Highlights of 2003 (http://www.nig.ac.jp/hot/index.html)

1. Conserved Role of nanos Proteins in Germ Cell Development.
Tsuda, M., Sasaaka, Y., Kisa, M.,Abe, K.,Haraguchi, S., Kobayoshi, S. and Saga, Y.
Science 307, 1239-1241, 2003.
2. Tracking FACT and the RNA polymerase II elongation complex throughchromatin in vivo.
Saunders, A, Werner, J. Andrulis, E. D., Nakayama, T, Hirose, S., Reinberg, D. andlis, J T
Science 301, 1094-1096, 2003.

3. The MSP 1 gene is necessary to restrict the number of cells entering into maleand female sporogenesis and to initiate anther wall formation in rice.
Nonomura, K., Miyoshi, K., Eiguchi, M., Suzuki, 1, Miyao, A, Hirochika, H. and
Kurata, N.
PlantCell 75,1728-1739,2003.
4. GINS, a novel multi-protein complex required for chromosomal DNA
replication in budding yeast.
Takayama, Y., Kamimura, Y., Okawa, M., Muramatsu, S., Sugino, A and Araki, H.
Genes & Dev., 77, 1153-1165, 2003.
A novel ring-like complex of Xenopus proteins essential for the initiation of
DNA replication.
Kubota, Y., Takase, Y., Komori, Y., Hashimoto, Y., Arata, T, Kamimura, Y., Araki, H. andTakasawa, H.
Genes & Dev., 77, 1 141-1152, 2003.
5. SDF-9, a protein tyrosine phosphatase-like molecule, regulates the L3/dauer
developmental decision through hormonal signaling in C. elegans.
Ohkura, K., Suzuki, N., Ishihara, T and Kalsura, I.
Development, 130, 3237-3248, 2003.

6. Drosophila FACT contributes to Hox gene expression through physical andfunctional interaction with GAGA factor.
Shimojima, 1, Okada, M., Nakayama, T, Ueda, H., Okawa, K., Iwamatsu, A, Handa,
H. and Hirose, S.
Genes & Dev., 77,1605-1616,2003.
7. Role of CG and non-CG methylation in immobilization of transposons

In

Arobidopsis.
Kato, M., Miura, A, Bender, J., Jacobsen, S.E. and Kakutani, T
Curro BioI., 13, 421-426, 2003

8. The genome sequence and structure of rice chromosome 1.
Sasaki, 1, Matsumoto, T, Yamamoto, K., Sakata, K., Baba, T, Katayose, Y., Wu, J.
Niimura, Y., Cheng, Z., Nagamura, Y., Antonio, B. A, Kanamori, H., Hosokawo, S.,
Masukawa, M., Arikawa, K., Chiden, Y., Hayashi, M., Okamoto, M., Ando, T, Aoki,
H., Arita, K., Hamada, M., Harada, c., Hijishila, S., Honda, M., Ichikawa, Y.,
Idonuma, A, lijima, M., Ikeda, M., Ikena, M., Ito, S., Ito, 1, 110, Y., Ito, Y., Iwabuchi,
A, Kamiya, K., Karasawa, W., Katagiri, 5., Kikuta, A, Kobayashi, N., Kono, I.,
Machita, K., Maehara, T, Mizuno, H., Mizubayashi, T, Mukai, Y., Nagasaki, H.,
Nakashima, M., Nakama, Y., Nakamichi, Y., Nakamura, M., Namiki, N., Negishi, M.,
Ohta, I., Ono, N., Saji, S, Sakai, K., Shibala, M, Shimokawa, T, Shomura, A, Song,J. Takazaki, Y., Terasawa, K., Tsuji, K., Waki, K., Yamagata, H., Yamane, H., Yoshiki,S, Yoshihara, R., Yukawa, K., Zhong, H, Iwama, H., Endo, T, Ito, H., Hahn J H."
Kim, H.-I., Eun, M.-Y., Yano, M., Jiang, J and Goiobori, T
Nature, 420, 312 - 316, 2002.

CONTENTS
Introduction····································································································STAFF
COUNCIL
ADVISORY COMMITTEE

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A. DEPARTMENT OF MOLECULAR GENETICS

A-a. Division of Molecular Genetics
Tatsuo Fukagawa Group
Assembly of kinetochores in higher vertebrate cells
Yoshikazu Mikami, Tetsuya Hori, Mitsuko Yoshikawa, Atsushi Fukushima and Tatsuo
Fukagawa
8
Molecular analysis of the Nuf2-Hecl complex that transiently localizes to centromere duringmitosisTetsuya Hori, Atsuto Uenoyama, Kazuko Suzuki and Tatsuo Fukagawa
8Functional roles of the RNAi machinery in verterbrate centromeres
Masahiro Nogami, Tomoko Motohashi, Mitsuko Yoshikawa and Tatsuo Fukagawa......................................................................................................... 8
A-b. Division of Mutagenesis
Fumiaki Yamao Group
Ubiquitin Pathway Regulating Recombination and DNA Damage Repair in Fission Yeast.
9
Joon-Hyun Park, Toyoaki Natsume and Fumiaki Yamao
A-c. Molecular Mechanism Laboratory
Hiroshi Mitsuzawa Group
Identification of proteins that interact with the Rpb7 subunit of RNA polymerase II
Hiroshi Mitsuzawa
10
A-c. Molecular Mechanism Laboratory
Hiroaki Seino Group
Ubiquitin-conjugating enzymes involved in polyubiquitination of mitotic cyclin
Hiroaki Seino

10

A-d. Division of Nucleic Acid Chemistry
Saburo Aimoto Group
Development of a strategy that enables to use the thioester method and the native chemical
ligation method for the synthesis of a single protein
Saburo Aimoto
11
Development of a photoremovable ligation auxiliary for an extended native chemical ligation
methodSaburo Aimoto
11
Synthetic studies of G protein-coupled receptor, opioid receptor like-l (ORL-l)
Saburo Aimoto

12

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

12

A-d. Division of Nucleic Acid Chemistry
Tsutomu Katayama Group
Transcriptional control for initiation of chromosomal replication in Escherichia coli: fluctuationof origin transcription ensures timely initiation
Masayuki Su'etsugu, Akiko Emoto, Kazuyuki Fujimitsu, Kenji Keyamura and Tsutomu

CONTENTS

Katayama

13

Structural analysis of the DnaA DNA-binding domain complexed with DNA: Structural basisof replication origin recognition by the DnaA protein
Norie Fujikawa, Hitoshi Kurumizaka, Osamu Nureki, Takaho Terada, Mikako Shirouzu,
Tsutomu Katayama and Shigeyuki yokoyama·············································· 13
Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in
Escherichia coli: Binding of DnaA to the sliding clamp-loaded DNA and binding of thesliding clamp to Hda
Masayuki Su'etsugu, Makoto Takata, Toshio Kubota and Tsutomu Katayama
14
B. DEPARTMENT OF CELL GENETICS

B-a. Division of Cytogenetics
Tamotsu Yoshimori Group
Membrane traffic: the intracellular transport network organized by proteins and membranes
Tamotsu Yoshimori
15
Identification of a new component of a large protein complex associated with the autophagic
membrane
Noboru Mizushima, Akiko Kuma, Yoshinori Kobayashi, Akitsugu Yamamoto, Masami
Matsubae, Toshifumi Takao, Tohru Natsume, Yoshinori Ohsumi and Tamotsu
Yoshimori
15
Involvement ·of autophagy in degradation of unfolded proteins causing diseases
Shisako Shouji, Takahiro Kamimoto, David H. Perlmutter, Akira Kakizuka and Tamotsu
Yoshimori
15
Analysis of autophagic response to invasion of pathogenic bacteria into cells
Kayoko Tsuda, Ichiro Nakagawa, Atsuo Amano and Tamotsu Yoshimori
Reconstitution of early step of autophagy by using semi-intact cells
Shunsuke Kimura, Atsuki Nara and Tamotsu Yoshimori
Functional analysis of SKDl by RNAi
Atsuki Nara, Masako Sakai and Tamotsu YoshimoriInteraction of Toml with Tollip, ubiquitin, and clathrin
Megumi Yamakami, Tamotsu Yoshimori and Hideyoshi Yokosawa
Regulated transport of yeast amino acid permease
Kyohei Umebayashi and Akihiko Nakano

16
16
16
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16

Ubiquitin-dependent protein sorting in the endocytic pathway
Kyohei Umebayashi, Min-Soo Kim, Yasue Hatanaka and Tamotsu Yoshimori ...... 17
B-b. Division of Microbial Genetics
Hiroyuki Araki GroupThe interaction between replication proteins for the initiation of DNA replication
18
Yoichiro Kamimura and Hiroyuki Araki
Molecular mechanism of the initiation of DNA replication regulated by cyclin-dependent
kinase (CDK)
Yon-Soo Tak, Yoichiro Kamimura, Seiji Tanaka and Hiroyuki Araki·
19
Functional analysis of the Dpbll protein
Sachiko Muramatsu and Hiroyuki Araki······················································ 19Counteractions of DNA polymerase E and yCHRAC for epigenetic inheritance of telomere
position effect variegation (TPE)
Tetsushi Iida and Hiroyuki Araki······························································· 20

CONTENTSB-b. Division of Microbial Genetics
Seiichi Yasuda Group
Mechanism of DnaA interaction with phospholipids
Seiichi yasuda······················································································ 20
B-c. Division of Cytoplasmic Genetics
Takeo Yoshikawa Group
Linkage disequilibrium (LD) scanning of functional psychoses
Takeo yoshikawa·················································································· 21
Genetic and functional analyses of candidate genes
Takeo yoshikawa·················································································· 21
Studies on animal models
Takeo Yoshikawa····························22
C. DEPARTMENT OF DEVELOPMENTAL GENETICS

C-a. Division of Developmental Genetics
Yasushi Hiromi GroupLongitudinal axon guidance in the Drosophila CNS
25
Masaki Hiramoto and Yasushi Hiromi
Cell-autonomous axonal patterning in Drosophila neurons
Takeo Katsuki, Masaki Hiramoto and Yasushi Hiromi
25
How is the positional information reorganized?
Tsuyoshi Nagasaka, Masaki Hiramoto and Yasushi Hiromi
25
Nuclear receptor Seven-up is essential for the temporal switching of Neuroblast fate in the
Drosophila CNS.
26
Makoto Kanai, Masataka Okabe and Yasushi Hiromi
A search for Seven-up-interacting proteins
Takayuki Hondoh and Yasushi Hiromi
26
Diversification and differentiation of lateral glia in the Drosophila CNS
Yoshihiro Yuasa and Yasushi Hiromi

26

Development of a novel fluorescent probe to visualize caspase activity in a living organism
Shu Kondo and Yasushi Hiromi
27
The establishment of the stem cell fate in the Drosophila female germline: A cell lineage study
Miho Asaoka and Haifan Lin···································································· 27
C-a. Division of Developmental Genetics
Toshitaka Fujisawa Group
Compartments of Neuron Subtypes Revealed by in situ Hybridization of Neuropeptide Genesin Hydra.
Toshitaka Fujisawa, Chiemi Fujisawa, Yasuharu Takaku, Yukihiko Noro, Shiho
Hyayakawa and Takashi Gojobori
28
Evolutionary Origin of Autonomic Nervous System in Hydra.
Hiroshi Shimizu, Masataka Okabe and Toshitaka Fujisawa .... ·· .... ·.... · .. ·.......... ·28
Motility of Endodermal Epithelial Cells Plays a Major Role in Reorganizing Endodermal and
Ectodermal Tissue Layers.
Yasuharu Takaku, Takahiko Hariyama and Toshitaka Fujisawa
28
C-b. Division of Gene Expression
Susumu Hirose Group
Role of GAGA factor-dependent chromatin remodeling in epigenenetic gene expression
Tsukasa Shimojima, Masahiro Okada, Takahiro Nakayama, Hitoshi Veda, Kenichi
Nishioka, Hiroshi Handa, Tadashi Wada and Susumu Hirose···························· 29

CONTENTSTracking of FACT and RNA polymerase nelongation complex through chromatin in vivo
Abbie Saunders, Janis Werner, Erik D. Andrulis, Takahiro Nakayama, Susumu Hirose,
Danny Reinberg and John T. Lis
30
Role of DNA topology in the formation of active chromatin
Kuniharu Matsumoto, Hirofumi Furuhashi, Youhei Ogasawara, Hitoshi Veda, Kazuma
Hanai, Koji Akasaka and Susumu Hirose····················································· 30
Functional analysis of transcriptional coactivator MBFl
Qin-Xin Liu, Marek Jindra, Hitoshi Veda, Masataka Okabe, Yasushi Hiromi and Susumu
Hirose···········································30
Mechanism of transcriptional regulation of the FTZ-Fl geneYasuo Agawa, Masa-aki Yamada, Susumu Hirose and Hitoshi Veda··················· 31
Establishment of a new mutant screening system in the silkworm Bombyx mori
Hitoshi Veda, Yuko Nagashima and Toshiki Tamura
31
Histone lysine methylation: a signature for chromatin function
31
Robert J. Sims, ill, Kenichi Nishioka and Danny Reinberg
Facile synthesis of site-specifically acetylated and methylated histone proteins: Reagentsfor evaluation of the histone code hypothesis
Shu He, David Bauman, J amaine S. Davis, Alejandra Loyola, Kenichi Nishioka, Jennifer
L. Gronlund, Danny Reinberg, Fanyu Meng, Neil Kelleher and Dewey G. Mccafferty...................................................... ···················································31
Methods and tips for the purification of human histone methyltransferases
Kenichi Nishioka and Danny Reinberg

32

Construction of a replication-selective adenovirus for ovarian cancer therapy
Katsuyuki Hamada, Shohei Kohno, Mari Iwamoto, Hiroko Yokota, Masato Okada,
Masatoshi Tagawa, Susumu Hirose, Kenshi Yamasaki, Yuji Shirakata, Koji Hashimotoand Masaharu Ito32
Coco Division of Molecular and Developmental Biology
Koichi Kawakami Group
Gene trap approaches in zebrafish
33
Koichi Kawakami, Yasuyuki Kishimoto and Ghislaine Morvan
Genetic analysis of zebrafish maternal-effect mutations affecting early embryogenesis
Yasuyuki Kishimoto, Koichi Kawakami, Sumito Koshida, Makoto Furutani-Seiki and
Hisato Kondoh
34
Transposition of the Tol2 element in mouse embryonic stem cells.
Koichi Kawakami and Tetsuo Noda

35

The Fugu EST projects
Melody S. Clark, Yvonne J.K. Edwards, Dan Peterson, Sandra W. Clifton, Amanda J.
Thompson, Masahide Sasaki, Yutaka Suzuki, Kiyoshi Kikuchi, Shugo Watabe, Koichi
35
Kawakami, Sumio Sugano, Greg Elgar and Stephen L. Johnson
Characterization of the hagoromo gene in East African cichlids
Yohei Terai, Naoko Morikawa, Koichi Kawakami and Norihiro Okada
35
Cod. Division of Physiological Genetics
Haruhiko Koseki Group
Functional and biochemical analyses of mammalian Polycomb-group proteins
Haruhiko Koseki
36
Cod. Division of Physiological Genetics
Yukiko Gotoh Group
Regulation of neural development and cell death
Koji Oishi, Maiko Higuchi, Fuminori Tsuruta, Jun Sunayama, Yoko Ogawara, Norihisa

CONTENTSMasuyama and Yukiko Gotoh·············38

D. DEPARTMENT OF POPULATION GENETICS

D-a. Division of Population Genetics
Naruya Saitou Group
Construction of a gorilla fosmid library and its PCR screening system
Choong-Gon Kim, Asao Fujiyama and Naruya Saitou

40

Netview: Aplication software for constructing and visually exploring phylogenetic networks· .. ·40
Kirill Kryukov and Naruya Saitou
Understanding the dynamics of Spinocerebellar Ataxia 8 (SCA8) locus through a comparative
genetic approach in humans and apes
Aida M. Andres, Marta Soldevila, Naruya Saitou, Victor Volpini, Francese Calafell andJ aume Bertranpetit
40
Evolution of the cystatin B gene: implications for the origin of its variable dodecamer tandem
repeat in humans
Motoki Osawa., Mika Kaneko, Hidekazu Horiuchi, Takashi Kitano, Yoshi Kawamoto,
Naruya Saitou and Kazuo Umetsu
40
Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability
and synthesis of the receptor
Jubao Duan, Mark S. Wainwright, Josep M. Cameron, Naruya Saitou, Alan R. Sanders,
41
Joel Gelernter and Pablo V. Gejman
Mitochondrial DNA polymorphisms in nine aboriginal groups of Taiwan: implications forthe population history of aboriginal TaiwaneseAtsushi Tajima, Takafumi Ishida, Naruya Saitou and Satoshi Horai
41
Sequence polymorphisms of the mitochondrial DNA control region and phylogenetic analysis
of mtDNA lineages in the Japanese population
Sayaka Maruyama, Kiyoshi Minaguchi and Naruya Saitou
42
Microsatellite variation in Japanese and Asian horses and their phylogenetic relationshipusing a European horse outgroup
Teruaki Tozaki, Naoko Takezaki, Telhisa Hasegawa, Nobushige Ishida, Masahiko
Kurosawa, Motowo Tomita, Naruya Saitou and Harutaka Mukoyama
42
The diversity of bovine MHC class n DRB3 genes in Japanese Black, Japanese Shorthorn,
Jersey and Holstein cattle in Japan.
Shin-noske Takeshima, Naruya Saitou, Mitsuo Morita, Hisetoshi Inoko and Yoko Aida...................................................... ···················································42
D-a. Division of Population Genetics
Toshiyuki Takano Group
Non-random association between variants at the Drosophila olfactory and gustatory receptor
genes
Akira Kawabe, Yuriko Ishii, Masanobu Itoh, Nobuyuki Inomata, Rumi Kondo, Noriko···44
Nanba, Yutaka Inoue and Toshiyuki Takano-Shimizu .. · .. ·DNA variation at Odorant binding protein genes in Drosophila
Aya Takahashi and Toshiyuki Takano-Shimizu
45
Searching for genes involved in sexual isolation between Drosophila simulans and Drosophilamelanogaster
Aya Takahashi, Kuniaki Takahashi, Ryu Ueda and Toshiyuki Takano-Shimizu ..... 45
Genetic and molecular basis of the within- and between-species variation in the sex comb
teeth number in Drosophila.
45
Haruki Tatsuta, Yuriko Ishii and Toshiyuki Takano-Shimizu

CONTENTS

D-b. Division of Evolutionary Genetics
Toshimichi Ikemura GroupA novel bioinformatics strategy for unveiling hidden genome signatures
Takashi Abe, Yuta Ichiba and Toshimichi Ikemura

46

Self-organizing map reveals sequence characteristics of 90 prokaryotic and eukaryotic genomes
on a single map
Takashi Abe, Atushi Fukushima,Yoko Kosaka and Toshimichi Ikemura
46
Novel bioinformatics developed for phylogenetic classifications of genomic sequences fromuncultured microorganisms in environmental samples
Takashi Abe, Yoko Kosaka, Jian-Ping Song and Toshimichi Ikemura
46
Inter- and intraspecies characterizations of eukaryotic genome sequences and in silico
prediction of genetic signal sequences
Takashi Abe, Yoko Kosaka, Yuta Ichiba, Kiyomi Kita and Toshimichi Ikemura ..... 47
E. DEPARTMENT OF INTEGRATED GENETICS

E-a. Division of Human Genetics
Hiroyuki Sasaki Group
Analyses of the distal imprinted domain on mouse chromosome 7 and its orthologue onchicken chromosome 5
Hiroyuki Sasaki, Takaaki Yokomine, Hisao Shirohzu, Chikako Suda, Wahyu
Purbowasito, Masaoki Tsuzuki, Atsushi Toyoda, Masahira Hattori and Yoshiyuki Sakaki...................................................... ············································· .. ····48
Imprinting mechanisms of the mouse Igf2/ H19 subdomain
Hiroyuki Sasaki, Yuzuru Kato, Ko Ishihara, Melanie Ehrlich and Mitsuyoshi Nakao...................................................... ···················································48
Establishment and maintenance of genomic imprinting in the germline and in early embryos
Hiroyuki Sasaki, Masahiro Kaneda, Masaki Okano, Kenichiro Hata, Takashi Sado, Naomi
Tsujimoto, En Li, Kenji Kumaki, Hiroyasu Furuumi, Tomohiro Suzuki, Shigeharu
Wakana and Toshihiko Shiroishi
48
Computer-assisted search for sequence features common to imprinted DMRs
Hiroyuki Sasaki, Hisato Kobayashi, Takashi Abe and Toshimichi Ikemura
49
Molecular pathology of ICF syndrome
Hiroyuki Sasaki, Hiroyasu Furuumi and Takeo Kubota··································· 49
Investigation on the possibility of Z chromosome dosage compensation in chicken
Hiroyuki Sasaki, Takaaki Yokomine, Asato Kuroiwa, Yoichi Matsuda and Masaoki
Tsuduki········································49
Development of a universal DNA chip system applicable to any organism
Hiroyuki Sasaki, Shin-ichi Mizuno, Tadafumi lino, Hidetoshi Ozawa, Teruhisa Otsuka,
49
Kosuke Tashiro and Takashi Gojohbori
Role of de novo DNA methyltransferases in X chromosome inactivation
Takashi Sado, Masaki Okano, En Li and Hiroyuki Sasaki ........ ·...... ·...... · ........ · 49
Effect of Tsix disruption on Xist expression
Takashi Sado and Hiroyuki Sasaki
50
X-inactivation in mouse embryos deficient for histone methyltransferase G9a
Tatsuya Ohhata, Makoto Tachibana, Hiroyuki Sasaki, Yoichi Shinkai and Takashi Sado....................................................... ··················································50
Role of Dnmt3L in spermatogenesis and in genomic imprinting during oogenesis
Kenichiro Hata, En Li and Hiroyuki Sasaki

CONTENTS

50

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 and Masaomi Kato
Hodetoshi Saze
52
Epigenetic control of transposons and their behavior in natural population
Asuka Miura, Masaomi Kato, Kazuya Takashima, Yuki Kinoshita and Tetsuji Kakutani...................................................... ···················································52
Inheritance of epigenetic developmental abnormality.
Yuki Kinoshita, Asuka Miura, Tetsu Kinoshita and Tetsuji Kakutani
Epigenetic control of FWA gene expression in endosperm
Tetsu Kinoshita, Asuka Miura, Yuki Kinoshita and Tetsuji Kakutani
E-b. Division of Agricultural Genetics
Keiichi Shibahara Group
Mechanism of nucleosome assembly during DNA replication
Tomohiro Kubo, Akira Shimizu and Keiichi Shibahara"
Physiological implications of CAF-l and ASFI in Arabidopsis
Hidetaka Kaya, Takashi Araki and Keiichi Shibahara"
Identification of a novel regulatory element in the X- inactivation Center (Xic)Yuya Ogawa and Jeannie T. Lee

52
53

53
54
54

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
55
Chronotopic Organization of Olfactory Bulb Axons in the Lateral Olfactory Tract
Hitoshi Yamatani, Yasufumi Sato, Hajime Fujisawa and Tatsumi Hirata·············· 55
E-d. Division of Applied Genetics
Hirohiko Hirochika Group
Target site specificity of the Tos17retrotransposon shows a preference for insertion withingenes and against insertion in retrotransposon-rich regions of the genome
Akio Miyao, Katsuyuki Tanaka, Kazumasa Murata, Hiromitsu Sawaki, Shin Takeda,
Kiyomi Abe, Yoriko Shinozuka, Katsura Onosato and Hirohiko Hirochika
56
Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesisin the secondary wall
Katsuyuki Tanaka K, Kazumasa Murata, Muneo Yamazaki, Katsura Onosato, Akio
57
Miyao and Hirohiko Hirochika
Transcriptional activation mediated by binding of a plant GATA-type zinc finger protein
AGPI to the AG-motif (AGATCCAA) of the wound-inducible Myb gene NtMyb2.
57
Kazuhiko Sugimoto, Shin Takeda and Hirohiko Hirochika
Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system
Ki-Hong Jung, Junghe Hur, Choong-Hwan Ryu, Youngju Choi, Yong-Yoon Chung, Akio
Miyao, Hirohiko Hirochika and Gynheung An
57
The MSPl gene is necessary to restrict the number of cells entering into male and female
sporogenesis and to initiate anther wall formation in rice
Ken-ichi Nonomura, Kazumaru Miyoshi, Mitzugu Eiguchi, Tcizunu Suzuki, Akio Miyao,
Hirohiko Hirochika and Nori Kurata
58
Loss-of-Function Mutations of the Rice GAMYB Gene Impaira -Amylase Expression in
Aleurone and Flower Development.

CONTENTSMiyuki Kaneko, Yoshiaki Inukai, Miyako Ueguchi-Tanaka, Hironori Itoh, Takeshi Izawa,
Yuhko Kobayashi, Tsukaho Hattori, Akio Miyao, Hirohiko Hirochika, Motoyuki Ashikari
and Makoto Matsuoka············································································ 58
E·d. Division of Applied Genetics
Kunio Shiota GroupDNA methylation profiles of CpG islands in normal tissues
Jun Ohgane, Naka Hattori, Satoshi Tanaka and Kunio Shiota
59
Preference of DNA methyltransferases for CpG islands in normal cells
Naka Hattori, Tetsuya Abe, Masako Suzuki, Tomoki Matsuyama, Shigeo Yoshida, En
Li and Kunio Shiota
59
Methyl-CpG-binding protein, MeCP2, is a target molecule for maintenance DNA
methyltransferase, Dnmtl
Hiromichi Kimura and Kunio Shiota
59
Transcription of mouse DNA methyltransferase 1 (Dnmtl) is regulated by both
E2F-Rb-HDAC-dependent and -independent pathways
Hiromichi Kimura, Takahisa Nakamura, Tomoya Ogawa, Satoshi Tanaka and Kunio
Shiota···········································60
The Sa1l3locus is an epigenetic hotspot of aberrant DNA methylation associated withplacentomegaly of cloned mice
Jun Ohagane, Teruhiko Wakayama, Sho Senda, Yukiko Yamazaki, Kimiko Inoue, Atsuo
Ogura, Joel Marh, Satoshi Tanaka, Ryuzo Yanagimachi and Kunio Shiota
60
F. GENETIC STRAINS RESEARCH CENTER
F -a. Mammalian Genetics Laboratory
Toshihiko Shiroishi Group
Genetic Incompatibility between X-linked Loci and Two Autosomal Regions Causes Hybrid
Breakdown between Two Mouse Subspecies
Ayako Oka, Akihiko Mita, Yoichi Mizushina, Noriko Sakurai·Yamatani, Hiromi
Yamamoto, Kazuo Moriwaki and Toshihiko Shiroishi
62
Functional analysis of GsdmA -3 using mutant mice with aberrant epidermal morphogenesis
Shigekazu Tanaka, Masaru Tamura, Aya Aoki and Toshihiko Shiroishi
62
Identification and characterization of novel gene family, Gasdermin
Tomoaki Fujii, Shigekazu Tanaka, Aya Aoki, Masaru Tamura and Toshihiko Shiroishi...................................................... ···················································63
F-b. Mammalian Developmental Laboratory
Yumiko Saga Group
Molecular mechanism of somite segmentation
Yu Takahashi, Kaoru Mitsui, Mitsuru Morimoto and Yumiko Saga
63
Regulation of Mespl and Mesp2 expression
Yukuto Yasuhiko, Masayuki Oginuma and Yumiko Saga
64
Heart morphogenesis and Notch signaling
Hiroki Kokubo, Yusuke Watanabe, Yoshiaki Okamura, Wataru Saito and Yumiko Saga...................................................... ···················································64
Gene hunting possibly involved in somitogenesis
Satoshi Kitajima, Aki Ishikawa and Yumiko Saga
Functional analysis of mouse nanos genes
Masayuki Tsuda, Yumiko Sasaoka, Makoto Kiso and Yumiko Saga

CONTENTS

65
65

F-c. Mouse Genomics Resource Laboratory
Tsuyoshi Koide Group
Quantitative traits analyses of sensitivities to heat evoked pain assayed by tests of hot plate
and tail flick
Tamio Furuse, Toshihiko Shiroishi and Tsuyoshi Koide
66
QTL analysis of home-cage spontaneous activity using hyper- and hypoactive mouse strains
Juzoh Umemori, Tamio Furuse, Toshihiko Shiroishi and Tsuyoshi Koide
67
Different effect of apomorphine, dopamine agonist, on open-field activities in wild derived
mouse strains
Akinori Nishi, Toshihiko Shiroishi and Tsuyoshi Koide
67
Descriptive analysis of the open-field behavior in wild mice strains
Aki Takahashi, Toshihiko Shiroishi and Tsuyoshi Koide

67

F-d. Plant Genetics Laboratory
Nori Kurata Group
Characterization of sporogenesis defects in sterile rice mutants
Ken-ichi Nonomura, Mutsuko Nakano, Mitsugu Eiguchi, Akio Miyao, Hirohiko Hirochika
and Nori Kurata··········68
Structural and functional analysis of rice OsHAP3 genes
Kazumaru Miyoshi, Thiruvengadam Thirumurugan, Yukihiro Ito and Nori Kurata...................................................... ···················································68
Functional analysis of PLASTOCHRONI in rice
Kazumaru Miyoshi, Yukihiro Ito and Nori Kurata
Regulation of expression of KNOX family class 1 homeobox genes of rice
Yukihiro Ito and Nori Kurata

69·····69

Search for nuclear protein genes having regulatory activity in rice development
Kazuki Moriguchi, Mitsugu Eiguchi and Nori Kurata

69

Intruduction of rice artificial chromosomes into riceTadzunu Suzuki, Ken-ichi Nonomura and Nori Kurata

70

Generation and screening of enhancer trap lines of rice
Yukihiro Ito and Nori Kurata

70

Positional cloning of a segregation distortion gene detected in a progeny of a cross between
J aponica and Indica rice
70
Yoshiaki Harushima and Nori Kurata
F-e. Microbial Genetics Laboratory
Akiko Nishimura Group
Demonstration of native dynamics of FtsZ in the growing Escherichia coli single cell by on-chip
microculture·· .. ·· .. 71
Ippei Inouye, Kenji Yasuda and Akiko Nishimura
A complete set of E. coli genes/ORFs on a mobile plasmid: successful use in the isolationof all cell division mutants
Kimiko Saka, Ken Nishikawa, Hideaki Sugawara, Noriko Matsumoto, Hiroshi Fujishima
and Akiko Nishimura·············································································· 72
F-f. Invertebrate Genetics Laboratory
Ryu Ueda GroupRNAi mutant fly bank for comprehensive analyses of gene function in Drosophila
Ryu Ueda, Misako Taniguchi, Kaoru Saigo and Kuniaki Takahashi
72

CONTENTSF-g. Laboratory for Frontier Research
Takako Isshiki Group
Search for the factors involved in temporal specification within Drosophila neuroblast lineage
Kusano Ayumi and Takako Isshiki··············································74
G.CENTER FOR GENETIC RESOURCE INFORMATION

G-a. Genetic Informatics Laboratory
Yukiko Yamazaki GroupSHIGEN project

PEe
Takehiro Yamakawa, Junichi Kato and Yukiko Yamazaki

76

CARDR-DB
Takehiro Yamakawa, Hideki Kato, Naomi Nakagata, Kenichi Yamamura and Yukiko
Yamazaki76
JMSR
Takehiro Yamakawa and Yukiko Yamazaki

76

OryzabaseTakehiro Yamakawa, Shingo Ueno, Nori Kurata, Atsushi Yoshimura, Hikaru Sato, Yasuo
76
Nagato and Yukiko Yamazaki
KOMUGI
Takehiro Yamakawa, Takashi Endo, Yasunari Ogihara, Hitoshi Tsujimoto, Taihachi
Kawahara, Tetsuro Sasakuma and Yukiko Yamazaki
··
·
·
·77
G-b. Genome Biology Laboratory
Yuji Kohara Group
NEXTDB: The nematode expression pattern map database
Tadasu Shin-I, Ikuko Sugiura-Muramatsu, Masumi Obara, Wakako Shimizu, Yuko
Suzuki, Aya Hamakawa, Jean Thierry-Mieg, Danielle Thierry-Mieg, Sachiko Takahashi,
Kyoko Nakata, Hiroko Uesugi, Takayo Hamanaka, Yasuko Sugiyama and Yuji Kohara...................................................... ···················································78
Systematic antibody raising and maternal gene expression analysis in C.elegans
Yumiko Ueta, Masahiro Ito, Hiroko Ochi, Chihiro Hijikata, Sachiko Takahashi, Keiko
Hirono, Yoshinori Ukai, Midori Shinohara, Hisayoshi Torii, Emi Shimizu, Miki Takenaka,
Yoshitaka Iba, Yoshikazu Kurosawa and Yuji Kohara
78
Protein phosphatase 2A (PP2A), encoded by let-92 and paa-l, is required for early cleavageof C. elegans
Ken-ichi Ogura, David L. Baillie, Sachiko Takahashi and Yuji Kohara
79
Systematic analysis of cell-specific enhancers in C. elegans
Hiroshi Kagoshima, Akiko Kamamoto and Yuji Kohara
79
Synergistic effect of T-box transcription factor tbx-9 and Hox cofactor Hth!Meis orthologue
unc-62 on C. elegans embryonic morphogenesis
Yoshiki Andachi
80
Physical Modeling of Cellular Arrangement in Early Embryos of C.elegans
Atsushi Kajita, Masayuki Yamamura and Yuji Kohara
80
SPI: A tool for incorporating gene expression data into a four-dimensional database of C.
elegans embryogenesis
81
Yohei Minakuchi, Masahiro Ito and Yuji Kohara
Semi-automatic system for creation of cell shape model in C. elegans embryogenesis
Hideaki Hiraki and Yuji Kohara
Towards comparative genomics: Genome/EST sequencing

CONTENTS

81

Kazuko Oishi, Shinobu Haga, Hisayo Nomoto, Masako Sano, Fumiko Ohta, Sachiko
Miura, Tomomi Morishita, Tomoko Endo, Motoyo Tamiya, Keiko Nogata, Akiko Hase,
Tomoharu Tandoh, Yoshiki Mochizuki, Hiromitsu Miyauchi, Noriko Hasegawa, Masumi
Mizukoshi, Etsuko Yokoyama, Nanayo Ishihara, Junko Miyamoto, Shigeko liyama,
Tadasu Shin-I, Toshinobu Ebata, Shigeru Saito, Masaki Itoh, Masayo Nakagawa, Kumiko
Kawaguchi, Naoko Sakamoto, Yasuko Sugiyama and Yuji Kohara
82
G-c. Publicity and Intellectual Property Unit
Munehiro Tomikawa Group
2003 Annual Publicity and Intellectual Property Unit Report83
Acquirement and utilization of Intellectual Property Rights···································· 83
Acceleration of public relations activities··· 84
Social Action Work····················································································· 84
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·· .. ···85
Visualization and quantitative analysis of dynamics of single lipid rafts in vivo
Kumiko Sakata-Sogawa, Sho Yamasaki, Takashi Saito and Makio Tokunaga········ 85
RNG105: A Novel Regulatory Protein in Neuronal RNA Granules Responsible for
Stimulation-Dependent Local Translation
Nobuyuki Shiina, Kazumi Shinkura and Makio Tokunaga································ 85
Single Hydrogen Bonds of DNA Base Pairs Detected in Unzipping Force by Intermolecular
Force Microscopy
Michio Hiroshima and Makio Tokunaga·· 86
A Novel in vitro Assay System of Nucleocytoplasmic Transport
Atsuhito Okonogi, Michio Hiroshima, Nobuyuki Shiina, Naoko Imamoto and Makio
Tokunaga···························································································· 86
Molecular Imaging of translation initiation factors in neuronal dendrites
86
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

87

Structural dynamics of a 70 and its extra roles in vivo
Shoji Yagi, Usha Padmanabhan, Hiroki Nagai, Taciana Kasciukovic, Richard S. Hayward,
Yumiko Sato and Nobuo Shimamoto
88
Applicability of thermodynamics to equilibria in biology
Nobuo Shimamoto and Jyun-ichi Tomizawa

89

H-c. Multicellular Organization Laboratory
Isao Katsura Group
Analysis of synthetic dauer-constitutive mutants in the nematode Caenorhabditis elegans
Tomoko Yabe, Kiyotaka Ohkura, Takeshi Ishihara and Isao Katsura
90
Molecular genetic studies on sensory integration and behavioral plasticity in' C. elegans
Takeshi Ishihara, Yuichi lino, Akiko Mohri, Ikue Mori, Keiko Gengyo-Ando, Shohei
Mitani and Isao Katsura
91
C. elegans mutants in the "associative" learning with odorants and food

CONTENTS

Ichiro Torayama, Takeshi Ishihara and Isao Katsura

92

Behavioral analysis with "uncomfortable" stimuli of the nematode Caenorhabditis elegans
Kotaro Kimura and Isao Katsura
93
Fluoride-resistant mutants of the nematode Caenorhabditis elegans
Akane Oishi, Takeshi Ishihara and Isao Katsura

93

H-d. Biomolecular Structure Laboratory
Yasuo Shirakihara Group
Crystallographic Study of F1-ATPase
Yasuo Shirakihara and Aya Shiratori

95

Crystallization of ATP synthaseYasuo Shirakihara and Aya Shiratori

96

Comprehensive Crystallographic Study of Transcription factors and Genome-partitioning
Factors from E. coli
Yasuo Shirakihara, Aya Shiratori, Hisako Inoue and Megumi Seki
96
Crystallographic Study of Transcription factors from Pseudomonas aeruginosaYasuo Shirakihara, Aya Shiratori, Hisako Inoue and Megumi Seki
97
Crystallization of TRAF6, a transducer in CD40 signaling pathway
Yasuo Shirakihara, Megumi Seki and Aya Shiratori
97
Structural analysis of glutaminase from Micrococcus luteus K·3
Yasuo Shirakihara and Aya Shiratori
98
I. CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPANI-a. Laboratory for DNA Data Analysis
Takashi Gojobori GroupThe origin and evolution of porcine reproductive and respiratory syndrome viruses
Kousuke Hanada, Yoshiyuki Suzuki and Takashi Gojobori
99
The discovery of the genes relating to the evolution of an eyeless cave dwelling form froman eyed surface dwelling form of Mexican tetra, Astyanax mexicanus, by cDNA
mIcroarrays
Nobuhiko Tanaka, Shozo Yokoyama, Kazuho Ikeo and Takashi Gojobori
99
Evolutionary mechanisms of duplicated genes
Takashi Makino, Yoshiyuki Suzuki and Takashi Gojobori
99
Study on conserve upstream noncoding sequences between human and mouse
Hisakazu Iwama and Takashi Gojobori
100
Data analysis on Rice Genome Project
100
Hisakazu Iwama and Takashi Gojobori
Biased biological functions of horizontally transferred genes on 324,653 open reading framesof 116 prokaryotic complete genomes
Yoji Nakamura, Takeshi Itoh, Hideo Matsuda and Takashi Gojobori
100Comparative complete genome sequence analysis of the amino acid replacements responsible
for the thermostability of Corynebacterium efficiens
Yousuke Nishio, Yoji Nakamura, Yutaka Kawarabayasi, Yoshihiro Usuda, Eiichiro
Kimura, Shinichi Sugimoto, Kazuhiko Matsui, Akihiko Yamagishi, Hisashi Kikuchi,
100
Kazuho Ikeo and Takashi Gojobori
The genome stability in Corynebacterium species due to lack of the recombinational repair
system101
Yoji Nakamura, Y.osuke Nishio, Kazuho Ikeo and Takashi Gojobori
Identification of !-cell specific genes of hydra and its evolutionary implication]. Shan Hwang, Chiemi Nishimiya-Fujisawa, Toshitaka Fujisawa, Kazuho Ikeo and

CONTENTS

Takashi Gojobori

101

The evolutionary origin of long-crowing chicken based on the molecular phylogeny
Tomoyoshi Komiyama, Kazuho Ikeo and Takashi Gojobori

101

The analysis to the evolutionary relationship between duplication and alternative splicing
in terms of increasing functional diversity
102
Lihua Jin, Kazuho Ikeo, Yoshiyuki Suzuki and Takashi Gojobori
Comparative evolutionary study of the single cell-based gene expression profiles from theascidinan blastmeres •Katsuhiko Mineta, Kazuho Ikeo, Hiroaki Yamamoto, Yuzuru Tanaka and Takashi
Gojobori
102
Molecular evolutionary analyses of host-parasite interactions
Yoshiyuki Suzuki

102

Molecular basis of convergent evolution of camera eye between octopus and humanAtsushi Ogura, Kazuho Ikeo and Takashi Gojobori
Genomic diversification of bilaterian animals
Atsushi Ogura, Kazuho Ikeo and Takashi Gojobori

103
103

I-b. Laboratory for Gene-Product Informatics
Ken Nishikawa Group
Unique amino acid composition of proteins in halophilic bacteria
Satoshi Fukuchi, Kazuaki Yoshimune, Mitsuaki Moriguchi and Ken Nishikawa···· 105
Compositional changes in RNA, DNA and proteins for bacterial adaptation to higher andlower temperatures
Hiroshi Nakashima, Satoshi Fukuchi and Ken Nishikawa
106
Prediction of catalytic residues in enzymes based on known tertiary structure, stability profile,and sequence conservation
Motonori Ota, Kengo Kinoshita and Ken Nishikawa· .. ···
··
·
· 106
I-c. Laboratory for Gene Function Research
Yoshio Tateno Group
Parallel evolution of ligand specificity between LacVGalR family repressors and periplasmic
sugar-binding proteins
Kaoru Fukami-Kobayashi, Yoshio Tateno and Ken Nishikawa
107
CIBEX: Center for Information Biology Gene Expression Database
Kazuho Ikeo, Jun Ishi-i, Takurou Tamura, Takashi Gojobori and Yoshio Tateno .. 107
Highly differentiated and conserved sex chromosome in fish species (Aulopus japonicus:
Teleostei, Aulopidae)
Kinya Ota, Yoshio Tateno and Takashi Gojobori
107
DNA Data Bank of Japan (DDBJ) in XML
Satoru Miyazaki, Hideaki Sugawara, Takashi Gojobori and Yoshio Tateno
108
Characterization of folding pathways of the type-1 and type-2 periplasmic binding proteins
MglB and ArgT
Kenji Kashiwagi, Kiyataka Shiba, Kaoru Fukami- Kobayashi, Tetsuo Noda, Ken
Nishikawa and Hiroshi Noguchi······························································· 108
Standardization of Microarray Data
Brazma Alvis, Kazuho Ikeo and Yoshio Tateno
108
DNA Data Bank of Japan as an Indispensable Public Database
Satoru Miyazaki and Yoshio Tateno
I-d. Laboratory for Research and Development of Biological Databases
Hideaki Sugawara Group
Information systems for molecular biology and its related disciplines

109

110

CONTENTS

Infonnation systems on microbes

110

Applications of IT to the International Nucleotide Sequence Database····················· III
I-e. Laboratory for Gene-Expression Analysis
Kousaku Okubo Group
Expression profiling of human genes
112
Knowledge encoding and computation with gene functions (BOB): "Are you confident inyour massive data interpretation?"
Kousaku Okubo, Koichi Itoh and Osamu Ogasawara
113
J. RADIOISOTOPE CENTER

Identification of a cis-acting site for bipolar positioning of orie on the E. coli chromosome
Yoshiharu Yamaichi, Katsunori Yata and Hironori Niki
115
Subcellular localization of Escherichia coli proteins in living cells
Toshiyuki Hatano, Yasuyuki Ogata and Hironori Niki

115

Stationary phase-induced illegitimate recombination in Escherichia coli
Yasuyuki Ogata and Hideo Ikeda
·

· 115

K. EXPERIMENTAL FARM

Preparation of wild rice accessions for comparative genomics
Toshie Miyabayashi, Mitsugu Eiguchi and Nori Kurata
L. TECHNICAL SECTION

BIOLOGICAL SYMPOSIUM 2003
FOREIGN VISITORS IN 2003

CONTENTS

117···························118··················119121

Introduction
The paradigm of genetics is changing rapidly through the increasing rate of genome sequencings. It was
symbolized by the completion of human genome project, to which our DDBJ (DNA Databank of Japan) contributed
as one of the three database centers of the world. Our sequencing center is also producing genome data of various
model organisms, including that of chordate. The ever-increasing accumulation of such genome-wide knowledge
about many biological species, combined with the rapid progress in computer and information sciences, isrevolutionizing the study of genetics. Weare supporting DDBJ and its related research activities, as well as its
educational programs in bioinformatics. Another power of NIG is in the research activities using many modelorganisms; they include phage, bacteria, yeast, nematode, Drosophila, zebra fish, medaka, mouse and man. Riceand Arabidopsis research groups are also very active. Less popular but increasingly important organisms studied
here include planaria, hydra and chordate. The combination of such a variety of scientific research will provide
us an additional impetus in the post-genome sequence era. In this sense, NIG has an advantage in the "scientificturmoil". NIG's mottoes are, "From Molecules to Individuals", "From Development to Evolution" and "FromExperiment to Theory".
Japanese Diet passed a law to make a reform of national universities and institutes that belong to Ministryof Education, Culture, Science, Sports and Technology (MEXT). NIG is going to collaborate with other three
institutes to form Research Organization of Information and Systems (ROIS) from April 2004. The unique functionsof our institutes as inter-university collaboration system will remain as usual, but we would like to add a new
function to create new scientific paradigm by the collaboration with institutes specialized for informatics. We
believe that such a reorganization, although it was originally started as a part of national financial reform plan,
will eventually prove to be the right decision to promote and contribute to the current revolution in life sciences.
Yoshiki Hotta, Director-General

Introduction

1

STAFF (as of December 31,2003)
Director-General

HOTTA, Yoshiki, D. Med.
Vice-Director

KOHARA, Yuji, D. Sc.
Members

1. Department of Molecular Genetics

SHIMAMOTO, Nobuo, D. Sc., Head of the Department
Division of Molecular Genetics
FUKAGAWA, Tatsuo, D.Sc., Associate Professor
Division of Mutagenesis
YAMAO, Fumiaki, D. Sc., Professor
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, D. Med.
TANAKA, Seiji, D. Sc.
Division of Cytoplasmic Genetics
YOSHIKAWA, Takeo, D. Med., Adjunct Professor
YANAGIDA, Mitsuhiro, D. Sc., Professor (Adjunct)
3. Department of Developmental Genetics
HIROSE, Susumu, 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
UEDA, Hitoshi, D. Ag., Associate Professor
YAMADA, Masa-aki, D. Ag.

2

STAFF

NISHIOKA, Kenichi, D. Med.
Division of Molecular and Developmental Biology
KAWAKAMI, Koichi, D. Sc., Associate Professor
KISHIMOTO, Yasuyuki, D. Sc.
Division of Physiological Genetics
KOSEKI, Haruhiko, D. Med., Professor (Adjunct)
GOTOH, Yukiko, Associate Professor(Adjunct)
4. Department of Population Genetics
IKEMURA, Toshimichi, D. Sc., Head of the Department
Division of Population Genetics
SAITOU, Naruya, Ph. D., Professor
T AKANO, Toshiyuki, D. Sc., Associate Professor
SUMIYAMA, Kenta, D. Sc.
TAKAHASHI, Aya, D. Ag.
Division of Evolutionary Gentics
IKEMURA, Toshimichi, D. Sc., Professor
Division of Theoretical Genetics
FUJIYAMA, Asao, D. Sc., Professor(Adjunct)
TAKAGI, Toshihisa, D. Eng., Professor (Adjunct)
5. Department of Integrated Genetics
SASAKI, Hiroyuki, D. Med., Head of the Department
Division of Human Genetics
SASAKI, Hiroyuki, D. Med., Professor
SADO, Takashi, D. Sc.
HATA, Kenichiro, D. Med.
Division of Agricultural Genetics
KAKUTANI, Tetsuji, D. Sc., Associate Professor
SHIBAHARA, Kei-ichi, M. D., Ph. D., Associate Professor
KINOSHITA, Tetsu, D. Sc.
OGAWA, Yuya, Ph. D.
Division of Brain Fanction
HIRATA, Tatsumi, D. Med., Associate Professor
KAWASAKI, Takahiko, D. Sc.
Division of Applied Genetics
HIROCHIKA, Hirohiko, D. Sc., Adjunct Professor
SHIOTA, Kunio, D. Ag., Professor(Adjunct)
6. Genetic Strains Research Center
SHIROISHI, Toshihiko, D. Sc., Head of the Center
Mammalian Genetics Laboratory
SHIROISHI, Toshihiko, D. Sc., Professor
TAMURA, Masaru, D. Sc.
Mammalian Development Laboratory
SAGA, Yumiko, D. Sc., Professor
KOKUBO, Hiroki, D. Sc.
MITSUI, Kaoru, D. Med.

STAFF

3

Mouse Genomics Resource Laboratory
KOIDE, Tsuyoshi, D. Med., Associate Professor
Model Fish Genomics Resource Laboratory
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.
Laboratory 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
FUJITA, Masaya, D. Eng.
Genome Biology Laboratory
KOHARA, Yuji, D. Sc., Professor
ANDACHI, Yoshiki, D. Sc.
Publicty and Intellectual Property UnitTOMIKAWA, Munehiro, D. Pharm, Professor
8. Structural Biology Center

KATSURA, 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.
Multicelluar Organization Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.
KIMURA, Koutarou, D. Ag.
Biomolecular Structure Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate Professor
Gene Network Laboratory
9. Center for Information Biology and DNA Data Bank of JapanGOJOBORI, Takashi, D. Sc., Head of the Center
Laboratory for DNA Data Analysis
GOJOBORI, Takashi, D. Sc., Professor
IKEO, Kazuho, D. Sc., Associate Professor
SUZUKI, Yoshiyuki, M. D., Ph. D.

4

STAFF

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
Laboratory for the Research and Development of Biological Databases
SUGAWARA, Hideaki, D. Eng., Professor
MIYAZAKI, Satoru, D. Sc., Associate Professor
Gene-Expression Analysis
OKUBO, Kousaku, M. D., Ph. D., Professor
ITOH, Koichi, M. D., Ph. D.

10. Radioisotope Center
NIKI, Hironori, D. Med., Associate Professor Head of the Center
OGATA, Yasuyuki, D. Phann. Sci.
11. Experimental Farm
KURATA, Nori, D. Ag., Head of the FannNONOMURA, Ken-ichi, D. Ag.
12. Technical Section
ISHII, Yuriko, Chief of the Section
13. Department of Administration
ISHIKAWA, Kenji, Head of the Department
ISHIDA, Yuzo, Chief of the General Affairs Section
KAWAGUCHI, Kenji, Chief of the Finance Section

STAFF

5

COUNCIL (as of December 31,2003)
Chairman
SUGIMURA, Takashi; President Emeritus, National Cancer Center
Vice-chairman
OSAWA, Shozo; Professor Emeritus, The University of Nagoya
Members (Alphabetical order)
HIROBE, Masaaki; President,University of Shizuoka
ISHII, Shiro; Vice-Derector, Japan Society for the Promotion of Science, Research Center for Science System
IWATSUKI, Kunio; Professor, University of the Air
KATSUKI, Motoya; Director-General, National Institute for Basic Biology
KODAIRA, Keiichi; President, The Graduate University for Advanced Studies
KURODA, Reiko; Professor, Graduate School of Arts and Science, The University of Tokyo
MATSUBARA, Ken-ichi; President, DNA Chip Research Inc.
MATSUO, Minoru; President, Nagoya University
MIURA, Kin-ichiro; President, Proteios Research Inc.
OKADA, Masukichi; Vice-Director, International Institute for Advanced Studies
OSAKI, Hitoshi; Director-General, Center for National University Finance
OTSUKA, Eiko; Fellow, National Institute of Advanced Industrial Science and Technology
SASAKI, Kazuo; President, Okazaki National Research Institutes
SHINJI, Isoya; President, Tokyo University of Agiculture
TOYOSHIMA, Kumao; Director-General, SNP Reserch Center, The Institute of Physical and Chemical Research
TSUNEWAKI, Koichiro; President, Fukui Prefectural University
YAMANOUCHI, Kazuya; Senior Scientific Staff, Nippon Institute for Biological Science

6

COUNCILADVISORY COMMITTEE (as of December 31, 2003)
Chairman
KOHARA, Yuji; Professor, National Insutitute of Genetics
Vice-chairman
SEKIGUCHI, Mutsuo; Director, Biomolecular Engineering Research InstituteOutside Members (Alphabetical order)GO, Michiko; Dean, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology
HANAOKA, Fumio; Professor, Graduate School of Frontier Biosciences, Osaka University
ISONO, Katsumi; Director, National Institute of Technology and Evaluation
ITO, Koreaki: Professor, Institute for Virus Research, Kyoto University
MATUURA, Etsuko; Professor, Faculty of Science, Ochanomizu University
OGAWA, Tomoko; Vice-President, Iwate College of Nursing
SASAZUKI, Takehiko; Director-General, International Medical Center of Japan Research Institute
SHINOZAKI, Kazuo; Chief Scientist, RIKEN Tsukuba Institute
T AlIMA, Fumio; Professor, Graduate School of Science, The University of Tokyo
Inside Menbers (Alphabetical order)
ARAKI, Hiroyuki; Professor, National Insutitute of Genetics
GOJOBORI, Takashi; Professor, National Insutitute of Genetics
HIROMI, Yasushi; Professor, National Insutitute of Genetics
HIROSE, Susumu; Professor, National Insutitute of Genetics
IKEMURA, Toshimichi; Professor, National Insutitute of Genetics
KATSURA, Isao; Professor, National Insutitute of Genetics
NISHIKAWA, Ken; Professor, National Institute of Genetics
SASAKI, Hiroyuki; Professor, National Insutitute of Genetics
SHIMAMOTO, Nobuo; Professor, National Insutitute of Genetics
SHIROISHI, Toshihiko; Professor, National Insutitute of Genetics

ADVISORY COMMITTEE

7

A. DEPARTMENT OF
MOLECULAR GENETICS

A-a. Division of Molecular Genetics
Tatsuo Fukagawa Group

CENP-H interacts with Heel, which transiently
associate with centromeres during mitosis. Heel alsointeract with Nuf2. We carried out several analyses
concerning interaction between CENP-H and theNuf2-Heel complex. We then propose that the
Nuf2-Heel complex may act to connect between theouter and inner kinetochores.
(2) Molecular analysis of the Nuf2-Heel complex thattransiently localizes to centromere during mitosis

Tetsuya Hori, Atsuto Uenoyama, Kazuko Suzuki and
Tatsuo Fukagawa

RESEARCH ACTIVITIES(1) Assembly of kinetochores in higher vertebrate cells

Yoshikazu Mikami, Tetsuya Hori, Mitsuko Yoshikawa,Atsushi Fukushima 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 of homologous recombination in DT40 cells permit efficient targeted
disruption of genes of interest. We also generated human artificial mini-chromosomes using the telomeredirected breakage method in DT40 cells. We havecombined human artificial mini-chromosomes andDT40 cells to study chromosome segregation. We have
improved this system and have created several celllines with conditional knockouts of several centromere
proteins to investigate the molecular mechanism ofcentromere assembly and function. In this year we
focused on analysis of CENP-H (Fukagawa et al.,EMBO ]., 2001; Nishihashi et al., Dev. Cell, 2002). We
identified functional domain of CENP-H usinga CENP-H-deficient cells. Yeast two-hybrid analysis
with the CENP-H functional domain revealed that

8

DEPARTMENT OF MOLECULAR GENETICS

Nuf2 and Heel are evolutionarily conserved
centromere proteins. To clarify the functions of these
proteins in vertebrate cells, we characterized themin chicken DT40 cells. We generated GFP fusion
constructs of Nuf2 and Heel to examine in detaillocalization 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 thecentrosomes during Gland S phases and then moves
through the nuclear membrane to the centromere in
G2 phase. During mitosis, this complex is localized tothe centromere. We also created conditionalloss-offunction mutants of Nuf2 and Heel. In both mutants,
the cell cycle arrested at prometaphase, suggesting that
the Nuf2-Heel complex is essential for mitotic
progression. The inner centromere proteins CENPA, -C, and -H and checkpoint protein BubRI werelocalized to chromosomes in the mutant cells arrested
at prometaphase, but Mad2 localization was abolished.
Furthermore, photobleaching experiments revealed
that the Nuf2-Heel complex is associated stably withthe centromere and that interaction of this complex
with the centrosome is dynamic. We also took theproteomics approach to identify proteins that interact
with the Nuf2-Heel complex. We identified several
components that localizes centrosome and centromere
and are now characterizing these components.
(3) Functional roles of the RNAi machinery inverterbrate centromeres

Masahiro Nogami, Tomoko Motohashi, Mitsuko
Yoshikawa 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-mediated genesilencing 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 in vertebrate cells. To examine the biological function of theRNAi-related pathway in vertebrate cells, we generated
a conditionalloss-of-function mutant of Dicer in achicken-human hybrid DT40 cell line that contains
human chromosome 21. Loss of Dicer leads to cell
death with accumulation of abnormal mitotic cells thatshow premature sister chromatid separation. Aberrant
accumulation of transcripts from Q: satellite sequences,which consist of human centromeric repeat DNAs,
was detected in Dicer-deficient cells. Cytologicalanalysis revealed abnormal ties in localization ofheterochromatin proteins, but core kinetochore
proteins such as CENP-A and -C were normal. We
conclude that Dicer-related RNAi machinery is involvedin formation of the heterochromatin structure in higher
vertebrate cells.
Publications
Papers

1. Hori, T., Haraguchi, T., Hiraoka, Y., Kimura, H.
and Fukagawa, T. (2003). Dynamic behavior of
Nuf2-Heel complex that localizes to the centrosome
and centromere and is essential for mitotic progressionin vertebrate cells. J. Cell Sci. 116,3347-3362.
2. Regnier, V., Novelli,]., Fukagawa, T., Vagnarelli,
P. and Brown, W. (2003). Characterization of chicken
CENP-A and comparative sequence analysis ofvertebrate centromere-specific histone H3-like proteins.
Gene 316, 39-46.
EDUCATION(in Japanese).
2. Dr. Fukagawa was invited a seminar on"Centromere Assembly" at Kyushu University,
Fukuoka, August, 2003 (in Japanese).

