., Kubota, T., Matsuda,
Y and Katayama, T. (2004). Molecular mechanism of
DNA replication-coupled inactivation of the initiator
protein in Escherichia coli: Interaction of DnaA withthe sliding clamp-loaded DNA and the sliding
clamp-Hda complex. Genes Cells, 9,509-522.
1. Fujimitsu,

Reviews

5. J1l1J $frh (2004) r::klmif~-@'1*~~O)OOMl C: ~0)1I11j:fii'I!1c:OOvQAAA+ ;7..-/'\-7 7 ~ 1) --7 ://'\7jt~jE.{t'¥76, pp.1440-1443.
Books

6. J1l1J $frh,*~:i.E~,JiIJ::JAlr(2004) r::k 1m if 7' /ADNA~~ifu, 7' / ~ 7;7.. C: 7°P7'"::t'::: 7 ;7..O)*JrmkOO"'-':'E. ~ti!fiR O)Mt!f C: J;tffl "'-' (.:r. ~ • 7'" -1 - • .:r.;7.., 4- $ J~ 1i
Iliif~) pp.57-64.

SOCIAL ACTIVITIES AND OTHERS

1. Dr. Katayama was awarded a Junior Research
Leader of Kyushu University.

16

Division of Nucleic Acid Chemistry

B. DEPARTMENT OF
CELL GENETICS

We previously reported that rat LC3, a homologueof yeast AtgS, localizes to autophagosomal membranes
after post-translational modifications. The C-terminal
fragment of LC3 is cleaved immediately following synthesis to yield a cytosolic form, LC3-I. A subpopulation of LC3-I IS further converted to an
autophagosome-associating form, LC3-II by theubiquitylation-like conjugation system. In this study,
we showed that 4CJ-ethanolamine was preferentiallyincorporated into LC3-II, suggesting that LC3-II is a
phosphatidylethanolamine (PE)-conjugated form likea membrane-binding form of yeast Atg81). LC3-II canbe a substrate of mammalian Atg4B, a homologue of
yeast Atg8-PE deconjugase, supporting the idea that
LC3-II is LC3-PE.
There are at least two other homologues of yeast
AtgS, y -aminobutyric-acid-type-A-receptor-associatedprotein (GABARAP) and Golgi-associated ATPaseenhancer of 16kDa (GATE16) in mammals. We foundthat they also generate form II and its generation
correlated with autophagosomal association of
GABARAP and GATE161). The results suggest that all
mammalian Atg8 homologues receive a common
modification to associate with autophagosomal
membrane as the form II. GABARAP and GATE16
have been suggested to be involved in the
GABAA-receptor transport and an intra-Golgi transport, respectively. However, it remains a possibility thatthey participate in autophagy in addition to or insteadof their functions originally described.

rB-a. Division of Cytogenetics
Tamotsu Yoshimori GroupRESEARCH ACTIVITIES

Tamotsu Yoshimori
Most of membrane-bound organelles inside
eukaryotic cells are linked each other by dynamic
membrane trafficking regulated by a set of specificproteins. Membrane traffic is essential not only tosurvival of each cell but also to various functionsorganizing the multi-cellular system, e.g., formation ofcell polarity and intercellular communication. We aim
to unravel molecular mechanisms of membrane traffic
and their roles in physiological functions and diseases
in animals, which must produce knowledge contributing clinical medicine. Weare now focusing on two
trafficking routes; autophagy and the endosomal
system. Autophagy is membrane traffic delivering
cytoplasmic components to lysosomes for bulk
degradation. The process is mediated by the formationof the double membrane-bound autophagosomes.
Endosomes receive macromolecules taken up by
endocytosis from outside. The cargo is then either
sorted to lysosomes or recycled back to the plasma
membrane.
(1) Three mammalian homologues of yeast Atg8localize to autophagosomal membrane depending on
post-translational processing
Yukiko Kabeya\ Noboru Mizushima2, Akitsugu
Yamamoto3, Satsuki Oshitani-Okamoto\ Yoshinori
Ohsumi1 and Tamotsu Yoshimori CNational Institutefor Basic Biology, 2Tokyo Metropolitan Institute ofMedical Science, 3Nagahama Institute of Bio-Scienceand Technology)(2) Autophagy plays a pivotal role in survival during
the early neonatal starvation period
Akiko Kuma\ Masahiko Hatano 2, Makoto Matsui\
Akitsugu Yamamoto3, Haruaki Nakaya2, Tamotsu
Yoshimori, Yoshinori Ohsumi\ Takeshi Tokuhisa2
and Noboru Mizushima1 CTokyo Metropolitan
Institute of Medical Science, 2Chiba University,
3Nagahama Institute of Bio-Science and Technology,
4National Institute for Basic Biology)
At birth the trans-placental nutrient supply issuddenly interrupted, and neonates face severe
starvation until supply can be restored through milk
nutrients. We showed that neonates adapt to thisadverse circumstance by inducing autophagy todegrade cytoplasmic constituents within lysosomes 61•
The level of autophagy in mice, which was monitored

DEPARTMENT OF CELL GENETICS

17

by GFP-LC3 expressed in the transgenic mice l ),remains low during embryogenesis; however,autophagy is immediately upregulated in various
tissues after birth and is maintained at high levels for
3-12 h before returning to basal levels within 1-2 days.
Mice deficient for Atg5, which is essential for
autophagosome formation, appear almost normal atbirth but die within 1 day of delivery. The survival timeof starved Atg5-deficient neonates ( 12 h) is much
shorter than that of wild·type mice ( 21 h) but can be
prolonged by forced milk feeding. Atg5-deficientneonates exhibit reduced amino acid concentrations
in plasma and tissues, and display signs of energy
depletion. These results suggest that the productionof amino acids by autophagic degradation of 'self'
proteins, which allows for the maintenance of energy
homeostasis, is important for survival during neonatal
starvation.
(3) Autophagic machinery provides a cellular defense
system against invasion by group A Streptococcus

Ichiro Nakagawa\ Takahiro Kamimoto, Akitsugu
Yamamot0 2, Noboru Mizushima3 , Kayoko Tsuda,
Atsuki Nara, Junko Funao\ Masanobu Nakata\ Atsuo
Amano\ Shigeyuki Hamadal and Tamotsu Yoshimori
COsaka University, 2Nagahama Institute of Bio-Scienceand Technology, 2Tokyo Metropolitan Institute ofMedical Science)
A Gram-positive bacteria, group A Streptococcus(GAS) is the etiological agent for a diverse collectionof human diseases. GAS invades non-phagocytic cells,
but the destination of GAS after internalization is notwell understood. We found that the autophagicmachinery could effectively eliminate GAS within
non-phagocytic cells 7). GAS that had invaded HeLa
cells or MEF was first trapped by endosomes and thensuccessfully escaped to the cytoplasm using bacterial
cytolysin SLO. Then, the cytoplasmic GAS was
selectively sequestered by large, unique compartments
bearing LC3, the formation of which was specificallyinduced by bacterial infection and required the Atg5
gene. The numbers of living GAS decreased to lessthan 10% at 4 h of post·infection and this reduction wascompletely suppressed in autophagy deficient Atg5-1cells. In these cells, GAS survived, multiplied, and werereleased from the cells. The compartments trapping
GAS eventually fused with lysosomes, in which

18

Division of Cytogenetics

enzymes degraded and killed the GAS. The autophagicmachinery could only recognize GAS that had esc;lpedfrom endosomes to the cytoplasm. Based on theseresults, we concluded that the autophagic machinery
can act as an innate defence system against invading
pathogens.
(4) Intracellular Shigella escapes from Autophagy

Michinaga Ogawa\ Tamotsu Yoshimori, ToshihikoSuzuki\ Hiroshi Sagara\ Noboru Mizushima2 andChihiro Sasakawal eUniversity of Tokyo, 2Tokyo
Metropolitan Institute of Medical Science)

Shigella is a group of Gram-negative bacteriacausing Shigellosis. The invasion of Shigella into thecolonic epithelium initiates the disease. IcsB, one of the
Shigella flexneri effectors, is secreted via the type III
secretion system of cytoplasmic bacteria and locatedon the bacterial surface. The icsB mutant is fully
invasive, but defective in spreading within host cells.
To clarify the role of IcsB in promoting infection, weinvestigated the intracellular behaviors of the icsB
mutanel. As a result, we found that the mutant
bacteria were trapped by autophagy in the cytoplasm.
IcsB did not directly inhibit autophagy. Rather, Shigella
VirG, a protein required for intracellular actin-based
motility, induced autophagy by binding to Atg5. In
non-mutant Shigella, this binding is competitively
inhibited by IcsB binding to VirGo Thus, in contrast
to GAS, Shigella in the cytoplasm is able to escapefrom attack of autophagy by secreting IcsB which act
as an inhibitor of recognition of the bacteria by Atg5.
(5) The intracellular inclusions containing mutanta I-antitrypsin Z are propagated in the absence ofautophagic activity

Takahiro Kamimoto, Shisako Shoji, Noboru
Mizushima\ Kyohei Umebayashi, Tunda Hidvegi 2,
David H. Perlmutter and Tamotsu Yoshimori eTokyo
Metropolitan Institute of Medical Science, 2Universityof Pittsburgh)
Formation of intracellular inclusions comprisedof terminally misfolded proteins is the most obvious
hallmark of diseases that are collectively termed
conformational disease. Mutant a I-Antitrypsin Z(a I·ATZ) protein accumulates within the endoplasmic

reticulum (ER) of the liver cells as an aggregatedpolymer and is associated with the development ofchronic liver injury and hepatocellular carcinoma in
hereditary a I-antitrypsin (a I-AT) deficiency. Previous
studies have suggested that efficient intracellulardegradation of a I-ATZ is correlated with protection
from liver disease in a I-AT deficiency, and that theubiquitin proteasome system accounts for a major, but
not sole, route of a I-ATZ disposal. Yet autophagy has
also been implicated in the pathophysiology of a I-AT
deficiency. In this study, to provide genetic evidence
that autophagy can mediate disposal of a I-ATZ, we
used autophagy deficient Atg5-1- cells. The results
showed that in the absence of autophagy, degradationof a I-ATZ was retarded and that a I-ATZ
accumulated in characteristic cellular inclusions
colocalized with calnexin and ubiquitin. These data
provide definitive evidence that autophagy canparticipate in the quality controVdegradative pathway
for a I-ATZ and suggest that autophagic degradation
plays a fundamental role in preventing the formationof characteristic cytoplasmic inclusions.
(6) Analysis of invasion of Porphyromonas gingivalis
into cells by using beads coated with the bacterial vesicles

Kayoko Tsuda, Ichiro Nakagawal , Kyohei
Umebayashi, Atsuo Amand and Tamotsu Yoshimori
eOsaka University)

Porphyromonas gingiva/is, Gram-negative
anaerobic bacterium, is considered to be a bona fide
pathogen of adult periodontitis, which is the mostprevalent chronic disease among human over theworld. P. gingiva/is can internalize into primary
gingival epithelial cells and other cell types. To clarify
the mechanisms underlying the invasion and itineraryof the pathogen within host cells, we have usedfluorescent beads coated with vesicles secreted by P.
gingiva/is. The beads efficiently entered into cells
through membrane trafficking, while the internalization did not occur at all when beads were coated with
BSA or the heat-inactivated vesicles. We have been
investigating cellular components involved in the beads
internalization and fate of the internalized beads.

(7) Reconstitution of initiation of autophagosorne
fonnation by using semi-intact cells

Shunsuke Kimura, Atsuki Nara, Yoshitaka Nagai l andTamotsu Yoshimori eOsaka University)

In vitro reconstitution of membrane trafficking byusing the semi-intact cell systems is a powerful tool
to resolve its molecular machinery. To elucidate
mechanisms underlying autophagic membrane
dynamics, we established semi-intact cells by treatmentof cultured cells with a bacterial toxin, Streptolysin 0,
which forms micro pores on the plasma membranes
and allow us to access the cytoplasm directly. We
succeeded to reconstitute formation of the small vesicle
decorated with GFP-Atg5, which is a precursor of
autophagosome in the semi-intact cells by adding thecytosol fraction and ATP-regenerating system. The
cytosol isolated from starved cells was more effective
than that from cells cultured in nutrient-rich condition.
Since autophagy is known to be induced by starvation,
the result indicates that the starved cell cytosol
contains factor(s) triggering autophagosome formation.
Moreover, we found that adding of the recombinant
protein containing expanded polyglutamine fragment,
which causes a class of inherited neurodegenerative
diseases, so-called polyglutamine diseases, including
Huntington's disease, induced autophagy in thissystem. Weare screening the cellular molecules
recognizing the starvation signal or the expanded
polyglutamine fragment to induce autopagy.
(8) Ubiquitin-dependent sorting of the epidennal growth
factor receptor in the endocytic pathway

Kyohei Umebayashi and Tamotsu Yoshimori
When plasma membrane proteins are ubiquitinated, they follow the endocytic pathway tolysosomes. In the case of the epidermal growth factor
receptor (EGFR), the Cbl E3 ligase is responsible forligand-dependent ubiquitination. It has been considered that the ubiquitination of the receptor occurs in
the plasma membrane, however, we found that Cbl is
localized to early endosomes when cells are stimulatedwith EGF. Early endosomes are composed of distinct
membrane domains, which may represent varioustransport directions from this organelle. Hrs sorts
ubiquitinated cargoes to lysosomes, and is localized

Division of Cytogenetics

19

to specific domains of early endosomal membrane. The
localization patterns of Cbl and Hrs were overlappedvery well, suggesting that Cbl is localized to earlyendosomal subdomains where cargoes destined forlysosomes are concentrated. It is known that
expression of a dominant negative form Cbl(C381A)
abolishes the ubiquitination of EGFR Strikingly, we
found that Cbl(C381A) does not inhibit theinternalization of EGF from the plasma membrane.
EGF could reach endosomes where it was colocalizedwith Cbl(C381A). Thus, ubiquitination of EGFR is not
obligatory for the internalization process. Both theubiquitination and the ubiquitin-dependent sorting of
EGFR may occur in early endosomal subdomains.
Tracking the localization after EGF stimulationsuggested that Cbl binds EGFR in the plasma
membrane, remains associated in early endosomes,
and then becomes separated from the receptor. We
have obtained an implication that an AAA ATPase
SKDl regulates the ubiquitinated status of EGFR, andwill investigate the molecular mechanisms in detail.
PUBLICATIONSFYVE domain-containing pro'tein associated withprotein granules and autophagic membranes.]. Cell.
Sci. 117, 4239-4251.
6. Kuma, A, Hatano, M., Matsui, M., Yamamoto, A.,
Nakaya, H., Yoshimori, T., Ohsumi, Y., Tokuhisa, T.
and Mizushima, N. (2004). Role of autophagy during
the early neonatal starvation period. Nature 432,
1032-1036.
7. Nakagawa, I., Amano, A., Mizushima, N.,
Yamamoto, A, Yamaguchi, H., Kamimoto, T., Nara,A, Funao,]., Nakata, M., Tsuda, K, Hamada, S. andYoshimori, T. (2004). Autophagy defenses cells against
invading group A Streptococcus. Science 306, 1037-1040.
8. Ogawa, M., Yoshimori, T., Suzuki, T., Sagara, H.,
Mizushima, N. and Sasakawa, C. (2004). Escape ofIntracellular Shigella from Autophagy. Sciencepublished online 2 December [DOl: 10.1126/
science.1106036] .
9. Nakatsukasa, K, Okada, S., Umebayashi, K,
Fukuda, R, Nishikawa, S. and Endo, T. (2004). Rolesof O-mannosylation of aberrant proteins in reductionof the load for endoplasmic reticulum chaperones inyeast. ]. BioI. Chern. 279, 49762-49772.

Papers

Reviews

1. Mizushima, N., Yamamoto, A., Matsui, M.,
Yoshimori, T. and Ohsumi, Y (2004). In vivo analysis
of autophagy in response to nutrient starvation usingtransgenic mice expressing a fluorescent
autophagosome marker. Mol. BioI. Cell 15, 1101-1111.
2. Prentice, E.W., Jerome, G., Yoshimori, T.,
Mizushima, N. and Denison, M.R (2004). Coronavirus
Replication Complex Formation Utilizes Componentsof Cellular Autophagy. J. BioI. Chern. 279, 10136-10141.
3. Kabeya, Y, Mizushima, N., Oshitani-Okamoto, S.,
Ohsumi, Y. and Yoshimori, T. (2004). LC3, GABARAP
and GATE16 localize to autophagosomal membrane
depending on form-II formation. J. Cell Sci. 117,2805-2812.
4. Fujita, H., Umezuki, Y, Imamura, K, Ishikawa,D., Uchimura, S., Nara, A, Yoshimori, T., Hayashizaki,
Y., Kawai,]., Ishidoh, K, Tanaka, Y. and Himeno, M.
(2004). Mammalian class E Vps proteins, SBPI andmVps2/CHMP2A, interact with and regulate thefunction of anAAA-ATPase SKDlNps4B.J. Cell Sci.
117, 2997-3009.
5. Birkeland, H.C.G., Simonsen, A, Gillooly, D.].,
Mizushima, N., Kuma, A, Yoshimori, T., Slagsvold,
T., Brech, A. and Stenmark, H. (2004). Alfy, a novel

10. Yoshimori, T. (2004). Autophagy: a regulated bulk
degradation process inside cells Biochem. Biophys. Res.
Commun. 313, 453-458.
11. s~ 1~iH2004H§:8,«jdl[m5}fW~.A7"A.!::L--C(J)20

Division of Cytogenetics~-~77~-:~G~K~0-C~k~.~~~.ft.

m8 '«~~M~1tflJ~ fiFIBJff!H;::;to ~t Q m8 ,«(J)-~J 49,
1029-1032.
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Ji.211, 147-151.
13. :f'fjj;#$-¥ (2004) .A 7" t1 -}v.!::.::z..~· ~T /'-~.A ~ ::I}v~-c:'(J) -7 /' /'\ -7 ,«~JjIJ~J2S ~;::;to ~t Qf9:jIflJ- B *Jf£~1t'¥:~tt; 78, 39-41.
EDUCATION

1. Dr. T. Yoshimori gave a lecture at Division of
Pharmaceutical Science, Graduate School of Natural
Science and Technology, Kanazawa University,
January, 2004 (in Japanese).
2. Dr. T. Yoshimori gave a lecture at Kakegawa-nishiHigh School, Shizuoka-ken, March, 2003 (in Japanese).
3. Dr. T. Yoshimori gave a lecture at Graduate Schoolof Integrated Science, Yokohama City University,
March, 2004 (in Japanese).

4. Dr. T. Yoshimori was invited to give a seminar at
Institute for Frontier Medical Science, Kyoto
University, March, 2004 (in Japanese).
5. Dr. T. Yoshimori gave a lecture at Graduate Schoolof Pharmaceutical Science, The University of Tokyo,
July, 2004 (in Japanese).
6. Dr. T. Yoshimori was invited to give a seminar at
Geneva University Sciences II, Geneva, September,
2004.
7. Dr. T. Yoshimori was invited to give a seminar at
The Institute of Medical Science, The University ofTokyo, September, 2004 (in Japanese).
8. Dr. K. Umebayashi was invited to give a seminarat the Norwegian Radium Hospital, Oslo, September,
2004.
SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. T. Yoshimori was appointed for a councillorof the Japan Society for Cell Biology.
2. Dr. T. Yoshimori was appointed for an associate
editor of "Cell Structure and Function".
3. Dr. T. Yoshimori was appointed for a member ofeditorial board of "Autophagy".
4. Dr. T. Yoshimori organized a symposium "Sorting
and Selection in Membrane Traffiking" at The 57th
Annual Meeting of Japan Society for Cell Biology,
Osaka, May, 2004.

B-b. Division of Microbial Genetics
Hiroyuki Araki GroupRESEARCH ACTIVITIESWe have been studying on eukaryotic
chromosomal DNA replication and its regulation by
the cell cycle. For this purpose, we have employedbudding yeast, Saccharomyces cerevisiae, as a model
system of eukaryotic cells. Using strong genetics ofbudding yeast, we have identified novel factors involving in chromosomal DNA replication and revealed theirfunctions in chromosomal DNA replication. We havebeen also interested in chromosome instability causedby replication defect.

(1) The interaction between replication proteins for the
initiation of DNA replication

Sachiko Sakamoto, Kazuyuki Hirai, Y oichiro
Kamimura and Hiroyuki Araki
At replication origins in eukaryotes, the pre-RC(pre-Replicative Complex) forms from late M to Gl
phases and other replication proteins including DNA
polymerases assemble when CDK activity increases
from Gl/S boundary. We have reported three yeast
complexes, Sld2-Dpbll, Sld3-Cdc45 and GINS, all ofwhich associate with origins in a mutually dependentand the pre-RC dependent manner. Dpbll has four
BRCT repeats and form a complex with the Sld2
protein phosphorylated by CDK. The Sld3-Cdc45
complex is observed throughout the cell cycle. The
GINS complex consisting of Sld5, Psfl, Psf2 and Psf3
subunits first associates with origins and then moves
with replication forks.
To elucidate how the proteins assemble,weinvestigated the complex formation between GINS andother proteins by co-immunoprecipitation assay. GINS
coprecipitated with Pol E throughout the cell cycle andwith Mcm, a component of the pre-RC, in the S phase.
Moreover, when we treated the cells with thecross-linking agent before precipitation, Sld3, Sld2 andDpbll also coprecipitated with GINS. Even when
S-CDK activity increased without the pre-RC in Cdc6depleted cells, Sld2-Dpbll and Pol E coprecipitatedwith GINS, suggesting that they form a complex
without origin-association. We therefore propose thepre-Landing Complex (pre-LC) containing Sld2, Dpbll,
Pol E and GINS, which is formed before associatingwith origins. The pre-LC seems to associate with the
pre-RC via Sld3 since the C-terminal portion of Sld3
interacts with GINS whereas the N-terminal portion
interacts with Cdc45. The pre-LC complex is observed
only when CDK activity increases. Since the complex
formation between Sld2 and Dpbll depends on
CDK-phosphorylation of Sld2 (see below), CDK
regulates the pre-LC formation through the Sld2-Dpbll
complex formation.
In parallel with the in vivo analysis, we have tried
to purify the proteins related to the pre-LC to know
their function. First, we expressed all the subunits of
GINS in insect cells and purified GINS to be a near
homogeneity. We also expressed Dpbll and Sld3 inEscherichia coli and partially purified them. The

Division of Cytogenetics

21

purified GINS complex binds to the purified Dpbll andSld3, consistent with two hybrid assay and in vivo
co-immunoprecipitation. We will further extend thisanalysis to all the components of the pre-LC.
(2) Regulatory mechanism of the complex formation
between Sld2 and Dpbll by cyclin-dependent kinase(CDK)

Yon-Soo Tak, Yoshimi Tanaka, Yoichiro Kamimuraand Hiroyuki Araki
The initiation of chromosomal DNA replication in
eukaryotes requires the CDK activity. However, how
CDK regulates the initiation of DNA replication is notwell elucidated since the substrates of CDK in DNA
replication have not been identified except the Sld2
protein. Sld2 has a cluster of eleven CDK-dependent
phosphorylation sites (S/T-P), six of which are
preferred CDK phosphorylation sites. Phosphorylationof ,.Sld2 enhanced its binding to Dpbll, which appears
to be necessary for onset of DNA replication. We havestudied how the complex formation between Dpbll
and Sld2 is regulated by CDK activity. Dpbll has four
copies of the BRCT domain, which is recently reported
as a phospho-peptide binding module. A C-terminal
pair of BRCT domains of Dpbll binds to a shortstretch of Sld2.
We purified the truncated Sld2 containing the
Dpbll-binding stretch and ll-CDK phosphorylationsites and the C-terminal pair of BRCT domains of
Dpb11. Using these purified proteins, we demonstrated
that CDK-phosphorylation enhances the complex
formation between Sld2 and Dpb11. To this in vitro
binding reaction, we challenged various synthesizedpeptides with or without phosphorylation. It revealed
that the 20-aa stretch with phosphorylation at
threonine followed by proline (CDK-catalyzed
phosphorylation site) competes with the complex
formation between phosphorylated Sld2 and Dpb11.
Thus, the 20-aa stretch with the phosphorylation
functions as a binding stretch to Dpb11. Consistent
with these observations, an alanine substitution of thisthreonine reduced the binding activity to Dpbll in in
vitro binding assay as well as two-hybrid assay.
Furthermore, this alanine-substitution confers defectof cell growth.
We previously reported that the simultaneous
alanine substitutions of all the canonical (preferred)

22

Division of Microbial Genetics

CDK-phosphorylation sites of Sld2 confers defect ofcell growth as well as reduced affinity to Dpbll(Masumoto et al., Nature 415, 651, 2002). The 20-aa
stretch does not contain these canonical phosphorylation sites. Moreover, the phosphomimetic replacementof this threonine by asparagine overrides the defect of
the simultaneous substitutions of canonicalphosphorylation-sites in a two-hybrid assay. Theseobservations suggest that phosphorylation of canonical
CDK-sites affects the phosphorylation of the threonine
in the 20-aa stretch.
CDK is consisted of a catalytic subunit and acyclin. Budding yeast has one catalytic subunit, Cdc28
and nine different cyclins, and thus 9 species of CDK.
We expressed these CDK subunits in E. coli andpurified typical CDK species. Using these purified
CDKs, we found that Cdc28-Clb5, S-phase CDK mostefficiently phosphorylates the Sld2 protein. We also
identified efficient phosphorylation sites by CDK in
both in vivo and in vitro using various Sld2 proteinwith mutations in CDK-phosphorylation sites.
According to this assay, the threonine in the 20-aa
stretch is an inefficient phosphorylation site. Moreover,the simultaneous mutations in canonical phosphorylation sites, some of which are actually efficientphosphorylation sites, reduced severely the phosphorylation of the threonine in the 20-aa stretch. We
therefore propose that the threonine in the 2O-aa stretchof Sld2 is phosphorylated cooperatively with otherefficient phosphorylation sites and then Sld2 binds to
Dpb11.
(3) CDK targets in the initiation of DNA replication

Seiji Tanaka and Hiroyuki Araki
The initiation of eukaryotic DNA replication istriggered by two essential kinases, Cdc7 and CDK.
Requirement of Cdc7 in initiation can be bypassed witha bobl-l mutation, which is allelic to the one of thesubunit of proposed replicative helicase, MCM5.
Mutations that can bypass requirement of CDK,
however, are not known so far. We have recently
identified Sld2 as an essential substrate of CDK in theinitiation of DNA replication. Phosphorylation of Sld2
by CDK enhances the interaction between Sld2 and its
binding partner, Dpbll, which is likely to be important
for association of the Sld2-Dpbll complex to replication
origins (Masumoto et al. Nature 415,651-655,2002).

To understand the individual processes in theinitiation of DNA replication, we have tested whether
Sld2 phosphorylation by CDK is sufficient for
initiation. We have constructed a Sld2 mutant whichmight mimic a phosphorylated status of wild type Sld2.
Although this mutant could support cell growth, CDK
activity was still required for initiation. This suggeststhat CDK regulates multiple targets in the initiationof DNA replication. In order to identify these CDK
target(s) other than Sld2, we have set a screening ofmutants that show synthetic lethality withphosphomimetic sld2. The rationale of the screeningis that cells might to be a lethal because of untimely
DNA replication when all of the CDK targets in theinitiation process including Sld2 are freed from strict
regulation of CDK. Although we have not beensuccessful to isolate that kind of mutants so far, we
will continue the screening.
(4)

are good substrates for recombination and contributeto generating genomic instability. To test this, we haveintroduced a genomic sequence that is known to block
the progression of replication fork and found that thisinduce higher rate of GCR. Weare now proceeding thedetailed analysis on this.
PUBLICATIONSPapers

1. Iida, T. and Araki, H. (2004). Non-competitive
counteractions of DNA polymerase E and ISW2/yCHRAC for epigenetic inheritance of telomereposition effect in Saccharomyces cerevisiae. Mol. Cell.
BioI. 24,217-227.
2. Mimura, S., Seki, T., Tanaka, S. and Diffley, ].F.x.
(2004). Phophorylation-dependent binding of mitotic
cyclins to Cdc6 contributes to DNA replication control.
Nature 431, 1118-1123.

Mechanisms for generating genomic instability
Books

Seiji Tanaka and Hiroyuki Araki"

3. Jit>i>:31.z (2004) ~~~~O)~~IjfJM3BtJ;t, DNA~~ .
{~1i;6~:b;6~Q CffiWilJtftt*,;$),

Although genomic instability is a hallmark ofhuman cancer cells, the mechanisms by which genomic
instability is generated and selected for duringoncogenesis remain obscure. In most human cancers,
the pathway leading to the activation of the G1 cyclins
is deregulated. Previously we hypothesized that G1
cyclin deregulation could cause genomic instability
because the lack of a proper 'low Cdk' period in G1
may reduce numbers of functional pre-RCs that confer
replication competence to cells. Using budding yeast
as a model, we have shown that overexpression of the
Gl cyclin, Cln2, inhibits the assembly of pre-RCs andinduces gross chromosome rearrangements (GCRs)
such as translocation, deletion of a chromosome arm,
interstitial deletions and inversions. These results
suggest that deregulation of G1 cyclins, selected for in
oncogenesis because it confers clonal growthadvantage, may also provide an important mechanism
for generating genomic instability by inhibiting
replication licensing. However, we still do not know
how reducing origin licensing/firing contributes togenomic instability. We have focused on this question.
Reduced numbers of replication forks and slow
DNA replication as a result may cause broken
chromosomes in M phase or stalled replication forks
that persist for longer periods. We have asked if these

4.

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m*31.z(2004)DNAO)~~,/<;/f'T"T:7 /0-/-0)tc~O)£~7t-T~~~(*~,~~~ . ~t*M1V<. . JJf:~~ .

iE)lltIlE'W), {t~IPJ.A..
EDUCATION

1. Dr. H. Araki gave a lecture at Medical School,
University of Tokyo, February, 2004 (in Japanese).
2. Dr. H. Araki gave a seminar at Paterson Instituteat Manchester, UK, April, 2004.
3. Dr. H. Araki gave a seminar at Clare Hall
Laboratories, Cancer Research UK at South Mims, UK,
April, 2004.
4. Dr. H. Araki gave a seminar at National Instituteof Environmental Health Sciences, Research Triangle
Park, NC, USA, July, 2004.
5. Dr. H. Araki gave a lecture at Shizuoka University,
October, 2004 (in Japanese).
6. Dr. H. Araki gave a lecture and a seminar at Tokyo
Institute of Technology, November, 2004 (in Japanese).

Division of Microbial Genetics23

B-b. Division of Microbial Genetics
Seiichi Yasuda GroupRESEARCH ACTIVITIES(1) Mechanism of DoaA interaction with phospholipids

Seiichi Yasuda
The replication of chromosome in Escherichia coli
is initiated at a unique chromosomal locus, oriC. A
protein called DnaA catalyzes the first step in theinitiation by binding and opening double-stranded
DNA at an AT-rich region in oriC. The opened
single-stranded region serves as the site of assemblyof other proteins to form a replication complex.
Comparative studies on the dnaA genes of many
bacterial strains have established that DnaA is madeof four functional domains. Among them, domain 3 is
responsible for ATP-binding, and domain 4 forsequence-specific binding to oriC DNA. It has been
known that acidic phospholipids such as cardiolipin
release bound ATP and ADP from DnaA. Thisphenomenon was suggested to be involved in
rejuvenation of used DnaA. It has been postulated that
DnaA interacts with phospholipids at a site near the
C-terminal portion of domain 3 because this site hasan amino acid sequence that can form an alpha helix
having a hydrophobic surface.
Previous studies with mutant DnaA proteins inthis laboratory showed that the interaction of DnaAwith phospholipids depends on the presence of the
DNA-binding domain of DnaA, but not on that of theputative mem-brane-interaction site in domain III. Thiswas further confirmed by quantitative measurementsof DNA-binding and phospholipid-induced
ATP-release using mutant DnaA proteins. DnaA
mutants used were DnaADelM that has a deletion of
the membrane-interaction site, DnaADelV that has adeletion of C-terminal 14 amino acids of the
DNA-binding domain, and DnaADelS that has adeletion of almost all of the DNA-binding domain butthat retains the membrane-interaction site. ATf'binding of all these mutant proteins was essentiallythe same as that of wild type DnaA. Dissociation
constants of DNA-binding were measured and foundto be 5.75, 16.5, 115, and 825 nM for wild type DnaA,
DnaADelM, DnaADelV and DnaADeIS, respectively.

24

Division of Microbial Genetics

The rate of ATP release versus cardiolipin
concentration was determined and it was found thatthe order of cardiolipin sensitivity of these DnaA
proteins was exactly reverse to that of the DNA binding
constants. This suggests strongly that cardiolipininteracts with DnaA at the DNA-binding domain
leading to the release of ATP from the protein.
During the course of this work it was found thatcardiolipin inactivates the ATP, and DNA-binding
activities of DnaA, but not the replication activity as
measured in the presence of oriC and a crude protein
fraction. The crude protein fraction was found toreactivate DnaA that had been inactivated bycardiolipin. Characterization of the reactivating factor
is under way.

B-c. Division of Cytoplasmic Genetics
Kazuto Kobayashi GroupRESEARCH ACTIVITIES(1) Mechanisms of Neural Circuitry Underlying Control
of Behaviors

Kazuto Kobayashi
Control of behaviors is based on the mechanismsthat mediate processing and regulation of information
through the complex neural circuitry in the brain.
Dysfunction in the neural circuitry is involved in the
etiology and pathogenesis of some neural diseases. Our
research group is interested in understanding the roleof specific neurons or neural pathways that mediate
motor control, memory, and emotion as well as thefunction of the signaling molecules that are involvedin these behaviors. In the present year, we developed
a Cre-loxPrecombination system to study the role ofsignaling pathways in the function and developmentof the neural circuitry2). Using this system, we
identified a critical role of Rho/Rho-kinase signaling
pathway in the survival of developing motor
neuronsl, 8). In addition, we reported behavioral andphysiological roles of the neural pathway originatingfrom the striatum by using immunotoxin-mediated celltargeting6,9). Immunotoxin-mediated cell targeting was
also used to generate an animal model for autonomic
diseases1ol • Furthermore, we identified subunit
composition of GABAA receptors in midbraindopamine neurons by using the transgenic mousestrain that expresses GFP in these neurons 3). Thistransgenic strain was also utilized for modelexperiments of cell transplantation for Parkinson's
disease therapl' 5, 7).
PUBLICATIONSPapers

1. Kobayashi, K., Takahashi, M., Matsushita, N.,
Miyazaki, ].-1., Yaginuma, H., Osumi, N., Kaibuchi, K.
and Kobayashi, K. (2004). Survival of developing motor
neurons by Rho GTPase signaling pathway throughRho-kinase.]. Neurosci. 24, 3480-3488.
2. Matsushita, N., Kobayashi, K., Miyazaki, J. andKobayashi, K. (2004). Fate of transient
catecholaminergic cell types revealed by site-specificrecombination in transgenic mice. J. Neurosci. Res. 78,
7-15.
3. Okada, H., Matsushita, N., Kobayashi, K. andKobayashi, K. (2004). Identification of GABAA receptor
subunit variants in midbrain dopaminergic neurons.
]. Neurochem. 139, 7-14.
4. Saino-Saito, S., Sasaki, H., Volpe, B.T., Kobayashi,K., Berlin, R. and Baker, H. (2004). Differentiation of the
dopaminergic phenotype in the olfactory system of
neonatal and adult mice.]. Compo Neurol. 479,389-398.
5. Yoshizaki, T., Inaji, M., Kouike, H., Shimazaki, T.,
Sawamoto, K., Ando, K., Date, I., Kobayashi, K.,
Suhara, T., Uchiyama, Y. and Okano, H. (2004).
Isolation and transplantation of dopaminergic neurons
generated from mouse embryonic stem cells. Neurosci.
Lett. 363,33-37.

EDUCATION~*'¥/~~mtoo~O)~.~~~-r-

1. Dr. K. Kobayashi gave a lecture at Kobe University
School of Medicine, June, 2004 (In Japanese).
2. Dr. K. Kobayashi was invited to give a seminaron "Functional dissection of the neural circuitry thatmediates dopamine-dependent behavior" atWallenberg Neuroscience Center of Lund University,
Lund, Sweden, September, 2004.
3. Dr. K. Kobayashi was invited to give a seminaron "Functional dissection of the neural circuitry thatmediates dopamine-induced behavior" at Rutger
University, New Jersey, USA, October, 2004.
m~~~,*~~~O)~~~O)~~~~

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5JL~~~B't~*'¥#~~mmtOO~A M.·~.ATAm~mt. OO~)!f~'¥m~JiJT ~ ~~B't

B-c. Division of Cytoplasmic Genetics
Masayuki Yamamoto GroupRESEARCH ACTIVITIES(1) Fission yeast Mes1p ensures the onset of meiosis II
by blocking degradation of eyelin Cdc13p 2l

Masayuki Yamamoto
Reviews

6. Ij\;j:*fPA (2004) -1 A / t-:\'- ~ /,{f.HJJl1QmBr.J~ : :f!I!i!\I@]
1m ;( :7J .:::.:J.:A O)m#f~. ffll621 7, 195-200.
7. IN,-*fpA (2004) :f!I!i!\I~O)mt~:jI}j::.ro...O)J7"P -T.
!!I!'¥~~'¥31, 448-450.
8. IN,-*Jll:j(, Ij\;.f%fpA (2004) Cre-loxP~.A T A : ::I / '4 ~ 3 -r IVfJ:~f~-=f~~tt1J\lj. ffll621 7, 312-315.
9. {tr:!I!HtHf~, Ij\;.f%fpA (2004HJHRf*t:\tM1m~1l'T Q~
L-1t\:f!I!i!\I@]1m;(7J.:::.:J.:A. Clinical Neuroscience 22, 732.

or

Books

10. Kobayashi, K. and Nagatsu, T. (2004). Transgenic
strategies in autonomic research. In The Primer on theAutonomic Nervous System, 2nd edition, Robertson, D.
ed. (London: Elsevier Academic Press), pp.435-438.

Meiosis is a special form of nuclear division to
generate eggs, sperm, and spores in eukaryotes.
Meiosis consists of the first (MI) and the second (MIl)meiotic divisions, which occur consecutively. MI is
reductional, in which homologous chromosomes
derived from parents segregate. MIl is equational, in
which replicated sister-chromatids separate as inmitosis. MIl is generally considered to mimic mitosis
in mechanism. However, fission yeast Meslp isessential for MIl but dispensable for mitosis. We havedemonstrated that Meslp is a factor that suppresses
destruction of cyclin Cdcl3p at anaphase I. Meslp
inhibits the activity of APC/C (anaphase promoting
complex/cyclosome) to degrade Cdcl3p, thereby

Division of Cytoplasmic Genetics

25

playing a key role in saving a sufficient level of MPF(M-phase promoting factor) activity required for the
execution of MI{2).
(2) The pI50-Glued Ssm4p regulates microtubular
dynamics and nuclear movement in fission yeast4)

aberrant chromatin structure, small nucleoli, absence of the cytoplasmic core, and precocious
cellularization 3). It appears that DAZ-l in C. elegans
plays essential roles in female pre-meiotic and early
meiotic germ cells, probably via regulating thetranslational activity of specific target genes required
for the progression of oogenesis.

Masayuki Yamamoto
PUBLICATIONS

During vegetative growth of the fission yeast,
microtubules nucleate from multiple MTOCs in thevicinity of the nucleus, polymerizing until they reachthe end of the cell and then shrinking back to the cell
middle. In response to mating pheromone, fission yeast
undergoes a morphological switch from a vegetative
to a shmooing growth pattern. The switch in growthmode is paralleled by a switch in microtubular
dynamics. Microtubules nucleate mostly from a single
MTOC and pull on the ends of the cell to move thenucleus back and forth. This movement continues after
cellular and nuclear fusion in the zygote and isimportant to ensure correct chromosome pairing,recombination and segregation during meiosis. We
have demonstrated that Ssm4p, a pl50-glued protein,
is induced specifically in response to pheromone andis required for this nuclear movement. Ssm4p is
associated with the cytoplasmic dynein complex andregulates dynein heavy chain localization. We have also
shown that Ssm4p functions in establishing theshmooing microtubular array4).
(3) C. elegans DAZ-l directs proper nuclear organization and cytoplasmic core formation during oogenesis3)

Papers

1. Inoue, T., Sugimoto, A., Suzuki, Y., Yamamoto, M.,

Tsujimoto, M., Inoue, K., Aoki, J. and Arai, H.(2004).
Type II platelet-activating factor-acetyl hydrolase isessential for epithelial morphogenesis in Caenorhabditis
elegans. Proc. Natl. A cad. Sci. USA. 101, 13233-13238.
2. Izawa, D., Goto, M., Yamashita, A., Yamano, H.
and Yamamoto, M. (2005). Fission yeast Meslp ensures
the onset of meiosis II by blocking degradation of
cyclin Cdc13p. Nature 434, 529-533.
3. Maruyama, R., Endo, S., Sugimoto, A. andYamamoto, M. (2005). C. elegans DAZ-l is expressedin proliferating germ cells and directs proper nuclear
organization and cytoplasmic core formation during
oogenesis. Dev. Bioi. 277, 142-154.
4. Niccoli, T., Yamashita, A., Nurse, P. andYamamoto, M. (2004). The pl50-Glued Ssm4p regulates
microtubular dynamics and nuclear movement infission yeast. J Cell Sci. 117, 5543-5556.
5. Yamashita, A., Sato, M., Fujita, A., Yamamoto, M.
and Toda, T. (2005). The roles of fission yeast Asel inmitotic cell division, meiotic nuclear oscillation andcytokinesis checkpoint signaling. Mol. BioI. Cell 16,1378-1395.

Masayuki Yamamoto
SOCIAL CONTRIBUTIONS AND OTHERSThe Deleted in Azoospermia (DAZ) family genes
encode potential RNA-binding proteins that are
expressed exclusively in germ cells in a wide range ofmetazoans. Mutations in daz-1, the only DAZ familygene in Caenorhabditis elegans, cause pachytene stagearrest of female germ cells, but do not affectspermatogenesis. We have demonstrated that DAZ-lprotein is most abundantly expressed in proliferating
female germ cells. DAZ-l is dispensable in males, but
it is expressed also in male mitotic germ cells. By
detailed phenotypic analyses we have found that lossof daz-1 function causes multiple abnormalities as earlyas the onset of meiotic prophase, which include

26

Division of Cytoplasmic Genetics

President: The Molecular Biology Society of Japan
Editor: Genes to Cells
Editorial Board: Current Genetics, YEAST

same molecular cassette multiple times.

c. DEPARTMENT OF
DEVELOPMENTAL GENETICS

C-a. Division of Developmental Genetics
Yasushi Hiromi GroupRESEARCH ACTIVITIES(1) Multi-dimensional axon network formation

Masaki Hiramoto and Yasushi Hiromi
A key feature of the CNS that allows efficient
information processing is its repetitive modular
structure. Such structure is found in the invertebrate
ventral nerve cord, the vertebrate rhombomere or thecolumnar structures of the cortex. An individual
module contains the complement of cell types andguidance cues required to form a neural circuit within
the module. To construct a neural network, however,multiple modules must be interconnected by axons
exiting the module border and then re-entering the next
module. Such "boundary crossing" requires axons to
adopt navigation strategies that are different from therules that shaped their trajectories within the module
unit. In bilateralians a boundary crossing problem
occurs at the midline, the axis of symmetry. In animals
with segmentally repeated body plan, an additionalboundary crossing problem resides at the border of therepeat unit, segment boundary. In contrast to thewell-studied midline crossing event, segmetal boundary
crossing problem has never been subjected to an
analysis, either in the vertebrate or the invertebrate
CNS. We showed that two evolutionary conserved axon
guidance molecules, secreted ligand Netrin and a
guidance receptor ROBO, which are known to play
important role in axon guidance across the midline,
are also used as a molecular cassette to licenselongitudinal axons to cross the segmental boundary,
thereby connecting modular structures in the CNS.
This finding demonstrates that the dimension of themulti-modular structure can be increased by using the(2) Intrinsic sub-axonal patterning in Drosophila
neurons

Takeo Katsuki, Masaki Hiramoto and Yasushi Hiromi
During the development of the nervous system,neurons extend their axons over a long distance to theirtargets with the assistance of guidan<;e cues andguidance receptors. Although a number of moleculesthat play instructive role in axonal navigation havebeen discovered, little is known about how the
distributions of such guidance molecules are regulated
in space and time. Immunohistochemical studies haverevealed that the spatial distribution of guidance
receptors in vivo is often restricted to specific segments
ofaxons. Is such sub-axonal localization of guidance
receptors generated by the intrinsic properties ofneurons or by extrinsic signals? And how is the
localization of transmembrane receptors maintain6din the axonal membrane despite the fact that themembrane is continuous and fluid? Using a primarycell culture system of Drosophila, we demonstrated thatisolated neurons possess an intrinsic property togenerate sub-axonal localization of guidance molecules.
FRAP analysis provided evidence that an underlying
mechanism for the sub-axonal localization iscompartmentalization of the axonal membrane by adiffusion barrier. We propose that the regulated
expression of guidance receptors in vivo is based on
the intrinsic sub-axonal patterning property of neurons.
(3) Identification of BPI02 antigen which shows specificsub-axonal localization

Tohru Umemura, Takeo Katsuki and Yasushi Hiromi
A striking feature of the neuron is its regionalized
cellular organization. Cellular compartments such asaxon and dendrite have distinct molecular signaturesthat enable specific physiological functions. Axons
themselves have regional specifications, such as thegrowth cone and the distal end and the initial segment
at the base, which differ in cytoskeletal organizations
that influence the dynamics of the axonal membrane
and the movement of molecules within. Although theaxonal shaft is seemingly uniform, a number ofmolecules are localized to specific sub-segments of

DEPARTMENT OF DEVELOPMENTAL GENETICS

27

axons in vivo. Such sub-axonal localization of molecules
is likely based on intrinsic patterning ability of theneuron that regionalizes the axon to sub-axonal"compartments" (see above). One of the moleculesthat exhibit intrinsic sub-axonal localization is theantigen of the monoclonal antibody BPI02, whichstains many axons in the Drosophila CNS highlighting
the ladder-like axonal scaffold. In primary culture thisantibody stains the middle or proximal segment of theaxon, sugge,sting the existence of distinct sub-axonal
compartments. Weare trying to identify the BPI02
antigen to investigate how it organizes itself to such
sub-axonal compartments. We found that the antigen
is a membrane protein of about lOOkD, and requires
glycosilation to be recognized by the antibody.
(4) Lamina-specific connection in mouse hippocampus

Fumikazu Suto, Hajime Fujisawa1 and Yasushi
Hiromi eNagoya University)
In many part of central nervous system, distinct
populations ofaxons confine their terminal arbors andsynapses to different subsets of laminae. Theselamina-specificities can be divided into two categories,
cellular and subcellular. Several molecules that are
involved in lamina-specific projection at thecellular-specific level have recently been identified in
the Drosophila visual system. However, little is known
about the molecular mechanisms underlying theconnection to a specific subcellular region, a processthat requires recognition of a subcellular compartment.
To understand the molecular mechanisms for thesubcellular-specific connection, we are using mouse
hippocampus as a model system. In the hippocampus,
dentate granule cells (DGCs) project their axons (mossy
fibers; MFs) mainly to the proximal-most part of apicaldendrites of CA3 pyramidal cells and partly to theproximal basal dendrites in subcellular-specificmanner. We found that axon guidance moleculeSema6A was localized on the dendritic field of CA3
pyramidal cells, and had repulsive activity for MFs,
suggesting that Sema6A might be the signal thatspecify subcellular specificity of the MF projection. We
thus analyzed the roles of Sema6A receptors, Plexin-A2
and Plexin-A4, on MF projection using mutant andnormal explants. plexin-A4-deficient DGCs projected
MFs abnormally to all parts of the apical and basal
dendrites of genotypically normal CA3 pyramidal cells.

28

Division of Developmental Genetics

In contrast, CA3 pyramidal cells that are mutant forplexin-A2failed to receive projections from genotypically normal MFs in the apical dendritic field, andreceived only in the proximal basal dendrite. These
results suggest that during subcellular-specific MFs
projection, Plexin-A4 mediates Sema6A repulsive
activity in MFs, and Plexin-A2 regulates Sema6A
activity and/or distribution in the CA3 field,(5) seven-up controls switching of transcription factorsthat specify temporal identities of Drosophila
neuroblasts

Makoto Kanai, Masataka Okabe and Yasushi Hiromi
Drosophila neuronal stem cell neuroblasts
constantly change character upon division to produce
a different type of progeny at the next division.
Transcription factors Hunchback (HB), Kriippel (KR),Pdm (PDM), etc. are expressed sequentially in thedevelopmental history of each neuroblast and act asdeterminants of birth-order identity. How neuroblast
switches its expression profile from one transcription
factor to the next is poorly understood. We showed thatthe HB-to-KR switch is directed by the nuclear receptor
Seven-up. Seven-up expression is confined to atemporally restricted subsection within theneuroblast's lineage. Loss of seven-up function causes
an increase in the number of HB-positive cells withinseveral neuroblast lineages, whereas misexpression ofseven-up leads to the loss of these early-born neurons.
Lineage analysis provided evidence that seven-up isrequired to switch off HB at the proper time. Thus,
seven-up modifies the self-renewal stem cell program
to allow chronological change of cell fates, thereby
generating neuronal diversity.
(6) A search for Seven-up-interacting molecules

Takayuki Hondoh and Yasushi Hiromi
Seven-up is a transcription factor containing a
DNA binding domain and an evolutionarily-conserved
domain that has homology to the ligand binding
domains of nuclear receptors. The spatio-temporal
expression pattern of Seven-up is tightly linked to thecell fate; in the developing Drosophila eye, seven-up isexpressed in only four of the eight photoreceptor
neurons, which are transformed to another neuronalfate in seven-up mutant. In the embryonic CNS seven-up
expression is temporally restricted to a subsection of
the neuroblast lineage. The loss-of function mutation
causes a "temporal transformation" of the fate of theneuroblast and its progeny (see above). Although
Seven-up plays instructive roles in many cell fatedecisions, how this transcription factor accomplishes
such cell fate choices is not understood. We have
prepared antibodies against Seven-up that would
enable us to probe the endogenous Seven-up molecule.
Using IP and ChIP methods, we are investigating themolecular environment around Seven-up.
(7) Glial-expression of Prospero integrates inputs frommultiple signaling

Yoshihiro Yuasa and Yasushi Hiromi
Glial cells III the nervous system aremorphologically and functionally diverse, performing
various functions such as providing a scaffold foraxonal migration, insulation ofaxons, and supporting
neuronal survival. In Drosophila, interface glia such as
longitudinal glia enwraps the longitudinal axon and,in some ways, resembles oligodendrocytes invertebrates. Longitudinal glia also plays a key role in
the formation of longitudinal axon pathway. Tenlongitudinal glial cells that reside in each hemisegment
are generated from a single precursor that divides
while migrating towards the midline. Theselongitudinal glial cells likely comprise multiple glial
sub-types, because some of them have specificassociation with distinct axon pathways. One moleculethat is expressed in a subset of the longitudinal glia
is a homeodomain transcription factor Prospero(PROS). PROS is expressed in six out of ten
longitudinal glial cells, and is required for thegeneration of the longitudinal axonal scaffold andneuronal survival. In order to understand the gene
regulatory cascade leading to the specification of
longitudinal glial cells, we analyzed how PROS
expression is regulated in the longitudinal glia. We
found that the combinational expression of threetranscriptional factors and Notch signaling is essential
for the PROS expression in the longitudinal glia.

(8) A screening for genes involving germline stem cell
establishment and niche formation in Drosophila

Miho Asaoka, Shuji ShigenobuI, Satoru KobayashPand Yasushi Hiromi eNIBB)
Stem cells playa central role in generating andmaintaining most adult tissues in higher organisms.
They are defined by their ability to self-renew and to
produce numerous differentiated daughter cells. Stemcells in the adult tissue reside in a special
microenvironment called "niche", which are thought
to produce signals that support and maintain stem cell
specification and function. However, how stem cell fateand niche are initially established during tissue
development is currently unknown. In Drosophila,
germline stem cells are derived from a subset ofprimordial germ cells (PGCs). We have previously
shown that only the PGCs contacting somatic cells in
the anterior half of embryonic gonad will become
germline stem cells3l• We also found that the somatic
cells in the anterior half of the embryonic gonad
already differ from the posterior somatic cells andlikely constitute the niche in the adult. These results
suggest that somatic cells in the anterior half of theembryonic gonad playa key role in the germline stem
cell fate establishment and niche formation. As the firststep for understanding molecular mechanisms ofgermline stem cell establishment and niche formation,
we are focusing on genes that are expressed in thesomatic cells in the anterior half of the embryonic
gonad. Through EST and micro array analyses we
identified 187 genes which are expressed only in thesomatic cells in the embryonic gonad. In situ
hybridization analyses show that some of these genes
are expressed only in the anterior somatic cells in theembryonic gonad. We plan to test the function of thesecandidate genes in germline stem cell establishment
and niche formation, using RNAi methods.
(9) The origin of the parathyroid gland

Masataka Okabe and Anthony Graham! eMRC,
Centre for Developmental Neurobiology, King's College
London)It has long been held that the parathyroid glands
and parathyroid hormone evolved with the emergenceof the tetrapods, reflecting a need for new controls on

Division of Developmental Genetics

29

calcium homeostasis in terrestrial, rather than aquatic,
environments. Developmentally, the parathyroid glandis derived from the pharyngeal pouch endoderm, andstudies in mice have shown that its formation is under
the control of a key regulatory gene, Gcm-2. We haveused a phylogenetic analysis of Gcm-2 to probe theevolutionary origins of the parathyroid gland. We showthat in chicks, as in mice, Gcm-2 is expressed in thepharyngeal pouches and the forming parathyroid
gland. We find that Gcm-2 is present not only intetrapods but also in teleosts and chondrichthyans, andthat in these species, Gcm-2 is expressed within thepharyngeal pouches and internal gill buds that derivefrom them in zebrafish (Danio rerio), a teleost, anddogfish (Scyliorhinus canicula), a chondrichthyan. We
further demonstrate that Gcm-2 is required for theformation of the internal gill buds in zebrafish. We also
have identified parathyroid hormone 1/2-encoding
genes in fis9 and show that these genes are expressedby the gills. We further show that the gills express thecalcium-sensing receptor, which is used in tetrapods
to monitor serum calcium levels. These results indicatethat the tetrapod parathyroid gland and the gills of fish
are evolutionarily related structures, and that theparathyroid likely came into being as a result of thetransformation of the gills during tetrapod evolution 4).

EDUCATION

1. Dr. Y. Hiromi gave a lecture course at Tokyo

University of Agriculture and Technology. December,2004 (in Japanese).
SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. Y. Hiromi served as an editor for Development,
Growth & Differentiation.
2. Dr. Y. Hiromi served as a member of the councilof The Genetics Society of Japan.
3. Dr. M. Okabe developed "Barrier-free presentationthat is friendly to colorblind people", and enhanced its
public awareness through web pages:
CDflliJ$lE~, ftti1i~ http://www.nig.ac.jp/color!
®Jkabe, M. and Ito, K. http://jfly.iam.u-tokyo.ac.jp/
color!
4. Dr. M. Asaoka contributed in making a DVD/
Videor~*HIIffl!~':::''YT-~*--c~t:::~~*HIIffl!OY9.7fn~-J(i1iUW : tif~OOiU, 1i?:00i: rft;;.~~«*),~Jf~~, rft1mftf~, rft!*JP,3C7'6)'~f~: -¥~,~~~,

C-a. Division of Developmental Genetics
Toshitaka Fujisawa GroupPUBLICATIONSRESEARCH ACTIVITIESPapers(1) Systematic identification of peptide signaling
molecules in Hydra

1. Niwa, N., Hiromi, Y. and Okabe, M. (2004). A

conserved developmental program for sensory organ
formation in Drosophila melanogaster. Nat. Genet. 36,
293-297.
2. Jindra, M., Gaziova, I., Uhlirova, M., Okabe, M.,
Hiromi, Y. and Hirose, S. (2004). Coactivator MBFl
preserves the redox-dependent AP-l activity duringoxidative stress in Drosophila. EMBO J. 23,3538-3547.
3. Asaoka, M. and Lin, H. (2004). Germline stem cells
in the Drosophila ovary descend from pole cells in theanterior region of the embryonic gonad. Development
131, 5079-5089.
4. Okabe, M. and Graham, A. (2004). The origin of
the parathyroid gland. Proc. Nat. Acad..Sci. USA 101,
17716-17719.
5. flliJ$lE~ (2004) @.jt/'\ 1) 77 1) - . *~~llRf-I.~~
tR187, 3-9.

30

Division of Developmental Genetics

Toshitaka Fujisawa, Chiemi Fujisawa, Eisuke
Hayakawa, Yukihiko Noro, Akemi Hayashiuchi,
Hiroshi Shimizu and Toshio TakahashP CSuntory
Institute for Bioorganic Research, Osaka, Japan)
Systematic purification and structural
determination has been carried out by using HPLC,
peptide sequencing and mass spectrometry. About 50
new peptides were purified and subjected to structural
determination. The peptide sequences were blasted
against cDNA database we have created and identified
7 new peptide genes and 3 novel neuropeptides. Outof 3 neuropeptides, the first one belonged to the alreadyknown GLWamide family, the second belonged to theHym-176 family and the last (Hym-65) to the hitherto
unknown family. The Hym-65 gene presumably encode
another peptide with a FRamide C-terminal motif.

Whole mount in situ hybridization indicated thatHym-65 gene is expressed in a subpopulation ofneurons throughout the body column. Hym-65 peptide
induced body column contraction of epithelial polypthat is only made of epithelial cells and gland cells but
has no other cells in the interstitial cell lineage
including neurons. Thus, this peptide appears to act
directly to the muscle cells presumably as aneurotransmitter at the neuro-muscular junctions. In
contrast, the new Hym-176 family peptide strongly
evoked contraction of ectodermal muscle of normal
polyp but not of epithelial polyp suggesting that this
peptide does not function as a neurotransmitter at theneuro-muscular junctions but acts via other neurons.
PUBLICATIONSPapers

1. Horibata, Y., Sakaguchi, K., Okino, N., !ida, H.,
Inagaki, M., Fujisawa, T., Hama, Y and Ito, M. (2004).
Unique catabolic pathway of glycosphingolipids in ahydrozoan, Hydra magnipapillata, involving endoglycoceramidase. J. BioI. Chern. 279(32), 33379-33389.
2. Shimizu, H., Koizumi, 0., and Fujisawa, T. (2004).
Three digestive movements in Hydra regulated by the
diffuse nerve net in the body column. ]. Compo PhysioI.
A 190 (8), 623-630.
3. Hayakawa, E., Fujisawa, C. and Fujisawa, T. (2004).
Involvement of Hydra achaete-scute gene, CnASHin the
differentiation pathway of sensory neurons in thetentacles. Development, Genes and Evolution 214,486-492.
4. Alexopoulos, H., Boettger, A, Fisher, S., Levin, A,
Wolf, A, Fujisawa, T., Hayakawa, S., Gojobori, T.,
Davies, J.A., David, C.N. and Bacon, J.P. (2004).
Evolution of gap junctions: the missing link? Current
Biology 14 (20), 879-880.
5. Takaku, Y, Hariyama, T. and Fujisawa, T. (2005).
Motility of endodermal epithelial cells plays a majo role
in reorganizing the two epithelial layers in Hydra.
Mech. Dev. 122, 109-122.
SOCIAL CONTRIBUTIONS AND OTHERS,g.f:m$'t.
The Zoological Society Prize for Toshitaka Fujisawa

C-b. Division of Gene Expression
Susumu Hirose GroupRESEARCH ACTIVITIES(1) Role of histone modifications and chromatin
remodeling in epigenetic gene expression

Takahiro Nakayama, Tsukasa Shimojima, Kazuma
Hanai, Kenichi Nishioka, Koji Akasaka1 and Susumu
Hirose (IDepartment of Biological Science, Universityof Tokyo, Tokyo, Japan)
In multicellular organisms some patterns of gene
expression are remembered in the chromatin structure
and maintained through many rounds of cell cycle.
These are termed epigenetic gene expression. Twotypes of epigenetic gene expression ar~ known in
Drosophila melanogaster: (1) maintenance of Hox gene
expression governed by Polyomb (Pc) and trithorax(trx) group genes, and (2) position effect variegation(PEV). Trithorax-like (Trl) encoding GAGA factor isinvolved in the both types of regulation. Thus Trl isa member of trx group and Trl mutation is an enhancerof PEV. We have shown that GAGA factor recruits
F ACT, a heterodimer of dSPT16 and dSSRP1,
facilitates chromatin remodeling around its bindingsite and contributes to the maintenance of Hox gene
expression.
When an actively transcribed white (w) gene isjuxtaposed with heterochromatin by chromosome
rearrangement such as uf"4, its expression is subject
to variable but heritable silencing. This is PEV. We
found that GAGA factor-FACT complex binds to a site
just down stream of w, facilitates chromatin
remodeling, and plays an important role in themaintenance of wexpression against heterochromatinsilencing.
RSF (remodeling and spacing factor) has been first
purified from human cells as a heterodimer of RSF1
and SNF2H (a human counterpart of Drosophila ISWI),
which can assemble regularly spaced nucleosome
arrays in vitro. To investigate in vivo role of RSF, weanalyzed its Drosophila counterpart, a heterodimer ofdRSF1 and ISWI. Genetic studies implicate Drosophila
RSF in PEV through facilitating the spreading of silent
chromatin.

