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Highlights of 2005 (http://www.nig.ac.jp/hot/index.html)

1. A novel RNA-binding protein
machinery for local translationIn

neuronal RNA granules: Regulatory

Shiina N, Shinkura K, Tokunaga M.

J. Neurosci, 25 (I7), 4420-4434,2005
2. The transcription factor Mesp2 establishes segmental borders by suppressing
Notch activity.
Morimoto M, Takahashi Y, Endo M, Saga Y.
Nature, 435

3. Tsix Silences Xist through Modification of Chromatin Structure
Takashi Sado, Yuko Hoki, and Hiroyuki Sasaki
Developmental Cell, 9(1},in press,2005

4. Generation of neuronal diversity through switching genes expression in neuralstem cells
Kanai MI, Okabe M, Hiromi Y.
Developmental Cell, Bt2) 203-213, 2005

CONTENTS
Introduction···································································································· 1
STAFF
. 2
ADVISORY COMMITTEE················································································ 6
ADVISORY BOARD························································································· 7
A. DEPARTMENT OF MOLECULAR GENETICS

A-a. Division of Molecular Genetics
Tatsuo Fukagawa Group
Identification and characterization of the CENP-H-I complex proteins in higher vertebrate
cells
Masahiro Okada, Tetsuya Hori, Mi-Sun Kwon, Aussie Suzuki, Mayumi Takahashi,
and Tatsuo Fukagawa

8Functional analysis of CENP-50 that is required for recovery from spindle damage
Tetsuya Hori, Masahiro Okada, Tomoko Motohashi, Kazuko Suzuki,
and Tatsuo Fukagawa

8Functional analysis of the CENP-C complex, which associates with the Mis12 complex
Mi-Sun Kwon, Masahiro Okada, Tetsuya Hori, and Tatsuo Fukagawa
Functional roles of the RNAi machinery in verterbrate centromeres
Tomoko Motohashi, and Tatsuo Fukagawa
A-b. Division of Mutagenesis
Fumiaki Yamao GroupMultifunctional role of Mcll/Slr3 in DNA replication, repair and chromatin structure
Toyoaki Natsume, Yasuhiro Tsutsui and Fumiaki Yamao
A-c. Molecular Mechanism Laboratory
Hiroshi Mitsuzawa Group
Regulatory roles of the Rpb7 subunit of eukaryotic RNA polymerase II
Hiroshi Mitsuzawa
Identification of ammonium transporter genes in S. pombe
Hiroshi Mitsuzawa
A-c. Molecular Mechanism Laboratory
Hiroaki Seino GroupAn in vitro ubiquitination assay of mitotic cyclin
Hiroaki Seino
Regulation of the DNA damage checkpoint by the ubiquitin pathway
Hiroaki Seino
A-d. Division of Nucleic Acid Chemistry
Tokuko Haraguchi Group
Dynamics of nuclear structure and function
Tokuko Haraguchi

9
9

10

11
11

12
12

13

A-d. Division of Nucleic Acid Chemistry
Hiroshi Iwasaki Group
Functional overlap between RecA and MgsA (RarA) in the rescue of stalled replication
forks in Escherichia coli
Hiroshi Iwasaki····················································································· 14
Genetic and physical interactions between Schizosaccharomyces pombe Mcll and Rad2,
DnaZ and DNA polymerase alpha: evidence for a multifunctional role of Mcll in DNA

CONTENTS

replication and repair
Hiroshi Iwasaki····················································································· 14
Structue-function analysis of the three domains of RuvB motor protein
Hiroshi Iwasaki····················································································· 14
Role of the Schizosaccharomyces pombe F-box DNA helicase in processing recombinationintermediates
Hiroshi Iwasaki····················································································· 14
B. DEPARTMENT OF CELL GENETICS

B-a. Division of Cytogenetics
Tamotsu Yoshimori Group
Intracellular inclusions containing mutant 0: i-antitrypsin Z are propagated in the absenceof autophagic activity
Takahiro Kamimoto, Shisako Shoji, Tunda Hidvegi, Noboru Mizushima, Kyohei
Umebayashi, David H. Perlmutter, and Tamotsu Yoshimori
16
LC3 interacts with dynein motor and has an important role in microtubule-dependent
autophagosome movement
Shunsuke Kimura and Tamotsu Yoshimori
16
Molecular dissection of internalization of Porphyromonas gingivalis by cells using fluorescentbeads coated with bacterial membrane vesicle
Kayoko Tsuda, Atsuo Amano, Kyohei Umebayashi, Hiroaki Inaba, Ichiro Nakagawa,Yoshinobu Nakanishi, Tamotsu Yoshimori
17
Ubiquitin-dependent sorting of the epidermal growth factor receptor in the endocytic
pathway
Kyohei Umebayashi and Tamotsu Yoshimori
17
B-b. Division of Microbial Genetics
Hiroyuki Araki GroupThe pre-Landing complex of replication proteins formed in a CDK-dependent manner
Sachiko Sakamoto, Kazuyuki Hirai, Yoichiro Kamimura, and Hiroyuki Araki
18
Regulatory mechanism of the complex formation between Sld2 and Dpbll by
cyclin-dependent kinase (CDK)
Yon-Soo Tak, Yoshimi Tanaka, Yoichiro Kamimura and Hiroyuki Araki············ 19
CDK targets in the initiation of DNA replication
19
Seiji Tanaka and Hiroyuki Araki",B-c. Division of Cytoplasmic Genetics
Kazuto Kobayashi Group
Mechanisms of Neural Circuitry Underlying Behavioral Control
Kazuto Kobayashi·················································································· 20
B-c. Division of Cytoplasmic Genetics
Masayuki Yamamoto Group
HrslpIMcp6p on the meiotic SPB organises astral microtubule arrays for oscillatory nuclear
movement
21
Masayuki Yamamoto
The C. elegans Homologue of Deleted in Azoospermia is Involved in the Sperm/Oocyte Switch
22
Masayuki Yamamoto

C. elegans CPB-3 interacts with DAZ-l and functions in multiple steps of germline
development
22
Masayuki Yamamoto

CONTENTS

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

C-a. Division of Development Genetics
Yasushi Hiromi GROUP
Control of ligand trapping for the recognition of spatial cues
Masaki Hiramoto and Yasushi Hiromi

23

Intrinsic sub-axonal compartmentalization in Drosophila neurons
Takeo Katsuki, Masaki Hiramoto and Yasushi Hiromi

23

The relationship between sub-axonal localization of proteins and glycosylation
Tohru Umemura, Takeo Katsuki and Yasushi Hiromi
23
Neutralization of repulsive-axon guidance cue on target neurons
Fumikazu Suto, Hajime Fujisawa and Yasushi Hiromi
23
Exploring the factors that regulate temporal gene expression profile in neuronal precursors
Makoto Kanai and Yasushi Hiromi
24
Developmental context for Notch-dependent PROS expression in longitudinal glial cells
Yoshihiro Yuasa and Yasushi Hiromi
24
Stem cell formation in Drosophila germline requires gonadal somatic cells
24
Miho Asaoka, Satoru Kobayashi, Ryu Ueda and Yasushi Hiromi
C-a. Division of Development Genetics
Toshitaka Fujisawa GROUP
Systematic identification of peptide signaling molecules in Hydra
Toshitaka Fujisawa, Chiemi Fujisawa, Eisuke Hayakawa, Yukihiko Noro,
Akemi Hayashiuchi, Hiroshi Shimizu and Toshio Takahashi···························· 25
Aboral end of Hydra and oral end of higher organisms share a common ancestral origin
26
Hiroshi Shimizu, Yasuharu Takaku & Toshitaka Fujisawa
C-b. Division of Gene Expression
Susumu Hirose Group
Role of histone modifications and chromatin remodeling in epigenetic gene expression
Takahiro Nakayama, Kenichi Nishioka, Yi-Xin Dong, Tsukasa Shimojima,
Kazuma Hanai, Takashi Yamamoto, Koji Akasaka and Susumu Hirose"
26
Chromatin transcription
Mikage Nakajima, Kenichi Nishioka, Toshiharu Komori, Tadashi Wada, Hiroshi Handa,
27
and Susumu Hirose
Role of DNA topology in the formation of active chromatin
Hirofumi Furuhashi, Kuniharu Matsumoto, Mikage Nakajima, Youhei Ogasawara
and Susumu Hirose······································

27

Linker histone variants control chromatin dynamics during early embryogenesis
Hideaki Saeki, Keita Ohsumi, Hitoshi Aihara, Takashi Ito, Susumu Hirose, Kiyoe Ura
and Yasufumi Kaneda
28
Solution structure of the HMG-box domain in the SSRP subunit of FACT
Nobuyuki Kasai, Yasuo Tsunaka, Izuru Ohki, Susumu Hirose, Kosuke Morikawa
and Shin-ichi Tate················································································· 28
Roles of the heterotrimeric G proteins in Drosophila gastrulation
Naoyuki Fuse, Takuma Kanesaki and Susumu Hirose
28
Anterior epidermis-specific expression of the cuticle gene EDG84A is controlled by many
cis-regulatory elements in Drosophila melanogasterYasunori Kayashima, Susumu Hirose and Hitoshi Ueda
29
Molecular mechanisms of Sonic hedgehog mutant effects in holoprosencephaly
Tapan Maity, Naoyuki Fuse and Philip A. Beachy" .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... 29

CONTENTSC-c. Division of Molecular and Developmental Biology
Koichi Kawakami Group
Transposon tools and methods in zebrafish
Koichi Kawakami
Expression pattern-driven insertional mutagenesis in zebrafish
Saori Nagayoshi, Eriko Hayashi, Kazuhide Asakawa, Akihiro Urasaki,
Kazuki Horikawa, Hiroyuki Takeda and Koichi Kawakami

30

30

Development of the Gal4-enhancer trap system using the Tol2 transposable element
Kazuhide Asakawa, Akihiro Urasaki, Tomoya Kotani, Saori Nagayoshi,
Yasuyuki Kishimoto, Masahiko Hibi and Koichi Kawakami······························ 31
misty somites, a maternally expressed gene identified by the transposon-mediated gene trap
method in zebrafish, is required for somite boundary formation
Tomoya Kotani, and Koichi Kawakami
31
Remobilization of integrated transP9sonS: the jump starter system in zebrafish
Akihiro Urasaki, Kazuhide Asakawa and Koichi Kawakami .... · .. · .......... · .... · .. · .. · 32
The transposon-mediated Gal4 gene trapping identified genes expressing in unique patterns
in the adult zebrafish
Kanta Mizusawa, Kazuhide Asakawa, Akihiro Urasaki, Tomoya Kotani,
Saori Nagayoshi, Yasuyuki Kishimoto, Shigeru Kondo and Koichi Kawakami········ 32
The zebrafish bobtail mutation identifies the MOCSl (the molybdenum cofactor synthesis
step-I) gene that is required for the posterior body formation as an essential elementof the Fgf/ERK signaling
Yasuyuki Kishimoto, Sumito Koshida, Makoto Furutani-Seiki, Atsushi Kawakami,]. Reiss, G. Schwarz, Yuji Kohara, Hisato Kondoh and Koichi Kawakami············ 33
The Zebrafish pob Gene Encodes a Novel Protein Required for Survival of Red Cone
Photoreceptor Cells
Michael R. Taylor, Satoshi Kikkawa, Vishvanathan Ramamurthy, Koichi Kawakami,
and Susan E Brockerhoff
33
C-d. Division of Physiological Genetics
Fumitoshi Ishino Group~~.~m~~~~~~~~*~~y~~~m/~~~.

EPIGENETIC VIEW OF MAMMALIAN DEVELOPMENT/FUMITOSHI ISHINO

34

C-d. Division of Physiological Genetics
Yukiko Gotoh Group
The Wnt-B-catenin pathway directs neuronal differentiation of cortical neural precursor
cells
Yusuke Hirabayashi, Yasuhiro !toh, Norihisa Masuyama and Yukiko Gotoh ........ 35
Hes binding to STAT3 mediates crosstalk betweenNotch and JAK-STAT signaling
Sachiko Kamakura, Koji Oishi, Takeshi Yoshimatsu, Norihisa Masuyama,
Yukiko Gotoh
36
Notch promotes survival of neural precursor cells via mechanisms distinct from thoseregulating neurogenesis
Koji Oishi, Sachiko Kamakura, Yuko Isazawa, Takeshi Yoshimatsu, Keisuke Kuida,········36
Norihisa Masuyama and Yukiko Gotoh
JNK antagonizes Akt-mediated survival signals by phosphorylating 14-3-3
Jun Sunayama, Fuminori Tsuruta, Yasunori Mori, Norihisa Masuyama
and Yukiko Gotoh

CONTENTS

36

D. DEPARTMENT OF POPULATION GENETICS

D-a. Division of Population Genetics
Naruya Saitou GroupApe Genome Project Silver
Saitou Naruya, Sumiyama Kenta
Molecular evolution of developmental regulation
Sumiyama Kenta, Kim Hyung-Cheol, Saitou Naruya

38············38

Analysis of evolution of closely related populations
Saitou Naruya, Ishibashi Minaka

38

Evolution of blood group genes
Saitou Naruya

38

Large scale evolutionary analysis of many gene sequences
Saitou Naruya, Ezawa Kiyoshi

38

Development of new methods for the study of gene evolution
Saitou Naruya Kirill Kryukov

38

D-a. Division of Population Genetics
Toshiyuki Takano Group
Detecting single-generation selection acting on inversion polymorphism in Drosophila
Toshiyuki Takano-Shimizu, Nobuyuki Inomata, Masanobu Itoh, Rumi Kondo,
Miki Ohshima and Yutaka Inoue······························································ 40
A high frequency null mutant of an odorant-binding protein, Obp57e, in Drosophilamelanogaster40
Aya Takahashi and Toshiyuki Takano-Shimizu
Analyses of assortative mating and morphological differentiations in populations ofDrosophila melanogaster
Aya Takahashi and Toshiyuki Takano-Shimizu ·············································40
Spontaneous mutation spectrum in Drosophila melanogasterYutaka Watanabe, Aya Takahashi and Toshiyuki Takano-Shimizu
41
Nucleotide variation and linkage disequilibrium in 150-kb region around the Scr gene in
D. melanogaster and D. simulans
Yutaka Watanabe, Haruki Tatsuta and Toshiyuki Takano-Shimizu···41
Fluctuation in linkage disequilibrium scale in Drosophila melanogaster genome
Toshiyuki Takano-Shimizu, Masanobu Itoh, Rumi Kondo, Nobuyuki Inomata,
41
Noriko Nanba, Masako Hasegawa, Miki Ohshima and Yutaka Inoue
D-c. Division of Theoretical Genetics
Sumio Sugano Group
Functional Genomics Based on the full-length cDNA collection
42
Sumio Sugano
E. DEPARTMENT OF INTEGRATED GENETICS

E-a. Division of Human Genetics
Hiroyuki Sasaki Group
Establishment and maintenance of DNA methylation imprints in the germline and earlymouse embryos
Hiroyuki Sasaki, Yuzuru Kato, RyutaroHirasawa, Kenji Kumaki, Hiroyasu Furuumi,
Masahiro Kaneda, Masaki Okano, En LI, Mizue Hisano, Masami Nozaki, Tomohiro
Suzuki, Shigeharu Wakana, and Toshihiko Shiroishi
45
Comparative analyses of the distal imprinted domain on mouse chromnosome 7 and the

CONTENTS

orthologous domain on chicken chromosome 5
Hiroyuki Sasaki, Takaaki Yokomine, Yuzuru Kato, Wahyu Purbowasito, Hisao
Shirohzu, Hisakazu Iwama, Kazuho Ikeo, Tetsuya Hori, Masaoki Tsuzuki, Shigeki
Mizuno, Yo-ichi Matsuda, Atsushi Toyoda, Masahira Hattori, and Yoshiyuki Sakaki........................................................ ····· .. ·····································45Computer-assisted search for the sequence features common to the imprinted DMRs
Hiroyuki Sasaki, Hisato Kobayashi, Takashi Abe, Yuji Kohara and Toshimichi Ikemura...................................................... ··············································45
Antisense regulation at the Xist locus by Tsix
Takashi Sado, Yuko Hoki, Hiroyuki Sasaki .. ·

·

·

· 46

Tsix-independent mechanism for Xist silencing
Takashi Sado, Tatsuya Ohhata, Yuko Hoki, Hiroyuki Sasaki···························· 46
Role of Dnmt3L in gametogenesis
Kenichiro Hata, Maki Kusumi, Takaaki Yokomine, En LI and Hiroyuki Sasaki····· 46
Role of Dnmt3L in placentation
Kenichiro Hata, Takahiro Arimal, Maki Kusumi, Satoru Tanaka, Takaaki Yokomine,
Kunio Shiota, Norio Wake, Masaoki Tsudzuki and Hiroyuki Sasaki·················· 47
E-b. Division of Agricultural Genetics
Tetsuji Kakutani Group
Developmental abnormalities induced by DNA methylation mutation of Arabidopsis
Tetsuji Kakutani, Tetsu Kinoshita, Asuka Miura, Yuki Kinoshita, Masaomi Kato,
Miyuki Nakamura, Hodetoshi Saze48
Epigenetic behavior of CACTA transposon
Asuka Miura, Masaomi Kato, Miyuki Nakamura, Kazuya Takashima, Yuki Kinoshita,
Tetsuji Kakutani
48
Inheritance of epigenetic developmental abnormality
Yuki Kinoshita, Asuka Miura, Tetsu Kinoshita, Tetsuji Kakutani
49
Epigenetic control of FWA gene expression in endosperm
Tetsu Kinoshita, Asuka Miura, Yuki Kinoshita, Tetsuji Kakutani
49E-b. Division of Agricultural Genetics
Kei-ichi Shibahara Group
Mechanism of nucleosome assembly during DNA replication
Tatsuya Ono, Yasunari Takami, Fumiyuki Sanematsu, Tatsuo Nakayama,
and Kei-ichi Shibahara··········································································· 50
Physiological implications of CAF-l and CAF-l-dependent nucleosome assembly in higher
eukaryotes
Tatsuya Ono, Hidetaka Kaya, Shin Takeda, Tetsuji Kakutani, Takashi Araki,Kei-ichi Shibahara·················································································· 50
Histone macroH2A-mediated formation of transcriptionally repressed states of chromatin
Yuya Ogawa, Hitoshi Nishijima, and Kei-ichi Shibahara .... · .... · .. · ...... · ........ · .... · 50
Analysis of S. pombe RanGAP SpRnal protein in the heterochromatin assembly via histone
H3
Hitoshi Nishijima, Ayumi Kusano, Tomoyuki Ohba, Hideo Nishitani, Kei-ichi Shibahara,
Takeharu Nishimoto··············································································· 51
E-c. Division of Brain Function
Tatsumi Hirata Group
Regulation of ventral tangential migration of guidepost neurons in the lateral olfactory
tract
Takahiko Kawasaki, Keisuke Ito and Tatsumi Hirata· .. · · .. ··· 51

CONTENTS

Identification of the Antigens Recognized by Monoclonal Antibodies Raised against theDeveloping Lateral Olfactory Tract
Takahiko Kawasaki, Yoshiko Takagi, Hitoshi Yamatani, and Tatsumi Hirata······ 52
E-d. Division of Applied Genetics
Kunio Shiota Group
Stage-by-stage change in DNA methylation status of Dnmtllocus during mouse early
development
Yeoung-Gyu Ko, Koichiro Nishino, Naoko Hattori, Yoshikazu Arai, Satoshi Tanaka
and Kunio Shiota
52
DNA methylation-dependent epigenetic regulation of dimethylarginine
dimethylaminohydrolase 2 gene in trophoblast cell lineage
Junko Tomikawa, Kazumi Fukatsu, Satoshi Tanaka and Kunio Shiota
53
DNA methylation profiles of donor nuclei cells and tissues of cloned bovine fetuses
Kremenskoy Maksym, Kremenska Yuliya, Masako Suzuki, Kei Imai, Seiya Takahashi,
Kazuyoshi Hashizume, Shintaro Yagi, Kunio Shiota
53
Epigenetic characterization of CpG islands of bovine leptin and POU5Fl genes in cloned
bovine fetuses
Kremenskoy Maksym, Kremenska Yuliya, Masako Suzuki, Kei Imai, Seiya Takahashi,
Kazuyoshi Hashizume, Shintaro Yagi, Kunio Shiota
53
Proliferation related acidic leucine rich protein PAL3l functions as a caspase-3 inhibitor
Weiyong Sun, Hiromichi Kimura, Naka Hattori, Satoshi Tanaka, Shigemi Matsuyama,
and Kunio Shiota
54
Dimethyl Sulfoxide(DMSO) Increases Expression of Dnmt3as and Affects Genome-wide
DNA Methylation Profiles in Mouse Embryoid Body
Misa Iwatani, Kohta Ikegami, Yuliya Kremenska, Naka Hattori, Satoshi Tanaka,
Shintaro Yagi and Kunio Shiota
54
E-d. Division of Applied Genetics
Takashi Araki Group
Physiological roles of chromatin assembly factor-l (CAF-l) in epigenetic control of meristem
activity during post-embryonic development in plants
Takashi Araki, Mitsutomo Abe, Hidetaka Kaya, Yuya Ogawa, Masaomi, Kato, Tetsuji
Kakutani, Kei-ichi Shibahara···································································· 55
Physiological roles of anti-silencing function 1 (ASFl) in embryonic and post-embryonicdevelopment in plants
Takashi Araki, Ken-ichi Kurotani, Mitsutomo Abe, Hidetaka Kaya, Tetsuji Kakutani,
Kei-ichi Shibahara·················································································· 55
Genes involved in the integration of various genetic pathways for regulation of the floraltransition
Takashi Araki, Mitsutomo Abe, Sumiko Yamamoto, Yasufumi Daimon, Ayako
Yamaguchi, Yoko Ikeda, Michitaka Notaguchi, Masaki Kobayashi·······55
Molecular basis of late-flowering phenotype in epialleles of FWA in Arabidopsis
Takashi Araki, Yoko Ikeda, Mitsutomo Abe················································ 56

F. GENETIC STRAINS RESEARCH CENTER

F-a. Mammalian Genetics Laboratory
Toshihiko Shiroishi GroupA novel long-range enhancer to specify the Sonic hedgehog (Shh) expression in the oralcavity morphogenesis

CONTENTS

Tomoko Sagai, Takanori Amano, Yoichi Mizushina, Hiromi Yamamoto, Noriko
Sakurai-Yamatani, Ayaka Okagaki, Masaru Tamura and Toshihiko Shiroishi ...... 58
Chromosomal dynamics of a long-range cis-regulator for limb bud-specific Shh expression
Takanori Amano, Tomoko Sagai, Hideyuki Tanabe and Toshihiko Shiroishi
58
Molecular mechanisms of epithelium formation of skin and gastrointestinal tract in mice
Masaru Tamura, Shigekazu Tanaka, Tomoaki Fujii, Hiromitsu Komiyama
and Toshihiko Shiroishi
59
Transgenesis with a modified-BAC DNA harboring the Rim3-type GsdmA3 mutation
recaptures the mutant skin phenotype
Shigekazu Tanaka, Masaru Tamura, Aya Aoki, Tomoaki Fujii, Hiromitsu Komiyama,
Toshihiko Shiroishi
59
Disruption of genetic interaction between two autosomal regions and the X chromosome
causes hybrid breakdown in a mouse intersubspecific cross
Ayako Oka, Toshihiro Aoto, Yoshikazu Totsuka, Riichi Takahashi, Masatsugu Ueda,
Akihiko Mita, Noriko Sakurai-Yamatani, Hiromi Yamamoto Nobuo Takagi, Kazuo
Moriwaki and Toshihiko Shiroishi
60
QTL analysis of hereditary predisposition of obesity based on inter-subspecific cross of
C57BLl6J and MSM/Ms strains
Akiteru Maeno, Hideko Watanabe, Akihiko Mita, Ayako Oka
and Toshihiko Shiroishi
60
Genetic dissection of obesity using mouse consomic strains
Toyoyuki Takada, Akiteru Maeno and Toshihiko Shiroishi
F-b. Mammalian Development Laboratory
Yumiko Saga Group
Molecular mechanism of somite segmentation
Mitsuru Morimoto, Makoto Kiso and Yumiko Saga
Regulation of Mespl and Mesp2 expression
Masayuki Oginuma, and Yumiko Saga
Search for target genes of Mesp2 transcription factor
Yoshiro Nakajima and Yumiko Saga

61

62
62
63

Functional analyses of Hesr1 and Hesr2 in the cardiovascular development
Hiroki Kokubo, Raul VizcardoSakoda, and Yumiko Saga································ 63
Analyses toward understanding the function of Protein O-fucosyltransferase1 in the Notch
signaling
63
Yoshiaki Okamura, and Yumiko Saga
Analyses of nanso2 expression and function during mouse germ cell development
Atushi Suzuki, Kaoru Mitsui, and Yumiko Saga .. · .. ·· .. ··

64

Functional analysis of nanos3
Hitomi Suzuki, Makoto Kiso and Yumiko Saga· .. · .. ·.. .. .. .. .. .. . .. .. .. . .. .. .. .. .. . .. .. ... 64
F-c. Mouse Genomics Resource Laboratory (MGRL)
Tsuyoshi Koide GroupA systematic analysis of genetic factors associated with the spontaneous activity in mice
Akinori Nishi, Toshihiko Shiroishi and Tsuyoshi Koide
64
Genetic analyses of psychological traits using consomic mouse strains of C57BLl6 and MSM
65
Aki Takahashi, Akinori Nishi, Toshihiko Shiroishi, Tsuyoshi Koide
Genetic factors responsible for difference in spontaneous home-cage activity between KJR
and C57BLl6J
Juzouh Umemori and Tsuyoshi Koide
65

CONTENTS

F -d. Model Fish Genomics Resource
Noriyoshi Sakai Group
Gene targeting system with RNA interference (RNAi) in ~ebrafish
Minori Shinya, Kimiko Saka, Kayo Kobayashi and Noriyoshi Sakai···················· 67
Analysis of functionally distinctive testicular cell lines of zebrafish to support male germ
cell development
Aki Masuda and Noriyoshi Sakai······························································· 67
Culture condition for zebrafish spermatogonial stem cells
Kenji Saito and Noriyoshi Sakai································································ 68
Analysis of the ability of cultured embryonic cells derived from different developmental
stages to induce the anterior-posterior axis
Megumi Hashiguchi, Minori Shinya and Noriyoshi Sakai·································· 68
F-e. Plant Genetics Laboratory
Nori Kurata Group
Cytogenetic and molecular analysis of rice meiotic mutants
Ken-ichi Nonomura, Mutsuko Nakano, Akane Morohoshi, Mitsugu Eiguchi, Akio Miyao,
69
Hirohiko Hirochika and Nori Kurata
Structural and functional analysis of rice OsHAP3 genes
Yukihiro Ito, Thiruvengadam Thirumurugan and Nori Kurata

70

Regulation of expression of KNOX family class 1 homeobox genes of rice
Yukihiro Ito, Katsutoshi Tsuda and Nori Kurata

70

Analysis of a genetic program in embryosac/ovary formation
Shinichiro Yamaki, Yasuo Nagato and Nori Kurata

70

Establishment of rice TILLING system and mutant pools
Tadzunu Suzuki, Mitsugu Eiguchi, Toshihiro Kumamaru, Hikaru Satohand Nori Kurata

71

Positional cloning and statistical analysis of segregation distortion genes between Japonica
and Indica rice
Yoshiaki Harushima, Satoshi Kuriki, Youko Mizuta, Hironori Fujisawa,
and Nori Kurata
71
Comparative and evolutional studies for cultivated and wild rice species
Ken-ichi Nonomura, Yukie Sano, Akane Morohoshi and Nori Kurata
Detection of genetic/expression variation among rice species
Yoshiaki Harushima, Youko Horiuchi, Masanori Kawakita, Shinto Eguch,and Nori Kurata

71

72

Rice genetic resource project in NBRP and Oryzabase
Nori Kurata, Toshie Miyabayashi, Shinic;hiro Yamaki and Ken-ichi Nonomura
F-f. Microbial Genetics Laboratory
Hironori Niki Group
Guidance of Actively Partitioning Plasmid by a Helical Structure of an ATPase, SopA
Toshiyuki Hatano and Hironori Niki
Analysis of Dynamics of ATPase, PI ParA in Partitioning of PI Plasmid
Toshiyki Hatano and Hironori Niki

72

73
74

Characterization of proteins that are interact with a bacterial centromere site, migS
Yashuyuki Ogata, Toshiyuki Hatano and Hironori Niki·······74
F-g. Invertebrate Genetics Laboratory
Ryu Ueda GroupRNAi mutant fly bank for comprehensive analyses of gene function in Drosophila
Ryu Ueda, Yukiko Sado, Kazuko Fujitani, and Kuniaki Takahashi···················· 75

CONTENTS

F -h. Laboratory for Frontier Research
Takako Isshiki Group
Analysis of temporal specification within late phases of Drosophila neuroblast lineage
Takako Isshiki

77

Investigation of molecular functions of Castor in Development of the CNS
Horiuchi Takayuki

77

Temporal change, quiescence, and reactivation of neuroblasts
Katsutomo Okamura, Takako Isshiki

77

G. CENTER FOR GENETIC RESOURCE INFORMATION

G-a. Genetic Informatics Laboratory
Yukiko Yamazaki Group
Wheat mitochondrial genome analysis.
Ogihara, Y., Yamazaki, Y, Murai, K., Kanno, A., Terachi, T., Shiina, T., Miyashita,N., Nasuda, S., Nakamura, C., Mori, N., Takumi, S., Murata, M., Futo, S.
and Tsunewaki, K,
78
CARD R-BASE
Takehiro Yamakawa, Hideki Kato, Naomi Nakagata, Kenichi Yamamura
and Yukiko Yamazaki
Guide dog database
Kazuhiro Oogushi, Yukiko Yamazaki, Yoshinori Suwa and Hiroshi Suzuki
Rice
Koji Watanabe, Nori Kurata, Atsushi Yoshimura, Yasuo Nagato
and Yukiko Yamazaki

78
78

79

SHIGEN project
Takahiro Yamakawa, Koji Watanabe, Rie Tsuchiya, Sumiko Yano, Kazuhiro Oogushi,
Shingo Sakaniwa, Mutsumi Saito, Sharoh Yip, Toru Watanabe, Miharu Yoshioka
and Yukiko Yamazaki'
80
Featured contents of each database in 2005
Mouse Polymorphism Database' . . . . . . . .. . . .. . .. . . . . . . . . . . . . . .. . . . .. . . .. . . .. . . .. . . . . . .. . . . . . . . . .. . .. 80
Medaka Atlas
80
Drosophila
80
Wheat
LegumeE.coli················································808181G-b. Genome Biology Laboratory
Yuji Kohara Group
Extension of the nematode expression pattern map database (NEXTDB) towards
comparative genomics
Tadasu Shin-i, Hiroshi Kagoshima, Kaori Ikeda, Ikuko Muramatsu-Sugiura, Masumi
Obara, Wakako Shimizu, Aya Hamakawa, Sachiko Takahashi, Takayo Hamanaka,
Yasuko Sugiyama, Yumiko Ueta, Hiroko Ochi, Chihiro Hijikata, Yuji Kohara ....... 82
The C. elegans RUNX transcription factor MAB-2/RNT-1 is required for asymmetrical cell
division of the T blast cell
Hiroshi Kagoshima, Hitoshi Sawa, Shohei Mitani, Thomas R. Burglin,
Katsuya Shigesada, Yuji Kohara
82
POLO like kinase, PLK-1,is required for the establishment and the maintenance ofanterior-posterior cell polarity in C. elegans zygotes
Eisuke Sumiyoshi and Yuji Kohara
83

CONTENTSStudies on localization mechanisms of pos-l mRNA in C.elegans embryos
Kouki Noguchi, Yuji Kohara

83

Effects of poly (A) elongation on the translational activation of maternally transcribed mRNAin C. elegans
Yoshiki Andachi, Shuichi Onami, Yuji Kohara···84
A physical model for C. elegans embryo: towards computer simulation of gastrulation
Atsushi Kajita, Masayuki Yamamura, Yuji Kohara
84
Semi-automatic system for the creation of cell shape models in C. elegans embryogenesis
Hideaki Hiraki, Yuji Kohara
85
Towards comparative genomics: Genome/EST sequencing
Takanori Narita, Kazuko Oishi, Fumiko Ohta, Tomomi Morishita, Tomoko Endo, Keiko
Nogata, Akiko Hase, Hisayo Nomoto, Noriko Hasegawa, Masumi Mizukoshi, Etsuko
Yokoyama, Nanayo Ishihara, Junko Miyamoto, Shigeko liyama, Tadasu Shin-i,
Toshinobu Ebata, Yohei Minakuchi, Kumiko Kawaguchi, Naoko Sakamoto, Yasuko
Sugiyama, Yuji Kohara
85
H. STRUCTURAL BIOLOGY CENTER

H-a. Biological Macromolecules Laboratory
Makio Tokunaga Group
Single Molecule Imaging, Quantitative Analysis and Model Simulation of Nuclear Transport
in Cells
87
Makio Tokunaga and Naoko Imamoto
Identification and Characterization of Associated Proteins with RNG105, An RNA-Binding
Protein Responsible for Neuronal Local Translation
Nobuyuki Shiina, Kazumi Shinkura and Makio Tokunaga····87Single Hydrogen Bonds of DNA Base Pairs Detected in Unzipping Force by Intermolecular
Force Microscopy·· 88Michio Hiroshima and Makio Tokunaga
Single Molecule Detection of Mechanical Unfolding of Proteins
Akihiro Fukagawa, Michio Hiroshima and Makio Tokunaga

88

Visualization of initiation of T cell activation
Kumiko Sakata-Sogawa, Michio Hiroshima, Tadashi Yokosuka, Sho Yamasaki,
Takashi Saito, and Makio Tokunaga·
88
Three-dimensional Multi-color Visualization and Quantitative Analysis of Nulcear Fine
Structures
Michio Hiroshima, Kumiko Sakata-Sogawa, Kazumi Shinkura, Kyoichi Isono, Haruhiko
Koseki, and Makio Tokunaga···································································· 89
Asymmetric nucleocytoplasmic transport revealed by a novel assay system using plannerreconstituted nuclear envelope
Atsuhito Okonogi, Michio Hiroshima, Nobuyuki Shiina, Shingo Kose, Naoko Imamoto
and Makio Tokunaga·············································································· 89
Molecular Imaging of Local Translation in Neuronal Dendrites by LTP Electrical Stimulation
Hiraku Miyagi, Kumiko Sakata-Sogawa, Michio Hiroshima
and Makio Tokunaga········································································ 89
Development and Optimization of Molecular Microscopy and Software
Makio Tokunaga, Michio Hiroshima and Kumiko Sakata-Sogawa
90

CONTENTS

H-b. Molecular Biomechanism Laboratory
Nobuo Shimamoto Group
Regulatory Mechanism of transcription initiation by switching conformations of RNApolymerase: a homogeneous protein works inhomogeneously
Motoki Susa, Takashi Miyamoto, and Nobuo Shimamoto
90
Applicability of thermodynamics to equilibria in biology
Nobuo Shimamoto and Jyun-ichi Tomizawa
91
Systematic search for promoters encoded in the genomic DNA sequences of E. coli
Nobuo Shimamoto, Hideki Nakayama and Hironori Aromaki
H-c. Multicellular Organization Laboratory
Isao Katsura Group
Enhancement of chemotaxis by pre-exposure to odorants and food in the nematodeC. elegans
Ichiro Torayama, Hiroshi Ichijo, Kotaro Kimura, Takeshi Ishihara
and Isao Katsura
Genetic analysis of plasticity of avoidance behaviors in C. elegans
Kotaro Kimura and Isao Katsura

92

93
94

Class 1 flr mutants of the nematode Caenorhabditis elegans
Yuri Kobayashi, Kotaro Kimura and Isao Katsura
94
Class 2 flr mutants of the nematode Caenorhabditis elegans
Akane OISHI, Kotaro Kimura, Takeshi Ishihara and Isao Katsura
95
Analysis of synthetic dauer-constitutive mutants in the nematode Caenorhabditis elegans
Tomoko Yabe, Norio Suzuki, Tatsuo Furukawa, Takeshi Ishihara
and Isao Katsura
95
H-d. Biomolecular Structure Laboratory
Yasuo Shirakihara Group
Crystallographic Study of ATP synthaseYasuo Shirakihara and Aya Shiratori

96

Comprehensive Crystallographic Study of Transcription factors and Genome-partitioning
Factors from E.coli
Yasuo Shirakihara and Aya Shiratori
97
Structure-based functional studies on transcription factors
Hiroshi Itou and Yasuo Shirakihara

97

Expression and crystallization Study of Eukaryotic Transcription Factors
Hiroshi Itou and Yasuo Shirakihara

98

Expression and crystallization Study of Transcription factors from Pseudomonas aeruginosaYasuo Shirakihara and Aya Shiratori
98
Crystallization of Kid, a chromosome mover
Yasuo Shirakihara and Aya Shiratori

98

Structural analysis of glutaminase from Micrococcus luteus K-3
Yasuo Shirakihara and Aya Shiratori

99

H-e. Gene Network Laboratory
Emiko Suzuki Group
Searching for cell-surface and secreted proteins controlling motor axon targeting andsynaptogenesis
Mitsuhiko Kurusu, Kai Zinn and Emiko Suzuki··
99
High-resolution in vivo analysis of synaptogenesis in Drosophila neuromuscular system
Franklin Carrero-Martinez, Akira Chiba and Emiko Suzuki···························· 100
Formation of topological axon layer in Drosophila mushroom body

CONTENTSMitsuhiko Kurusu, Kai Zinn and Emiko Suzuki··················'" 101
Molecular studies on the diacylglycerol kinase function in Drosophila photoreceptor neurons
Takuma Yamada and Emiko Suzuki··........ 102
I. CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPANI-a. Laboratory for DNA Data Analysis
Takashi Gojobori Group
Various adaptations for the perpetual darkness in the cave-dwelling population of Mexican
tetra, Astyanax mexicanus, by eDNA microarrays
Nobuhiko Tanaka, Shozo Yokoyama, Kazuho Ikeo and Takashi Gojobori
104
Compensatory change of interacting amino acids in the coevolution of transcriptional
coactivator MBF1 and TATA-box binding protein TBP
Qing-Xin Liu, Naomi Nakashima-Kamimura, Kazuho Ikeo, Susumu Hirose
and Takashi Gojobori
104
Targets analysis of the Drosophila tdf by using oligonucleotide microarrays
Qing-Xin Liu, Kazuho Ikeo, Yasushi Hiromi, Susumu Hirose
and Takashi Gojobori

104

Functional analysis of Drosophila Midline during the neuronal development
Qing-Xin Liu, Masaki Hiramoto, Hitoshi Ueda, Takashi Gojobori, Yasushi Hiromi
and Susumu Hirose···················· .. . . . .. . . . . . . . .. . . . .. . . . . ... . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . 105
A possible regulatory role of cis sense-antisense mRNAs in gene expression
Naoki Osato, Yoshiyuki Suzuki, Kazuho Ikeo and Takashi Gojobori
105
The Rice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp. japonica genome
information
105
Hajime Ohyanagi, Yasumasa Shigemoto, Kazuho Ikeo and Takashi Gojobori
Intracellular transporters evolve slowly
Hajime Ohyanagi and Takashi Gojobori
106
An analysis of gene expression for sexual dimorphism in a mouse brain
Kazuya Yuge, Kazuho Ikeo and Takashi Gojobori

106

On the origin of mechanosensory systemlung Shan Hwang, Shiho Hayakawa, Kazuho Ikeo, Toshitaka Fujisawa
and Takashi Gojobori

106

To search the origin of neural tube: Gene expression analysis of ciliary band in sea urchin
embryoSonoko Kinjo, Kazuho Ikeo and Takashi Gojobori
107
Evolutionary studies of the nervous system based upon comparative genomics
Akiko Ogura Noda

107

I-b. Laboratory for Gene-Product Informatics
Ken Nishikawa Group
Predicting absolute contact numbers of native protein structure from amino acid sequence
Akira R. Kinjo, Katsuhisa Horimoto and Ken Nishikawa·················· .. . .. . . . .. . . .. 110
Recoverable one-dimensional encoding of three-dimensional protein structures
Akira R. Kinjo and Ken Nishikawa··
110
Genome-wide survey of transcription factors in prokaryotes reveals many bacteria-specificfamilies not found in archaea
Yoshiaki Minezaki, Keiichi Homma and Ken Nishikawa' . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . 110
Predicting secondary structures, contact numbers and residue-wise contact orders of native
protein structure from amino acid sequence using critical random networks
Akira R. Kinjo and Ken Nishikawa·· .. ·
111

CONTENTSIntrinsically disordered loops inserted into the structural domains of human proteins
Satoshi Fukuchi, Keiichi Homma, Yoshiaki Minezaki and Ken Nishikawa·········· 111
I-c. Laboratory for Gene Function Research
Yoshio Tateno Group
Genomic evolution of MHC class I region in primates
Fukami-Kobayashi, K, Shiina, T., Anzai, T., Sano, K, Yamazaki, M., Inoko, H
and Tateno, Y
112
Development of a spot evaluation score for DNA micro arrays
Matsumura, Y, Shimokawa, K, Hayashizaki, Y, Ikeo, K, Tateno, Y.,
and Kawai, J

112

Large-scale analysis of human alternative protein isoforms: pattern classification andcorrelation with subcellular localization signals
Nakao M, Barrero RA, Mukai Y, Motono C, Suwa M, and Nakai K
113
Evolution of vitamin B6 (pyridoxine) metabolism by gain and loss of genes
Tanaka T, Tateno Y, Gojobori T
113
DDBJ in collaboration with mass-sequencing teams on annotation
Tateno Y, Saitou N, Okubo K, Sugawara H, Gojobori T
113
Investigation of protein functions through data-mining on integrated human transcriptome
database, H-Invitational database
Yamasaki C, Koyanagi KO, Fujii Y, Itoh T, Barrero RA, Tamura T, Yamaguchi-Kabata
Y, Tanino M, Takeda J, Fukuchi S, Miyazaki S, Nomura N, Sugano S, Imanishi T, andGojobori T
114
Novel mammalian microRNA targets identified by an extensive transcriptome-wide affinity
screening
Barrero RA, Tamura T, Sakurai H, Hayakawa S, Tateno Y, Ikeo K, Imanishi T
and Gojobori T
114
Development of a method for constructing a phylogenetic tree by using many different
orthologs
Tokumasa Horiike, Daisuke Miyata, Kazuo Hamada, Satoshi Saruhashi,
Takao Shinozawa, Yoshio Tateno
114
The analysis of the evolutionary relationship between duplication and alternative splicing
Jin LH., Ikeo K, Suzuki Y and Gojobori T
115
Evolutionary study of small-RNA-mediated gene silencing pathways by investigating theevolution of Rnase III enzymes
115
Jin LH., Ikeo K, Suzuki Y and Gojobori T
A Novel Arabidopsis Gene Causes Bax-like Lethality in Saccharomyces cerevisiae
Kawai-Yamada M., Saito Y" Jin LH., Ogawa T., Kim KM., Jin LH., Tone Y,
Hirata A., Umeda M., and Uchimiya H

115

Collaboration with Prof. Tadao Saito of Tohoku University and his laboratory on thefunction and evolution of glucosidase and galactosidase genes in Lactobcillus.
Saito, T, Yamazaki Y and Tateno Y
115
Collaboration with Prof. Shintou Eguchi of the Institute of Mathematical Statistics andhis laboratory on the statistical analyses of SNP and gene expression data.
Eguchi S, Ikeo K and Tateno Y
116
Collaboration with Dr. Kaoru Fukami-Kobayashi of RIKEN on evolutionary analysis of
MHC genes in primates
116
Fukami-Kobayashi K and Tateno Y
Collaboration with Prof. Naoko Takezaki of Kagawa Medical College on the evolution ofgenes expressed in brains

CONTENTSTakezaki Nand Tateno Y

116

Collaboration with AHG-Evolutional Curation System teams on annotation of expressed
human genes
Ikeo K., Suzuki Y, Ohta S., Jin LH., Yamazaki c., Suzuki M., Kanno M., Saichi N.,
Sanbonmatsu R., Koyanagi K., Sakate R., Itoh T., and Imanishi T
116
I-d. Laboratory for Research and Development of Biological Databases
Hideaki Sugawara Group
Information systems for molecular biology and its related disciplines
From Web services to a Bioportal
Yasumasa Shigemoto, Haruka Sakai, Takshi Abe, Satoru Miyazaki,
117
Hideaki Sugawara
Expansion of Genome Information Broker (GIB)
Masaki Hirahata, Naoto Tanaka, Takashi Abe, Satoru Miyazaki,
Hideaki Sugawa

117

Development of Genome Information Broker for Viruses genome (GIB-V)
Masaki Hirahata, Takashi Abe, Naoto Tanaka, Hideaki Sugawa
118
Development of Genome Information Broker for Insertion sequence (GIB-IS)
Takehide Kosuge, Takashi Abe, Yoshio Tateno, Hideaki Sugawara
118
Information systems on microbes and biodiversity
WFCC-MIRCEN World Data Centre for Microorganisms (WDCM)
Yasumasa Shigemoto, Junko Nagaya, Hideaki Sugawara
118
Development of an e-Workbench for Biological Classification and Identification (InforBIO)
Naoto Tanaka, Kouji Koorikawa, Takashi Abe, Satoru Miyazaki,
118
Hideaki Sugawara
An information system for pathogenic microorganisms················
118
Global Biodiversity Information Facility (GBIF)
Hideaki Sugawara, Motomi Itoh and Keiichi Matsuura··································· 119
Applications of IT to the International Nucleotide Sequence Database
Development of Open Annotation System
Satoru Miyazaki, Takashi Abe, Hideaki Sugawara
119
Development of an e-Workbench for comparative genome analysis (G-InforBIO)
Naoto Tanaka, Kouji Koorikawa, Takashi Abe, Satoru Miyazaki,
Hideaki Sugawara
119
Exhaustive evaluation of the annotation data obtained by simultaneous annotations of wholebacterial genomes
Takehide Kosuge, Toshihisa Okido, Yasumasa Shigemoto, Masaki Hirahata,
119
Naoto Tanaka, Takashi Abe, Satoru Miyazaki, Hideaki Sugawara
Genomics
A large-scale Self-Organizing Map (SOM) unveils sequence characteristics of a wide rangeof eukaryote genomes
Takashi Abe, Toshimichi Ikemura, Hideaki Sugawara
120
Phylogenetics Analyses of Environmental Samples on the Basis of Self-Organizing Map(SOM)
Takashi Abe, Toshimichi Ikemura Hideaki Sugawara
120
I-e.

Laboratory for Gene-Expression Analysis
Kosaku Okubo Group
BodyMap-Xs: Anatomical breakdown of 17 million animal ESTs for cross-species
comparison of gene expression
Ogasawara 0, Watanabe K, Imamura H, Iizuka T, Okubo K
122

CONTENTS

Identification of differentially expressed genes in psoriasis using expression profiling
approaches
122
Comparison of Gene Expression Patterns between human and mice brain
Otsuji M, Okubo K

122

A Theoretical Model for the Accumulation of Gene Expression Divergence
Ogasawara 0, Okubo K

122

J. RADIOISOTOPE CENTER

Environmental genetics: Stationary phase-induced genome rearrangements
Yasuyuki Ogata

124

Archaeological genetics: Isolation of ancient bacteria from Antarctic iceberg
Yasuyuki Ogata' . .. . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . .. . . . .. .. . . . . . . . .. . . . . .. . . . . . . .. 124
Field genetics: Obtaining of Antarctic gene resources without contamination
Yasuyuki Ogata

124

K. EXPERIMENTAL FARM

Examination of genetic characters for accessions of wild rice core collection
Ken-Ichi Nonomura, Yukie Sano, Toshie Miyabayashi, Mitsugu Eiguchi,
and Nori Kurata

125

L. INTELLECTUAL PROPERTY UNIT

Munehiro Tomikawa
Acquirement and utilization of Intellectual Property Rights·································· 127
Acceleration of public relations activities························································ 127
Collaboration Research with academia, industry and government,
and Social Action Work128
M. TECHNICAL SECTION····· . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . .. . . . . .. . . . . . . .. . . . . . . . .. . . . . 129

BIOLOGICAL SYMPOSIUM 2005
FOREIGN VISITORS IN 2005

CONTENTS

130
132

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

Introduction

1

STAFF (as of December 31, 2005)
Director-General

KOHARA, Yuji, D. Sc.
Vice- Director

HIROSE, Susumu, D. Sc.
Member
1. Department of Molecular Genetics

YAMAO, Fumiaki, D. Sc., Head of the Department
Division of Molecular Genetics
FUKAGAWA, Tatsuo, D. Sc., Associate Professor
OKADA, Masahiro, D. Sc.
Division of Mutagenesis
YAMAO, Fumiaki, D. Sc., Professor
TSUTSUI, Yasuhiro, D.Med.
Molecular Mechanism Laboratory
SEINO, Hiroaki, D. Sc.
Division of Nucleic Acid Chemistry
HARAGUCHI, Tokuko, D. Med., Adjunct Professor
IWASAKI, Hiroshi, D. Med., Adjunct Associate Professor
2. Department of Cell Genetics
ARAKI, Hiroyuki, D. Sc., Head of the Department
Division of Cytogenetics
YOSHIMORI, Tamotsu, D. Med., Professor
UMEBAYASHI, Kyohei, D. Ag.
Division of Microbial Genetics
ARAKI, Hiroyuki, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate Professor
KAMIMURA, Yoichiro, Dc. Med.
TANAKA, Seiji, D. Sc.
Division of Cytoplasmic Genetics
KOBAYASHI, Kazuto, Ph. D., D. Ag ., Adjunct Professor
YAMAMOTO, Masayuki, D. Sc., Adjunct Professor
3. Department of Developmental Genetics
HIROMI, Yasushi, D. Sc., Head of tq.e Department
Division of Developmental Genetics
HIROMI, Yasushi, D. Sc., Professor
FUJISAWA, Toshitaka, Ph. D., Associate Professor
SHIMIZU, Hiroshi, D. Eng.
ASAOKA, Miho, D. Sc.
Division of Gene Expression
HIROSE, Susumu, D. Sc., Professor
NISHIOKA, Kenichi, D. Med.
FUSE, Naoyuki, D. Sc.