A-b. Division of Mutagenesis
Fumiaki Yamao GroupRESEARCH ACTIVITIES(1) Ubiquitin Pathway Regulating Recombination and
DNA Damage Repair in Fission Yeast.
Joon-Hyun Park, Toyoaki Natsume and Fumiaki
Yamao
In appropriate or unregulated homologous recombination can have deleterious effects and lead togenomic instability. In diploid eukaryotes, homologous
recombination is essential during meiosis, but it issuppressed during mitosis by an as yet unknown
mechanism. We identified and characterized Ubc7, oneof the 14 ubiquitin-conjugating enzymes
in Schizosaccharomyces pombe. Mutants defective inUbc7 have enhanced mitotic recombination between
tandem-repeated segments at matl or ade6. The
increased mitotic recombination was observed only inubcr cells which are competent for DSB-induced
homologous recombination. Furthermore, Ubc7
mutants are hyper-resistant to UV radiation and
MMS-induced DNA damage in cells carrying wild typeRhp51. Genetic recombination is a two-step reactionin eukaryotes; the initiation step establishes competence
for homologous recombination and DNA exchange,
while the second step mediates the recombination
itself. This study demonstrates that Ubc7 negatively
regulates the latter step during mitosis in S. pombe.
Publications
PapersSeino, H., T. Kishi,. H. Nishitani. and F. Yamao.
(2003). Two Ubiquitin-Conjugating Enzymes, UbcPll
Ubc4 and UbcP4/Ubell, Have Distinct Functions for
Ubiquitination of Mitotic Cyclin. Mol Cell Bioi 23,
3497-505

1. Dr. Fukagawa gave several lectures at The
Graduate University for Advanced Studies, Hayama

Division of Molecular Genetics

9

EDUCATION

1. Dr. Yamao was invited to give a seminar on"Ubiquitin Regulates Recombination and DNA
Damage Repair" at Virus Institute of Kyoto University,
June 2003 (in Japanese).
2. Dr. Y amao gave a lecture at Civil Education
Course of Numazu City, July 2003 (in Japanese)
3. Dr. Yamao gave a lecture at Department of
Veterinary Medicine, Yamaguchi University, December
2003 (in Japanese).

A-c. Molecular Mechanism Laboratory
Hiroshi Mitsuzawa GroupRESEARCH ACTIVITIES(1) Identification of proteins that interact with the Rpb7
subunit of RNA polymerase n

cerevisiae Nrd1. Further support for the functional
conservation came from the observation that Nrdl
interacted with Rpb7. We also found, throughsite-directed mutagenesis, that the Glu-166 and/or
Asp-167 of S. pombe Rpb7, residues near the C-terminusof the I72-amino acid protein, were important for its
interaction with Seb1.
X-ray crystal structure of the I2-subunit pol II (thelO-subunit core plus Rpb4-Rpb7) was reported in 2003
by two groups. In the pol II structure, Rpb4-Rpb7 islocated in the vicinity of the linker to the CTD, withwhich RNA processing factors are known to associate.
Because the CTD is likely to be mobile, the interactionof Sebl with. Rpb7 as well as the CTD may function
to anchor the putative processing factor to the pol II
apparatus, thereby processing machinery on the CTD
becomes close proximity to nascent RNA transcript.
In summary, our results, together with the recent
structural data, suggest that the Rpb7 subunit of pol
II has a role in coupling RNA processing to transcription in addition to in initiating transcription.

Hiroshi Mitsuzawa
Publications

RNA polymerase II (pol II) in eukaryotes, theenzyme responsible for synthesis of all mRNAs andsome of small nucleolar and small nuclear RNAs,
comprises twelve subunits, Rpbl to Rpbl2. Two ofthem, Rpb4 and Rpb7, form a heterodimer dissociablefrom pol II core. The Rpb4-Rpb7 heterodimer isdispensable for RNA synthesis but required fortranscription initiation. In the budding yeast Saccharomyces cerevisiae, deletion mutants of Rpb4 are
viable under optimal growth conditions, and growthdefects of the mutants are suppressed by overexpression of Rpb7. In addition, the high level of Rpb7 allows
its interaction with the pol II core in the absence of
Rpb4. These results suggest that Rpb7 is the criticalcomponent of the Rpb4-Rpb7 heterodimer.
To gain insight into the function of the Rpb7
subunit of pol II, we performed a two-hybrid screen for
Rpb7-interacting proteins using the fission yeast
Schizosaccharomyces pombe as a model organism. l ) The
screen identified a protein (named Sebl for sevenbinding) that is 28% identical to S. cerevisiae Nrdl, an
RNA-binding protein implicated in 3'-end formationof small nucleolar and small nuclear RNAs transcribedby pol II. Conservation of functional domains between
Sebl and Nrdl suggests that S. pombe Sebl is involvedin processing of pol II transcripts as in the case of S.

10

Division of Mutagenesis

PapersMitsuzawa, H., Kanda, E. and Ishihama, A. (2003). Rpb7
subunit of RNA polymerase II interacts with an
RNA-binding protein involved in processing oftranscripts. Nucleic Acids Res. 31, 4696-4701.

A-c. Molecular Mechanism Laboratory
Hiroaki Seino GroupRESEARCH ACTIVITIES(1) Ubiquitin-eonjugating enzymes
polyubiquitination of mitotic eyelin

involved

inHiroaki Seino
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. The ubiquitin-proteasome system is involvedin this process, requiring APC/C (anaphase-promoting
complex/cyclosome) as a ubiquitin ligase. In Xenopus
and clam oocytes, the ubiquitin-conjugating enzymes

that function with APC/C have been identified as
two proteins, UBC4 and UBCx/E2-C. Previously wereported that the fission yeast ubiquitin-conjugating
enzyme UbcP4/ubcll, a homologue of UBCxlE2-C, isrequired for mitotic transition. Here we show that theother fission yeast ubiquitin-conjugating enzyme,
UbcPl/Ubc4, which is homologous to UBC4, is also
required for mitotic transition in the same manner as
UbcP4lUbcll. Both ubiquitin-conjugating enzymes are
essential for cell division and directly required for the
degradation of mitotic cyclin Cdcl3. They function
nonredundantly in the ubiquitination of Cdcl3 becausea defect in ubcPl/ubc4+ cannot be suppressed by highexpression of UbcP4/Ubcll and a defect in ubcP4/
ubcll + cannot be suppressed by high expression of
UbcPlIUbc4. In vivo analysis of the ubiquitinated stateof Cdcl3 shows that the ubiquitin chains on Cdcl3
were short in ubcPl/ubc4 mutant cells while
ubiquitinated Cdcl3 was totally reduced in ubcP4/
ubcll mutant cells. Taken together, these results
indicate that the two ubiquitin-conjugating enzymes
play distinct and essential roles in the degradation of
mitotic cyclin Cdcl3, with the UbcP4lUbcll-pathway
initiating ubiquitination of Cdcl3 and the UbcPl/
Ubc4-pathway elongating the short ubiquitin chainson Cdcl31).
Publications
Papers5eino, H., Kishi, T., Nishitani, H. and Yamao, F.
(2003) Two Ubiquitin-Conjugating Enzymes, UbcPl/
Ubc4 and UbcP4/Ubcll, Have Distinct Functions for
Ubiquitination of Mitotic Cyclin. Mol Cell BioI 23,
3497-3505.
EDUCATION

Dr. H. 5eino was invited to gave a seminar on "Theregulatory mechanism of cell cycle by ubiquitination"at the National Institute for Basic Biology, Okazaki,
June, 2003 (in Japanese)

A-d. Division of Nucleic Acid Chemistry
Saburo Aimoto GroupRESEARCH ACTIVITIES(1) Development of a strategy that enables to use thethioester method and the native chemical ligation method
for the synthesis of a single protein
5aburo Aimoto (Institute for Protein Research, Osaka
University)
A thioester method and the native chemical
ligation method are useful for polypeptide synthesis.
Though both methods use peptide thioesters asbuilding blocks, no route was known to use thethioester method after the peptide bond formation
reaction by the native chemical ligation method. In thethioester method, the 5H 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 5H groups 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 a protectiong group that is easily
introduced to free 5H groups in polypeptide under m.ild
conditions and stable in the presence of silver ions. The
protecting group we found was a thiosulfonate group.
This group was quite easily introduced to 5H groups
in polypeptide only by mixing with Naz5406. The
resulting 5-5503 - was stable toward silver ions. Hence
this protecting group was ideal for 5H protection
produced by the native chemical ligation. In conclusion,
the use of 5503 - make it possible to employ the native
chemical ligation method and thioester method for thesynthesis of a single polypep-tide. 3)(2) Development of a photoremovable ligation auxiliary
for an extended native chemical ligation method
5aburo Aimoto (Institute for Protein Research, Osaka
University)
Most of synthetic strategies in peptide chemistry
use acid for the removal of protecting groups to obtainfree peptides. This procedure is true in extended native

Molecular Mechanism Laboratory

11

chemical ligation strategies, too. The ligation auxiliarygroups are removed by acid treatment at the final stageof synthesis. The acids frequently used for this purpose
are, for example, trifluoroacetic acid or anhydroushydrogen fluoride. This constitutes the restriction insynthesis of peptides with acid -labile modifications
and also in the development of synthetic methodologies
in which locally folded peptide segments are used asbuilding blocks. To develop a procedure to remove
auxiliary groups under mild conditions, we designed aphotoremovable ligation auxiliary group, 2-mercapto1-(2-nitrophenyl)ethyl group, based on the frame work
developed Dawson et. al. G. Offer, P. E. Dawson, Org
Lett., 2, 23-6 (2000)). A model synthetic experiment using
two decapeptides as building blocks showed that
ligation proceeded and the photoremoval of theligation auxiliary group proceeded under the irradiation of 365 nm light. Each reaction step is now under
optimization. 2)(3) 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 proteins and is the target of most pharmaceuticals. Much of the infonnation concerning thestructure 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-tenninal 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 has been optimized to give adesired product. The second coupling was carried out
by the thioester method to give ORL-l(288-270). Wearecontinuously searching the route to accomplish thetotal synthesis of ORL-1.

12

Division of Nucleic Acid Chemistry(4) 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-I), 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.
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-I in a manner analogous to thehuman immunodeficiency virus type-l protease inacquired immunodeficiency syndrome. In the design
potent protease inhibitors for this virus, the knowledgeof the characteristics of HTLV -1 PR itself and its
substrate specificities is critical. Then, we synthesized
HTLV-l PR and examined its substrate specificities.
Based on the obtained date we designed HTLV-lprotease inhibitors containing hydroxyethylamine
dipeptide isostere. l )Publications
Papers

1. Akaji, K., Teruya, K. and Aimoto, S. (2003).
Solid-phase synthesis of HTLV-l protease inhibitors
containing hydroxyethylamine dipeptide isostere. ].
Org. Chern., 68, 4755,4763.
2. Kawakami, T. and Aimoto, S. (2003). A photoremovable ligation auxiliary for use in polypeptide synthesis.
Tetrahedron Lett., 44, 6059-6061.
3. Sato, T. and Aimoto, S. (2003). Use of thiosulfonate
for the protection of thiol groups in peptide ligationby the thioester method. Tetrahedron Lett. 44,8085-8087.
Reviews

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Books

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Jt~fij.~'/!J -:xV. B*ft~~iii. OIV~: 1vi'f) ,pp.321-325.

EDUCATION

1. Dr. S. Aimoto was invited to give a lecture at Seoul
National University, Korea, November, 2003.
2. Dr. S. Aimoto was invited to give a seminar on"Perspective on chemical synthesis of protein" at theannual meeting of the Japanese Chemical Society,
Tokyo, April, 2003 (in Japanese).
3. Dr. Aimoto, S. was invited to give a seminar on"The history of chemical synthesis of proteins" at aforum of the Japanese Peptide Society, Okayama,
November, 2003 (in Japanese).
SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. S. Aimoto organized a symposium entitlted''Perspective on the Peptide and Protein Preparation",
Osaka, November, 2003. (in Japanese).
2. Dr. S. Aimoto, director of the Japanese Peptide
Society(B*~7TF~~:f!Il.ln

A-d. Division of Nucleic Acid Chemistry
Tsutomu Katayama GroupRESEARCH ACTIVITIESTranscriptional control for initiation of chromosomal
replication in Escherichia coli: fluctuation of origin
transcription ensures timely initiation!)(1)

Masayuki Su'etsugu, Akiko Emoto, Kazuyuki
Fujimitsu, Kenji Keyamura and Tsutomu Katayama
During the cell cycle, the initiation of chromosomal
replication is carefully controlled. In E. coli, the initiator DnaA and the replication origin oriC are major
targets for this regulation. Here, we assessed the roleof transcription of the mioCgene, which reads throughthe adjacent oriCregion. This mioC-oriCtranscription
is regulated in a replication cycle-coordinated manner
to be activated after initiation and to be repressed before initiation. We for the first time isolated a strain
bearing a mioC promoter mutation that causes constitutive mioC-oriCtranscription on the chromosome.
In this mutant fluctuations of the transcription wereabolished. Introduction of this mutation suppressed
the growth defect of an overinitiation-type dnaAcos

mutant, and severely inhibited growth of initiationdefective dnaA mutants at semi-permissive temperatures in a dnaA allele-specific manner. These results
suggested that the mioC-oriC transcription inhibits
initiation at orie. Indeed, flow cytometry analysis andquantification of DNA replication in synchronizedcultures revealed that the mioC promoter mutation
reduces initiation activity of chromosomal replication,
resulting in alterations in cell cycle-dependent initiation
control such as delay in time. These results suggestthat the transcriptional regulation of the mioC geneis required for cell cycle-coordinated initiation ofchromosomal replication.
(2) Structural analysis of the DnaA DNA-binding
domain complexed with DNA: Structural basis of
replication origin recognition by the DnaA protein2)

Norie Fujikawa, Hitoshi Kurumizaka, Osamu
Nureki, Takaho Terada, Mikako Shirouzu, Tsutomu
Katayama and Shigeyuki Yokoyama

Escherichia coli DnaA binds to 9 bp sequences(DnaA boxes) in the replication origin, oriC, to form
a complex initiating chromosomal DNA replication. In
the present study, we determined the crystal structureof its DNA-binding domain (domain IV) complexedwith a DnaA box at 2.1 A resolution. DnaA domain IV
contains a helix-tum-helix motif for DNA binding. One
helix and a loop of the helix-tum-helix motif are
inserted into the major groove and 5 bp (3' two-thirdsof the DnaA box sequence) are recognized throughbase-specific hydrogen bonds and van der Waals
contacts with the C5-methyl groups of thymines. In theminor groove, Arg399, localized in the loop adjacentto the motif, recognizes three more base pairs (5'one-thirds of the DnaA box sequence) by base-specifichydrogen bonds. DNA bending by about 28" was also
observed in the complex. These results well coincidewith the data of solution structure of this complex
obtained using NMR. 3) Base-specific interactions
revealed here explain how DnaA exhibits higher
affinity for the strong DnaA boxes (Rl, R2 and R4) than
the weak DnaA boxes (R3 and M) in the replication
origin.

Division of Nucleic Acid Chemistry13

(3) Molecular mechanism of DNA replication-coupled
inactivation of the initiator protein in Escherichia coli:
Binding of DnaA to the sliding clamp-loaded DNA andbinding of the sliding clamp to Hda
Masayuki Su'etsugu, Makoto Takata, Toshio Kubota
and Tsutomu Katayama
In Escherichia coli, the ATP-DnaA protein initiates
chromosomal replication. After the DNA polymerase
ill 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 illsliding clamp subunit, which results in yielding of
ADP-DnaA, an inactivated form for initiation. This
regulatory DnaA-inactivation is required to repress
extra initiation events. In this study, in vitro replication intermediates and structured 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 withclamps were not associated with DnaA-ATP
hydrolysis, and duplex DNA provided in trans did not
rescue this defect. Moreover, the DnaA-ATP hydrolysis
was inhibited by excessive supply in trans of a DnaA
box-bearing oligonucleotide that tightly binds DnaA
These results imply that the DnaA-ATP hydrolysis
involves the interaction of ATP-DnaA with duplex
DNA flanking the sliding clamp. Furthermore, Hda
protein formed a stable complex with the sliding
clamp, suggesting that this complex is an important
structure for interaction with ATP-DnaA These
proposals provide a molecular basis in mechanismsof the DnaA-inactivation that depends on chromosomal
DNA replication in vivo.
Publications
Papers

1. Su'etsugu, M., Emoto, A, Fujimitsu, K, Keyamura,

K and Katayama, T. (2003) Transcriptional control for
initiation of chromosomal replication in Escherichia
coli: fluctuation of the level of origin transcription
ensures timely initiation. Genes Cells 8, 731-745.
2. Fujikawa, N., Kurumizaka, H., Nureki, 0., Terada,
T., Shirouzu, M., Katayama, T. and Yokoyama, S.
(2003) Structural basis of replication origin recognitionby the DnaA protein. Nucleic Acid Res. 31, 2077-2086.

14

Division of Nucleic Acid Chemistry

3. Yoshida, Y, Obita, T., Kokusho, Y, Ohmura, T.,
Katayama, T., Ueda, T. and Imoto, T. (2003) Identification of the region of Escherichia coli DnaA protein
required for specific recognition of the DnaA box. Cell.
Mol. Life Sci. 60, 1998-2008.
4. Higuchi, K, Katayama, T., Iwai, S., Hidaka, M.,
Horiuchi, T. and Maki, H. (2003) Fate of DNA replication fork encountering a single DNA lesion during orie
plasmid DNA replication in vitro. Genes Cells 8, 437-449.
Reviews

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EDUCATION

1. Dr. Katayama gave a lecture for extension courses
at Kyushu University, Fukuoka, July, 2003 (inJapanese).
2. Dr. Katayama gave a lecture at Tochiku HighSchool, Fukuoka, October, 2003 (in Japanese).
3. Dr. Katayama and others gave a poster presentation in "Genome Square 2003 in Fukuoka", Fukuoka,
November, 2003 (in Japanese).
SOCIAL CONTRIBUTIONS AND OTHERS

A best paper award in the 75th annual meeting of the
Genetic Society of Japan was conferred on thepresentation by Kawakami, Su'etsugu and Katayama.

30saka University, 4National Institute of Advanced
Industrial Science and Technology)

B. DEPARTMENT OF
CELL GENETICS

B-a. Division of Cytogenetics
Tamotsu Yoshimori GroupRESEARCH ACTIVITIESMembrane traffic: the intracellular transport networkorganized by proteins and membranes
Tamotsu Yoshimori
Most of membrane-bound organelles in eukaryotic
cells are linked each other by dynamic membranetrafficking regulated by proteins. Mem brane traffic is
involved not only in survival of each cell but also invarious functions required for organizing themulti-cellular system, e.g., formation of cell polarity
and intercellular communication. We aim to unravelmolecular mechanisms of membrane traffic and theirroles in higher 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 received macromolecules taken up by
endocytosis from outside. The cargo is then either
sorted to lysosomes or recycled back to the plasma
membrane.
(1) Identification of a new component of a large protein
complex associated with the autophagic membrane
Noboru Mizushima\ Akiko Kumat, Yoshinori Kobayashit,
Akitsugu Yamamoto2, Masami Matsubae3, Toshifumi
Takao 3, Tohru N atsume 4, Yoshinori Ohsumi 1 andTamotsu Yoshimori CNational Institute for Basic Biology,~agahama Institute of Bio-Science and Technology,

We previously reported that association of the
Atg12-Atg5 conjugate with the small precursor
membranes is essential in autophagosome formation.
In this work, we demonstrated that the mouse
Atg12-Atg5 conjugate forms a "-'800 kDa protein
complex2l• Moreover, we identified a novel WD-repeat
protein as a component of the large protein complex2).
We have designated it mouse Atg16-like protein(Atg16L), since its N-terminal region has homology withyeast Atg16. However, C-terminal WD domain of
Atg16L is absent from the yeast homologue, suggesting
possible higher-eukaryote-specific function of thecomplex. Atg16L binds to both Atg5 and additional
Atg16L monomers. These interactions are, however,independent of the WD-repeat domain. Atg16L,
together with Atg12-Atg5 conjugate, is associated withthe membrane during formation of autophagosome.
The membrane targeting of Atg16L requires Atg5 but
not Atg12. Existence of the WD-repeat domain, which
provides a platform for protein-protein interactions,suggests further interaction of Atg16L with unidentified protein(s) in mammalian cells.
(2) Involvement of autophagy in degradation of
unfolded proteins causing diseases
Shisako Shouji, Takahiro Kamimoto, David H.
Perlmutter\ Akira Kakizuka 2 and Tamotsu
Yoshimori eUniversity of Pittsburgh, 2Kyoto
University)
Intracelluar accumulation and aggregation ofabnormal unfolded protein are linked to many
diseases, including neurodegeneration. It has been
believed that cells avoid accumulating potentially toxic
aggregates by degradation of unfolded protein mainly
via ubiquitin-proteasome system. We hypothesizedthat autophagy is also involved in degradation of suchprotein, since induction of autophagy has been often
observed in the unfolded protein diseases. To examine
the possibility, we have used disease models ofcultured cells; we overexpressed the expanded polyQ
fragment, which causes Huntington disease, or mutant
an-antitrypsin Z (ATZ), which causes degeneration ofliver cells. Atg5-deficient ES cells that we previously
established shows defect of autophagosome formation.

DEPARTMENT OF CELL GENETICS

15

In these cells, both polyQ and ATZ was accumulated
and aggregated more than in the normal ES cells. The
phenotype was suppressed by co-transfection of
wild-type Atg5 eDNA but not by the KR-mutant Atg5
which cannot form autophagosomes. A pulse-chaseexperiment demonstrated that ATZ degradation was
retarded in the Atg5-KO cells. The results suggest that
autophagy is involved in degradation of at least thetwo distinct unfolded proteins.

formatrion, in cells by adding the cytosol fraction andATP-regenerating system. The cytosol isolated fromstarved 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. We will identified
such factor(s) by using the reconstitution system.
(5) Functional analysis of SKDl by RNAi

(3) Analysis of autophagic response to invasion ofpathogenic bacteria into cells

Kayoko Tsuda, Ichiro Nakagawa', Atsuo Amano' andTamotsu Yoshimori COsaka University)

Atsuki Nara, Masako Sakai and Tamotsu Yoshimori
We previously demonstrated that the E235Q
mutant of mouse SKDl AAA ATPase lacking ATPhydrolysis activity has dominant negative effect ontransport from endosomes to both the plasma
membrane and to lysosomes, suggesting that SKDl isinvolved in sorting and transport out of cargos inendosomes. To address precise role of SKDl, we
decreased the endogenous protein by RNA interference
and showed significant inhibition of the endosometo-lysosome transport. We are examining othertransports including recycling and transport to the
TGN. Moreover, wefound that overexpression of SKDl
suppressed transport defect in some lipid storage
disease.

Some pathogenic bacteria invade into animal cells
and survive or proliferate inside cells. Such bacteria
often internalize to the membrane-bound compartments such as phagosomes and modulate them toavoid digestion in lysosomes. It was recently reported
that some bacteria appears in autophagosomes afterinvasion. We confirm this about two pathogenic
bacteria. They were colocalized with LC3, an
autophagosome membrane marker that we reported
previously. Autophagy was induced by infection ofboth bacteria in cultured cells. One of them significantly multiplied in the Atg5-KO cells, suggesting that
autophagy functions in cellular protection against
invaded bacteria.

(6) Interaction of Toml with Tollip, ubiquitin, andclathrin(4) Reconstitution of early step of autophagy by usingsemi-intact cells

Megumi Yamakami, Tamotsu Yoshimori and
Hideyoshi Yokosawal ('Hokkaido University)

Shunsuke Kimura, Atsuki Nara and Tamotsu
YoshimoriWe investigated protein-protein interaction ofToml, a member of the VHS domain-containing
protein family. Although proteins belonging this family
have been implicated in membrane trafficking, the roleof Toml is unclear. We found that Toml binds toubiquitin chains and Tollip, which is a mediator ofinterleukin-l signaling71 • Toml was also capable ofbinding to clathrin heavy chain 71• Based on the results,
we suggest that Toml plays a role in link ofpolyubiquitinated receptors to clathrin in endocytosis.

In vitro reconstitution of membrane trafficking byusing the semi-intact cell systems is a powerful tool
to resolve its molecular machinery; application of thetechnique to secretory pathway has provided many
crucial information about it. To elucidate mechanisms
underlying autophagic membrane dynamics, weestablished semi-intact cells by treatment of 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 toreconstitute binding of GFP-Atg5 to the precursor
membranes, the initial step of autophagosome

16

Division of Cytogenetics(7) Regulated transport of yeast amino acid permease

Kyohei Umebayashi and Akihiko Nakand CMolecular
Membrane Biology Laboratory, RIKEN)

Although it can be speculated that nutrient uptake
into cells is tightly regulated, there has been limited
experimental evidence. We have studied intracellulartransport of Tat2p, the yeast tryptophan permease, andshown that uptake of tryptophan is controlled bysorting of Tat2p to the plasma membraneS). When cells
are grown in high tryptophan medium, Tat2p is nottargeted to the plasma membrane. Instead, it is sorted
to the vacuole for degradation. In contrast, Tat2p istargeted to the plasma membrane when cells are
subjected to low tryptophan. Our detailed analysis
indicates that the sorting occurs in early endosomes
and that ubiquitination of Tat2p is the key determinant
for the sortint). Tat2p is polyubiquitinated on its way
to the vacuole, and inhibiting the polyubiquitination
results in efficient plasma membrane sorting. In
addition to ubiquitination, the sorting of Tat2p isdependent on sterols in the membraneS). Associationof Tat2p with a sterol-rich membrane domain, whichis called "lipid raft", is required for its plasma
membrane delivery. Failure in raft association resultsin missorting to the vacuole, and the sorting of Tat2p
is drastically affected by changes in sterol compositionof the membrane. These results lead to the hypothesisthat polyubiquitinated cargo proteins are excluded
from lipid rafts to be sorted to the vacuole.
(8) Ubiquitin-dependent protein sorting in the endocytic

pathway

Kyohei Umebayashi, Min-Soo Kim\ Yasue Hatanaka
and Tamotsu Yoshimori (tTokyo Metropolitan
Institute of Medical Science)
Epidermal growth factor (EGF) receptor (EGFR)
is ubiquitinated by the E3ligase Cbl, and it has been
considered that the ubiquitination is required forreceptor internalization from the plasma membrane.
We found, however, that Cbl is localized to earlyendosomes after cells are stimulated with EGF. Under
this condition, coimmunoprecipitation of EGFR with
Cbl has been confirmed. These results raise thepossibility that ubiquitination and sorting of EGFR
occurs also in endosomes. Weare now examining thefunctions of SKD1 in the receptor ubiquitination.
Recently, the concept has been emerging that receptor
signaling can occur in endosomes. We will examine thepossibility that EGFR can transmit the signal inendosomes.

Publications

Papers

1. Fujita, H., Yamanaka, M., Imamura, K, Tanaka,
Y, Nara, A, Yoshimori, T., Yokota, S. and Himeno,
M. (2003). A dominant negative form of the AAA
ATPase SKD1NPS4 impairs membrane trafficking out
of endosomal/lysosomal compartments: Class E vps
phenotype. ]. Cell Sci. 116,401-414.
2. Mizushima, N., Kuma, A., Kobayashi, Y.,
Yamamoto, A., Matsubae, M., Takao, T., Natsume, T.,
Ohsumi, Y and Yoshimori, T. (2003). Mouse Apg16L,
a Novel WD Repeat Protein, Targets to the Autophagic
Isolation Membrane with the Apg12-Apg5 Conjugate.
]. Cell Sci. 116, 1679-1688.
3. Saeki, K, Hong, Z., Nakatsu, M., Yoshimori, T.,
Kabeya, Y, Yamamoto, A, Kaburagi, Y. and You, A.
(2003) on line. Insulin-dependent signaling regulates
azurophil granule-selective macroautophagy in human
myeloblastic cells. ]. Leukoc. BioI. 74, 1108-1116.
4. Ohashi, M., Mizushima, N., Kabeya, Y. andYoshimori, T. (2003). Localization of Mammalian
NAD(P)H Steroid Dehydrogenase-like Protein on Lipid
Droplets. ]. BioI. Chern. 278, 36819-36829.
5. Katoh, K, Shibata, H., Suzuki, H., Nara, A.,
Ishidoh, K, Kominami, E., Yoshimori, T. and Maki,
M. (2003). The ALG-2-interacting protein Alix
associates with CHMP4b, a human homologue of yeast
Snf7 that is involved in multivesicular body sorting.
]. BioI. Chern. 278, 39104-39113.
6. Kanazawa, C., Morita, E., Yamada, M., Ishii, N.,
Miura, S., Asao, H., Yoshimori, T. and Sugamura, K(2003). Effects of deficiencies of STAMs and Hrs,
mammalian class E Vps proteins, on receptor
downregulation Biochem. Biophys. Res. Commun. 309,
848-856.
7. Yamakami, M., Yoshimori, T. and Yokosawa H.
(2003) on line. Tom 1, a VHS domain-containing
protein, interacts with Tollip, ubiquitin, and clathrin.
]. BioI. Chern. 278, 52865-72.
8. Umebayashi, K and Nakano, A (2003). Ergosterol
is required for targeting of tryptophan permease to theyeast plasma membrane.]. Cell BioI. 161, 1117-1131.
Reviews

9. Mizushima, N., Yoshimori, T. and Ohsumi, Y.
(2003). Role of the Apg12 conjugation system inmammalian autophagy. Int. ]. Biochem. Cell BioI. 35,
553-561.

Division of Cytogenetics

17;jlRj** $-'¥, s~

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Books

12. Yoshimori, T. and Mizushima, N. (2003) Mammalian homologues of Yeast Autophagy Proteins. In
Autophagy. Klionsky D. ]. ed. (Georgetown, Texas,
USA: Eurekah.com/Landes Bioscience), pp.204-21O.
13. s~ f!>iH2003) ~ ~ ~-A/::fJv:/#:/""Jv;t!f:-~~-A/KDELW7IJ/PDI/r~!m~H+-ATP7--e/j]~;t~://~~~D~./77,,/v-~-~~~D~•7}-T~~~'~~~!f:--?-F~~~2~

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EDUCATION

1. Dr. T. Yoshimori gave a lecture at Graduate Schoolof Medicine, Osaka University, May, 2003 (in Japanese).
2. Dr. T. Yoshimori gave a lecture at Division of
Pharmaceutical Science, Graduate School of Natural
Science & Technology, Kanazwa University, July, 2003
(in Japanese).
3. Dr. T. Yoshimori gave a lecture at Graduate Schoolof Bioagricultural Sciences, Nagoya University, July,
2003 (in Japanese).
4. Dr. T. Yoshimori gave a lecture at Graduate Schoolof Medicine, The University of Tokushima, November,2003 (in Japanese).
SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. T. Yoshimori is a councillor of the Japan
Society for Cell Biology.
2. Dr. T. Yoshimori gave a lecture on Open Day of
National Institute of Genetics, Mishima, April 2003.
3. Dr. T. Yoshimori organized a symposium entitled"Membrane trafficking to endosomesllysosomes: Thelong and winding roads" at the 76th Annual Meetingof the Japanese Biochemical Society, Yokohama,August 2003.

18

Division of Cytogenetics

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 employed budding yeast,
Saccharomyces cerevisiae, as a model system of
eukaryotic cells. Using strong genetics of budding
yeast, we have identified novel factors involving inchromosomal DNA replication and revealed theirfunctions in chromosomal DNA replication. We havebeen also interested in the S-phase checkpoint thatmonitors the status of DNA replication and arrest thecell cycle when DNA replication is inhibited since theDpbll protein we found seems to be related to thecheckpoint. Furthermore, we have extended our study
to duplication mechanism of chromatin structureaccompanying DNA replication.
(1) The interactions between replication proteins for the
initiation of DNA replication

Yoichiro Kamimura and Hiroyuki Araki
The initiation of eukaryotic chromosomal DNA
replication requires two steps; pre-replicative complex(pre-RC) formation at replication origins from late M
to G1 phase and replisome formation that is theassembly of factors involved in DNA synthesis in S
phase. In budding yeast, Dpbll forms a complex withessential DNA polymerase E (Pol E) and is required
for assembly of DNA polymerases with origins. Thus,
Dpbll functions for replisome formation in chromosomal
DNA replication. To know the molecular mechanismof replisome formation, we have identified Sld2,
Sld3-Cdc45 and GINS (see below) as factors geneticallyinteracting with Dpb11. All these factors function for
chromosomal DNA replication. In summary, Sld2
forms a complex with Dpbll and this complex is
involved in assembly of DNA polymerases with origins.
On the other hand, Sld3-Cdc45 functions as acomplex and the association of Sld3-Cdc45 with replication origins in the chromatin immunoprecipitation(ChIP) assay precedes Dpbll-Sld2 association withorigins. We recently identified the new complex
composed of Sld5, Psfl, Psf2 and Psf3 and named thiscomplex GINS (Go, !chi, Ni, San; Five, One, Two, Threein Japanese).!I) The GINS complex was also identified
in Xenopus egg extracts and found to function for DNA
replication.(2) We analyzed the replication intermediates by N/N 2D agarose gel electrophoresis andfound that the initiation frequencies of chromosomal
DNA replication reduces in sld5-12and psIl-l cells. The
ChIP assay showed that Psfl associates with replication origins at the same timing with Pol E in S phaseand the association of Psfl, Sld3 and Dpbll withorigins are mutually dependent. These results suggestthat Sld3-Cdc45, GINS, and Pol E-Dpbll-Sld2 arerequired for rep Iisome formation through someinteractions. Indeed, Psfl interacts with not only Dpbll
but also Sld3 in a two hybrid assay. We therefore
investigate the interaction between Sld3 and GINS in
detail. The C-terminal portion of Sld3 interacts with
Psfl (GINS). The sld3-1 mutation, which was originallyisolated by the synthetically lethal screening with
dpbll-l, occurred in the C-tenninal portion of Sld3 andSld3-1 proteins reduce the interaction with Psfl. These
data suggest that the interaction between Sld3 and
GINS is important for chromosomal DNA replication.
From these results we propose the following model as
to replisome fonnation. First, Dpbll-Sld2 interacts withPol Ein S phase. Next, Pol E -Dpbll-Sld2 is competent
to fonn a complex with GINS. Finally, this complex
is loaded onto replication origins via the protein
interaction between GINS and Sld3.
(2) Molecular mechanism of the initiation of DNA
replication regulated by cyclin-dependent kinase (CDK)

Yon-Soo Tak, Yoichiro Kamimura, Seiji Tanaka and
Hiroyuki Araki

independent of Cdc45 and Cdc7 kinase, both of which
are required for the initiation of DNA replication. Thus,
the complex formation seems to occur without anyother replication proteins. In yeast two-hybrid assay,
we mapped the interaction region of Sld2 and Dpbll
using various truncated forms of the proteins. Dpbll
has four copies of the BRCT domain, which isimportant for protein-protein interaction. Two BRCT
domains at C terminal of Dpbll bound to specificregion of Sld2. We also delimitated a binding domain
of Sld2 for Dpbll into a 39-amino acids stretch,partially overlapping with a cluster of the phosphorylation sites. While a 39-aa stretch binds to Dpbll in
phosphorylation-independent manner, the binding of
Dpbll and Sld2 depended on phosphorylation when
this 39-aa stretch was connected to a cluster of thephosphorylation sites of Sld2.
We then developed the in vitro binding assay of
Sld2 and Dpbll. This in vitro assay demonstrated that
the Sld2 protein directly interact with Dpbll andCDK-dependent phosphorylation of Sld2 (39 aa plus
a cluster of phosphorylation sites) enhances its binding
to Dpbll. Therefore, we propose that a cluster ofphosphorylation sites in Sld2 regulates binding of the
39-aa stretch to Dpbll.
The Sld2 protein is a good candidate for thesubstrate of CDK to regulate the initiation of DNA
replication. We therefore constructed the phosphomimetic form of Sld2, in which all the SP and TP
sites are substituted by DP. While this mutant protein
is functional, it could not bypass the requirement of
the CDK activity for the initiation of DNA replication.
It suggests that there are other important CDKsubstrates to initiate DNA replication.
(3) Functional analysis of the Dpbll protein

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 interaction with Dpbll, whichappears to be necessary for onset of DNA replication.
We thus studied how the interaction between Dpbll
and Sld2 is regulated by CDK activity.
We showed that Sld2 fonns a complex with Dpbll,

Sachiko Muramatsu and Hiroyuki Araki
To elucidate the function of Dpbll further, we
explored defect in dpbll-l cells. Previous studies showthat Dpbll is required for the origin association of
DNA polymerases at the initiation step of DNA
replication and moves rapidly away from origins in
ChIP assay, suggesting that Dpbll functions only forthe initiation of DNA replication. However, dpbll-l cells
that were arrested by hydroxyurea (HU), an inhibitorof DNA replication, and released at the restrictive
temperature lost their viability quickly. Moreover, theDivision of Microbial Genetics

19

cells that were released in medium with Nocodazole,
an inhibitor of mitosis, from HU at the restrictive
temperature did not restore viability. These results
suggest two possibilities. First, Dpb11 plays roles not
only before HU block but also after HU block in S
phase. Second, the origin firing before HU block is notenough to complete DNA replication. In the presenceof HU, cells released from Gl arrest activate only theorigins firing in early S phase. Thus, replication forks
travels longer distance than those in wild-type cells,
which may lead to collapse of replication forks.
We therefore examined collapse of replication
forks in dPbll-l cells. For this purpose, we observed
focus formation of Ddc2-GFP. Ddc2 is a damagecheckpoint protein that forms a focus at the damaged
DNA. We then found that the frequency of focus
formation of Ddc2 increases in dPbll-l. This result
suggests that Dpb11 is solely required for the initiation
step of DNA replication and its defect causes collapseof replication forks indirectly.
(4) Counteractions of DNA polymerase E and yCHRAC
for epigenetic inheritance of telomere position effectvariegation (TPE)13)

We therefore propose that inheritance of specificepigenetic states of telomere requires at least two
counteracting regulators.
Publications
Papers

1. Takayama, Y., Kamimura, Y., Okawa, M.,
Muramatsu, S., Sugino, A. and Araki, H. (2003). GINS,a novel multi-protein complex required for chromosomal
DNA replication in budding yeast. Genes & Dev. 17,
1153-1165.
2. Kubota, Y, Takase, Y., Komori, Y, Hashimoto,Y, Arata, T., Kamimura, Y, Araki, H. and Takisawa,
H. (2003). A novel ring-like complex of Xenopus proteins
essential for the initiation of DNA replication. Genes &Dev. 17, 1141-1152.
3. !ida, T. and Araki, H. (2004). Non-competitive
counteractions of DNA polymerase E and ISW21yCHRAC for epigenetic inheritance of telomereposition effect in Saccharomyces cerevisiae. Mol. Cell.
BioI. 24, 217-227.
Reviews

4.

Tetsushi !ida and Hiroyuki Araki
Relocation of euchromatic genes near heterochromatin
region often results in mosaic gene silencing. In
budding yeast, cells with the genes inserted attelomeric heterochromatin-like regions show
phenotypic variegation known as telomere-position
effect (TPE), and the epigenetic states are stably
inherited to following generations. Here we show thatthe epigenetic states of the telomere gene are not stably
inherited in cells either bearing a mutation in a
catalytic subunit (PoI2) of replicative DNApolymeraseE(PoIE) or lacking one of non-essential andhistone-fold motif-containing subunits of Pol E, Dpb3
and Dpb4. We also report a novel and putative
chromatin-remodeling complex, ISW2/yCHRAC that
contains Isw2, ltd, Dpb3-like subunit (Dlsl) and Dpb4.
Using the single-cell method developed in this study,
we demonstrated that without Pol E and ISW21yCHRAC, the epigenetic states of telomere are
frequently switched. Furthermore, we revealed that Pol
E and ISW2/yCHRAC function independently: Pol E
operates for stable inheritance of a silent state, while
ISW2/yCHRAC works for that of an expressed state.

20

Division of Microbial Genetics

m*51.z (2003) S M-tJ--1-7 !J /*ff'I1~T--t.n: J:~.~~ • • ~., ~.~~~, ~3$~

5. J::*t~-~~, m*5kz(2003) • • ~ -7 t1~7/.~,'-7 t1 ~7 / ~ J!~~mtfl~*,J.J (:f;@~lE~&ljiD, ~ .:L 7°!J/j)'- . 7:r. 77--7JIOj~, pp.121-129.

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

Seiichi Yasuda
DnaA is a protein essential for the initiation of
replication that occurs at a unique locus, oriC, on thechromosome in Escherichia coli. DnaA binds to the oriC
DNA and opens the double stranded DNA at an
AT-rich region within oriC. The opened singlestranded region serves as the site of assembly of otherproteins to form a replication complex. DnaA is madeof four functional domains. Among them, domain 3 is
responsible for ATP-binding, and domain 4 forsequence-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. To determine whether
this site is really involved in the DnaA-phospholipid
interaction, several mutant DnaA proteins wereconstructed and the release of bound ATP bycardiolipin treatment was measured. All mutant DnaA
had ATP-binding activity that was comparable to thewild type DnaA, implying that the function of domain
3 is intact in these mutants. One mutant protein thathas a complete deletion of the putative membraneinteraction site was found to release bound ATP as
efficiently as wild type DnaA upon cardiolipin
treatment. OriC DNA bound the protein normally andinhibited the nucleotide release almost completely aswith wild type DnaA. Another mutant DnaA that has
a deletion of almost all of domain 4 but that retains
the membrane-binding site was also prepared. Thismutant DnaA did not bind oriC DNA and showed norelease of bound ATP at all upon cardiolipin treatment.
These results suggest that the putative membranebinding site may not be involved in the cardiolipininduced release of nucleotides from DnaA. Recent
analyses of DnaA structure revealed the presence ofa large cavity between domain 3 and domain 4, wherebound DNA is located. They also showed that manyhydrogen bonds are formed between amino acid side
chains of DnaA and phosphate moiety of DNA.
Considering the partial structural resemblance between
DNA and cardiolipin in which two phosphate groups
are separated by three carbon atoms of glycerol, it
might be possible to suppose that cardiolipin interacts
with DnaA protein by binding to the DNA-binding site.

B-c. Division of Cytoplasmic Genetics
lakeo Yoshikawa GroupRESEARCH ACTIVITIES(1) Linkage disequilibrium (LD) scanning of functional
psychoses

Takeo Yoshikawa
We are continuing to collect human samples fromfamilies and unrelated individuals with schizophrenia
and mood disorder (particularly bipolar disorder) andcontrols. For sample recruitment and genetic analyses,
we are working closely with nation-wide collaborative
networks for schizophrenia (JSSLG: Japanese
Schizophrenia Sib-Pair Linkage Group)2) and mooddisorders (JGIMD: Japanese Genetics Initiative for
Mood Disorders). In 2003, we completed an LD scanof schizophrenia pedigrees using 444 genome-wide
microsatellite markers 151 • For chromosome 18p, we
have completed LD mapping of schizophrenia families
with a marker density of less than 1 cM, revealing
C180rfl as a tempting candidate gene, and publishedthis result9). 101• Currently, we are focusing on chromosome 6p where the dysbindin gene has been mapped
as a schizophrenia susceptibility gene and chromosome
8p where the neuregulin 1 and calcineurin genes arelocated. Both genes are reported to confer a risk-for
schizophrenia. For the study of endophenotypes, weare preparing to use the measurement of prepulse
inhibition (PPI) in schizophrenia.
(2) Genetic and functional analyses of candidate genes

Takeo Yoshikawa
Dysregulation of catecholaminergic neurotransmission has been proposed as a common pathophysiological basis for schizophrenia and mood disorders. In
2003, we analyzed PMX2B, a protein involved in the development and differentiation of midbrain dopaminergic and adrenergic neurons. PMX2B is also involvedin the development of brain motor neurons that control
the extraocular muscles for eye movement. This
protein functions as a transcription factor and contains
two alanine homopolymeric stretches. In general, thelength of alanine tracts in transcription factors is

Division of Microbial Genetics

21

strictly conserved, and alterations in the number ofalanine residues usually result in serious phenotypic
defects. In the C-terminal alanine stretch canonicallyconsisting of 20 alanine residues, we have foundnon-rare length polymorphisms that show association
with schizophrenia, especially in those patients whocarry ocular misalignments l4l •Another well established hypotheses for schizophrenia etiology, is that of hypoglutamatergic
neurotransmission via NMDA receptors. We analyzed
the NR2A subtype gene (GRIN2A) of the NMDA
receptor complex, because its ontogenic expression
corresponds to the peri-pubertal period of schizophrenia onset in humans and because it is abundantly
expressed in the cortices, anatomical regions relevant
to disease. We identified a polymorphic GT repeat in
the promoter region of GRIN2A, and published thefollowing results in 2003: (i) the longer the (GT)nstretch, the lower the promoter activity, (ii) the lengthof the (GT)n inversely correlates with MK80l bindingsites (MK80l specifically binds to NMDA receptors)
in cortical regions of postmortem brains, (iii) the mean(GT)n length is longer in schizophrenia than in control
samples and this length correlated with the severityof chronic outcome in schizophrenia7).
Continuing from 2003 and into 2004, we havereported on NR4A28) and we are studying the netrin
Gl and G2 genes.
For mood disorders, in 2003 we examined thegenes PMX2B and GRIN2A6 ) mentioned above, as well
as AKTl13 ) and NCAMll). Weare also investigating additional genes in collaboration with other laboratories.
We have performed a genome-wide expression
analysis of mRNA from the postmortem brains ofbipolar disorder subjects, which were provided by ourcollaborator, Dr. Brian from Australia. Weare now
correlating gene mRNA changes with protein expression levels. Once this has been completed, the genes
showing consistent changes will be treated as
candidates for population genetic analysis.
Myo-inositol monophosphatase 2 (IMPA2) isthought to be a target for lithium, a therapeutic drug
for mood swings, and it maps to 18pl1.2 where linkage
to both bipolar disorder and schizophrenia has beenreported. Weare currently determining the enzymaticfunction of IMPA2 and also making gene-manipulated
animals.

22

Division of Cytoplasmic Genetics(3) Studies on animal models

Takeo Yoshikawa
We studied gene expression profiles in the frontal
cortex and hippocampus of brains from learned
helplessness (LH) rats, a reliable depression model, andfound that Limkl is the gene of most dramaticallyreduced in the frontal cortex of LH rats ll ). Based on thisresult, we have obtained Limkl knockout mice andperformed extensive behavioral analyses of theseanimals during the year 2003. We have obtained datasuggesting that the gene-disrupted mice display
anxiety-related behaviors that are associated with
deficient serotonergic neurotransmission.
Previously, by performing QTL analysis, wereported the mouse chromosomal regions that control
immobility times for the forced swim test and tailsuspension test, behavioral paradigms for depression
models. In 2003, we have attempted to identify
responsible genes in these QTL, using two approaches:(i) continuing the preparation of consomic mice strains,(ii) narrowing down candidate genes by combining
information on differential expressions between
progenitor mouse strains, revealed by GeneChip
experiments, and information on the positions of thesegenes (i.e. whether these gene are located in QTL
regions or not). The latter strategy has led to the ideathat genes involved in actin turnover may underlie thepathophysiology of depression.
Deficits in prepulse inhibition (PPI) have beenreported in schizophrenics and first degree relatives
suggesting that it may represent an endophenotype forschizophrenia. PPI is one of the few behavioral
measures that can be examined by the same paradigm
in both rodents and humans. Based on the assumptionthat genes controlling PPI should overlap with thosefor schizophrenia susceptibility, we have performed
QTL analysis using mice. We first tested several
inbred strains for their intrinsic PPI, and found that
C57BLl6N (B6) mice showed the highest percentage of
PPI and the C3WHeN (C3) strain displayed the lowest.
During 2003, we prepared 1000 F2 mice from B6 andC3 progenitor strains, scored their PPI and genotyped
them using 80 genome-wide microsatellite markers.
Interestingly, we found that one QTL for PPI mapped
to the syntenic region for human chromosome 6p, awell documented linkage region for schizophrenia.

Publications
Papers

1. Arai, M., Itokawa, M., Yamada, K, Toyota, T.,
Arai, M., Haga, S., Djike, H., Sora, I., Ikeda, K and
Yoshikawa, T. (2004). Association of neural celladhesion molecule 1 gene polymorphisms with bipolar
affective disorder in Japanese. BioI. Psychiatry. 55:
804-810.
2. Arinami, T, Ishiguro, H., Minowa, Y, Ohtsuki, T,
Tsujita, T., Imamura, A., Yoshikawa, T., Toyota, T.,
Yamada, K, Shimizu, H., Yoshitsugu, K, Shibata, H.,
Fujii, Y, Fukumakil, Y, Tashiro, N., Inada, T., Iijima,
Y, Kitao, Y, Furuno, T., Someya, T., Muratake, T.,
Kaneko, N., Tsuji, S., Mineta, M., Takeichi, M., Djike,
H., Takehisa, Y, Tanaka, Y., Nakata, K, Kitajima, T.,
Nishiyama, T., Yamanouchi, Y, Iwata, N., Ozaki, N.,
Ohara, K, Ohara, K, Suzuki, Y, Shibuya, H., Ohmori,0., Shinkai, T., Hori, H., Nakamura, J., Kojima, T.,
Takahashi, S., Tanabe, E., Yara, K., Nanko, S., Yoneda,
H., Kusumi, I., Kameda, K, Koyama, T, Fukuzako, H.,
Hashiguchi, T., Tanabe, K and Okazaki, Y (2003).
Initial genome-wide scan for linkage with schizophrenia in the Japanese Schizophrenia Sib-pair Linkage
Group aSSLG) families. Am. J. Med. Genet. 120B: 22-28.
3. Ebihara, M., Ohba, H., Hattori, E., Yamada, K and
Yoshikawa, T (2003). Transcriptional activities ofcholecystokinin promoter haplotypes and theirrelevance to panic disorder susceptibility. Am. J. Med.
Genet. Neuropsychiatric Genet. 118B: 32-35.
4. Ebihara, M., Ohba, H., Kikuchi, M. and Yoshikawa,T. (2004). Structural characterization and promoter
analysis of human potassium channel Kv8.1 (KCNV1)
gene. Gene 325: 89-96.
5. Itokawa, M., Kasuga, T., Yoshikawa, T. andMatsushita, M. (in press). Identification of a maleschizophrenic patient carrying a de novo balanced
translocation, t(4;13)(pI6.1;q21.31). Psychiatry Clin.
Neurosci.
6. Itokawa, M., Yamada, K, Iwayama-Shigeno, Y.,
Ishitsuka, Y, Detera-Wadleigh, S.D. and Yoshikawa,T. (2003). Genetic analysis of a functional GRIN2A
promoter (GT)n repeat in bipolar disorder pedigrees
in humans. Neurosci. Lett. 345: 53-56.
7. Itokawa, M., Yamada, K, Yoshitsugu, K, Toyota,
T., Suga, T., Ohba, H., Watanabe, A., Hattori, E.,
Shimizu, H., Kumakura, T., Ebihara, M., Meerabux,].M.A., Toru, M. and Yoshikawa, T. (2003). A
microsatellite repeat in the promoter of the NMDA

receptor 2A subunit (GRIN2A) gene suppresses
transcriptional activity and correlates with chronic
outcome in schizophrenia. Pharmacogenetics, 13:271-278.
8. Iwayama-Shigeno, Y., Yamada, K, Toyota, T.,
Shimizu, H., Hattori, E., Yoshitsugu, K, Fujisawa, T.,
Yoshida, Y, Kobayashi, T., Toru, M., Kurumaji, A.,
Detera-Wadleigh, S. and Yoshikawa, T. (2003). Distribution of haplotypes derived from three common
variants of the NR4A2 gene in Japanese schizophrenic
patients. Am. J. Med. Genet. Neuropsychiatric Genet.
118B: 20-24.
9. Kikuchi, M., Yamada, K, Toyota, T, Itokawa, M.,
Hattori, E., Yoshitsugu, K, Shimizu, H. and Yoshikawa,T. (2003). Two-step association analyses of thechromosome 18pl1.2 region in schizophrenia detect alocus encompassing C18orfl. Mol. Psychiatry 8: 467-469.
10. Kikuchi, M., Yamada, K., Toyota, T. andYoshikawa, T. (2003). C180rf located on chromosome
18pl1.2 may confer susceptibility to schizophrenia. J.
Med. Dent. Sci. 50: 225-229.
11. Nakatani, N., Aburatani, H., Nishimura, K, Semba,
J. and Yoshikawa, T. (2004). Comprehensive expression
analysis of a rat depression model. Pharmacogenomics
J. 4: 114-126..
12. Segurado, R., Detera-Wadleigh, S.D., Levinson,
D.F., Lewis, C.M., Gill, M., Nurnberger, J.I.]r., Craddock,N., Raymond, DePaulo, ]., Baron, M., Gershon, E.S.,
Ekholm,]., Cichon, S., Tureck, G., Claes, S., Kelsoe, J.R,
Schofield, P.R., Badenhop, RF., Morissette, ]., Coon,
H., Blackwood, D., Curtis, D., Mcinnes, LA., Foroud,T., Edenberg, H.]., Reich, T., Rice, J.P., Goate, A.,
Mcinnis, M.G., McMahon, F.]., Badner, J.A., Goldin,
L.R, Bennett, P., Willour, V.L., Zand, P.P., Jianjun, Liu,]., Gilliam, C., Juo, S-H., Wade, H., Berrettini, W.H.,
Yoshikawa, T., Peltonen, L., Lonndnqvist,]., Nothen,M.M., Schumacher, J., Windemuth, C., Rietschel, M.,
Propping, P., Maier, W., AIda, M., Grof, P., Rouleau,
G.A., Del-Favero,]., Van, Broeckhoven, C., Mendlewicz,]., Adolfsson, R., Spence, M.A., Luebbert, H., Adams,L.]., Donald, J.A., Mitchell, P.B., Barden, N., Shink, E.,
Byerley, W., Muir, W., Visscher, P.M., Macgregor, S.,
Gurling, H., Kalsi, G., McQuillan, A., Escamilla, M.A.,
Reus, V.I., Leon, P., Freimer, N.B., Ewald, H., Kruse,
T.A., Mors, 0., Radhakrishna, D., Blouin, J-L.,
Antonarakis, S.E. and Akarsu, N. (2003). Genome scan
meta-analysis of schizophrenia and bipolar disorderpart ill: bipolar disorder. Am. J. Hum. Genet. 73: 49-62.
13. Toyota, T., Yamada, K., Detera-Wadleigh, SD.