Division of Developmental Genetics31(2) Chromatin transcription

Mikage Nakajima, Kenichi Nishioka, Tadashi Wada\
Hiroshi Handa' and Susumu Hirose ('Faculty of
Bioscience and Biotechnology, Tokyo Institute ofTechnology, Nagatsuda, Yokohama, Japan)
SPT6 is a transcription factor that is conserved
across species from yeast to human. While biochemical
studies on human SPT6 have revealed it to be atranscription elongation factor, genetic studies on )feast
SPT6 have suggested its role in the recovery of
nucleosome structure from deformation due to thepassage of RNA polymerase II through a nucleosome.
To study in vivo role of SPT6 in mulicellular organism,
we started genetic study on Drosophila SPT6. We also
continued studies on SPT6 and FACT using chromatintranscription in vitro in collaboration with Drs. T.
Wada and H. Handa.
(3) Role of DNA topology in the formation of active
chromatin

Kuniharu Matsumoto, Hirofumi Furuhashi, Youhei
Ogasawara and Susumu Hirose
Bulk DNA within the eukaryotic genome istorsionarily relaxed. However unconstrained negative
supercoils of DNA have been detected in few local
domains of the genome through preferential bindingof psoralen. To make genome-wide survey for suchdomains, we introduced biotinylated psoralen into
Drosophila salivary glands and visualized it on polytene
chromosomes with fluorescent streptavidin. We
observed bright psoralen signals on many transcriptionally active interbands and puffs. Upon heat shock,the signals appeared on heat-shock puffs. The signals
were resistant to RNase treatment but disappeared or
became faint by prior nicking of DNA or inhibition oftranscription with Cl: -amanitin. These data demonstrate that transcription-coupled, unconstrained
negative supercoils of DNA exist in approximately 150
loci within the interphase genome!)
Supercoiling factor (SCF) is a protein capable ofintroducing negative supercoils into DNA in conjunction with topoisomerase II. Localization of SCF on
puffs of polytene chromosomes has suggested its rolein the formation of active chromatin. Knocking downof the SCF function in vivo by RNAi implicated SCF

32

Division of Gene Expression

in twice activation of genes on male X chromosome.
In good agreement with this, overexpression of SCF
resulted in bloated appearance of the male X
chromosome.
(4) Nucleosomal histone kinase-!

Hitoshi Aihara', Takeya Nakagawa', Kiyoshi Yasui',Tsutomu Ohta2, Susumu Hirose, Naoshi Dhomae3,
Koji Takio\ Mayumi Kanek0 5, Yukio Takeshima5,
Masami Muramatsu6 and Takashi Ito' CDepartmentof Biochemistry, Nagasaki University School of
Medicine, Nagasaki, Japan; 2Medical Genomic Center,
National Cancer Center Research Institute, Tokyo,
Japan; 3The Institute of Physical and Chemical
Research, Wako-shi, Saitama, Japan; 4RIKEN Harima
Institute, Mikazuki-cho, Hyogo, Japan; 5Second
Department of Pathology, Hiroshima University Schoolof Medicine, Hiroshima, Japan; 6Saitama Medical
School Research Center for Genomic Medicine, Hidaka,
Saitama, Japan)
Posttranslational histone modifications areimportant for the regulation of many biologicalphenomena. Here, we show the purification andcharacterization of nucleosomal histone kinase-l(NHK-I). NHK-I has a high affinity for chromatin andphosphorylates a novel site, Thr 119, at the C terminusof H2A. Notably, NHK-I specifically phosphorylates
nucleosomal H2A, but not free H2A in solution. In
Drosophila embryos, phosphorylated H2A Thr 119 isfound in chromatin, but not in the soluble core histone
pool. Immunostaining of NHK-I revealed that it goesto chromatin during mitosis and is excluded fromchromatin during S phase. Consistent with the
shuttling of NHK-I between chromatin and cytoplasm,
H2A Thr 119 is phosphorylated during mitosis but not
in S phase. These studies reveal that NHK-I-catalyzed
phosphorylation of a conserved serine/threonine
residue in H2A is a new component of the histone codethat might be related to cell cycle progression.')(5) Tips in analyzing antibodies directed against specifichistone tail modifications

Kavitha Sarma', Kenichi Nishioka and Danny
Reinberg' ~Howard Hughes Medical Institute, Divisionof Nucleic Acids Enzymology, Department ofBiochemistry, University of Medicine and Dentistry ofNew Jersay, Piscataway, NJ, USA)
Histone methylation has been known to exist forover 40 years but the enzymes that catalyze thisreaction have remained elusive until the discovery thatSuv39H1 methylates histone H3 specifically at lysine
9. This discovery was followed by a bevy of papers
describing other methyltransferases specific fordifferent residues and their apparent function in vivo.
Histones are methylated as lysine as well as arginine
residues. Lysines can be mono-, di-, or tri-methylated
tn VlVO.

Studies on the effect of these modifications on gene
regulation have been greatly facilitated by the production of antibodies "specific" for the modified state. The
need to carefully characterize antibodies raised against
methylated histone peptides stems from the observation by several laboratories that these antibodies canbe promiscuous depending on several factors such asconcentration, peptide context, substrate, etc. Due tothis, several papers have been subject to scrutiny inrecent months as the specificity of the antibodies used
was questionable.
In this article, we present several parameters to
be taken into consideration and some useful hints for
systematic characterization of antibodies raised against
methylated histone peptides. Although we havefocused on antibodies against methylated residues:
H3-K4, H3-K27, and H4-K20, the methods andprocedures described herein are applicable for any
antibody directed against the histone tail modification,
including arginine methylation and lysine acetylation,
among other modifications. G)(6) Functional analysis of transcriptional coactivator
MBFl

Marek Jindra\ Ivana Gaziova\ Mirka Uhlirova\
Masataka Okabe2, Yasushi Hirome, Kenichi Tsuda3,
Toshiro Tsuji3, Ken-ichi Yamazake and Susumu
Hirose ~Department of Molecular Biology, Universityof South Bohemia and Institute of Entomology ASCR,
Ceske Budejovice, Czech Republic; ~ivision of
Developmental Genetics, National Institute of Genetics;
3Laobratory of Environmental Molecular Biology,
Graduate School of Environmental Earch Science,
Hokkaido University, Sapporo, Japan)

dimeric transcription factor AP-1, essential for celldifferentiation and immune and antioxidant defenses.
AP-l activity is controlled, in part, by the redox stateof critical cysteine residues within the basic regionsof Jun and Fos. The redox control is necessary since
replacement of these cysteines contributes to oncogenic
potential of Jun and Fos. How cells maintain theredox-dependent AP-1 activity at favorable levels is notknown. We show that the conserved coactivator MBF1
is a positive modulator of AP-l. Via a direct interaction
with the basic region of DrosophilaJun (D-Jun), MBF1
prevents an oxidative modification (S-cystenylcystenylation) of the critical cysteine and stimulates
AP-l binding to DNA. Cytoplasmic MBFl translocatesto the nucleus together with a transfected D-Junprotein, suggesting that MBF1 protects nascent D-Jun
also in Drosophila cells. Consistent with the role of AP-1
in antioxidant defense, mbfl-null mutants exhibit
shorter life than mbfr control in the presence ofhydrogen peroxide (H202). An AP-l-dependent processof epithelial closure becomes sensitive to H202 in flies
lacking mbfl. These results indicate that by preserving
the redox-sensitive AP-1 activity, MBFl provides anadvantage during oxidative stress. 3)
Multiprotein bridging factor 1 (MBF1) is known
to be a transcriptional co-activator that mediates
transcriptional activation by bridging between an
activator and a TATA-box binding protein (TBP). We
demonstrated that expression of every three MBF1
from Arabidopsis partially rescues the yeast mbfl
mutant phenotype, indicating that all of them function
as co-activators for GCN4-dependent transcriptional
activation. We also report that each of their subtypes
shows distinct tissue-specific expression patterns andresponses to phytohormones. These observations
suggest that even though they share a similar
biochemical function, each MBFl has distinct roles invarious tissues and conditions.7)

Basic leucine zipper proteins Jun and Fos form theDivision of Gene Expression33

(7) Differential functions of G protein and Baz-aPKC
signaling pathways in Drosophila neuroblast asymmetric
division
Yasushi Izumi\ N ao Ohta\ Asako Itoh-Furuya\
Naoyuki Fuse and Fumio Matsuzaki' ~Laboratoryfor
Cell Asymmetry, Center for Developmental Biology,
Institute of Physical and Chemical Research, andCREST, Japan Science and Technology Corporation,
Kobe, Japan)

Drosophila melanogaster neuroblasts (NBs)
undergo asymmetric divisions during which cell-fate
determinants localize asymmetrically, mitotic spindles
orient along the apical-basal axis, and unequal-sized
daughter cells appear. We identified here the first
Drosophila mutant in the Gy 1 subunit of heterotrimeric
G protein, which produces Gy llacking its membraneanchor site and exhibits phenotypes identical to thoseof G/3 13F, including abnormal spindle asymmetry andspindle orientation in NB divisions. This mutant fails
to bind G /3 l3F to the membrane, indicating anessential role of cortical Gl·G/3l3F signaling in asymmetric divisions. In Gy 1 and Gjj 13F mutant NBs,
Pins-G a i, which normally localize in the apical cortex,
no longer distribute asymmetrically. However, the otherapical cotpponents, Bazooka-atypical PKC-Part>-Inscuteable,
still remain polarized and responsible for asymmetric
Miranda localization, suggesting their dominant rolein localizing cell-fate determinants. Further analysis
of Gjjy and other mutants indicates a predominantrole of Partner of Inscuteable-G a i in spindleorientation. We thus suggest that the two apicalsignaling pathways have overlapping but different
roles in asymmetric NB division. 2)(8) Introduction ofp16'NK4a inhibits telomerase activity
through transcriptional suppression of human telomerase
reverse transcriptase expression in human gliomas
Masahiro Saito\ Kou Nakagawa\ Katsuyuki
Hamada2, Susumu Hirose, Hironobu Haradat, Shohei
Kohno\ Shigeyuki Nagato' and Takanori Ohnishi'.
(Department of (Neurosurgery and 20bstetrics andGynecology, Ehime University School of Medicine,
Ehime Japan)
The pl6 and p53 tumor suppressor proteins, which
are frequently altered in malignant gliomas, have been

34

Division of Gene Expression

noted as regulators of telomerase activity. However,the link between telomerase regulation and thesesuppressor proteins has not been adequately clarified.
In the present study, we demonstrated that pl6, as wellas p53, suppress telomerase activity through transcriptional regulation of human telomerase reverse
transcriptase (hTERT) in malignant glioma. To
examine the effect of pl6 and p53 on telomerase
activity, we utilized wild-type pl6 or p53 expression
plasmid and three human glioma cell lines differingin their PS3 and p16status. Restoring P16_significantly
reduced the level of telomerase activity of glioma cells.
Furthermore, cotransfection of the P16 gene with
5'-deletion constructs of the hTERTpromoter carrying
Spl binding sites, repressed the transcriptional activityof hTERTpromoter in pl6-deleted cells. In addition,
electrophoretic mobility shift assay revealed that pl6
expression inhibited the binding of Spl to theconsensus Spl responsive element, indicating that therecruitment of Spl to the h TERT proximal core
promoter is inhibited by pl6 protein. These results
were similar to those from a PS3 transfection study in
pS3-mutated cells. These findings implicate p16in thetranscriptional regulation of telomerase activity byinhibiting the function of Spl in human malignant
gliomas. 5)
PUBLICATIONSPapers

1. Aihara, H., Nakagawa. T., Yasui, K., Ohta, T.,

Hirose, S., Dhomae, N., Takio, K., Kaneko, M.,
Takeshima, Y., Muramatsu, M. and Ito, T. (2004).
Nucleosomal histone kinase-l phosphorylates H2A Thr
119 during mitosis in the early Drosophila embryo.
Genes Dev., 18, 877-888.
2. Izumi, Y., Ohta, N., Itoh·Furuya, A., Fuse, N. andMatsuzaki, F. (2004). Differential functions of G protein
and Baz-aPKC signaling pathways in Drosophila
neuroblast asymmetric division. f Cell BioI. 164,729-738.
3. Jindra, M., Gaziova, I., Uhlirova, M., Okabe, M.,
Hiromi, Y, Tsuda, K., Tsuji, T., Yamazaki, K. andHirose, S. (2004). Coactivator MBFl preserves theredox-dependent AP-l activity during oxidative stressin Drosophila. EMBO f, 23, 3538-3547.
4. Matsumoto, K. and Hirose, S. (2004). Visualizationof unconstrained negative supercoils of DNA on
polytene chromosomes of Drosophila. J Cell Sci., 117,

3797-3805.
5. Saito, M., Nakagawa, K., Hamada, K., Hirose, S.,
Harada, H., Kohno, S., Nagato, S. and Ohnishi, T. (2004).
Introduction of p1f1NK4a inhibits telomerase activity
through transcriptional suppression of humantelomerase reverse transcriptase expression in humangliomas. Int.]. Oncol., 24, 1213-1220.
6. Sarma, K., Nishioka, K. and Reinberg, D. (2004).
Tips in analyzing antibodies directed against specifichistone tail modifications. Methods Enzymol. 376,
255-269.
7. Tsuda, K., Tsuji, T., Hirose, S. and Yamazaki, K.
(2004). Three Arabidopsis MBFl homologs with distinct
expression profiles play roles as transcriptional
co-activators. Plant Cell Physiol., 45, 225-231.

SOCIAL CONTRIBUTION AND OTHERS

*f~

1.

/:1:Hjj*~:

2004-315398, ~IlA(1)~#J\ : JrlO)Jmt>"lt1v

DNAO)~ili~,~~~:JA*~'~*OO~,iliDA:*?¥~~~m~~~A._·~.A'TAa~~.

C-c. Division of Molecular and Developmental Biology
Koichi Kawakami GroupRESEARCH ACTIVITIES(1) Gene trap and enhancer trap approaches in zebrafish

Reviews
8. ~iIF.f;ij(~, j:JIjli4Ji{-(2004) 'HoxJl~-=ffH:::to~tQ
xl::V:;r.*'T-1 Y~.Aililj:mO)5}-=f,j~\H_J*BIJlfgI?¥, 23,1
155-1161.
9. j:JIjli4Ji{- (2004) 'xl:: v: ;r. -* 'T -1 Y ~ .A $IJ:mU~i::to~t:Q;(TJv1te.A ~/'O)~~~J/~~~?¥, 22,1361-1370.
10. JA* ~ (2004) '*;(:i""'T -1 Y ~ j!f~-=fO)~:m$lJ:mJ~~~?¥, 22, 1371-1375.
11. JA* ~(2004) 'MBFl ~~ittJ~?¥O)<b~Jr.,
211, 769.
Books
12. j:JIjli4JJl{- (2004) ';( T JHt e.A ~ /' Ii: J: Q ~ 0 '?T/'.~O)xl::V:;r.-*'T-1~.A.:m~.J"xl::V:;r.-*'T-1~.A". 1ti:k*mziH~.:L7"!J/,jJ-·7;r.77-~*

JiO, pp31-50.
13. JA* ~(2004) '4ii¥::I77~?<'-J "::t--7- F""f:f!l1.
MTQ4ii¥-17.A ~'?Y7"". E8f;j~IlAi3(~±*±), pp52-58.
EDUCATION

1. Dr. S. Hirose was invited to give a seminar on"Chromatin remodeling as a barrier against theepigenetic silencing" at National Institute for Basic
Biology, Okazaki, July, 2004 (in Japanese).
2. Dr. S. Hirose was invited to give a seminar on"GAGA factor-FACT complex as a boundary against
the spreading of silent chromatin" at Tokyo Instituteof Technology, Nagatsuda, August, 2004 (in Japanese).

Koichi Kawakami, Yasuyuki Kishimoto, Kazuhide
Asakawa, Tomoya Kotani, Saori Nagayoshi andAkihiro Urasaki
In order to understand the genetic basis fordevelopmental processes in vertebrate, we have beenusing a small tropical fish, the zebrafish, as a model
animal. Because it is practically possible to breed andmaintain very large numbers of fish in the lab, andbecause zebrafish embryos develop in wateli and aretransparent, forward genetic approaches (i.e., collecting
a large number of mutations affecting developmental
processes and analyzing genes responsible for the
mutant phenotypes) are feasible in the fish. The Tol2
element is a transposable element identified from thegenome of the medaka fish. Previously we found that
the Tol2 element encodes a fully functional transposase
and showed that the Tol2element can transpose into
the zebrafish genome in the germ lineage. To date, theTol2 element is the only natural transposon invertebrate for which an autonomous element has been
identified. We have been interested in developing novel
genetic methodologies in zebrafish using the Tol2
element. Using this Tol2transposon system, we haveconstructed various gene trap and enhancer trap
vectors, that contain either a splice acceptor or a basal
promoter and a promoterless fluorescent reporter gene(i.e., GFP, RFP, etc). The plasmid DNAs containing
these transposon vectors were coinjected in zebrafish
fertilized eggs with the transposase mRNA, andoffspring from the injected fish were analyzed forexpression of the reporter gene in specific tissues andDivision of Gene Expression

35

organs. The integration site of the transposon vector
and the gene trapped by the vector insertion werecloned rapidly by PCR-based techniques such as
inverse PCR and 5'RACE. These approaches should
facilitate our understanding of vertebrate morphogenesis and organogenesis.
(2) Genetic analysis of zebrafish maternal-effectmutations affecting early embryogenesis

Yasuyuki Kishimoto, Sumito Koshida\ Makoto
Furutani-Seike, Atsushi Kawakami 3, Hisato Kondoh 2.4
and Koichi Kawakami ~National Institutes of Natural
Sciences, 2Kondoh Differentiation Signaling Project,
JST, 3University of Tokyo, 40saka University)
Maternal-effect genes play essential roles in early
embryogenesis in many animals. We have carried out
a genetic screen for mutations affecting suchmaternal-effect genes employing an F3 screen strategy,
identifying six recessive mutations out of 60
mutagenized genomes (ref. 6). Among them, four
mutations were kept and analyzed. Three of themutations (acytokinesis mutations: ack"t5, ack"t62 andack"tll~ prohibited the cell cleavage in the embryosfrom homozygous females without affecting nuclear
divisions. These embryos are defective in generating
contractile rings; the ack"t62 mutation abolished theorganization of cortical F-actin, while other mutations
caused abortive contractile ring-like structures at
ectopic sites. Defects in contractile ring formation leads
to the absence of microtubule arrays at the prospective
cleavage plane. Thus, these mutations reveal thesequence of events associated with cytokinesis. It isremarkable that in all acytokinetic embryos, daughter
nuclei after mitosis are arranged in spatially normal
positions, and maternal vasa mRNAs accumulate in
the prospective planes of the first and the second cellcleavages despite complete loss of cytokinesis. This
indicates that the basic cell architectures of early
embryos are largely established by the autonomous
activities of the mitotic apparatus, without any
dependence on the cell cleavage machinery. The fourth
mutation, bobtail (btl), caused a strong reduction of
the tailbud outgrowth. The expression of myoD insomites and evel in tailbud is reduced in the mutant
embryos, whereas that of ntl in notochord and pax2.1
in pronephros is comparable between wild-type andmutant embryos, suggesting that the btlgene product

36

Division of Molecular and Developmental Biology

may regulate gene expression involved in myogenesis
and posterior mesodermal development. We also foundthat the third ventricle in the tectal region is inhibitedin the btl mutant embryo. In btl mutant embryos,
expression of MHB genes such as wntl, IgfS and pax2.1
are once activated at the 8-somite stage, but eliminatedfrom the MHB region afterward. Thus, the btl geneproduct is important for maintenance of the MHB gene
expression. btlhas been mapped on two BAC and PAC
clones on chromosome 17. We are currently focusing
on identification of the btl gene from these genomic
clones.
(3) Transposition of the Tol2 element in mouseembryonic stem cells

Koichi Kawakami and Tetsuo Nodal CTohoku
University)
It has not been known whether Tol2can transposein vertebrates other than fish. We investigatedtransposition of Tol2 in mouse embryonic stem (ES)cells. We constructed a transposon donor plasmid
containing a nonautonomous Tol2 element with theneomycin resistance gene and a helper plasmid capableof expressing the transposase, and introduced thedonor plasmid with various amounts of the helperplasmid by electroporation into mouse ES cells. The
number of G418-resistant ES colonies increased as theamount of helper plasmid was increased, in adose-dependent manner, indicating that thetransposase activity elevated the integration efficiency.
These G418-resistant ES colonies were cloned and thestructure of the junction of the integrated Tol2 element
and the genomic DNA was analyzed by inverse PCR.
In those clones, Tol2 was surrounded by mouse
genomic sequences and an 8-bp direct repeat was
created adjacent to the both ends of To12, indicatingthat Tol2 was integrated in the genome throughtransposition. The Tol2 transposon system is thus
active in mouse as well as in fish. We propose that itshould be used as a genetic tool to develop novel genetransfer, transgenesis and mutagenesis methods inmammals (ref. 1).

(4) A transposon-mediated gene trap approach identifies
developmentally regulated genes in zebrafish

Koichi Kawakami, Hisashi Takeda\ Noriko
Kawakami, Makoto Kobayashe, Naoto Matsudal andMasayoshi Mishinal eUniversity of Tokyo, and2Tsukuba University)
We developed a novel gene trap method inzebrafish using the Tol2transposon system. First, weestablished a highly efficient transgenesis method inwhich a plasmid DNA containing the Tol2transposon
vector and the transposase mRNA synthesized in vitro
were coinjected into one-cell stage embryos. The
transposon vector inserted in the genome could betransmitted to the Fl progeny at high frequencies, andregulated gene expression by a specific promoter could
be recapitulated in transgenic fish. Then weconstructed a transposon-based gene trap vector
containing a splice acceptor and the GFP gene,performed a pilot screen for gene trapping, andobtained fish expressing GFP in temporally andspatially restricted patterns. We confirmed theendogenous transcripts were indeed trapped by theinsertions, and the insertion could interfere withexpression of the trapped gene. We propose our genetrap approach should facilitate studies of vertebrate
development and organogenesis (ref. 2, 5).
(5) Excision of the Tol2 transposable element of the
medaka fish Oryzias lalipes in Xenopus laevis andXenopus tropicalis

Koichi Kawakami, Kosuke Imanaka\ Mari Itoh) and
Masanori Tairal CUniversity of Tokyo)
We demonstrated transposase-dependent excisionof the Tol2 element in Xenopus laevis and Xenopus(Silurana) tyopicalis embryos. We coinjected a plasmid
DNA containing a non autonomous Tol2 element andthe transposase mRNA synthesized in vitro into
two-cell-stage embryos, and analyzed DNA extracted
from the injected embryos by polymerase chain
reaction (PCR). We demonstrated that the Tol2 element
could be excised from the plasmid DNA in both X.
laevis and X tyopicalis only when it was coinjected withthe transposase mRNA. In most cases, a complete lossof the Tol2 sequence was accompanied by addition ofa short DNA sequence to the target sequence,indicating that transposase-dependent excisionoccurred. While these footprints were characteristic
to those created upon excision of transposons of thehAT family, the additional bases found in Xenopus
were longer and their structures were more
complicated than those detected upon excision inzebrafish. This may reflect differences in the activities
of host factors involved in either transposition, repair,or both between fish and frog. Our present study
suggests that the Tol2 transposon system should be
used as a novel genetic tool to develop transgenesis
and mutagenesis methods in Xenopus (ref. 3).
(6) The status, quality, and expansion of the NIH
full-length eDNA project: the Mammalian Gene Collection(MGC)

Gerhard D. S. and 113 others (The MGC project team).
The National Institutes of Health's Mammalian
Gene Collection (MGC) project was designed togenerate and sequence a publicly accessible cDNA
resource containing a complete open reading frame(ORF) for every human and mouse gene. The project
initially used a random strategy to select clones froma large number of cDNA libraries from diverse tissues.
Candidate clones were chosen based on.5'-EST
sequences, and then fully sequenced to high accuracy
and analyzed by algorithms developed for this project.
Currently, more than 11,000 human and 10,000 mousegenes are represented in MGC by at least one clone
with a full ORF. The random selection approach is now
reaching a saturation point, and a transition to
protocols targeted at the missing transcripts is now
required to complete the mouse and human collections.
Comparison of the sequence of the MGC clones toreference genome sequences reveals that most cDNA
clones are of very high sequence quality, although it
is likely that some cDNAs may carry missense variants
as a consequence of experimental artifact, such as
PCR, cloning, or reverse transcriptase errors. Recently,
a rat cDNA component was added to the project, andongoing frog (Xenopus) and zebrafish (Danio) cDNA
projects were expanded to take advantage of thehigh-throughput MGC pipeline (ref. 4). We have madea contribution to this project by constructing thezebrafish full-length cDNA library.

Division of Molecular and Developmental Biology

37

PUBLICATIONS

Fumitoshi Ishino

PapersGenomic imprinting memories persist in somatic
cells after fertilization, and need to be erased andre-established during germ cell development to reflect
the sex of the individual. For the study on there-establishment process of genomic imprinting
memory in paternal germ cell development, weanalyzed the patterns of each imprinted genes'
expression and DNA methylation of DMRs in 3
paternally imprinted regions using embryonic day 10
fetuses each derived from several stage of gonocytes
by nuclear transfer, and then could make a detailed
profile on the procedure of paternal genomic
imprinting memory establishing (in preparation).
Recently, establishment of germline stem (GS) cells
and ES-like cells from postnatal mouse testis has beenachieved by Kanatsu-Shinohara et a1. 21• Analysis of thepaternally imprinted regions in these cell lines showedthat the former had complete paternal imprinting
status but the latter had intermediate status between
paternal type and somatic cell type, indicating DNA
demetylation occurred in the process from GS cells to
ES-like cells 2l •1. Kawakami, K and Noda, T. (2004). Transpositionof the Tol2 element, an Ac-like element from theJapanese medaka fish Oryzias latipes, in mouseembryonic stem cells. Genetics 166,895-899.
2. Kawakami, K, Takeda, H., Kawakami, N.,
Kobayashi, M., Matsuda, N. and Mishina, M. (2004). A
transposon-mediated gene trap approach identifies
developmentally regulated genes in zebrafish.
Developmental Cell 7, 133-144.
3. Kawakami, K, Imanaka, K, Itoh, M. and Taira,M. (2004). Excision of the Tol2 transposable elementof the medaka fish Oryzias latipes in Xenopus laevis andXenopus tropicalis. Gene 338, 93-98.
4. Gerhard, D. S. and 113 others (The MGC projectteam). (2004). The status, quality, and expansion of the
NIH full-length eDNA project: The mammalian genecollection (MGC). Genome Research 14,2121-2127.
5. Kawakami, K (2004). Transgenesis and gene trap
methods in zebrafish by using the Tol2transposable
element. Methods in Cell Biology 77, 201-222.
6. Kishimoto, Y, Koshida, S., Furutani-Seiki, M. andKondoh, H. (2004). Zebrafish maternal-effect mutations
causing cytokinesis defect without affecting mitosisor equatorial vasa deposition. Mech Dev. 121, 79-89.

PUBLICATIONSPapers

Reviews

7.

JIIl:~-(2004)~'7'7

7 -1 'Y ~ ::1.(1)77 /7 ~ 3 T/V7 / ~ 7.:A.. f7 / ~ ~.:A. . 7'oT:::t~ ~ .:A.(1)~~OOJ
A,-$J~fTiiH~(.:r:.~ ·7-1- . .:r:..:A.),

8.

!JY~bl-th,

pp362-372.
-1 'Y ~ ~(1)Ili'f~)lll:~-(2004)-lt7'77

rll'*f~B"Jf.tm:f~-T~:m. mBJ{~~M~49, 2111-2116.

SOCIAL CONTRIBUTIONS AND OTHERS

1. K. Kawakami served as a Chair of the 6thInternational Conference on Zebrafish Development
and Genetics held at Madison, U.S.A. on July, 2004.

1. Kai, M., Irie, M., Okutsu, T., Inoue, K, Ogonuki,N., Miki, H., Yokoyama, M., Suzuki, R., Muguruma, K,
Fujita, H., Kohda, T., Ogura, A., Kaneko-Ishino, T. and
Ishino, F. (2004). The novel dominant mutation Dspd
leads to a severe spermatogenesis defect in mice. BioI.
Reprod. 70 (5), 1213-1221.
2. Kanatsu-Shinohara, M., Inoue, K, Lee, J.,
Yoshimoto, M., Ogonuki, N,. Miki, H., Kato, T., Shiro,B., Kazuki, Y., Toyokuni, S., Toyoshima, M., Niwa, 0.,Oshimura, M., Heike, T., Nakahara, T., Ishino, F.,
Ogura, A. and Shinohara, T. (2004). Generation ofpluripotent stem cells from neonatal mouse testis. Cell
119 (7), 1001-1012.

C-d. Division of Physiological
Fumitoshi Ishino Group

Reviews

RESEARCH ACTIVITIES~,'!-iai!tc~~ ~~(1)~~Ji.

3.

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1fi:%-~~~-T(2004)7/.Lv(/7'1)/

T-1/~-W.ft~~-T~:m(1).~4. 1fi:%-~~~-T,*f. ~¥~.~
211 (2) 177-181.

¥(Bf;!,], ~~~~(2004).:r:. ~~ ~~~~~OO~~~(1).:r:.~~~*7-1'Y~B"J~m/~~~~*7-1'Y~~.K~~7/A~n(1)m~ft

Epigenetic view of mammalian development

R(1)N~m~K~~k_~.~m~ft~~(1)f~=XA

38

Division of Molecular and Developmental Biology*f. ~m~Mi~~

22 (1) 34-38.

5. ~1Jr.-1J!l!fW-=f,

1J!l!fi£B.&:(2004)1lffl~~*f~B"J?'/

A~~~Ll~h~tt~~-=f~~~m*f~~~~K~kl<~t~~u~~~~~~:@:Htr~ ~ -t-~**?'/AM~*HIJffllI~~mB"JM

23 (4), 433-438.

Books
6. ~1Jr.-1J!l!fW-=f, $EBrJ!,1, 1J!l!fi£B.&:(2004)Springer
Review"S/!J - ;<r.:r. t::~ .:c.*~-{ ~ ~J3. ?' J A-1 /'7°!J/'~ -{ /' // (.fti:)nt(:mz~)Springer-Verlarg-Tokyo
pp.109-116.
7. ~1Jr.-1J!l!fW-=f, 1J!l!fi£B.&:(2004).:r.t::~.:c.*~-{ ~~(J);fjrl'l~~t§~5}ip~~Mi~~"S/!J - ;<rr±§~.:r. t::~.:c.
*~-{~~~b~~-?'/A~ • • ·m~R.~~.~~ti·lt, ,,*,~ ~ ~~b l) J (¥Ilt-tJ'Ht~) $±f± pp.97-102.

Cod. Division of Physiological Genetics
Yukiko Gotoh GroupRESEARCH ACTIVITIES(1) The Wnt-13 -catenin pathway directs neuronal differentiation of cortical neural precursor cells
Yusuke Hirabayashi, Yasuhiro Hoh, Norihisa
Masuyama and Yukiko Gotoh
Neural precursor cells (NPCs) have the ability to
self-renew and to give rise to neuronal and glial
lineages. The fate decision of NPCs between
proliferation and differentiation determines thenumber of differentiated cells and the size of eachregion of the brain. However, the signals that regulate
the timing of neuronal differentiation remain unclear.
Here we show that the Wnt signaling inhibits
self-renewal capacity of mouse cortical NPCs, andinstructively promotes their neuronal differentiation.
Overexpression of Wnt7a or of a stabilized form of{3 -catenin in mouse cortical NPC culture induced
neuronal differentiation even in the presence of FGF2,
a self-renewal-promoting factor in this system.
Moreover, blockade of the Wnt signaling led toinhibition of neuronal differentiation of cortical NPCs
in the developing mouse neocortex. Furthermore, the{3-catenin/TCF complex appears to directly regulate
the promoter of neurogeninl, a gene implicated incortical neuronal differentiation. Importantly, stabilized{3-catenin did not induce neuronal differentiation ofcortical NPCs at earlier developmental stages,
consistent with previous reports, suggestingstage-specific functions of the W nt signaling.
Collectively, these results reveal pivotal physiologicalroles for the W nt signaling in neuronal differentiation.
(2) Res binding to STAT3 mediates crosstalk between
Notch and JAK-STAT signaling
Sachiko Kamakura, Koji Oishi, Takeshi Yoshimatsu,
Norihisa Masuyama and Yukiko Gotoh
Although the Notch and JAK-STAT signaling
pathways fulfill overlapping roles in growth anddifferentiation regulation, no coordination mechanism
has been proposed to explain their relationship. Here
we show that STAT3 is activated in the presence ofactive Notch as well as the Notch effectors Hesl andHes5. Hes proteins associate with J AK2 and STAT3,
and facilitate complex formation between JAK2 andSTAT3, thus promoting STAT3 phosphorylation andactivation. Furthermore, suppression of endogenous
Hesl expression reduces growth factor induction of
STAT3 phosphorylation. STAT3 appears to be
essential for maintenance of radial glial cells anddifferentiation of astrocytes by Notch in the developing
central nervous system. These results imply that direct
protein-protein interactions coordinate cross-talkbetween the Notch-Hes and JAK-STAT pathways.
(3) Notch promotes survival of neural precursor cells
via mechanisms distinct from those regulatingneurogenesis
Koji Oishi, Sachiko Kamakura, Yuko Isazawa, Takeshi
Yoshimatsu, Keisuke Kuida, Norihisa Masuyama andYukiko Gotoh
During development of the mammalian brain,
many neural precursor cells (NPCs) undergo apoptosis.
The regulation of such cell death, however, is poorly
understood. We now show that the survival of mouseembryonic NPCs in vitro was increased by culture ata high cell density and that this effect was attributableto activation of Notch signaling. Expression of an active
form of Notchl thus markedly promoted NPC survival.
Hes proteins, key effectors of Notch signaling ininhibition of neurogenesis, were not sufficient for thesurvival-promoting effect of Notchl. This effect ofDivision of Physiological39

Notchl required a region of the protein containing theRAM domain and was accompanied by up-regulationof the anti-apoptotic proteins Bel-2 and Mel-I.
Moreover, knockdown of these proteins by RNA interference resulted in blockade of the Notchl-induced
survival. These results reveal a new function of Notch,
the promotion of NPC survival.
(4) JNK promotes Bax translocation to mitochondria
through phosphorylation of 14-3-3 proteins

Fuminori Tsuruta, Jun Sunayama, Yasunori Mori,
Norihisa Masuyama and Yukiko Gotoh
Targeted gene disruption studies have establishedthat the c-Jun NH2-terminal kinase GNK) is required
for the stress-induced release of mitochondrial
cytochrome c and apoptosis, and that the Bax
subfamily of Bel-2-related proteins is essential for
JNK-dependent apoptosis. However, the mechanism
by which JNK regulates Bax has remained unsolved.
Here we demonstrate that activated JNK promotes Baxtranslocation to mitochondria through phosphorylationof 14-3-3, a cytoplasmic anchor of Bax. Phosphorylationof 14-3-3 led to dissociation of Bax from this protein.
Expression of phosphorylation-defective mutants of
14-3-3 blocked JNK-induced Bax translocation to
mitochondria, cytochrome c release and apoptosis.
Collectively, these results have revealed a key
mechanism of Bax regulation in stress-induced
apoptosis.
PUBLICATIONS

Cell. BioI., 24, 4207-4220.
4. Oishi, K, Kamakura, S., Isazawa, Y, Yoshimatsu,
T., Kuida, K, Nakafuku, M., Masuyama, N. and Gotoh,
Y (2004). Notch promotes survival of neural precursor
cells via mechanisms distinct from those regulatingneurogenesis. Dev. BioI., 276, 172-184.
5. Tsuruta, F., Sunayama, ]., Mori, Y, Shimizu, S.,
Tsujimoto, Y, Yoshioka, K, Masuyama, N. and Gotoh,
Y. (2004). JNK promotes Bax translocation to
mitochondria through phosphorylation of 14-3-3
proteins. EMBO]. 23, 1889-1899.
6. Sunayama,]., Ando, Y, Itoh, N., Tomiyama, A.,
Sakurada, K, Sugiyama, A., Kang, D., Tashiro, F.,
Gotoh, Y, Kuchino, Y and Kitanaka, C. (2004). Physicaland functional interaction between BH3-only protein
Hrk and mitochondrial pore-forming protein p32. Cell
Death Differ. 11, 771-781.
7. Ogihara, T., Asano, T., Katagiri, H., Sakoda, H.,
Anai, M., Shojima, N., Ono, H., Fujishiro, M.,
Kushiyama, A., Fukushima, Y., Kikuchi, M., Noguchi,N., Aburatani, H., Gotoh, Y, Komuro, I. and Fujita, T.
(2004). Oxidative stress induces insulin resistance by
activating the nuclear factor-B pathway and disrupting
normal subcellular distribution of phosphatidylinositol
3-kinase. Diabetologia 5, 794-805.
Reviews

8. :kU-J.l=. f~!JiE8~-r (2004) ij&l~~ (: *HIJm~, ~~iflflJ, 22 (11), ppI634-1639.
9. :k;fifi¥¥-fI~, f~!JiE8~-r(2004)~1¥:/~-rJv, ~1*
O)W,¥:, 55 (5), pp450-451.
10. lilUt¥-r . -SfliijUii\;;, f~!JiE8~-r (2004) ;pjl~~*HIJmO)~'¥:~~~ilfIJff!lJTQNotch-Hes~Jm(:JAK-STAT3~JmO):7

A I- -:7, ~~~,¥:, 22 (15), pp2167-2170.
Papers

1. Hirabayashi, Y., Itoh, Y, Tabata, H., Nakajima,
K, Akiyama, T., Masuyama, N. and Gotoh, Y (2004).
The Wnt-beta-catenin pathway directs neuronaldifferentiation of cortical neural precursor cells.
Development, 131,2791-2801.
2. Kamakura, S., Oishi, K, Yoshimatsu, T.,
Nakafuku, M., Masuyama, N. and Gotoh, Y (2004). Hes
binding to STAT3 mediates crosstalk between Notch
and JAK-STAT signaling. Nat. Cell BioI. 6,547-554.
3. Miyagi, S., Saito, T., Mizutani, K, Masuyama, N.,
Gotoh, Y, lwama, A., Nakauchi, H., Masui, S., Niwa,
H., Nishimoto, M., Muramatsu, M. and Okuda, A.
(2004). The sox-2 regulatory regions display theiractivities in two distinct multipotent stem cells. Mol.

40

Division of Physiological Genetics

SOCIAL CONTRIBUTIONS AND OTHERS

Oncogene (Editor)
Journal of Biochemistry (Associate Editor)
Cell Structure and Function (Associate Editor)~'Jt~'Jt
B*fIllj,¥:~~JjjJJ'Jt~'Jt

B*3c'jg:W'¥:1!f0)~~JjjJJ'Jt ~'Jtp(2) Human specific amino acid changes found in 103
protein coding genes. Molecular Biology and Evolution

D. DEPARTMENT OF
POPULATION GENETICS

D-a. Division of Population Genetics
Naruya Saitou GroupRESEARCH ACTIVITIES

Saitou Naruya, Professor(1) DNA sequence and comparative analysis ofchimpanzee chromosome 22

The International Chimpanzee Chromosome 22
Consortium [Hidemi Watanabel • 2, Choong-Gon Kim,
Satoshi Gota, Takashi Kitano, Yuji Kohara, Naruya
Saitou and Yoshiyuki Sakaki 2]
ara Institute of
Science, and Technology, 2RIKEN,Genomic Sciences
Center)eN

Human-chimpanzee comparative genome researchis essential for narrowing down genetic changes
involved in the acquisition of unique human features,such as highly developed cognitive functions,bipedalism or the use of complex language. Here, wereport the high-quality DNA sequence of 33.3
megabases of chimpanzee chromosome 22. Bycomparing the whole sequence with the humancounterpart, chromosome 21, we found that 1.44% of
the chromosome consists of single-base substitutionsin addition to nearly 68,000 insertions or deletions.
These differences are sufficient to generate changes
in most of the proteins. Indeed, 83% of the 231 coding
sequences, including functionally important genes,
show differences at the amino acid sequence level.
Furthermore, we demonstrate different expansionof particular subfamilies of retrotransposons between
the lineages, suggesting different impacts ofretrotranspositions on human and chimpanzee
evolution. The genomic changes after speciation andtheir biological consequences seem more complex thanoriginally hypothesized. For details, see ref. (1)Takashi Kitano, Yuhua Liu\ Shintaroh Ueda2 andNaruya Saitou (IThe Jackson Laoratory, USA,
2Department of Biological Sciences, Graduate Schoolof Science, University of Tokyo)
We humans have many characteristics that are
different from those of the great apes. These
human-specific characters must have arisen throughmutations accumulated in the genome of our direct
ancestor after the divergence of the last common
ancestor with chimpanzee. Gene trees of human andgreat apes are necessary for extracting thesehuman-specific genetic changes. We conducted asystematic analysis of 103 protein-coding genes forhuman, chimpanzee, gorilla, and orangutan. Nucleotide
sequences for 18 genes were newly determined for thisstudy, and those for the remaining genes were retrieved
from the DDBJ/EMBLIGenBank database. The total
number of amino acid changes in the human lineage
was 147 for 26,199 codons (0.56%). The total number ofamino acid changes in the human genome was, thus,
estimated to be about 80,000. We applied theacceleration index test and Fisher's synonymous/
nonsynonymous exact test for each gene tree to detect
any human-specific enhancement of amino acidchanges compared with ape branches. Six and two
genes were shown to have significantly higher
nonsynonymous changes at the human lineage fromthe acceleration index and exact tests, respectively. We
also compared the distribution of the differences of thenonsynonymous substitutions on the human lineage
and those on the great ape lineage. Two genes weremore conserved in the ape lineage, whereas one gene
was more conserved in the human lineage. These
results suggest that a small proportion ofprotein-coding genes started to evolve differently in thehuman lineage after it diverged from the ape lineage.
For details, see ref. (2)(3) Genetic variation versus recombination rate in a
structured population of mice

Aya Takahashi, Yuhua Liu l and Naruya Saitou ~The
Jackson Laoratory, USA)The correlation between genetic variation and

DEPARTMENT OF POPULATION GENETICS

41

recombination rate was investigated in a structured
mouse population. Nucleotide sequence data from 19
autosomal DNA loci from eight inbred strains of mouse(Mus musculus) sampled from three major subspecies
were analyzed. The recombination rate was estimatedfrom the comparison of genetic and physical map
distances between markers flanking a lO-cM region ofeach locus. The strains were categorized into fourgroups (subpopulations) based on geography. By
partitioning the genetic diversity into within-group andamong-group variation, we detected a positive
correlation between the recombination rate andnucleotide diversity within groups. The level of
nucleotide differentiation among groups (G(ST))
showed a negative correlation with the rate ofrecombination. There was no significant correlation
between recombination rate and nucleotide diversity
when data from different subpopulations were pooled.
No correlation was detected between recombination
rate and nucleotide divergence of M. musculus and M.
spicilegus. These patterns deviate from the strict
neutral expectation under the constant nucleotide
substitution rate, and they are likely to have been
formed either by a hitchhiking effect of positively
selected mutants or by background selection ofdeleterious mutants occurring in a subdivided
population. Our series of comparisons show that
because a real population always has some structure,
incorporation of its information is important in
detecting non-neutral evolution. For details, see ref. (3)(4) Phylogenetic analysis of proteins associated in four
major energy metabolism systems: photosynthesis,oxidative phosphorylation, nitrogen metabolism andsulfur metabolism

Takeshi Tomiki and Naruya Saitou
The four electron transfer energy metabolism
systems, photosynthesis, aerobic respiration,
denitrification, and sulfur respiration, are thought tobe evolutionarily related because of the similarity ofelectron transfer patterns and the existence of somehomologous proteins. How these systems have evolved
is elusive. We therefore conducted a comprehensive
homology search .using PSI-BLAST, and phylogenetic
analyses were conducted for the three homologous
groups (groups 1-3) based on multiple alignments ofdomains defined in the Pfam database. There are five

42

Division of Population Genetics

electron transfer types important for catalytic reaction
in group 1, and many proteins bind molybdenum.
Deletions of two domains led to loss of the functionof binding molybdenum and ferredoxin, and thesedeletions seem to be critical for the electron transfer
pattern changes in group 1. Two types of electrontransfer were found in group 2, and all its member
proteins bind siroheme and ferredoxin. Insertion of thepyridine nucleotide disulfide oxidoreductase domain
seemed to be the critical point for the electron transfer
pattern change in this group. The proteins belonging
to group 3 are all flavin enzymes, and they bind flavinadenine dinucleotide (FAD) or flavin mononucleotide(FMN). Types of electron transfer in this group aredivergent, but there are two common characteristics.
NAD(P)H works as an electron donor or acceptor, and
FAD or FMN transfers electrons from/to NAD(P)H.
Electron transfer functions might be added to thesecommon characteristics by the addition of functional
domains through the evolution of group 3 proteins.
Based on the phylogenetic analyses in this study andprevious studies, we inferred the phylogeny of theenergy metabolism systems as follows: photosynthesis(and possibly aerobic respiration) and the sulfur/
nitrogen assimilation system first diverged, then thesulfur/nitrogen dissimilation system was produced
from the latter system. For details, see ref. (4)(5) Mitochondrial DNA Genealogy of Chimpanzees in
Nimba Mountains and Bossou, West Africa

Makoto Shimada, Sachiko Hayakawat, Tatyana Humle2,
Shiho Fujita3, Satoshi Hirata4, Yukimaru Sugiyama5
and Naruya Saitou (lPrimate Research Institute, Kyoto
University, 2University of Stirling United Kingdom,
3Faculty of Agriculture, Gifu University, 4Hayashibara
Biochemical Laboratories Inc., 5Tokai-Gakuen
University)
The chimpanzee populations of the Bossou andNimba regions in West Africa were geneticallysurveyed to 1) reveal the genetic relationship between
the Bossou and Nimba populations, and 2) elucidate
the evolutionary relationship between the BossouNimba and other West African populations. The
chimpanzee group at Bossou is characterized by its
small population size, no evidence of contact withneighboring populations, and no female immigration.
It is believed that most females and adolescent males

emigrate from this population. To reveal the genetic
signature of these characteristics, we examined thegenetic diversity of Bossou and two neighboring
populations (Seringbara and Yeale) in the Nim ba
Mountains by sequencing approximately 605 bp of themitochondrial DNA (mtDNA) control region. A totalof 20 distinct mtDNA variants were observed from 56
sequences of noninvasively collected, anonymous
samples. Nucleotide diversity in the Nimba Mountainpopulations was 0.03-0.04, and did not differ
significantly from that in the Bossou population. Veryfew mitochondrial variants are shared among the sites
sampled, which suggests that there is little gene flow
involving mtDNA. Nevertheless, no clear population
structures were revealed in either population. A
comparison with published sequences from West
African chimpanzees (Pan troglodytes verus) indicatesthat the variants observed in the Bossou and Nimba
regions are scattered throughout the subspecies, rather
than clustered according to geographic region. Thissuggests that the Bossou-Nimba populations derived
only recently from the common ancestral populationof the West African chimpanzees, and did not passthrough a bottleneck. For details, see ref. (5)(6) Evolution of 0 alleles of the human ABO bloodgroup geneFrancis Roubinet!, Stephanie Despiaut, Francesc
Calafe1l2, Fen Jin 3, Jaume Bertanpetie, Naruya Saitou
and Antoine Blanche~ ePaul Sabatier University,
Rangueil Hospital, 2Universitat Pompeu Fabra, 3The
Institute of Genetics and Developmental Biology)
To date, at least 40 different alleles 0 have beencharacterized on the basis of exon 6 and exon 7
sequences but not always for intron 6. STUDYDESIGN AND METHODS: Among 415 individuals,
from four continents (Africa, Europe, South America,and Asia), studied for exon 6 and exon 7 sequences,
we selected 46 individuals (of respective phenotypeso [39], AB [3], B [3], or A [1]) for sequencing 1800-bp
amplicons spanning exon 6, intron 6, and exon 7. The
amplicons were characterized either by direct
sequencing or after cloning when required. We defined
14 new intron 6 0 allele sequences, including fourrecombinant alleles. Based on sequence comparison,a phylogenetic network was constructed. It confirmed
recombinant allele origins and that most 0 alleles arederived by point mutations from the two worldwide
distributed alleles 001 and 002. CONCLUSION: Alleleo phylogenetic analysis suggests that the mostfrequent silencing mutation (deletion of a G in exon
6) appeared once in human evolution in the ancient 002
allele lineage and that allele 001 resulted from aninterallele exchange between 002 and AlOl. Assuming
constancy of evolutionary rate, diversification of therepresentative alleles of the three human ABO lineages(AI01, BIOI, and 002) was estimated at 4.5 to 6 million
years ago. For details, see ref. (6)(7) Comparative genetics of functional trinucleotide
tandem repeats in humans and apes

Aida Andres!, Marta Soldevilat, Oscar Lao!, Victor
VolpinP, Nayuya Saitou, Howard Jacobs!, Ikuo
Hayasaka2, Francesc CalafelP and Jaume Bertranpetie('Universitat Pompeu Fabra, 2Kumamoto Primate
Park, Sanwa Kagaku Kenkyusho)
Several human neurodegenerative disorders arecaused by the expansion of polymorphic trinucleotide
repeat regions. Many of these loci are functional shorttandem repeats (STRs) located in brain-expressed
genes, and their study is thus relevant from both amedical and an evolutionary point of view. The aimsof our study are to infer the comparative pattern ofvariation and evolution of this set of loci in order toshow species-specific features in this group of STRs
and on their potential for expansion (therefore, aninsight into evolutionary medicine) and to unravel
whether any human-specific feature may be identified
in brain-expressed genes involved in human disease.
We analyzed the variability of the normal range ofseven expanding STR CAG/CTG loci (SCA1, SCA2,SCA3-MJD, SCA6, SCA8, SCA12, and DRPLA) and twononexpanding polymorphic CAG loci (KCNN3 andNCOA3) in humans, chimpanzees, gorillas, andorangutans. The study showed a general conservationof the repetitive tract and of the polymorphism in thefour species and high heterogeneity among locidistributions. Humans present slightly larger alleles
than the rest of species but a more relevant diference
appears in variability levels: Humans are the species
with the largest variance, although only for the
expanding loci, suggesting a relationship between
variability levels and expansion potential. The
sequence analysis shows high levels of sequence

Division of Population Genetics

43

conservation among species, a lack of correspondence
between interruption patterns and variability levels,and signs of conservative selective pressure for someof the STR loci. Only two loci (SCAI and SCA8) show
a human specific distribution, with larger alleles than
the rest of species. This could account, at the sametime, for a human-specific trait and a predisposition
to disease through expansion. For details, see ref. (7)(8) Polymorphisms in the Trace Amine Receptor 4(TRAR4) Gene on Chromosome 6q23.2 Are Associatedwith Susceptibility to Schizophrenia

Jubao Duan!, Alan Sanders!, Cuiping Hou!, Naruya
Saitou, Takashi Kitano and Pablo Gejman! Wepartmentof Psychiatry and Behavioral Sciences, Northwestern
University)
Several linkage studies across multiple populationgroups provide convergent support for a susceptibility
locus for schizophrenia-and, more recently, for bipolar
disorder-on chromosome 6qI3-q26. We genotyped 192
European-ancestry and African American (AA)pedigrees with schizophrenia from samples that
previously showed linkage evidence to 6qI3-q26,
focusing on the MOXDl-STX7-TRARs gene cluster at
6q23.2, which contains a number of prime candidate
genes for schizophrenia. Thirty-one screening
single-nucleotide polymorphisms (SNPs) were selected,
providing a minimum coverage of at least 1 SNP/20
kb. The association observed with rs4305745 (P=.0014)
within the TRAR4 (trace amine receptor 4) generemained significant after correction for multiple
testing. Evidence for association was proportionallystronger in the smaller AA sample. We performed
database searches and sequenced genomic DNA in a
30-proband subsample to obtain a high-density mapof 23 SNPs spanning 21.6 kb of this gene. Single-SNP
analyses and also haplotype analyses revealed thatrs4305745 and/or two other polymorphisms in perfectlinkage disequilibrium (LD) with rs4305745 appear tobe the most likely variants underlying the associationof the TRAR4 region with schizophrenia. Comparative
genomic analyses further revealed that rs4305745 and/or the associated polymorphisms in complete LD withrs4305745 could potentially affect gene expression.
Moreover, RT-PCR studies of various human tissues,
including brain, confirm that TRAR4 is preferentiallyexpressed in those brain regions that have been

44

Division of Population Genetics

implicated in the pathophysiology of schizophrenia.
These data provide strong preliminary evidence that
TRAR4 is a candidate gene for schizophrenia;
replication is currently being attempted in additional
clinical samples. For details, see ref. (8)Kenta Sumiyama, Assistant Professor(9) Evolutionary functional genomic analysis of
cis-regulation in mammalian Dlx gene system

Kenta Sumiyama
Dlx genes are important regulators for mammalian
early development. Mammalian Dlx genes has been
duplicated through evolution and resulted in 6 genes
in 3 clusters that have overlapping but also distinct
functions. In branchial arches three Dlx gene clusters
are expressed in nested pattern that is essential toestablish positional information in jaw patterning. This
nested expression pattern is found only in higher
vertebrates so that studying genomic regulation
mechanism through cis-regulatory elements would beof great interest in mammalian jaw evolution. Recent
expansion of genome sequence information enables us
to compare intergenic noncoding genomic sequencesamong different species. Although intergenic regions
have been sometimes called "junk DNA" due to lackof knowledge about their functional importance, a large
number of sequence conservations have been foundin intergenic regions through comparative genomic
sequence analysis. Such conservations are likely to befunctional elements that are repressing, initiating ormaintaining gene expressions. I focus on genomic
analysis of cis-regulatory elements in Dlx3-7 geneclusters by using comparative genomics and further
functional analysis by using transgenic mouse system.
PUBLICATIONSPapers

1. The International Chimpanzee Chromosome 22
Consortium [Watanabe, H., Fujiyama, A, Hattori, M.,
Taylor, T.D., Toyoda, A, Kuroki, Y., Noguchi, H., Ben,
Kahla, A, Lehrach, H., Sudbrak, R., Kube, M., Taenzer,
S., Galgoczy, P., Platzer, M., Scharfe, M., Nordsiek, G.,
Blocker, H., Hellmann, L, Khaitovich, P., Paabo, S.,
Reinhardt, R., Zheng, H.-J., Zhang, X.-L., Zhu, G.-F.,
Wang, B.-F., Fu, G., Ren9, S.-X., Zhao, G.-P., Chen, Z.,

Lee, Y.-S., Cheong, J.-E., Choi, S.-H., Wu, K-M., Liu,
T.-T., Hsiao, K-J., Tsai, S.-F., Kim, C.-G., Oota, S.,
Kitano, T., Kohara, Y., Saitou, N., Park, H.-S., Wang,
S.-Y., Yaspo, M.-L. and Sakaki, Y.] (2004). DNA
sequence and comparative analysis of chimpanzee
chromosome 22. Nature, 429, 382-388.
2. Kitano, T., Liu, Y.-H., Ueda, S. and Saitou, N. (2004).
Human specific amino acid changes found in 103
protein coding genes. Molecular Biology and Evolution.
21, 936-944.
3. Takahashi, A., Liu, Y.-H. and Saitou, N. (2004).
Genetic variation versus recombination rate in a
structured population of mice. Molecular Biology and
Evolution, 21, 404-409.
4. Tomiki, T. and Saitou, N. (2004). Phylogenetic
analysis of proteins associated in four major energy
metabolism systems: photosynthesis, oxidative
phosphorylation, nitrogen metabolism and sulfur
metabolism. Journal of Molecular Evolution, 59,
158-176.
5. Shimada, M., Hayakawa, S., Hamle, T., Fujita, S.,
Hirata, S., Sugiyama, Y. and Saitou, N. (2004).
Mitochondrial DNA Genealogy of Chimpanzees in
Nimba Mountains and Bassou, West Africa. American
Journal of Primatology, 64,261-275.
6. Roubinet, F., Despiau, S.-P., Calafell, F., Jin, F.,
Bertanpetit, J., Saitou, N. and Blancher, A. (2004).
Evolution of 0 alleles of the human ABO blood groupgene. Transfusion, 44, 707-715.
7. Andres, A.M., Soldevila, M., Lao, 0., Volpini, V.,
Saitou, N., Jacobs, H.T., Hayasaka, I., Calafell, F. andBertranpetit, ]. (2004). Comparative genetics offunctional trinucleotide tandem repeats in humans andapes. Journal of Molecular Evolution, 59, 329-339.
8. Duan,]., Martinez, M., Sanders, A.R., Hou, C.,
Saitou, N., Kitano, T., Mowry, B.]., Crowe, R.R.,
Silverman, ].M., Levinson, D.F. and Gejman, P.V. (2004).
Polymorphisms in the Trace Amine Receptor 4(TRAR4) Gene on Chromosome 6q23.2 Are Associatedwith Susceptibility to Schizophrenia. American Journalof Human Genetics, 75, 624-638.
9. Abe, K, Noguchi, H., Tagawa, K, Yuzuriha, M.,
Toyoda, A., Kojima, T., Ezawa, K, Saitou, N., Hattori,
M., Sakaki, Y., Moriwaki, K and Shiroishi, T. (2004).
Contribution of Asian mouse subspecies Mus
musculus molossinus to genomic constitution of strain
C57BL/6J, as defined by BAC end sequence-SNP
analysis. Genome Research, 14, 2239-2247.
10. Imanishi, T. and many other authors including

Saitou, N. and Sugano, S. (2004). Integrative annotationof 21,037 human genes validated by full-length cDNA
clor~es. PLoS Biology, 2, 1-20.
11. Nakajima, T., Wooding, S., Sakagami, T., Emi, M.,
Tokunaga, K, Tamiya, G., Ishigami, T., Umemura, S.,
Munkhbat, B., Jin, F., Guan-Jun, J., Hayasaka, I., Ishida,T., Saitou, N., Pavelka, K, Lalouel, ].M., Jorde, L.B. andInoue, I. (2004). Natural selection and population history
in the human angiotensinogen gene (AGT): 736
complete AGT sequences in chromosomes fromaround the world. American Journal of Human
Genetics, 74, 898-916.
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EDUCATION

Division of Population Genetics45

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Toshiyuki Takano GroupRESEARCH ACTIVITIES(1) Inter-locus nonrandom association ofpolymorphisms
in Drosophila chemoreceptor genes

Toshiyuki Takano-Shimizu, Yuriko Ishii, Akira
Kawabet, Masanobu Itoh 2, Nobuyuki Inomata3, Rumi
Kondo· and Yutaka Inoue5~University of Edinburgh,
2Kyoto Institute of Technology, 3Kyushu University,·Ochanomizu University, 50saka University of Foreign
Studies)
Some forms of multilocus selection with epistasis,such as truncation selection, can effectively reduce themutation load. Many quantitative characters, including
complex genetic diseases, are likely to be subje~t tothese types of selection. However, direct measurementof selection in natural populations is difficult and the46

effect of epistasis on within-species variations remains
unclear. Epistatic interaction in the fitness effect cangenerate linkage disequilibrium (LD). Therefore, we
may detect the action of natural selection from its
amount and pattern. We found a large number ofinter-locus nonrandom associations between
polymorphisms in 98 Drosophila chemoreceptor genes.
LD was examined in two fly samples collected at thesame location, but in different seasons. The amountof LD was much larger in the spring sample than in
the autumn one. This between-sample difference wasmuch more striking for the replacement polymorphisms than for the silent polymorphisms. The
difference between the replacement and silent
polymorphisms could not be attributed to differencesin the mean marker distances. We also found asignificant excess of associations between one frequentand one less common allele for the replacement
polymorphisms, but not for the silent polymorphisms.
It is unlikely that a simple seasonal bottleneck could
explain all these differences in the scale of LD between
the samples and between the replacement and silent
polymorphisms. Natural selection is suggested to playa significant role in shaping the pattern of LD observedin this study. For details, see Paper 4.