2

STAFF

Division of Molecular and Developmental Biology
KAWAKAMI, Koichi, D. Sc., Associate Professor
KISHIMOTO, Yasuyuki, D. Sc.
Division of Physiological Genetics
ISHINO, Fumitoshi, D. Sc., Adjunct Professor
GOTOH, Yukiko, D. Sc., Adjunct Professor

4. Department of Population Genetics
SAITOU, Naruya, Ph. D., 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 Theoretical Genetics
FUJIYAMA, Asao, D. Sc., Adjunct Professor
SUGANO, Sumio, D. Med., Adjunct Professor
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.
NISHUIMA, Hitoshi, D. Sc.
Division of Brain Fanction
HIRATA, Tatsumi, D. Med., Associate Professor
KAWASAKI, Takahiko, D. Sc.
Division of Applied Genetics
SlOTA, Kunio, D. Ag., Adjunct Professor
ARAKI, Takashi, D. Sc., Adjunct Associate Professor
6. Genetics 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.
Mouse Genomics Resource Laboratory
KOIDE, Tsuyoshi, D. Med., Associate Professor
Model Fish Genomics Resource Laboratory
SAKAI, Noriyoshi, Ph. D., Associate Professor

STAFF

3

SHINYA, Minori, D. Sc.
Plant Genetics Laboratory
KURATA, Nori, D. Ag., Professor
ITO, Yukihiro, D. Ag.
Microbial Genetics Laboratory
NIKI, Hironori, D. Med., Professor
Invertebrate Genetics Laboratory
UEDA, Ryu, D. Sc., Professor
TAKAHASHI, Kuniaki, D. Sc.
Labolatory for Frontier Research
ISSHIKI, Takako, D. Sc., Associate Professor
HORIUCHI, Takayuki, D. Sc.
7. Center for Genetic Resource Information
SHIROISHI, Toshihiko, D. Sc., Head of the Center
Genetic InformaUcs Laboratory
YAMAZAKI, Yukiko, D. Sc., Associate Professor
Genomu Biology Laboratory
KOHARA, Yuji, D. Sc., Professor
ANDACHI, Yoshiki, D. Sc.
8. Structral 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
KIMURA, Koutarou, D. Ag.
Biomolecular Structure Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate Professor
ITO, Hiroshi, D. Sc.
Gene Network Laboratory
SUZUKI, Emiko, D. Med., Associate Professor
KURUSU, Mitsuhiko, D. Sc.
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.
Laboratory for Gene-Product Informatics
NISHIKAWA, Ken, D. Sc., Professor
FUKUCHI, Satoshi, D. Sc.
KINjO, Akira, D. Sc.

4

STAFF

Laboratory for Gene Function Research
T ATENO, Yoshio, Ph. D., D. Sc., Professor
BARRERO, Robert A., D. Sc.
Laboratory for the Research and Development of Biological Databases
SUGAWARA, Hideaki, D. Eng., Professor
ABE, Takashi, D. Sc.
Gene-Expression Analysis
OKUBO, Kousaku, M. D., Ph. D., Professor
10. Radioisotope Center

NIKI, Hironori, D. Med., Head of the Center
OGATA, Yasuyuki, D. Pharm. Sci.
11. Experimental Farm

KURATA, Nori, D. Ag., Head of the FarmNONOMURA, Ken-ichi, D. Ag.
12. Intellectual Property UnitTOMIKAWA, Munehiro, D. Pharm., Director
13. Technical Section

HIROSE, Susumu, Deputy Chief of the Section
YAT A, Katsunori, Assistant Chief of the Section
14. Department of Administration

MARUYAMA, Ken-ichi, Head of the Department
SAKAMOTO, Nagao, Chief of the General Affairs Section
YAMADA, Yoshihiro, Chief of the Finance Section

STAFF

5

ADVISORY COMMITTEE (as of December 31, 2005)
Chairman
HIROSE, Susumu; Professor, Department of Developmental Genetics, National Institute of Genetics
Vice-chairman
SEKIGUCHI, Mutsuo; Adjunct Professor, Fukuoka Dental College
Outside Members (Alphabetical order)

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

Inside Members (Alphabetical order)

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

6

ADVISORY COMMITTEEADVISORY BOARD (as of December 31, 2005)
Members ( Alphabetical order)

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

7

A. DEPARTMENT OF
MOLECULAR GENETICS

A-a. Division of Molecular Genetics
Tatsuo Fukagawa Group
Research Activities(1) Identification and characterization of the CENP-H-I
complex proteins in higher vertebrate cells.

Masahiro Okada, Tetsuya Hori, Mi-Sun Kwon, Aussie
Suzuki, Mayumi Takahashi, and Tatsuo Fukagawa
The centromere plays a fundamental role inaccurate chromosome segregation during mitosis andmeiosis in eukaryotes. Its functions include sister
chromatid adhesion and separation, microtubule
attachment, chromosome movement and mitotic
checkpoint control. Although chromosome segregation
errors cause genetic diseases including some cancers,
the mechanism by which centromeres interact withmicrotubules of the spindle apparatus during cell
division is not fully understood.
To understand the function of the centromere, wewere led to develop a genetic method that utilizes thehyper-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 have improved this system and havecreated several cell lines with conditional knockouts
of several centromere proteins to investigate themolecular mechanism of centromere assembly andfunction. We have proposed a model for kinetochore
assembly in vertebrate cells (Fukagawa et aI., EMBO
J., 2001; Nishihashi et aI., Dev. Cell, 2002; Fukagawa,Exp. Cell Res., 2004; Regnier et aI., Mol. Cell. BioI., 2005;
Mikami et aI., Mol. Cell. BioI., 2005). In this model we
explained that many unidentified proteins could be
involved in kinetochore assembly. Therefore, we tried
to identify new centromere proteins that associate with
CENP-H or CENP-I using the proteomics approach.

8

DEPARTMENT OF MOLECULAR GENETICS

We prepared DT40 cell lines in which expression of
CENP-H was replaced by expression of CENP-H-Flagor CENP-H-GFP (Mikami et aI., Mol. Cell. BioI., 2005).
We also prepared DT40 cell lines in which expressionof CENP-I was replaced by expression of CENP-I-Flagor CENP-I-GFP. We extracted chromosome fraction
from these cell lines and performed immunoprecipitation with anti-Flag or anti-GFP antibodies. Proteinsco-precipitated with antibodies were analyzed by mass
spectrometry, and we identified several polypeptides
and cloned the corresponding cDNAs. As we confirmedthat these proteins constitutively localize tocentromeres in DT40 cells, we named them CENPs. We
also performed immunoprecipitation experiments withhuman cells and identified similar proteins. In total,
our purifications in chicken and human cells identified
11 interacting proteins (CENP-K, CENP-L, CENP-M,
CENP-N, CENP-O, CENP-P, CENP-Q, CENP-R,
CENP-50, CENP-H and CENP-I) that localize
constitutively to centromeres as components of the
CENP-H-I associated complex. Our analysis of these
proteins using chicken DT40 knockouts and RNAi
analysis in human cells demonstrates that the
CENP-H-I complex can be divided into three functional
sub-complexes, each of which is required forchromosome alignment and faithful chromosome
segregation (Okada et aI., Nature Cell Biology, 2006).
(2) Functional analysis of CENP-50 that is required forrecovery from spindle damage.

Tetsuya Hori, Masahiro Okada, Tomoko Motohashi,
Kazuko Suzuki, and Tatsuo Fukagawa
We identified CENP-50 as a kinetochore
component. We found that CENP-50 is a constitutive
component of the centromere that colocalizes with
CENP-A and CENP-H throughout the cell cycle invertebrate cells. To determine the precise function of
CENP-50, we examined its role in centromere function
by generating a loss-of-function mutant in the chicken
DT40 cell line. The CENP-50 knockout was not lethal;
however, the growth rate of cells with this mutation
was slower than that of wild-type cells. We observedthat the time for CENP-50-deficient cells to complete
mitosis is longer than that for wild-type cells.
Centromeric localization of CENP-50 was abolished inboth CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that

CENP-50 interacts with the CENP-H-I complex and theNuf2-Heel complex in chicken DT40 cells. We also
observed severe mitotic defects in CENP-50-deficient
cells with apparent premature sister chromatid
separation when the mitotic checkpoint was activated,indicating that CENP-50 is required for recovery fromspindle damage (Minoshima, Hori et aI., Mol. Cell. BioI.,
2005).
Although the CENP-50 knockout was not lethal atcellular level, it is interesting to investigate functionof CENP-50 at individual level. Therefore, we aremaking and characterizing knockout mice of CENP-50.
We also discovered that expression of a brain specificgene was reduced in CENP-50-deficient cells.
(3) Functional analysis ofthe CENP-C complex, whichassociates with the Mis12 complex.
Mi-Sun Kwon, Masahiro Okada, Tetsuya Hori, andTatsuo Fukagawa
CENP-C, an essential inner kinetochore
component, is essential for faithful chromosome
segregation. We previously reported a creation of
conditional knockout DT40 cell line of CENP-C witheastergen receptor (ER) system. Since a target protein
is inactivated by post-translational regulation in the
ER system, it would be better to use the system inwhich a target protein is inactivated at expression levelsuch as a tetracycle-inducible system. In this project
we created a CENP-C conditional knockout cell linewith the tetracycle-inducible system. Consistent withprior results, CENP-C inactivation cause mitotic delay.
However, living cell analysis showed that CENP-C
knockout cells progressed to next cell cycle withoutnormal cell division following the mitotic delay andinterphase cells had di-nuclei before subsequent cell
death. We detected significant reduction of signal
intensities of the Mis12 complex proteins and the Nuf2
complex proteins at centromeres in CENP-C-deficient
cells. We also found ""'50% of CENP-C-deficient cells
had no Mad2 signals even after treatment of a spindle
poison, suggesting that CENP-C-deficient cells
compromise spindle checkpoint function. We also tried
to identify CENP-C interacting proteins and isolated
some candidates. Weare now characterizing them.

(4) Functional roles of the RNAi machinery inverterbrate centromeres

Tomoko Motohashi, and Tatsuo Fukagawa
RNAi-mediated silencing of gene expression occurs
when double-stranded RNAs (dsRNAs) are cleaved by
Dicer into 21- to 23-nt small interfering RNAs (siRNAs).
These siRNAs guide a multicomponent nuclease,
RNA-induced silencing complex (RISC), to degrade
specific mRNAs. Although the Dicer-mediatedgene-silencing is evolutionarily conserved system, thebiological functions of the RNAi machinery are notfully understood. Genetic strategies have been used to
examine the biological functions of the RNAi
machinery in C. elegans, Arabidopsis, Drosophila andfungi. There are also many reports that the RNAi
machinery via RNA-induced initiation of transcriptional silencing (RITS) complex is related to heterochromation formation and chromosome segregation in fission
yeast. However, it was unclear whether the RNAi
machinery is associated with chromosome segregationin vertebrate cells. To examine the biological functionof the RNAi-related pathway in vertebrate cells, we
generated and characterized a conditional loss-offunction mutant of Dicer in a chicken-human hybrid
DT40 cell line that contains human chromosome 21(Fukagawa et aI, Nature Cell BioI., 2004). Loss of Dicer
leads to cell death with accumulation of abnormal
mitotic cells that show premature sister chromatid
separation. Aberrant accumulation of transcripts froma-satellite sequences, which consist of humancentromeric repeat DNAs, was detected in Dicerdeficient cells. We then started to examine expression
profile of non-coding region using high-density
DNA-microarray, when expression of Dicer was lost.
We identified several non-coding RNAs from thismethod. Weare characterizing biological function theseRNAs. We also created conditional knockout cells forother components involved in RNAi machinery suchas Ago-family proteins. Phenotype analyses of
Ago-deficient cells were started. We would like tocomprehensive understand relationships RNAi
machinery with centromere function.

Division of Molecular Genetics

9PUBLICATIONS

A-b. Division of Mutagenesis
Fumiaki Yamao Group

Papers1. Mikami, Y., Hori, T., Kimura, H., Fukagawa T.
(2005) The functional region of CENP-H interacts with
the Nuf2 complex that localizes to centromere duringmitosis, Mol. Cell. BioI., 25, (5) 1958 - 1970
2. Minoshima, Y., Hori, T., Okada, M., Kimura, H.,
Haraguchi, T, Hiraoka, Y., Bao, Y. C., Kawashima, T.,
Kitamura, T, Fukagawa, T. (2005) The constitutive
centromere component CENP-50 is required forrecovery from spindle damage, Mol. Cell. BioI. 25,
10315 - 10328
3. Motohashi, T., Shimojima, T., Fukagawa, T.,
Maenaka, K., Park, E. Y. (2005) Efficient large-scaleprotein production of larvae and pupae of silkworm
by Bombyx mori nuclear polyhedrosis virus bacmid
system, Biochem. Biophys. Res. Common., 326, (3)564-

569
4. Regnier, V., Vagnarelli, P., Fukagawa, T, Zerjal,T., Bums, E., Trouche, D., Earnshaw, W., Brown, W.
(2005) CENP-A is required for accurate chromosome
segregation and sustained kinetochore association of
BubR1, Mol. Cell. BioI. 25, 3967 - 3981
5. Yamauchi, A., Ichimiya, T., Inoue, K., Taguchi, Y.,
Matsunaga, N., Koyanagi, S., Fukagawa, T, Aramaki,
H., Higuchi, S., Ohdo, S. (2005) Cell-cycle-dependent
pharmacology of methotrexate in HL-60, J. Pharmacol.
Sci., 99, 335 - 341
EDUCATION

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10

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Division of Molecular Geneticsy~

The higher-order structure of chromatin essential
for the chromosomal integrity is maintained via a
variety of the post-translational modifications. The
preservation of the chromatin structure during
replication of chromosomes is another aspect of theepigenetic inheritance of the structural information.
We focused on a replication factors involved in thechromatin structure maintenance, possibly by
regulating the modifications of the chromatin proteinsduring the chromosomal duplication.
Research Activities(1) Multifunctional role of Mcll/Slr3 in DNA replication,
repair and chromatin structure.
Toyoaki Natsume, Yasuhiro Tsutsui and Fumiaki
Yamao
Slr3 in fission yeast is a member of eukaryotic
family of WD repeat proteins and important for
genome stability. slr3 gene was originally identified as
mutants showing synthetic lethality with Rad2 defect.
Fission yeast Rad2 is a FEN-1 family nucleaseprocessing Okazaki fragment during lagging strand
DNA synthesis. Since recombination activity isessential for survival of cells defective in Rad2 function,
genes that show, when mutated, synthetic lethality with
Rad2-defect was expected to be involved inrecombination or recombination repair of damaged
DNA. Actually slr3 mutants are sensitive to DNA
damage reagents such as MMS, UV, bleomycin andhydroxyurea. Null mutant cell is temperature sensitivefor growth, and shows severe growth retardation evenat the permissive temperature. The mutant cells arrest
at G2 phase with 2C DNA content at the restricted
temperature, showing that the bulk DNA synthesis
occurred. However, DNA double-strand breaks were
found to accumulate in the arrested cells, which wasa similar phenotype caused by defect of Okazaki
fragment processing in such as dna20r cdc17mutants.
It was further shown that Slr3 interacts not only
genetically but also physically with DNA polymerase.
These results strongly suggest that Slr3 functions inlagging strand synthesis and Okazaki fragmentprocessing in addition to DNA repair. On the otherhand, slr3 gene was found allelic to mell that isrequired for proper chromosomal segregation.
Concomitantly slr3mutant cells were sensitive to TBZ.
Consequently, highly frequent chromosome loss
observed in mell mutant cells was attributed to
defective cohesion of sister chromatids in centromere
region(Tsuitsui et al.). Recently, we also found that Slr3
affects heterochromatin structure, which is now underan extensive investigation (Natsume et al. Unpublishedresults). Thus, Slr3/Mcll protein plays multifunctionalrole to maintain chromosomal stability by connecting
DNA replication, recombination, damage repair,cohesion and heterochromatin structure. This work
was collaborated with H. Shinagawa's and H. Iwasaki's
group in Osaka University and Yokohama-City
University, respectively.
PUBLICATIONSPapers

1. Kotani, T., Nagai, D., Asahi, K., Suzuki, H., Yamao,
F., Kataoka, N. and Yagura, T. (2005) Antibacterial
Properties of Some cyclic Organo bismuth (III)Compounds, Antimicrob. Agents Chemother., 49,2729 2734
2. Tsutsui, Y., T. Morishital, T. Natsume, K.
Yamashital, H. Iwasaki, F. Yamao and H. Shinagawa(2005) Genetic and Physical Interactions between
Schizosaccharomyces pombe Mcll and Rad2, DnaZ and
DNA polymerase IX: Evidence for a Multifunctional
Role of Mcll in DNA Replication and Repair., Cnrr.
Genet., 48,34- 43
EDUCATION

1.

UJ~)ca)}ff~fl~)RESEARCH ACTIVITIES(1) Regulatory roles ofthe Rpb7 subunit of eukaryotic
RNA polymerase IT
Hiroshi Mitsuzawa
The RpM and Rpb7 subunits of RNA polymerase
II (Pol II) form a dissociable subcomplex. The RpM/
Rpb7 heterodimer is dispensable for RNA synthesis
but is required for promoter-dependent transcription
initiation. Rpb4lRpb7 can thus be viewed as a generaltranscription factor that associates tightly with Pol II.
To elucidate the role of Rpb7 subunit in Pol IItranscription, proteins were identified that interact with
Rpb7 of the fission yeast Schizosaccharomyces pombe(Mitsuzawa et al. 2005). Identification of glyceraldehyde-3-phosphate dehydrogenase and actin as
Rpb7-interacting proteins has suggested regulatory
roles for Rpb7 in both the activation and initiation of
Pol II transcription. Interestingly, RpM/Rpb7-related
subunits have recently been identified in RNA
polymerases I (Imazawa et al. 2005) and III. Rpb4lRpb7
and their homologues seem to play diverse regulatory
roles in eukaryotic cells.
(2) Identification of ammonium transporter genes in S.
pombe
Hiroshi Mitsuzawa

1:.~f-I.¥!f,f~ilfiJ~ (~~f&{~MJi ~?r /' /~:7

~1N¥~*¥' !1I!I¥liJf~f-I.

2. UJ~)c~A-c. Molecular Mechanism Laboratory
Hiroshi Mitsuzawa Group~~1:.~¥~~UJO*¥·.¥$

Ammonium is an important source of nitrogen foryeast, and its availability has substantial effects on
nitrogen metabolism and development of yeast cells.
Transport of ammonium across the cell membrane ismediated by members of the AmtlMep family, proteins
with eleven transmembrane domains. Threeammonium transporter genes of S. pombe, named
amtl, amt2, and amt3, were identified by sequencehomology. A series of amt deletion strains wereconstructed and tested for growth on low ammonium,
resistance to the toxic ammonium analog
methylammonium, and ammonium removal from thegrowth medium. The results have suggested that theamt genes encode functional transporters with different

Division of Mutagenesis

11uptake capacities.
PUBLICATIONSPapers
1. Imazawa, Y., Hisatake, K., Mitsuzawa, H.,
Matsumoto, M., Tsukui, T., Nakagawa, K., Nakadai,T., Shimada, M., Ishihama, A., Nogi, Y. (2005) The
fission yeast protein Kerlp is an ortholog of RNApolymerase I subunit A14 in Saccharomyces cerevisiae
and is required for stable association of Rrn3p and
RPA21 in RNA polymerase I, J. BioI. Chern., 280 (12)11467 - 11474
2. Mitsuzawa, H., Kimura, M., Kanda, E., Ishihama,A. (2005) Glyceraldehyde-3-phosphate dehydrogenase
and actin associate with RNA polymerase II andinteract with its Rpb7 subunit, FEBS Lett., 579 (1) 4852

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

Hiroaki 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 by a ubiquitin-proteasome system.
Previously we reported that two ubiquitinconjugating enzymes, UbcPl/Ubc4 and UbcP4lUbc11,
were responsible for degradation of mitotic cyclin
Cdc13 in fission yeast. Each of these two ubiquitinconjugating enzymes is essential for cell viability andresponsible for degradation of Cdc13. These results
suggest that the functions of these two ubiquitinconjugating enzymes are not redundant and they havedistinct functions for ubiquitination of Cdc13.
Furthermore, we found that ubiquitin chains of Cdc13
were totally reduced in ubcll mutant cells, whereas
ubiquitin chains were short and not reduced in ubc4
mutant cells. Thus, we proposed a hypothesis thatUbcll might be involved in initiation of ubiquitination,and Ubc4 might be involved in elongation of ubiquitin

12

Molecular Mechanism Laboratory

chains of Cdc13. However, this hypothesis has not been
elucidated yet.
To clarify the functional differences between Ubc4
and Ubc11 for degradation of Cdc13, development of
an in vitro assay system for ubiquitination for Cdc13
by using fission yeast components is required.
Currently, I am attempting to develop this assay
system for Cdc13. A ubiquitin-activating enzyme, these
two ubiquitin-conjugating enzymes and substrate
Cdc13 were expressed as recombinant proteins inbacterial cells and purified. One component of
Cdc13-specific ubiquitin ligase anaphase promoting
complex/cyclosome (APC/C) was tagged and expressedin fission yeast cells, and APC/C was purified fromfission yeast cells. Now I am examining the conditions
for reconstitution of a ubiquitination reaction of Cdc13.
(2) Regulation of the DNA damage checkpoint by theubiquitin pathway

Hiroaki Seino
The mutant strain of ubcll+ gene showed thedefect in mitotic progression. Additionally, this strain
showed the cell elongation phenotype. It was shownthat this cell elongation phenotype was caused by thedelay of G2 phase by cell synchronization experiments.
This phenotype is dependent on the DNA damage
checkpoint but not the DNA replication checkpoint.
When ubc1r was inactivated, the checkpoint kinase
Chkl was activated. It is possible that the ubiquitin
pathway involving Ubc11 degrades the regulator(s) of
the DNA damage checkpoint. It is important to identify
the target protein(s) to understand the relationshipbetween this ubiquitin pathway and the DNA damage
checkpoint. I am attempting the establishment of themethod of screening the target proteins of a specificubiquitin pathway.

A-d. Division of Nucleic Acid Chemistry
Tokuko Haraguchi GroupRESEARCH ACTIVITIES(1) Dynamics of nuclear structure and function

T okuko Haraguchi
The cell nucleus is a structure that functions toautonomously decode the genetic information encoded
in DNA. Its structure changes in response to biologicalevents such as development, cell growth,differentiation, aging, and the cell's reaction toenvironmental factors. To study these changes innuclear structure, we have developed fluorescencemicroscope systems capable of recording the dynamic
behavior of molecular components in living cells. Using
these microscope systems, we study the functional andtemporal organization of nuclear structures, andattempt to propose a model for the genome operating
system which supports fundamental biologicalactivities.
Toward such research goals, we have studied
molecular mechanisms of cell division by focusing on
the function of centromere proteins. Cdc42 works inmetaphase by regulating bi-orient attachment of
spindle microtubuIes to kinetochores. We reported thatoverexpression of dominant-negative mutants of Ect2
(a Rho GTPase guanine nucleotide exchange factor)
and MgcRacGAP (GTPase activating protein), or
depletion of Ect2 suppresses the level of GTP-Cdc42
in mitosis, and caused defects in microtubule
attachment to kinetochores. In this study, we
concluded that Ect2 and MgcRacGAP regulate theactivation and function of Cdc42 in mitosis(Oceguera-Yanez et aI, 2005). We also studied functionof a newly identified kinetochore protein, CENP-50, in
mitosis, and found that CENP-50 is a constitutive
component of the centromere. To determine the roleof CENP-50, we generated a loss-of-function mutant
in the chicken DT40 cell line. The CENP-50 knockout
was not lethal; however, the time required for
CENP-50-deficient cells to complete mitosis was longer
than that for wild-type cells. We also observed severe
mitotic defects in CENP-50-deficient cells with apparent
premature sister chromatid separation when the
mitotic checkpoint was activated, indicating that

CENP-50 is required for recovery from spindle damage(Minoshima et aI, 2005). In fission yeast, centromeres
remain clustered at the spindle-pole body (SPB) duringmitotic interphase whereas those dissociate from the
SPB during meiotic prophase. We examined the.
behavior of centromere proteins in living meiotic cellsof fisson yeast. We show that the Nuf2-Ndc80 complex
proteins (Nuf2, Ndc80, Spc24, and Spc25) disappear
from the centromere in meiotic prophase when thecentromeres are separated from the SPB. We also showthat mutation of Nuf2 causes the release ofcentromeres from the SPB in patl mutant cells,suggesting that the Nuf2-Ndc80 complex connects
centromeres to the SPB. In this paper, we proposed that
the Nuf2-Ndc80 complex may be important in
remodeling of the kinetochore for meiotic chromosome
segregation (Asakawa et aI, 2005).
PUBLICATIONSPapers

1. Asakawa, H., Hayashi, A., Haraguchi, T., Hiraoka,
Y. (2005) Dissociation of the Nuf2-Ndc80 complex
releases centromeres from the spindle-pole body during
meiotic prophase in fission yeast., Mol. BioI. Cell, 16(5) 2325 - 2338
2. Oceguera-Yanez, F., Kimura, K., Yasuda, S.,
Higashida,C., Kitamura,T., Hiraoka, Y., Haraguchi, T.
and Narumiya, S. (2005). Ect2 and MgcRacGAP
regulate the activation and function of Cdc42 inmitosis. J. Cell BioI. 168, 221 - 232.
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Division of Nucleic Acid Chemistry13

A-d. Division of Nucleic Acid Chemistry
Hiroshi Iwasaki Group.RESEARCH ACTIVITIES(1) Functional overlap between RecA and MgsA (RarA)in the rescue of stalled replication forks in Escherichia
coli
Hiroshi Iwasaki (Yokohama City University)

Escherichia coli RecA protein plays a role in DNA
homologous recombination, recombination repair, andthe rescue of stalled or collapsed replication forks. The
mgsA (rarA) gene encodes a highly conserved
DNA-dependent ATPase, whose yeast orthologue,
MGS1, plays a role in maintaining genomic stability.
In this study, we show a functional relationshipbetween mgsA and recA during DNA replication. The
mgsA recA double mutant grows more slowly and has
lower viability than a recA single mutant, but they are
equally sensitive to UV-induced DNA damage.
Mutations in mgsA and recA cause lethality in DNA
polymerase I deficient cells, and suppress thetemperature-dependent growth defect of dnaE486 (PolIII alpha-catalytic subunit). Moreover, recAS25P, anovel recA allele identified in this work, does notcomplement the slow growth of d mgsA d recA cells
or the lethality of polA12 d recA, but is proficient in
DNA repair, homologous recombination, SOS
mutagenesis and SOS induction. These results suggestthat RecA and MgsA are functionally redundant in
rescuing stalled replication forks, and that the DNA
repair and homologous recombination functions of
RecA are separated from its function to maintainprogression of replication fork (Shibata et aI., 2005).
(2) Genetic and physical interactions between
Schizosaccharomyces pombe Mcll and Rad2, Dna2 andDNA polymerase alpha: evidence for a multifunctionalrole of Mcll in DNA replication and repair.

to methyl methanesulfonate as single mutants. One ofthese mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1(mcll-101) is allelic to mcl1(+), which is required forchromosome replication, cohesion and segregation.
mcll-101 is temperature-sensitive for growth and ishighly sensitive to DNA damage. mcll cells arrest with
2C DNA content and chromosomal DNA double-strand
breaks accumulate at the restrictive temperature.
Mcllp, which belongs to the Ctf4p/SepBp family,interacts both genetically and physically with DNA
polymerase alpha. Mutations in rhp51 and dna2enhance the growth defect of the mcll-101 mutant.
These results strongly suggest that Mcllp is afunctional homologue of Saccharomyces cerevisiae Ctf4p
and plays a role in lagging-strand synthesis andOkazaki fragment processing, in addition to DNA
repair (Tsutsui et aI., 2005).
(3) Structue-function analysis of the three domains of
RuvB motor protein
Hiroshi Iwasaki (Yokohama City University)
RuvB protein forms two hexameric rings that bindto the RuvA tetramer at DNA Holliday junctions. The
RuvAB complex utilizes the energy of ATP hydrolysis
to promote branch migration of Holliday junctions.
The crystal structure of RuvB from Thermus
thermophilus (Tth) HB8 showed that each RuvB
monomer has three domains (N, M, and C). This study
is a structure-function analysis of the three domainsof RuvB. The results show that domain N is involvedin RuvA-RuvB and RuvB-RuvB subunit interactions,domains Nand M are required for ATP hydrolysis andATP binding-induced hexamer formation, and domain
C plays an essential role in DNA binding. The side
chain of Arg-318 is essential for DNA binding and may
directly interact with DNA. The data also provide
evidence that coordinated ATP-dependent interactions
between domains N, M, and C play an essential role
during formation of the RuvAB Holliday junction
ternary complex (Ohnishi et aI., 2005).

Hiroshi Iwasaki (Yokohama City University)

Schizosaccharomyces pombe rad2 is involved in
Okazaki fragments processing during lagging-strand
DNA replication. Previous studies identified several
sir mutants that are co-lethal with rad2d and sensitive

14

Division of Nucleic Acid Chemistry(4) Role of the Schizosaccharomyces pombe F-box
DNA helicase in processing recombination intermediates
Hiroshi Iwasaki (Yokohama City University)

In an effort to identify novel genes involved inrecombination repair, we isolated fission yeast
Schizosaccharomyces pombe mutants sensitive to methyl
methanesulfonate (MMS) and a synthetic lethal withrad2. A gene that complements such mutations was
isolated from the S. pombe genomic library, andsubsequent analysis identified it as the fbh1 geneencoding the F-box DNA helicase, which is conservedin mammals but not conserved in Saccharomyces
cerevisiae. An fbhl deletion mutant is moderately
sensitive to UV, MMS, and gamma rays. The rhp51(RAD51 ortholog) mutation is epistatic to fbhl. fbhl isessential for viability in stationary-phase cells and in. the absence of either Srs2 or Rqh1 DNA helicase. In
each case, lethality is suppressed by deletion of therecombination gene rhp57. These results suggested
that fbhl acts downstream of rhp51 and rhp57.
Following UV irradiation or entry into the stationary
phase, nuclear chromosomal domains of the fbhl Llmutant shrank, and accumulation of somerecombination intermediates was suggested bypulsed-field gel electrophoresis. Focus formation of
Fbh1 protein was induced by treatment that damages
DNA. Thus, the F-box DNA helicase appears to process
toxic recombination intermediates, the formation ofwhich is dependent on the function of Rhp51 (Morhisita
et aI., 2005).

Y, Iwasaki H, and Shinagawa H. J. BioI. Chern. (2005)
26:280 (34): 30504-30510.

PUBLICATIONSPapers

1. Functional overlap between RecA and MgsA
(RarA) in the rescue of stalled replication forks inEscherichia coli. ShibataT, Hishida T, Kubota Y, Han
YW, Iwasaki H, and Shinagawa H. Genes Cells. (2005)
10:181-191.
2. Genetic and physical interactions between
Schizosaccharomyces pombe Mcll and Rad2, Dna2 andDNA polymerase alpha: evidence for a multifunctionalrole of Mcll in DNA replication and repair. Tsutsui Y,
Morishita T, Natsume T, Yamashita K, Iwasaki H,
Yamao F, Shinagawa H. Curr Genet. (2005) 48:34-43
3. Role of the Schizosaccharomyces pombe F-box DNA
helicase in processing recombination intermediates.
Morishita T, Furukawa F, Sakaguchi C, Toda T, Carr
AM, Iwasaki Hand Shinagawa H. Mol. Cell. BioI.
(2005) 25:8074-883.
4. Structue-function analysis of the three domainsof RuvB motor protein. Ohnishi T, Hishida T, Harada

Division of Nucleic Acid Chemistry15

B. DEPARTMENT OF
CELL GENETICS

B-a. Division of Cytogenetics
Tamotsu Yoshimori GroupRESEARCH ACTIVITIES

Most of membrane-bound organelles inside
eukaryotic cells are linked each other by dynamic
membrane trafficking regulated by a set of specificproteins. Membrane traffic is essential not only tosurvival of each cell but also to various functionsorganizing the multi-cellular system, e.g., formation ofcell polarity and intercellular communication. We aim
to unravel molecular mechanisms of membrane traffic
and their roles in physiological functions and diseases
in animals, which must produce knowledge
contributing clinical medicine. Weare now focusing
on two trafficking routes; autophagy and theendosomal system. Autophagy is membrane trafficdelivering cytoplasmic components to lysosomes forbulk degradation. The process is mediated by theformation of the double membrane-boundautophagosomes. Endosomes receive macromolecules
taken up by endocytosis from outside. The cargo isthen either sorted to lysosomes or recycled back to theplasma membrane.
(1) Intracellular inclusions containing mutant aIantitrypsin Z are propagated in the absence of autophagic
activity
Takahiro Kamimoto, Shisako Shoji, Tunda Hidvegi\
Noboru Mizushima2, Kyohei Umebayashi, David H.
Perlmutter', and Tamotsu Yoshimori ('University ofPittsburgh, 2Tokyo Metropolitan Institute of Medical
Science)
Mutant aI-antitrypsin Z (al-ATZ) protein, which
has a tendency to form aggregated polymers as it
accumulates within the endoplasmic reticulum of the16

DEPARTMENT OF CELL GENETICS

liver cells, is associated with the development ofchronic liver injury and hepatocellular carcinoma in
hereditary aI-antitrypsin (aI-AT) deficiency. Previous
studies have suggested that efficient intracellulardegradation of al-ATZ is correlated with protection
from liver disease in a [-AT deficiency and that theubiquitin-proteasome system accounts for a majorroute, but not the sole route, of a [-ATZ disposal. Yetanother intracellular degradation system, autophagy,
has also been implicated in the pathophysiology of
aI-AT deficiency. To provide genetic evidence for
autophagy-mediated disposal of a l-ATZ, here we usedcell lines deleted for the Atg5 gene that is necessary
for initiation of autophagy. In the absence ofautophagy, the degradation of al-ATZ was retarded,
and the characteristic cellular inclusions of al-ATZ
accumulated. In wild-type cells, colocalization of the
autophagosomal membrane marker GFP-LC3 andal-ATZ was observed, and this colocalization wasenhanced when clearance of autophagosomes wasprevented by inhibiting fusion between autophagosome
and lysosome. By using a transgenic mouse withliver-specific inducible expression of a I-ATZ matedto the GFP-LC3 mouse, we also found that expressionof al-ATZ in the liver in vivo is sufficient to induce
autophagy. These data provide definitive evidence that
autopahgy can participate in the quality control/
degradative pathway for al-ATZ and suggest that
autophagic degradation plays a fundamental role inpreventing toxic accumulation of a [-ATZ. (Kamimoto,T., et al., 2005)(2) LC3 interacts with dynein motor and has animportant role in microtubule-dependent autophagosome
movement
Shunsuke Kimura and Tamotsu Yoshimori
Time-lapse video microscopy revealed that
autophagosomes labeled with GFP-LC3 moved in thecytosol. Nocodazole inhibited the motion, whereas
other cytoskeletal inhibitors did not inhibit it.
Immunofluorescent microscopic study showed that
endogenous LC3 arranged along the tracks of
microtubules. These findings indicate that
autophagosome moves along microtubules. Then, we
examined the motor protein involved in this movement.
Overexpression of dynamitin, which is known to
disrupt dynein- and dynactin-dependent organelle

movement, inhibited autophagosome movements. In
addition, endogenous LC3 colocalized with dyneindynactin complex. Coimmunoprecipitation and GST
pull-down assay showed that LC3 directly interacts
with the intermediate chain of cytoplasmic dynein,
which is a subunit of dynein motor complex. These
results indicate that autophagosomes are linked withthe dynein-dynactin complex through the interaction
between LC3 and dynein intermediate chain.
To clarify the involvement of LC3 in autophagosome
movement, microinjection experiments of anti-LC3
antibodies were performed. While injection of neither
anti-GFP nor control IgG had effect on autophagosome
movement, injection of anti-LC3 antibodies inhibitedthe movement. Moreover the anti-LC3 peptide
antibodies raised against the N-terminal 1-15 residues,
which is known as a microtubule binding region, also
inhibited the movement. These results suggest thatinteraction of microtubule with LC3 is important forthe movement. Finally, using FRAP assay, thetransport and fusion of autophagosomes to lysosomes
were measured. The transport and fusion weresignificantly inhibited by the injection of anti-LC3
antibody, suggesting that microtubule-dependent
movement is necessary for targeting of peripherallyformed autophagosomes to lysosomes localizing toperinuclear region. From these results, we concluded
that 1) autophagosome movement is dependent on
microtubule and dynein/dynactin motor complex, 2)
this movement is necessary for targeting of
autophagosomes to lysosomes in mammalian cells, and
3) LC3 plays an important role in assembly of thetransport machinery. LC3 may link dynein motor,
microtubule and autophagosome. This is the firstreport elucidating the function of LC3 in autophagy
in mammalian cells.
(3) Molecular dissection of internalization ofPorphyromonas gingivalis by cells using fluorescentbeads coated with bacterial membrane vesicle
Kayoko Tsuda, Atsuo Amano\ Kyohei Umebayashi,
Hiroaki Inaba\ Ichiro Nakagawa\ Yoshino bu
Nakanishi 2, Tamotsu Yoshimori COsaka University,
2Kanazawa University)

Porphyromonas gingivalis is one of the causative
agents of adult periodontitis, and has been reported
to be internalized by nonphagocytic epithelial cells.

However, the mechanism for the internalization
remains unclear. In the present study, we addressed
this issue using fluorescent beads coated with bacterial
membrane vesicles (MVs) that retain surface
components of P. gingivalis. We established an assay
system in which we could easily quantify the beadinternalization to cells. MVs-coated beads wereinternalized by HeLa cells in kinetics similar to thatof living bacteria. The internalization depended on
dynamin but not clathrin. The beads were internalized
through the actin-mediated pathway that is controlledby phospatidylinositol (PI) 3-kinase. The dynamics of
microtubule assembly and disassembly was also
required. Further, the treatement of cells withcholesterol-binding reagents significantly inhibitedbead internalization, and the internalized beads wereapparently colocalized with ganglioside GMI andcaveolin-l, which suggest the involvement of the lipid
raft in the process. These results suggest that P.
gingivalis accomplishes its internalization utilizing
membrane lipid raft and cytoskeletal functions of thetarget cells. (Tsuda, K., et al., 2005)(4) Ubiquitin-dependent sorting of the epidermal growth
factor receptor in the endocytic pathway
Kyohei Umebayashi and Tamotsu Yoshimori
When plasma membrane proteins are
ubiquitinated, they follow the endocytic pathway tolysosomes. In the case of the epidermal growth factor
receptor (EGFR), the Cbl E3 ligase is responsible forligand-dependent ubiquitination. It has been
considered that the ubiquitination of the receptor
occurs in the plasma membrane. However, we foundthat Cbl was relocated to early endosomes when cells
were stimulated with EGF. Analyses of colocalization
as well as coimmunoprecipitation indicated two modesof EGFR-Cbl binding: Cbl was initially targeted to
EGFR in the plasma membrane, dissociated after awhile, and again bound to the receptor in earlyendosomes. Notably, Cbl was localized to earlyendosomal subdomains marked by Hrs, a ubiquitin
binding protein required for multivesicular body(MVB) sorting. At longer chase periods after the ligand
stimulation, Cbl was not colocalized with EGF, strongly
suggesting that Cbl becomes separated from thereceptor prior to the MVB sorting. We have obtained
evidence that an AAA ATPase SKDl is required for

Division of Cytogenetics

17the final dissociation of Cbl from EGFR. Expressionof the dominant-negative SKD1(E235Q) resulted in
prolonged EGFR-Cbl binding and excess ubiquitination of the receptor. Concomitantly, EGFR transport
was blocked at aberrant endosomes where the receptor
was preferentially colocalized with Hrs. Consideringthat the functions of SKD1 and its homologues are todisassemble protein complexes, we propose that SKD1
terminates the receptor ubiquitination by dissociating
Cbl from EGFR. Termination of ubiquitination andsubsequent deubiquitination would be required forproper sorting of cargo proteins into the MVBs.
PUBLICATIONS

B-b. Division of Microbial Genetics
Hiroyuki Araki GroupRESEARCH ACTIVITIESWe have been studying on eukaryotic
chromosomal DNA replication and its regulation by
the cell cycle. For this purpose, we have employedbudding yeast, Saccharomyces cerevisiae, as a model
system of eukaryotic cells. Using strong genetics ofbudding yeast, we have identified novel factors
involving in chromosomal DNA replication andrevealed their functions in chromosomal DNA
replication.

Papers

1. Boya, P., Gonzalez-Polo, R.-A., Casares, N.,
Perfettini, l-L., Dessen, P., Larochette, N., Metivier, D.,
Meley, D., Souquere, S., Yoshimori, T., Pierron, G.,
Codogno, P., Kroemer, G. (2005) Inhibition ofmacroautophagy triggers apoptosis, Mol. Cell BioI., 25(3) 1025 - 1040
2. Hoshino, M., Yoshimori, T., Nakamura, S. (2005)
Small GTPase proteins Rin and Rit bind to Par6
GTP-dependently, and regulate cell transformation, l
BioI. Chern., 280 (24) 22868 - 22874
3. Mizuno, E., Iura, T., Mukai, A., Yoshimori, T.,
Kitamura, N., Komada, M. (2005) Regulation of
epidermal growth factor receptor down-regulation by
UBPY-mediated deubiquitination at endosomes, Mol.
BioI. Cell, 16 (11) 5163 - 5174

4. Towns, R., Kabeya, Y., Yoshimori, T., Guo, C.,
Shangguan, Y., Hong, S., Kaplan, M., Klionsky, D. ].,Wiley, J. W. (2005) Sera from patients with type 2
Diabetes and Neuropathy Induce Autophagy and
Colocalization with Mitochondria in SY5Y cells,
Autophagy, 1, 163 - 170
5. Tsuda, K, Amano, A., Umebayashi, K, Inaba, H.,
Nakagawa, I., Nakanishi, Y., Yoshimori, T. (2005)
Molecular dissection of internalization ofPorphyromonas gingivalis by cells using fluorescentbeads coated with bacterial membrane vesicle, Cell
Struct. Funct., 30 (2) 81 - 91
6. Yamada, T., Carson, A. R., Caniggia, I.,
Umebayashi, K, Yoshimori, T., Nakabayashi, K,
Scherer, S. W. (2005) Endothelial nitric oxide synthase
antisense (NOS3AS) gene encodes an autophagyrelated protein (APG9-like2) highly expressed introphoblast, J. BioI. Chern., 280 (18) 18283 - 18290

18

Division of Cytogenetics(1) The pre-Landing complex of replication proteins
formed in a CDK-dependent manner

Sachiko Sakamoto, Kazuyuki Hirai, Yoichiro
Kamimura, and Hiroyuki Araki
At replication origins in eukaryotes, thepre-Replicative Complex (pre-RC) forms from late M
to G1 phases in the absence of CDK (Cyc1in-dependent
kinase) activity and other replication proteins including
DNA polymerases assemble when CDK activity
increases from G1/S boundary. We have reported threeyeast complexes, Sld2-Dpbll, Sld3-Cdc45 and GINS,
all of which associate with origins in a mutuallydependent and the pre-RC dependent manner. Dpbll
has four BRCT repeats and form a complex with the
Sld2 protein phosphorylated by CDK The Sld3-Cdc45
complex is observed throughout the cell cycle. The
GINS complex consisting of Sld5, Psfl, Psf2 and Psf3
subunits first associates with origins and then moves
with replication forks.
To elucidate how the proteins assemble, weinvestigated the complex formation between GINS andother proteins by co-immunoprecipitation assay. GINS
coprecipitated with Pol E and with Mcm, a componentof the pre-RC, in the S phase. Moreover, when wetreated the cells with the cross-linking agent before
precipitation, Sld3, Sld2 and Dpbllalso coprecipitatedwith GINS. Without the pre-RC in Cdc6-depleted cells,
Sld2-Dpbll and Pol E coprecipitated with GINS,
suggesting that they form a complex without originassociation. In the same condition, we could precipitate
tagged one of Sld2, Dpbll and Pol E with remainingthree proteins. Furthermore, there were strong genetic
interactions between Sld2, Dpb11, Pol E and GINS. We
therefore propose that Sld2, Dpb11, Pol E and GINS
constitute the pre-Landing Complex (pre-LC), which
is formed before associating with origins.
To initiate chromosomal DNA replication, twoprotein kinases, CDK and DDKlCdc7 (Dbf4-dependent
kinase) are required. The pre-LC formation appeared
to depend on CDK but not DDK Using several mutant
cells, we revealed that the Pol E -GINS and the
Sld2-Dpb11 complexes are mainly connected bybinding of Pol E and Sld2 in the presence of CDK
activity. The pre-LC seems to associate with the pre-RC
via Sld3 since the C-terminal portion of Sld3 interacts
with GINS whereas the N-terminal portion interacts
with Cdc45.
In parallel with the in vivo analysis, we havepurified the proteins involved in the pre-LC toreconstitute it. We expressed all the subunits of GINSin insect cells and purified GINS to be a near
homogeneity. We also purified Dpb11, Sld2 and Pol E
from yeast cells. Preliminary result showed that they
form a complex in vitro without other proteins. Thus,
although we do not know all the components, at least
Dpb11, Sld2, Pol E and GINS are core components ofthe pre-LC.
(2) Regulatory mechanism of the complex formation
between Sld2 and Dpbll by eyelin-dependent kinase(CDK)

Yon-Soo Tak, Yoshimi Tanaka, Yoichiro Kamimuraand Hiroyuki Araki
Phosphorylation often regulates protein-protein
interactions to control biological reactions. The Sld2
and Dpb11 proteins of budding yeast form aphosphorylation-dependent complex that is essential
for chromosomal DNA replication. The Sld2 protein
has a cluster of 11 cyclin-dependent kinase (CDK)phosphorylation-motifs (Ser/Thr-Pro), six of whichmatch the canonical sequences Ser/Thr-Pro-X-Lys/Arg,
Lys/Arg-Ser/Thr-Pro and Ser/Thr-Pro-Lys/Arg.
Simultaneous alanine-substitution for serine or
threonine in all the canonical CDK-phosphorylation
motifs severely reduces complex formation between
Sld2 and Dpbll, and inhibits DNA replication. We
show that phosphorylation of these canonical motifs
does not playa direct role in complex formation but

rather regulates phosphorylation of another residue,
Thr84. This constitutes a non-canonical CDKphosphorylation motif within a 28-amino acid sequencethat is responsible, after phosphorylation, for bindingof Sld2 to Dpb11. We further suggest that CDKcatalysed phosphorylation of sites other than Thr84
renders Thr84 accessible to CDK From these results,
we argue that this novel mechanism sets a thresholdof CDK activity for formation of the essential
Sld2-Dpb11 complex and therefore prevents premature
DNA replication.
(3) CDK targets in the initiation of DNA replication

Seiji Tanaka and Hiroyuki Araki
In eukaryotic cells, CDKs playa pivotal role in theregulation of cell cycle progression. Periodic activationand inactivation of stage-specific CDK ensures the cell
cycle to progress in one way at the correct timing. Of
the cellular processes regulated by CDK, chromosomal
DNA replication is one of the most important. Because
the failure in chromosomal DNA replication iscatastrophic for cells and organisms, !=ell has theregulatory mechanism that ensures the chromosomal
DNA replication to happen exactly once per cell cycle.
In this mechanism, CDKs play a central role byregulating two mutually exclusive steps. First, CDKs
trigger the initiation of DNA replication, and at thesame time, CDKs inhibit formation of the pre-RCs,
which is required for the initiation of DNA replication_
Because of this inhibitory action of CDK, the pre-RCs
are formed at the origins of DNA replication only inlate M and G1 in the cell cycle when CDK activity islow. Once CDK is activated, it trigger DNA replication(S phase) by activating the pre-RC. Importantly,
re-replication is strictly inhibited at this time because
CDK blocks pre-RC re-formation at the fired origins.
During following G2-M phase, CDK activity is high andthus keeps to inhibit re-replication. When cells finish
M phase and enter into the next G1, CDK activity
rapidly decreases and then cells can form the pre-RC
again.
To understand how CDK activates pre-RC and
how inhibits pre-RC formation has been a major
challenge of the research field of DNA replication.
Recent studies including ours revealed that CDK
inhibits pre-RC formation by downregulating all ofknown pre-RC components. In contrast, target(s) of

Division of Microbial Genetics

19

CDK in pre-RC activation had been elusive for a longtime. Recently, we have reported that Sld2 is an
essential target of CDK for the initiation in budding
yeast. We first asked whether Sld2 is a sole target of
CDK in the initiation. To test the possibility, we
simultaneously substituted aspartic acid for all ofserine or threonine residues at CDK phosphorylation
motifs in Sld2. This sld2mutant (sld2-11D) supported
cell growth, suggesting that Sld2-11D mimics a
phosphorylated active form of Sld2. In this condition,
CDK was still required for the initiation of DNA
replication. Therefore, we concluded that CDK has
essential targets other than Sld2 in the initiation
process. To identify unknown target(s) of CDK, we
performed a genetic screening and finally isolated amutant which can initiate DNA replication without
CDK activity. The mutant, JETl-1 (lumping CDK
requirement with ~even D mutant of SLD-Iwo), could
initiate the CDK-free DNA replication only whenSld2-11D was expressed. Further analyses revealed that
Jetl-1 and Sld2-11D can replace the role of CDK in
initiation. These results suggest that CDK regulates
at least two essential processes in the initiation of DNA
replication.
PUBLICATIONSPapers

1. Ohya, Y., Sese,]., Yukawa, M., Sano, F., Nakatani,Y., Saito, T. L., Saka, A., Fukuda, T., Ishihara, S., Oka,S., Suzuki, G., Watanabe, M., Hirata, A., Ohtani, M.,
Sawai, H., Fraysse, N., Latge, 1. P., Francois,]. M., Aebi,
M., Tanaka, S., Muramatsu, S., Araki, H., Sonoike, K.,
Nogami, S., Morishita, S. (2005) High-dimensional andlarge-scale phenotyping of yeast mutants, Proc. Natl.
Acad. Sci. USA, 102 (52) 19015 - 19020
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EDUCATION

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Division of Microbial Genetics

B-c. Division of Cytoplasmic Genetics
Kazuto Kobayashi GroupRESEARCH ACTIVITIES(1) Mechanisms of Neural Circuitry Underlying
Behavioral Control
Kazuto Kobayashi
Control of behaviors is based on the mechanismsthat mediate processing and regulation of information
through the complex neural circuitry in the brain.
Dysfunction in the neural circuitry is involved in the
etiology and pathogenesis of some neural diseases. Our
research group is interested in understanding the roleof specific neurons or neural pathways that mediate
motor control, memory, and emotion as well as thefunction of the signaling molecules that are involvedin these behaviors. In the present year, we analyzed
the mechanism by which the subthalamic nucleus,
which is a key component of basal ganglia, regulates
motor behavior in the neural circuitry. Conditional
ablation of the subthalamic neurons by immunotoxinmediated cell targeting revealed that these neurons act
to coordinate motor behavior through differential
neural pathways depending on the state of dopaminetransmission (Yasoshima et al., 2005). In addition, wedeveloped a new system for physiological studies ofidentified neurons in cell culture by using transgenic
mice expressing green fluorescent protein in tyrosinehydroxylase-positive neurons Gomphe et aI., 2005). Thissystem was used for studying the functional propertiesof the olfactory bulb dopamine neurons (Pignatelli etal., 2005) and the receptor desensitization/internalization in the locus coeruleus noradrenaline neurons(Arttmangkul et al., 2006). The system was also usefulfor examining the role of some factors in thedevelopment and survival of midbrain dopamine
neurons (Rawal et al., 2006; Meurer et al., 2006).
Furthermore, we described the mutations of tyrosinehydroxylase gene that are involved in human inherited
diseases, including dystonia and movement disorders(Kobayashi and Nagatsu, 2005).