Division of Cytoplasmic Genetics

23and Yoshikawa, T. (2003). Analysis of a cluster ofpolymorphisms in AKTl gene in bipolar pedigrees: afamily-based association study. Neurosci. Lett. 339: 5-8.
14. Toyota, T., Yoshitsugu, K, Ebihara, M., Yamada,K, Ohba, H., Fukasawa, M., Minabe, Y., Nakamura,
K, Sekine, Y., Takei, N., Suzuki, K, Itokawa, M.,
Meerabux, ].M.A., Iwayama-Shigeno, Y., Tomaro, Y,
Shimizu, H., Hattori, E., Mori, N. and Yoshikawa, T.
(2004). Association between schizophrenia with ocular
misalignment and polyalanine length variation in
PMX2B. Hum. Mol. Genet. 13: 551-561.
15. Yamada, K, Iwayama-Shigeno, Y., Yoshida, Y,
Toyota, T., Itokawa, M., Hattori, E., Shimizu, H. andYoshikawa, T. (in press). Family-based association
study of schizophrenia with 444 markers and analysis
of a new susceptibility locus mapped to 11qI3.3. Am.
J. Med. Genet. (Neuropsychiatr. Genet.)
16. Yamada, K, Watanabe, A., Iwayama-Shigeno, Y
and Yoshikawa, T. (2003). Evidence of association
between gamma-aminobutyric acid type A receptor
genes located on 5q34 and female patients with mooddisorders. Neurosci. Lett. 349: 9-12.
17. Yoshitsugu, K, Meerabux, J.M.A., Asai, K and
Yoshikawa, T. (2003). Fine mapping of an isodicentric
Y chromosomal breakpoint from a schizophrenic
patient. Am. J. Med. Genet. Neuropsychiatric Genet.
116B, 27-31.
EDUCATION

1. Dr. T. Yoshikawa gave a lecture at the 37th
Training Seminar for Psychiatric Medical Staff ofNagano Prefecture, under the title of "PsychiatricIllnesses and Genes", March, 2003 (in Japanese).

2.

**~Wmf--t *¥~¥$~F1ItiJJ~Mj

SOCIAL CONTRIBUTIONS AND OTHERS

Psychiatry and Clinical Neuroscience-editorial board
B *Wjtlfl . fT~)!{~~¥~:E!I!$24

Division of Cytoplasmic Genetics

c. DEPARTMENT OF
DEVELOPMENTAL GENETICS

C-a. Division of Developmental Genetics
Yasushi Hiromi GroupRESEARCH ACTIVITIES(1) Longitudinal axon guidance in the Drosophila CNS

Masaki Hiramoto and Yasushi Hiromi
The anterior-posterior organization of the animal
body plan requires axons to interconnect successive
segments. How the longitudinal axons that initiate theirtrajectory using intra-segmental guidance signals
interpret segmentally-repeated cues along the entire
length of the body is poorly understood. Roundabout(robo) is a gene required for the correct axonal
guidance, named after its mutant phenotype in
Drosophila; longitudinal axons fail to enter the next
segment and instead project towards the midline, as
if running along a roundabout (rotary). This phenotype
implies that there is a pathway choice point at thesegme.nt boundary: whether or not to observe an
attractive signal towards the midline. While the roleof ROBO as a receptor for the midline repellant Slitis well established, neither the nature of the attractive
field that generates the "roundabout" phenotype nor
the function of ROBO in the pathway choice is known.
We showed that in robo mutant longitudinal axons aresteered toward midline by responding to Netrin, an
evolutionary conserved midline attractant. Whilst a
gradient of Netrin can attract axons in vitro, Netrin that
misguides longitudinal axons is presented on thecommissural axons by the Netrin receptor Frazzled,thereby exerting a long-range effect. ROBO appears tocounteract Netrin signaling, allowing longitudinalaxons to enter the next segment.

(2) Cell-autonomous axonal patterning in Drosophila
neurons

Takeo Katsuki, Masaki Hiramoto and Yasushi Hiromi
Expression of axon guidance receptors in an axon
is temporally and spatially regulated, presumably
because it has a significant influence on growth conenavigation. In the Drosophila central nervous system,
members of the Roundabout (ROBO) receptor family(ROBO, ROB02 and ROB03) play crucial roles in axon
tract formation. Notably, ROBO proteins are accumulated specifically on the longitudinal axon segment but
remain at undetectable levels on commissures. This
raises a question of how neurons create such spatiallyregulated patterns of expression within axons. Can
neurons autonomously generate an intra-axonal
pattern, or are extrinsic factors required, such as
signals provided by midline cells? To address thisissue, we used a low-density cell culture system where
neurons grow axons in vitro, devoid of cell-cell contacts.
In cultured neurons, ROB02 and ROB03 werelocalized to the distal segment ofaxons, consistent
with the patterns observed in vivo. In contrast, ROBO
was uniformly distributed along the axon. These
results indicate that neurons possess an ability toregulate the localization of axon guidance receptors
cell-autonomously. Furthermore, the differences
observed among ROBO family members suggest thatthe localization is regulated in a highly moleculespecific manner.
(3) How is the positional information reorganized?

Tsuyoshi Nagasaka, Masaki Hiramoto and Yasushi
Hiromi
Positional information that guides organogenesis
is temporally multi-layered, changing its pattern andcharacter as development proceeds. Even a single
protein that provides positional information can beused many times during organogenesis, in different
developmental processes. For example, Netrin, asecreted axon guidance molecule, has its first rolein attracting commissural axons to the midline.
Subsequently, Netrin is captured by its receptor
Frazzled, and is presented
on the surface of theIcommissural axons as a pmjitional information for thepathfinding of longitudinal axons (Nature 406: 886,

DEPARTMENT OF DEVELOPMENTAL GENETICS

25

2000). Since axons that are initially guided by Netrin
are also those that later present Netrin, there might
be a causal link between these two processes. We aretrying to understand how Netrin secreted at one place
travels to the finalloca1,:ion for presentation as a local
guidance cue. To identify components that arenecessary for the correct positioning of Netrin within
the Drosophila CNS, we are screening a set of deletionstrains that lack a part of the genome, focusing on therelationship between the pathfinding ofaxons and the
distribution patterns of Netrin and other guidance
molecules. We find three typical abnormal distribution
patterns of Netrin: (1) Netrin completely absent, (2)Netrin absent on commissural axons, while present incells that secrete Netrin, (3) Netrin localized ectopicallyin the CNS. These phenotypes likely represent distinct
steps in Netrin expression, secretion and localization.
(4) Nuclear receptor Seven-up is essential for the
temporal switching of Neuroblast fate in the Drosophila
CNS.

Makoto Kanai, Masataka Okabe and Yasushi Hiromi
A critical step in generating neuronal diversity in
the Drosophila CNS is the production of various
neuronal and glial cell types by the sequential divisionof the precursor cell, neuroblast. Neuroblast undergoes
anum ber of asymmetric divisions generating a chainof secondary precursor cells. Rather than self-renewing
itself like a typical stem cell, neuroblasts change theircharacter upon division, so that they can produce
different type of progeny at the next division. Moleculardeterminants of the temporal character of theneuroblast are transcription factors HE, KR, PDM, etc.,
which are expressed sequentially in the developmental
history of each neuroblast. How neuroblasts switch its
expression profile from one transcription factor to thenext one is poorly understood. We focused on the geneseven-up, which encode a nuclear receptor class oftranscription factors, and demonstrated that it has an
essential function for a switch from the HB+, KR +state to the KR + state. While seven-up is expressedin almost all neuroblasts, it expression is restricted tothe subsection within the neuroblast lineage. Loss offunction mutant of seven-up increases the number ofcells of the early cell type (HB+, KR+) within several
neuroblast lineages. Lineage analysis provide theevidence that in mutant embryo neuroblasts fail to26

Division of Developmental Genetics

switch off HB+ at proper timing. These findings
indicate that seven-up is a fundamental regulator ofneuroblast switching.
(5) A search for Seven-up-interacting proteins

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, and in seven-up mutant these neurons aretransformed into another neuronal fate (Cell 60: 211,
1990). In the embryonic CNS seven-up expression is
temporally restricted to a subsection of the neuroblast
lineage, and its absence appears to cause a "temporaltransformation" of the fate of the neuroblast and its
progeny (see above). This suggests that seven-up
performs an instructive role in choosing particular cellfates. Misexpression of seven-up interferes with manycell fate decisions, causing transformation towards a
different fate, which depends on the cell that seven-up
is misexpressed (Development 118: 1123, 1993, Development 121: 1361, 1995). Interestingly, many of suchphenotypes induced by misexpression do not require
the DNA binding domain of Seven-up, suggesting thatthey are caused by titration of proteins that interact
with the ligand binding domain of Seven-up. By
studying the mechanism of such unusual cell fatecontrol, we expect to better understand the signallingcascade that occur downstream of Seven-up. Toward
this goal, we are searching Seven-up interacting
proteins by biochemical strategies. Such efforts should
also lead to identification of components of other, yet
unknown, cell fate switches.
(6) Diversification and differentiation of lateral glia in
the Drosophila CNS

Yoshihiro Yuasa and Yasushi Hiromi
Glial cells are important elements of the nervous
system, comprising more than 90% of the cells in thevertebrate brain. In the Drosophila embryo, there areapproximately 60 glial cells per abdominal neuromere.

Majority of these are "lateral glia", whose differentiation is regulated by the homeodomain protein REP0 2•
Lateral glia is a heterogeneous population of cells,consisting of various glial subtypes such as longitudinal glia, subperineural glia and channel glia,
classified based on the location, cell shape and lineage.
Very little is known about the processes of thediversification and differentiation of lateral glia cell
types. In order to identify genes involved in thedevelopment of lateral glia, we are screening
enhancer-trap lines for those that are expressed in thelateral glial cells. In addition to genes previously known
to be expressed in lateral glia, such as loco and spinster,
we identified eleven lines that are expressed in subsetsof lateral glia.
(7) Development of a novel fluorescent probe tovisualize caspase activity in a living organism

Shu Kondo and Yasushi Hiromi
Visualization of intracellular events in a livingorganism has discovered various amazing phenomenathat were unanticipated from in vitro experiments.
During animal development, many unnecessary cells
are removed by apoptosis. This is especially importantfor constructing fine structures such as the central
nervous system. However, compared to the current
knowledge of apoptosis in vitro, little is known about
naturally occurring cell death during development.
This is largely due to the lack of tools to detect
apoptosis in its early phase. To overcome this problem,
we created a genetically encoded fluorescent probe thatallows in vivo detection of the activity of caspase, asequence-specific protease that executes the final stepof apoptosis. The efficacy of this probe was validatedby time-lapse analysis of apoptosis in cultured cells.
Transgenic Drosophila harboring this probe weregenerated and several cases of apoptosis weresuccessfully visualized in live samples.
(8) The establishment of the stem cell fate in the
Drosophila female germline: A cell lineage study

daughter cells. These properties enable stem cells toplaya central role in generating and maintaining most
adult tissues in higher organisms. A fundamental yet
unexplored question in stem cell biology is how
the stem cell fate is initially determined during
development. In Drosophila germline, stem cells
descend from a subset of primordial germ cells (PGCs)
at the onset of oogenesis. However, it remains unknown
when and how this subpopulation is selected. It is
possible that the stem cell fate is determined at theonset of oogenesis. Alternatively, stem cell fate
determination may start earlier, as early as the
beginning of the expansion of PGC population. As afirst step to understand the mechanisms underlying
germline stem cell (GSC) fate determination, we dida lineage tracing analysis for individual PGCs from theembryonic stage to the adult stage. We found that at
the embryonic stage, PGCs located in the anterior partof the gonad produced PGC population that became
GSCs in the adult ovary. In contrast, PGCs in theposterior part of the gonad produced PGC populationthat differentiate directly to produce eggs. These
results suggest that GSC fate determination starts
before PGC proliferation. The anterior half of thesomatic embryonic gonads may provide a niche for theestablishment of GSCs.
Publications
Papers

1. Liu, Q-X., Jindra, M., Ueda, H., Hiromi, Y. andHirose, S. (2003). Drosophila MBF1 is a coactivator for
Tracheae Defective and contributes to the formationof tracheal and nervous systems. Development 130,719-728.
2. Yuasa, Y, Okabe, M., Yoshikawa, S., Tabuchi, K.,
Xiong, W.-C., Hiromi, Y. and Okano, H. (2003).
Drosophila homeodomain protein REPO controls glial
differentiation by cooperating with ETS and BTB
transcription factors. Development 130, 2419-2428.
3. Yamada, T., Okabe, M. and Hiromi, Y. (2003).
EDLIMAE regulates EGF-mediated induction byantagonizing Ets transcription factor Pointed.
Development 130, 4085-4096.

Miho Asaoka and Haifan Lin l (IDept. Cell Biology,
Duke University)

EDUCATION

Stem cells are characterized by their ability to
self-renew and to produce numerous differentiated1. Dr. Y. Hiromi gave a lecture course at Tokyo
University of Agriculture and Technology. January,

Division of Developmental Genetics

27

2003 (in Japanese).
2. Dr. Y. Hiromi gave a two-day lab course on"Genetic transformation" at Gyoshu High School.
December, 2003.

compartment seem to have a distinct function specificto the compartment.
(2) Evolutionary Origin of Autonomic Nervous System
in Hydra.

SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. Y. Hiromi served as an associate editor for

Genes to Cells.
2. Dr. Y. Hiromi served as an editor for Development,
Growth & Differentiation.
3. Dr. Y. Hiromi served as a member of the councilof The Genetics Society of Japan.
4. Dr. M. Okabe developed "Barrier-free presentationthat is friendly to colorblind people", and enhanced its
public awareness through web pages:
http://www.nig.ac.jp/color!
http://jfly.iam.u-tokyo.ac.jp/color!

C-a. Division of Developmental Genetics
Toshitaka Fujisawa GroupRESEARCH ACTIVITIES(1) Compartments of Neuron Subtypes Revealed by insitu Hybridization of Neuropeptide Genes in Hydra.

Toshitaka Fujisawa, Chiemi Fujisawa, Yasuharu
Takaku, Yukihiko Nora, Shiho Hyayakawal and
Takashi GojoborP eDNA Data Analysis laboratory,
Center for Information Biology)
We have identified new members of several
neuropeptide gene families in the Hydra ESTs. The insitu hybridization analysis revealed that some of thesegenes were expressed in particular regions along the
body column. Double in situ hybridization showed thatthe subpopulations of neurons expressing different
neuropeptide genes did not overlap thereby forminga sharp boundary between two regions. Meinhardt(2000) incorporating expression patterns of various axis
forming genes suggested that the Hydra body column
can be divided into several distinct regions. He further
suggested that the Hydra body could be an ancestralform of brain. The present results are consistent withhis view and suggest that the Hydra nervous system(or brain) is composed of several compartments.
Furthermore, the neuropeptides present in each

28

Division of Developmental Genetics

Hiroshi Shimizu, Masataka Okabe and Toshitaka
Fujisawa
Autonomic Nervous System (ANS) is a subpopulation of peripheral nervous system in mammals, which
regulates physiological activities of organs. The
mechanism of action of the ANS has been well studied,however, it remains unraveled when and how thisnervous system appeared in evolution and developed.
We propose that the most primitive form of ANS wasinvented in lower metazoan phyla such as Cnidaria
and Plathyhelminthes. On capturing prey, mammals
change their behavioral features from so-called "Fightor Flight stage" to "Rest and Digest stage". The heart
rate is reduced whereas digestion is activated after
feeding. Although defined solely in mammals, similarchanges of behavior and physiological activities canbe found also in other vertebrates or even in
invertebrates. We examined the physiological activities
of Hydra and found that Hydra and mammals show
similar changes after feeding, the motility andcirculation being reduced whereas digestion elevated.
We interpret these similarities to demonstrate that themost primitive form of ANS was invented in lower
metazoa and developed later with evolution.
(3) Motility of Endodermal Epithelial Cells Plays a
Major Role in Reorganizing Endodermal and Ectodermal
Tissue Layers.

Yasuharu Takaku, Takahiko Hariyama l andToshitaka Fujisawa CHamamatsu Univ. School of
Medicine, Hamamatsu)
Hydra has a simple body plan with only two layersof epithelial cells. It exhibits a strong regenerative
capacity from dissociated single cells. When dissociated cells are allowed to form aggregates, the firstevent to occur is sorting of cells to establish two cell
layers. In this study, we examined double-layer
formation under various situations. (1) When theaggregate of separated ectodermal cells and that ofendodermal cells were placed in direct contact, theendodermal cells migrated into the ectodermal~egate until entirely covered up by ectodermal cells(epiboly). This process was completely inhibited bycytochalasin B although initial firm attachment
between the two aggregates was not blocked. In
addition, cytochalasin pretreatment of only an
endodermal aggregate delayed the onset of epiboly. (2)A single endodermal epithelial (endoepi-) cell placedin contact with an ectodermal aggregate, actively
extended pseudopod-like structures and migrated
toward the center of the ectodermal aggregate. In
contrast, an ectoepi-cell remained in contact with anendodermal aggregate and never exhibited theingressive behavior. The cytochalasin B treatment of
only endodepi-cells abolished the ingress movements.
(3) Single endoepi- and/or ectoepi-cells were adhered
one by one up to 4-cell aggregates. Endoepi-cells
showed high motility that can be attributed to theingress movement described above. Finally, visualization of actin bundles with rhodamine-phalloidin always
correlated with pseudopod formation except for thelater stage of aggregate regeneration where actin
bundles were incorporated into smooth muscle fibers
at the base of epithelial cells and no longer sensitive
to cytochalasin. These results indicate that the highmotility of endoepi-cells plays an important role inreorganizing endodermal and ectodermal tissue layers.
Publications
Papers

1. Shimizu, H. and Fujisawa, T. (2003). Genesis 36,

182-186.
2. Takahashi, T., Kobayakawa, Y., Muneoka, Y.,
Fujisawa, Y., Mohri, S., Shimizu, H., Fujisawa, T.,
Sugiyama,T., Takahara, M. and Koizumi, O. (2003).
Comparative Biochemistry and Physiology 135,309-324.
3. Morishita, F., Nitagai, Y., Furukawa, Y.,
Matsushima, 0., Takahashi, T., Hatta, M., Fujisawa,T., Tsunamoto, S. and Koizumi, O. (2003). Peptides 6452,
1-10.
Reviews

4. Fujisawa, T. (2003). Developmental Dynamics 226,
182-189.

C-b. Division of Gene Expression
Susumu Hirose GroupRESEARCH ACTIVITIES(1) Role of GAGA factor-dependent chromatin remodeling in epigenenetic gene expression

Tsukasa Shimojima, Masahiro Okadat, Takahiro
Nakayama, Hitoshi Veda, Kenichi Nishioka, Hiroshi
Handa2, Tadashi Wada2and Susumu Hirose CPresent
address, Division of Molecular Genetics; 2Faculty of
Bioscience and Biotechnology, Tokyo Institute ofTechnology, Nagatsuda, Midori-ku, Yokohama,226-8501, Japan)
There are two types of epigenetic gene expression
in Drosophila. One is maintenance of Hox gene
expression governed by Polycomb (Pc) and trithorax(trx) group genes. The other is position effectvariegation (PEV). Trithorax-like (Trl) encoding GAGA
factor plays a role in the both types of regulation: Trl
is a member of trx group and Trl mutation is anenhancer of PEV.
We found that Drosophila FACT, a heterodimerof dSPT16 and dSSRP1, is associated with GAGA
factor through its dSSRPl subunit, binds to anucleosome and faciltitates GAGA factor-directed
chromatin remodeling. Immunostaining of polytene
chromosomes revealed colocalization of GAGA factor
and FACT in many specific loci. Mutation in Trlabolished the colocalization, indicating that GAGA
factor recruits FACT to the specific loci in the genome.
Genetic interactions between Trl and spt16implicate
the GAGA factor-FACT complex in the expression ofHox genes. Chromatin immunoprecipitation experiments indicated presence of the GAGA factor-FACT
complex in the bxd region of Ultrabithorax and the iab-6
element of Abdominal-B. These data illustrate a crucialrole of the GAGA factor-FACT complex in themodulation of chromatin structure for the maintenanceof Hox gene expression against Pc silencing;)lO).
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 (PEV). We found thatthe silencing of wwas enhanced by spt16mutation andfurther enhanced by Trl, spt16 double mutation.

Division of Developmental Genetics

29Chromatin immunoprecipitation assays revealed
presence of the GAGA factor-FACT complex on a site
just downstream of the coding region of w+ in
euchromatin. However, we observed significant
reduction in the occupancy of the complex on the samesite in tif'4 and further decrease in tif'4, Trl/ +. These
results indicate that the GAGA factor-FACT complex
contributes to maintenance of wexpression against
heterochromatin silencing. We also continued studies
on chromatin transcription in collaboration with Drs.
T. Wada and H. Handa.
Tracking of FACT and RNA polymerase II
elongation complex through chromatin in vivo
(2)

Abbie Saunders\ Janis Werner\ Erik D. Andrulis\
Takahiro Nakayama, Susumu Hirose, Danny
Reinber!f and John T. Lis! CMolecular Biology andGenetics, Biotechnology Building, Cornell University,
Ithaca NY 14850, USA; 2Howard Hughes Medical
Institute, Department of Biochemistry, Robert WoodJohnson Medical School, University of Medicine andDentistry of New Jersey, Piscataway, NJ 08854, USA).
RNA polymerase II (Pol ll) transcription throughnecleosomes is facilitated in vitro by the protein
complex FACT (Facilitates Chromatin Transcription).
We showed that FACT is associated with activelytranscribed Pol II genes on Drosophila polytene
chromosomes. Immunostaining of polytene chromosomes
and chromatin immunoprecipitation experiments
revealed that FACT displays kinetics of recruitment
and of chromosome tracking in vivo similar to Pol II
and elongation factors Spt5 and Spt6. Interestingly,
FACT does not colocalize with Pol ill-transcribedgenes, which are known to undergo nucleosometransfer rather than disassembly in vitro. Our
observations are consistent with FACT being restricted
to transcription that involves nucleosome disassembly
mechanisms 5l •(3) Role of DNA topology in the formation of active
chromatin

Kuniharu Matsumoto, Hirofumi Furuhashi, Youhei
Ogasawara, Hitoshi Ueda, Kazuma Hanai\ Koji
Akasaka l and Susumu Hirose CGraduate Universityof Gene Science, Hiroshima University, Kagamiyama,
Higashi-Hiroshima 739-8526, Japan)

30

Division of Gene Expression

Recent studies have established that chromatin
structure is important for transcriptional regulation.
However, our knowledge on conformation of chromatin
DNA was elusive due to lack of proper probes foranalyses of DNA topology in vivo. To circumvent thesituation, we have developed a method for visualizationof negatively supercoiled DNA within the interphasegenome. Using the methods, we observed transcriptioncoupled, unconstrained negative supercoils of DNA inapproximately 150 loci on polytene chromosomes ofDrosophila melanogaster.
Supercoiling factor (SCF) is a protein capable ofintroducing negative supercoils into DNA in conjuction
with DNA topoisomease ll. We have identified a fly line
carrying non-sense mutation within the coding regionof SCF. RNAi and genetic studies revealed that SCF
is involved in dosage compensation of X chromosome
in Drosophila.
We also made studies on a Drosophila counterpartof sea urchin insulator binding protein in collaboration
with Dr. K. Akasaka.
(4) Functional analysis of transcriptional coactivator
MBFl

Qin-Xin Liu, Marek Jindra, Hitoshi Ueda, Masataka
Okabe\ Yasushi Hiromi l and Susumu Hirose
CDivision of Developmental Genetics)
During gene activation, the action of sequence-specific regulators is transmitted to RNA polymerase bymeans of coactivators. Although many coactivators
have been reported, their developmental roles arepoorly understood. We conducted molecular andgenetic dissection of an evolutionarily conserved but
unique coactivator, Multiprotein Bridging Factor
1(MBF1) in Drosophila. The results of these studies
demonstrate that MBFl serves as a coactivator of abZIP protein Tracheae defective/Apontic during
development of the tracheal and central nervous
systems 3l •Despite the evolutionary conservation of MBFl
suggests an essential role for the protein, null mutants
lacking MBFl proved to be viable under laboratory
conditions. However, MBFl is essential during stresssituation encountered in the real world; mbfl mutants
are sensitive to oxidative stress induced by H202. A
comparative advantage provided by MBFl under the
stress condition is thus likely cause of its evolutionaryconservation.
(5) Mechanism of transcriptional regulation of the
FTZ-Fl geneYasuo Agawa, Masa-aki Yamada, Susumu Hirose andHitoshi Ueda
Insect FTZ-F1 is a unique transcription factor,
because it is induced after a pulse exposure of
ecdysteroids and is expressed just before hatching,larval ecdysis and pupation in a stage specific manner.
The stage specific expression of FTZ-F1 is necessaryfor embryogenesis, larval ecdysis and metamorphosis,
indicating that temporary precise regulation of the geneis important for the development of Drosophila.
Transcription factor p170 has been identified as asequence specific binding factor that is present duringhigh ecdysteroid periods and that binds to a cisregulatory region of the FTZ-F1 gene. To examinefunction of p170 during development, we first observed
loss of function phenotype. RNAi of p170 inducedpremature expression of FTZ-F1, suggesting that p170
works as a repressor for the FTZ-F1 gene. On the other
hand, over expression of p170 during high ecdysone
period from heat shock promoter-p170 cDNA fusion
gene resulted in super induction of FTZ-F1 after
decline of ecdysone level, indicating that the factor
potentiates expression of the FTZ-F1 gene during highecdysone periods. Transgenic fly reporter assays
supported the observations. From these results, we
propose p170 to be a novel type transcriptional
regulator.
(6) Establishment of a new mutant screening system inthe silkworm Bombyx mori

Hitoshi Ueda, Yuko Nagashima and Toshiki Tamura!
CNational Institute of Agrobiological Science)
Silkmoth, Bombyx mori is a lepidopteran insect
and has been used for genetical, biochemical,
endocrinological, neurobiological and pathologicalresearches as a useful research organism due to its
character that have not present in other insects.
However, molecular genetic analyses were delayedcompared with other organisms because of lacking a
convenient system to analyze established mutants. To
overcome the problem, we started a project to collectmutants induced by a transposon in the silkworm. To
make a mutant screen easily in the silkworm, wedeveloped new piggyBac transposone vectors that havea system to distinguish homozygous and heterozygous
mutants easily. Usefullness of the system wasconfirmed in Drosophila melanogaster and now is
testing in the silkworm in collaboration with Toshiki
Tamura.
(7) Histone lysine methylation: a signature for chromatinfunction
Robert]. Sims, li\ Kenichi Nishioka and Danny
Reinberg! CHoward Hughes Medical Institute, Divisionof Nucleic Acids Enzymology, Department ofBiochemistry, University of Medicine and Dentistry of
New Jersey, Robert Wood Johnson Medical School,
Piscataway, NJ 08854, USA)The rapid progress in deciphering the processesof covalent histone modifications has broadened ourunderstanding of transcriptional regulation. Histonelysine methylation, along with DNA methylation,
establishes the framework for long-term epigenetic
maintenance. Recent studies of the mechanisms ofspecific histone lysine methylation have revealed acomplex process that controls aspects of short- andlong-term transcriptional regulation, in addition to the
propagation of bulk chromosome structure andstability. In this article, we review the functional
properties of histone lysine methylation and the
enzymes that catalyze this covalent modification 7).
(8) Facile synthesis of site-specifically acetylated andmethylated histone proteins: Reagents for evaluation of
the histone code hypothesis

Shu He!, David Bauman!, Jamaine S. Davis!, Alejandra
Loyola2, Kenichi Nishioka, Jennifer L. Gronlund\
Danny Reinber~, Fanyu Ment, Neil Kellehet and
Dewey G. Mccafferty! CJohnson Research Foundation
and Department of Biochemistry and Biophysics,
University of Pennsylvania School of Medicine,Philadelphia, PA 19104-6059; ~oward Hughes Medical
Institute and Department of Biochemistry, Universityof Medicine and Dentistry of New JerseylRobert WoodJohnson Medical School, Piscataway, NJ 08854; and3Department of Chemistry, University of Illinois at
Urbana-Champaign, Urbana, IL 61801)

Division of Gene Expression

31

The functional capacity of genetically encoded
histone proteins can be powerfully expanded byposttranslational modification. A growing body ofbiochemical and genetic evidence clearly links theunique combinatorial patterning of side chain
acetylation, methylation, and phosphorylation mainly
within the highly conserved N termini of histones H2A,
H2B, H3, and H4 with the regulation of gene expression
and chromatin assembly and remodeling, in effectconstituting a "histone code" for epigenetic signaling.
Deconvoluting this code has proved challenging given
the inherent posttranslational heterogeneity of histone
proteins isolated from biological sources. Here we
describe the application of native chemical ligation to
the preparation of full-length histone proteins containingsite-specific acetylation and methylation modifications.
Peptide thioesters corresponding to histone N termini
were prepared by solid phase peptide synthesis using
an acid labile BodHF assembly strategy, then subsequently ligated to recombinantly produced histone
C-terminal globular domains containing an engineered
N-terminal cysteine residue. The ligation site is thenrendered traceless by hydrogenolytic desulfurization,
generating a native histone protein sequence. Synthetic
histones generated by this method are fully functional,
as evidenced by their self-assembly into a higher order
H3IH4 heterotetramer, their deposition into nucleosomes
by human ISWI-containing (Imitation of Switch) factor
RSF (Remodeling and Spacing Factor), and by
enzymatic modification by human Sirt1 deacetylase
and G9a methyltransferase. Site-specifically modified
histone proteins generated by this method will proveinvaluable as novel reagents for the evaluation of thehistone code hypothesis and analysis of epigenetic
signaling mechanisms 2l .
(9) Methods and tips for the purification of humanhistone methyltransferases

Kenichi Nishioka and Danny Reinbergl CHoward
Hughes Medical Institute, Division of Nucleic Acids
Enzymology, Department of Biochemistry, Universityof Medicine and Dentistry of New Jersey, Robert WoodJohnson Medical School, Piscataway, NJ 08854, USA)
Recently developed biochemical techniques haveenabled researchers to study histone modifications
more easily and accurately. One of these modifications,
histone lysine methylation, has been shown to be

32

Division of Gene Expression

highly stable and to represent an epigenetic alteration.
Extensive biochemical analyses have led to discoveries
about the nature and functions of this modification,
thus accelerating our understanding of this crucial
epigenetic event. Here we describe basic methods for
purification and biochemical analysis of lysinedirected, histone methyltransferases from HeLa
cell-derived extracts. In the section on substratepreparation, we describe a simple method for the
preparation of recombinant substrates, although we
recommend using native substrates for initial detectionof the activities. The purification protocols for several
histone methyltransferases have been streamlined sothat those researchers with a basic understanding of
biochemistry can perform them. We also describe
many tips and provide suggestions to avoid common
pitfalls in the biochemical analysis of histone
methyltransferases 41 .
(10) Construction of a replication-selective adenovirus
for ovarian cancer therapy

Katsuyuki Hamada\ Shohei Kohn0 2, Mari Iwamoto\
Hiroko Yokota\ Masato Okada4, Masatoshi Tagawa5,
Susumu Hirose, Kenshi Yamasaki3, Yuji Shirakata3,
Koji Hashimot03 and Masaharu ltd CDepartmentsof Obstetrics and Gynecology 2Neurosurgery and3Dermatology, School of Medicine, Ehime University,
Ehime 791-0295, Japan 4Division of Oncogene Research,
Research Institute for Microbial Diseases, Osaka
University, Osaka 565-0871, Japan. 5Division of
Pathology, Chiba Cancer Center Research Institute,
Chiba 260-8717, Japan)
Little is known concerning promoters or genetherapy specific for ovarian cancer. To explore thepotential use of IAl3B isolated from ovarian cancer
cells in gene therapy for ovarian cancer, we identified
the promoter region of the IAl3B gene and created areplication-selective adenovirus, AdE3-IAI3B, driven
by the promoter. Transient transfection experiments
showed that the DNA segment located between -1816
and -1 bp resulted in preferential expression in ovariancancer cells with negligible expression in squamouscell carcinoma and normal cells. The promoter activityof IAl3B was almost the same as that of cytomegalovirus
and an order of magnitude higher than those of
midkine and cyclooxygenase-2 in ovarian cancer cells.
AdE3-IAI3B replicated as efficiently as the wild-typeadenovirus and caused extensive cell killing in a panelof ovarian cancer cells in vitro. In contrast, squamouscell carcinoma and normal cells were not able tosupport AdE3-IAL3B replication. In animal studies,
AdE3-IAL3B administered to flank and i.p. xenograftsof ovarian cancer cells led to a significant therapeutic
effect. These results demonstrate the usefulness of the
IAL3B promoter for generation of ovarian cancerspecific adenoviral vectors and provide a potential forthe development of ovarian cancer-specific oncolytic
viral therapies. I)
Publications
Papers

1. Hamada, K, Kohno, S., Iwamoto, M., Yokota, H.,
Okada, M., Tagawa, M., Hirose, S., Yamasaki, K.,
Shirakata, Y., Hashimoto, K and Ito, M. (2003).
Identification of the human IAI.3B promoter element
and its use in the construction of a replication-selective
adenovirus for ovarian cancer therapy. Cancer Res., 63,
2506-2512.
2. He, S., Bauman, D., Davis, 1.S., Loyola, A, Nishioka,
K, Gronlund, 1.L., Reinberg, D., Meng, F., Kelleher, N.,
McCafferty, D.G. (2003) Facile synthesis of sitespecifically acetylated and methylated histone proteins:
reagents for evaluation of the histone code hypothesis.
Proc Nat! Acad Sci USA. 100, 12033-12038.
3. Liu, Q.-X., Jindra, M., Ueda, H., Hiromi, Y. andHirose, S. (2003). Drosophila MBF1 is a co-activator for
Tracheae Defective and contributes to the formationof tracheal and nervous systems. Development, 130,719-728.
4. Nishioka, K. and Reinberg, D. (2003) Methods and tips
for the purification of human histone methyltransferases.
Methods. 31, 49-58.
5. Saunders, A., Werner, J.. Andrulis, E. D.,Nakayama, T., Hirose, S., Reinberg, D. and Lis, 1. T.
(2003). Tracking FACT and RNA polymerase II
elongation complex through chromatin in vivo. Science,
301, 1094-1096.
6. Simojima, T., Okada, M., Nakayama, T., Ueda, H.,
Okawa, K., Iwamatsu, A, Handa, H. and Hirose, S.
(2003). Drosophila FACT contributes to Hox gene
expression through physical and functional interactions
with GAGA factor. Genes Dev. 17, 1605-1616.
7. Sims, R.1. 3rd, Nishioka, K. and Reinberg, D. (2003)
Histone lysine methylation: a signature for chromatinfunction. Trends Genet. 19, 629-639.

8. Markova, E. P., Ueda, H., Sakamoto, K, Oishi, K,
Shimada, T. and Takeda, M. (2003) Cloning of Cyc(Bmal-1) homolog in Bombyx morT, Structural analyses
and the tissue specific distributions. Comp.Biochem.
Physiol. B. 134, 535-542.
Reviews

9.

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re;A t-:/H4!J V::/200);(71HI:

1;:,t9-:7 0-;:"7:/~~~~O)iliIJ1iflJJ*IBIffllI¥22,669-674.
10. JAm! ~ (2003) r -:7 0 -;:"7:/ !J "E-f!J :/ -:7° C GAGAE§ -f-J 1::1*OYf-!.¥ 55, 193-196.
11. JAm! ~(2003) r:/ 3 '7 v: 3'7 /{",:::r..0)-:7 0-;:"7:/ CjI-fi;-f-ililj1iflJJ "-:7 0-;:"7:/cjI-fi;-f-;I~U~fliIJ1iflJ". tIiil~lE~
m(:/ ::L 7' !J :/ jJ - . 7::x:. 7 '7 - -:7 :~IOjO, pp233-240.
12. l:.EE ~ (2003) r Ee.!R~1::Ji~H~H;::Jo It 9 *lv"E- :/fliIJ
1iflJ1;:,t 9J1-fi;-f-:1J ;AJr- FJmBJ!i~~ 48, 2254-2260.

EDUCATION

1. Dr. S. Hirose was invited to give a seminar on"Chromatin remodeling as a boundary against
silencing" at the Institute of Molecular and Cellular
Biology, The University of Tokyo, Tokyo, June, 2003
(in Japanese).
2. Dr. S. Hirose gave a lecture at University of
Shizuoka, Shizuoka, December, 2003 (in Japanese).
3. Dr. S. Hirose was invited to give a seminar on"Chromatin remodeling serves as boundary against
silencing" at Department of Medicine, Okayama
University, Okayama, November, 2003 (in Japanese).
SOCIAL CONTRIBUTIONS AND OTHERS*f~

1. l±lIJiifl:%: 2003-146059, ~1l~0)15#J\ : jr{O)ImG-ltA,
DNA~l±l~~,~~~:JAm!

~'~*OO~,l±lOA:

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

Koichi Kawakami, Yasuyuki Kishimoto and Ghislaine
MorvanDivision of Gene Expression

33

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 water 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. Taking
advantage of these merits, large-scale chemicalmutagenesis screens in zebrafish had been successfully
carried out and hundreds of mutants defective in every
aspect of early developmental processes had beencollected. It has, however, not been easy to identify themutated genes since most of the chemically induced
mutations were point mutations and time-consuming
positional cloning approaches are required to clone thegenes. Therefore it is important to develop insertionalmutagenesis methods in zebrafish, such as the P
transposon system in Drosophila.
The Tol2 element is a transposable element
identified from the genome of the medaka fish.
Previously we have found that the Tol2 element
encodes a fully functional transposase and have shownthat the Tol2element can transpose into the zebrafish
genome in the germ lineage. To date, the Tol2element
is the only natural transposon in vertebrate,. that has
ever been shown to encode a functional transposase,and thereby from which an autonomous element has
been identified. We have been interested in developing
novel insertional mutagenesis methods in zebrafish
using the Tol2 element. Using this Tol2 transposon
system, we successfully established a protocol togenerate transgenic zebrafish very efficiently. We thenmade an exon trap construct with a promoter-less GFP
gene using the Tol2 transposon, and generated
hundreds of insertions of the exon trap construct inthe zebrafish genome. 10% of the insertions yielded"specific" GFP expression, indicating that the gene(exon) trap method using the Tol2transposon system
is successfully working. In those fish, GFP wasexpressed the heart, forebrain, midbrain, midbrainhindbrain boundary, hindbrain, notochord, floorplate,etc. Our current goal is to establish methods to identify
genes expressed in a spatially and temporally regulated
fashion and play an important role in development of
the structure or the organ where the GFP expression
is detected. Efforts are in progress toward this goal.

34

Division of Molecular and Developmental Biology(2) Genetic analysis of zebrafish maternal-effectmutations affecting early embryogenesis

Yasuyuki Kishimoto, Koichi Kawakami, Sumito
Koshida 2, Makoto Furutani-Seiki 1 and Hisato
Kondoh 1•3 eKondoh differentiation project (ERATO)
JST; 2Center for Integrative Bioscience, Okazaki
National Research Institute; 30saka University)
Maternal-effect genes play essential roles in early
embryogenesis particularly before activation of thezygotic genes. We previously carried out a genetic
screen for mutations affecting such maternal-effectgenes employing an F3 screen strategy, identifying 6
recessive mutations out of 60 mutagenized genomesl .
4 mutations exhibiting characteristic phenotype wereanalyzed. Three of the mutations (acytokinesis
mutations: acltt.~ aclt t62 and aclt t119)caused absence ofcell cleavage, without affecting nuclear divisions. These
embryos are defective in generating contractile rings.
acltl62 mutation abolishing reactions to organize
cortical F-actin, while other mutations causing abortive
contractile ring-like structures at ectopic sites. Defectof contractile ring formation in the affected embryos
leads to the absence of microtubule arrays at theprospective cleavage plane. Thus, these mutations
reveal the sequence of events associated with cytokinesis,in particular, the cortical actin dynamics. 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 second cellcleavages in the total absence of cytokinesis. This
indicates that the basic cell architectures of early
embryos are largely established by the autonomous
activities of the mitotic apparatus, without muchdependence on the cell cleavage machinery.
The bobtail (btl) mutant exhibited a recessive
maternal effect causing a strong reduction of the
tailbud outgrowth and abnormal posterior mesodermal
development. Whole mount in situ hybridization
analysis revealed that, in btl mutant embryos, myoD
expression in adaxial and paraaxial mesoderm isstrongly reduced without affecting the level of no tailexpression in axial mesoderm. btl is mapped on
chromosome 17, and positional cloning of the genemutated in btl is under way.

(3) Transposition of the Tol2 element in mouseembryonic stem cells.

Koichi Kawakami and Tetsuo Nodal eTohoku
University)
The To12 transposable element of the Japanese
medaka fish belongs to the hAT family of transposons
including ho bo of Drosophila, Ac of maize, and Tam3of snapdragon. To date, To12 is the only naturaltransposon in vertebrate that has ever been shown to
encode a fully functional transposase. It has not been
known, however, whether To12 can transpose invertebrates other than fish. We demonstrated transposition of To12 in mouse embryonic stem (ES) cells.
We constructed a transposon donor plasmid containing a nonautonomous To12element with the neomycin
resistance gene and a helper plasmid capable ofexpressing the transposase, and introduced the donor
plasmid with various amounts of the helper plasmidby electroporation into mouse ES cells. The numberof G418-resistant ES colonies increased as the amountof helper plasmid was increased, in a dose-dependent
manner, indicating that the transposase activity
elevated the integration efficiency. These G418-resistant
ES colonies were cloned and the structure of thejunction of the integrated To12 element and the
genomic DNA was analyzed by inverse PCR. In thoseclones, To12 was surrounded by mouse genomic
sequences and an 8-bp direct repeat was createdadjacent to the both ends of To12, indicating that To12
was integrated in the genome through transposition.
The To12 transposon system is thus active in mouse
as well as in fish. We propose that it should be used
as a genetic tool to develop novel gene transfer,transgenesis and mutagenesis methods in mammals.
(4) The Fugo EST projects

Melody S. Clark" Yvonne J.K. Edwards" Dan
Peterson2, Sandra W. Clifton2, Amanda]. Thompson\
Masahide Sasaki3, Yutaka Suzuki 3, Kiyoshi Kikuchi\
Shugo Watabe\ Koichi Kawakami, Sumio Sugan03,
Greg Elgar and Stephen L. Johnson 2CMRC RosalindFranklin Centre for Genomics Research, UK;
2Washington University Medical School, USA; 3The
Institute of Medical Science, University of Tokyo;
4Graduate School of Agricultural and Life Sciences,
University of Tokyo)

The draft Fugu rubripes genome was released in
2002, at which time relatively few cDNAs were available
to aid in the annotation of genes. We sequenced andanalyzed 24,398 expressed sequence tags (ESTs)
generated from 15 different adult and juvenile Fugu
tissues, 74% of which matched protein databaseentries. Analysis of the EST data compared with theFugu genome data predicts that approximately 10,116
gene tags have been generated, covering almost
one-third of Fugu predicted genes. This represents aremarkable economy of effort. Comparison with the
Washington University zebrafish EST assemblies
indicates strong conservation within fish species, but
significant differences remain. This potentiallyrepresents divergence of sequence in the 5' terminalexons and UTRs between these two fish species,
although clearly, complete EST data sets are notavailable for either species. This project provides new
Fugu resources, and the analysis adds significant
weight to the argument that EST programs remain an
essential resource for genome exploitation andannotation. This is particularly timely with theincreasing availability of draft genome sequence fromdifferent organisms and the mounting emphasis on
gene function and regulation.
(5) Characterization of the hagoromo gene in East
African cichlids

Yohei Terait, Naoko Morikawa" Koichi Kawakami
and Norihiro Okada l eGraduate School of Bioscienceand Biotechnology, Tokyo Institute of Technology)
The adaptive radiation of cichlid fishes in the lakesof East Africa is a prime example of speciation. The
choice of cichlid mates on the basis of a variety ofcoloration represents a potential basis for speciationthat led to adaptive radiation. We characterized thecichlid homolog of the zebrafish hagoromo (hag) genethat was recently cloned and characterized from apigmentation mutant. Although only one hagmRNAwas reported in zebrafish, cichlids express ninedifferent hag mRNAs resulting from alternative
splicing. The hagmRNAs are expressed between the
myotome and the epidermis where pigment cells arelocated, suggesting the cichlid hag gene is involved inpigmentation. The hag mRNA splicing pattern does
not fluctuate among individuals from each of two
species, suggesting that alternative splice site choice

Division of Molecular and Developmental Biology

35

is fixed within species. Furthermore, cichlids inlineages that underwent explosive speciation expresseda greater variety of hagmRNAs than those in lineages
that did not undergo such a degree of speciation,suggesting that species in the explosively speciatedlineage acquired a complex regulatory mechanism ofalternative splicing over a very short evolutionary
period. Thus we provide an example in whichalternative splicing may playa role in mate choice,leading to cichlid speciation through diversification ofgene function by production of multiple mRNAs froma single gene.
Publications
Papers

1. 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.
2. 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.
3. Clark, M.S., Edwards, Y.].K, Peterson, D., Clifton,
S.W., Thompson, A.J., Sasaki, M., Suzuki, Y, Kikuchi,
K, Watabe, S., Kawakami, K, Sugano. S., Elgar, G. andJohnson, S.L. (2003). Fugu ESTs: New resources fortranscription analysis and genome annotation. Genome
Research 13, 2747-2753.
4. Terai, Y., Morikawa, N., Kawakami, K and Okada,N. (2003). The complexity of alternative splicing ofhagoromo mRNAs is increased in an explosively
speciated lineage in East African cichlids. Proc. Nat!.
Acad. Sci. USA 100, 12798-12803.
EDUCATIONK Kawakami gave a lecture at Sars Center for Marine
Molecular Biology, Norway, March, 2003.
SOCIAL CONTRIBUTIONS AND OTHERS

K. Kawakami organized a symposium at 26th themolecular biology society of Japan annual meeting,
Ko be, Dec. 2003.

C-d. Division of Physiological Genetics
Haruhiko Koseki GroupRESEARCH ACTIVITIES(1) Functional and biochemical analyses of mammalian
Polycomb-group proteins
Haruhiko Koseki
1) Association of mammalian PcG proteins at Hoxb8
gene in mice
Mammalian RinglB is involved in at least two
disticnt multimeric protein complexes both are closely
related to Drosophila Polycomb reppressive complex-l(PRC1). We have previously shown that RinglE
mediates transcriptional repression of Hox cluster
genes (Suzuki et al., 2002). To understand how RinglE
functions on Hox cluster, we have compared thebinding pattern of RinglE on Hoxb7/8/9 region byusing chromatin immunoprecipitation with 500 to 1000
bp resolution between cranial and caudal regions of
11.5 dpc embryos where Hoxb7/8/9 are repressed ortranscribed, respectively. RinglB turned out toassociate predominantly to promotor and enhancer
regions in cranial region where Hoxb7/8/9 aretranscriptionally inactive. Closely related protein
RinglA distribution was very similar to RinglE,
however, other PcG components, Rae28/Mphl andM33, were associated with Hoxb7/8/9 region in bothcranial and caudal regions. This implies Polycomb
complexes are associated with chromatin in not onlytranscriptionally inactive but also active region. We
further examined degree of HistonH3 acetylation andmethylation on Hoxb7/8/9 regions. Acetylated HistonH3
predominantly associated to Hoxb7/8/9 regions in thecaudal region. In addition, we found colinearity
between gene order and acetylation of HistonH3. We
did not find out significant methylation of HostonH3.
These observations suggest that inversely gradedassociations of mammalian polycomb RinglB andacetylated HistoneH3 associations upon Hoxb7/8/9
region is involved in the maintenance of anterior
boundaries of their expression.
2) Role of PcG proteins during organ development
We have isolated Mph2 (Mouse polyhomeotic-2)
as an interacter of Me118, a homologue of Drosophila

36

Division of Molecular and Developmental Biologyposterior sex combs (Psc) and demonstrated that Mph2
is an component of mammalian PcG complexes. We
have previously reported Me118 mutants show defects
in lymphoid development that include subtle hypoplasia
of Peryer' s patches (PPs). Mph2 single deficient mice
showed posterior transformations in the axial skeleton
but did not exhibit obvious defects in lymphoiddevelopment. Mph2 mutation, however, was shown toenhance defects in PP development observed in Me118
mutants. In Mph2/Me118 doubly deficient mice,
accumulation of haematopoietic cells into prospective
PP anlagen may be significantly affected and subsequently condensation of PP anlagen were clearly
impaired. Either number and proliferation of PP
inducer cells in the developing intestine or theirmigratory activity upon BLC signaling was not
significantly affected in double mutants. Immunohistochemical analysis revealed BrdU-Iabeled CD45positive cells were reduced exclusively in PP anlagenin gene-dosage dependent manner. It is thus suggested
PcG multimeric complexes may be essential for
proliferation of lymphoid-lineage cells immigrated into
PP anlagen. Mechanisms underlying this defect iscurrently addressed.
Publications
Papers

1. Shirasawa, T, Izumizaki, M., Suzuki, YI., Ishihara,
A, Shimizu, T., Tamaki, M., Huang, F., Koizumi, KI.,
Iwase, M., Sakai, R., Tsuchida, E., Ueshima, K, Inoue,
H., Koseki, H., Senda, T., Kuriyama, T and Homma,
I. (2003) Oxygen affinity of hemoglobin regulates 02
consumption, metabolism, and physical activity. J BioI
Chem 278, 5035-5043.
2. Fukuda, K, Yoshida, H., Sato, T., Furumoto,T., Mizutani-Koseki, Y., Suzuki, Y., Saito, Y.,
Takemori, T., Kimura, M., Sato, H., Nakayama,T., Taniguchi, M., Nishikawa, S. and Koseki, H.
(2003) Mesenchymal expression of Foxl1, a winged helix
transcriptional factor, regulates generation andmaintenance of gut associated lymphoid organs. Dev.
BioI. 255, 278-289.
3. Yamada, R, Mizutani-Koseki, Y, Hasegawa, T.,
Osumi, N., Koseki, H. and Takahashi. N. (2003).
Requirement for Mab2111 during development ofmurine eye and preputial gland. Development 130,
1759-1770.
4. Horie, A., Isono, K and Koseki, H. (2003)

Generation of a monoclonal antibody against themouse Sf3b1 (SAP155) gene product for U2 snRNP
component of spliceosome. Hybridoma and Hybridomics
22,117-119.
5. Kaneko, T., Miyagishima, H., Hasegawa, T.,
Mizutani-Koseki, Y, Isono, K and Koseki, H. (2003)
Mouse YAF2 gene generates two distinct transcripts
and is expressed in pre- and postimplantationembryos. Gene 315, 183-192.
6. Miyagishima, H., Isono, K, Fujimura, Y., Iyo, M.,
Takihara, Y., Masumoto, H., Vidal, M. and Koseki, H.
(2003) Dissociation of mammalian Polycomb-groupproteins, RinglE and Rae28/Ph1, from the chromatincorrelates with configuration changes of the chromatinin mitotic and meiotic prophase. Ristochem Cell. BioI
120, 111-119.
7. Nakamura, Y, Ozaki, T, Koseki, R., Nakagawara,
A and Sakiyama, S. (2003) Accumulation of p27(KIP1)
is associated with BMP2-induced growth arrest andneuronal differentiation of human neuroblastomaderived cell lines. Biochem Biophys Res Commun. 307,206-213.
8. Shin, T., Kennedy, G., Gorski, K, Tsuchiya, H.,
Koseki, H., Azuma, M., Yagita, H., Chen, L., Powell, J.,
Pardoll, D. and Housseau, F. (2003) Cooperative B7-1/
2 (CD80/CD86) and B7-DC Costimulation of CD4+ T
Cells Independent of the PD-1 Receptor. J Exp Med.
198,31-38.
9. Nakajima, M., Yuasa, S., Ueno, M., Takakura, N.,
Koseki, R. and Shirasawa, T. (2003) Abnormal bloodvessel development in mice lacking presenilin-l. Mech
Dev. 120, 657-667.
10. Koike,]., Wakao, H., Ishizuka, Y., Sato, TA,
Hamaoki, M., Seino, K., Koseki, H., Nakayama, T. andTaniguchi, M. (2004) Bone Marrow Allograft Rejection
Mediated by a Novel Murine NK Receptor, NKG2I. J
Exp Med.199,137-144.
11. Okazaki, N., Kikuno, R, Ohara, R, Inamoto, S.,
Koseki, H., Hiraoka, S., Saga, Y, Nagase, T., Ohara,
O. and Koga, H. (2003) Prediction of the coding
sequences of mouse homologues of KIAA gene: lli. thecomplete nucleotide sequences of 500 mouse KIAAhomologous cDNAs identified by screening of terminal
sequences of cDNA clones randomly sampled fromsize-fractionated libraries. DNA Res.10,167-80.
12. D, Graaff, W., Tomotsune, D., Oosterveen, T.,
Takihara, Y., Koseki, H. and Deschamps, J. (2003)
Randomly inserted and targeted Hoxlreporter fusions
transcriptionally silenced in Polycomb mutants. Proc

Division of Physiological Genetics

37Natl Acad Sci USA. 100, 13362-13367.
13. Hoki, Y., Araki, R., Fujimori, A., Ohhata, T.,
Koseki, H., Fukumura, R., Nakamura, M., Takahashi,
H., Noda, Y, Kito, S. and Abe, M. Growth retardationand skin abnormalities of the Recql4-deficient mouse.
Hum Mol Genet.12, 2293-2299.
14. Murata, K, Inami, M., Hasegawa, A., Kubo, S.,
Kimura, M., Yamashita, M., Hosokawa, H., Nagao, T.,
Suzuki, K, Hashimoto, K, Shinkai, H., Koseki, H.,
Taniguchi, M., Ziegler, SF. and Nakayama, T. (2003)
CD69-null mice protected from arthritis induced with
anti-type ncollagen antibodies. Int Immuno1.15, 987·992.
15. Yamamoto, K, Hamada, H., Shinkai, H., Kohno,
Y, Koseki, H. and Aoe, T. (2003) The KDEL receptor
modulates the endoplasmic reticulum stress response
through mitogen-activated protein kinase signalingcascades. J BioI Chern. 278, 34525-34532.
16. Wang, T., Tamakoshi, T., Uezato, T., Shu, F.,
Kanzaki-Kato, N., Fu, Y., Koseki, H., Yoshida, N.,
Sugiyama, T. and Miura, N. Forkhead transcriptionfactor Foxf2 (LUN)-deficient mice exhibit abnormal
development of secondary palate. Dev Biol.259, 83-94.
Books

17. "i5~ a.JV~r~~. *DIJff!!7N~)@Jf~H;::to~t.Q.:r: 1:::"::/;1:.*
7 4 'Y 7 t~~1f:*,Jm(J)f9:W!JJ, 7 t:1"'<'T /' C Jlf~r~~*,Jm(~;:I. 7!J /'jJ- . 7;1:. 77-7 . #tlI~:iE~*W), 187-195,(2003) .
EDUCATION

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Cod. Division of Physiological Genetics
Yukiko Gotoh GroupRESEARCH ACTIVITIESRegulation of neural development and cell death

Koji Oishi, Maiko Higuchi, Fuminori Tsuruta, Jun
Sunayama, Yoko Ogawara, Norihisa Masuyama andYukiko Gotoh
During neural development, a large number of cells
undergo programmed cell death to eliminate damaged
cells and developmentally unwanted cells, mostly
through a process called apoptosis. Caspases, a familyof cysteine proteases, playa central role in inductionof apoptosis. Gene disruption of caspase-9 results in
exencephaly due to a marked increase of neuralprecursor cells (NPCs), suggesting the cell death/survival regulation of NPCs is critical for neuraldevelopment. We found that NPCs prepared frommouse forebrain neuroepithelial cultures survived
better under high-density conditions. The densitydependent survival-promoting effect was likely torequire the Notch signaling, since a gamma-secretaseinhibitor, which inhibits the Notch processing,
suppressed it. Expression of an active form of Notch
was sufficient for promotion of NPC survival.
Importantly, Hesl and Hes5, two effectors of the Notch
signaling in inhibition of neuronal 9ifferentiation, could
not promote NPC survival. Therefore, Notch appears
to have two independent functions in NPCs, namely,
Hes-dependent inhibition of neuronal differentiationand promotion of survival through unknown
mechanisms.
This year we have also found that phosphorylationof 14-3-3 proteins by the stress-activated kinase JNK
is a key event in regulation of cell death and that Akt
plays a crucial role in regulation of neurite
morphogenesis during neuronal differentiation.
Publications
Papers

1. 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. In press.