Division of Population Genetics(2) A high frequency null mutant of an odorant-binding
protein, Obp57e, in Drosophila melanogaster

Aya Takahashi and Toshiyuki Takano-Shimizu
We have found a null mutant of anodorant-binding protein, Obp57e, in Drosophilamelanogaster. This frameshift mutation, which is a 10
bp deletion in the coding region, is at a high frequency
in the Kyoto population, and is also present in Taiwan
and Africa. We have sequenced an about 1.5kb region
including the tandemly duplicated gene, ObP57d, from
16 inbred lines sampled in Kyoto, Japan. The analyses
showed a peak of nucleotide diversity and strong
linkage disequilibrium around this mutation. Thispattern suggests an elevated mutation rate or an
influence of balancing selection in this region. The levelof nucleotide divergence between D. melanogasterand
D. simulans does not support the former possibility.
Thus, this presence/absence polymorphism may be
due to balancing selection, which takes advantage ofthe relatively weak functional constraint in membersof a large gene family. In addition, the ObP57d gene

region showed an excess of high frequency derived
mutants that is consistent with a pattern predictedunder positive natural selection.
(3) Analyses of the mechanism and genetics of matechoice by Drosophila melanogaster males

Aya Takahashi and Toshiyuki Takano-Shimizu
We surveyed multiple strains of D. melanogasterand D. simulans for differences in heterospecificcopulation frequencies, and identified two D.
melanogaster inbred lines between which mating
frequencies of males against D. simulans females differ
by a large extent. One line was originated in West
Africa and the other was collected in Taiwan. We also
performed an intraspecific mating experiment andfound that males from these two lines have different
mating preference against conspecific females ofdifferent lines. A cuticular hydrocarbon transfer
experiment revealed that this preference is likely to beaffected by the differences in cuticular hydrocarbon
components of the females. These two lines have been
subjected to over 20 generations of sib-mating, and aresuitable materials for genetic mapping. Our genetic
analyses showed that the third chromosome has amajor effect on these behavioral differences of males.
(4) Null alleles at Drosophila chemoreceptor genes

Akira Kawabel , Yuriko Ishii and Toshiyuki
Takano-Shimizu eUniversity of Edinburgh)
We determined a total of about 85-kb sequence ofno chemoreceptor genes of seven D. melanogasterstrains and found 16 putative null alleles at 13 loci: 4
nonsense mutations, 7 small insertions/deletions
causing frame shifts, and 5 long deletions encompassing parts of coding sequence. The seven strains
analyzed in the present study had at least one nullallele and, in total, 27 null alleles. Because wedetermined only one-half of the coding sequences, there
might be eight null alleles per haploid genome of D.
melanogaster. Importantly, the ratio (7tH/ 7ts) of theaverage nucleotide diversity (7t) at the replacementsites to that at the synonymous sites suggested thatthe null alleles did not necessarily appear only in very
low constraint loci, although the Or85e seems to be a
pseudogene. On the other hand, the frequencies of mostof these putative null alleles in a natural population
are not very low: 36% of a deletion in Or98b, 30% of an
insertion in Gr2a, 1% of a nonsense mutation in Gr22b,
10% of a nonsense mutation in Gr36c, 39% of a deletionin Gr39aA, 8% of a nonsense mutation in Gr47a, 12%of a nonsense mutation in Gr58a and 4% of a deletionin Gr85a. Then, the frequencies of null homozygotes
are not expected to be very low. In sum, the results
suggested that selection is weak but not completely
relaxed in the Or and Gr genes. This condition may
allow rapid evolution of the chemoreceptor genes. We
found a single putative null mutation in 54 loci of asingle D. simulans strain, which did not include Or85e.
The expected number of null alleles at the chemoreceptor genes in D. simulans was comparable to that in D.
melanogaster.
(5) Genetic and molecular basis of sex-comb-toothnumber variation in Drosophila

Haruki Tatsuta\ Yuriko Ishii and Toshiyuki
Takano-Shimizu ~National Institute of Environmental
Studies)
The sex comb on the forelegs of Drosophila males
is a secondary sexual trait, and the number of teethon these combs varies greatly within and between
species. To understand the relationship between
the intra- and interspecific variations, we did quantitative trait loci (QTL) analyses of intra-specificsex-comb-tooth-number variations in five mapping
populations with two inbred Drosophila simulans
strains of large and small numbers of sex-comb teeth(whole-genome F?, two second-chromosome substitution
strains, and two third-chromosome congenic strains).
From these mapping studies, we identified threemajor QTLs on the third chromosome and one ofthem likely matched the QTL responsible for thesimulans-mauritiana differences. We focused ouranalyses on this region. Linkage disequilibrium survey
for association study is now under way.
PUBLICATIONSPapers
1. Kobayashi, S., Noro, Y., Nagano, H., Yoshida, K.,
Takano-Shimizu, T., Kishima, Y. and Sano, Y.
Evidence for an evolutionary force that prevents
epigenetic silencing between tail-to-tail rice genes with

Division of Population Genetics47

a short spacer. Gene (in press).
2. Takahashi, A., Liu, Y.-H. and Saitou, N. (2004).
Genetic variation versus recombination rate in a
structured population of mice. Mol. BioI. Evol. 21,
404-409.
3. Takahashi, A. and Ting, C.-T. (2004). Genetic basisof sexual isolation in Drosophila melanogaster. Genetica
120, 273-284.
4. Takano-Shimizu, T., Kawabe, A., Inomata, N.,
Nanba, N., Kondo, R., Inoue, Y. and Itoh, M. (2004).
Inter-locus nonrandom association of polymorphisms
in Drosophila chemoreceptor genes. Proc. Natl. Acad.
Sci. USA 101, 14156-14161.
EDUCATION

1. Dr. T. Takano gave a lecture at The Graduate
University for Advanced S'tudies, April, 2004 (inJapanese).
2. Dr. T. Takano was invited to give a lecturein "Shonan Lecture: Diversity and Evolution ofPopulations" at The Graduate University for Advanced
Studies, August, 2004.
3. Dr. T. Takano organized a Collaborative Research
Meeting at the National Institute of Genetics, Mishima,
June, 2004.
4. Dr. T. Takano gave a two-day training course forMishima Kita Junior High School students, October,
2004.
SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. T. Takano served as a member of the steering committee of National Bio-Resource Project"DROSOPHILA".
2. Drs. T. Takano and A. Takahashi served as
members of the election administration committee of
the Genetics Society of Japan.
3. Dr. T. Takano served as a member of theexamination specialist committee for National
Personnel Authority.

48

Division of Population Genetics

D-b. Division of Evolutionary Genetics (Until May)
Toshimichi Ikemura GroupRESEARCH ACTIVITIES(1) Phylogenetic classification of environmental DNA
sequences including those from the Sargasso Sea and of
rDNA sequences with Self-Organization Map (SOM)

Takashi Abe, Jian-Ping Song and Toshimichi Ikemura
The self-organizing map (SOM) is a powerful toolfor clustering and visualizing high-dimensional
complex data on a two-dimensional plane.
Oligon ucleotide freq uency is an example ofhigh-dimensional data, and we developed an SOM asa novel bioinformatics strategy to capture and visualize
phylogenetic diversity of a wide variety of microbial
genomic sequences obtained from an environmental
sample. First we constructed SOMs for tetranucleotide
frequencies in approximately 200,000 5-kb sequence
fragments obtained from 1500 prokaryotes for which
at least 10 kb of genomic sequence has been depositedin DDBJ/EMBLIGenBank (a total of 1.05 Gb). The
sequences could be classified primarily according to
25 phylogenetic groups without information regarding
the species. The classification was possible without
orthologous sequence sets, and therefore, is especially
useful for phylogenetic classification of novel
seq uences from poorly characterized species inenvironmental and clinical samples. We used the SOM
method to classify 810,000 sequences recently reported
by Venter et al. for pooled DNA samples from theSargasso Sea near Bermuda. Phylogenetic diversity andnovelty of the Sargasso Sea sequences was visualized
on a single map, and sequences that were derived froma single genome but cloned independently could bereconstructed in silico.
Phylogenetic classification of genomic sequence
fragments from a groundwater metagenome library
and a human gut library was conducted using thetetranucleotide SOM. (For details, see Nature
Biotechnology, 2005: Canadian Journal ofMicrobiology,
2005)
Because 16S rDNA sequences were highly
conserved during evolution, their sequences have beenused for detailed phylogenetic classification ofprokaryotic species, including uncultured prokaryotes.

Approximately 20,000 16S rDNA sequences longer than
1 kb from 6,100 known prokaryotic species have been
compiled in DDBJ/EMBLIGenBank. We constructed
a tetranucleotide SaM with these 16S rDNA sequences
after normalization for the sequence lengths. Clear
clustering according to phylogenetic group wasobserved; 97% of sequences were classified into thecorrect group territory on the tetranucleotide SaM. The
finding that the hit level of 16S rDNA classification into
the correct group territory was higher than that ofgenomic sequences may indicate that the occurrenceof horizontal transfer of rDNAs, if present, is lower
than that of other genome portions. Combination of
SOMs for genomic and 16S rDNA sequences will
provide a tool for detailed phylogenetic studies ofgenomic sequence fragments from environmental
uncultured prokaryotes.
(2) 80M classification of mammalian genomic and cDNA
sequences according to function
Takashi Abe, Yoko Kosaka and Toshimichi Ikemura
In addition to protein-coding sequences (CDSs), 5'
and 3' untranslated regions (UTRs) and transcription
regulatory regions of eukaryotic genes have attracted
wide attention because of their crucial roles intranscriptional and post-transcriptional regulation ofgene activity. We constructed SOMs of tri- andtetranucleotide frequencies in all 1-kb sequences
derived from human or mouse genome. Whensequences of 5' and 3' UTRs, CDSs, and introns, as wellas 1-kb upstream regions from transcriptional startsites, were mapped on these SOMs, a major portionof the sequences were clustered primarily according
to the functional categories. This showed that SaM
could detect sequence characteristics specific to the
distinct functional categories. Importantly, the territoryof each functional category was divided into multiple
zones. Furthermore, when we constructed tetra- andpentanucleotide SOMs for human and mouse eDNA
sequences, protein-coding and -noncoding eDNA
sequences tended to be separated from each other.
Because no information other than oligonucleotide
frequencies is required for the map generation, SaM
is a novel in silico method useful for identifying
characteristic and diagnostic sequences for individualfunctional categories. Function-unknown sequences
colocalized in a zone where sequences of known

functions are clustered can be assumed presumably
to have similar functions.
(3) Compilation of "GenomeWordDictionary"Takashi Abe, Yoko Kosaka, Kiyomi Kita andToshimichi Ikemura
In order to know the biological meaning ofcharacteristic oligonucleotide sequences specified by
SaM, it is important to systematically refer toliteratures focusing on experimental studies of theoligonucleotide sequences. With regard to genomes on
which experimental studies have advanced, manysignal and motif sequences with functional activity are
known. Referring to the SaM data, the signal and motif
sequences that are not experimentally identified may
be presumed newly. Furthermore, based on the SaM
data, exploration of signal and motif candidates on the
genomes of which experimental studies have notadvanced may be possible. It should also be noted thata large amount of exploration information itself whichis accumulated in the process of the above literature
exploration should become significant and valuable
data sets. We have compiled a new database called"GenomeWordDictionary" in the form of collection of
the exploration results of papers which describe
experimental facts regarding each oligonucleotide
sequence. In the dictionary, oligonucleotides being
composed of 4 letters (A, T, G, and C) are arranged in
the alphabetical order, and the abstract of the paper
reporting the experimental results are compiled. Since
we have created the database using Oracle and Postgres
relational-database systems, we can extract a
dictionary for a particular phylogenetic group; e.g. Rice
GenomeWordDictionary and Fly GenomeWordDictionary.
PUBLICATIONSPapers

1. Uchiyama, T., Abe, T., Ikemura, T. and Watanabe,
K. (2005). Substrate-induced gene-expression screeningof environmental metagenome libraries for isolationof catabolic genes, Nature Biotechnology, 23, 88-93.
2. Abe, T., Kanaya, S., Kinouchi, M. and Ikemura, T.
(2004). Genome informatics for unveiling hidden
genome signatures. Proceedings of the Institute of
Statistical Mathematics, 52,207-215.
3. Abe, T., Ikemura, T., Kanaya, S., Kinouchi, M. andDivision of Evolutionary Genetics

49

Sugawara, H. (2004). Novel genome informatics forunveiling hidden signatures in genome sequences:self-organizing map (SOM) of oligonucleotide
frequencies, Proceedings ofInformation-Based Induction
Sciences, 94-99.
4. Hayashi, H., Abe, T., Sakamoto, M., Ohara, H.,
Ikemura, T., Sakka, K. and Benno, Y (2005). Directcloning of genes encoding novel xylanases from humangut, Canadian Journal of Microbiology (in press).

cDNA clones. This includes, Zebrafish3), red alga7), andmalaria1~. We also helped isolating various full-length
cDNA clones1)5) 8) 9) 10) 17) 181.
Finally, we made very interesting finding thatsome of the small open reading frames (ORF) within
5' un-translated region (UTR) of mRNA do make small
proteins. About half of the mRNAs have small ORF
in their 5' UTR. Thus, there is a possibility that
unexpectedly high number of small proteins istranslated from 5' UTR.

Journal Editor

5. GENE (Elsevier)
6. DNA Sequence (Harwood Academic Publisher)

PUBLICATIONSPapers

D-c. Division of Theoretical Genetics
Sumio Sugano GroupRESEARCH ACTIVITIES(1) Functional Genomics Based on the full-length cDNA

collection

Sumio Sugano
We have been constructing full-length cDNA
libraries from various human tissues and cells using
oligo-capping method. From these libraries, we havebeen isolating a number of clones and determining
their 5' end sequences. Using these 5' end sequencedata, we have selected about 30,000 clones anddetermined entire sequences. Part of the result of thisactivity and initial analysis of the fully sequenced
clones were published this yearll ). We also gathered
an international consortium, H-invitational, for the
annotation of human full-length cDNAs with Takashi
Gojobori and Nomura Nobuo and made H-invitational
database6).
The 5' end sequence data of full-length cDNAs areindispensable for identifying transcription start sites(tss). We made and are maintaining database of tss,
DBTSS, with Kenta Nakai 15). Using this database, we
compared human and mouse tss and devised andevaluated promoter prediction programs13)14) 2).
Furthermore, we collaborated with Shin-Ichi
Hashimoto and developed a method to isolate a 5' tagsof mRNAs 4).
We also collaborated with various groups to makefull-length cDNA libraries and to collect full-length

50

Division of Evolutionary Genetics

1. Adachi, S., Jigami, T., Yasui, T., Nakano, T.,
Ohwada, S., Omori, Y., Sugano, S., Ohkawara, B.,
Shibuya, H., Nakamura, T. and Akiyama, T., (2004).
Role of a BCL9-related beta-catenin-binding protein,
B9L, in tumorigenesis induced by aberrant activationof Wnt signaling. Cancer Res. 64,8496-8501.
2. Bajic, VB., Tan, SL., Suzuki, Y and Sugano, S.
(2004). Promoter prediction analysis on the wholehuman genome. Nat Biotechnol. 22, 1467-1473.
3. Gerhard, DS., Wagner, L., Feingold, EA, Shenmen,
CM., Grouse, LH., Schuler, G., Klein, SL., Old, S.,
Rasooly, R., Good, P., Guyer, M., Peck, AM., Derge, JG.,
Lipman, D., Collins, FS., Jang, W., Sherry, S., Feolo, M.,
Misquitta, L., Lee, E., Rotmistrovsky, K., Greenhut, SF.,
Schaefer, CF., Buetow, K., Bonner, TI., Haussler, D.,
Kent, J., Kiekhaus, M., Furey, T., Brent, M., Prange, C.,
Schreiber, K., Shapiro, N., Bhat, NK., Hopkins, RF.,Hsie, F., Driscoll, T., Soares, MB., Casavant, TL.,
Scheetz, TE., Brown-stein, MJ., Usdin, TB., Toshiyuki,
S., Carninci, P., Piao, Y., Dudekula, DB., Ko, MS.,
Kawakami, K., Suzuki, Y, Sugano, S., Gruber, CE.,
Smith, MR., Simmons, B., Moore, T., Waterman, R.,
Johnson, SL., Ruan, Y., Wei, CL., Mathavan, S.,
Gunaratne, PH., Wu, J., Garcia, AM., Hulyk, SW., Fuh,
E., Yuan, Y., Sneed, A, Kowis, C., Hodgson, A, Muzny,DM., McPherson, J., Gibbs, RA, Fahey, J., Helton, E.,
Ketteman, M., Madan, A, Rodrigues, S., Sanchez, A,
Whiting, M., Madari, A, Young, AC., Wetherby, KD.,
Granite, SJ., Kwong, PN., Brinkley, CP., Pearson, RL.,
Bouffard, GG., Blakesly, RW., Green, ED., Dickson,
MC., Rodriguez, AC., Grimwood, J., Schmutz, J., Myers,
RM., Butterfield, YS., Griffith, M., Griffith, OL.,
Krzywinski, MI., Liao, N., Morrin, R., Palmquist, D.,
Petrescu, AS., Skalska, U., Smailus, DE., Stott, JM.,
Schnerch, A, Schein, JE., Jones, SJ., Holt, RA., Baross,Ao, Marra, MA, Clifton, So, Makowski, KA, Bosak, So
and Malek,]. (2004)0 MGC Project Teamo The status,
quality, and expansion of the NIH full-length cDNA
project: the Mammalian Gene Collection (MGC)o
Genome Reso 14,2121-21270
4o Hashimoto, SI., Suzuki, Y, Kasai, Y, Morohoshi,
K, Yamada, To, Sese, Jo, Morishita, So, Sugano, So andMatsushima, K (2004)0 5'-end SAGE for the analysisof transcriptional start siteso Nat BiotechnoI. 22,
1146-1149, Epub Aug 8o
50 Huang, DY, Kuo, YYo, Lai, JSo, Suzuki, Yo, Sugano,
So and Chang, ZFo (2004)0 GATA-l and NF-Y cooperate
to mediate erythroid-specific transcription of Gfi-lB
geneo Nucleic Acids Reso 32,3935-39460
6o Imanishi, To, Itoh, To, Suzuki, Y, O'Donovan, Co,
Fukuchi, So, Koyanagi, KOo, Barrero, RAo, Tamura, To,
Yamaguchi-Kabata, Y, Tanino, Mo, Yura, K, Miyazaki,
So, Ikeo, K, Homma, K, Kasprzyk, A, Nishikawa, To,
Hirakawa, Mo, Thierry-Mieg, Jo, Thierry-Mieg, Do,
Ashurst, Jo, Jia, L., Nakao, Mo, Thomas, MA, Mulder,
No, Karavidopoulou, Yo, Jin, L., Kim, So, Yasuda, To,
Lenhard, B., Eveno, Eo, Suzuki, Yo, Yamasaki, Co,
Takeda, JI., Gough, Co, Hilton, Po, Fujii, Yo, Sakai, Ho,
Tanaka, So, Amid, Co, Bellgard, Mo, Bonaldo, Md, Mo,
Bono, Ho, Bromberg, SK, Brookes, AJo, Bruford, Eo,
Carninci, Po, Che1ala, Co, Couillault, Co, Souza, SJo,
Debily, MA, Devignes, MDo, Dubchak, I., Endo, To,
Estreicher, A, Eyras, E., Fukami-Kobayashi, K, R,
Gopinath, Go, Graudens, Eo, Hahn, Yo, Han, Mo, Han,
ZGo, Hanada, K, Hanaoka, Ho, Harada, Eo, Hashimoto,
K, Hinz, Do, Hirai, Mo, Hishiki, To, Hopkinson, I.,
Imbeaud, So, Inoko, Ho, Kanapin, Ao, Kaneko, Yo,
Kasukawa, To, Kelso,]., Kersey, Po, Kikuno, R, Kimura,K, Koro, B., Kuryshev, Vo, Makalowska, I., Makino, To,
Mano, So, Mariage-Samson, R, Mashima,]., Matsuda,
Ho, Mewes, HWo, Minoshima, So, Nagai, K, Nagasaki,
Ho, Nagata, No, Nigam, R, Ogasawara, 0o, Ohara, 0o,
Ohtsubo, Mo, Okada, No, Okido, To, Oota, So, Ota, Mo,
Ota, To, Otsuki, To, Piatier-Tonneau, Do, Poustka, A,
Ren, SXo, Saitou, No, Sakai, K, Sakamoto, So, Sakate,
R, Schupp, I., Servant, Fo, Sherry, So, Shiba, R, Shimizu,
No, Shimoyama, Mo, Simpson, No, Soares, Bo, Steward,
Co, Suwa, Mo, Suzuki, Mo, Takahashi, A, Tamiya, Go,
Tanaka, H., Taylor, To, Terwilliger, JDo, Dnneberg, Po,
Veeramachaneni, Vo, Watanabe, So, Wilming, L.,
Yasuda, No, Yoo, HSo, Stodolsky, Mo, Makalowski, Wo,
Go, Mo, Nakai, K, Takagi, To, Kanehisa, Mo, Sakaki,
Y, Quackenbush,]., Okazaki, Yo, Hayashizaki, Yo,
Hide, Wo, Chakraborty, R, Nishikawa, K, Sugawara,

Ho, Tateno, Yo, Chen, Zo, Oishi, Mo, Tonellato, Po,
Apweiler, R., Okubo, K, Wagner, L., Wiemann, So,
Strausberg, RL., Isogai, To, Auffray, Co, Nomura, No,
Gojobori, To and Sugano, S. (2004)0 Integrative
Annotation of 21,037 Human Genes Validated by
Full-Length cDNA Cloneso PLoS BioI. Apr 20 [Epubahead of print] °7o Matsuzaki, Mo, Misumi, 0o, Shin-I, To, Maruyama,
So, Takahara, Mo, Miyagishima, SYo, Mori, To, Nishida,
K, Yagisawa, Fo, Nishida, K, Yoshida, Yo, Nishimura,
Y, Nakao, So, Kobayashi, To, Momoyama, Yo,
Higashiyama, To, Minoda, A, Sano, Mo, Nomoto, Ho,
Oishi, K, Hayashi, Ho, Ohta, Fo, Nishizaka, So, Haga,
So, Miura, So, Morishita, To, Kabeya, Yo, Terasawa, K,
Suzuki, Yo, Ishii, Yo, Asakawa, So, Takano, Ho, Ohta,
No, Kuroiwa, H., Tanaka, K, Shimizu, No, Sugano, So,
Sato, No, Nozaki, H., Ogasawara, No, Kohara, Y andKuroiwa, To (2004)0 Genome sequence of the ultrasmall
unicellular red alga Cyanidioschyzon merolae 10Do
Natureo 428,653-6570
8o Niida, Ao, Hiroko To, Kasai, Mo, Furukawa, Yo,
Nakamura, Y, Suzuki, Yo, Sugano, So, Akiyama, To(2004)0 DKKl, a negative regulator of Wnt signaling,
is a target of the beta-cateninlTCF pathwayo Oncogeneo
23, 8520-85260
9o Noma, To, Yoshizawa, 1o, Sugano, So, Ogawa, No and
Kawano, Yo (2004)0 Somatropin promotes
dermatophagoides farinae-specific IgE generation
independently of IL-4 and IL-lOo Asian Pac J Allergy
Immunol. 22, 133-1420
10o Nonaka, Ho, Sugano, So and Miyajima, A (2004)0
Serial analysis of gene expression in sinusoidal
endothelial cells from normal and injured mouse livero
Biochem Biophys Res Communo 324, 15-240
11. Ota ,To, Suzuki, Yo, Nishikawa, To, Otsuki, To,
Sugiyama, To, Irie, R, Wakamatsu, Ao, Hayashi, K,
Sato, Ho, Nagai, K, Kimura, K, Makita, H., ~ekine, Mo,
Obayashi, Mo, Nishi, To, Shibahara, To, Tanaka, To,
Ishii, So, Yamamoto,]., Saito, K, Kawai, Yo, Isono, Yo,
Nakamura, Yo, Nagahari, K, Murakami, K, Yasuda,
To, Iwayanagi, To, Wagatsuma, Mo, Shiratori, A, Sudo,
Ho, Hosoiri, To, Kaku, Yo, Kodaira, Ho, Kondo, Ho,
Sugawara, Mo, Takahashi, Mo, Kanda, K, Yokoi, To,
Furuya, To, Kikkawa, Eo, Omura, Yo, Abe, K, Kamihara,
K, Katsuta, No, Sato, K, Tanikawa, Mo, Yamazaki, Mo,
Ninomiya, K, Ishibashi, T., Yamashita, Ho, Murakawa,
K, Fujimori, K, Tanai, Ho, Kimata, Mo, Watanabe, Mo,
Hiraoka, So, Chiba, Yo, Ishida, So, Ono, Yo, Takiguchi,
So, Watanabe, So, Yosida, Mo, Hotuta, To, Kusano, Jo,

Division of Theoretical Genetics

51

Kanehori, K., Takahashi-Fujii, A, Hara, H., Tanase,TO., Nomura, Y., Togiya, S., Komai, F., Hara, R.,
Takeuchi, K., Arita, M., Imose, N., Musashino, K.,
Yuuki, H., Oshima, A., Sasaki, N., Aotsuka, S.,
Yoshikawa, Y., Matsunawa, H., Ichihara, T., Shiohata,N., Sano, S., Moriya, S., Momiyama, H., Satoh, N.,
Takami, S., Terashima, Y., Suzuki, 0., Nakagawa, S.,
Senoh, A., Mizoguchi, H., Goto, Y., Shimizu, F.,
il\.Wakebe, H., Hishigaki, H., Watanabe, T., Sugiyama,A., Takemoto, M., Kawakami, B., Yamazaki, M.,
Watanabe, K., Kumagai, A, Itakura, S., Fukuzumi, Y.,
Fujimori, Y., Komiyama, M., Tashiro, H., Tanigami,A, Fujiwara, T., Ono, T., Yamada, K., Fujii, Y., Ozaki,
K., Hirao, M., Ohmori, Y., Kawabata, A, Hikiji, T.,
Kobatake, N., Inagaki, H., Ikema, Y., Okamoto, S.,
Okitani, R., Kawakami, T., Noguchi, S., Hoh, T.,
Shigeta, K., Senba, T., Matsumura, K., Nakajima, Y.,
Mizuno, T., Morinaga, M., Sasaki, M., Togashi, T.,
Oyama, M., Hata, H., Watanabe, M., Komatsu, T.,
Mizushima-Sugano,]., Satoh, T., Shirai, Y., Takahashi,Y., Nakagawa, K., Okumura, K., Nagase, T., Nomura,N., Kikuchi, H., Masuho, Y., Yamashita, R., Nakai, K.,
Yada, T., Nakamura, Y., Ohara, 0., Isogai, T. and
Sugano, S. (2004). Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat Genet.
36,40-45.
12. Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi,
T., Natsume, T., Isobe, T. and Sugano, S. (2004).
Analysis of small human proteins reveals thetranslation of upstream open reading frames ofmRNAs. Genome Res. lOB, 2048-2052.
13. Suzuki, Y., Yamashita, R., Shirota, M., Sakakibara,Y., Chiba,]., Mizushima-Sugano, J., Kel, AE., Arakawa,T., Caminci, P., Kawai,]., Hayashizaki, Y., Takagi, T.,
Nakai, K. and Sugano, S. (2004). Large-scale collection
and characterization of promoters of human andmouse genes. In Silico Bioi. 4 (3), 0036 [Epub aheadof printJ.
14. Suzuki, Y., Yamashita, R., Shirota, M., Sakakibara,Y., Chiba,]., Mizushima-Sugano,]., Nakai, K. and
Sugano, S. (2004). Sequence comparison of human andmouse genes reveals a homologous block structure in
the promoter regions. Genome Res. 14, 1711-1718.
15. Suzuki, Y., Yamashita, R., Sugano, S. and Nakai,
K. (2004). DBTSS, DataBase of Transcriptional Start
Sites: progress report 2004. Nucleic Acids Res. 32,
D78-81.
16. Watanabe,]., Sasaki, M., Suzuki, Y. and Sugano,S. (2004). Full-malaria 2004: an enlarged database for

52

Division of Theoretical Genetics

comparative studies of full-length cDNAs of malaria
parasites, Plasmodium species. Nucleic Acids Res. 32,
D334-338.
17. Yamada, S., Ohira, M., Horie, H., Ando, K.,
Takayasu, H., Suzuki, Y., Sugano, S., Hirata, T., Goto,
T., Matsunaga, T., Hiyama, E., Hayashi, Y., Ando, H.,
Suita, S., Kaneko, M., Sasaki, F., Hashizume, K.,
Ohnuma, N. and Nakagawara, A (2004). Expression
profiling and differential screening between
hepatoblastomas and the corresponding normal livers:
identification of high expression of the PLK1 oncogene
as a poor-prognostic indicator of hepatoblastomas.
Oncogene. 23,5901-5911.
18. Yu, L., Frith, MC., Suzuki, Y., Peterfreund, RA,
Gearan, T., Sugano, S., Schwarzschild, MA, Weng, Z.,
Fink, JS. and Chen, JF. (2004). Characterization of
genomic organization of the adenosine A2A receptor
gene by molecular and bioinformatics analyses. Brain
Res. 1000, 156-173.
Databases

19. http://dbtss.hgc.jp/
20. https://www.jbirc.aist.go.jp/hinv/index.jsp
SOCIAL CONTRIBUTIONS AND OTHERS~~mgw

1. HUGO: Council
2. HUGO Pacific: Co-Chair
3. j!~-Ti11~~~: JW~~

E. DEPARTMENT OFINTEGRATED GENETICS

E-a. Division of Human Genetics
Hiroyuki Sasaki GroupRESEARCH ACTIVITIES(1) Establishment and maintenance of genomic
imprinting in the germline and in early embryos
Hiroyuki Sasaki, Masahiro Kaneda, Ryutaro Hirasawa,
Kenichiro Hata, Maki Kusumi, Takashi Sado, Kenji
Kumaki, Hiroyasu Furuumi, Masaki Okano\ Naomi
Tsujimoto2, En Li 2, Tomohiro Suzuki3, Shigeharu
Wakana3 and Toshihiko Shiroishi3 CCDB, RIKEN;
2Harvard Med. Sch.; 3GSC, RIKEN)
DNA methylation serves as an important
gene-marking mechanism for discrimination of theparental alleles of imprinted genes. Although de novo
DNA methyltransferases of the Dnmt3 family are
implicated in maternal imprinting, the lethality of
conventional Dnmt3a and Dnmt3b knockout miceprecluded further studies. We have disrupted Dnmt3a
and Dnmt3b in male and female germ cells, leaving
them intact in somatic cells, by conditional geneknockout technology21.31• Offspring from the Dnmt3a
conditional mutant females died in utero and lacked
methylation and allele-specific expression at maternallyimprinted loci. The Dnmt3a conditional mutant males
showed impaired spermatogenesis and a lack ofmethylation at paternally imprinted loci inspermatogonia. Although these defects closely
resembled those of Dnmt3L knockout mice, exact
contribution of Dnmt3a and Dnmt3L to paternal
imprinting varied from locus to locus. By contrast, theDnmt3b conditional mutants and their offspring
showed no phenotype. These results indicate that
Dnmt3a is the critical enzyme responsible for bothpaternal and maternal imprintin~)'31. We also study
how the primary imprints are maintained in

preimplantation mouse embryos, and set out to screen
ENU-treated mutant mouse stocks for mutations that
affect the establishment of germ line imprints incollaboration with a group in GSC, RIKEN.
(2) Comparative analyses of the distal imprinted domain
on mouse chromnosome 7 and the orthologous domain
on chicken chromosome 5
Hiroyuki Sasaki, Takaaki Y okomine, Wahyu
Purbowasito\ Hisao Shirohzu, Chikako Suda, Takashi
Sado, Hisakazu Iwama, Kazuho Ikeo, Tetsuya Hori,
Masaoki Tsuzuki 2, Shigeki Mizuno3, Yo-ichi Matsuda4,
Chiyoko Sato5, Katsuzumi OkumuraS, Tsunehiro
Mukai 6, Mohamad Zubair7, Ken TsutsuiS, Reiko Kato9,
Atsushi Toyoda9, Masahira Hattori9 and Yoshiyuki
Sakaki9 CKyushu Univ.; 2Hiroshima Univ.; 3Nihon
Univ.; 4Hokkaido Univ.; 5Mie Univ.; 6Saga Univ.; 7NIBB;
SOkayama Univ.; 9GSC, RIKEN)Genomic imprinting, an epigenetic gene-marking
phenomenon in the germline, causes parent-of-originspecific monoallelic expression of a subset of
mammalian genes. Imprinted genes tend to form
clusters in the genome (imprinted domains), which may
be related to the mechanism of imprinting or to theevolution of imprinting. As a step to understand thestructural and functional characteristics of theimprinted domains, we have cloned and sequenced a
1-Mb imprinted domain in mouse chromosome 7F4/
F5 and its orthologous domain in chicken chromosome
5 (0.5 Mb). Using the mouse YAC clone that we haveisolated, Cerrato et al. found that this domain is in factcomposed of two autonomous subdomains1). We thenfound that the genes of the chicken domain are notimprinted and, furthermore, that the chicken domain
lacks the unique tandem repeat cluster of 0.2 Mb, theH19 gene, and the imprinting control elements, all ofwhich are present in the mouse. The results indicatethat the mammalian imprinted genes were alreadyclustered in the common ancestors of mammals andbirds and that the imprinting mechanism, which canaffect multiple genes in the cluster, came in later during
mammalian evolutionS),91. We also mapped a total of
52 nuclear matrix attachment regions (MARs) in theimprinted mouse domain. We found that the MARs
are unevenly distributed in the domain and that there
is a large MAR cluster in the boundary region of two
imprinted subdomains61 .

DEPARTMENT OF INTEGRATED GENETICS53

(3) Imprinting mechanisms of the mouse Igf2/H19
sub-domain

Goto 2 and Takeo~ amanashi Univ.)

Hiroyuki Sasaki, Yuzuru Kato, Ko Ishihara\ Melanie
Ehrlich2, Walter Reith3 and Mitsuyoshi Nakad
eKumamoto Univ.; 2Tulane Univ.; 3Univ. Geneva)

We previously studied two Japanese families withICF (immunodeficiency, centromeric instability, facial
anomalies) syndrome, an autosomal recessive disorderwith hypomethylation of satellite DNA, and found thatthey have mutations in the de novo DNA
methyltransferase gene DNMT3B. We have now
studied a new Japanese ICF case and found that thispatient does not have any mutation in DNMT3B. Thissuggests that the molecular pathology of ICF syndromeis heterogeneous and that a gene other than DNMT3B
can affect the methylation of pericentromeric satellite
DNNI.

The imprinted mouse 7F4/F5 domain contains two. linked imprinted genes Igf2 and H19 near its
centromeric boundary: Igf2is paternally expressed andH19 maternally expressed. It is known that thepaternal-specific methylation of the differentiallymethylated region (DMR) upstream of H19 is theprimary signal for the Igf21H19 imprinting. We foundthat a winged-helix type DNA-binding protein called
RFX1 or MDBP binds to the conserved sequences
within the DMR. Interestingly, this protein binds to thetarget sequence preferentially when they are
methylated at CpG sites. We are currently looking at
RFX knockout mice to see whether these proteins playa role in Igf21H19 imprinting. We also wrote a review
on the interactions between the DMRs by chromatin
loopingIO).
(4) Computer-assisted search for sequence featurescommon to imprinted DMRs

Hiroyuki Sasaki, Hisato Kobayashi, Takashi Abe andToshimichi Ikemural eSOKENDAI)
Although the imprinted DMRs, which show
differential methylation depending on parental origin,often play crucial roles in imprinting, features common
to the DMRs have not been identified. We therefore
set out to look for sequence features common to the
DMRs by computer-assisted programs. We firstlocated the DMR sequences on self-organizing maps
(SOMs) produced from the mouse genome sequences
for di-, tri- and tetra-nucleotides. We found that most
DMRs are located in the periphery of the SOMs: they
are more CpG-rich than most of the genome but less
CpG-rich than the CpG islands. More detailed studies
on the DMR sequences are ongoing.
(5) Molecular pathology of ICF syndrome

Hiroyuki Sasaki, Hiroyasu Furuumi, Tadashi Kajii,
Tomohiro Kamoda!, Nobuaki Iwasaki!, Naomi
Tobita\ Nobuko Fujiwara\ Akira matsui\ Yu-ichi

54

Division of Human Genetics

Kubota2 eUniv.

Tsukuba;(6) Development of a universal DNA chip systemapplicable to any organism

Hiroyuki Sasaki, Shin-ichi Mizuno!, Tadafumi Iino!,
Hidetoshi Ozawa!, Kosuke Tashirol and Takashi
Gojohbori eKyushu Univ.)
We carried out a collaborative research projectwith groups in Kyushu University to develop auniversal DNA chip system that can be used to study
expression of any gene in any organism. We
established the basic chip design and protocols for thisinnovative chip system (patent application no.
2004-278122). Development of the universal DNA chipsystem for practical use is underway.
(7) Antisense regulation at the Xist locus

Takashi Sado, Tatsuya Ohhatat, Yuko Hoki1andHiroyuki Sasaki ePRESTO, JST)
Xist(X-inactive specific transcript), which does not
encode a protein, is essential for X-inactivation to occur
in cis. Expression of Xist is negatively regulated in cis
by its antisense gene Tsix. Disruption of Tsix, therefore,
induces upregulation of Xist in cis. We have been
studying the molecular mechanism of how Tsix
regulates Xist expression in a cis-limited manner bygene targeting technology. We examined chromatin at
the Xist locus on the Tsix deficient X chromosome inembryos. We found that in the absence of Tsix,
methylation levels of CpG sites were reduced andchromatin structure became open at the Xist locus. Inaddition, histones of this region were modified to be
characteristic of active chromatin. These results
suggest that Tsix plays a role in establishment ofrepressive chromatin at the Xist and silence the Xist
gene.
(8) X-inactivation in mouse embryos deficient for histone
methyltransferase G9a

Tatsuya Ohhata, Makoto Tachibana\ Hiroyuki
Sasaki, Yoichi Shinkai 1 and Takashi Sado CKyoto
Univ.)
Accumulating evidence suggests that methylationof histone H3 at lysine 9 (K9) and 27 (K27) is implicatedin X-inactivation. Histone methyltranferase G9a is the
enzyme that catalyzes methylation of K9 and perhaps
K27 in euchromatic region. We studied X-inactivationin mouse embryos deficient for G9a, which die aroundthe early somite stage. RNA-FISH revealed that Xist
was appropriately regulated in both males and females.
Taking advantage of X-linked GFP transgenes, effects
of functional loss of G9a on the maintenance of
X-inactivation were analyzed. We did not observe
reactivation of the hitherto inactivated GFP transgenes
in both the embryonic and extraembryonic tissues,suggesting that X-inactivation is stably maintained in
G9a-null embryos. The same result was obtained using
X-linked LacZ transgenes located more distal to the
X-linked GFP transgenes, indicating that inactive stateof these transgenes was stably maintained regardlessthe distance from the X-inactivation center, from which
X-inactivation initiates and spreads. The results
suggest that the X-inactivation proce~s is properly
regulated in the absence of G9a5l • It is likely thatmethylation of histone H3 at K9 and K27 on theinactive X chromosome is mediated by an enzyme(s)other than G9a.
Role of Dnmt3L in spermatogenesis and genomic
imprinting during oogenesis(9)

Kenichiro Hata, Maki Kusumi, En Lit and Hiroyuki
Sasaki CHarvard Med. Sch.)
Dnmt3L (DNA cytosine-5-methyltransferase 3-Like)
encodes a protein of 421 amino acid residues andharbors a putative zing finger domain that shares ahigh degree of homology with the PHD-like domain of

DNA methyltransferases Dnmt3a and Dnmt3b. The
C-terminal part of Dnmt3L is related to DNA
cytosine-5-methyltransferase, but it does not possess
critical motifs for methyltransferase activity. We havegenerated Dnmt3L-deficient mice by gene targeting.
While Dnmt3L- 1- female mice grew normally, allembryos from pregnant Dnmt3L -1- mothers diedaround ElO.5. The maternally methylated imprinted
genes, e.g. Igf2r and Pegl, were hypomethylated inembryos derived from Dnmt3L -1- females x
Dnmt3L +1+ males, but paternally methylated
imprinted genes were unaffected. Also, Dnmt3L- 1male mice showed severe defects in spermatogenesis,
which is similar to, but severer than, the phenotype
displayed by Dnmt3a-- mice. We speculate that
Dnmt3L functions via interactions with Dnmt3a and/or Dnmt3b to control DNA methylation in developing
germ cells. Dnmt3L may be involved in not only theestablishment of genomic imprinting but also DNA
methylation of other regions.
PUBLICATIONSPapers

1. Cerrato, F., Sparago, A., Zou, X., Dean, W.,

Bruggemann, M., Sasaki, H., Reik, W. and Riccio, A.
(2005) The two domain hypothesis in BeckwithWiedemann syndrome: autonomous imprinting of thetelomeric domain of the distal chromosome 7 cluster.
Hom. Mol. Genet. (in press).
2. Kaneda, M., Okano, M., Hata, K., Sado, T.,
Tsujimoto, N., Li, E. and Sasaki, H. (2004). Essential
role for de novo DNA methyltransferase Dnmt3a inpaternal and maternal imprinting. Nature 429,900-903.
3. Kaneda, M., Sado, T., Okano, M., Hata, K.,
Tsujimoto, N., Li, E. and Sasaki, H. (2004). Role of denovo DNA methyltransferases in initiation of genomic
imprinting and X-chromosome inactivation. Cold
Spring Harbor Symp. Quant. BioI. (in press).
4. Kubota, T., Furuumi, H., Kamoda, T., Iwasaki, N.,
Tobita, N., Fujiwara, N., Goto, Y, Matsui, A., Sasaki,
H. and Kajii, T. (2004). .ICF syndrome in a girl with
DNA hypomethylation but without detectable
DNMT3Bmutation. Am. J. Med. Genet. 129A, 290-293.
5. Ohhata, T., Tachibana, M., Tada, M., Tada, T.,
Sasaki, H., Shinkai, Y. and Sado, T. (2004).
X-inactivation is stably maintained in mouse embryos
deficient for histone methyltransferase G9a. Genesis
40, 151-156.

Division of Human Genetics55

6. Purbowasito, W., Suda, C., Yokomine, T., Zubair,
M., Sado, T., Tsutsui, K. and Sasaki, H. (2004).
Large-scale identification and mapping of nuclear
matrix-attachment regions in the distal imprinted
domain of mouse chromosome 7. DNA Res. 11,391-407.
7. Sado, T., Okano, M., Li, E. and Sasaki, H. (2004).
De novo methylation is dispensable for the initiationand propagation of X chromosome inactivation.
• Development 131, 975-982.
8. Shirohzu, H., Yokomine, T., Sato, C., Kato, R.,
Toyoda, A., Purbowasito, W., Suda, C., Mukai, T.,
Hattori, M., Okumura, K., Sakaki, Y. and Sasaki, H.
(2004). A 210-kb segment of tandem repeats andretroelements located between imprinted subdomains
o'f.mouse distal chromosome 7. DNA Res. 11, 325-334.
9. Yokomine, T., Shirohzu, H., Purbowasito, W.,
Toyoda, A., Iwama, H., Ikeo, K., Hori, T., Mizuno, S.,
Tsudzuki, M., Matsuda, Y., Hattori, M., Sakaki, Y. and
Sasaki, H. (2005). Structural and functional analysis of
a 0.5-Mb chicken region orthologous to the imprinted
mammalian Asclz!Mash2-Igf2-H19 region. Genome Res.
(in press).
Reviews

10. Kato, Y. and Sasaki, H. (2005). Imprinting andlooping: epigenetic marks control interactions between
regulatory elements. BioEssays (in press).
11. ~®:*, ft,:k*mZ, *~:J'{;~q2004) -7 O~T/-{/~~-~-~•• ~.ft. b~~~.~~~~-~

B:§~::r:.l:::/.:c.;f:.7-1'-7 ~;6~bip~63-70.

12.

~EH:iE3b,ft,:iJt1lDc, ft,:k*mZ (2004) JI{~+f;::,\jllj fJ

Jb~ntd2.1i-*BIJffl!7t{t~DNA~}Tlv{t·If / A-{ /7' ~ /7 -1' /~. 1':~~f-I.~ iI{~58, 69-74.
13. ~EH:iE3b,ft,:k*mZ(2004)HOT PRESS: DNA}T Iv{tM~'(mnmt3a;6~1':~~~J'"('1f / A,\jllj fJ Jb.7f (-{ /70~ /7 -1' /~) ~fT?
*BIJffl!I~23, 934-935.
U n*.~, ft,:k*mZ. •*BIJffl!~If/A-{/7'~/
7-1' /~. /-"-{;t"-{ /~.~ !- ~ -(~,\jlljr:f-!)'
15. ft,:k*mz(2004)::r:.l:::/.:c.-*7-1' -7 ~~OC~'oMolecular
Medicine 4l(ffiflJ~) I::: !-If / A-I::: !-~f:!I!m~~m~Sl
n1B32-338.
16. ft,:k*mZ(2005). If / A-{ /7'~ /7-1' /-7', *JTJ...,
11 \iii/.t~b tr. G J..., tr.~ -/~-.::c7 Iv (Overview). Molecular
Medicine(~,\jllj*).
17. ~(2004)X~'@.f*)!{~+:litlfjft. If / A~¥4, 51-57.
18. ft,:iJt1lDc, * ffiI;fMili(2005). ~ tJ ~JI1S1*~m~lU;:::tMt ~-( /7'~ / !-~X~'@.1*~m1t{t. Molecular Medicine(~,\jllj*).

19.

~.-a~, ft,:k*mZ(2005) : 1':~~7IjfctMt~-{ /56Division of Human Genetics

7' ~ / !- 1JX;:lL••• Molecular Medicine(~,\jlljr:j:t).
20. l5'7fij3bJ*, ft,:k(2004) ~ ~ - ~~*JT~¥~J* .
'i~Iz;;)+12. ::r:.l:::/.:c.-*7'"-1' -7 ~1liljm~~~~1it. 1i;J*~1t48, 1673-1679.

*mZ

Books

21. Sasaki, H. (2005). DNA methylation in epigenetics,
development and imprinting. In: Encyclopedia of
Genetics, Genomics, Proteomics and Bioinformatics (eds.
Dunn, M., Jorde, L., Little, P. & Subramaniam, S.), John
Wiley & Sons, Chichester (in press).
22. ft,:k*mz~lijj(2004)Springer Reviews ~ ~ -~-::r:.
l:::/.:c.-*7-1'-7~,~.:z.7°~/jJ-· 7.:c.7'7--7JIOj~.

23. ft,:iJt1lDc (2004) 'ttE~'@.1*~JI{~+:litlfj11t Springer
Reviews~ ~ - ~-::r:.l:::/ .:c.;f:.7-1' -7 ~ (ft,:k*mzW),
pp.117-127, ~.:z.7'~ /jJ-. 7.:c.7'7--7**.
24. ~.-a~(2004)1':~, ~1':~~1it~::r:.I:::0:/.:c.;f:.7'"-1'-7 ~. Springer Reviews~ ~ - ~-::r:.l:::/ .:c.-*7'" -1' -7 ~(ft,:k*mZW),pp.183-190, ~.:z.7°~ /jJ_. 7.:c.7'7-7**.
25. -'jZ$11'Ej:a~, ft,:k*mZ. DNA} Tlv{t. :jl}1':~.
~f-I.~~ ~ -~, ~ 3 ~r:jl}1':~.~tr.Ilt>~7t+1':~~({~!l!fW: • $¥fi!~*~{~) J (~,\jllj*).
26. ili'fi3bJ*, ft,:k*mZ(2004)nm~L~~} /7Ivjl{~
T ~::r:.l:::/ .:c. -* 7'" -1' -7 ~. Springer Reviews ~ ~ - ~­::r:.l:::/ .:c.-*7'"-1' -7 ~ (ft,:k*mZW), pp.129-134, ~.:z. 7'~ /jJ- . 7.:c. 7'7--7**.
27. ft,:iJt1lDc (2004) (B *~U~) Hall, LM., Grewal, S.LS.
Structure and Function of Heterochromatin: Imprication fro Epigenetic Gene Silencing and Genome
Organization. In "RNAi: A guide to gene silencing" (Ed.
Hannon, GJ.)(B*~}ffi{~{~ r:j:t*;J-~-), pp.199-223,}7-1'~~·~-{::r:./~·-{/~-~~3~~

EDUCATION

1. Prof. H. Sasaki was invited to give a seminar"Essential role for de novo DNA methyltransferase
Dnmt3a in paternal and maternal imprinting" at the
Hospital for Sick Children, Toronto, Canada, June,
2004.
2. ft,:k*mZ: W~*¥~¥$~., JS:m.tIHT, 20041F
6ft
3. ~.-a~: NHK~~~J*r1':~J, 20041F10~1&~.
4. ft,:k~t7fiji!!*¥~¥$~., *L~, 20041F

*mZ :

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5. ft,:k*mZ: ~tm.*~f:!I!¥$~~, *lU~®:, 20041F
10~.

SOCIAL CONTRIBUTIONS AND OTHERS
1.

*ffft±lD1i%: 2004-278122, ~IlJJ 0)4"t~ : ~~"7-1~D7~-1&O~~~D-~0)&~~~~OK~~~.

(2) Epigenetic behavior of GAGTA transposon

Asuka Miura, Masaomi Kato, Kazuya Takashima,
Yuki Kinoshita and Tetsuji Kakutanit±l~~,~M*:&k*mZ.44"t,t±lDA:ftffl*~
1f~ .

"S/ A T .b.1iJF~.;fi\J.

2. &k*mZ: (lIt)JI~'¥:~&it'~ti!'l.

a ••-M, &k*mZ:~W.~~-1~~-~"S/y~ 2 4"t~~t An, 2004~ 7 ~.

4. &~~, &k*mZ:I~~~~A,¥:~D~7.b.1
4"t~~tAn,

2004~8~.

~ &k*mZ:.~.ft.~~~~~~-1iJF~mJl~~M~*-~~'tC~~!'lit'~!'l.

E-b. Division of Agricultural Genetics
Tetsuji Kakutani GroupRESEARCH ACTIVITIES(1) Developmental abnormalities induced by DNA
methylation mutation of Arabidopsis

Tetsuji Kakutani, Tetsu Kinoshita, Asuka Miura, Yuki
Kinoshita, Masaomi Kato and Hodetoshi Saze
Arabidopsis provides a genetically tractablesystem to learn role of DNA methylation, since viablemutants with reduced genomic DNA methylation areavailable. Arabidopsis METI (METHYLTRANSFERASE
1, ortholog of mammalian DNA methyltransferase
Dnmtl) is necessary for maintaining genomic cytosine
methylation at 5'-CG-3' sites. Arabidopsis additionallymethylates non-CG sites using CHROMOMETHYLASE3(CMT3). A third Arabidopsis gene necessary for DNA
methylation is DDMl (DECREASE IN DNA
METHYLA TIONj, which encodes a chromatin
remodeling factor (J eddeloh et al., 1999, Nat. Genet 22,94-). The ddml mutation affects both CG and non-CG. methylation. A striking feature of ddml mutation isthat it induces a variety of developmental abnormalities by causing heritable change in other loci(Kakutani et al., 1996, PNAS 93, 12406-). The molecularbasis has been clarified in two of the loci directly
causing the developmental abnormalities (see below).
In addition, we are starting genetic characterizationof other types of developmental abnormalities, suchas bonsai (Kakutani et al., 2005).

Through genetic characterization of one of the
ddml-induced developmental abnormalities, we
identified a novel endogenous Arabidopsis transposon,named CACTAl. This transposon transposes andincreases in the copy number in DNA hypomethylation
background in ddml mutant (Miura et al., 2001, Nature
411, 212-). Loss of DNA methylation seems to be
sufficient for mobilization of CACTA1, because it wasmobilized in mutants of DNA methyltransferase genes,
METl and CMT3. High frequency transposition .df
CACTA elements was detected in cmt3-metl double
mutants. Single mutants in either metl or cmt3 weremuch less effective in mobilization. Thus CG andnon-CG methylation systems redundantly function forimmobilization of transposons (Kato et al., 2003 Curr
BioI 13, 421-426). CMT3gene and non-CG methylationin plants may have evolved as an additional epigenetic
tag dedicated to transposon control.
CACTAl activated by the ddml mutation
remained mobile in the presence of the wild type
DDMl gene, suggesting that de novo silencing is notefficient for the defense of the genome against CACTA
movement. The defense depends on maintenance oftransposon silencing over generations (Kato et al., 2004).
Differentiation of gene-rich and transposon-rich(gene-poor) regions is a common feature in plant
genomes. That may be due to preferential integrationof transposons to gene-poor regions or may be due topurifying selection against transposon insertion to
gene-rich regions in natural populations. To evaluate
the possible contribution of natural selection to theformation of transposon distribution pattern, we
examined the distribution of the CACTA transposons
in genomes of 19 natural variants (ecotypes) andcompared that to integration induced in laboratory inthe ddml mutants. Sequences similar to mobile
CACTAl copy distribute among the ecotypes andshow high polymorphism in genomic localization.
Despite the high polymorphism, the copy number was
low in all the examined ecotypes and they localized
preferentially in pericentromeric and transposon-richregions (Miura et aI, 2004). This contrasts totransposition induced in laboratory, in which the
integration sites are less biased and the copy number

Division of Human Genetics

57frequently increases. In addtion, transposition inducedin DDM1 wild type background also showed unbiased
integration sprcificity (Kato et aI., 2004). The differencesin the integration sites may be due to natural selection
against deleterious insertion into chromosomal arm
regions.
(3) Inheritance of epigenetic developmental abnormality

Yuki Kinoshita, Asuka Miura, Tetsu Kinoshita and
Tetsuji Kakutani
Another developmental abnormality, late flowering
trait, was induced by ectopic expression of FWA geneassociated with hypomethylation of tandem repeat
upstream of the coding region (Soppe et aI., 2000, Mol
Cell 6, 791-). Interesting thing is that change innucleotide sequence was also not observed in fwa-1 and
fwa-2 alleles isolated by conventional mutagenesis. In
both cases, over-expression associated with thehypomethylation resulted in the phenotypes.
Combining ddm1 mutation and linkage analysis isuseful for identifying epigenetically regulated genes
affecting plant development (Kakutani, 1997, Plant].,
12,1447-). The ddm1-induced late flowering trait as wellas the hypomethylation and ectopic expression of the
FWA gene were stably inherited over generations evenin the presence of the wild type DDM1 copy
(manuscript in preparation).
(4) Epigenetic control of FWA gene expression inendosperm

Tetsu Kinoshita, Asuka Miura, Yuki Kinoshita and
Tetsuji Kakutani
Although FWA is ectopically expressed in theepigenetic alleles stated above, the role of FWA geneproduct in normal development remained unknown.
To understand why the FWA gene is epigeneticallycontrolled, we further examined expression of this geneduring normal development in wild type. Results of
GFP reporter system and direct detection of thetranscript both suggest that FWA is expressed
specifically in the endosperm. Endosperm is a planttissue analogous to mammalian placenta; it serves as
nutritional support to the embryo. Furthermore, theFWA gene was expressed in parent-of-origin-specificmanner; only maternal gene is expressed. The FWA

58

Division of Agricultural Genetics

imprint depends on the maintenance DNA
methyltransferase MET1, as is the case in mammals.
Unlike mammals, however, the FWA imprint is notestablished by allele-specific de novo methylation. It
is established by maternal gametophyte-specific geneactivation, which depends on a DNA glycosylase gene,
DEMETER. Since endosperm does not contribute to
the next generation, the activated FWA gene need not
be silenced again. Double fertilization enables plants
to use such 'one-way' control of imprinting and DNA
methylation in endosperm (Kinoshita et aI, 2004).
PUBLICATIONSPapers

1. Kinoshita, T., Miura, A, Choi, Y., Kinoshita, Y,
Cao, X., Jacobsen, SE., Fischer, RL. and Kakutani, T.
(2004). One-way control of FWA imprinting in
Arabidopsis endosperm by DNA methylation. Science
303, 521-523.

2. Miura, A, Kato, M., Watanabe, K., Kawabe, A,
Kotani, H. and Kakutani, T. (2004). Genomic
localization of endogenous mobile CACTA familytransposons in natural variants of Arabidopsis thaliana.
MGG 270, 524-532.
3. Kato, M., Takashima, K and Kakutani, T. (2004).
Epigenetic control of CACTA transposon mobility in
Arabidopsis thaliana. Genetics 168, 961-969.
4. Kakutani, T., Kato, M., Kinoshita, T. and Miura,
A (2005). Control of Development and Transposon
Movement by DNA Methylation in Arabidopsis
thaliana. Cold Spring Harbor Symposia on
Quantitative Biology. 69 in press.
5. Bachmair, A., Garber, K, Takeda, S., Sugimoto,K, Kakutani, T. and Hirochika, H. (2004). Biochemical
analysis of long terminal repeat retrotransposons.
Methods in Molecular Biology 260, 73-82.
EDUCATION

1. JlJ~W:f=
2. JlJ~W:f=))1'DR*~~f-I.~ijf~FJT"1:'~., 2oo4~ 5 jj
*tIl*~.~$"1:'~I:f:l~.,2004~ 7 Ji

3. JlJ~W:f=***~.~$"1:'~I:f:l~.,2004~l1Ji

E-b. Division of Agricultural Genetics
Keiichi Shibahara GroupRESEARCH ACTIVITIES(1) Mechanism of nucleosome assembly during DNA
replication
Yasunari Takamit, Tatsuya Ono, Tatsuo Nakayamal
and Kei-ichi Shibahara CDepartment of Biochemistry,Miyazaki Medical College, University of Miyazaki)
A newly replicated DNA is assembled into
nucleosome soon after the passage of replication fork.
This rapid formation of nucleosome is functionallylinked to DNA replication machineries and plays acritical role for the maintenance of genome integrityand epigenetic states of chromatin in proliferating cells.
Two histone binding proteins, CAF-l (Chromatin
Assembly Factor-I) and ASFI (Anti-Silencing
Functionl), are involved in some process of thisnucleosome assembly reaction.
In collaboration with Takami and Nakayama in
University of Miyazaki, using CAF-lpI50-or p60deficint chicken DT40 cells, we recently showed thatwithout CAF-l function, S-phase progression was
delayed and a rapid nucleosome assembly during DNA
replication was disturbed in vivo. This is the first direct
evidence for the involvement of CAF-l in a rapid
nucleosome assembly during DNA replication in vivo.
In addition, CAF-l was suggested in involving in
Chkl-dependent checkpoint pathway after thetreatment with HU.

caf1 mutants displayed severely disturbed cellular andfunctional organization of both meristems (Cell 104,131-142.2(01). We recently showed that transcriptional
gene silencing (TGS) of endogenous CACTA
transposons was released infrequently in a stochastic
manner in las, without decreasing DNA methylation.
Other endogenous silent genes at different
chromosomal sites were also transcriptionallyactivated non-concomitantly with each other.
Furthermore, TGS of the silent transgene {3glucuronidase (GUS) was also de-repressed randomly
in las mutant plants, irrespectively of developmental
abnormalities, and the activated state of GUS was
maintained during growth to produce clusters of cells
expressing GUs. We suggest that CAF-l contributes
to the stable inheritance of epigenetic states throughmultiple rounds of cell divisions and that defects in
CAF-l functions explain the stochastic occurrence ofpleiotropic phenotypes in las mutants.
In collaboration with]. Paszkowski in Universityof Geneva, we analyzed bru1 mutants and foundstriking phenotypic similarities to las mutants.
Epistatic analysis of bru1 and las mutants suggeststhat BRUI and CAF-l have overlapping functions.
BRUI encodes a novel nuclear protein containing two
protein-protein interaction domains. However, themolecular function of BRUI and the molecular link
between BRUI and CAF-l is still unknown. The future
analysis of BRUI would provide yet another clue tounderstanding the mechanisms of epigenetic
maintenance.
(3) Role of histon macroH2A in gene repression
Yuya Ogawa, Tatsuya Ono and Kei-ichi Shibahara(2) Physiological implications of CAF-l and CAF-ldependent nucleosome assembly in higher eukaryo'tes
Tatsuya Ono, Hidetaka Kaya\ Shin Takeda2, Jerzty
Paszkowski 2, Takashi Araki l and Kei-ichi Shibahara
CDepartment of Botany, Graduate School of Science,
Kyoto University, 2Department of Plant Biology,
University of Geneva)
The genetic approach of Arabidopsis is a powerfultool to see physiological implications of CAF-l and
CAF-ldependent nucleosome assembly in higher
eukaryotes. We analyzed loss-of-function mutants ofcal-1 (fasciata) in Arabidopsis and showed that those

The unusual histone variant macroH2A (mH2A)
is associated with transcriptional repression as it
appears to be enriched in the inactive X chromosome
by immunostaining. In vitro analysis showed that
non-histone region of mH2A in carboxy-terminal
prevents transcription factor from interacting withchromatin reconstituted with mH2A, but, the molecularfunction of mH2A in modulating chromatin structures
remains unknown.
To reveal the roles of mH2A, we purified
mH2A-containing nucleosome by affinity purification
with anti-epitope tag antibodies, and eventually, we
found mono-ubiquitinated form of mH2A.

Division of Agricultural Genetics

59Mono-ubiquitination of H2A and H2B has been shown
to be important in regulating chromatin structure andtranscription. Weare currently trying to identify theubiquitinated site of mH2A and reveal its implications
in modulation of chromatin structures andtranscriptional regulations.
(4) Differential methylation of Xite and CTCF sites inTsix in mouse X-inactivation

Shibahara, K. Submitted
Reviews

4. !Jd*~*, !;!j!§Jjj(!l- (2004)DNA~~;&V'?< -7 v;;t-rF.*• • K#5?<-7v;;tY-A~~ b~~W.~~
rDNA~~ c!:: DNAf~.J.