PUBLICATIONSEDUCATION, SOCIAL CONTRIBUTIONS AND OTHERS:

Papers~*~/W~~OO~~~.~~~T-

1. Arttamangkul, S., Torrecilla, M., Kobayashi, K,
Okano, H, and Williams, ].T. (2006) Separation of mu
opioid receptor desensitization and internalization:
endogenous receptors in primary neuronal cultures.
J. Neurosci. in press.

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2. Jomphe, C., Bourque, M.-]., Fortin, G., St-Gelais,F., Okano, H., Kobayashi, K, and Trudeau, L.-E. (2005)
Use of TH-GFP transgenic mice as a source ofidentified dopaminergic neurons for physiologicalstudies in postnatal cell culture, J. Neurosci. Methods,
146 (1) 1 - 12
3. Kobayashi, K and Nagatsu, T. (2005) Molecular
genetics of tyrosine 3-mono- oxygenase and inherited
diseases, Biochem. Biophy. Res. Commun., 338 (1)267270
4. Meuer, K, Teismann, P., Peters, K, Goricke, B.,
Lingor, P., Schlachetzki, ]., Kobayashi, K, Dietz, G.
P.H, Schulz, lB., Bahr, M., Schneider, A., andWeishaupt, ]. H. (2006) Granulocyte-colony stimulating
factor is neuroprotective in a model of Parkinson's
disease. J. Neurochem. in press.
5. Pignatelli, A., Kobayashi, K, Okano, H., andBelluzzi, O. (2005) Functional properties of
dopaminergic neurones in the mouse olfactory bulb,
J. Physiol., 564 (2) 501 - 514
6. Rawal, N., Castelo-Branco, G., Sousa, K M.,
Kobayashi, K, Okano, H., and Arenas, E. (2006)
Dynamic temporal and cell type-specific expression of
Wnt signaling components in the developing midbrain.
Exp. Cell Res. in press.
7. Yasoshima, Y., Kai, N., Yoshida S., Shiosaka, S.,
Koyama, K, Kayama, F., and Kobayashi, K (2005)
Subthalamic neurons coordinate basal ganglia function
through differential neural pathways, J. Neurosci., 25(34) 7743 - 7753
Reviews

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~~~~

B-c. Division of Cytoplasmic Genetics
Masayuki Yamamoto GroupRESEARCH ACTIVITIES(I) Hrslp/Mcp6p on the meiotic SPB organises astral

microtubule arrays for oscillatory nuclear movement

Masayuki Yamamoto
Microtubules and the motor protein dynein play
pivotal roles in the movement and positioning of thenucleus and cytoplasmic organelles in a cell. In fission
yeast, oscillatory movement of the nucleus termed
horsetail nuclear movement (HNM), has been observed
during meiotic prophase. HNM is led by an astral
microtubule array emanating from the spindle polebody (SPB), a centrosome equivalent organelle inyeasts, aided by the dynein-dynactin complex, and is
proposed to facilitate the alignment of homologous
chromosomes necessary for efficient meiotic
recombination. We have shown that a meiosis-specific
SPB component Hrs1p (also known as Mcp6p) is a key
molecule to remodel microtubules into thehorsetail-astral array (HAA). Deletion of Hrs1p
impaired HAA formation, leading to compromised
HNM. Ectopic expression of Hrs1p during the mitotic
cell cycle resulted in the formation of a HAA-like astral

Division of Cytoplasmic Genetics

21

microtubule array, which drove an oscillatory nuclear
movement in interphase cells. Hrs1p interacted withcomponents of the y -tubulin ring complex (y -TuRC)
as well as with a meiotic SPB component. Thus, we
propose that Hrs1p facilitates formation of the HAA,
responsible for the vigorous HNM, by stabilisingconnection between the SPB and minus ends ofmicrotubules (Tanaka, K., et al., 2005).
(2) The C. elegans Homologue of Deleted in
Azoospermia is Involved in the Spenn/Oocyte Switch
Masayuki Yamamoto
The Deleted in Azoospermia (DAZ) gene family
encodes putative translational activators that are
required for meiosis and other aspects ofgametogenesis in animals. The single C. elegans
homologue of DAZ, daz-1, is an essential factor for
female meiosis. We have clarified that daz-1 isimportant for the switch from spermatogenesis tooogenesis (the sperm/oocyte switch), which is an
essential step for the hermaphrodite germline to
produce oocytes. RNA interference of the daz-1
orthologue in a related nemetode C. briggsae resulted
in a complete loss of the sperm/oocyte switch. The C.
elegans hermaphrodite deficient in daz-1 also revealed
a failure in the sperm/oocyte switch if the genetic
background was conditional Mog (masculinization of
germline). DAZ-1 could bind specifically to mRNAs
encoding the FBF proteins, which are translational
regulators for the sperm/oocyte switch and germ stem
cell proliferation. Expression of the FBF proteins
seemed to be lowered in the daz-1 mutant at the stage
for the sperm/oocyte switch. Conversely, a mutationin gld-3, a gene that functionally counteracts FBF, could
partially restore oogenesis in the daz-1 mutant. Taken
together, we propose that daz-1 plays a role upstreamof the pathway for germ cell sex determination (Otori,M., et al., 2006).
(3) C. elegans CPB-3 interacts with DAZ·l andfunctions in multiple steps of gennline development

Masayuki Yamamoto
Cytoplasmic polyadenylation element-binding
proteins (CPEBs) are well-conserved RNA-binding
proteins, which regulate mRNA translation mainly

22

Division of Cytoplasmic Genetics

through control of poly(A) elongation. We have shownthat CPB-3, one of the four CPEB homologs in C.
elegans, positively regulates multiple aspects of oocyte
production. CPB-3 protein was highly expressed inearly meiotic regions of the hermaphrodite gonad.
Worms deficient in cpb-3were apparently impaired ingerm cell proliferation and differentiation including
sperm/oocyte switching and progression of female
meiosis. We also show that cPb-3 is likely to promote
the meiotic entry in parallel with gld-3, a componentof one of the redundant but essential genetic pathways
for the entry to and progression through meiosis.
Altogether, CPEB appears to have a conserved role in
the early phase of meiosis and in the sperm/oocyte
specification, in addition to its reported function during
meiotic progression. (Hasegawa, E., et al., 2006).
PUBLICATIONSPapers

1. Izawa, D., Goto, M., Yamashita, A., Yamano, H.,
Yamamoto, M. (2005) Fission yeast Mes1p ensures theonset of meiosis II by blocking degradation of cyclin
Cdcl3p, Nature, 434, 529 - 533
2. Maruyama, R., Endo, S., Sugimoto, A., Yamamoto,M. (2005) C. elegans DAZ-1 is expressed in proliferating
germ cells and directs proper nuclear organization andcytoplasmic core formation during oogenesis., Dev.
BioI., 277, 142 - 154
3. Tanaka, K., Kohda, T., Yamashita, A., Nonaka, N.,
Yamamoto, M. (2005) Hrs1pIMcp6p on the meiotic SPB
organizes astral microtubule arrays for oscillatory
nuclear movement, Curro Biol.,15,1479 - 1486
4. Yamashita, A., Sato, M., Fujita, A., Yamamoto M.,
Toda, T. (2005) The roles of fission yeast Ase1 inmitotic cell division, meiotic nuclear oscillation andcytokinesis checkpoint signaling, Mol. BioI. Cell, 16,1378 - 1395
EDUCATION, SOCIAL CONTRIBUTIONS AND OTHERS

Editor: Genes to Cells; Protein, Nucleic Acid andEnzymeEditorial Board: Current Genetics; YEAST

c. DEPARTMENT OF
DEVELOPMENTAL GENETICS

C-a. Division of Development Genetics
Yasushi Hiromi GROUPRESEARCH ACTIVITIES(1) Control of ligand trapping for the recognition of
spatial cues

Masaki Hiramoto and Yasushi Hiromi
When secreted molecules guide cell migration or
navigation of cellular processes, guidance molecules
are "trapped" by receptors that are expressed in the
motile cells. We have previously shown that trappingof a secreted axon guidance molecule Netrin by its
receptor Frazzled can result in the redistribution ofNetrin and subsequent presentation to other axons.
Here we show that trapping of Netrin by another
Netrin receptor, UNC5, has a different consequence;
it abolishes Frazzled-mediated Netrin presentation on
the cell surface. This activity of UNC5 was achievedby the extracellular domain of UNC5, and was
specifically associated with the ability to recognize thedirection of navigation, and not with the simple
inhibition of the extension. This implies that thecontrol of the ligand trapping is an crucial factor in
the recognition of spatial cues.
(2) Intrinsic sub-axonal compartmentalization in
Drosophila neurons

Takeo Katsuki, Masaki Hiramoto and Yasushi Hiromi
In the developing nervous system of vertebrates
and invertebrates, distribution of membrane molecules,
especially axon guidance receptors, is often restrictedto specific segments ofaxons. For example, a repulsive
axon guidance receptor ROBO and its family members
are expressed in distal axon segments, but largely

excluded from the proximal regions. It is not known
how such sub-axonal distribution patterns are
generated and maintained within individual neurons.
Using a low-density primary culture system ofDrosophila, we found that segmental localization ofmolecules along axons can be generated in the absenceof cell-cell contacts, which suggests that the localizationis due to an intrinsic property of neurons. Furthermore,
fluorescence recovery after photobleaching (FRAP)
analysis revealed that the mobility of transmembrane
molecules is restricted at the boundary of the localized
region, indicative of a barrier mechanism. We proposethat axons of Drosophila neurons are compartmentalized into sub-axonal regions by a barrier mechanism,and that this compartmentalization is achieved
cell-intrinsically.
(3) The relationship between sub-axonal localization ofproteins and glycosylation

Tohru Umemura, Takeo Katsuki and Yasushi Hiromi
A majority of cell surface proteins are modified
by the addition of carbohydrate moieties, which play
important roles in cell-cell interactions and protein
sorting. As a part of an effort to understand how
membrane proteins are targeted to specific sub-axonal
compartments, we examined whether or not sub-axonal
localization patterns correlate with modification via
a particular sugar moiety. A battery of 20 lectins werescreened by staining primary culture of Drosophila
embryonic neurons. One of the lectins exhibited astriking staining pattern; localization to either theproximal or middle segment of an axon. This pattern
was indistinguishable from the staining pattern witha monoclonal antibody BPI02, whose epitope is likely
a carbohydrate. These results raise a possibility thatglycosylation may mark the protein to be delivered toa particular sub-axonal compartment. On Western
blots both the lectin and BP102 antibody recognizedglycoproteins with molecular weights of about 75kD
and lOOkD. Weare trying to identify the BP102 antigens
to investigate how they are targeted to sub-axonal
compartments.
(4) Neutralization of repulsive-axon guidance cue on
target neurons

Fumikazu Suto, Hajime Fujisawa' and Yasushi Hiromi

DEPARTMENT OF DEVELOPMENTAL GENETICS

23

CNagoya University)
Axon guidance molecules are loosely classified in
two categories, attractive and repulsive. While it is easy
to see how an attractive guidance molecule may
provide a local guidance cue, how repulsive signals
pattern axonal trajectories is not well understood. We
are using mouse hippocampus, where its dendritic fieldreceives "layer-specific" inputs from multiple neuronal
types, to investigate the function and regulation of arepulsive guidance signal Sema6A. One neuronal typethat project in a layer-specific fashion is mossy fibers,
which mainly connect on the proximal-most part of theapical dendrite of CA3 pyramidal neurons. Although
cultured mossy fibers are repelled by Sema6A, Sema6A
was strongly localized in the dendritic field of CA3
pyramidal cells, suggesting that there is a mechanism
to counteract its repulsive activity to allow mossy fiber
projection. We found that Plexin-A2, a receptor forSema6A, was expressed in CA3 pyramidal cells, andexerted an "attractive" effect on mossy fiber projection;in plexin-A2 mutant mice, mossy fibers avoided thenormal target layer and were redirected to the basal
dendrite. This avoidance behavior likely involves
Sema6A, because it was suppressed by reducing
Sema6A levels. We propose that the target neurons
neutralizes the repulsive activity of Sema6A byexpressing its receptor on their dendrites, to licenselayer-specific projections within the dendritic field.
(5) Exploring the factors that regulate temporal gene

expression profile in neuronal precursors

Makoto Kanai and Yasushi Hiromi
One of the elements that directs developmental
events is the precise temporal regulation of gene
expression. Neuroblasts (NBs), neuronal precursors in
the Drosophila embryonic CNS, are an excellent model
to study the temporal regulation of gene expression,
because these cells undergo shifts in gene expression
profile over time, to generate neuronal diversity of theirprogeny. A determinant for such temporal specificity
is a transcription factor Seven-up (SVP). SVP is
expressed in a temporally restricted subsection within
the NB's lineage, and this restricted expression
directs "switching" of gene expression profile within
the NB; in svp mutants, NBs maintain gene expression
patterns of a younger stage. Weare trying to

24

Division of Developmental Genetics

understand how the temporally restricted expressionof svp is regulated. Preliminary results indicate thatcell cycle progression and cytokinesis might affect theexpression of svp.
(6) Developmental context for Notch-dependent PROS
expression in longitudinal glial cells

Yoshihiro Yuasa and Yasushi Hiromi
Notch signaling is utilized in choosing binary cellfates in many aspects of organogenesis. The outcomeof Notch activation is context-dependent, resulting in
the activation of different target genes in eachdevelopmental process. The molecular nature of thedevelopmental context that provides target specificity
is poorly understood. One process that Notch activity
determines a particular cell type in the nervous systemis the subtype specification of longitudinal glia in the
Drosophila embryonic central nervous system. In eachhemisegment, ten longitudinal glial cells are generated
from a single precursor and are positioned on thelongitudinal axon bundle. These glial cells comprise
two subtypes, because six of them activate Notch
signaling and express a homeodomain transcription
factor Prospero (PROS), whereas the other four are
PROS-negative. PROS expression is dependent on
Notch activation, and conversely, when Notch isartificially activated all longitudinal glial cells become
PROS-positive. To identify the developmental context
for Notch-dependent glial subtype specification, we are
analyzing the regulation of PROS expression in thelongitudinal glia. We found that PROS expression in
the longitudinal glia requires three transcriptional
factors: homeodomain protein REPO, ets transcription
factor Pointed and AT-rich binding protein Dead
RingerlRetain (DRI). An enhancer element of the pros
gene that recapitulates the expression pattern of PROSin the longitudinal glial cells includes the consensus
binding sites for all three transcriptional factors. These
results suggest that these three transcriptional factors
provide a context for Notch-dependent PROS
expression in the longitudinal glia.
(7) Stem cell formation in Drosophila germline requires

gonadal somatic cells

Miho Asaoka, Satoru KobayashiI, Ryu Ueda2 andYasushi Hiromi CNIBB, 2Invertebrate Genetics

Laboratory)

EDUCATION

Stem cells in the adult tissue are maintained bya special microenvironment called a "niche", which
produces signals that maintain stem cell specificationand function. However, how stem cell fate is initiallyestablished during organogenesis is currently
unknown. Stem cell. fate may be determined by acell-autonomous mechanism. Alternatively, it may be
induced by a niche-like microenvironment (induction
hypothesis). To test these hypotheses, we use
Drosophila germline stem cells (GSCs) as a model
system. Our previous lineage analysis has shown that
primordial germ cells (PGCs) contacting somatic cells
in the anterior half of embryonic gonad give rise to
GSCs in the adult ovary, whereas PGCs in the posterior
half of the gonad differentiate directly to cystoblasts.
These results suggested that anterior somatic cells in
the embryonic gonad may play an inductive role in
GSC formation. Here we isolated six genes that are
required in the somatic gonad for GSC formation.
These genes were identified in our screen involving
microarray, in situ hybridization, and RNAi-mediatedgene knockdown. During embryogenesis all six genes
are preferentially expressed in the somatic cells of the
gonads, one of them only in the anterior half of thesomatic gonad. Gene knock-down in the gonadal
somatic cells resulted in the absence or decrease of
GSC in adult ovaries. These results suggest thatsomatic embryonic gonad is required for GSC
formation, and support the somatic induction
hypothesis.

1. r.t:m •/" ;A.(J)*~J

PUBLICATIONSPapers

1. Iwanami, M. Hiromi, Y. and Okabe, M. (2005)
Cell-type specific utilization of multiple negative
feedback loops generates developmental constancy.
Genes Cells, 10, 743 - 752.
2. Kanai, M. I., Okabe, M. and Hiromi, Y. (2005)
seven-up controls switching of transcription factors thatspecify identities of Drosophila neuro blasts.
Developmental Cell, 8, 203 - 213.
3. r.t:m .(2005) c..(J)JI~.:t-···tt;h:?? A~f;:"Gt~1v
ttJl~.:t-(J):i5Hr1, {t~, 60, 28 - 29.

~-g~~(J) IJRf.II!

J!lH~~~ '7 -17 -lj- -1 ;I:.

~flijJ~~~i1rt-e /"17 -, ~jJ-lj--rJ

2. Yasushi Hiromi Intra-axonal patterning: its
mechanism and implications. IGBMC, Strasbourg
3. Yasushi Hiromi Intra-axonal patterning: its
mechanism and implications. University of Southern
California
4. Yasushi Hiromi Intra-axonal patterning: its
mechanism and implications. Caltech
5.

r.t:m •

~E~(J)Mm~JI~.:t-~m~~~~

JI~1i1f-~000~

r.t:m •

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7. ~flijJ~~ ;/ 3 1/ ~ 3 1/ /",;I:.1::~~f;:~ft~~E~;/ ;A. T .b. "" ~ E Jj?U~ Jjj(; (J) tc ~ (J) ~ I J\!:Jit:fl"", *%i't1i1f?E::k~~::k~""1i1f?E~~Jjj(;(J)tc~(J)1i1f?Ec~~""~~::k~!:Jit:fl1*.~$

8. ~flijJ~~;/ 3 1/~ 3 1//",;I:.1::~~f;:~vt~~E~;/;A. T .b.""~E~~Jjj(;(J)tc~(J)1l'tt/N~:fl""~~::k~*%

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9.

~flijJ~~;/ 3 1/~ 3 1//",;I:.~~~f;:~vt~~E~;/;A.T.b.""~E~~Jjj(;(J)tc~(J)1l'tt~!:Jit:fl"".~::k~~~~~1i1f?EFJTSOCIAL CONTRIBUTIONS AND OTHERS~~ftSt1J

r.t:m •

Dr. Y. Hiromi served as an editor for
Development, Growth & Differentiation.
r.t:m • Dr. Y. Hiromi served as a member of thecouncil of The Genetics Society of Japan.
r.t:m • Dr. Hiromi served as a member of the
Gender Equality Committee of The Molecular Biology
Society of Japan.

C-a. Division of Development Genetics
Toshitaka Fujisawa GROUPRESEARCH ACTIVITIES(1) Systematic identification of peptide signaling
molecules in Hydra
Toshitaka Fujisawa, Chiemi Fujisawa, Eisuke
Hayakawa, Yukihiko Noro, Akemi Hayashiuchi,

Division of Developmental Genetics

25

Hiroshi Shimizu and Toshio Takahashi!)1) Suntory Institute for Bioorganic Research, Osaka,
Japan
Systematic purification and structural
determination of peptide signaling molecules has been
carried out by using HPLC, peptide sequencing andmass spectrometry. About 50 new peptides were
purified and subjected to structural determination. The
peptide sequences were blasted against Hydra EST andgenome databases. We have also carried out
data-mining to identify peptide genes. One of theoutcome was the identification of a PW gene. We
already identified 4 PW peptides, all of them are shortand have a L(I)PW motif at the C-terminus. The
encoding gene had been long sought for without
success. The EST data showed that the deduced
precursor protein contained all 4 PW peptides, one
newly purified peptide, LHW and 3 putative PW
peptides. Most of the peptides were preceded by alysine residue and followed by dibasic amino acids,
typical processing sites for bioactive peptides. Bothimmunostaining using anti-AALPW antibody and insitu hybridization showed that PW peptides werelocalized in ectodermal epithelial cells. PW peptides
inhibited neuron differentiation and counteracted byHym-355, a neuropeptide that enhances neuron
differentiation. We have also identified one geneencoding neuropeptide Y like peptides and 3 genes
encoding insulin-like peptides.

end connecting the interior with the exterior of theanimal. Genetic resemblance came from the fact thatpeduncle tissue of Hydra that expresses CnNk-2, ahydra homologue of Nkx-2.5, shows pumping
movement. This is comparable to pharyngeal tissueof nematodes that expresses ceh-22, a Nkx-2.5
homologue, while showing pumping movement.
Functional resemblance came from the observationthat there is inward and outward fluid transfer throughthe aboral pore. Taken together, we interpret theresemblance listed above to demonstrate that aboral
pore of Hydra and oral opening of higher organisms
share common ancestral origin although the functionof aboral pore is more diverse.
PUBLICATIONSPapers

1. Takahashi, T., Hatta, M., Yum, S., Koizumi, 0.,
Kobayakawa, Y, Gee, L., Ohtani, M., Fujisawa, T. andBode, H. R. (2005) Hym-30l, a novel peptide, regulates
the number of tentacles formed in hydra., Development,
132,2225 - 2234
2. Takaku, Y, Hariyama, T. and Fujisawa, T. (2005)
Motility of endodermal epithelial cells plays a majorole
in reorganizing the two epithelial layers in Hydra.,
Mechanisms of Development, 122, 109 - 122

C-b. Division of Gene Expression
Susumu Hirose Group(2) Aboral end of Hydra and oral end of higherorganisms share a common ancestral origin

RESEARCH ACTIVITIESHiroshi Shimizu, Yasuharu Takaku & Toshitaka
Fujisawa

Role of histone modifications and chromatin
remodeling in epigenetic gene expression

Oral opening of metazoans tends to be formed
around the anterior end. Polyps of phylum Cnidaria
is exceptional since oral opening is located at the siteof Wnt-3a homologue expression that usually takes
place at the posterior end of developing embryos oradults. Why this odd location of oral opening relative
to anterior-posterior axis occurs in this phylum has
remained unknown. We provide a solution to thisoddness by showing that aboral end of Hydra bears
structural, genetic and functional resemblance to theoral end of higher organisms. Structural resemblancecomes from the fact that there is a pore at the aboral

Takahiro Nakayama, Kenichi Nishioka, Yi-Xin Dong,
Tsukasa Shimojima, Kazuma Hanai, Takashi
Yamamoto\ Koji Akasaka 2 and Susumu Hirose
CGraduate Department of Gene Science, Hiroshima
University, Higashi-Hiroshima, Japan; 2Department of
Biological Science, University of Tokyo, Tokyo, Japan)

26

Division of Developmental Genetics(1)

Epigenetic maintenance of the expression state of
the genome is important for development. There are
two types of epigenetic gene expression in Drosophila.
One is maintenance of Hox gene expression governedby Polycomb and trithorax group genes. The other isposition effect variegation (PEV). Trithorax-like (Trl)encoding GAGA factor is involved in the both typesof regulation: Trl is a member of trithorax group and
Trl mutation is an enhancer of PEV.
Previous study from this laboratory has
demonstrated that the GAGA factor recruits FACT,
facilitates chromatin remodeling around its binding
site, and contributes to the maintenance of Hox gene
expression. To further understand the GAGA factorFACT complex-dependent chromatin remodeling, wesearched for remodeling factors that are associatedwith the GAGA factor, and found PBAP complex as
well as NURF. Two types of Brm chromatin
remodeling complex are known in Drosophila. Theseare BAP complex containing Osa, and PBAP complex
containing Polybromo in addition to shared subunits.
Our study showed that the GAGA factor recruits the
PBAP but not the BAP complex to the regulatory
regions of Hox.
We also found that the GAGA factor-FACT
complex on a site just downstream of the white geneis crucial for PEV. The GAGA factor facilitates
chromatin remodeling and replacement of histone H3
by its variant H3.3, and maintains white expression
under the heterochromatin environment.
RSF (remodeling and spacing factor) is achromatin remodeling factor consisting of RSFI and
SNF2H (a human counterpart of Drosophila ISWI). The
factor has a unique activity that can assemble regularly
spaced nucleosome arrays in the absence of any
histone chaperone. To investigate in vivo role of RSF,
we carried out genetic analyses on its Drosophilacounterpart, a heterodimer of dRSFl and ISWI, andfound that it contributes to PEV through facilitating
the spreading of silent chromatin and replacement ofhistone H2A by its variant H2AV.
(2)

Chromatin transcription

Mikage Nakajima, Kenichi Nishioka, Toshiharu
Komori\ Tadashi Wada\ Hiroshi Handa\ andSusumu Hirose CFaculty of Bioscience andBiotechnology, Tokyo Institute of Technology,
Nagatsuda, Yokohama, Japan)
Spt6 is an evolutionarily conserved transcription
factor. Through biochemical studies on human SPT6,
Handa's group has shown it to be a transcriptional
elongation factor. To understand in vivo role of SPT6

in multicellular organism, we carried out genetic
studies on Drosophila SPT6. Knockdown of the SPT6
function by heritable RNAi showed some interesting
phenotypes that are our target of in depthcharacterization.
Drosophila Ashl is a member of trithorax groupand is known to present as a multi-protein complex.
However, molecular nature of the complex remains
unknown. We started to isolate the Ashl complex froma transgenic fly line expressing epitope-tagged Ashl.
Role of DNA topology in the formation of active
chromatin(3)

Hirofumi Furuhashi, Kuniharu Matsumoto, Mikage
Nakajima, Youhei Ogasawara and Susumu Hirose
Supercoiling factor (SCF) is a protein capable ofgenerating negative supercoils into DNA in conjunction
with topoisomerase II. To clarify in vivo role of SCF,
we carried out genetic studies on Drosophila SCF.
Knockdown of SCF by heritable RNAi revealed its rolein dosage compensation of the X chromosome. SCF
co-localizes with the MSL complex along the male X
chromosome. Upon overexpression of SCF, the male
X chromosome showed a bloated appearance. Thisphenotype was suppressed by simultaneous
overexpression of ISWI. Furthermore, a similar
phenotype has been reported for a loss of function
mutant of ISWI. These findings suggest that twice
activation of the male X chromosome is achieved
through a balance between two activities, SCF forming
an open and active chromatin and ISWI working
toward a compact and silent chromatin.
We have developed a method to visualize
negatively supercoiled DNA domains within a cell,which relies on preferencial binding of psoralen tonegatively supercoiled DNA. Upon beat shock ofDrosophila larvae, we observed strong signals ofpsoralen on heat shock puffs of salivary gland polytene
chromosomes. Furthermore, scs and scs' sequences arenecessary to detect the strong signals on the hsP70
gene, suggesting formation of a topologically closed
domain through the scs and scs'.
Transcription and supercoiling of the template
DNA are closely related each other. DNA supercoiling
affects transcription and transcription affects
supercoiling of the template DNA. Furthermore,
packaging of genomic DNA into chromatin in

Division of Gene Expression

27

eukaryotes raises another type of relation. DNA
supercoiling can affect transcription throughmodulation of the chromatin structure (Hirose, S. andMatsumoto, K., 2005).
(4) Linker histone variants control chromatin dynamics
during early embryogenesis

Hideaki Saeki\ Keita Ohsumi2, Hitoshi Aihara 2,
Takashi Ito\ Susumu Hirose, Kiyoe Ural andYasufumi Kaneda l ~Division of Gene Therapy, Osaka
University Graduate School of Medicine, Suita, Osaka,
Japan; 2Cell and Developmental Biology, Tokyo
Institute of Technology Graduate School of Bioscienceand Biotechnology, Nagatsuda, Yokohama, Japan;
3Department of Biochemistry, Nagasaki University
School of Medicine, Sakamoto, Nagasaki, Japan)
Complex transitions in chromatin structure
prod uce changes in genome function during
development in metazoa. Linker histones, the lastcomponent of nucleosomes to be assembled into
chromatin, comprise considerably divergent subtypes
as compared with core histones. In all metazoa studied,their composition changes dramatically during early
embryogenesis concomitant with zygotic geneactivation, leading to distinct functional changes thatare still poorly understood. Here, we show that early
embryonic linker histone B4, which is maternallyexpressed, is functionally different from somatic
histone HI in influencing chromatin structure anddynamics. We developed a chromatin assembly systemwith nucleosome assembly protein-l as a linker histone
chaperone. This assay system revealed that maternal
histone B4 allows chromatin to be remodeled by
ATP-dependent chromatin remodeling factor, whereas
somatic histone HI prevents this remodeling.
Structural analysis shows that histone B4 does not
significantly restrict the accessibility of linker DNA.
These findings define the functional significance ofdevelopmental changes in linker histone variants. We
propose a model that holds that maternally expressedlinker histones are key molecules specifying nuclear
dynamics with respect to embryonic totipotency (Saeki,
H. et al., 2005).

28

Division of Gene Expression(5) Solution structure of the HMG-box domain in the
SSRPI subunit of FACT

Nobuyuki Kasai', Yasuo Tsunaka\ Izuru Ohki\
Susumu Hirose, Kosuke Morikawal and Shin-ichi
Tatel CDepartment of Structural Biology, BiomolecularEngineering Research Instirute, Suita, Osaka, Japan)
The FACT proteins, which are highly conservedin all eukaryotes, form heterodimers consisting of two
subunits, structure-specific recognition protein-l(SSRPl) and SPT16 (also known as CDC68). The
smaller SSRPI subunit contains a High Mobility Group(HMG)-box domain. The FACT HMG-box domain(FACT-HMG) is categorized in the non-sequencespecific HMG-box protein group, which includes
HMGBl, HMGB2 and NHP6A. The intact FACT
heterodimers also interact with nUcleosomes and DNA
in a sequence independent manner. The solution
structures of free yeast homolog NHP6A and the
NHP6A-DNA complex, which shared the FACT
function, were already reported. However, the structureof the HMG-box domain belonging to SSRPI has notbeen determined yet. Here, we report the solution
structure of the HMG-box domain (dFACTHMG) in
the SSRPI subunit (residues 555-624) of FACT from
Drosophila melanogaster (Kasai, N. et al., 2005).
(6) Roles of the heterotrimeric G proteins in Drosophila
gastrulation

Naoyuki Fuse, Takuma Kanesaki and Susumu Hirose
In gastrulation of a Drosophila embryo,presumptive mesodermal cells undergo sequential cell
movements, and invaginate into the ventral furrow. It
has been shown that the concertina, encoding a G ex
subunit of the heterotrimeric G protein (G protein), is
involved in an initial step of the cell movements.
However, mechanisms that govern various cell
movements during gastrulation are largely unknown.
To address this issue, we investigated roles of the Gproteins in gastrulation.
We utilized Moesin-GFP to characterize cell
movements in living embryos and classified three steps
for the mesoderm invagination. First, mid-ventral cells
among presumptive mesodermal cells coordinately
constrict their apical cell surfaces. Secondly, remaining
mesodermal cells on the side extend filopodia-likeprotrusions toward the mid-ventral and actively
migrate into furrow. Thirdly, cells, which migrate fromeach side and meet at the mid-ventral, immediately
adhere and cease movement. The concertina mutant
perturbed the first and the second steps, however,mutant for another G ex subunit, G ex i, compromised
the second and the third steps. These observations
suggest that differential G protein signaling organize
various cell movements during gastrulation.
(7) Anterior epidermis-specific expression of the cuticle
gene EDG84A is controlled by many cis-regulatory
elements in Drosophila melanogasterYasunori Kayashima, Susumu Hirose and Hitoshi
Ueda1 CThe Graduate School of Natural Science andTechnology, Okayama University, Tsushima-naka,
Okayama, Japan)
During insect metamorphosis, a pulse of
ecdysteroids induces many different morphologicalchanges depending on different parts of the body. In
Drosophila, although a number of transcription factors
are expressed in a stage-specific manner in responseto an ecdysteroid pulse, little is known on theregulatory mechanism for space-specific gene
expression during metamorphosis. The EDG84A geneencoding pupal cuticle protein is one of the targets ofecdysteroid-inducible transcription factor {3 FTZ-Fl
and is expressed only in anterior epidermis of the bodyduring mid- to late prepupal period, whereas {3 FTZ-Fl
is expressed in almost all tissues. To address theregulatory mechanism of the tissue-specific expressionof the EDG84A gene, we established transgenic flylines which carry various upstream regions of the gene
fused to the LacZ gene and examined the expression
pattern of the reporter gene. Results of the transgenic
fly reporter assays showed that the space-specificexpression is controlled by at least four positive andtwo negative elements within a 263-bp region near thetranscription start site, and at least three of themshowed space-specific effects to the anterior bodytrunk. These results suggest that both high expression
level and differential expression are achieved throughmany cis-regulatory elements (Kayashima, Y et al.,2005).

(8) Molecular mechanisms of Sonic hedgehog mutant
effects in holoprosencephaly
Tapan Maity\ Naoyuki Fuse and Philip A.
Beachy! CHoward Hughes Medical Institute,
Department of Molecular Biology and Genetics, The
Johns Hopkins University School of Medicine,
Baltimore, Maryland, USA)
Holoprosencephaly (HPE), a human developmental
brain defect, usually is also associated with varying
degrees of midline facial dysmorphism. Heterozygous
mutations in the Sonic hedgehog (SHH) gene are themost common genetic lesions associated with HPE,
and loss of Shh function in the mouse produces
cyclopia and alobar forebrain development. The
N-terminal domain (ShhNp) of Sonic hedgehog protein,
generated by cholesterol-dependent autoprocessing andmodification at the C terminus and by palmitate
addition at the N terminus, is the active ligand in the
Shh signal transduction pathway. Here, we analyze
seven reported missense mutations (G31R, D88V,
QIOOH, N115K, W117G, W117R, and E188Q) that alter
the N-terminal signaling domain of Shh protein, andshow that two of these mutations (QlOOH and E188Q),
which are questionably linked to HPE, produce nodetectable effects on function. The remaining fivealterations affect normal processing, Ptc binding, andsignaling to varying degrees. These effects include
introduction of a recognition site for furin-likeproteases by the G31R alteration, resulting in cleavageof 11 amino acid residues from the N terminus of
ShhNp and consequent reduced signaling potency.
Two other alterations, W117G and W117R, causetemperature-dependent misfolding and retention in thesterol-poor endoplasmic reticulum, thus disrupting
cholesterol-dependent autoprocessing (Maity, T. et al.,2005).
PUBLICATIONSPapers

1. KASAl, N., TSUNAKA, Y, OHKI, I., HIROSE, S.,

MORIKAW A, K. and TATE, S. (2005) Solution
Structure of the HMG-box domain in the SSRPI
subunit of FACT, J. Biomol. NMR, 32,83 - 88
2. KAWASAKI, H., HIROSE, S. and UEDA, H. (2005)
A simple and quick method to isolate nuclear extracts
from pupae of Drosophila melanogaster, Cytotechnology,

Division of Gene Expression29

49,67 - 70
3. KAYASHIMA, Y., HIROSE, S. and UEDA, H.
(2005) Anterior epidermis-specific expression of the
cuticle gene EDG84A is controlled by many cisregulatory elements in Drosophila melanogaster, Dev.
Genes Evol., 215, 545 - 552
4. MAlTY, T., FUSE, N. and BEACHY, P. A. (2005)
Molecular mechanisms of sonic hedgehog mutant
effects in holoprosencephaly, Proc. Nat!. Acad. Sci.
USA, 102, 17026 - 17031
5. SAEKI, H., OHSUMI, K, AIHARA, H., ITO, T.,
HIROSE, S., URA, K, and KANEDA, Y. (2005) Linker
histone variants control chromatin dynamics during
early embryogenesis, Proc. Natl. Acad. Sci. USA, 102,5697 - 5702
6. J.tlfi .il (2005)*IBJffllIl2:Il~Jt*-Q-707T/, 1:fitr#t~.y-TJV,

46

7. J.tlfi .il (2005) ~1tA r- v AJ;t,~~JtIC-t Q:3 7-7 T-"'-?i' -MBFl, malt . ~l!5t • iW~, 50, 136 - 140
8. J.tlfi .il (2005H~:!~H:~bQ?i'>/-\-7ff,1:-1t~,
77, 1417 - 1419
Books
9. HIROSE, S. and MATSUMOTO, K (2005) Possibleroles of DNA supercoiling in transcription, DNA
Conformation and Transcription, (OHYAMA, T.),
Landes Bioscience· Texas, USA, 138 - 143

C-c. Division of Molecular and Developmental Biology
Koichi Kawakami GroupRESEARCH ACTIVITIES(1) Transposon tools and methods in zebrafish
Koichi Kawakami
Zebrafish is an excellent model animal to study
vertebrate development by genetic approaches.
Hundreds of mutations affecting various processes ofdevelopment have been isolated by chemicalmutagenesis and insertional mutagenesis using a
pseudotyped retrovirus. However, useful transposon
tools and methods had not been available in zebrafish.
This is mainly because no active transposable element
has been found from the zebrafish genome. Recently,
we have developed efficient transgenesis, gene trap andenhancer trap methods in zebrafish by using the Tol2transposon systems. These methods should increase
the usefulness of zebrafish as a model vertebrate andfacilitate the study of developmental biology, genetics
and genomics (Kawakami. K, 2005).
(2) Expression pattern-driven insertional mutagenesis
in zebrafish

EDUCATION

J.tlfi .il-707T/m~~~~~~ W~*~

2. J.tlfi .il-707T/m~~~~~~ ~W*~

1.

Saori Nagayoshi, Eriko Hayashi\ Kazuhide Asakawa,Akihiro U rasaki, Kazuki Horikawa 1, Hiroyuki
Takeda 1 and Koichi Kawakami (1 University ofTokyo)SOCIAL CONTRIBUTIONS AND OTHERS~~~.±ftJ.tlfi .il, m*~~OOli~8~~~m~~~-707

T/Y~T~-7A~~/A.n~. ~.

HIROSE, S. and SHINKAI, Y. The 28th Annual
Meeting of the Molecular Biology Society of Japan,
Symposium Chromatin dynamics FUKUOKA*f~*fIDl2005-091475, ifl. O)m ~ it IvDNAO)~I±l¥!( 3 ), J.tlfi~ ~*OOM,*~#~~m.~¥!AM.·~A~A

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30

Division of Gene Expression

Although gene and enhancer trap methods usingtransposons and a retrovirus have been recently
described in zebrafish, no insertional mutation has
been created by these methods and it is not clear
whether those are applicable to insertionalmutagenesis. We report here the development of an
efficient enhancer trap method in zebrafish using the
Tol2transposable element and the identification of adevelopmental mutant created by this method. We
constructed a transposon vector containing thezebrafish hsP70 promoter upstream of the GFP gene.
Transgenic fish carrying chromosomal insertions of
the construct expressed GFP in various tissues andorgans at normal temperatures, indicating that
locus-specific enhancers activated the hsP70 promoter.

More than half of the insertions gave rise to unique
GFP expression patterns, suggesting that the hsP70
promoter is highly capable of responding tochromosomal enhancers. 16 enhancer trap lines witha single insertion were established and characterized.
These insertions were frequently found withinprotein-coding genes; i.e., 13 out of 16 were inserted
within a gene. Furthermore, three were located within
an exon. One of them expressed GFP strongly in the
median fin folds and the pectoral fin. The insertion
disrupted the first exon of the tcf7gene, which encoded
a transcription factor involved in the Wnt signaling
pathway. Homozygous embryos exhibited anomalies
in the median fin folds and the pectoral fin, where GFP
expression was detected. Thus, we demonstrated that
our enhancer trap method can generate an insertion
mutation in a developmentally important gene, which
is identifiable by observing GFP expression patterns
in living vertebrate embryos.
(3) Development of the Gal4-enhancer trap system using
the Tol2 transposable element
Kazuhide Asakawa, Akihiro Urasaki, Tomoya Kotani,
Saori Nagayoshi, Yasuyuki Kishimoto, Masahiko Hibi1
and Koichi Kawakami CRIKEN CDB)First, by using the Tol2transposable element, weconstructed an enhancer trap vector hsp-gaI4, whichcontains the yeast transcription factor GAL4 geneplaced under the heat shock promoter, and reporter
vectors, UAS-EGFP and UAS-DsRED. Then, weestablished transgenic zebrafish lines carrying thesetransposon vectors by Tol2-mediated transgenesis. We
found that, upon heat shock, embryos obtained fromcrosses between the hsp-gal4 fish and these reporterlines could express EGFP and DsRed ubiquitously,suggesting that Gal4 expressed from hsp-gal4 can
induce expression of a gene placed under UAS. Next,
to determine whether Gal4-enhancer trapping isfeasible, we created random insertions of the hsp-gal4
vector in the genome. In a pilot screen, we identified
30 lines that showed unique Gal4 expression patterns
during embryonic stages by crossing the hsp-gal4 fishwith the reporter lines. Inverse PCR and the following
database search allowed us to map rapidly theinsertion sites of hsp-gal4 on the genome and to identify
candidate genes regulated by the trapped enhancer.
These Gal4lines should be used to express a gene ofinterest in the specific organs and tissues.

misty somites, a maternally expressed gene
identified by the transposon-mediated gene trap methodin zebrafish, is required for somite boundary formation(4)

Tomoya Kotani, and Koichi Kawakami
Maternal factors, which are transmitted frommother to offspring through eggs, play important roles
during early development. Although chemicalmutagenesis screens to isolate maternal effect mutants
have been performed, it is difficult to identify genes
responsible for maternal effect mutants by positional
cloning. We have been developing the gene trap
method in zebrafish using the Tol2 transposon. The
insertions of the Tol2-based gene trap vector cause
GFP-expression III unique patterns during
development. We hypothesized that when theinsertions were integrated in genes expressedmaternally and captured their transcripts, GFP should
be expressed in eggs. In fact, we isolated transgenic
fish expressing GFP in one-cell stage embryos. Then,we generated females homozygous for the insertions
and analyzed embryos from these females formorphological defects during embryonic development.
By screening 16 homozygous females, we found thatembryos from one homozygous female exhibited
defects in somite boundary formation at 13 hpf (the6-somite stage). The maternal insertional mutation wasnamed misty somites (mys) and further analyzed. In
the mys mutant, the gene trap construct was integrated
within an intron of a novel gene, and captured its
transcript. The in situ hybridization analysis using aprobe for the mys gene revealed that the mys mRNA
was indeed accumulated in one-cell stage embryos. To
prohibit expression of the Mys protein completely, we
injected an antisense morpholino oligonucleotide (MO)that blocks the translation initiation site of the mys
gene to the mys homozygous embryos. The MO
injection resulted in a phenotype severer than that of
the mys mutant and in embryonic lethality, indicatingthat the mys mutant phenotype is hypomorph. In themorphants, the genes expressed during somitogenesis,
such as fgf8, deltaC, herl, tbx24 and mespb, wereexpressed and somite boundaries were observable at
16 hpf (the 14-somite stage). At 20 hpf (the 20-somite
stage), however, the boundaries were disappeared in
the MO-injected embryos. Thus, the mys gene encodes

Division of Molecular and Developmental Biology

31

a maternal factor required for the maintenance of thesomite boundaries during vertebrate development.
(5) Remobilization of integrated transposons: the jump
starter system in zebrafish

Akihiro Urasaki, Kazuhide Asakawa and Koichi
Kawakami
We have been developing the gene and enhancer
trap methods using the Tol2transposon, and creatingtransgenic zebrafish expressing GFP in specific tissues
and organs. Currently we are making transposon
insertions in the genome by microinjection of both atransposon-donor plasmid and the transposase mRNA
into fertilized eggs. Creating a number of zebrafish
lines with different transposon insertions would beeasier if the jump-starter system, which enables
creation of new transposon insertions without
microinjection, was developed. Toward this goal, first
we tested whether integrated transposons can beremobilized. We injected transposase mRNA into
embryos homozygous for a single transposon insertionin the hoxc3a and the pax6b gene. The integrated
transposons were excised from these two loci, andinserted into different loci efficiently. 11 out of 13
reintegration loci mapped on the genome were locatedon different chromosomes, and two were located on
the same chromosome, only 100-kb and 550-kb fromthe original locus. Thus, integrated transposons canbe mobilized by supplying the transposase activity,
and, upon remobilization, both genome-wide global
hopping and local hopping, predominantly global
hopping, were observed. Then, we constructedtransgenic fish carrying the transposase eDNA under
the control of the zebrafish hsP70 promoter. Doubletransgenic fish carrying the transposase eDNA and an
insertion of the Tol2 enhancer trap construct, causing
specific GFP expression, were heat-shocked repeatedly,
and their offspring were screened for new patterns
during embryonic development. We found that thegerm cells of the heat-shocked fish became highly
mosaic, and a pair of the male and the female could
generate more than 11 new patterns at the freq uency
of approximately one pattern per 20 embryos. Southernblot analysis and inverse PCR revealed that these new
GFP expression patterns indeed resulted fromtransposon insertions at new loci. In these cases, global
hopping was also predominant. These results indicate

32

Division of Molecular and Developmental Biologythat the jump starter system we established here canbe used to create transposon insertions in new lociefficiently in the germ cells.
(6) The transposon-mediated Gal4 gene trapping
identified genes expressing in unique patterns in the adultzebrafish

Kanta Mizusawa, Kazuhide Asakawa, Akihiro Urasaki,
Tomoya Kotani, Saori Nagayoshi, Yasuyuki
Kishimoto, Shigeru Kondo and Koichi Kawakami
Gene-trap and enhancer trap methods haverecently been developed in zebrafish. The previous
studies have shown that these methods are powerfulto study the function of developmental genes. However,these methods have not been applied to the study ofgenes functioning in the adult stages. To identify andcharacterize genes that are expressed in spatiallyrestricted patterns in the adult and play importantroles to maintain the adult system, we have developed
the Gal4-gene trap system in zebrafish and performeda screen for unique Gal4 expression patterns in theadult. First, 247 fish injected with the Gal4 gene traptransposon construct-donor plasmid and transposasemRNA were mated with the UAS-GFP reporter fish,
and the offspring were screened for unique GFP
expression during embryonic development. 154 unique
GFP expression patterns were identified, and the F1
fish were raised. Then, 1252 F1 fish with these 154
embryonic patterns were analyzed for unique GFP
expression at the adult stages. We observed unique
GFP expression in various organs; i.e., eye, nose, jaw,
fin, skin, gill, muscle, skeleton and part of the brain,
and, in total, 121 unique GFP expression patterns wereidentified. To determine whether the prescreen at theembryo stages enriched fish with unique GFP
expression patterns at the adult stage, we performed
the adult screen without the embryonic prescreen. A
total of 1984 F1 fish from 99 injected fish were analyzed
at the adult stage, and 79 unique GFP expression
patterns were identified. Thus, the adult screen withthe embryonic prescreen (79%:121/154) was comparablewith that without the prescreen (80%:79/99), suggestingthat the adult and the embryonic patterns may beirrelevant. Weare now focusing on fish lines exhibiting
unique Gal4 expression in different parts of the adultbrain (telencephalon, diencephalon, and cerebellum)
and in the pigment cells on the skin (the inter-stripeand the stripe boundary regions). Further studies ofthese lines should disclose genes functioning theseorgans. Also, these Gal4 lines will be useful to modify
the activities of those cells in the adult zebrafish.
(7) The zebrafish bobtail mutation identifies theMOCS} (the molybdenum cofactor synthesis step-I) genethat is required for the posterior body formation as an
essential element of the Fgf/ERK signaling
Yasuyuki Kishimoto, Sumito Koshidal , Makoto
Furutani-Seiki2, Atsushi Kawakami 3, ]. Reiss\ G.
Schwarz5, Yuji Kohara, Hisato Kondoh 2 and Koichi
Kawakami CNational Institute of Natural Science,
2ERATO/SORST ]ST, 3Tokyo Institute of Technology,
4Goettingen Univ., 5Univ. of Cologne)
The formation of vertebrate posterior body andtail requires coordinated growth, differentiation andmovement of multipotent progenitor cells located in
the tailbud, and is dependent upon the Fgf signaling.
Here we describe the zebrafish bobtail (btl) mutationthat exhibits a recessive maternal effect, causing astrong reduction of the tail region and abnormal somite
patterning. Through positional cloning, we show thatthe gene mutated in the btl mutant is a zebrafish
homologue of the molybdenum cofactor synthesis
step-l (MOGS1) gene. In humans, mutations in MOGS1
have been described for a lethal genetic disorder,
molybdenum cofactor deficiency. MOGS1 catalyzes
conversion of GTP to cyclic pyranopterin monophosphate (cPMP), a biosynthetic intermediate formolybdenum cofactor. Injection of cPMP at the one-cellstage completely rescued the btl mutant phenotype,demonstrating that the defects in the btl mutant arecaused by the lack of cPMP biosynthesis. Furthermore,
we found that phosphorylation of the ERK protein in
the tail bud region, which has been shown to be
regulated through the Fgf signaling, strongly decreased
during gastrulation and segmentation stages in the btl
mutant embryos. Implantation of Fgf-soaked beads
could not induce ectopic ERK phosphorylation in the
btl mutant, indicating that MOCSI is required for
Fgf-dependent ERK activation. Our results suggest amolecular link between molybdenum cofactor
biosynthesis and FgflERK signaling during thedevelopment of the vertebrate posterior structures.