38

Division of Physiological Genetics

2. 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.
3. Tsuruta, F., Sunayama, J., 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 J. 23, 1889-1899.
4. Miyagi, S., Saito, T., Mizutani, K, Masuyama, N.,
Gotoh, Y, Iwama, 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.
Cell. BioI. In press.
5. Higuchi, M., Onishi, K, Masuyama, N. and Gotoh,
Y (2003). The phosphatidylinositol-3 kinase (PI3K)-Aktpathway suppresses neurite branch formation in
NGF-treated PC12 cells. Genes Cells 8, 657.
6. Suzawa, M., Takada, I., Yanagisawa, J., Ohtake,F., Ogawa, S., Yamaguchi, T., Kadowaki, T., Takeuchi,
Y., Shibuya, H., Gotoh, Y, Matsumoto, K and Kato,
S. (2003). Cytokines suppress adipogenesis and
PPAR-gamma function through the TAKI/TABIINIK
cascade. Nat. Cell BioI. 5, 224-230.
7. Fujishiro, M., Gotoh, Y, Katagiri, H., Sakoda, H.,
Ogihara, T., Anai, M., Onishi, Y., Ono, H., Abe, M.,
Shojima, N., Fukushima, Y, Kikuchi, M., Oka, Y andAsano, T. (2003). Three Mitogen-Activated Protein
kinases inhibit Insulin signaling by different
mechanisms in 3T3-Ll adipocytes. Mol. Endocrinology
17,487-497.
8. Sakoda, H., Gotoh, Y, Katagiri, II., Kurokawa, M.,
Ono, H., Onishi, Y, Anai, M., Ogihara, T., Fujishiro,
M., Fukushima, Y, Abe, M., Shojima, N., Kikuchi, M.,
Oka, Y, Hirai, H. and Asano, T. (2003). Differing rolesof Akt and SGK in glucose metabolism, DNA synthesis
and oncogenic activity. J. BioI. Chern. 278, 25802-25807.
9. Katome, T., Obata, T., Matsushima, R.,
Masuyama, N., Cantley, L.C., Gotoh, Y., Kishi, K,
Shiota, H. and Ebina, Y. (2003). Use of RNAinterference-mediated gene silencing and adenoviraloverexpression to elucidate the roles of AKT/
PKB-isoforms in insulin actions. J. BioI. Chern. 278,
28312-28323.

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Editor:Oncogene
Journal of Biochemistry (Associate Editor)
Cell Structure and Function (2001)
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Division of Physiological Genetics

39

D. DEPARTMENT OF
POPULATION GENETICS

D-a. Division of Population Genetics
Naruya Saitou GroupRESEARCH ACTIVITIES(1) Construction of a gorilla fosmid library and its PCR
screening systemlChoong-Gon Kim, Asao Fujiyama and Naruya Saitou
CNational Institute of Infonnatics)
A gorilla fosmid library of 261,120 independent
clones was constructed and characterized. The fosmid
vector is similar to the cosmid in average insert sizeof ca. 40 kb but contains the F factor for replication,and it is more resistant to recombination. This clone
library represents about 3.7 times coverage of thegorilla genome. A simple screening system by PCR was
established, and we successfully found 9 clones thatcover the entire Hox A gene cluster of the gorilla
genome. This gorilla fosmid DNA library is a useful
resource for comparative genomics of human and apes.
For details, see ref. (1).
(2) Netview: Aplication software for constructing andvisually exploring phylogenetic networks

network for accurate representation. Parallel
substitutions are also sources of non-tree networks.
For the purpose of understanding such history wedeveloped a program Netview, which enables
constructing phylogenetic network based on thesequence data. For details, see ref. (2).
(3) Understanding the dynamics of Spinocerebellar
Ataxia 8 (SCA8) locus through a comparative genetic
approach in humans and apes

Aida M. Andres\ Marta Soldevila\ Naruya Saitou,
Victor Volpinit, Francese CalafelP and J aume
Bertranpetie C Unitat de Biologia Evolutiva,
Facultat de Ciencies de la Salut i de la Vida,
Universitat Pompeu Fabra)
Spinocerebellar Ataxia 8 (SCA8) is a neurodegenerative disorder caused by expansion of a trinucleotide
repeat. We undertake a comparative genetic analysis
among human populations and primate species in thenormal variation range, where forces that shaped
present diversity can be recognised. We determinate
number of repeats of the short tandem repeat throughallele length sizing and sequencing methods. Humanallele distributions are very similar among populations,ruling out ethnicity as a genetic risk for allele expansion.
Primate comparison shows human-specific features,with longer human alleles due to a novel variable
trinucleotide repeat, not present in non-humanprimates, which increased the disease-eausing expansion likelihood. SCA8 seems to be a human specificdisease. For details, see ref. (3).
(4) Evolution of the cystatin B gene: implications forthe origin of its variable dodecamer tandem repeat inhumans

Kirill Kryukov and Naruya Saitou
Reconstructing evolutionary history of a group of
species is a major task in biological study. Many
methods exist for reconstructing such history or
phylogeny, but most of them are based on anassumption that evolutoin of given gene family can be
represented as a tree. However some families of genes
may have alternative historical structure that cannot
be represented as a tree. Such history can result fromevents such as recombination, gene conversion andhorizontal gene transfer, and require not tree but

40

DEPARTMENT OF POPULATION GENETICS

Motoki Osawa.t, Mika Kanekot, Hidekazu Horiuchi\
Takashi Kitano, Yoshi Kawamoto 2, Naruya Saitou andKazuo Umetsu I CYamagata University School of
Medicine, 2Kyoto University)
The human cystatin B gene contains a variable
number of 12-bp tandem repeats in its promoter region,of which the common alleles contain two or threecopies and unusual expansion causes progressive
myoclonus epilepsy of the Unverricht-Lundborg type.
We undertook a comprehensive analysis of thegenomic sequence to address the evolutionary eventsof this variable repeat. By examination of a contiguous
genome sequence spanning 5.0 kb and linkage analysis
of detected polymorphic changes, we identified six
major intragenic haplotypes in unrelated Japanese
subjects. The number of normal repeats was closely
correlated with these alleles, indicating that changes
in the array should be comparatively rare events
during human evolution. To examine the origin of therepeat array further, we also analyzed five primate
genomes. Repetitive polymorphism was unlikely inhominoids, and the array originated with thedodecamer itself in the course of primate evolution.
The variability conceivably developed after the
separation to humans. For details, see ref.(4).
(5) Synonymous mutations in the human dopamine
receptor D2 (DRD2) affect mRNA stability and synthesisof the receptor

Jubao Duant, Mark S. Wainwrighe, Josep M.
Comeron\ Naruya Saitou, Alan R. Sanders\ Joel
Gelernter! and Pablo V. Gejman! CDepartment of
Psychiatry, The University of Chicago, Jules F. Knapp
Medical Research Center)
Although changes in nucleotide sequence affecting
the composition and the structure of proteins are wellknown, functional changes resulting from nucleotide
substitutions cannot always be inferred from simple
analysis of DNA sequence. Because a strong synonymous codon usage bias in the human DRD2 gene,suggesting selection on synonymous positions, wasrevealed by the relative independence of the G+C
content of the third codon positions from the isochoric
G+C frequencies, we chose to investigate functional
effects of the six known naturally occurring synonymous changes (C132T, G423A, T765C, C939T, C957T,
and G11OlA) in the human DRD2. We report here thatsome synonymous mutations in the human DRD2 havefunctional effects and suggest a novel genetic
mechanism. 957T, rather than being 'silent' , altered
the predicted mRNA folding, led to a decrease inmRNA stability and translation, and dramaticallychanged dopamine-induced up-regulation of DRD2
expression. 1101A did not show an effect by itself but
annulled the above effects of 957T in the compoundclone 957T/1101A, demonstrating that combinationsof synonymous mutations can have functional

consequences drastically different from those of eachisolated mutation. C957T was found to be in linkage
disequilibrium in a European-American population
with the -141C Ins/Del and TaqI 'A' variants, whichhave been reported to be associated with schizophrenia
and alcoholism, respectively. These results call into
question some assumptions made about synonymous
variation in molecular population genetics a~d genemapping studies of diseases with complex inheritance,
and indicate that synonymous variation can haveeffects of potential pathophysiological and pharmacogenetic importance. For details, see ref. (5).
(6) Mitochondrial DNA polymorphisms in nineaboriginal groups of Taiwan: implications for the
population history of aboriginal TaiwaneseAtsushi Tajima\ Takafumi Ishida 2, Naruya Saitou andSatoshi HoraP CDepartment of Biosystems Science,
Graduate University for Advanced Studies 2
Department of Biological Sciences, Graduate Schoolof Science, University of Tokyo)
Mitochondrial DNA (mtDNA) polymorphisms in
the D-loop region and the intergenic COHltRNA(Lys)
9-bp deletion were examined in 180 individuals fromall nine aboriginal Taiwanese groups: Atayal, Saisiat,
Bunun, Tsou, Rukai, Paiwan, Ami, Puyuma, and Yami.
A comparison of 563-bp sequences showed that there
were 61 different sequence types, of which 42 types
were specific to respective aboriginal groups. D-loop
sequence variation and phylogenetic analysis enabled
the 180 aboriginal lineages to be classified into eightmonophyletic clusters (designated C1-C8). Phylogeographic
analysis revealed that two (C2 and C4) of the eightclusters were new characteristic clusters of aboriginal
Taiwanese and accounted for 8.3% and 13.9% of theaboriginal lineages, respectively. From the estimatedcoalescent times for the two unique clusters, the
mtDNA lineages leading to such clusters were inferredto have been introduced into Taiwan approximately
11,000-26,000 years ago, suggesting ancient immigrations of the two mtDNA lineages. Genetic distances,
based on net nucleotide diversities between populations,revealed three distinct clusters that were comprisedof northern mountain (Atayal and Saisiat), southern
mountain (Rukai and Paiwan), and middle mountain!
east coast (Bunun, Tsou, Ami, Puyuma, and Yami)
groups, respectively. Furthermore, phylogenetic

Division of Population Genetics

41

analysis of 16 human populations (including six other
Asian populations and one African population)
confirmed that the three clusters for aboriginal
Taiwanese had remained largely intact. Each of theclusters (north, south, and middle-east coast) wascharacterized by a high frequency of a particularlineage (C4, C2, and 9-bp deletion, respectively). This
may result from random genetic drift among theaboriginal groups after a single introduction of all the
mtDNA lineages into Taiwan, but another plausible
explanation is that at least three genetically distinct
ancestral populations have contributed to the maternal
gene pool of aboriginal Taiwanese. For details, see ref.
(6).

(7) Sequence polymorphisms of the mitochondrial DNA
control region and phylogenetic analysis of mtDNA
lineages in the Japanese population

Sayaka Maruyamat, Kiyoshi Minaguchi! and Naruya
Saitou CDepartment of Forensic Odontology, Tokyo
Dental College)
Sequence polymorphisms of the hypervariablemitochondrial DNA (mtDNA) regions HVI and HVII,
and coding region polymorphisms were investigatedin 211 unrelated individuals from the Japanese
population. Sequence comparison of the HVI and HVII
regions led to the identification of 169 mitochondrial
haplotypes defined by 147 variable positions. Amongthem 145 types were observed in only 1 individual; theother 24 types were shared by 2 or more individuals.
The gene diversity was estimated at 0.9961, and theprobability of two randomly selected individuals fromthe population having identical mtDNA types was
0.86%. We also established phylogenetic haplogroups
in the Japanese population based on the coding andcontrol region polymorphisms and compared thehaplotypes with those in other Japanese, Korean andChinese populations. As a result, three new subhaplogroups, G4a, G4b, and N9b, and several
haplotypes specific for the Japanese and Korean
populations were identified. The present database canbe used not only for personal identification but also
as an aid for geographic or phenotype (race) estimation
in forensic casework in Japan. For details, see ref. (7).

42

Division of Population Genetics(8) Microsatellite variation in Japanese and Asian horses
and their phylogenetic relationship using a European
horse outgroup

Teruaki Tozaki!, Naoko Takezaki 2, Telhisa
Hasegawat, Nobushige Ishidat, Masahiko Kurosawat,
Motowo Tomita\ Naruya Saitou and Harutaka
Mukoyama! CDepartment of Molecular Genetics,
Laboratory of Racing Chemistry, 2Max Planck
Institute, Tubingen)
The genetic relationships of seven Japanese andfour mainland-Asian horse populations, as well as two
European horse populations, were estimated using datafor 20 microsatellite loci. Mongolian horses showed thehighest average heterozygosities (0.75-0.77) in all
populations. Phylogenetic analysis showed the existence of three distinct clusters supported by highbootstrap values: the European cluster (Anglo-Araband thoroughbreds), the Hokkaido-Kiso cluster, andthe Mongolian cluster. The relationships of theseclusters were consistent with their geographicaldistributions. Basing our assumptions on thephylogenetic tree and the genetic variation of horse
populations, we suggest that Japanese horses
originated from Mongolian horses migrating throughthe Korean Peninsula. The genetic relationship ofJapanese horses corresponded to their geographicaldistribution. Microsatellite polymorphism data wereshown to be useful for estimating the genetic
relationships between Japanese horses and Asian
horses. For details, see ref. (8).
(9) The diversity of bovine MHe class n DRB3 genes
in Japanese Black, Japanese Shorthorn, Jersey andHolstein cattle in Japan.

Shin-noske Takeshima\ Naruya Saitou, Mitsuo
Morital, Hisetoshi Inoko 2and Yoko Aida! CRetrovirus
Research Unit, RIKEN, 2Tokai University)
We sequenced exon 2 of the major histocompatibility complex (MHC) class II DRB3 gene from 471
individuals in four different Japanese populations of
cattle (201 Japanese Black, 101 Holstein, 100 Japanese
Shorthorn, and 69 Jersey cattle) using a new method
for sequence-based typing (SBT). We identified the 34
previously reported alleles and four novel alleles. These
alleles were 80.0-100.0% identical at the nucleotide level

and 77.9-100.0010 identical at the amino acid level to thebovine MHC (BoLA)-DRB3 cDNA clone NR1. Amongthe 38 alleles, eight alleles were found in only one breedin this study. However, these alleles did not form
specific clusters on a phylogenetic tree of 236-base pairs(bp) nucleotide sequences. Furthermore, these breeds
exhibited similar variations with respect to averagefrequencies of nucleotides and amino acids, as well as
synonymous and non-synonymous substitutions, in all
pairwise comparisons of the alleles found in this study.
By contrast, analysis of the frequencies of the various
BoLA-DRB3 alleles in each breed indicated that DRB3
* 1101 was the most frequent allele in Holstein cattle(16.8%), DRB3 * 4501 was the most frequent allele inJersey cattle (18.1%), DRB3*1201 was the most frequent
allele in Japanese Shorthorn cattle (16.0%) and DRB3
*1001 was the most frequent allele in Japanese Black
cattle (17.4%), indicating that the frequencies of alleles
were differed in each breed. In addition, a population
tree based on the frequency of BoLA-DRB3 alleles ineach breed suggested that Holstein and Japanese Black
cattle were the most closely related, and that Jersey
cattle were more different from both these breeds thanJapanese Shorthorns. For details, see ref. (9).
Kenta Sumiyama, Assistant Professor
Evolutionary study of cis-element in mammaliandevelopment
Kenta Sumiyama studied evolution of genetic
regulatory mechanism of developmental controllinggenes, including Dlx and Hox genes. Dlx genes areindispensable factors for forming proper jaw structure.
We have identified branchial arch specific cis-element
by using both large scale genomic clone and transgenic
mouse technologies (ref. 10). Gene duplication andsubsequent functional diversification was analyzed on
three Dlx clusters in mammal (ref. 11). Hoxc8 gene isregulated by an element named "hoxc8 early enhancer"
located intergenic region of Hox cluster. We haveanalyzed evolutionary change in the Hoxc8 earlyenhancer throughout many mammalian species (ref.
12).
Publications
Papers

1. Kim, C.-G., Fujiyama, A. and Saitou, N. (2003)

Construction of a Gorilla Fosmid Library and its PCR
Screening System. Genomics, 82, 571-574.
2. Kryukov, K and Saitou, N. (2003) Netview: pplication Software for Constructing and Visually Exploring
Phylogenetic Networks. Genome Informatics. 14,
280-281.
3. Andres, A.M., Soldevila, M., Saitou, N., Volpini, V.,
Calafell, F. and Bertranpetit, ]. (2003) Understanding
the dynamics of Spinocerebellar Ataxia 8 (SCA8) locus
through a comparative genetic approach in humans
and apes. Neuroscience Letters, 336, 143-146.
4. Osawa, M., Kaneko, M., Horiuchi, H., Kitano, T.,
Kawamoto, Y., Saitou, N. and Umetsu, K. (2003)
Evolution of the cystatin B gene: implications for theorigin of its variable dodecamer tandem repeat inhumans. Genomics, 81, 78-84.
5. Duan,]., Wainwright, M.S., JComeron, ].M., Saitou,N., Sanders, A.R., Gelernter,]. and Gejman, P.V. (2003)
Synonymous mutations in the human dopamine
receptor D2 (DRD2) affect mRNA stability andsynthesis of the receptor. Human Molecular Genetics,
12, 205-216.
6. Tajima, A., Cheih-Shan, Sun, c.-S., Pan, I-H.,
Ishida, T., Saitou, N. and Horai, S. (2003) Mitochondrial
DNA polymorphisms in nine aboriginal groups of
Taiwan: implications for the population history ofaboriginal Taiwanese. Human Genetics, 113, 24-33.
7. Maruyama, S., Minaguchi, K and Saitou, N. (2003)
Sequence polymorphisms of the mitochondrial DNA
control region and phylogenetic analysis of mtDNA
lineages in the Japanese population. International
Journal of Legal Medicine, 117, 218-225.
8. Tozaki, T., Takezaki, N., Hasegawa, T., Ishida, N.,
Kurosawa, M., Tomita, M., Saitou, N. and Mukoyama,
R. (2003) Microsatellite variation in Japanese and Asian
horses and their phylogenetic relationship using a
European horse outgroup. Journal of Heredity, vol. 94,
no. 5, pp.374-380.
9. Takeshima, S., Saitou, N., Morita, M., Inoko, H. andAida, Y. (2003) The diversit¥ of bovine MHC class II
DRB3 genes in Japanese Black, Japanese Shorthorn,
Jersey and Holstein cattle in Japan. Gene 316,111-118.
10. Sumiyama, K and Ruddle, F.H. (2003). Regulationof Dlx3 gene expression in visceral arches by
evolutionarily conserved enhancer elements. Proceedings of the National Academy of Sciences of the UnitedStates of America 100, 4030-4034.
11. Sumiyama, K, Irvine, S.Q. and Ruddle, F.R. (2003).
The role of gene duplication in the evolution andDivision of Population Genetics

43

function of the vertebrate distal-lessIDlx bigene cluster.
Journal of Structural and Functional Genomics 3,
151-159.
12. Kim, C.B., Shashikant, C.S., Sumiyama, K.,
Amemiya, c.T., Wang, W.C.R. and Ruddle, F.R. (2003).
Phylogenetic Analysis of the Mammalian Hoxc8
Non-coding Region. Journal of Structural andFunctional Genomics 3, 195-199.
13. ~niPX:-t!~Jj~~ilR{:/ 3 /':/ 3 - • -Y-7 7 77/':!f, +m
fp~ . +¥iiJ~r&ilR} (2003) W:II:-=fJ1fHtl :#;.tl.I±lIt&.
14. ~niPX:ili(2003Hf~4.: J1!~-=f0)~~'1!. gglEfrJ~ .
{tcni~I'HTIii:!fnJf L, 1t\f,{(~O)TT~ . ~!lm~~, 73-88Jt.

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EDUCATION

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SOCIAL CONTRIBUTIONS AND OTHERS

1. Molecular Biology and Evolution, Associate Editor
2. B *JI~~~ ~~t~.

3. B *ntft~~ f.ft~~.
4. ft1ffij~AJI~~-tt&~ ~.

44

Division of Population Genetics

D-a. Division of Population Genetics
Toshiyuki Takano GroupRESEARCH ACTIVITIES(1) Non-random association between variants at the
Drosophila olfactory and gustatory receptor genes

Akira Kawabe, Yuriko Ishii, Masanobu Itoh l ,Nobuyuki Inomata2, Rumi Kondo 3, Noriko Nanba\
Yutaka Inoue 4 and Toshiyuki Takano-Shimizu
CKyoto Institute of Technology, 2Kyushu University,
30 c hanomizu University, 40saka University of Foreign
Studies)Multilocus selection, besides genetic drift and geneflow, can generate linkage disequilibrium. Many
previous allozyme analyses of Drosophila have failed
to detect long-distance linkage disequilibrium, but thisdoes not prove a general lack of linkage disequilibrium.
Indeed, previous Drosophila studies have demonstrated
the existence of recom bination load, meaning thatsome natural variants are not randomly combined in
an individual, but in linkage disequilibria because ofadvantageous and disadvantageous combinations of
natural variants. With the availability of the complete
DNA sequence, we are now in a position to be able tomeasure the degree of linkage disequilibria at thewhole-genome level. As a first step, we took agenome-wide analysis of sequence variation in theolfactory and gustatory receptor families, identifying
about 1700 nucleotide segregating sites including morethan 300 replacement variations. What is more, we
found twelve different putative null alleles at ten of 104
loci analyzed. By extrapolation, a single fly of D.
melanogaster may have eight or more null alleles forthese genes.
Using the variations identified, we genotyped
about 400 wild-caught flies, 800 chromosomes from twonatural populations of D. melanogaster. In contrast to
the previous studies, many pairs of marker sites at theolfactory and gustatory receptor genes were insignificant linkage disequilibrium. Indeed, about 50
pairs of polymorphic sites showed significant linkage
disequilibrium at the 0.5% level and 15 pairs of sites
did with Bonferroni correction for the whole tests (atthe 0.0017%) in both samples. Significant linkage
disequilibria were not necessarily between polymor-

phisms at the different olfactory receptor genes or
between those at the gustatory receptor genes, but they
were also observed between an olfactory receptor gene
polymorphism and a gustatory receptor gene
polymorphism, suggesting functional relationshipbetween these two classes of genes. Furthermore theobserved linkage disequilibrium between replacementsite polymorphisms showed a significant excess of
haplotypes composed of one frequent and one rarer
allele. This was not observed for marker pairs consisting of two silent-site polymorphisms or one replacement polymorphism and one silent polymorphism.
This finding suggested synergistic deleterious effects
of replacement changes.
(2) DNA variation at Odorant binding protein genes in
Drosophila

Ten two-day-old virgin females of one species and ten
two-day-old males were placed in a food vial for one
day. The mating frequency was assessed by the
number of females with sperm in their spermathecae
and/or ventral receptacles. The data showed large
variation among lines in mating frequencies between
the two sibling species, suggesting multiple gene
involvement. Two D. melanogaster lines of high and low
heterospecific mating frequencies were chosen for QTL
mapping of the genes responsible for the differences
in interspecific mating behavior.
As a candidate gene for sexual isolation, yellow
gene has long been one of them. Its mutant females
have repeatedly been observed to show higher mating
frequency against males of other species. Strains for
inducible RNAi of this gene were constructed. Several
aspects of mating behavior are being tested in theyellow knock-down mutants by RNAi.

Aya Takahashi and Toshiyuki Takano-Shimizu
Odorant binding proteins (OBPs) are lowmolecular-weight proteins that are known to bind andsolubilize hydrophobic odorants. The specific functionof OBPs in olfaction is still not known. However, they
are found in olfactory and gustatory organs and seemto play an important role in odor detection by restricting the odorants accessible to specific receptors.
Among around 40 OBP genes annotated in the
Drosophila genome, we have focused on ObPS7d andObpS7e genes because of their specific expression attarsi. DNA sequences of these two genes were obtained
from 18 inbred lines of D. melanogaster collected in
Kyoto, 4 D. melanogaster lines collected around theworld, and 1 D. simulans line. The sequence variations
showed a pattern that deviates from the neutral
expectation, thus indicating a signature of natural
selection.
(3) Searching for genes involved in sexual isolation
between Drosophila simulans and Drosophilamelanogaster
Aya Takahashi, Kuniaki Takahashi\ Ryu Ueda! andToshiyuki Takano-Shimizu CInvertebrate Genetics
Laboratory)
The interspecific mating frequencies between D.
melanogasterand D. simulanswere surveyed in sevenlines of D. melanogasterand seven lines of D. simulans.

(4) Genetic and molecular basis of the within- andbetween-species variation in the sex comb teeth number
in Drosophila.
Haruki Tatsuta, Yuriko Ishii and Toshiyuki TakanoShimizu
Sex comb of Drosophila melanogasteris a specificrow of enlarged bristles on male foreleg and a good
representative of sexually dimorphic characters. In
general, such characters are likely driven by sexual
selection. Using two inbred Drosophila simulans linesof the large (Tananarive) and the small (Sim3) numberof sex-comb teeth, we made QTL analyses for thesex-comb-tooth-number variation in five mapping
populations of whole genome F2, two secondchromosome substitution strains (STSS and STTT),
and two third-chromosome congenic strains. While no
QTL was detected on the X chromosome, we foundthree QTL on the third chromosome and two of themwere detected in both the whole genome F2and Sim3
backgrounds. Notably, we found strong background
dependency of second-chromosome QTL identification:
two QTL in the whole genome F 2background, a single
QTL in the Sim3 background (STSS), and three QTL
in the Tananarive background (STTT). Although thetwo QTL in STTT were possibly identical with the
QTL detected in the other backgrounds, the remaining
two QTL identified were background-specific, implying
extensive inter-chromosomal epistatic interactions.
I

Division of Population Genetics

45Indeed, we found a significant interaction between thesecond-chromosome QTL and the third-chromosome
one. Background dependency of second-chromosome
QTL effects but independence of third-chromosome
QTL effects may indicate the gene-action order of the
QTL involved: the third-chromosome QTL act upstream of the second-chromosome QTL. For finemapping, we are now studying the level and patternof linkage disequlibrium in the candidate gene region.
SOCIAL CONTRIBUTION
OO*0~j!~~(2) Self-organizing map reveals sequence characteristicsof 90 prokaryotic and eukaryotic genomes on a single
map~~:w:r~~j!

D-b. Division of Evolutionary Genetics
Toshimichi Ikemura GroupRESEARCH ACTIVITIES(1) A novel bioinfonnatics strategy for unveiling hidden
genome signatures

Takashi Abe, Yuta Ichiba and Toshimichi Ikemura
In addition to protein-coding information, genome
sequences contain a wealth of information of interestin many fields of biology from molecular evolution to
genome engineering and biomedicine. G+C% is used
as a fundamental characteristic of individual genomes
but too simple a parameter to differentiate a wide
variety of genomes. Because oligonucleotide frequencies vary significantly among genomes, they can beused to distinguish genomes and genomic regions. We
used an unsupervised neural network algorithm, aself-organizing map (SOM), to analyze di-, trio, andtetranucleotide frequencies in a wide variety ofprokaryotic and eukaryotic genomes. The SOM, which
can cluster complex data efficiently, was shown to be
an excellent tool for analyzing global characteristics
of genome sequences and for revealing key combinations of oligonucleotides representing individual
genomes. From analysis of 1-kb and 1O-kb genomic
sequences derived from 65 bacteria (a total of 170 Mb)
and from 6 eukaryotes (460 Mb), clear species-specificseparations of major portions of the sequences wereobtained with the di-, trio, and tetranucleotide SOMs.
The unsupervised algorithm could recognize, in most
1O-kb sequences, the species-specific characteristics(key combinations of oligonucleotide frequencies) that46

are signature features of each genome. We were able
to classify DNA sequences within one and between
many species into subgroups that corresponded tobiological categories. Because the classification poweris very high, the SOM is an efficient and fundamental
bioinformatic strategy for extracting a wide range ofgenomic information from a vast amount of sequences.
(Abe et al. Genome Research, 13, 693-702, 2003).

Division of Population Genetics

Takashi Abe, Atushi Fukushima,Yoko Kosaka andToshimichi Ikemura
With the increasing amount of available genome
sequences, novel tools are needed for comprehensive
analysis of species-specific sequence characteristics
for a wide variety of genomes. An unsupervised neuralnetwork algorithm, Kohonen's self-organizing map(SOM), is an effective tool for clustering and visualizing
high-dimensional complex data on a two-dimensional
array of weight vectors. We adapted SOM to genome
informatics, making the learning process and resulting
map independent of the order of data input. We used
the SOM to characterize oligonucleotide frequencies
in a total of 1.4 Gb derived from 90 prokaryotic andeukaryotic genomes for which complete genomic
sequences are available. SOMs classified 140,000 10-kb
sequences from the 90 genomes mainly according to
species and could unveil hidden sequence characteristics of each genome. Because the classification poweris very high, SOM is an efficient and fundamental
bioinformatic strategy for extracting a wide range ofgenomic information from a vast amount of sequence
data. (Abe et a!., Workshop 2003 on Self-Organizing
Maps, pp. 95-100, 2003)(3) Novel bioinfonnatics developed for phylogeneticclassifications of genomic sequences from uncultured
microorganisms in environmental samples

Takashi Abe, Yoko Kosaka, Jian-Ping Song andToshimichi Ikemura
Self-organizing map (SOM) is a powerful tool forclustering and visualizing high-dimensional complex
data on a two-dimensional plane. Oligonucleotidefrequency is an example of high-dimensional data. We
constructed SOMs of tri- and tetranucleotide frequencies in 1- and 5-kb sequence fragments from bacterial
genomes for which complete sequences are available.
The sequence fragments could be classified primarily
according to species and thus to 11 major bacterialgroups without information regarding the species.
SOM also classified 16S rRNA gene (16S rDNA)
sequences accurately into the phylogenetic groups. For
example, approximately 97% of rDNA sequences and
80% of non-rDNA sequences longer than 1 kb wereclassified into the correct phylogenetic groups. Because
the classification power is very high, SOM is a powerfultool for phylogenetic classification of sequence
fragments obtained from mixed genomes of uncultured
environmental microorganisms. With this method, all
rDNA and non-rDNA sequences including sargasso sea
sequences in GenBank that were from unidentified
bacteria and longer than 1 kb were classified into 11
major bacterial groups. The result indicated that themethod is also useful for survey of pathogenic
microorganisms causing novel and unclear infectious
diseases.
(4) Inter- and intraspecies characterizations ofeukaryotic genome sequences and in silico predictionof genetic signal sequences

Takashi Abe, Yoko Kosaka, Yuta Ichiba, Kiyomi Kita
and Toshimichi Ikemura

sequences from 2.8 Gb of human sequences identified
oligonucleotides with frequencies characteristicallybiased from random occurrence predicted from themononucleotide composition, and lO-kb sequences richin these oligonucleotides were self-organized on a map.
Because these oligonucleotides often corresponded togenetic signals or the constituent elements, we propose
an in silico method that should be useful for identification of genetic signal sequences in genomes forwhich large amounts of sequence data are available
but additional experimental data are lacking.
Publications
Papers

1. Abe, T., Kanaya, S., Kinouchi, M., Ichiba, Y.,

Kozuki, T. and Ikemura, T. (2003). Informatics forunveiling hidden genome signatures. Genome Research
13, 693-702.
2. Abe, T., Kozuki,T., Kosaka, Y., Fukushima, A.,
Nakagawa, S. and Ikemura T. (2003). Self-organizing
map reveals sequence characteristics of 90 prokaryotic
and eukaryotic genomes on a single map. Workshop
2003 on Self-Organizing Maps 3, 95-100.
3. Kato, M., Onishi, Y., Wada-Kiyama, Y, Abe, T.,
Ikemura, T., Kogan, S., Bolshoy, A., Trifonov, E.N. andKiyama, R. (2003). Dinucleosome DNA of human K562
cells: experimental and computational characterizations.
]. Mol. BioI. 332, 111-125.
SOCIAL CONTRIBUTIONS AND OTHERS

Novel tools are needed for comprehensive
comparisons of inter- and intraspecies characteristics
of massive amounts of increasingly available genome
sequences. An unsupervised neural network algorithm,
self-organizing map (SOM), is an effective tool forclustering and visualizing high-dimensional complex
data on a single map. We generated SOMs for trio,
tetra-, and pentanucleotide frequencies in 300,000 10-kb
sequences from 13 eukaryotes for which almost
complete genomic sequences are available (a total of
3 Gb). SOM recognized in most 10-kb sequences
species-specific characteristics (key combinations ofoligonucleotide frequencies), permitting species-specificclassification of sequences without information
regarding the species. Because the classification poweris very high, SOM is an efficient and powerful tool for
extracting a wide range of genomic information. SOM
constructed with oligonucleotide frequencies in lO-kb!fiffF4. I±lU*~: 2003-343862, ~fl~(7)~~: ""<'-1-7 07v-1~~~G~Ghkf-~.~(7)~~~~M~:~M~ji . 3i'i~m~~#~~·mB3

5.

. g§~-I:!!' frlB3{@zf)- . !J\~JJjfi~~ .

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I±lU*~: 2003-328845, ~fl~(7)~~: ij[~~~JO)7.t~~A7A~lVT~~~-7vTT~I±l~~~(7).~~A7A, ~M~:~M
mA~ji, I±lUA:OO~~~~mR

Division of Evolutionary Genetics47

E. DEPARTMENT OFINTEGRATED GENETICS

E-a. Division of Human Genetics
Hiroyuki Sasaki GroupRESEARCH ACTIVITIES(1) Analyses of the distal imprinted domain on mouse
chromosome 7 and its orthologue on chicken chromosome
5

Hiroyuki Sasaki, Takaaki Yokomine, Hisao Shirohzu,
Chikako Suda, Wahyu Purbowasito\ Masaoki
Tsuzuki 2, Atsushi Toyoda3, Masahira Hattori3 andYoshiyuki Sakaki3eKyushu Univ.; 2Hiroshima Univ.;
3GSC, RIKEN)Genomic imprinting, an epigenetic gene-marking
phenomenon in mammals, causes parent-of-originspecific monoallelic expression of a subset of genes.
Imprinted genes tend to form clusters in the genome(imprinted domains), which may be related to themechanism of imprinting or to the evolution of
imprinting. As a step to understand the structural andfunctional characteristics of the imprinted domains,
we have characterized a loMb imprinted domain inmouse chromosome 7F4/F5 and its orthologous
domain in chicken chromosome 5 ('"'-'D.5 Mb). We foundthat the genes of the chicken domain are not imprinted
and, furthermore, that the chicken domain lacks theunique tandem repeat cluster of '"'-'D.2 Mb, the H19 gene,and the imprinting control elements, all of which arepresent in the mouse. The results indicate that the
mammalian imprinted genes were already clusteredin the common ancestors of mammals and birds andthat the imprinting mechanism, which can affectmultiple genes in the cluster, came in later during
mammalian evolution (in preparation). We also
mapped a total of 52 nuclear matrix attachment regions(MARs) in the imprinted mouse domain. We foundthat the MARs are unevenly distributed within the48

DEPARTMENT OF INTEGRATED GENETICS

domain and that there is a large MAR cluster in theboundary region of two imprinted subdomains (inpreparation).
(2) Imprinting mechanisms of the mouse Igf2/H19
subdomain

Hiroyuki Sasaki, Yuzuru Kato, Ko Ishihara!, Melanie
Ehrlich 2 and Mitsuyoshi Nakao! eKumamoto Univ.;
2Tulane Univ.)
The imprinted mouse 7F4IF5 domain contains twolinked imprinted genes Igf2 and H19 near its centromericboundary: Igf2 is paternally expressed and H19maternally expressed. It is known that the paternalspecific methylation of the differentially methylated
region (DMR) located upstream of H19is the primarycause for the Igf2/ H19 imprinting. We found that awinged-helix type DNA binding protein called RFXl
or MDBP binds to the conserved sequences within the
DMR. Interestingly, this protein binds to the target
sequence preferentially when they are methylated at
CpG sites. We are currently knocking out the target
sequences within the DMR in mice to see whether these
proteins playa role in Igf2/ H19 imprinting.
(3) Establishment and maintenance of genomic
imprinting in the germline and in early embryos

Hiroyuki Sasaki, Masahiro Kaneda, Masaki Okanot,
Kenichiro Hata, Takashi Sado, Naomi Tsujimoto2, En
Li 2, Kenji Kumaki, Hiroyasu Furuumi, Tomohiro
Suzuki3, Shigeharu Wakana3 and Toshihiko Shiroishi3
eCDB, 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 mice has
precluded further studies. We have disrupted Dnmt3a
and Dnmt3b in male and female germ cells, leaving
them intact in somatic cells, by conditional knockout
technology. Offspring from the Dnmt3a conditional
mutant females died in utero and lacked methylation
and allele-specific expression at maternally imprintedloci. The Dnmt3a conditional mutant males showed
impaired spermatogenesis and a lack of methylation

at paternally imprinted loci in spermatogonia.
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, the Dnmt3b conditional mutants
and their offspring showed no phenotype. These
results indicate that Dnmt3a is the critical enzyme
responsible for both paternal and maternal imprinting(submitted). We also study how the primary imprints
are maintained in preimplantation mouse embryos,
and set out to screen ENU-treated mutant mousestocks for mutations that affect the establishment ofgermline imprints in collaboration with a group in
GSC, RIKEN.

geneous and that a gene other than DNMT3B canaffect the methylation of pericentromeric satellite DNA.
(6) Investigation on the possibility of Z chromosome
dosage compensation in chicken
Hiroyuki Sasaki, Takaaki Yokomine, Asato Kuroiwa\
Yoichi Matsudal and Masaoki Tsuduki 2 CHokkaido
Univ.; ~iroshima Univ.)(4) Computer-assisted search for sequence featurescommon to imprinted DMRs

We studied whether chicken Z chromosome issubject to inactivation for gene dosage compensationin ZZ males. It was found that Z-linked genes arebialle1ically expressed in ZZ males (ref. 2). The results
suggest that sex-chromosome inactivation of a
mammalian X-chromosome type does not occur in thechicken.

Hiroyuki Sasaki; Hisato Kobayashi, Takashi Abel andToshimichi Ikemural CDiv. Evo!. Genet., NIG)

(7) Development of a universal DNA chip systemapplicable to any organism

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. Wefound 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.

Hiroyuki Sasaki, Shin-ichi Mizunol, Tadafumi Iino\
Hidetoshi Ozawa\ Teruhisa Otsuka\ Kosuke Tashird
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. Development of the universal
DNA chip system for practical use is now underway.
(8) Role of de novo DNA methyltransferases in X chromosome inactivation(5) Molecular pathology of ICF syndrome
Hiroyuki Sasaki, Hiroyasu Furuumi and Takeo
Kubotal CYamanashi Univ.)
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 1UJVO DNA methyltransferase
gene DNMT3B. We have now studied a new Japanese
ICF case and found that this patient does not have any
mutation in DNMT3B(submitted). This suggests thatthe molecular pathology of ICF syndrome is hetero-

Takashi Sado, Masaki Okano\ En Le and Hiroyuki
Sasaki CCDB, RIKEN; 2Harvard Med. Sch.)

Xist (X-inactive specific transcript) plays a criticalrole in X-inactivation. This non-coding RNA becomes
upregulated on the X chromosome that is to beinactivated upon differentiation. Previous studies
revealed that although maintenance-type DNA
methylation is not essential for X-inactivation to occur,
it is required for the stable repression of Xist indifferentiated cells. However, it is unknown whether
differential de novo methylation at the Xist promoter,
which is mediated by Dnmt3a and/or Dnmt3b, is a

Division of Human Genetics

49cause or a consequence of monoallelic expression ofXist. In this study, we showed that Xist expression wasappropriately regulated in the absence of Dnmt3a andDnmt3b and that a single X chromosome underwent
proper inactivation in mutant females. Our results
indicate that a mechanism(s) other than DNA methylation plays a principal role in initiating X-inactivation.
We also demonstrated that delayed upregulation ofXist did not induce X-inactivation, consistent with acritical developmental window for the chromosomal
silencing (ref. 5).
(9) Effect of Tsix disruption on Xist expression

Takashi Sado and Hiroyuki Sasaki

Xist and its antisense partner, Tsix, are the key
players in X inactivation, both of which encode a
non-coding RNA. Targeted disruption of Tsixcauses
ectopic expression of Xistin the extraembryonic tissues
upon maternal transmission, which subsequently
causes embryonic lethality due to inactivation of both
X chromosomes in females and a single X chromosome
in males. Tsix, therefore, plays a crucial role inmaintaining Xist silencing in cis and in the regulationof imprinted X-inactivation in the extraembryonictissues. We examined the effect of Tsix-disruption on
Xist expression in the embryonic lineage using
embryonic stem (ES) cells as a model system. Upon
differentiation, Xistis ectopically activated in a subset
of nuclei of male ES cells harboring the Tsix-deficient
allele on the single X chromosome. Such ectopic
expression, however, ceased during prolonged culture.
Weare tempted to speculate that surveillance by thecounting mechanism somehow shuts off ectopic Xist
expression before that X chromosome undergoes
inactivation. Counting does not seem to be impairedin Tsix-deficient ES cells (ref. 1).
(10) X-inactivation in mouse embryos deficient for histone
methyltransferase G9a

Tatsuya Ohhata, Makoto Tachibanat, Hiroyuki
Sasaki, Yoichi Shinkai l 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 the50

Division of Human Genetics

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 was 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 results suggest that theX-inactivation process is properly regulated in theabsence of G9a. It is likely that methylation of histone
H3 at K9 and K27 on the inactive X chromosome ismediated by an enzyme(s) other than G9a.
(11) Role of Dnmt3L in spermatogenesis and in genomic
imprinting during oogenesis

Kenichiro Hata, En U 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- / - female mice grew normally, allembryos from pregnant Dnmt3L-/- mothers diedaround ElO.5. The maternally methylated imprinted
genes, e.g. Igf2r and Pegl, were hypomethylated inembryos derived from Dnmt3L -/- females x
Dnmt3L + / + males, but paternally methylated
imprinted genes were unaffected. Also, Dnmt3L- /male 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.

Publications

Books

Papers

15. ftil!lt]t, ftik*mZ : ·1!~'@.~~#ltJ1e:~fI~. Springer
Reviews:/ V-:X -7 ""'"7 /' e: .{~-r~fI~l!fljm (:I1ffi~lE~*!iii), 15-24,:/;:J.. ~V /'jJ- . 7;r. 7'7-7**(2003).

1. Sado, T., Li, E. and Sasaki, H. (2002). Effect of
Tsix-disruption on Xist expression in male ES cells.
Cytogenet. Genome Res. 99, 115-118.
2. Kuroiwa, A, Y okomine, T., Sasaki, H., Tsudzuki,M., Tanaka, K, Namikawa, T. and Matsuda, Y. (2002).
Biallelic expression of Z-linked genes in male chickens.
Cytogenet. Genome Res. 99, 310-314.
3. Ishida, C., Ura, K, Hirao, A, Sasaki, H., Toyoda,
A, Sakaki, Y, Niwa, H., Li, E. and Kaneda, Y (2003).
Genomic organization and promoter analysis of theDnmt3b gene. Gene 310, 151-159.
4. Gribnau, J., Hochedlinger, K, Hata, K, Li, E. andJaenisch, R. (2003). Asynchronous replication timingof imprinted loci is independent of DNA methylation,
but consistent with differential subnuclear localization.
Genes Dev. 17, 759-773.
5. 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.

°

ffi • • • m, ftik*mZ:~/A~/'~V/'~~/'~~Jfg1t. Springer Reviews:/ V- :X-~ / AipGhte.~!lm~?!>-~'tte:Jfg1tGi{~i!lil.*!iii), 164-170,:/;:J.. 7°V /,jJ- .

7;r. 7'7-7**(2003).
17. ftik*mziii: SpringerReviews:/V-~-;c.t"'S/;r.
*' ~ ~ 7 A, :/ ;:J.. ~ V/' jJ - . 7 ;r. 7 '7 -7 ** (2004).
18. ftil!lt]t: ·I!~'@.~~.{~-r::l;fmfl. Springer Reviews:/ V- ;(-;c. t"'S/ ;r.-*~ ~ 7 A (ftik*mz*!iii) , 117-127,: / ;:J.. ~ V/' jJ . 7;r. 7 '7 - 7 ** (2004).
19. 1l~5.£."*, ftik*mZ: 1Iffl1L~~j /'71V.{~T~;c. t" 'S/ ;r. -* ~ ~ 7 A. Springer Reviews:/ V- ~ -;c. t"'S/ ;r.-*~ ~ 7 A (ftik*mz*!iii) , 129-134, :/;:J.. ~ V /,jJ- .
7;r. 7'7-7**(2004).
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Springer Reviews:/ V- :X-;c.I:::0'S/ ;r.-*~ ~ 7 A (ftik*m~, 183-19O,:/;:J.. ~V /,jJ- . 7;r. 7'7-7*J?-(2004).
EDUCATION

1. • • -D:~.-r~~~;c.t"'S/;r.*'~~7A.ftffl
Reviews*~~~~m~~ffl~~T-, m~,

6. Hata, K and Sasaki, H. (2003). Genomic imprinting:
Mechanisms, significance and evolution. ]. Mamm. Ova
Res. 20, 64-68.
7. 1i'iE8lE5.£., {tck*mZ : DNAj71v{te: 7 O""'"7/'2.

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54, 206-210(2003).

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ftik*mZ:7o-/'tt~~m~.

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6. ftik*mZ: ~t.m*~:EII!~$~it, flJ~}Jj(, 1O~.
7. Sasaki, H.: Role of de novo DNA methyltransferases
in genomic imprinting revealed by conditional geneknockout in mice. Seminar in the Department of
Genetics, UniversiUi.t of Saarland, Saarbriicken,
Germany, November.

/'-7 lV~. :mft~~ 35, 965-970(2003).
9. 1i'iE8lE~, ~*.~, ~k*mZ:~/A~/'~V/'~~ /'~. Molecular Medicine 40(±lflj~)¥J~~~,
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/'~~ /,7·. ~!lm~f-4~ .{~ 58, 69-74(2004).
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j

Division of Human Genetics

51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 and Masaomi Kato 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 Geddeloh et al., 1999, Nat. Genet 22,94-). The ddmlmutation 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).

mutants. Single mutants in either metl or cmt3weremuch less effective in mobilization. Thus CG andnon-CG methylation systems redundantly function forimmobilization of transposons (Kato et al., 2003). CMT3
gene and non-CG methylation in plants may have
evolved as an additional epigenetic tag dedicated totransposon control.
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. This contrasts to transposition induced inlaboratory, in which the integration sites are less biased
and the copy number frequently increases. The
differences in the integration sites may be due tonatural selection against deleterious insertion into
chromosomal arm regions (Miura et aI, 2004).
(3) Inheritance of epigenetic developmental abnormality.

(2) Epigenetic control of transposons and their behavior
in natural population

Asuka Miura, Masaomi Kato, Kazuya Takashima,
Yuki Kinoshita and Tetsuji Kakutani
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 of
CACTA elements was detected in cmt3-metl double

52

Division of Agricultural Genetics

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 al., 2000, Mol
Cell 6, 791-). Interesting thing is that change innucleotide sequence was also not observed in fwa-l andfwa-2 alleles isolated by conventional mutagenesis.
In both cases, over-expression associated with thehypomethylation resulted in the phenotypes. Combining ddml mutation and linkage analysis is useful for
identifying epigenetically regulated genes affecting
plantdevelopment (Kakutani, 1997, Plant]., 12, 1447-).
The ddml-induced late flowering trait as well as thehypomethylation and ectopic expression of the FWA
gene were stably inherited over generations even in thepresence of the wild type DDMl copy (manuscript in
preparation).

Kotani, H. and Kakutani, T. (2004) Genomic localizationof endogenous mobile CACTA family transposons innatural variants of Arabidopsis thaliana. MGG, 270,524-532. (online publication, Nov 8, 2003).

(4) Epigenetic control of FWA gene expression inendosperm

EDUCATION

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
imprint depends on the maintenance DNA methyltransferase
MET1, as is the case in mammals. Unlike mammals,
however, the FWA imprint is not established byallele-specific de novo methylation. It is established bymaternal gametophyte-specific gene activation, 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 methylationin endosperm (Kinoshita et aI, 2004).
Publications
Papers
1. Kato, M., Miura, A., Bender, J., Jacobsen, S.E.
and Kakutani, T. (2003) Role of CG and non-CG
methylation in immobilization of transposons in
Arabidopsis. Current Biology 13, 421-426.
2. 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. (online publication, Nov 20, 2003).
3. Miura, A., Kato, M., Watanabe, K., Kawabe, A.,

1. Kakutani T, a seminar at Cold Spring Harbor
Laboratory, August, 2003
2. Kakutani T, a seminar at UCLA, August, 2003
3. Kakutani T, a seminar at California Institute ofTechnology, August, 2003
4. Kinoshita T, a seminar at UC Berkeley, August,2003
5. ~:e:Hiltf=~t~Ji*'¥fII1'¥$-C:#;IfIil#~,

2003~ 2

Jj

E-b. Division of Agricultural Genetics
Keiichi Shibahara GroupRESEARCH ACTIVITIES(1) Mechanism of nucleosome assembly during DNA
replication

Tomohiro Kubo', Akira Shimizu' and Keiichi
Shibahara2 ('Graduate School of Biostudies, Kyoto
University, 2Japan Science and Technology Agency)
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 found to be involved in some processof this nucleosome assembly reaction.
We recently developed an in vitro reconstitution
system with human cell extract to analyze a mechanism of the DNA replication-dependent nucleosome
assembly. The nucleosome assembly reaction wasformerly analyzed in an ongoing replication reactionof SV 40 in vitro system. This replication-coupledsystem has proven particularly useful in identifying
CAF-1. However, its use to identify other activities
involved in the replication-dependent nucleosome

Division of Agricultural Genetics

53

assembly has been limited, because biochemical
fractionations of the human cell cytosolic extract (theSlOO extract) used in the SV40 DNA replication (whichcontains all the necessary DNA replication factors,
histones, and other chromatin assembly factors) often
resulted in a severe loss of DNA replication activity.
In order to overcome these limitations, we successfully
used replicated DNA purified by a gel filtration spincolumn as a substrate for the nucleosome assembly
reaction. The newly replicated and separated DNA wascompetent for the replication-dependent nucleosome
assembly even after separation from free protein
fractions in the Sloo extract because the DNA remains
marked, at least by PCNA (Cell 96, 575-585, 1999).
With this "two step system", we showed that
unidentified activities in the Sloo extract were required
for the nucleosome assembly in addition to CAF-1 andhistones. We are now on the way to identify additional
activities required for the DNA replication-dependent
nucleosome assembly.
(2) Physiological implications of CAF-I and ASF1 in
Arabidopsis
Hidetaka Kaya l • 2, Takashi Araki l and Keiichi
Shibahara3 (IDepartment of Botany, Graduate Schoolof Science, Kyoto University, 2Japan Society for the
Promotion of Science, 3Japan Science and Technology
Agency)The genetic approach of Arabidopsis was apowerful tool to see physiological implications of
CAF-1 and ASF1. We analyzed loss-of-function
mutants of cal-i (fasciata: las) in Arabidopsis andshowed that those las mutants displayed severely
disturbed cellular and functional organization of bothshoot apical meristem and root apical meristem (Cell104,131-142.2001). Those mutants also showed a varied
pattern of distorted expression of both WUS and SCR,
which play key roles in the organization of SAM and
RAM, respectively. Recently, we found that genesilencing of CACTA and TSL which expressions are
repressed in wild type plants, was de-repressed in asmall number of cells, and the percentage of the cells
express those genes was increased with growth of theplants. These observations suggested that silent statusof gene expression cannot be maintained stably in las
mutants and thus those cells express CACTA and TSI. accidentally appear in a stochastic manner.

54

Division of Agricultural Genetics

We proposed several models to explain thoseobservations, and the most plausible is that CAF-1
ensures stable propagation of epigenetic states ofchromatin through facilitating nucleosome assembly
during DNA replication. Rapid re-formation of
nucleosomes onto newly replicated DNA by CAF-1
would prevent DNA binding factors like transcriptional
regulators from being targeted to the DNA in a
non-regulated manner, thereby preventing randomchanges in gene expression patterns in daughter cells.
(3) Identification of a novel regulatory element in theX- inactivation Center (Xic)Yuya Ogawa and Jeannie T. Leel eMassachusetts
General Hospital, Harvard Medical School and Howard
Hughes Medical Institute)During X-chromosome inactivation in mammalian
female, one X-chromosome is randomly silenced by
Xist. X-chromosome choice is determined byasymmetric expression of Tsixwhose antisense action
represses Xist. In mice, choice is also regulated by themodifier, Xce. We identified a novel element in the X
inactivation center (Xic) region near Xist and Tsix that
regulates allelic choice. Termed Xite, the element
harbors two clusters of intergenic transcription startsites and series of DNase I hypersensitive sites withallelic differences. Deleting Xite skews choice to favor
the mutated X for inactivation, but truncating thetranscripts associated with Xite does not abolish thiseffect. Evidence suggests that, at the onset of XCI, Xite
enables the persistence of Tsix expression on the future
active X. We propose that Xite is a candidate for the
Xce and promotes allelic inequality by biasing Tsix
expression.
Publications
Papers
1. Ogawa, Y. and Lee, J. T. (2003). Xite, X-inactivationintergenic transcription elements that regulate theprobability of choice, Mol Cell 11, 731-743.
2. Takeda, S., Tadele, Z., Hofmann, I., Angelis, K.J.,
Kaya, H., Araki, T., Mengiste, T., Scheid, OM, Probst,
AV., Shibahara, K-i., Scheel, D. and Paszkowski, J.:
BRUi, a novel link between responses to DNA damageand epigenetic gene silencing in Arabidopsis. (2004)
Genes & Dev. 18. 782-793

Reviews

3. lj\Jllm-tlH2003), ~m1't1I:X~@'f*O)JlfR : Xite4lJi~
I~ J::.Q TsixO)~llJljfJm, ~~~~21, 1653-1656.
4. ~1*~ltk, ~~,.-: DNA~~MO)?<~ v::t/'-A
7-e ~7"!J -, ~~~~jfflJ';c to::; :r.;f:.-r -1 'Y ~ .A t:it
-8-i'-~llJljfJmJ, VOL,21 No.H, 24-28, 2003.
5. ~1*~*, ~~,.- : DNA~~:&.vt?< ~ v::t'f- F~"*1~:fl1~1~'5?< ~ v::t/'-A~JJX;, :bip.Q~~~~'DNA~~tDNA1~:flJ, 2004. 88-92.

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

Hirokazu Tozaki and Tatsumi Hirata
The olfactory sensory neurons arranged on theolfactory epithelium express odorant receptors (ORs),
which capture odorous chemicals in the air. The ORs
are encoded by about 1000 genes in mice. Among thelarge repertoires, however, only one allele of the single
OR gene is expressed in individual sensory neurons.
The neurons expressing the same OR are randomly
scattered over the olfactory epithelium, but converge
their axons on a few glomeruli, specialized synaptic
structures, connecting with the second-order neurons.
For the glomerular convergence, ORs play an
indispensable role. Deletion of an OR coding sequenceby genetic disruption destroys the glomerularconvergence by sensory neurons that are committed
to express the OR. OR swap experiments in which one
OR coding sequence is replaced by another induce a
positional shift of the glomerulus targeted by theOR-expressing neurons from the endogenous glomeruli
defined by either of the donor or acceptor ORs. These
results suggest that the ORs instruct the axons where
to converge.
How do the ORs instruct the convergence ofolfactory axons? Several studies have hypothesized amodel in which ORs presented on axon terminals
re~ognize positional cues in the olfactory bulb as a
guidance receptor. Another possibility is activitydependent guidance of olfactory axons through theORs. Activity-dependency is one of the fundamental
mechanisms involved in the construction of neuralnetworks, and basically represented by the Hebbianrule that simultaneous electrical activities between preand post-synaptic cells reinforce their synaptic
connections, whereas incoherent activities do not.
With regard to the olfactory projection, the activitydependent versus -independent issue is still muchcontroversial.
We computationally simulated how olfactoryaxons target if activity-dependent mechanisms wereconsidered. We modified Tanaka's activity-dependent
self-organization model, which is based on the Hebbianrule. In many nervous systems, spatially neighboring
relationships of the presynaptic neurons are the basis
for the correlated activities. By contrast, olfactory
sensory neurons with adjacent cell bodies on theolfactory epithelium do not necessarily mean theircorrelated activities, but it is more likely thatrecognition of the same OD as a common ligand
triggers correlated activities in the neurons. Therefore,
we formulated the OD-evoked activities of olfactory
sensory neurons and evaluated them as the force thatorganizes glomeruli. This model successfully generated
a glomerular map, which represents various aspectsof the glomerular layer in vivo. Furthermore, our model
explained wide range of perplexing phenomena andresults reported in the olfactory system. For example,
local fluctuation of glomerular positioning inindividuals, failure of olfactory targeting in OR-gene
knockout mice, segregated projection of olfactory axons
in OR-gene transgenic mice, and seemingly inconsistent targetings of olfactory axons in the knockout micefor activity-evoking ion channel genes, are simply
reproducible if only activity-dependent mechanisms
are taken into consideration. Thus, we propose theactivity-dependent targeting of olfactory axons as asimple probable mechanism of glomerular formation.
(2) Chronotopic Organization of Olfactory Bulb Axonsin the Lateral Olfactory Tract

Hitoshi Yamatani, Yasufumi Sato, Hajime Fujisawa*and Tatsumi Hirata (*Nagoya University Graduate
School)The olfactory system senses volatile chemicals in
the air with high sensitivity, compromising spatial
resolution of the stimuli. Odorous chemicals are first

Division of Agricultural Genetics

55

perceived by odorant receptors expressed on olfactory
sensory neurons of the olfactory epithelium. Because
the sensory neurons expressing the same odorant
receptor converge their axons onto a few topographically fixed glomeruli, the axons creates a stereotyped
spatial map on the OB that represents odor codes
extracted from molecular features of odorants. Thisodor map on the OB is subsequently transmitted tothe higher-order centers through mitral and tufted cells,
the output neurons of the OB. The primary projectionsof these neurons collectively construct the lateralolfactory tract (LOT). The LOT axons give offcollateral branches outside, which eventually makesynaptic connections with third-order neurons in theolfactory target areas such as the anterior olfactory
nucleus, the piriform cortex and the olfactory tubercle.
The principle underlying this projection remains
obscure. Axonal tracing studies have failed to find apoint-to-point topographic relationship between thespatial map on the OB and any of the olfactory target
areas. Thus, the stereotyped odor map on the OB is
believed to be completely disorganized in thehigher-order centers. Furthermore, it is evident thatthe spatial representation of the OB is already lost at
the level of the LOT, in which mitraVtufted cell axons
are intermingled randomly regardless of positions of
their cell bodies.
We isolated monoclonal antibody (mAb) H2C7 thatrecognizes a segregated subset of LOT axons at eachdevelopmental stage. The antigen recognized by mAb
H2C7, which was identified as c-kit receptor tyrosine
kinase, was only transiently expressed by newborn
mitraVtufted cells that were radially migrating in theintermediate zone of the OB. These findings reveal thechronotopic nature of the organization of LOT axons;the mitral/tufted cells that differentiate at the samedevelopmental stage assemble their axons together
within the LOT, regardless of positions of their cellbodies in the OB. Because newly arriving axons weresequentially added to the ventral superficial part of the
tract, displacing the older axons, there seems to be adevelopmental gradient in organization of LOT axons
from the dorsal depth to ventral surface. This special
assembly of LOT axons explains the seemingly
disordered arrangement of LOT axons that has beenreported previously, and could influence thesubsequent target selection by collateral branches
projected from the LOT.