~~f*f1iQ2005-65442, J!f~-r11lA1J~, :;/- '/ -7 -If 'Y::;- -( '/~1J~;&V'~7'/A:;/~~'Y-7~~~~~1J~,!;!j!§Jjj(.-

Rebecca Maxfield BoumiP' 2. 3, Yuya Ogawa, Bryan K
Sun l • 2• 3 and Jeannie T. Lee l • 2• 3 eMassachusetts
General Hospital, ~arvard Medical School and3Howard Hughes Medical Institute)
In X-chromosome inactivation (XCI) in mammalian
female cells, one of two X-chromosomes is inactivatedin the imprinted or stochastic manner. Although someloci influencing the choice decision have been
genetically identified, the primary marks for imprinting
and random choice remain undefined. We examined
the role of DNA methylation, a mechanism known toregulate imprinting in autosomal loci, in XCI. We
found differentially methylated domains (DMDs) in
Tsix and Xite, two genes previously shown to influence
choice. The DMDs occured in gametes and disappearedin ES cells with two active X chromosomes. Because
the DNA methylation pattern reflects events of XCI,
we propose that DMDs in Tsix and Xite are candidates
for the primary marks of epigenetic regulation in XCI.
PUBLICATIONSPapers

1. Takeda, S., Tadele, Z., Hofmann, I., Probst, A.V.,

Angelis, KJ., Kaya, H., Araki, T., Mengiste, T.,
Mittelsten Scheid, 0., Shibahara, K., Scheel, D. andPaszkowski, J. (2004). BRUl, a novel link between
responses to DNA damage and epigenetic genesilencing in Arabidopsis. Genes Dev 18, 782-93.
2. Chromatin Assembly Factor-l Dependent
Nucleosome Assembly Coupled with DNA Replication,is Essential for Cell Proliferation in Vertebrate Cells.
Takami, Y, Ono, T., Fukagawa, T., Shibahara, K. andNakayama. T. Under revision in Mol Cell Bioi.
3. Mutations in CAFI of Arabidopsis releasetranscriptional gene silencing in a stochastic manner.
Ono, T., Kaya, H., Takeda, S., Abe, M., Ogawa, Y.,
Kato, M., Kakutani, T., Scheid, O.M., Araki, T. and60

Division of Agricultural Genetics

E-c. Division of Brain Function
Tatsumi Hirata GroupRESEARCH ACTIVITIES(1) Theoretical Consideration of Olfactory Axon
Targeting with an Activity-Dependent Neural Network
Model

Hirokazu Tozaki and Tatsumi Hirata
Olfactory sensory neurons that express a given
odorant receptor target their axons onto a few specificglomeruli in the olfactory bulb. Although the odorant
receptor plays an indispensable role in olfactory axon
targeting, the mechanisms underlying this guidance
are largely unknown. In particular, there is muchcontroversy regarding the involvement of activitydependent mechanism in the targeting process. In thisstudy, we developed an activity-dependent selforganization model of the glomerular layer in theolfactory bulb and simulated the targeting of olfactoryaxons onto the layer). Our model successfully
constructed discrete glomeruli that received olfactoryaxons expressing a common odorant receptor throughodorant-evoked neural activities. Furthermore, ourmodel explained the perplexing experimental resultsthat have been reported in olfactory axon targeting. For
example, the segregated projection of the axons that
express the same odorant receptor transcribed fromthe different alleles was successfully simulated if thegenetically modified allele was assumed to express asmaller amount of the receptor protein. The activitydependent model even explained the inconsistenteffects of disruption of the activity-evoking ion channel
on axons expressing different odorant receptors,
although some of these results were regarded asevidence for activity-independency of the olfactorytargeting. Taken together, the activity-dependent
targeting of olfactory axons seems to be a simple
probable mechanism that can provide a unified
explanation of glomerular formation.
(2) Systematic Screening and Identification of the
Antigens Recognized by Monoclonal Antibodies Raised
against the Developing Lateral Olfactory Tract

This special assembly of the axons explains the
non-topographical relationships between the olfactory
bulb and the lateral olfactory tract axons that havebeen described in previous studies, and could possibly
influence the subsequent selection of the olfactorytarget areas by these axons.
PUBLICATIONSPapersTakahiko Kawasaki, Yoshiko Takagi, Hitoshi
Yamatani and Tatsumi Hirata
During development, olfactory bulb axons navigate
the complex microenvironment composed of myriads
of molecules in the telencephalon to construct a bundle
called the lateral olfactory tract. The axons, themselves,also express thousands of different molecules during
the navigation. In this study, we produced andcharacterized six monoclonal antibodies that label thelateral olfactory tract and the surroundings in a uniquepattem 2). The labeling profiles suggested that the
antigen molecules recognized by each antibody areheterogeneously distributed around the developing
lateral olfactory tract. We developed an efficient
screening method to identify the antigen molecules bycombining expression of a cDNA library in COS-7 cells
and the subsequent immunohistochemical staining of
the cells. The systematic screening successfully
identified the specific cDNA clones for all of themonoclonal antibodies, which highly probably code forthe antigen molecules, and therefore unveiled themolecular nature of local components that embrace thedeveloping lateral olfactory tract.
(3) Chronotopic Organization of Olfactory Bulb Axonsin the Lateral Olfactory Tract

Hitoshi Yamatani, Yasufumi Sato and Tatsumi Hirata
The arrangement ofaxons in a tract can have a
specific consequence on the organization of functional
neuronal circuits. We found that olfactory bulb axons
are chronologically arranged in the lateral olfactory
trace). Newly-differentiated projection neurons over thewhole olfactory bulb are similarly marked withtransient expression of c-kit protein. Their axons areassembled together and project into the ventral
superficial part of the tract, displacing the older axons.

1. Aizawa, H., Sato, Y., Maekawa, M., Fujisawa, H.,
Hirata, T. and Yuasa, S. (2004). Development of theamygdalohypothalamic projection in the mouseembryonic forebrain. Anat. Embryol. 208,249-264.
2. Kawasaki, T., Takagi, Y., Yamatani, H. and Hirata,
T. (2004). Systematic screening and identification of theantigens recognized by monoclonal antibodies raised
against the developing lateral olfactory tract. ].
Neurobiol. 62,330-340.
3. Tozaki, H., Tanaka, S. and Hirata, T. (2004).
Theoretical consideration of olfactory axon projection
with an activity-dependent neural network model. Mol.
Cell. Neurosci. 26, 503-517.
4. Yamatani, H., Sato, Y., Fujisawa, H. and Hirata,
T. (2004). Chronotopic organization of olfactory bulb
axons in the lateral olfactory tract. ]. Compo Neurol.
475,247-260.

E-d. Division of Applied Genetics
Takashi Araki GroupRESEARCH ACTIVITIES(1) Physiological roles of chromatin assembly factor-lin epigenetic control of meristem activity during
post-embryonic development in plants

Takashi ArakP· 2, Mitsutomo Abel. 3, Hidetaka Kaya4
and Kei-ichi Shibahara eGraduate School of Science,
Kyoto University, 2]apan Science and Technology
Agency, 3pROBRAIN, 4Genome & Drug ResearchCenter, Tokyo University of Science)
Loss-of-function mutants of chromatin assembly
factor-l (CAF-l) in Arabidopsis, fasciata (fas), show awide variety of morphological abnormalities andunique defects in the expression of WUSCHEL and
SCARECROWgenes in meristems (Cell 104, 131-142.

Division of Brain Function

61

2001). We found that transcriptional gene silencing(TGS) of endogenous CACTA transposons was
released in a stochastic manner in las, without
decreasing global levels of DNA methylation. Other
endogenous silent genes at different chromosomal
sites, a DNA transposon AtMul and a hypotheticalgene T5L23.26 at a heterochromatin knob, were alsotranscriptionally activated. Activation of the threedifferent silent loci occurred non-concomitantly to eachother. Furthermore, TGS of a silent 13 -glucuronidase(GUS) transgene was also de-repressed randomly inlas plants, without apparent correlation with
developmental abnormalities. Activated state of GUS
was maintained during growth to produce clusters ofcells expressing GUS. Based on these observations, we
propose a model that CAF-1 contributes to the stable
inheritance of epigenetic states through multiple
rounds of cell divisions. Defect of CAF-1 function
explains the stochastic occurrence of pleiotropic
morphological phenotypes observed in las mutants(Ogawa, Kaya, and others, submitted). We also
investigated other genes involved in epigenetic controlof meristem activity, such as BRUSHYI (BRUl] andANTI-SILENCING FUNCTION1 (ASFl) (in preparation).
(2) Genes involved in the integration of various genetic
pathways for regulation of the floral transition

Takashi Araki l. 2, Mitsutomo Abel. 3, Sumiko
Yamamotd,2, Yasufumi Daimon\ Ayako Yamaguchi\
Yoko Ikeda\ Michitaka Notaguchi l and Masaki
KobayashiI CGraduate School of Science, Kyoto
University, 2]apan Science and Technology Agency,
3pROBRAIN)
Floral transition in Arabidopsis is regulated byseveral pathways which converge on thetranscriptional regulation of floral pathway integrators
including FT. FTis a direct target of CO and encodesa protein with similarity to mammalian proteins(PEBP/RKIP) involved in cellular signaling. FTtranscription is immediately induced in cotyledon andleaf vascular tissues upon transfer from short-day to
inductive long-day photoperiods. Promotion of
flowering by FT requires the activity of anotherflowering-time gene FD which encodes a bZIP
transcription factor preferentially expressed in theshoot apex. FD is involved in transcriptional activationof the floral meristem identity genes API and CAL

62

Division of Applied Genetics

redundantly with LFY. It; lfy and Id; lfy double mutants
are very similar in severe reduction of API mRNAlevels and strong defects in floral specification. Lossof FTfunction suppresses ectopic up-regulation of API
in seedlings by FDover-expression. Mutant forms of
FD which lack a C-terminal potential phosphorylationsite cannot interact with FT in yeast cells and fail tocomplement Id late-flowering phenotype even by
over-expression. These and other evidences suggestthat FT and FD are inter-dependent in promotion offloral transition and activation of API expression (inpreparation). Since the activity of FD, which ispreferentially expressed in shoot apex, seems to require
protein/protein interaction with FT, shoot apex is likely
the site of action of FT protein. Consistent with this,restoration of FTfunction in whole region or outermost
layer (Ll) of the shoot apex can rescue It late-flowering.
These raise an interesting possibility that the FT
protein may represent a long-distance signal generated
in photoperiodically-induced leaves (mainly invascular tissues) and act at the shoot apex to initiate
floral development. Arabidopsis genome has a homologof FT, called TWIN SISTER OF FT (TSF). We foundthat TSFacts redundantly with FTas a floral pathway
integrator (Yamaguchi et al., submitted).
PUBLICATIONSPapers

1. Takeda, S., Tadele, Z., Hofmann, I., Probst, A.V"
Angelis, KJ" Kaya, H., Araki, T., Mengiste, T., Scheid,a.M., Shibahara, K, Scheel, D. and Paszkowski, ].
(2004). BRUl, a novel link between responses to DNA
damage and epigenetic gene silencing in Arabidopsis.
Genes Dev 18, 782-93.
Reviews. 2. m*~ (2004) ":tEpx;~*,JmT -r:>Jlf~Iz;Ff C: ~~E1SI-f­O);flllitpjf]", Wf1[~(1)~~kt%Fc:~1!\U~PX;(1)~ 0 A 1'~~, :WOOJ¥fHM· 3i1F.i-EiHiiD, ~.:z.7"1) '.,,/jj-. 7::r.;7-~Jlf*,

pp.57-63.

Books

3. ti~(2004) "m24. :tEPX;(1)w.afm", naiz & Zeiger
f1[~1:::f!I!¥

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!!In, pp.568-601.

EDUCATION

1. m*~*~*~*~~:E!I!~liJf~f-I.· :E!I!~$ (~t!DIIj]~~

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SOCIAL CONTRIBUTIONS AND OTHERS

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cloned mice and the genomic loci frequently
associated with the epigenetic error have been exploredin the cloned animals. We identified the Spalt-like gene
3 (Sa1l3) locus as a hypermethylated region in theplacental genome of cloned mice. The Sa1l310cus has
a CpG island containing a T-DMR. The T-DMR
sequence is conserved in the human genome at the
SALL3 locus of chromosome 18q23, which has beensuggested to be involved in the 18q deletion syndrome.
Intriguingly, larger placentas were more heavily
methylated at the Sa1l3 locus in cloned mice. Thisepigenetic error was found in all cloned mice examined
regardless of sex, mouse strain and the type of donor
cells. In contrast, the placentas of in vitro fertilized(IVF) and intracytoplasmic sperm injected (ICSI) mice
did not show such hypermethylation, suggesting thataberrant hypermethylation at the Sa1l3 locus isassociated with abnormal placental development
caused by nuclear transfer of somatic cells. Thus theSa1l310cus is the area with frequent epigenetic errors
in cloned mice. These data suggest that there exists
at least genetic locus that is highly susceptible toepigenetic error caused by nuclear transfer.

In

2004~(~Hl@]) B*fi[~1:::E!I!~~~)c1t(#

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2. :W"*~ Plant and Cell Physiology (editorial board)
3. m*~ B *fi[~1:::E!I!~~ (iW~~)

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P.J<:l?Jit~*~~J* (ft~ : %J!l!fBB-*~*~1'llf~~) ~RiP,
2004~ 4 -lOJi

E-d. Division of Applied Genetics
Kunia Shiota GroupRESEARCH ACTIVITIES(1) The Sa113locus is an epigenetic hotspot of aberrant
DNA methylation associated with placentomegaly ofcloned mice

Jun Ohagane\ Teruhiko Wakayama 2. 3, Sho Senda\
Yukiko Yamazaki 3, Kimiko Inoue2, Atsuo Ogura2, Joel
Marh3, Satoshi Tanaka\ Ryuzo Yanagimashi 3 andKunio Shiota! CUniv. Tokyo; 2RIKEN; 3Hawaii Univ.)
Cloned offspring develop a variety of abnormal
phenotypes such as increased body weight (large fetus
syndrome), pulmonary hypertension, placental
overgrowth, respiratory problems and early death. We
had identified several aberrantly methylated loci in thetissues of full-term cloned fetuses. Interestingly, eachcloned animal has a different DNA methylation pattern
and the extent of hyper- or hypo-methylation varies
among the individuals (Ohgane et aL, Genesis 30, 45-50,
2001). In contrast, overgrowth of the placenta is one of
the commonly observed symptoms in all cloned mice
regardless of the sex and strain of animal and the typeof donor cell. Thus, there may be genomic lociassociated with the abnormal placental development(2) Epigenetic control of mouse Oct-4 gene expressionin embryonic stem cells and trophoblast stem cells

Naoko Hattori\ Koichiro Nishino!' 2, Yeoung-Gyu Ko\
Naka HattorP' 2, J un Ohgane\ Satoshi Tanaka! andKunio Shiota1 CUniv. Tokyo; 2Bio-oriented Technology
Research Advancement Institution)
The first cell differentiation event in mammalianembryogenesis segregates inner cell mass lineage fromthe trophectoderm at the blastocyst stage. Oct-4, amember of the POU family of transcription factors, isnecessary for the pluripotency of the inner cell masslineage. Embryonic stem (ES) cells, which contributeto all of embryonic lineages, express the Oct-4 gene.
Trophoblast stem (TS) cells, which have the ability todifferentiate into trophoblast lineage in vitro, nevercontribute to embryonic proper tissues in chimeras anddifferentiate only into trophoblastic cells in theplacenta. Expression of the Oct-4 gene wasundetectable and severely repressed in trophoblastic
lineage, including the stem cells. We found that theculture of TS cells with 5-aza-2' -deoxycytidine ortrichostatin A caused the activation of the Oct-4 gene.
Analysis of the DNA methylation status of mouse Oct-4

Division of Applied Genetics

63gene upstream region revealed that Oct-4 enhancer/
promoter region was hypomethylated in ES cells but
hypermethylated in TS cells. Furthermore, in vitro
methylation suppressed Oct-4 enhancer/promoter
activity in reporter assay. In the placenta of
Dnmtl(n/n) mutant mice, most of the CpGs in theenhancer/promoter region were unmethylated, andOct-4 gene expression was aberrantly detected.
Chromatin immunoprecipitation assay revealed that
Oct-4 enhancer/promoter region was hyperacetylated
in ES cells compared with TS cells, thus demonstratingthat DNA methylation status is closely linked to thechromatin structure of the Oct-4 gene. Here Wi:- proposethat the epigenetic mechanism, consisting of DNA
methylation and chromatin remodeling, underlies thedevelopmental stage- and cell type-specific mechanismof Oct-4 gene expression.
(3) DNA methylation-mediated control of Sry gene
expression in mouse gonadal development
Koichiro Nishind' 2, Naoko Hattori!, Satoshi Tanakal
and Kunio Shiotal CUniv. Tokyo; 2J3io-oriented
Technology Research Advancement Institution)
DNA methylation at CpG sequences is involvedin tissue-specific and developmentally regulated gene
expression. The Sry (sex determining region on the Y
chromosome) gene encodes a master protein for
initiating testis differentiation in mammals, and its
expression is restricted to gonadal somatic cells at
10.5-12.5 days post coitum (dpc) in the mouse. We foundthat in vitro methylation of the 5'-flanking region of the
Sry gene caused suppression of reporter activity,
implying that Sry gene expression could be regulated
by DNA methylation-mediated gene silencing. Bisulfite
restriction mapping and sodium bisulfite sequencing
revealed that the 5'-flanking region of the Sry gene was
hypermethylated in the 8.5 dpc embryos in which the
Sry gene was not expressed. Importantly, this region
was specifically hypomethylated in the gonad at 1l.5
dpc, while the hypermethylated status was maintainedin tissues that do not express the Sry gene. We
concluded that expression of the Sry gene is under thecontrol of an epigenetic mechanism mediated by DNA
methylation.

64

Division of Applied Genetics(4) Skewed X-inactivation in cloned mice
Sho Senda\ Teruhiko Wakayama2, Yukiko Yamazake,lun Ohgane\ Naka Hattori!, Satoshi Tanaka\ Ryuzo
Yanagimachi 2 and Kunio Shiotal CUniv. Tokyo;
2Hawaii Univ.)
In female mammals, dosage compensation for
X-linked genes is accomplished by inactivation of oneof two X chromosomes. The X-inactivation ratio (apercentage of the cells with inactivated maternal X
chromosomes in the whole cells) is skewed as aconsequence of various genetic mutations, and has
been observed in a number of X-linked disorders. We
previously reported that phenotypically normal
full-term cloned mouse fetuses had loci withinappropriate DNA methylation. Thus, cloned mice areexcellent models to study abnormal epigenetic eventsin mammalian development. In the present study, we
analyzed X-inactivation ratios in adult female cloned
mice (B6C3F1). Kidneys of eight naturally produced
controls and II cloned mice were analyzed. Althoughvariations in X-inactivation ratio among the mice wereobserved in both groups, the distributions weresignificantly different (Ansary-Bradley test, P<O.01).
In particular, 2 of II cloned mice showed skewed
X-inactivation ratios (19.2% and 86.8%). Similarly, inintestine, 1 of 10 cloned mice had a skewed ratio (75.7%).
Skewed X-inactivation was observed to various degrees
in different tissues of different individuals, suggestingthat skewed X-inactivation in cloned mice is the resultof secondary cell selection in combination withstochastic distortion of primary choice. The present
study is the first demonstration that skewed
X-inactivation occurs in cloned animals. This findingis important for understanding both nuclear transfer
technology and etiology of X-linked disorders.
(5) Non-coding RNA directed DNA demethylation of
Sphkl CpG island
Takuya Imamura\ Soshi Yamamoto\ lun Ohgane\
Naka Hattori l , Satoshi Tanakal and Kunio Shiotal(IUniv. Tokyo)
The formation of DNA methylation patterns is oneof the epigenetic events that underlie mammaliandevelopment. The Sphk1 CpG island is a target for
tissue-dependent DNA methylation as well as atemplate for generating multiple subtypes. The numberof mammalian non-coding RNA genes is rapidly
expanding. In this study, we found endogenous
antisense transcripts, Khpsl subtypes with different
sizes (600-20,OOOnt). A subtype, Khpsla, was a 1290-bp,
non-coding, 5'-capped and 3'-polyadenylated RNA thatoriginated from the CpG island and overlapped witha tissue-dependent differentially methylated region(T-DMR) of Sphk1. Intriguingly, overexpression of two
fragments of Khpsl caused demethylation of CG sites
in the T-DMR. Furthermore, this RNA-directed
demethylation was associated with DNA methylation
at three CC(A/T)GG sites in the T-DMR. The link
between the RNA-directed CG demethylation andnon-CG methylation provides a novel mechanism of
epigenetic regulation and potential tool for epigenetic
manipulation of mammalian cells.
(6) Preference of DNA methyltransferases for CpG
islands in normal cells

Naka Hattori\ Tetsuya Abe\ Masako Suzuki\
Tomooki Matsuyama2, Shigeo Yoshida2, En Li 3 andKunio Shiotal CUniv. Tokyo; 2RIKEN; 3Massachusets
General Hospital)
In vitro studies indicated that DNA methyltransferasel(Dnmtl) prefers hemimethylated DNA compared tounmethylated DNA, while Dnmt3a/3b methylate CpG
dinucleotides without preference for hemimethylatedor unmethylated DNA. Collectively, the fact that Dnmtl
is localized to DNA replication foci and associated withthe methyl-CpG binding protein, MeCP2, which directs
DNA methyltransferase activity to hemimethylated
DNNl, implies that Dnmtl is involved in maintenancemethylation in vivo to preserve methylation patterns
in genomic DNA, and that Dnmt3a/3b function as denovo methyltransferases_ However, a direct association
between DNMTs was reported, and it wasdemonstrated that DNMTl and DNMT3B function
cooperatively for maintenance methylation in thehuman cancer cell line. Therefore, categorizing Dnmts
into maintenance and de novo DNA methylation may
not be appropriate when attempting to characterize invivo mechanisms involved in determining or
establishing DNA methylation profiles in the genome.
To address the question how the T-DMR of CpG
islands are regulated by Dnmts in vivo, we analyzed
the genome-wide DNA methylation pattern focussing

on CpG islands and found that each Dnmt has target
preferences depending the genomic component (inpreparation).
(7) Stage-by-stage change in DNA methylation status
of DNA methyltransferase 1 (Dnmtl) locus during mouseearly development

Yeoung-Gyu Ko\ Koichiro Nishino\ Naoko Hattori\
Yoshikazu Arai\ Satoshi Tanakal and Kunio Shiotal
eUniv. Tokyo)
Methylation of DNA is involved in tissue-specificgene control, and establishment of DNA methylation
pattern in the genome is thought to be essential forembryonic development. Three isoforms of DNA
methyltransferase 1 (Dnmtl) transcripts, Dnmtls,
Dnmtlo and Dnmtlp, are produced by alternative
usage of multiple first exons. Dnmtls is expressed insomatic cells. Dnmtlp is found only in pachytene
spermatocytes, whereas Dnmtlo is specific to oocytes
and preimplantation embryos. Here we determinedthat there is a tissue-dependent differentiallymethylated region (T-DMR) in the 5' region of Dnmtlo
but not in that of the Dnmtls/lp. The methylation
status of the Dnmtlo T-DMR was distinctively
different in the oocyte from that in the sperm and adultsomatic tissues, and changed at each stage fromfertilization to blastocyst stage, suggesting that active
methylation and demethylation occur during
preimplantation development. The T-DMR was highly
methylated in somatic cells and ES cells. Analysis
using Dnmt-deficient ES cell lines revealed that eachof Dnmtl, Dnmt3a and Dnmt3b is partially responsible
for maintenance of methylation of Dnmtlo T -DMR. In
particular, there are compensatory and cooperativeroles between Dnmt3a and Dnmt3b. Thus, theregulatory region of Dnmtlo, but not of Dnmtls/lp,
appeared to be a target of DNA methylation. The
present study also suggested that the DNA methylation
status of the gene region dynamically changes during
embryogenesis independently of the change in the bulk
DNA methylation status.
PUBLICATIONSPapers

1. Ohgane,]., Wakayama, T., Senda, S., Yamazaki,
Y., Inoue, K., Ogura, A., Marh, ]., Tanaka, S.,

Division of Applied Genetics

65Yanagimachi, R. and Shiota, K (2004). The Sa1l3locus
is an epigenetic hotspot of aberrant DNA methylation
associated with placentomegaly of cloned mice. Genes
to Cells, 9, 253-260.
2. Hattori, N., Nishino, K., Ko, Y.G., Ohgane, J.,
Tanaka, S. and Shiota, K (2004). Epigenetic control ofmouse Oct-4 gene expression in embryonic stem cells
and trophoblast stem cells. ]. BioI. Chern., 279,
17063-17069.
3. Nishino, K, Hattori, N., Tanaka, S. and Shiota, K(2004). DNA methylation-mediated control of Sry gene
expression in mouse gonadal development. ]. BioI.
Chern., 279, 22306-22313.
4. Senda, S., Wakayama, T., Yamazaki, Y, Ohgane,]., Hattori, N., Tanaka, S., Yanagimachi, R. and Shiota,
K (2004). Skewed X-inactivation in cloned mice.
Biochem. Biophys. Res. Commun., 321, 38-44.
5. Imamura, T., Yamamoto, S., Ohgane, ]., Hattori,N., Tanaka, S. and Shiota, K (2004). Non-coding RNA
directed DNA demethylation of Sphk1 CpG island.
Biochem. Biophys. Res. Commun., 322, 593-600.
6. Hattori, N., Abe, T., Suzuki, M., Matsuyama, T.,
Yoshida, S., Li, E. and Shiota, K (2004). Preference ofDNA methyltransferases for CpG islands in mouseembryonic stem cells. Genome Res., 14, 1733-1740.
7. Ko, Y G., Nishino, K, Hattori, N., Arai, Y, Tanaka,
S. and Shiota, K (2005). Stage-by-stage change in DNA
methylation status of DNA methyltransferase 1(Dnmtl) locus during mouse early development. ]. BioI.
Chern., in press.

EDUCATION

1. Dr. K Shiota was invited to give a seminar on"Cell- and tissue-specific DNA methylation profiles inmammalian genome: Epigenetics of Embryo
Development", at Canadian Workshop on "Human
Reproduction & Reproductive Biology", Ottawa,
Canada, May 3-5, 2004.
2. Dr. K Shiota was invited to give a lecture on "DNAmethylation profiles of CpG islands during cellulardifferentiation and development in mammals." At
McGill University, Reproductive and Developmental
Biology Seminar, Montreal, Canada, May 6, 2004.
3. Dr. K Shiota was invited to give a lecture on "DNAmethylation profiles of CpG islands for cellulardifferentiation and development in mammals", at 13th
Conference of the International Society ofDifferentiation, Honolulu, USA, September 5-9,2004.
4. Dr. K Shiota was invited to give a seminar on"Epigenetic marks by DNA methylation specific to cell
types", at IX International Congress of Reproductive
Immunology, Hakone, Japan, October 11-15, 2004.
5. Dr. K Shiota was invited to give a seminar on"DNA methylation profiles for evaluation of epigeneticrisk", at International Symposium on Environmental
Endocrine Disrupters 2004, Nagoya, Japan, December
15-17,2004.
SOCIAL CONTRIBUTIONS AND OTHERS

1. *~*'¥*'¥~.'¥!f.:~W'¥~~W
Reviews

8. Shiota, K (2004). DNA methyaltion profiles of CpG
islands for Cellular differentiation and development
in mammals. Cytogenetic and Genome Res., 105,
325-334.
9. Ohgane, J., Hattori, N. and Shiota, K (2004).
Analysis of tissue-specific DNA methylation during
development. Methods Mol. BioI., 289, 371-382.
10. :I&*~~, ~JE~~~ (2004) ~!f.:7"o:7'7 A bm• .
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66

Division of Applied Genetics

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F. GENETIC STRAINSRESEARCH CENTER

F-a. Mammalian Genetics Laboratory
Toshihiko Shiroishi GroupRESEARCH ACTIVITIES(1) Elimination of a long-range cis-regulatory modulecauses complete loss of limb-specific Shh expression andtruncation of the mouse limb

Tomoko Sagai, Masaki Hosoya, Youichi Mizushina,
Masaru Tamura and Toshihiko Shiroishi
Point mutations in a conserved non-coding region
in intron 5 of the Lmbrllocus, which is 1 Mb awayfrom the sonic hedgehog (Shh) coding sequence, areresponsible for mouse and human preaxial polydactyly
with mirror-image digit duplications. In the mousemutants, ectopic Shh expression is observed in theanterior mesenchyme of limb buds. Previously we
showed that the conserved intronic sequence functions
as a cis-acting regulator for limb specific expressionof Shh. The phylogenetic studies have also shown that
this sequence is highly conserved among tetrapods,
and even in teleost fishes. Paired fins of teleost fishes
and tetrapod limbs have evolved from common
ancestral appendages, and polarized Shh expression
is commonly observed in fins and limbs. Recently wefound that this conserved sequence motif is also
physically linked to the Shh coding sequence in teleostfish, medaka, by homology search of a newly available
genomic sequence database. On the other hand, thissequence has been lost in certain limbless species ofreptiles and amphibians, such as snakes and a limblessnewt.
For the precise functional analysis of the intronic
sequence, we eliminated it from mouse genome bymeans of ES targeting. The targeted mutant mice
showed complete loss of Shh expression in the limb
buds and truncation of skeletal elements distal to thestylopod/zeugopod junction. The result revealed thatthe sequence contains a major limb-specific Shhenhancer necessary for distal limb development. All
results suggested that the conserved intronic sequence
evolved in a common ancestor of fishes and tetrapods
to control polarized expression of Shh in fins andlimbs. It is also possible that loss of the conserved
intronic sequence represents one way by which
limblessness may have evolved in vertebrate species.
In addition, in order to find other putative
cis-regulators in the intervening sequence between the
Shh and Lmbrl genes, we conducted comparative
genomics to search for conserved sequence in the
genomes of medaka fish and mammals. We foundthree clustering blocks of conserved non-coding
sequences between medaka fish and mammals, andthe medaka sequences are located in the same scaffold
as the Shh coding sequence. The ordering of the threeblocks is conserved between medaka fish andmammals, with the most distant block found in intron
5 of Lmbrl. The function analysis for these conserved
non-coding sequences is also underway.
(2) Genetic incompatibility between X-linked loci andtwo autosomal regions causes hybrid breakdown between
two mouse subspecies

Ayako Oka, Nobuo Takagi\ Kiyotaka Toshimori2,
Toshiyuki Takan03, Akihiko Mita, Yoichi Mizushina,
Noriko Yamatani, Hiromi Yamamoto, Kazuo
Moriwaki 4 and Toshihiko Shiroishi eDepartment of
economics, Hokusei Gakuen University, ~epartmentof Anatomy and Developmental Biology (Gl) Graduate
School of Medicine, Chiba University, 3Division ofPopulation Genetics, NIG, 4RIKEN, BRC)
Hybrid breakdown is a type of reproductive failurethat appears after F2 generation of crosses between
different species or subspecies. It is caused byincompatibility between alleles of interacting genes.
We have studied the hybrid breakdown using a
consomic strain, C57BLl6j-XMSM , in which the X
chromosome of C57BLl6j (derived mostly from Mus
musculus domesticus) is substituted by the X chromosome of the MSMIMs strain (M m. molossinus). Malesof this consomic strain are sterile, whereas Fl hybrids
between C57BU6j and MSMIMs are completely fertile.
This finding implies that incompatibility between
X-linked gene(s) and other chromosomal gene(s) causes

GENETIC STRAINS RESEARCH CENTER

67

the hybrid breakdown observed in C57BL/6J-XMSM• The
previous our study revealed that at least three X-linked
QTLs are responsible for the spenn head abnonnality
and male sterility of C57BLl6J-XMSM •In this study, we conducted the whole-genome
QTL analysis to detect loci that interact with the
X-linked genes. The result suggested that one locus in
Chr 1 and two loci in Chr 11 interact with the X-linked
genes for proper male reproduction in parental strains,and disruption of this interaction causes the hybrid
breakdown. Phenotype characterization showed thatsperms of C57BL/6J _XMSM fail to penetrate the zona
pellucida of egg, and is responsible for disability tofertilize. This result implies that the causative genes
located in X-chr, Chr 1 and Chr 11 orchestrate spenn
function to penetrate zona pellucida.
(3) Comparison of BAC-end genome sequences ofJapanese wild mice-derived MSM/Ms strain with thewhole genome sequence of standard laboratory strain
C57BL/6J detected vast amount of SNPs

Kuniya Abet, Hideki Noguchi 2, Keiko Tagawa3, Misako
Yuzurihat, Atsushi Toyoda 2, Toshio Kojima2, Kiyoshi
Ezawa4, Naruya Saitou4, Masahira Hattori 2, Yoshiyuki
Sakaki2, Kazuo MoriwakP and Toshihiko Shiroishi
eRIKEN BRC, 2RIKEN GSC, 3Inst. Mol. Embryology,
Kumamoto Univ., 4Div. of Population Genet., NIG)
MSMIMs is an inbred strain established from theJapanese wild mouse, Mus musculus molossinus in
National Institute of Genetics, Mishima. Inbreeding
generation numbers of this strain reached to F73 asof the end of 2004. It is believed that subspecies
molossinus has substantially contributed to the genome
constitution of common laboratory strains of mice,although the majority of their genome is derived fromthe west European M m. domesticus. Information on
the molossinus genome is thus essential not only for
genetic studies involving molossinus but also for characterization of common laboratory strains. We
constructed an arrayed BAC library from male MSM!
Ms genomic DNA, covering ---..11 X genome equivalent.
Both ends of 176,256 BAC clone inserts were sequenced,
and 62,988 BAC end-sequence (BES) pairs were mapped
onto the C57BLl6J genome (NCBI mouse Build 30),
covering 2,228,164 kbp or 89% of the total genome.
Taking advantage of the BES map data, we established
a computer-based clone screening system. Comparison

68

Mammalian Genetics Laboratoryof the MSM/Ms and C57BLl6J sequences revealed
489,200 candidate SNPs in 51,137,941 bp sequenced. The
overall nucleotide substitution rate was as high as
0.0096. The distribution of SNPs along the C57BLl6J
genome was not unifonn: the majority of the genome
showed a high SNP rate, and only 5.2% of the genome
showed an extremely low SNP rate (percentage
identity = 0.9997); these sequences are likely derived
from the molossinus genome. This result indicatedusefulness of the MSMIMs strain in genetical analysis
particularly in cross expreiments with domesticus
derived standard inbred strains.
(4) Characterization of M00745 mutants resembling
Rim3 mutants

Shigekazu Tanaka, Hiroshi Masuya\ Shigeru
Wakana\ Masaru Tamura and Toshihiko Shiroishi
eRIKEN Genomic Sciences Center)A dominant skin mutant M00745 was generated
by the ENU-mutagenesis project of RIKEN Genomic
Sciences Center, and it exhibits scarring alopecia
resembling the phenotype of Rim3 and Re don• First, we
carried out linkage analysis based on totally 70
backcross progeny generated from a backcross of(MOO745/ + x JF1) Fl to JF1. This inter-subspecificbackcross showed tight linkage of M00745to a marker
DllMitl24 and the GsdmA cluster. Sequencing of theM00745 mutant revealed a point mutation in the
C-terminus of GsdmA -3, which is the causative genefor Rim3 and Re don• This single base substitution leads
to a nonsense mutation at the amino acid residue
conserved among all members of the Gsdm family. It
suggested that the conserved C-terminus of GsdmA-3
is important for regulation of proliferation anddifferentiation of epidermal cells.
To elucidate phenotypic difference of two
mutations, Rim3 and M00745, we employed histological analysis of the two mutant mice. Degenerated hairfollicles, atrophy of sebaceous glands, hyperproliferation of epidermis and hair follicles were commonly
observed in the both mutants, but epidermis of
M00745/ + mice was much thicker than that of the
Rim3l+ mouse. Another difference observed between
Rim3/ + and MOO745/ + mice was late-onset corneal
opacity. Rim3l+ mice exhibited corneal opacity after
3 months of age, whereas MOO745!+ mice did not evenafter 10 months of age. We found that meibomianglands of the Rim3/ + mouse were cystic and seemed
to be dysfunctional, but M00745/ + mouse has cells
containing meibum. It indicated that meibum lipid isdepleted in Rim3/ +, but not in MOO 745/ +. The
affected structure of the meibomian glands may
explain the late-onset corneal opacity specificallyobserved in Rim31 + mice. Difference of the phenotypes
between Rim3/ + and MOO 745/ + mice is possibly
attributable to difference of the mutation type in the
C-terminus of GsdmA-3, although we can not exclude
another possibility that different genetic backgroundof the Rim3/ + and MOO 745/ + is responsible for thedifference.
PUBLICATIONSPapers
1. Abe, K, Noguchi, H., Tagawa, K, Yuzuriha, M.,
Toyoda, A., Kojima, T, Ezawa, K, Saitou, N., Hattori,
M., Sakaki, Y, Moriwaki, K and Shiraishi, T. (2004).
Contribution of Asian mouse subspecies Mus musculus
molossinus to genomic constitution of strain C57BL/
6], as defined by BAC-end sequence-SNP analysis.
Genome Res. 14,2439-2447.
2. aka, A., Mita, A., Sakurai-Yamatani, N.,
Yamamoto, H., Takagi, N., Takano-Shimizu, T.,
Toshimori, K, Moriwaki, K and Shiraishi, T. (2004).
Hybrid breakdown caused by substitution of the X
chramosome between two mouse subspecies. Genetics.
166,913-924.
3. Nemoto, M., Morita, Y, Mishima, Y., Takahashi,S., Nomura, T, Ushiki, T., Shiraishi, T., Kikkawa, Y.,
Yonekawa, H. and Kominami, R. (2004). Ahl3, a third
locus on mouse chromosome 17 affecting age-related
hearing loss. Biochem Biophys Res Commun. 324,
1283-1238.
4. Sagai, T., Masuya, H., Tamura, M., Shimizu, K,
Yada, Y., Wakana, S., Gondo, Y., Noda, T. and
Shiroishi, T. (2004). Phylogenetic conservation of alimb-specific, cis-acting regulator of Sonic hedgehog(Shh). Mamm Genome. 15, 23-34.
5. Sagai, T., Hosoya, M., Mizushina, Y., Tamura, M.
and Shiraishi, T.(2005). Elimination of a long-range
cis-regulatory module causes complete loss oflimb-specific Shh expression and truncation of themouse limb. Development 132,797-803.
6. Sakai, T, Miura, I., Yamada-Ishibashi, S., Wakita,Y., Kohara, Y., Yamazaki, Y., Inoue, T., Kominami, R.,
Moriwaki, K, Shiroishi, T., Yonekawa, H. andKikkawa, Y. (2004). Update of mouse microsatellite
database of Japan (MMDBJ). Exp Anim. 53,151-154.
Books
7. #!~~~(2004) r!ll!M'"?7 Ao:>Jf / A1:tili'):-~' ') L-C~(:El~!h?~Jl') O:>7h I:: t- Jf / A . ~~~ A 7" Ao:>l'!I!M C:
12f'¥.ro...o:>J.iF7l'J, Molecular Medicine Vo1.411:!tb~±lflJ.
EDUCATION

1. #!~~~r!l!f~'"?7 A~flJm L-teJf / A#\l~'~M#fJJf/ A V'bli2004 inmllij, mllij, 8 J'I, 2004. (in Japanese).

2. Dr. T. Shiroishi organized a satellite symposium
at the 36th Annual Meeting of The Japanese Societyof Genetics, Osaka, May, 2004.
3. Dr. T. Shiroishi gave a lecture at the Tohoku
University, Sendai, September, 2004 (in Japanese).
4. Dr. T. Shiroishi was invited to give a seminar on"Mouse Limb development" at the Tohoku University,
Sendai, September, 2004 (in Japanese).
5. Dr. T. Shiroishi organized a satellite symposium
at the 27th Annual Meeting of the Molecular Biology
Society of Japan, Kobe, December, 2004.
SOCIAL CONTRIBUTION AND OTHERS

Dr. T. Shiraishi served as an editor for Mammalian
Genome.
Dr. T. Shiroishi served as an editor for Genes andGenetic Systems.
Dr. T. Shiraishi served as a member of the council of
The Genetics Society of Japan.
Dr. T. Shiraishi served as a member of the council ofJapanese Association for Laboratory Animal Science.
Dr. T. Shiroishi is a project director of Mouse
Functional Genomics Research Group, Genome
Science Center, RIKEN.

F-b. Mammalian Development Laboratory
Yumiko Saga GroupRESEARCH ACTIVITIES(1) Molecular mechanism of somite segmentation

Mitsuru Morimoto, Yu Takahashi! and Yumiko SagaeN ational Institute of Health Sciences)

Mammalian Genetics Laboratory

69

The somite is the first morphologically distinct
segmental unit formed in a vertebrate embryo andgives rise to metameric structures such as vertebrae,ribs and skeletal muscles. A 'clock and wavefront'
model has been proposed to explain the underlying
mechanism, in which the periodicity is generated by
a 'clock' in the posterior PSM and this temporal
periodicity is then translated into the segmental units
in the 'wavefront'. The wavefront is thought to exist
in the anterior PSM and progress backwards at aconstant rate. The majority of the oscillating genes are
related to Notch-signaling pathway. However, an
important qu·estion is whether the level of Notch
activity really oscillates and how such oscillation istranslated into a segmental pattern in the anterior
PSM. We have succeeded in visualizing the levels of
Notchl-activity in mice, by using an antibody against
an activated form of Notchl, and show that it oscillates
in the posterior PSM but is arrested in the anterior
PSM. Detailed analyses of the distribution of anactivated form of Notchl and Mesp2 protein in theanterior PSM demonstrate that somite boundaries areformed at the interface between Notchl-activatedand -repressed domains and that Mesp2 protein
localizes in the Notchl-respressed domain. Thus,
Mesp2 plays a crucial role in translation of thetemporal oscillation of Notch activity into theformation of regularly-spaced somites.
Somitogenesis is not only an attractive example
of metameric pattern formation but is also a goodmodel system for studies of morphogenesis,
particularly epithelial-mesenchymal interconversion
in vertebrate embryos. Mespl and Mesp2 arehomologous bHLH transcription factors that areco-expressed in the anterior presomitic mesoderm(PSM) just prior to somite formation. Analysis ofpossible functional redundancy of Mespl and Mesp2
has been prevented by the early developmental arrestof Mespl/Mesp2 double-null embryos. We performed
chimera analysis using either Mesp2-null cells or
Mespl/Mesp2 double-null cells, to clarify (1) possiblefunctional redundancy and the relative contributionsof both Mespl and Mesp2 in somitogenesis and (2) thecell autonomy of Mesp functions in several aspects ofsomitogenesis. Both Mesp2-null and Mespl/Mesp2
double-null cells fail to form initial segment borders
and to acquire rostral properties, confirming that thecontribution of Mespl is trivial in these aspects. In
contrast, Mespl/Mesp2 double-null cells contribute to70

Mammalian Development Laboratory

neither epithelial somite nor dermomyotome formation
while Mesp2-null cells partially contribute toincomplete somites and dermomyotome. This indicatesthat Mespl has a significant role in the epithelializationof somitic mesoderm. We have found that the roles ofthe Mesp genes in epithelialization and establishing
rostral properties are cell autonomous. However, we
also found that epithelial somite formation with normal
rostro-caudal patterning by wild-type cells was severely
disrupted by the presence of Mesp mutant cells,
showing non-cell autonomous effects and supporting
our previous hypothesis that Mesp2 is responsible forthe rostro-caudal patterning process itself in theanterior PSM via cellular interaction.
(2) Regulation of Mespl and Mesp2 expression

Masayuki Oginuma, Yukuto Yasuhiko l and Yumiko
Saga CNational Institute of Health Sciences)

Mespl and Mesp2 are both expressed in the early
mesoderm and presomitic mesoderm (PSM) just before
segmentation. In order to analyze global transcriptional
regulation of both Mespi and Mesp2, we started to use
BAC transgenic strategy. Mespl and Mesp2 are locatedin head to head orientation and separated only by 16
kb. Since the expression pattern is very similar, wehave introduced ALPgene in the Mespllocus and LacZ
gene in the Mesp2 locus of a BAC clone using
homologous recombination in bacteria. By introducing
this BAC transgene, we were able to reproduce bothgene expressions in a single embryo. We havegenerated several BAC constructs that have deletionsin the possible regulatory region. Using thosetransgenes, we will find regulatory regions required
for faithful expression of Mespl and Mesp2 in vivo.
For Mesp2 specific enhancer, we have alreadydetermined the minimum sequence required for PSM
expression. Further analyses revealed that theexpression was regulated by both Tbx6 and Notch
signaling. Although we have identified the directbinding of Tbx6, no RBP-}k-binding is found,indicating that another mechanism is involved in the
Mesp2 regulation, which is mediated via Notch
signaling.
(3) Search for target genes of Mesp2 transcription factor

Kaoru Mitsui, Yoshiro Nakajima and Yumiko SagaMesp2 transcription factor is critically importantfor both segment border formation and establishing
rostro-caudal patterning of somites. However, the directtarget genes are not identified yet. In order to obtain
information of the target sequences, we have employed
SELEX method. Random nucleotide oligomers with
PCR primers were mixed with Mesp2 protein and thepossible binding sequences were identified. However,the sequence is different from so-called E-box or N-box
sequences that are known to be consensus sequences
for bHLH-type transcription factor. However, thesimilar sequence is found in the promoter region of
Delta-like 1 gene that is known to be negatively
regulated by Mesp2. Weare currently examining thesequence by generating transgenic mice.
In addition, the analyses of Mesp2-knockout
mouse and the genetic studies have identified several
genes affected by the Mesp2 during somitogenesis. Oneof genes that are downregulated in the Mesp2-knockout
mouse is EphA4. We are expecting that the EphA4
might be a direct target of Mesp2 protein since theexpression domain is very similar to that of Mesp2 andthe expression disappears in the Mesp2-nullbackground. To address this question, we havesearched the enhancer sequence of EphA4. Using
transgenic mouse strategy, we have succeeded to
identify the minimum sequence of EphA4 enhancer.
The enhancer contains multiple E-box sequences andthe deletion of some of them results in the loss ofenhancer activity, which strongly indicates that these
E-boxes are responsible for the activation of EphA4
by Mesp2.
(4) Cardiovascular development and Notch signaling

Hiroki Kokubo, Yusuke Watanabe, Yoshiaki
Okamura, Wataru Saito and Yumiko Saga
Notch signaling is required for multiple aspectsof cardiovascular development, including arterialvenous differentiation, septation and cushion
formation. Despite recognition of the importance of the
Notch pathway in normal cardiovascular development,
the proximate downstream effectors are not yet known.
Likely candidate effectors are members of the hesr
(hairy and enhancer of split related) family of bHLH
transcription factors. However, mutational analysis ofindividual hesrgenes has so far failed to elucidate theirrole in all Notch-mediated cardiovascular signaling

events. An example of this is evident for mutants ofgridlock, the zebrafish counterpart of mouse hesr2,
which have vascular defects, whereas mouse hesr2
mutants have only cardiac defects. One possible
explanation for these differences could be functional
redundancy between hesr family members. Micelacking the hesrl gene are viable and fertile, whereas
knockout mouse of both hesrl and hesr2is embryoniclethal at 11.5 days postcoitum (dpc) and recapitulates
most of the known cardiovascular phenotypes of
disrupted Notch pathway mutants including defects
in arterial-venous specification, septation and cushion
formation. Taken together, our results demonstrate a .•requirement for hesrl and hesr2 in mediating Notch
signaling in the developing cardiac and vascularsystems. In addition, we have tried to find out arterial
specific enhancer of hesrl gene. There are at least 5
RBP-Jk binding sites in the upstream region of hesrl
gene and disruption of these sites resulted in the great
reduction of hesrl expression. However, further detailedtransgenic analyses revealed that another enhancer
sequence might be involved in the regulation of hesrl
in the artery.
Weare also studying Notch function in heartmorphogenesis using transgenic mouse, which has
activated Notchl after floxed CAT gene under thecontrol of CAG promoter. We can achieve forced Notch
activation by intercrossing the transgenic mouse with
Cre-expressing mouse. Since Mespl is expressed in theheart precursor cells, we can drive Notch activation
only in Mespl-lineage using Mespl-cre mouse. The
trans-heterozygous mouse exhibits heart abnormality,
which is characterized by abnormal myocardialtrabeculation and AVC formation. To know thedownstream genes involved in the abnormal
morphogenesis, we have conducted GeneChip analysis,by which ectopic induction of Wnt2, BMP6, llet-l was
detected in addition to hesrl. Since hesrl is known to
be a direct target of Notch signaling, we have first
asked whether these changes in gene expression ismediated by hesrl or not by activating Notchl in theabsence of hesr1. Interestingly, the changes in gene
expression were observed even in the absence of hesrl,
indicating that these genes are up-regulated by ahesrl-independent Notch signaling pathway.

Mammalian Development Laboratory71

(5) Functional analysis of mouse nanos genes

Masayuki Tsuda, Atushi Suzuki, Hitomi Suzuki,
Makoto Kiso and Yumiko Saga
Previously we have isolated three mouse nanos
genes (nanos1, nanos2 and nanos3). Among them, we
focus on function of nanos2 and nanos3 since these are
specifically expressed and play important roles ongerm cell development.
We have shown that nanos2 is expressed in thegerm cells in both embryonic and adult testes anddisruption of nanos2 resulted in a complete loss ofgerm cells in the testis. To understand the molecularmechanism leading to the loss of germ cells, we haveto know the direct targets of nanos2 since it is known
that nanos protein works as a translational repressor
in the Drosophila germ cells. To achieve this, we havefirst tried to generate good antibodies against nanos2and naons3 to be used for immunoprecipitation. Using
purified nanos2 and nanos3 protein expressed in E.coli,
we have succeeded to generate antibodies for bothproteins. The nanos2 antibody can be used for
immunoprecipitation of nanos2 protein fromembryonic testes. Therefore, this antibody would be
useful to identify not only the target genes but also
proteins interacting with nanos2 in future studies. We
are also interested in the regulation of nanos2
eKpression. The nanos2 expression starts in the PGC
after entering male gonad and the expression is
testis-specific and not observed in any other tissues.
Using transgenic mouse, we have identified a coreenhancer region required for the testis specificexpression of nanos2. The identification of the
upstream signal would be a key to understand amechanism of early male germ cell specification.
PUBLICATIONSPapers

1. Haraguchi, S., Saga, Y., Naito, K., Inoue, H. andSeto, A. (2004). Specific gene silencing in the
pre-implantation stage mouse embryo by an siRNA
expression vector system. Mol Reprod Dev. 68,17-24.
2. Kokubo, H., Miyagawa-Tomita, S., Tomimatsu, H.,
Nakashima, Y., Nakazawa, M., Saga, Y. and Johnson,
RL. (2004). Targeted disruption of hesr2 results inatrioventricular valve anomalies that lead to heart
dysfunction. Circ Res. 95,540-7.

72

Mammalian Development Laboratory

3. Okazaki, N., Kikuno, R, Ohara, R, Inamoto, S.,
Koseki, H., Hiraoka, S., Saga, Y., Seino, S., Nishimura,M., Kaisho, T., Hoshino, K., Kitamura, H., Nagase, T.,
Ohara, O. and Koga, H. (2004). Prediction of the coding
sequences of mouse homologues of KIAA gene: IV.
The complete nucleotide sequences of 500 mouse
KIAA-homologous cDNAs identified by screening ofterminal sequences of cDNA clones randomly sampledfrom size-fractionated libraries. DNA Res. 11,205-18.
4. Okazaki, N., Kikuno, R, Ohara, R, Inamoto, S.,
Koseki, H., Hiraoka, S., Saga, Y., Kitamura, H.,
Nakagawa, T., Nagase, T., Ohara, 0., Koga, H. (2004).
Prediction of the coding sequences of mouse
homologues of FLJ genes: the complete nucleotide
sequences of 110 mouse FLJ-homologous cDNAs
identified by screening of terminal sequences of cDNA
clones randomly sampled from size-fractionatedlibraries. DNA Res. 11, 127-35.
5. Kii, I., Amizuka, N., Shimomura,]., Saga, Y., Kudo,
A. (2004). Cell-cell interaction mediated by cadherin-11
directly regulates the differentiation of mesenchymal
cells into the cells of the osteo-lineage and thechondro-lineage. J Bone Miner Res. 19, 1840-9.
6. Ishikawa, A., Kitajima, S., Takahashi, Y., Kokubo,
H., Kanno, J., Inoue, T. and Saga, Y. (2004). Mouse
Nkdl, a Wnt antagonist, exhibits oscillatory gene
expression in the PSM under the control of Notch
signaling. Mech Dev. 121, 1443-53.
Books

7.

i1l~m-~-T (2005) Notch ~(~f1J),//-r !vO)~1:jJH1, ~~~¥

r~j::. 5Ht . fl}j::UJf~2005J 23, 64-72.

EDUCATION

1. Dr. Y. Saga gave a lecture at the Fujita Health
University, January, 2004 (in Japanese).
2. Dr. Y. Saga gave a lecture at the Keio University,
Aprile, 2004 (in Japanese).
3. Dr. H. Kokubo gave a lecture at the Tokyo
University of Science on "Functions of hesrl and hesr2
in the heart morphogenesis", September, 2004 (inJapanese).

F-c. Mouse Genomics Resource Laboratory
Tsuyoshi Koide GroupRESEARCH ACTIVITIES(1) Identification of genetic factors responsible fordifference in spontaneous home-cage activity between
KJR and C57BL/6
Juzoh Umemori and Tsuyoshi Koide

It has been previously reported that mice move
spontaneously in the habituated home-cage according
to a light/dark rhythm, and most mice are active during
the dark period. This kind of activity is called aspontaneous home-cage activity. In a variety of strains,mice of C57 were relatively hypoactive, in contrast to
mice of KJR were especially hyperactive in habituatedhome-cage. We studied the spontaneous home-cage
activity in terms of the ethological components,
genetics factors and biological functions.
For the ethological and genetic studies, F2 progeny(BKF2) were made between C57BLl6J and KJR strains,
and analyzed. Ethological studies showed that total
home-cage activity (THA) could be divided to two
independent components, active time (AT) thatindicated temporal elements of spontaneous home-cage
activity, and average activity (AA) that indicatedquantitative elements. A little correlation between AT
and AA suggested that AA and AT are independentmeasurement and share only a few common genetic
factors.
In order to study the genetic bases responsible forthe differences in spontaneous home-cage activity
between KJR and C57BLl6J, we conducted quantitative
trait loci (QTL) analyses using BKF2 progeny. QTL
analyses showed three significant QTLs associatedwith the spontaneous home-cage activity. These QTLs
were designated as hyperlocomotive activity related
QTLl (Hylaql), Hylaq2 and Hylaq3. (All Hylaq locicontrolled THA that was defined by summing up
effects of the AT and the AA.) Hylaql was located ina middle region on chr2 and mainly controlled activity
time. Hylaq2was located on the distal side of Hylaqlon
chr2 and controlled both of AT and AA. Hyaq3 was
located near the telomeric region on chrlO, andcontrolled mainly AA.
It has been reported that dopamine (DA) system

in basal ganglia in brain is associated with movement
and spontaneous locomotive activity. To search forcandidate genes in the above QTL region, and tounderstand the biological mechanism that is associatedwith the spontaneous home-cage activity, straincomparative analyses on the DA system have been
conducted with pharmacological and biochemical
methods. The results showed that difference inhome-cage activity might be associated with neither
DA synthesis, release nor uptake in presynapse butwith downstream pathway in DA system following
activation of DA receptors in dopaminergic neuron. In
particular, function of Dl DA pathway that has roleof suppressing the spontaneous activity is possibly
reduced in KJR.
(2) A systematic analysis of genetic factors associatedwith the diversity of mouse behavior
Akinori Nishi, Toshihiko ShiroishP and Tsuyoshi
Koide (lMammalian Genetics Laboratory)
A great variation of behavioral pattern is observedin a variety of animals, such as among individuals,
populations and species. A number of studies indicatedthat the behavioral diversity is greatly influenced bygenetic factors. The aim of our study is to identify thegenetic factors associated with the diversity of
behavioral traits.
In order to study genetic mechanism associatedwith the behavioral diversity, we are using mousestrains, C57BLl6 and MSM. C57BLl6 is a widely usedlaboratory strain. The other strain, MSM, wasestablished from Japanese wild mice after the 20
generations of brother-sister breeding. These twostrains are genetically different to a great extent andknown to exhibit a behavioral difference in a variety
of behavioral tests.
Recently, consomic strains, in which one of thechromosomes of C57BL/6 was replaced with thecorresponding chromosome from MSM, were
established for a series of chromosomes. These strainsare expected as powerful tools for investigatingcomplex traits, which are influenced by multiple
genetic factors. In this project, we applied a systematic
test battery on the consomic strains to reveal genetic
factors associated with behavioral traits. As abehavioral test battery, the home cage test, novel
home-cage test and light/dark box test were applied.

Mouse Genomics Resource Laboratory73

In the tests, behavioral traits in a habituated condition
were measured by the home cage test, the activity inthe novel place and habituation for the novel situation
were evaluated by the novel home cage test, andanxiety related place preference behavior wasmeasured by using the light/dark box test.
As the result of these tests, we found that
B6-6CMSM consomic strain, which carries MSMderived chromosomal segment from D6Mit166 to
D6Mit12 on chromosome six, exhibited significantly
lower activity in the home cage test and the novel home
cage test, and indicated higher anxiety related behavior
in the light/dark box test comparing to C57BLl6.
(3) Descriptive and Temporal Analysis of the Open-field
Behavior in Wild-derived Mouse Strain

Aki Takahashi, Toshihiko Shiroishi' and Tsuyoshi
Koide ('Mammalian Genetics Laboratory)

C57BLl6 and MSM. These parental strains exhibited
great difference in the open-field behavior, C57BLl6
tended to show locomotion and leaning in the later
session but MSM exhibited grooming and freezing.
Each consomic mouse strain has same genetic
background as C57BLl6 except for one chromosome
which is introduced from MSM. By examining a seriesof strains\ we will be able to reveal the chromosomesthat are associated with the behavioral difference
between C57BLl6 and MSM. A half of those strains
have been so far analyzed, and some of the strains
exhibited different behavior comparing to the parental
strain C57BL/6. One strain carrying chromosome 17
from MSM, B6-17MSM, showed reduced locomotion
and rearing, and extended risk assessment behavior,
stretching. In order to find genetic locus responsible
for this behavioral pattern, we will conduct further
genetic analysis using B6-17MSM and C57BLl6 strains.
PUBLICATIONS

The open-field is one of the widely used methods
for measuring emotionality. In this test, an animal issimply placed into a novel, brightly lit, arena fromwhich escape is barred. It is known that genetic factors
make a large contribution to phenotypic variation invarious measures of open-field behavior. Most study
have used two major variables, ambulation anddefecation, that totaled per test. However, mice actuallyshow more various behaviors such as sniffing, rearing,and grooming in the field, and frequency of thosebehaviors change with time. In this point of view,
detailed analysis of the open-field behavior isimportant to understand psychological status of mice.
We therefore described detail of open-field behavior
with 12 ethograms (sniffing, locomotion, stretching,leaning, rearing, grooming, face-washing, digging,
gnawing, jumping, pausing, freezing) by counting
presence of those behaviors in 5 seconds. We
previously examined descriptive and temporal analysis
of open-field behavior in genetically diverse mousestrains: ten wild-derived mouse strains (PGN2, BFW
2, HMI, CASTlEi, NJL, BLG2, CHD, SWN, KJR, MSM),
one fancy strain JF1, and one laboratory strain C57BLI
6. It was revealed that those strains had a great
diversity in the temporal changes of those open-field
behaviors.
To elucidate genetic mechanisms underlying theopen-field behavior, we have been performing thisdescriptive study on consomic mouse strains made by74

Mouse Genomics Resource Laboratory

Reviews

1.

tl~m1:.,

INillli1U (2004) tJI1t~~1tO)iOO-f*i~H~ipipb9j1{~$B"Jm;j:}f. :t"1::":t"-1 FUJf~O)~?ffic:1i~, 155-170.

~tJI.·:t"I::":t"-1FUJf~~m*:t"~~~~~

2. INiI !fijU(2004) !l!f1:.E!3*"'<' t7 7-~*1f~m1r\tL § Jejj!
gw'I1O)fTgw)!~$B"Jm;j:}f. iIJ~IN!I!$UJf~54, 59-66.
EDUCATION

1. Dr. T. Koide gave a seminar at Tokyo Institute of
Psychiatry, February, 2004 (in Japanese).
2. Dr. T. Koide gave a lecture at Institute of
Psychology, University of Tsukuba, October, 2004 (inJapanese).