(8) The Zebrafish pob Gene Encodes a Novel Protein
Required for Survival of Red Cone Photoreceptor Cells
Michael R. Taylor\ Satoshi Kikkawa\ Vishvanathan
Ramamurthy\ Koichi Kawakami, and Susan E
Brockerhoffl CUniversity of Washington)
The zebrafish mutant, partial optokinetic responseb (pob), was isolated using an n-ethyl n-nitrosourea(ENU)-based screening strategy designed to identify
larvae with defective optokinetic responses in red but
not white light. Previous studies showed that red-lightblindness in pob was due to the specific loss oflong-wavelength photoreceptor cells via an apoptotic
mechanism. Here, we used a positional cloning strategy
to identify the mutated pob gene. We find that pob
encodes a highly conserved 30kDa protein of unknown
function. Pob contains putative transmembraneregions and protein sorting signals, and is localized to
intracellular compartments in COS-7 cells and theinner segment and synapse in photoreceptor cells. To
demonstrate that the correct gene was isolated, weused the Tol2 transposon system to generatetransgenic animals and rescue the mutant phenotype.
We also show that the degeneration of red cone .
photoreceptors in the mutants is independent of light.
Based on these findings, we propose that Pob is not
involved in phototransduction but rather plays anessential role in protein sorting and/or trafficking(Taylor, M. et al., 2005).
PUBLICATIONSPapers

1. Kawakami, K (2005) Transposon tools andmethods in zebrafish, Developmental Dynamics, 234 (2)244 - 254
2. Miskey, c., Izsvak, Z., Kawakami, K., Ivies, Z. (2005)
DNA transposons in vertebrate functional genomics,
Cellular and Molecular Life Sciences, 62, 629 - 641
3. Taylor, M. R., Kikkawa, S., Diez-]uan, A.,
Ramamurthy, V., Kawakami, K, Carmeliet, P.,
Brockerhoff, S. E. (2005) The zebrafish pob geneencodes a novel protein required for survival of red
cone photoreceptor cells, Genetics, 170, 263 - 273
EDUCATION

1. Kawakami, K

Transposon-mediated gene

Division of Molecular and Developmental Biology

33trapping and enhancer trapping in zebrafish. Johns
Hopkins School of Medicine
2. Kawakami, K Transposon-mediated genetrapping and enhancer trapping in zebrafish. Peking
University
SOCIAL CONTRIBUTIONS AND OTHERS