56

Division of Brain Function

E-d. Division of Applied Genetics
Hirohiko Hirochika GroupRESEARCH ACTIVITIES(1) Target site specificity of the Tos17retrotransposonshows a preference for insertion within genes and against
insertion in retrotransposon-rich regions of the genome

Akio Miyao 1, Katsuyuki Tanaka2, Kazumasa Murata2,
Hiromitsu Sawaki2, Shin Takeda\ Kiyomi Abe\
Yoriko Shinozuka2, Katsura Onosato2 and Hirohiko
Hirochika1 eNational Institute of Agrobiological
Sciences and .2Institute of the Society for Technoinnovation of Agriculture, Forestry and Fisheries)
Because retrotransposons are the major component of plant genomes, analysis of the target site
selection of retrotransposons is important forunderstanding the structure and evolution of plant
genomes. Here, we examined the target site specificityof the rice retrotransposon Tos17, which can be
activated by tissue culture. We have produced 47,196
Tos17-induced insertion mutants of rice. This mutant
population carries approximately 500,000 insertions.
We analyzed >42,000 flanking sequences of newlytransposed Tos17 copies from 4316 mutant lines. Morethan 20,000 unique loci were assigned on the ricegenomic sequence. Analysis of these sequences showedthat insertion events are three times more frequent ingenic regions than in intergenic regions. Consistent
with this result, Tos17was shown to prefer gene-dense
regions over centromeric heterochromatin regions.
Analysis of insertion target sequences revealed apalindromic consensus sequence, ANGTT-TSDAACNT, flanking the 5-bp target site duplication.
Although insertion targets are distributed throughout
the chromosomes, they tend to cluster, and 76% of theclusters are located in genic regions. The mechanismsof target site selection by Tos17, the utility of themutant lines, and the knockout gene database are
discussed.

(2) Three distinct rice cellulose synthllBe catalytic subunit
genes required for cellulose synthesis in the secondarywall
Katsuyuki Tanaka K\ Kazumasa Murata\ Muneo
Yamazaki 2, Katsura Onosato\ Akio Miyao 2 andHirohiko Hirochika 2 eInstitute of the Society forTechno-innovation of Agriculture, Forestry and
Fisheries and 2National Institute of Agrobiological
Sciences)
Several brittle culm mutations of rice (Oryza
sativa) causing fragility of plant tissues have been
identified genetically but not characterized at amolecular level. We show here that the genes
responsible for three distinct brittle mutations ofrice, induced by the insertion of the endogenous
retrotransposon Tos17, correspond to CesA (cellulose
synthase catalytic subunit) genes, OsCesA4, OsCesA 7
and OsCesA9. Three CesA genes were expressed inseedlings, culms, premature panicles, and roots but not
in mature leaves, and the expression profiles were
almost identical among the three genes. Cellulose
contents were dramatically decreased (8.9%-25.5% of
the wild-type level) in the culms of null mutants of thethree genes, indicating that these genes are notfunctionally redundant. Consistent with these results,
cell walls in the cortical fiber cells were shown to bethinner in all the mutants than in wild-type plants.
Based on these observations, the structure of acellulose-synthesizing complex involved in thesynthesis of the secondary cell wall is discussed.
(3) Transcriptional activation mediated by binding ofa plant GATA-type zinc finger protein AGPI to theAG-motif (AGATCCAA) of the wound-inducible Myb gene
NtMyb2.
Kazuhiko Sugimoto, Shin Takeda and Hirohiko
Hirochika (National Institute of Agrobiological
Sciences)
NtMyb2 is a regulator of the tobacco retrotransposon
Ttol and the defense-related gene phenylalanine
ammonia lyase (PAL), which are induced by various
stress stimuli such as wounding or elicitor treatment.
NtMyb2 is also induced by wounding or elicitor
treatment and is regulated at the transcriptional level.
In this study, mutational analysis of the promoter of

NtMyb2 and gain-of-function analysis in vivo showedthat the sequence AGATCCAA, named the AG-motif,
is a cis-element sufficient to confer responsiveness towounding and elicitor treatment. Furthermore, byusing the south-western method, we cloned cDNAs
encoding a GATA-type zinc finger protein, which can
specifically bind to the AG-motif, named AG-motif
binding Protein (AGP1). Domain analysis revealed that
not only the GATA-type zinc finger region but also thedownstream His2 motif of AGPI is required forbinding activity, showing that the AGP has a novel
GATA-type zinc finger domain. AGPI can activate
expression from promoters containing the AG-motif
in tobacco protoplasts, indicating that AGPI is apositive regulator of NtMyb2. We also found that the
AGPI binding activity is highly enhanced by adenine
methylation of the AG-motif by bacterial dam
methylase.
(4) Characterization of a rice chlorophyll-deficientmutant using the T-DNA gene-trap system
Ki-Hong Jung\ Junghe Hur\ Choong-Hwan Ryu\
Youngju Choi\ Yong-Yoon Chung\ Akio Miyao 2,
Hirohiko Hirochika2 and Gynheung Ani ePohang
University of Science and Technology and 2National
Institute of Agrobiological Sciences)
We have previously generated a large pool of
T-DNA insertional lines in rice. In this study, we
screened those T-DNA pools for rice mutants that had
defective chlorophylls. Among the 1,995 lines examinedin the T2 generation, 189 showed a chlorophyll-deficientphenotype that segregated as a single recessive locus.
Among the mutants, 10 lines were beta-glucuronidase(GUS)-positive in the leaves. Line 9-07117 has aT-DNA
insertion into the gene that is highly homologous to
XANTHA -F in barley and CHLH in ARABIDOPSIS:This OsCHLH gene encodes the largest subunit of therice Mg-chelatase, a key enzyme in the chlorophyll
branch of the tetrapyrrole biosynthetic pathway. In theT2 and T3 generations, the chlorina mutant phenotypes
are co-segregated with the T-DNA. We have identified
two additional chlorina mutants that have a Tos17
insertion in the OsCHLH gene. Those phenotypes wereco-segregated with Tos17in the progeny. GUS assays
and RNA blot analysis showed that expression of the
OsCHLH gene is light inducible, while TEM analysis
revealed that the thylakoid membrane of the mutant

Division of Applied Genetics

57

chloroplasts is underdeveloped. The chlorophyll
content was very low in the OschlH mutants. This is
the first report that T-DNA insertional mutagenesis
can be used for functional analysis of rice genes.
(5) The MSPI gene is necessary to restrict the numberof cells entering into male and female sporogenesis andto initiate anther wall formation in rice

Ken-ichi Nonomura!, Kazumaru Miyoshi!, Mitzugu
Eiguchi!, Tazunu Suzuki!, Akio Miya0 2, Hirohiko
Hirochika2 and Nori Kurata l eNational Institute of
Genetics and 2National Institute of Agrobiological
Sciences)
The function of the novel gene MSPl (MUL TIPLE
SPOROCYTEJ, which controls early sporogenic
development, was elucidated by characterizing a
retrotransposon-tagged mutation of rice. The MSPl
gene encoded a Leu-rich repeat receptor-like protein
kinase. The mspl mutation gave rise to an excessive
number of both male and female sporocytes. In
addition, the formation of anther wall layers was
disordered and the tapetum layer was lost completely.
Although the mutation never affected homologous
chromosome pairing and chiasma maintenance, thedevelopment of pollen mother cells was arrested atvarious stages of meiotic prophase I, which resulted incomplete male sterility. Meanwhile, plural megaspore
mother cells in a mutant ovule generated several
megaspores, underwent gametogenesis, and produced
germinable seeds when fertilized with wild-type pollen
despite disorganized female gametophytes. In situ
expression of MSPl was detected in surrounding cellsof male and female sporocytes and some flower tissues,
but never in the sporocytes themselves. These results
suggest that the MSPI product plays crucial roles in
restricting the number of cells entering into male andfemale sporogenesis and in initiating anther wall
formation in rice.
(6) Loss-of-Function Mutations of the Rice GAMYBGene Impair a -Amylase Expression in Aleurone andFlower Development.

Miyuki Kaneko!, Yoshiaki Inukai!, Miyako UeguchiTanaka!, Hironori Itoh!, Takeshi Izawa2, Yuhko
Kobayashii, Tsukaho Hattori\ Akio Miya0 2, Hirohiko
Hirochika2, Motoyuki Ashikari l and Makoto

58

Division of Applied Genetics

Matsuokal eNagoya University andNational Instituteof Agrobiological Sciences)
GAMYB was first isolated as a positive transcriptional regulator of gibberellin (GA)-dependent alphaamylase expression in barley aleurone cells, and its
molecular and biochemical properties have been wellcharacterized. However, the role of GAMYB elsewherein the plant is not well understood. To investigate themolecular function of GAMYB outside of the aleurone
cells, we isolated loss-of-function mutants from a panelof rice mutants produced by the insertion of a
retrotransposon, Tosl7. Through PCR screening using
primers for rice GAMYB (OsGAMYBJ and Tosl7, we
isolated three independent mutant alleles that
contained Tos17 inserted in the exon region. Noalpha-amylase expression in the endosperm was
induced in these mutants in response to GA treatment,
indicating that the Tos17 insertion had knocked out
OsGAMYBfunction. We found no significant defects
in the growth and development of the mutants at thevegetative stage. After the phase transition to thereproductive stage, however, shortened internodes anddefects in floral organ development, especially a defectin pollen development, were observed. On the other
hand, no difference was detected in flowering time.
High-level OsGAMYBexpression was detected in thealeurone cells, inflorescence shoot apical region, stamen
primordia, and tapetum cells of the anther, but only
low-level expression occurred in organs at thevegetative stage or in the elongating stem. These
results demonstrate that, in addition to its role in the
induction of alpha-amylase in aleurone, OsGAMYB
also is important for floral organ development andessential for pollen development.
Publications
Papers1. Miyao, A., Tanaka, K., Murata, K., Sawaki, H.,
Takeda, S., Abe, K., Shinozuka, Y, Onosato, K. andHirochika, H. (2003) Target site specificity of the Tos17
retrotransposon shows a preference for insertion withingenes and against insertion in retrotransposon-richregions of the genome. Plant Cell 15: 1771-1780
2. Tanaka, K., Murata, K. Y, Onosato, K., Miyao, A.
and Hirochika, H. (2003) Three distinct rice cellulose
synthase catalytic subunit genes required for cellulose
synthesis in the secondary wall. Plant PhysioI133: 73-83

3. Sugimoto, K, Takeda, S. and Hirochika, H. (2003)
Transcriptional activation mediated by binding of aplant GATA-type zinc finger protein AGP1 to theAG-motif (AGATCCAA) of the wound-inducible Myb
gene NtMyb2. Plant]. 35: 550-564.
4. Hong, Jung., Junghe, Hur., Choong-Hwan, Ryu.,
Young-Joo, Choi., Yong-Yoon, Chung., Akio, Miyao.,
Hirohiko, Hirochika. and Gynheung, An. (2003)
Characterization of a Rice Chlorophyll-Deficient
Mutant Using the T-DNA Gene-Trap System. Plant
Cell Physiol 44: 463-472.
5. Nonomura, K, Miyoshi, K, Eiguchi, M., Suzuki,T., Miyao, A, Hirochika, H. and Kurata, N. (2003) The
MSPl gene is necessary to restrict the number of cells
entering into male and female sporogenesis and toinitiate anther wall formation in rice. Plant Cell 15:
1728-1739.
6. Kaneko, M., Inukai, Y, Ueguchi-Tanaka, M., Itoh,H., Izawa, T., Kobayashi, Y, Hattori, T., Miyao, A,
Hirochika, H., Ashikari, M. and Matsuoka, M. (2003).
Loss-of-function mutations of the rice GAMYB gene
impair the a -amylase expression in aleurone andflower development. Plant Cell 16: 33-44.

E-d. Division of Applied Genetics
Kunio Shiota GroupRESEARCH ACTIVITIES(1) DNA methylation profiles of CpG islands in normal
tissuesJun Ohgane" Naka Hattori" Satoshi Tanaka! andKunio Shiota! eUniv. Tokyo)
Methylation of DNA is involved in various
biological phenomena through gene silencing,stabilizing chromosomal structure and suppressing themobility of retrotransposons. Most CpGs are
methylated in the mammalian genome; the value varies
between 60-90% depending on the report. Overall levels
of DNA methylation reflect the hypermethylation
status of intergenic regions such as repeated sequences
rather than gene encoding regions, because a large
amount of the genome is composed of non-genic
repetitive elements. Although most CpG islands wereonce considered to be unmethylated regions in normal
tissue, there are many CpG islands with tissue-

dependently and differentially methylated regions(T-DMRs) in normal cells or tissues, and the methylation profile of the CpG islands is unique in each tissue
or cell type41 (Shiota et al., Genes to Cells 7, 961-969,
2002). Abnormal DNA methylation profiles associatewith various abnormal phenotypes ll .
(2) Preference of DNA methyltransferases for CpG
islands in normal cells
Naka Hattori\ Tetsuya Abe\ Masako Suzuki\ Tomoki
Matsuyama 2, Shigeo Yoshida2, En Li 3 and Kunio
Shiota! eUniv. Tokyo; 2RIKEN; :lMassachusets General
Hospital)

In vitro studies indicated that DNA methyltransferase1(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
DNN>, implies that Dnmtl is involved in maintenancemethylation in vivo to preserve methylation patterns
in genomic DNA, and that Dnmt3a/3b function as
de novomethyltransferases. However, a direct association between DNMTs was reported, and it wasdemonstrated that DNMTI 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
in vivo 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).
(3) Methyl-CpG-binding protein, MeCP2, is a target
molecule for maintenance DNA methyltransferase, Dnmtl
Hiromichi Kimura! and Kunio Shiota! eUniv. Tokyo)
Dnmtl preferentially methylates hemimethylated
DNA During mammalian cell division, DNA methyla-

Division of Applied Genetics

59tion patterns are transferred accurately to the newly
synthesized DNA strand. This depends on maintenance DNA methyltransferase activity. Methyl-CpG
binding proteins (MBDs) bind methylated CpG
dinucleotides. Dnmtl does not contain a methyl-CpG
binding domain common to the MBD family, raising
the question how this enzyme is recruited tohemimethylated DNA and how its replicates the
methylation pattern. We found that MeCP2 interacts
directly with the maintenance DNA methyltransferase,
Dnmtl. The region of MeCP2 that interacts with Dnmtl
corresponds to the transcription repressor domain,which can also recruit histone deacetylases (HDACs)
via a corepressor, mSin3A. Although Dnmtl can form
complexes with HDACs as well as MeCP2, the
MeCP2-Dnmtl complex does not contain the HDACl.
Thus, Dnmtl takes the place of the mSin3A-HDACl
complex, indicating that the MeCP2-interacting Dnmtl
does not bind to HDACl. Further, we demonstrated
that MeCP2 could form a complex with hemimethylated as well as fully methylated DNA. Immunoprecipitated MeCP2 complexes show DNA
methyltransferase activity to hemimethylated DNA.
These results suggest that Dnmtl associates with
MeCP2 in order to perform maintenance methylationin vivo. We propose that genome-wide and/or -specificlocal DNA methylation may be maintained by the
Dnmtl-MeCP2 complexes, bound to hemimethylatedDNA. Dnmtl may be recruited to targeted regions via
multiple steps that mayor may not involve histone
deacetylases 2).
(4) Transcription of mouse DNA methyltransferase
1 (Dnmtl) is regulated by both E2F-Rb-HDACdependent and -independent pathways

Hiromichi Kimura!, Takahisa Nakamura!, Tomoya
Ogawa2, Satoshi Tanaka! and Kunio Shiota! CUniv.
Tokyo; 2RIKEN)
Abnormal expression of Dnmtl in vivo induces
cellular alterations such as transformation, and anincrease in Dnmtl mRNA plays a causal role in c-fos-,
ras- and SV40 large T antigen-induced transformationof fibroblasts in vitro. Thus, Dnmtl expression mustbe tightly regulated during normal cell growth. The
expression of many replication-related genes, such as
PCNA, cye/ine E, pola, E2F-l and cdc6, is regulated by
E2F family of transcription factors. For each target

60

Division of Applied Genetics

gene, the pathways regulating transcription areclassified as either HDAC dependent or independent.
We have investigated the regulation of Dnmtl
transcription and identified the promoter region andmajor transcription start sites of mouse Dnmtl andfound two important cis-elements within the core
promoter region. One is an E2F binding site, and theother is a binding site for an as yet unidentified factor.
Point mutations in the two cis-elements decreased
promoter activity in both non-transformed andtransformed cells. Thus, both sites playa critical role
in regulation of Dnmtl transcription in proliferating
cells. Treatment with trichostatin A, a specific inhibitorof histone deacetylase, increased Dnmtl promoter
activity in GO/G1-arrested NIH 3T3 cells. Furthermore,
the decrease in promoter activity induced by expression of E2F-1 and Rb was reversed by trichostatin A
treatment of Saos-2 cells. Taken together, these data
indicate that transcription of Dnmtl is regulated in acomplex fashion by E2F and other transcription factors through E2F-Rb-HDAC-dependent and -independent pathways. These findings suggest that Dnmtl isa target gene of these pathways in cell proliferation,
cell transformation and tumorigenesis 31 .
(5) The Sa1l3locus is an epigenetic hotspot of aberrant
DNA methylation associated with placentomegaly ofcloned mice

Jun Ohagane!, Teruhiko Wakayama2,3, Sho Senda!,
Yukiko Yamazaki 3, Kimiko Inoue 2, Atsuo Ogura2, Joel
Marh3, Satoshi Tanaka!, Ryuzo Yanagimache andKunio Shiota1 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
in 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 (Salt3) 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 Salt3 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 Salt3 locus isassociated with abnormal placental development
caused by nuclear transfer of somatic cells. Thus theSall310cus 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!).
Publications
Papers
1. Ohgane,]., Wakayama, T., Send a, S., Yamazaki,
Y., Inoue, K., Ogura, A., Marh, ]., Tanaka, S.,
Yanagimachi, R. and Shiota, K. (2004). The Sa1l310cus
is an epigenetic hotspot of aberrant DNA methylation
associated with placentomegaly of cloned mice. Genes
to Cells, 9, 253-260.
2. Kimura, H. and Shiota, K. (2003). Methyl-CpGbinding protein, MeCP2, is a target molecule formaintenance DNA methyltransferase, Bnmt1. ]. BioI.
Chern. 278, 4806-4812.
3. Kimura, H., Nakamura, T., Ogawa, T., Tanaka, S.
and Shiota, K. (2003). Transcription of mouse DNA
methyltransferase 1 (Dnmtl) is regulated by both
E2F-Rb-HDAC-dependent and -independent pathways.
Nuci. Acids Res. 31, 3101-3113.

mi!t;>j>, Jl:i~~7.~ '/ 7~-m-~. Animus~ 8 ~20-25.
7. j§!I!f:J't~f!~, ±gl33nf!~(2003). DNA}71HtfC.J:.o361'::10/;"7 A. ~~~'¥:mflJli} 21, 1499-1507.
8. miHfll~*t~, ±gl33nf!~ (2003).361': . 1':Ji ~.:r:.J::":;.:r:.
T ~ '/ /; 7.. fJ1.ft~~ 35, 977-984.

*'

EDUCATION

1. Dr. K. Shiota gave a lecture at Tokyo Universityof Pharmacy and Life Science, Tokyo, May, 2003 (inJapanese).
2. Dr. K. Shiota was invited to give a seminar on"DNA methylation" at Yamaguchi University, Ube,
August, 2003 (in Japanese).
3. Dr. K. Shiota was invited to give a seminar on"DNA methylation" at the National Livestock Breeding
Center, Shirakawa, October, 2003 (in Japanese).
4. Dr. K. Shiota was invited to give a seminar on"Epigenetics and DNA methylation" at Yamagata
University, Yamagata, October, 2003 (in Japanese).
5. Dr. K. Shiota gave a lecture at The University ofTokyo, Graduate School of Veterinary Medical
Sciences, Tokyo, October, 2003 (in Japanese).
6. Dr. K. Shiota was invited to give a seminar on"DNA methylation of normal and cloned animal
development" at Yunsei University, Seoul, October,
2003.
SOCIAL CONTRIBUTIONS AND OTHERS

1. ***'¥:*'¥:~m'¥:1':i!tw'¥:~~W ~~2. ***'¥:*'¥:~~'¥:1':i!tw'¥:~~w~m~~w'¥:W

J&: WJ&:::ti£
3. B*'Hit1':~'¥:~

fJl!1J

4. 1':Ji*r5t'¥:~ JW~~
5. R *1!f:l\[!if'¥:~1':fJI! . 1::i~'¥:5tfiJ-~ ~*Reviews
4. Shiota, K. (2004) DNA methyaltion profiles of CpG
islands for Cellular differentiation and development
in mammals. Cytogenetic and Genome Res., In press.
5. INE ~El3~, ±g133::pIH!~ (2003). .:r:.J::":;.:r:. T ~ '/ /;7. ~ ilm-t*~1': . *1Il!lli5ti~. 5t~*IIlM1tL¥f:l~ 2, 469-475.
6. &)II)IIn~, ±gl33nf!~(2003). -t**IIlM?l./; o-/IJ:f~1f*'

Division of Applied Genetics

61

breakdown.

F. GENETIC STRAINSRESEARCH CENTER

F-a. Mammalian Genetics Laboratory
Toshihiko Shiraishi GroupRESEARCH ACTIVITIES(1) Genetic Incompatibility between X-linked Loci and
Two Autosomal Regions Causes Hybrid Breakdown
between Two Mouse Subspecies

Ayako Oka, Akihiko Mita, Yoichi Mizushina, Noriko
Yamatani, Hiromi Yamamoto, Kazuo Moriwaki 1 andToshihiko Shiroishi CRIKEN BioResource Center)
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 due to the abnormal
sperm head morphology, whereas F. hybrids between
C57BLl6J and MSM/Ms are completely fertile. Thisfinding implies that incompatibility between X-linked
genets) and other chromosomal genets) causes the
hybrid breakdown observed in C57BLl6J-XMSM • The
previous our study revealed that at least three X-linked
QTLs are responsible for the sperm head abnormality
and the hybrid breakdown.
In this study, we conducted the whole-genome scan
to detect autosomal and/or Y chromosomal regions,which interact with the X-linked QTLs and underlie
the genetic incompatibility that results in the hybrid
breakdown. The result suggested that Chromosome
1 and 11 interact with the X-linked QTLs for propermale reproduction in parental MSM/Ms strain, anddisruption of this interaction causes the hybrid

62

GENETIC STRAINS RESEARCH CENTER(2) Functional analysis of GsdmA -3 using mutant micewith aberrant epidermal morphogenesis

Shigekazu Tanaka\ Masaru Tamura, Aya Aoki 2andToshihiko Shiroishi CGrad. Univ. Advanced Studies,
2Juntendo Univ. Medical School/Tokyo Metropolitan
Hiroo General Hospital)
Epidermis is maintained by the balance between
proliferation of stem cells and cell death following
terminal differentiation. Epidermal stem cells reside
in both basal layer and upper region of hair follicles,
which is known as bulge. While the stem cells in thebasal layer differentiate into epidermal keratinocytes,
those in the bulge give rise to not only hair follicles but
also epidermal keratinocytes and sebocytes in thesebaceous gland. However, the molecular mechanismof epidermal stem cells growth and differentiation ispoorly understood. To elucidate this mechanism, ourlaboratory has analyzed several mutant mice in skin.
Two dominant mouse skin mutants, Recombinantinduced mutation 3 (Rim3) and Rex denuded (R(/en),
exhibit hyperkeratosis and alopecia, which arose
spontaneously in independent strains. Both mutants
have a mutation in GasderminA-3 (GsdmA-3), which
is a member of novel gene family, Gsdm. The Gsdm
family has leucine-rich domain of C-terminus, but noknown motif.
In this study, to characterize the function of
GsdmA -3 in the epidermal morphogenesis, we
performed the expression analysis of GsdmA -3 andmore detailed analysis of Rim3 phenotype. RT-PCR
and in situ hybridization analysis showed that
GsdmA -3 is expressed in epidermis and/or hair follicles
from postnatal stage. We conducted histologicalanalysis to examine whether hair follicles aredegenerated depending on the hair cycle in Rim3 over
several months of ages. As a result, developmental
stages of all hair follicles were synchronized in wild
type mouse over any month of the ages. By contrast,
developmental stages were desynchronized in Rim3
mice. This result indicates that the hair cycle isdisturbed in Rim3. Furthermore, BrdU labeling andimmunohistochemical analysis with epidermal
cells-specific markers demonstrated hyperproliferationand misdifferentiation of the upper follicular epidermis
in Rim3. All data suggest that GsdmA3 controls

proliferation and differentiation of epidermal
keratinocytes.
(3) Identification and characterization of novel genefamily, Gasdermin

Tomoaki Fujii\ Shigekazu Tanaka\ Aya Aoki 2,
Masaru Tamura and Toshihiko Shiroishi CGrad. Univ.
Advanced Studies, 2Juntendo Univ. Medical School!
Tokyo Metropolitan Hiroo General Hospital)
We report a novel gene family, named Gasdermin(Gsdm), which is composed of seven genes, Gsdm A -1,
A -2, A -3, C-1, C-2, C-3 and GsdmD in the mouse.
GsdmA-3 was originally identified as a causative genefor the two mouse mutantations, Rim3 and Rele", which
exhibit epidermal hyperplasia. GsdmA and GsdmC
genes form a cluster in the mouse chromosome 11 and
15, respectively, and GsdmD was mapped to mouse
chromosome 15. Comparison of amino acids sequences
revealed that members of the Gsdm family sharesimilar sequences in the N- and C-terrninus. Especially,in the C-terminus, all members of the Gsdm family
share a well-conserved novel leucine-rich motif.
Gsdm family genes were expressed mainly in thegastrointestinal tract, and some of genes were in theskin. Expression of GsdmC-2, -3 and GsdmD wereobserved in the small intestine and colon, but not inupper gastric tract. By contraries, GsdmA -1 andGsdmA -2 were expressed in the upper gastrointestinal
tract, but not in small intestine and colon. Moredetailed analysis of the upper gastric tract revealed that
expression of GsdmA -1 is specific to the squamous
epithelium (esophagus and fore stomach) and that of
GsdmA -2 was in the glandular epithelium (glandular
stomach).
In-depth characterization of mouse mutants, Rim3
and Rele ", and analysis of the gene expression patterns
suggested that the members of the Gsdm family are
involved in regulation of proliferation anddifferentiation of the epithelial cells in a tissue specificmanner.

Wakabayashi, Y., Shiroishi, T., Kominami, Rand
Yonekawa, H. Mutations in a new scaffold protein
Sans cause deafness in Jackson shaker mice. Hum.
Mol. Genet. 12, 453-461, 2003.
2. Floyd,]., Gold, D., Concepcion, D., Poon, T, Wang,
X., Keithley, E., Chen, D., Ward, E., Chinn, S.B.,
Friedman, R A, Yu, H-T, Moriwaki, K., Shiroishi, T.
and Hamilton, B. A. A natural allele of Nxj1/T AP
suppresses retrovirus insertion mutations. Nature
Genet. 35, 221-228, 2003.
3. Kikkawa, Y, Oyama, A, Ishii, R, Miura, I., Amano,T., Ishii, Y, Yoshikawa, Y, Masuya, H., Wakana, S.,
Shiroishi, T., Taya, C. and Yonekawa, H. A Small
Deletion Hotspot in the Type II Keratin Gene mK6irsl/
Krt2-6g on Mouse Chromosome 15, a Candidate for
Causing the Wavy Hair of the Caracul (Ca) Mutation.
Genetics 165, 721-33, 2003.
4. Katoh, H., Watanabe, Y, Ebukuro, M., Muguruma,
K., Takabayashi, S. and Shiroishi, T Chromosome
mapping of the peroneal muscular atropy (pma) genein the mouse. Exp. Anim. 52, 433-436, 2003.
5. Takeya, C., Esumi, M., Shiroishi, T. andYamamoto, T. Multiple single-nucleotide polymorphism in the methylenetetrahydrofolate reductase andits truncated pseudogene of 23 inbred strains of mice.
Biochem. Biophys. Res. Commun. 312, 480-486, 2003.
6. Sagai, T., Masuya, H., Tamura, M., Shimizu, K.,
Yada, Y., Wakana, S., Gondo, Y., Noda, T. and
Shiroishi, T. Phylogenie conservation of a cis-acting
regulator that controls polarized expression of Sonic
hedgehog (Shh) in limb buds. Mammal. Genome 15,
23-34, 2004.
7. Oka, A, Mita, A., Sakurai-Yamatani, N., Yamamoto,H., Takagi, N., Takano-Shimizu, T., Toshimori, K.,
Moriwaki, K. and Shiroishi, T. Hybrid Breakdown
Caused by Substitution of X Chromosome between
Two Mouse Subspecies. Genetics 166, 913-924, 2004.

F-b. Mammalian Developmental Laboratory
Yumiko Saga GroupRESEARCH ACTIVITIES

Publications(1) Molecular mechanism of somite segmentation

Papers
1. Kikkawa, Y, Shitara, H., Wakana, S., Kohara, Y,
Takada, T., Okamoto, M., Taya, C., Kamiya, K.,
Yoshikawa, Y, Tokano, H., Kitamura, K., Shimizu, K.,

Yu Takahashi (N ational Institute of Health Sciences),
Kaoru Mitsui, Mitsuru Morimoto and Yumiko Saga

Mammalian Genetics Laboratory

63

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. Each somite is subdivided
into anterior (rostral) and posterior (caudal) compartments that differ in their properties and gene
expression. The rostro-caudal polarity of a somite isestablished within the PSM prior to segmentation. Our
aim is to understand the mechanisms underlying
generation of a segmental pattern within the PSM. By
the genetic analysis, we have shown that bHLH-typetranscription factor, Mesp2 plays a critical role in establishment of rostro-caudal polariy of a somite by
regulating Dill expression via Notch signaling
pathways. We have shown that there are two Notch
signaling cascades; one is Psen1-dependent Dlll-Notch
pathway and results in activation of Dlll and the otheris Psen1-independent Dil3-Notch pathway toantagonize the Dlll-Notch pathway to suppress Dlll
expression 3• Mesp2 is also a strong suppressor for Dlll
expression. However, Mesp2 acts as like a transcriptional activator for rostral genes, Cerl and EphA4. To
understand the mechanism of Mesp2 function and to
identify direct targets of Mesp2, we started to analyze
molecular nature of Mesp2. Since Mesp2expression is
temporally restricted, we have analyzed the stabilityof Mesp2 protein and found that Mesp2 could be
regulated by a proteosome-dependent pathway. By
analyzing a series of deletion constructs, we haveidentified amino acid sequences required for the
instability of Mesp2. Furthennore, Mesp2 is heavily
phosphorylated. We have constructed mutant Mesp2
molecules and are planning to knock in these
constructs in Mesp2 locus to know in vivo function ofthese sequences.
(2)

Regulation of Mespl and Mesp2 expression

Yukuto Yasuhiko (National Institute of Health
Sciences), Masayuki Oginuma and Yumiko Saga
Mespl and Mesp2 are both expressed in the early
mesodenn and presomitic mesodenn (PSM) just before
segmentation. We have previously identified anenhancer required for Mesp2 expression in the PSM.
Using yeast one-hybrid screening, we have found thatTbx6 is a factor that specifically binds to the enhancer
sequences. Actually the enhancer region contains
consensus sequence known as a T -box binding motif.

64

Mammalian Developmental Laboratory

Using in vitro binding assay, we confinned the specificbinding of Tbx6 on the enhancer. Tbx6 is expressed
in PSM and partially overlapped with the Mesp2
expression domain. However, the expression domain
of Tbx6 is much wider than that of Mesp2. Thus it isunlikely that the Tbx6 is an only factor to regulate
Mesp2 expression. For the further analysis, we areplanning to generate transgenic mouse expressing
Flag-tagged Tbx6.
In order to analyze global transcriptional
regulation of both Mespl and Mesp2, we use BAC
transgenic strategy. Mespl and Mesp2 are located inhead to head orientation and separated only by 16 kb.
Since the expression pattern is very similar, we need
to use different reporters for each gene. To introduce
LacZ and ALP genes, we have constructed targeting
vectors for homologous recombination in bacteria. So
far LacZ gene has been introduced either Mespl or
Mesp210cus by homologous recombination.
(3)

Heart morphogenesis and Notch signaling

Hiroki Kokubo, Yusuke Watanabe, Yoshiaki
Okamura, Wataru Saito and Yumiko Saga
Notch signaling is crucial for the cardiovasculardevelopment. We have cloned three hesr (hairy andenhancer of split related) transcription factors, hesr1,
hesr2 and hesr3 as possible downstream targets ofNotch signaling. We have shown that hesr2 mutant
mice are highly lethal in early postnatal period withcongenital heart defects, featured by the enlarged
hearts. Although hesrl gene is also expressed in thedeveloping cardiovascular system, hesrl single
knockout mouse does not show any abnormality.
However, mice lacking both hesrl and hesr2 die atembryonic day (E) U.5 and showed not only cardiac
defect but also vascular malfonnations. The hesrl andhesr2double homozygous hearts exhibit hypoplastic
development of myocardial trabeculation of theventricle due to apoptosis, and few of the cardiac
endothelial cells in the atrio-ventricular cushion
undergo epithelial-mesenchymal transfonnation. Our
results highlight the importance of hesrl and hesr2inthe developing cardiac and vascular system.
We are also studying Notch function in heartmorphogenesis using tr~nsgenic mouse, which has
activated Notch1 after floxed CAT gene under thecontrol of CAG promoter. We can achieve forced Notch

activation by intercrossing the transgenic mouse witha Cre-expressing mouse. Since Mespl is expressed in
the heart 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. When we compared Notch activation
between wild-type and the transgenic mouse, ectopic
Notch activation was observed in myocardium in
addition to endocardium in which Notch is normally
activated. Furthermore, hesrl expression was abnormally induced in the myocardium. This system may
provide a key to elucidate hesrl function in heartmorphogenesis.
(4)

Gene hunting possibly involved in somitogenesis

Satoshi Kitajima (National Institute of Health Sciences),
Aki Ishikawa and Yumiko Saga
In order to obtain possi ble downstream target
genes of Mesp2, and to get more information aboutmolecular mechanism underlying the formation andmaturation of PSM cells required for the somite
segmentation, we generated two subtractive cDNA
libraries and screened them by in situ hybridization
screening method. After screening, we identified morethan 30 clones that showed the expression in somite
and/or PSM region. Among them, we selected 4 genes
for further expression and functional analyses. One ofthem, nkdl that is a negative regulator for Wnt
signaling, exhibits interesting expression patternduring somitogenesis. The expression showed oscillation in the PSM. Several genes are known to becontrolled by a molecular clock. The expression of suchgenes changes during somitogenesis; it starts from tailbud and the expression domain progressively moves
to the anterior region. The one cycle is perfectly
matched with a cycle for somite formation. Obviously
Notch signaling is oscillating, since expressions ofmany gene involved in Notch signal cascade are
oscillating. In addition, W nt signal might be also
oscillating. So far only one oscillating gene, axin2 hasbeen reported. So nkdl is a second gene found to be
oscillating. Interestingly, the oscillation phase between
axin2 and nkdl is not synchronized, indicating under
the different regulation. To know the function of nkdl
during somitogenesis, we are generating the geneknockout mouse.

(5)

Functional analysis of mouse nanos genes

Masayuki Tsuda, Yumiko Sasaoka, Makoto Kiso andYumiko Saga
Previously we have isolated three mouse nanos
genes (nanosl, nanos2 and nanos3p. Among them, we
focus on functions of nanos2 and nanos3 since theseare specifically expressed and play important roles ongerm cell developmene.
Studies on Zebrafish and medaka nanos haveindicated that the 3' UTR of nanos gene is important
for their localization and own translation. Accordingly,
we have generated two types of knock-in genes fornanos210cus; one is LacZ gene with SV40 poly A signal
and the other is LacZwith own nanos23' -UTR. When
we compared the expression pattern between them, we
noticed B-gal activity is much higher in the mouse withendogenous nanos2 3' -UTR. However, the expression
level of their transcripts is unchanged, indicating
different translational efficacy. In contrast, maternal
expression of nanos2 is lower in the mouse withendogenous nanos2 3' -UTR. These observations
indicate that the expression of nanos protein isregulated both in the transcription and translation and
3' -UTR plays an important role on the translationl
control.
For nanos3, we tried to determine the time for
initial expression. By collaborating with Dr. Saito (CDB,
Kobe), we have successfully detected the nanos3
expression in presumptive PGC cells in 7.25 dpc
embryos. The expression was accompanied withexpressions of other PGC markers, stella and fragillis.
Importantly, no expression was observed in any
somatic cells. Therefore it is formally proved thatnanos3 is an earliest gene that is activated in PGC andplays an important role.
Publications
Papers

1. Rhee,]., Takahashi, Y, Saga, Y, Wilson-Rawls,]. and Rawls, A. (2003) The protocadherin papc is
involved in the organization of the epithelium along
the segmental border during mouse somitogenesis. Dev
BioI. 254, 248-261.
2. Haraguchi, S., Tsuda, M., Kitajima, S., Sasaoka,
Y, Nomura-Kitabayashi, A., Kurokawa, K. and Saga,
Y (2003). nanosl: a mouse nanos gene expressed in the

Mammalian Developmental Laboratory65

central nervous system is dispensable for normal
development. Mech Dev. 120, 721-731.
3. Takahashi, Y, Inoue, T., Gossler, A. and Saga, Y(2003). Feedback loops comprising Dlll, Dll3 and Mesp2,
and differential involvement of Psen1 are essential for
rostrocaudal patterning of somites. Development 130,4259-4268.
4. Tsuda, T., Sasaoka, Y., Kiso, M., Abe, K.,
Haraguchi, S., Kobayashi, S. and Saga, Y. (2003).
Conserved role of nanos proteins in germ cell
development. Science 301, 1239-1241.
5. Mitsui, K., Tokuzawa, Y, Segawa, K., Murakami,
M., Itoh, H., Takahashi, K., Maeda, M. and Yamanaka,
S. (2003). A novel homeobox gene ECAT4 is required
and sufficient for self-renewal of mouse pluripotent
cells. Cell 113, 631-642.
6. Takahashi, K., Mitsui, K. and Yamanaka, Y (2003).
Important role of E-Ras in tumor-like properties ofmouse embryonic stem cells. Nature 423, 541-545.
7. Tokuzawa, Y., Kaiho, E., Maruyama, M., Takahashi,K., Mitsui, K., Maeda, M., Niwa, H. and Yamanaka, S.
(2003). Fbx15 is a novel target of Oct3/4 but isdispensable for ES cell self-renewal and mouse
development. Mol Cell Bioi. 23, 2699-2708.
8. Okazaki, N., Kikuno, R., Ohara, R., Inamoto, S.,
Koseki, H., Hiraoka, S., Saga, Y, Nagase, T., Ohara,
O. and Koga, H. (2003). Prediction of the coding
sequences of mouse homologues of KIAA gene: m. thecomplete nucleotide sequences of 500 mouse KIAAhomologous cDNAs identified by screening of terminal
sequences of cDNA clones randomly sampled fromsize-fractionated libraries. DNA Res. 10, 167-80.
Books

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1::~iUIII~~pX;~f!I!1C.:tQIt 9 N anosil)~fj'~, *HIn1!!I'¥, 22,1065-1068, 2003.
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pp.24-29, 2003.

EDUCATION

1. Dr. Y. Saga gave a lecture at the University of. Tokyo, March, 2003 (in Japanese).
2. Dr. Y. Saga gave a seminar on "Molecularmechanism leading to the establishment of rostrocaudal polarity of somites" at UCLA, USA, August,2003.
3. Dr. K. Mitsui was invited to give a seminar on

66

Mammalian Developmental Laboratory"Maintenance of pluripotency in mouse embryonicstem cell" at the Institute of Life Science, Kurume
University, Fukuoka, July, 2003 (in Japanese).

F-C. Mouse Genomics Resource Laboratory
Tsuyoshi Koide GroupRESEARCH ACTIVITIES(1) Quantitative traits analyses of sensitivities to heatevoked pain assayed by tests of hot plate and tail flick

Tamio Furuse, Toshihiko Shiroishi 1) and Tsuyoshi
Koide CMammalian Genetics Laboratory)
In animal, heat stimulation is transmitted tocentral nervous system (CNS) via sensory neuron, andis recognized as heat or pain. Various levels of heatsensations activate the multi functional pain receptors,
TRPV channels that expressed in sensory ganglia.
Many behavioral tests had been established to measure
the pain sensitivity in rodents by investigating
behavioral response from the aversive pain stimuli. Hot
plate test displays the ability of pain recognition
system or analgesic system based on upper CNS. The
tail flick test displays the pain sensitivity based on
nociceptive reflex. In the present study, we conducted
QTL analyses to characterize genetic loci involved in
the different sensitivity to heat evoked pain between
two mouse strains, C57BLl6 and KJR. In the study, weapplied hot plate test and tail flick test to assess thetwo types of pain sensitivity of BKF2 progeny
generated form F1 progeny of C57BLl6 and KJR. In theanalyses, we detected three significant loci for hot plate
test, Hpql, Hpq2 and Hpq3 and two significant loci for
tail flick test, Tfql and Tfq2. The pain associatedquantitative traits loci, Hpql, Hpq2, Hpq3, Tfql, andTfq2were located on the chromosomes, 2,10,18,8 and
10, respectively. Hpq2 and Tfq1 were overlapped on
the same region of chromosome 10. These datasuggested that genetic mechanism for strain differenceof pain sensitivities between C57BLl6 and KJR thatmediated by upper CNS and reflection system aremostly different but partly shared.

(2) QTL analysis of home-cage spontaneous activity
using hyper- and hypoactive mouse strains]uzoh Umemori, Tamio Furuse, Toshihiko Shiroishi')and Tsuyoshi Koide ('Mammalian Genetics Laboratory)
It has been previously reported that mice move
spontaneously in the home cage according to a light!
dark rhythm, and most mice are active during the dark
period. This behavior is called as spontaneous activity,
which is quite different among various mouse strains
including wild-derived and laboratory strains. Amongthese strains, activity of K]R strain were significantly
high comparing to that of BLG2 and C57BLl6]. The
Fl females from the cross between K]R and either
BLG2 or C57BLl6] exhibited the similar levels of
spontaneous activity as K]R mice strain. We haveconducted genetic analyses to elucidate the genetic
basis of the spontaneous activity in the home cage. To
obtain QTLs involved in spontaneous locomotive
activity, we conducted QTL analysis by using F2
populations (BKF2), which were made by intercrossof Fl mice between K]R and C57BLl6], In the QTL
analysis of BKF2, we found that two QTLs, positioned
on chromosomes 2 and 10, are associated with thedifferent level of spontaneous activity of K]R and
C57BLl6. These loci are different to previously
identified QTLs, locol and loco2, which are associatedwith different level of activity between KJR and BLG2.
These findings suggested existence of complex genetic
mechanism for controlling spontaneous activities
among strains.
(3) Different effect of apomorphine, dopamine agonist,

C57BLl6JJcl. Different dose of apomorphine, 0.2, 1.0,
2.0, 5.0mg/kg, were administrated to these mice strains.
We found that sensitivities to apomorphine were significantly different among these strains. In case of thevehicle administration, the locomotor and rearing
activities of N]L and MSM are significantly lower thanthat of KJR and C57BLl6. In contrast to the behavioral
response of locomotion and rearing, number ofgrooming of N]L and MSM was significantly higher
than that of N]L and K]R. The activities andbehavioral traits of these strains were changed byapomorphine injection. N]L indicated high locomotor
activity at 2mg/kg of apomorphine injection, while
0.2-1.0mg/kg of apomorphine injection exhibited noremarkable effect on this strain. Comparing to thevehicle injection, administration of 0.2mg/kg of
apomorphine reduced locomotor activity of KJR andelevated grooming behavior. The locomotor activity ofthis strain was increased with further dose of
apomorphine injection (1-5mg/kg). Locomotion of MSM
was increased from lmg/kg of apomorphine injection
while grooming behavior was decreased withincreasing dose of apomorphine. Comparing to wildderived strains, C57BLl6 exhibited striking reductionof locomotor activity and rearing behavior by apomorphine injection (l-5mg/kg). As a result of thisexperiment, we showed that dopaminergic systems aredifferent among these strains.
(4)

Descriptive analysis of the open-field behavior inwild mice strains

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

on open-field activities in wild derived mouse strains

Akinori Nishi, Toshihiko Shiroishi') and Tsuyoshi
Koide ('Mammalian Genetics Laboratory)
It is known that the dopaminergic system play
important roles in controlling locomotor activity ofmice. Apomorphine hydrochloride is known as a mixed
Dl/D2 dopamine receptor agonist, which causes
behavior changes in locomotor activity and climbing
behavior, and frequently induces stereotypic behavior
in rodents. In this study, we investigated and compared
the effect of apomorphine on open-field behavior inwild derived mouse strains, N]LlMs, K]R/Ms and
MSM/Ms, and common laboratory inbred strain,

Open-field is one of the apparatuses for measuring
emotionality. The open-field behavior was believed to
be influenced by genetic factors. Recent genetic studies
have reported that some QTLs for open-field emotionality
involved in the behavior. Although most of those study
have used ambulatory movement and defecation as the
open-field measurements, mice actually show morevarious behavior in the open-field such as scout andrear to look around, groom their body, or run in thefield. We therefore described detail of open-field
behavior with 12 behavioral patterns (sniffing,
locomotion, stretching, leaning, rearing, grooming,
face-washing, digging, gnawing, jumping, pausing,freezing) by counting presence of those behaviors in

Mouse Genomics Resource Laboratory67every 5 seconds. Behavioral frequency of each patternis calculated in every 1 min for 10 min test period. We
used females and males of 10 wild mouse strains(Mishima battery), a laboratory strain C57BLl6, anda fancy mouse JFl. The 12 strains of mice showedapparent difference in the temporal changes of thosebehavioral patterns without any significant sex
difference. We then applied the principal component
analysis for those behavioral patterns to find thestructure of the factors lurk behind of the open-field
behavior. Four independent components explaining
66.8% of the total variance were found. Component 1(PC1) acquired high loading from variables related toactivity (locomotion, leaning and sniffing for positive,
freezing for negative). Variables not related to
locomotion (stretching and sniffing for positive, rearing
and grooming for negative) loaded highly on Component 2 (PC2), might relate to exploration or habituation.
The PCl and PC2 were thought to be major components for open-field behaviors because they hadlarge variance. We therefore used PCl and PC2 tocompare the 12 mouse strains. In the analysis, common
temporal changes were observed; they first exhibitedhigh score for both PCl and PC2 (stretching, freezing),
but both reduced with time (locomotion, leaning,
rearing, grooming). KJR and SWN, belonging to same
subspecies, showed exactly same characters in thescores and the temporal change patterns in PCl andPC2. However, most strains showed various charactersin PCl and PC2. These findings indicated that PCl andPC2 were influenced by multiple genetic mechanisms.
Further analysis for characterizing these genetic factors
will provide useful information for understanding
mechanism of regulating open-field behavior in mice.
Publications

F-d. Plant Genetics Laboratory
Nori Kurata GroupRESEARCH ACTIVITIES(1) Characterization of sporogenesis defects in sterile
rice mutants
Ken-ichi Nonomura, Mutsuko Nakano, Mitsugu
Eiguchi, Akio Miyao*, Hirohiko Hirochika* and Nori
Kurata (*Natl. Inst. Agrobiol. Resour.)This study aims to dissect genetic machinery forcontrolling sporogenesis of higher plants. We screened
insertion mutant lines tagged by the Tosl7, an endogenous retrotransposon of rice, and succeeded toisolate three sterile mutants: pairl, pair2 and mspl. The
PAIRI (HOMOLOGOUS EAIRING !lBERRA TION IN WCE MEIOSIS 1) gene encodes a putative nuclear
protein of 492 amino acids with the coiled-coil structure,and expresses in both male and female meiocytes(submitted). The PAIR2encodes a protein of 610 aminoacids, homologous to the yeast HOPI and Arabidopsis
ASYl, both of those play an important role in establishing the synaptonemal complex between homologous
chromosomes. The loss-of-function mutation of PAIRI
and PAIR2genes resulted in the defect of homologous
chromosome pairing and division. The MSPI (MULTIPLE SPOROCYTES 1) gene encodes a putative
LRR receptor-like protein kinase of 1294 amino acids
and expresses in the nurse cells surrounding the maleand female sporocytes. The loss-of-function mutation
resulted in aberrant increase of sporocyte numbers,suggesting a major function of MSPI to be therestriction of surrounding cells not entering into
sporogenous pathway.

PapersFuruse, T., Miura, Y, Yagasaki, Y, Shiroyishi, T. andKoide, T. (2003). Identification of QTLs for differential
capsaicin sensitivity between mouse strains KJR and
C57BLl6. Pain 105, 169-175.

(2) Structural and functional analysis of rice OsHAP3
genes
Kazumaru Miyoshi, Thiruvengadam Thirumurugan,
Yukihiro Ito and Nori Kurata

EDUCATION

Dr. T. Koide gave a lecture at Faculty of Science,
University of Tokyo, April, 2003 (in Japanese).

68

Mouse Genomics Resource Laboratory

We identified 11 genes (OsHAP3A to OsHAP3K)
which encode a HAP3/NF-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 inchloroplast biogenesis by an antisense and RNAiapproach1). Considering that LEC1 and L1L, Arabidopsis members of the HAP3INF-YB subunit, are criticalregulators of embryogenesis, plant HAP3INF-YB genes
may have diverse functions ranging from chloroplastbiogenesis to embryogenesis depending on each gene.
We examined the function of other two members,
OsHAP3E and OsHAP3D, which are most closely
related to LEC1 and L1L, respectively, by in situ
hybridization and by generating transgenic rice plantsof overexpression or knock down. The overexpressing
transgenic plants showed various developmental
abnormalities such as dwarfism, small erected leaves
and malformed flowers. RNAi, antisense or repressormediated knock down plants are awaiting for theirphenotypes.
(3) Functional analysis of PLASTOCHRONI in rice

Kazumaru Miyoshi, Yukihiro Ito and Nori Kurata
Rice plants with loss-of-function mutations of
PLASTOCHRON1 (PLA1) show pleiotropic effects on
the rice development, giving short plastochron (rapid
emergence of leaves), small erected leaves, dwarfism
and elongati~m of vegetative development. We showedthat PLA1 encodes a member of a cytochrome P450
family and is expressed in young leaves, but not in the
SAM2l. In plants, cytochrome P450s are involvedin various metabolic pathways including phytohormone biosynthesis such as auxin, gibberellins andbrassinosteroids.
To understand a molecular function of PLA1, we
generated transgenic rice plants overexpressing the
PLAl. Regenerated shoots showed severe dwarfism
with twisted leaves. Initiation and/or elongation ofroots were also inhibited. Metabolome analysis showedthat the synthesis of auxin- and gibberellin-related
compounds were affected in the overexpressing plants.
RT-PCR analysis showed that OsDWARFand OsBRIl,
which encode a brassinosteroid biosynthesis enzyme
and a brassinosteroid receptor, respectively, and aretargets of a negative feedback control, were downregulated in the overexpressing plants. These results
suggest that PLA1 is involved in phytohormone
synthesis.

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

Yukihiro Ito and Nori Kurata
KNOX family classl homeobox genes playa vitalfunction for shoot apical meristem (SAM) formationand maintenance. We found that cytokinin, which
is necessary for shoot regeneration, induced the expression of OSH1, a member of the KNOX family, inthe callus upon regeneration. It was assumed thatthe cytokinin-induced gene expression is under contol
of a two-component system which consists of ahistidine kinase, a phosphotransmitter protein and a
response regulator, like as revealed in Arabidopsis.
To examine this possibility, we searched cytokinin
signal transducers in rice and examined one of histidine kinase homologues named COS3. We generated
COS3-overexpressing rice calli and detected OSH1
expression in it, even on a medium lacking thecytokinin. This suggests that the two-component
system is involved in the cytokinin-induced OSH1
expression in the callus. We have also prepared an
examination of OSH1 expression induced by several
possible dominant negative mutations of COS3.
(5) Search for nuclear protein genes having regulatory
activity in rice development

Kazuki Moriguchi, Mitsugu Eiguchi and Nori Kurata
We applied NTT (nuclear transportation trap)system to collect rice nuclear protein genes from threedevelopmental stages; young panicle, flowering panicle
and regenerating calli. We identified over 500 genes
including many novel proteins and many transcription
factors (submitted). A subset of those genes were
examined for their nuclear localization by introducing
the genes fused with GFP 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 (submitted). Some of these genes wererevealed to express at specific stages of rice development by RT-PCR and in situ hybridization. To
characterize the unknown gene function, generationof transgenic rice which overexpress or being knocked
down l4FPB077 protein is in progress.