F-d. Model Fish Genomics Resource
Noriyoshi Sakai GroupRESEARCH ACTIVITIES(1) Gene targeting system with RNA interference (RNAi)in zebrafish

Minori Shinya, Kimiko Saka and Noriyoshi Sakai
Gene silencing via short interfering RNAs (siRNAs)
has proved to be a useful tool in studying gene function

in plants, invertebrates and mammalian systems. To
date, gene silencing effects of siRNAs have confirmedin the zebrafish, which is an emerging model fordevelopmental and diseases analysis. However, theeffects were temporal (only in early developmental
stages) and sometimes mosaic in an embryo becauseof the method injecting siRNAs into the one-cell stageof embryos. We recently succeeded in the productionof transgenic zebrafish from in vitro-cultured sperm l ).
The advantage of this technique is that the mosaicism
inherent in other conventional transgenic methods"isavoided. Our aim in the present study is theestablishment of a rapid system with cultured sperm
to generate transgenic zebrafish for gene silencing by
siRNA. To achieve this, we first selected the targeted
genes that we have already known the silenced
phenotype and also the phenotype can be easily
recognized. Then, we established the zebrafi.sh cultured
cells expressing the targeted genes, in order todetermine the best sequence of siRNA for specificsuppression of the gene by transfecting several
possible designed siRNAs. After finding out the bestsiRNA, we will construct the retroviral vector of DNA
vector-based siRNA constructs and then infect to thecultured spermatogonia with the virus. By in vitro
insemination with the sperm derived from the infected
spermatogonia, we can obtain the transgenic zebrafish
producing siRNA in all the cells. Once the above
system is developed, the inducible siRNA in thespecific cells or organs will be easily conducted, andthus, the technique leads to a high-throughput system
for genome-wide loss-of-function studies.
(2) Analysis of functionally distinctive testicular cell linesof zebrafish to support male germ cell development

Kayoko Kurita, Kataaki Okubo!, Masaru Matsuda\
Yoshitaka Nagahamal and Noriyoshi Sakai
eLaboratory of Reproductive Biology, National
Institute for Basic Biology)

expression remain unclear. We recently established
two testicular cell lines of zebrafish with distinct
functions to support the development of male germ
cells 2). Twelve cell lines were established bysingle-colony isolation from tumor-like testis-derived
ZtA6 cells. Multiple features characteristic of Sertoli
cells such as phagocytic activity and transcription
specific genes (sox9a and Wilms' tumor suppressor
WTl) were analyzed in the lines. The lines, ZtA6-2 andZtA6-12, showed almost the same characteristics as
Sertoli cells, but exhibited distinctive features whenmale germ cells were co-cultured with each line as
feeders. The in vitro fertilization by the culture of germ
cells with ZtA6-12 produced more embryos than thatwith ZtA6-2. In contrast, ZtA6-2 gave rise to significantly larger clumps of germ cells after a 12-day culturecompared to the ZtA6-12 cell line. Expression of vas,strongly expressed in spermatogonia and premeiotic
spermatocytes, was prolonged in the culture using
ZtA6-2 feeders, while it was reduced with the germ cellson the ZtA6-12 feeders. Compared with the previous
results obtained on the original ZtA6 cells, these results
suggested that the function of the ZtA6-2 cells was
directed to stimulate the proliferation of spermatogonia,
and ZtA6-12 to stimulate the differentiation into sperm.
These cell lines will facilitate investigation of
Sertoli cell molecules that contribute to theproliferation and differentiation of spermatogonia. We
are currently working on the molecular cloning ofgenes expressed specifically in each line. 205 clones and256 clones were isolated by the subtraction experimentswith ZtA6-2 eDNA minus ZtA6-12 eDNA and vice versa.
Several have already been confirmed by screening withdot blot hybridization and virtual Northernhybridization to eDNA of each line. In situ
hybridization for sections of a testis showed that someof the cDNAs were expressed in Sertoli cells. Analysis
of other cDNAs is under investigation.
(3) Culture condition for zebraflsh spermatogonial stem
cells

Sertoli cells are important to germ cells in everything from male sex-determination to spermatogenesis.
In spermatogenesis, Sertoli cells interact directly with
germ cells in the testis to induce the complex process
required for the production of functional sperm. Thesecells mediate the production of many molecules as wellas cell junctions and adhesion. The function of manyof these molecules and the regulation of gene

Kenji Saito and Noriyoshi Sakai
Spermatogonial stem cells (SSCs) maintain
spermatogenesis by continuous production of thedaughter cells and cells that differentiate into
spermatozoa. Sertoli cells are the only type of somatic
cells that closely interact with SSCs to create aModel Fish Genomics Resource

75

favorable environment inter testis. However, theregulatory mechanisms are still unclear because of the
difficulty of identifying and manipulating an individual
SSC and the surroundings.
To establish in vitro system in which SSCs
proliferate continuously, we performed the isolation
procedure of A type spermatogonia and determineda culture condition to proliferate A type spermatogonia
on a Sertoli cell feeder layer. When zebrafish weretreated with busulfan reagent, we found the decline ofdifferentiating germ cells, such as spermatocytes andspermatids, while A type spermatogonia increased
after 4 days of treatment. Then, on day 7 germ cellsof the testis were consisted of only A type spermatogonia and sperm. In teleosts, a single spermatogonium that enclosed within germinal cysts ofSertoli cells is defined as A type. Furthermore, we
observed that the A type spermatogonia proliferate
rapidly after the damage with busulfan reagent. The
definition and the observation suggest that A typespermatogonia are SSCs of a teleost. When enzymatically dissociated testicular cells containing A typespermatogonia were co-cultured on ZtA6-6 Sertoli cellline, proliferation of A type spermatogonia wasobserved without differentiation by a BrdU
incorporation experiment after 14 days of culture.
These results indicate that A type spermatogonia
self-renew in the culture condition that might represent
testicular microenvironments to maintain SSCs.
(4) Analysis of the ability of cultured embryonic cells
derived from different developmental stages to induce
the anterior-posterior axis

Megumi Hashiguchi and Noriyoshi Sakai
One of the most fascinating phenomena in primary
embryonic induction is the regional specificity of theneural structures that are produced. Primary embryonic induction can be divided into three major
components; head specific, trunk specific and tailspecific induction.
We established the condition to culture zebrafish
embryonic cells continuously without any artificial
immortalization treatment. When cultured cells weretransplanted into a blastulae embryo, the cells fromdifferent developmental stages had different abilities
to induce specific second axes. Cells derived fromearlier stage embryos (gastrula stage) induce complete

76

Model Fish Genomics Resource

anterior structure. Cells from the pharyngula stage(embryos segmented) induce anterior structures eitherwith cyclopia or without an eye. Cells from later stages(early larva) induce posterior structure with otic
vesicles and a heart. Interestingly, cells from laterstages had low induction efficiency, but the efficiency
increased when proliferation of the cells was arrested
with mitomycin C. Whole-mount in situ hybridizationfor emx-1 (telencephalon marker), krox 20 (rhombomere
marker), and shh (notochord marker) indicated similar
patterns of gene expression to the specific induction.
Cultured cells derived from embryos at various
developmental stages, therefore, change theirproperties to induce the second axis from an anterior
to a posterior orientation according to their developmental stage. In addition, cells may secrete more
inducer(s) when proliferation is stopped, particularly
in later stages. These cultured cells can be used to findcellular factors involved in anterior-posterior specificinduction.
PUBLICATIONSPapers

1. Kurita, K., Burgess, S.M. and Sakai, N. (2004).

Transgenic zebrafish produced by retroviral infectionof in vitro-cultured sperm. Proc. Natl. Acad. Sci. USA
101, 1263-1267.
2. Kurita, K. and Sakai, N. (2004). Functionallydistinctive testicular cell lines of zebrafish to support
male germ cell development. Mol. Reprod. Dev. 67,
430-438.
3. Matsumoto, T., Yukawa, W., Nozaki, Y.,
Nakashige, R., Shinya, M., Makino, S., Yagura, M.,
Ikuta, T., Imanishi, T., Inoko, H., Tamiya, G. andGojobori, T. (2004). Novel algorithm for automatedgenotyping of microsatellites. Nucleic Acids Res 32,
6069-6077.
Reviews

4. Sakai, N. (2004). Genetically modified sperm in fish.
ISB News Report April, 1-3.
EDUCATION
f1Bj;::~/~~~O)~~-\">-t:!2.-r-

1. Dr. N. Sakai gave a seminar on "Geneticallymodified sperm in fish" at Shizuoka University, March,
2004 (in Japanese).

2. Dr. N. Sakai gave a course of lectures at the
Department of Marine Bioscience of Fukui Prefectural
University, April-August, 2004 (in Japanese).
3. Dr. N. Sakai was invited to give a seminar on"Genetically modified sperm in fish" at National
Institute of Child Health and Human Development of
NIH, Bethesda, USA, July, 2004.

F-e. Plant Genetics Laboratory
Nori Kurata Group

proteins were removed from chromosomes, but notfrom the centromeric regions. The centromere-specifichistone H3 variant always colocalized with the PAIR2
through early to late meiosis I. In addition, our study
revealed that the rice centromeres paired preceding tousual homologous chromosome pairing. We are now
underway to make it sure that the centromere pairs
at early meiosis I prior to pairing of other chromosomal
regions in rice meiocytes.
(2) Structural and functional analysis of rice OsHAP3
genes

RESEARCH ACTIVITIES(1) Molecular cytogenetics of plant meiosis analyzed
with rice mutants

Ken-ichi Nonomura, Mutsuko Nakano, Mitsugu
Eiguchi, Akio Miyao*, Hirohiko Hirochika* and Nori
Kurata (*Natl. Inst. Agrobiol. Sci.)
This study aims to dissect genetic machinery
controlling meiosis and sporogenesis of higher plants.
By screening of insertional mutant lines tagged by theTos17, the endogenous retrotransposon of rice, we
could identify and isolate two meiotic genes of interest;
PAIRl (HOMOLOGOUS EAIRING !1BERRA TION IN RICE MEIOSIS 1) (Nonomura et al. 2004a) andPAIR2 (Non omura et al. 2004b). The PAIR2 geneencodes a protein of 610 amino acids, homologous to
the yeast HOPi and Arabidopsis ASYl. We raised apolyclonal anti body against recombinant P AIR2
protein and identified the PAIR2 expression in ricemeiocytes by western blotting and immuno-fluorescentanalyses. The PAIR2 protein began to accumulate in
the nucleus of meiocytes following the onset ofpre-meiotic DNA synthesis, and disappeared at
metaphase I. In early meiosis I, the PAIR2 antibody
stains the lateral elements, which are components ofchromosomal axes and play important roles inmediating homologous chromosome pairing. The
PAIR2 protein was removed from the chromosomes
as soon as the homologous chromatins aligned andpaired. This observation suggests the PAIR2 function
in recruiting the central components, which areimportant to establish a rigid structure between lateralelements of homologous partners.
The immuno-cytological analysis at diakinesis,
prior to metaphase I, revealed that the most PAIR2

Yukihiro Ito, Thiruvengadam Thirumurugan,
Kazumaru Miyoshi and Nori Kurata
We identified 11 genes (OsHAP3A to OsHAP3K)
which encode a HAP3INF-YB subunit of CCAAT-box
binding complex in rice by cDNA screening and a
database search. We showed that three genes,
OsHAP3A, OsHAP3B and OsHAP3C, are involved inchloroplast biogenesis (Miyoshi et al. 2003). We next
examined the function of OsHAP3E, which is mostclosely related to LECl and LlL. The LECl and LlL
are Arabidopsis members of the HAP3INF-YB subunit
and are critical regulators of embryogenesis. The
OsHAP3Ewas also expressed in developing embryoand young panicle. The antisense plants of OsHAP3E
showed slightly increased plant height, and its
overexpressing plants showed dwarfism, erected leaves
and abnormal inflorescence with the increased numberof spikelets. Microarray analysis showed thatbrassinosteroid-related genes and MADS-box genes
were affected in the overexpressing plants. These
results suggested that OsHAP3E may be involved inbrassinosteroid-related response and panicle development
Yeast two-hybrid analysis showed that OsHAP3E
interacted with several specific members of OsHAP2
and OsHAP5, which are other subunits of heterotrimeric
CCAAT-box binding complex. These results suggested
that several different combination of HAP complexes
with OsHAP3E can be formed in rice cells. Consideringthat LECl and LlL are critical regulators of embryogenesis
in Arabidopsis, OsHAP3A, Band C are controlling
factors of chloroplast biogenesis and OsHAP3E isthought to regulate hormonal and/or influorescencedevelopment pathways, plant HAP3INF- YB genes aresuggested to have diverse functions depending on eachgene.

Model Fish Genomics Resource77

(3) Regulation of expression of KNOX family class 1
homeobox genes of rice

Yukihiro Ito and Nori Kurata
KNOX family class1 homeobox genes playa vitalfunction for shoot apical meristem (SAM) formationand maintenance. We found that cytokinin, which isnecessary for shoot regeneration, induced theexpression of OSHl, a member of the KNOX family,in the callus. Expression of OSHl was observed anhour after cytokinin treatment, whereas expression of
ORR6, a homologue of a cytokinin early response gene,
was detected within 30 min. It was assumed that thecytokinin-induced gene expression is under control ofa histidine phosphotransfer system which consists ofa histidine kinase, a phosphotransmitter protein anda response regulator. To examine this possibility, we
generated transgenic plants overexpressing COS3
histidine kinase gene, OHP2phosphotransmitter geneor ORRl response regulator gene. These transgenic
plants showed enhanced expression of OSHl upontreatment with cytokinin. This suggested that thesegenes are involved in the cytokinin-induced OSHl
expression. Analysis of transgenic plants withdominant negative constructs of these genes isunderway.
(4) Generation and screening of enhancer trap lines ofrice

Yukihiro Ito and Nori Kurata
To isolate tissue-specific enhancers and valuablemutants defective in various steps of rice development
and to clone the corresponding genes, we are
generating enhancer trap lines of rice. We employed
an enhancer trap system based on the AdDs
two-element system and the GUS reporter gene. We
already have established the system by which Ds-GUS
transposants are efficiently identified in rice (Ito et al.
2004).
This year we screened about 2,000 transposed lines
and identified 146 lines with tissue-specific GUS
activity. GUS expression was observed in variousorgans including embryo (7 lines), leaf (59 lines), root
(22 lines), panicle (11 lines), flower (22 lines), seed (18lines) and shoot apex (7 lines). We also determined theDs-GUS insertion site of the 27 GUS-positive lines. We

78

Plant Genetics Laboratory

identified a trapped gene by a Ds-GUS transposition
in a line P1772, which expressed the GUS on a leaf
lesion after wounding, as an example. In this line theDs-GUS was shown to be inserted near a full-length
cDNA AK073621 on chromosome 12, and this gene wasexpressed transiently in the leaf after wounding. This
indicated that the enhancer of this gene was trappedin P1772 and that our trap system can usefully work
not only on enhancer selection but also on molecular
identification of the trapped genes. Cloning of othertrapped enhancers is underway. These data will be
opened through Oryzabase(http://www.shigen.nig.ac.jp/rice/oryzabase/top/
top.jsp).
(5) Positional cloning of a segregation distortion genedetected in a progeny of a cross between Japonica andIndica rice

Yoshiaki Harushima and Nori Kurata
The aim of this study is isolation of the mostprominent barrier on chromosome 3 detected in the F2of Nipponbare-Kasalath hybrid by positional cloning,and elucidation of the molecular nature of theindividual reproductive barrier. We have clarified thepollen with Kasalath genotype at the gametophyte gene
preferentially fertilize eggs by 94% probability in thematernal plant that is heterozygote or Kasalathhomozygote at the interactive locus on chromosome 6.
For fine mapping the gametophyte gene, we haveselected plants with recombination in the candidate
region from 5691 F2 and 473 backcross plants andmapped it by their selfed progeny test. A prominentcausal gene is an A rgonaute gene. Expression of thisArgonaute gene in both Nipponbare pollen and
Kasalath pollen was confirmed. RT-PCR and microarray analysis showed there was no difference in theexpression level. However, high frequency of nonsynonymous substitutions, 8 out of 9 single nucleotidechanges in a coding region, was detected. This suggests
rapid evolution that is common feature of reproductive
barriers. There are two possibilities to explain Kasalathpollen preferential fertilization; one is Kasalath pollen
gene accelerates fertilization, another is Nipponbare
pollen gene retards it in pollen competition. For
complementation test, two kinds of Nipponbare nearisogenic lines, both the aimed gametophyte gene andthe interactive gene regions are Kasalath homozygote

and only the interactive gene region is Kasalathhomozygote, were transformed by the Nipponbare or
Kasalath genomic fragment containing the Argonautewith GFP. These transform ants are now growing andtheir selfed seeds will be tested for the transmissionof introduced Argonaute and GFP(6) Identification of nuclear proteins showing various
sub-nuclear distribution in riceTadzunu Suzuki, Kazuki Moriguchi and Nori Kurata
We screend rice nuclear proteins from threedevelopmental stages; young panicle, flowering panicle
and regenerating calli by using NTT (nuclear
transportation trap) system and identified over 500
genes including many novel proteins and manytranscription factors. A subset of these genes were
examined for their nuclear localization by introducing
GFP-fusion constructs into onion epidermal cells. A
variety of protein localization in the cell; for instance,
nuclear peripheral, foci-like, speckle-like, chromatinassociated and matrix-associated distribution, wereobserved (Moriguchi et al. 2005). Some of these genes
were revealed to be expressed at specific stages of ricedevelopment by RT-PCR and in situ hybridization. Oneof these which we designated OsAHPI was further
characterized for its tissue specificity and sub-nuclear
localization by detecting with an antibody produced.
The results showed that this protein is expressedtransiently in the floral tissue and co-localized withchromatins in the nucleus (Moriguchi et al. 2005).
(7) Comparative genomics among cultivated and wild
rice species

Yukie Sano, Hiroyuki Kanamori*, Nobukazu Namiki*,
Yukiko Yamazaki and Nori Kurata (*STAFF institute,
Tsukuba)
We have started to analyze gene structure andexpression differences between cultivated rice, Oryza
sativa (AA genome species), and wild rice of 0.
punctata (BB genome) and O. officinalis (CC genome).
Total of 4,000 cDNA clones randomly picked up fromthe libraries of SAM (shoot apical meristem) or very
early influorescence were subjected to EST sequencing
and blast search analyses. Some clones were further
analyzed for full length clone sequences. Totally about

2,900 clones have been qualified in their sequences.
Blast searches showed about 70% of the clones had
highly identical sequences with rice cDNA or genomic
sequences. Around 3% (from 7% to 0%) base substitution and in frame or out of frame insertion/deletion
were detected between cultivated and wild rice in theclones which showed identities with rice sequences.
Simultaneous examination for expression profilingusing 22K microarrays revealed scores of genes whichshowed quite large differences in their expression
between cultivated and wild rice. Weare going to
identify gene structure of clones having no similarity
with rice and showing much higher expression in either
cultivated or wild rice.
Rice genetic resource project in NBRP and
Oryzabase(8)

Nori Kurata, Ken-ichi Nonomura, Yukihiro Ito andYukiko Yamazaki
The National Bioresource Project (NBRP) was
organized in 2002 to conserve and distribute biologicalresources for scientific communities. Rice is one of theorganisms whose resources are required tq amplify,
conserve and distribute to the scientific community as
genetic resources. In the NIG, we are dealing 2,000 wild
rice accessions covering 10 genomes and 23 species
collected all over the world for over 50 years. Four ricesub-centers in other institutions dealing with different
kinds of genetic materials are joined to this project.
Resource materials and their information can beaccessed at; http://shigen.lab.nig.ac.jp/rice/oryzabase/nbrpStrains/ aboutNbrp.jsp;jsessionid =9AD3EACD512AlDA03ABB65874CF638lD.tomcat4_6 in
the Oryzabase.
The Oryzabase is a comprehensive rice biological
database composed of IS sections of information.
Major data characteristic in the Oryzabase are;mutants and their trait genes collection, wild ricecollection and development/anatomy of rice, together
with genetic maps, physical maps, basic biological dataand so on. The rice genetic resources committee
centered in the NIG is responsible to collect and curate
the contents of information and construction, andmaintenance of those data is carried out by the genetic
informatics laboratory. The DB is accessible at;
http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp

Plant Genetics Laboratory792. :£fB3(J) ~ (2005)/'~7/~@..f*~*, 7 o.::cV-A :ff[~~@'.f*liJf9Ei'1d~ft1it, m* . rfiJ* . ~r::HI) m,\jltl$.

Publications
Papers1. Miyoshi, K, Ahn, B-O., Kawakatsu, T., Ito, Y., Itoh,
J-L, Nagato, Y. and Kurata, N. (2004). PLASTOCHRON1,EDUCATION

a timekeeper of leaf initiation in rice, encodes
cytochrome P450. Proc. Natl. A cad. Sci. USA 101,875-880.
2. Ito, Y., Chujo, A, Eiguchi, M. and Kurata, N. (2004).
Radial axis differentiation in a globular embryo ismarked by HAZ, a PHD-finger homeobox gene of rice.
Gene 331, 9-15.
3. Nonomura, KI., Nakano, M., Murata, K, Miyoshi,
K, Eiguchi, M., Miyao, A, Hirochika, H. and Kurata,
N. (2004). The insertional mutation of rice PAIR2gene,
the ortholog of Arabidopsis ASY1, caused a defect inhomologous chromosome pairing in meiosis. Mol.
Genet. Genomics 271, 121-129.
4. Nonomura, KL, Nakano, M., Fukuda, T., Eiguchi,M., Miyao, A, Hirochika, H., Kurata, N. (2004). The
novel gene HOMOLOGOUS PAIRING ABERRA TION
IN RICE MEIOSIS 1 of rice encodes a putative
coiled-coil protein required for homologous
chromosome pairing in meiosis. Plant Cell 16,
1008-1020.
5. Salina, E.A, Adonina, I., Vatolina, T. and Kurata,
N. (2004). A comparative analysis of the compositionand organization of two subtelomeric repeat familiesof Aegilops speltoides Tausch.and related species.
Genetica 122,227-237.
6. Moriguchi, K, Suzuki, T., Ito, Y., Yamazaki, Y.,
Niwa, Y. and Kurata, N. (2005). Functional isolation ofnovel nuclear proteins showing a variety of sub-nuclear
localizations. Plant Cell 17, 389-403.

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Reviews

7. Itoh, ].1., Nonomura, KI., Ikeda, K, Yamaki, S.,
Inukai, Y., Yamagishi, H., Kitano, H. and Nagato, Y.
(2005). Rice plant development: from zygote to spikelet.
Plant Cell Physiol. 46, 23-47.
8. Kurata, N., Miyoshi, K, Nonomura, KL, Yamazaki,
Y. and Ito, Y. (2005). Rice mutants and genes related
to organ development, morphogenesis and physiological traits. Plant Cell Physiol. 46,48-62.
Book

*Jf /

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~JF~m . t~flldtHi)' 90-102.

80

Plant Genetics Laboratory

SOCIAL CONTRIBUTIONS

1. :£fB3(J)~ B*~~~~n~~~m~~
2. :£f B3 (J) ~ B *n~~~~$
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4. :£fB3(J)~:1:.~~~~~-{*~~R~~~~
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6. :£fB3(J) ~ : Rice Genetics Newsletter Editor
7. :£fB3 (J) ~ : NSF project advisory committee
memberF-f. Microbial Genetics Laboratory
Akiko Nishimura GroupRESEARCH ACTIVITIESDynamics of bacterial tubulin: coupling model
between DNA replication and cell division(1)

Ippei Inoue, Kenji Yasuda and Akiko Nishimura
The most approaches understanding cell division
have asked whether cell division occurs coupling with
DNA replication. To solve this problem, we examinedthe dynamics of FtsZ, an essential key protein ofEscherichia coli cell division, in relation to the stage of
DNA replication by creating a fusion protein containing
FtsZ and a green fluorescent protein (GFP). FtsZ
started to assemble at potential division site coincident
with initiation of DNA replication, all FtsZ assembled
in the ring structure coincident with termination of
replication, and the ring constricted after nucleoids
separation. These results suggest that cell divisionoccurs in association with DNA replication.

(2) A complete set of Escherichia coli open reading
frames in mobile plasmids and their successful application
to the systematic identification of cell division mutant(fts) genes
Kimiko Saka, Maki Tadenuma, Shinsuke Nakade,
Noriko Tanaka, Hideaki Sugawara, Ken Nishikawa,Nobuyuki Ichiyoshi, Masanari Kitagawa, Hirotada
Mori, Naotake Ogasawara and Akiko Nishimura
To facilitate genetic studies of Escherichia coli, weconstructed a complete set of mobile plasmid clones
of intact open reading frames (ORFs). The vector formobile plasmid clones carries the following genes andsequences; (i) bam, rom, and mob of ColEl, whichenable the plasmid to transfer from F+ to F- by
F-mediated conjugation; (ii) Ptac/lacr, which enables
control of the expression of the cloned ORF by IPTG;(iii) the ColEl replication origins, ori and pri, tomaintain a low plasmid copy number in the cell undernormal growth condition; (iv) the ampicillin resistantgene, bla, for selection; (v) the unique multi-cloning siteof pJF1l8HE; (vi) universal primer recognition
sites -21Ml3 and SP6 for confirmation of the cloned
ORF by sequencing; and (vii) rrnB with a transcriptional terminator which prevents read-through. Since
replication of the plasmid in vivo does not require
protein synthesis, the plasmid can be enriched during
inhibition of protein synthesis that leads to inhibitionof chromosomal replication without blocking theplasmid replication. To clone an ORF with its native
SD into pNT3, we amplified the predicted ORF alongwith 20 bp of the upstream region by PCR using primer
sets designed to contain the appropriate restriction
sites. With this procedure, native products should be
produced even if fragments longer than necessary arecloned. The plasmids carrying each ORF wereintroduced into an F+ recA strain and stored in 96-well
microtiter plates. In this way, 96 clones can betransferred simultaneously to F- bacteria using the
conjugative system. In order to simplify the screening
method, we investigated the possibility of searchingfor positive clones from a mixed pool of unrelated
clones. We first made a test stock composed of
subpools of 48, 24, 12, or 6 clones within single wellsof a 96-well microtiter plate. A complementation test
was carried out using ftsA, fts!, fts W; and ftsZ mutants.
All four mutants were complemented by the subpoolsof 48, 24, 12, or 6 clones, or by a single clone. There werefewer and smaller colonies in the ftsZ-positive patcheson the 1 mM IPTG plate than on the 0.1 mM IPTG orno IPTG plates. The results are supported by the facts
that low-level expression of ftsZ (less than two times)
can complement the ftsZ mutant, but high level
expression of ftsZ (more than four times) causes
inhibition of growth of theftsZmutant. The size of theftsA-positive patches was the largest on the 1 mM
IPTG-supplemented plate and very small on a plate
without IPTG. TheftsA mutants might need high-level
expression of FtsA for normal growth when it isprovided from plasmid-bomftsA. The results for fts!
were similar. to ftsA. Changes in IPTG concentration
did not affect the size of the fts Wpositive patches.
These results indicate that the 48-clone pool is effective
for complementation studies if we use the threeselection plates containing 0, 0.1, and 1 mM of IPTG.
Having demonstrated that we can identify the positive
ORF within a mixture of 48 clones, we created two
types of stock: a single microtiter plate containing poolsof 48 clones in each well, and a second stock composedof 45 microtiter plates containing the individual clones.
In summary, to find the clone that complements themutant, we first identify the positive mixed pool andthen determine which clone in the mixture
complements the mutant. This provides a convenient
procedure for systematic identification of ORFs thatsuppress or complement mutations.
(3) Global regulation of cell division: Isolation of awhole set of cell division mutants
Kimiko Saka, Noriko Matsumoto and Akiko
Nishimura
The entire nucleotide sequence of E. coli has been
analyzed, and 4311 ORFs have been demonstrated, but
the functions of more than half of these ORFs remain
unknown. The greater part of these ORFs are
considered to be involved in coordinating cellproliferation. To thoroughly analyze the hierarchy andnetwork responses in expression of cell division genes,
as one of the model cases for post-genome analysis,
we have identified a whole set of cell division genes
using above mobile plasmid clone sets by their ability
to complement a filament-temperature-sensitive (Its)cell division mutants. A total of 339 fts strains fromthe Hirota thermosensitive (Ts) mutant bank, which
form multi-cellular filaments at 41"C without immediate

Microbial Genetics Laboratory81arrest of DNA synthesis or an increase in cell mass
were tested. We found that 278 Its mutants werecomplemented by 403 of ORF clones. Of these, 69% of
the Its mutants were complemented by one ORF each.
Sequence analysis of genomic DNA of 10 of these Its
mutants showed that all had missense mutation in thecorresponding ORF. However, 15% of the Its mutants
were complemented by two ORFs, and 16% by three
ORFs. These may contain the allele of the Its mutant
gene and a high-dosage suppressor gene(s). Sequence
analysis of five of these Its mutants showed that all
five had missense mutation corresponding to one ORF
and no mutation in the remaining ORF(s). The cog
database (http://www.ncbi.nlm.nih.gov/COG/new/) was
consulted for functional annotation of individual genes.
From the functional annotation analysis, known cell
division genes were found in only 6% of the Its mutants,
while 30% were unknown. Of all genes identified, 24%
were involved in a basic process, such as DNA
replication or protein synthesis, despite the fact thatthe mutants involved in this category had presumably
been excluded by the process of selection of the Its
mutants. 19% were involved in cellular processes, suchas ion transport and signal transduction; and 21% wereinvolved in metabolism. These results suggest thatvarious intracellular reactions are coordinated with cell
division and that the E. coli cell cycle is the result of
the coordination of multiple independent processes,
so that each daughter cell receives a complete copy ofcell components. Weare currently planning to purify
these mutations in cells with wild-type background,and thoroughly analyze the hierarchy and network
responses in expression of cell division genes using
DNA chip technologies along with these mutants, as
one of the models for post-genome analysis for global
cellular regulation in E. coli.
PUBLICATIONSPapers

1. Saka, K., Tadenuma, N., Nakade, S., Tanaka, N.,
Sugawara, H., Nishikawa, K., Ichiyoshi, N., Kitagawa,M., Mori, H., Ogasawara, N. and Nishimura, A. (2004).
A complete set of Escherichia coli open reading frames
in mobile plasmids and their successful application
to the systematic identification of cell division mutant(Its) genes. (DNA res., imprinting).

82

Microbial Genetics Laboratory

Database

2. http://www.shigen.nig.ac.jp/ecoli/strain/top.jsp
SOCIAL CONTRIBUTIONS AND OTHERS~~

1. mB.~*~, v~~~~~~~-~,~M~~'