1. KKawakami was served as a Chair of the 3rd
Annual International Conference on Transposition andAnimal Biotechnology at Minneapolis, U.S.A. on June,2005.
2. K Kawakami was a lecturer in EMBO practicalcourse: enhancer detection, gene trapping, and
TILLING in zebrafish held at Bergen, Norway on
August, 2005.
3. K Kawakami had a seminar at Johns Hopkins
School of Medicine, Baltimore, U.S.A. on September,2005.
4. K Kawakami had a seminar at Peking University,
China on November, 2005.
5. K Kawakami organized a workshop entitled"Functional genomics using transposable elements"
in the 28th annual meeting of the molecular biology
society of Japan at Fukuoka on December, 2005.
~~~±:fl:Mead, P., Hackett, P., McIvor, S., Kawakami, K.,
Fahrenkru, S. The 3rd Annual International
Conference on Transposition and Animal
Biotechnology Germline Mutagenesis. Minneapolis,
Minnesota
Kawakami, K, Becker, T., Burgess, S., Cuppen, E.
EMBO Practical Course: Enhancer Detection, Gene
Trapping, and TILLING in Zebrafish. Bergen, Norway
JJll:l'1!f-, ¥raJiOO= ~28@]8*5t-f-1::~~~~~7­~~3Y~~

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Cod. Division of Physiological Genetics
Fumitoshi Ishino GroupRESEARCH ACTIVITIES~~.~m~~1::~x~~x*~~y~~~m/~~~~EPIGENETIC
VIEW
OF
MAMMALIAN
DEVELOPMENTIFUMITOSHI ISHINO

34

Division of Molecular and Developmental Biology

fa~1) Genomic imprinting in an Australian marsupial
species, tammar wallaby

We demonstrated that IGF2 and PEGl!MESTin the
tammar wallaby showed paternal expressions as wellas humans and mice (eutherians). However, there
found no DMR (differentially methylated regions) in
the wallaby PEGl!MES T (Suzuki, S., et aL 2005). It waspreviously reported that IGF2R in a South American
marsupial species, grey-tail opossum, was imprinted
without apparent DMR. As DMR is the central
regulator of genomic imprinting in the eutherians,
these results suggest the differential regulatory
mechanisms between the marsupials and eutherians
although existence of their common imprinted genes(regions).
2) Abnormal gene expression profiles in somatic cloned
mice

Comprehensive gene expression analysis ofsomatic cloned mice clearly demonstrated that they
were epigenetically heterologous creatures in spite of
their genetic identity. Although we analyzed several
normal-looking somatic cloned mice, up to 40 % ofgenes showed over two fold changes compared withnormal mice produced from sexual mating.
Interestingly, some gene clusters sensitive to somatic
cloning process were identified (Kohda, T. et at. 2005).
3) A retrotransposon-derived gene essential for
mammalian development

We previously identified PeglO (paternallyexpressed 10) as a Sushi-ichi retrotransposon derived
gene. Recently, it turned out that PeglO was amammal-specific gene that was conserved in alleutherian mammals registered in GenBank. Genetargeting experiment showed that PeglOKO embryos
were early embryonic lethal due to severe placental
defects. This is the first demonstration that an
acquired gene from a retrotransposon has essentialfunction in mammalian development (Ono, R. et aL Nat
Genet 2005 online).

PUBLICATIONSPapers

1. Isotani, A, Nakanishi, T., Kobayashi, S., Lee,].,Chuma, S., Nakatsuji, N., Ishino, F. and Okabe, M.
(2005) Genomic imprinting of XX spermatogonia andXX oocytes recovered from XX - XY chimeric testis,
Proc. Natl. Acad. Sci. U. S. A, 102 (11) 4039 - 4044
2. Kagami, M., Nishimura, G., Okuyama, T.,
Hayashidani, M., Takeuchi, T., Tanaka, S., Ishino, F.,
Kurosawa, K, Ogata, T (2005) Segmental and fullpaternal isodisomy for chromosome 14 in threepatients: narrowing the critical region and implication
for the clinical features., Am. J. Med. Genet., 138 (A)127 - 132
3. Kanatsu-Shinohara, M., Ogonuki, N., Iwano, T,
Lee,]., Kazuki, Y., Inoue, K, Miki, H., Takehashi, M.,
Toyokuni, S., Shinkai, Y., Oshimura, M., Ishino, F.,
Ogura, A. and Shinohara, T. (2005) Genetic andepigenetic properties of mouse male germline stem
cells during long-term culture., Development, 132 (18)4155 - 4163
4. Kohda, T, Inoue, K, Ogonuki, N., Miki, H., Naruse,
M., Kaneko-Ishino, T., Ogura, A, and Ishino, F. (2005)
Variation in gene expression and aberrantly regulated
chromosome regions in cloned mice., BioI. Reprod., 73(6) 1302 - 1311
5. Miki, H, Inoue, K, Kohda, T, Honda, A, Ogonuki,N., Yuzuriha, M., Mise, N., Matsui, Y, Baba, T., Abe,K, Ishino, F. and Ogura, A (2005) Birth of Mice
Produced by Germ Cell Nuclear Transfer., Genesis, 41(2) 81 - 86
6. Ono, R., Nakamura, K., Inoue, K., Naruse, M.,
Us ami, T., Wakisaka-Saito, N., Hino, T.,
Suzuki-Migishima, Ogonuki, N., Miki, H, Kohda, T.,
Ogura, A., Yokoyama, M., Kaneko-Ishino, T., andIshino, F. (2005) Deletion of PeglO, an imprinted geneacq uired from a retrotransposon, causes early
embryonic lethality., Nature Genetics, online, 11 Dec
7. Shiura, H., Miyoshi, N., Konishi, A.,
Wakisaka-Saito, N., Suzuki, R., Muguruma, K, Kohda,
T., Wakana, S., Yokoyama, M., Ishino, F. andKaneko-Ishino, T. (2005) Meg1/Grb10 overexpression
causes postnatal growth retardation and insulin
resistance via negative modulation of the IGF1R andIR cascades., Biochem. Biophys. Res. Common., 329 (3)909 - 916
8. Suzuki, S., Renfree, M. B., Pask, A., Shaw, G.,
Kobayashi, S., Kohda, T, Kaneko-Ishino, T and Ishino,

F. (2005) Genomic imprinting of IGF2, P57KIP2 andPEG1/MEST in a marsupial, the tammar wallaby.,
Mechanisms of Development, 122 (2) 213 - 222
9. U"!I!f5!:*, 1fi:% - U"!I!f 9;o-=f (2005) If / A -1 /7' !J /7~/~~!J7'o~7~/~~~~~~~~~~, ~~~~!lfll, 23 (18) 33 - 39
10. 1fi:% - U"!I!f 9;0-1-, U"!l!f5!:* (2005) If / A -1 /7°!J /7 ~ /~. ~~. ~ J1M~, Molecular Medicine, 42 (2) 181 188
11. 1fi:% - U"!I!f 9;o-=f, U"!l!f5!:* (2005) {mJf*~~ ~:r. t." ~;r.
*7~ ~ A, Hormone Frontier in Gynecology, 12 (4)54 - 60
12. 1fi:YC-U"!I!f 9;0-1-, U"!l!f5!:* (2005) Peg/Meg "'-1/
7'!J/7~/~~~#k~"'U~~M~k.~-=f,~~~~,

23 (12) 1952 - 1954

Books

13. U"!I!f5!:*, 1fi:%-U"!I!f 9;o-=f(2oo5) If / A-1 /7°!J /7 ~ / -Y' 'i¥UJf~~rj:!"'?' A?t /"\-1 ::tUJf~-y A?t~!J -;;( (-*83*, fp83Ji!~±, IlJl=H:t.), $±1±.
J?:, 134 - 143*

Cod. Division of Physiological Genetics
Yukiko Gotoh GroupRESEARCH ACTIVITIES(1) The Wnt-B-catenin pathway directs neuronaldifferentiation of cortical neural precursor cells
Yusuke Hirabayashi, Yasuhiro Itoh, Norihisa
Masuyama and Yukiko Gotoh
Neural precursor cells (NPCs) have the ability to
self-renew and to give rise to neuronal and glial
lineages. The fate decision of NPCs between
proliferation and differentiation determines thenumber of differentiated cells and the size of eachregion of the brain. However, the signals that regulate
the timing of neuronal differentiation remain unclear.
Here we show that the Wnt signaling inhibits
self-renewal capacity of mouse cortical NPCs, andinstructively promotes their neuronal differentiation.
Overexpression of Wnt7a or of a stabilized form ofB-catenin in mouse cortical NPC culture induced
neuronal differentiation even in the presence of FGF2,
a self-renewal-promoting factor in this system.
Moreover, blockade of the Wnt signaling led toDivision of Physiological Genetics

35

inhibition of neuronal differentiation of cortical NPCs
in the developing mouse neocortex. Furthermore, theB-catenin/TCF complex appears to directly regulate
the promoter of neurogenin1, a gene implicated incortical neuronal differentiation. Importantly, stabilized
B-catenin did not induce neuronal differentiation ofcortical NPCs at earlier developmental stages,
consistent with previous reports, suggestingstage-specific functions of the Wnt signaling.
Collectively, these results reveal pivotal physiologicalroles for the W nt signaling in neuronal differentiation.
(2) Hes binding to STAT3 mediates crosstalk
betweenNotch and JAK-STAT signaling
Sachiko Kamakura, Koji Oishi, Takeshi Yoshimatsu,
Norihisa Masuyama, Yukiko Gotoh
Although the Notch and JAK-STAT signaling
pathways fulfill overlapping roles in growth anddifferentiation regulation, no coordination mechanism
has been proposed to explain their relationship. Here
we show that STAT3 is activated in the presence ofactive Notch as well as the Notch effectors Hes1 and
HesS. Hes proteins associate with JAK2 and STAT3,
and facilitate complex formation between JAK2 andSTAT3, thus promoting STAT3 phosl?horylation andactivation. Furthermore, suppression of endogenous
Hes1 expression reduces growth factor induction of
STAT3 phosphorylation. STAT3 appears to be
essential for maintenance of radial glial cells anddifferentiation of astrocytes by Notch in the developing
central nervous system. These results imply that direct
protein-protein interactions coordinate cross-talkbetween the Notch-Hes and JAK-STAT pathways.
(3) Notch promotes survival of neural precursor cells
via mechanisms distinct from those regulatingneurogenesis
Koji Oishi, Sachiko Kamakura, Yuko Isazawa, Takeshi
Yoshimatsu, Keisuke Kuida, Norihisa Masuyama andYukiko Gotoha high cell density and that this effect was attributableto activation of Notch signaling. Expression of an active
form of Notch1 thus markedly promoted NPC survival.
Hes proteins, key effectors of Notch signaling ininhibition of neurogenesis, were not sufficient for thesurvival-promoting effect of NotchI. This effect ofNotch1 required a region of the protein containing theRAM domain and was accompanied by up-regulationof the anti-apoptotic proteins Bel-2 and Mel-I.
Moreover, knockdown of these proteins by RNA
interference resulted in blockade of the Notch1-induced
survival. These results reveal a new function of Notch,
the promotion of NPC survival.
(4) JNK antagonizes Akt-mediated survival signals byphosphorylating 14-3-3
Jun Sunayama, Fuminori Tsuruta, Yasunori Mori,
Norihisa Masuyama and Yukiko Gotoh
Life and death decisions are made by integratinga variety of apoptotic signals and survival signals inmammalian cells. Therefore, there is likely to be acommon mechanism that integrates multiple signals
adjudicating between the alternatives. We propose inthis study that 14-3-3 represents such an integration
point. A number of proapoptotic proteins commonly
become associated with 14-3-3 upon phosphorylation
by survival-mediating kinases such as Akt. We
reported previously that cellular stresses induce
JNK-mediated 14-3-3' phosphorylation at Ser184. Here
we show that phosphorylation of 14-3-3 by JNK releases
the proapoptotic proteins Bad and FOX03a from 14-3-3,
and antagonizes the effects of Akt signaling. As a resultof dissociation, Bad is dephosphorylated, andtranslocates to the mitochondria, where it associateswith Bel-2/Bcl-xL • Since Bad and FOX03a share the14-3-3 binding motif with other proapoptotic proteins,
we propose that this JNK-mediated phosphorylationof 14-3-3 regulates these proapoptotitc proteins inconcert and makes cells more susceptible to apoptotic
signals.
PUBLICATIONS

During development of the mammalian brain,
many neural precursor cells (NPCs) undergo apoptosis.
The regulation of such cell death, however, is poorly
understood. We now show that the survival of mouseembryonic NPCs in vitro was increased by culture at

36

Division of Physiological Genetics

Papers

1. Hirabayashi, Y. and Gotoh, Y. (2005)

Stage-dependent fate determination of neural precursor
cells in mouse forebrain.

Neurosci. Res. 51, 331-336.
2. Sunayama, J.,Tsuruta, F., Masuyama, N. and
Gotoh, y. (2005)
JNK antagonizes Akt-mediated survival signals byphosphorylating 14-3-3.
J. Cell. BioI. 170, 295-304.
3. Takada, T., Suzuki, H., Gotoh, Y. and Sugiyama,
Y. (2005)
Regulation of the cell surface expression of human
BCRP/ABCG2 by the phosphorylation state of Akt inpolarized cells.
Drug Metab. Dispos. 33, 905-909.
3. Yoshimatsu, T., Kawaguchi, D., Oishi, K., Takeda,
K., Akira, S., Masuyama, Nand Gotoh, Y. (2006)
Non-cell-autonomous action of STAT3 in maintenanceof neural precursor cells in the mouse neocortex.
Development, 133, 2553-2563. (2006)
4. ~Jiii'$11!i, f&JiiEl3~-T(2005)t$il\I* Jltr!~IHIBJl?U:: n~n:;1v~;$Jl?!, Medical Science Digest 31, 353-357
5. li'J>UJi1MI, ~EEX., f&JiiEl3~-f-(2005);$Jl?!~f¥~ ~~lT~~~~~T~~X~~~K~~~~~~• • ~~~¥, 23, 2006-2011
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SOCIAL CONTRIBUTIONS AND OTHERS
Oncogene (Editor)

Journal of Biochemistry (Associate Editor)
Cell Structure and Function (Associate Editor)
Journal of Histochemistry and Cytochemistry (Editorial
Board Member)~~jjSliJ

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Division of Physiological Genetics

37

D. DEPARTMENT OF
POPULATION GENETICS

D-a. Division of Population Genetics
Naruya Saitou Group(1) Ape Genome Project Silver

Saitou Naruya, Sumiyama Kenta

In search of genetic changes responsible for humanuniqueness, we are determining genomic sequences ofchimpanzee and gorilla that are phylogenetically close
to human, and do molecular evolutionary analyses. In
2005, we have three publications on this subject.
(i) We examined evolutionary conservation of 5'
upstream sequence of nine genes between human andgreat apes (Kitano, T., et.al. , 2005).
(ii) We compared nucleotide sequences of chromosome
rearrangement on human chromosome 12 andcorresponding ape chromosomes (Shimada, M., et al.,2005).
(iii) We reviewed on evolution of hominoids and searchfor genetic basis for creating humanness (Saitou, N.,2005).
(2)

Molecular evolution of developmental regulation

hade two publications on this subject.
(i) Phylogenetic relationship of the populations within
and around Japan was examined by using 105 shorttandem repeat polymorphic loci (Li, S. L., et al., 2005).
(ii) During ancient DNA investigation in China, wediscovered bronze weapon based on an embedded
fragment in a 3000-year-old skull (Kurosaki, K., et al.,2005).
(4)

Evolution of blood group genes

Saitou Naruya
Blood group antigens are expressed on cell surface,and have a higher chance of being affected by bacteria
or virus. Therefore, their genes may have undergone
positive selection. We are studying evolution of ABO
and Rh blood group genes for humans and non-humanprimates. Several papers are now in preparation.
(5) Large scale evolutionary analysis of many genesequences

Saitou Naruya, Ezawa Kiyoshi
We do genome-wide comparison of many protein
coding and non-protein coding nucleotide sequencesamong various organisms to elucidate evolutionary
pattern. In 2005, we conducted systematic analysis ofmouse and rat genome on gene conversion. Paper was
accepted and is published in 2006 (Ezawa, K., et al., Mol
BioI Evol)(6) Development of new methods for the study of gene
evolution

Sumiyama Kenta, Kim Hyung-Cheol, Saitou Naruya
Saitou Naruya Kirill Kryukov
Weare studying cis-control elements of thedevelopmental genes by sequence analysis and genetransfer experiments of large scale genomic clones, inorder to elucidate relationship between evolution ofcis-elements and body plan.
(3) Analysis of evolution of closely related populations

Saitou Naruya, Ishibashi Minaka
We are mainly interested III evolutionary
relationship of various human populations. In 2005, we

38

DEPARTMENT OF POPULATION GENETICS

We developed new algorithm for multiple alignment,
named MISHIMA (Method to Infer Sequence History
In terms of Multiple Alignment), and successfully
applied this method to 100 mammalian whole
mitochondorial genome sequences and 4 closely related
bacterial genome sequences. Paper is now in
preparation.

PUBLICATIONSPapers

1. Kitano, T. and Saitou, N. (2005) Evolutionary
conservation of 5' upstream sequence of nine genes
between human and great apes, Genes and Genetics
Systems, 80 (3) 225 - 232
2. Kurosaki, K, Wang, L., Tang, J.. Wang, W., Saitou,N., Endo, T., and Ueda, S. (2005) Identification of abronze weapon based on an embedded fragment in a
3000-year-old skull, Forensic Science International, 151(1) 105 - 108
3. Li, S. L., Yamamoto, T., Yoshimoto, T., Uchihi, R.,
Mizutani, M., Kurimoto, Y., Tokunaga, K., Jin, F.,
Katsumata, Y., and Saitou, N. (2005) Phylogenetic
relationship of the populations within and around
Japan using 105 short tandem repeat polymorphic loci,
Hnman Genetics, 118 (6) 695 - 707
4. Nakajima, T., Wooding, S., Satta, Y, Jinnai, N.,
Goto, S., Hayasaka, 1., Saitou, N., Guan-Jun, ].,Tokunaga, K, Jorde, L. B., Emi, M., and Inoue, 1. (2005)
Evidence for natural selection in the HAVCR1 gene:high degree of amino-acid variability in the mucindomain of human HAVCR1 protein, Genes
Immunology, 6 (5) 398 - 406
5. Saitou, N. (2005) Evolution of hominoids and thesearch for a genetic basis for creating humanness,
Cytogenetic and Genome Research, 108 (1-3) 16 - 21
6. Shimada, M., Kim, C.-G, Kitano, T., Ferell, R. E.,
Kohara, Y, and Saitou, N. (2005) Nucleotide sequencecomparison of a chromosome rearrangement on thehuman chromosome 12 and corresponding ape
chromosomes, Cytogenetic and Genome Research, 108,
83 - 90
7. Tateno, Y, Saitou, N., Okubo, K, Sugawara, H.,
and Gojobori, T. (2005) DDBJ in collaboration with
mass-sequencing teams on annotation, Nucleic Acids
Research,33,25 - 28
8. ~~!I!ftr, Ji\f§JJX:ili (2005) l:: I-- ~ ~A~0)1' / Alt~t,pGb;6~.:o~~, ?t~~*,~~, 5(4)381-385
9. Ji\f§JJX:ili(2005):I1ii~!ffi:fUJ;J31·0)1' / AJ!{~f4~fJT, f{l'!I[f-j.~, 43 (12) 70 - 76
10. Ji\f§JJX:ili(2005) r:p:lLJ!{~T.:o1' / A, f{l'!I[f-j.~, 43(9) 70 - 76
11. Ji\f§JJX:ili(2005) 7 / AO)J1!H~~(0MfJT, f{l'!I[f-j.~,
43 (7) 64 - 70
12. Ji\f§JJX:ili (2005) 7 / Af4~fJTO)~i'!, f{l'!I[f-j.~, 43 (6)77 - 83
13. Ji\f§JJX:ili (2005) 7/ A ~ Ii, f{l'!I[f-j.~, 43 (4) 68 - 75

14. Ji\f§JJX:ili (2005) *mtE*, ')' I-- IJ -7 ~fflv\fd!j~:fUri'1'~
f*O)*3'J!, f{l'!I[f-j.~, 43 (3) 60 - 66
15. Ji\f§JJX:ili (2005) ~1tl/j\~7 -?,;6~ G *mtE;JM ~ftJJX:T.:0 1Jtk., f{l'!I[f-j.~, 43 (1) 64 - 72
16. IlMwf~:t:, Ji\f§fJX:ili(2005)~;j;fUPJ~O)lt~1' / A :JI~~ri'1'~Ji~O)J.:~~MfJT, *18Jff!lI~, 24 (10) 1108 - 1111
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tc~*~~ ~-cO)e 1--, mBfU~.M~, 50 (16) 20782082
Books

18. ~~!I!ytr, Ji\f§JJX:ili (2005) DDBJO)*-A.r.::- y ~ fflv\
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ig~, lNJJr:~~ii1)' ~J!ttlltR, 158 - 29
19. Ji\f§JJX:ili(2005)DNA;6~G~tcB*A, m••m
20. Ji\f§JJX:ili (2005) Jf!f~~;6~ Gm1.:o,7' /'7 Iv?' - Iv
AO)lEf*, (~~llOCiiI), ~B~llfJt±, 141-164
21. Ji\f§JJX:ili (2005) AfmJ: ~ G Ii, Afm~~O)~jJi'~~r", 5, (ttiR~~ii1), AX.Im, 468 - 486*'

EDUCATION

1. Ji\f§JJX:ili

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Division of Population Genetics

39

D-a. Division of Population Genetics
Toshiyuki Takano GroupRESEARCH ACTIVITIES(1) Detecting single-generation selection acting on
inversion polymorphism in Drosophila
Toshiyuki Takano-Shimizu, Nobuyuki Inomata,Masanobu hoh, Rumi Kondo, Miki Ohshima and
Yutaka Inoue
Signatures of past natural selection have beenfound in DNA sequences. However, detecting ongoing
selection has been difficult so far. Such selection are
particularly relevant to the future status of populations.
Furthermore, there is very little evidence for strong
natural selection. Here, we developed a new method
to detect viability selection acting on inversion
polymorphisms in extant natural populations. The
central issue of the method is a difference in theamount and pattern of molecular variation between
two samples of inversion-free (standard) chromosomes:
one from standard-chromosome homokaryotype andthe other fromstandard and mverSlOn
heterokaryotype. Under no selective differential among
genotypes, we expect no difference in the scale ofvariation between these two samples. Alternatively,
under models with selection, some differences are
expected. We applied this method to In(2L)tinversion
polymorphism in a natural population of D.
melanogaster. The number of haplotypes on standard
chromosomes from the heterokaryotype wassignificantly smaller than that from thehomokaryotype. This result suggests that strong
selection currently acting on inversion polymorphism
in Drosophila. Our method is applicable to a wide
variety of sequence data, especially for humans, which
are accumulating very fast. Our findings also imply
the evolutionary significance of multi-locus
interactions. Strong selection may be more common
than we have believed.
(2) A high frequency null mutant of an odorant-binding
protein, Obp57e, in Drosophila melanogaster
Aya Takahashi and Toshiyuki Takano-Shimizu

40

Division of Population Genetics

We found a null mutant of an odorant-binding
protein, ObpS7e, in Drosophila melanogaster. This
frameshift mutation, which is a 10 bp deletion in thecoding region, was at a high frequency in the Kyoto
population, and was also present in Taiwan and Africa.
We analyzed an about 1.5kb DNA sequence including
the tandemly duplicated gene, ObpS7d, from 16 inbredlines sampled in Kyoto,]apan. The analyses suggested
that this presence/absence polymorphism is underbalancing selection (Takahashi and Takano-Shimizu,
2005).
In order to assess if there is overdominanceselection affecting this locus, we genotyped 211
wild-caught individuals from the same population
using RFLP (Restriction Fragment Length
Polymorphism), which can distinguish wild-type anddeletion type alleles. These data did not indicate
significant deviation from the genotype frequencies
expected under Hardy-W ein berg eq uili briurn.
Therefore, the locus is probably under other type ofbalancing selection such as frequency dependent
selection or selection caused by environmental
heterogeneity.
(3) Analyses of assortative mating and morphologicaldifferentiations in populations of Drosophilamelanogaster
Aya Takahashi and Toshiyuki Takano-Shimizu
We surveyed multiple strains of D. melanogasterand D. simulans for differences in heterospecificcopulation frequencies, and identified two D.
melanogaster inbred lines between which mating
frequencies of males against D. simulans females differ
by a large extent. One line was originated in West
Africa (MeI6) and the other was collected in Taiwan(Twn). We also performed an intraspecific mating
experiment and found that these two lines show
assortative mating. Especially, males from these twolines have different mating preference againstconspecific females of different lines. These two lines
have been subjected to over 20 generations ofsib-mating, and are suitable materials for genetic
mapping. Our genetic analyses showed that the third
chromosome has a major effect on these behavioral
differences of males. We have constructed 70 third
chromosome recombinant lines between the two inbredlines and have been testing their mating behavior.

We noticed that these two lines differ in thorax
pigmentation pattern as well. The thorax trident issharply pigmented in Twn individuals whereas it is
not obvious in Me16. We mapped this phenotypic
differences using the recombinant lines, and narrowed
down the genetic region responsible for this phenotype
to 75kb. We have been analyzing a candidate gene inthis region.
Spontaneous mutation spectrum in Drosophilamelanogaster(4)

Yutaka Watanabe, Aya Takahashi and Toshiyuki
Takano-Shimizu
Mutation is among the most fundamental of all
evolutionary biology. Its rate and pattern often vary
greatly among different individuals, species, and evenamong different chromosomal regions in a single
genome; however, so little is known about themechanisms of such surprising variation. Mutation
rate cannot be estimated from a comparison of
nucleotide sequences from different species since manyfactors such as selection, population structure, andrandom genetic drift would change mutant frequency
and then the information obtained provides only apartial picture of the range of spontaneous mutations.
On the other hand, direct studies on spontaneous
mutation have been limited to a few model organisms.
The fruit fly (D. melanogastef; is one of them. However,
the issue of spontaneous mutation rate is still far fromresolved partly because many internal and external
factors affecting the rate are not controlled adequately
to estimate rates in natural conditions and partly
because there are large variation in rate amongindividuals. We have started large mutation screen
experiments aimed at understanding the nature of
spontaneous mutation and obtaining the picture of thestandard mutation spectrum in natural populationsof D. melanogaster. We have designed an experimental
scheme to screen mutations that occur in germ cellsof the first generation offspring of wild-caught
inseminated·females. The offspring were produced
from crosses occurred in nature and therefore carried
genetic compositions close to that of flies in nature.
Male and female F1 offspring were individually crossed
to mutant stocks carrying three or four visible recessive
mutations, and the F2 offspring were screened formutations. From more than 800,000 F2 flies, we

obtained eight independent mutants and are currently
investigating the nature of these mutations at
molecular level.
Nucleotide variation and linkage disequilibrium in
150-kb region around the Scr gene in D. melanogasterand D. simulans(5)

Yutaka Watanabe, Haruki Tatsuta and Toshiyuki
Takano-Shimizu
The sex comb is a specific row of enlarged bristles
on the foreleg of male flies and a recently derived trait
that is found almost exclusively in the melanogasterand obscura species groups in the subgenus
Sophophora. Its structure varies greatly among species
with respect to the numbers of sex combs and theirteeth, and orientation of the sex comb. We performed
QTL analyses of sex-comb tooth-number variation
within Drosophila simulans, and identified, in total,seven QTL. One of candidate regions is the
Antennapedia Complex (ANTC), which consists of the
Hox genes including the Sex com b reduced (Scr) gene.
The Hox genes are well known as selector genes, but
may also be involved more directly in the formationof morphological structures. Despite many functionaland comparative studies, the gene regulation
mechanisms of the HOX genes have remained elusive.
As a first step in identifying QTN for the sex-comb
tooth number variation, we surveyed nucleotide
variation in about 150kb region surrounding the Scrgene in both D melanogaster(n= 10) and D. simulans(n = 10). The amount of nucleotide variation within
species (nucleotide diversity = 1t) was lower than the
typical values in both species. This may reflect low
recombination rate at ANTC, but the degree of linkage
disequilibrium was not so strong in both species.
Further analysis is currently in progress.
Fluctuation in linkage disequilibrium scale inDrosophila melanogaster genome(6)

Toshiyuki Takano-Shimizu, Masanobu Itoh, Rumi
Kondo, Nobuyuki Inomata, Noriko Nanba, Masako
Hasegawa, Miki Ohshima and Yutaka Inoue
LinMge disequilibrium (LD) is a complex measureand infhlenced by many factors, of which relative
effects are largely unknown. Nevertheless, it is not only

Division of Population Genetics

41useful to infer population history but also necessary
to describe the genetic structure of populations in
multi-locus context. By using two fly samples from thesame collection site, we have analyzed linkage
disequilibria between polymorphisms at about 100
Drosophila chemoreceptor genes, finding larger LD ina spring sample than in an autumn sample and asignificant excess of associations between one frequentand one less common allele in the replacement
polymorphisms. To gain a finer picture of LD in
Drosophila genome, we extended our analysis tofurther samples and genes. Despite the seasonal
fluctuation in the scale of LD between the
chemoreceptor genes, the amount of LD between
randomly chosen genes did not differ so much among
samples of different collection sites and seasons.
Seasonal change in the amount of LD may generally
be smaller than we may have expected from the census
number. By using a spring sample of a different
collection year, we confirmed a significant excess of
haplotypes composed of one frequent and one rarer
allele in replacement polymorphisms. At the sametime, we found a significant bias in the opposite
direction in silent polymorphisms. Further surveys on
LD are under way for a quantitative assessment of
natural selection in shaping patterns of LD.
PUBLICATIONSPAPERS

1. Kobayashi, S., Noro, Y, Nagano, H., Yoshida, K.
T., Takano-Shimizu, T., Kishima, Y Sano, Y (2005)
Evidence for an evolutionary force that prevents
epigenetic silencing between tail-to-tail rice genes witha short spacer., Gene, 346, 231 - 240
2. Takahashi, A., Takano-Shimizu, T. (2005) A highfrequency null mutant of an odorant-binding protein
gene, Obp57e, in Drosophila melanogaster., Genetics, 170,709 - 718
EDUCATION

1. Dr. Takano gave a two-day training course forMishima Kita Junior High School students, October,
2004.
SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. Takano served as a member of the steering

42

Division of Population Genetics

committee of National Bio-Resource Project"DROSOPHILA."

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

Sumio Sugano
We have been constructing full-length cDNA
libraries from various tissues and cells using
oligo-capping method. From these libraries, we havebeen isolating a number of clones and determining
their 5' end sequences l5 ,. Furthermore, we collaborated
with Shin-Ichi Hashimoto and are isolating 5' sequencetags of mRNAs 1l. Using these 5' end sequence data, wemade and are maintaining database of transcriptional
start sites, DBTSS, the We also gathered aninternational consortium, H-invitational, for the
annotation of human full-length cDNAs with Takashi
Gojobori and Nomura Nobuo and made H-invitational
database l2 '. Using this database, we compared humanand cynomolgus monkey 5' UTR regionS' and devisedand evaluated tissue specific expression and promoter
motifs 6 '. Our data also contributed to make dbQSNP,
that provides sequence and allele frequency
information for single-nucleotide polymorphisms
located in the promoter regions of human genes l1 ,.
We also collaborated with various groups to makefull-length cDNA libraries and to collect full-length
cDNA clones. This includes, Dictyostelium
discoideum 4', red cynomolgus monkey6', and malaria IO).
We also helped isolating various full-length cDNA
clones and provided varIOUS clones foranalysis 2'3'5) 7) 9) 13'16).
Finally, we performed mass spectrometry analysis
of surface proteins of ES cells and found 59 clusters
of differentiation-related molecules and more than 80
components of multiple cell signaling pathways thatare characteristic of a number of different cell lineages.

PUBLICATIONSPapers

1. Eichinger L, PachebatJA, Glockner G, Rajandream
MA, Sucgang R, Berriman M, Song J, Olsen R,
Szafranski K, Xu Q, Tunggal B, Kummerfeld S,
Madera M, Konfortov BA, Rivero F, Bankier AT,
Lehmann R, Hamlin N, Davies R, Gaudet P, Fey P,
Pilcher K, Chen G, Saunders D, Sodergren E, Davis P,
Kerhornou A, Nie X, Hall N, Anjard C, Hemphill L,
Bason N, Farbrother P, Desany B, Just E, Morio T, Rost
R, Churcher C, Cooper J, Haydock S, van Driessche N,
Cronin A, Goodhead I, Muzny D, Mourier T, Pain A,
Lu M, Harper D, Lindsay R, Hauser H, James K, Quiles
M, Madan Babu M, Saito T, Buchrieser C, Wardroper
A, Felder M, Thangavelu M, Johnson D, Knights A,
Loulseged H, Mungall K, Oliver K, Price C, Quail MA,
Urushihara H, Hernandez J, Rabbinowitsch E, Steffen
D, Sanders M, Ma J, Kohara Y, Sharp S, Simmonds M,
Spiegler S, Tivey A, Sugano S, White B, Walker D,
Woodward], Winckler T, Tanaka Y, Shaulsky G,
Schleicher M, Weinstock G, Rosenthal A, Cox EC,
Chisholm RL, Gibbs R, Loomis WF, Platzer M, Kay
RR, Williams J, Dear PH, Noegel AA, Barrell B, Kuspa
A. The genome of the social amoeba Dictyostelium
discoideum. Nature. 435: 43-57, 2005.
2. Hayashi H, Matsuzaki 0, Muramatsu S, Tsuchiya
Y, Harada T, Suzuki Y, Sugano S, Matsuda A, Nishida
E. Centaurin-alpha 1 is a PI3K dependent activator of
ERKI /2 map kinases. J Bioi Chern. 281: 1332-1337,2005.
3. Kamei Y, Aoyama Y, Fujimoto T, Kenmotsu N,
Kishi C, Koushi M, Sugano S, Morohashi K, Kamiyama
R, Asakai R. A steroidogenic cell line withdifferentiation potential from mouse granulosa cells,
transfected with Ad4BP and SV40 large T antigen
genes. J Endocrinol. 185: 187-195, 2005.
4. Kasai Y, Hashimoto SI, Yamada T, Sesel, Sugano
S, Matsushima K, Morishita S. 5'SAGE: 5'-end Serial
Analysis of Gene Expression database. Nucleic Acids
Res. 33: D550-D552, 2005.
5. Lan NT, Yamaguchi R, Uchida K, Sugano S,
Tateyama S. Growth Profiles of Recent Canine
Distemper Isolates on Vero Cells Expressing Canine
Signalling Lymphocyte Activation Molecule (SLAM).
J Comp Pathol. 133: 77-81, 2005.
6. Miyajima N, Watanabe M, Ohashi E, Ohmori K,
Mochizuki M, Nishimura R, Ogawa H, Sugano S,
Sasaki N. Identification and characterization of acanine highly similar to retinoic acid receptor alpha.

DNA Seq. 16: 7-15, 2005.
7. Nakagawa T, Watanabe M, Ohashi E, Uyama R,
Takauji S, Mochizuki M, Nishimura R, Ogawa H,
Sugano S, Sasaki N. Cyclopedic protein expression
analysis of cultured canine mammary glandadenocarcinoma cells from six tumours. Res Vet Sci.
80: 317-323 (2006) 2005 Sep 19;[Epub ahead of print]
8. Nunomura K, Nagano K, Itagaki C, Taoka M,
Okamura N, Yamauchi Y, Sugano S, Takahashi N,
Izumi T, Isobe T. Cell surface labeling and mass
spectrometry reveal diversity of cell-surface markers
and signaling molecules expressed in undifferentiatedmouse embryonic stem cells. Mol Cell Proteomics. 4:
1968-1976 (2005) 2005 Sep 20; [Epub ahead of print]
9. Osada N, Hirata M, Tanuma R, Kusuda J, Hida
M, Suzuki Y, Sugano S, Gojobori T, Shen CK, Wu CI,
Hashimoto K. Substitution Rate and Structural
Divergence of 5'UTR Evolution: Comparative Analysis
Between Human and Cynomolgus Monkey cDNAs.
Mol BioI Evol. 22: 1976-1982,2005.2005 Jun 8; [Epubahead of print]
10. Otsuki T, Ota T, Nishikawa T, Hayashi K, Suzuki
Y, Yamamoto J, Wakamatsu A, Kimura K, Sakamoto
K, Hatano N, Kawai Y, Ishii S, Saito K, Kojima S,
Sugiyama T, Ono T, Okano K, Yoshikawa Y, Aotsuka
S, Sasaki N, Hattori A, Okumura K, Nagai K, Sugano
S, Isogai T. Signal Sequence and Keyword Trap insilico for Selection of Full-Length Human cDNAs
Encoding Secretion or Membrane Proteins from
Oligo-Capped cDNA Libraries. DNA Res. 12: 117-126,
2005.
11. Scott A, Pantoja-Uceda D, Koshiba S, Inoue M,
Kigawa T, Terada T, Shirouzu M, Tanaka A, Sugano
S, Yokoyama S, Guntert P. Solution structure of the
Src homology 2 domain from the human feline
sarcoma oncogene Fes. J Biomol NMR. 31: 357-361, 2005.
12. Shibui A, Shiibashi T, Nogami S, Sugano S,
Watanabe]. A novel method for development ofmalaria vaccines using full-length cDNA libraries.
Vaccine. 23: 4359-4366, 2005.
13. Tahira T, Baba S, Higasa K, Kukita Y, Suzuki Y,
Sugano S, Hayashi K. dbQSNP: a database of SNPs
in human promoter regions with allele frequency
information determined by single-strand conformation
polymorphism-based methods. Hum Mutat. 26: 69-77,
2005.
14. Yamaguchi R, Kojimoto A, Sakai H, Uchida K,
Sugano S, Tateyama S. Growth characteristics of
canine distemper virus in a new cell line CCT cells

Division of Theoretical Genetics

43

originated from canine malignant histiocytosis. J Vet
Med Sci. 67: 203-206, 2005.

15. Yamasaki C, Koyanagi KO, Fujii Y, Itoh T, Barrero
R, Tamura T, Yamaguchi-Kabata Y, Tanino M,
Takeda]l, Fukuchi S, Miyazaki S, Nomura N, Sugano
S, Imanishi T, Gojobori T. Investigation of proteinfunctions through data-mining on integrated humantranscriptome database, H-Invitational database(H-InvDB). Gene. 364: 99-107 (2005). 2005 Sep 23;[Epubahead of print]
16. Yamashita R, Suzuki Y, Sugano S, Nakai K.
Genome-wide analysis reveals strong correlation
between CpG islands with nearby transcription startsites of genes and their tissue specificity. Gene. 350:
129-136, 2005.
Databases
http://dbtss.hgc.jp/
https:!/www.jbirc.aist.go.jp/hinv/index.jsp
SOCIAL CONTRIBUTIONS AND OTHERS~~5!iIJHUGO: Council
HUGO Pacific: Co-Chair
J1t~-=t-ra~'¥:~:~~~

44

Division of Theoretical Genetics

E. DEPARTMENT OFINTEGRATED GENETICS

E-a. Division of Human Genetics
Hiroyuki Sasaki GroupRESEARCH ACTIVITIES(1) Establishment and maintenance of DNA methylation
imprints in the germline and early mouse embryos
Hiroyuki Sasaki, Yuzuru Kato, Ryutaro Hirasawa,
Kenji Kumaki, Hiroyasu Furuumi, Masahiro Kaneda\
Masaki Okan0 2, En LP, Mizue Hisan0 4, Masami
Nozaki 4, Tomohiro Suzuki 5, Shigeharu Wakana5, andToshihiko Shiroishi5eGurdon Inst. Cancer Dev. BioI.;
2CDB, RIKEN; 3Novartis; 40saka Univ.; 5GSC, RIKEN)Genomic imprinting, an epigenetic gene-marking
phenomenon in the germline, causes parent-of- .
origin-specific monoallelic expression of a subset of
mammalian genes in offspring. DNA methylation
serves as an important mechanism to mark theparental alleles of the imprinted genes. Evidence
suggests that the imprinted genes are regulated by
nearby differentially methylated regions (DMRs). We
previously showed by conditional gene targeting thatthe de novo DNA methyltransferase gene Dnmt3a, but
not Dnmt3b, is essential for the establishment of themethylation imprints in both male and female
germlines. Weare now studying in detail how the
methylation imprints are affected in germ cells of theconditional mutants. Another question is how the
methylation imprints are maintained in cleavage stage
mouse embryos, in which the rest of the genome isextensively demethylated. Weare therefore studying
cleavage stage embryos lacking either Dnmtl, Dnmt3a,
Dnmt3b, or combinations of these to see whichmethyltransferase(s) maintains the imprints. Lastly,
we have also set out to screen ENU-treated mutant
mouse stocks for new mutants that have a defect inestablishment of the imprints.

(2) Comparative analyses of the distal imprinted domain
on mouse chromnosome 7 and the orthologous domain
on chicken chromosome 5
Hiroyuki Sasaki, Takaaki Yokomine, Yuzuru Kato,
Wahyu Purbowasito, Hisao Shirohzu, Hisakazu
Iwama, Kazuho Ikeo, Tetsuya Hori, Masaoki Tsuzuki\
Shigeki Mizun0 2, Yo-ichi Matsuda3, Atsushi Toyoda 4,
Masahira Hattod, and Yoshiyuki Sakaki4eHiroshima
Univ.; 2Nihon Univ.; 3Hokkaido Univ.; 4GSC, RIKEN)
Imprinted genes tend to form clusters in the
genome (imprinted domains), which may be related tothe mechanism of imprinting or to the evolution of
imprinting. We have cloned and sequenced a 1-Mb
imprinted domain in mouse chromosome 7F4IF5 andits orthologous domain in chicken chromosome 5 (0.5
Mb). Using one of the mouse YAC clones, we showedthat this domain is in fact composed of two
autonomous subdomains (Cerrato et al., 2005). We thenfound that the genes of the chicken domain are notimprinted and, furthermore, that the chicken domain
lacks the unique tandem repeat cluster of 0.2 Mb, theH19 gene, and the imprinting control elements, all ofwhich are present in the mouse. The results indicatethat the mammalian imprinted genes were alreadyclustered in the common ancestors of mammals andbirds and that the imprinting mechanism, which
affects multiple genes in the cluster, came in later
during mammalian evolution (Yokomine et al., 2005).
We also published a review on the interactions between
the DMRs within this domain by chromatin looping(Kato and Sasaki, 2005).
(3) Computer-assisted search for the sequence featurescommon to the imprinted DMRs
Hiroyuki Sasaki, Hisato Kobayashi, Takashi Abe, Yuji
Kohara and Toshimichi Ikemud eSOKENDAI)
Although the imprinted DMRs, which show
differential methylation depending on parental origin,often play crucial roles in imprinting, features common
to the DMRs have not been identified. We therefore
set out to look for the sequence features common to
the DMRs by computer-assisted programs. We first
determined the extent of each mouse DMR bybisulphite sequencing in 12.5-day embryos. We thenlocated the DMR sequences on self-organizing maps

DEPARTMENT OF INTEGRATED GENETICS

45

(SOMs) produced from the mouse genome sequences
for di-, tri- and tetra-nucleotides. We found that most
DMRs are located in the periphery of the SOMs: they
are more CpG-rich than most of the genome but less
CpG-rich than the CpG islands. Furthermore, thepaternally methylated DMRs contain less CpGs thanthe maternally methylated DMRs (Kobayashi et al. inpress). These findings provide a basis for the further
characterization of the DMRs.
(4) Antisense regulation at the Xist locus by Tsix
Takashi Sado, Yuko Hoki, Hiroyuki Sasaki
X-inactivation is controlled by Xist and its
antisense gene, Tsix, neither of which encodes aprotein. Xist is essential for X-inactivation to occur incis and its differential expression is a key event in theinitiation of X-inactivation. Xist and Tsix are imprinted
in the extraembryonic tissues of mouse embryos, sothat they are expressed from the paternal and maternal
X, respectively, resulting in the preferential inactivationof the paternal X. Targeted disruption of Tsix causes
ectopic expression of Xist, suggesting that Tsix
negatively regulates Xist in cis. However, the molecularmechanism of this antisense regulation remains
unknown. We have demonstrated that Tsixtranscriptionally silences Xist in both embryonic andextraembryonic tissues of mouse embryos. Moreover,
we have shown that disruption of Tsix impairs
establishment of repressive epigenetic modifications
and chromatin structure at the Xist locus. We proposethat Tsix silences Xist through modification of thechromatin structure (Sado et al., 2005).
(5)

Tsix-independent mechanism for Xist silencing

Takashi Sado, Tatsuya Ohhata, Yuko Hoki, Hiroyuki
SasakiDifferential induction of the X-linked non-coding
Xistgene is a key event in the process of X-inactivation
occurring in female mammalian embryos. Xist isnegatively regulated in cis by its antisense gene Tsix
through modification of the chromatin structure. The
maternal Xist allele, which is normally silent in theextraembryonic lineages, is ectopically activated whenTsix on the same chromosome is disrupted, andsubsequently the maternal X chromosome undergoes

46

Division of Human Genetics

inactivation in the extraembryonic lineages even inmales. However, it is still unknown whether the single
Tsix-deficient X chromosome (X T.i-') in males is also
inactivated in the embryonic lineage. Here, we showthat both male and female embryos carrying amaternally derived X JTsix could survive if theextraembryonic tissues were complemented bywild-type tetraploid cells. In addition, Xiston the X jT.ix
was properly silenced and methylated at CpG sites inadult male somatic cells. These results indicate thatthe embryonic lethality caused by the maternal X jT.ix
jis solely attributable to the defects in theextraembryonic lineages. X jT.ix does not seem toundergo inactivation in the embryonic lineage in males,suggesting the presence of a Tsix-independent silencing
mechanism for Xistin the embryonic lineage (Ohhata
et al., in press).
(6) Role of Dnmt3L in gametogenesis
Kenichiro Hata, Maki Kusumi, Takaaki Yokomine, En
LP and Hiroyuki Sasaki CNovartis)
The Dnmt3L (DNA cytosine-5-methyltransferase
3-Like) gene encodes a protein of 421 amino acidresidues and harbors a putative zing finger domainthat shares a high degree of homology with the
PHD-like domains of Dnmt3a and Dnmt3b. The
C-terminal part of Dnmt3L is related to Dnmts, but it
does not possess critical motifs for methyltransferase
activity. We have generated Dnmt3L-deficient mice bygene targeting. While Dnmt3L- j - mice grew normally,both males and females are infertile. All embryos frompregnant Dnmt3L
mothers died around ElO.5. The
maternally methylated imprinted genes, e.g. Igf2r andPegl, were hypomethylated in embryos derived from
Dnmt3L- j - females x Dnmt3L+ j + males, butpaternally methylated DMRs were unaffected.
Dnmt3L- j - male mice showed azoospermia. Theseabnormal germ cells have methylation defects inpaternally imprinted DMRs and lAP retrotransposon
sequences (Hata et al., in press). We speculate that
Dnmt3L functions via interactions with Dnmt3a and!
or Dnmt3b to control de novo methylation of theimprinted DMRs and other sequences including
retrotransposons in male germ cells.
-j-(7) Role of Dnmt3L in placentation
Kenichiro Hata, Takahiro Arima\ Maki Kusumi 2,
Satoru Tanaka2, Takaaki Yokomine, Kunio Shiota2,
Norio Wake1 , Masaoki Tsudzuki3 and Hiroyuki
Sasaki CKyushu Univ., 2Tokyo Univ., 3Hiroshima
Univ.)
Although the Dnmt3L protein lacks DNA
methyltransferase activity, it probably cooperates withDnmts to establish methylation imprints. Oogenesis
apparently proceeds normally in female Dnmt3L.-/mice but their heterozygous offspring (Dnmt3Lmat-/- 1
die before midgestation probably due to a lack ofimprints. The Dnmt3Lmat-/- embryos also showed
defective formation of the labyrinth, reduced formationof the spongiotrophoblast layer, excess trophoblast
giant cells and insufficient attachment between thechorion layer and ectoplacental cone. Cells of theextraembryonic tissues were arrested but not apoptotic.
Dnmt3Lmat-/- trophoblastic stem cells showed adisturbed cell fate in vitro. It was also suggested thata maternal methylation imprint is required for Mash2
imprinting. (Arima et al., submitted). Intriguingly, ourcomparative studies showed that the presence of theDnmt3L gene is restricted to the species that have
imprinting. The acquisition of Dnmt3L by a common
ancestor of eutherians and marsupials might have beenclosely related to the evolution of imprinting(Yokomine et al., in press). These findings indicate thatthe maternal imprints established by de novo
methylation are required for placentation.
PUBLICATIONS

Imprinted X inactivation in the preimplantation mouseembryos., Hum. Mol. Genet., 14 (Spec.1) 59 - 64
5. Yokomine, T., Shirohzu, H., Purbowasito, W.,
Toyoda, A., Iwama, H., Ikeo, K., Hori, T., Mizuno, S.,
Tsudzuki, M., Matsuda, Y, Hattori, M., Sakaki, Y and
Sasaki, H. (2005) Structural and functional analysis of
a 0.5-Mb chicken region orthologous to the imprinted
mammalian Ascl2/Mash2-lgf2-HI9 region., GenomeRes., 15 (1) 154 - 165
6. l'f~*.¥a, {tek*mZ.(2005)~*"!Jmc-7' /./:..-1/7°!J /7--1 /7', /'\-1::t"-1 /?t:A
22 (2) 29 - 34
7. 1tck*miZ. (2005) -7' / ./:..-1 /7°!J /7 --1/ Y : *JT L1t\i1mE~'h tc. G L-tc.:A-/-\-"E-Tlv(Overview) , Molecular
Medicine, 429, 146 - 148
8. {tek *miZ. (2005);r: l::::0:;:r. *' 7 --1 7:A C ~J~ -I"i \;IJOIC., ~~(J)GIi>l1:J7f., 215 (105)
9. {tei1tfj)(, ::fdllltMttt (2005) '"'<' tJ :AJB:;-f*>tUlI..IC.:tOl'T.Q-1/7°!J / H~':!x~g.,-f*~mtl:11:::, Molecular Medicine,42, 173 - 180
10. ~.-~~, {tek*miZ.(2005)1::~~7IH;::f-Ht.Q-1 /7°!J / t- nX:.ll..m, Molecular Medicine, 42"1) -,Books

11. Sasaki, H. (2005) DNA methylation in epigenetics,
development and imprinting. Encyclopedia of Genetics,
Genomics, Proteomics and Bioinformatics (Dunn, M.,
Jorde, L., Little, P. & Subramaniam, S.) John Wiley &Sons, Chichester, 285 - 291
12. {tek*miZ.(2005);r:l::::°:;:r.*'7--17 :AAr~-~=B~ta~ • • ~~5~~.Q~~ {tek*miZ., ~• • rn
EDUCATION

1. {tek*miZ.
{tek*miZ.

~

~~$~~

W~*~~~$~~;r:~:;:r.*,7--17:Ac~M~ .~~Papers~*~~*~~W~~~~~/~-

1. Cerrato, F., Sparago, A., Zou, X., Dean, W.,
Bruggemann, M., Sasaki, H., Reik, W. & Riccio, A.
(2005) The two domain hypothesis in BeckwithWiedemann syndrome: autonomous imprinting of thetelomeric domain of the distal chromosome 7 cluster.,
Hum. Mol. Genet., 14 (4) 503 - 511
2. Kato, Y. and Sasaki, H. (2005) Imprinting andlooping: epigenetic marks control interactions between
regulatory elements., BioEssays, 27 (1) 1 - 4
3. Sado, T., Hoki, Y and Sasaki, H. (2005) Tsix
silences Xist through modification of chromatin
structure., Dev. Cell, 9 (1) 159 - 165
4. Sado, T. and Ferguson-Smith, A. C. (2005)

3. {tek*miZ.

-7'/./:..-1/7!J/T--1/~~~~~~~-r- CAJ!Ji~~~~I:!;\i;J*~{i;~~gfE~*1j~~~~~ ~~~{~~ . 1:!;\i;J*~{i;:w:r~~*Ij~~~~)

4. {tek*miZ. Jt~~~ Mtr.nJ*~, ~;f~~f-:t*~,
Mtr.nJYf-,.ll.*~, ffiI.ll.~~~~~FJT

5. {tek *miZ. f!I!~$~~ ~t.!l:!*~f!I!~$SOCIAL CONTRIBUTIONS AND OTHERS~~fiSlIJ{tek*miZ. M)~~~tf:&.~iW~~{tek*miZ. 8*1::~~~;r:/:;~7!J/Y~~~m~A

Division of Human Genetics

47~O)fth(2) Epigenetic behavior of CA CTA transposon

ftr:k *m,Z.

/~/'qJJ? -y-yfJJ ? ~~*liJf~. 7" v.:c-CUff!

0)1t*±)

ftr:k*m,Z.

.R~~.R~~~~~-liJf~m.~~mAsuka Miura, Masaomi Kato, Miyuki Nakamura,Kazuya Takashima, Yuki Kinoshita, Tetsuji Kakutani~;t~~*~~~~~~

ftr:k*m,Z.

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Bang! (NTT-B],y lv-7°J1;:$IH;i;)E-b. Division of Agricultural Genetics
Tetsuji Kakutani GroupRESEARCH ACTIVITIES(1) Developmental abnormalities induced by DNA
methylation mutation of Arabidopsis

Tetsuji Kakutani, Tetsu Kinoshita, Asuka Miura, Yuki
Kinoshita, Masaomi Kato, Miyuki Nakamura,
Hodetoshi Saze
Arabidopsis provides a genetically tractablesystem to learn role of DNA methylation, since viablemutants with reduced genomic DNA methylation areavailable. Arabidopsis MET1 (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 TION), which encodes a chromatin
remodeling factor (Jeddeloh et al., 1999, Nat. Genet 22,94-). The ddml mutation affects both CG and non-CG
methylation. A striking feature of ddml mutation isthat it induces a variety of developmental
abnormalities by causing heritable change in other loci(Kakutani et al., 1996, PNAS 93,12406-). The molecularbasis has been clarified in two of the loci directly
causing the developmental abnormalities (see below).
In addition, we are starting genetic characterizationof other types of developmental abnormalities(Kakutani et al., 2004, Cold Spring Harbor Symp Quant
BioI 69, 139-).

48

Division of Human Genetics

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
41~, 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
mutants. Single mutants in either metl or cmt3 weremuch less effective in mobilization. Thus CG andnon-CG methylation systems redundantly function forimmobilization of transposons (Kato et al., 2003 Curr
BioI 13, 421-). CMT3gene and non-CG methylation inplants may have evolved as an additional epigenetic
tag dedicated to transposon control.
CACTAl activated by the ddml mutation
remained mobile in the presence of the wild type
DDMl gene, suggesting that de novo silencing is notefficient for the defense of the genome against CACTA
movement. The defense depends on maintenance oftransposon silencing over generations (Kato et al., 2004,
Genetics 168, 961-).
Differentiation of gene-rich and transposon-rich(gene-poor) regions is a common feature in plant
genomes. That may be due to preferential integrationof transposons to gene-poor regions or may be due topurifying selection against transposon insertion to
gene-rich regions in natural populations. To evaluate
the possible contribution of natural selection to theformation of transposon distribution pattern, we
examined the distribution of the CACTA transposons
in genomes of 19 natural variants (ecotypes) andcompared that to integration induced in laboratory inthe ddml mutants. Sequences similar to mobile
CACTAl copy distribute among the ecotypes andshow high polymorphism in genomic localization.
Despite the high polymorphism, the copy number was
low in all the examined ecotypes and they localized
preferentially in pericentromeric and transposon-richregions (Miura et aI, 2004, MGG 270, 524-). This
contrasts to transposition induced in laboratory, inwhich the integration sites are less biased and the copy
number frequently increases. In addition, transposition
induced in DDMl wild type background also showed
unbiased integration specificity (Kato et aI., 2004,
Genetics 168,961-). The differences in the integrationsites may be due to natural selection against
deleterious insertion into chromosomal arm regions.
(3) Inheritance of epigenetic developmental abnonnality.
Yuki Kinoshita, Asuka Miura, Tetsu Kinoshita, Tetsuji
Kakutani
Another developmental abnormality, late flowering
trait, was induced by ectopic expression of FWA geneassociated with hypomethylation of tandem repeat
upstream of the coding region (Soppe et aI., 2000, Mol
Cell 6, 791-). Interestingly, this region is similar to SINE
retroelement in the sequences (Lippman et al., Nature
430,471-). We examined if DNA methylation of thisSINE-related region is really critical for the silencingof FWA using RNA-directed DNA methylation (RdDM)
strategy (Mette et aI., EMBO J. 19, 5194-) and theddml-induced gain-of-function epigenetic FWA allele.
Re-silencing of the epigenetic FWA allele was induced
when the SINE-related region was methylated de novo.
The silent state is heritable and associated withmethylation at CG sites (manuscript submitted).
(4) Epigenetic control of FWA gene expression inendospenn
Tetsu Kinoshita, Asuka Miura, Yuki Kinoshita, 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 notestablished by allele-specific de novo methylation. It
is established by maternal gametophyte-specific geneactivation, which depends on a DNA glycosylase gene,
DEMETER. Since endosperm does not contribute to
the next generation, the activated FWA gene need not
be silenced again. Double fertilization enables plants
to use such 'one-way' control of imprinting and DNA
methylation in endosperm (Kinoshita et al, 2004 Science
303,521-).
In order to identify factors involved in theestablishment of imprinted FWA expression, we
screened mutant defective in this process. A transgenic
line expressing FWA-GFPwas mutagenized and themutants with defective FWA expression were selectedby monitoring the GFP signal. Among 1200
EMS-mutagenized M, plants, we obtained several
mutant candidates. Further genetic and molecularcharacterization of them is in progress.
PUBLICATIONSPapers

1. Ono T, Kaya H, Takeda S, Abe M, Ogawa Y, Kato
M, Kakutani T, Mittelsten Scheid 0, Araki T, andShibahara K (2006). Chromatin assembly factor 1
insures th~ stable maintenance of silent chromatinstates in Arabidopsis. Genes Cells 11, 153-162.
2. Jullien P, Kinoshita T, Ohad N, Berger F (2006)
Maintenance of DNA methylation during the
Arabidopsis life cycle is essential for parental
imprinting. Plant Cell in press.
3. Takamiyama T, Hosobuchi S, Asai K, Nakamura
E, Tomioka K, Kawase M, Kakutani T, Paterson A,
Murakami Y, and Okuizumi H (2006) Restriction
landmark genome scanning method using
isoschizomers (MspIlHpaIl) for DNA methylation
analysis. Electrophoresis in press.
4. 1J~jJijJ)(li!, ~~~f=(2005)RNAU:::DNA;o(TJHtl(:
J:.g7 / AO)~;jitti\1fJm, 1t~~1::.~, 43, 542 - 547
EDUCATION

1. ~~~f=~rp~~***~fI~~~SOCIAL CONTRIBUTIONS AND OTHERS~fi§'t.

*l'"!f : B *~-8~:@'>~JJJh.~.
Division of Agricultural Genetics

49

*Tffi~, ~oo~BW,*Tg, ~~~t:B*~8~~~77@]*~BestPaper]t

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

Tatsuya Ono, Yasunari Takami\ Fumiyuki
Sanematsu, Tatsuo Nakayamat, and Kei-ichi
Shibahara
IDepartment of Biochemistry, Miyazaki Medical
College, University of Miyazaki
A newly replicated DNA is assembled into
nucleosome soon after the passage of replication fork.
This rapid formation of nucleosome is functionallylinked to DNA replication machineries and plays acritical role for the maintenance of genome integrity
in proliferating cells. Two histone binding proteins,
CAF-l (Chromatin Assembly Factor-I) and ASFI
(Anti-Silencing Functionl), are involved in someprocess of this nucleosome assembly reaction.
In collaboration with Dr. Nakayama in Universityof Miyazaki, using CAF-l and ASFl-deficint chicken
DT40 cell lines, we recently showed that without CAF-l
and/or ASFI function, S-phase progression was
delayed and a rapid nucleosome assembly during DNA
replication was disturbed in vivo. (Sanematsu, F, et al.,
JBiol. Chem. in press, 2006). This is the first direct
evidence for the involvement of CAF-l and ASFI ina rapid nucleosome assembly during DNA replicationin vivo. In addition, we obtained some interesting
evidence for that CAF-l is involved in Chkl-dependent
checkpoint pathway after the treatment with HU(Takami, Y, et al., submitted).
(2) Physiological implications of CAF-I and
CAF-I-dependent nucleosome assembly in higher
eukaryotes

Tatsuya Ono, Hidetaka Kaya\ Shin Takeda2, Tetsuji
Kakutani, Takashi Araki', Kei-ichi Shibahara
lDepartment of Botany, Graduate School of Science,
Kyoto University

50

Division of Agricultural Genetics

2 Department of Plant Biology, University of Geneva,
Switzerland

The genetic approach of Arabidopsis is a powerfultool to see physiological implications of CAF-l and
CAF-ldependent nucleosome assembly in higher
eukaryotes. We have analyzed loss-of-function mutantsof call (fasciata :fas) in Arabidopsis and have shownthat the las mutants displayed severely disturbed
cellular and functional organization of both meristems(Kaya et al., Cell, 2001; Takeda et al., Genes Dev., 2004).
We recently showed that transcriptional gene silencing(TGS) of endogenous CACTA transposons was
released infrequently in a stochastic manner in las,without decreasing DNA methylation. Other
endogenous silent genes at different chromosomal sites
were also transcriptionally activated non-concomitantlywith each other. Furthermore, TGS of the silent
transgene {3 -glucuronidase (GUS) was alsode-repressed randomly in las mutant plants,
irrespectively of developmental abnormalities, and theactivated state of GUS was maintained during growth
to produce clusters of cells expressing GUS (Ono et al.,Genes Cells, 2006). Taken together, we strongly suggestthat CAF-l ensures stable inheritance of epigenetic
states through multiple rounds of cell divisions andthat defects in CAF-l functions explain the stochastic
occurrence of pleiotropic phenotypes in the lasmutants.
(3) Histone macroH2A-mediated formationtranscriptionally repressed states of chromatinof

Yuya Ogawa', Hitoshi Nishijima, and Kei-ichi
Shibahara, Department of Genetics, Harvard Medical School,
USA
The unusual histone variant macroH2A(macroH2A) is predicted to be functionally associatedwith transcriptional repression as it appears to beenriched in the inactive X chromosome byimmunostaining. However, molecular function ofmacroH2A in modulating chromatin structures
remains unknown. To reveal the roles of macroH2A,
we purified macroH2A-containing nucleosome byaffinity purification with anti-epitope tag antibodies,and eventually, we found mono-ubiquitinated form of
maroH2A and determined the ubiquitinated sites inmacroH2A (Ogawa, Y, et al., 2005). Weare currently
clarifying the function of these modifications andtrying to isolate the molecules enriched in andassociate with macroH2A-containing nucleosomes.

E-c. Division of Brain Function
Tatsumi Hirata GroupRESEARCH ACTIVITIES(4) Analysis of S. pombe RanGAP SpRnal protein in
the heterochromatin assembly via histone H3.
Hitoshi Nishijima, Ayumi Kusano\ Tomoyuki Ohba\
Hideo Nishitani', Kei-ichi Shibahara, Takeharu
Nishimoto'
1: Department of Molecular Biology, Graduate Schoolof Medical Science, Kyushu University
We have isolated temperature-sensitive mutantsof the RanGAP homologue, Sprnal, in S. pam be.
Spranl ts strains display a strong defect in mitotic
chromosome segregation (Kusano, A., et al., Mol. BioI.
Cell, 2004). This phenotype is suppressed by theoverexpression of Clr4, which methylates Lys 9 ofhistone H3 (H3K9). We found that histone H3 controls
the RanGAP activity of SpRna1 to prevent disruptionof the RanGTP/GDP gradient and that SpRna1enhances the activity of the Clr4-dependent
methylation of histone H3K9 (Nishijima, H., et al.,Mol.Biol.Cell, in press). In addition, we had isolated
another suppressor, Snf2SR, which is likely to be anovel member of the SNF2 family involved in the Ran
GTPase cycle (Ohba, T., et al., in preparation).
PUBLICATIONSPapersOgawa, Y., Ono, T., Wakata, Y., Okawa, H., Tagami,
H. and Shibahara, K-i. (2005) Histone variant
macroH2A1.2 is mono-ubiquitinated at its histone
domain., Biochem. Biophys. Res. Common., 336, 204 209
EDUCATION~~~-

7~~~~~~~W~~~~:~~~~*T~Y*~~~~

OOli~~~~~ffl'~~T-~(I) Regulation of ventral tangential migration ofguidepost neurons in the lateral olfactory tract
Takahiko Kawasaki, Keisuke Ito and Tatsumi Hirata
In the developing nervous system, many neurons
migrate for a long distance from their birthplace to thefinal destination. We found a ventral tangential
migration stream of early-generated neurons in themouse telencephalon (Tomioka et aI., 2000). Although
this migration mode had been overlooked for a longtime, this is the most common migration mode in theearly telencephalon when the neurogenesis has juststarted, and therefore has immeasurable impact on all
the following developmental processes. One of theimportant function of this migration is to convey a
specific type of neocortex-born neurons called lot cells
to the future pathway of the lateral olfactory tract(LOT), where the cells construct a tight cellular arrayand guide the following projection of LOT axons.
We developed an organotypic culture system toinvestigate the regulation of this cell migration in thedeveloping telencephalon (Kawasaki et aI., 2006). Using
the system, we found that two types of signals govern
the ventral tangential migration. One is the signal
contained in the neocortex, which directs the neurons
ventrally. The other is the repulsive signal in the
ganglionic eminence, a ventral structure of thetelencephalon, which excludes neocortex-born neurons