Plant Genetics Laboratory69

(6) Introduction of rice artificial chromosomes into riceTadzunu Suzuki, Ken-ichi Nonomura and Nori Kurata
Since the transfer methods of huge size DNAs into
rice cells have not been established, two methods oflipofection and particle bombardment system were
examined for transferring the candidate DNAs into
rice. Though lipofection can reduce damages of DNAsof huge size, it had rarely been used for plant celltransformation. This method was applied for rice
protoplasts isolated from rice calli. Results indicatedthe effectiveness of the lipofection method for rice
protoplasts, at least with a small size DNAs. Anotherattempt to introduce larger size BACs possessing ricecentromeric sequences into rice by particle bombardment could generate a few lines of calli and regenerated
plants. RT-PCR analysis showed that they expressed
the marker gene. However, Southern analysis indicatedthat the tranfered DNAs should be integrated into the
genome in the transform ants.
(7) 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 twoelement system and the GUS reporter gene. We alreadyhave established the system by which Ds-GUS
transposants are efficiently identified in rice (in press).
This year we screened about 1,000 transposed lines
and identified 62 lines with tissue-specific GUS activity.
GUS expression was observed in various organs
including embryo, leaf, root, flower, tiller and shootapex. Some lines expressed the GUS protein upon
wounding. We cloned a trapped gene by aDs-GUS
insertion in a line P2503, which expressed the GUS in
the wounded portion of a leaf, as an example. The
Ds-GUSwas shown inserted between two genes, one
was OsLEA3 and the other was an unknown gene.
OsLEA3 is a member of a late embryogenesis
abundant protein gene family and is expressed in roots
by drought or ABA treatment. To examine which
enhancer of the two genes is trapped by the Ds-GUS,

70

Plant Genetics Laboratory

we examined the expression of these genes by RT-PCR.
OsLEA3 was expressed transiently in the leaf after
wounding, while the unknown gene was expressed
without wounding. This indicates that the enhancerof OsLEA3 was trapped in P2503. Cloning of othertrapped enhancers is underway. These data will be
opened through Oryzabase (http://www.shigen. nig.
ac.jp/rice/oryzabase/top/top.jsp).
(8) 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 theaimed gene was a male gametophyte gene that interact
with a maternal locus on chromosome 6. In otherwords, the pollen with Kasalath genotype at thegametophyte gene preferentially takes place infertilization of eggs by 94% probability in the maternal
plant that is heterozygote or Kasalath homozygote 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. The
genotypes of the target gene in the recombinants were
determined by segregation analyses of selfed progenies
showing heterozygous genotype for the interactive
region. In case the target gene locus of a recombinantis heterozygous, segregation of heterozygous marker
in selfed progeny is 0:1:1 for Nipponbare homozygote(N): heterozygote (H): Kasalath homozygote (K). In caseof homozygous of the gene, segregation would be 1:2:1
as Mendelian expectation. Progeny of one of theselected plants that recombined Kasalath homozygote
to heterozygote at the vicinity of an Argonaute gene
showed unexpected segregation, 1:3:2 for N: H: K. One
possible explanation is that the causal cistron in thisrecombinant is chimera that has half ability to fertilize
comparing to Kasalath homogeneous cistron and the
Argonaute gene is the causal gene. Expression of thisArgonaute gene in both Nipponbare pollen and
Kasalath pollen was confirmed. RT-PCR and microarray analysis and showed there was no difference inthe expression level. However, 14 single-base changes
between Nipponbare and Kasalath genome sequenceswere detected in the 6.9 kb Argonaute region. Nine outof the 14 changes were in the peptide-coding region;
one and eight were synonymous and nonsynonymous
substitutions, respectively. High frequency of nonsynonymous substitutions suggests rapid evolutionthat is common feature of reproductive barriers. We
are preparing complementation test for this barrier.

functional genomics in rice. Mol. Genet. Genomics: inpress.

Publications

1. ifEEO) l') r ~ *f.4:fi[~~{7U C: lJ~~ / A b1MtO)~J
jf~~~nii~, jf{~~ijf9Effl-~0~, 200M¥ 4 ~2. ifEEO) l') r ~ / A;6:>G~k{):fi[~O)j::ff~III§J:='~mPapers1. Miyoshi, K, Ito, Y, Serizawa, A and Kurata, N.
(2003). OsHAP3 genes regulate chloroplast biogenesis
in rice. Plant J 36, 532-540.
2. Miyoshi, K, Ahn, B-O., Kawakatsu, T., Ito, Y.,
Itoh, J-L, Nagato, Y. and Kurata, N. (2004). PLASTOCHRON1, a timekeeper of leaf initiation in rice,
encodes cytochrome P450. Proc. Nat!. Acad. Sci. USA
101, 875-880.
3. Nonomura, KL, Miyoshi, K, Eiguchi, M., Suzuki,T., Miyao, A, Hirochika, H. and Kurata, N. (2003). The
MSP1 gene is necessary to restrict the number of cells
entering into male and female sporogenesis and toinitiate anther wall formation in rice. Plant Cell IS,
1728-1739.
4. Suzuki; T., Nonomura, K-L, Takeda, N.,
Murayama, Y and Kurata, N. (2003). Application ofenhanced lipofection to rice transformation. Rice Genet,
Newslet. 20, 116-118.
5. Nonomura, KI., Nakano, M., Murata, K, Miyoshi,
K, Eiguchi, M., Miyao, A, Hirochika, H. and Kurata,
N. (2003). 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.
6. Nonomura, K-I., Nakano, M., Fukuda, T., Eiguchi,M., Miyao, A, Hirochika, H. and }\urata, N. (2004). The
novel gene HOMOLOGOUS PAIRING ABERRA TION
IN RICE MEIOSIS1 of rice encodes a putative
coiled-coil protein nequired for homologous chromosome pairing in meiosis. Plant Cell 16, 1008-1020.
7. 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
8. Ito, Y., Eiguchi, M. and Kurata, N. Establishmentof an enhancer trap system with Ds and GUS forBook

9. Kurata, N. and Fukui, K·(2003). Chromosome
research in genus Oryza. 'Monograph in genus Oryza.'
Nanda JS ed. Science Publisher. pp 213-261.
EDUCATION~~Mf~,,*, :='~mj::HI~~~ /')1-, 20041f11~

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1tl'1W1iHi:w:r~~~,j::~JI{~~~~ */J\~ ~~~ ~~)F-e. Microbial Genetics Laboratory
Akiko Nishimura GroupRESEARCH ACTIVITIES(1) Demonstration of native dynamics of FtsZ in thegrowing Escherichia coli single cell by on-chip microculture
Ippei Inouye, Kenji Yasuda and Akiko Nishimura
FtsZ, first identified genetically by Lutkenhaus isessential key protein acting at an early stage ofcytokinesis, structural and functional homologue ofeukaryotic tubulin, and highly conserved over probably
all prokaryotes including archaea, chloroplasts, andsome mitochondria. Precise timing of the FtsZ-ring
dynamics in a cell cycle has become one of theimportant problems to make clear since Bi and

Plant Genetics Laboratory

71

Lutkenhaus demonstrated that FtsZs are scatteredthrough the cytoplasm during most of the cell cycle
but assemble into a microtuble-like cytokinetic ringon the inner membrane of the potential divisionsite in
the early stages of septum apparatus constriction in
E. coli. Despite the exhilarating advances in role of FtsZ
in bacterial cell division, it is not well known when andhow FtsZs change their location in a growing cell cycle.
We have succeeded to demonstrate the timing of native
dynamics of FtsZ such as assembly, constriction, anddisassembly in the exponentially growing single cellsof E. coli, using an on-chip microculture system. In thesystem, single bacterium was grown in an arrayedmicrochamber arranged on glass slide and separated
by semi-permeable membrane from cover chamberwith circulating medium, and the real time dynamics
of FtsZ-GFP localization was observed by fluorescent
microscopy. Fluorescence intensity of FtsZ-GFP wasmeasured by NIH image. Constructed strain expressed
FtsZ-GFP, with long flexible linker to minimize theobstruction of GFP, under the control of native ftsZ
promoters on chromosomal DNA, and divided/grew
completely similar as the normal tube cultures. As theresults, we proposed new model for dynamics of FtsZ
and timing of cell division (in preparation).

Further, to minimize the difficulties in identifying a
positive clone, we evaluated pools of 48 clones, which
we found worked well for complementation studies.
We created two types of clone sets: the original set
stored as individual clones in 45 microtiter plates, thesecond set made up of pools of 48 clones stored in asingle microtiter plate. Using these two kinds of clone
sets, we have identified 388 genes that can correct thetemperature sensitive defect in cell division (fts). We
have further analyzed these genes systematically andidentified up to hundred novel genes involved in thedivision process. We also demonstrated the global
network coordinating cellular events in a cell cycle
through cell division.
Publications
Papers

1. Makinoshima. H., Aizawa. S., Hayashi. H., Miki.
T., Nishimura. A. and Ishihama. A. (2003). Growthphase-coupled alterations in cell structure and functionof Escherichia coli. J. Bacteriol, 185: 1338-1345.
Database

http://www.shigen.nig.ac.jp/ecoli/strain/top.jsp(2) A complete set of E. coli genes/ORFs on a mobile
plasmid: successful use in the isolation of all cell divisionmutants

Social contributions and others

Kimiko Saka, Ken Nishikawa, Hideaki Sugawara,
Noriko Matsumoto, Hiroshi Fujishima and Akiko
Nishimura7v-~, ~~*:~#~r·~~~X, I±l~A:OOli*f~1f

1. I±lffliJl1i~: 2003-359534, ~IlJlO)i;f:J\ : -y IVT tJ ::r. Iv~B~~~ffl~, ~ff~Y7A~V-T~/~

2.

I±lffliJl1ii7: 2003-405263, JeIlA(7)i;f:J\ : -y IVTtJ ::r.lv

7v-~, ~~*:~#~r·m~.X, I±l~A:OO~

To facilitate genetic studies of Escherichia coli, weconstructed a complete set of mobile plasmid clonesof W31l0 open reading frames (ORFs). The plasmid
could be transferred from F+ to F- strains and carries
a multi-cloning site as well as ampicillin resistancegene. Expression of the cloned ORF is strictly
controlled by Ptac/lacIq• The plasmids carrying each
ORF were introduced in an F+ recA strain and storedin 96-well microtiter plates. In this way, 96 clones ofthe plasmid can be transferred concurrently to Fbacteria using the conjugative system and without
making competent cells. This provides a convenient
procedure for screening the ability of a cloned ORF to
suppress or complement E. coli mutations, and, thus,
for the systematic identification of mutant genes.

72

Microbial Genetics Laboratory

mB~~~ffl~, ~ff~Y7A~V-T~/~~~;~IIJ

1. Drs. Y. Kohara, A. Nishimura, and Y. Yamazakiorganized a satellite poster session "Bio-resource" at26th Annual Meeting of the Molecular Biology Societyof Japan, Kobe, December, 2003 (in Japanese).
2. Dr. A. Nishimura was appointed for a memberof editorial board of "Microbiology and Culture
Collections".

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

Ryu Ueda, Misako Taniguchi, Kaoru Saigo 1 andKuniaki Takahashi CGraduate School of 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 13,500
genes. Considering there is such a small number of fly
genes, each one of them may have an essential functionin fly development and behavior. In other words, it may
be easy to detect and analyze gene function in the fly
by reverse genetics because the abnormal phenotype
will frequently appear when knocking down a target
gene whose function is unknown. We are planning toinvestigate the function of fly genes comprehensively
as a suitable model for studying the functional
genomics of multicellular organisms.
How does one investigate the function of all 13,500
protein-coding genes in the fly? We use RNA interference (RNAi) to knock down the activity of the target
gene. RNAi is one of the emerging technologies withwhich to investigate gene function in multicellularorganisms. When introduced into the cell, double
stranded RNA (dsRNA) works as a specific mutagen
for each gene. That is, dsRNA recognizes host mRNAand digests it in a sequence-specific manner, andconsequently brings a loss-of-function mutation
phenotype to the host cell. The detailed mechanismof this RNAi phenomenon has not yet been elucidated,
but it works efficiently in many multicellularorganisms, 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 togene function analysis in C. elegans. It was then also
found to be effective in fly genetics. By making a
UAS-tI:ansformation 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.
Weare 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 13,500
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 2003, over 4000 transformation
vectors had been constructed, 3600 of which have beensuccessfully introduced into the fly. Considering thepace of this work, the construction of vectors for over
11,000 genes (accounting for 8oo!o of the entire genome)
should be accomplished during the next fiscal year.
Completion of the transformation will require about
one additional year. Clearly then, we will be able toobtain a powerful tool with which to determine thefunction of the entire fly genome in the near future.
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 theInvertebrate Genetics Laboratory73

Act5C-GAL4 fly. The Act5C-GAL4 induces the
VAS-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. We are currently collaborating with 40
groups. The usefulness of inducible RNAi forinvestigating gene function in Drosophila is being
revealed in many aspects. We published 2 papers using
RNAi flies 2),3) and 1 paper on RNAi mechanism!) in
2003.
This work was supported in part by financial
assistance to Dr. Veda from the Mitsubishi Kagaku
Institute of Life Sciences (MITILS).

EDUCATION

1. R Veda gave a seminar on RNAi mutant fly bankat the ZoeGene Corporation on February, 2003.
2. R Veda gave a lecture on RNAi at the Mitsubishi
Pharma Corporation on April, 2003.
3. R Veda gave a seminar on a database of RNAimutant fly bank at the Mitsubishi Chemical Corporation on October, 2003.
SOCIAL CONTRIBUTIONS AND OTHERS

1. R. Veda was invited to the Mitsubishi Kagaku
Institute of Life Sciences as a Visiting Researcher andlead mutant fly bank project in collaboration with NIG
lab.
2. R Veda joined organizing committee for biennial
meeting of Japan Drosophila Research Conference heldat Tokyo on July.

F-g. Laboratory for Frontier Research
Takaka Isshiki Group

Publications

RESEARCH ACTIVITIESPapers

1. Doi, N., Zenno, S., Veda, R., Ohki-Hamazaki, H.,
Vi-Tei, K and Saigo, K (2003). Requirement of Dicerand elF2C translation initiation factors for shortinterfering-RNA-mediated gene silencing in mammalian cells. Curro BioI. 13, 41-46.
2. Kamiyama, S., Suda, T., Veda, R., Suzuki, M.,
Okubo, R, Kikuchi, N., Chiba, Y, Goto, S., Toyoda, T.,
Saigo, K, Watanabe, M., Narimatsu, H., Jigami, Y andNishihara, S. (2003). Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter.
J. BioI. Chern., 278, 25958-25963.
3. Takemae, H., Veda, R, Ohkubo, R, Nakato, H.,
Izumi, S., Saigo, K and Nishihara, S. (2003). Proteoglycan
UDP-galactose:beta-xylose betal,4 galactosyltransferase
I is essential for viability in Drosophila melanogaster.
J. BioI. Chern., 278, 15571-15578.
Reviews

6.

~JJ¥-~-=f.

1::~?¥ J

J:EB~ (2003)

r:/ 3 ? ~ 3 ? /'\.:r.(7).~mB itf¥(~M~2oo3{P 6 ~ ~1tflJ. r.~mtfjb.

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1064-1071Jt. jt:ftl±llflj. *J?:.

74

Invertebrate Genetics Laboratory. ~*flJ]~m.

(1) Search for the factors involved in temporal
specification within Drosophila neuroblast lineage

Kusano Ayumi and Takako Isshiki
During development, neural progenitors often
generate diverse cell types in an invariant order,
changing their property over time. Yet we know
relatively little about the molecular mechanisms of how
different cell fates are generated in order, especiallyin vertebrate, because of the lack of any particularvertebrate neural progenitor lineage which can be
reproducibly identified in vivo. The Drosophila central
nervous system (CNS) is an excellent model system tostudy temporal specification of cell fates within alineage. All of the progenitors of the Drosophila
embryonic CNS, called neuroblasts, can be easily
identified, and each has a unique and invariant lineage.
Moreover, we have shown that Drosophila neuroblasts
sequentially express the transcription factors
Hunchback, Kriippel, Pdm and Castor over time. The
identification of these transcription factors enables us
to investigate the molecular mechanisms of neuroblast

lineage development in detail.
To identify yet unrevealed factors involved in
temporal specification within neuroblast lineage, weare searching a public dataset of the expression profilesof the Drosophila gene transcripts. So far, we found thatseveral Drosophila homologs of the Cclegans heterochronic
genes are expressed widely in the CNS in temporally
regulated manner. The heterochronic genes control
developmental timing in Cclcgans larvae, and mutations in these genes cause defects in temporal specification within certain cell lineages. Recent studies
have shown that Cclcgans hunchback homolog (hbl-l)
is actually one of the heterochronic genes.
We have been focusing on studying the three
Drosophila homologs of heterochronic genes, lin-28,
lin-4l, and lin-29. lin-28 encodes a putative wellconserved RNA binding protein, although its molecularfunction is largely unknown. We have revealed thatlin-28mRNA and Lin-28 protein are expressed only in
most, if not all, Hunchback positive neural precursors
and neurons, and absent from neuroblasts. Furthermore, misexpression of hunchback leads the ectopic
expression of Lin-28 in hunchback mis-expressed
precursors and neurons. Thus, lin-28 is a very goodcandidate of differentiated cell specific target of
Hunchback. Asymmetric division of neuroblasts
generates cell fate difference between neural precursor
and neuroblast. Weare interested in investigating how
asymmetric division and Hunchback coordinately
regulate Lin-28 expressions in the future.
Lin-41 is also a RNA binding protein, but its
molecular functions are barely understood even in
Cclcgans. Drosophila lin-41 mRNA is expressedin neuroblasts from the early to mid stage ofembryogenesis. Genetic analysis of Drosophila lin-41
function is now underway.

neuroblasts. We are trying to reveal the precise orderof Lin-29, Kriippel, Seven-up, and Castor expressions
in individual neuroblasts during late embryogenesis.
This analysis should provide the basic knowledge forelucidating the molecular mechanisms for the latesteps of lineage development which are not neuroblast
understood well at present.

(2) Neuroblast lineage development at late period inembryogenesis.

Takako Isshiki and Kusano Ayumi
Another heterochronic gene homolog, lin-29,
encodes a Kriippel type zinc finger protein. We havefound that Lin-29 is expressed in the CNS from the lateperiod of embryogenesis. In addition, we have foundthat Kriippel and Seven-up, which are once expressed
and tur:ned off at relatively early time point inneuroblast lineage, are re-expressed afterward inLaboratory for Frontier Research

75

Kenichi Yamamura and Yukiko Yamazaki

G. CENTER FOR GENETICRESOURCE INFORMATION

G-a. Genetic Informatics Laboratory
Yukiko Yamazaki GroupRESEARCH ACTIVITIESSHIGEN project(1) PEC

Takehiro Yamakawa, Junichi Kato and Yukiko
Yamazaki
PEC (Profiling of Escherichia coli Chromosome)database is an integrated public resource databasewhich provides an useful information that could help
to characterize the gene function of Escherichia coli.
A unique characteristic of the PEC is the geneclassification based on essentiality for cell growth. Allof the Escherichia coli genes were classified into threegroups, (1) gene essential for cell growth (essential), (2)those dispensable for cell growth (non-essential), and(3) those unknown to be essential or non-essential(unknown), mainly using information from deletionmutants and journal articles. Another uniquecharacteristic is the comparative gene analysis among
bacterial genomes. PEC provides the list of numbersof homologues genes found in other bacteria genomes
analyzed three times with different e-values, so thatspecies-specific genes and/or orthologous genes areeasily detected. In the database, each gene has link to
the relevant strain stocks publicly available. The
database is based on ObjectStore (Object Oriented
Database Management System) and is released witha comprehensive interface.
PEC database can be accessed through the
SHIGEN server (http://www.shigen.nig.ac.jp/ecoli/pec/).

CARD R-DB is a genetically engineered mice
database of Center for Animal Resources andDevelopment (CARD), Kumamoto University, established as a core center for production, cryopreservationand supply of those mice.
The database system has been implemented witha direct online data submission and validation system.
Each entry belongs to at least one group and one entry
consists of strain-specific and/or gene specificinformation. Although the data type of most items is
free text type, strain and gene names strictly follow theinternationally controlled vocabulary. We havedeveloped data submission and validation tools by
which user can easily submit their data through theinternet and administrator can validate the informationright after the submission and then automatically
update the database on the same web screen. The
database is managed in relational database system(Oracle) with Java servlet applications. The CARD
R-DB is accessible at http://cardb.cc.kumamoto-u.ac.jp/
transgenic/.
(3) JMSR

Takehiro Yamakawa and Yukiko Yamazaki
JMSR (Japan Mouse Strain Resource) databaseprovides a common gateway of mouse genetic
resources from different organizations. Current
database compiles information from four organizations
including Kumamoto University, National Institute of
Genetics, Tokushima University and Japan Animal
Strain Committee. Each data consists of minimal items
such as (1) Strain name, (2) Strain group, (3) Stock type,(4) Genes, (5) Model disease name and/or application
field and (6) URL address from where full informationof the certain strain can be retrieval. We use the free
PostgreSQL relational database in order to make thesystem freely available under an Apache open source
license. JSMR expects new members to join in the near
future. Data distribution in XML format with theassociated document type definition (DTD) file is also
ongoing project. JMSR provides a web access at http://
www.shigen.nig.ac.jp/mouse/jmsr/.

(2) CARD R-DB

(4) Oryzabase

Takehiro Yamakawa, Hideki Kato, Naomi Nakagata,

Takehiro Yamakawa, Shingo Ueno, Nori Kurata,
Atsushi Yoshimura, Hikaru Sato, Yasuo Nagato and76

CENTER FOR GENETIC RESOURCE INFORMATION

Yukiko Yamazaki

nig.ac.j pi riceloryzabase/.

Oryzabase represents an integrated rice science
database, containing.
(1) Rice Genetic Resources, (2) Mutant Phenotype
Collection, (3) Gene Dictionary, (4) Genetic Maps, (5)Developmental stage and gene expression, (6) Basic
Information and (7) Useful site information.
Current database compiles ca. 11000 strains
maintained by 25 stock centers. Oryzabase also provide
the "Core Collection of Wild Rice" to show representative wild rice accessions, which cover 9 different
genomes (AA, BB, CC, BBCC, CCDD, EE, FF, GG, and
HHJJ) and 18 different species. All collections havebeen characterized and presented with image data.
Mutant phenotype collection is currently classified into
5 groups such as Coloration, Heterochrony, Reproductive organ, Seed, and Vegetative organ. Oryzabase also
provide web-based data submission system by whichusers can upload the mutant phenotype image files andsubmit them to the database.
The gene information of Oryzabase is based on
the report of the Committee on Gene symbolization,
Nomenclature and Linkage Groups.
Ontology is one of powerful idea to specify acertain concept and useful especially to compare theconcept common between different fields. Among
many biological ontology projects recently established,
the gen~ ontology (GO) project is most popular andwidely spread because major databases such as
Flybase, SGD, MGI, T AIR, WormBase, PomBase, RGD,
EEl, TIGR have already joined. Recently Gramene
database maintained by Cornell University has joined
the GO (Gene Ontology) project with leading of Rice
databases. Gramene database now proposes three riceontologies, gene ontology (GO), traits ontology (TO)and plant ontology (PO) that defines the plant anatomy
and/ or plant developmental stages. Coordinating theGramene's efforts, all genes of the current dictionary
were assigned to the above ontologies (GO/TO) in the
Oryzabase. Assignment of GO for newly coming genes
obtained from genomic sequences as well as PO
assignment is ongoing project in collaboration with rice
researchers and other rice databases. To make thiscollaboration easier, comprehensive viewer called"GOALL", that will give researchers overview of thecurrent GO and help them understand how ontology
works and how it is useful, was developed.
Oryzabase is available at http://www.shigen.

(5)

KOMUGI

Takehiro Yamakawa, Takashi Endo, Yasunari
Ogihara, Hitoshi Tsujimoto, Taihachi Kawahara,
Tetsuro Sasakuma and Yukiko Yamazaki
KOMUGI was originally established as a wheat
genetic resource database by collecting strain stock
information from several Japanese universities andgrew up to an integrated wheat information database.
Current version of KOMUGI database consists of (1)strain resource information, (2) EST and DNA resource
information, (3) wheat gene catalogue, (4) composite
maps, (5) wheat information service (WIS), and (6) DNA
sequence collection of wheat (Triticeae, Avena, Secale
and..). The gene catalogue is published every 5 years
and only supplemental data is annually published. To
make the up-to-date gene catalogue available throughthe Internet, we ve developed the management systemof the gene catalogue called "MacGene" by which thecatalogue keeper can easily submit and/or update thedata followed by opening the database to the publicthrough the Internet automatically. KOMUGI databaseis available at http://www.shigen.nig.ac.jp/wheat/komugil.
Publicatons
Papers

1. Ogihara, Y., Mochida, K., Nemoto, Y., Murai,
K., Yamazaki, Y., Shin-I, T. and Kohara, Y. (2003).
Correlated clustering and virtual display of gene
expression patterns in the wheat life cycle bylarge-scale statistical analyses of expressed sequencetags. The Plant Journal. 33, 1001-1011.
2. Mochida, K., Yamazaki, Y. and Ogihara, Y. (2003).
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.
Reviews3.

wilffij-El3*c.-r r ~,I! -r7"Jvl!J1o/JT"- -7 /'(- AJ [2f~0) lib

tP.7:f. Vol.204 No.6, 463-467 (2003).

Genetic Informatics Laboratory

77

Databases
4. Oryzabase http://www.shigen.nig.ac.jp/rice/oryzabase
5. KOMUGI http://www.shigen.nig.ac.jp/wheat/komugi/6. PEC http://www.shigen.nig.ac.jp/eco/pec/
7. Barley EST http://www.shigen.nig.ac.jp/barley/
8. C. elegans http://www.shigen.nig.ac.jplc.elegans/
9. WGR http://www.shigen.nig.ac.jp/shigen/wgr
EDUCATION

1. Y. Yamazaki gave a lecture at the Kyoto
University, March, 2003 (in Japanese).
2. Y. Yamazaki was invited to give a seminar on ")I
f~~~.:r--7 A:- ;A.O):j!i}*;f:fUtf' at University, KyotoI~.*l*, September, 2003 (in Japanese).

SOCIAL CONTRIBUTIONS AND OTHERS

1. ~fiIlj~~~B~~liJf~jt~z& ~2. NBRPjJ!-gz& ~ : ~ -\,,::J ~~l-iT . ~'-1 ;;:(, ::J A =¥, ;t;tA=¥, ~~, ES*BIII1!!, :I-;lj]
3. JWflJliz&~: ~~~~liJf~fiJTBRC
4. :f!I!liJfBRC.:r--7A:- ;A.~Wz&~~z&~ :G-b. Genome Biology Laboratory
Yuji Kohara GroupRESEARCH ACTIVITIES(1) NEXTDB: The nematode expression pattern map
database

Tadasu Shin-I, Ikuko Sugiura-Muramatsu, Masumi
Obara, Wakako Shimizu, Yuko Suzuki, Aya
Hamakawa, Jean Thierry-Mieg\ Danielle ThierryMieg\ Sachiko Takahashi, Kyoko Nakata, Hiroko
Uesugi, Takayo Hamanaka, Yasuko Sugiyama andYuji Kohara CNCBI, NIH)
We have developed a WWW-based database"NEXTDB" to integrate all the information of ESTs,
gene expression patterns and gene functions ofC.elegans which are being produced and analyzed inthis laboratory.
In order to integrate NEXTDB, we applied ahierarchical model to arrange all the clones and cDNA

78

Genetic Informatics Laboratorygroups; 1) chromosome, 2) cosmid clone, 3) CDS, 4)
cDNA group, and 5) cDNA clone. The cosmid and CDS
map which connects 1), 2) and 3) are constructed fromthe genome and cosmid data obtained from theSanger Centre and the WormPep database. All theinformation, such as tag sequences, homologies of thecDNA, in situ images, and RNAi phenotypes, arearranged based on the corresponding cDNA clones.
They are integrated with the genome map based on
WWW, and the information of maps and theirrelations are displayed visually by use of JAVA
applets.
The database contained the information of abo1,1t
135,000 cDNA clones, which classified into about 14,000
groups, corresponding to about two-thirds of the total
gene number. About 11,400 groups hit to the predicted
CDSs in the genomic sequences, and about 2,600
groups hit to the non CDS regions. We compared some
ESTs and the corresponding genomic sequences
strictly and found the presence of alternative splicingsand differential termination. We also found that some
CDSs were predicted incorrectly and some genes were
not predicted even there were the corresponding ESTs.
Therefore, close comparison of ESTs and the genomic
sequences is very important to identify genes precisely.
NEXTDB also incorporates in situ images of about
9,200 cDNA groups, RNAi phenotype images of 300
groups. Most of in situ images have been annotatedpreliminarily.
The latest version is available through thefollowing URL. http://nematode.1ab.nig.ac.jp
Many collaborations based on NEXTDB have been
conducted (See Ref.l)(2) Systematic antibody raising and maternal gene
expression analysis in C.elegans

Yumiko Ueta, Masahiro Ito, Hiroko Ochi, Chihiro
Hijikata, Sachiko Takahashi, Keiko Hirono, Yoshinori
UkaF, Midori Shinohara\ Hisayoshi Torii 2, Emi
Shimizu2, Miki Takenaka2, Yoshitaka Iba3, Yoshikazu
Kurosawa3 and Yuji Kohara CInst. for Antibodies,
2MBL, 3Fujita Health Univ.)
The accumulation of mRNA expression patterndata of C.elegans that have been produced in this lab
provided unique sets of genes that are expressed in
specific developmental stages, cell-lineages, tissues andso on. We are particularly interested in earlyembryogenesis. Thus, we focus on a set of genes whosemRNA are maternally supplied and disappear by earlystage of gastrulation or localize to specific cells in early
embryos, since developmentally important genes areenriched in the subset. We found 1107 (about 10.8%)
such genes when the expression analysis of 10290 genes(more than the half of all the genes) was finished. We
are analyzing the subset of genes systematically withrespect to the distribution of the protein products sincetranslation and localization are important for theunderstanding of maternal gene functions. We started
to raise rat or mouse antibodies against the 880
maternal gene products using bacterially expressed
partial proteins in a systematic manner. Until now, we
obtained antisera against 282 gene products, andimmuno-stained embryos and gonads using theantisera. As to a part of the antibodies, phage display
forms were constructed, which easily lead tomonoclonal forms. The spatial immunostaining
patterns were as follows; nucleus (33%), cytoplasm(27%), plasma membrane (17%), nuclear membrane(11 %) and P-granules (18%). The temporal staining
patterns were as follows; the staining signals were firstobserved from the mitotic region in gonad (27%),
oogenesis (11%), and from 2-cell stage (16%). We
confirmed the specificity of some of the antibodies by
testing if the staining signals disappeared in RNAitreated specimen.
Weare trying to extract consensus regulatory
sequence for translation and cellular localization. We
will integrate the results of the immunostaining withthe results of functional analysis by RNAi, ultimately
aiming at understanding of the gene network of early
embryogenesis in C.elegans.
(3) Protein phosphatase 2A (PP2A), encoded by let-92
and paa-l, is required for early cleavage of C. elegans

Ken-ichi Ogura, David L. Baillie\ Sachiko Takahashi
and Yuji Kohara eSimon Fraser Univ.)
The serine/threonine protein phosphatase 2A(PP2A) is implicated in many cellular functions,
including cell division and cell fate determination. Here
we revealed that the catalytic subunit of PP2A
(PP2A-C) of C. elegans is encoded by the gene let-92.
We found that let-92 embryos showed incorrect
positioning of chromosomes, uncoordinated cytokinesis,
and inhibition of chromosome duplication. Furthermore,

let-92 caused the disappearance of P granules in early
em bryos, indicating a role for PP2A in the properorganization of P granules. We found that thephenotype of RNAi of a PR65/regulatory A subunit of
PP2A, named paa-l, was identical to that of let-92
embryos, supporting the idea that PP2A-C and PP2A-A
form the core complex of PP2A. LET-92IPP2A-C wasabundant in the cytoplasm of the gonad and all theblastomeres. (See Ref.3)(4) Systematic analysis of cell-specific enhancers in C.
elegans

Hiroshi Kagoshima, Akiko Kamamoto and Yuji
KoharaThere are so many promoter::GFP reporters
showing cell-specific expression in particular set ofcells in C. elegans. It would be reasonable to expect thatsome of the genes expressed in the same cell might
be controlled by the same mechanism. To examine
the mechanism of the cell-specific transcriptional
regulation, we first collect many GFP reporter
constructs expressed in a certain cell. We next
construct a series of deletion promoter:: GFP reporters,
and checked the expression pattern in the transgenic
worms. Lastly we can obtain numbers of minimized
enhancer for the cell-specific expression. Not all, butsome of them sould share the common sequence in thesize around 5"-'10 bp. Most probably it would be thebinding site for upstream transcription factor.
Weare currently analyzing AFD thermosensory
neuron-specific promoters. Thus far, we could havenarrowed down 7 promoters for the expression in the
AFD neuron; for example, 50 bp sequence of gcy-8(guanylyl cyclase), 120 bp sequence of nhr-38 (nuclear
hormone receptor), etc. Interestingly, in these sequences
we have found numbers of putative binding sites forthe transcription factor OTX1, which has importantroles for head development in fly and vertebrate.
Eventually, it has been revealed that ttx-l (OTX1homolog of C. elegans) regulates both gcy-8 and nhr-38
expression in AFD. We confirmed direct binding abilityof TTX-1 to the 50 bp sequence in the minimized gcy-8
promoter/enhancer by gel shift assay. We have recently
obtained the data indicating that another transcription
factor, LIM homeobox gene ceh-14, might involve in
AFD specific expression.

Genome Biology Laboratory79

(5) Synergistic effect of T-box transcription factor tbx-9
and Hox cofactor Hth/ Meis orthologue unc-62 on C.
elegans embryonic morphogenesis

Yoshiki Andachi
T-box transcription factors play important rolesin embryonic development of metazoans. Aiming at
understanding the regulation of gene expression in C.
elegans embryogenesis, we have been studying two
T-box genes, tbx-9 and tbx-S, that are phylogeneticallymost related to each other among 20 T-box genes inthis organism. tbx-9 is expressed in a subset ofembryonic cells that are precursors of the intestine,
body-wall muscle, and hypodermis. The expression
pattern of tbx-S is markedly similar to that of tbx-9.
Both tbx-9mutants and tbx-Smutants show incomplete
penetrant morphogenetic defects in embryogenesis, but
the malformations of the tbx-9 and tbx-S mutants areobserved in different parts of their bodies. In embryoswith both tbx-9 and tbx-S inactivated, the bodystructure is severely disorganized, more so than thesum of the separate mutant phenotypes. Further
analysis shows that the hypodermis and body-wallmuscle show abnormalities at the site of morphogenetic defects of these mutants. Together, thesedata indicate that tbx-9 and tbx-S do not only contributeindividually to formation of the hypodermis andbody-wall muscle, but also suggests functional
redundancy between tbx-9 and tbx-S in embryonicmorphogenesis.
To find TBX-9 binding proteins, especially those
implicated in gene expression along with TBX-9, we
employed the yeast two-hybrid system. One of thegenes isolated by the screening is unc-62, an orthologueof Drosophila Homothorax (Hth) and vertebrate Meisthat encode cofactors of Hox transcription factors. invitro pulldown assay confirmed weak binding between
TBX-9 and UNC-62. The transcripts of both tbx-9 andunc-62 were detected in several cells of early-stageembryos. unc-62 mutants also show defects in embryonic morphogenesis that result from abnormality inthe hypodermis. unc-62(e644) is a weak allele: not morethan half worms of this mutant show the phenotype
of malformation. In tbx-9(ms31J, unc-62(e644) the
phenotype was observed in most of worms, andenhancement of the phenotype was also found in
tbx-S(RNAiJ, unc-62(e644j. To know that the functionof unc-62 for embryonic morphogenesis is required in80

Genome Biology Laboratory

the cells that also express tbx-9, unc-62 cDNA was
induced by the tbx-9promoter. Partial suppression of
the phenotype of malformation was observed in
tbx-9(ms31); unc-62(e644); Ex[ tbx-9promoter::unc-62].
These results suggest that the T-box factor and theHox cofactor work collaboratively. It is speculated thatthese factors function as an enhanceosome complex
in the same enhancer of their target genes or that the
Hox cofactor functions as a cofactor for the T -box
factor to regulate their target genes. (See Ref.12)(6) Physical Modeling of Cellular Arrangement in Early
Embryos of C. elegans

Atsushi Kajita, Masayuki Yamamura' and Yuji
Kohara CTokyo Institute of Technology)
Cellular arrangements are important for development. In C. elegans, cell-cell interactions are essential
for cell-fate determination in early embryos. The
arrangement of cells is largely restricted by physicalconditions including the force and the direction of cell
division, the existence of a hard egg shell, and so on.
We created a physical model for cellular arrangement
as a first step towards a platform of virtual experiments. Currently this model is based solely on
dynamics of cells and, therefore, includes neither
chemical reaction networks nor gene-regulated
networks within the cells, in order to make the model
as simple as possible. The physical model of anembryo consisted of a constant size eggshell andviscoelastic cells. The cell was represented by a meshsurface of mass particles connected by springs anddampers. Cell division was implemented with theprocesses of elongation of a cell and contraction of acontractile ring.
Using this model we performed computer simulations of wild-type embryos up to the 4-cell stage. We
found a range of parameters at which the simulationsof embryos successfully generated the same cellulararrangement, called diamond-shape, as real embryos.
We also found that the simulations satisfied thecellular arrangements during the process of earlycleavage, by comparing of the characteristic values of
the cellular arrangements with those of real embryosthat were extracted from the 4D microscopic images.
Next, we performed simulations up to the8-cellstage. Using the same parameters as above, thesimulations also generated the same cellular arrange-ments as real embryos at the 8-cell stage. However, astatistical bias was not observed in the simulations,while it was observed in real embryos at the 6-cellstage. The statistical bias is that the left-side granddaughter cells (ABxl) of AB cell are arranged at moreanterior than the right-side ones (ABxr). This meansthat the model lacks something to show the statistical
flexibility at the stage, although it reconstructedsuccessfully the restriction of cell movements by theeggshell. Weare currently improving our model byimplementing other phenomena in early cleavages. (See
Ref.2)

embryo contour. The detection accuracy of embryocontours was 92.1% when applied to 2- to 28-cell-stageembryos. The accuracy of the developmental stage
prediction method was 81.2% for 2-8-cell stage embryos.
We manually judged only the later stage em bryos
because the accuracy for embryos at the later stages
was unsatisfactory due to experimental noise effects.
Finally, our system chose the optimal CG andperformed the superposition and assignment of geneproduct distribution. We established an initial4D gene
expression database with 56 maternal gene products.
(See Ref.H)(7) SPI: A tool for incorporating gene expression data
into a four-dimensional database of C. elegans
embryogenesis(8) Semi-automatic system for creation of cell shapemodel in C. elegans embryogenesis

Hideaki Hiraki and Yuji Kohara
Yohei Minakuchi, Masahiro Ito and Yuji Kohara
A comprehensive gene expression database isessential for computer modeling and simulation of
biological phenomena, including development. Development is a four-dimensional (4D; 3D structure
and time course) phenomenon. We are constructing
a 4D database of gene expression for the early
embryogenesis of the nematode C. elegans. As aframework of the 4D database, we have constructedcomputer graphics (CG), into which will incorporate
the expression data of a number of genes at thesubcellular level. However, the assignment of 3D
distribution of gene products (protein, mRNA) ofembryos at various developmental stages is bothdifficult and tedious. We need to automate this process.
For this purpose, we developed a new system, named
SPI, for superimposing fluorescent confocal microscopic data onto a CG framework.
The scheme of this system comprises thefollowing: (1) acquirement of serial sections (40 slices)of fluorescent confocal images of three colors (DAPI
for nuclei, Cy-3 for the internal marker, which is agermline-specific protein POS-l (Tabara et al, 1999) andCy-5 for the gene product to be examined); (2)identification of several features of the stained
embryos, such as contour, developmental stage andposition of the internal marker; (3) selection of CG
images of the corresponding stage for templatematching; (4) superimposition of serial sections onto
the CG; (5) assignment of the position of superimposed
gene products. The Snakes algorithm identified theCells change their shapes and arrangements
during embryogenesis. 4Dmicroscopy is useful toobserve the process of C. elegans. Time-lapse recordingof multiple optical sections with Nomarski DIC microscope and tools to analyse the results have beendeveloped and used successfully, especially for celllineage analysis because cell nuclei of C. elegans areevident in DIC images. Although cell shape models ofvery early embryo can be reconstructed from DIC
images, delineating cell boundaries become harder as
the cell number increases. Since cell boundaries canbe directly visualized by confocal fluorescent microscopy with vital stain of plasma membranes, we havebeen developing a computer system to create cell shapemodels from a time series of confocal images of plasma
membranes.
In the system, cell boundaries are automaticallycalculated by a seeded region growing algorithm froma 3D image and a set of seed point coordinates. The
algorithm segment the image volume to enclosed
regions one per seed. Here a cell boundary is detected
as ridges of high fluorescent signal between two seedpoints. In many cases, the seed points can be
automatically placed using results of another time
point. But manual editing of the seed sets is necessary
where signal to noise ratio is low. The systemconstructs a pseudo cell lineage from the overlay
relationships among the segmented regions over thetime course. The inconsistency in the pseudo lineage
indicates where editing is required and the editing is
assisted by the graphical user interface.

Genome Biology Laboratory81

We have tested the system on the "dub" data set
kindly provided by Bill Mohler, DW-Madison, that isof N2 embryo in 24-200 cell stages. Changes in physicalparameters of cell shapes and arrangements, such ascell volumes and cell-to-cell contacting areas, could be
estimated from the resulting shape model. We areplanning to apply this system to compare mutant
embryos and the embryos from other species closely
related to C. elegans.
(9) Towards comparative genomics: Genome/EST
sequencing

Kazuko Oishi, Shinobu Haga, Hisayo Nomoto, Masako
Sano, Fumiko Ohta, Sachiko Miura, Tomomi
Morishita, Tomoko Endo, Motoyo Tamiya, Keiko
Nogata, Akiko Hase, Tomoharu Tandoh, Yoshiki
Mochizuki, Hiromitsu Miyauchi, Noriko Hasegawa,
Masumi Mizukoshi, Etsuko Yokoyama, Nanayo
Ishihara, Junko Miyamoto, Shigeko Iiyama, Tadasu
Shin-I, Toshinobu Ebata, Shigeru Saito, Masaki Itoh,
Masayo Nakagawa, Kumiko Kawaguchi, Naoko
Sakamoto, Yasuko Sugiyama and Yuji Kohara

Japanese morning glory (Eiji Nitasaka, Kyushu U.).
High quality sequences were obtained with 70-90% of
the reads (depending on the samples) and depositedin DDBJ. The analyzed cDNA clones are also
distributed from this laboratory. The EST sequencing
was also supported by the National BioResource
Project.
(b) Genome (BAC, fosmid clones): nematodePristionchus pacifficus (Yuji Kohara, NIG and Ralf
Sommer, MPI, Germany), fruit fly Drosophila sechelia
Drosophila simulans, Drosophila parabipectinata,
Drosophila virilis, Drosophila yakuba (Toshiro Aigaki,
Tokyo Metoropolitan D.), Acidian Ciona intestinalis(Nori Satoh, Kyoto D.), medaka fish Oryzias latipes(Hiroshi Hori, Nagoya D. and Hiroyuki Takeda, D.
Tokyo), Chimpanzee (Naruya Saitou, NIG), Gorilla(Naruya Saitou, NIG), liverwort Marchantia polymorpha
L Y-chromosome (Kanji Ohyama, Kyoto D.).
(c) Genome (whole genome): read algae Cyanidioschyzon merolae 16Mb (Tsuneyoshi Kuroiwa, U. Tokyo),
Ascidian Ciona intestinalis 160Mb (Nori Satoh, Kyoto
D.) The whole genome shotgun sequencing of Medaka
fish Oryzias latipes (800Mb) is completing. (See Ref. 4,
5, 6, 7, 8, 9, 10)

As a core facility of the group grant "Genome"(supported by MEXT and consisted of about 300 labs),
we have established a DNA sequencing center.
Currently our capacity is 12 million reads per year.
Thus far, we have performed the followings incollaboration with the members of the group grant (inthe parenthesis) ;(a) EST: the nematode Caenorhabditis elegans andDiploscapter sp. (Yuji Kohara, NIG), Ascidian Ciona
intestinalis (Nori Satoh, Kyoto D.), Ascidian Ciona
savignyo (Nori Satoh, Kyoto U.), Ascidian Halocynthia
roretzi (Kazuhiro Makabe, Kyoto D.), Hagfish
Eptatretus burgeri (Masanori Kasahara, the Graduate
D. for Advanced Studies), Medaka fish Oryzias latipes(Hiroyuki Takeda, U. Tokyo and Akira Kudo, Tokyo
Inst. Tech.), ciclid fish Haplochromis chilotes (Norihiro
Okada, Tokyo Insti. Tech.), loach Misgurnus anguillicaudatus (Yasufumi Emori, U. Tokyo), frog Xenopus
laevies (Makoto Motii, NIBB), slime mold Dictyostelium
discodeum (Yoshimasa Tanaka, Tsukuba U.), red algae
Cyanidioschyzon merolae (Tsuneyoshi Kuroiwa, D.
Tokyo), moss Physcomitrella patens subsp. patens(Mitsuyasu Hasebe, NIBB), barley (Kazuhiro Satoh,
Okayama D.), wheat Triticim aestivum cv. Chinese
Spring (Yasunari Ogiwara, Yokohama City D.),82

Genome Biology Laboratory

Publication
Papers

1. Ichimiya, H., Hino, 0., Kohara, Y. and Ishii, N.

(2003). VBP-l is necessary for morphogenesis in
Caenorhabditis elegans. Oncology Reports 10, 293-295.
2. Kajita, A., Yamamura, M. and Kohara, Y. (2003).
Computer Simulation of the Cellular Arrangement in
Early Cleavage of the Nematode C. elegans. Bioinformatics 19, 704-716.
3. Ogura, K-I., Kishimoto, N., Mitani, S., GengyoAndo, K and Kohara, Y. (2003) Translational controlof maternal glP-l mRNA by POS-l and its interacting
protein SPN-4 in Caenorhabditis elegans. Development
130, 2495-2503.
4. Ohta, N., Matsuzaki, M., Misumi, 0., Miyagishima,S., Nozaki, H., Tanaka, K, Shin-I, T., Kohara, Y. andKuroiwa, T. (2003). Complete sequence and analysisof the plastid genome of the unicellular red alga
Cyanidioschyzon merolae. DNA Research 10, 67-77.
5. Ogihara, Y., Mochida, K, Nemoto, Y., Murai, K,
Yamazaki, Y., Shin-I, T. and Kohara, Y. (2003).
Correlated clustering and virtual display of gene
expression patterns in the wheat life cycle bylarge-scale statistical analyses of expressed sequencetags. The Plant Journal 33, 1001-1011.
6. Nishiyama, T., Fujita, T., Shin-I, T., Seki, M.,
Nishide, H., Uchiyama, I., Kamiya, A., Carninci, P.,
Hayashizaki, H., Shinozaki, K, Kohara, Y. and Hasebe,M. (2003). Comparative genomics of the Physcamitrella
gametophytic transcriptome and Arabidapsis genome:
Implication for the land plant evolution. Proc. Natl.
Acad. Sci., USA 100, 8007-8012.
7. Muramoto, T., Suzuki, K, Shimizu, H., Kohara, Y,
Kohriki, E., Obara, S., Tanaka, Y and Urushihara, H.
(2003). Construction of a gamete-enriched gene pool andRNAi-mediated functional analysis in Dictyastelium
discaideum. Mechanisms of Development 120, 965-975.
8. Satou, Y., Imai, KS., Levine, M., Kohara, Y.,
Rokhsar, D. and Satoh, N. (2003). A genomewide surveyof developmentally relevant genes in Giana intestinalis
I. Genes for bHLH transcription factors. Development
Genes and Evolution 213, 213-221.
9. Wada, S., Tokuoka, M., Shoguchi, E., Kobayashi,
K, Di Gregorio, A., Spagnuolo, A., Branno, M., Kohara,
Y, Rokhsar, D., Levine, M., Saiga, H., Satoh, N., Satou,
Y. (2003). A genomewide survey of developmentally
relevant genes in Giana intestinalis 11. Genes forhomeobox transcription factors. Development Genes
and Evolution 213, 222-234.
10. Yagi, K., Satou, Y., Mazet, F., Shimeld, S.M.,
Degnan, B., Rokhsar, D., Levine, M., Kohara, Y, Satoh,N. (2003). A genomewide survey of developmentally
relevant genes in Giana intestinalis m. Genes for Fox,
ETS, nuclear receptors and NFkB. Development Genes
and Evolution 213, 235-244.
11. Minakuchi, Y., Ito, M. and Kohara, Y (2004). SPI:
A tool for incorporating gene expression data into afour-dimensional database of C. elegans embryogenesis.
Bioinformatics 20, 1097-1109.
12. Andachi, Y (2004). Gaenarhabditis elegans T-box
genes tbx-9 and tbx-8 are required for formation of
hypodermis and body-wall muscle in embryogenesis.
Genes to Cells, 9, 331-344.
Database

13. NEXTDB: URL. http://nematode.lab.nig.ac.jp/

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

2003 Annual Publicity and Intellectual Property Unit
ReportThis unit consisting of Professor Munehiro
Tomikawa, Ms. Nozomi Yokoyama and Ms. Kiyomi
Kuwabara makes various efforts to return the outcomeof 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 has devoted
all her energy to this work from starting up. From
August to November, Ms. Mika Ogawa had assisted,
especially by creating the MTA (Material Transfer
Agreement) form and getting it off the ground. In
December, Ms. Kiyomi Kuwabara took over from her.
The results of our work in 2003 are shown as follows.
(1) . Acquirement ~d utilization of Intellectual Property
RightsFirst of all, we were engaged in promoting a senseof entitlement in the scientists belonging to NIG by
giving them speeches on why entitlement is necessary
to the outcome of basic research. Also, in visiting
laboratories, monitoring the scientific progress andthinking of each scientist, we have explained how
intellectual property rights can be gained from theirresults. We have also established an efficient patenting
procedure. We have prepared an invention exposition
format to minimize the scientists' burden in informing
our unit of their scientific results. Our unit helps thescientists avoid the troublesome task of acquiring ofpatent rights to the practical applications of their

Genome Biology Laboratory83inventions.
In tenns of patent applications, we try to promotenationalization of the patent rights so the NIG scientist
does not need to bear the costs necessary for the
preparation of patent documents. At this time, eight
government patents have been applied for and six
patents are under preparation, of which 12 will beapplied for as government patents (including joint
applications). Also, two international patents have been
applied for. One patent has already come into
marketing as of November 2003.
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, two kinds of agreements, depending on thepositioning of biological resources as intellectual
property, have been created and are in use. Each has
a domestic version and an international version.
Despite not having been authorized yet, a total of 30
agreements (seven are domestic and twenty three areinternational) have been signed.
In the near future, preparation of a third agreement
format will be necessary to correspond to specificmaterials such as yeast, wonns and flies.
In addition, we participated in the ShizuokaTLO-Yaramaika (STLO) campaign, which facilitates
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 poiIJts. 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 10 groups of visitors in 2003:
technical school students, legal apprentices, high school
students, schoolteacher interns, members of the
judiciary, elderly community college students andothers. An overview of this institute, genetic scienceand current laboratory experiments was given to thevisitors.
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 the84

Publicity and Intellectual Property Unit

Japanese Cell Biology Meeting (in Ootsu), the JapaneseSociety for Developmental Biology meeting (in Sapporo),and the Japanese Molecular-Biology meeting (in Kobe).
Thirdly, we presented five specifically excellent
research results in the Hot News column on the website
as an introduction of research results to the public. We
have also had three research results inserted inmain-stream newspapers through cooperation with themedia. Furthennore, we introduced two scientists tocompanies which were interested in their work, andfortunately, this effort led to further business-academia
collaboration.
(3) Social Action Work

The scientists at NIG have played central roles inthe struggle to create barrier-free color vision. Theirwork has been shifted to the Association for
Propagation of Genetic Knowledge in an attempt tobring it to full fruition. So far, however, we have notgotten completely satisfactory results, as not all groups
participating in the project are in full agreement withthe objectives and methodology.
Recently, the participation of public facilities, suchas universities and NIG, in regional revitalization isincreasing. In December I attended a regional
revitalization symposium as a panelist on behalf of
NIG, which was held as part of the events commemorating the 80th anniversary of Numazu's
incorporation as a city. It was quite meaningful to beable to develop relationships with those involved.
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 management of NIG, after turning into
inter-university research institute organization.

Medicine)

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 1• 2 CGene
Network Laboratoy, 2Riken)
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
Jnclined and 1aminated Qptical sheet (HILa) microscopy.
Very little is known about the interactions between
transport molecules and the assembled nuclear porecomplex (NPC) because of its large supramolecular
structure. Obtained single-molecule video images
inside cells are very clear, therefore kinetic parametersof the molecular interactions in cells are obtained
through quantitative analysis. We have discovered how
many molecules interact, how strong the interactions
are, and which molecular interaction is the open
sesame to nuclear import. We have opened up a new
way to quantify molecular dynamics and interactions
at the single-cell level, which is 'open sesame' toward
quantitative molecular and system biology.
(2) Visualization and quantitative analysis of dynamics
of single lipid rafts in vivo

Kumiko Sakata-Sogawa\ Sho Yamasaki 2, Takashi
Saitd' 2 and Makio Tokunaga CResearch Center forAllergy and Immunology, RIKEN, 2Dept of Molecular
Genetics, Chiba University Graduate School of

Lipid rafts are submicroscopic sphingolipid- andcholesterol-rich domains in which many membraneproteins are assembled. This assembly is supposed toenhance the interaction between membrane proteins
and increase of the efficiency of signal transduction
cascades. Although accumulating evidence frommicroscopic studies shows the existence of lipid rafts,
the important features of their dynamics still remain
to be elucidated.
Aiming at visualization of the dynamics of lipid
rafts in vivo, we constructed LAT IGFP, a chimeric
protein of GFP and LAT, which is known as araft-localized protein, and established transgenic miceexpressing LAT/GFP ubiquitously. This chimeric
protein retains the consensus raft localization signal
sequence but not the functional tyrosine residues.
Indeed, LAT/GFP localized mainly in biochemicallyidentified lipid rafts in various types of cells from thetransgenic mice. We prepared bone marrow derived
mast cells from the mice and observed them at 3TC
C using a single-molecule TIRF (Totally internalreflection fluorescence) microscope. The lateral andtransverse motions of the fluorescent clusters 'were
imaged, reflecting the movements of single rafts. Single
molecule images of LAT/GFP were visualized andtheir movements showed the same behavior as single
rafts. The results of the quantitative analysis of the
dynamics of lipid rafts confirmed the existence of lipid
rafts and revealed their quick movement both lateraland transverse.
(3) RNGI05: A Novel Regulatory Protein in Neuronal
RNA Granules Responsible for Stimulation-Dependent
Local Translation

Nobuyuki Shiina, Kazumi Shinkura and Makio Tokunaga
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 RNGl05 (RNA granule protein 105) as acomponent of RNA granules, which play central rolesin the transport of mRNAs to the dendrites. The
RNGl05-localizing RNA granules contain mRNAs,
such as CaM kinase II alpha, CREB and BDNF
mRNAs, whose translational products play key roles

STRUCTURAL BIOLOGY CENTER85

in synaptic plasticity.
In this year, we have found that RNG105 contains
an RNA-binding motif, and shown that RNG105 is aregulatory protein for local translation in dendrites ofhippocampal neurons. RNG 105 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 RNG 105 is
significantly correlated with the induction of local
translation of the mRNAs near the granules. Thesefindings indicate that RNG105 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.
(4) Single Hydrogen Bonds of DNA Base Pairs Detected
in Unzipping Force by Intennolecular Force Microscopy

Michio Hiroshima and Makio Tokunaga
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 feed back 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 toreduce 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 kaT, in other words, about 1.3-fold of the thermalenergy. This is the first report to detect the force of
single hydrogen bonds in biological macromolecules.

86

Biological Macromolecules Laboratory(5) A Novel in vitro Assay System of Nucleocytoplasmic
Transport

Atsuhito Okonogi, Michio Hiroshima, Nobuyuki
Shiina, Naoko Imamotd· 2 and Makio Tokunaga
eGene Network Laboratoy, 2Riken)
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. At first, agarose plates weremodified with glutation. Then glutation-agarose plates
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 byobserving import of fluorescently labeled proteins. This
new cell-free system has marked advantages: 1)
Solutions in both sides, pseudo-cytoplasmic and pseudo
nucleoplasmic sides, can be replaced independently. 2) The
shape of the nuclear envelope can be changed as one
likes, for example, a vertical plane and a horizontal
plane. 3) The system does' nt contain no organelle orcellular structures except the nuclear envelope. Using
the in vitro system together with single molecule
techniques or fluorescence resonance energy transfer
should provide an innovative and powerful tool toinvestigate molecular mechanisms of transport.
(6) Molecular Imaging of translation initiation factors
in neuronal dendrites

Hiraku Miyagi, Nobuyuki Shiina and Makio Tokunaga
Local protein synthesis in neuronal dendrites isgradually revealed to be required for synaptic
plasticity. In order to investigate when and where thetranslation is initiated locally in the dendrites, we
visualized eukaryotic translation initiation factors (elF)
4E and elF4G in dendrites of rat hippocampal neurons.
Binding of elF4E and elF4G is known to trigger
translation initiation. Immunostaning of rat hippocampal primary neurons showed that most of eIF4E andelF4G were localized in distinct granular structures,
whereas some of them were colocalized in somegranular structures in the dendrites. Immunostaningof rat hippocampal slices showed essentially the same

results as in the case of the primary cultures.
Stimulation by brain-derived neurotrophic factor(BDNF) increased colocalization of eIF4E and eIF4G
in rat hippocampal slices and hippocampal primaryneurons. Immunostaning of rat hippocampal slices for
eIF4G and PSD-95, a marker protein for postsynapses,
showed that colocalization of eIF4G and PSD-95 wasincreased by BDNF stimulation. These results indicated that translation in the neuronal dendrites isinhibited by separating spatially eIF4E and eIF4G, andtranslation is induced by BDNF stimulation at
postsynaptic areas.
eIF4E and eIF4G were fused to GFP, CFP and YFPproteins to be used for real time imaging in living cells.
These fusion proteins were bound to endogenous
eIF4G and eIF4E respectively in CHO cells, indicatingthat these fusion proteins are functional in the cells.
In cultured hippocampal neurons, eIF4E-CFP and
YFP-eIF4G were observed as distinct granularstructures under the thin-layer illumination fluorescence microscopy. We are trying to observe changes
in dynamics of the fusion proteins induced by BDNF
stimulation.