11Ht~)(, *~#[iij5fIJm~~l$A'lf¥R ~ A7" AliH~~#Ii .
~~~tln~Y7A~~-T4~~

2. ~ff2004-195247, jjqntID!v JV~~ ~Jv:1~- ~ (()~
JiIl$, ~M~~' 'i'JII*1t . r§i5='$, *~#[iijfIJm~~l$A.¥R~A7"AliH~~#Ii·.'$~• • ~~tl~~5%tI.J

Dr. A. Nishimura was appointed for a member ofeditorial board of "Microbiology and Culture
Collections".

F -g. Invertebrate Genetics Laboratory
Ryu Ueda GroupRESEARCH ACTIVITIES(1) RNAi mutant fly bank for comprehensive analysesof gene function in Drosophila

Ryu Veda, Misako Taniguchi, Yukiko Sado\ Kaoru
Saig02and Kuniaki Takahashi eJST, 2Graduate Schoolof Science, University of Tokyo)
Genome sequencing projects have revealed thenumber of genes for several model organisms forgenetics. The small worm Caenolabditis elegans, whichis composed of only 959 cells, has 19,000 genes in its
genome. On the other hand, Drosophila melanogaster,which has a long and sophisticated alimentary canal,
a tubular heart that circulates hemolymph, and a large
brain composed of over 104 cells, harbors only 13,800
protein-coding genes. Considering there is such a small
number of fly genes, each one of them may have an
essential function in fly development and behavior. In
other words, it may be easy to detect and analyze genefunction in the fly by reverse genetics because theabnormal phenotype will frequently appear whenknocking down a target gene whose function is
unknown. Weare planning to investigate the functionof fly genes comprehensively as a suitable model forstudying the functional genomics of multicellularorganisms.
How does one investigate the function of all 13,800
genes in the fly? We use RNA interference (RNAi) to
knock down the activity of the target gene. RNAi is oneof the emerging technologies with which to investigate
gene function in multicellular organisms. Whenintroduced into the cell, double stranded RNA (dsRNA)
works as a specific mutagen for each gene. That is,
dsRNA recognizes host mRNA and digests it in asequence-specific manner, and consequently brings aloss-of-function mutation phenotype to the host cell.
The detailed mechanism of this RNAi phenomenon
has not yet been elucidated, but it works efficiently inmany multicellular organisms, including humans.
We coupled the RNAi with the GAL4-UAS gene
expression system to induce a conditionalloss-offunction mutation in the fly. The GAL4-UAS system
is a binary system for inducing transgene expression,in which two fly lines are used. One is the GAL4 driver
fly line, which expresses yeast transcription factor,
GAL4, in a specific cell/tissue or at a specificdevelopmental stage in favor of the GAL4 transgene.
The other fly line harbors a transgene on thechromosome, in which an appropriate gene to be
expressed is fused to the UAS promoter, the GAL4
target. When these two fly lines are crossed with eachother, we can observe in the fly progeny that the GAL4
protein induces target transgene expression in adriver-specific conditional fashion. In this GAL4-UAS
system, when we use a UAS-transgene having an
inverted repeat (IR) sequence, the transcribed RNA
may form a dsRNA in the cell and induce aloss-of-function mutation by the RNAi mechanism.
Such inducible RNAi caused by the transcription of
an IR sequence was first successfully adopted to genefunction analysis in C. elegans. It was then also foundto be effective in fly genetics. By making a
UAS-transformation vector containing an IR sequenceof the gene predicted by the fly genome project, andby introducing it into a fly line (IR fly), a mutant
phenotype of the gene can be easily observed in any
cell or at any developmental stage of the progeny,
whenever the IR fly is crossed to an appropriate GAL4
driver fly.
We are expanding this inducible RNAi to thewhole genome of the fly. This process involves two
major procedures.
1) in vitro construction of transformation vectors
containing an IR sequence from each of the 13,800

predicted genes.
2) Transformation of IR vectors by injecting theminto fly eggs and establishment of IR fly lines bytraditional genetic methods.
As of the end of 2004, over 6,900 transformation
vectors had been constructed, 6,300 of which have beensuccessfully introduced into the fly. We will continue
this work in the next year, and may be able to add up
more than 2,000 IR fly lines to our fly bank.
Along with the establishment of IR fly lines, basic
characterization of the target genes is conducted using
these fly lines. All of the IR fly lines are crossed to the
Act5C-GAL4 fly. The Act5C-GAL4 induces the
UAS-transgene in all cells at all developmental stages.
Thus, if the gene targeted by RNAi has functions thatare indispensable for fly development, the progeny of
IR and GAL4 flies should die before the adult flies
emerge. Among the 1954 genes tested, 51.1%of the flylines showed lethality. This value is rather highcompared to that obtained by classical genetics (25%),
while the fact that many of the genes tested here wereconsidered to have important functions in various
aspects of fly development by our collaborators may
bring about such a high score. Detailed analyses on
known genes and greater accumulation of data arenecessary. Weare currently collaborating with 70
groups. The usefulness of inducible RNAi forinvestigating gene function in Drosophila is being
revealed in many aspects. We published 5 papers using
RNAi flies 3)-7) and 2 papers on RNAi mechanism!)' 2) in
2004.
This work was supported in part by financial
assistance to Dr. Ueda from the Mitsubishi Kagaku
Institute of Life Sciences (MITILS).
PUBLICATIONSPapers

1. Ui-Tei, K, Naito, Y., Takahashi, F., Haraguchi, T.,
Ohki-Hamazaki, H., ]uni, A., Ueda, R. and Saigo, K(2004). Guidelines for the selection of highly effective
siRNA sequences for mammalian and chick RNAinterference. Nucleic Acids Res., 32, 936-948.
2. Ui-Tei, K, Ueda, R., Zenno, S., Takahashi, F., Doi,N., Naito, Y., Yamamoto, M., Hashimoto, N.,
Takahashi, K, Hamada, T., Tokunaga, T. and Saigo,
K (2004). RNA interference induced by transient or
stable expression of hairpin structures of double
stranded RNA in Drosophila and mammalian cells.

Invertebrate Genetics Laboratory83Molekulyamaya biologiya, 38, 228-238.
3. Pili-Floury, S., Leulier, F., Takahashi, K, Saigo,
K, Samain, E., Ueda, Rand Lemaitre, B. (2004). In vivo
RNA interference analysis reveals an unexpected rolefor GNBP1 in the defence against Gram-positivebacterial infection in Drosophila adults. J, BioI. Chern.
279, 12848-12853.
4. Ichimiya, T., Manya, H., Ohmae, Y, Yoshida, H.,
Takahashi, K, Ueda, R, Endo, T. and Nishihara, S.
(2004). The twisted-abdomen phenotype of Drosophila
POMT1 and POMT2 mutants coincides withtheirheterophilic protein O-mannosyltransferase activity.
J. BioI. Chern., 279,42638-42647.
5. Kamimura, K, Rhodes, J.M., Ueda, R, McNeely,
M., Shukla, D., Kimata, K, Spear, P.G., Shworak, N.W.
and Nakato, H. (2004). Regulation of Notch signaling
by Drosophila heparan sulfate 3-0 sulfotransferase. J,
Cell BioI., 166, 1069-1079.
6. Koganeya, A. K, Sasamura, T., Oshima, E., Suzuki,
E., Nishihara, S., Ueda, Rand Hirabayashi, Y (2004).
Drosophila glucosylceramide synthase: A negative
regulator of cell death mediated by proapoptotic
factors. J, BioI. Chern., 279, 35995-36002.
7. Ishimaru, S., Ueda, R, Hinohara, Y, Ohtani, M.
and Hanafusa, H. (2004). PVR plays a critical role via
JNK activation in thorax closure during Drosophila
metamorphosis. The EMBO J" 23, 3984-3994.
Reviews

8. Nishihara, S., Ueda, R, Goto, S., Toyoda, H., Ishida,
H. and Nakamura, M. (2004). Approach for functional
analysis of glycan using RNA interference. Glycoconj]., 21, 63-68.
EDUCATION

1. RUeda was invited to give a seminar on "RNAimutant fly bank" at Keio University, Tokyo, Mar., 2004
(in Japanese).
2. RUeda was invited to give a seminar on "RNAitechnology" at the satellite meeting of the annual
meeting of Japan society for Bioscience, Biotechnology,
and Agrochemistry, Hiroshima, Mar., 2004 (inJapanese).
3. RUeda was appointed as an adjunct lecturer at
Tsukuba University, Department of biological sciences
to give a course on Genetics.

84

Invertebrate Genetics Laboratory

SOCIAL CONTRIBUTIONS AND OTHERS

1. R. Ueda joined advisory committee of National

Institute of Agrobiological Sciences at Tsukuba.

F-h. Laboratory for Frontier Research
Takako Isshiki GroupRESEARCH ACTIVITIES(1) Analysis of temporal specification within late phasesof Drosophila neuroblast lineage

Takako Isshiki and Ayumi Kusano
During development, neural progenitors often
generate diverse cell types in an invariant order,
changing their property over time. Althoughsubstantial progresses have been made in understanding the molecular mechanisms of how different cellfates are generated in order, many parts of themechanisms still remain unknown, especially invertebrate. We utilize the Drosophila central nervous
system (CNS) as an excellent model system to study
temporal specification of cell fates within a lineage. We
previously showed that Drosophila neuroblasts
sequentially express the transcription factors
Hunchback, Kriippel, Pdm and Castor over time.
However, most neuroblasts divide additional 10 times
on average after they start expressing Castor. Thus,
there must be subsequent mechanisms for temporal
specification.
To identify yet unidentified factors involved in
temporal specification within neuroblast lineage, we
searched a public dataset of the expression profiles of
the Drosophila gene transcripts. We found severaltranscription factors expressed later than Castor inmost neuroblasts. Next, we have investigated andelucidated the precise order of their expression.
Furthermore, we start investigating how neuroblastschange their property during larval stages. Recently,
it has been reported that a couple of mouse homologsof these factors are also expressed in cerebral cortex
in temporally regulated manner. This fact suggeststhat the mechanisms we are studying might beconserved across species.

(2) Investigation of molecular functions of Knippel typezinc finger proteins in Development of the eNS

2. -f5~-=f (2004) *'IJ*U~~Jffl! ~ A 7" A f~ J: G *'IJ~~Jffl!
1j;¥~1t(J)7t-=f~mm B f{~DI,H~ (ijHIj) 49, 228-233.

Ayumi Kusano
A C. elegans heterochronic gene, lin-29, encodes a
Krtippel type zinc finger protein. Last year, we reported
that Drosophila melanogaster Lin-29 is expressedtransiently in neuroblast at very late stages ofembryogenesis and subsequently in their late-bornprogeny. We have been investigating molecularfunctions of DmLin-29, by conducting genetic analysis.
Our analysis revealed that DmLin-29 and Krtippel havedistinct functions, although they share high homology
in their zinc fingers, that are thought to bind DNA
directly. This fact suggests that not their zinc fingers
but other parts of the proteins are mainly responsible
for making the differences in their function. Aiming
to elucidate the molecular mechanism that makes thedifferences, we have start searching proteins that canform a complex with either only one of Kriippel or
DmLin-29.
(3)

In situ Detection of microRNA

Katsutomo Okamura
Hundreds of microRNAs are found in each animal,
so microRNAs are thought to have a fundamental role
in regulating gene expression. Northern blot analysesof their expression strongly suggest that many
microRNAs have their function in development.
However, the lack of a conventional method in
detecting spacio-temporal distribution of microRNA
makes it very difficult to study their functions indevelopment. Okamura has developed an efficientmethod for detecting mature form of microRNA in situ
in Drosophila embryos. So far, he has identified a few
microRNAs expressed in specific cells in the nervous
system. Future analyses on neural microRNAs should
provide us comprehensive understanding of gene
networks that control neural cell fate specification, andmay reveal novel roles for microRNAs.
PUBLICAliONS
Reviews

1. Isshiki, T. and Doe, CQ. (2004). Maintaining Youthin Drosophila Neural Progenitors. Cell Cycle. 3,296-299.

Laboratory for Frontier Research85

G. CENTER FOR GENETICRESOURCE INFORMATION

genomes to researchers.
PEC database can be accessed through the
SHIGEN server (http://www.shigen.nig.ac.jp/ecoli/ped).
1.2 CARD R-DB and IMSR (International Mouse Strain
Resources)
Takehiro Yamakawa, Hideki Kato, Naomi Nakagata,
Kenichi Yamamura and Yukiko Yamazaki

G-a. Genetic Informatics Laboratory
Yukiko Yamazaki GroupRESEARCH ACTIVITIES

1 SHIGEN project
1.1 E coli Databases-E. coli strain DB and PEC(Profiling of Escherichia coli Chromosome) database.
Takehiro Yamakawa, Junichi Kato, Akiko Nishimura
and Yukiko Yamazaki
As a result of the National BioResource Project,
a lot of new E. coli resources were accumulated. These
resources include mutant strains established fromresearch activities of individual researchers, Genomic
DNA plasmid such as Cosmid collection and pLC
plasmid, and deletion clones for each ORF (mobile
plasmid clones and deletion mutants). E. coli strain
database was rebuilt in order to compile theseresources and allow users to request them through theinternet directly from the list page after searching.
Gene information for each ORF in the strain database
was linked to the GenoBase (E. coli W3110 strain
Genome Database) and to the PEC (Profiling of E. coli
MG1655 chromosome). Since newly developed large
scale chromosomal deletion mutants of E. coli that lack
2.4% to 29.7% of the parental chromosome (Ref.5) areavailable for use, "Essential Genes and Minimal
Genome" section was added to PEC and a minimal
genomic view and detailed information of each deletionmutants were also provided. By successfully reducing
the genome size of E. coli, the number of genes whichwere classified as "unknown" decreased. Gene's
essential information as well as huge amount of
resource collections would become very useful toprecede E. coli researches. PEC not only collects latest
public information of MG1655 but also gives
comparative genomics platform among microbe

86

CENTER FOR GENETIC RESOURCE INFORMATIONCARD R-BASE is a database of transgenic mousestrains established and deposited by individual
researchers. All resources are stored as frozen embryos
and available on request. The database contains
genetic backgrounds, genes destroyed/transferred, andrelevant human diseases for each strain. In this year,
we introduced new links from CARD R-BASE to thegene details page of the Mouse Genome Informatics(MGI). CARD-R-BASE became a member ofInternational Mouse Strain Resources (IMSR) since a
new version was implemented in July 2004. IMSR isa searchable online database of mouse strains andstocks available worldwide and maintained by JacksonLaboratory. Before merging data set of CARD R-BASE
into IMSR, manual annotation on nomenclature ofstrain type, mutant type and gene/allele name was
required. In order to encourage researchers, who
deposit mouse strains, to get used to the controlled
vocabulary of strain name, a new tool named"GODFATHER" was developed. GODFATHER is a
simple naming tool with web-interface and it is divided
into three levels depending on the level of user's
knowledge. By filling necessary information such as
background mouse name, gene name and laboratorycode, the user can obtain a candidate name for themouse strain. An English version of GODFATHER is
also planned to be developed. CARD R-BASE also
made bilateral linking to the Exchangeable Gene Trap
Clones (EGTC) which provides trapped gene sequence
information and its homology search results.
The International Federation of Mouse Resource(IFMR) established in 2004 is a collaborating group of
Mouse Repository and Resource Centers worldwide
whose collective goal is to archive and provide strainsof mice as cryopreserved embryos and gametes, ES celllines, and live breeding stock to the researchcommunity. CARD R-BASE as well as RIKEN BRC(BioResource Center) are members of IFMR. The goalsof the IFMR are 0) to coordinate repositories andresource centers, (2) to establish consistent, highest
quality animal health standards in all resource centers,(3) to provide genetic verification and quality controlof genetic background and mutations, and (4) toprovide resource training to enhance user ability toutilize cryopreserved resources. IMSR database would
be a good start of IFMR database.
The CARD R-DB is accessible at http://
cardb.cc.kumamoto-u.ac.jp/transgenic/.
1.3 Mouse polymorphism database

Takehiro Yamakawa, Eri Kibukawa, Toshihiko
Shiroishi and Yukiko Yamazaki
Mouse microsatellite database (MMDBJ) provided
SSLPs among different mouse strains and cSNPs
between B6 and MSM mouses based on their fulllength cDNA sequence information. Since a hugeamount of MSM BAC end sequences as well as B6/
MSM gSNPs are now available for use, the name ofthe database was changed from MMDBJ to Mouse
Polymorphism Database and all information was put
together in a genomic viewer. We applied the Ensembl,
which is freely provided by EEl, as a genome browser
and added original data of MSM BAC ends, MSM!B6
cSNPs and microsatellite polymorphism as DAS(Distributed annotation source) and then customized
the browser. Although keeping the Ensembl data
up-to-date is not a simple task, adding original data
as DAS sources to Ensembl instead of developing an
original genomic browser from scratch was quicker toimplement. All data are accessible through the internet
now. Then we started developing our genomic browser(shigen genomic browser) to overcome several
functional limitations of Ensembl.
Mouse Polymorphism Database is released at
http://shigen.lab.nig.ac.jp/mouse/polymorphism/
1.4 Oryzabase and Plant Ontology

Takehiro Yamakawa, Nori Kurata and Yukiko
Yamazaki
Oryzabase is a comprehensive rice database tocollect as much as public knowledge information andprovide them with a user friendly interface to support
rice research activities. As a member of the National
BioResource Project, large amount of new rice

resources were collected and ready to be distributed
to researchers through the Oryzabase. We developed
an online resource order system which allows users
to send e-mails to order from each resource center
directly from the resource list page. The system selects
an appropriated Material Transfer Agreement (MTA)
form depending on which resource center maintains
the requested resource, and a reply mail is
automatically sent back to senders as an attached file.
Oryzabase started to provide genomic sequence
information using shigen-genome-browse.
POC (Plant Ontology Consortium) is a consortiumthat aims to develop, curate and share controlled
vocabulary that describe plant growth stages andstructures (anatomy). Japanese rice researchers
independently started to clarify organ specificdevelopmental stage and collected stage specifically
expressed genes and their mutants from journal
articles. Oryzabase integrated these data into thealready existing genes and mutant collections. We havebeen developing multiple ontology platform in order
to integrate several different ontologies andvocabularies consistently. Three dimensional matrixes
consisting of "time" (development/growth), "space"(location/structure/anatomy), and "features"(characteristics) was conceptually build at the early
developmental stage. Recently Zebrafish group (ZFIN)
started to establish the PATO (Phenotype And Trait
Ontology) to describe mutant phenotypes. PATO is
described with a combination of terms and their
attribute and has a more general concept than our"features" which means that most species can share
this concept. Therefore, Oryzabase started to apply
PATO and is replacing "feature" with PATO type
expression. As a result of collecting all available
information as much as possible and as soon as
possible, the current Oryzabase have a lot ofinconsistencies on genes, mutants and phenotypes. Our
next task will be to resolve these problems.
Oryzabase is available at http://www.shigen.
nig.ac.jp/rice/oryzabase/.
1.5 KOMUGITakehiro Yamakawa, Takashi Endo, Yasunari
Ogihara, Hitoshi Tsujimoto, Taihachi Kawahara,
Tetsuro Sasakuma and Yukiko Yamazaki
KOMUGI is a wheat resource database and is

Genetic Informatics Laboratory87

unique as a wheat specific database in the world.
Bioresources of KOMUGI contains wild strains;
Landraces; experimental strains such as chromosome
lines, cytoplasm substitution lines, and mutants; andDNA resources such as EST clones and array. The
KOMUGI is also a member of the National
BioResource Project and the e-mail resource ordersystem has been implemented. Array data was newly
incorporated into KOMUGI database and is usedinternally at the moment. The task of adding mostrecent information to KOMUGI gene dictionary
remained although the "Macgene" system wasdeveloped for this purpose last year. Since MacGene
is a stand alone type system instead of a web-based
platform, there are still problems for dictionary keepers
to share a common reference number when they work
individually at different locations. Making cross linking
between sequence accessions and KOMUGI genes isalso a task that will be carried out next.
KOMUGI database is available at http://
www.shigen.nig.ac.jp/wheat/komugi/.
1.6 NBRP·databases

As an information center of the National
BioResource Project, we intensively support theconstruction of resource databases of each resource
center including the following species so that all
resource centers could make their resources public.
1.6.1 NBRP·C. elegans

Miharu Ikizawa, Shohei Mitani, Hiroshi Kagoshima,
and Ylikiko Yamazaki
NBRP-C. elegans provides deletion mutants onrequest after isolating targeted gene among mutant
pool. Users can see the status of mutant screening on
the web page and resource curator also manage thescreening processes using a web-interface. This system
also gives results of statistic analysis. The databasehas bilateral cross-linkings to WormBase, a worldwide
famous C. elegans database. Promoter information is
also part of this database and it is maintained byseveral volunteers. The common search site forresource database and promoter database are now
available.

88

Genetic Informatics Laboratory

1.6.2 NBRP·Silkwonn

Miharu Ikizawa, Yutaka Banno, Hiroshi Fujii, andYukiko Yamazaki
NBTP-Silkworm database consists of the following
three parts, (1) gene dictionary, (2) references, and (3)strain phenotypes. Since the traditional classificationof phenotypes and resources was not consistentenough to construct an electronic database, manual
annotation was performed intensively. As there is nosilkworm gene database in the world, this database is
the first gene dictionary of silkworm and it will be very
useful for scientific researches.
Weare planning to incorporate genomic
information when it is opened to the public.
1.6.3 NBRP·Legurne (Lotus and Glycine) database

Takehiro Yamakawa, Shoko Isobe, Masatsugu
Hashiguchi, Ryo Akashi, Satoshi Tabata, and Yukiko
yamazaki
NBRP-LegumeBase is a resource ordering siteshared by Lotus japonica resource database andGlycine max. resource database. Lotus japonica
database provides wild accessions, root culture,
Rebominant Inbred Lines as resources. Some strains
were characterized in phenotype when growing in
northern areas and southern areas. Although Glycine
max database currently only contains wild species,
Glycine soja, RIL as well as DNA resources such as
EST, full length cDNA clones are expected to be added.
Legume follows rice (monocot model plant) andarabidopsis (dicot model plant) as the third model
plant.
1.6.4 NBRP·Medaka database

Takehiro Yamakawa, Yuko Wakamatsu, and Yukiko
Yamazaki
The NBRP Medaka site web server was moved
from Nagoya University to National Institute of
Genetics this year. Medaka Genomic Information has
been incorporated into the construction of the Medaka
resource database.
The Medaka site also prepares the web-based
protocols "Medaka Book" in collaboration with several
Medaka researchers using PukiWiki software. The

Medaka web-atlas is also among the attractive contents
available and the brain part is now under construction.
Interactive interface and glossary will be integrated
into the atlas images and illustrations.
Medaka group applied the Ze brafish P ATO
ontology to describe mutant phenotypes so that the
database would be designed to handle the PATO data.

and Kato, ]. (2005). Cell size and nucleoid organizationof engineered Escherichia coli cells with a reduced
genome. Mol. Microbiol 55 (1), 137-149.
6. Moriguchi, K, Suzuki, T., Ito, Y, Yamazaki, Y,
Niwa, Y. and Kurata, N. (2005). Functional Isolation ofNovel Nuclear Proteins Showing a Variety ofSubnuc1ear Localizations. The Plant Cell 17, 389-403.

1.6.5 NBRP-Drosophila database

Reviews/Books

Takehiro Yamakawa, Masatoshi Tomaru, Masatoshi
Yamamoto, Ryu Ueda, and Yukiko Yamazaki
NBRP Drosophila consists of four organizations
and there are 4 individual databases. In order to makethese databases more useful without changing thecurrent system, we developed an one-stop-shop site
"Flystock" from where user can search resources forfour databases at once and make order directly fromthe resource list page.
We are planning to develop a maintenance system
to manage each database and to make connections
between Flystock and each database.
PUBLICATION
Papers

1. Mochida, K, Yamazaki, Y and Ogihara, Y. (2004).
Discrimination of homologous gene expression inhexaploid wheat by SNP analysis of contigs groupedfrom a large number of expressed sequence tags. Mol
Gen. Genomics. 270,371-377.
2. Sakai, T., Miura, I., Yamada-Ishibashi, S., Akita,Y, Kohara, Y., Yamazaki, Y., Inoue, T., Kominami, R.,
Moriwaki, K., Shiroishi, T., Yonekawa, H. andKikkawa, Y (2004). Update of Mouse Microsatellite
Database of Japan (MMDBJ). Exp. Anim. 53 (2), 151-154.
3. Kawai, Y., Ishii, Y., Arakawa, K, Uemura, K,
Saitoh, B., Nishimura, ]., Kitazawa, H., Yamazaki, Y,
Tateno, Y, Itoh, T. and Saito, T. (2004). Structural andFunctional Differences in Two Cyclic Bacteriocins with
the Same Sequences Produced by Lactobacilli, Appliedand Environmental Microbiology, 70 (5),2906-2911.
4. Yamazaki, Y, Nagato, Y. and Kurata, N. (2004).
Oryzabase (Integrated Rice Database) in 2004, Rice
Genetics Newsletter, 20, 9-10.
5. Hashimoto, M., Ichimura, T., Mozoguchi, H.,
Tanaka, K, Fujimitsu, K, Keyamura, K, Ote, T.,
Yamakawa, T., Yamazaki, Y., Mori, H., Katayama, T.

7. 'iff.
lit, l1rilffiJEl3wc-r(2004) r:kJm§?, / .A:r-3J.r(-AJ?' / ~~A· 7"t1T:;t~~AO)f)TJ.mIJfJ,.::c.~· T-{ .
15-2l.
8. l1rilffiJEl3wc-r(2004) r/,\-1:;t!J ~-At/3J-JmBJt~l§tM~Vo1.49 No.ll, 1956-1963.
9. l1rilffiJEl3wc-r(2004) r.:r:-:rJvJ!lJ~O)fF~U~*ftJ;lfJJJIJ-fIIt,
1-17.
.::c.~,

Database

10. JMSR http://www.shigen.nig.ac.jp/mouse/jmsr/
11. Mouse Polymorphism DB http://www.shigen.
nig.ac.jp/mouse/mmdbj/
12. CARD R-BASE http://cardb.cc.kumamoto-u.ac.j p/transgenic/
13. FlyStock http://218.44.182.89/%7Eflystock/html/
indexj.html
14. NigFly http://www.shigen.nig.ac.jp/fly/
15. Oryzabase http://www.shigen.nig.ac.jp/rice/oryzabase/
16. KOMUGI http://www.shigen.nig.ac.jp/wheat/komugi/17. BarleyGermplasm
Database
http://
www.shigen.nig.ac.jp/barley/
18. PEC http://www.shigen.nig.ac.jp/ecoli/pec/
19. E.coli Strain Database http://www.shigen.nig.ac.jp/
ecoli/strain/
20. GRW http://www.shigen.nig.ac.jp/shigen/grw/
21. WGR http://www.shigen.nig.ac.jp/shigen/wgr/
22. NBRP-ChrysanthemumDatabase http://www.
shigen.nig.ac.jp/kiku/
23. NBRP -Algae DB http://www.shigen.nig.ac.jp/algae!
24. NBRP-Silkworm http://shigen.1ab.nig.ac.jp/
silkwormbasel
25. NBRP-Legume http://shigen.1ab.nig.ac.jp/legume!
legumebase/
26. NBRP-C.elegans http://www.shigen.nig.ac.jp/cele!
27. NBRP http://www.nbrp.jp/

Genetic Informatics Laboratory89

(1) Acquirement and utilization of Intellectual Property
RightsEDUCATION**#f:ffJl}]~~~1f-e~T-7::t-7A3Jj

ff~**~~~~~SOCIAL CONTRIBUTIONS AND OTHERS~ftg~~J!f~~#n:liJf?E)t~~ ~~m~~:~~~~~

~.a

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iPJEfiHi~ ~ : ~!jo/.J~#n:liJf?EfflBRC~M~ ~ : :E!l!liJfBRC

G-b. Publicity and Intellectual Property Unit
Munehiro Tomikawa GroupRESEARCH ACTIVITIES

2004 Annual Publicity and Intellectual Property Unit
ReportThis unit makes various efforts to return theoutcome of research generated by NIG to the industrial
world and the public while respecting the scientists'priorities in terms of scientific ideas, partnership andpublications.
To be more precise, what we have done is toestablish scientific results as intellectual property,
explain the property rights to the industrial world
intelligibly, seek collaboration with commercial
industries for actualizing those results and establish
partnerships. This shows the importance of carryingout both public relations and the acquisition of
intellectual property.
This unit was set up in March 2003. Since April
2003, a secretary, Ms. Nozomi Yokoyama and Ms.
Kiyomi Kuwahara have devoted all their energy to thiswork from starting up, and had assisted by creating
the MTA(Material Transfer Agreement) form, andgetting it off the ground. They have also been
supporting various works such as planning andpromotion of publicity activities and procedures ofapplication for intellectual property rights. The resultsof our work in 2004 are shown as follows.

90

Genetic Informatics Laboratory

We have continued to promote a sense ofentitlement in the scientists belonging to NIG andDivision of Life Science in the Graduate University for
Advanced Studies (Sokendai) by giving them speecheson why entitlement is necessary to the outcome of basic
research. Also, in visiting laboratories, monitoring thescientific progress and thinking of each scientist, wehave explained how intellectual property rights can begained from their results. We have also established anefficient patenting procedure. We have prepared aninvention exposition format to minimize the scientists'burden in informing our unit of their scientific results.
Our unit helps the scientists avoid the troublesometask of acquiring of patent rights to the practicalapplications of their inventions.
In terms of patent applications, we have tried topromote acquirement of the patent rights so the NIG
scientist does not need to bear the costs necessary forthe preparation of patent documents. At this time, eightdomestic patents have been applied for and four
patents are under preparation, of which 15 as a wholewill be applied for as NIG patents (including nine joint
applications and three international patents) in the
Research Organization of Information and Systems(ROIS). One patent has already come into marketing
as of November 2003. We got the royalty with a small
amount from its first business at April 2004.
Secondly, we have been involved in making
guidelines for the Material Transfer Agreement which
stipulates ownership, ethical codes and compensation
for biological resources based on research results. At
present, three kinds of agreements, depending on thepositioning of biological resources as intellectual
property, have been created and are in use. Each has
a simple version corresponding to specific materials. in the research field such as yeast, worms and flies(Form 1) and an intermediate version without provision
for sharing of industrial property rights (Form 2) anda complete version with provision for sharing ofindustrial property rights (Form 3). They have beenauthorized and, a total of 272 agreements(approximately 80% of them are international) havebeen signed by the end of this year.
In addition, we have participated in the
Shizuoka-TLO-Yaramaika (STLO) campaign, whichfacilitates putting patents owned by the NIG into

practical use.
(2)

Acceleration of public relations activities

Ideally, this kind of work should be developed overa broad spectrum, however, we have focused ourattention on the following main points. We have given
priority to our response to visitors, promotion atacademic conferences, introduction of research results
to the public, and the search for potential collaboration.
Firstly, we had 9 groups of visitors (total 225
persons) in 2004: technical school students, legal
apprentices, high school students, schoolteacher
interns, members of the judiciary, elderly community
college students and others. An overview of thisinstitute, genetic science and current laboratory
experiments was given to the visitors.
Secondly, promotion at academic conferences has
been carried out in order to obtain more excellent
students and scientists. We introduced the current
research results obtained from NIG scientists at theJapanese Cell Biology Meeting (in Osaka), the JapaneseSociety for Developmental Biology meeting (in Nagoya),and the Japanese Molecular-Biology meeting (in Kobe).
Thirdly, we presented fourteen specifically
excellent research results in the Hot News column on
the website as an introduction of research results andacademic awards for life science to the public. We havealso had fourteen research results inserted inmain-stream newspapers through cooperation with themedia. Furthermore, we introduced two scientists tocompanies which were interested in their work, andfortunately, this effort led to further business-academia
collaboration.

on higher order structure of chromatin DNA thisOctober.
Recently, the participation of public facilities, suchas universities and NIG, is increasing in the
Government-Industry-University-Research Cooperation
Conference. In June, We attended first Third
Government-Industry-University Cooperation Conference as a panelist on behalf of NIG, which was heldat Kyoto. It was quite meaningful to be able to develop
a relationship with industrial people who wereinterested in our patent and products.
In conclusion, acquiring intellectual property andengaging in returning excellent technology fromscientific results to the industrial world through public
relations will be important factors in gaining wide
public recognition of NIG. These efforts are expected
to facilitate the financial management of NIG, after
turning into independent administrative entities.

(3) Collaboration Research with academia, industry andgovernment, and Social Action WorkI have been conducting research of Fuji Pharma
Valley Cancer Diagnostics Study Group consisting ofacademia, industry and government. The aim of thisproject is to increase Research and Development
potential in the Mt. Fuji area and its cities of Numazu,
Mishima, Fujinomia, Fuji and Nagaizumi and to study
together with companies operating in the field of
medicine as well as the pharmaceutical industry on thedevelopment of cancer diagnostics through genomics
and proteomics approaches. Fortunately, we could
apply one patent regarding cancer diagnostics based

Publicity and Intellectual Property Unit

91'.

H. STRUCTURAL BIOLOGYCENTER

H-a. Biological Macromolecules Laboratory
Makio Tokunaga GroupRESEARCH ACTIVITIES(1) Single Molecule Imaging and Quantitative Analysis
of Nuclear Transport in Cells using Highly Inclined andLaminated Optical sheet microscopy

Makio Tokunaga and Naoko Imamoto! CCellular
Dynamics Laboratory, Riken)
What is a key to enter the nuclei through nuclear
pores? This question has been answered by visualizing
single molecules inside cells. Clear video images ofsingle molecules translocating into the nuclei are
obtained using novel fluorescence microscopy, Highly
Inclined and 1.aminated Optical sheet (HILO)
microscopy. Very little is known about the interactions
between transport molecules and the assembled
nuclear pore complex (NPC) because of its large
supramolecular structure. Obtained single-molecule
video images inside cells are very clear, therefore
kinetic parameters of the molecular interactions in cells
are obtained through quantitative analysis. We havediscovered how many molecules interact, how strong
the interactions are, and which molecular interactionis the open sesame to nuclear import. We have opened
up a new way to quantify molecular dynamics andinteractions at the single-cell level, which is 'open
sesame' toward quantitative molecular and system
biology.
(2) RNGI05: A Novel Regulatory Protein in Neuronal
RNA Granules Responsible for Stimulation-Dependent
Local Translation

Nobuyuki Shiina, Kazumi Shinkura and Makio
Tokunaga

92

STRUCTURAL BIOLOGY CENTER

mRNA translocation and subsequent local
translation in neuronal dendrites are important bases
for long-term synaptic plasticity, but responsible
molecules have not been fully identified. We previously
identified RNGI05 (RNA granule protein 105) as acomponent of RNA granules, which play central rolesin the transport of mRNAs to the dendrites. The
RNG105-localizing RNA granules contain mRNAs,
such as CaM kinase II alpha, CREB and BDNF
mRNAs, whose translational products play key roles
in synaptic plasticity.
In this year, we have found that RNG105 contains
an RNA-binding motif, and shown that RNGI05 is aregulatory protein for local translation in dendrites ofhippocampal neurons. RNGI05 has an ability torepress translation in vivo, consistent with the findingthat the RNA granule is translationally arrested in thebasal conditions. Dissociation of RNG 105 from the
RNA granules is induced by BDNF (brain-derived
neurotrophic factor), a growth factor responsible forsynaptic plasticity. The dissociation of RNGI05 is
significantly correlated with the induction of local
translation of the mRNAs near the granules. Thesefindings indicate that RNGI05 is responsible for the
local translational regulation in neuronal dendrites, andsuggest its implication in the regulation of local
synaptic plasticity in a stimulation-dependent manner.
(3)· Single Hydrogen Bonds of DNA Base Pairs Detected
in Unzipping Force by Intermolecular Force Microscopy

Michio Hiroshima! and Makio Tokunaga eSingle
Molecule Immunoimaging, Research Center for Allergy
and Immunology, RIKEN)
Single hydrogen bonds of DNA base pairs havebeen measured by unzipping double-stranded DNA
oligomers with an intermolecular force microscope(IMF). To detect such ultrafine forces, high resolutionof force as well as high accuracy in controlling theprobe position is required. IFM has achieved the force
resolution of subpico-newton using ultrasensitive
cantilevers. The probe position is controlled withnanometer accuracy using a feedback system, whichuses laser radiation pressure to reduce thermal
fluctuation of the cantilever.
Force vs. extension curves showed repeated force
peaks of 1O-15pN. Auto- or cross-correlation analysis
and averaging of force curves were carried out toreduce noises in the force curve. The previous studies
showed that the force for separating poly(G-C) DNA
was 1.5 to 2 times stronger than that for poly(A-T).
However, no difference was found in the force between
individual G-C and A-T base pairs. The force curveof individual G-C and A-T pairs showed three and two
peaks, respectively, which are assigned to singlehydrogen bonds. The force is variable but the work
is constant. The work to break single hydrogen bonds
is 1.3 kB . T, in other words, about 1.3-fold of thethermal energy. This is the first report to detect theforce of single hydrogen bonds in biologicalmacromolecules.
(4) Mechanism for passive force generation ofinvertebrate connectin revealed by single molecule measurement

Michio Hiroshima\ Atushi Fukuzawa2, Koscak
Maruyama2, Sumiko Kimura2 and Makio Tokunaga
eSingle Molecule Immunoimaging, Research Center
for Allergy and Immunology, RIKEN, ~epartment of
Biology, Chiba University)
Connectin is an elastic protein in muscle and isthought to keep the thick filament at the center ofsarcomere or protect a myofibril from damages byextraordinary loads. Invertebrate connectin (I-connectin)
is a 1960 kDa elastic protein linking the Z line to thetip of the myosin filament in the giant sarcomere ofcrayfish claw closer muscle. There are several
extensible regions in I-connectin: two long PEVK
regions, one unique sequence region and Ser-, Glu- andLys-rich 68 residue-repeats called SEK repeats.
The force measurements of the single recombinant
polypeptide including SEK or PEVK regions was
performed by intermolecular force microscopy (IFM).
The force versus extension curves were well fit to thewormlike chain (WLC) model. The obtained persistence lengths were 0.38±O.lnm (n=63) of SEK peptide
and 3.07 ± 1.0nm (n =35) of PEVK peptide. The
persistence lengths well explain the elastic behaviorof SEK and PEVK regions in muscle fiber. The valueof O.38nm indicates that the SEK region is a random
coil for full length. This is the first observation of anentropic elasticity of fully random coil region
contributing to the physiological function of invertebrate connectin.

(5) Asymmetric nucleocytoplasmic transport revealed
by a novel assay system using planner reconstituted
nuclear envelope

Atsuhito Okonogi, Michio Hiroshima\ Nobuyuki
Shiina, Shingo Kose2, Naoko Imamotd and Makio
Tokunaga eSingle Molecule Immunoimaging, RCAI,
Riken, 2Cellular Dynamics Laboratory, Riken)
We have developed a novel in vitro assay systemof nucleocytoplasmic transport. We aim at applicationof the method to new single-molecule experiments,
imaging and nano- or force-measurement. Nuclear
envelope was formed on a planar surface of a small
agarose plate. Agarose plates were modified withglutation, and were coated with GST-RanGDP fusion
protein. Nuclear envelope was formed onto the
RanGDP-coated surface using extracts from Xenopus
laevis frog eggs. Formation of Nuclear Pore Complexes
was confirmed by observing import of fluorescently
labeled proteins. Import assays using fluorescently
labeled proteins showed that the reconstituted NPCs
retain the transport activity. The dependence of theimport activity on RanGTP concentration showed
cooperativity.
Import and export of importin a was examined in
the absence of RanGTP and other soluble factors using
the present cell-free in vitro assay system. Asymmetry
between import and export was discovered in theinteraction of importin a and the NPC in the absenceof Ran-GTP and other soluble factors. In the apparent
equilibrium, import of importin a showed a differencein the concentration between at the cytoplasmic side
and at the nucleoplasmic side, whereas export ofimportin a showed no difference. The concentrationdifference of importin a is found to correlate with theamount of binding of importin a with the NPC. The
asymmetry between import and export was also foundin the transport of importin a in the presence of NLS
cargo molecules. In the presence of NLS-protein,
importin a showed no biding with the NPC. The
present finding shows that the NPC has an asymmetricfeature in the interactions with cargo molecules andtransport factors between import and export.

Biological Macromolecules Laboratory93

(6) Molecular Imaging of translation initiation factors
in neuronal dendrites

EDUCATION~*~/~~~~~~~.~t~7-

Hiraku Miyagi, Nobuyuki Shiina and Makio Tokunaga
Local protein synthesis in neuronal dendrites isrequired for synaptic plasticity, which is associatedwith long-term memory storage. This protein synthesis
is reported to be induced at activated postsynaptic
sites. In order to investigate when and where thetranslation is initiated locally in the dendrites, we
visualized eukaryotic translation initiation factors (elF)
4E and eIF4G in dendrites of rat hippocampal neurons.
Immunostaning of rat hippocampal primaryneurons showed that most of eIF4E and eIF4G werespreaded in distinct granular structures, and few ofthem were colocalized in some granular structures inthe dendrites. Immunostaning of rat hippocampal
slices showed essentially the same results as in the caseof the primary cultures. Stimulation by brain-derived
neurotrophic factor (BDNF) increased colocalizationof eIF4E and eIF4G in both rat hippocampal slices andprimary neurons. As the association of eIF4E andeIF4G is known to trigger translation initiation, theincrease of their colocalization suggests that translation
is induced. Immunostaning of rat hippocampal slices
and primary neurons for eIF4G and PSD-95, a marker
protein for postsynapses, showed that colocalizationof eIF4G and PSD-95 was increased by BDNF
stimulation. Colocalization of eIF4E and PSD-95 is also
increased by same stimulation. In GFP-expressedprimary neurons, colocalization of eIF4E and eIF4G
in spines is increased by BDNF stimulation. These
results indicate that translation in the neuronaldendrites is inhibited by separating spatially eIF4E andeIF4G, and translation is induced by BDNF stimulation
at postsynaptic areas.
PUBLICATIONS

1. Dr. M. Tokunaga gave a lecture at Tokyo Instituteof Technology, Department of Bioscience andBiotechnology, August, 2004 (in Japanese).
2. Dr. M. Tokunaga gave a Seminar at the Universityof Tokyo, Department of Applied Chemistry, Schoolof Engineering, September, 2004 (in Japanese).
SOCIAL CONTRIBUTIONS AND OTHERS
~fi~~

1.

1t!*]J¥~/,,-{:::t'T -7 / 0 ~-~Jfl1tf,1li~~*Jl-fr

r*BIJmfi'J* 'Y !' 17--7 ~y -17 ~ A'AMtTtHiIli~JflJ~~~Jfl~ffi!

2.

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1. Dr. M. Tol;<.unaga organized a symposium entitled"Frontier of Single Molecule, GFP and Bioimaging"supported by Grant-in-Aid for Scientific Research on
Priority Areas "Molecular Imaging" from MEXT,Tokyo International Forum, March, 2004.

H-b. Molecular Biomechanism Laboratory
Nobuo Shimamoto GroupRESEARCH ACTIBITIES(1) The branched mechanism of transcription initiationin E. coli

Motoki Susa\ Shouji Yagil and Nobuo Shimamoto leStructural Biology Center, National Institute of
Genetics and and Department of Genetics, School ofLife Science, The Graduate University for Advanced
Studies)

Papers

1. Kitamura, K., Tokunaga, M., Esaki, S., Iwane, AH.

and Yanagida, T. (2004). Mechanism of muscle
contraction based on stochastic mechanical featuresof single actomyosin motors observed in vitro.
BIOPHYSICS, in press.

94

Biological Macromolecules Laboratory

For several decades, the mechanism oftranscription initiation has been assumed to be asequence of three essential steps: formation of acomplex between RNA polymerase and a promoter(closed complex), formation of another complex withpartially melted DNA duplex to form phosphodiester
bonds (open complex and chemical reaction), andescape of RNA polymerase from the promoter

associated with the progress of RNA elongation(promoter clearance). There is a process called"abortive initiation", an iterative synthesis and releaseof oligo-RNA molecules. This process is often excluded
from the mainstream of the mechanism, because itsrole as well as its occurrence in vivo has been
unknown. However, this process has been observed invitro with all prokaryotic and eukaryotic RNA
polymerases so far isolated. The products of theprocess, abortive transcripts, are typically 2 to 15
nucleotides in length. On the assumption that theseshort transcripts are "unsuccessful precursors" of the
full-length transcript, abortive synthesis has been
considered to precede promoter clearance.
We have been clarifying for several years that thissequential mechanism is not the case and the initiation
follows branched pathways, one of which contains themoribund complex, being defined as a complex that
produces only abortive and no full-length transcripts.
Followings are its characteristics. 1. The moribund
complex, as 'well as the productive complex thatsynthesizes full-length product, are formed from thesame homogeneous fraction of enzyme molecules, anddissociation of the molecules from the promoter DNA
cancels any difference between them. 2. Structuraldifferences between these complexes have been
demonstrated. 3. At some promoters, a moribund
complex is converted into a dead-end complex that stillretains a short transcript but has no elongation activity.
Therefore, the initiation pathway is branched into theconventional productive pathway and the abortive
pathway that can lead towards a dead end. 4. The fatesof a moribund complex are either inactivation as adead-end complex, dissociation from the DNA, or directconversion into a productive complex, and the ratesof these reactions vary with the promoter. 5. There arefactors that affect the fate of the moribund complex
in a manner that depends on the promoter.
To examine the existence and significance of the
branched pathway in vivo, we selected GreA and GreB
for clues. At the ,U~AL promoter these factors
enhance conversion of the moribund complex into the
productive one, in the presence of high concentrationsof initiating nucleoside triphosphate in vitro. If the
branched mechanism exists in vivo, absence of the Gre
factors should result in reduction of productive
transcription from promoters at which the moribund
complex is susceptible to these factors. We constructeda double-disruptant of E.coli, 11 greAI1 greB, and thenarbitrarily selected 10 genes from among those whoselevels of transcripts in the mutant strain were foundto be lower than those in the parental greA + greIr
strain. Finally, the promoter for three of these genes,
atpC (uncC), cspA, and rpsA, passed a further
conventional test which confirmed that they displayeda branched initiation pathway in a reconstituted
transcription system composed of purified
components. The results obtained prove that thebranched initiation pathway exists in vivo and isutilized in regulation of transcription initiation fromsome promoters, through modulation of the fractionof polymerase-promoter complexes entering eachbranch of the pathway.
In this year, we examined various physiologicalconditions that affect on the levels of the Gre factors.
The determination of the level of GreB was observed
to be constitutive. The level of GreA remained the same
through the growth phase, and did not respond muchto the richness of the culture media. However, it
decreased into half in aerobic conditions, indicatingthat some genes are regulated by GreA. Therefore, theregulatory circuit responding to the levels of proteins
involving GreA, namely the branched pathway
mechanism, is working in cells.
(2) Applicability of thermodynamics to equilibria inbiologyNobuo Shimamotol and Jyun-ichi Tomizawal
CStructural Biology Center, National Institute of
Genetics and Department of Genetics, School of Life
Science, The Graduate University for Advanced
Studies)
Most DNA-binding proteins are biologicallyfunctional as a specific complex, one containing a
special short DNA segment. Such a complex is usuallyassumed as a state tenable for thermodynamic analysisof binding equilibrium. Thus, forward and backward
reactions should balance at equilibrium in every
pathway, and the affinity should be independent of thelength of DNA. However, we have found that thebalance at equilibrium is broken for some proteins by
their sliding along DNA during association but notdissociation and that their affinities for their specificsites dependent on the length of DNA harboring thesites. This seeming disagreement is explained by anindeliberate use of the state of specific complex in

Molecular Biomechanism Laboratory95thermodynamics. In the presence of sliding, the state
does not satisfy the second law (the ergodic condition)and thus is disqualified for thermodynamic analysis.
A general treatment of binding equilibrium, while
maintaining the specific complex as a distinct state,is proposed on the base of the master equation or
chemical kinetics.
(3) Systematic search for promoters encoded in the
genomic DNA sequences of E. coli

Books

4.

~,l%*{$H:t (2004) /<;-1 ::t7' -7 / 0 ~ -~ -}- /7' -7 / 0~-(J)/Ml~, 1f!.fmlE*~~~tt.ttlJJX:ijfgeffl.

EDUCATION

Dean of School of Life Science, Graduate School of
Advansed Studies

Papers

Lectures in other universities and institutes
1. Shimamoto, N. "RNA polymerase and a secret ofnano-biomachines", Center For DNA Fingerprinting
and Diagnostics (CDFD), Hyderabad, India January,
2004.
2. Shimamoto, N. "The firm start-point of
nanobiology and biological nanotechnology;
Understanding the characteristics of biologicalnanomachines" (in Japanese,r-}- / /<;-1 ::t(J)m.Q ~f~v'
m~,8j::1*(J)-}-/"? ~ /,(J)*f-@.J), Graduate School ofEngeneering, The university of Tokyo, June 2004.
3. Shimamoto, N. "Chemical and Biologicalconsequences of one-dimensional diffusion of proteins
along DNA School of Chemistry", Seoul National
University, Seoul, Korea, September 2004.
4. Shimamoto, N. "RNA polymerase and a secret ofnano-biomachines", Department of Life Sciences, Seoul
National University, Seoul, Korea, September 2004.
5. Shimamoto, N. "Proteins as biologically functional
molecules in Fundamental Course of Molecular andCellular Biology (KAST)" (in J apanesel!i@ip G~.s:;
KAST7t-1-)fflJJmj:: ~~ =:r - 7- : ;fI13~7t-1- ~ ~ ~ (J) -7 /' /~-7lt) The Institute of Medical Science, The Universityof Tokyo October 2004.
6. Shimamoto, N.
"Acey-deucy biologicalnanotechnology: Truths and ghosts in nano-biologicalscience and technology (in Japanese,r3iip~ip? : -}/ /<;-1 ::t~Ji ~ ~ .w..Qf!l.~ ~ tt*J(J)mr~J), The Osaka
Cabinet of Commerce and Industry (*IIRjt'ijI~~ffl),
November 2004.

1. Sakata-Sogawa, K. and Shimamoto, N. (2004). RNApolymerase can track a DNA groove during promoter
search. Proc. Nat. Sci. U.S.A. 101, 14731-14735.

Organization of International Meeting

Nobuo Shimamoto\ Hideki Nakayama1 andHironori Aromaki 2 CStructural Biology Center,
National Institute of Genetics and Department of
Genetics, School of Life Science, The Grad uate
University for Advanced Studies. ~aiichi College of
Pharmaceutical Sciences)
Irrespective of a pile of compiled sequences
identified as promoters for E. coli vegetative RNA
polymerase holoenzyme, a 70 holoenzyme, there is no
successful methods to predict strength of a promoter.
To construct such prediction method, we started todesign a functional SELEX to select promoter
sequences. The complexity of random oligo-DNA
available in a lab scale, 412- 14 is too small to cover the
all-possible promoters. Therefore we limited thecandidate sequences to those involved in the genomic
sequence. In order to construct and select a library, wetransferred and amplified parts of the genomic
sequence with PCR with a single primer. As adrawback of the use of single primer, the constructedlibrary contains DNA fragments generated from invitro recombination. A theoretical method to diminish
the effect of such recombinants has been developed.
PUBLICATIONS

Reviews2.

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96

Molecular Biomechanism Laboratory

SOCIAL CONTRIBUTIONS AND OTHERS

1. Shimamoto, Nand Shivashankar, G. V.
(Organizers),
Nakayama, Hand Susa, M. (Organizing Members)
JSPS-DST Asia Academic Seminar:
NIG-NCBS International Workshop on Singlemolecule Biophysics, Bangalore, India January, 2004.
(25 International Invitees from US, France, Swiss,Israel, Korea, Japan, India and 30 Asian Trainees from
India, Japan, China, Korea, Malaysia, Singapore)
2. Member of international Committee of Asian
Conference of Transcription (ACT), the delegate ofJapan. (Organizing members of ACT8, Bangkok,
November, 2004)

H-c. Multicellular Organization Laboratory
Isao Katsura GroupRESEARCH ACTIVITIES(1) Analysis of synthetic dauer-constitutive mutants inthe nematode Caenorhabditis elegans
~alf

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1. Member of international Committee of Asian
Conference of Transcription(ACT), the delegate of
J apan.(Organizing members of ACT8, Bangkok,
November, 2004)

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If newly hatched larvae of C. elegans are in a
crowded state and given a limited food supply, theysense the environmental signal of pheromone and foodwith a head sensory organ called amphid, and deviate
from the normal life cycle, ending in non-feeding larvae
called dauer larvae. Since the assay of dauer larva
formation is much less time-consuming than behavioral assays such as chemotaxis assays, we are
analyzing the pathway of sensory signals in theamphid neural circuit by detecting dauer larva
formation as the output. We found that mutations inmore than 50 known genes show synthetic dauerconstitutive (synDaf) phenotypes, i.e., they induce dauer
larva formation in certain mutant backgrounds,
regardless of the environmental conditions. The
synthetic nature of the phenotype, we think, is basedon the pathway of sensory signals. Namely, the signals
are transmitted through parallel routes, and therefore
two mutations are required to block them. Weare
determining the combinations of mutations for the
synDaf phenotype and the pattern of suppression of
the synDaf phenotype by various suppressor
mutations. In this way we hope to elucidate thepathway of sensory signals for dauer regulation.
Furthermore, to identify new genes required forthe sensory signal transduction, we have isolated andmapped 44 mutations that show the synDaf phenotype
in the unc-31(e169) background, where unc-31 geneencodes CAPS protein, which acts in secretion fromdense core vesicles. Eight of the mutations map in 4
known genes, but most of the remaining 36 mutations,which map at least in 13 genes, seem to be located in
novel genes, which we named sdfgenes. Of these genes,
sdJ-9, sdJ-13, and sdJ-14 have been cloned. sdJ-9 geneencoded a protein tyrosine phosphatase-like molecule,was expressed in a pair of neuron-associated cells
called XXXLlR, and regulated dauer larva formation

Molecular Biomechanism Laboratory97

in the steroid honnone signaling pathway (Ohkura, K.
et al.: Development 130,3237-3248,2(03). sdj-13 encodeda homologue of the transcription factors Tbx2
andTbx3, ~as expressed in AWB, Awe, and AS]
sensory neurons as well as many pharyngeal neurons,• and controlled olfactory adaptation in Awe and dauer
larva formation in cells other than Awe (possiblyAS]))). sdf-14 gene was the same as mrp-1 gene, which
was fonnerly identified by its homology to multidrug
resistance-associated protein genes in mammals. A
functional sdj-14::GFP fusion gene was expressed inmany tissues including neurons, pharyngeal muscles
and intestinal cells. Experiments with extrinsic
cell-specific promoters revealed that expression in two,but not one, of the three tissues rescue the mutant
phenotype efficiently. Interestingly, human MRPI
could substitute for C. elegans MRP-l in dauer larva
regulation, and an inhibitor of the MRPI export activity
impaired this function, showing that the export activity
is required for nonnal dauer larva regulation.
In the year 2004, we found by epistatis analysis thatsdj-14 acts neither in the cGMP nor TGF- {3 signaling
pathway among the four signaling pathways involved
in dauer larva regulation. The result is consistent withthat of the cell-specific expression experiments, because
genes in these two pathways act only in neurons. We
found that sdj-14 mutations strongly enhance thedauer-constitutive phenotype of the daj-2(e1370)
mutation, which confers resistance to many environmental stresses. Furthennore, sodium arsenite, which
is a substrate of human MRPl, as well as hightemperature (21C) enhanced the dauer larva fonnationof the unc-31(e169) mutant, which is known to blockthe daf-2insulin signaling pathway. Thus, sdf-14 gene
may be involved in heat and heavy metal stressresponses. The proof of this possibility as well as its
relation to dauer larva fonnation remains to be studied.
C. elegans mutants in the "associative" learning withodorants and food(2)

Ichiro Torayama, Hiroshi Ichijo, Kotaro Kimura,Takeshi Ishihara and Isao Katsura
The nematode C. elegans provides a good system
for the study of learning with a combination of two
stimuli. However, the mechanism of this learning looks
different from that of classical conditioning, because(a) the unconditioned stimulus is usually limited to98

Multicellular Organization Laboratory

food or starvation, and because (b) the learning isefficient, if the conditioned stimulus is presented at thesame time as but not before the unconditioned
stimulus. To elucidate the molecular mechanism ofsuch "associative" learning in C. elegans, we are
isolating and characterizing mutants that showabnonnality in the learning with butanone and food/starvation. It is known that butanone attracts wild-type
animals without conditioning. Conditioning withbutanone and starvation decreased the efficiency ofchemotaxis to butanone, while conditioning withbutanone and food increased it. We isolated mutantsin these behaviors, some of which showed decrease in
the efficiency of chemotaxis after conditioning withfood and butanone, while others are attracted
efficiently by butanone only after conditioning withfood and butanone. Of those mutants, ut305 and ut306,
which belong to the fonner category, have been studied
in detail. ut305, but not ut306, showed abnonnality inadaptation to isoamyl alcohol and benzaldehyde, which
are sensed by the same type of sensory neurons (AWe)as butanone. ut305 showed inefficient chemotaxis tobutanone at low concentration, although it showednonnal chemotaxis at the concentration used in thelearning assay. ut305 gene encoded a novel protein
containing predicted transmembrane domains andshowing limited homology to the Drosophila Raw
protein.
In the year 2004, we examined the rescue of ut305
mutant phenotypes by expressing the wild-type ut305
gene in various cells, using extrinsic promoters. The
mutant phenotypes were rescued, if the wild-type ut305
gene was expressed in neurons including Awe sensory
neurons, although we fonnerly detected the fluorescence of a functional ut305::GFP fusion gene in AlA
interneurons and many pharyngeal neurons, but not
in Awe neurons. Furthennore, str-2.:GFP fusion gene,which is nonnally expressed in only one of the AweLl
R neurons, was not expressed in either of them in the
ut305mutant, while it was expressed in both of themwhen the wild-type ut305 gene was overexpressed in
Awe neurons. These results indicated that ut305 geneacts in Awe neurons for the learning with butanone
and food, for the adaptation to benzaldehyde andisoamyl alcohol and for the regulation of str-2
expression. We are now making specific antibodies tothe ut305 protein to examine whether the ut305 protein
is present in Awe neurons. We are also detennining
the position of ut305 gene in the cascade of theregulation of str-2expression, in which Ca2+ signaling
and the ASKI MAP kinase cascade are involved. We
also cloned ut306 gene and analyzed the mutant
phenotypes. The study will be continued in 2005.
(3) Molecular genetic studies on sensory integration andbehavioral plasticity in C. elegans

Takeshi Ishihara, Yuichi lind, Akiko Mohd, Ikue
Mori 2, Keiko Gengyo-Ando 3, Shohei Mitani3 and Isao
Katsura CMolecular Genetics Laboratory, Universityof Tokyo, 2Division of Biological Science, Nagoya
University, 3Department of Physiology, Tokyo
Women's Medical University School of Medicine)
Animals receive environmental cues, select andintegrate necessary information, and make proper
responses, while all these steps can be modified byexperience or memory. In C. elegans, many behavioral
mutants defective in chemotaxis and thermotaxis, forinstance, have been isolated and analyzed, and themolecular mechanisms of sensation have been
elucidated. On this basis and as a next step, we are
analyzing mutants that show abnormality in thelearning and selection (evaluation) of sensory signals,
to elucidate novel mechanisms of higher order sensory
signal processing.
C. elegans shows avoidance of copper ion andchemotaxis to odorants by receiving these stimuli withdifferent sensory neurons in the head. We developed
a behavioral assay for the interaction of two sensory
signals: aversive copper ion and attractive odorant,
diacetyl. Wild-type animals change their preference.
between the responses, depending on the relative
concentration of copper ion and odorants. On the basisof the C. elegans neural circuitry, the result suggeststhat the two sensory signals interact with each otherin a neural circuit consisting of about 10 pairs ofneurons. While well-fed animals are usually used forthis assay, we found that animals starved for 5 hours
tend to prefer chemotaxis to odorants. The change isdue to the desensitization of copper ion avoidance by
starvation, and can be suppressed by serotonin, whichmimics the effect of food. This desensitization isadvantageous in natural environment, because starved
animals can search for food over a wider area.
To elucidate the mechanism of sensory integrationin the neuronal circuit, we are isolating and analyzing
mutants that show abnormality in this assay. The

hen-] mutants showed much weaker tendency to crossthe Cu2+ barrier when migrating toward attractive
odorants than the wild type, although that the hen-]mutants had defects neither in the chemotaxis toward
the attractive odorant nor in the avoidance of Cu2+ ion
per se.
To elucidate molecular mechanisms for thesensory integration, we cloned the hen-] gene andfound that it encodes a secretory protein with an LDL
receptor ligand binding domain, LDLa. This domain
in HEN-1 is most similar to that domain of Drosophila
signaling molecule Jeb, which regulates migration anddifferentiation of visceral mesodermal precursor cells.
Immunostaining by using antibody against recombinant HEN-l protein revealed that the gene productis localized in the axon and cell body of each one pairof sensory and inter-neurons. The localization in theaxon was abolished in unc-]04 (kinesin KIFIA
homologue) mutants, which show defects in thetransport of synaptic vesicles. Expression studies withvarious promoters showed that this gene acts
non-cell-autonomously in the mature nervous system.
The hen-] mutants also show abnormality inlearning by paired presentation of starvation and NaCl(collaboration with Dr. lino, University of Tokyo) andby paired presentation of starvation and temperature(collaboration with Ms Mohri and Dr. Mori, Nagoya
University). Wild-type animals show chemotaxis to
NaCI under a well-fed condition, although they avoid
NaCI after conditioned with starvation and NaCl. The
hen-] mutants show a weaker behavioral change than
the wild type after the conditioning, although theyshow normal chemotaxis to NaCI under a well-fed
condition. Wild-type animals prefer the cultivation
temperature under a well-fed condition, while they
avoid that temperature after conditioned in the absenceof food at the same cultivation temperature. Although
the hen-] mutants show normal thermotaxis under awell-fed condition, they do not avoid the cultivation
temperature after conditioned in the absence of food.
Since starvation was used to induce plasticity in bothlearning assays, we analyzed whether hen-] animals
can sense starvation, but we could not find any
abnormality in the behavior after simple starvation.
These results indicate that the hen-] show defects inthe behavioral plasticity after paired presentation of
starvation and NaCI or starvation and temperature,
although it responds normally to each of these stimuli.
Molecular genetic analyses of HEN-1 suggest thatMulticellular Organization Laboratory99

HEN-1 functions as a neuronal modulator for sensory
integration and learning. To elucidate the molecularmechanisms of this neuromodulation, we started
investigating the protein interacting with the HEN-I
protein. First, we developed a binding assay for
identification of receptors for HEN-I. By using a
HEN-I-alkaline phosphatase fusion protein as a ligand,which was expressed by HEK293 cells, we found that
HEN -1 specifically binds a subpopulation of the
primary culture cells in C. elegans, suggesting thatreceptors for HEN-1 exist in these cells.
Recently, Jeb in Drosophila was reported toregulate development of mesodermal cells through thereceptor tyrosine kinase DAlk, which is a homologueof human proto-oncogene Alk. Since the LDLa domain
in Jeb is similar to that in HEN-I, we started to analyze
the scd-2 gene, which encodes a receptor tyrosine kinasesimilar to DAIk. Expression analyses by using an scd-2
promoter-GFP construct suggested that SCD-2 isexpressed in several sensory neurons and intemeurons.
scd-2mutants showed the same behavioral defects as
hen-1 mutants in paradigms for sensory integrationand learning. The expression of SCD-2 in scd-2mutants
driven by neuron specific promoters and a heatshock
promoter suggested that SCD-2 functions in the mature
nervous system, like HEN-I. These results suggest that
SCD-2 may be a receptor for HEN-I. Determination of
the cells where SCD-2 functions for sensory integrationand learning may reveal the center of informational
processing in C. elegans.
(4) Genetic analysis of plasticity of avoidance behaviors
in C. elegans

Kotaro Kimura and Isao Katsura
C. ele'gans avoid many toxic and/or hazardous
signals, such as high osmotic strength, acidic pH and
SDS, as well as several volatile and soluble small
compounds. In general, preceded stimulation(s) ofanimal's sensory neuron lead to a reduction in themagnitude of its response (adaptation), and C. elegans
has been shown to exhibit adaptation to attractive
odors or to body touch after pre-exposure to thestimulus. However, plasticity of the avoidance
behaviors of C. elegans to the signals mentioned above
is poorly understood. We wondered whether the
avoidance behaviors of C. elegans are modulated bypreceded experience of the same stimulus.

100 Multicellular Organization Laboratory

We found that pre-exposure to 2-nonanone, oneof the repellent odors, enhances the avoidance behaviorof the animals to the odor. For example, the avoidance
behavior to 10% (v/v) 2-nonanone of the wild-type
animals was significantly enhanced after pre-exposure.
The magnitude of the enhanced avoidance behavior
was comparable to the behavior to 30% 2-nonanone ofpreviously unexposed animals, suggesting thatpre-exposure may enhance the sensitivity to
2-nonanone by about 3-fold.
Enhancement after pre-exposure was also
observed for l-octanol, another repellent odor, but notfor osmolarity as the repellent stimulus, suggestingthat only certain type(s) of repellent stimuli trigger thisphenomenon, and that it requires specific molecular
and/or neuronal mechanisms.
Weare interested in the sensitization because of
the following reasons: (1) Not all types of the repellent
stimuli trigger the sensitization. (2) Sensitization to
repellent odors and tastes has not been reported in C.
elegans to our knowledge. (3) The molecularmechanisms of sensitization to odor, taste or even lightin other animals are poorly understood. We believethat genetic analysis of the sensitization may reveal
novel molecular mechanism of regulation of signal
sensation.
(5) Class 1 flrmutants of the nematode Caenorhabditis
elegans

Yuri Kobayashi, Kotaro Kimura, Takeshi Ishihara andIsao Katsura
Class 1 flr mutants of C. elegans, which map in
flr-1, flr-3 and flr-4, were originally isolated byresistance to 0.4 mg/ml NaF (Katsura, 1. et at.: Genetics
136, 145-154, 1994). They also show many otherphenotypes including slow growth, short defecation
cycle periods, frequent skip of the expulsion step ofdefecation, synthetic abnormality in dauer larva
formation, weak tendency to stay on food, andhypersensitivity to serotonin. The flr-1 gene encodes
an ion channel belonging to the DEGIENaC (c. elegans
degenerins and mammalian amiloride-sensitive
epithelial sodium channels) superfamily, while flr-4 andflr-3 code for a novel Ser/Thr protein kinase and akinase-like molecule, respectively, both having ahydrophobic domain on the carboxyl terminus. A
functional flr-1::GFP fusion gene is expressed only inthe intestinal cells from the comma stage of embryos
to the adult stage, while a functional flr-4::GFP isexpressed in the intestinal cells from the loS-fold stage,in the isthmus of the pharynx from the 3-fold stage andin a pair of head neurons called AVA from Lllarvae
to adults. Moreover, the expression of various flr-3::lacZand flr-3::GFP fusion genes is detected only in theintestine. Temperature-shift experiments of aflr-4(ts)mutant showed that the activity of FLR-4 is requiredat the time of defecation assay (i.e., young adults) for
normal defecation cycle periods. We therefore thinkthat class 1 flr genes constitute a regulatory systemthat acts in the differentiated intestinal cells.
In 2004, we confirmed using an extrinsic promoter
that expression of the wild-type flr-4 gene in theintestine of flr-4 mutants is sufficient for the wild -type
phenotypes in growth, defecation cycle periods,
expulsion, and dauer regulation 31 • This is consistent
with our old experiments showing that killing of AVA
neurons in wild-type animals and flr-4 mutant animals
does not change their defecation phenotypes. Thus, we
could show that the three class 1 flr genes act in theintestinal cells to regulate various functions. Weare
studying how they interact with one another.
(6) Class 2 nrmutants of the nematode Caenorhabditis
elegans

Akane Oishi, Kotaro Kimura, Takeshi Ishihara andIsao Katsura
Class 2 fir mutations were isolated mostly as
suppressors of the slow growth or serotoninhypersensitivity of class 1 flrmutations. Besides these
phenotypes, they also suppress the dauer larva
formation abnormality and weak tendency to stay on
food, but not the defecation abnormalities or strong
fluoride-resistance. By themselves, class 2 flrmutations
show the phenotypes of weak resistance to NaF andshort average longevity as compared with wild-type
animals. The phenotypes suggest two possibilities on
the relationship between class 1 and class 2 flrgenes.
(a) Class 2 flr genes may act downstream of the class
1 regulatory pathway. At the downstream, theregulatory pathway bifurcates into two branches, thegrowthldauer branch and the defecation branch, whileclass 2 genes act in the former branch and not thelatter. (b) Class 2 flrgenes may act antagonistically toclass 1 genes, while the threshold of the phenotypes

is different between the growthldauer phenotypes andthe defecation phenotypes.
Class 2 mutations map in four genes, flr-2, flr-5,
flr-6 and flr-7, of which only flr-2has been cloned. flr-2
encodes a secretory protein belonging to the gremlin!
DAN/cerberus family. A functional flr-2:GFP fusion
gene was expressed in some neurons in the head andthe tail as well as many pharyngeal neurons.
FLR-2::alkaline phosphatase fusion protein molecules
bound specifically to the intracellular compartment ofa limited number of C. elegans primary culture cells.
We therefore screened for C. elegans cDNA clones
whose expression in COS cells enabled the cells to bindto FLR-2::alkaline phosphatase. By screening 264 poolsof cDNA, where one pool consists of about 1000 clones,
we obtained a single positive clone (ZK20.1) encoding
a secretory protein.
In 2004, we carried out cell-specific expression
experiments, using extrinsic promoters. Although afunctional flr-2:GFP fusion gene was expressed onlyin neurons, expression of the wild-typeflr-2gene in the
body wall muscle and intestine, respectively, rescued
the flr-2 mutant phenotype, as assayed by the recoveryof the slow grow.th phenotype of flr-2; flr-1 double
mutants. Rescue with neuronal promoters was
successful, only when we used low concentrations of
the construct DNA for transformation. Thus, it seems
that although FLR-2 is expressed and secreted by someneurons under natural conditions, body wall muscles
and intestinal cells also have the ability to secrete it.
Besides these experiments, we are working on thefunctional interaction between flr-2 and ZK20.1, by
isolating a deletion mutant in the ZK20.1 gene andlooking for its phenotype.