from invading this ventral area. These two types ofsignals appear to cooperatively function to arrange thelot cells in a packed cellular array at the boundary
between the neocortex and the ganglionic eminence.
To investigate molecular mechanisms that control
the ventral tangential migration, we examined effects
of several signaling molecules on the migration lot cells
in the organotipic culture and found that netrin 1, anaxon guidance factor, attracts these cells (Kawasaki
et aI., 2006). Furthermore, we demonstrated that
mutations in the genes encoding netrin 1 and its
functional receptor Dcc lead to inappropriate
distribution of lot cells and subsequent partial
disruption of LOT projection. These results suggest

Division of Agricultural Genetics

51

that netrin 1 regulates the migration of lot cells and
LOT projections, possibly by ensuring the correct
distribution of these guidepost neurons.

EDUCATION(2) Identification of the Antigens Recognized by
Monoclonal Antibodies Raised against the Developing
Lateral Olfactory Tract

2. -'JZfijtC0lj.

1. -'JZfijk0lj.

~~.~O)$lb~~~~ff~~~~• •JA9h*'¥'I'¥$

Takahiko Kawasaki, Yoshiko Takagi, Hitoshi
Yamatani, and Tatsumi Hirata
During development, olfactory bulb axons navigate
the complex microenvironment composed of myriads
of molecules in the telencephalon to construct a bundle
called the lateral olfactory tract. The axons, themselves,also express thousands of different molecules during
the navigation. Previously, we have produced many
monoclonal antibodies that label the lateral olfactory
tract and the surroundings in a unique pattern. The
labeling profiles suggested that the antigen molecules
recognized by each antibody are heterogeneously
distributed around the developing lateral olfactory
tract. We developed an efficient screening method to
identify the antigen molecules by combining expressionof a cDNA library in COS-7 cells and the subsequent
immunohistochemical staining of the cells (Kawasaki
et aI., 2005). The systematic screening successfully
identified the specific cDNA clones for many of themonoclonal antibodies, which highly probably code forthe antigen molecules, and therefore unveiled themolecular nature of local components that embrace thedeveloping lateral olfactory tract.
PUBLICATIONSPapers

1. Kawasaki, T., Takagi, Y, Yamatani, H. and Hirata,
T (2005) Systematic screening and identification of theantigens recognized by monoclonal antibodies raised
against the developing lateral olfactory tract, J.
Neurobiol., 62, 330 - 340
2. Koma, Y, Ito, A., Watabe, K., Hirata, T, Mizuki,
M., Kitamura, T, Kanakura, Y and Kitamura, Y (2005)
Distinct role for c-kit receptor tyrosine kinase andSgIGSF adhesion molecule in attachment of mast cells
to fibroblasts., Lab. Invest., 85, 426 - 435
3. -'JZfijtC0lj. (2005) n~0)~JftJ!X¥.f~1~ ~ $lb~:~O~JJX, 1$~6Jf~O)ift~, 49, 77 - 83

52

Division of Brain Function.~~~'¥¥.fJ}lJilJIl:iJ;*~~*,¥. ~'¥$E-d. Division of Applied Genetics
Kunio Shiota GroupRESEARCH ACTIVITIES(1) Stage-by-stage change in DNA methylation statusof Dnmtllocus during mouse early development

Yeoung-Gyu Ko, Koichiro Nishino, Naoko Hattori,
Yoshikazu Arai, Satoshi Tanaka and Kunio Shiota(The University of Tokyo)
Methylation of DNA is involved in tissue-specificgene control, and establishment of DNA methylation
pattern in the genome is thought to be essential forembryonic development. Three isoforms of Dnmtl(DNA methyltransferase 1) transcripts, Dnmtls,
Dnmtlo, and Dnmtlp, are produced by alternative
usage of multiple first exons. Dnmtls is expressed insomatic cells. Dnmtlp is found only in pachytene
spermatocytes, whereas Dnmtlo is specific to oocytes
and preimplantation embryos. Here we determinedthat there is a tissue-dependent differentiallymethylated region (T-DMR) in the 5' region of Dnmtlo
but not in that of the Dnmtls/lp. The methylation
status of the Dnmtlo T-DMR was distinctively
different in the oocyte from that in the sperm and adultsomatic tissues and changed at each stage fromfertilization to blastocyst stage, suggesting that active
methylation and demethylation occur during
preimplantation development. The T-DMR was highly
methylated in somatic cells and embryonic stem cells.
Analysis using Dnmt-deficient embryonic stem celllines revealed that Dnmtl, Dnmt3a, and Dnmt3b areeach partially responsible for maintenance of
methylation of DnmtloT-DMR. In particular, there arecompensatory and cooperative roles between Dnmt3a
and Dnmt3b. Thus, the regulatory region of Dnmtlo,
but not of Dnmtls/lp, appeared to be a target of DNA
methylation. The present study also suggested that the
DNA methylation status of the gene region dynamicallychanges during embryogenesis independently of thechange in the bulk DNA methylation status. (Ko, et al.,2005).
(2) DNA methylation-dependent epigenetic regulationof dimethylarginine dimethylaminohydrolase 2
gene in trophoblast cell lineage
Junko Tomikawa, Kazumi Fukatsu, Satoshi Tanaka
and Kunio Shiota(The University of Tokyo)
Trophoblast cell lineage is established through thefirst cellular differentiation in mammalianembryogenesis, and its developmental potential isrestricted to the extraembryonic tissues contributing
solely to the placenta. Several lines of evidence suggest
a relative lack of importance of DNA methylation ingene regulation in the extraembryonic tissues
compared to embryonic ones. Here we analyzed the
dynamics of epigenetic status in the upstream regionof mouse Ddah2 gene, which was found to be
specifically repressed in a stem cell population oftrophoblast cell lineage. We found a tissue-dependent
differentially methylated region in the regulatory regionof Ddah2 gene. This region was hypermethylated introphoblast stem cells and was hypomethylated indifferentiated cells both in vivo and in vitro. Thischange was well correlated with Ddah2 expression. In
addition, in vitro methylation confined to thedifferentially methylated region was sufficient torepress promoter activity in the reporter assay.
Furthermore, a repressive pattern of histone
modifications was formed around the differentiallymethylated region in undifferentiated TS cells withrepressed Ddah2. Our data suggest that DNA
methylation-mediated chromatin remodeling is
involved in the regulation of Ddah2 gene expression,and thus is important even in trophoblast cell lineage(Tomokawa, et al., 2006).
(3) DNA methylation profiles of donor nuclei cells andtissues of cloned bovine fetuses
Kremenskoy Maksym\ Kremenska Yuliya\ Masako
Suzuki\ Kei Imai 2, Seiya Takahashi3, Kazuyoshi
Hashizume\ Shintaro Yagi\ Kunio Shiota1(tThe University of Tokyo, 2National Livestock
Breeding Center, 3National Institute of Livestock andGrassland Science, 4Iwate University)
Methylation of DNA in CpG islands plays animportant role during fetal development anddifferentiation, because in mammalian genomic DNA,CpG islands are preferentially located at upstream
regions including the transcription start site ofhousekeeping genes, as well as associated withtissue-specific genes. Somatic nuclear transfer (NT)technology has been used to generate live clones innumerous mammalian species, but only a low
percentage of nuclear transferred animals develop toterm. Abnormal epigenetic changes in CpG islands ofdonor nuclei after nuclear transfer could contribute toa high rate of abortion during early gestation andincrease perinatal death; these changes have yet to be
explored. Thus, we investigated genome-wide DNA
methylation profiles of CpG islands in nuclei donor
cells and NT animals. Using Restriction Landmark
Genomic Scanning (RLGS), we showed for the firsttime, the epigenetic profile formation of tissues from
NT bovine fetuses produced from cumulus cells. Fromapproximately 2600 unmethylated NotI sites visualizedon the RLGS profile, at least 35 NotI sites showed
different methylation status. Moreover, we proved that
fetal and placental tissues from artificially inseminatedand cloned cattle have tissue-specific differences ingenome-wide methylation profiles of CpG islands. We
also found that possible abnormalities occurred in fetal
brain and placental tissues of cloned animals(Maksym, et. al., 2006a).
(4) Epigenetic characterization of CpG islands of bovine
leptin and POU5FI genes in cloned bovine fetuses
Kremenskoy Maksym\ Kremenska Yuliya1, Masako
Suzuki\ Kei Imai 2, Seiya Takahashi3, Kazuyoshi
Hashizume 4, Shintaro Yagi\ Kunio Shiota1 • CThe
University of Tokyo, 2National Livestock Breeding
Center, 3National Institute of Livestock and Grassland
Science, 4Iwate University)
Production of nuclear transferred animals
concerns with number of abnormal development andfetal loss during postimplantation period. Abnormal
DNA methylation is one of the reasons of poor survivalof cloned animals. In mammalian genome DNA, CpG
islands are preferentially located at the start oftranscription of housekeeping genes and they are also

Division of Applied Genetics

53

associated with tissue-specific genes. The correct andconsecutive mechanisms of DNA methylation in CpG
islands are necessary for selective gene expressions
that determine the properties of individual cells, tissues
or organs. In this study we investigated CpG islands
methylation status in fetal and placental tissues ofbovine Leptin and POU5Fl genes in fetuses produced
by artificial insemination (AI) and nuclear transfer(NT) at days 48 and 59 of pregnancy. Altered DNA
methylation was observed in the normal and cloned
fetal, placental and endometrial tissues using bisulfite
sequencing and pyrosequensing. Tissue-specificdifferently methylated region in bovine Leptin gene and
POU5Fl gene shows variable methylation status in NT
fetuses compare to AI control (Maksym, et. at., 2006b).
(5) Proliferation related acidic leucine rich protein
PAL31 functions as a caspase-3 inhibitor
Weiyong Sun1,2. Hiromichi Kimura 2• Naka Hattod'
Satoshi Tanaka2, Shigemi Matsuyama b. and Kunio
Shiota1.* (" The University of Tokyo, b Case Western
Reserve University)
Proliferation related acidic leucine rich protein
PAL31 (PAL3l) is expressed in proliferating cells andconsists of 272 amino acids with a tandem structureof leucine-rich repeats in the N-terminus and a highly
acidic region with a putative nuclear localization signal
in the C-terminus. We previously reported that PAL31
is required for cell cycle progression. In the present
study, we found that the antisense oligonucleotide of
PAL31 induced apoptosis to the transfected Nb2 cells.
Stable transfectants, in which PAL31 was regulatedby an inducible promoter, were generated to gain
further insight into the signaling role of PAL31 in theregulation of apoptosis. Expression of PAL31 resulted
in the marked rescue of Ratl cells from etoposide andUV radiation-induced apoptosis and the cytoprotection
was correlated with the levels of PAL31 protein. Thus,
cytoprotection from apoptosis is a physiological
function of PAL31. P AL31 can suppress caspase-3
activity but not cytochrome c release in vitro, indicatingthat PAL31 is a direct caspase-3 inhibitor. In
conclusion, PAL31 is a multifunctional protein working
as a cell cycle progression factor as well as a cell
survival factor (Sun, et al., 2006).

(6) Dimethyl Sulfoxide(DMSO) Increases Expression of
Dnmt3as and Affects Genome-wide DNA Methylation
Profiles in Mouse Embryoid Body
Misa Iwatani, Kohta Ikegami, Yuliya Kremenska, Naka
Hattori, Satoshi Tanaka, Shintaro Yagi and Kunio
Shiota (The University of Tokyo, Tokyo, Japan)
Dimethyl sulfoxide (DMSO), an amphipathic
molecule, is widely used not only as a solvent forwater-insoluble substances, but also as a
cryopreservant for various types of cells. Exposure to
DMSO sometimes causes unexpected changes in cellfates. Because mammalian development and cellulardifferentiation are controlled epigenetically by DNA
methylation and histone modifications, DMSO likely
affects the epigenetic system. The effects of DMSO on
transcription of three major DNA methyltransferases(Dnmts) and five well studied histone modification
enzymes were examined in mouse embryonic stem
cells (ES cells) and embryoid bodies (EBs) by RT-PCR.
Addition of DMSO (0.02-1.0%) to EBs in culture induced
an increase in Dnmt3a mRNA level with increasing
dosage. Increased expression of two subtypes ofDnmt3a in protein level was confirmed by Western
blotting. Southern blot analysis revealed that DMSO
caused hypermethylation of two kinds of repetitive
sequences in EBs. Furthermore, Restriction Landmark
Genomic Scanning (RLGS), by which DNA methylation
status can be analyzed on thousands of loci in genic
regions, revealed that DMSO affected DNA methylation
status at multiple loci, inducing hypomethylation as
well as hypermethylation depending on the genomic
loci. In conclusion, DMSO has an impact on theepigenetic profile: upregulation of Dnmt3as' expression
and alteration of genome-wide DNA methylation
profiles with phenotypic changes in EBs.
PUBLICATIONSPapers

1. Ko, YG, Nishino, K, Hattori, N, Arai, Y, Tanaka,
S, Shiota, K (2005) Stage-by-stage change in DNA
methylation status of Dnmtllocus during mouse early
development., J. BioI. Chern., 280 (10) 9627 - 9634
2. :W~~tJ>. *£iiM. :liiEEn~~ (2005) Jfi1:: . :5Ht ~.:r.
t'-/ :r.*,T-1' 7 A. ?' / A~~. 5. 25 - 30
3. :liiEEn~~ (2005Hj!Ujlj-1::~f-j.~0)~tc.t.J:/"' '7 ?t-{ A :.:r.l::' -/ :r.

54

Division of Applied Genetics*'

T -1' A 7. ~~~~. 23. 2096 - 2099

4.

EB~~, ±fj[EBn~~ (2005) DNA;( T Jv{t'lf~n~G.lj.tc.

1JIij~[J~riij:E~~ ~ *BIJI?!5t1t, ~~I2f~,

23, 2100 - 2106

E-d. Division of Applied Genetics
Takashi Araki GroupRESEARCH ACTIVITIES

1-(1). Physiological roles of chromatin assembly factor-l(CAF-1) in epigenetic control of meristem activity during
post-embryonic development in plants

Takashi Arakp,2, Mitsutomo Abe l ,3, Hidetaka Kaya\
Yuya Ogawa, Masaomi, Kato, Tetsuji Kakutani,
Kei-ichi Shibahara CGraduate School of Science, Kyoto
University, 2]apan Science and Technology Agency,
3pROBRAIN, 4 Genome & Drug Research Center,
Tokyo University of Science)
Loss-of-function mutants of chromatin assembly
factor-1 (CAF-1) in Arabidopsis,fasciata (fas), show awide variety of morphological abnormalities andunique defects in the expression of WUSCHEL and
SCARECROW genes in meristems (Kaya et al., Cell
104,131-142.2001). We found that transcriptional genesilencing (TGS) of endogenous CACTA transposons
was released in a stochastic manner in fas, without
decreasing global levels of DNA methylation. Other
endogenous silent genes at different chromosomal
sites, a DNA transposon AtMul and a hypotheticalgene T5L23,26 at a heterochromatin knob, were alsotranscriptionally activated. Activation of the threedifferent silent loci occurred non-concomitantly to eachother. Furthermore, TGS of a silent B-glucuronidase(GUS) transgene was also released randomly in fas
plants, without apparent correlation with
developmental abnormalities. Activated state of GUS
was maintained during growth to produce clusters ofcells expressing GUS, Analysis of patterns in
cotyledons suggested that release from TGS may
sometimes occur during endoreduplication and/or
post-mitoptically. Based on these observations, we
proposed a model that CAF-1 contributes to the stable
inheritance of epigenetic states through multiple
rounds of cell divisions. Defect of CAF-1 function
explains the stochastic occurrence of pleiotropic
morphological phenotypes observed in fas mutants(Ono et al., Genes to Cells 11,153-162.2006; issue cover).

1-(2). Physiological roles of anti-silencing function 1(ASF1) in embryonic and post-embryonic developmentin plants

Takashi Araki l ,2, Ken-ichi Kurotani, Mitsutomo Abe l ,3,Hidetaka Kaya4, Tetsuji Kakutani, Kei-ichi Shibahara
CGraduate School of Science, Kyoto University, 2]apan
Science and Technology Agency, 3pROBRAIN,
4 Genome & Drug Research Center, Tokyo Universityof Science)

Arabidopsis has two genes for a histone chaperone,anti-silencing function 1 (ASF1), ASF1a and ASF1b.
Weare interested in physiological role of ASF1 inplant, especially functional overlap and differentiation
between ASF1 and CAF-l. To address these questions,loss-of-function mutations of ASF1a, ASF1b, andCAF-1 (represented by fas2lacking the second largest
subunit) are being analyzed in various combinations.
Biochemical work of ASF1 is also being performed.
asfla; asflb double mutants were viable as are
CAF-1 mutants, but with much more subtle
abnormality in gross morphology. asfla; fas2 and asfl b;
fas2 had more severe morphological defect than fas2
single mutant indicating the functional redundancy,
Pollen formation was arrested at the tetrad stage
causing complete male sterility in the double mutants
suggesting that either meiosis or pollen maturation isseverely perturbed. asfla; asfl b; fas2J + plants gave rise
to about a quarter aborted seeds which are likely asfla;
asflb;fas2triple homozygotes. Embryos in these seeds
were arrested at the late globular stage. Therefore, inplants histone chaperon activity represented by CAF-1
or ASF1 is indispensable for completion of embryonicdevelopment.
1-(3). Genes involved in the integration of various genetic
pathways for regulation of the floral transition

Takashi Araki l ,2, Mitsutomo Abe l ,3, Sumiko
Yamamotd,2, Yasufumi Daimon\ Ayako Yamaguchi\
Yoko Ikeda\ Michitaka Notaguchi\ Masaki
Kobayashii CGraduate School of Science, Kyoto
University, 2]apan Science and Technology Agency,
3pROBRAIN)
Floral transition in Arabidopsis is regulated byseveral pathways which converge on thetranscriptional regulation of floral pathway integrators

Division of Applied Genetics

55

including FT. FTis a direct target of CO and encodesa protein with similarity to mammalian proteins(PEBPIRKIP) involved in cellular signaling. Functionof FT as a potent promoter of flowering is conserved
among various plant species (Kojima et al., Plant Cell
Physiol. 43, 1096-1105. 2002; Endo, T. et al. 2005).
FTtranscription was immediately induced incotyledon and leaf vascular tissues upon transfer fromshort-day to inductive long-day photoperiods. We
found that the promotion of flowering by FTrequires
the activity of another flowering-time gene FDwhich
encodes a bZIP transcription fac;:tor preferentiallyexpressed in the shoot apex. FT-FD protein interaction
was observed in yeast cells, in vitro, and in planta. FD
is involved in transcriptional activation of the floral
meristem identity genes API and CAL redundantlywith LFY. ft; lfy and fd; lfy double mutants were very
similar in severe reduction of API mRNA levels andstrong defects in floral specification. Loss of FT
function expression suppressed ectopic up-regulationof API in seedlings by FD over-expression. Mutant
forms of FD which lack a potential phosphorylationsite at the C-terminus did not interact with FTin yeast
cells and failed to complement fd late-flowering
phenotype even by over-expression. These and otherevidences suggest that FT and FD are inter-dependent
in promotion of floral transition and activation of API
expression. Since the activity of FD, which ispreferentially expressed shoot apex, seems to require
protein/protein interaction with FT, shoot apex is likely
the site of action of FT protein. Consistent with this,restoration of FTfunction in the whole region or theoutermost layer (L1) of the shoot apex rescued
late-flowering phenotype of ft. Based on theseobservations we proposed a hypothesis that the FT
gene products (protein and/or mRNA) represent a
long-distance signal generated in photoperiodicallyinduced leaves (mainly in vascular tissues) and act at
the shoot apex to initiate floral development (Abe etal., 2005).
We also found the involvement of short-distancesignal(s) in regulation of FTby phytochrome B (phyB)
which senses light quality. Cell type-specific restoration
experiments of phyB function demonstrated that phyBin mesophyll cells is essential for down-regulation of
FT in phloem tissue. These suggest that phyB in
mesophyll cell generate signal(s) which are transported
to the phloem companion and parenchyma cells whichis the site of FT expression (Endo, M. et al., 2005).

56

Division of Applied Genetics

Arabidopsis genome has a homolog of FT, called
TWIN SISTER OF FT (TSF). We demonstrated that
TSF acts redundantly with FT as a floral pathway
integrator. Interestingly, regions of expression of FT
and TSF did not overlap (FT was expressed incotyledons and leaves, whereas TSFwas expressed in
hypocotyls), although they shared the same
tissue-specificity in phloem. Based on these and otherobservations we proposed a model in which various
regulatory pathways of flowering are integrated in thephloem tissue to activate transcription of FTand TSF,
products of which in turn act as the long-distancesignals (Yamaguchi et al., 2005).
1-(4). Molecular basis of late-flowering phenotype in
epialleles of FWA in Arabidopsis
Takashi ArakF,2, Y oko Ikeda\ Mitsutomo Abel,aCGraduate School of Science, Kyoto University, 2Japan
Science and Technology Agency, apROBRAIN)
Dominant late-flowering mutantation fwa isan "epigenetic mutation" that causes ectopic
expression of FWA due to hypomethylation of direct
repeats in the promoter (Soppe et al., Mol. Cell 6,
791-802, 2000). We have shown that FWA is not
expressed in seedlings and that ectopically-expressed
FWA inhibits floral transition by interfering with the
FTfunction through protein interaction. Since FWA
protein is a HD-ZIP protein which is likely atranscription factor, we further tested the possibility
that FWA inhibits flowering through thetranscriptional mis-regulation of its target genes. We
performed microarray analysis using two fwa epialleles(fwa-2 and fwa-IOID) and 35S::FWA. The
transcriptional profiles of these plants suggest that
late-flowering phenotype of fwa is unlikely due to themis-regulation of transcription. We also investigated
the site of action of FT protein using FWA protein asa specific inhibitor of the FTfunction. FWA expressedin shoot apex by the FD promoter delayed flowering,whereas FWA expression in vascular tissues did not.
These support the notion that shoot apex is the siteof action of FT protein (Ikeda et al., in preparation).
PUBLICATIONSPapers

1. Abe, M., Kobayashi, Y., Yamamoto, S., Daimon,

Y., Yamaguchi, A., Ikeda, Y., Ichinoki, H., Notaguchi,M., Goto, K., Araki, T. (2005) FD, a bZIP protein

mediating signals from the floral pathway integrator
FT at the shoot apex., Science, 309 (5737) 1052 - 1059
2. Endo, M., Nakamura, S., Araki, T., Mochizuki, N.,
Nagatani, A. (2005) Phytochrome B in the mesophylldelays flowering by suppressing FLOWERING
LOCUS T expression in Arabidopsis vascular bundles.,Plant Cell, 17 (7) 1941 - 1952
3. Endo, T., Shimada, T., Fujii, H., Kobayashi, Y.,
Araki, T., Omura, M. (2oo5)Ectopic expression of an
FT homolog from Citrus confers an early flowering
phenotype on trifoliate orange (Poncirus trifoliata L.
Raf.)., Transgenic Res., 14 (5) 703 - 712
4. Yamaguchi, A., Kobayashi, Y., Goto, K., Abe, M.,
Araki, T. (2005) TWIN SISTER OF FT (TSF) acts asa floral pathway integrator redundantly with FT., Plant
Cell Physiol., 46 (8) 1175 - 1179
Books
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EDUCATION

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Division of Applied Genetics

57

F. GENETIC STRAINSRESEARCH CENTER

F-a. Mammalian Genetics Laboratory
Toshihiko Shiroishi GroupRESEARCH ACTIVITIES(1) A novel long-range enhancer to specify the Sonic
hedgehog (Shh) expression in the oral cavity
morphogenesis

Tomoko Sagai, Takanori Amano, Yoichi Mizushina,
Hiromi Yamamoto, Noriko Sakurai-Yamatani, Ayaka
Okagaki, Masaru Tamura and Toshihiko Shiroishi
Shh is a secreted glycoprotein that is essential forthe fundamental morphogenesis in vertebrate
embryogenesis. In developing embryos, Shh expresses
in various organogenesis processes including head,lung, heart and limbs fonnation. Mutant mice in which
nonnal Shh expression is affected in the organogenesis
exhibit severe defects in the relevant organs. The Shh
expression in a particular organogenesis is controlledby specific cis-acting regulatory elements. Recently,
such several long-range enhancers have been identified
by cross species comparison of genome sequences andexploration of conserved sequences among
evolutionary remote species. A noncoding sequence,
MFCS1, localized in the 1Mb upstream of the Shhcoding sequence is one of such cis-acting elements,
which includes limb bud-specific Shh enhancer (Sagai
et al., 2005).
Here we report another novel long-range enhancer
involving in morphogenesis of mouse oral cavity.
MFCS4 is a phylogenetically conserved noncoding
sequence residing in the 800kb upstream region of the
Shh coding. In order to test whether the sequence acts
as cis-acting regulatory element, first we carried outtransgenic assay with a LacZ-reporter gene flanked by
the MFCS4 fragment. In the transgenic mice, we foundthat MFCS4 induces expression of the reporter gene

58

GENETIC STRAINS RESEARCH CENTER

specifically in the oral epithelium of palate, epiglottis
and arytenoid. Next, we conducted ES targeting to
generate MFCS4 knockout (KO) mice. The miceheterozygous for the MFCS4 KO did not show any
defect. By contrast, the KO homozygotes caused
hypoplastic defects in the palate and epiglottis
surrounding the naso-and oropharyngeal opening.
Because of this defect, airway of the homozygotes was
disturbed, and eventually it resulted in neonatal
lethality.
In the oral cavity of wild-type embryos,
endogenous Shh expression was detected in palatal
and tongue epithelium and partially overlapped theexpression domain of the reporter gene in thetransgenic mice. Then, we examined the Shh
expression in the MFCS4 KO homozygotes. Section insitu hybridization with a Shh riboprobe showed thatin the MFCS4 KO homozygotes, Shh did not expressin the oral epithelium where the signal of transgenic
reporter gene was detected. Finally, in order to confinn
that MFCS4 is a cis-acting element, we carried out atest cross of the Shh-coding KO and the MFCS4 KO
mice. From the above cross, we generated compound
heterozygotes of the both KO alleles. We observed that
all compound heterozygotes exhibited the phenotype
almost identical to that of the single MFCS4 KO
homozygotes. Thus, all data demonstrated that MFCS4
is a cis-acting element including naso-and oropharyngeal specific long-range enhancer of Shh.
(2) Chromosomal dynamics of a long·range cis-regulator
for limb bud-specific Shh expression

Takanori Amano, Tomoko Sagai, Hideyuki Tanabel
and Toshihiko Shiroishi (lThe Graduate University for
Advanced Studies)
MFCSI (mammal-fish conserved sequence) is
located at the intron 5 of Lmbrllocus, which is 1 Mb
apart from the Shh coding sequence. We have reported
that elimination of MFCSI causes a defect of Shh
expression and distal truncation of skeletal elements
of mouse limb. It is suggested that MFCSI is a limb
bud-specific cis-acting regulatory element of Shh (Sagai
et al., 2005). This fact raises a question of how MFCSI
controls a transcription of Shh beyond such a long
distance. To address this question, we paid attention
on higher-order chromosomal structure surrounding
the Shh locus.

To examine topological relationship of the Shhcoding region and MFCSl, we performed 3D-FISH
analysis using hapten-labeled probes that hybridize
with these two genomic regions. We dissected anterior,intermediate and posterior portions of developing limb
buds at EI0.5 embryos. The posterior portion of limb
bud is referred to as zone of polarizing activity (ZPA)
and it expresses Shh exclusively in wild-type mice. We
immediately prepared single cell suspension from theeach dissected specimen, and used for the FISH
analysis. After capturing 3D images of the two
fluorescent signals, physical distance between the Shhcoding region and MFCSI was computationallycalculated. This result clearly indicated that the mean
distance in the posterior limb bud cells was
significantly shorter than that in the intermediate limb
bud cells, suggesting that MFCSI tends to approach
the Shh locus in the Shh-expressing cells. Notably, themean Shh-MFCSI distance in the anterior limb budcells was somewhat smaller than that in the posteriorlimb bud cells, but the value was close to that in theposterior limb bud cells. It is known that many mousemutants exhibit ectopic Shh expression at the anterior
margin of limb buds, and it leads to preaxial
polydactyly. It is, therefore, conceivable that theanterior limb bud cell potentially has a competence toexpress Shh. Thus, adjoining of physically distant two
regions, MFCSI and the Shh coding, implies that thecells have a competence for expressing Shh.
To further investigate the chromatin dynamism
surrounding the Shh coding and MFCSI regions, we
adopted a method of chromosome conformation
capture (3C). Limb buds from E1O.5 embryos, which
express Shh at very high level, were used for the
analysis. We used limb buds from E12.5 embryo, in
which Shh expression already ceased, as a control. As
a result, we captured an expected positive signal onlyin the limb buds from E1O.5 embryos, suggesting that
MFCSI physically interacts with the Shh coding regionin Shh-expressing cells.
(3) Molecular mechanisms of epithelium formation ofskin and gastrointestinal tract in mice

Masaru Tamura, Shigekazu Tanaka, Tomoaki Fujii,
Hiromitsu Komiyama! and Toshihiko Shiroishi
CGraduate School of Medicine, Juntendo University)
Epithelial cells are tightly connected to one anotherin sheets and tubes, and form epidermis and mucosal
epithelium of gastrointestinal tracts. Homeostasis of
the epithelial linings is controlled by the balance
between proliferation of the stem cells and apoptosis
following terminal differentiation. Disruption of thisbalance may cause abnormal cell proliferation andseveral diseases like as cancers. However, ourknowledge on the molecular basis underlying theepidermis development and homeostasis is still limited.
We have overcome this problem using the forward andreverse genetics approaches. We performed threefundamental studies: (i) Screening and phenotype
analysis of mutant mice, which exhibit abnormal
epidermal development; (ii) identification of causative
gene of these mutant mice; (iii) analysis of these genefunctions using the transgenic and gene knockout
technique.
We identify five novel genes during the analysis
of just one mutant mouse. Four of five these genes are
clustering in the mouse chromosome 15, and thesegenes have several conserved sequences and novel
functional motifs. Each of these genes is expressed in
the epidermis and gastrointestinal tract (GI) in a tissue
specific manner, and expression patterns and levels
were depended heavily on proximal-distal axis in the
GI tract. Weare now examining the function of thesegenes using the reverse genetics approach.
Transgenesis with a modified-BAC DNA harboring
the Rim3-type GsdmA3mutation recaptures the mutant
skin phenotype(4)

Shigekazu Tanaka, Masaru Tamura, Aya AokP,
Tomoaki Fujii, Hiromitsu Komiyama2, Toshihiko
Shiroishi CDepartment of Neurosurgery, Tokyo
Metropolitan Hiroo Hospital 2Graduate School of
Medicine, Juntendo University)
In our previous study, we showed that aspontaneous dominant skin mutant, Rim3, has a point
mutation in Gasdermin A3 (GsdmA3). The Rim3
mutation has a single base substitution, 1124 G to A,
which results in a missense change, alanine 348
threonine. To confirm that the genetic alteration foundin the GsdmA3gene causes the mutant phenotype, inthis study we carried out transgenesis using amodified-BAC DNA containing the Rim3-type GsdmA3
mutation (alanine 348 threonine). A BAC clone
RP23-438D7 contains 160 kb genomic region that

Mammalian Genetics Laboratory59

includes GsdmA cluster except for GsdmAl and most
likely contains the promoter and the cis-regulatory
elements of GsdmA3gene within its 115 kb upstream
fragment of GsdmA3. We introduced the Rim3-type
mutation into the exon 9 of GsdmA3 in the RP23-438D7
clone by means of BAC modification technology. The
modified BAC construct was transferred into genomeof wild-type (DBN2J x C57BLl6J)F, mice. We selectedtransgenic mice by genotyping for the vector sequence,and established three transgenic founder lines carrying
the Rim3-type GsdmA3mutation. We found that these
all three lines exhibit skin phenotype similar to thatof Rim3. Histological analysis revealed that thetransgenic mice have degenerated hair follicles at 10
months of age as was observed in Rim3mutant mice.
In contrast, transgenic mice that express wild-type
GsdmA3 did not exhibited any phenotype in the skin.
The result clearly demonstrated that GsdmA3 is thecausative gene for Rim3.
(5) Disruption of genetic interaction between two
autosomal regions and the X chromosome causes hybrid
breakdown in a mouse intersubspecific cross
Ayako aka, Toshihiro Aoto', Yoshikazu Totsuka',
Riichi Takahashi', Masatsugu Ueda', Akihiko Mita,
Noriko Sakurai-Yamatani, Hiromi Yamamoto Nobuo
Takage, Kazuo Moriwaki3 and Toshihiko Shiroishi
CThe YS Institute, Inc., 2Hokusei Gakuen University,
3RIKEN BioResource Center)
Reproductive isolation that initiates speciation is
likely caused by incompatibility between multiple lociin organisms belonging to genetically diverging
populations. Laboratory C57BLl6J mice, which
predominantly originated from Mus musculus
domesticus, and a MSM/Ms strain derived fromJapanese wild mice (M m. molossinus) are reproductively isolated. These two strains exhibit hybrid
breakdown: F, hybrids are fertile, but succeeding
intercrosses result in male sterility. A consomic strain,
C57BLl6J-ChrXMsM , which carries the X chromosomeof MSMIMs in the C57BLl6J background, shows malesterility, suggesting genetic incompatibility of the
MSM/Ms X chromosome and other C57BLl6J
chromosome(s). In this study, genome-wide linkage
analysis and subsequent QTL mapping successfully
detected significant QTLs on chromosomes 1 and 11
that interact with the X chromosome and are

60

Mammalian Genetics Laboratory

responsible for the hybrid breakdown. This was
confirmed by the observation that introduction of the
MSM/Ms chromosomes 1 and 11 into the C57BLl6JChrXMsM background partially restored fertility. Thus,
genetic interactions between the two autosomal regions
and the X chromosome have a crucial role in proper
sperm differentiation, and their disruption causes
hybrid breakdown. Intracytoplasmic sperm injection
and zona-free in vitro fertilization showed that the
C57BLl6J _ChrX MsM spermatozoa have a defect inpenetration of the extracellular zona pellucida.
Moreover, two-dimensional electrophoretic proteome
study of whole proteins from sperms provided
considerable candidates responsible for the sterilityof C57BLl6J-ChrXMsM males.
(6) QTL analysis of hereditary predisposition of obesity
based on inter-subspecific cross of C57BL/6J and MSM/
Ms strains
Akiteru Maeno, Hideko Watanabe, Akihiko Mita,
Ayako aka and Toshihiko Shiroishi
Obesity is a genetically complex trait, and morethan hundreds of potential loci have been identified
so far. In order to elucidate the genetic determinantsof this disease, we are taking approach to use mousemodels for heritable obesity. In this study, we
conducted genetic analysis of obesity-related
quantitative traits based on the cross between astandard laboratory mouse strain C57BLl6J (B6) anda Japanese wild mouse-derived strain MSMIMs (MSM).
The quantitative traits that we analyzed in this study
are followings: body weight, body weight that excludes
total fat pads weights (lean body weight), percentageof the weight of each of fat pad surrounding threevisceral tissues (gonad, retroperitoneum andmesenterium) to lean body weight, percentage of thetotal weight of the above three visceral fad pads to leanbody weight, percentage of total subcutaneous fad
pads weight to lean body weight and total fat pads
weights to lean body weight (adiposity index: A.I.).
MSM has higher A.I. value than that of B6 at ten
weeks of age, and this tendency became more
prominent as age proceeded. This suggests that MSM
shows higher predisposition to obesity as comparedwith B6. For the quantitative trait loci (QTL) analysis,
we crossed B6 and MSM mice and generated FI
hybirds, and subsequently intercrossed the F, mice andobtained totally 272 F2 progeny. Then, we carried out
QTL analysis with the F2 progeny at 10 weeks of age
to identify loci responsible for eight obesity-related
traits described above. First, single-locus genome scans
analysis identified four QTLs on Chr 2, 6, 9 and 13, allof which control A.I. value. All these QTLs showed
sex-dependent effect. For the three QTLs on Chr 6, 9
and 13, MSM alleles increase A.I. value, while B6 allele
increases the A.I. value for the QTL on Chr 2. Because
all four QTLs explain relatively small proportion of
the variance observed in the F2 progeny (only 9 to 11%of the total variance), many other minor QTLs andepistatic interaction of two independent loci were
expected to be contributed to the remaining genetic
variance. Therefore, we implemented pairwise genome
scan analysis for the same data set of the 272 F2progeny. As a result, we identified a significant
epistatic interaction between two loci on Chr 9 and 13.
QTL analyses with Multiple Interval Mapping(MIM) and Bayesian Interval Mapping (ElM) arethought to be more suitable for the cases in which
multiple QTLs are responsible for complex traits. We
need further studies using the above analyses to reveal
hidden QTLs that were not detected in this study.
Genetic dissection of obesity using mouse consomic
strains(7)

Toyoyuki Takada, Akiteru Maeno and Toshihiko
Shiroishi
Obesity is a common disease and a major risk todevelop type 2 diabetes mellitus and metabolic
syndrome. Genetic obesity is generally thought to be
multifactorial disease, which is caused by multiple
genetic determinants and influence of environmental
factors. To understand pathology of obesity, genetic
analysis of responsible genes for this complex trait has
been prominent issue as in many common humandiseases. Weare taking approach to use mouse models
for obesity. C57BL/6J-Ch~sM consomic strains wereconstructed by transferring individual chromosomesfrom MSM/Ms strain, derived from Japanese wild
mouse, Mus musuculs molossinus, into the genetic
background of a standard laboratory strain C57BLI
6J, which was derived from West European wild
mouse, Mm. domesticus. These consomic strains are
strikingly useful for chromosome mapping of various
phenotypes, because above two mouse strains showed

extremely large extent of phenotypic variations basedon vast amount of genomic differences (,,-, 1% SNPs).
Our preliminary phenotyping of the consomic strains
indicated a variety of range in susceptibility or
resistance to obesity or obesity-related phenotypes
among the strains. Based upon the strain difference,
we intend to explore genes and genetic pathways
underlying the polygenic obesity. At the first step, now
we are conducting more detailed characterization of
the phenotype of each consomic strain using following
tests: 1) measurement of consumption of foods (normal
chaw and high fat diet, etc.) and body weight, 2)
detection of metabolism-related parameters (activity,
body fat content, blood and hepatic lipid content,
plasma insulin and leptin levels, etc), 3) radiographicalmeasurement of adipose tissue mass using computer
tomography (CT) scanning, 4) transcriptome analysisof metabolism-related tissue of all consomic strains.
PUBLICATIONSPapers

1. Ogasawara, M., Imanishi, T., Moriwaki, K.,
Gaudieri, S., Tsuda, H., Hashimoto, H., Shiroishi, T.,
Gojobori, T., Koide, T.(2005)Length variation of CAGICAA triplet repeats in 50 genes among 16 inbred mousestrains, Gene, 349, 109 - 117
2. Sagai, T., Hosoya, M., Mizushina, Y, Tamura, M.,
Shiroishi, T. (2005) Elimination of a long-range
cis-regulatory module causes complete loss oflimb-specific Shh expression and truncation of themouse limb, Development, 132 (4) 797 - 803
3. Sakai, T., Kikkawa, Y., Miura, I., Inoue, T.,
Moriwaki, K., Shiroishi, T., Satta, Y., Yonekawa, H.
(2005) Origins of mouse inbred strains deduced fromwhole-genome scanning by polymorphic microsatellite
loci, Mammal. Genome, 16 (1) 11 - 19
4. Taguchi, Y, Koide, T., Shiroishi, T., Yagi, T. (2005)
Molecular evolution of cadherin-related neuronal
receptor/protocadherin (alpha) (CNR/Pcdh (alpha))gene cluster in Mus musculus subspecies, Mol Bioi
Evol, 22 (6) 1433 - 1443

5. :j]jn:l{~~(2005)iI'Ii{~O~it(l)JI~~!i!i""'?? 7,(l)!T /.b.*,~n~G1*itOO~JI~r~~Q, msli • ~i'Ht • ~~,50 (16) 2184 - 2190

Mammalian Genetics Laboratory

61

SOCIAL CONTRIBUTIONS AND OTHERS~~5lSi1J:lJiX:£" {~~ J!fi;~~{tjjl!j.~~$:lJiX:£" {~~ '!1if~JW~~~~~~ j'i:lJiX:£" {~~ Editorial boad of Mammalian Genome:lJiX:£" {~~ Editor of Gene & Genetic Systems~{~~"The Scientific World" Associate editor for

Geneticsof O){tB:lJiX:£"~~

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jjI!j~ffil~~ffil:lJiX:£"{~~(M)JIIff:R1itN~~~~~mvertebrate embryos. Mesp1 and Mesp2 arehomologous bHLH transcription factors that areco-expressed in the anterior presomitic mesoderm(PSM) just prior to somite formation. Analysis ofpossible functional redundancy of Mesp1 and Mesp2
has been prevented by the early developmental arrestof Mesp1/Mesp2 double-null embryos. We performed
chimera analysis using either Mesp2-null cells or
Mesp1/Mesp2 double-null cells, to clarify (1) possiblefunctional redundancy and the relative contributionsof both Mesp1 and Mesp2 in somitogenesis and (2) thecell autonomy of Mesp functions in several aspects ofsomitogenesis. Both Mesp2-null and Mesp1/Mesp2
double-null cells fail to form initial segment borders
and to acquire rostral properties, confirming that thecontribution of Mesp1 is trivial in these aspects. In
contrast, Mesp1/Mesp2 double-null cells contribute toneither epithelial somite nor dermomyotome formation
while Mesp2-null cells partially contribute toincomplete somites and dermomyotome. This indicatesthat Mesp1 has a significant role in the epithelializationof somitic mesoderm (Takahashi et al. 2005).
in

fJI!$

F-b. Mammalian Development Laboratory
Yumiko Saga Group(2) Regulation of Mespl and Mesp2 expression
Masayuki Oginuma, and Yumiko SagaRESEARCH ACTIVITIES(1) Molecular mechanism of somite segmentation
Mitsuru Morimoto, Makoto Kiso and Yumiko Saga
The somite is the first morphologically distinct
segmental unit formed in a vertebrate embryo and giverise to metameric structures such as vertebrae, ribs andskeletal muscles. A 'clock and wavefront' model has
been proposed to explain the underlying mechanism,
in which the periodicity is generated by a 'clock' in theposterior PSM and this temporal periodicity is thentranslated into the segmental units in the 'wavefront'.
The wavefront is thought to exist in the anterior PSM
and progress backwards at a constant rate. Mesp2
plays a crucial role in translation of the temporaloscillation of Notch activity into the regularly-spaced
somite (Morimoto et al. 2005).
Somitogenesis is not only an attractive example
of metameric pattern formation but is also a goodmodel system for studies of morphogenesis,
particularly epithelial-mesenchymal interconversion

62

Mammalian Genetics Laboratory

Mespl and Mesp2encoding bHLH-type transcription factors Mesp1 and Mesp2, locate close to eachother on chromosome 7. The both genes areco-expressed in the anterior presomitic mesoderm(PSM) just prior to somite formation. In our previous
study, we identified the enhancer for the PSM in the
185bp upstream region of Mesp2, and called it theP2PSME (Mesp2 PSM enhancer). To identify global
transcriptional regulation of both Mespl and Mesp2,
we conducted enhancer analyses using BAC transgenic
system. Using BAC recombination system, we first
constructed a BAC-P1AP-P2Ztransgenic vector that
has inserted Alp gene in Mespllocus and inserted nlacZ
gene in Mesp2locus. BAC-P1AP-P2Ztransgenic mouseshowed similar expression pattern to that ofendogenous Mespl and Mesp2. Next, we constructed
the BAC-P1AP-P2Z6. (P2PSME) vector which lacked
P2PSMEfrom the BAC-P1AP-P2Zvector. Transgenic(TG) embryos that have BAC-P1AP-P2Z6. (P2PSME)
did not express Mesp2 in the PSM but the Mespl
expression was intact. We also identified the enhancer
for the PSM in the 4kb upstream region of Mespl, andcalled it the P1PSME (Mespl PSM enhancer). To know
the enhancer specificity, we constructed a BACP1AP-P2ZtJ. (P1PSME) which lacked P1PSMEfrom
the BAC-P1AP-P2Zvector. TG embryos that have
BAC-P1APP2ZIJ. (P1PSME) did not express Mespl in
the PSM but the Mesp2 expression was intact. These
results indic~te that Mespl and Mesp2 use different
enhancers for the expression in the PSM. By thecomparative analysis of these enhancer sequences, we
identified conserved motives for several transcription
factors.
(3) Search for target genes of Mesp2 transcription factor

Yoshiro Nakajima and Yumiko Saga
Somites provide the basic body plan for metamericaxial structures in vertebrates and establish thesegmental features thorough the sequential gene
expression in the presomitic mesoderm (PSM). A
critical gene for segment border formation is the bHLH
transcription factor Mesp2, the expression of which is
restricted to the anterior PSM. A gene candidate thatis activated by Mesp2 is EphA4, since its expression
pattern resembles Mesp2 and is absent in Mesp2-nullembryos. We have analyzed the enhancer region of
EphA4, which is responsible for its expression in theanterior PSM, and identified an E-box containing
region. Subsequent transgenic and transient luciferase
analyses successfully determined that the presence ofrepeated E-box sequences is a minimum essential
requirement for faithful EphA4 expression. We also
show that Mesp2 directly binds the enhancer sequenceof EphA4. Furthermore, the forced expression of Mesp2
in somitic cells results in the activation of EPhA4 andrepression of the caudal gene Uncx4.1, which may
trigger the events leading to the formation of abnormal
somites and rostralized vertebra. In addition, ectopic
Mesp2 expression induces abnormally epithelialized
structures, which support to the idea that Mesp2
induces the formation of segmental borders byactivating genes, which play roles in cellularepithelialization.
(4) Functional analyses of Hesrl and Hesr2 in the
cardiovascular development

Hiroki Kokubo, Raul VizcardoSakoda, and Yumiko
Saga

Establishment of atrial and ventricular chamber
specificity is initial requirement for complex cardiac
morphogenesis and function of the heart. We haveshown the hesrl and hesr2 genes, which encode the
Hairy and enhance 0/ split (HesJ-related bHLH
transcription repressors, are required for the normal
cardiovascular development (Kokubo et al. 2(05). They
are expressed specifically in the atria or the ventricle,
implicating to function in the chamber specification.
We found that the forced expression of Hesrl or Hesr2
in the entire cardiac lineage resulted in the reduction
or loss of the atrioventricular (AV) canal. In the
Hesrl-activated embryos, gene expressions
demarcating boundaries between chambers and AV
canal appeared to be vague on either side of atria andventricle, and the expression of specific marker for AV
myocardium, Bmp2 or Tbx2, was weakened orundetected. Similarly in the Hesr2-activated embryos,
gene expression revealed as like that the atrium andventricle seems to be directly connected, and neither
expression of Bmp2nor Tbx2was detected in the AV
canal region. These data suggest that Hesrl and Hesr2
are involved in the AV boundary formation.
(5) Analyses toward understanding the function ofProtein O-fucosyltransferasel in the Notch signaling

Yoshiaki Okamura, and Yumiko Saga
Notch signaling is an evolutionarily conserved
signaling pathway, which plays key roles in regulatingcell fate decisions and morphogenesis. The
glycosylation of Notch receptor, especiallyO-fucosylation by Protein O-fucosyitransferasel(Po/utl), is indispensable for signal activation.
However, it remains unclear how the Pofutl acts on
Notch signaling in mouse embryogenesis. Using
conditional strategy in the mouse, we could delete
Po/utl alleles specifically in cardiovascular system,
which showed the defects in the cardiogenesis
including valve formation. Moreover, we have alreadyshown that the forced expression of the activatedNotchl (actNI) in cardiovascular system causes the
myocardial defect. Taking advantage of this system,
we have examined the epistasis between Pofutl and
actNI on Notch signaling. Both in the control and
Pofutl mutant embryos with forced expression of
actNI, the myocardial defect was observed. This result
suggests that the Pofutl acts upstream of the actNI.

Mammalian Development Laboratory63

(6) .Analyses of nanso2 expression and function during
mouse germ cell development
Atushi Suzuki, Kaoru Mitsui, and Yumiko Saganever seen in nanos;rl-male mice. The adultnanos;rl- bax-1-female mice had ovaries whichcontained morphologically normal oocytes although
the number was considerably low. In the nanos3-bax-1-embryo, a few PGCs reached gonads and weremaintained until adulthood. These observations
suggest that one reason for PGC loss in nanos;rl-miceis BAX-dependent apoptosis but nanos3 is responsible
for the other pathway to maintain germ cells.

Mouse nanos proteins, nanos2 and nanos3 arerequired for germ cell development and both proteinsshare highly conserved zinc finger domain. However,
the expression pattern during development is different
each other. nanos3 expression starts in the primordial
germ cell (PGC)s just after their formation and the lackof this protein results in germ cell-less phenotype inboth sexes. However, nanos2 expression starts in themale PGC only after entering to the genital ridge andthe lack of nanos2 results in the lack of male germ cells
irrespective of the co-expression of nanos3. These
results indicate that these proteins have distinct
function depending on the time and place of theirexpression. We addressed the question by producing
transgenic mouse lines which express nanos2 under
the control of Oct4 ~ PE promoter and examined thenanos2 function in the nanos3-null genetic background.
The ectopically produced nanos2 protein rescued thelack of nanos3 and germ cells developed in both sexes.
This result indicates that the nanos2 can substitutenanos3 function in the early PGC development.

RL. (2005) Mouse hesrl and hesr2 genes are
redundantly required to mediate Notch signaling in thedeveloping cardiovascular system, Dev. BioI., 278,301 309
2. Morimoto M, Takahashi Y, Endo M, Saga Y (2005)
The transcription factor Mesp2 establishes segmental
borders by suppressing Notch activity., Nature, 435,
354 - 359
3. Takahashi, Y, Kitajima S, Inoue T, Kanno,]., Saga
Y (2005) Differential contributions of Mespl and Mesp2
to the epithelialization and rostrocaudal patterning ofsomites., Development, 132, 787 - 796(7) Functional analysis of nanos3

Books

Hitomi Suzuki, Makoto Kiso and Yumiko Saga

4. ·Hr.rfHfiH· (2005) Notch'/7"TJv<1)~~'~,~~
12["¥: (mflJ) , r~~. 5Ht . ¥}~liJf9E2005J, 23, 64 - 72

nanos3 is one of nanos genes essential for germ cell
development. nanos3 expression starts in PGCs
(primordial germ cells) just after their formation andcontinues during migration until reaching the gonads.
In the nanos3-null mice, the number of PGCs rapidly
decreased during their migration, indicating thatnanos3 is responsible for the maintenance of PGCs
rather than their generation. There are two possibilities
for the loss of PGCs, apoptotic cell death or abnormal
differentiation to somatic cells. By the immunostaining,
we detected apoptosis in migrating PGCs of nanos;rlmice, which prompted us to rescue the loss of PGC by
preventing apoptosis in PGCs. We have then generated
double knockout mice of nanos3 and a pro-apoptotic
factor Bax that is known to be involved in the germ
cell development. Although adult nanos;rl- bax-1-malemice were sterile and showed the similar phenotype
to bax-1-male mice, they had germ cells that were64

Mammalian Development Laboratory

PUBLICATIONSPapers

1. Kokubo H, Miyagawa-Tomita S, Saga Y, Johnson

F -C. Mouse Genomics Resource Laboratory (MGRL)
Tsuyoshi Koide.GroupRESEARCH ACTIVITIES(1) A systematic analysis of genetic factors associatedwith the spontaneous activity in mice
Akinori Nishi, Toshihiko Shiroishi! and Tsuyoshi
Koide C Mammalian Genetics Laboratory)
A great variation of behavioral pattern is observedin a variety of animals. A number of studies indicatedthat the behavioral diversity is greatly influenced bygenetic factors. The aim of our study is to identify thegenetic factors associate with the diversity of
behavioral traits.

In order to study genetic mechanism associate withthe behavioral diversity, we are using mouse strains,
C57BLl6, which is mostly derived from Mus musculus
domesticus and MSM, which belongs to M m.
molossinus, and the consomic strains. The consomic
strains were established by replacing eachchromosome of C57BLl6 by corresponding
chromosome of MSM. These strains are expected as
powerful tools for investigating complex traits, which
are influenced by multiple genetic factors. In the resultof systematic test battery on the consomic strains, wefound that B6-6CMSM consomic strain, which carries
MSM-derived chromosomal segment from D6Mit166
to D6Mit12 on chromosome six, exhibited significantly
lower activity in the home cage test and the novel home
cage test, and indicated higher anxiety related behavior
in the light/dark box test comparing to C57BLl6.
To identify the quantitative trait loci (QTLs)
associated with the behavioral differences between
C57BLl6 and B6-6CMSM, I performed the QTL
analysis by use of the F2 population establishedbetween these two strains. In the result of QTL
analysis, I detected the several QTLs. The QTL locatedon centromere side mainly associated with generalactivity and emotionality related traits and the QTL
located on telomere side associated with generalactivity.

characters. Such as, B6-3MSM and B6-9MSM showed
higher locomotion in the open-field than B6, andB6-3MSM exhibited jumping behavior andcharacterized by vertical movement while B6-9MSM
never showed jumping and tend to avoid central partof the open-field. This result suggested that many
genetic components that have variety effect associatedto anxiety-related behavior. Also, we performed social
interaction test in those consomic mouse strains, andfound some chromosomes associated with social oraggressive behavior.
One strain B6-17MSM, which have MSM derived
chromosome 17, showed reduced locomotion in thenovel situation, increased fear responses, andrisk-assessment behavior, and thus it is thought that
there is a locus/loci related to the emotionality. To
identify the gene/genes on the chromosome 17, we are
currently making subconsomic strains that havenarrower part of chromosome 17 from MSM. By
analyzing some of those subconsomic strains, it has
been suggested that a locus/loci is in the telomericregion.
(3) Genetic factors responsible for difference inspontaneous home-cage activity between KJR and
C57BL/6J

Juzouh Umemori and Tsuyoshi Koide(2) Genetic analyses of psychological traits using
consomic mouse strains of C57BL/6 and MSM

Aki Takahashi, Akinori Nishi, Toshihiko Shiroishi\
Tsuyoshi Koide eMammalian Genetics Laboratory)
It is thought that genetic variation that underlies
emotionality is complex and is regulated by loci thathave quantitative effect on the phenotype. We havebeen examining to reveal those genetic mechanisms
by using B6-MSM consomic mouse strains. It is known
that the laboratory strain C57BLl6 (B6) and thewild-derived strain MSM have a wide genetic diversity
and behavioral differences. B6-MSM consomic strains
have same genetic background as B6 except for one
chromosome from MSM. By examining bunch of
consomic strains, it was found that over a half ofconsomic strains changed anxiety-related behavior
from B6. Detailed observations of behavior in the
open-field, assumed as anxiety-evoked situation,
revealed that some of consomic strains have specific

It has been previously reported that mice move
spontaneously in the habituated home-cage according
to a light/dark rhythm, and that most mice are active
during the dark period. This kind of activity is called
as spontaneous home-cage activity. In a variety ofstrains, mice of C57BLl6J were relatively hypoactive,
in contrast to mice of KJR those were especially
hyperactive in the habituated home-cage. We studied
the spontaneous home-cage activity in terms of theethological comRonents and biological functions.
F2 progeny (BKF2) were made between C57BLl6J
and KJR strains to be analyzed for the ethological andgenetic studies. Ethological studies showed that total
home-cage activity (THA) could be divided to two
independent components, active time (AT) thatindicated temporal elements of spontaneous home-cage
activity, and average activity (AA) that indicatedquantitative elements. A little correlation between AT
and AA suggested that AA and AT are independentmeasurement.

Mouse Genomics Resource Laboratory65In order to study the genetic bases responsible forthe differences in spontaneous locomotive activity
between K]R and C57BL/61, we conducted quantitative
trait loci (QTL) analysis using BKF2 progeny. QTL
analysis showed three significant QTLs associatedwith the spontaneous home-cage activity. These QTLs
were designated as hyperlocomotive activity related
QTLI (Hylaql), Hylaq2 and Hylaq3. All Hylaq locicontrolled THA that was defined by summing up
effects of the AT and the AA Hylaql was located ina middle region of chr2 and mainly controlled activity
time. Hylaq2was located on the distal side of Hylaql
on chr2 and controlled both of AT and AA. Hyaq3was
located near the telomeric region on chr10, andcontrolled mainly AA In order to analyze the single
gene effect more precisely, we are currently making
congenic strains that carry KJR allele at Hylaq loci in
BLG2 background. They will be used to conduct
further fine mapping and phenotypic analyses. We
have currently made N5 generation of the congenic
strains.
It has been reported that dopamine (DA) system
in basal ganglia in brain is associated with movement
and spontaneous locomotive activity. To search forcandidate genes in the above QTL region, and tounderstand the biological mechanism that is associatedwith the spontaneous home-cage activity, straincomparative analyses on the DA system have been
conducted with pharmacological and biochemical
methods. The pharmacological study suggested thatthe function of D1-like DA pathway might be reducedin K]R while that of D2-like pathway might be highly
suppressive for the activity only in C57BLl6J.
Expression analyses and microdialysis analyses
indicated that there was no significant difference inDA synthesis, release and uptake in presynaptic
dopaminergic neuron in striatum. These results
suggested that C57BLl6] and KJR have difference inthe downstream pathways following activation of the
DA receptors.
PUBLICATIONSPapers

1. Esumi, S., Kakazu, N., Taguchi, Y, Hirayama, T,
Sasaki, A, Hirabayashi, T, Koide, T., Kitsukawa, T,
Hamada, S., Yagi, T. (2005) Monoallelic yet
combinatorial expression of variable exons of the
protocadherin-a gene cluster in single neurons., Nature

66

Mouse Genomics Resource Laboratory

Genetics, 37, 171 - 176
2. Ogasawara, M., Imanishi, T., Moriwaki, K.,
Gaudieri, S., Tsuda, H., Hashimoto, H., Shiroishi, T.,
Gojobori, T, Koide, T. (2005) Length variation of CAG/
CAA triplet repeats in 50 genes among 16 inbred mousestrains., Gene, 349, 107 - 119
3. Taguchi, Y, Koide, T, Shiroishi, T, Yagi, T (2005)
Molecular evolution of Cadherin-related neuronal
receptor / protocadherin a (CNRlPcdha) gene clusterin Mus musculus subspecies., Molecular Biology and
Evolution, 22, 1433 - 1443
EDUCATION

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2. Koide, T. System genetics: Toward identificationof genetic factors for behavioral diversity in mice
University of Strasbourg, France
3. Koide, T System genetics: Toward identificationof genetic factors for behavioral diversity in mice
University of Zurich, Swiss
4. IN±lIlIJU J!f1:.Ei3*"'" t7 ArM1EO)J!~I'J"J~t~H1:~t.5ffl~kff~O)J!~~I'J"JM~~WdM~tt~*~

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F-d. Model Fish Genomics Resource
Noriyoshi Sakai GroupRESEARCH ACTIVITIES(1) Gene targeting system with RNA interference (RNAi)in zebrafish
Minori Shinya, Kimiko Saka, Kayo Kobayashi andNoriyoshi Sakai
Gene silencing via small interfering RNAs(siRNAs) has proved to be a useful tool in studying
gene function in plants, invertebrates and mammaliansystems. To date, gene silencing effects of siRNAs haveconfirmed in the zebrafish, which is an emerging modelfor developmental and diseases analysis. However, theeffects were temporal (only in early developmental
stages) and sometimes mosaic in an embryo becauseof the method injecting siRNAs into the one-cell stageof embryos. We recently succeeded in the productionof transgenic zebrafish from in vitro-cultured sperm.