SOCIAL CONTRIBUTIONS AND OTHERS
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3. Dr. M. Tokunaga organized a symposium at the
16th Symposium on Bio-Analytical Sciences 2003,
Fujiyoshida, August, 2003.

H-b. Molecular Biomechanism Laboratory
Nobuo Shimamoto Group

Publications

RESEARCH ACTIVITIESPapers(1) The branched mechanism of transcription initiationin E. coli

1. Hirakawa, Y., Suzutoh, M., Ohnishi, H., Shingaki,T., Erying, E.M., Tokunaga, M. and Masujima, T.
(2003). Observation and analysis of single DNA
nano-kinetics by pin-fiber video scope. Analyst 128,676-680.
EDUCATION~~~/~~~OO~~~~~~~T-

1. Dr. M. Tokunaga gave a lecture at the Universityof Tokyo, Faculty of Engineering, May, 2003 (inJapanese).
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Motoki Susa\ Shouji Yagi) and Nobuo Shimamotd
In elucidating of the mechanism of a biologicalprocess, one tends to assume a simple sequence ofessential steps at the cost of ignoring steps withunknown roles. The mechanism of transcription
initiation in prokaryotes provides an example. For
several decades, the mechanism has been assumed to
be a sequence 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 r~action), andescape of RNA polymerase from the promoter
associated with the progress of RNA elongation(promoter clearance). This ignores the process called"abortive initiation", which is an iterative synthesis andrelease of oligo-RNA molecules. This process has been
observed in vitro with all prokaryotic and eukaryotic
RNA polymerases so far isolated, but its role as well

Biological Macromolecules Laboratory87

as its occurrence in vivo has been unknown. The
products of abortive initiation, abortive transcripts, aretypically 2 to 15 nucleotides in length. On theassumption that these short 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 ;L PNAL 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
factprs 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, j greA j 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+greB+
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

88

Molecular Biomechanism Laboratorytranscription system composed of purified components.
The results obtained prove that the branched initiation
pathway exists in vivo and is utilized in regulation oftranscription initiation from some promoters, throughmodulation of the fraction of polymerase-promoter
complexes entering each branch of the pathway.
In this year, we examined various physiologicalconditions that affect on thew levels of the Gre factors.
The determination of the level of GreB was difficult
because of the excess amount of GreA that has asimilar molecular weight and cross-react with antiGreB. The level of GreA remained the same throughthe growth phase. It did not respond much to therichness of the culture media. However, it decreased
into half in aerobic conditions, indicating that some
genes are regulated by the branched pathway mechanism.
We have tested the possibility of formation of dead-end
complex in vivo. In the cell homogenate of the jgreA
jgreB strain, but not its parental strain with the gregenes, we have found a significant fraction of RNApolymerase remain bounded to chromosomal DNA
after washing with high salt buffer. Since this salt
resistence is the feature of dead-end complex, thisevidence is consistent with the existence of the
branched pathway mechanism in vivo. Because of thisfraction, the yield of RNA polymerase from of thej greA j greB strain is small. We have solved thisproblem by combining differential precipitation of
DNA-RNA polymerase complex with sonication,
establishing the purification procedure of RNApolymerase free from the Gre factors.
(2) Structural dynamics of (J" 70 and its extra roles in vivo

Shoji Yagi, Usha Padmanabhan\ Hiroki Nagai\
Taciana Kasciukovic 2, Richard S. Hayward 2, YumikoSato 1 and Nobuo Shimamoto l CNat. Inst. of Genet.!
Grad. School of Adv. Stud., 22Inst Of Cell and Malec.
BioI., Edinburgh Univ., Scotland)
Several years ago, we found that 0 70, the major 0
factors of E. coli, forms aggregate in vivo and in vitro
at a high temperature within physiological condition.
The oligomeric 0 70 was confirmed to have little
transcriptional activity and the oligomerization in vitro
showed a sharp temperature dependence. We haveconstructed a strain with a disrupted rpoD (0 70 ), andplasmid born 0 70 supports its growth. We observed apositive correlation between the intracellular concen-

tration of (J70 and upper limit of growing temperature,suggesting a possibility that (J70 is a molecular t~er­
mometer controlling the upper limit of the growthtemperature.
The major (J factors of proteobacteria mostly havea big spacer region between the conserved regions 1
and 2 which is not conserved in eubacteria. In E. coli
(J70 this region includes extensive acidic patches but
its role was not known. The rpoD-disrupted strain wasused for testing the roles of this region. The mutant(J70 lacking the spacer region of aa130-374 (IlSR) complemented the disruption at 25"-'30 DC, but not at 35°C
or above, proving that the region is not essential for
growth at low temperature. At all tested temperature
this protein predominantly exists as aggregate whichis in equilibrium with a small fraction of monomer.
Therefore, the role of the spacer region is the maintenance of active monomeric form.
The subunit (J70 of RNA polymerase has been
evidenced in vitro to be dissociated during transcription initiation. However, this conclusion was challenged
by two recent studies: one using FRET assay, claimingthat (J 70 is never dissociated, and the other biochemical
study shows that (J 70 gets dissociated at log phase but
only partially at stationary phase. To examine what
actually occurs in cells, we are developing an in vivo
assay using fluorescent anisotropy that is more reliable
than FRET. We have constructed a strain lacking thegene of (J 70 on chromosome but the essential factor isexpressed from the gene on a plasmid as a form fusedwith GFP. To eliminate the possibility of generatingsemi-intact (J 70 by cleavage in cells, GFP is buried
among the domains of (J 70.
(3) Applicability of thermodynamics to equilibria inbiologyNobuo Shimamoto and Jyun-ichi Tomizawa
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 inthermodynamics. 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.
Publications
Papers

1. Shimamoto, N. (am). Movements of RNA polymerase
along DNA. In Methods Enzymology, Vo1.371, RNApolymerases and associated facrors, Part C, pp. 50-70.
Elsevier Science, San Diego.
Reviews

2. ~~*f$~ft(2003). !J *~~. RNA~!J j. '.7-~'.

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EDUCATION~~~/*~m~~O)~~~~~T-

1. Shimamoto, N., Physiological Significance ofprotein sliding along DNA: A new general regulatory
mechanism suggested by two bacterial repressors,
June 27, Department of Biochemistry, University of
Wisconsin-Madison, Madison, Wisconsin, USA.
2. ~,~*f$~t. -j-//<;-1;;t77/t1~-c:OJT7-::\'-Jv7­?,/,?,·'-F. -j-//<;-1;;t7;:t-'.7.A. 7JH68. 1f!f11iEE
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3. Shimamoto, N., Physiological Significance ofprotein sliding along DNA: A new general regulatory
mechanism suggested by two bacterial repressors,
September 10, National Yang-Ming University, Tiwan.
4. Shimamoto, N., Nanobiomachines as the target of
nanobiology and nano-biotechnology of Japan: an
example of transcription complex, September 12,
National Health Research Institutes, Taiwan.

*

Molecular Biomechanism Laboratory89

5. ~*i$1t,

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6. Shimamoto, N. A revision of the mechanism oftranscription initiation: The branched pathway
mechanism and a molecular memory in transcription
complex of E. coli. December 9, Ecole Normale Superieur de Cachan, Paris, France.
7. Shimamoto, N. Nanobiology of DNA-protein
interaction: Protein sliding along DNA and its
significance in biology and physics, December 12,
Padova University, Padova, Italy.
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90

Molecular Biomechanism Laboratory

H-c. Multicellular Organization Laboratory
Isao Katsura GroupRESEARCH ACTIVITIES(1) Analysis of synthetic dauer-constitutive mutants inthe nematode Caenorhabditis elegans
Tomoko Yabe, Kiyotaka Ohkura, Takeshi Ishihara
and Isao Katsura
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 formation
is much less time-consuming than behavioral assays
such as chemotaxis assays, we are analyzing thepathway of sensory signals in the amphid neuralcircuit by detecting dauer formation as the output. We
found that mutations in more than 50 known genes
show synthetic dauer-constitutive (Sdf-c) 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 based on the pathway of sensory signals.
Namely, the signals are transmitted through parallel
routes, and therefore two mutations are required toblock them. We are determining the combinations ofmutations for the Sdf-c phenotype and the pattern ofsuppression of the Sdf-c phenotype by various
suppressor mutations. In this way we hope to elucidate
the pathway 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 Sdf-c phenotype

in the unc-31(e169j 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. Some of thesegenes, sdf-13, sdj-9, and sdj-14, have been cloned andcharacterized at the molecular level, as shown below.
sdf-13 gene encodes a homologue of the mammalian Tbx2 and Tbx3 transcription factors. The two
sdf13 mutants that we isolated are missense hypomorphs,

and show abnormality in adaptation to odorants
sensed by Awe sensory neurons. This gene isexpressed in AWB, Awe, AS] and many pharyngeal
neurons, but expression in Awe neurons is sufficientfor normal adaptation. The Awe neurons in the
mutants retain their characteristic morphology andmany marker gene expressions, suggesting that the
mutants are abnormal in neural functions rather thanneuronal differentiation. The results imply that the
mammalian Tbx2 and Tbx3 genes, which play important roles in embryonic development, may also control
functions like neural plasticity in differentiatedneurons.
sdf9 gene encodes a tyrosine phosphatase-likemolecule. When cholesterol is removed from theculture medium, the mutants form dauer-like larvaethat resemble those of daf9 and daf12 dauer-constitutive
mutants. Epistasis analyses revealed that sdf-9 geneacts in the daf9/ daf-12 steroid hormone pathway.
SDF-9 is expressed in two head cells in which DAF-9
is expressed. By genetic mosaic experiments, we
identified these cells as XXXL!R cells, which were
known as embryonic hypodermal cells and whosefunction at later stages was unknown. Killing of XXXL/
R cells in the wild type first-stage larva induces
formation of the dauer-like larva. Thus, XXXL!R cells
seem to playa key role in the metabolism or functionof a steroid hormone that acts in dauer regulation.
sdf14 gene is identical to mrp-l gene, which wasformerly identified by its homology to multidrug
resistance protein genes in other organisms. Thus, we
discovered a function of mrp-l concerning dauer larva
formation. Three different SDF-14 proteins are produced
from sdf14 gene by alternative splicing, and two of thecDNAs, but not the third one, rescue the Sdf phenotypeof the mutants when expressed under the control ofthe sdf14 promoter. An sdf14::GFP fusion gene thatcan rescue the sdf14 mutant phenotype is expressedin neurons, pharyngeal muscles and intestinal cells.
Expression of sdf14 gene in each of the three tissues
using extrinsic promoters results in partial suppressionof the Sdf phenotype, while expression in two of them
results in more efficient suppression. It seems that
expression in each of the three tissues contributes tothe wild type phenotype.
In the year 2003, we found that SDF-14 protein canbe replaced by human MRP1 protein in C. elegans
without loosing the function. The synthetic dauerconstitutive phenotype of sdf14 mutants was rescuedby the wild-type human MRP1 cDNA (but not theLOdm missense mutant cDNA of MRP1) driven by thesdf14 promoter. This rescue could be reversed by theaddition of drugs that inhibit the transport activity ofhuman MRPl. We are also investigating in which
pathway sdf14 gene works. The daf2(e1370) mutant
produces no dauer larvae at 20°C, but the daf2;sdf14
double mutant produces many dauer larvae at thesame temperature. We are testing various dauerdefective mutations for the ability to suppress the latterphenotype. Furthermore, we found that sdf-14 mutants
are not hypersensitive to dauer pheromone. Wearecontinuing such experiments to locate sdf-14 gene indauer regulatory pathways.
(2) Molecular genetic studies on sensory integration andbehavioral plasticity in C. elegans

Takeshi Ishihara, Yuichi Iino\ Akiko Mohd, Ikue
Mori 2, Keiko Gengyo-Ando 3, Shohei Mitani 3 and Isao
Katsura CMolecular Genetics Laboratory, Universityof Tokyo, 2Division of Biological Science, Nagoya
University, anepartment 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 the learning andselection (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 forMulticellular Organization Laboratory91this 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 sehsory 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-I 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-I 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-cellautonomously in the mature nervous system.
The hen-] mutants also show abnormality inlearning by paired presentation of starvation and NaCI
(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 NaC!. 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

92

Multicellular Organization Laboratory

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 that
HEN-I 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-I specifically binds a subpopulation of the
primary culture cells in C. elegans, suggesting thatreceptors for HEN-1 exist in these cells.
In 2003, Jeb protein in Drosophila was reported toregulate development of visceral mesodermal cells
through the tyrosine kinase receptor, DAIk. Since the
LDLa domain in Jeb is similar to that of HEN-I, we
started to analyze the scd-2 gene of C.elegans, which
encodes a receptor tyrosine kinase similar to DAIk.
Expression analyses by using the scd-2promoter GFP
fusion gene suggested that SCD-2 is expressed inseveral sensory neurons and interneurons. In addition,an scd-2 mutant shows behavioral defects same as
hen-I mutants in paradigms for sensory integrationand learning. These results suggest that SCD-2 is acandidate for the HEN-I receptor.
(3) C. elegans mutants in the "associative" learning withodorants and foodIchiro Torayama, 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 tofood 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 ofsuch "associative" learning in C. elegans, we are
isolating and characterizing mutants that show
abnormality in the learning with butanone and food/stravation. It is known that butanone attracts wild typeanimals 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 decreased theefficiency of chemotaxis after conditioning with foodand butanone, while others are attracted efficiently bybutanone only after conditioning with food andbutanone. Of those mutants, ut305 and ut306, which
belong to the former category, have been studied indetail, because they showed strongest abnormality.
Interestingly, ut305, but not ut306, showed abnormality
in adaptation to isoamyl alcohol and benzaldehyde,
which are sensed by the same type of sensory neurons(AWC) as butanone. Mapping with SNP markers
revealed that the ut305 mutation is located in theright-hand part of the linkage group X, while ut306
near the center of the linkage group V.
In the year 2003, we found that ut305, but not
ut306, shows abnormality in chemotaxis to butanone
at low concentration, although ut305 shows normal
chemotaxis at the concentration used in the learning
assay. It is known that serotonin has similar effects
as food in various behaviors in C. elegans. The ut305
mutant showed normal responses in the inhibition of
locomotion by food (dopamine-dependent), and in
further inhibition of locomotion on food by pre-starvation(serotonin-dependent). Also in the learning assay withbutanone and food, serotonin had an effect similar tofood in. both wild type and ut305 animals. However,a deletion mutant in tph-l (tryptophan hydroxylase)
gene, in which serotonin is not synthesized, wasessentially normal in the learning, showing that thelearning of wild type animals with butanone and foodoccurs via serotonin-dependent and independent
pathways.
We cloned ut305 gene, which encoded a novel
protein containing predicted transmembrane domains
and showing limited homology to the Drosophila Raw
protein. A functional GFP fusion gene was expressedin AlA interneurons as well as many pharyngeal
neurons. Laser killing of AlA neurons in wild typeanimals resulted in learning abnormality similar to but
somewhat weaker than that of the ut305 mutant. We
are now carrying out rescue experiments using

extrinsic promoters to learn the cells in which theexpression of the wild type ut305 gene is sufficient fornormal learning.
(4) Behavioral analysis with "uncomfortable" stimuliof the nematode Caenorhabditis elegans

Kotaro Kimura and Isao Katsura
C. elegans as well as many other animals escapefrom uncomfortable stimuli, such as repulsive tastes
and odorants, noxious heat, acid and mechanicalstimuli, to seek for better conditions to grow andreproduce. However, what if the animals cannot escapefrom those stimuli? Long or repetitive exposure(s) torepulsive stimulus ("fear conditioning" for example)
makes vertebrates and invertebrates in some cases togive up escaping from the stimulus, and causes strange
behavior and/or unbalanced homeostasis. What about
C. elegans?
We hypothesized that the mechanism to causesuch behavior and/or status with the repulsive
stimulus might be evolutionarily conserved, and thatgenetic analysis of C. elegans could reveal the genes
involved in the mechanism.
C. elegans' avoidance behavior is reported to be
induced by several volatile or soluble compounds, or
nociceptive stimuli, which are sensed by 3 different
classes of sensory neurons. However, they are notstudied in detail enough compared to attractive stimuli,
and not at all in terms of long exposure. To understand
how long exposure to repulsive stimulus affects C.
elegans' behavior, we are currently optimizing experimental conditions to analyze avoidance behavior
induced by l-octanol (sensed via ADL sensory neuron),
2-nonanone (AWB sensory neuron), or high osmolarity(ASH polymodal sensory neuron). We will firstexamine whether the avoidance behavior is alteredwith long exposure before the assay, and then examine
effects on other behaviors.
(4) Fluoride-resistant mutants of the nematode Caenorhabditis elegans

Akane Oishi, Takeshi Ishihara and Isao Katsura
Fluoride-resistant lj7r)mutations of C. elegans arerecessive and grouped into two categories: class 1
mutations, which map in flr-l, flr-3 and flr-4, and classMulticellular Organization Laboratory93

2 mutations, which map in flr-2 and flr-S (Katsura, I.
et al.: Genetics 136, 145-154, 1994). Class 1 flrmutations
show diverse phenotypes: slow growth, short defeca. tion cycle periods, frequent skip of the expulsion stepof defecation, and synthetic abnormality in dauer
formation, besides strong resistance to fluoride ion.
The flr-l gene encodes an ion channel belonging to the
DEG/ENaC (c. elegans degenerins and mammalianamiloride-sensitive epithelial sodium channels) superfamily,
while flr-4 and flr-3 code for a novel Ser/Thr protein
kinase and a kinase-like molecule, respectively, bothhaving a hydrophobic domain on the carboxyl-terminal
side. A functional flr-l::GFP fusion gene is expressed
only in the intestinal cells from the comma stage ofembryos to the adult stage, while flr-4::GFP isexpressed in the intestinal cells from the 1.5-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. We therefore think that class 1 flr genes
constitute a regulatory system that acts in the intestineand that controls many food·related functions. Class
2 fir mutations, which confer weak resistance tofluoride ion, suppress the slow growth and dauer
formation abnormality but not the defecation abnormality and strong fluoride-resistance of class 1 fir
mutations. Hence, it seems that the regulatory pathway
bifurcates after class 1 genes, and that class 2 genes
control only one of the two branches. Of the class 2
genes, 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 and the tail as well as many pharyngeal
neurons. On the basis of these results, we have madea model in which a signal controlled by class 1 genes
is transmitted from the intestine and represses theaction of class 2 gene products in the head nervous
system.
In the year 2003, we obtained the following results.
1) Both flr-2 and flr-S mutants had shorter average
longevity than wild type animals by about one week.
Since this abnormality in flr-2mutants is rescued by
the wild typeflr-2gene, we think the same abnormality
in the flr-S mutant may be used for the cloning of flr-S
gene.
2) Although a functional flr-2::GFP fusion gene wasexpressed in neurons, the expression of the wild typeflr-2 gene with myo-3 or ges-l promoters, which drive

94

Multicellular Organization Laboratory

expression in the body wall muscle and intestine,
respectively, was sufficient for the rescue of the mostobvious flr-2phenotype, namely, suppression of slow
growth caused by flr-l mutations. In contrast, rescue
was not observed with H2O, tph-l, and che-2promoters,
which drive expression in all neurons, serotonergic
neurons and ciliated sensory neurons, respectively.
3) To identify proteins that interact with FLR-2, we
produced FLR-2::alkaline phosphatase fusion protein
molecules in HEK293 cells, and added them to primarycell cultures of C. elegans. The results showed that thefusion protein molecules bound specifically to theintracellular compartment of a limited number of theculture cells as detected by alkaline phosphataseactivity. We then screened for C. elegans cDNA clones
whose expression in COS cells enables the cells to bindto FLR-2::alkaline phosphatase. By screening 264 poolsof eDNA, where one pool consists of about lOOO clones,
we obtained a single positive clone encoding a secretory
protein, which we are now characterizing.
4) Since only a few alleles of class 2flrmutants had
been isolated, we carried out a new screen for the class
2 mutants using the phenotype of suppressing serotoninhypersensitivity of the flr-l(utll) mutant. Eight independent mutants were obtained by screening about
12,800 genomes. All the mutations also suppressed theslow growth of flr-l(utll) mutant. By complementation
tests, we found that four of them seem to map in flr-2,
whereas the other four probably map in two new genes.
Publications
Papers

1. Ohkura, K., Suzuki, N., Ishihara, T. and Katsura,
I. (2003). SDF-9, a protein tyrosine phosphatase-likemolecule, regulates the L3/dauer developmental
decision through hormonal signaling in C. elegans.
Development 130, 3237-3248.
2. Miyahara, K., Suzuki, N., Ishihara, T., Tsuchiya,
E. and Katsura, I. (2003). TBX2/TBX3 transcriptional
factor homologue controls olfactory adaptation in
Caenorhabditis elegans. 1. Neurobiol. Published Online:
3 Nov 2003.
Reviews

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Yasuo Shirakihara GroupWl~l~

RESEARCH ACTIVITIESEDUCATION(1) Crystallographic Study of F1-ATPase
1. Dr. T. Ishihara was invited to give a seminar at

the Center for Developmental Biology (Riken), February
2003 (in Japanese).
2. Dr. T. Ishihara gave a lecture at Kobe University
Graduate School of Medicine, February 2003 (inJapanese).
3. Dr. I. Katsura gave a lecture at Kyoto University,
Graduate School of Science, April 2003 (in Japanese).
4. Dr. I. Katsura gave a lecture at the University ofTokyo, Graduate School of Arts and Sciences, July 2003
(in Japanese).
5. Dr. I. Katsura gave a lecture at Gakushuin
University, Graduate School of Science, July 2003 (inJapanese).
6. Dr. I. Katsura was invited to give a seminar on"Molecular biological analysis on the behavior of thenematode C. elegans" at Gakushuin University,
Graduate School of Science, July 2003 (in Japanese).
7. Dr. T. Ishihara was invited to give a seminar at
Tokyo Metropolitan Institute of Neuroscience,
September 2003 (in Japanese).
8. Dr. I. Katsura gave a lecture at Kyoto University,
Faculty of Medicine, November 2003 (in Japanese).
SOCIAL CONTRIBUTIONS AND OTHERS

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Newsletter,*"(flHiJf.*fJE~:R~RNA1#~~1l~ 0)~~rI'f9'Y "?-~)Vol. 2, No.1, pp72-75, August, 2003.
3. t£ ~ r~.!R "'t'f,j1Q jt~-=f ~ fTJilJJ ~liJf*:; '" -7 Iv~i'lliJfge*~Im*"F No.4, pp6-9, 2003tk.
4. Dr. I. Katsura served as one of the associate editorsof the journal "Genes to Cells.".

Yasuo Shirakihara and Aya Shiratori
FI-ATPase is the catalytic sector of ATP synthasethat is responsible for ATP production in living cells.
ATP synthase is buried in the energy conversion
membrane, such as that of mitochondria, using Fo
portion (subunit composition of ab2c8-12) and has FI
as a soluble and protruding portion. FI has molecularmass of 380,000 dalton and a subunit composition of
CX3/33Y(JE . The unique rotational catalysis mechanismof FI includes rotation of the rod-like y subunit, whichis thought to control the conformations of the threecatalytic /3 subunits in a cyclic manner by its rotation.
We have moved to the structural study of the a3/33YE
sub-assembly of thermophilic FI, after solution of thestructure of the m/33 sub-assembly and committed
attempts to crystallize the m/33Y -sub-assembly.
Employing the a3/33YE sub-assembly that showcatalytic properties very similar to those of FI, we aim
to detect structural changes caused by different
nucleotide occupancy, which should provide withstructural basis for understanding the rotational
catalysis mechanism. Last year, we solved the structureof the m/33Y Esub-assembly with Mg-free ADP boundto only one /3-subunit and examined its biologicalsignificance4)•
The difficulty in interpreting the above structure
resided in that the molecule has no bound Mg, whichis essential for catalysis, and that little biochemical
evidence on the Mg-free state was available. We
therefore moved to the structure analysis of the
Mg-bound form. Previous difficulties in dealing with
Mg-bound form have gone as described last year, anda data set from a MgADP bound form was collected
for the first time, to a resolution of about 5.5 A at
Spring8 BL44 (Protein Institute, Osaka Univ.). The
MgADP bound form was crystallized in the presenceof 25mM Mg and 0.5mM ADP. The structure wassolved by molecular replacement. Among the various
available models (with zero, one, two or three nucleotide
bound /3 -subunits), one nucleotide bound form (i.e. theMulticellular Organization Laboratory95

PS3 CX3/33YE sub-assembly with Mg-free ADP bound to
only one /3-subunit) fitted best with the lowest R-factor
that is 5% lower than those for other models. The map,using the model correctly positioned in the crystal unit
cell, was carefully examined paying attention to apossible problem of model bias. The resultant map
was closely similar to that for the adopted model(single Mg free ADP bound form), and therefore
suggests that Mg has no significant effect on the
structure as far as the present low resolution structuralfeatures are concerned. However, at the same time, it
raises an open question why there is only one
nucleotide-bound /3 -subunit in the structure where atleast two nucleotide-bound /3 -subunits are normally
expected.
These structure studies were done in collaboration
with Toshiharu Suzuki and Masasuke Yoshida, atResearch Laboratory of Resource Utilization, Tokyo
Institute of technology.
(2) Crystallization of ATP synthaseYasuo Shirakihara and Aya Shiratori
ATP synthase, the mother complex of Fl, is stilla challenging target for crystallization, in view of only
a few solved structures of the membrane proteins so
far. We restarted the crystallization study by examining the purification procedure for the ATP synthase
extracted with dodecylmaltoside from the PS3 membrane,
and found that an array of Q-sepharose high performance column, Superdex prep grade 200 column and thesecond Q-sepharose high performance column yieldeda preparation of crystallization grade. The preparation
gave small crystals in screening experiments using
polyethylene glycol as a precipitant and MgADP as anadditive. Further refinement of the crystallization
conditions is in progress. Also a number of detergents
have been examined for good extraction capability inthe preparation procedure, and in addition to dodecylmaltoside described above, both decylglucoside anddedcylmaltoside have been identified as promising
candidates.
The crystallization study was done in collaboration
with Satoshi Murakami at Institute of scientific andindustrial research, Osaka University.

96

Biomolecular Structure Laboratory(3) Comprehensive Crystallographic Study of Transcription factors and Genome-partitioning Factors from E.coli

Yasuo Shirakihara, Aya Shiratori, Hisako Inoue andMegumi Seki
More than 160 transcription factors have been
identified in E.coli, and they control transcription of
their target gene(s) in either positive or negative
manners, by binding to both their specific DNA
sequence and the transcription apparatus. Sixty-fivesuch transcription factors have been purified in one
step Ni-NTA column chromatography using carefully
constructed over-expression systems in Ishihama
laboratory. In Niki laboratory, a number of novel
proteins, that are judged to play roles in the genome
and plasmid partitioning from their localization
patterns, have been prepared using similarly constructed over-expression systems. Setting thesetranscription and genome partitioning factors astargets for structural study, we have started comprehensive structure determination in the Protein 3000
project (the sub-field of 'transcription and translation').
Last year, which was the first year of the project, we
examined 34 proteins for their crystals but identified
only two proteins that gave crystals although the
crystals were not satisfactory.
Following the lessons from the unsatisfactory
crystallization success rate in the last year, we initiallytried to crystallize transcription factors in a form ofcomplex with their cognate DNA. We examined six
transcription factors (KdpE, Mlc, IlvY, RcsB, ModE,
and SdiA) out of fifteen factors with known binding
DNA sequence. Four transcription factors (KdpE, Mlc,
IlvY and RcsB) crystallized in the form of suchcomplex, thus with much higher success rate than thatfor protein alone crystallization. However, the best co
crystal that was obtained from KdpE showed diffraction patterns of just 5 A resolution at a Spring 8 beamline BL38Bl. Partly due to high cost for large amountof DNA oligomer, we have suspended the co crystal
approach.
We continued protein-alone crystallization conditions search against both the old targets provided lastyear and new targets (this year) comprising fifteentranscription factors (ModE, Hns, YedW, b238l, ArsR,
CadC, CheY, CrI, DicA, PutA, Usg, YfgB, YafC, YagI,
HupA, YigT, YebG) and seven partioning factors(MukF, PutA, Malk, FadD, 269#3). Among the newtargets, only CadC and HupA gave microcrystalline
aggregates. Among the old targets, Mlc were retried
after finding that the provided turbid preparation,
which had looked unsuitable to a crystallization
experiment, gave fairly high protein concentration in
the supernatant after centrifuge. Mlc gave tiny crystals
from Ammonium sulfate solution in the screeningstage, and larger but aggregate-looking crystals in therefinement stage. The aggregating tendency was
reduced by changing pH (pH7 -) pH5.8) or temperature (25° C -) 25° ).
We did some biochemistry on the partioning
factors. In examination with a gel filtration column,
MukF, MalK and RecA appeared exclusively in thevoid, which may be consistent with failure of
crystallization of these proteins due to preformed
aggregates. We purified further the provided FadD
preparation, which is composed of the intact protein
and the two fragments; all three species bound to a NiNTA resin. Hydrophobic, ion exchange and hydroxyapatitecolumns worked in separation of those, but the
separated components were not soluble whichhindered the crystallization study.
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).

of which resin was chosen empirically depending upon
the protein. PhzR was an exception, ands was purified
with Q-sepharose high performance, Butyl-toyopearl
and hydroxyapatite columns, because the protein did
not bind to aNi-NTA column. A gel filtration analysis
and the subsequent x-ray crYstal analysis showed that
the PhzR molecules form a tetramer, suggesting thatthe His-tag attached C-terminus of the molecule may
be buried in the tetramer thus explaining no attachment of PhzR to Ni-NT A.
The preparations were then subjected to
crystallization study. All proteins gave signs of crystals
in the screening stage, however, the diffraction grade
crystals were obtained only from PhzR so far. Phz
crystals were obtained from polyethylene glycol at pH
4.7, and with a typical size of 0.2 x 0.2 x 0.05 mm. They
diffracted to about 3.0 A resolution in the Spring8 BL38
beam. MAD data were 'collected from HgCl2 derivative
using the same beam line as above. The data were
processed with MOSFLM and CCP4 program suite.
The unit cell is a=74.4A b=242.9A c=129.3A (spacegroup C222), and its asymmetric unit contains 4
molecules of PhzR. MAD analysis using programs
SOLVE and Sharp is in progress, revealing a novel
mode of tetramer assembly of the PhzR.
This work has been done in collaboration with
Hironori ARAMAKI (Daiichi Pharmaceutical College).

(4) Crystallographic Study of Transcription factors from
Pseudomonas aeruginosa(5) Crystallization of TRAF6, a transducer in CD40
signaling pathway

Yasuo Shirakihara, Aya Shiratori, Hisako Inoue andMegumi Seki

Yasuo Shirakihara, Megumi Seki and Aya Shiratori

Pseudomonas aeruginosa is the well-known opportunistic bacterial pathogen, and a number of transcription factors responsible for the pathogenicity have been
identified. Among those, we chose PtxR, PtxS, PhzR,
and PA3547 and have started their structure biology.
Initially, over expression system was constructedby incorporating the provided expression plasmids,
which were designed to have 6 His-tags at The Cterminus, into host cells (BL21 (DE3), B834 (DE3), ]MI09(DE3)) and by examining which conditions, characterized by temperature (31C, 25'C) and inducer presence(plus, minus), produce the target protein in a soluble
fraction. Fortunately good conditions were found for
all the proteins. Three proteins out of the four were
purified by the Ni-NTA column and an extra column,

CD40 signalings play crucial roles in B-cellfunction. Signals through CD40 rescue B cells from
apoptosis induced by cross-linking of the surface
immunoglobulin complex and also induce B cells todifferentiate. In the down stream, the signal trans~ucers,
TRAF6 (TNF receptor associated factor 6) and TRAF5,
interact with a cytoplasmic tail of CD40. We tried to
crystallize the TRAF6 in order to understand themechanism of signal transduction employing CD40 and
TRAF6. TRAF6 was expressed in insect cells as
His-tagged protein, and was purified with Ni-NTA andion exchange columns. The crystallization condition
search was done using the recently developed method
for comprehensive crystallization of transcription
factors as described above. Small crystals wereobtained from MPD, but further study may be

Biomolecular Structure Laboratory97

necessary for its establishment.
The crystallization study was done in collaboration
with Jun-ichiro Inoue, Ayaka Moriya at the Instituteof medical science, the university of Tokyo.
(6) Structural

analysisofglutaminasefromMicrococcus [uteus K-3
Y a~mo Shirakihara and Aya Shiratori
Glutaminase (EC 3.5.1.2) catalyzes the hydrolytic
degradation of L-glutamine to L-glutamic acid. The
salt-tolerant glutaminase from marine Micrococcus
luteus K-3 has an unusual property that its maximum
activity is observed at about 2M salt, and also has
industrial relevance that this enzyme may be more
efficient than the conventional ones in the soy sauce
fermentation that is carried out in the high-salt
environments. Previously MAD data using the Se-Met
derivatives for the intact form (2.6 A resolution) andthe truncated form (2.4 A resolution) had been collected!)
and a structural analysis of the truncated form was
attempted.
The structure of the truncated form (2.4 A resolution)of the enzyme was refined successfully this year, withfree R factor of 23% and satisfactory geometry. The
structure solution of the intact enzyme has been
attempted by molecular replacement using the structure of the truncated form as a model. There has beena difficulty in such calculation, because the asymmetricunit in the intact crystal contains more than one
molecule of the enzyme.
The structural study was done in collaboration
with Kazuaki Yoshimune and Mitsuaki Moriguchi at
Oita University, and Mamoru Wakayama at Ritsumeikan University.
Publications
Papers1. Chantawannakul, P., Yoshimune, K, Shirakihara,
Y, Shiratori, A., Wakayama, M. and Moriguchi, M.
(2003). Crystallization and preliminary X-ray crystallographic studies of salt-tolerant glutaminase fromMicrococcus luteus K-3, Acta Crystallogr D 59, 566-568.
2. Hosaka, T., Meguro, T., Yamato, I. and Shirakihara,
Y. (2003). Crystal Structure of Enterococcus hirae
enolase at 2.8 A Resolution. J. Biochem (Tokyo), 133
817-823.

98

Biomolecular Structure Laboratory

3. Shiroishi, M., Tsumoto, K, Amano, K, Shirakihara,Y., Colonna, M., Braud, V. M., Allan, D. S. ]., Azure,S., Makadzange, Jones. R., Willcox, B., Jones, E. Y, vander Merwe, P. A., Kumagai, I. and Maenaka, K (2003).
Human inhibitory receptors Ig-like transcript 2 (ILT2)
and ILT4 compete with CD8 for MHC class I binding
and bind preferentially to HLA-G. Proc. Natl. Acad. Sci.
100: 8856-8861.
4. Suzuki, T., Murakami, T., lino, R., Suzuki,]., Ono,
S., Shirakihara, Y. and Yoshida, (2003). FO-FI-ATP asel
Synthase is geared to the synthesis mode by conformational rearrangement of e subunit in response to
proton motive force and ATP/ ADP balance. J. BioI.
Chem. 278, 46840-46846.
EDUCATION

1. Y Shirakihara was invited to give a seminar on"Strucrural analysis of FI-ATPase to reveal its
rotational catalysis mechanism" at the Protein Instituteof Osaka University., Osaka, Janauary 2003 (inJapanese).
2. Y. Shirakihara was invited to give a seminar on"Structural analysis of FI-ATPase to reveal its
rotational catalysis mechanism" at Tokyo University,
Tokyo, July 2003 (in Japanese).
3. Y Shirakihara was invited to give a seminar on"Structural analysis of FI-ATPase to reveal its
rotational catalysis mechanism" at Tokyo Institute ofTechnology, Yokohama, September 2003 (in Japanese).

by altering the transmembrane regions.

I. CENTER FOR INFORMATION BIOLOGYAND DNA DATA BANK OF JAPAN(2) The discovery of the genes relating to the evolutionof an eyeless cave dwelling form from an eyed surface
dwelling form of Mexican tetra, Astyanax mexicanus,
by cDNA microarrays
Nobuhiko Tanaka, Shozo Yokoyama, Kazuho Ikeo
and Takashi Gojobori

I-a. Laboratory for DNA Data Analysis
Takashi Gojobori GroupRESEARCH ACTIVITIES(1) The origin and evolution of porcine reproductive andrespiratory syndrome viruses
Kousuke Hanada, Yoshiyuki Suzuki and Takashi
Gojobori
Porcine reproductive and respiratory syndrome
viruses (PRRSV) are divided into North American andEuropean types, which show about 40% difference
between the amino acid sequences. The divergencetime of these two types has been considered to bearound 1987 from the epidemiological data. It follows
that PRRSV have evolved at a higher evolutionary rate(order of 1O-2/site/year) compared with other RNA
viruses because the evolutionary rates of most RNA
viruses are the orders of 10.3 - 10-5• Here, to test theevolutionary history of PRRSV speculated by the
epidemiological background, we estimated thedivergence time and evolutionary rate of PRRSV withmolecular evolutionary analysis. As a result, thedivergence time (1986±1.8) estimated corresponded wellto that estimated by the epidemiological data, and therate (7.7x10-~ of PRRSV was indeed the highest among
RNA viruses so far reported. Furthermore, to examine
how PRRSV have adapted to swine since they emergedin 1987, we inferred important sites for the adaptation.
The adaptive sites were located not only in the epitopes
related to immunity, but also in the transmembraneregions including a signal peptide. In particular, theadaptive sites in the transmembrane regions wereconsidered to affect compatibility to the host cell
membrane. Therefore, we conclude that PRRSV haverecently been transmitted from another host species
to swine in the middle of the 1980' s, adapting to swine

The understanding of the evolution of genediversification should be simplified by studying asingle species which is in the process of evolutionary
diversification. Mexcan tetra (Astyanax mexicanus), a
single species showing eyed surface and eyeless cavedwelling forms, is a suitable model species to study
the evolutionary diversification process. "How docavefishes cope with the dark environment?" and"What types of genes are involved in these changes?"
To solve these evolutionary problems, we tried to
discover the genes relating to the evolution from thesurface form to the cave form by cDNA microarrays.
3070 kinds of clones from an eyed adult fish were
finally used for the construction of microarrays. As aresult of hybridizations, crystalline genes wererelatively highly expressed in surface fish, whereastransferrin genes were relatively highly expressed incave fish. The transferrin proteins are related to theoxygen transportation in the blood. As the variationand quantity of phytoplanktons and plants in the dark(cave) environment must be less than those in the light(surface) environment, the concentration of oxygen in
the water of the dark (cave) environment must be lower
than that of light (surface) environment. Accordingly,
to effectively transport oxygen in the blood of cavefish,transferrin genes must be required to have relatively
highly expression in the cavefish.
(3) Evolutionary mechanisms of duplicated genes
Takashi Makino, Yoshiyuki Suzuki and Takashi
Gojobori
It is proposed that gene duplication produces agene with novel function along with the evolution of
the asymmetrical copies. It is quite interesting in theperspective of understanding how gene duplication contributes to the evolution of novel functions. Therefore, we
analyzed duplicated genes from evolutionary rate and

CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPAN99

functional differentiation for understanding evolutionary mechanisms of that in Saccharomyces cerevisiae.
When the genes of S. cerevisiaewere categorized alongwith the fitness effects, the fitness effect pairs ofduplicated genes were almost dispensability/ indispensability pairs or dispensability/ dispensability pairs,
and rarely indispensability/ indispensability pairs. The
evolutionary rate ratio of the duplicated gene pairs
without common protein-protein-interaction partners
or common functions was higher than that of others.
In particular, the evolutionary rate ratio of dispensability/ indispensability pairs was higher than thatof dispensability/ dispensability pairs. In the case ofdispensability/ indispensability pairs, dispensablegenes evolved faster and expressed higher than indispensable genes. In the case of dispensability/ dispensability pairs, however, there were not the differences
observed in dispensability/ indispensability pairs. The
results indicated that the asymmetric evolution of
duplicated genes was specific in dispensability/
indispensability pairs.
(4) Study on conserve upstream noncoding sequences
between human and mouse

Hisakazu Iwama and Takashi Gojobori
With the aim of elucidating what functional
category of genes tend to be conserved in their upstream regions, we systematically estimated the degreeof upstream sequence conservation of 4,074 genes along
the 4-kb stretch and categorized the gene function
according to the Gene Ontology. We found that thehighly upstream-conserved genes are dominantly
transcription factor (TF) genes. In particular, developmental process-related TF genes showed higher
conservation of upstream sequences.
(5) Data analysis on Rice Genome Project

Hisakazu Iwama and Takashi Gojobori
We have participated in the Rice Genome Research
Program in Japan and conducted computational
analyses on the rice genome. We studied the functionalclassification of all the predicted proteins encoded in
the rice genome.

100 Laboratory for DNA Data Analysis(6) Biased biological functions of horizontally transferredgenes on 324,653 open reading frames of 116 prokaryotic
complete genomes

Yoji Nakamura, Takeshi Hoh, Hideo Matsuda andTakashi Gojobori
Horizontal gene transfer is one of the majormechanisms contributing to microbial genome
diversification. To clarify the overall picture ofinterspecific gene flow among prokaryotes, we havedeveloped a new method for detecting horizontallytransferred genes (HT genes) and their possible donors
by Bayesian inference. As a result, we discovered that46,759 (14%) out of 324,653 open reading frames (ORFs)
in 116 prokaryotic complete genomes have been
subjected to horizontal transfer. Based on this dataset,for the first time, we quantitatively revealed that thebiological functions of HT genes except mobile element
genes are biased to three categories; cell surface, DNAbinding, and pathogenicity-related functions. Thus, itsuggests that transferability of genes depends heavily
upon their function.
(7) Comparative complete genome sequence analysis of
the amino acid replacements responsible for thethermostability of Corynebacterium efficiens

Yousuke Nishio, Yoji Nakamura, Yutaka Kawarabayasi,
Yoshihiro Usuda, Eiichiro Kimura, Shinichi Sugimoto,
Kazuhiko Matsui, Akihiko Yamagishi, Hisashi
Kikuchi, Kazuho Ikeo and Takashi Gojobori

Corynebacterium efficiens is the closest relative ofC. glutamicum, a species widely used for the industrial
production of amino acids. C. efficiens but not C.
glutamicum can grow above 40°C. We sequenced thecomplete C. efficiens genome to investigate the basisof its thermostability by comparing its genome withthat of C. glutamicum. The difference in GC content
between the species was reflected in codon usage andnucleotide substitutions. Our comparative genomic
study clearly showed that there was tremendous bias
in amino acid substitutions in all orthologous ORFs.
Analysis of the direction of the amino acid substitutions suggested that three substitutions: from lysine
to arginine, serine to alanine, and serine to threonine,
are important for the stability of the C. efficiens
proteins. Our results strongly suggest that the accu-

mulation of these three types of amino acid substitutions correlates with the acquisition of thermostability
and is responsible for the greater GC content of
C. efficiens 51 •(8) The genome stability in Corynebacterium species
due to lack of the recombinational repair system

Yoji Nakamura, Yosuke Nishio, Kazuho Ikeo andTakashi Gojobori

Corynebacterium species are members of grampositive bacteria closely related to Mycobacterium
species, both of which are classified into the sametaxonomic order Actinomycetales. Recently, threecorynebacteria, C. efficiens, C. glutamicum, and C.
diphtheriae have been sequenced independently. We
found that the order of orthologous genes in these
species has been highly conserved though it has been
disrupted in Mycobacterium species. This syntenysuggests that corynebacteria have rarely undergone
extensive genome rearrangements and have maintained ancestral genome structures even after thedivergence of corynebacteria and mycobacteria. Thisis the first report that the genome structures have beenconserved in free-livin~ bacteria such as C. efficiens andC. glutamicum, although it has been reported thatobligate parasites such as Mycoplasma and Chlamydia
have the stable genomes. The comparison of recombinational repair systems among the three corynebacteria and M tuberculosis suggested that the absenceof recBCD genes in corynebacteria be responsible forthe suppression of genome shuffling in the species. The
genome stability in Corynebacterium species will give
us hints of the speciation mechanism with the nonshuffled genome, particularly the importance ofhorizontal gene transfer and nucleotide substitution
in the genomelli .
(9) Identification of I-cell specific genes of hydra andits evolutionary implication]. Shan Hwang, Chiemi Nishimiya-Fujisawa, Toshitaka
Fujisawa, Kazuho Ikeo and Takashi Gojobori
Epithelial hydra, which is derived from Hydra
magnipapillata (strain 105), possesses no interstitialstem cell and its subsequent differentiation products
such as nerve cells and nematocytes, but maintains its

abilities to grow, bud and regenerate into a complete
polyp. Using cDNA microarray analysis, we compared
the expression profiles between epithelial hydra normal
hydra (strain 105). The cDNA microarray was constructed by a total number of 6,588 non-redundant
clones which is generated from our Hydra EST project.
As a result, we have obtained 200 genes that are
differentially expressed in the normal but not inepithelial hydra. To further study whether these genes
are essentially specific for the interstitial stem celllineage, we are now in process of performing whole
mount in situ hybridization to localize their expression
patterns in hydra. Our preliminary results indicatedthat the gene expressions were found specifically ineither the nematocyte lineage or the nerve cell lineage.
Given the fact that nematocyte is unique to the phylum
Cnidarian, it is interesting to examine if the genetic
network controlling nematocyte pathway is also
exclusive to Cnidarian or it is evolutionarily conserved
among metazoans. Moreover, the study of neuronspecific genes that were identified by microarray would
address the question of what molecular basic isessential to form the origin of nervous system inmetazoan.
(10) The evolutionary origin of long-crowing chicken
based on the molecular phylogeny

Tomoyoshi Komiyama, Kazuho Ikeo and Takashi
Gojobori
We tried to elucidate the origin and the domestication process of Naganakidori using the mitochondrial
DNA D-Ioop region sequences. In Japan, three varietiesof chickens can be found that have been specificallybred to develop an exceptionally long crow for over 20
seconds. Such characteristics of long-crowing chickens
are also as popular as the fighting cock among
breeders. The long crowing chicken has been mainly
kept for the purpose of beauty and its long crow. It is
not found only in Japan but also in other Asian
countries such as Indonesia and China. However, thedomestication process of long-crowing chicken iscurrently unclear. Our study is to elucidate this issue
by using mitochondrial DNA D-loop region sequences.
For this purpose, we collected blood sample of 9long-crowing chickens and 74 other ornamental
chickens from the 14 varieties of Japanese domesticatedchickens. Then, we also obtained 2 DNA sequence data

Laboratory for DNA Data Analysis

101

of wild chicken from the International DNA databank(DDBJ/ EMBLI GenBank) as the out-group. A
phylogenetic tree was constructed by using both theneighbor-joining method CNJ) and the maximum
likelihood method(ML). The results indicated that the
14 varieties of Japanese chickens were clearly separated
into three different groups. One group contained thesamples from Okinawan fighting cocks, the second
group consisted of the long-crowing chickens, and thirdgroup is composed of the fighting cocks from Honshu
and Kyushu in Japan. This result suggested that thelong-crowing chicken was descened from the fighting
cock, Shamo for cockfighting. In addition, we foundthat the three varieties of chickens share the common
origin in spite of conspicuously different characters.
(11) The analysis to the evolutionary relationshipbetween duplication and alternative splicing in terms ofincreasing functional diversity

Lihua Jin, Kazuho Ikeo, Yoshiyuki Suzuki and Takashi
Gojobori
Living things increase their biological diversity byvariable evolutionary mechanisms. Among thesemechanisms, gene duplication and alternative splicing(A. S.) are the two major evolutionary routes that canbring the functional variation by increasing genediversification. Our research interest is to study about
the evolutionary relationship between the two different
phenomena by utilizing available all data resources.
The results of this study showed that the percentage
of A.S. locus in the singleton gene group was less thanthat in the duplicated gene group and the duplicate
genes tended to have more A. S. isoforms thansingleton genes. In spite of these results, there was noapparent tendency of increasing isoform numbers ofalternative splicing when family size increased in thegroup of duplicates genes. Our results suggested thatgene duplication would induce more alternative
splicing events on the duplicate genes than on
singletons by reducing the functional constraint on
those duplicates, and the degree of reduction offunctional constraint was significantly large whensingleton duplicated into two homologous genes.

102 Laboratory for DNA Data Analysis(12) Comparative evolutionary study of the single
cell-based gene expression profiles from the ascidinan
blastmeres

Katsuhiko Mineta, Kazuho Ikeo, Hiroaki Yamamoto,
Yuzuru Tanaka and Takashi Gojobori
Since the multicellular organism is composed ofcells at the most basal portion, to understand theevolutionary diversification of organisms, we took an
approach to examine the genes expressed at thevarious types of cells. As a first target, we focused on
the cells of ascidians (Ciona intestinalis). The celllineage of ascidians is well-documented, and their
phylogenetic position is near the root of the Chordata,
which is suitable for the analysis of the evolutionary
process toward the vertebrates. Successfully, we could
isolate and collect the cells (blastomeres) from the 1,
2,4 and 8 cell early developmental stages of ascidians,
and then cDNA libraries were constructed for each typeof cells. We are sequencing ESTs from those libraries
and comparing them with EST of the other cells
sequenced. As a result, we sequenced, first, ""'2,400
ESTs from six different types of blastomeres (A3 andB3 from 4-cell stage, A4.1, a4.2, B4.1 and b4.2 from the
8-cell stage) and then obtained 291""'1266 non-redundant
clones. Weare also developing a 3D database for theintuitive management of the expression data. By useof the database and comparing genes identified at eachsingle cell, we found that on average 7% of clones werecommonly expressed between blastomeres. This result
showed that the divergence of genes expressed at thesecells was large, suggesting the characteristics of a cell
as a whole might have already begun to be establishedat these stages.
(13) Molecular evolutionary analyses of host-parasite
interactions

Yoshiyuki Suzuki
In order to identify and characterize genetic
polymorphism of the swine major histocompatibility
complex (Mhc: SLA) class I genes, RT-PCR productsof the second and third exons of the three SLA classicalclass I genes, SLA-l, SLA-2 and SLA-3 were subjected
to nucleotide determination. These analyses allowed
the identification of four, eight and seven alleles at the
SLA-l, SLA-2 and SLA-3Ioci, respectively, from threedifferent breeds of miniature swine and one mixed
breed. Among them, 12 alleles were novel. Constructionof a phylogenetic tree using the nucleotide sequencesof those 19 alleles indicated that the SLA-l and -2 genes
are more closely related to each other than to SLA-3.
Selective forces operating at single amino acid sites of
the SLA class I molecules were analyzed by the
Adaptsite Package program. Ten positive selection
sites were found at the putative antigen recognition
sites (ARSs). Among the 14 positively selected sites
observed in the human MHC (HLA) classical class I
molecules, eight corresponding positions in the SLA
class I molecules were inferred as positively selected.
On the other hand, four amino acids at the putative
ARSs were identified as negatively selected in the SLA
class I molecules. These results suggest that selective
forces operating in the SLA class I molecules arealmost similar to those of the HLA class I molecules,
although several functional sites for antigen andcytotoxic T-Iymphocyte recognition by the SLA class
I molecules may be different from those of the HLA
class I molecules\ We also analyzed the evolution offeline immunodeficiency virus l2l , and Haemophilus
influenzae20l • The methodologies for analyzing hostparasite interactions were also summarized211221 24l.
(14) Molecular basis of convergent evolution of camera
eye between octopus and humanAtsushi Ogura, Kazuho Ikeo and Takashi Gojobori
It is well known that the camera eye of octopus
is very similar to that of human. However, a phylogenetic
consideration has suggested that their camera eyes
have been acquired independently during evolution.
Thus, the camera eye structures of octopus and humanhave been known as a typical example of convergent
evolution. To examine if convergent evolution has
really taken place at the molecular level, I conducted
the comparative analysis of gene expression in thecamera eye between human and octopus. In practice,
I sequenced about 16,234 ESTs of an octopus eye, andcompared these EST sequences with the complete
genome sequences of nematode, fly and human as wellas the gene expression data of human eye. As a result,
I found that 729 genes were expressed commonly
between the camera eye of human and octopus.
Conducting comprehensive homology search using
octopus ESTs with non-redundant protein database,

I found that 933 genes had already existed at thecommon ancestor of Protostomes and Deuterostomes.
I also found that 875 out of 1,019 genes were conserved
between human and octopus, although nematode andfly are closely related to octopus than to human. Thus,
genes expressed in the eye of octopus are highly sharedwith human. This suggests that the evolution of thecamera eye of human and octopus is not subjected tothe convergent evolution.
(15) Genomic diversification of bilaterian animals
Atsushi Ogura, Kazuho Ikeo and Takashi Gojobori
To understand the process of bilaterian evolution,
we estimated ancestral gene sets at the divergence ofbilaterian animals and the split of the plant-animalfungus using the currently available gene sequence
data. Using these ancestral gene sets, we examined how
the number of gene members in the gene clusters haschanged during evolution. As a result, the ancestralgene set of the bilaterian animals and the plantanimal-fungus were estimated to be 6,577 and 2,469
gene clusters, respectively. Thus, we found 2.5-foldincrease in the number of gene clusters during theperiod of the time from the evolutionary split of theplant-animal-fungus to the divergence of bilateriananimals. Moreover, when we compared those numbersof the ancestral gene clusters with those the extant
animals such as nematode, fly, mouse and human, we
found that the extant bilaterian animals retained mostof the ancestral gene set. It implies that diversificationof bilaterian animals must have been attained bydifferently networking of the available gene set rather
than producing a lot of functionally new genes.
Publications
Papers

1. Ando, A., Kawata, H., Shigenari, A., Anzai, T., Ota,
M., Katsuyama, Y., Sada, M., Goto, R., Takeshima,
S.-N., Aida, Y, Iwanaga, T., Fujimura, N., Suzuki, Y,
Gojobori, T. and Inoko, H. (2003). Genetic polymorphism of the swine major histocompatibility complex(SLA) class I genes, SLA-l, -2 and -3. Immunogenetics
55,583-93.
2. Anzai, T., Shiina, T., Kimura, N., Yanagiya, K,
Kohara, S., Shigenari, A., Yamagata, T., Kulski,]. K,
Naruse, T. K, Fujimori, Y, Fukuzumi, Y., Yamazaki,

Laboratory for DNA Data Analysis

103M., Tashiro, H., Iwamoto, C., Umehara, Y, Imanishi,T., Meyer, A, Ikeo, K, Gojobori, T., Bahram, S. andInoko, H. (2003). Comparative sequencing of human andchimpanzee MHC class I regions unveils insertions/deletions as the major path to genomic divergence.
Prac. Nat!. Acad. Sci. USA 100: 7708-13.
3. Hotta, K, Takahashi, H., Ueno, N. and Gojobori,T. (2003). A genome-wide survey of the genes for planar
polarity signaling or convergent extension-related genes
in Ciona intestinalis and phylogenetic comparisons ofevolutionary conserved signaling Components. Gene
317,165-85.
4. Ikeo, K, Ishi-i, ]., Tamura, T., Gojobori, T. andTateno, Y. (2003). CIBEX: Center for Information
Biology Gene Expression Database. C.R. BioI. 326,
1079-82.
5. Ishibashi, T., Nakazawa, M., Ono, H., Satoh, N.,
Gojobori, T. and Fujiwara, S. (2003). Microarray
analysis of embryonic reinoic acid target genes in the
ascidian Ciona intestinalis. Dev. Growth Differ249-259.
6. Komiyama, T., Ikeo, K and Gojobori, T. (2003).
Where is the origin of the Japanese gamecocks? Gene
317: 195-202.
7. Mineta, K, Nakazawa, M., Cebria, F., Ikeo, K,
Agata, K and Gojobori, T. (2003). Origin andevolutionary process of CNS elucidated by comparative
genomics analysis of planarian ESTs. Proc. Nat!. Acad.
Sci. USA 100: 7666-7671.
8. Miyazaki, S., Sugawara, H., Ikeo, K, Gojobori, T.
and Tateno, Y. (2003). DDBJ in the stream of various
biological data. Nucleic Acids Res. 32, D31-4.
9. Miyazaki, S., Sugawara, H., Gojobori, T. andTateno, Y (2003). DNA Data Bank of Japan (DDBJ) in
XML. Nucleic Acids Res. 31(1): 13-6.
10. Nakamura, Y., Nishio, Y, Ikeo, K and Gojobori,T. (2003). The genome stability in Corynebacterium
species due to lack of the recombinational repair
system. Gene 317: 149-55.
11. Nakamura, K, Suzuki, Y, Ikeo, K, Ikeda, Y., Sato,E., Nguyen, N. T. P., Gojobori, T., Mikami, T. andMiyazawa, T. (2003). Phylogenetic analysis of Vietnamese isolates of feline immunodeficiency virus: genetic
diversity of subtype C. Arch. Viral. 148 783-91.
12. Nakazawa, M., Cebria, F., Mineta, K., Ikeo, K,
Agata, K and Gojobori, T. (2003). Search for theevolutionary origin of a brain.; Planarian brain
characterized by micro array. Mol. BioI. Evol. 20(5):784-791.
13. Niimura, Y., Terabe, M., Gojobori, T. and Miura,

104

Laboratory for DNA Data Analysis

K(2003). Comparative analysis of the base biases at
the gene terminal portions in seven eukaryote genomes.
Nucleic Acids Res. 31: 5195-201.
14. Nishio, Y., Nakamura, Y, Kawarabayashi, Y.,
Usuda, Y., Kimura, E., Sugimoto, S., Matsui, K,
Yamagishi, A, Kikuchi, H., Ikeo, K and Gojobori, T.
(2003). Comparative complete genome sequence analysisof the amino acid replacements responsible for thethermostability of Corynebacterium efficiens. Genome
Res. 13: 1572-1579.
15. Ohshima, K, Hattori, M., Yada, T., Gojobori, T.,
Sasaki, Y. and Okada, N. (2003). Whole-genome
screening indicates a possible burst of formation ofprocessed pseudogenes and Alu repeats by particular
L1 subfamilies in ancestral primates. Genome BioI. 4:
R74.
16. Ota, K, Tateno, Y and Gojobori, T. (2003). Highly
differentiated and conserved sex chromosome in fish
species (Aulopus japonicus: Teleostei, Aulopidae). Gene
317: 187-93.
17. Pignatelli, S., Dal Monte, P., Rossini, G., Chou, S.,
Gojobori, T., Hanada, K., Guo, n., Rawlinson, W., Britt,W., Mack, M. and Landini, MP. (2003). Humancytomegalovirus glycoprotein N (gpUL73-gN) genomic
variants: identification of a novel subgroup, geographical distribution and evidence of positive selective
pressure. J Gen. Viral. 2003 Mar; 84(Pt3), 647-655.
18. Yajima, I., Endo, K, Sato, S., Toyoda, R., Wada,
H., Shibahara, S., Numakunai, T., Ikeo, K, Gojobori,T., Goding, C. R. and Yamamoto, H. (2003). Cloning andfunctional analysis of ascidian Mitf in vivo: insights
into the origin of vertebrate pigment cells. Mech Dev.
120: 1489-504.
Reviews

ill $H~~ _*_~ E • • • :~~7~~~~(-{ /" 7 Iv.:r: /" -If;f;!j! 00) . B *Ii;E*. B *Ii;E**±. 2003.
61. 716-721.
Books

20. Suzuki, Y and Gojobori, T. (2003). Analysis ofcoding sequences. In The Phylogenetic Handbook: A
Proctical Approach to DNA and Protein Phylogeny.
Salemi, M., and Vandamme, A-M. eds. (Cambridge,
UK: Cambridge University Press), pp. 283-303.
21. i1!!~-ft!.
Ef~ijl .~~ (2003) r j::iIt_*_$.