PUBLICATIONSPapers1. Miyahara, K, Suzuki, N., Ishihara, T., Tsuchiya,
E. and Katsura, I. (2004). TBX2/TBX3 transcriptional

factor homologue controls olfactory adaptation in
Caenorhabditis elegans. ]. Neurobiol. 58, 392-402.
2. Kimura, KD., Miyawaki, A., Matsumoto, K andMori, I. (2004). The C. elegans thermosensory neuron
AFD responds to warming. Curro BioI. 14, 1291-1295.
3. Take-uchi, M., Kobayashi, Y., Kimura, K, Ishihara,
T. and Katsura, I. (2005). FLR-4, a novel Serine/I
Threonine protein kinase, rero.tlates defecation rhythm
in Caenorhabditis elegans. Mol. BioI. Cell, 16, 1355-1365.

Multicellular Organization Laboratory

101

Reviews4. ~)Jj( (@(2004) r~!E..¥f.!fT~O)7t-T)!~'¥: J!i'i'¥f.!$JJmJAfi;-TJMSfl . ~ftt . M~49, 450-455.
5. tE~ (2005) r~!R C. elegans<J)~~ . 7tfI::UJf9E.!:=

if / A1W¥R/~~I!f'¥itf~J23 OJ, 157-163.
EDUCATION

1. Dr. I. Katsura gave a lecture at Kyoto University,
Graduate School of Science, June 2004 (in Japanese).
2. Dr. I. Katsura gave a lecture at Kyushu University
Graduate School, Faculty of Sciences, June 2004 (inJapanese).
3. Dr. I. Katsura was invited to give a seminar on"Molecular biological analysis of the behavior of thenematode C. elegans" at Kyushu University Graduate
School, Faculty of Sciences, June 2004 (in Japanese).
4. Dr. I. Katsura gave a lecture at the University ofTokyo, Graduate School of Arts and Sciences, July 2004
(in Japanese).
SOCIAL CONTRIBUTION AND OTHERS

1. *fJH,Q2004-314935 r \::: "MRPUJl~~JO)7-7 !J -=:..'./1/
l'!J~aA:lf : ~$~-T, f!
~,~)Jj( {@, ~*~a~,I±llllA : *'¥~lWJfIJm.Ml'!A1W¥R . ;/ 7- T AUJf9E.~.
2. Dr. I. Katsura served as one of the associate editorsof the journal "Genes to Cells".
3. =€.fIJ3%'£t, ~ ~ftfT, *FtJJt=, r:pj!!f~l1m, "T ~~, iWi;fjlE~, tE~,tt!!12~/iWi~'¥13t~~ IT J ~~1it2004.

H-d. Biomolecular Structure Laboratory
Yasuo Shirakihara GroupRESEARCH ACTIVITIES(1) Crystallographic Study of ATP synthaseYasuo Shirakihara and Aya Shiratori
ATP synthase is responsible for ATP production
in living cells, and is a membrane protein located in
the energy conversion membrane. ATP synthase
consists of a channel Fo portion (about 100,000 dalton,
subunit composition of ab2C8-12) and a large soluble
catalytic F1 portion (380,000 dalton, £x 3 (3 3'YOE ). The
unique rotational catalysis mechanism of F1 includes

102 Multicellular Organization Laboratory

rotation of the rod-like 'Y subunit which is thought tocontrol the conformations of the three catalytic{3 -subunits in a cyclic manner by its rotation. Startingfrom elucidation of the £X3{33 sub-assembly structureof the thermophilic F1, we have been moving up to thehigher sub-assembly. The £X3{33'Y sub-assembly wasdifficult to crystallize, but the £X3{33'YE sub-assembly
gave crystals that allowed to see a novel conformationof F1. Weare now dealing with the holo-enzyme, ATPsynthase.
The membrane protein ATP synthase is still achallenging target for a structural study, in view ofrelatively few solved structures of the membraneproteins so far. After an initial trial for preparation andcrystallization of the protein made last year, we havecontinued examination of both aspects extensively.
Detergents such as decy-maltoside, undecyl-maltoside,
dodecyl-maltoside, tridecyl-malltoside were good in
preparation, but others like alkyl glucosides were not.
Among various columns examined, useful ones were
Q-sepharose high performance, Superdex prep grade
200 columns. An array of Q-sepharose high performance,
Superdex prep grade 200 and the second Q-sepharosehigh performance columns yielded a preparation of
crystallization grade, but a smaller array of Superdex
prep grade 200 and Q-sepharose columns only was
indistinguishable from the first array. Initial tiny
crystals are now replaced by crystals with typicaldimensions of 0.2mm, 0.2mm, O.Olmm after extensive
and systematic crystallization condition search. Amongthe detergents mentioned above, only decy-maltosideand dodecyl-maltoside gave diffracting crystals, though
all of the four detergents gave similar-looking crystals.
ADP is the most favorite ligand, as others like
AMPPNP, an ATP analogue, gave crystals thatdiffracted poorly. Other factors such as temperaturein crystallization, kind and concentration ofmonovalent salts and those of divalent salts have been
optimized. ATP synthase in our crystals contains all
the 8 subunits, in contrast to yeast ATP synthase thatlacks a and b subunits in crystals. Our crystals diffract
to a resolution of about 7 A. Further refinement of thecrystallization conditions to get better diffracting
crystals is in progress.
The preparation and crystallization study wasdone in collaboration with Satoshi MURAKAMI at
Institute of scientific and industrial research, Osaka
University, Toshiharu SUZUKI and Masasuke
YOSHIDA, at Research Laboratory of ResourceUtilization, Tokyo Institute of technology.
(2) Comprehensive Crystallographic Study of Transcription factors and Genome-partitioning Factors from E.coli

Yasuo Shirakihara and Aya Shiratori
In E.coli, more than 160 transcription factors
control transcription of their target gene(s) by binding
to both their specific DNA sequence and thetranscription apparatus. Sixty-five such transcription
factors had been purified in Ishihama laboratory. In
Niki laboratory, a number of novel proteins, that are
judged to play roles in the genome and plasmid
partitioning, have been prepared. Setting thesetranscription and genome partitioning factors astargets for a structural study, we are doing comprehensive structure determination in the Protein 3000
project (the sub-field of 'transcription and translation').
In the previous first two years, we examined 65 proteinsfor their crystals but identified only three proteins thatgave crystals of diffraction quality. This year, we haveconcentrated to refine the crystals obtained so far.
Mlc, an E.coli transcription factor, was found todiffract to about 3 A resolution. The pattern wasrecorded after reducing the aggregating tendency of
the Mlc crystals. Phasing was hampered by either
unavailability of appropriate heavy atom derivatives
or inability of a Se-methionine substituted form of theprotein to form crystals.
YmcB, a protein believed to be involved in the
genome and plasmid partitioning, was found to form
extremely good crystals, diffracting beyond 2 A evenwith the laboratory beam source, after a sustained
crystallization condition search. We did some
expression experiments on YmcB, as the delivered
preparation ran out and the subsequent expression in
our hands turned out to be difficult. By changing
expression conditions from the previous one(BL21(pLys), 30° incubation, IPTG induction) to newones (BL21, 25" or 20° incubation, no induction), we got
two preparations: from 25° incubation, a preparationof slightly shorter fragment of the molecule: from 20°
incubation, a preparation of the intact molecule. The
initial delivered preparation was a mix of the two, andthe species responsible for crystals was found to be
the fragment. Collection of diffraction data andanalysis of the data is in progress.
This work has been done in collaboration with

Akira ISHIHAMA, Emi KANDA (Nippon Institute for
Biological Science), Hironori NIKI, Rie INABA,
Katsynori YATA, Yasushi OGATA (isotope center).
(3) Comprehensive structure-based functional studies ontranscription factors

Hiroshi Itou and Yasuo Shirakihara
Genomic-DNA information on number oforganisms is now available. Using the information,
comprehensive structure analysis of transcription
factors for their structure-based functional understanding is in progress. Our research targets are transcription factors (including putative ones) from hyperthermophilic archaean Pyrococcus horikoshii, mesophilic
bacteria Corynebacterium glutamicum and vertebrates
Homo sapience. 162 genes were selected as target, and146 of 162 were cloned, over-expressed (125 of 142) andpurified (49 of 125). These purified proteins were tried
crystallization for X-ray crystal structure analysis, andten initial crystals were 0 btained. We had alreadysucceeded in structure analysis of three of crystals,
PH1l61 and PH1932 from Phorikoshii and CGL2612
from C.glutamicum. PH1l61 protein is a homologue ofbacterial transcriptional activator TenA, and PH1932
and CGL2612 are homologue of transcriptional
repressor protein ArsR and QacR, respectively. As afunctional approach, target DNA sequences for these
proteins were determined using SELEX (Systematic
Evolution of Ligand by EXponential enrichment)
method. This led to a tentative identification of thetarget genes for these transcription factors. This
combination of tertiary structure analysis and binding
DNA sequence determination by SELEX method is a
powerful tool that provides us with key information
on biological functions of transcription factors newly
discovered by genomic analysis.
This work has been done in collaboration with Ui
OKADA and Isao TANAKA at Division of Biological
Sciences, Graduate School of Science, Hokkaido
University.
(4) Crystallographic Study of Transcription factors from
Pseudomonas aeruginosaYasuo Shirakihara and Aya Shiratori

Pseudomonas aeruginosa is the well-known

Biomolecular Structure Laboratory103

opportunistic bacterial pathogen, and a number oftranscription factors responsible for the pathogenicity
have been identified. Among those, we are doing
structural study of PtxR, PtxS, PhzR, and PA3547.
Last year, we analyzed the MAD data from PhzR
crystals and got an electron density map which showed
clear helical densities in some part. However, inspiredby a lesson from a failing expression experiment of aplant cell-death related protein SAG12 (molecular massof about 30k) where SAG12 was not expressed but
lactamase from a vector was instead expressed, C
terminus amino acid sequence of PhzR (26K) expressedin our hands was examined. The sequence corresponded to that of C terminus residues of lactamasefrom Staphylococcus aureus, not to that of PhzR. Thereis a strong suspect that our PhzR crystals were notgenuine. Diffraction data have been re-examined in thisregard.
This work has been done in collaboration with
Hironori ARAMAKI (Daiichi Pharmaceutical College).

medical science, the university of Tokyo.
PUBLICATIONSPapers
1. Itou, H., Yao, M., Watanabe, N. and Tanaka, I.
(2004). Structure analysis of PH1161 protein, atranscriptional activator TenA homologue from thehyperthermophilic archaean Pyrococcus horikoshii. Acta
Cryst. D60, 1094-1100.

H-e. Gene Network Laboratory
Emiko Suzuki GroupRESEARCH ACTIVITIES(1) Molecular and fine morphological studies on thephototransduction in Drosophila

Emiko Suzuki(5) Crystallization of Kid, a chromosome mover

Yasuo Shirakihara and Aya Shiratori
Kid is involved in spindle formation andchromosome movements in mitosis/meiosis. Kidconsists of three domains: an N-terminal kinesin-likemotor domain (35-370), a C-terminal chromosomebinding domain (594-647) and a connecting domain(371-593) including a coiled-coil region. Our Previous
trials to crystallize the N-terminal motor and the
C-terminal chromosome-binding domains had failed,partly because both preparations contained a GST-tag
that was attached for expression and easy preparation.
We tried this year to crystallize a longer N-terminal
fragment (1-462), and got small crystals from jeffamine
or isopropanol. The crystals were formed only in thepresence of MgADP. Further refinement of thecrystallization conditions is in progress.
We also tried to express the full-length Kid using
our favorite E. coli expression system described lastyear. B834(DE3), JMI09(DE3) and Rosetta produced theprotein, though each in slightly different conditions.
Expression was checked by a Western blot analysis.
Efforts are being made to get a pure preparation in
10mg scale.
The study was done in collaboration with Noriko
TOKAI and Jun-ichiro INOUE at the Institute of104

Biomolecular Structure Laboratory

Phototransduction in Drosophila photoreceptor
cells is a G-protein coupled phosphoinositide(PI)-signaling cascade. It is the most rapid process
among G-protein coupled cascade known to date(receptor potential is generated within "-"20milliseconds after light stimulation), and the
responsiveness is dynamically regulated.We are
studying how such elaborated signaling cascade isaccomplished, from the viewpoint of intracellularorganization of the molecules involved in this system.
Our recent studies have shown that the core components of this cascade, phospholipase C (NORPA),
protein kinase C (INAC) and TRP channel protein,
form a macromolecular complex "transducisome" inthe photoreceptive microvilli, by binding to a scaffold
protein, INAD (collaboration with Dr. Charles Zuker's
group in U.c. San Diego). The rapidness ofphototransduction is dependent on this topologicalcoupling of signaling molecules. In addition to thesecore components, there are several regulatory proteins,and proteins involved in the supporting system ofphototransduciton. This year, we studied some of these
protein functions. As for the back-up system of PI
metabolism, we have found that many of the enzymes
for PI-regeneration are localized on subrhabdomeric
cisternae (SRC), the specialized smooth endoplasmic
reticulum adjacent to the photoreceptive microvilli.

Among these, we focused on the diacylglycerol kinase
2 (RDGA) that is essential not only for the regulationof the TRP channel activity but also for the structural
maintenance of photoreceptors. We asked which
domain of RDGA protein molecule is important for the
intracellular targeting and/or its function. By theexpression studies of modified RDGA proteins inphotoreceptors, we found that the cysteine-rich regionof RDGA molecule is essential for the protein
localization and enzyme antivities. The western
blotting analysis showed that the RDGA protein
lacking the cysteine-rich region is unstable. Thissuggests that the proper targeting of RDGA protein is
important for the protein stability.
Besides PI metabolism, rhodopsin metabolism isimportant for the regulation of phototransduction. This
year we studied Sunglass (SUN) protein that appears
to be involved in rhodopsin degradation. This project
was done in collaboration with Dr. Craig Montell'sGroup atJohns Hopkins University School of Medicine.
We found that SUN protein co-localizes with rhodopsin
in the photoreceptor cell organelles involved in protein
degradation, and found that sun mutant is resistantto the rhodopsin degradation induced by bright lighe l.
(2) Fine morphological studies of the synaptic target
recognition(3) Fine morphological analysis of the function of a Ser/Thr kinase, UNC51 in Drosophila nervous system

Hirofumi Toda*, Hiroaki Mochizuki*, Emiko Suzuki
and Katsuo Furukubo-Tokunaga* (*University ofTsukuba)
UNC51 has been known as a Ser/Thr kinase
necessary for the axonal formation in C. elegans andmice. We have identified Drosophila gene orthologue
of unc51 in an attempt to study the detailed molecularfunction of UNC51 in axonal formation by use of
Drosophila molecular genetics. We localized the UNC51
protein to the vesicular structures in axons andsynaptic terminals by immuno-electron microscopy.
Further, we found that the unc51 gene mutation affects
axonal transport of vesicular cell organelles, by the
immuofluorescence microscopy of synaptotagmin andelectron microscopic analysis. Studies on genetic
interactions showed that UNC51 regulates axonal
elongation cooperatively with TRIO and Rae GTPases.
These results suggest that UNC51 is involved in axonal
development through interaction with the proteins that
regulate axonal membrane trafficking.
PUBLICATIONSPapersEmiko Suzuki
The neuromuscular junction of Drosophila
embryonic body wall musculature is one of the ideal
models for studying development and function ofsynapses at the single cell level. Each hemisegment ofan embryo/larva has only 30 muscle cells, innervatedby about 40 motoneurons. The neuromuscular
projection forms in the late embryos. During thesestages, one can fillet dissect the embryos and observeor manipulate the development of neuromuscular
networks under a light microscope. Our previous
studies on this system using combination of single celllabeling and electron microscopy, have revealed thetarget-specific interaction of pre- and post-synaptic
cells with filopodia. This year we studied this processby immuno-scanning electron microscopy we havedeveloped recently. With this technique, we could
observe the intimate interaction of clustered muscle
filopodia (myopodia) and neuro-filopodia, that is notrecognizable by fluorescence microscopy.

1. Xu, H., Lee, S.-]., Suzuki, E., Dugan, K.D., Stoddard,A., Li, H.-S., Chodosh, L.A. and Montell, C. (2004). A
lysosomal tetraspanin associated with retinaldegeneration identified via a genome-wide screen.
EMBO ]. 23, 811-822.
2. Kohyama-Koganeya, A., Sasamura, T., Oshima,E., Suzuki, E., Nishihara, S., Ueda, R. and Hirabayashi,y. (2004). Drosophila glucosylceramide synthase. ]. BioI.

Chern. 279, 35995-36002.
Review

3.

~*;;tlj.~ (2004)~nX:tj#li:J*HIJffll,'~i¥J~~m&7t~n~ G~tc 'Y-r7°;A36, 66-69.

Gene Network Laboratory105

(2) Molecular hierarchy in neurons differentiated frommouse ES cells containing a single human chromosome

I. CENTER FOR INFORMATION BIOLOGYAND DNA DATA BANK OF JAPANI-a. Laboratory for DNA Data Analysis
Takashi Gojobori GroupRESEARCH ACTIVITIES(1) Comparative analysis of gene expression forconvergent evolution of camera eye between octopus andhuman
Atsushi Ogura, Kazuho Ikeo and Takashi Gojobori
Although the camera eye of the octopus is very
similar to that of humans, phylogenetic andembryological analyses have suggested that theircamera eyes have been acquired independently. It has
been known as a typical example of convergent
evolution. To study the molecular basis of convergent
evolution of camera eyes, we conducted a comparative
analysis of gene expression in octopus and humancamera eyes. We sequenced 16,432 ESTs of the octopus
eye, leading to 1052 nonredundant genes that havematches in the protein database. Comparing these 1052
genes with 13,303 already-known ESTs of the humaneye, 729 (69.3%) genes were commonly expressed
between the human and octopus eyes. On the contrary,when we compared octopus eye ESTs with humanconnective tissue ESTs, the expression similarity wasquite low. To trace the evolutionary changes that arepotentially responsible for camera eye formation, we
also compared octopus-eye ESTs with the completed
genome sequences of other organisms. We found that1019 out of the 1052 genes had already existed at thecommon ancestor of bilateria, and 875 genes wereconserved between humans and octopuses. It suggeststhat a larger number of conserved genes and theirsimilar gene expression may be responsible for theconvergent evolution of the camera eye.

21
Chi Chiu Wang, Mitsutaka Kadota, Ryuichi Nishigaki,
Yasuhiro Kazuki, Yasuaki Shirayoshi, Michael Scott
Rogers, Takashi Gojobori, Kazuho Ikeo and Mitsuo
Oshimura
Defects in neurogenesis and neuronal differentiation in the fetal brain of Down syndrome (DS) patients
lead to the apparent neuropathological abnormalities
and contribute to the phenotypic characters of mental
retardation, and premature development of Alzheimer's
disease, those being the most common phenotype in
DS. In order to understand the molecular mechanism
underlying the cause of phenotypic abnormalities inthe DS brain, we have utilized an in vitro model of
TT2F mouse embryonic stem cells containing a single
human chromosome 21 (hChr21) to study neuron
development and neuronal differentiation by
microarray containing 15K developmentally expressed
cDNAs. Defective neuronal differentiation in thepresence of extra hChr21 manifested primarily the
post-transcriptional and translational modification,such as MrpllO, SNAPC3, Srprb, and SF3a60 in theearly neuronal stem cell stage, and Mrpsl8a, Eeflg, andUbce8 in the late differentiated stage. Hierarchicalclustering patterned specific expression of hChr21 genedosage effects on neuron outgrowth, migration, anddifferentiation, such as Syngr2, Dncic2, Eif3sf, and
Peg3.
(3) Strong positive selection and recombination drive
the antigenic variation of the PilE protein of the humanpathogen Neisseria meningitidis
Daniel T. Andrews and Takashi Gojobori
The PilE protein is the major component of the
Neisseria meningitidis pilus, which is encoded by thepilE/pilS locus that includes an expressed gene andeight homologous silent fragments. The silent genefragments have been shown to recombine through geneconversion with the expressed gene and thereby
provide a means by which novel antigenic variants of
the PilE protein can be generated. We have analyzed
the evolutionary rate of the pilE gene using thenucleotide sequences of two complete pilE/pilS loci.

106 CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPANThe very high rate of evolution displayed by the PilE
protein appears driven by both recombination andpositive selection. Within the semivariable region of
the pilE and pilS genes, recombination appears tooccur within multiple small sequence blocks that lie
between conserved sequence elements. Within thehypervariable region, positive selection was identified
from comparison of the silent and expressed genes.
The unusual gene conversion mechanism that operates
at the pilE/pilS locus is a strategy employed by N
meningitidis to enhance mutation of certain regions of
the PilE protein. The silent copies of the geneeffectively allow "parallelized" evolution of pilE, thus
enabling the encoded protein to rapidly explore a large
area of sequence space in an effort to find novel
antigenic variants.
(4) Difference of organelles involved in membrane traffic

Hajime Ohyanagi and Takashi Gojobori
Contrary to prokaryotes, eukaryotes have anumber of membranous organelles involved inmembrane traffic in cells. Therefore, the gain of
membrane systems could be one of the most epochal
events in the evolution of eukaryotes. However, details
about the evolution of a membrane system are still
unclear. Each protein involved in a membrane system
must have been evolved with its own characteristics,
so that proteins localized in the same position in cells
may show the same characteristics in the context ofmolecular evolution. With the aim of giving insight tothe evolutionary studies of membrane systems, we areconducting the following analyses. First, we obtained
subcellular localization data of yeast proteins that havealready published (http://yeastgfp.ucsf.edu/). Second,in order to classify these yeast proteins into groups
according to their evolutionary emergence times, we
performed the BLAST reciprocal best hit analysis ofyeast proteome against the proteomes of other species.
From these analyses, we observed that protein sets inendoplasmic reticulum and Golgi apparatus, major
components of the membrane traffic system, clearly
showed evolutionary emergence times different fromeach other. Thus, it suggests that endoplasmic
reticulum and Golgi apparatus appeared independently
in the evolution of eukaryotes.

(5) Highly conserved upstream sequences fortranscription factor genes and implications for the
regulatory network

Hisakazu Iwama and Takashi Gojobori
Identifying evolutionarily conserved blocks inorthologous genomic sequences is an effective way todetect regulatory elements. In this study, with the aimof elucidating the architecture of the regulatory
network, we systematically estimated the degree ofconservation of the upstream sequences of 3,750
human-mouse orthologue pairs along 8-kb stretches.
We found that the genes with high upstream
conservation are predominantly transcription factor(TF) genes. In particular, developmental processrelated TF genes showed significantly higher
conservation of the upstream sequences than other TF
genes. Such extreme upstream conservation of thedevelopmental process-related TF genes suggests thatthe regulatory networks involved with developmental
processes have been evolutionarily well conserved inboth human and mouse lineages.
(6) Computational prediction of microRNA (miRNA)target gene in human and its experimental varidation

Hitomi Sakurai, Roberto Barrero, Shiho Hayakawa,
Takuro Tamura, Yoshio Tateno, Ikeo Kazuho,
Imanishi Tadashi and Takashi Gojobori
MicroRNAs (miRNAs) form a novel class of small
RNA genes of 21-25 nucleotides derived from highly
conserved hairpin precursors (pre-miRNAs) present
from mollusc to mammals. miRNAs act as
post-transcriptional repressors of target transcripts via
mRNA degradation or translation inhibition. We
present a novel large scale RNA-affinity screening tool
combined with the evaluation of RNA secondary
structures to identify putative microRNA recognition
elements (MREs) on target mRNAs. We identified 1,476
MREs for 115 known mammalian miRNAs. Eleven of
the top predicted MREs for Let-7a, miR-20, miR-97andmiR-182a were analyzed using a reporter assay. As aresult, we found that seven MREs mediated inhibitionof reporter expression. It suggests that our target
prediction algorithm, RNAFFY, identifies highly
reliable miRNA targets.

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107

(7) Detection of apoptosis during planarian regenerationby the expression of apoptosis-related genes and TUNEL
assay

Jung Shan Hwang, Chiyoko Kobayashi, Kiyokazu
Agata, Kazuho Ikeo and Takashi Gojobori
Apoptosis is a tightly organized cell death processthat plays a crucial role in metazoan development, but
it has not yet been revealed whether apoptotic events
are involved in the process of regeneration. Here, we
tried to detect apoptotic cells during planarianregeneration using the TdT-mediated dUTP nick-end
labeling (TUNEL) assay as well as the expression of
apoptosis-related genes. Three novel cDNAs wereisolated from a planarian eDNA library and shown to
be closely related to other metazoan caspases at theamino acid sequence level. One of these cDNAs,
Caspase-like gene 3 (DjClg3), was expressed primarily
in apoptotic cells by double detections with the TUNEL
assay. Whole mount in situ studies indicated that
DjClg3 was expressed in the cells of the mesenchymal
space and also around the pharynx of the intact body.
Its expression in the regenerating head piece was seenin the blastema and less significantly in the brain,
while in the regenerating tail piece, DjClg3 expression
was detected uniformly throughout the entire region.
In parallel experiments, we performed in situ TUNEL
assays to localize the regions where cell death occurred
during regeneration and comparable results to the
DjClg3 expression patterns were obtained. This is thefirst report to show that planarians have apoptosisrelated genes, and the results suggest that the apoptotic
mechanism probably takes place to a large extent innormal intact worms as well as during theirregeneration. We hypothesize that the presence of
apoptosis in planarians may have a role in controllingcell numbers, eliminating unnecessary tissues or cells
and remodeling the old tissues of regenerating bodyparts.
(8) Analysis of sexual dimorphism of gene expression
in mouse brain

Kazuya Yuge, Kazuho Ikeo and Takashi Gojobori
There are sexual differences in morphologicalfeatures of the mammalian brain. The so-called sexual
dimorphism of mammalian brains is thought to be

108 Laboratory for DNA Data Analysis

determined by the gonadal hormones; this view iscalled "gonadal origin theory". However, recent studies
suggest that differences in gene expression of sex-liked
genes in neuronal cells directly generate sexual
dimorphism in the brain. To examine this possibility
we conducted microarray experiments to screen thegenes that were expressed differentially in the brain
between male and female mice before gonadal
hormone secretion. In this study we have identified 57
female enhanced genes and 24 male enhanced genes
in brains at embryonic day 10.5 (E 10.5). These results
indicate that sexual differences in gene expression inneuronal cells before gonadal hormone secretion would
play an important role in sexual dimorphism in thebrain.
(9) Seeking for signes of aging in Hydra, a primitive
metazoan

Kengo Yoshida, Hwang Jung Shang, Chimi Fujisawa,
Toshitaka Fujisawa, Kazuho Ikeo and Takashi
Gojobori
Aging occurs in a variety of organisms including
yeast, nematode, fly, and mammals. However, hydra
belonging to phylum Cnidaria has been considered to
be immortal (Martinez 1998). This apparent immortality was observed when hydra were maintained so thatthey underwent asexual reproduction, budding. Do theyremain immortal if they undergo sexual reproduction?
A previous study using Hydra Oligactis showed thatthe depression occurred after sexual reproduction(Littlefield 1985). To seek for possible signs of aging
in hydra, we studied the process of the depression indetail. As a result, reduction in the capacity of foodcapture, digestive movements and contractile
movements was all observed. Moreover, exponential
increase in mortality rate of population was also
observed. From these observations, we conclude thataging-like phenomenon occurs in hydra. Aging
research on hydra, one of the most primitive
metazoans, has potential for providing further insight
into the understanding of evolution of aging.
(10) A large variation in the rates of synonymous
substitution for RNA viruses and its relationship to adiversity of viral infection and transmission modes

Kousuke Hanada, Yoshiyuki Suzuki and Takashi

GojoboriRNA viruses successfully adapt to various
environments by repeatedly producing new mutants,
often through generating a number of nucleotide
substitutions. To estimate the degree of variation in
mutation rates of RNA viruses and to understand thesource of such variation, we studied the synonymous
substitution rate because synonymous substitution is
exempt from functional constraints at the protein level,
and its rate reflects the mutation rate to a great extent.
We estimated the synonymous substitution rates fora total of 49 different species of RNA viruses, and we
found that the rates had tremendous variation by 5orders of magnitude (from 1.3 x 10-7 to 6.2 X 10-2 /synonymous site/year). Comparing the synonymous
substitution rates with the replication frequencies andreplication error rates for the RNA viruses, we foundthat the main source of the rate variation was
differences in the replication frequency because therates of replication error were roughly constant overdifferent RNA viruses. Moreover, we examined a
relationship between viral life strategies andsynonymous substitution rates to understand which
viral life strategies affect replication frequencies. The
results show that the variation of synonymous
substitution rates has been influenced most by either
the difference in the infection modes or the differencesin the transmission modes. In conclusion, the variationof mutation rates for RNA viruses is caused bydifferent replication frequencies, which are affected
strongly by the infection and transmission modes.
(11) Evolutionary processes of gene splicing and genesilencing

LihuaJin, Yoshiyuki Suzuki, Kazuho Ikeo and Takashi
Gojobori
Both gene splicing and gene silencing have become
known to contribute significantly to functional
diversification of genes and gene networks. For gene
splicing, we conducted an evolutionary study of therelationship between gene duplication and alternative
splicing. For gene silencing, we studied theevolutionary process of small RNA-guided pathways,
focusing on the Rnase III family enzymes. For the first
topic, we mentioned that gene duplication andalternative splicing (AS) were the two major

evolutionary mechanisms that could bring thefunctional variation through the diversification of genes
and gene products. The purpose of this research is tounderstand the evolutionary relationship between thetwo different mechanisms, utilizing the available data
resources. The results of this study showed that the
proportion of the AS loci in the singleton gene group
was less than that in the duplicated gene group.
Moreover, we found that the duplicated genes tended
to have more AS isoforms than singleton genes. These
results suggest that gene duplication would induce
more alternative splicing events on duplicated copies
than on singletons possibly by reducing the functional
constraints on the duplicates. For the second topic, wepointed out that the RNA-mediated gene silencing
pathways were evolutionarily conserved processes. It
highlights a fundamental role of short RNAs in
eukatyotic gene regulation and antiviral defense.
Recently, three distinct small RNA-directed silencing
pathways were observed, such as the destruction ofmRNA via siRNA, inhibition of mRNA translation via
miRNA, and epigenetic gene silencing via siRNA. It
was also found that in these pathways, the membersof ribonclease III family played important roles indiverse RNA maturation and decay. Here we
investigated the evolution of Rnase ill nucleases, Dicer
as representative, to further figure out the evolutionary
relationship among the three gene silencing pathways.
With the advantage of using genomic sequences as the
subjects of homology search in the annotated andun-annotated genomic regions, we were able to detect
possible candidates for a gene of dicer and its twofunctional domains. Moreover, we found that
representative prokaryotes including eubacteria andarchbacteria lacked completely the PAZ domain ofDicer. These results clearly show the taxonomydependent evolution of the RNA-mediated genesilencing pathways. The results obtained in this study
provide the information for the understanding of theevolutionary origin and relationships of the threepathways. The information may also be useful for the
conducting of the relevant experiments. As a summary,these results suggested that changes in the genome andregulatory network were closely related evolutionary
events, implying that gene multiplication andfunctional constraints were the two important sources
for the change in gene function in the networks oftranscription and gene regulation in evolution.

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109

(12) The bioinformatics challenges in comparative
analysis of cereal genomes-an overview

Matthew Bellgard, Jia Ye, Takashi Gojobori and Rudi
AppelsComparative genomic analysis is the cornerstoneof in silica-based approaches to understanding
biological systems and processes across cereal species,
such as rice, wheat and barley, in order to identify
genes of agronomic interest. The size of the genomic
repositories is nearly doubling every year, and this has
significant implications on the way bioinformatics
analyses are carried out. The concepts and technology
underpinning bioinformatics as applied to comparative
genomic analysis are discusse as the overview.
(13) Various adaptations for the perpetual darkness inthe diversification process from an eyed surface-dwelling
form to an eyeless cave-dwelling form of Mexican tetra,
Astganax mexican us, by eDNA rnicroarrays

Nobuhiko Tanaka, Shozo Yokoyama, Kazuho Ikeo andTakashi Gojobori
Mexican tetra (Astyanax mexicanus), a single
species with eyed surface and eyeless cave dwelling
forms, gives a unique opportunity of the evolutionary
process of gene diversification during a short periodof time. In this study, we tried to find genes related tothe evolution of eyeless cave form from the eyedsurface form by cDNA microarrays, each of which has
3,070 non-redundant clones from the entire body of aneyed adult fish. Target genes for the microarrays weremade from the entire body of each an eyed fish andan eyeless fish. On the basis of the results ofcomparative microarray analysis for 716 functionallyknown genes, relatively highly expressed genes in thesurface fish and in the cavefish were listed up,
respectively. In the surface fish, we for at that there
were relatively highly expression of eye formationrelated genes, digestive system-related genes, energy
metabolism-related genes, lipocalin-type prostaglandin
D synthase gene, elastase genes. The eye formationrelated genes may be highly expressed because surface
fish have well-developed eyes whereas cavefish havedegenerated eyes. The digestive system-related andenergy metabolism-related genes may be also highly
expressed because the surface fish are more active than

110 Laboratory for DNA Data Analysis

the cavefish. The lipocalin-type prostaglandin D
synthase protein is related to the regulation of
circadian sleep-wake cycles. The highly expression of
the protein gene corresponds to the regressive
circadian activity in the cavefish. The elastase digests
elastin fibers in the skin, when the ultraviolet (UV) lightreaches the dermis in the skin. It is suggested that theelastase genes is highly expressed because the surface
fish has been exposed to sufficiently sunny conditions
compared with the cavefish. On the other hand, in the
cavefish, relatively highly expression of transferrin
genes, lipid metabolism-related genes, sexdifferentiation-related genes were observed.
Transferrin proteins are related to the oxygen
transportation in the blood. To effectively transportoxygen in the blood of the cavefish living in the waterwith the lower concentration of oxygen at the dark caveenvironments, transferrin genes may be required tohave relatively highly expression in the cavefish. The
highly expression of lipid metabolism-related genes
would be due to efficient acquirement of the energy
under circumstances of at least temporary shortness
in food. The highly expressions of the sex
differentiation-related genes (diazepam binding
inhibitor gene and granulin 1 gene) in the cavefish
would be related to negative sex differentiation becauseof the lack of any kind of display/courtship in the
cavefish. In conclusion, various adaptations for theperpetual darkness were evolutionarily raised in thediversification process from the surface fish to the
cavefish. This is the first report that differences of gene
expression between the surface fish and the cavefish
are extensively examined.
(14) Evolutionary analysis of transcriptional coactivator

MBFl
Qing-Xin Liu, Kazuho Ikeo, Susumu Hirose andTakashi Gojobori
Transcriptional coactivators playa crucial role ingene expression by communicating between regulatory
factors and the basal transcription machinery. Howa coactivator evolves was poorly understood. We havetaken a phylogenetic approach to analyze an
evolutionarily history of coactivator MBF1 and TATA
element-binding protein TBP. We found the following
points. (1) MBF1 and TBP are evolutionarily conserved
from Archaea to human to achieve transcription

initiation. (2) Archaeal MBFl carries a DNA-binding
domain consisting of a Zn-ribbon which is absent fromits eukaryotic counterparts. Thus, Eukaryotic MBFl
can interact with various regulatory factors whilearchaeal MBFl has a single partner. (3) The interaction
between MBFl and TBP is also conserved from
Archaea to human. In Archaea, MBFl binds to TBP
through Lysine or Asparagine to Glutamic acidinteraction. In eukaryotes, MBFl binds to TBP throughAsparatic acid or Glutamic acid to Glutamineinteraction. (4) The phylogenetic tree of MBFl is very
similar to that of TBP, indicating that MBFl coevolved
with TBP.
(15) Pigment cell lineage-specific expression activity of
the ascidian tyrosinase-related gene
Reiko Toyoda, Akiko Kasai, Shigeru Sato, Shuichi
Wada, Hidetoshi Saiga, Kazuho Ikeo, Takashi
Gojobori, Takaharu Numakunai and Hiroaki
Yamamoto
Solitary ascidian tadpole larvae develop two typesof black pigment cells in the major sensory organs of
the brain. Such pigment cells have been demonstrated
to express the melanogenic genes, tyrosinase andTyrp/TRP (tyrosinase-related protein). To understandthe genetic and developmental mechanisms underlying
the differentiation of chordate pigment cells, we
examined the function of the promoter region of Tyrp/
TRP gene, an ascidian (Halocynthia roretzi) tyrosinase
family gene. The expression of the gene in pigment celllineage starts at the early-mid gastrula stages. To
identify the transcriptional regulatory region of thegene allowing cell-type-specific expression, a deletion
series of the HrTyrp 5' flanking region fused to a lacZ
reporter gene was constructed and microinjected into
ascidian fertilized eggs. The region of 73 bp in HrTyrp
was identified as sufficient for expression in pigment
cell-precursors of tailbud stage embryos. It isnoteworthy that there is no M-box element highly
conserved in the promoters for vertebrate tyrosinase
family genes such as tyrosinase, Tyrpl/TRP-l andTyrp2/TRP-2 (Dct). Although the regulatory systemof ascidian pigment-cell development is likely tocontain most factors critical to vertebrate pigment-celldevelopment, there might be critical differences in themode of regulation, such as the developmental timingof interactions of factors, proteins and genes, involvedin pigment cell differentiation and pigmentation.
(16) DDBJ in the stream of various biological data
Satoru Miyazaki, Hideaki Sugawara, Kazuho Ikeo,
Takashi Gojobori and Yoshio Tateno
In the past year we at DDB] (http://www.ddbj.nig.
ac.jp) have made a steady increase in the number ofdata submissions with a 50.6% increment in thenumber of bases or 46.5% increment in the number of
entries. Among them the genome data of man, ascidian
and rice hold the top three. Our activity has extended
to providing a tool that enables sequence retrieval
using regular expressions, and to launching our SOAP
server and web services to facilitate the acquisition ofproper data and tools from a huge number of biological
data resources on websites worldwide. We have also
opened our public gene expression database, CIBEX.
(17) Note on the maximum likelihood estimation of
haplotype frequencies
Shuhei Mano, Norikazu Yasuda, Toru Katoh, Kenichi
Tounai, Hidetoshi Inoko, Tadashi Imanishi, Gen
Tamiya and Takashi Gojobori
The maximum likelihood estimation (MLE) is oneof the most popular ways to estimate haplotype
frequencies of a population with genotype data whose
linkage phases are unknown. The MLE is commonly
implemented in the use of the ExpectationMaximization (EM) algorithm. It is known that the EM
algorithm carries the risk that an estimator may
converge erroneously to one of the local maxima or
saddle points of the likelihood surface, resulting inserious errors in the MLE of haplotype frequencies. In
this note, by theoretical treatments we present thenecessary and sufficient conditions that the local
maxima or saddle points on the likelihood surface
appear. As a rule of thumb, that the difference between
the coupling and repulsive haplotype frequencies inphase known individuals is 3/2 times larger than thefrequency of phase ambiguous individuals is thesufficient condition that the likelihood surface isunimodal. Moreover, we present the analytic solution
to the biallelic two-locus problem, and construct a
general algorithm to obtain the global maximum.

Laboratory for DNA Data Analysis

111

(18) The evolutionary rate of a protein influenced byfeatures of the interacting partners

Takashi Makino and Takashi Gojobori
We focused upon how the evolutionary rates ofproteins were influenced by the characteristic featuresof PPIs. Because the rt;cent advancement of moleculartechnologies enables us to understand actual featuresof protein to protein interactions (PPIs), it becomes
possible to make objective descriptions about thecharacteristic features of the proteins in the PPI
networks. In this analysis, we defined a protein having
a larger number of PPI partners of the same functionalclass as the SF (Same Function) protein, and a protein
having a larger number of PPI partners of different
functional classes as the DF (Different Function)
protein. We also classified proteins in the PPI networks
into respective proteins in dense and sparse parts of
the PPI network, denoting these proteins as the DP(Dense part) and SP (Sparse Part) proteins, respectively.
Because these two classifications were independent ofeach other, we classified the proteins in PPIs further
into the four categories, the SF-DP, SF-SP, DF-DP and
DF-SP proteins. Then, we compared the evolutionary
rates between the SF and DF proteins, between the DP
and SP proteins, and among the four categories. As
a result, we found that the DF proteins evolved at a
slower rate than the SF proteins. We also found that
the SP proteins evolved at a slower rate than the DP
proteins. In particular, we pointed out that the DF-SP
proteins evolved at the slowest rate in the proteinsexamined. Because all these differences in theevolutionary rates are statistically significant, it issuggested that the proteins with their PPI partners
belonging to different functional classes and occupyinga sparse part of the PPI network are under strong
functional constraints. It follows that those proteinsare very important for the maintenance and survivalof the PPI network.
(19) Integrative annotation of 21,037 human genes
validated by full-length cDNA clones

Tadashi Imanishi, (154 authors), Takashi Gojobori and
Sumio Sugano
The human genome sequence defines our inherent
biological potential; the realization of the biology

112

Laboratory for DNA Data Analysis

encoded therein requires knowledge of the function ofeach gene. Currently, our knowledge in this area is still
limited. Several lines of investigation have been used
to elucidate the structure and function of the genes in
the human genome. Even so, gene prediction remains
a difficult task, as the varieties of transcripts of a genemay vary to a great extent. We thus performed an
exhaustive integrative characterization of 41,118
full-length cDNAs that capture the gene transcripts ascomplete functional cassettes, providing an unequivocal report of structural and functional diversity at thegene level. Our international collaboration has
validated 21,037 human gene candidates by analysis
of high-quality full-length cDNA clones throughcuration using unified criteria. This led to theidentification of 5,155 new gene candidates. It also
manifested the most reliable way to control the qualityof the cDNA clones. We have developed a human genedatabase, called the H-Invitational Database (H-InvDB;
http://www.h-invitational.jp/). It provides the following:
integrative annotation of human genes, description ofgene structures, details of novel alternative splicing
isoforms, non-protein-coding RNAs, functional
domains, subcellular localizations, metabolic pathways,
predictions of protein three-dimensional structure,
mapping of known single nucleotide polymorphisms(SNPs), identification of polymorphic microsatellite
repeats within human genes, and comparative resultswith mouse fUll-length cDNAs. The H-InvDB analysis
has shown that up to 4% of the human genome
sequence (National Center for Biotechnology
Information build 34 assembly) may containmisassembled or missing regions. We found that 6.5%of the human gene candidates (1,377 loci) did not havea good protein-coding open reading frame, of which
296 loci are strong candidates for non-protein-coding
RNA genes. In addition, among 72,027 uniquely
mapped SNPs and insertions/deletions localized withinhuman genes, 13,215 nonsynonymous SNPs, 315
nonsense SNPs, and 452 indels occurred in coding
regions. Together with 25 polymorphic microsatellite
repeats present in coding regions, they may alter
protein structure, causing phenotypic effects or
resulting in disease. The H-InvDB platform represents
a substantial contribution to resources needed for the
exploration of human biology and pathology.

(20) Japanese domesticated chickens derived from Sharno
traditional fighting cocks

Tomoyoshi Komiyama, Kazuho Ikeo, Yoshio Tateno
and Takashi Gojobori
With the aim of elucidating the evolutionary origin
of Japanese domesticated chickens, we examined 85
chicken mtDNA sequences. Thirty-four various
ornamental chickens, 42 fighting cocks (Shamo), andnine long-crowing chickens (Naganakidorz) were
included in these samples. Of the Shamo, 18 were
sampled from Okinawa, while the remaining 24 werecollected in other islands around Japan. In addition,
three Southeast Asian Junglefowls were used as areference to determine the common ancestor of from
Okinawa that clearly diverged from the other Japanese
domesticated chickens studied. We found that all
Japanese domesticated chickens, including theornamental varieties and Naganakidori, were derived
from the ancestors of the Shamoin Okinawa. To create
novel varieties of ornamental chickens, intensive
artificial selection is imposed on ancestral Shamo
population, resulting in profoundly differentiation ofJapanese domesticated chickens.
(21) The evolutionary origin of long-crowing chicken: its
evolutionary relationship with fighting cocks disclosedby the mtDNA sequence analysis

Tomoyoshi Komiyama, Kazuho Ikeo, Yoshio Tateno
and Takashi Gojobori
Chickens with exceptionally long crow are often
favored all over the world, and connoisseur breeders
have bred certain types of chicken exclusively for thistrait. In Japan, three chicken varieties have been
specifically bred to develop an exceptionally long crowof over 15 seconds. Although these three long-crowing
chickens, Naganakidori, are honored as heritage
varieties of Japan, the domestication process andgenealogical origin of long-crowing chickens remain
unclear. The purpose of this study is to clarify theseissues using nucleotide sequences of the mitochondrial
DNA D-Ioop region. Blood samples from a total of nine
long-crowing chickens and 74 chickens from 11
Japanese native varieties were collected. DNA sequence
data of two Junglefowl species were also collected fromthe International DNA database (DDBJ /EMBLIGenBank) for use as the outgroup. A phylogenetic tree
was then constructed revealing that all three
Naganakidori varieties were monophyletic andoriginated from a fighting cock, a Shamo, for
cockfighting. These results suggest that these three
long-crowing chickens share a common origin in spiteof their conspicuously different characters, and thathuman cultures favoring long-crowing chickens might
have been preceded by a tradition of cockfighting.
Moreover, these long-crowing varieties first separated
from the fighting cocks of Okinawa, which isgeographically closer to Southern China and Indochina
than Mainland Japan (HonshulKyushu). This implies
that Japanese long-crowing chickens were first brought
to Mainland Japan as fighting cocks from thesurrounding regions of Southern China or Indochina
and through Okinawa.
(22) Novel algorithm for automated genotyping of
microsatellites

Toshiko Matsumoto, Wataru Yukawa, Yasuyuki
Nozaki, Ryo Nakashige, Minori Shinya, Satoshi
Makino, Masaru Yagura, Tomoki Ikuta, Tadashi
Imanishi, Hidetoshi Inoko, Gen Tamiya and Takashi
Gojobori
Microsatellites or short tandem repeats (STRs) areabundant in the human genome with easily assayedpolymorphisms, providing powerful genetic tools formapping both Mendelian and complex traits.
Microsatellite genotyping requires detection of the
products of polymerase chain reaction (PCR)
amplification by electrophoresis, and analysis of thepeak data for discrimination of the true allele. A
high-throughput genotyping approach requires
computer-based automation at both the detection andanalysis phases. In order to achieve this, complicatedpeak patterns from individual alleles must be
interpreted in order to assign alleles. Previous methods
consider limited types of noise peaks and cannot
provide enough accuracy. By pattern recognition ofvarious types of noise peaks, such as stutter peaks andadditional peaks, we have achieved an overall average
accuracy of 94% for allele calling in our actual data. Ouralgorithm is crucial for a high-throughput genotyping
system for microsatellite markers by reducing manual
editing and human errors.

Laboratory for DNA Data Analysis

113

(23) Evolution of vitamin b6 (pyridoxine) metabolism by
gain and loss of genes
Tsuyoshi Tanaka, Yoshio Tateno and Takashi
Gojobori
Vitamin B(6) (VB6) functions as a cofactor of many
diverse enzymes in amino acid metabolism. Threemetabolic pathways for pyridoxaI5'-phosphate (PLP;
the active form of VB6) are known: the de novo
pathway, the salvage pathway, and the fungal type
pathway. Most unicellular organisms and plants
biosynthesize VB6 using one or two of these threebiosynthetic pathways. However, animals such as
insects and mammals do not possess any of thepathways and, thus, need to intake VB6 in their dietto survive. It is conceivable that breakdowns of thesepathways occurred in the evolutionary lineages of
insects and mammals, and one of the major reasonsfor this would be the loss of pertinent genes. We
studied the evolution of VB6 biosynthesis from theview of the gain and loss of 10 pertinent genes in 122
species whose genome sequences were completely
determined. The results revealed that each gene in thepathways was lost more than once in the entire
evolutionary lineages of the 122 species. We also found
the following three points regarding the evolution of
PLP biosynthesis: (1) the breakdown of the PLP
biosynthetic pathways occurred independently at least
three times in animal lineages, (2) the de novo pathway
was formed by the generation of pdxB in gammaproteobacteria, and (3) the order of the gene loss in VB6
metabolism was conserved among differentevolutionary lineages. These results suggest that the evolutionof VB6 metabolism was subject to gains and frequentlosses of related genes in the 122 species examined.
This dynamic nature of the evolutionary changes musthave been responsible for the breakdowns of thepathways, resulting in profound differentiation ofheterotrophy among the species.
(24) Biased biological functions of horizontallytransferred genes in prokaryotic genomes
Yoji Nakamura, Takeshi Itoh, Hideo Matsuda andTakashi Gojobori
Horizontal gene transfer is one of the main
mechanisms contributing to microbial genome

114 Laboratory for DNA Data Analysis

diversification. To clarify the overall picture ofinterspecific gene flow among prokaryotes, wedeveloped a new method for detecting horizontallytransferred genes and their possible donors by
Bayesian inference with training models for nucleotide
composition. Our method gives the average posterior
probability (horizontal transfer index) for each genesequence, with a low horizontal transfer index
indicating recent horizontal transfer. We found that
14% of open reading frames in 116 prokaryotic complete
genomes were subjected to recent horizontal transfer.
Based on this data set, we quantitatively determinedthat the biological functions of horizontally transferredgenes, except mobile element genes, are biased to three
categories: cell surface, DNA binding andpathogenicity-related
functions.
Thus,
the
transferability of genes seems to depend heavily on
their functions.
(25) False positive selection identified by ML-based
methods: examples from the SigI gene of the diatom
Thalassiosira weissflogii and the tax gene of a humanT-cell lymphotropic virus
Yoshiyuki Suzuki and Masatoshi Nei
Sexually induced gene 1 (Sigl) in the centric
diatom Thalassiosira weissflogii is considered to encode
a gamete recognition protein. Sorhannus (2003)
analyzed nucleotide sequences of Sigl using parsimony
analysis and the maximum-likelihood (ML)-based
Bayesian method for inferring positive selection at
single amino acid sites and reported that positively
selected sites were detected by the latter method but
not by the former. He then concluded that for this typeof study the ML-based method is more reliable thanparsimony analysis. Here we show that his results
apparently represent false-positive cases of theML-based method and that there is no solid evidence
that this gene contains positively selected sites. We
further demonstrate that in the tax gene of humanT-celllymphotropic virus type I (HTLV-I) all codonsites, including invariable sites, can be inferred as
positively selected sites by the ML-based method.
These observations indicate that the ML-based method
may produce many false-positive sites. One of the main
reasons for the occurrence of false positives is that in
the ML-based method codon sites are grouped into
several categories with different non synonymous/

synonymous rate ratios (w 's) on a purely statisticalbasis and positive selection is inferred indirectly byexamining whether the average w for each category
is greater than 1 or not. In parsimony analysis,
however, the evolutionary change of nucleotides at eachcodon site is examined. For this reason, parsimonybased methods rarely produce false positives and aresafer than ML-based methods for detecting positive
selection at individual codon sites, though a large
number of sequences are necessary.
(26) Negative selection on neutralization epitopes ofpoliovirus surface proteins: implications for predictionof candidate epitopes for immunization

Yoshiyuki Suzuki
For development of effective vaccines against
viruses, it is of importance to choose appropriate
epitopes as the target for immunization. These epitopes
should eventually be determined experimentally, but
it would be helpful if we could predict candidate
epitopes computationally because it accelerates theentire process. To predict candidate epitopes for
immunization, it is of great interest to characterize thetarget epitopes of poliovirus vaccine, which has
empirically proven to be the most effective among all
vaccines available. Here I show that almost all aminoacid sites of poliovirus surface proteins VP1, VP2, and
VP3 including neutralization epitopes are negatively
selected and no site is under positive selection. These
results, together with those obtained in previous
studies, indicate that vaccines directed against epitopes
which consist of negatively selected sites protect
vaccinees more effectively than those directed against
epitopes which contain positively selected sites. Theseobservations suggest that candidate epitopes for
immunization are predicted by the molecular
evolutionary analysis of viral protein (and its coding
nucleotide) sequences, as the epitopes which consist
exclusively of negatively selected amino acid sites.
(27) New Methods for Detecting Positive Selection atSingle Amino Acid Sites

Yoshiyuki Suzuki
Inferring positive selection at single amino acidsites is of particular importance for studying

evolutionary mechanisms of a protein. For thispurpose, Suzuki and Gojobori (1999) developed amethod (SG method) for comparing the rates ofsynonymous and nonsynonymous substitutions at
each codon site in a protein-coding nucleotide
sequence, using ancestral codons at interior nodes ofthe phylogenetic tree as inferred by the maximum
parsimony method. In the SG method, however,selective neutrality of nucleotide substitutions cannot
be tested at codon sites, where only termination codons
are inferred at any interior node or the number of
equally parsimonious inferences of ancestral codons
at all interior nodes exceeds 10,000. Here I present a
modified SG method which is free from these
problems. Specifically, I use the distance-based
Bayesian method for inferring the single most likely
ancestral codon from 61 sense codons at each interior
node. In the computer simulation and real data
analysis, the modified SG method showed a higher
overall efficiency of detecting positive selection thanthe original SG method particularly at highly
polymorphic codon sites. These results indicate thatthe modified SG method is useful for inferring positive
selection at codon sites where neutrality cannot be
tested by the original SG method. I also discuss thatthe p-distance is preferable to the number ofsynonymous substitutions for inferring thephylogenetic tree in the SG method, and present a
maximum likelihood method for detecting positive
selection at single amino acid sites, which produced
reasonable results in the real data analysis.
(28) Three-dimensional window analysis for detecting
positive selection at structural regions of proteins

Yoshiyuki Suzuki
Detection of natural selection operating at theamino acid sequence level is important in the studyof molecular evolution. Single site analysis andone-dimensional window analysis can be used to detectselection when the biological functions of amino acidsites are unknown. Single site analysis is useful whenselection operates more or less constantly overevolutionary time, but less so when it operates
temporarily. One-dimensional window analysis is moresensitive than single site analysis when the functionsof amino acid sites in close proximity in the linear
sequence are similar although this is not always theLaboratory for DNA Data Analysis

115

case. Here I present a three-dimensional window
analysis method for detecting selection given thethree-dimensional structure of the protein of interest.
In the three-dimensional structure, the window isdefined as the sphere centered on the a -carbon of anamino acid site. The window size is the radius of thesphere. The sites whose a-carbons are included in thewindow are grouped for the neutrality test. The
window is moved within the three-dimensional
structure by sequentially moving the central site along
the primary amino acid sequence. To detect positive
selection, it may also be useful to group thesurface-exposed sites in the window separately.
Three·dimensional window analysis appears to be not
only more sensitive than single site analysis andone-dimensional window analysis, but also provides
similar specificity for inferring positive selection in theanalyses of the hemagglutinin and neuraminidase
genes of human influenza A viruses. This method,
however, may fail to detect selection when it operates
only on a particular site, in which case single siteanalysis may be preferred although a large number ofsequences is required.
(29) Evolutionary process of amino Acid biosynthesis incorynebacterium at the whole genome level
Yousuke Nishio, Yoji Nakamura, Yoshihiro Usuda,
Shinichi Sugimoto, Kazuhiko Matsui, Yutaka
Kawarabayasi, Hisashi Kikuchi, Takashi Gojobori and
Kazuho Ikeo

Corynebacterium glutamicum, which is the closest
relative of Corynebacterium efficiens, is widely used forthe large scale production of many kinds of aminoacids, particularly glutamic acid and lysine, by
fermentation. Corynebacterium diphtheriae, which iswell known as a human pathogen, is also closely
related to these two species of Corynebacteria, but itlacks such productivity of amino acids. It is an
important and interesting question to ask how those
closely related bacterial species have undergone suchsignificant functional differentiation in amino acidbiosynthesis. The main purpose of the present study
is to clarify the evolutionary process of functional
differentiation among the three species of Corynebacteria by conducting a comparative analysis of genome
sequences. When Mycobacterium and Streptomyces
were used as out groups, our comparative study

116 Laboratory for DNA Data Analysis

suggested that the common ancestor of Corynebacteria
already possessed almost all of the gene sets necessary
for amino acid production. However, C. diphtheriaewas
found to have lost the genes responsible for amino acidproduction. Moreover, we found that the common
ancestor of C. efficiens and C. glutamicum haveacquired some of genes responsible for amino acidproduction by horizontal gene transfer. Thus, weconclude that the evolutionary events of gene loss andhorizontal gene transfer must have been responsible
for functional differentiation in amino acidbiosynthesis of the three species of Corynebacteria.
PUBLICATIONSPapers

1. Nakamura, Y, Itoh, T., Matsuda, H. and Gojobori,T. (2004). Biased biological functions of horizontallytransferred genes on 324,653 open reading frames of116 prokaryotic complete genomes. Nature Genetics
36,760-6.
2. Iwama, H. and Gojobori, T. (2004). Highly conserved
upstream sequences for transcription factor genes andimplications for the regulatory network. Proc. Natl.
Acad. Sci. USA 101, 17156·61.
3. Imanishi, T. and other 152 authors, *Gojobori, T.
and Sugano S. (2004). Integrative annotation of 21,037
human genes validated by full-length cDNA clones.
PLoS Biology 2, 1-21. *Correspondence Author
4. Ogura, A., Ikeo, K. and Gojobori, T. (2004).
Comparative analysis of gene expression forconvergent evolution of camera eye between octopus
and human. Genome Res. 14, 1555-61.
5. Andrews, T.D. and Gojobori, T. (2004). Strong
positive selection and recombination drive theantigenic variation of the PilE protein of the humanpathogen neisseria meningitidis. Genetics 166, 25-32.
6. Hanada, K., Suzuki, Y. and Gojobori, T. (2004). A
large variation in the rates of synonymous substitution
for RNA viruses and its relationship to a diversity ofviral infection and transmission modes. Mol Bioi Evol.
21, 1074-80.
7. lung Shan, H., Kobayashi, C., Agata, K., Ikeo, K.
and Gojobori, T. (2004). Detection of apoptosis during
planarian regeneration by the expression of
apoptosis-re1ated genes and TUNEL assay. GENE 333,
15-25.
8. Komiyama, T., Ikeo, K. and Gojobori, T. (2004). The
evolutionary origin of long-crowing chicken: itsevolutionary relationship with fighting cocks disclosedby the mtDNA sequence analysis. GENE 333,91-99.
9. Komiyama, T., Ikeo, K., Tateno, Y. and Gojobori,T. (2004). Japanese domesticated chickens have been
drived from Shamo, traditional fighting cocks. Mol.
Phylogenet. Evo. 33, 16-21.
10. Mano, S., Yasuda, N., Katoh, T., Tounai, K., Inoko,
H., Imanishi, T., Tamiya, G. and Gojobori, T. (2004).
Notes on the maximum likelihood estimation of
haplotype frequescies. Ann Hum Genet. 68,257-64.
11. Matsumoto, T., Yukawa, W., Nozaki, Y.,
Nakashige, R., Shinya, M., Makino, S., Yagura, M.,
Ikuta, T., Imanishi, T., Inoko, H., Tamiya, G. andGojobori, T. (2004). Novel algorithm for automatedgenotyping of microsatelites. Nucleic Acids Res. 32,
6069-77.
12. Tanaka, T., Tateno, Y and Gojobori, T. (2004).
Evolution of vitamin B6(Pyridoxine) metabolism by
gain and loss of genes. Mol. Bioi. Evol.
13. Alexopoulos, H., Bottger, A, Fischer, S., Levin, A,
Wolf, A, Fujisawa, T., Hayakawa, S., Gojobori, T.,
Davies, J., David, C. and Bacon,]' (2004). Evolution ofgap junctions: the missing link? CUrT. Bioi. 14 (20),R879-80.
14. Bellgard, M., Ye, ]., Gojobori, T. and Appels, R.
(2004). The bioinformatics challenges is comparative
analysis of cereal genomes-an overview. Funct. Integr.
Genomics 4, 1-11.
15. Kadota, M., Nishigaki, R., Wang, C.C., Toda, T.,
Shirayoshi, Y, Inoue, T., Gojobori, T., Ikeo, K., Rogers,
M.S. and Oshimura, M. (2004). Containing a single
human chromosome 21 in neuronal differentiation: anin vitro model of Down syndrome. Neuroscience 129(2),325-35.
16. Miyazaki, S., Sugawara, H., Ikeo, K., Gojobori, T.
and Tateno, Y (2004). DDBJ in the stream of various
biological data. Nucleic Acids Res 32, D31-4.
17. Nishio, Y, Nakamura, Y, Usuda, Y, Sugimoto, S.,
Matsui, K., Kawarabayashi, Y., Kikuchi, H., Gojobori,T. and Ikeo, K. (2004). Evolutionary process of theamino acids biosynthesis in Corynebacterium at thewhole genome level. Mol. Bioi. Evol. 21, 1683-91.
18. Suzuki, Y. (2004). Negative selection on
neutralization epitopes of poliovirus surface proteins:
implications for prediction of candidate epitopes for
immunization. Gene 328,127-133.
19. Suzuki, Y. (2004). New methods for detecting
positive selection at single amino acid sites. J. ofMol.
Evol. 59, 11-19.

20. Suzuki, Y. and Nei, M. (2004). False positive
selection identified by ML-based methods: examples
from the Sig1 gene of diatom Thalassiosira weissflogii
and the tax gene of a human T-celllymphotropic virus.
Molecular Biology and Evolution 21, 914-921.
21. Suzuki, Y. (2004). Three-dimensional window
analysis for detecting positive selection at structural
regions of proteins. Mol Bioi and Evol 21, 2352-2359.
22. Toyoda, R., Kasai, A, Sato, S., Wada, S., Saiga, H.,
Ikeo, K., Gojobori, T., Numakunai, T. and Yamamoto,H. (2004). Pigment cell lineage-specific expression
activity of the ascidian tyrosinase-related gene. GENE
332,61-69.
23. Wang, C.C., Kadota, M., Nishigaki, R., Kazuki, Y.,
Shirayoshi, Y, Rogers, M.S., Gojobori, T., Ikeo, K. andOshimura, M. (2004). Molecular hierarchy in neurons
differentiated from mouse ES cells containing a single
human chromosome 21. Biochem. Biophys. Res.
Commun. 314, 335-50.
24. fjtiJi Il/ilU, nf~#iiiif: (2004) r if J ~ -7 A . 7° 0 TT
~ -7 AO)~J.iOOJm 5 Wlt~if J A (~1t)~~~O)if J A~1tm#r, ;c?(. T-{ . ;cA, 897-903.
25. IJv~)JKf1fB*, 11. f~#iiiif: (2004) rmJJl!~~ fcto ft.:5 7}-f-~~~-m-$16. /"'\-{ T-{ /'7 ;t-"'<'T-{ -7 AJ WmJJl!
~ ~J*J~lKfmflJ-'%, 91-97.
26. 11.f~#iiiif: (2004) r~~1t~ iitf-4~ ~ l, -C 0)~ft~1t~ J (~mi i3) n'-t~~10Jj -,%, 1203.
27. nf~#iiiif:(2004) r/"'\-{T-{/'7;t-"'<'T-{-7A-~iit0)~;fitE~~1tf(:£0<~iitf-.l.~0)*1E~1tf(:tfft-CJ W~

UJf*:/-\"'-Tlj..-~5-'%, 26-28.
28. rn rp Il/ilU, 11. f~#iiiif: (2004) r*¥~ 7}-f-~1t~0)~
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241-6.
29. 1ErnfJl:1l-, nf~#iiiif: (2004) r if J AO):j4;ijjjJJX:Jm-.
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30. ilLl:~5!:., 1ErnfJl:1l-, 11. f~#iiiif: (2004) r 7 -{ Iv A'~)jf~ (J::.) J S *~,,*62~, 76-80.

Reviews

31. nf~#iiiif:' Hwang Jung Shan . rnrp@~, rt H(::tMt.:5 t F7J1~-f-J, ~f*(7)f-.l.~m55~ 5 -,%, 2004~10Jj
EDUCATION

1. Y Suzuki "Methods for detecting positive selection
at single amino acid sites" The Graduate Universityof Advanced Studies, Hayama, April, ~004.

Laboratory for DNA Data Analysis

117

I-b. Laboratory for Gene-Product Informatics
Ken Nishikawa GroupRESEARCH ACTIVITIES(1) Eigenvalue analysis of amino acid substitution
matrices reveals a sharp transition of the mode ofsequence conservation in proteins

Akira R. Kinjo and Ken Nishikawa
The pattern of amino acid substitutions andsequence conservation over many structure-basedalignments of protein sequences was analyzed as afunction of percentage sequence identity. The statisticsof the amino acid substitutions were converted into theform of log-odds amino acid substitution matrices towhich eigenvalue decomposition was applied. It was
found that the most important component of thesubstitution matrices exhibited a sharp transition at
the sefluence identity of 30-35%, which coincides withthe twilight zone. Above the transition point, the mostdominant component is related to the mutability ofamino acids and it acts to disfavor any substitutions,whereas below the transition point, the most dominantcomponent is related to the hydrophobicity of aminoacids and substitutions between residues of similarhydrophobic character are positively favored.
Implications for protein evolution and sequence
analysis are discussed. See Ref. 1 for details.
(2) Estimation of the number of authentic orphan genes
in bacterial genomes

Satoshi Fukuchi and Ken Nishikawa
Genome annotation produces a considerable
number of putative proteins lacking sequencesimilarity to known proteins. These are referred toas "Orphans". The proportion of orphan genes varies
among genomes, and is independent of genome size.
In the present study, we show that the proportion oforphan genes roughly correlates with the isolationindex of organisms (110), an indicator introduced in
the present study, which represents the degree of
isolation of a given genome as measured by sequencesimilarity. However, there are outlier genomes withrespect to the linear correlation, consisting of those

118

Laboratory for Gene-Product Informatics

genomes that may contain excess amounts of orphan
genes. Comparisons of genome sequences amongclosely related strains revealed that some of theannotated genes are not conserved, suggesting thatthey are ORFs occurring by chance. Exclusion of these
non-conserved ORFs within closely related genomes
improved the correlation between the proportion oforphan genes and the 110 values. Assuming that thecorrelation holds in general, this relationship was used
to estimate the number of "authentic" orphan genes
in a genome. Using this definition of authentic orphan
genes, the anomalies arising from over-assignments,
e.g., the percentages of structural annotations, werecorrected for 16 genomes, including those of fivearchaea. See Ref. 2 for details.
(3) Alternative splice variants encoding unstable protein
domains exist in the human brain

Keiichi Homma, Reiko F. Kikuno, Takahiro Nagase,
Osamu Ohara and Ken Nishikawa
Alternative splicing has been recognized as amajor mechanism by which protein diversity is
increased without significantly increasing genome sizein animals and has crucial medical implications, asmany alternative splice variants are known to cause
diseases. Despite the importance of knowing what
structural changes alternative splicing introduces tothe encoded proteins for the consideration of its
significance, the problem has not been adequately
explored. Therefore, we systematically examined thestructures of the proteins encoded by the alternative
splice variants in the HUGE protein database derived
from long (>4 kb) human brain cDNAs. Limiting ouranalyses to reliable alternative splice junctions, wefound alternative splice junctions to have a slighttendency to avoid the interior of SCOP domains anda strong statistically significant tendency to coincidewith SCOP domain boundaries. These findings reflect
the occurrence of some alternative splicing events thatutilize protein structural units as a cassette. However,
50 cases were identified in which SCOP domains are
disrupted in the middle by alternative splicing. In sixof the cases, insertions are introduced at the molecularsurface, presumably affecting protein functions, whilein 11 of the cases alternatively spliced variants were
found to encode pairs of stable and unstable proteins.
The mRNAs encoding such unstable proteins aremuch less abundant than those encoding stable
proteins and tend not to have corresponding mRNAs
in non-primate species. We propose that most unstable
proteins encoded by alternative splice variants lacknormal functions and are an evolutionary dead-end.
See Ref. 3 for details.
(4) Construction and characterization of chimeric
proteins composed of type-1 and type-2 periplasmic
binding proteins MglB and ArgT

Kenji Kashiwagi, Kaoru Fukami-Kobayashi, Kiyotaka
Shiba and Ken Nishikawa
The respective type-1 and type-2 periplasmic
binding proteins (PBPs) MglB and ArgT are believed
to have evolved from a common ancestor into siblings
showing topological differences in their main chain
connectivity. At first glance, they show similar
structure. But, more detailed examination reveals thatthe chain connectivity of ArgT is more convoluted thanthat of MglB. Reflecting that complexity, the foldingof ArgT is complicated and involves intermediate folds.
On the other hand, the folding of MglB is a simple
two-state transition. In the present study, weconstructed and characterized several chimeras madeup of various subdomains of MglB and ArgT with theaim of gaining insight into the evolution of protein
folding and protein structure. Although these chimeras
did not fold as compactly as their parental proteins,some did exhibit cooperative folding, which suggeststhat novel proteins with new connectivity and new
folding pathways could have emerged at a fairly highrate throughout the evolution of proteins. See Ref. 4