The advantage of this technique is that the mosaicism
inherent in other conventional transgenic methods isavoided. Our aim in the present study is theestablishment of a rapid system with cultured sperm
to generate transgenic zebrafish for gene silencing by
siRNA.
As targeted genes, sonic hedgehog (shh), signaltransducers and activators of transcription 3 (stat3) andgreen fluorescent protein (gfp) were selected becauseof their clear silenced phenotypes. To determine thebest sequence of siRNA for the specific suppressionof each gene, we planned two kinds of assays: one is
the injection of siRNA into one-cell stage embryos andthe other is the transfection of siRNA to the zebrafish
cultured cells. In the first assay, we tested four
sequences of siRNA for shh, two for stat3 and one forgfp. Northern blot analyses of the siRNA-injected
embryos revealed that one siRNA for shh (sishh) and
one for gfP (sigfp) suppressed each transcript
efficiently. However, sishh-injected embryos showed
not only the shh null mutant phenotype but also themalformed development and cell death. Further
analyses e.g. precise dosage test, are required. In
contrast, sigfp-injected embryos were developed
normally. In addition, sigfp injection gave us an insightin the effective time of siRNA in zebrafish embryos.

Northern blot analysis of sigfp-injected embryos inevery two hours revealed that sigfp suppressed thetranscript only for 2-4 hours. The result suggests thatstable expression of a short hairpin RNA(shRNA)
would be required for efficient and constant genesilencing in zebrafish. Weare currently working on theconstruction of a plasmid DNA encoding a shRNA
which is expected to generate sigfp in vivo throughprocessing by Dicer, double strand RNA-specificendonuclease. Using retroviral system and the shRNA
construct, we will produce the cultured sperm withforeign DNA encoding the shRNA.
The second assay, the transfection of siRNA to thezebrafish cultured cells, is easier and faster than theinjection to embryos. Thus, the establishment of thisassay will be a great help to build a high-throughput
system for genome-wide loss-of-function studies. For
the assay, we established the zebrafish cultured cells
expressing shh and stat3, and are trying to isolate thecell line expressing GFP stably. As there was no
method for the efficient transfection to the zebrafish
cultured cells (less than 1% transfection efficiency),
several known transfection methods were tested invarious condition. So far, we found to achieve 30-40%
introduction of foreign DNA into the cells by someelectroporation protocols.
(2) Analysis of functionally distinctive testicular cell linesof zebrafish to support male germ cell development
Aki Masuda and Noriyoshi Sakai
Sertoli cells are important to germ cells ineverything from male sex-determination tospermatogenesis. In spermatogenesis, Sertoli cells
interact directly with germ cells in the testis to induce
the complex process required for the production offunctional sperm. These cells mediate the productionof many molecules as well as cell junctions andadhesion. The function of many of these molecules andthe regulation of gene expression remain unclear. We
recently established two testicular cell lines of zebrafish
with distinct functions to support the development ofmale germ cells.
The lines, ZtA6-2 and ZtA6-12, showed almost thesame characteristics as Sertoli cells, but exhibited
distinctive features when male germ cells were
co-cultured with each line as feeders: the function of
the ZtA6-2 cells was directed to stimulate theModel Fish Genomics Resource67

proliferation of spermatogonia, and ZtA6-12 tostimulate the differentiation into sperm.
These cell lines facilitate investigation of Sertoli
cell molecules that contribute to the proliferation anddifferentiation of spermatogonia. The micro array
analysis for approximately 20,000 genes showed that45 genes were expressed 3 times higher in ZtA6-2 cells
than ZtA6-12 cells, and that 156 genes in ZtA6-12 cells
than ZtA6-2 cells. Interestingly, several steroid-related
genes were found in up-regulated genes of ZtA6-12
cells. In a zebrafish testis, Sertoli cells constitute a cyst
structure in which single A-type spermatogonium
develops and differentiates synchronously. This
characteristic makes it easy to determine the stagesof Sertoli cells corresponding to the stages of germ
cells. To investigate the stage specific expression ofthese genes in Sertoli cells, we developed a protocol
for frozen sections of a zebrafish testis. The section
kept the testicular structure enough to determine eachdevelopmental stage of germ cells. In situ hybridizationof the profiled cDNAs to the frozen sections is underinvestigation.
(3) Culture condition for zebrafish spermatogonial stem
cells

testes on day 7, the germ cells proliferated on newly
isolated Sertoli cell line, ZtA6-6. Morphology of thegerm cells resembles to the SSC of paraffin section,and did not change during the culture. Furthermore,
the cells expressed a germ cell marker gene vasa, andun-synchronous proliferation of the germ cells wasobserved by BrdU incorporation experiments. These
results indicate that SSCs of zebrafish proliferate underthese culture conditions. Growth of the SSCs did notseem to recover the amount of lost cells at re-plating
every 7 days, however, we were not able to maintainthe SSCs more than 1 month. Towards better culture
conditions, we are now investigating the effect ofgrowth factors on the proliferation of zebrafish SSCs,
such as glial cell line-derived neurotrophic factor(GDNF) which is an essential growth factor for mouse
SSCs, basic fibroblast growth factor (bFGF), epidermal
growth factor (EGF), and insulin-like growth factor-l(IGF-l).
(4) Analysis of the ability of cultured embryonic cells
derived from different developmental stages to induce
the anterior-posterior axis.
Megumi Hashiguchi, Minori Shinya and Noriyoshi
Sakai

Kenji Saito and Noriyoshi Sakai
In zebrafish, a culture system using primarycultures of male germ cells on a Sertoli cell line, inwhich the differentiation from spermatogonia tofunctional sperms can occur in vitro, allows us togenerate transgenic zebrafish lines rapidly through asimple in vitro fertilization using transfected sperm.
However, the proliferation of spermatogonial stem cells(SSCs) was not observed under the culture conditions.
Here, we studied the enrichment procedure of SSCs inzebrafish testis by in vivo treatment with busulfan
reagent, and a culture condition to proliferate SSCs
with newly isolated Sertoli cell line as feeder cells.
When zebrafish were soaked in busulfan solution
for 24 hr, we observed decline in the amount ofdifferentiating germ cells, such as spermatocytes andspermatids, and increase of SSCs on day 4 after
treatment. Germ cells in the testis consisted of only
SSCs and sperm on day 7. After 14 days, type B
spermatogonia appeared, and after 1 month the testis
became normal and the fish became fertile. When weused enzymatically dissociated cells of busulfan-treated68

Model Fish Genomics Resource

One of the most fascinating phenomena in primary
embryonic induction is the regional specificity of theneural structures that are produced. Primary
embryonic induction can be divided into three major
components: head specific, trunk specific and tailspecific induction.
We established the culture condition for zebrafish
embryonic cells that continuously proliferate without
any artificial immortalization treatment. When
cultured cells were implanted into a blastula embryo(3.5 hours post fertilization, hpf), the cells from different
developmental stages showed different abilities to
induce specific second axes. Cells derived from theearly gastrula stage (6 hpf) induced complete anterior
structure. Cells from the pharyngula stage (embryos
segmented, 24 hpf) also induced anterior structureswith cyclopia or two eyes. However, cells from laterstages (protruding mouth larva, 72 hpf, and swimminglarva, 120 hpf) induced posterior structure with otic
vesicles and a heart. Interestingly, cells from laterstages had low induction efficiency, but the efficiency
increased when proliferation of the cells was arrested

by mitomycin C. Whole-mount in situ hybridizationfor emx-1 (telencephalon marker), krox 20 (rhombomere
marker), and shh (notochord marker) indicatedappropriate gene expression in the observed specificstructure of induction. Cultured cells derived fromembryos at various developmental stages, therefore,change their properties to induce the second axis froman anterior to a posterior orientation according to theirdevelopmental stage. In addition, cells may secrete
more inducer(s) when proliferation is stopped,
particularly in later stages.
Following four reasons indicate that these
induction of the secondary axis results from inductionof organizer. (1) A organizer marker, goosecoid wasectopically expressed around the cultured cells at theearly gastrula stage. (2) Cultured cells formed clumps
without differentiation, while notochord was induced.
(3) Growth-arrested cells by mitomycin C still have theinduction activity. (4) When cultured cells were
implanted into an early gastrula embryo, we did notobserve the secondary axis. Implanting cultured cells
into maternal-zygotic one-eyed pinhead mutants that
defect Nodal co-receptor revealed that secondary axis
was induced without Nodal signaling. These cultured
cells would be used to find new cellular factors
involved in induction of organizer.
PUBLICATIONSPapers
1. Kimura, T., Yoshida, K., Shimada, A., lindo, T.,
Sakaizumi, M., Mitani, H., Naruse, K., Takeda, H.,
Inoko, H., Tamiya, G., Shinya, M. (2005) Genetic linkage
map of medaka with polymerase chain reaction length
polymorphisms. Gene 363, 24 - 31.
2. Tamiya, G., Shinya, M., Imanishi, T., Ikuta, T.,
Makino, S., Okamoto, K., Furugaki, K., Matsumoto, T.,
Mano, S., Ando, S., Nozaki, Y., Yukawa, W., Nakashige,R., Yamaguchi, D., Ishibashi, H., Yonekura, M.,
Nakami, Y., Takayama, S., Endo, T., Saruwatari, T.,
Yagura, M., Yoshikawa, Y., Fujimoto, K., Oka, A.,
Chiku, S., Linsen, S. E., Giphart, M. ]., Kulski, ]. K.,
Fukazawa, T., Hashimoto, H., Kimura, M., Hoshina,
Y., Suzuki, Y., Hotta, T., Mochida,]., Minezaki, T.,
Komai, K., Shiozawa, S., Taniguchi, A., Yamanaka, H.,
Kamatani, N., Gojobori, T., Bahram, S., Inoko, H. (2005)
Whole genome association study of rheumatoid
arthritis using 27 039 microsatellites. Hum. Mol. Genet.
14 (16), 2305 - 2321

EDUCATION

Sakai, N. *H!Jl§:I:fjtfI"F tljHfw.:li*"F*"F~· jc:~~~"FliJf9Ef-j.

F-e. Plant Genetics Laboratory
Nori Kurata GroupRESEARCH ACTIVITIES(1) Cytogenetic and molecular analysis of rice meiotic
mutants
Ken-ichi Nonomura, Mutsuko Nakano, Akane
Morohoshi, Mitsugu Eiguchi, Akio Miyao*, Hirohiko
Hirochika* and Nori Kurata (*Natl Inst. Agrobiol. Sci.)
This study aims to dissect genetic machinery
controlling meiosis and sporogenesis of rice (Oryza
sativa L.). We have identified four meiotic genes andtheir insertional mutants to date; PAIR1(HOMOLOGOUS EAIRING AJ3ERRA TION IN weE
MEIOSIS 1), PAIR2, PAIR3 and MEAL (MEIOSISARRESTED AT ~EPTOTENE). The PAIR1 andPAIR2 genes were found to encode a putative
DNA-binding protein and a HORMA-domain protein,respectively. Identification of PAIR3 and MEAL genes
is in progress.
The P AIR2 is an important protein to complete
synaptonemal complex (SC) formation by associatingtransiently with chromosomal axial elements (AEs) andrecruiting transverse filaments and central elements
between homologous AE pairs. Most of PAIR2 proteinsdissociate from AEs at pachytene, but small amountsof them continue to associate with centromeric regions
until nuclear envelope breakdown. We found that
non-disjunction of chromosomes was occurred inmeiocytes of pair2 mutant, but absence of P AIR2
proteins from the centromere did not affect any
centromere functions such as sister-chromatid
cohesion and kinetochore assembly in rice meiosis(Nonomura et al., ]. Cell Sci. (in press)).
The meal mutant showed an interesting phenotype
and its candidate gene was expressed in a specificpattern that have not been reported yet. In male andfemale meiocytes of the mutant, meiosis was almost
arrested at early stages around at leptotene andzygotene. In addition, the size and shape of meiocytes

Model Fish Genomics Resource69became irregular, indicating that the meal mutation
affected not only meiosis progression but alsopre-meiotic mitosis of germ cells. In situ hybridization
revealed that expression of the meal candidate mRNA
is only observed in germ cells in a specific pattern.
Analyses of the MEAL gene will shed light on the
germ-cell lineage development of higher plants.
(2) Structural and functional analysis of rice OsHAP3
genes

Yukihiro Ito, Thiruvengadam Thirumurugan and Nori
Kurata
We identified 11 genes (OsHAP3A to OsHAP3K)
which encode a HAP3INF-YB subunit of CCAAT-box
binding complex in rice by cDNA screening and a
database search. We showed that three genes,
OsHAP3A, OsHAP3B and OsHAP3C, are involved inchloroplast biogenesis (Miyoshi, et al. 2003), and
OsHAP3Emight be involved in brassinosteroid-related
responses and panicle development. We also revealed
that several specific members of OsHAP2 and OsHAP5
interacted with OsHAP3A and OsHAP3E, indicating
specific combinations of OsHAP3, OsHAP2 and
OsHAP5 in the formation of heterotrimeric
CCAAT-box binding complex.
This year we examined expression patterns of
OsHAP2 and OsHAP5 genes by RT-PCR. Among ten
HAP2 genes, five (OsHAP2A to OsHAP2E) showedubiquitous expression, while remaining five showedvarious or gene-specific expression patterns. OsHAP5
genes also showed ubiquitous expression (OsHAP5A,
B, D, and E) or organ-specific expression (three genes).
For example, OsHAP2G and OsHAP2Hwere mainly
expressed in shoot apex and callus, respectively, and
OsHAP5Fand OsHAP5Gwere expressed ~n callus androot, respectively. These results suggest that subunit
interaction of a HAP complex could be controlled by
specificities of gene expression as well as the
combination of protein-protein interaction.
(3) Regulation of expression of KNOX family class 1
homeobox genes of rice

Yukihiro Ito, Katsutoshi Tsuda and Nori Kurata
KNOX family classl homeobox genes, which show
SAM-specific expression, playa vital function for shoot70

Plant Genetics Laboratory

apical meristem (SAM) formation and maintenance.
We found that cytokinin, which is necessary for shootregeneration, induced the expression of OSH1, amember of the KNOX family, in callus. Overexpressionof homologues of cytokinin signalling genes such asa histidine kinase gene OHK3, histidine-containing
phosphotransmitter gene OHP2 and response regulator
gene ORRl enhanced the cytokinin-induced OSHl
expression, and conversely, expression of a dominantnegative of OHK3 reduced it. In situ hybridization
showed overlapped expression of these three genes inregenerating callus. Yeast two-hybrid analysis showedinteraction of OHK3 and OHP2, and OHP2 and ORRl.
These results indicate that these genes mediatecytokinin-induced expression of OSHl in regenerating
callus.
Analyses of dominant mutants of KNOX genes inmaize and barley, and our transgenic analyses of rice
KNOX genes showed that introns and exons havecis-regulatory elements of their expression. To identify
trans-factors that bind to the cis-elements and control
the OSHl expression, we started yeast one-hybrid
screening. We also searched and identified a recessive
mutant showing ectopic expression of OSHl in leaf andresembling overexpressors of OSHl. The mutant shootwas very small, and no leaf was formed after
germination. The mutant was seedling-lethal probably
due to a failure of the SAM maintenance. These results
indicate that this gene is required for the SAM
maintenance and the suppression of the OSHl
expression in leaf. Detail analyses of the mutant andthe one-hybrid screening are underway.
(4) Analysis of a genetic program in embryosac/ovary
formation

Shinichiro Yamaki, Yasuo Nagato* and Nori Kurata(*Univ. Tokyo, Agr. Sci)
Ovule is a female reproductive organ wheremegasporogenesis and megagametogenesis take place.
To elucidate the mechanism of rice ovule development,
the gypsy embryo (gym) mutant was found andanalysed. The mutant showed an interesting
phenotype of apically displaced embryos. Observationof the mutant indicated that the apically displaced
embryo came from the apically displaced egg cells
caused by incomplete ovule curvature. Apicallydisplaced egg cells resulted in low fertility comparedto the normally positioned egg cells. This suggests thatthe normal ovule needs to be curved for allocating an
egg cell to the position advantageous for fertilization.
In situ hybridization analysis revealed that OSHl
expression, a marker of indeterminate cells, wasectopically remained in ovule primordium. In contrast,
OsMADS13 expression, a marker of ovule, wasrestricted to a smaller region than that of wild type.
These results indicated that the primary function of
GYM gene is establishment of ovule identity bysuppressing an indeterminate state of primordial cells.
This suggestion was supported by the result of double
mutant analysis that combined gym with other related
mutations ·(Yamaki et aI. 2005). We have mapped the
GYM gene within 30 kb region on the short arm ofchromosome 2. Cloning and identification of the GYM
gene is now in progress.
(5) Establishment of rice TILLING system and mutant
pools
Tadzunu Suzuki, Mitsugu Eiguchi, Toshihiro
Kumamaru*, Hikaru Satoh* and Nori Kurata(*Kyushu Univ., Faculty of Agr.)
Many resources originated from rice insertion
mutagenesis have been stacked for functional genomic
studies in the world. These insertion lines, however,are difficult to cover knockout mutants for all genes
because of the integration of DNA or transposons in
the preferential target sites. In contrast, chemicallymutagenized population is thought to cover all
mutations for every gene. We collected MNU-treatedmutant M2 population for screening mutations.
To establish a system for rice mutant screening,
we applied a "Targeting Induced Local Lesions IN
Genomes (TILLING)" method with a few modifications(Suzuki, et aI., 2005). The modified TILLNG method,in which non-labeled primers for PCR and rapid
capillary gel electrophoresis are used in place offluorescence primers and polyacrylamide gelelectrophoresis, was applied to screen mutations in the
MNU-induced mutant population of T65 rice. Morethan 700 M2 lines were used to search basesubstitutions in two genes of known function, andmany mutations were successfully detected by themodified TILLING system. The result suggested thataround ten different mutant lines would be expected
in 1,000 lines for 1 kb gene region. Thus, the

MNU-induced mutant pool could be a useful resource
for the reverse genetics approach and to compensate
other mutant populations that cannot cover all genes.
(6) Positional cloning and statistical analysis ofsegregation distortion genes between Japonica and Indica
rice
Yoshiaki Harushima, Satoshi Kuriki *, Youko Mizuta,
Hironori Fujisawa*, and Nori Kurata (*The Instituteof Statistical Mathematics)
Reproductive barrier is a genetic mechanism toisolate "species". The aim of this study is tounderstand molecular mechanisms of reproductive
barriers by mapping genes, isolating genes, andobserving phenotypes relating to its isolation
mechanisms. Asian cultivated rice 0. sativa can bedivided into two ecotype groups, Japonica and Indica,and many reproductive barriers had been identified
in their crosses (Harushima et aI. 2002). Reproductive
isolation in the crosses is known to be resulted fromgene interactions. In 2005, we have been developing astatistical method to detect interactive locus/gene pairsof reproductive isolation in an F2 population. In thedevelopment of the method, a gene pair of malegametophytic reproductive barrier was detected on
chromosome 1 and 6 using an F2 population of
Nipponbare x Kasalath, and was confirmed reciprocal
backcross populations. Positional cloning of the genepair has been started.
(7) Comparative and evolutional studies for cultivatedand wild rice species
Ken-ichi Nonomura, Yukie Sano, Akane Morohoshi
and Nori Kurata
The genus Oryza is composed of 23 species, ofwhich two species of 0. sativa and 0. glaberrima are
cultivated and the rest are wild relatives. An F1 hybrid
can be produced in almost all cross-combinations
among Oryza species. However, F2 seeds showed
complete sterility in many hybrids. This F1 sterility
is often accompanied by aberrant meiosis andgametogenesis caused by reproductive isolation
mechanisms. Elucidation of genetic diversity among
Oryza species especially in genes related to meiosis/
gametogenesis events could help to reveal a

Plant Genetics Laboratory71reproductive isolation mechanism that will then be
used for overcoming reproductive barriers.
The PAIRl gene plays an important role inhomologous
chromosome
recognition
andcondensation in rice meiosis. It is possible that the
PAIRl might be one of the candidate genes working
in the reproductive isolation process. The PAIRlmRNA was expressed in young flowers from several
wild relatives. In tetraploid species, two types of PAIRlmRNAs were detected. Sequencing of genomic DNA
and cDNA from wild relatives revealed that the PAIRl
locus possesses high diversity in several important
domains. To examine relationship between F1 sterility
and PAIRl sequence diversity among species, acomplementation test of pairl mutation of cultivatedspecies with PAIRl genes of wild relatives is inprogress.
We also performed comparison of ESTs (expression
sequence tags) from shoot and panicle between acultivated species and wild relatives (BB and CC
genome species) as described in the report ofExperimental Farm. A total of 2300 cDNAs from BB
and CC genomes analyzed so far showed about 3%
base substitutions with AA genomes and scores ofhighly diverged genes and several wild specific genes
have been identified. A detailed characterization ofthese divergent genes is now underway. This approach
will bring us useful information to have knowledge
about global molecular diversity in genus Oryza.
(8) Detection of genetic/expression variation among ricespecies

Yoshiaki Harushima, Youko Horiuchi, Masanori
Kawakita*, Shinto Eguch *, and Nori Kurata (*The
Institute of Statistical Mathematics)

In the preliminary micro array comparative
analysis between wild and cultivated rice, we saw
abnormally higher expression of a set of cultivated ricegenes. The higher expression was thought to comefrom sequence diversity between two rice species. The
aim of this study is discriminate genetic and expression
variation among rice species using Affymetrix Rice
Genome array that contains 631,066 25mer-probes totarget 49,824 transcripts mainly from Japonica rice. To
distinguish genetic diversity from expression variation,
we are going to develop a combinational analyticalmethod. The method is composed of a comparison

72

Plant Genetics Laboratory

between experimental hybridization and in silico
hybridization of the probe sequences of Nipponbare(J aponica) and 93-11 (Indica), whose sequences areavailable and averaged differences were 3.0 SNP/kb
in coding region and 4.5 SNP/kb in 3'UTR. The method
would become indispensable in the expression analysis
of genetically diverged wild rice genes on the array ofcultivated rice genes.
Rice genetic resource project in NBRP and
Oryzabase(9)

Nori Kurata, Toshie Miyabayashi, Shinichiro Yamaki
and Ken-ichi Nonomura
The National Bioresource Project (NBRP) works
to conserve and distribute genetic and biologicalresources for scientific communities. In the NIG, we
are dealing 2,000 wild rice accessions covering 10
genomes and 23 species collected all over the world forover 50 years. Four rice sub-centers in other institutions
deal with different kinds of genetic materials and work
together in the project. Resource materials and theirinformation can be accessed at;
http://www.shigen.nig.ac.jp/rice / oryzabase /nbrpStrains/aboutNbrp.jsp
To evaluate the NIG Oryza collection in amolecular level, we measured DNA contents of 39
accessions from 20 species by flow cytometric analysis.
The karyotype analysis is also performed to confirm
the ploidy level. Flow cytometric analysis revealed thatDNA contents of the accessions involved in theofficinalis complex (CC genome species) are various.
In addition, 0. granulata and 0. meyeriana, assigned
as GG genome species, possessed two-fold or more
DNA contents than that of 0. sativa. Karyotype
analysis revealed that the accession W1525 thatbelongs to the species of officinalis complex andpreviously assigned as a diploid species of 0. eichingeri-,possessed a tetraploid chromosome set, indicating
contamination in the process of maintenance.
Identification of each accession with species specific
DNA markers is also in progress.
In addition, we observed morphological featuresof vegetative and reproductive architectures of 44
Rank1 core accessions of wild rice. These observations
revealed some relationships of each feature. First, theaccessions with many tillers per plant tended todifferentiate few spikelets per panicle and vice versa.

Second, seed weight was also in inverse proportion to
the number of spikelets per panicle. Third, the numberof panicles per plant had positive correlation with seedfertility. Because it is supposed that seed fertility is
involved in pereniality, the number of panicles perplant may also be involved in pereniality. The intimate
analyses of these relationships are underway.
The Oryzabase is a comprehensive rice biological
database composed of 15 sections of information.
Major data characteristic to the Oryzabase are;mutants and their trait genes collection, wild ricecollection and development/anatomy of rice, together
with genetic maps, physical maps, basic biological dataand so on. In 2005, we finalized an important sectionof rice developmental stages that is served as one of
the bases of plant ontology activities in cereal plants.
NBRP data have also been added. The rice genetic
resources committee centered in the Plant Genetics
Laboratory is responsible for collecting and managing
the contents of information. Construction andmaintenance of the DB have been carried out by the
Genetic Informatics Laboratory. The DB is accessibleat;http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp

rice mutant screening, Rice Genet. Newslet., 22, 89 - 90
7. Xu, ]-H., Kurata, N., Akimoto, M., Ohtsubo, H. and
Ohtsubo, E. (2005) Identification and characterizationof Australian wild rice strains of Oryza meridionalis
and Oryza rufipogon by SINE insertion polymorphism,
Genes Genet. Syst., 80, 129 - 134
8. Yamaki, S., Satoh, H. and Nagato, Y (2005) Gypsy
embryo specifies ovule curvature by regulating ovule /integument development in rice, Planta, 222, 408 - 417
Books

9. fjtJli¥t~ (2005).::r::/ /'\ :/-lj--, .::r::/ /'\ :/-lj-- " '7 'Y7, ") /j T -{ A.-:,,- 3 :/ ~~:/ /j. ff[~fffl~~¥~ (B*nfl~~)±g JittB

EDUCATION

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SOCIAL CONTRIBUTIONS

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1. Ito, Y, Takaya, K and Kurata, N. (2005) Expressionof SERK family receptor-like protein kinase genes inrice, Biochim. Biophys. Acta, 1730, 253 - 258

5. ~~~ ~ : Rice Genetics Newsletter Editor
6. ~~~ ~ : NSF project advisory committee member

2. Ito, Y. and Kurata, N.(2005) Identification ofenhancer trap lines with tissue-specific GUS activities
in rice, Rice Genet. Newslet., 22, 32 - 33
3. Itoh,]. I., Nonomura, K I., Ikeda, K, Yamaki, S.,
Inukai, Y, Yamagishi, H. and Nagato, Y (2005) Rice
plant development: from zygote to spikelet, Plant Cell
Physiol, 46,23 - 47
4. Kurata, N., Miyoshi, K., Nonomura, K. I.,
Yamazaki, Y. and Ito, Y (2005) Rice mutants and genes
related to organ development, morphogenesis andphysiological traits, Plant Cell Physiol, 46, 48 - 62
5. Moriguchi, K, Suzuki, T., Ito, Y, Yamazaki, Y,
Niwa, Y and Kurata, N. (2005) Functional isolation ofnovel nuclear proteins showing a variety of subnuclear
localizations, Plant Cell, 17, 389 - 403
6. Suzuki, T., Eiguchi, M., Satoh, H., Kumamaru, T.
and Kurata, N. (2005) A modified TILLING system for

F-f. Microbial Genetics Laboratory
Hironori Niki GroupRESEARCH ACTIVITIES(1) Guidance of Actively Partitioning Plasmid by a
Helical Structure of an ATPase, SopA
Toshiyuki Hatano and Hironori Niki
In a bacterial cell, replicated chromosomal
segments including the replication origin and the Fplasmid actively move from midcell toward cell poles
and are located at cell quarter positions. The Sop ABC
partitioning system of F plasmid has beencharacterized genetically and biochemically to elucidate

Plant Genetics Laboratory73

molecular mechanism of DNA partitioning. The
SopABC system consists of two proteins, SopA
ATPase and SopB, which binds to centromere site,
sopc. The SopA protein is a hypothetical motor protein
because of its walker-type ATPase activity. We havepreviously constructed an active SopA-YFP and
observed oscillation of YFP fluorescence from one cellpole to the other with a helical structure extending
along the long axis of a cell. Simultaneously, migrationof the plasmid DNA was observed fluorescent labelingtechnique with lacO array and Lacl-CFP. The analyses
of movement of both the fluorescence suggest that
SopA could be a motor protein to govern "direction"of movement of the plasmid DNA.
We further analyzed dynamics of movement of
SopA-YFP. SopA-YFP oscillated between the "nucleoid
borders" in a cell. The result indicates that SopA may
recognize and cluster at the positions of nucleoid
borders during oscillation. To test this possibility, weobserved localization of SopA-YFP in anucleated cells(DNA less cells). Surprisingly, SopA-YFP showeddiscrete foci and oscillated in anucleated cells.
Statistical analysis revealed that the positions of fociwere corresponding to the potential positions of thenucleoid borders. Furthermore, we observed helicalstructure of SopA-YFP in anucleated cells. These
results indicate that SopA can assemble at the specificsubcellular positions where overlap with nucleoid
boader.
Furthermore, we found the helical structure of
SopA-YFP kept constant length in a cell duringoscillation. In E. coli, a bacterial action like filamentous
protein maintains cell morphology. The cytoskeletal
protein, MreB makes a helical structure along the longaxis of a cell To test the possibility that the bacterial
cytoskeleton protein might be required for the helicalstructure of SopA, we observed the localization of
SopA-YFP in a cell lacking MreB. SopA-YFP formed
filamentous structure in the deletion mutant cells, andthe foci were oscillated. In addtiton, we found that F
plasmids are stably maintained in JmreBCDmutant
cells. These results indicate that the bacterial actin,
MreB is not required for F plasmid partitioning itself
and movement of SopA. SopA may govern apartitioning system independent from nucleoid
structure and cytoskeleton.

74

Microbial Genetics Laboratory(2) Analysis of Dynamics of ATPase, PI ParA in
Partitioning of PI Plasmid

Toshiyki Hatano and Hironori Niki
ParA/SopA family proteins are conserved andinvolved in partitioning of a variety of plasmids andbacterial chromosomes. PI ParA protein is a memberof ParA/SopA family and essential for stablepartitioning of PI phage plasmids. The PI Par system
consists of two proteins, ParA ATPase and ParB,
which binds to centromere site, parS. Because PI ParA
protein also contains a walker type ATPase motif, PI
ParA may have function as motor for migration of PI
plasmids like the SopA protein.
We have been constructed a PI ParA-YFP fusion
protein that sustained biological function for theplasmid partitioning. Interestingly, in spite of similarity
with SopA as walker ATPase, the PI ParA-YFP fusion
protein did not show oscillation in a cell, but showed
localization spread on nucleoid with weak foci at cell
quarter positions. Comparison of the dynamics
between SopA and ParA in a living cell may elucidate
conserved or diverse functions of the ATPase motor
involved in plasmid partitioning. Especially, differenceof dynamics between them may explain difference ofincompatibility of partitioning between F and PI
plasmids.
(3) Characterization of proteins that are interact witha bacterial centromere site, migS

Yashuyuki Ogata, Toshiyuki Hatano and Hironori Niki
The replicated Ori domains migrates towards
opposite cell poles. This indicates that a putative
cis-acting site for bipolar migration of the oriC segment
is located within the domain. A new method to splita bacterial chromosome into two sub-chromosomes
has enabled to identify the cis-acting site. In the end,
we found that a 25 bp sequence, migS, is responsible
for bipolar migration of the oriC segment. Although
rapid bipolar migration of the oriC segments isinhibited, the migS is not a defect in chromosome
segregation. This suggests that a redundantmechanism to compensate for the migS pathway might
be involved in bacterial chromosome partitioning. To
examine this idea, synthetic lethal mutations with themigS deletion were screened in a library with insertion

mutations of a transposon. Weare investigating
isolated mutations at the present time.
We would like to focus on study of the molecularmechanism for bipolar migration of DNA in a bacterialcell. Although actin-like proteins have been found inprokaryotes, a motor function to partition
chromosomes into daughter cells has not yet been
identified on bipolar migration of the chromsome.
Presumably, a motor protein works on the process togenerate a driving force. Investigation of the molecularmechanism of migS dependent migration is providing
vital new clues as how the putative motor protein
contributes to chromosome partitioning. Proteomics
approaches have already been identified two proteins
as migS binding proteins. Further investigation of thegenes that encode the migS binding proteins havealready been carried out, and the results indicates thegenes would be involved in bipolar migration of mig5.
We investigate on property of the proteins for bipolar
migration of mig5.
EDUCATION1.

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F-g. Invertebrate Genetics Laboratory
Ryu Ueda GroupRESEARCH ACTIVITIES(1) RNAi mutant fly bank for comprehensive analysesof gene function in Drosophila
Ryu Ueda, Yukiko Sado1 , Kazuko Fujitani, andKuniaki Takahashi CJST)Genome sequencing projects have revealed thenumber of genes for several model organisms forgenetics. The small wonn 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 10 1 cells, harbors only 14,066
protein-coding genes. Considering there is such a small
number of fly genes, each one of them may have an
essential function in fly development and behavior. In
other words, it may be easy to detect and analyze genefunction in the fly by reverse genetics because theabnormal phenotype will frequently appear whenknocking down a target gene whose function is
unknown. Weare planning to investigate the functionof fly genes comprehensively as a suitable model forstudying the functional genomics of multicellularorganisms.
How does one investigate the function of all 14,066
genes in the fly? We use RNA interference (RNAi) to
knock down the activity of the target gene. RNAi is oneof the emerging technologies with which to investigate
gene function in multicellular organisms. Whenintroduced into the cell, double stranded RNA (dsRNA)
works as a specific mutagen for each gene. That is,
dsRNA recognizes host mRNA and digests it in asequence-specific manner, and consequently brings aloss-of-function mutation phenotype to the host cell.
The detailed mechanism of this RNAi phenomenon
has not yet been elucidated, but it works efficiently inmany multicellular organisms, including humans.
We coupled the RNAi with the GAL4-UAS gene
expression system to induce a conditionalloss-offunction mutation in the fly. The GAL4-UAS system
is a binary system for inducing transgene expression,in which two fly lines are used. One is the GAL4 driver
fly line, which expresses yeast transcription factor,
GAL4, in a specific cell/tissue or at a specificdevelopmental stage in favor of the GAL4 transgene.
The other fly line harbors a transgene on thechromosome, in which an appropriate gene to be
expressed is fused to the UAS promoter, the GAL4
target. When these two fly lines are crossed with eachother, we can observe in the fly progeny that the GAL4
protein induces target transgene expression in adriver-specific conditional fashion. In this GAL4-UAS
system, when we use a UAS-transgene having an
inverted repeat (IR) sequence, the transcribed RNA
may form a dsRNA in the cell and induce a lossof-function mutation by the RNAi mechanism. Such
inducible RNAi caused by the transcription of an IR
seq uence was first successfully adopted to geneMicrobial Genetics Laboratory

75

function analysis in C. elegans. It was then also foundto be effective in fly genetics. By making a
UAS-transformation vector containing an IR sequenceof the gene predicted by the fly genome project, andby introducing it into a fly line (lR fly), a mutant
phenotype of the gene can be easily observed in any
cell or at any developmental stage of the progeny,
whenever the IR fly is crossed to an appropriate GAL4
driver fly.
We are expanding this inducible RNAi to thewhole genome of the fly. This process involves two
major procedures.
1) in vitro construction of transformation vectors
containing an IR sequence from each of the 14,066
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 2005, over 8,200 transformation vectors
had been constructed, 7,300 of which have beensuccessfully introduced into the fly to establish 12,000
IR fly lines. We will be continuing this work to the endof the next year, and may be able to add up to 9,000
vectors and 14,000 IR fly lines to our fly bank.
Along with the establishment of IR fly lines, basic
characterization of these fly lines is conducted. All of
the IR fly lines are crossed to the Act5C-GAL4 fly. The
Act5C-GAL4 induces the UAS-transgene in all cells at
all developmental stages. Thus, if the gene targeted byRNAi has functions that are indispensable for fly
development, the progeny of IR and GAL4 flies should
die before the adult flies emerge. Among the 1,954 genes(3,908 IR fly lines) tested, 51.1%of the fly lines showedlethality. This value is rather high compared to thatobtained by classical genetics (25%), while the fact thatmany of the genes tested here were considered to haveimportant functions in various aspects of fly
development by our collaborators may bring aboutsuch a high score. Detailed analyses on known genes
and greater accumulation of data are necessary.
Weare currently collaborating with over 100
research groups in the world with our IR flies. The
usefulness of inducible RNAi for investigating genefunction in Drosophila is being revealed in many
biological phenomena, such as morphogenesis(Adachi-Yamada, T., et al., 2005; Hirota, Y., et al., 2005),
immunity (Kleino,A., et al., 2005), behavior Oshimoto,H., et al., 2005; Usui-Aoki, K, et al., 2005), circadian76

Invertebrate Genetics Laboratory

rhythm, brain development, cell proliferation, DNA
repair, glycobiology, neurodegenerative disease, sex
determination, etc.
Through feedback from these collaborations, weconvinced the efficacy of our fly bank. Thus, a part ofthe bank has been opened to the community (http://
www.shigen.nig.ac.jp/fly/nigfly/) with a support ofNational Bio-Resource Project in Japan. Significant partof the bank will be opened in the next year after
re-inspection and integration of many associated datawith these flies.
This work was supported in part by financial
assistance from the Mitsubishi Kagaku Institute of Life
Sciences (MITILS) to Dr. Ueda.
PUBLICATIONSPapers1. Adachi-Yamada, T., Harumoto, T., Sakurai, K,
Ueda, R, Saigo, K, O'Connor, M. B., and Nakato, H.
(2005) Wing-to-Ieg homeosis by Spineless causes anapoptosis regulated by Fish-lips, a novel Leucine-Rich
Repeats transmembrane protein., Mol. Cell. BioI., 25(8) 3140 - 3150
2. Hirota, Y., Sawamoto, K, Takahashi, K, Ueda, R,
and Okano, H. (2005) The transmembrane protein,
Tincar, is involved in the development of thecompound eye in Drosophila melanogaster., Dev. Genes
Evol., 215, 90 - 96
3. Ishimoto, H., Takahashi, K, Ueda, R, andTanimura, T. (2005) G-protein gamma subunit 1 isrequired for sugar reception in Drosophila., The EMBO
J., 24, 3259 - 3265
4. Kleino, A., Val anne, S., Ulvila, ]., Kallio, ].,Myllymaki, H., Enwald, H., Stoven, S., Poidevin, M.,
Ueda, R, Hultmark, D., Lemaitre, B., and Ramet, M.
(2005) Inhi bitor of apoptosis 2 and T AK1- binding
protein are components of the Drosophila Imd
pathway, The EMBO J., 24, 3423 - 3434
5. Usui-Aoki, k., Matsumoto, K, Koganezawa, M.,
Kohatsu, S., Isono, K, Matsubayashi, H., Yamamoto,M., Ueda, R, Takahashi, K, Saigo, K, Mikoshiba, K,
and Yamamoto, D. (2005) TARGETED EXPRESSION
OF IP3 SPONGE AND IP3DSRNA IMPAIRES SUGARTASTE SENSATION IN DROSOPHILA, J.
Neurogenet., 19, 123 - 141
6. t§i:§.fY&fl~, J:1E~(2005) ~ 3?S/ 3 ?/"\::r:.ij' / A(J)f*~i¥Jm!~W¥t1A---~1::. 5}{t(J)J!{~i'-~.A 7" AW¥t1A I;:~ It

-c, m8W~~M~ 5JUW r if J ,bip;1':1flr~A7" ,b"'J,
50 (16) 2146 - 2152

pivotal role in orchestrating temporal change ofneuroblasts at the late stages of embryonicdevelopment.

EDUCATION

1. J::B3~
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JI{~-f-O)IIJ~ ~liJf~i" ~JI~~7:t" -'7 AJ?:fIl2005(2) Investigation of molecular functions of Castor in
Development of the CNS
Horiuchi Takayuki

J?:fIlr1J· It . ~ . ~ 7°'7-1fJ?:fIl
SOCIAL CONTRIBUTIONS AND OTHERS-f-(J)ft.l2
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F-h. Laboratory for Frontier Research
Takako Isshiki GroupRESEARCH ACTIVITIES(1) Analysis of temporal specification within late phasesof Drosophila neuroblast lineage
Takako Isshiki
During development, neural progenitors often
generate diverse cell types in an invariant order,
changing their property over time. Althoughsubstantial progresses have been made inunderstanding the molecular mechanisms of how
different cell fates are generated in order, many partsof the mechanisms still remain unknown. We utilize
the Drosophila central nervous system (CNS) as an
excellent model system to study temporal specificationof cell fates within a lineage. We previously showedthat Drosophila neuroblasts sequentially express thetranscription factors Hunchback, Kriippel, Pdm andCastor over time. However, most neuroblasts divide
additional 10 times on average after they start
expressing Castor. Thus, there must be subsequentmechanisms for temporal specification.
We have found, to date, several transcription
factors expressed later than Castor in mostneuroblasts, and elucidated the precise order of theirexpression. This year we investigated functions ofthese factors by genetic analyses. These analyses
revealed that, among these factors, Castor plays a

Our genetic study indicated that Castor is one of
crucial transcription factors for temporal change ofneuroblasts, although it remains mostly unknown whatmolecular mechanisms enable Castor to performfunction. Aiming to elucidate the molecular mechanism,
we searched the proteins that directly bind to Castor
by using Yeast 2 Hybrid screening. So far, we could
obtain 4 good candidates that might bind to Castor invivo. Weare now investigating whether each caudidate
has any effect on Castor by genetic analyses.
(3) Temporal change, quiescence, and reactivation ofneuroblasts
Katsutomo Okamura, Takako Isshiki
At the end of embryogenesis, most, not all,abdominal neuroblasts cease cell division and undergo
apoptosis eventually. In contrast, thoracic and brainneuroblasts go into the quiescent state, and then get
reactivated at the larval stages. Upon the reactivation,neuroblasts change their property dramatically. For
example, larval neuroblasts require glial niche andcontinue cell growth, which enables them to generate
50-100 neurons per a single neuroblasts during a longlarval period. However the mechanisms of thequiescence, the reactivation and the transformation ofneuroblasts are poorly understood. We have started
addressing the questions by observing the expression
pattern of the temporal transcription factors just before
entering the quiescence and during the larval andpupal stages.

Invertebrate Genetics Laboratory77

(2) CARD R-BASE

G. CENTER FOR GENETICRESOURCE INFORMATION

G-a. Genetic Informatics Laboratory
Yukiko Yamazaki GroupRESEARCH ACTIVITIES(1) Wheat mitochondrial genome analysis

Ogihara, Y., Yamazaki, Y., Murai, K., Kanno, A.,
Terachi, T., Shiina, T., Miyashita, N., Nasuda, S.,
Nakamura, c., Mori, N., Takumi, S., Murata, M., Futo,
S. and Tsunewaki, K..
The application of a new gene-based strategy forsequencing the wheat mitochondrial genome shows its
structure to be a 452528 bp circular molecule, andprovides nucleotide-level evidence of intra-molecular
recombination. Single, reciprocal and double
recombinant products, and the nucleotide sequencesof the repeats that mediate their formation have been
identified. The genome has 55 genes with exons,
including 35 protein-coding, 3 rRNA and 17 tRNA
genes. Nucleotide sequences of seven wheat genes havebeen determined here for the first time. Nine genes
have an exon-intron structure. Gene amplification
responsible for the production of multicopy
mitochondrial genes, in general, is species-specific,
suggesting the recent origin of these genes. About 16,
17, 15, 3.0 and 0.2% of wheat mitochondrial DNA(mtDNA) may be of genic (including introns), open
reading frame, repetitive sequence, chloroplast andretro-element origin, respectively. The gene order ofthe wheat mitochondrial gene map shows little synteny
to the rice and maize maps, indicative that thoroughgene shuffling occurred during speciation. Almost all
unique mtDNA sequences of wheat, as compared withrice and maize mtDNAs, are redundant DNA. Featuresof the gene-based strategy are discussed, and amechanistic model of mitochondrial gene amplification
is proposed. (Ogihara Y. et al. 2005)78

CENTER FOR GENETIC RESOURCE INFORMATION

Takehiro Yamakawa, Hideki Kato, Naomi Nakagata,
Kenichi Yamamura and Yukiko Yamazaki
CARD R-BASE is a database of transgenic mousestrains established and deposited by individual
researchers. All resources are stored as frozen embryos
and are available on request. The database mainlycontains genetic backgrounds, destroyed / transferredgenes, relevant human diseases and journal articles
for each strain. In this year, we developed an on-line
application system. This system not only supports
depositors to submit data of their mice but also allows
mouse maintainers, data reviewer and databasemanager to carry out their own tasks on-line while
taking an overview of whole processes.
CARD R-BASE became a member of FIMRe(Federation of International Mouse Resources) and has
been periodically sending data files to the IMSR, adatabase of FIMRe. The FIMRe established in 2005 isa collaborating group of Mouse Repository andResource Centers worldwide whose collective goal is
to archive and provide strains of mice as cryopreserved
embryos and gametes, ES cell lines, and live breeding
stock to the research community. CARD group also
started collaboration with Asian mouse archiving
centers.
(3) Guide dog database

Kazuhiro Oogushi, Yukiko Yamazaki, Yoshinori Suwaand Hiroshi Suzuki
There are 900 guide dogs in Japan but the numberof people requiring guide dogs exceeds this number
by five times and there are 10 times more people whowish to own a guide dog. In addition, only 30% of thedogs which undergo strict training manage to pass theaptitude test necessary for them to become guide dogs.
Whether or not the inborn characteristics of a dog meetthe conditions required for a guide dog is also an
important factor. Since the dog's training process only
begins after its contraception or castration, a guide dog
cannot reproduce even if it is found to be of a superior
breed. Therefore, a dog can only be either a guide dog
or a breeding dog, disabling a guide dog to be used asa breeding dog at a later stage. If the number ofbreeding dogs decreases, the offspring will become
genetically closer, and this may cause various problems
in the future. At present, it is also difficult to importguide dogs from abroad.
In order to overcome the shortage of guide dogs,
the project entitled "Reproduction engineering researchon breeding of superior guide dogs" has been started.
This project aims to establish "K9 BioBank" wherein
the genetic resources (sperms, embryos, ovaries, etc.)
of superior guide dogs are frozen and stored, and the
reproduction of superior guide dogs can be realized,
if needed.
The database construction of the K9 BioBank is
one of the pillars of this project. We have constructeda trial database of individual dogs information (bloodline, case history, existence of etiologic gene, character
evaluation result) this year and plan to construct asystem that facilitates the sharing of information
between related organizations and consequently fulfill
requests from users.
The Asia GuideDogs Breeding Network (AGBN)
was established in 2002 with eight facilities in Japan,
one in South Korea, and two in Taiwan. The
construction of this database is expected to lead tofurther cooperation among these facilities.
(4)

Rice

Koji Watanabe, Nori Kurata, Atsushi Yoshimura,Yasuo Nagato and Yukiko Yamazaki
Oryzabase is a comprehensive rice databaseintegrating biological data derived from various studies
on morphology, physiology and ecology with moleculargenomic information. The data in Oryzabase are
divided into 15 sections: development / anatomy,
mutants, trait genes, linkage maps, physical maps,
comparative maps, references, basic biological data,
DNA sequence, BLAST search, chloroplast andmitochondrion, tools and protocols, strains, stock
centers and wild rice.
The biological data incorporated in Oryzabaseinclude morphological and gene expression
characteristics of rice at different developmental stages
and in various mutants. These data are classified into
four sections, namely, development / anatomy, trait
genes, mutants, and basic biological information. The
development / anatomy is the most distinguishingfeature of Oryzabase. The database contains

information on 1,698 trait genes and .136 unclassified
genes that has been identified from a mutant or naturalvariant Each entry (trait gene) is provided with a genesymbol, gene name, chromosome (location if identified),mutant class name, GO and / or trait ontology number.
The Oryzabase is also a repository for about 20,000
rice strains including wild rice accessions, cultivars,
mutant lines, chromosome substitution lines,
recombinant inbred lines and marker gene lines in
conjunction with the National Bioresource Project(NBRP) in Japan. The wild rice strains include 23
species from the AA, BB, CC, BBCC, CCDD, EE, FF,
GG and HHJJ genomes. A core collection of 289
accessions from wild species was chosen and ranked
for convenience in accessing. The crossed lines include
RILs of four japonica x indica crosses, chromosome
substitution lines with japonica backgrounds crossedwith other AA genome species. A collection of morethan 6,000 mutants induced by N-methyl-N-nitrosourea(MNU) and which has been classified into 12 classesof visible phenotypes is also available.
As to data on rice genomics, Oryzabase provides
access to genome maps, sequences and comparative
mapping resources. The four basic linkage maps ofrice, namely, the classical linkage map with 571
phenotypic genes, the integrated linkage map with 83
RFLP markers and 40 phenotypic markers, therecombinant inbred (RI) map with 375 RFLP markers,
and the high-density Nipponbare / Kasalath linkage
map with 2,275 DNA markers were integrated using
commonly mapped markers. A genome viewer
showing the physical maps of the 12 rice chromosomeswith windows ranging from 250 kb to 1 Mb and 10 to
100 kb has been added recently. The genome sequences
represent the latest submissions in DDBJ / EMBL /GenBank. The comparative maps include barley clones
and wheat ESTs mapped in the rice genome.
Oryzabase also aims to establish a comprehensive
ontology for all morphological features and trait genes
in rice. All trait genes are manually annotated andassigned with gene ontology (GO) IDs in agreementwith the central GO database at Gramene (http:! /www.gramene.org/). A major priority is to develop,
curate and share controlled vocabularies describing
various morphological structures of the rice plant at
different stages of growth and development in line with
the
Plant
Ontology
Consortium(http://
www.plantontology.org/) to establish a widely acceptedplant ontology (PO) and plant and trait ontology

Genetic Informatics Laboratory

79

(PATO) framework for rice. An integrated viewer, 03,will soon be incorporated in the database to correlate
the concept of ontology with various biological data.
Therefore Oryzabase can be a source of bothinformation and resources that will provide thefoundation for rice functional genomics.
(5) SHIGEN project

Takahiro Yamakawa, Koji Watanabe, Rie Tsuchiya,
Sumiko Yano, Kazuhiro Oogushi, Shingo Sakaniwa,
Mutsumi Saito, Sharoh Yip, Toru Watanabe, Miharu
Yoshioka and Yukiko Yamazaki
In 2002 the National Bioresource Project wasstarted in Japan to reinforce the system for collecting,
maintaining, and providing research resources andtheir information effectively. Twenty-four resource
centers, each of which is representative organizationof a species and one information center were
established.
Our missions as the information center arecollection of resource information and provision of thedatabases in hopes of promoting effective use of theresources. Twenty-three individual databases are now
available for use through the internet and both
intra-species integration such as genetic resources andgenomic resources integration, and inter-species
integration using ontology have been proceeding. We
are developing a system, the BioResource WorId, bywhich resource maintainer could upload and provide
their information through the online database without
having to maintain a database. This system also allows
resource users to search several different resource
databases simultaneously.
According to the access logs, nearly 40000 visits
per month and over 100 feedbacks from users wererecorded each month in 2005.
Featured contents of each database in 2005
5-1: Mouse Polymorphism Database

Mouse Polymorphism Database provides SSLPs
among different mouse strains, cSNPs between B6 andMSM mice based on their full length cDNA sequence
information and gSNPs between B6 / MSM mice basedon BAC end sequences. We appfied the Ensembl,
which is provided for free by EBI, as a genome browser.

80

Genetic Informatics Laboratory

The original data of MSM BAC ends, MSM / B6 cSNPs
and microsatellite polymorphism was added to thebrowser as DAS (Distributed annotation source). We
also developed an original browser, the SHIGEN
genomic browser, to overcome several functional
limitations of Ensembl. Sequence alignment and SNP
positions as well as genomic position can be displayedby using the SHIGEN browser.
5-2: Medaka Atlas

The on-line medaka atlas was developed on thebases of an original atlas published in an article. The
content of the first release was the adult brain of awild-type inbred strain (HNI). This year, new content
of the blood vessel was added. The system was
implemented using flash technology and presents a
detailed characterization of the vascular anatomy ofa developing medaka embryo from the stage 24
through stage30 with dorsal-, dorsolateral- andventrolateral views.
5-3: Drosophila

Information of over 2000 RNAi-mutant flies wasadded to the NIG-Fly database. Each stock has basic
information (STOCK ID, CG No, Synonyms, Accession
No. Inserted chromosome number, phenotype andreferences) and sequences including primers, fragment
sequences, assemble data, and off-target genecandidates. FLYSTOCK database is a one-stop-shopof Drosophila resources and compiles all available
drosophila resources maintained by four organizations,
Kyoto DGRC, NIG, KYORIN and Ehime.
5-4: Wheat

Wheat database, KOMUGI, provided an online
journal submission and editorial system and started
distribution of the electronic newsletter for wheat
researchers"eWIS" this year. This activity was carriedout by the Japanese wheat geneticist community.
KOMUGI collected EST end sequences from various
tissues and stress conditions. Sequence analysis was
performed by the NBRP project and all clones areavailable through the on-line order system. Wheat genedatabase was updated by using MacGene2003, 2004-,
2005-supplement data. We applied the CMAP tool todisplay markers on maps instead of incorporatingthem into the gene database as we used to.
5-5: Legume

NBRP-LegumeBase is a resource ordering siteshared by Lotus japonica resource database andGlycine max. resource database. Lotus japonica
database provides wild accessions, root culture,
Recombinant Inbred Lines (RILs), BAC I T AC clones,
and eDNA clones as resources. Some strains werecharacterized by phenotype when growing in northernareas and southern areas. Glycine max database
contains wild species, Glycine soja, RILs and mutant
strains. Legume follows rice (monocot model plant) andarabidopsis (dicot model plant) as the third model
plant.

Subnuclear Localizations., Plant Cell, 17 (2) 389 - 403
5. Ogihara, Y, Yamazaki, Y., Murai, K, Kanno, A.,
Terachi, T., Shiina, T., Miyashita, N., Nasuda, S.,
Nakamura, c., Mori, N., Takumi, S., Murata, M., Futo,
S., Tsunewaki, K (2005) Structural dynamics of cereal
mitochondrial genomes as revealed by complete
nucleotide sequenceing of the wheat mitochondrial
genome., Nucleic Acid Res., 33 (19) 6235
6. Yamazaki, Y. and Jaiswal, P. (2005) Biological
Ontologies in Rice Databases., Plant and Cell
Physiology, 46 (1) 63 - 68
Books

7. 11p'F.JEMc.-r- (2005) Gene Ontology (GO) 7- ~.;{.­
7-., *HlJmI~, 1207
Database

5-6: E.coli

Sixty-four hundreds transposon insertion
disruptants were added to the E. coli strain database
after the analysis of insertion position on E. coligenome. A collection of cloning vectors of E. coli wasincorporated into this E. coli strain database.
Essentiality of genes based on different criteria as wellas huge amount of resource collections would become
very useful for E. coli researchers and acceleratefunctional genomics studies.
PUBLICATIONSPapers

1. Hashimoto, M., Ichimura, T., Mozoguchi, H.,
Tanaka, K, Fujimitsu, K, Keyamura, K, Ote, T.,
Yamakawa, T., Yamazaki, Y, Mori, H., Katayama, T.,
Kato, ]. (2005) Cell size and nucleoid organization ofengineered Escherichia coli cells with a reduced
genome., Mol. Microbiol., 55 (1) 137
2. Kawaura, K, Mochida, K, Yamazaki, Y, Ogihara,
Y (2005) Transcriptome analysis of salinity stressresponses in common wheat using a 22k oligo-DNA
micro array., Funct Integr Genomics, 132 - 142
3. Kurata, N., Miyoshi, K, Nonomura, K, Yamazaki,
Y., Ito, Y (2005) Rice mutants and genes related toorgan development, morphogenesis and physiologicaltraits., Plant and Cell Physiology, 46 (1) 48 - 62
4. Moriguchi, K, Suzuki, Y, Ito, Y, Yamazaki, Y,
Niwa, Y, Kurata, N. (2005) Functional Isolation ofNovel Nuclear Proteins Showing a Variety of

8. Mouse Genetic Resources (July, 2005) http;11
shigen.lab.nig.ac.jp/mouse/strainl
9. Mouse Polymorphism DB (2005) http:! I
shigen.lab.nig.ac.jp/mouse/polymorphism/top/top.jsp
10. CARD R-BASE 2005 http://cardb.cc.kumamoto-u.
ac.jp/transgenicl
11. Japan Mouse Strain Resources (JMSR 2005)
http://shigen.lab.nig.ac.jp/mouse/jmsrI
12. National BioResource Project MEDAKA 2005
http://shigen.lab.nig.ac.jp/medaka/
13. National BioResource Project XENOPUS 2005
http://shigen.lab.nig.ac.jp/xenopus/top.jsp
14. National BioResource Project ZEBRAFISH 2005
http://shigen.lab.nig.ac.jp/zebra/
15. NIG-FLY 2005 http://shigen.lab.nig.ac.jp/fly/
nigflyl
16. Flystock 2005 http://218.44.182.89/%7Eflystock/
htmllindexj.html
17. C. elegans MUTANTS 2005 http://shigen.lab.nig.
ac.jplc.eleganslmutantsl
18. SilkwormBase 205 http://shigen.lab.nig.ac.jp/
silkwormbasel
19. KOMUGI2005 http://shigen.lab.nig.ac.jp/wheat/komugi/20. Oryzabase 2005 http://shigen.lab.nig.ac.jp/rice/oryzabasel
21. BARLEY DB 2005 http://shigen.lab.nig.ac.jp/
barleyl
22. NBRP Chrysanthemum 2005 http://shigen.lab.nig.
ac.j pichrysanthem uml
23. NBRP LegumeBase 2005 http:/ I shigen.lab.nig.
ac.jp/legume/legumebasel

Genetic Informatics Laboratory8124. ~~,{!(iJJDB~ A:;- 1.>.2005 http://shigen.lab.nig.
ac.jp/algae/
25. NBRP E.coli Strain 2005 http://shigen.lab.nig.
ac.jp/ecolilstrain/
26. Profiling of E.coli Chromosome (PEC) 2005 http}/
shigen.lab.nig.ac.jp/ecolil peel
27. National BioResource Project YEAST 2005 http}/
yeast.lab.nig.ac.jp/nig/
28. National BioResource Projectm}Jj(~1:.!jo/.}2005
http://wdcm.nig.ac.jp/byogen/