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No.4, 364-365.
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Satoshi Fukuchi, Kazuaki Yoshimune\ Mitsuaki
Moriguchil and Ken Nishikawa CDept. Appl. Chern.,
Oita Univ.)

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Unique amino acid composition of proteins in
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SOCIAL CONTRIBUTIONS AND OTHERS

n!!~-li,Suzuki), and a secretary (Ms. Sugiyama). We aim attheoretical work on protein molecules, such asprediction of three-dimensional structures from aminoacid sequences, theoretical basis of protein stability,
and molecular dynamics of proteins. Recently we haveextended our research fields to genome analysis basedon protein three-dimensional structures. We havedeveloped two databases, Protein Mutant Database(PMD) and Genomes to Protein Structures andFunctions (GTOP), and three computer tools foranalysis of amino acid sequences and structures ofproteins, LIBRA (Protein structure-sequence compatibility analysis), MATRAS (Protein tertiary structurecomparison), and SSThread (Prediction of protein
secondary structure using threading).

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2J], 2003.

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1P-fi'[t£lTJf~~~, == lflJ, 12J], 2003.

I-b. Laboratory for Gene·Product Informatics
Ken Nishikawa GroupRESEARCH ACTIVITIESAt present the Nishikawa laboratory consist of 15
members: Professor Ken Nishikawa, Assistant Professors Satoshi Fukuchi and Akira Kinjo, three postdoctral research fellows (Drs. Homma,Nagashima and
Minezaki), 8 technical assistants (Mses_ Mimura, Ito,
Yamamoto, Kuromaru, Abe, Hongo, Komatsubara and

The amino acid compositions of proteins from
halophilic archaea were compared with those from
non-halophilic mesophiles and thermophiles, in termsof the protein surface and interior, on a genome-wide
scale. As we previously reported for proteins fromthermophiles, a biased amino acid composition also
exists in halophiles, in which an abundance of acidic
residues was found on the protein surface as comparedto the interior. This general feature did not seem todepend on the individual protein structures, but wasapplicable to all proteins encoded within the entire
genome. Unique protein surface compositions arecommon in both halophiles and thermophiles. Statistical tests have shown that significant surface
compositional differences exist among halophiles,
non-halophiles, and thermophiles, while the interior
composition within each of the three types of organisms does not significantly differ. Although
thermophilic proteins have an almost equal abundanceof both acidic and basic residues, a large excess ofacidic residues in halophilic proteins seems to becompensated by fewer basic residues. Aspartic acid,lysine, asparagine, alanine, and threonine significantly
contributed to the compositional differences of halophiles
from meso- and thermophiles. Among them, however,
only aspartic acid deviated largely from the expected

Laboratory for DNA Data Analysis

105amount estimated from the dinucleotide compositionof the genomic DNA sequence of the halophile, which
has an extremely high G+C content (68%). Thus, theother residues with large deviations (Lys, Ala, etc.) fromtheir non-halophilic frequencies could have arisen
merely as "dragging effects" caused by the compositional shift of the DNA, which would have changed toincrease principally the fraction of aspartic acid alone.
See Ref. 4 for details.
(2) Compositional changes in RNA, DNA and proteinsfor bacterial adaptation to higher and lower temperatures
Hiroshi Nakashima\ Satoshi Fukuchi and Ken Nishikawa
CSchool of Health Science, Kanazawa Univ.)
It is known that in thermophiles the G+C content
of ribosomal RNA linearly correlates with growthtemperature, while that of genomic DNA does not.
Although the G+C contents (singlet) of the genomic
DNAs of thermophiles and methophiles do not differ
significantly, the dinucleotide (doublet) compositionsof the two bacterial groups clearly do. The averageamino acid compositions of proteins of the two groups
are also distinct. Based on these facts, we here analyzed
the DNA and pro~ein compositions of various bacteria
in terms of the optimal growth temperature (OGT).
Regression analyses of the sequence data for theill1ophilic,
mesophilic and psychrophilic bacteria revealed good
linear relationships between OGT and the dinucleotide
Icompositions of DNA, and between OGT and theamino acid compositions of proteins. Together withthe above-mentioned linear relationship between
ribosomal RNA and OGT, the DNA and protein
compositions can be regarded as thermostability
measures for RNA, DNA and proteins, covering a wide
range of temperatures. Both the DNA and proteins of
psychrophiles apparently exhibit characteristics
diametrically opposite to those of theill1ophiles. The
physicochemical parameters of dinucleotides suggested
that supercoiling of DNA is relevant to its theill1ostability.
Protein stability in thermophiles is realized primarily
through global changes that increase charged residues(i.e., Glu, Arg, and Lys) on the molecular surface of all
proteins. This kind of global change is attainablethrough a change in the amino acid composition
coupled with alterations in the DNA base composition.
The general strategies of theill10philes and psychrophiles
for adaptation to higher and lower temperatures,

106 Laboratory for Gene-Product Informatics

respectively, that are suggested by the present study
are discussed. See Ref. 5 for details.
(3) Prediction of catalytic residues in enzymes based onlmown tertiary structure, stability profile, and sequenceconservation
Motonori Otat, Kengo Kinoshita2 and Ken Nishikawa
CGlobal Scientific Infoill1ation and Computing Center,
Tokyo Institute of Technology, 2Graduate School ofIntegrated Science, Yokohama City Univ.)
The catalytic or functionally important residuesof a protein are known to exist in evolutionarily
constrained regions. However, the patterns of residue
conservation alone are sometimes not very infoill1ative,
depending on the homologous sequences available fora given query protein. Here, we present an integrated
method to locate the catalytic residues in an enzyme
from its sequence and structure. Mutations of functional residues usually decrease the activity, but
concurrently often increase stability. Also, catalytic
residues tend to occupy partially buried sites in holes
or clefts on the molecular surface. After confirming
these general tendencies by carrying out statisticalanalyses on 49 representative enzymes, these data
together with amino acid conservation were evaluated.
This novel method exhibited better sensitivity in theprediction accuracy than traditional methods thatconsider only the residue conservation. We applied it
to some so-called "hypothetical" proteins, with known
structures but undefined functions. The relationships
among the catalytic, conserved, and destabilizing
residues in enzymatic proteins are discussed. See Ref.
6 for details.
Publications
Papers

1. Fukami-Kobayashi, K., Tateno, Y. and Nishikawa,
K. (2003). Parallel evolution of ligand specificity
between LacVGalR family repressors and periplasmic
sugar-binding proteins. Molecular Biology and
Evolution, 20, 267-277.
2. Kinjo, A.R. and Takada, S. (2003). Competition
between protein folding and aggregation with molecular chaperones in crowded solutions: Insight frommesoscopic simulations. Biophysical Journal, 85,
3521-3531.

3. Kashiwagi, K., Shiba, K., Fukami-Kobayashi, K.,
Noda, T., Nishikawa, K. and Noguchi, H. (2003).
Characterization of folding pathways of the type-I andtype-2 periplasmic binding proteins MglB and ArgT.
J Biochem. 133, 371-376.
4. Fukuchi, S., Yoshimune, K., Wakayama, M.,
Moriguchi, M. and Nishikawa, K. (2003). Unique aminoacid composition of proteins in halophilic bacteria. J
Mol. Bioi., 327, 347-357.
5. Nakashima, H., Fukuchi, S. and Nishikawa, K.
(2003). Compositional changes in RNA, DNA andproteins for bacterial adaptation to higher and lower
temperatures. J Biochem., 133, 507-513.
6. Ota, M., Kinoshita, K. and Nishikawa, K. (2003).
Prediction of catalytic residues in enzymes based on
known tertiary structure, stability profile, and sequenceconservation. J. Mol. BioI., 327, 1053-1064.
7. Kashiwagi, K., Fukami-Kobayashi, K., Shiba, K.
and Nishikawa, K.: Construction and characterizationof chimeric proteins composed of type-I and type-2
periplasmic binding proteins MglB and ArgT. Biosci.
Biotec. Biochem., in press.

family evolved by acquiring the DNA-binding domain
in the N-terminal of an ancestral periplasmic binding
protein (PBP). Phylogenetic analysis of the LacVGalR
family repressors and their PBP homologues revealed
that the acquisition of the DNA-binding domain
occurred first in the family, and ligand specificity thenevolved. The phylogenetic tree also indicates that theacquisition occurred only once before the divergenceof the major lineages of eubacteria, and that the LacV
GalR and the PBP families have since undergone
extensive gene duplication/loss independently along
the evolutionary lineages. Multiple alignments of therepressors and PBPs furthermore revealed that
repressors and PBPs with the same ligand specificity
have the same or similar residues in their binding sites.
This result, together with the phylogenetic relationship,demonstrates that the repressors and the PBPs
individually acquired the same ligand specificity byhomoplasious replacement, even though their genes
are encoded in the same operon. 11(2) CIBEX: Center for Information Biology Gene
Expression Database

Reviews

8. rft,l;l/Ai!;, m:l1!!ftc4i!;, ImJIIJl, rW~1i(J)~*)E'tt",(J)~III3-J,

W?' / .An~GJi.te1:.¥!.J(J)~;ji'ttbjMt~ Clif~

Kazuho Ikeo, lun Ishi-i, Takurou Tamura, Takashi
Gojobori and Yoshio Tateno

mm ~ ='- '1'!J /7J- . 7::r.. 7'7-'i'!JK.$.)pp.41-49C2003).
I-c. Laboratory for Gene Function Research
Yoshio Tateno GroupRESEARCH ACTIVITIES(1) Parallel evolution of ligand specificity between LacI/
GalR family repressors and periplasmic sugar-binding
proteins

Kaoru Fukami-Kobayashi, Yoshio Tateno and Ken
Nishikawa
The bacterial Lad/GaiR family repressors suchas lactose operon repressor (Lad), purine nucleotide
synthesis repressor (PurR), and trehalose operon
repressor (TreR) consist of not only the N-terminal
helix-tum-helix DNA-binding domain but also the
C-terminalligand-binding domain that is structurally
homologous to periplasmic sugar-binding proteins.
These structural features imply that the repressor

We describe the current status of the gene
expression database CIBEX (Center for Information
Biology gene EXpression database, http://cibbex.
nig.ac.jp), with a data retrieval system in compliancewith MIAME, a standard that the MGED Society has
developed for comparing and data produced in
micro array experiments at different laboratories
worldwide. CIBEX serves as a public repository for awide range of high-throughput experimental data ingene expression research, including microarraybased
experiments measuring mRNA, serial analysis of gene
expression (SAGE tags), and mass spectrometry
proteomic data. 2l(3) Highly differentiated and conserved sex chromosome
in fish species (Aulopus japonicus: Teleostei,
Aulopidae)

Kinya Ota, Yoshio Tateno and Takashi Gojobori
While highly differentiated and long-conserved sex
chromosomes such as XY and ZW chromosomes are

Laboratory for Gene-Product Informatics

107

observed, respectively, in mammalian and avian
species, no counterparts to such chromosomes wereobserved in fish until we reported in the previous study
that well-conserved and highly differentiated ZW sex
chromosomes existed in the family of Synodontidae.
Then, the problem was if the evolutionary history of
the fish ZW chromosomes was long enough to becomparable to the mammalian and avian counterparts.
To tackle the problem, we had to extend our findingof the fish sex chromosomes further than a family
alone. For this purpose, we chose Aulopus japonicus
that belonged to one of the related families to Synodontidae.
Our cytogenetic and fluorescence in situ hybridization(FISH) analyses have clearly demonstrated that A.
japonicus also has ZW chromosomes. We have also
found that 5S rDNA clusters are located on the Z and
W chromosomes in this species. Using nontranscribedintergenic sequences in the 5S rDNA clusters as peRprimers, we successfully amplified a 6-kb-longfemale-specific sequence on the W chromosome. The
6-kb-long sequence contained one transposable element
and two tRNA sequences. The function of the sequence
remains to be studied. Our Southern blot analysis
confirmed that the 6-kb sequence was located only on
the W chromosome.Therefore, it is now said thathighly differentiated ZW chromosomes have beenconserved over two fish families. As these families werereported to have been diverged 30-60 million years ago,the fish ZW chromosomes have an evolutionary
history corresponding to the history of the families.
This is perhaps the first case that fish sex chromosomes
are shown to have such a long evolutionary lineage. 3)(4) DNA Data Bank of Japan (DDBJ) in XML

Satoru Miyazaki, Hideaki Sugawara, Takashi Gojobori
and Yoshio Tateno
The DNA Data Bank of Japan (DDBJ, http://
www.ddbj.nig.ac.jp) has collected and released more
entries ane). bases than last year. This is mainly due
to large-scale submissions from Japanese sequencing
teams on mouse, rice, chimpanzee, nematoda and otherorganisms. The contributions of DDBJ over the past
year are 17.3% (entries) and 10.3% (bases) of the
combined outputs of the International Nucleotide
Sequence Databases (INSD). Our complete genome
sequence database, Genome Information Broker (GIB),
has been improved by incorporating XML. It is now

108 Laboratory for Gene Function Researchpossible to perform a more sophisticated databasesearch against the new GIB than the ordinary BLAST
or FASTA search. 41(5) Characterization of folding pathways of the type-l
and type-2 periplasmic binding proteins MglB and ArgT

Kenji Kashiwagi, Kiyataka Shiba, Kaoru FukamiKobayashi, Tetsuo Noda, Ken Nishikawa and Hiroshi
Noguchi
The family of periplasmic binding proteins (PBPs)
is believed to have arisen from a common ancestor
and to have differentiated into two types. At first
approximation, both types of PBPs have the same foldpattern, reflecting their common origin. However, theconnection between the main chains of a type 2 PBP
is more complicated than a type 1 PBP's. We have beeninterested in the possibility that such structuralchanges affect the folding of PBPs. In this study, we
have characterized the folding pathways of MglB (atype 1 PBP) and ArgT (a type 2 PBP) by using urea
gradient gel electrophoresis, fast protein size-exclusion
liquid chromatography and hydrophobic dye ANS
binding assay. We found a distinct difference in folding
between these two proteins. The folding of MglB
followed a simple two-state transition model, whereas
the folding of ArgT was more complicated. 5)(6) Standardization of Microarray Data

Brazma Alvis, Kazuho Ikeo and Yoshio Tateno
It is now a routine practice to conduct microarrayexperiments in order to study gene expression undervarious biological, chemical and physical conditions.
However, there is a serious problem with microarraydata produced at different laboratories. As no unified
protocol for the microaray experiment exists, the data
produced at one laboratory cannot be compared to that
produced at another. The data are not like DNA
sequence data. To alleviate this problem, someconcerned researchers organized the Microarray Gene
Expression Data Society (MGED) and have worked toformulate the Minimum Information About Microarray
Experiments (MIAME) that makes it possible for one
to compare two sets of the data produced at two
different laboratories. We describe MIAME particularly to relevant Japanese researchers in the hope that

they understand it and cooperate with MGED. 6) (inJapanese)

Characterization of folding pathways of the type-l andtype-2 periplasmic binding proteins MglB and ArgT.,
Journal of Biochemistry 133, 371-376.

(7) DNA Data Bank of Japan as an Indispensable Public
Database& 7~ij~~=~~~v, ~~-& .~.~, v~-7 07v~T-?t0)~~1~,~Bj!f~~ff~48:

280-285(2003) .
Satoru Miyazaki and Yoshio Tateno
Books

The International Nucleotide Sequence Databases(INSD), which are composed of the DNA Data Bank ofJapan (DDBJ), EMBL Bank, and GenBank, have served
innumerable researchers worldwide as the public
repository of DNA sequence data and the related
information for more than 15 years. INSD have steadily
been recognized and acknowledged, and become now
indispensable for research in bioscience and biotechnology.
To serve researchers in these fields more effectively
and efficiently, DDBJ has developed a number ofdevices in collaboration with the two partners. One ofthem is to form an XML format for data presentation.
The data in the traditional flat file format is straightforward so that one can easily glimpse the data
contents. Recently, however, one entry has tended tocontain more and more pieces of information. Therefore,
it is often difficult for one to extract the pieces of
information of one's interest from such entries. To
make it easier to cope with such a situation, we
adopted XML and developed the DDBJ-XML.71
Publications
Papers

1. Fukami-Kobayashi, K., Tateno, Y. and Nishikawa,
K. (2003). Parallel evolution of ligand specificity
between LacVGalR family repressors and periplasmic
sugar-binding proteins. Mol BioI Evol 20, 267-277.
2. Ikeo, K., Ishi-i, ]., Tamura, T., Gojobori, T. andTateno, Y. (2003). CIBEX: Center for Information
Biology Gene Expression Database. C.R.Biologies 326,
1079-1082.
3. Ota, K., Tateno, Y. and Gojobori, T. (2003) Highly
differentiated and conserved sex chromosomes in fish
species (Aulopus japonicus: Teleostei, Aulopidae) Gene
317C, 187-193.
4. Miyazaki, S., Sugawara, H., Gojobori, T. andTateno, Y. (2003). DNA Data Bank of Japan (DDBJ) in
XML, Nucleic Acids Res 31,13-16.
5. Kashiwagi, K., Shiba, K., Fukami-Kobayashi, K.,
Noda, T., Nishikawa, K. and Noguchi, H. (2003).

7. Miyazaki, S. and Tateno, Y. (2003) DNA Data Bankof Japan as an Indispensable Public Database, In:
Populations and Genetics, B. M. Knoppers ed, pp115-121,
Martinus Nijhoff Publishers, Leiden/Boston.
Database

8. DDBJ is operated by the DNA Data Analysis, Gene
Function Research, Gene-Product Informatics, Research and Development of Biological Databases, andGene Expression Laboratories and Division of Population Genetics. DDBJ collects, annotates and publishes
DNA sequence data on the daily basis. In addition, it
edits the data published by DDBJ, EMBL and GenBank
together four times a year and publishes as a release.
In 2003 DDBJ published four releases as follows.
Release 53 March, 03 23,250,813 entries
29,711,299,332 bases
Release 54 June, 03
25,149,821 entries
32,162,041,177 bases
Release 55 Sept., 03
27,753,140 entries
34,280,225,489 bases
Release 56 Dec., 03
30,405,173 entries
36,079,046,032 bases
9. Dr. Y. Tateno attended the 16th International
Nucleotide Sequence Databases Collaborative Meeting,and the 14th International Nucleotide Sequence
Databases Advisors Meeting held at NLM, NIH,
Bethesda, U.S.A., May, 2003.
10. Dr. Y. Tateno attended the H-Invitational 2,
Leaders' Meeting, Odaiba, Tokyo, November, 2003.
EDUCATION

1. Dr. Y. Tateno gave a lecture on "Genetics, Environments and Health" at the Mishima Welfare Association
Meeting, Mishima, Shizuoka, February, 2003 (inJapanese).
2. Dr. Y. Tateno gave a lecture on "Population
Genetics" at Tokyo Medical and Dental University,
February, 2003 (in Japanese).
3. Dr. Y. Tateno was invited to give a seminar on

Laboratory for Gene Function Research

109

"Comparative genomics" for The 2nd Korea- Japan
Bioinformatics Training Course at KAIST, Daejeon,
Korea, August, 2003.
4. Dr. Y. Tateno gave a lecture on "Evolution ofGenes and Proteins" at Chiba University, November,2003 (in Japanese).
SOCIAL CONTRIBUTIONS AND OTHERS
1. Dr. Y. Tateno served the International Genetics

Federation as Treasurer (1999-2003), and attended the19th International Congress of Genetics, Melbourne,
Australia, July, 2003.
2. Dr. Y. Tateno serves the Microarray Gene Expression Data Society as an Advisory Board Member(2001-), and attended the Board Member Meeting,Colorado, USA, February, 2003, and that, Axan-Provance,
France, September, 2003.
3. Dr. Y. Tateno served the Society of Evolutionary
Studies, Japan as Treasurer and General Secretary(2003).
4. Dr. Y. Tateno serves the Society of Evolutionary
Studies as a Member of the Committee of Genetics
under the Science Council of Japan.
5. Dr. Y. Tateno serves the Genetics Society of Japanas a Member of Editorial Board.

I-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

The publicly available bioinformatics resources,
comprising databases 2• 5 and analytical toolst, have
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 have overlapping functions. We need an information environment

110 Laboratory for Gene Function Researchthat is conducive to efficient and appropriate bioinformatics resource utilization for a wide range of users.
Therefore, the Center for Information Biology and DNA
Data Bank of Japan, in alliance with the National
Institute of Informatics (NIl) and the Mitsubisi
Research Institute, Inc. (MRI) have started a three years
long project, "Research and Development of the New
Generation of Bio-portal", to enhance the information
environment for the relevant user communities. In thisproject, the Laboratory for Research and Developmentof Biological Databases is responsible for the following
tasks:-Development of a meta-database by analyzing thespecifications of major information resources that arelisted in the database issue of the journal "Nucleic
Acids Research".
-Development of an electronic workbench, by use of
web services3 , a workflow description language, andGRID4 technology.
-Development of a semantic web prototype, in collaboration with NIl.
In the meantime, we recognized the need to process
a long job and a large data file to cope with the tsunami
of biological data. However, the SOAP server that we
set up using tools in the public domain could notsatisfy the requirements. Therefore, we have enhanced
the SOAP server for the web services to process a longjob and a large scale data 3•2) Expansion of Genome Information Broker (GIB)

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
150 strains as of April 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)

WFCC and MIRCEN stand for World 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 62 countries
and also the list of their holdings. Any culturecollection is able to register, update and delete theinformation at http://www.wdcm.orgl. We also used
XML technology to organize the Web page. The
WDCM Web site also provides a parallel search engine
named AHMII6•
2) Development of an e- Workbench for BiologicalClassification and Identification (InforBIO)

We continued the development of an e-Workbench
named InforBIO by use of JAVA, XML and a relational
database management system in the public domain.
We have distributed InforBIO to several laboratories
that study microbes and improved the utility androbustness of InforBIO based on the feedback fromthem.
3) An information system for pathogenic microorganisms

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.
( *) The information system on pathogenic microorganisms has been supported by Special Coordination
Funds for Promoting Science and Technology.
(3) Applications of IT to the International Nucleotide
Sequence Database1) Development of Open Annotation ~ystem

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 Agency aST)

2) Exhaustive evaluation of microbial genome information by use of GRID

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 120 microbial genomes
that were stored in the Genome Information Broker asof May, 20034 • This scheme is being applied to morethan 300 thousands ORFs of genomic sequences of 124
microbial species.
Publications
Papers1. Chenna, R, Sugawara, H., Koike, T., Lopez, R,
Gibson, T.]., Higgins, D. G. and Thompson,]. D. (2003).
Multiple sequence alignment with the Clustal seriesof programs. Nucleic Acids Research 31(13), 3497-3500.
2. Miyazaki, S.,Sugawara, H., Gojobori, T. andTateno, Y (2003). DNA Data Bank of Japan (DDBJ) in
XML. Nucleic Acids Research31(1), 13-16.
3. Shigemoto, Y, Ymaguchi, M. Miyzazakim, S. and
Sugawara, H. (2003). Enhancement of the SOAP Serverin DDBJ Web Services to Process Tsunami of Biological Data. Genome Informatics 14, 669-670.
4. Sugawara, H., Nakamura, Y, Ikeo, K., Miyazaki,S., Gojobori, T., Satou, K. and Konagaya, A. (2003).
Analysis of Hrizontal Gene Transfer and Clusteringof Microbial ORFs by Use of a GRID Environment.
Genome Informatics 14, 567-568.
5. Sugawara, H., Gojobori, T., Konno, T., Shigemoto,
Y and Ymaguchi, M.(2oo3). A Prototype of PubMed
Central Japan.Genome Informatics 14, 671-672.
6. Sugawara, H. and Miyazakim, S. (2003). AHMII:
Agent to Help Microbial Information Integration.
Nucleic Acids Research31(13), 3727-3728.
7. ffJJJOffIlJ'}. (2003). 1::: 1"-7' / A 7-?r O)lli)l . 1!!t~¥f000c~n:a-b6<··G~~1fi. mBJ{**~M~48(13), 1857-1862.
Books

8. Satoru, Miyazaki. and Yoshio, Tateno. (2003). DNA
Data Bank of Japan as an Indispensable Public
Database, Bartha maria Knoppers eds. (Martinus
Nijhoff Publishers), pp.115-121.
9. ffJJfJffIlJ'}. (2003). j::trrf-l.'¥: fctMr G *mf:l ~<A T A :1f.~WV,-~f~r -:.nipGO)*'¥:~HJf~1ii!i~

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Laboratory for Research and Development of Biological Databases

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pp.10-3-1O-9.
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I-e. Laboratory for Gene-Expression Analysis
Kousaku Okubo Group

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RESEARCH ACTIVITIES(1) Expression profiling of human genes(Ia) Data integration (BodyMapII): "Do you know how
much of our genes have reliable expression patterns on
the net?"SOCIAL CONTRIBUTIONS AND OTHERS

Osamu Ogasawara and Kousaku Okubo
1. Prof. H. Sugawara organized the International
Conference on Interoperability, Tokyo, November, 2003.
2. Prof. H. Sugawara organized a symposium of the
Frontier Studies and International Networking of
Genetic Resources in Pathogenic Microorganisms,
Tokyo, December, 2003.
3. Prof. H. Sugawara organized the conference of theInteroperability of Biological Data Resources, Mishima,
December, 2003 (in Japanese).
4. Prof. H. Sugawara organized a symposium at the
WFCC workshop of Commercial Use of Microbial
Diversity, Melborne, December, 2003.
5. Task Force of Biological Resource Centers, OECD
Working Party for Biotechnology (Vice-chair).
6. World Federation for Culture Collections, Executive board member and journal editor Genome
Informatics 2004 (Program committee).
7. fiIIIH~1::~~4i;(t>/ZMl~)

8. B *1I¥1l9;o~ ~4i; (.f1Ir$)

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 ofsuch data opened up opportunities to explore thedependence of constitutive expression patterns onother 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
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 JBIRC. (httpsJlwww.jbirc.aist.go.jp/
hinv/h-angel)(Ib) Data generation and analysis; "Are you satisfied by
the present resolution of anatomical expression data?"
Makiko Otsuji and Koichi Itoh
In order to functionally relate genes in coexpression cl\,lster or to deduce promoter sequences
through alignment of upstream regions of them, the112

Laboratory for Research and Development of Biological Databases

resolution of expression pattern should be at the levelof cells or homogeneous cell populations. The vast
majority of the public data do not meet this criterion,
except for those from induction experiments with celllines. Moreover, the majority of the target genes for
drug development such as receptors for signaling
molecules and channels localize in the minority cells
in the complex 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 Itoh 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 for system·
atic and efficient hypothesis generation.
There are three steps in automation of interpre·
tation 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, experts' 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 chemical compounds.
These limitations make the effectiveness of 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
experts' 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 adisease' and 'molecular mechanism of an organs
function.' If there is a book covering essentially all
fundamental topics in medical biology, and if eachpage contains a list of all relevant genes, interpretation
would be achieved by computing fitness of a given genecluster to each page (meaningfulness) and by returning
the title 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 repre·

Laboratory for Gene-Expression Analysis

113

sentation 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. 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
epithelium-specific cytosolic phospholipase A2,
cPLA2delta, induced in psoriatic skin. J BioI Chem.,
279, 12890-12897.
2. Ogasawara, 0., Kawamoto, S. and Okubo, K (2003).
Zipf's law and human transcriptomes: an explanation
with an evolutionary model. C R BioI., 326, 1097-110l.
3. Wakimoto, K, Chiba, H., Michibata, H., Seishima,M., Kawasaki, S., Okubo, K. Mitsui, H., Torii, H. andImai, Y. (2003). A novel diacylglycerol acyltransferase(DGAT2) is decreased in human psoriatic skin andincreased in diabetic mice. Biochemical and Biophysical Research Communications., 310, 296-302.
4. Kawasaki, S., Kawamoto, S., Yokoi, N., Connon,C., Minesaki, Y., Kinoshita, S. and Okubo, K (2003).
Up-regulated gene expression in the conjunctivalepithelium of patients with Sjogren's syndorome. Exp
Eye Res., 77, 17-26.

5. Kaimori, JY., Takenaka, M., Nakajima, H.,
Hamano, T., Horio, M., Sugaya, T., Ito, T., Hori, M.,
Okubo, K and Imai, E. (2003). Induction of glia
maturation factor-beta in proximal tubular cells leads
to vulnerability to oxidative injury through the p38
pathway and changes in antioxidant enzyme activities.
]. BioI. Chern., 278, 33519-33527.
Books

6. Okubo, K and Hishiki, T. (2003) Knowledge
Discovery from the Human Transcriptome, In Introduction to Bioinformatics. Krawetz, SA. and Womble,
D.D. ed. (Human Press).

114 Laboratory for Gene-Expression Analysis

SOCIAL CONTRIBUTIONS AND OTHERS

1. Databese: http://www.jbirc.aist.go.jp/hinv/h-angel
2. Databese: http://bodymap.ims.u-tokyo.ac.jp
3. ACL2003 (41st Annual Meeting of the Association
for Computational Linguistics) Program committee
4. JST~J{Mm1~7"t1-7'7Ar 1/ J A ~ §mJ :3;t---JJT-1-tf-

able to migrate from mid -cell to bipolar positions. We
conclude that the 25 bp sequence with a stem-loop
structure is involved in bipolar localization of orie.

·J. RADIOISOTOPE CENTER(2) Subcellular localization of Escherichia coli proteinsin living cells

Toshiyuki Hatano, Yasuyuki Ogata and Hironori Niki

RESEARCH ACTIVITIES

Identification of a cis-acting site for bipolar
positioning of oriC on the E. coli chromosome(1)

Yoshiharu Yamaichi, Katsunori Yata and Hironori
Niki
We have already reveled subcellular localizationof different DNA segments on the E. coli chromosome
during the cell division cycle by using fluorescence insitu hybridization. These chromosome segments arelocated within the cell in the same order as a genetic
map. A large chromosomal region including replication
origin, oriC shows bipolar localization after replication,
which we call the Ori domain. Then, if a cis-acting site
is involved in bipolar positioning of oriC, the site will
located within the Ori domain.
To identify the cis-acting site, we have constructed
a new mutant, in which a circular chromosome wassplit into two circular chromosomes (Yamaichi and
Niki, 2004). In the mutants, we were able to study aneffect of a cis-acting segment on chromosome
segregation. One of the chromosome-split mutants, inwhich 740 kb of the chromosome region [84.7-89.4 min]in the Ori domain is separated from the original
chromosome, showed irregular localization of nucleoids
and defective in bipolar migration of the chromosomal
segment including orie. These results suggest that thecis-acting site for bipolar positioning of oriCis locatedwithin this chromosomal segment. Furthermore, wehave analyzed that nucleoid localization and oriC
migration in a series of chromosome-split mutants, anddeletion mutants. Finally, we identified the site thatwas responsible for bipolar migration of the oriC
segment was a 25 bp sequence on 89.1 min of the E.
coli map. In addition, multicopy of the sequence in cells
affected on bipolar migration of the oriC segment on
the chromosomes. A plasmid with the sequence wasEscherichia 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. coliW31l0
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 also found that the genes encoding thefilamentous proteins contain a novel genes that
contributes to cell division and the genes of unknown
function.
(3) Stationary phase-induced illegitimate recombinationin Escherichia coli

Yasuyuki Ogata and Hideo Ikeda (Institute of
Molecular Genetics, Medinet Inc.)
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,

RADIOISOTOPE CENTER

115

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 illegit~ate 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 Abio-transducing
phage. Moreover, we found that illegitimate recombination increased in the tag alkAl double mutant inearly-stationary phase, thereby implying that thealkylated lesion may be responsible for the stationary
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.
Since illegitimate recombination results inchromosomal rearrangements including deletion,
duplication, insertion, or translocation, stationary
phase may induce chromosomal rearrangements.
Publications
Paper
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,
doi: 10.1038/sj .em boj. 7600028 Pu blished online 18
December 2003.
Reviews

2. 'J\1JJ*.¥., f=**~, r/-\'l' T!J 7 f;:~ft Q*HIlm./i'iJM
f;:/Z t; tc?t /' /'\ 'l' f{ C: ~~O)gjI)lY-JtJ:f,iJ:(£~1l:J ~ 1l:'¥,

m

75~,

m2~, 2003~ 2 JL 137-143.

3. Ikeda, H., Shiraishi, K. and Ogata, Y. (2004).
Illegitimate recombination induced by double-strand
break and end-joining. Adv. BioPhys. in press.
EDUCATION

Dr. Y. Ogata gave a lecture at the University of Tokyo,
Tokyo, June, 2003 (in Japanese).

116 RADIOISOTOPE CENTER

Publications

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. Onlya preliminary topic in the wild rice research is shown
here.
(1) Preparation of wild rice accessions for comparative
genomics

Toshie Miyabayashi, Mitsugu Eiguchi and Nori Kurata
For making rice genetic stocks good resources, wedeveloped core collection of wild rice. Out of 2,000
accessions 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 Rnak1,
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 has been started_ Searches for structural andfunctional diversity between genes of different genomes
are performed by cDNA clone sequencing and by
microarray expression profile analysis. Highly divergent genes between the genomes may explain the wayof evolution and the gerie function.

Papers1. Miyoshi, K, Ito, Y, Serizawa, A and Kurata, N.
(2003)_ OsHAP3genes regulate chloroplast biogenesis
in rice. Plant J 36, 532-540.
2. 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. Nat!. Acad. Sci. USA
101, 875-880.
3. Nonomura, KI., Miyoshi, K, Eiguchi, M., Suzuki,T., Miyao, A, Hirochika, H. and Kurata, N. (2003). The
MSPl gene is necessary to restrict the number of cells
entering into male and female sporogenesis and toinitiate anther wall formation in rice. Plant Cell 15,
1728-1739.
4. Suzuki, T_, Nonomura, K-I., Takeda, N.,
Murayama, Y and Kurata, N. (2003). Application ofenhanced lipofection to rice transformation. Rice Genet,
Newslet. 20, 116-118.
5. Nonomura, KI., Nakano, M., Murata, K, Miyoshi,
K, Eiguchi, M., Miyao, A, Hirochika, H. and Kurata,
N. (2003). 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.
6. Nonomura, K-I., Nakano, M., Fukuda, T., Eiguchi,M., Miyao, A, Hirochika, H_ and Kurata, N. (2004). The
novel gene HOMOLOGOUS PAIRING ABERRA TION
IN RICE MEIOSIS1 of rice encodes a putative
coiled-coil protein nequired for homologous chromosome pairing in meiosis. Plant Cell 16, 1008-1020.
7. 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
8. Ito, Y, Eiguchi, M. and Kurata, N. Establishmentof an enhancer trap system with Ds and GUS forfunctional genomics in rice. Mol. Genet. Genomics: inpress.
Book

9. Kurata, N. and Fukui, K (2003). Chromosome
research in genus Oryza. 'Monograph in genus Oryza.'
Nanda JS ed. Science Publisher. pp 213-261.

Publications and activities shown in the Plant
Genetics Lab. were all done with the members of theExperimental Farm.

EXPERIMENTAL FARM

117

L. TECHNICAL SECTION

The 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 E. coli 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 thetechnical officials.
Publications
Papers1. Chihara, T., Kato, K, Taniguchi, M., Ng, J., andHayashi, S. (2003). Rac promotes epithelial cell rearrangement during tracheal tubulogenesis in Drosophila.
Development 130(7),1419-1428.
2. Kato, M., Miura, A., Bender,]., Jacobsen, S.E., andKakutani, T. (2003). Role of CG and non- CG methylation in immobilization of transposons in Arabidopsis.
Current Biology 13, 421-426.
3. Kinoshita, T., Miura, A., Choi, Y, Kinoshita, Y,
Cao, X., Jacobsen, S.E., Fischer, R.L., and Kakutani, T.
(online publication Nov. 20, 2003). One-way control of
FWA imprinting in Arabidopsis endosperm by DNA
methylation. Science, in press.
4. Miura, A., Kato, M., Watanabe, K, Kawabe, A.,
Kotani, H., and Kakutani, T. (online publication Nov.
8,2003). Genomic localization of endogenous mobile
CACTA family transposons in natural variants ofArabidopsis thaliana. MGG, in press.
5. Nonomura, KI., Miyoshi, K, Eiguchi, M., Suzuki,T., Miyao, A., Hirochika, H., and Kurata, N. (2003). The
MSPl gene is necessary to restrict the number of cells
entering into male and female sporogenesis and toinitiate anther wall formation in rice. Plant Cell 15,
1728-1739.

118 TECHNICAL SECTION

6. Sugimura, K, Yamamoto, M., Niwa, R., Satoh, D.,
Goto, S., Taniguchi, M., Hayashi, S., and Demura, T.
(2003). Distinct developmental modes and lesioninduced reactions of dendrites of two classes ofDrosophila sensory neurons. J. Neurosci. 23(9),3752-3760.
7. Takayama, Y, Kamimura, Y, Okawa, M, Muramatsu,
S., Sugino, A., and Araki, H. (2003). GINS, a novel
multiprotein complex required for chromosomal DNA
replication in budding yeast. Genes Dev. 17(9),1153-1165.

BIOLOGICAL SYMPOSIUM 2003
Jan.7
Jan.14Jan.15Jan.15Jan.15Jan.17Jan.20Jan.21
Jan.21
Jan.23Jan.28Jan.29Jan.30
Feb.5
Feb.5
Feb.5
Feb.10Feb.17
Feb.17
Feb.19Feb.19Mar.5
Mar.6
Mar.13
Mar.19Mar.26Mar.27
Apr.23
Apr.25/''--{ TT / 11ii'itUjf:-r~JfH/\td'H~-rfPI5:E (Hideki Nakayama)
Segregation distortion caused by nuclear Ran GAP activity in Dro~ophila CAyumi Kusano)*Bl1ffil)1.1Z.:I+ C: 17'11£'11:7' p -7"7 A 1;:: J: Q t$*Ut-*IIIIffil(7)5t1~1I11j1fEll CKinichi Nakashima)*JT:JsHfli~f'tiJ~tll<Dynamical Biology CKenji Nagai)
1o.*Bllffilrc.:ro ft Q!! P '"'<'T /'i~mf14:(7)mIlJJ P:(.i] ft-C (Hiroshi Kimura)§:*5t-rn'U:f"~1t\ : ~Bf'f(7)*5:E'I1:, tlTtLtck7j., (~~"'-(7)~fll CfJUfB) CAkira Kinjo)~.(7)mM~~mKJ:Q~/AM~~-~(7)M~c:~m~oos~uO~~)

Molecular mechanism for signaling cytokinesis in animal cells CMaki Hori).:r../ F ~ -A-::::flvs;Mt~~J2H~(7)1o.fJI!~fl~ c: } jJ :=..'XA CMasato Ohashi)~mC.elegans(7)t$i\\l*-C~fl~TQ'1'/ A I) /~~f*:t§fPIJ!{~+daf-2

CKotaro Kimura)

Germ/Soma Distinction in C.elegans. CYingdee Unhavaithaya)
REGULATION OF p53-MEDIATED TRANSCRIPTION BY NONHISTONE CHROMATIN PROTEIN
CTapas K Kundu)
CDKf.: J: QDNA~iMII11j1fEll c: If / A (7)'tC5:E*!ft-fmf14: CSeiji Tanaka):Ii.L~~$;DNAf~1iK;JoftQ~Bf'fIZ.:l-r(7)f§1L{lpm(7)~~\!1j
CYoshihiro Matsumoto)DNAm~T.:r. 'Y !!;f-'1' / r- :::z / r- P - Iv C: ~(7)DNAm{JJ~~.~f14: CKatsunori Sugimoto)Novel Y-family DNA polymerases and their roles in mutagenesis CTakehiko Noumi)
ES*BlIffil(7)*5t1~'I1:*!ft-fC: ECAT4 CKaoru Mitsui):\'-'1' P ~ 3 '7 s; 3 '7 /"\.:r..f.:;Joft Q!! T!! 7 .@.t1~7l<~1H~':!0)5t-rmf14: C: ~(7)Jft1~ CAya Takahashi)h!!-!T//"~-lfT~m1t\k5t-r~I'fIJ!{~~. *IIIIffilJl~~~~9l: CAkira Kawabe)
Inductive Tissue Interactions: Looking through the eye into the heart CYasuhide Furuta)
T"- ~.rZ- AiJ~ G (7)~.tI!Il±J : m¥l1I±Mi3(7)t.lt~iJ~ G CGe Xijin)
Signature of balancing selection in Arabidopsis; Balancing selection and linkage disequilibrium of
RPS5 presence/absence polymorphism in Arabidopsis thaliana CHitoshi Araki)"Gene Finding in DNA Sequences: from Viruses to High Eukaryotes" CMark Borodovsky)
HARI ~1l' L- tc~1o. C: ~1'l~pj(;(7)~:!it~1I11j1fEll ~ AT A CMasayoshi Kawaguchi)
Study of DRB gene polymorphism in crab-eating monkey CMacaca fascicularis) (Antoine Blancher)
A Role for Ran-GTP and Crml in Blocking Re-Replication CRyuji Yamaguchi)
Axon Guidance in the developing Xenopus visual system CChristine Holt)*Bl1ffil1*J} /7- v / r- 77-1 'Y !! (7) ~-'1' T ~ !! A CAkihiko Nakano)*Bl1ffil1*J!J\Iffil~J2S*(7)f14:xg1o.~~: ARF C: (7):t§1L{!pm f.: J: Q 7" ?f7° ~ - ~ :.//~!! f'fGGA(7)TGNMt"'-(7) F 'Y:\'- / -7" CSoichi Wakatsuki)
May.12
May.19
May.26May.28May.28
Jun.6
Jun.23
Jun.25
Jun.25
Ju1.10Ju1.14
Ju1.22
Ju1.23} ?fjJ'I1:lk:5:EJ!{~-r(7);f- S;~ 3 T Iv!! P -:=../ -7" CMasaru Matsuda)
CYasumasa Bessho)
1o.~~~v':~TQ~.(7)tI!Il±JC:~{33~CToshihisa Takagi)
Xite, X-inactivation intergenic transcription elements that regulate the probability of choice(Yuya Ogawa)
MM:\'-T--e(7)~~C: £f'f(7)~~ (Katsuya Takenaka)
8tr.ll.Jl*$*f~~m~~flJm lJc ry !! T /~~ C: ,Jlij1Mo;f~~CTL(7)~1;f K J: Q *,~J!{~-rr&~ CKoichi Ito)
Building hearts: genetic perturbations, functional networks, and systems biology(Joseph H. Nedeau)~ 3'7 s; 3 '7 /"\.:r..fff-r~pj(;v':;JovtQMyosin V I (7)*BlIffilI*J~fj~v':01t\-C(7)m*JT CTatsuhiko Noguchi)
f*im~pj(;~5i:lfi2TQ 5t-r~~to) } jJ :=.. 'XA-e77 7 -1 'Y ~ .::z.~Mt5t{~v':~TQ~M~~f*(7)*~c:m*JT

CIchiro Masai)*BlIffilWJM~1I11j1fEllTQAurora-A:\'-T--e(7)mftE1~mf14:(Takeshi Urano)::L t:":\'-T /~ AT A (7)II1'J1fEIlmf14: C: -t-(7)1o.fJI!mfj~m*JT CTomoki Chiba)
Targeted Expression of Temperature-Sensitive Dynamin to Study Neural Mechanisms of Complex
Behavior in Drosophila CToshihiro Kitamoto)
The Intimate Association of Transposable Elements and Genes (Thomas Bureau)

BIOLOGICAL SYMPOSIUM 2003

119Aug.11
Aug.19Aug.22
Aug.25
Sep.I
Sep.lO
Sep.lO
Sep.24
Sep.29
Oct.1
Oct.8
Oct.9
Oct.15
Oct.16
Oct.16
Oct.20
Oct.22
Oct.23
Oct.23
Oct.27
Oct.30
Oct.30
Oct.31
Oct.31
Nov.5
Nov.6
Nov.7
Nov.12
Nov.13
Nov.13
Nov.20Nov.26
Nov.28
Dec.2
Dec.2
DecA

Dec.5
Dec.8
Dec.15
Dec.16
Dec.17
Dec.18~~~ C: L--c O)~~~~~ : ~~ . m!fj~;fl:j~O)mtlJlIr.I"Httc.~,7j. <Hiroshi Ito}

*DI1ffi!rJ;]3t~oo-O)7t.:r:li1t: :li1t~J3[J::~Ir.{f '5 7 ~ JD~~~!/~:7-:/O)m:#T (Takeshi Ito)
(Yuji Kageyama)~Jt'~¥R C: cGMP~ -7'-r }v*£ImIr. J:: Q~.ffiO)f*ffi: C: fr~O)fljfJm (Manabu Hujiwara)
Retargeting Retroviral Entry (Monica J. Roth)SPB asymmetry and spindle positioning in yeast <Hiromi Maekawa}DNA{~1i . 1l~ 1r.~-9-T Q7t~M£3:slnl{~.:rO)$1!it C: -{-O)m:#T (Yasuhiro Tsutsui)
The Nature Journals and the world of scientific publishing (Charles Jennings)*DI1ffi!rJ;]1t~&);t,0)~G ri' C: *DIn~m!fj~ (Tatsuo Shibata)
Adaptive Mutation (Susan Rosenberg)
Microarray Applications in Developmental Toxicology (Thomas B. Knudsen)
Migrating cells can not only receive signals from their environment, but also deliver signals to it(Norbert Konig)~1it*DIn~~tM~T Q 1i$ C: L- -c 0)3P § ~(8*D1Iffi!~ (Takashi Adachi)
Epigenetic inheritance in mammals (Emma Whitelaw)
Sequence and analysis of the Dictyostelium genome (William F. Loomis)
MITOTIC MODIFICATION OF TOPOISOMERASEII BY SUMO-2 (Yoshiaki Azuma)
Open reading frame categorization by cellular automata and genetic algorithms (Johannes Olson)
1r.;tolt'*tlJtO) j. t.J ::::XA : Ir.;tolt'it!!~;to J:: lff=j~~Mi;lJ' G O)m,~ (Naoshige Uchida)
Germline transgenesis of an ascidian Ciona intestinalis by a Tell mariner superfamily transposon
Minos. (Yasunori Sasakura)
Genetic and genomic approaches to the Drosophila immune response (Bruno Lemaitre)-e' 7"'7 7 -{ '/ ~ ;:l.~t~~*DIIffi!±'lH~~O)litftC: -{-O)!fj.~ Ir. J:: Q*~.:r"-7:7 - (Noriyoshi Sakai)-li7''7 7 -{ '/ ~ ;:l.~mlt'td&lrJ;]:Yt~~f*O)m:#T <Daisuke Kojima}*DI1ffi! ~ -7'-r !J :/ -7' (7)7t.:rJ€~JI:~~ (Atsushi Kawakami)j. .;It.J ~mlt'tc.~~f*O)f*~(8A -7 !J -.::.:/ -7·Ir. J:: Q lNf~iNIIlr.7&0 td$*£~O)~mUf%X;m!~O)m:#T(Makoto Furutani-Seiki)
ff*~~0)$mflll*£~0):li1t; 1l!f'm~~Y j. r7-r:¥~mlt'tc.7t.:rJ€~~(8m:#T (Yasunori Murakami)
Individual variation in aggression of feral rodent strains: a standard for the genetics of aggression
and violence? (Jaap M. Koolhaas)
Molecular mechanisms controlling the integrity of replicating chromosomes (Marco Foiani)~ 3 r7 :; 3 r7 /~.:r..flIl*£~ ~ m It' te.*DIn~m!fj~O)~*DI~t~~(8m:#T (Emiko Suzuki)
Redox regulation of germline and vulval development in Caenorhabditis elegans<Yukimasa Shibata)
Molecular mechanisms specifying muscle-identity and myotube number in the Drosophila adult(K. Vij ay Raghavan)
Environmental factors, DNA at-risk motifs and genetic defects that severely challenge genome
duplication. (Dmitry A. Gordenin)
Insulin signaling and growth control in Drosophila (Ernst Hafen)
DNA Replication Control and Genome Stability (John F.x. Diffley)
Natural Selection and Random Drift in Genome Evolution (Giorgio Bernardi)
C.elegansO)fIMf*(7):k ~ ~ O)fljfJmm!~O)m:#T (Yasumi Ohshima)Controlling the events of the cell cycle (Tim Hunt)'i~EJg.:rMybO)~(8J1:{~.:rO)FiiJJEC: ,i~fljfJmm!~O)m:#T (Masahiro Okada)
Studying the Cell Cycle by Functional Proteomics (Karim Labib)
Methylation crosstalk between histones and DNA in Neurospora (Hisashi Tamaru)
Forward Genetics of Vision-dependent Behaviors in Zebrafish (Akira Muto)
Transposable elements, RNA interference and the origin of heterochromatin (Robert Martienssen}
Sleeping Beauty: Evolution, Regulation and Genetic Applications of DNA Transposons in Vertebrates(Zsuzsanna Izsvak)~ 3 r7:; 3 r7 /~.:r..JI:{~.:r:litJl!1E~i'r1*O)~'@.1*i;g~m!~

120 BIOLOGICAL SYMPOSIUM 2003

FOREIGN VISITORS IN 2003
Jan.21
Jan.28Jan.29Jan.3D
Feb.5
Feb.19Mar.5
Mar.6
Mar.19Mar.26Mar.27May.28Jun.23
Jun.25
Ju1.22
Ju1.23
Sep.l
Sep.lO
Sep.24
Oct.1
Oct.8
Oct.9
Oct.16
Oct.16
Oct.2D
Oct.23
Oct.27
Oct.3D
Nov.6
Nov.7
Nov.13
Nov.26
Nov.28Maki Hori
Department of Physiology, University of Massachusetts Medical School
Yingdee Unhavaithaya Department of Cell Biology, University of Massachusetts Medical
School,Worcester
Tapas K Kundu
Transcription and Disease Laboratory
Jawaharlal Nehru Centre for Advanced Scientific Research ,Bangalore:560064
INDIA.
Seiji Tanaka
Cancer Reserach u.K.
Yoshihiro Matsumoto Department of Pharmacology, Fox Chase Cancer Center,Philadelphia, PA,
USA
Yasuhide Furuta
Department of Biochemistry & Molecular Biology University of Texas, M.D.
Anderson Cancer Center, Houston, TX, USA
Hitoshi Araki
Department of Ecology and Evolution, University of Chicago
Mark Borodovsky
Schools of Biology and Biomedical Engineering, Georgia Institute ofTechnology, Atlanta, Georgia, USA
Antoine Blancher
Laboratoire d'Immunogenetique Moleculaire, Universite Paul Sabatier,
Hopital Rangueil FRANCE
Ryuji Yamaguchi
University of California San Diego
Department of Anatomy, University of Cambridge
Christine Holt
Yuya Ogawa
Department of Molecular Biology,Massachusetts General Hospital,Harvard
Medical School
Case Western Reserve University, Cleveland OH, USA
Joseph H. Nedeau
Tatsuhiko Noguchi
Miller lab, Department of Biology, Washington university in St. Louis, MO,USA
Toshihiro Kitamoto
Department of Anesthesia, Roy J. and Lucille A. Carver College ofMedicine,University of Iowa
Department of Biology, McGill University
Thomas Bureau
Department of Biochemistry & Molecular Biology, University of Medicine
Monica J. Rothand Dentistry of New Jersey, Robert Wood Johnson Medical School, USA
Hiromi Maekawa
Paterson Institute for Cancer Research, UK
Executive Editor, Nature Research Journals
Charles Jennings
Department of Molecular and Human Genetics Baylor College of Medicine
Susan Rosenberg
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson
Thomas B. Knudsen
University
EPHE and INSERM, University of Montpellier 2, France.
Norbert Konig
Department of Biochemistry, University of Sydney, Australia
Emma Whitelaw
Division of Biological Sciences, UCSD
William F. Loomis
LGRD/NICHD, USA
Yoshiaki Azuma
Cold Spring Harbor Laboratory
Naoshige Uchida
Centre de Genetique Moleculaire, CNRS
Bruno Lemaitre
The Biological Laboratories, Harvard University
Daisuke Kojima
Department of Animal Physiology, Biological Center, University of Groningen,
J aap M. Koolhaas
The Netherlands
F.I.R.C. Institute of Molecular Oncology, Milan, Italy
Marco Foiani
National Centre for Biological Sciences, Tata Institute of Fundamental
K. Vij ay Raghavan
Research
Zoological Institute, University of Zurich
Ernst Hafen
Cancer Research UK, London Research Institute, Clare Hall Laboratories
John F.X. Diffley

FOREIGN VISITORS IN 2003

121

Dec.2
Dec.4
Dec.8
Dec.15
Dec.16
Dec.17
Dec.18Giorgio Bernardi
Tim Hunt
Karim Labib
Hisashi Tamaru
Akira Muto
Robert Martienssen
Zsuzsanna Izsvak

122 FOREIGN VISITORS IN 2003

President, Stazione Zoologica Anton Dohrn, Italy
Cancer Research UK London Research Institute Clare Hall Laboratories
Cancer Research U.K.,Paterson Institute for Cancer Research
University of Oregon, USA
Department of Physiology, University of California, San Francisco
Cold Spring Harbor Laboratory
Max Delbruck Center for Molecular Medicine, Berlin, GermanyAnnual Report of the National Institute of Genetics. No.54
Issued by Director-General; Yoshiki Hotta. D.Med.
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 Veda & Hironori Niki
Published by National Institute of Genetics