for details.

encoding unstable protein domains exist in the humanbrain. J Mol. BioI., 343, 1207-1220.
4. Kashiwagi, K, FUkami-Kobayashi, K, Shiba, K
and Nishikawa, K (2004). Construction andcharacterization of chimeric proteins composed of
type-1 and type-2 periplasmic binding proteins MglB
and ArgT. Biosci. Biotechnol. Biochem., 68 (4)808-813.
5. Imanishi, T., Itoh, T., Suzuki, Y, O'Donovan, C.
and Fukuchi, S. et al. (2004). Integrative annotation of
21,037 human genes validated by full-length cDNA
clones. PLoS Biology, 2 (6), 856-875.
6. Kinjo, A.R., Horimoto, K and Nishikawa, K (2005).
Predicting absolute contact numbers of native protein
structure from amino acid sequence. Proteins, 58,
158-165.
7. Kinjo, A.R. and Nishikawa, K Recoverable
one-dimensional encoding of protein three-dimensional
structures. Bioinformatics, in press.
Reviews

8. s*-~, fjitMti:4it, ~r::Fft~, i!9)J1}! (2004) r?r/' /~ 7Itip G 5!td~131Hl'~~~(1)~~jfiljJ;tlitt~ J, /~ ::t-iT-{.:r:./'.A~-{/'?r".A" 1)-, Vol.62,17-22.
9. fj±t!!{ft4it, i!9)J1}!(2004) r~BItmJiM#f7°t1 ~'7 A . :r-?rA.- .AJ~BIt~~M~±If1Jr/-\-{ ::tiWitt~~:!Hi . m~3H¥ifIJJfI'"'?'.::::.;:l. 7" ;VJ (/j\J;ij(lj)({iMliii, #lil±llWi)
Vol.49 (11), pp.1944-1948.
Database

GTOP(-7' / A~(1)?r /'/~7Itli1*mJiDB)
http://spock.genes.nig.ac.jp/-genome/gtop.html
PMD(~~?r /'/~7ItDB) :http://pmd.ddbj.nig.ac.jp/-pmd/pmd.html
TTDB(jjp.:~~~(1)~~lZSI-=fDB):
http://spock.genes.nig.ac.jp/-ttdb/

PUBLICATIONSEDUCATION

Papers

1. fj±t!!{ft4it ~ 9 @]DDBJing~1W~, *J?:, 3 JJ.
2. i!9)J1}!, fj±t!!{ft4it, ~:lpX$ f-4'¥::JsHf!j~J!.~1ill

1. Kinjo, A.R. and Nishikawa, K (2004). Eigenvalue
analysis of amino acid substitution matrices reveals
a sharp transition of the mode of sequenceconservation in proteins. Bioinformatics, 20 (16)2504-2508.
2. Fukuchi, S. and Nishikawa, K (2004). Estimationof the number of authentic orphan genes in bacterial
genomes. DNA Res., 11, 219-231.
3. Homma, K, Kikuno, R.F., Nagase, T., Ohara, O.
and Nishikawa, K (2004). Alternative splice variants~M7/AI)~'7~-~mr:r-?rA.-.A~~JfI~k~B

It (1) li1*mJirVlIJJ *J?:, 7 JJ.
3. i!9)J1}! li iltrg*'¥:.f!I!I '¥:$~ilttiUR,¥:f-I. -e ~ -r- ,11JJ .

Laboratory for Gene-Product Informatics

119I-c. Laboratory for Gene Function Research
Yoshio Tateno GroupRESEARCH ACTIVITY(1) Submission of microarray data to public repositories

Catherine A. Ball, Alvis Brazma, Helen Causton, Steve
Chervitz, Ron Edgar, Pascal Hingamp, John C. Matese,
Helen Parkinson, John Quacken bush, Martin
Ringwald, Susanna-Assunta Sansone, Gavin Sherlock,Paul Spellman, Chris Stoeckert, Yoshio Tateno, Ronald
Taylor, Joseph White and Neil Winegarden
What this work states is a change in the way inwhich we approach the publication of microarraybased studies. Both authors and journals have a
responsibility to assure that the requisite data are
available, and because submitting MIAME-compliant
data can take considerable time and effort, this process
shouiH be factored into review and publication
timelines. However, while this process may be time
consuming and painful at first, we believe that thebenefits of building an open repository of microarraydata will far outweigh any initial disadvantages. As
always, it is our sincere hope that these suggestions
stimulate discussion within the community and thattogether we can arrive at a consensus that ensures thatmicro array data are widely and easily accessible.
Finally we would like to urge the DDBJ, EBI, and NCBI
to work together towards exchanging all MIAMEcompliant microarray datal).
(2) The origin of eukaryotes is suggested as the
symbiosis of pyrococcus into proteobacteria byphylogenetic tree based on gene content

Tokumasa Horiike, Kazuo Hamada, Daisuke Miyata
and Takao Shinozawa
Attempts were made to define the relationshipamong the three domains (eukaryotes, archaea, andeubacteria) using phylogenetic tree analyses of 16SrRNA sequences as well as of other protein sequences.
Since the results are inconsistent, it is implied that the
eukaryotic genome has a chimeric structure. In ourprevious studies, the origin of eukaryotes to be thesymbiosis of archaea into eubacteria using the whole

120 Laboratory for Gene Function Research

open reading frames (ORF) of many genomes was
suggested. In these studies, the species participatingin the symbiosis were not clarified, and the effect ofgene duplication after speciation (in-paralog) was notaddressed. To avoid the influence of the in-paralog, wedeveloped a new method to calculate orthologous
ORFs. Furthermore, we separated eukaryotic inparalogs into three groups by sequence similarity toarchaea, eubacteria (other than -proteobacteria),
and -proteobacteria and treated them as individualorganisms. The relationship between the three ORF
groups and the functional classification was clarified
by this analysis. The introduction of this new method
into the phylogenetic tree analysis of 66 organisms (4eukaryotes, 13 archaea, and 49 eubacteria) based ongene content suggests the symbiosis of pyrococcus
into -proteobacteria as the origin of eukaryotes 2).
(3) Integrative annotation of 21,037 human genes
validated by full-length cDNA clones

Tadashi Imanishi, Takeshi Itoh, Yutaka Suzuki, Claire
O'Donovan, Satoshi Fukuchi, Kanako O. Koyanagi,
Roberto A. Barrero, Yoshio Tateno, Zhu Chen, Michio
Oishi, Peter Tonellato, Rolf Apweiler, Kousaku Okubo,
Lukas Wagner, Stefan Wiemann, Robert L. Strausberg,
Takao Isogai, Charles Auffray, Nobuo Nomura,
Takashi Gojobori and Sumio Sugano. et at.
The human genome sequence defines our inherent
biological potential; the realization of the biology
encoded therein requires knowledge of the function ofeach gene. Currently, our knowledge in this area is still
limited. Several lines of investigation have been used
to elucidate the structure and function of the genes in
the human genome. Even so, gene prediction remains
a difficult task, as the varieties of transcripts of a genemay vary to a great extent. We thus performed an
exhaustive integrative characterization of 41,118
full-length cDNAs that capture the gene transcripts ascomplete functional cassettes, providing an unequivocal report of structural and functional diversity at thegene level. Our international collaboration has
validated 21,037 human gene candidates by analysis
of high-quality full-length cDNA clones throughcuration using unified criteria. This led to theidentification of 5,155 new gene candidates. It also
manifested the most reliable way to control the qualityof the cDNA clones. We have developed a human genedatabase, called the H-Invitational Database (H-InvDB;
http://www.h-invitational.jp/). It provides the following:
integrative annotation of human genes, description ofgene structures, details of novel alternative splicing
isoforms, non-protein-coding RNAs, functional
domains, subcellular localizations, metabolic pathways,
predictions of protein three-dimensional structure,
mapping of known single nucleotide polymorphisms(SNPs), identification of polymorphic microsatellite
repeats within human genes, and comparative resultswith mouse full-length cDNAs. The H-InvDB analysis
has shown that up to 4% of the human genome
sequence (National Center for Biotechnology
Information build 34 assembly) may containmisassembled or missing regions. We found that 6.5%of the human gene candidates (1,377 loci) did not havea good protein-coding open reading frame, of which
296 loci are strong candidates for non-protein-coding
RNA genes. In addition, among 72,027 uniquely
mapped SNPs and insertions/deletions localized withinhuman genes, 13,215 nonsynonymous SNPs, 315
nonsense SNPs, and 452 indels occurred in coding
regions. Together with 25 polymorphic microsatellite
repeats present in coding regions, they may alter
protein structure, causing phenotypic effects or
resulting in disease. The H-InvDB platform represents
a substantial contribution to resources needed for the
exploration of human biology and pathology31•(4) Structural and functional differences in two cyclic
bacteriocins with the same sequences produced bylactobacilli

Yasushi Kawai, Yasuyuki Ishii, Kensuke Arakawa,
Koichiro Demura, Boku Saitoh, Junko Nishimura,
Haruki Kitazawa, Yukiko Yamazaki, Yoshio Tateno,
Takatoshi Itoh and Tadao Saito

Lactobacillus gasseri LA39 and L. reuteri LA6
isolated from feces of the same human infant were
found to produce similar cyclic bacteriocins (named
gassericin A and reutericin 6, respectively) that cannot
be distinguished by molecular weights or primaryamino acid sequences. However, reutericin 6 has anarrower spectrum than gassericin A. In this study,
gassericin A inhibited the growth of L. reuteri LA6, but
reutericin 6 did not inhibit the growth of L. gasseri
LA39. Both bacteriocins caused potassium ion efflux
from indicator cells and liposomes, but the amountsof efflux and patterns of action were different.
Although circular dichroism spectra of purified
bacteriocins revealed that both antibacterial peptides
are composed mainly of alpha-helices, the spectrapf
the bacteriocins did not coincide. The results of D- andL-amino acid composition analysis showed that two
residues and one residue of D-Ala were detected among
18 Ala residues of gassericin A and reutericin 6,
respectively. These findings suggest that the different
D-alanine contents of the bacteriocins may cause thedifferences in modes of action, amounts of potassium
ion efflux, and secondary structures. This is the firstreport that characteristics of native bacteriocins
produced by wild lactobacillus strains having the samestructural genes are influenced by a difference inD-amino acid contents in the molecules 41 •(5) Japanese domesticated chickens have been derived
from Shamo traditional fighting cocks

Tomoyoshi Komiyama, Kazuho Ikeo, Yoshio Tateno
and Takashi Gojobori.~,
With the aim of elucidating the evolutionary origin
of Japanese domesticated chickens, this study
evolutionarily analyzed 85 chicken mtDNA sequences.
Thirty-four various ornamental chickens, 42 fighting
cocks (Shamo), and nine long-crowing chickens(Naganakidori) were included. Of the Shamo, 18 were
sampled from Okinawa, while the remaining 24 werecollected in other islands around Japan. In addition,
three Southeast Asian Junglefowls were used as areference to determine the common ancestor ofJapanese domesticated chickens. A phylogenetic tree
was constructed for the 88 mtDNA sequences revealingthat the Shamo group from Okinawa clearly diverged
from the other Japanese domesticated chickens
studied. This strongly suggests that all Japanese
domesticated chickens, including the ornamental
varieties and Naganakidori, derived from the ancestorsof the Shamo in Okinawa. To create novel varieties ofornamental chickens, intensive artificial selection is
imposed on ancestral Shamo populations, resulting in
profoundly differentiated Japanese domesticatedchickens 51 •Laboratory for Gene Function Research121

(6) DDBJ in the stream of various biological data

Satoru Miyazaki, Hideaki Sugawara, Kazuho Ikeo,
T~kashi Gojobori and Yoshio Tateno
In the past year we at DDB] (http://www.ddbj.nig.
ac.jp) have made a steady increase in the number ofdata submissions with a 50.6% increment in thenumber of bases or 46.5% increment in the number of
entries. Among them the genome data of man, ascidian
and rice hold the top three. Our activity has extended
to providing a tool that enables sequence retrieval
using regular expressions, and to launching our SOAP
server and web services to facilitate the acquisition ofproper data and tools from a huge number of biological
data resources on websites worldwide. We have also
opened our public gene expression database, CIBEX61•(7) Molecular chaperones: proposal of a systematiccomputer-oriented nomenclature and construction of acentralized database

Haitham Sghaier, Thuy Le Huyen Ai, Tokumasa
Horiike and Takao Shinozawa
Molecular chaperones are a wide group of
unrelated protein families whose role is to assist others
proteins. Comparably, under environmental stress,
stress proteins behave as biocatalysts of protein
stabilization. Stress proteins include a large class ofproteins that were originally termed heat shockproteins (HSPs) due to their initial discovery in tissues
exposed to elevated temperatures. Many, but not all,stress proteins and HSPs are molecular chaperones.
Moreover, not all HSPs are derivable from stress. HSPs
are structurally diversified by the contribution ofvarious domains having specific roles. HSPs have beengrouped, mainly on the basis of their molecularmasses, into specific families that include small HSPs
(sHSPs)1 a -crystallins, HSPI0s, HSP40s, HSP60s,
HSP70s, HSP90s, HSP100s and HSPllOs. The namesof these major families are historical artefacts withlimited information content. Using the current
databases, names and proteic domains of many
molecular chaperones in different species wereanalyzed. Although traditional names of HSPs are
trivial, it is unrealistic to suggest replacing them,because they are preferred and widely used. Here wesuggest that these traditional names be chaperoned,

122 Laboratory for Gene Function Researchin silico, by a systematic nomenclature. Thus, forexample, with the same intent of use of [trioxygen: OJ
for ozone, we propose here C7HSP70[EhsaJER-Pll021
for GRP78 (78 kDa endoplasmic Human molecularchaperone in HSP70 superfamily with Pll021 as its
accession number in the database of the National
Center for Biotechnology Information (NCBI)). The
proposed systematic computer-oriented naming andclassification method is designed for HSPs and also
their partners based on the number of amino acids,
domain structure, phylogenetic domain, localizationin the cell and accession number as stated in the NCBI.
Arabidopsis thaliana was analyzed as a model, because
it contains a large number of various HSPs localized
in several organelles. Overall, this naming system helps
in building, optimizing and managing a novel online
database entirely devoted to HSPs. The purported
taxonomy, coupled with the newly constructed
database, can contribute to studies involving large
amounts of stored data on HSPs 7,.
(8) Extensive analysis of ORF sequences from two
different cichlid species in Lake Victoria provides
molecular evidence for a recent radiation event of theVictoria species flock Identity of EST sequences between
Haplochromis chilotes and Haplochromis sp.
"Redtailsheller"

Masakatsu Watanabe, Naoki Kobayashi, Tadasu
Shin-I, Tokumasa Horiike, Yoshio Tateno, Yuji Kohara
and Norihiro Okada
The Lake Victoria Cichlid fishes have diverged
very rapidly. The estimated 500 species inhabiting thelake are believed to have arisen within the last 14,000
years. The fishes' jaws and teeth have divergedmarkedly to adapt to different feeding behaviors andenvironments. To examine how the genomes of thesefishes differentiated during speciation, we performed
comparative analysis of expressed sequenced tag (EST)
sequences. We constructed cDNA libraries derived only
from the jaw portions of two cichlid species endemic
to Lake Victoria. We sequenced 17,280 cDNA clones
from Haplochromis chilotes and 9600 cDNA clones
from Haplochromis sp. "Redtailsheller" and obtained
543 different genes common to both species. Of thesegenes, 441 were essentially identical between species
and 102 contained base replacements in their open
reading frame (ORF) or untranslated (UTR) regions.

Comparative analysis of 7lselected sequences hasrevealed that while the degree of polymorphism is
O.OO54/site for H. chilotes and 0.OO47/site for H. sp. "Redtailsheller", genetic distance between the two
species is 0.OO31/site. The genetic distance particularly
indicates that the two species diverged about 890,000
years agd).
(9) International public gene expression database(CIBEX) and data submission

Yoshio Tateno and Kazuho Ikeo
We have opened our gene expression database,
CIBEX, to the public. CIBEX has been developed as
an international public database with the aim of thecollaboration with ArrayExpress at EBI and GEO atNCBL The collaboration mainly means to share theannotation manual and to exchange the data collected
and annotated among the three databases. The datacollection will hopefully be promoted by the open letter
issued by the MGED society to the editors of relevant
journals9l •(10) Analysis of biological networks in eukaryotes using
the whole genome sequences

Tsuyoshi Tanaka and Takashi Gojobori
Since the whole genome sequencing of
Haemophilus injluenzae was completed in 1995, thenumber of species whose genomes were completely
sequenced has steeply been increased. As of January
2005, the number of such species is more than 210 in
the Genome Information Broker (GIB) of the Center for
Information Biology and DNA Data Bank of Japan. The
information on the whole genome sequences enables
us to study the origins and evolutionary processes ofvarious biological networks such as metabolic
pathways and signal transductions. We analyzed
biological networks such as amino acid metabolic
pathways by conducting comparative analysis of thecomplete genome sequences of six eukaryotic species
including man, fly, nematode, yeasts and plant, andfound that a particular pathway had evolved
independently in multiple lineages of the species
studied 1ol •(11) Microarray gene expression database

Kazuho Ikeoand Yoshio Tateno•

As the international standardizations of
microarray data description and data sharing havebeen promoted by the MGED society, researchers
conducting microarray experiments are encouraged tosubmit their data to one of the international gene
expression databases, ArrayEpress, GEO and CIBEX.
CIBEX being developed by us is in compliance with
the international standard, MIAME, and equipped withseveral search functions lli •(12) Discovery and annotation of forty seven non-protein
coding human RNAs

Roberto A. Barrero, Inna Dubchak, Charles Auffray,
Laurens Wilming, Jun-ichi Takeda, Yutaka Suzuki,
Erimi Harada, Marie-Anne Debily, Esther Graudens,
John Quackenbush, Takuro Tamura, Dmitriy V.
Ryaboy, Sandrine Imbeaud, Kazuho Ikeo, Peter
Tonellato, Nobuo Nomura, Sumio Sugano, Tadashi
Imanishi, Takashi Gojobori and Libin Jia
Non-coding RNAs (ncRNAs) play crucial roles ina variety of procsses including replication, transcriptional regulation, splicing, dosage compensation,
genetic imprinting, translational regulation, andmodulation of protein function. Here we report thediscovery and annotation of ncRNAs from the humanfull-length cDNA dataset evaluated at the first
International Human Full-length cDNA Annotation
Meeting. A total of 1,485 cDNA transcripts, mapped
onto 1,300 loci on the human genome, were found to
encode putative open reading frames (ORFs) equal toor less than 80 amino acids (aa). To select putative
ncRNAs all cDNA sequences were mapped to thehuman genome to study the genomic neighbourhoodfor the presence of ab initio predicted genes andneighbouring genes, and compared to Expressed
I
Sequence Tag (EST) databases for supporting
evidence. This method yielded 296 putative ncRNAs
that were analyzed for conservation by determining
mouse DNA and RNA sequence similarities. Putative
ncRNAs with mouse ortholog support were further
analyzed using QRNA. We found 47 ncRNAs
containing a conserved RNA secondary structure. Of
these, 60% were found to be expressed in up to eight

Laboratory for Gene Function Research123human tissues, implying that ncRNAs are seemingly
tissue-specifically regulated.

our method to as many pertinent sequences as
possible.

(q) Evolutionary rate of enzymes in the metabolic(15) Weare in collaboration with Prof. Tadao Saito of
Tohoku University and his laboratory on the ftmction andevolution of glucosidase and galactosidase genes in
Lactobcillus

networkTsuyoshi Tanaka, Kazuho Ikeo and Takashi Gojobori
An enzyme interacts not only with the otherproteins but also with low-weight molecules called
substrates in the metabolic network. To understandan evolutionary process of interactions of enzymes, westudied the relationship between the evolutionary rateof the enzyme and these interacting partners. When
we focused on the 498 enzymes in Saccharomyces
cerevisiae that have orthologous pairs in Ashbya
gossypii, we discovered the significant negative
correlation between the evolutionary rate of the enzyme
and the number of interacting proteins (protein-protein
interaction; PPI). On the other hand, we found nocorrelation between the evolutionary rate of the enzyme
and the number of interacting substrates (proteinsubstrate interaction; PSI). Therefore, we conclude thatthe number of interacting proteins is the most affective
to the evolutionary rate of the enzyme compared withthat of the other interacting partners such as thesubstrates.
(14) Development of a method for constructing aphylogenetic tree using a comprehensive orthologous
gene cluster, and phylogenetic analysis of cyanobacteria

Tokumasa Horiike and Yoshio Tateno
Phylogenetic trees are constructed using DNA,RNA or amino acid sequences for estimating
evolutionary relationships of genes or species.
Currently, there are two problems with the tree
construction. One is that horizontal gene transfer
disturbs the estimation of the true relationships ofgenes or species. The other is that the construction
sometimes depends on the choice of sequences. We
occasionally observe that changing one sequence toanother erroneously alters the reconstructed tree.
Therefore, we are developing a method of the tree
construction which is to reduce the interference causedby the two problems. In the method we can incorporate
all available prokaryotic ORFs. We will then clarify
the phylogenetic position of cyanobacteria by applying

124

Laboratory for Gene Function ResearchTadao Saito, Yukiko Yamazaki and Yoshio Tateno(16) Weare in collaboration with Prof. Shintou Eguchiof the Institute of Mathematical Statistics and hislaboratory on the statistical analyses of SNP and gene
expression data

Shinto Eguchi, Kazuho Ikeo and Yoshio Tateno
PUBLICATIONSPapers

1. Ball, CA., Brazma, A., Causton, H., Chervitz, S.,
Edgar, R., Hingamp, P., Matese, lC., Parkinson, H.,
Quackenbush, J., Ringwald, M., Sansone, SA., Sherlock,G., Spellman, P., Stoeckert, C., Tateno, Y, Taylor, R.,
White, ]. and Winegarden, N. (2004). Submission of
microarray data to public repositories. PLoS BioI. 2,
1276-1277, also Microbiology 150,3522-3524 and Environ
Health Perspect 112, A666-A667.
2. Horiike, T., Hamada, K, Miyata, D. andShinozawa, T. (2004). The origin of eukaryotes issuggested as the symbiosis of pyrococcus
into y-proteobacteria by phylogenetic tree based ongene content. ]. Mol. Evol. 59, 606-619.
3. Imanishi, T., Itoh, T., Suzuki, Y., O'Donovan, C.,
Fukuchi, S., Koyanagi, KO., Barrero, RA., Tateno, Y,
Chen, Z., Oishi, M., Tonellato, P., Apweiler, R., Okubo,
K, Wagner, L., Wiemann, S., Strausberg, RL., Isogai,T., Auffray, c., Nomura, N., Gojobori, T. and Sugano,S. et at. (2004). Integrative annotation of 21,037 humangenes validated by full-length cDNA clones. PLoS BioI.
2,256-275.
4. Kawai, Y, Ishii, Y., Arakawa, K, Uemura, K.,
Saitoh, B., Nishimura, l., Kitazawa, H., Yamazaki, Y,
Tateno, Y, Itoh, T. and Saito, T. (2004). Structural andfunctional differences in two cyclic bacteriocins withthe same sequences produced by lactobacilli. Appl
Environ Microbiol. 70,2906-2911.
5. Komiyama, T., Ikeo, K, Tateno, Y and Gojobori,T. (2004). 1apanese domesticated chickens have beenderived from Shamo traditional fighting cocks. Mol
Phylogenet EvoI. 33, 16-21.
6. Miyazaki, S., Sugawara, H., Ikeo, K, Gojobori, T.
and Tateno, Y. (2004). DDBJ in the stream of various
biological data. Nucleic Acids Res. 32, D31-D34.
7. Sghaier, H., Ai, TL., Horiike, T. and Shinozawa,T. (2004). Molecular chaperones: proposal of asystematic computer-oriented nomenclature andconstruction of a centralized database. In Silico BioI.
16,0025.
8. Watanabe, M., Kobayashi, N., Shin-I, T., Horiike,T., Tateno, Y., Kohara, Y. and Okada, N. (2004).
Extensive analysis of ORF sequences from two
different cichlid species in Lake Victoria provides
molecular evidence for a recent radiation event of theVictoria species flock Identity of EST sequences
between Haplochromis chilotes and Haplochromis
sp. "Redtailsheller". Gene 343, 263-269.
9. ~!l!fjt~, i'lf!,ij1,-f!!( (2004) OO~0JtJ:l{~-r-~:m. 7?tA:-A(CIBEX) c7-?t(J)~~, mBJt~~M1U9,2679-2683.
10. IE J:j:tIlijIJ, Ii{~#iil~ (2004) If / A (J) J::~i\i:leWt#T C ?t-r-~1~, it#:(J)t--t~55, 241-246.
11. i'lf!,ij1,-f!!(, ~!l!fjt~ (2004) ~ -{ -7 t1 7' v -( m{~-r-~:m.7-?tA:-A, 1f/~Y-7A7t17~~Y-7A(J)~~~(4-J:j:t!~fi~{~), pp828-836, NTSl±lAA.
Database

12. DDBJ is operated by DNA Data Analysis, Gene
Function Research, Gene-Product Informatics,
Research and Development of Biological Databases andGene Expression Laboratories, and Division ofPopulation Genetics. DDBJ collects, annotates andpublishes DNA sequence data, and exchanges the datawith EMBL Bank and GenBank on a daily basis. In
addition, DDBJ edits the data published by DDBJ,
EMBL Bank and GenBank together four times a year
and publishes as a release. In 2004 DDBJ published thefollowing four releases.
32,693,678 entries
Release 57 March., 04
38,008,449,840 bases
34,917,581 entries
Release 58 June., 04
39,812,635,108 bases
37,926,117 entries
Release 59 Sept., 04
44,416,752,273 bases
40,583,945 entries
Release 60 Dec., 04
42,245,956,937 bases
13. Dr. Y. Tateno attended the 17th International
Nucleotide Sequence Databases Collaborative Meeting,and the 15th International Nucleotide Sequence
Databases Advisors Meeting held at EEl, Hinxton, UK,
May, 2004.
14. Dr. Y. Tateno attended the MGED Board Meeting,
Toronto, Canada, September, 2004.
15. Dr. Y. Tateno was invited to be a chairperson of
a session at the UniProt Meeting, Antibes, France,
September, 2004.
EDUCATION

1. Dr. Y. Tateno co-organized the 3rd Japan-Korea
Bioinformatics Training Course at NIG, March, 2004.
2. Dr. Y. Tateno co-organized the SOKENDAIinternational lecture and gave a lecture on DNA andProtein Databases at the Olympic Hotel, Shanghai,
China, October, 2004.
SOCIAL CONTRIBUTIONS AND OTHERS~~)311J

1. Dr. Y. Tateno serves the Microarray Gene
Expression Data Society as an Advisory Board Member(2001-), and attended the Board Member Meeting,
Toronto, Canada, September, 2004.
2. Dr. Y. Tateno serves the Society of Evolutionary
Studies as a Member of the Committee of Genetics
under the Science Council of Japan.
3. Dr. Y. Tateno serves the Genetics Society of Japanas a Member of Editorial Board.

1- d. Laboratory for Research and Developmentof Biological Databases
Hideaki Sugawara GroupRESEARCH ACTIVITIES(1) Information systems for molecular biology and its
related disciplines
1) From Web services to a Bioportal

Yasumasa Shigemoto t, Haruka Sakai, Takashi Abe,
Satoru Miyazaki t t and Hideaki Sugawara eHitachi
soft, t tTokyo Univ. of Sci.)
The publicly available bioinformatics resources,
comprising databases and analytical tools, have

Laboratory for Gene Function Research125expanded in recent years. While the information
environment for life sciences has gradually become
more abounding, it is still difficult to combine multiple,
heterogeneous bioinformatics resources for a specificresearch purpose. To set up and run an integrated
system, it is often necessary to write and update
custom programs. In addition, different researchgroups continually write programs that haveoverlapping functions. We need an information
environment that is conducive to efficient andappropriate bioinformatics resource utilization for awide range of users. Therefore, the Center for
Information Biology and DNA Data Bank of Japan, inalliance with the National Institute of Informatics (NIl)and the Mitsubisi Research Institute, Inc. (MRI) havestarted a three years long project since 2003, "Researchand Development of the New Generation of Bio-portal",
to enhance the information environment for the
relevant user communities. In this project, theLaboratory for Research and Development of Biological
Databases is responsible for the development of!lbiological Web services. The project site is open at
http://www.bioportal.jp/in 2004. From there, a Web
page for links to sites complete genome sequence andannotation are also prepared and accessible, in
addition to the biological Web services. The former isnamed "Genome Menu".
2) Expansion of Genome Information Broker (GIB)

Masaki Hirahata, Naoto Tanaka, Takashi Abe, Satoru
Miyazaki t and Hideaki Sugawa CTokyo Univ. of Sci.)
GIB was originally created for the retrieval andanalysis of E. coli genomic information in a set. We
implemented microbial genome data into GIB
whenever genome sequencing was completed and thedata is made open to the public. At the GIB Web page(http://gib.genes.nig.ac.jp/), key word search, homology
search, links to DBGET, KEGG and GTOP andvisualization of the data are available for more than
200 strains as of December 2004. We have utilized XML,CORBA and a distributed database in order to copewith the explosion of microbial genome information.

(2) Information systems on microbes!)
1) WFCC-MIRCEN World Data Centre for Microorganisms (WDCM)

Yasumasa Shigemoto t , Junko Nagaya and Hideaki
Sugawara eFujitsu)
WFCC and MIRCEN stand for WorId Federation
for Culture Collections and Microbial Resource Centers
network respectively. The laboratory is the host of
WDCM and maintains the World Directory of
microbial resource centers. The on-line World
Directory contains the detailed information of 469
centers in 65 countries and also the list of theirholdings. Any culture collection is able to register,
update and delete the information at http://
www.wdcm.orgl. WDCM could promote the update ofthe data by culture collections funded by the American
Society for Microbiology and UNESCO.
2) Development of an e-Workbench for BiologicalClassification and Identification (InforBIO)

Naoto Tanaka, Kouji Koorikawa t, Takashi Abe,
Satoru Miyazaki tt and Hideaki Sugawara eHitachi
soft, t'Tokyo Univ. of Sci.)
We continued the development of an e-Workbench
named InforBlO by use of JAVA, XML and a relational
database management system in the public domain.
We have distributed InforBlO to several laboratories
that study microbes and improved the utility androbustness of InforBIO based on the feedback (http://lilium.genes.nig.ac.jp/index_e.html).
3) An information system for pathogenic microorganisms

Masaki Hirahata, Naoto Tanaka, Yasumasa
Shigemoto t and Hideaki Sugawa eFujitsu)
We participated in a national project for the
resource center of pathogenic microorganisms. Our role
is to develop an information system for pathogenic
fungi and actinomycetes, and also a portal site forpathogenic microorganisms in general (http://
www.wdcm.org/byogen/).
(*) The information system on pathogenic microor-

126 Laboratory for Research and Development of Biological Databases

ganisms has been supported by Special Coordination
Funds for Promoting Science and Technology.

(4) Genomics
1) Development of the H-Invitational Database(3) Applications of IT to the International Nucleotide
Sequence Database2)

Yasumasa Shigemoto t, Satoru Miyazaki t t and
Hideaki Sugawara eFujitsu, t tTokyo Univ. of Sci.)

1) Development of Open Annotation System
Satoru Miyazaki t, Takashi Abe and Hideaki
Sugawara eTokyo Univ. of Sci.)
A number of the complete genome sequences havebeen submitted to INSD since 1995. The annotation
information, however, is not consistent among genome
sequencing teams. In addition, researchers outside of
the team might have more information and knowledge
on some genes and biological molecules. Therefore, it
is quite important to develop the system which allows
any expert to evaluate the annotation given by the teamto attach more valuable information. As a new featureof INSD, we develop so-called "Open Annotation
System (OASYS)" as an annotation editor in the
distributed environment on the Internet.
(*) OASYS project has been supported by BIRD ofJapan Science and Technology Corporation aST)
2) Exhaustive evaluation of microbial genome
information by use of GRID
Takehide Kosuge, Toshihisa Okido, Yasumasa
Shigemoto t, Masaki Hirahata, Naoto Tanaka, Yuzuru
Maruyama, Takashi Abe, Satoru Miyazaki tt and
Hideaki Sugawara eFujitsu, ttTokyo Univ. of Sci.)
Tsunami of biological data and multiple views of
the data analysis require an expandable and flexible
information environment. GRID computing is expected
to be the solution. We prepared a computational
environment composed of 5 sites in OBIGrid andsucceeded in analyzing horizontal gene transfer andclusters of ORFs of more than 100 microbial genomes
that were stored in the Genome Information Broker asof May, 2003. This scheme is being applied to morethan 300 thousands ORFs of genomic sequences of 124
microbial species. In 2004, we evaluated the results of
the analysis and have developed site to diffuse theresult to the public. We also applied the workflow to
all the microbial genome sequences that were publicly
available by September 2004.

We performed an exhaustive integrative
characterization of 41,118 full-length cDNAs that
capture the gene transcripts as complete functionalcassettes, providing an unequivocal report of structuraland functional diversity at the gene level. Ourinternational collaboration has validated 21,037 humangene candidates by analysis of high-quality full-length
cDNA clones through curation using unified criteria.
We have developed a human gene database, called the
H-Invitational Database (H-InvDB; http://www.hinvitational.jp/). The H-InvDB platform represents a
substantial contribution to resources needed for the
exploration of human biology and pathology.
l'2) Splicing Profile Based Protein Categorization
between Human and Mouse Genomes
Ake Vastermark t, Yasumasa Shigemoto t t, Takashi
Abe and Hideaki Sugawara eUniv. of Oxford,
ttFujitsu)
We compared gene structures of human andmouse to explore the relationships of functions of genes
and exon-intron structures. The central question iswhether protein function is more correlated withsplicing profiles than sequence similarity, or not. To
approach this question, a splicing profile similarity(SPS) index, which measures relative exon length
discrepancy, was devised. Arbitrary human proteins
were compared, in terms of SPS and amino acidsequence similarity, to their 1) mouse orthologues and2) human paralogues, which epitomise functional
equivalence and non-equivalence, respectively, tomethodically elucidate the global relationship betweena) biological function, b) splicing profile similarity, andc) sequence similarity. Protein function is morecorrelated with splicing profile similarity than sequencesimilarity as demonstrated by the fact that humanmouse orthologues (HMOs) display significantly higher
splicing profile similarity than do human-humanparalogues (HHPs), despite the mutual sequencesimilarity between these two categories. This finding

Laboratory for Research and Development of Biological Databases

127indicates that splicing profile-based
categorisation is biologically meaningful 41 .

protein3) Phylogenetics Analyses of Environmental Samples
on the Basis of Self-Organizing Map (SOM)

Takashi Abe, Toshimichi Ikemura t and Hideaki
Sugawara ('SOKEN-DAI)
Metagenomic approach, which is the genome
analysis on a mixture of uncultured microorganisms,
has been recently developed to search for novel andindustrially useful genes and to study microbial
diversity in a wide variety of environments. We
previously modified the conventional SOM for genome
informatics to make the learning process and resulting
map independent of the order of data inpue)· 61. In thepresent study, we developed the SOM as a novel
bioinformatics strategy to capture and visualize
micro bial diversity and relative abundance of
microorganisms within an environmental sample. First
we constructed SOMs of tri- and tetranucleotide
frequencies in 1- and 5-kb sequence fragments fromprokaryotic genomes for which complete sequence isavailable. The sequences could be classified primarily
according to species and to 11 major phylogenetic
groups without information regarding the species. For
example, 88% of 5-kb sequences were classified into thecorrect phylogenetic group. Importantly, theclassification could be done without orthologous
sequence sets, and therefore, SOM was especially
useful to analyze novel sequences from poorly
characterized species for industrial applications andscientific studies. With the SOM method, all non-rRNAsequences in the Database that were from unidentified
or uncultured bacteria and longer than 1 kb wereclassified into major phylogenetic groups71. The present
method can also be developed as a tool for surveys ofpathogenic microorganisms in environmental andclinical samples that can not be cultured easily andin sterilized samples.
PUBLICATIONSPapers

1. Sugawara, H., Abe, T., Tanaka, N. and Miyazaki,
S. (2004). Encounter of microbiology with the data
science in the phase called post-genome sequencing.
Soil microorganisms. 58 (2), 57-67.

2. Miyazaki, S., Sugawara, H., Ikeo, K, Gojobori, T.
and Tateno, Y (2004). DDBJ in the stream of various
biological data. Nucleic Acids Research. 32, D31-D34.
3. Imanishi, T., Hoh, T., Suzuki, Y., O'Donovan, C.,
Fukuchi, S., Koyanagi, K., Barrero, R., Tamura, T.,
Yamaguchi-Kabata, Y, Tanino, M., Yura, K, Miyazaki,
S., Ikeo, K, Homma, K, Kasprzyk, A., Nishikawa, T.,
Hirakawa, M., Thierry-Mieg, J., Thierry-Mieg, D.,
Ashurst, ]., Jia, L., Nakao, M., Thomas, M., Mulder, N.,
Karavidopoulou, Y., Jin, L., Kim, S., Yasuda, T.,
Lenhard, B., Eveno, E., Suzuki, Y., Yamasaki, C.,
Takeda,]" Gough, C., Hilton, P., Fujii, Y, Sakai, H.,
Tanaka, S., Amid, C., Bellgard, M., Bonaldo, Mde, F.,
Bono, H., Bromberg, S., Brookes, A., Bruford, E.,
Caminci, P., Chelala, C., Couillault, C., De Souza, SJ.,
Debily, M., Devignes, M., Dubchak, I., Endo, T.,
Estreicher, A., Eyras, E., Fukami-Kobayashi, K,
Gopinath, G.., Graudens, E., Hahn, Y, Han, M., Han,
Z., Hanada, K, Hanaoka, H., Harada, E., Hashimoto,
K, Hinz, D., Hirai, M., Hishiki, T., Hopkinson, I.,
Imbeaud, S., Inoko, H., Kanapin, A., Kaneko, Y.,
Kasukawa, T., Kelso,]" Kersey, P., Kikuno, R, Kimura,K, Kom, B., Kuryshev, V., Makalowska, I., Makino, T.,
Mano, S., Mariage-Samson, R, Mashima, ]., Matsuda,
H., Mewes, H., Minoshima, S., Nagai, K, Nagasaki, H.,
Nagata, N., Nigam, R., Ogasawara, 0., Ohara, 0.,
Ohtsubo, M., Okada, N., Okido, T., Oota, S., Ota, M.,
Ota, T., Otsuki, T., Piatier-Tonneau, D., Poustka, A.,
Ren, S., Saitou, N., Sakai, K, Sakamoto, S., Sakate, R,
Schupp, I., Servant, F., Sherry, S., Shiba, R, Shimizu,N., Shimoyama, M., Simpson, N., Soares, B., Steward,
C., Suwa, M., Suzuki, M., Takahashi, A., Tamiya, G.,
Tanaka, H., Taylor, T., Terwilliger, ]., Dnneberg, P.,
Veeramachaneni, V., Watanabe, S., Wilming, L.,
Yasuda, N., Yoo, H., Stodolsky, M., Makalowski, W.,
Go, M., Nakai, K, Takagi, T., Kanehisa, M., Sakaki,
Y, Quackenbush, J., Okazaki, Y., Hayashizaki, Y.,
Hide, W., Chakraborty, R, Nishikawa, K, Sugawara,
H., Tateno, Y., Chen, Z., Oishi, M., Tonellato, P.,
Apweiler, R, Okubo, K, Wagner, L., Wiemann, S.,
Strausberg, R, Isogai, T., Auffray, C., Nomura, N.,
Gojobori, T. and Sugano, S. (2004). Integrative
annotation of 21,037 human genes validated byfull-length cDNA clones. PLoS Biol., 2 (6), e162.
4. Vastermark, A., Shigemoto, Y., Abe, T. and
Sugawara, H. (2004). Splicing Profile-based Protein
Categorization between Human and Mouse Genome
by use of DDBJ Web Services. Genome Informatics 15,
13-20.

128 Laboratory for Research and Development of Biological Databases

5. Abe, T., Kanaya, S., Kinouchi, M. and Ikemura, T.
(2004). Genome Informatics for Unveiling Hidden
Genome Signatures. Proceedings of the Institute of
Statistical Mathematics 52, 207-215.
6. Abe, T., Kanaya, S., Kinouchi, M., Kosaka, Y. and
Ikemura, T. (2004). Novel bioinformatics for unveilinghidden characteristics in genome sequences andsearching in silico for genetic signal sequences.
Proceeding of The 8th Worid Multi-Conference on
Systemics, Cybernetics and Informatics.
7. Abe, T., Ikemura, T., Kanaya, S., Kinouchi, M. and
Sugawara, H. (2004). Novel genome informatics forunveiling hidden signatures in genome sequences:self-organizing map (SOM) of oligonucleotide
frequencies. Proceedings of Information-Based
Induction Sciences, 94-99.

of scientific system, Tokyo, August, 2004 (in Japanese).
~~)I5tI.J

1. Dr. H. Sugawara organized the 2nd International
Conference on Biodata Interoperability, Tokyo, June,
2004.
2. Dr. H. Sugawara organized International programcommittee of the International Congress for Culture
Collections, Tsukuba, October, 2004.
3. Dr, H. Sugawara organized Program committee ofGenome Informatics 2004, Yokohama, December, 2004.
4. Task Force of Biological Resource Centers, OECD
Working Party for Biotechnology (Vice-chair).
5. W orid Federation for Culture Collections,Executive board member and journal editor.

6. filllHJ%t1::~'¥~ (~~~)
7. B *'l1ffR9;o~,¥~ (f!I!$)Books

I-e. Laboratory for Gene-Expression Analysis

8. Sugawara, H. (2004). Tsunami of data: Data
resources and utilization. Microbial Genetic Resources
and Biodiscovery. Kurtboke, I. and Swings, J. ed.,(National Library of Australia), 40-56.

RESEARCH ACTIVITIES

Databases(1) Expression profiling of human genes

9. Japanese Bio-portal site (Jabion),
http://www.bioportal.jp/
10. Genome Information Broker,
http://gib.genes.nig.ac.jp/
11. WFCC-MIRCEN World Data Centre forMicroorganisms (WDCM), http://www.wdcm.org/
12. The portal site for pathogenic microorganisms,
http://www.wdcm.org/byogen/13. e-Workbench for Biological Classification and
Identification, http://lilium.genes.nig.ac.jp/index_e.html
14. H-Invitational Database,
http://www.h-invitational.jp/
EDUCATION

1. Dr. H. Sugawara was invited to give a lecture on"Databases are the key to bioinformatics" at the 2ndOpen Symposium of Joint Research with Wakayama
Pref., Wakayama, 2004 (in Japanese).
2. Dr. H. Sugawara was invited to give a lecture on"Invitation to information biology" at Campus system
Research Group of Private Universities, Hamanako,
August 2004 (in Japanese).
3. Dr. T. Abe was invited to give a lecture on"Genome analysis by PC-cluster." at Working group

Kousaku Okubo Group(1a) Data integration (BodyMap II): "Do you know how
much of our genes have reliable expression patterns on
the net?"Osamu Ogasawara and Kousaku Okubo
Unexpectedly small difference in gene numbers
among multicellular organisms, precisely determinedthough whole genome sequence supports the idea thatthe complexity in our body is evolutionary achievedby sophistication in expression controls of genes. The
anatomically comprehensive genome-wide gene
expression profile is key data to appreciate such
sophistication coded in our genome. Moreover,availability of such data opened up opportunities to
explore the dependence of constitutive expression
patterns on other features of genes and genomes, which
may eventually leads to the understanding of coding
principles in our genome.
Despite the frequent use of the term 'genome-wide
profiling' and wealth of expression data in the publicdomain, it is still not explicit what fraction of our genes
are provided with anatomical expression patterns(COVERAGE) and to what extent different data set

Laboratory for Research and Development of Biological Databases

129agrees in terms of tissue distribution (ACCURACY).
In order for rational design of studies of humantranscriptome as a whole, we started to integrate datafrom multiple different platforms on the frameworkof latest human genome. The preliminary data is
opened to the public in collaboration with integrated
database team at ]BIRC. (https://www.jbirc.aist.go.jp/
hinv/h-angel/)(1b) Data generation and analysis; "Are you satisfied by
the present resolution of anatomical expression data?"
Makiko Otsuji and Koichi Hoh
In order to functionally relate genes in co-expression
cluster or to deduce promoter sequences throughalignment of upstream regions of them, the resolutionof expression pattern should be at the level of cells or
homogeneous cell populations. The vast majority ofthe public data do not meet this criterion, except for
those from induction experiments with cell lines.
Moreover, the majority of the target genes for drug
development such as receptors for signaling moleculesand channels localize in the minority cells in thecomplex organs rather than in homogeneous cell
population. We are taking several different approaches
to generate such data in streamline in the organs withcomplex cell population such as brain and kidney.
(2) Knowledge encoding and computation with genefunctions (BOB): "Are you confident in your massive datainterpretation?"
Kousaku Okubo, Koichi Hoh and Osamu Ogasawara
With the advent in high throughput genome-widemeasurement, hypothesis generation on gene functions
by systematic and integrative interpretation of theaccumulating data is anticipated. For the last few
years, various statistical analysis techniques have been
employed in extraction of global patterns from themassive data, in the forms of gene clusters andnetworks. However, at present, even with intensive useof web-based knowledge bases, human interpretation
will not match machine-aided data production inneither speed nor scope. Automation of interpretation
process, at least in part, appears essential forsystematic and efficient hypothesis generation.
There are three steps in automation of interpretation

130

Laboratory for Gene-Expression Analysis

process; (1) encode biomedical knowledge into
computable form, (2) interpret data using encodedknowledge, and (3) represent the interpretation results.
The pioneering approaches in the step (1) were all
declarations (KEGG, GO). In general, some inherent
limitations are known in declarative approach. Firstof all, continuous revising and updating efforts by
experts are inevitable. Secondly, the manually declared
structure has usually low dimension and representation power is poor. For example GO provides only three'aspects' in the representation of functional relations
in genes. And thirdly, expert's declaration is apt to beinconsistent when target domain becomes wider.
Although such limitation may causes little problem inbiochemistry and cell biology, they become serious in
the field of medical biology where gene functions havebeen described by relating their roles with so many
types of concepts from behavior to chemicalcompounds. These limitations make the effectivenessof declaration approach in the medical problems still
elusive.
Rationale for our approach: A major process inbiomedical interpretation of these data is to determine
whether if there is a unique and common functionalfeature, within any part of data-driven structures. For
this purpose, experts recall features of genes fromevery aspect. Such a process appears so dependent on
expert's flexible recalling and thinking that machine
cannot possibly do. In medical biology, however, we
assumed that each aspect mostly represents a certain
topic in biomedicine, such as 'pathogenesis of a disease'and 'molecular mechanism of an organs function.' If
there is a book covering essentially all fundamental
topics in medical biology, and if each page contains
a list of all relevant genes, interpretation would beachieved by computing fitness of a given gene cluster
to each page (meaningfulness) and by returning thetitle of the page that fits best as a meaning of thecluster.
We aimed to create such a book, 'BOB (Biomedical
OminiBook)', by concept-based structuring of pages
in biomedical textbooks and description of genefunctions. We applied a concept based indexing
technique, latent semantic indexing (LSI), for thispurpose. LSI was developed to overcome the problems
in term-matching-based-document-searching causedby the fact that similar concept may represented by
texts with different term combinations and vice versa.
In LSI, terms and documents are structured in a'semantic' vector space, based on the global patterns
in term-document association data, beforehand. In
searching, users' queries are mapped onto this spaceas pseudo-documents. For each queries, the document
vectors having supra-threshold cosine value to thequery vector are returned.
In our application, we took textbooks in place of
documents to be searched. Started from term-page
association data, provided in the index sections of
textbooks, we created a high-dimensional vector space
wherein objects (term or page) are placed according
to their relevance. Then we prepared a gene vector, that
corresponds to a 'query vector' in original LSI, by
counting occurrence of textbook terms in corresponding molecular database entry including abstracts forcited papers. Thus prepared gene vectors were mapped
onto the textbook space similarly to the mapping ofusers queries. Resulted space contains vector representation of three different classes of objects; term
meanings, page contents, and gene functions, arranged
according to their conceptual relevance. Highdimensionality of this space is expected to allow
discriminative representation of many aspects ofrelations without inconsistency. Using this space, we
may scale the functional relevance of any pairs ofgenes, with which 'meaningfulness' of any given genecluster can be calculated. In addition, the title for the
page-vector nearest to the center of a gene cluster will
explain the biomedical 'meaning' of the meaningfulcluster.
Publications
Papers1. Michibata, H., Chiba, H., Wakimoto, K, Seishima,M., Kawasaki, S., Okubo, K, Mitsui, H., Torii, H. andImai, Y. (2004). Identification and characterization of
a novel component of the cornified envelope, cornifelin.
Biochem Biophys Res Commun., 318, 803-13.
2. Hishiki, T., Ogasawara, 0., Tsuruoka, Y. andOkubo, K (2004). Indexing anatomical concepts to
OMIM Clinical Synopsis using the UMLS Metathesaurus.
In Silico Bio!., 4, 31-54.
3. Chiba, H., Michibata, H., Wakimoto, K, Seishima,M., Kawasaki, S., Okubo, K, Mitsui, H., Torii, H. andImai, y. (2004). Cloning of a gene for a novel epitheliumspecific cytosolic phospholipase A2, cPLA2delta,
induced in psoriatic skin. J BioI Chern., 279, 12890-7.

Books

4. Kaimori,]., Takenaka, M. and Okubo, K (2004).
Quantification of Gene Expression in Mouse andHuman Renal Proximal Tubules., rLaser Capture
Microdissection Methods and ProtocolsJ (Grame I.
Murray and Stephanie Curran ed.) Methods in
Molecular Biology 293, Humana Press, 209-220.
5. *~ f*0rn:, )II*tr-f- (2004) '?" / A ~ft ~;t /' " t:l"/- ~1t\ 5150)%mL1:."¥--J1t1t~~"¥J,M
Vo!.74 No.lO,
1254-1257.
EDUCATION

1. Dr. Okubo gave a seminar on "The computational
interpretation of biological data and its significance"
at National Institute of Genetics, Oct., 2004 (inJapanese).
2. Dr. Okubo gave a seminar on "How can we(biologists) make good use of biological knowledge
available?" for DDBJing and TERAKOYA, Tokyo,
Mar., 2004 (in Japanese).
3. Dr. Okubo was invited to give a seminar on "Medical Science in the post-genome period- knowledge
engineering in medicine" for Department of
Biomolecular Sciences Saga Medical School Facultyof Medicine at Saga University, Mar., 2004 (inJ:=tpanese).
SOCIAL CONTRIBUTIONS AND OTHERS

1. BodyMap: http://bodymap.genes.nig.ac.jp/
2. Databese: http://www.jbirc.aist.go.jp/hinv/h-angeV
3. Databese: http://bodymap.ims.u-tokyo.ac.jp
4. JST~]i±iiX~~7" t:l ~. '7 A '?" / A ~ § ~ftJ ::I;t- jJj- ~

~.

-If.~
5. e*"¥~~~ ?"/A~"¥W~~~~~~ ~~6. (ff) /'\~ ;tRIt~1W~1t::I/' '/ - ~ 7" A ~ /' /'\ -7 j!{.

~nM~·mm~W~*~~~~ ~aLaboratory for Gene-Expression Analysis

131

J. RADIOISOTOPE CENTERRESEARCH ACTIVITIES(1) Chromosome partitioning mechanism of Escherichia
coli; Involvement of a cisacting site on the chromosome
Yoshiharu Yamaichi and Hironori Niki....~.

During replication of the Escherichia coli
chromosome, the replicated Ori domains migrate
towards opposite cell poles, suggesting that a cis-actingsite for bipolar migration is located in this region. To
identify this cis-acting site, a series of mutants wasconstructed by splitting subchromosomes from theoriginal chromosome. One mutant, containing a 720 kb
subchromosome, was found to be defective in the
bipolar positioning of orie. The creation of deletionmutants allowed the identification of migS, a 25 bp
sequence, as the cis-acting site for the bipolar
positioning of orie. When migS was located at the
replication terminus, the chromosomal segment
showe? bipolar positioning. migS was able to rescue
bipolar migration of plasmid DNA containing amutation in the SopABC partitioning system.
Interestingly, multiple copies of the migS sequence ona plasmid in trans inhibited the bipolar positioning oforie. Taken together, these findings indicate that migS
plays a crucial role in the bipolar positioning of orie.
In addition, real-time analysis of the dynamic
morphological changes of nucleoids in wild-type andmigSmutants suggests that bipolar positioning of thereplicated oriC contributes to nucleoid organization.
(2) Partitioning mechanism of F plasmid; elucidation of
motive force for bipolar migration of the plasmid DNA
Toshiyuki Hatano and Hironori Niki
In a bacterial cell, replicated chromosomal
segments including the replication origin and the Fplasmid actively move from midcell toward cell poles

132 RADIOISOTOPE CENTER

and are located at cell quarter positions. The Sop ABC
partitioning system of F plasmid has been characterized genetically and biochemically to elucidate
molecular mechanism of DNA partitioning. The SopA
protein is a hypothetical motor protein because of its
walker-type ATPase activity. Moreover, some proteinshomologous to SopA change subcellular location
periodically. We have constructed an active SopA-YFP
and observed oscillation of YFP fluorescence from one
cell pole to the other with an interval of a few minutes.
Simultaneously migration of the plasmid DNA wasdetected using fluorescent labeling technique with lacOarray and Lacl-CFP. The analyses of movement ofboth the fluorescence suggest that SopA should not
be the motor protein to move the plasmid DNA.
Another new possibility is that the SopA protein could
govern the direction of movement of the plasmid DNA.
(3) Subcellular localization of Escherichia coli proteinsin living cells
Yasuyuki Ogata, Toshiyuki Hatano and Hironori Niki
Bacterial cells are too small to see details of theirstructure using a light microscope because of theultimate limit to the resolution of the microscope set
by the wavelength of visible light. In recent years, thedevelopment of methods for the specific labeling andimaging of individual cell components and thereconstruction of their.three-demensional architecture
allows us to understand the structural organization ofbacterial cells. Cytoskeletal filaments that bacteria
were long thought to lack were found by means ofthese methods and visualization of DNA polymeraseof the living Bacillus subtilis cells by tagging thecatalytic subunit with fluorescent protein supported
the model in which the polymerase is stationary likea factory and DNA is pulled through.
Escherichia coli genome has been fully sequenced
and the genes of unknown function revealed. To
estimate their function, we used a plasmid library,archive clone library, that expresses fusion genes
containing open reading flame (ORF) of E. coli W3110
strain and green fluorescent protein (GFP) gene andwe observed their subcellular localizations in the living
E. coli cells. These proteins were classified into fivecategories: (i) at cell periphery, (ii) in whole cell, (iii)spot, (iv) filament, and (v) on nucleoid.
We subsequently investigated the localizations of

the proteins of known function and found thecorrelations between subcellular localization andprotein function. Most of the cell peripheral proteins
have either trans membrane elements or signal
peptides. Proteins with spots at cell quarter positions
like a replication factory include replication machineries
and recombination enzymes and proteins with several
spots participate in transcription. Some proteins witha ring at mid cell are involved in cell division. Nucleoid
proteins contain nucleoid constituents such as
histone-like proteins and various enzymes involved in
DNA-related events including DNA supercoiling,
recombination, and repair.
We further replaced stop codon of these genes with
gfP gene by the method of Warner and confirmed
subcellular localization of these proteins.
(4) Stationary phase-induced illegitimate recombinationin Escherichia coli

Yasuyuki Ogata and Hideo Ikeda
In nature, bacterial cells are usually in stationary
stages, in which the cells alter their genetic inheritance.
Adaptive mutations are induced when Escherichia coli
cells are exposed to conditions of stationary-phasestarvation, resulting in a variety of genetic changes,
some of which allow the cells to survive under suchconditions. Gene amplification of the lac operon of E.
coli also occurs during stationary-phase starvation.
Whether or not stationary-phase starvation induces
chromosomal rearrangements has remained obscure.
We found that illegitimate recombination is
induced when growth phase is changed from log tostationary phase. Illegitimate recombination occurred
more frequently in early-stationary phase than in
mid-log phase during formation of Ie bio-transducing
phage. Moreover, we found that illegitimate
recombination increased in the tag alkAl double
mutant in early-stationary phase, thereby implying thatthe alkylated lesion may be responsible for thestationary phase-induced illegitimate recombination.
When we did a quantitative analysis of deletion
formation using a miniF-based plasmid, we found thatthe stationary phase-induced illegitimate recombination can be also detected in this assay system. In
addition, we indicated that the frequency of deletionof the tag alkAl double mutant was higher than thatof its isogenic wild-type strain in early-stationary

phase.
Since illegitimate recombination results inchromosomal rearrangements including deletion,
duplication, insertion, or translocation, it seems likelythat stationary phase may induce chromosomal
rearrangements. Thus, when bacterial cells confront
unfavorable conditions for their growth, the cells seemto increase diverse mutations as if they struggle foradaptation to the environment. If the situation takes
a favorable turn, a suitable mutant clone is likely togrow preferentially, which may lead to evolution.
PUBLICATIONSPapers

1. Yamaichi, Y. and Niki, H. (2004). migS, a cis-actingsite that affects bipolar positioning of oriC on the
Escherichia coli chromosome. EMBO J 23,221-233.
2. Ishida, T., Akimitsu, N., Kashioka, T., Hatano, M.,
Kubota, T., Ogata, Y., Sekimizu, K. and Katayama, T.
(2004). DiaA, a novel DnaA-binding protein, ensures thetimely initiation of E. coli chromosome replication_ J
BioI. Chem., 279, 45546-45555_
3. Ikeda, H., Shiraishi, K. and Ogata, Y. (2004).
Illegitimate recombination mediated by double-strand
break and end-joining in Escherichia coli. Adv. Biophys.,
38,3-20.
Reviews4.

e**!1l! (2004) rJJf:W~~(1)~fg1*(1)5t~tQ,8J~ E3

j!{,

WD, M~49, 2017-2023.

Books

5.

e**!1l!, 7/~~A'7oT~~~A(1)~~~~~1ffR(1)M#f (: JJtffl, .:c~. T -{ . .:cA, 47-56.~.·RADIOISOTOPE CENTER

133

among genomes and among species would give somerelationships to the molecular nature of genetic
diversity.

K. EXPERIMENTAL FARMRESEARCH ACTIVITIES*"

Experimaental Farm is responsible for preparationof all kinds of rice strains necessary for the studies ofgenetic resources and of functional genomics. Allworks in the Experimental Farm have been carried out
as collaborative works with the Plant Genetics Lab.
For details, see the reports of plant genetics lab.
(1) Examination of genetic and phenotypic nature of
newly prepared core collections for wild rice comparative genomics

Toshie Miyabayashi, Mitsugu Eiguchi, Ken-Ichi
Nonomura and Nori Kurata~I

For making rice genetic stocks good resources, wedeveloped core collection of wild rice. Out of 2,000
accessiops composed of twenty-one wild rice species
in nine genomes, several accessions from each species
were selected to prepare core collection. About 300
selected lines had already sub-grouped to Rankl,
Rank2 and Rank3 categories, and a part of them werecharacterized for their phenotypes to record them in
the Oryzabase, a rice comprehensive database.
Comparative genomics among nine rice genomes
will reveal important aspects on evolution and genetic
diversity in rice. For finding out genome specific genes
or far-related genes different from AA, comparison of
BB and CC genome species with the cultivated AA
species have been started. Searches for structural andfunctional diversity between genes of different genomes
are performed by eDNA clone sequencing and by
microarray expression profile analysis. Some details
obtained in 2004 were presented in the report of Plant
Genet. Lab.
In addition, to estimate genome sizes of eachgenome and species, strains in the core collection were
examined for their DNA content by the simple
flow-cytometric analysis. Differences of DNA content

134 EXPERIMENTAL FARM

PUBLICATIONSPapers1. Miyoshi, K, Ahn, B-O., Kawakatsu, T., Ito, Y., Itoh,j-I., Nagato, Y and Kurata, N. (2004). PLASTOCHRON1,
a timekeeper of leaf initiation in rice, encodes
cytochrome P450. Proc. Natl. Acad. Sci. USA 101,875-880.
2. Ito, Y, Chujo, A., Eiguchi, M. and Kurata, N. (2004).
Radial axis differentiation in a globular embryo ismarked by HAZ, a PHD-finger homeobox gene of rice.
Gene 331, 9-15.
3. Nonomura, KI., Nakano, M., Murata, K, Miyoshi,
K, Eiguchi, M., Miyao, A., Hirochika, H. and Kurata,
N. (2004). The insertional mutation of rice PAIR2gene,
the ortholog of Arabidopsis ASY1, caused a defect inhomologous chromosome pairing in meiosis. Mol.
Genet. Genomics 271, 121-129.
4. Nonomura, KI., Nakano, M., Fukuda, T., Eiguchi,M., Miyao, A., Hirochika, H. and Kurata, N. (2004). The
novel gene HOMOLOGOUS PAIRING ABERRA TION
IN RICE MEIOSIS 1 of rice encodes a putative
coiled-coil protein required for homologous
chromosome pairing in meiosis. Plant Cell 16,
1008-1020.
5. Salina, E.A., Adonina, I., Vatolina, T. and Kurata,
N. (2004). A comparative analysis of the compositionand organization of two subtelomeric repeat familiesof Aegilops speltoides Tausch.and related species.
Genetica 122, 227-237.
6. Moriguchi, K, Suzuki, T., Ito, Y., Yamazaki, Y,
Niwa, Y and Kurata, N. (2005). Functional isolation ofnovel nuclear proteins showing a variety of sub-nuclear
localizations. Plant Cell, 17, 389-403.
Reviews
7. Itoh, 1.1., Nonomura, KI., Ikeda, K, Yamaki, S.,
Inukai, Y., Yamagishi, H., Kitano, H. and Nagato, Y.
(2005). Rice plant development: from zygote to spikelet.
Plant Cell Physiol. 46, 23-47.
8. Kurata, N., Miyoshi, K, Nonomura, KI., Yamazaki,
Y and Ito Y. (2005). Rice mutants and genes related
to organ development, morphogenesis andphysiological traits. Plant Cell Physiol. 46, 48-62.

Books5.

1!1#i51.J*, fftk *tGZ (2004) y !J - ;(~Jfil2f~~~I·&¥~~~:~~~x*~~~~~m~~~.#.~
J*~1!EVo1.48 No.13, 1673-1679.

L. TECHNICAL SECTION

6.

1!1#i51.J*, fftk*tGZ (2004) nm~~~ f /'7 JvJlf~

TQ~~~x*~~~~.fftk*tGZ~:~~~x*~~~~. Springer Reviewsy!J -;(, y;:1.. 7!J /'ff- . 7 x

77-~Jl:OR,

129-134.

RESEARCH ACTIVITIESThe Technical section supports the research
activity of NIG in many fields. For example, we support
the Radioisotope center and Experimental farm, and
the Genetic strain research center where genetic andtransgenic strains of mice, rice, flies, and fishes areproduced, maintained, and distributed to otherinstitutions. Members of the staff additionally support
many types of experiments in various laboratories,
such as developmental, cell, and molecular genetics.
We continually update our technical services throughattending the training and annual meetings for thetechnological staffs.

.".

PUBLICATIONSPapers

1. Kakutani, T., Kato, M., Kinoshita, T. and Miura,
A. (2005). Control of Development and Transposon
Movement by DNA Methylation in Arabidopsis
thaliana. Cold Spring Harbor Symposia on
Quantitative Biology 69 in press.
2. Kinoshita, T., Miura, A, Choi, Y., Kinoshita, Y.,
Cao, X., Jacobsen, S.E., Fischer, RL. and Kakutani, T.
(2004). One-way control of FWA imprinting in
Arabidopsis endosperm by DNA methylation. Science
303, 521-523.

3. Kubota, T., Furuumi, H., Kamoda, T., Iwasaki, N.,
Tobita, N., Fujiwara, N., Goto, Y, Matsui, A, Sasaki,
H. and Kajii, T. (2004). ICF syndrome in a girl with
DNA hypomethylation but without detectable
DNMT3B mutation. Am. J. Med. Genet. 129A, 290-293.
4. Miura, A., Kato, M., Watanabe, K., Kawabe, A,
Kotani, H. and Kakutani, T. (2004). Genomic
localization of endogenous mobile CACTA familytransposons in natural variants of Arabidopsis thaliana.
MGG 270, 524-532.

TECHNICAL SECTION

135

BIOLOGICAL SYMPOSIUM 2004
Jan.9
Jan.13
Jan.19Jan.21
Jan.28
Feb.3
Feb.26
Feb.26
Feb.27,.,

Mar.3
Mar.l5Mar.19Mar.23Mar.24Mar.24Mar.25
Apr.8
Apr.8
Apr.21
Apr.30
May.12
May.13May.l8May.24
May.31
Jun.3
Jul.5
Jul.7
JuLl4
JuLl5
Jul.27Aug.9
Aug.9
Aug.11
Aug.26

WMJ!-8-T . *,*Ijf{~-T . ~{l::jf-8-T : .:c-O)~vn: C: ~~1t (Nozomu Mori)
DNA Sequencing: A Tool for Biomedical Research (Shih-Feng Tsai)~~{M}IjC, -::n;O~ G{iIJ~liJf9'CT9? (Mitsuhiro Yanagida)
The Master Regulator for Entry into Sporulation in Bacillus subtilis becomes a Cell-Specific
Transcription Factor after Asymmetric Division (Masaya Fujita)
Mammalian micro RNA genes and their downstream targets (Roberto Barrero)
Sequencing the Rice Genome - Where do we go from here? (Rod, A. Wing)
Chemistry of Methylation: from DNA to Histone (Xiaodong Cheng)
H§')tJj;r.IIlJffllO)~'lt'i~C: lti!51l§1'tlm (Takashi Kadowaki)
Real time footprinting techniques reveal the mechanism of promoter recognition by E. coli RNA
polymerases (Malcolm Buckle)
B~f;j< . .Ifn.!NqiO)~~EJf,]~:t~~nX:~. C: .:c-O)~~",-O)J;tffl (Yuka Nagata):*Jmif ~:Rtl:fMf:lH~U"i*f*O)X ~*E~.JiIm#f (Satoshi Muarakami)
Cooperation between RNA polymerase molecules in transcription elongation (Evgeny Nudler)
SEEDLINGS, CHICKS AND CHILDREN: A DARWINIAN PEDIGREE FOR EUGENICS Games Moore}
Molecular genetic basis of the ABO system & expression profiling of glycosyltransfer ase genes(Fumiichiro Yamamoto)iXffl:f\;;J!{~-T~ffl.7' t:1 7 -{ - Ivm#frHirr : HiCEP (Masumi Abe)
Implications of Small Molecule Activator and Inhibitors of Histone Acetyltransferase p300 in Chromatin
Therapy (Tapas K. Kundu)
Meiotic chromosome morphogenesis and function in C. elegans (Kentaro Nabeshima)
Regulation of spindle orientation by apc tumor suppressor and centrosome in asymmetric stem cell
division in Drosophila (Yukiko Yamashita)~ :3 tJ:J :3 tJ /":r.O)MtH§''lt C: lIiiI.J:ftJ;t1§:,!tE : lIiiI.J.lIlltz:f'!tE~~f*O)*Mj'[ C: m#f (Shingo Umeda)~ :3 tJ:J :3 tJ /":r.*,*I~UIIJffllO)~~it#J\5}~'H::tolt 9"'-7 t:1 3 iif*G?' /' /":7 'ltO)f~i!llj (Naoyuki Fuse)
Licensing mitotic entry: mechanisms of concerted phosphorylation and down-regulation of Swel bymultiple kinases (Satoshi Asano)
Drosophila male GSCs (germline stem cells) I~:to It 9 BMP ~!lTIvO)f~i!fIJ (Eihachiro Kawase)
Follicle-Stimulating Hormone (FSH) and Testis Functions in Fish (Rudiger W. Schulz)
If / A ?' -{ !J /,~' 7 v -{ ~ ffllt \ tc. If / A ~:t~m#f (Katsuhiko Shirohige)
FGF signaling during mesoderm migration in the Drosophila gastrula (Amo Mueller):*Jmif~lI;A " v ;AJ;t1§:I~~:b9H~7't:177---e YaeL 0)~1i~ C: .:c-O)IDi\Jim (Yoshinori Akiyama):r. t" :J ;r. -* 7 -{ 'Y :7 t,J: 1':!t:.J~,!tE C: If / A -{ /' 7'!J /' 7 -{ /'!I (Kozo Mitsuya)
fli~O)~;O>m:h1Jrt;H~J;tl;-cJm~T9;( 1J .:::.XA (Masao Tasaka)~~~!!IH:'7!J /'!I C: .:c-O)lt1frt11f¥R~;A 7 A ",-O)J;tffl (Kazuyuki Aihara)
Navigating Massive Data Sets via Local Clustering (Michae E. Houle)
BRUl, a novel link between DNA damage response and epigenetic regulation in Arabidopsis(Shin Takeda)t ,,:to J:: V:;( Y1J I~:to It 9~i'i~'lt0)J!-8,¥EJf,]m#f (Minori Shinya)~&O)~~{I:: C: ~~~nX:I~:tolt9--e7'7 7 -{ 'Y ~.::r.. Vax ~~~-T0)~1i~ (Masaya Takeuchi)§ Mlt~ ~ t:1 -{ ?< T ':;(T;O>\: ~ 9 ~ \:"-' Y- tJ -{ >fJX~",-O)5}-T~ffiJ!{~'¥;O~ GO)m#f"-' (Kentaro Shimizu)

T /'.I" /':J- 221fi:~~f*C: O).l:t~l~ J:: 9 211fi:~~f*I~:tolt 91E0)j'j(Jtt<.:~J{:b!XO)iW~1it~fJ~JO)mfE : t "C:T/'/"/':J-~5}lt~-ctc.'bO)

Aug.30Sep.l
Sep.l(Satoshi Ota)
The origin and evolution of vertebrate mineralized skeleton (Kazuhiko Kawasaki)
77/ tJ -{ Iv;AA::7 ?'-I~J::9*,*I*HIJffllO)~ltB~~EJf,]J!-8-T11fA¥!~mlt\tc.,!N&I~nX:~.O)m#f(Mithsuhiro Hashimoto)In vivo imaging of Xenopus embryo early stages development using Quantum Dots encapsulated

136 BIOLOGICAL SYMPOSIUM 2004

Sep.13
Sep.l6
Sep.17
Sep.21
Sep.24
Sep.27
Sep.30
Oct.1
Oct.7
Oct.21
Nov.5
Nov.15
Nov.17
Nov.22
Nov.22
Nov.24
Nov.24
Nov.25
Nov.26
Dec.6
Dec.6
Dec.7
Dec.7
Dec.13
Dec.16
Dec.16
Dec.20
Dec.20in phospholipid micelles (Benoit Dubertret)
Tsukushi functions as a novel organizer inducer by inhibition of BMP activity in cooperation withchordin (Kunimasa Ota)
If;WJjIJ~llIifflH~ft.~(l)Jj§ft (Shigeru Kutaratani)-7- -{;A (-7"-{ X) fifJ:if1J!l-@.1I\;O~?-f!i~'""('I~H~H"J f.:~1At~ n:5 j] lv:::r /:Y / -7 --eJ!~-:;-(l) PTGS f.:'0
11 \ -c (Mineo Senda)
INlllf*&bff~:E!I!(l)7}-:;-:jj:fi\\' (Kazuhiro Nagata)
Dictyostelium and its genome project (Robert Kay).:.t~':\'-T/ft~;J:./ F-lT-{ !,-:Y;Af':J::5Mt.Ji~v-e7"?<'- Notch (l)ftIJlilJ (Shigeo Hayashi)
CDK ft(:.(¥H"J BRCA2 !J /~ftf': J: :5*ll.J;.~k {~1lftljlilJ (Fumiko Esashi)
Tbx J!{~-:;-M(l)Jj§fU::~1'!ur%x:;( j] .:::.XA(l)Jj§ft (Toshihiko Ogura):Y 3'7 Y 3'7 /'\;J:.f.:fMr :51::Mffj 'Y -e /7"!J - ~ 1::M*IIIJm~px::jj:fi\\' (Akira Nakamura)G9a / GLP ~ftf*f': J::5 in vivo 1:::;A !' / ;( T lvftftlJlilJ (Makoto Tachibana)Specification and assembly of locomotion in Drosophila (Michael Bate)lll~jJ-{ Y/;Af.::tQft:5*;A 77TYlv-{ /:Y !'-lv4 -!J /~5 -:\'--t---e(l)l~j§lJ (Masakazu Yamazaki)
Looking for the Genetic Origins of the Human Capacity for Language (Karl C. Diller)
iIiIlI~~1AtJ!{~-:;-lola ;O~~f*fJ:J!{~-:;-~~~1::J;.I±IT;( j] .:::.XA (Takayuki Horiuchi)
Geminin-Cdtl balance as a guardian against genomic instability (Anindya Dutta)-e 7" -7 -~T t:1 :Y/:\'--t--.-e' PVR ~ -t:-(l)rmt:y!T-t- lvfa~ f.: J::5 JNK (l)m'ltftU (Satoshi Ishimaru):Y 3 '7 Y 3 '7 /,\;J:.~fIJJij L-td$i1\l@)lm~~PX:(l)JI{~'¥:H"JM:#f (Mitsuhiko Kurusu)
If / AM:#f;O~ ~ ~tc1::M*IIIJm(l)*¥ft (Masami Nozaki)
RNAi i1\llm f.::tQ ft:5 Argonaute ~ Sf{ (l):jjfll§M:#f (Katsutomo Okamura)SPATIAL VARIATION IN GENE FREQUENCY (Aravinda Shakravarati)
Control of dendrite development (Yuh-Nung Jan)Histone Modifications in Polycomb Silencing, X-inactivation and Cancer (Yi Zhang)
Regulation of Nucleosome Assembly during DNA Replication (Alain Verreault)
Bi-orienting chromosomes on the mitotic spindle (Tomoyuki U. Tanaka)
Splicing profile-based protein categorization (Ake Johannes Vastermark)Cell cycle dependent acetylation of histone H3 lysine 56 contributes to double strand break repair .
in Saccharomyces cerevisiae (Hiroshi Masumoto)THE HUMAN SGTI - HSP90 COMPLEX IS REQUIRED FOR THE ASSEMBLY OF KINETOCHOREPROTEIN COMPLEXES (Katsumi Kitagawa)
The role of SMCl in DNA damage induced signaling pathways (Risa Kitagawa)

BIOLOGICAL SYMPOSIUM 2004

137

'.\"

FOREIGN VISITORS IN 2004•

Jan.13Shih-Feng Tsai

Jan.21
Jan.28Feb.3

Masaya Fujita
Roberto Barrero
Rod, A. Wing

Feb.26

Xiaodong Cheng

Feb.2?

Malcolm Buckle

Mar.19Evgeny Nudler

Mar.23Mar.24James Moore
Fumiichiro Yamamoto
Tapas K. Kundu

Apr.8
May.12
May.13
May.18May.31
Jun.?
JuLl5

Yukiko Yamashita
Satoshi Asano
Eihachiro Kawase
Rudiger W. Schulz
Arno Mueller
Masataka Okabe
Michae E. Houle

Jul.2?

Shin Takeda
Kentaro Shimizu

Aug.30
Sep.I
Sep.24
Sep.30Nov.5

Kazuhiko Kawasaki
Benoit DubertretRobert Kay
Fumiko Esashi
Michael Bate

Nov.11
Nov.l?
Nov.21
Nov.22
Nov.24
Nov.24
DecA
Dec.6

Hans R. Bode
Karl C. Diller
David Liberles
Anindya Dutta
Mitsuhiko Kurusu
Shozo Yokoyama
Antoine Blancher
Aravinda Shakravarati

Dec.6
Dec.?
Dec.?

Yuh-Nung Jan
Yi Zhang
Alan Verreault"'~t'Mar.25, '-if'•, Aug.11

138 FOREIGN VISITORS IN 2004

Division of Molecular & Genomic Medicine, National Health ResearchInstitutes, Taipei 115, TAIWAN
Department of Molecular and Cellular Biology Harvard University
Japan Biological Information Research Center, JBIRC-Tokyo
Arizona Genomics Institute, Department of Plant Science, The Universityof Arizona
Biochemistry Department Emory University School of Medicine Atlanta,
GA,USA
Enzymologie et cinet:ique structurale, LBPA, UMR 8113 du CNRS, Ecole
Normale Superieure Cachan cedex France
Department of Biochemistry, New York University School of Medicine,
New York, USA
The Open University UK
The Burnham Institute, La Jolla, CA, USA
Transcription and Disease Laboratory, Molecular Biology and Genetics
Unit, Jawaharlal Nehru Center for Advanced Scientific Research, India.
Stanford University
National Institutes of Health
Stowers Institute for Medical Research, Kansas City, USA
Dept Endocrinology, Fac Biology, University of Utrecht, The Netherlands
Institute for Genetics, Heinrich-Heine University, Dusseldorf
MRC Centre for Developmental Neurobiology, King's College London
National Institutes of Informatics Data mining (search, clustering,classification), design and analysis of algorithms, visualization,
combinatorial geometry
Lab of Plant Genetics University of Geneva Switzerland
North Carolina State University / Graduate School of Science, Kyoto
University
Department of Anthropology, Pennsylvania State University
Laboratoire d'Optique Physique, ESPCI, Paris, France
MRC Laboratory of Molecular Biology, Cambridge, England
Cancer Research UK, London Research Institute Clare Hall Laboratories
Department of Zoology Cambridge UK and National Centre for
Biological Sciences, Bangalore, India
Dept. of Developmental and Cell Biology, Univ. of California, Irvine
University of New Hampshire USA
Comptatioal biology unit, University of Bergen
University of Virginia Health Sciences Center
California Institute of Technology Kai Zinn Laboratory
Dept. of Biology Rollins Research Center Emory University
Laboratoire d'immunologie, Hopitale Rangueil, Toulouse France
Henry ]. Knott Professor & Director McKusick-Nathans Institute of
Genetic Medicine Johns Hopkins University School of Medicine
University of California, San Francisco
University of North Carolina at Chapel Hill NC, USA
Cancer Research UK, London Research Institute, Clare Hall Laboratories

Dec.13

Tomoyuki U. Tanaka

Dec.15Gerry WeinmasterDec.16
Dec.16
Dec.20

Ake Johannes Vastermark
Hiroshi Masumoto
Katsumi Kitagawa

Dec.20Risa Kitagawa

School of Life Sciences, University of Dundee, Wellcome Trust Biocentre
Dundee UK
Biological Chemistry Department David Geffen School of Medicine at
UCLA
Bioinformatics Group, Department of Statistics, University of Oxford
Cancer Research UK, London Research Institute, Clare Hall Laboratories
Department of Molecular Pharmacology, St. Jude Children's Research
Hospital, Memphis USA
Department of Hematology-Oncology St. Jude Children's Research
Hospital, Memphis U.S.A

j

FOREIGN VISITORS IN 2004139•

Annual Report of the National Institute of Genetics. No.55
Issued by Director-General: Yuji Kohara. D.Sc.
Research Organization of Information and Systems
National Institute of Genetics
Yata 1111 Mishima. Shizuoka-Ken. 411-8540 Japan
Tel. 055-981-6718
Fax. 055-981-6715
Edited by Ryu Ueda
Published by National Institute of Genetics