SOCIAL CONTRIBUTIONS AND OTHERS~~~±fI

Bruskiewich, R., Constanzo, M., Dolinski, K., Gojobori,T., Hide, W., Howe, D., May, S., Millburn, G., Misra,
S., Schaeffer, M., Rhee, S., Twigger, Simon, Yamazaki,
y. 1st International Biocurator Meeting Asilomar

G-b. Genome Biology Laboratory
Yuji Kohara GroupRESEARCH ACTIVITIESGenome Biology Laboratory, Center for Genetic
Resource Information(1) Extension of the nematode expression pattern map
database (NEXTDB) towards comparative genomics.

Tadasu Shin-i, Hiroshi Kagoshima, Kaori Ikeda, Ikuko
Muramatsu-Sugiura, Masumi Obara, Wakako
Shimizu, Aya Harnakawa, Sachiko Takahashi, Takayo
Hamanaka, Yasuko Sugiyama, Yumiko Ueta, Hiroko
Ochi, Chihiro Hijikata, Yuji KoharaWeareupdating
theWWW-based
database "NEXTDB" to integrate all the informationof ESTs, gene expression patterns and gene functionsof C.elegans and related nematodes which are being
produced and analyzed in this laboratory.
Based on 14,000 unique C.elegans cDNA speciesclassified from 130,000 clones, whole mount in-situ
hybridization of about 11,000 genes were carried out
and their images were loaded to the database. Thenthey were annotated by their developmental stages andexpression patterns. The images and their descriptionof RNAi phenotypes of 900 genes were also stored in82

Genetic Informatics Laboratory

the database. All the images and annotations arearranged based on the corresponding cDNA clones. All
the data are integrated with the genome map basedon WWW, and the information of maps and theirrelations are displayed visually. Based on the results,
we have carried out various collaborations (Kunitomo,
H. et al. 2005, Lazakovitch, E. et al., 2005). The latest
version is opened through the following URL. (http)/
nematode.lab.nig.ac.jp)
We have started EST/genome analysis of anothernematode Diploscapter sp. in collaboration with Dr.
Einhard Schierenberg at Univ. of Koeln. Weareinterested to compare gene expression and function
between different developmental systems, and thisnematode is one of the case, which has a different cellcleavage and arrangement pattern until gastrulation
stage. EST sequences got from about 70.000 cDNA
clones were classified and about 10,000 clusters wereidentified. About 5,800 clusters of them were found tohave homologs or orthologs to C.elegans. To identify
homologues systematically, we also did whole-genome
shotgun sequencing of Diploscapter sp. and got readsof xl.O amount of the genome size. They wereassembled preliminarily and the contigs werecompared to C.elegans peptides by use of WormPep.
We found additional 1,000 homologs in the contigs. All
the results were also incorporated to NEXTDB.
(2) The C. elegans RUNX transcription factor MAB.2/
RNT·l is required for asymmetrical cell division ofthe
T blast cell.

Hiroshi Kagoshima, Hitoshi Sawall, Shohei Mitani 2),
Thomas R. Burglin3), Katsuya Shigesada4), Yuji
Kohara1) RIKEN Center for Developmental Biology
2) School of Medicine, Tokyo Women's Medical
University3) Department of Biosciences, Karolinska Institutet,
Sweden4) Institute for Virus Research, Kyoto University
The RUNX genes encode conserved transcription
factors, which play vital roles in the development ofvarious animals and human diseases. Drosophila runt
is a secondary pair-rule gene, which regulates embryo
segmentation. Human RUNXl, previously known as
AMLl, is essential for hematopoiesis. C. elegans rnt-l
is co-orthologous to the human RUNX genes. We foundthat RNT-1::GFP is expressed in the HO-2, VI-6, andT blast cells in the embryo, and predominantly in theseam cells during larval to adult stages. rnt-l mutants
exhibit a loss of polarity in the asymmetrical T cell
division in hermaphrodites and abnormal raymorphology in the male tail. Genetic and molecularanalysis revealed that rnt-l is allelic to mab-2. Mutant
analysis suggested that mab-2/rnt-1 is involved inregulating T blast cell polarity in cooperation with theWnt signaling pathway. Expression studies of
GFP::POP-I and TLP-l::GFP reporters in mab-2/rnt-1
mutants indicated that this gene functions upstreamof tlp-l and downstream, or in parallel to, pop-l in thegenetic cascade that controls asymmetry of the T cell
division. All our data suggest that MAB-2/RNT-I
functions with POP-l to control the asymmetry of the
T cell division (Kagoshima, H. et al., 2005).
(3) POLO like kinase, PLK-l,is required for theestablishment and the maintenance of anterior-posteriorcell polarity in C. elegans zygotes
Eisuke Sumiyoshi and Yuji Kohara
Embryonic cell polarity is important to produce
diverse cell lineages from a single cell oocyte in C.
elegans. Establishment of embryonic cell polarity
requires PAR proteins and MEX-5. The asymmetriclocalization of these proteins is important fordiversification of cell lineage. We demonstrate that
PLK-I, a C. elegans POLO-like kinase, is required forthe asymmetric localization of PARol and themaintenance of the asymmetric distribution of MEX-5
and PIE-I in C. elegans zygotes. POLO-like kinase isa widely conserved ser/thr protein kinase and is
involved in the cell cycle progression in manyorganisms from yeast, fly, worm to vertebrate.
However, the role of POLO-like kinase in development
is largely unknown. RNA interference of plk-l caused
the aberrant distribution of P granules to the entire
cytoplasm and the expansion of the distribution of
MEX-5 and PIE-l to the posterior and the anterior
cytoplasm, respectively, after the completion ofpseudocleavage. RNAi of plk-l also caused the
localization of MEX-5 and PIE-I to P granules both in
the anterior and the posterior cytoplasm after thepseudocleavage furrow regressed. Furthermore, PAR-I,
which is localized to the posterior cortex in wild type,
is not localized to the posterior cortex in plk-l (RNAi)embryos. Therefore, plk-l seems to be involved in boththe establishment and the maintenance of cell polarity.
PLK-I is localized to the anterior cytoplasm in I-cellembryos, and to the cytoplasm of the somatic
blastomeres and the P granules in the germline
blastomeres in the later stage. The proper localizationof PLK-I to the anterior cytoplasm at I-cell stage
requires MEX-5 activity. Therefore, PLK-I and MEX-5
may compose a feedback loop that maintains thepolarity in the cytoplasm of C. elegans embryos.
(4) Studies on localization mechanisms ofpos-l mRNAin C.elegans embryos.
Kouki Noguchi, Yuji Kohara
Maternal pos-l mRNA localizes to posterior halfof em bryo at the 1st cleavage and localized to germ
lineage in early embryogenesis. We are trying toelucidate this localization mechanisms.
For this purpose, first, we injected various reporter
RNA into the gonad of adult hermaphrodite andexamined the RNA distribution in embryos by in situ
hybridization when the reporter RNA reached to theembryos (4 hours after the injection). When we useda RNA construct (Cap - pos-l 5'-UTR - lacZ - pos-l3'-UTR - polyA, using pJK370 vector kindly provided
by T. Evans), although the localization of the RNA was
not observed, the RNA signals disappeared after 1st
cleavage, probably due to degradation. Interestingly,
the dagaradation depended on the presence of pos-l3'-UTR. This suggest that a balance between
degradation and protection might produce the localized
distribution of pos-! mRNA. However, thisphenomenon may be caused by the nature of thereporter RNA that are supplied artificially intocytoplasm, therefore, secondly, we are trying to
examine the distribution of reporter RNA that aretranscribed from the genome in nuclei. For thispurpose, by the genomic co-injection method, we
obtained the transformant lines that expressed reporter
RNAs (expression vector are constructed from pJH4.52
vector kindly provided by G. Seydoux) maternally.
Although it took time to device to detect the low level
expression of the reporter construct, currently we are
analyzing these strains.

Genome Biology Laboratory83

(5) Effects of poly (A) elongation on the translational
activation of maternally transcribed mRNA in C. elegans

Yoshiki Andachi, Shuichi Onami l •2, Yuji Kohara CKeio
Univ., 2JST, BIRD)
Many proteins required for early embryogenesis
are produced by the translation of mRNAs that aretranscribed during oogenesis. Maternal gene glp-l, a
C. elegans Notch homolog, determines the fate of cells
derived from AB that is the anterior cell at the two-cellstage. Whereas glP-l mRNA is distributed in both cellsof two-cell stage embryos, the mRNA is translated onlyin the AB cell. The translational regulation is directedby 3' untranslated region (3'UTR) of the mRNA, where
two sites are present, Temporal Control Region
responsible for translational repression from gonad to
one-cell stage, and Spatial Control Region responsible
for translational repression in the posterior cell PI at
the two-cell stage. The 3'UTR interacts with threeprotein factors, GLD-l, POS-l and SPN-4, which workfor translational repression throughout the stages, fortranslational repression in the PI cell, and fortranslational activation in the AB cell, respectively.
Although the roles of poly(A) in translational regulation
have been studied in several model organisms,
ambivalent data are preventing to make clear
conclusion. We previously reported correlation between
translational activation and poly(A) length of glP-lmRNA, that is, the poly(A) length of glp-l mRNA wasshortened to 40 bases in oocytes, and was slightly
elongated to 70 bases in the PI cell and was highly
elongated to 160 bases in the AB cell.
To further confirm the correlation, we measuredpoly(A) length of glp-l mRNA in mutants where glP-ltranslation is affected. poly(A) length of glp-l mRNA
was 150 bases in pas-l mutant at the two-cell stage,
whereas the length was less than 30 bases in spn-4
mutant at the two-cell stage, indicating correlation
between translational activation and poly(A) length of
glP-l mRNA in these mutants. Next, we preparedsynthetic mRNA consisting of lacZreporter sequence,
glp-13'UTR sequence and poly(A) chain, and injected
it into gonad of wild type worms. Translation ofsynthetic mRNA whose poly(A) length was 30 bases
was regulated in the same manner as the endogenous
glP-l mRNA. On the other hand, synthetic mRNA
whose poly(A) length was 150 bases was activelytranslated in gonad, though translation of the mRNA

84

Genome Biology Laboratory

was restricted in AB lineage cells of the embryo. Sincepoly(A) length of the injected mRNA turned out to be
shortened by the onset of embryogenesis, we were notable to evaluate the effects of long poly(A) chain on theactivation oftranslation in embryo. Nevertheless, theresults suggest that long poly(A) chain can cancel thetranslational repression of glP-l mRNA in gonad.
(6) A physical model for C. elegans embryo: towards

computer simulation of gastrulation.

Atsushi Kajita, Masayuki Yamamurall , Yuji KoharaI) Tokyo Institute of Technology
Cellular arrangements are important for cell-fate
determination in development. In C. elegans embryos,
the arrangement of cells is largely restricted byphysical conditions: the existence of a hard egg shell;
the properties of membrane and cytoskeleton; thespecificity of cell adhesion; and the force and directionof cell division. In order to understand the mechanisms
underlying the dynamic cellular arrangements, we havestudied computer simulations of C. elegans early
embryos. We previously constructed a physical modelof cells for C. elegans embryo using deformable anddividable polyhedrons, in which we modeled cell
division as the main force generator. This modelingwas based solely on mechanics of cells in order tomake the model as simple as possible. We found thatthe model successfully simulated the cellulararrangements in wild type embryos up to the 8-cellstage, in embryos whose eggshell were removed, andin laser-ablated embryos.
To take the next step, we applied our model withsome extensions to mutant embryos and the embryos
at gastrulation. For this purpose, we added two
structures to the model: microtubules(MT) andcytoplasm. Here we report computer simulation resultsby the extended models.
First, we modeled MTs by thin spring rods which
compose a spindle. We found that the model could
generate the same cellular arrangement and cleavage
pattern at the I-cell stage as those of real embryos inwild type and zen-4 mutant. In zen-4 mutant, the center
connection of a spindle is not bridged between two
centrosomes, thus two centrosomes separate fasterthan those in wild type. This phenotype was simulatedby the model.
Second, we added cytoplasm as an elastic body,and implemented direct interactions between cells: thespindle elongation force is directly transferred froma cell to the neighboring cells, and the friction force
is directly exerted within a cell to the spindle that
contacts eggshell. Although the orientation of the two
daughter cells of E cell were still unsatisfactory, ourmodel simulated essentially the cellular arrangementsof embryos up to the 26-cell stage.
(7) Semi-automatic system for the creation of cell shapemodels in C. elegans embryogenesis

Hideaki Hiraki, Yuji Kohara.
Cell to cell interactions play critical roles in early
embryogenesis, therefore, it is very important to haveinformation about the arrangements of cells, cell
shapes and the contact among them. Early
embryogenesis of C. elegans can be analyzed utilizingtime-lapse recording of multiple optical sections witha Nomarski DIC microscope. Though cell shapemodels of early embryo can be reconstructed from DIC
images, delineating cell boundaries becomes harder as
the cell number increases. Conversely, cell shapes canbe directly visualized by confocal fluorescent
microscopy with vital stain of plasma membranes. We
have been developing a computer system to create cell
shape models from a time series of confocal imagesof plasma membranes.
In this system, cell shapes are automaticallycalculated by a seeded region growing algorithm froma 3D image and a set of seed point coordinates. Manual
editing of the seed coordinates is assisted by agraphical user interface. The region growing algorithm
segments the image volume in such a way that one
region contains one seed. A cell boundary is detected
as ridges of high fluorescent signal between two seedpoints. We tested the system on the "dub" data set in
WORM 4D CDs kindly provided by Bill Mohler, andobtained cell shape models successfully for the mostpart. However, we encountered a problem that some
contours of cells weren't delineated correctly where thesignal to noise ratio was low.
This time, we reinforced the system with thefollowing two methods to fix the problem. One is tomerge some regions after the region growing step. By
this method, a cell could be represented by a few
regions and the noise-derived false region boundary
became minimized. Another one is a correction method

based on the comparisons of the segmentation results
among neighboring timepoints. Even if the cell shapeis delineated incorrectly, the images of its neighboring
timepoints can be sometimes processed correctly.
However, because cells deform during development,
comparing cell shapes among the neighbor timepoints
is not straightforward. Therefore, we devised a
program to calculate a smooth deformation field
between two timepoints assuming that cells deformed
continuously. Some errors of the segmentation could
be corrected by voting among the candidate shapesthat were generated by deformation of thesegmentation results from the neighbor timepoints.
Weare improving the system more, and plan to
apply this system to compare the cellulararrangements and the cell-to-cell contacts among
mutant embryos and the embryos from other species
closely related to C. elegans.
(8) Towards comparative genomics: Genome/EST
sequencing

Takanori Narita, Kazuko Oishi, Fumiko Ohta,
Tomomi Morishita, Tomoko Endo, Keiko Nogata,
Akiko Hase, Hisayo Nomoto, Noriko Hasegawa,
Masumi Mizukoshi, Etsuko Yokoyama, N anayo
Ishihara, lunko Miyamoto, Shigeko liyama, Tadasu
Shin-i, Toshinobu Ebata, Yohei Minakuchi, Kumiko
Kawaguchi, Naoko Sakamoto, Yasuko Sugiyama, Yuji
KoharaAs a core facility of the group grant "Genome"(consisted of about 170 labs) and the National
BioResource Project, we are running a DNA sequencing
center. Currently our capacity is 12 million reads per
year. This year, we have carried out the sequencingof the followings;(a) EST: Ascidian Ciona savignyo (Nori Satoh, Kyoto
D.), Ascidian Halocynthia roretzi (Kazuhiro Makabe,
Tokushima D.), related species to Drosophilamelanogaster (Masatoshi Yamamoto, Kyoto Inst.
Tech), starfish (Takeo Kishimoto, Tokyo Inst. Tech.),Yeast Schizosaccharomyces pombe (Chikashi Shimoda,
Osaka City D.), tomato Lycopersicon esculentum(Hiroshi Ezura, Tsukuba D.), wheat Triticim aestivum
cv. Chinese Spring (Yasunari Ogiwara, Kyoto Pref. u.).
High quality sequences were obtained with 70-90% of
the reads (depending on the samples) and depositedin DDB]. The analyzed cDNA clones are also

Genome Biology Laboratory85

distributed from this laboratory. (Suzuki, T. et al., 2005;
Eichinger, L. et al., 2005; Kijimoto, T. et al., 2005;
Shoguchi, E. et al., 2005)(b) SAGE: Medaka fish Oryzias tatipes (Hiroyuki
Takeda, D. Tokyo),(c) Genome (BAC, fosmid clones): silkworm (Tohru
Shimada, Tokyo D.), platypus Orinihorhynchus
anatinus (Fumitoshi Ishino, Tokyo MDD), Acidian
Ciona intestinatis (Nori Satoh, Kyoto U.), medaka fishOryzias tatipes (Hiroyuki Takeda, U. Tokyo).(Harumi
Terasaki, H. et al., 2005)(d) Genome (whole genome): Medaka fish Oryzias
tatipes (800Mb) (Hiroyuki Takeda, Shin-ichi Morishita,
U. Tokyo), freshwater choanoflagellate Monosiga ovata(Naoyuki Iwabe, Kyoto u., Asao Fujiyama, NIl), mouse
MSM strain (Toshihiko Shiroishi, NIG)
PUBLICATIONSPapers
1. Elena Lazakovitch, John M Kalb, Reiko
Matsumoto, Keiko Hirono, Yuji Kohara, Richard M
Gronostajski (2005) nfi-l affects behavior and life-spanin C. elegans but is not essential for DNA replicationor survival, BMC Developmental Biology, 5, 24 - 24
2. Hirofumi Kunitomo, Hiroko Desugi, Yuji Kohara,
and Yuichi Iino (2005) Identification of ciliated sensory
neuron-expressed genes in Caenorhabditis elegans
using targeted pull-down of poly (A) tails, Genome
Biology, 6 (2 R) 17 - 17
3. Hiroshi KAGOSHIMA, Hitoshi SAWA, Shohei
MITANI, Thomas R. BURGLIN, Katsuya SHIGESADA
and Yuji KOHARA (2005) The C. elegans RDNXtranscription factor MAB-2/RNT-l is required forasymmetrical cell division of the T blast cell,
Developmental Biology, 287, 262 - 273
4. L. Eichinger, ]. A. Pachebat, G. Glockner, M.-A.
Raj andream, R. Sucgang, M. Berriman, ]. Song, R.
Olsen, K. Szafranski, Q. XU, B. Tunggal, S.
Kummerfeld, M. Madera, B. A. Konfortov, F. Rivero,
A. T. Bankier, R. Lehmann, N. Hamlin, R. Davies, P.
Gaudet, P. Fey, K. Pilcher, G. Chen, D. Saunders, E.
Sodergren, P. Davis, A. Kerhornou, X. Nie, N. Hall, C.
Anjard, L. Hemphill, N. Bason, P. Farbrother, B.
Desany, E. Just, T. Morio, R. Rost, C. Churcher, J.
Cooper, S. Haydock, N. van Driessche, A. Cronin, I.
Goodhead, D. Muzny, T. Mourier, A. Pain, M. Lu, D.
Harper, R. Lindsay, H. Hauser, K. James, M. Quiles,

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Genome Biology Laboratory

M. Madan Babu, T. Saito, C. Buchrieser, A. Wardroper,
M. Felder, M. Thangavelu, D. Johnson, A. Knights, H.
Loulseged, K. Mungal, K. Oliver, C. Price, M. A. Quai,
H. Drushihara, ]. Hernandez, E. Rabbinowitsch, D.
Steffen, M. Sanders, ]. Ma, Y. Kohara, S. Sharp, M.
Simmonds, S. Spiegler, A. Tivey, S. Sugano, B. White,
D. Walker, ]. Woodward, T. Winckler, Y. Tanaka, G.
Shaulsky, M. Schleicher, G. Weinstock, A. Rosentha,
E. C. Cox, R. L. Chisholm, R. Gibbs, W. F. Loomis, M.
Platzer, R. R. Kay,]. Williams, P. H. Dear, A. A. Noegel,
B. Barrell and A. Kuspa (2005) The genome of the social
amoeba Dictyostelium discoideum, Nature, 435 (7038)
43 - 57
5. Takashi Suzuki, Tadasu Shin-I, Asao Fujiyama"
Yuji Kohara and Masanori Kasahara (2005) Hagfish
Leukocytes Express a Paired Receptor Family with a
Variable Domain Resembling Those of Antigen
Receptors., J. Immunology, 174,2885 - 2891
6. Teiya Kijimoto, Masakatsu Watanabe, Koji
Fujimura, Masutni Nakazawa, Yasunori Murakami,
Shigeru Kuratani, Yuji Kohara, Takashi Gojobori and
Norihiro Okada (2005) cimpl, A Novel Astacin Family
Metalloproteinase Gene from East African Cichlids,
Is Differentially Expressed Between Species During
Growth, Mol. BioI. Evol, 22 (8) 1649 - 1660

H. STRUCTURAL BIOLOGYCENTER

H-a. Biological Macromolecules Laboratory
Makio Tokunaga Group(1) Single Molecule Imaging, Quantitative Analysis andModel Simulation of Nuclear Transport in Cells
Makio Tokunaga and Naoko Imamotd eCellular
Dynamics Laboratory, Riken)
What is a key to enter the nuclei through nuclear
pores? This question has been answered by visualizing
single molecules inside cells. Little is known about the
specific molecular interactions between the assembled
nuclear pore complex (NPC) and molecules innucleocytoplasmic transport. Using novel microscopy
we were able to clearly visualize single fluorescentmolecules during transport on the nuclear envelope inpermeabilized cells and in living cells. Kinetic
parameters of the interactions between translocating
molecules and NPCs were obtained. Two types ofbinding were found: weaker binding site gathers up
to '"100 molecules/NPC, concentrating su bstrates
locally; stronger binding site, where up to "'8molecules/NPC are bound, changes its affinity
drastically upon nuclear accumulation ability. In thepresence of RanGTP, the stronger binding was notfound and the active site emerges. Retention times bysingle molecule analysis and translocation rates bysingle nuclear analysis showed a correlation with acoefficient of "'8 molecules/NPC, which exhibits the
stoichiometry of the active site. Computer simulation
was carried out based on parameters obtained bysingle molecule analysis. Simulation based on a modelwith two types of multi-binding sites using theseparameters well explained the molecular kinetic
features in cells.

(2) Identification and Characterization of AssociatedProteins with RNGI05, An RNA-Binding Protein
Responsible for Neuronal Local Translation
Nobuyuki Shiina, Kazumi Shinkura and Makio
Tokunaga
We have identified and characterized RNG105associated proteins: Caprin-2, G3BP and RACKl.
Caprin-2 is a homolog of RNG 105, and has thecoiled-coil domain in the N-terminal and the RGG-box
in the C-terminal as well as RNG105. Caprin-2 showed
RNA-binding and translation-inhibition activities, andthe N-terminal coiled-coil domain was required for theactivities. These characteristics of Caprin-2 were very
similar to those of RNG105. Caprin-2 was expressed
highly in brain, and localized in granular structuresin neurons. Caprin-2 was colocalized with RNG105 insome of the granules in the dendrites, but was not
colocalized in other granules, especially in the cell body.
We have identified G3BP (RasGAP SH3-binding
protein) as an RNG 105-associated protein byimmunoprecipitation, and some papers also reported
that G3BP is associated with RNG105. G3BP is reported
to be an RNA-binding protein and localized in stressgranules in cultured cells, which are similar to the RNA
granules in neurons. We found that G3BP was bounddirectly to RNG105 in vitro, and that G3BP regulated
the RNA-binding specificity of RNG105 in vitro. G3BP
was highly expressed in brain. Its localization inneurons was mainly in the cell body, but some of G3BP
was localized with RNG105 in the dendritic RNA
granules.
RACKI (receptor for activated protein kinase C1) is reported to bind to ribosomal 40S subunit andregulate translation by controlling 40S-60S docking.
RACKI is also reported to be associated with G3BP
and RNG105 in polysome fractions. We found that
although RACKI was not bound directly to RNG105,
it was co-immunoprecipitated with RNG105 from rat
brain extracts. We transfected cultured cells with
RACK1-GFP fusion protein along with RNG105-RFP
fusion protein, and found that the localization of
RNG105 in the cytoplasmic granules was canceled by
the co-expression of RACKl. In rat hippocampal
neurons, RACKl was localized diffusely in the cellbody and the proximal dendrite, where
RNGl05-positive RNA granules were not detected
although they were located in the distal dendrites.

STRUCTURAL BIOLOGY CENTER

87

These results suggest that RACK1 may facilitate
RNG105 release from the RNA granules or RNA
granule disassembly.
(3) Single Hydrogen Bonds of DNA Base Pairs Detected

in Unzipping Force by Intennolecular Force Microscopy

Michio Hiroshima! and Makio Tokunaga CSingle
Molecule Immunoimaging, Research Center for Allergy
and Immunology, RIKEN)
Single hydrogen bonds of DNA base pairs havebeen measured by unzipping double-stranded DNA
oligomers with an intermolecular force microscope(IMF). IFM has achieved the force resolution of
subpico-newton using ultrasensitive cantilevers. The
probe position is controlled with nanometer accuracy
using a feedback system, which uses laser radiationpressure to reduce thermal fluctuation of the cantilever.
Previous methods of mechanical unfolding often
yield "stick-slip" processes, reducing the resolution.
The stick-slip processes are drops in the force caused
by a sudden increase in the extension of the unfolding
molecule. The feedback system eliminates the stick-slip
process by controlling the probe tip position by laser
radiation pressure.
DNA is bridged between a substrate and a beadbound to the tip of the cantilever. The duplex in thestem region was unzipped by pulling up the DNA end
bound to the cantilever tip. Force vs. extension curves
showed repeated force peaks of 1O-15pN. Auto- or
cross-correlation analysis and averaging of force curves
were carried out to reduce noises in the force curve.
The previous studies showed that the force forseparating poly(G-C) DNA was 1.5 to 2 times stronger
than that for poly(A-T). However, no difference wasfound in the force between individual G-C and A-T
base pairs. The force curve of individual G-C and A-T
pairs showed three and two peaks, respectively, which
are assigned to single hydrogen bonds. The force isvariable but the work is constant. The work to breaksingle hydrogen bonds is 1.3 kB . T, in other words,
about 1.3"fold of the thermal energy. This is the firstreport to detect the force of single hydrogen bonds inbiological macromolecules.

88

Biological Macromolecules Laboratory(4) Single Molecule Detection of Mechanical Unfoldingof Proteins

Akihiro Fukagawa, Michio Hiroshima! and Makio
Tokunaga CSingle Molecule Immunoimaging,Research Center for Allergy and Immunology, RlKEN)
Single molecule measurement of the mechanicalunfolding of staphylococcal nuclease (SNase) has been
carried out using the intermolecular force microscope(IFM). Cysteinyl residues at both the N- and C-terminiof SNase was attached to gold-coated surfaces of an
IFM probe and a glass substrate. Previous methodsof mechanical unfolding often yield "stick-slip"
processes, reducing the resolution. The stick-slip
process was eliminated by the feedback system withlaser radiation pressure. Therefore, force-extension
curves by IMF showed a clear breakdown of the force
due to structural changes. It has yielded more than 10times better resolution in the force-displacement
detection than previous methods.
Clear force-extention curves corresponding tosingle molecules were obtained. The extension distanceof approximately 55 nm showed accordance with thecalculated distance of the single-molecule extension,
54 nm (0.36 nm/residues by 149 residues). The
force-extension curves of single molecules showedsome peaks, which corresponding to unfolding of
subdomain structures. A few types of peak patterns
were found, which indicates some pathways in theunfolding. Weare demonstrating whether the finepeaks in the force-extention curves are attributed tosingle alpha-helices, single beta-sheets and single turns.
(5)

Visualization of initiation of T cell activation

Kumiko Sakata-Sogawat, Michio Hiroshimat, Tadashi
Yokosuka2, Sho Yamasake, Takashi Sait02, andMakio Tokunaga CSingle Molecule Immunoimaging,
2Cell Signaling, Research Center for Allergy and
Immunology, RIKEN)
Immunological synapse (IS) has been thought tomediate antigen recognition and activation of T cells.
Although recent studies suggest that the activation
signals precede IS formation and sustain for hours, thesites for initiating and sustaining TCR signals remain
elusive. We have developed a "time 0 method" andclearly visualized initiation of T cell activation fromthe moment of the activation. We reported that T cell
activation is initiated and sustained in TCR-containing
microclusters (MC) generated at the initial contact
areas and at the periphery of the mature IS. MCs
containing TCR, ZAP-70 and SLP-76 are continuously
generated in the periphery. MCs containing TCR,
ZAP-70 and SLP-76 migrate toward the cSMAC, but
SLP-76 disappeared completely and ZAP-70 was lostmostly before the MCs join the TCR rich cSMAC.
Tyrosine phosphorylation and Ca2+ influx was
induced as MCs formed in the initial contact sites.
These results suggest that TCR MCs initiate andsustain TCR signals.
(6) Three-dimensional Multi-color Visualization and

Quantitative Analysis of Nulcear Fine Structures

Michio Hiroshima\ Kumiko Sakata-Sogawa\ Kazumi
Shinkura, Kyoichi Ison0 2, Haruhiko Koseki 2, andMakio Tokunaga eSingle Molecule Immunoimaging,
2Developmental Genetics, Research Center for Allergy
and Immunology, RIKEN)
Using multi-color molecular imaging, finestructures of distributions of DNA and nuclear proteins
were visualized. Three kinds of fluorescent dyes were
simultaneously examined. Using DNA-labeling dyes
and fluorescently labeled antibodies, distributions of
nuclear proteins were visualized along the string andbeads structures of DNA in the nucleus.
Three-dimensional visualization and molecularquantification was carried out using GFP-fusion
Polycomb Group proteins by HILO spectroscopy
technique in living cells. Three-dimensional images
were reconstructed from a series of z-scanned images.
Defocus was eliminated using deconvolution technique.
The number of molecular clusters in single nuclei and,furthermore, the number of molecules in single clusters
are evaluated on the three-dimensional images. The
present technique opens a new way for quantificationsof three-dimensional distribution, dynamics and thenumbers of multi-molecules in living cells.
(7) Asymmetric nucleocytoplasmic transport revealed
by a novel assay system using planner reconstituted
nuclear envelope

Atsuhito Okonogi, Michio Hiroshima\ Nobuyuki
Shiina, Shingo Kose2, Naoko Imamot02 and Makio

Tokunaga CSingle Molecule Immunoimaging, RCAI,
Riken, 2Cellular Dynamics Laboratory, Riken)
Using a novel in vitro assay system of
nucleocytoplasmic transport, we found asymmetricmolecular interactions with the nuclear pore complexes
between import and export. Import and export ofimportin alpha was examined in the absence of
RanGTP and other soluble factors using the present
cell-free in vitro assay system. Asymmetry between
import and export was discovered in the interactionof importin alpha and the NPC in the absence ofRan-GTP and other soluble factors. In the apparent
equilibrium, import of importin alpha showed adifference in the concentration between at thecytoplasmic side and at the nucleoplasmic side,
whereas export of importin alpha showed nodifference. The concentration difference of importinalpha is found to correlate with the amount of bindingof importin alpha with the NPC. The asymmetry
between import and export was also found in thetransport of importin alpha in the presence of NLS
cargo molecules. In the presence of NLS-protein,
importin alpha showed no biding with the NPC. The
control molecules showed no asymmetry.
Re-addition of importin alpha to the apparently
steady distribution of importin alpha at different
concentrations between "inside" and "outside" ofnuclear envelope showed again the same asymmetric
distribution of importin alpha between "inside"and "outside". The present finding shows that the
NPC has an asymmetric feature in the interactions
with cargo molecules and transport factors between
import and export.
(8) Molecular Imaging of Local Translation in Neuronal
Dendrites by LTP Electrical Stimulation

Hiraku Miyagi, Kumiko Sakata-Sogawa\ Michio
Hiroshima! and Makio Tokunaga eSingle Molecule
Immunoimaging, RCAI, Riken)
Local protein synthesis in neuronal dendrites isrequired for synaptic plasticity, which is associatedwith long-term memory storage. This protein synthesis
is reported to be induced at activated postsynaptic
sites. In order to investigate when and where thetranslation is initiated locally in the dendrites, we
visualized local translation in dendrites of rat

Biological Macromolecules Laboratory89hippocampal neurons after LTP electrical stimulation.
Local translation was visualized by the fluorescenceof GFP translated from EGFP-CaMKIIa 3'UTR mRNA
Only in the vicinity of the tip of electrode, fluorescenceincrease, i.e. local translation was observed. It was
occurred at the root of spines. Shape change of spines
and sprouting of filopodia-like protrusion was also
found in the vicinity of the electrode tip after
simulation.
(9) Development and Optimization of Molecular
Microscopy and Software

Makio Tokunaga, Michio Hiroshima1and Kumiko
Sakata-Sogawa1 CSingle·Molecule Immunoimaging,
RCAI, Riken)
Weare presently developing and optimizing theHighly Inclined and Laminated Optical sheet (HILO)
microscopy technique for multi-color imaging. Stable
observation during long time was achieved by a novel
autofocusing system using total internal reflection withnear-red sensing light, whose resolution of the heightmeasurement of the specimen was higher than 0.1
micrometer. The resolution of images has been refined
especially by development of a high-sensitive camera
and an objective lens. Control system of the microscopewith a personal computer has been developed in both
hardware and software. Weare also developing asoftware system of single molecule imaging for image
analysis and quantification of molecular interactions
and kinetics.

activation by recruitment of Zap70 and SLP-76, Nature
Immunol, 6 (12) 1253 - 1262
Books

4. Tokunaga M
1i¥~(2005)(~;;tdt'!l1t:t· mjlj{}~~~

r5tr.:c-7 - J !Io/Jf1I!"¥*. fI:lJi.=:;fMW)~1fifJi5766 - 775
EDUCATION~~~/~~~OO~~~~~t~T-

1. Dr. M. Tokunaga gave a Seminar at Institute of
Molecular and Cellular Biosciences, the University ofTokyo, February, 2005 (in Japanese).
2. Dr. M. Tokunaga gave a Seminar at Yukawa
Institute, Kyoto University, December, 2005 (inJapanese).
~~f&~~

Tokunaga M

f4"¥:a-1ftf:J'{;"¥mi~~

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SOCIAL CONTRIBUTIONS AND OTHERS~Hi~~

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PUBLICATIONSPapers

1. Kitamura K., Tokunaga M., Esaki, S., Iwane A
H. & Yanagida T (2005) Mechanism of muscle
contraction based on stochastic properties of single
actomyosin motors observed in vitro, BIOPHYSICS,
1, 1 - 19
2. Shiina N., Shinkura K., Tokunaga M (2005) A novel
RNA-binding protein in neuronal RNA granules:
Regulatory machinery for local translation, J.
Neuroscience, 25 (17) 4420 - 4434
3. Yokosuka T., Sakata-Sogawa K., Kobayashi, W.,
Hiroshima, M., Hashimoto-Tane, A, Tokunaga M.,
Dustin, M. L., Saito, T, (2005) Newly generated T cell
receptor microclusters initiate and sustain T cell

90

Biological Macromolecules Laboratory

H-b. Molecular Biomechanism Laboratory
Nobuo Shimamoto GroupRESEARCH ACTIVITIES(1) Regulatory Mechanism of transcription initiation byswitching conformations of RNA polymerase: ahomogeneous protein works inhomogeneously.

Motoki Susa', Takashi Miyamoto\ and Nobuo
Shimamoto ' CStructural Biology Center, National
Institute of Genetics and and Department of Genetics,

School of Life Science, The Graduate University for
Advanced Studies)
For several decades, all RNA polymerase once
initiating formation of phosphodiester bonds at a
promoter (the initiation complex) has been supposed
to synthesize mature transcript. Accordingly, themechanism of transcription initiation has been
assumed to be a sequence of three essential steps:
formation of a complex of RNA polymerase and a
promoter (closed complex), isomerization into acomplex with a DNA bubbled duplex, (open complex),and escape of RNA polymerase from the promoter
associated with elongation of RNA (promoter
clearance). All RNA polymerases so far examined invitro are known to catalyze not only the synthesis offull-length RNA but also abortive synthesis, which isan iterative synthesis and release of larger amountsof oligo-RNA molecules, typically 2 to 15 nucleotidesin length. In the conventional sequential mechanism,abortive synthesis is often excluded from themechanism or supposed to be stochastic failure by thehomogeneous initiation complex. This sequential
model is based on the idea that a homogeneous
fraction of a protein should work homogeneously,
which is a basic hypothesis used in studies on various
biological systems.
However, we found that this idea is wrong at least
for transcription in E coli. We identified a new formof initiation complex, named moribund complex, which
exclusively synthesize abortive transcript. Since 1996,
we show it is
1. generated from homogeneous RNA polymerase[JMB 256, 449 ('96), Genes Cells 6, 389 ('01)]
2. the major source of abortive products on a APR
promoter and mitigated by GreAIB [the same refs in 1]
3. arrested at the promoter with a different structurefrom productive form [JBC 275, 10899 ('00)]
4. detected as persistent abortive synthesis after
completion of full-length synthesis [JBC 277, 15407 ('02),
the same refs in 1]
5. induced by force and could work as a safety valve
for misincorporation [NAR 25, 2640-47 ('97)]
In 2005, we finally proved that moribund complex
contributes transcriptional regulation in E coli cells
at least for three genes (atp, cspA, rpsA)ffief. 1 (2005)]
To examine the existence and significance of the
branched pathway in vivo, we selected GreA and GreB
for clues. At the A PRAL promoter 'these factors

enhance conversion of the moribund complex into the
productive one, in the presence of high concentrationsof initiating nucleoside triphosphate in vitro. If the
branched mechanism exists in vivo, absence of the Gre
factors should result in reduction of productive
transcription from promoters at which the moribund
complex is susceptible to these factors. We constructeda double-disruptant of Ecoli, 11 greA 11 greB, and thenarbitrarily selected 10 genes from among those whoselevels of transcripts in the mutant strain were foundto be lower than those in the parental greA+greIr
strain. Finally, the promoter for three of these genes,
atpC (uncC), cspA, and rpsA, passed a further
conventional test which confirmed that they displayeda branched initiation pathway in a reconstituted
transcription system composed of purified
components. The results obtained prove that thebranched initiation pathway exists in vivo and isutilized in regulation of transcription initiation fromsome promoters, through modulation of the fractionof polymerase-promoter complexes entering eachbranch of the pathway.
In this year, we examined various physiologicalconditions that affect on the levels of the Gre factors.
The determination of the level of GreB was observed
to be constitutive. The level of GreA remained the same
through the growth phase, and did not respond muchto the richness of the culture media. However, it
decreased into half in aerobic conditions, indicatingthat some genes are regulated by GreA. Therefore, theregulatory circuit responding to the levels of proteins
involving GreA, namely the branched pathway
mechanism, is working in cells.
(2) Applicability of thermodynamics to equilibria inbiology.

Nobuo Shimamotd and Jyun-ichi Tomizawa\
CStructural Biology Center, National Institute of
Genetics and Department of Genetics, School of Life
Science, The Graduate University for Advanced
Studies)
Most DNA-binding proteins are biologicallyfunctional as a specific complex, one containing a
special short DNA segment. Such a complex is usuallyassumed as a state tenable for thermodynamic analysisof binding equilibrium. Thus, forward and backward
reactions should balance at equilibrium in every

Molecular Biomechanism Laboratory91

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.
(3) Systematic search for promoters encoded in the
genomic DNA sequences of E. coli

Nobuo Shimamoto\ Hideki Nakayamal and Hironori
Aromake CStructural Biology Center, National
Institute of Genetics and Department of Genetics,
School of Life Science, The Graduate University for
Advanced Studies. 2Daiichi College of Pharmaceutical
Sciences)
Irrespective of a pile of compiled sequences
identified as promoters for E. coli vegetative RNA
polymerase holoenzyme, u 70 holoenzyme, there is no
successful methods to predict strength of a promoter.
To construct such prediction method, we started todesign a functional SELEX to select promoter
sequences. The complexity of random oligo-DNA
available in a lab scale,4lH4 is too small to cover the
all-possible promoters. Therefore we limited thecandidate sequences to those involved in the genomic
sequence. In order to construct and select a library, wetransferred and amplified parts of the genomic
sequence with PCR with a single primer. As adrawback of the use of single primer, the constructedlibrary contains DNA fragments generated from invitro recombination. A theoretical method to diminish
the effect of such recombinants has been developed.
PUBLICATIONSPapers1.
)1I0.~-f-, ~.\\*{$U:fH2005) j- J @]'iH;6~aJi;(p 1...- tr.
DN A~i!I-7 ://-\7'lt0)?¥~, Bionics, 2, 62 - 63+

92

Molecular Biomechanism LaboratoryBooks
2. ~.\\*{$H:l, Ed. (2005), j- J rC{:::tAr'.1 vols. Tokyo:-lj- -1 .:r. /' A f±.
3. ~.\\*{$U:l (2005). j- J /'\-1:::t7'" 7 J 0 :;- Ilfi~{t:Jt
J!IDl31E(j- J /'\-1:::t 0 - F""'<' 'Y 7°) $R~. NEDO~~,~~~15­

International
4. Miyamoto T.,Susa,M. and Shimamoto N. Coupling
between transcription initiation and DNA
damage, "The 9th Asian-Pacific Conference on
Transcription", Miaoli, Taiwan, 2005
5. Nakayama H., Shimamoto N., A system forsingle-molecule dynamics of translation "The 9th
Asian-Pacific Conference on Transcription", Miaoli,
Taiwan, 2005
6. Shimamoto N., Chemical and Biologicalconsequences of one-dimensional diffusion of proteins
along DNA "International Congress of Biorheology andInternational Conference on Clinical Hemorheolog",
Chongqing, 2005, 5/30
7. Shimamoto N., Chemical processes involved incontrol switches of transcription initiation, FASEB
Meeting "Prokaryotic Transcription Initiation", Saxton
Rever, VT., 2005, 6/21
8. Shimamoto N., Control switches of transcription
initiation (and big questions), "The 9th Asian-PacificConference on Transcription", Miaoli, Taiwan, 2005 12/15
9. Shimamoto N., Ceremonial speech for thepresident Chen-Wen Wu, "The 9th Asian-PacificConference on Transcription", Miaoli, Taiwan, 2005,
12/15
10. Suganthan Rajan Babu, Sato Y,Yagi S., Gyedu
Ampaabeng and Shimamoto N. Unexpected cleavage
occurred in modified sigma70 subunit of active E. coli
RNA polymerase, "The 9th Asian-Pacific Conference
on Transcription", Miaoli, Taiwan, 2005
~i7'J

11. Suganthan Raj an Babu, Sato, Y, Yagi, S., Gyedu
Ampaabeng and Shimamoto N., Unexpected cleavage
occurred in modified sigma70 subunit of active E. coli
RNA polymerase ~43@] B *1:.!jo/J!jo/J.f!I!~~, fLi1JYtm,
2005, 11/23
12. $ UJ *{j:, ~.\\*{$U:l ~ilR 0) 1 5t-f- -7" -1 j- ~ 'Y 7 AO)lJlIJfE~O)t1l~, RNAtiUIHIl!J~ 3 @]-lj-7'"7-1 "~-7'"-1/' 7', W:M, 2005
II $UJ*{j:, ~*~., W• • ~ilRo)l5t-f-Y1j-

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2005, 11/23

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EDUCATION

1. Dean of School of Life Science, Graduate Schoolof Advansed Studies
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SOCIAL CONTRIBUTIONS AND OTHERS
1MI~~

1. ~*{$:/;;t, Jt$M~ltI'M~ttfillj· ~fiIlj~ili~:w:r~~~2.

~.i*{$:/;;t, NEDO r ~ / /~~)jU~1b~~,*7" 7 /

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1. ember of international Committee of Asian
Conference of Transcription (ACT), the delegate ofJapan. (Organizing members of ACT8, Bangkok,
November, 2004)
2. ~.~*{$:/;;t, M~¥!A*lmM~ttfilljtbb~r~iIr~i1lIj~J*ll..~$(1) Enhancement of chemotaxis by pre-exposure toodorants and food in the nematode C. elegans

2005

g*1t!E, ~fti:j(W, ~.~*{$:/;;t, DNAO)UVm{~H~~~~&M

H-c. Multicellular Organization Laboratory
Isao Katsura Group

Ichiro Torayama, Hiroshi Ichijo, Kotara Kimura,Takeshi Ishiharal and Isao Katsura CKyushu
University)
The nematode C. elegans provides a good system
for the analysis of behavioral plasticity at the levels
from molecules to organisms. We have found thatpre-exposure to butanone and food enhances
chemotaxis to butanone but not to benzaldehyde,
although butanone and benzaldehyde are sensed by
the same olfactory neurons called AWe. Thisodor-specific plasticity, which we call butanone
enhancement, is different from the already known
plasticity in which food inhibits olfactory adaptation.
In the latter case, the chemotaxis index never exceedsthat of naive animals, and food can be replaced byserotonin, which is not true for butanone enhancement.
Butanone enhancement is beneficial to animals fortheir survival and proliferation, because it helps themfind food at higher probability. It resembles classicalconditioning in this aspect, but differs in the following
aspects. (a) Animals are attracted by the conditioned
stimulus (butanone) even without conditioning, while
conditioning only enhances the degree of attraction.
(b) The conditioned stimulus must be presented at thesame time as and not before the unconditioned
stimulus (food). To elucidate the molecular mechanism,
we isolated ten mutants that showed abnormality inthis plasticity. Of those mutants, olrn-l(ut305) andolrn-2(ut306), which showed the strongest abnormality,
were studied in detail. olrn-l gene encoded a novel
protein containing predicted transmembrane domains
and showing limited homology to the Drosophila Raw
protein. Expression of olrn-l in AWC sensory neurons
was sufficient for normal butanone enhancement. The
olrn-l(ut305) mutant showed abnormality in theneuronal asymmetry of AWC neurons: the wild-type
animal expresses str-2 gene in only one of the two
AWC neurons, but the olrn-l(ut305) mutant expresses
str-2 in neither of the AWC neurons. We also cloned

Molecular Biomechanism Laboratory93

olrn-2 gene, which was allelic to bbs-8 gene, one of theBardet-Biedl syndrome genes. The olrn-2(ut306)
mutant showed the wild-type phenotype in the AWC
asymmetry.
In 2005, we obtained the following results. (1) Since
the olrn-l mutant does not express str-2, we
hypothesized that the AWC ON neuron, which expresses
str-2, is required for butanone enhancement. Thishypothesis was proved by various experiments,
including the butanone enhancement assay of
AWCoN-killed wild type animals. (2) The olrn-2(ut306)
mutant showed structural defects in sensory cilia likeknown Bardet-Biedl syndrome mutants. We therefore
tested many cilium structure mutants for butanone
enhancement. The results revealed that all theBardet-Biedl syndrome mutants tested, olrn-2/ bbs-8,
bbs-l, and osm-12/ bbs-7, were abnormal, while othercilium structure mutants, including osm-3, dyj-l, daf-IO,
che-ll, osmol and che-2 mutants, were normal inbutanone enhancement. (3) We found that pre-exposure
to isoamyl alcohol and food enhances chemotaxis toisoamyl alcohol, and isolated mutants defective in thisbehavioral plasticity.
(2) Genetic analysis of plasticity of avoidance behaviors

in C. elegans

Kotaro Kimura and Isao Katsura
Although C. elegans exhibits several types of
behavioral plasticity, the plasticity of its avoidance
behaviors has not been studied enough. We havediscovered a novel type of plasticity in the avoidance
response of C. elegans. The avoidance response of theanimal to repellent odors was enhanced, rather thanbeing reduced, after preceded exposure to the odor. It
was somewhat surprising because almost all of thepreviously known stimuli (including other repellent
stimuli) to the animals cause adaptation, i.e., thered:uction of the sensory response after preceded
exposure. In general, adaptation has been extensively
studied using various experimental systems. However,
the enhancement of the sensory response has been far
less studied, particularly at the molecular level, withan exception of mammalian pain sensation andgill-withdrawal reflex in Aplysia. Therefore, analyzing
the enhancement of the avoidance response of C.
elegans may lead to the identification of a novel
regulatory mechanism of the sensory response

94

Multicellular Organization Laboratory

enhancement in general.
To gain insight into the molecular mechanism of
the enhancement of 2-nonanone avoidance behavior,
we have analyzed the phenotype of "'60 mutants ofgenes that may play roles in neuronal function, suchas ion channels, neurotransmitter receptors, andsignaling proteins. We found that several mutants
showed specific defects in the enhancement of
avoidance behavior, but not in naive avoidance
behavior itself. We are currently analyzing detail of thephenotype.
(3) Class 1 nrmutants of the nematode Caenorhabditis
elegans

Yuri Kobayashi, Kotaro Kimura and Isao Katsura
Class 1 flr mutants of C. elegans, which map in
fIr-I, flr-3 and flr-4, were isolated originally byresistance to 0.4 mg/ml N aF (Katsura, I. et al: Genetics
136, 145-154, 1994). They also show many otherphenotypes including slow growth, short defecation
cycle periods (Dec-s), frequent skip of the expulsion
step of defecation, synthetic abnormality in dauer larva
formation, weak tendency to stay on food, andhypersensitivity to serotonin. The flr-l gene encodesa cation channel of the DEGIEN aC superfamily, while
flr-4 and flr-3 code for a novel Ser/Thr protein kinase
and a kinase-like molecule, respectively, both having
a hydrophobic domain at the carboxyl terminus. They
are expressed essentially in the intestinal cells, exceptthat flr-4 is expressed also in the isthmus of thepharynx and in a pair of head neurons called AVA. We
consider that class 1 flrgenes constitute a regulatory
system that acts in differentiated intestinal cells.
In 2005, we found that animals expressing theFLR-1 ion channel without the C-terminal cytoplasmic
domain (FLR-lDC) show long defecation cycle periods(Dec-l phenotype). By carrying out RNAi of many genesthat are expressed in the intestine, we identified genes,
the RNAi of which suppresses the Dec-l phenotype of
FLR-lDC and the Dec-s phenotype of the flr-l(utll)
mutant, respectively. We think these genes act in theregulation of defecation cycle periods, and plan toanalyze them in detail.

(4) Class 2 nrmutants of the nematode Caenorhabditis
elegans

Akane OISHI, Kotaro Kimura, Takeshi Ishihara! andIsao Katsura CKyushu University)
Class 2 flrmutations were isolated first as a weakfluoride-resistant mutation and then as suppressorsof the slow growth or serotonin-hypersensitivity ofclass 1flrmutations. Besides these phenotypes, theyalso suppress the dauer larva formation abnormality
and weak tendency to stay on food, but not thedefecation abnormalities or strong fluoride-resistance.
By themselves, class 2 fir mutations show thephenotypes of weak resistance to NaF and shortaverage longevity as compared with wild-type animals.
The phenotypes suggest two possibilities on therelationship between class 1 and class 2 flr genes. (a)Class 2 flrgenes may act downstream of the class 1
regulatory pathway. At the downstream, the regulatory
pathway bifurcates into two branches, the growth/
dauer branch and the defecation branch, while class
2 genes act in the former branch and not the latter. (b)Class 2 flr genes may act antagonistically to class 1
genes, while the threshold of the phenotypes isdifferent between the growth/dauer phenotypes andthe defecation phenotypes.
Class 2 mutations map in four genes, flr-2, flr-5,
flr-6 and flr-7, of which only flr-2has been cloned. flr-2
encodes a secretory protein belonging to the gremlin!
DAN/cerberus family. A functional flr-2.:GFP fusion
gene was expressed in some neurons in the head andthe tail as well as many pharyngeal neurons. We also
found that FLR-2 interacts in vitro with a secretory
protein encoded by ZK20.1 gene, and isolated a deletionmutant in this gene.
In 2005, we obtained the following results. First,
we found that the ZK20.1 mutation increased
defecation cycle periods under certain genetic
backgrounds but not under the wild-type background.
For instance, the ZK20.1; flr-2(ut5) double mutant
showed longer defecation cycle periods than thewild-type or the flr-2(ut5) mutant. Furthermore, theZK20.1; flr-2(ut5) double mutation suppressed theshort defecation cycle periods and the expulsion defectof various flr-4 mutants (ut7, sa201, n2259). Second,although we tried to clone flr-5 gene by the positional
cloning method, we could not succeed because oftechnical difficulties. Therefore, we started to clone flr-6

and flr-7 genes.
(5) Analysis of synthetic dauer-constitutive mutants inthe nematode Caenorhabditis elegans

Tomoko Yabe, Norio Suzukit, Tatsuo Furukawa2,
Takeshi Ishihara3 and Isao Katsura CRiken CDB,
2Kagoshima University, 3Kyushu University)
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. Although mutations showing
abnormality in this phenomenon were isolated tosaturation, the regulation of dauer larva formation has
not been fully elucidated. We, therefore, areinvestigating synthetic dauer-constitutive (synDaf)
mutants, i.e., mutants that form dauer larva in a certain
mutant background even in the presence of plenty foodand at low population density. We found that
mutations in more than 50 known neural genes show
synDaf phenotypes. The synthetic nature of thephenotype, we think, is based on the pathway of
sensory signals. Namely, the signals are transmitted
through parallel routes, and therefore two mutations
are required to block them. Weare determining the
combinations of mutations for the synDaf phenotype
and the pattern of suppression of the synDaf
phenotype by various suppressor mutations. In thisway we hope to elucidate the detailed regulatory
pathway for dauer larva formation.
Furthermore, to identify new genes for the
regulation of dauer larva formation, we have isolated
and mapped 44 mutations that show the synDaf
phenotype in the unc-31(e169) background, where
unc-31 gene encodes CAPS protein, which acts insecretion from dense core vesicles. These mutations
mapped at least to 17 genes, of which sdf-9, sdj-13, andsdj-14 have been cloned. sdj-9 encoded a protein
tyrosine phosphatase-like molecule, was expressed ina pair of neuron-associated cells called XXXLlR, andregulated dauer larva formation in the steroid hormone
signaling pathway. sdj-13 encoded a homologue of thetranscription factors Tbx2 andTbx3, was expressed in
AWB, AWC, and AS] sensory neurons as well as many
pharyngeal neurons, and controlled olfactory
adaptation in AWC and dauer larva formation in cells

Multicellular Organization Laboratory95

other than AWC (possibly AS]). sdj-14 was allelic to
mrp-1, which was formerly identified by its homology
to multidrug resistance-associated protein genes inmammals. A functional sdj-14::GFP fusion gene wasexpressed in many tissues including neurons,
pharyngeal muscles and intestinal cells, and expression
in multiple tissues was required for the wild-type
phenotype. Interestingly, human MRP1 could
substitute for C. elegans MRP-1 in dauer larva
regulation, and an inhibitor of the MRP1 export activity
impaired this function, showing that the export activity
is required for normal dauer larva regulation. sdf-14
mutations strongly enhance the dauer-constitutive
phenotype of the daj-2(e1370) mutation, which confers
resistance to many environmental stresses.
Furthermore, sodium arsenite, which is a substrate ofhuman MRP1, enhanced dauer larva formation in the
background of the unc-31(e169) mutation, which
partially blocks the daj-2 insulin signaling pathway.
Thus, although we could not show direct evidence,
sdj-14 gene may be involved in stress responses.
In 2005, we wrote a paper on sdj-14 and publishedin the journal Development (Yabe et aI., 2005). Wearealso continuing epistasis studies to reveal theregulatory pathway.
PUBLICATIONSPapers

1. Take-uchi, M., Kobayashi, Y., Kimura, K. D.,
Ishihara, T., Katsura, 1.(2005) FLR-4, a novel Serine /Threonine protein kinase, regulates defecation rhythm
in Caenorhabditis elegans., Mol. BioI. Cell, 16, 1355 - 1365
2. Yabe, T., Suzuki, N., Furukawa, T., Ishihara, T.,
Katsura, I. (2005) Multidrug resistance-associatedprotein MRP-1 regulates dauer diapause by its export
activity in Caenorhabditis elegans., Development, 132,3197 - 3207
3. tE ~ (2005)~!R C. elegans(1)j£~ . 7t{t1iJf~.!::: Jf /A1t$R, ~~~~itflj, 23 (l) 157 - 163
EDUCATION

1. tE

~

~ittf:!l!I~$*fJ31j~1U~ I}fOj~I~*~·~ittf:!l!I~$

2. tE

~

~!Rc. elegansQ)fiJJJI1)7t-f-J!B~(f.]fW#f­

fiJJJ(1)$~(f.]~.~~I1)~.-.*I~*~·~ittf:!l!I~$

96

Multicellular Organization Laboratory

SOCIAL CONTRIBUTIONS AND OTHERSGenes to Cells, Associate Edditor
B*7t-f-~!In~4;.>, JA$R~.
B*~!fo/.J!fo/.Jf:!I!~4;.>, ~~H-d. Biomolecular Structure Laboratory
Yasuo Shirakihara GroupRESEARCH ACTIVITIES(1) Crystallographic Study of ATP synthaseYasuo Shirakihara and Aya Shiratori
ATP synthase is responsible for ATP production
in living cells, and is a membrane protein located in
the energy conversion membranes. ATP synthase
consists of a channel Fo portion (about 100,000 dalton,
subunit composition of ab2C8-12) and a large soluble
catalytic F1 portion (380,000 dalton, a3{33YO E). The
unique rotational catalysis mechanism of F1 includes
rotation of the rod-like y subunit, which is thought
to control the conformations of the three catalytic{3 -subunits in a cyclic manner by its rotation. Startingfrom elucidation of the a3{33 sub-assembly structureof the thermophilic F1, we have moved up to the higher
sub-assemblies. After the a3{33yand aa{33YE sub
assemblies, we have been working on the holo-enzyme,
ATP synthase.
ATP synthase is still a challenging target for astructural study, in view of relatively few solved
structures of the membrane proteins so far. Thoughlast year we were able to record the first diffraction
patterns from ATP synthase crystals to about 7 A, we
obviously had to improve quality of the crystals andthis was done this year.
Firstly, we have continued intensive examinationof the protein preparation by changing detergents
species, PS3 culture batches, kinds of nucleotide
present in the extraction step and various purification
parameters. Quality of each preparation was evaluatedby its crystallization capability and by diffraction
capability of the resultant crystals, if crystallized. This
procedure established the following. Decyl-maltosideand dodecyl-maltoside, both of which had been usefulfor crystallization but indistinguishable between thetwo, were shown to have their own features. Though
dodecyl-maltoside was much better than decymaltoside in a number of respects, we haveencountered a problem that crystals from dodecylmaltoside were produced less frequently. The problem
was cured partially by finding better PS3 cell stocks,
examining dodecyl-maltoside from different manufacturers and knowing properties of the used chromatographic
materials better.
Secondly, we did a laser experiment in a hope toget better crystals, because it was shown that a welldefined application of laser beam to crystallization
setups is useful for inducing crystallization andsometimes for getting better ordered crystals. Although
the laser beam dearly induced crystallization in theexperiment, they were small compared with crystals
grown without the laser beam application. We could
not record protein diffraction pattern from either ofthe crystals. Further efforts to optimize the target
crystallization conditions may be necessary for a
successful laser experiment.
Thirdly, we made further efforts to analyze thediffraction patterns obtained last year. The analysis
had been hampered by incorrect beam position
parameters supplied and a high mosaicity of crystals.
Data were recollected in a way that should allow
diffraction analysis, and an analysis is going with ourrefined procedures.
The preparation and crystallization study wasdone in collaboration with Satoshi MURAKAMI at
Institute of scientific and industrial research, Osaka
University, Toshiharu SUZUKI and Masasuke
YOSHIDA, at Research Laboratory of Resource
Utilization, Tokyo Institute of technology. The laser
experiment was done with Satoshi MURAKAMI andhis colleagues in Osaka University and in Sosho.

partitioning, have been prepared. Setting thesetranscription and genome partitioning factors astargets for a structural study, we have been doing
comprehensive structure determination in the Protein
3000 project (the sub-field of 'transcription andtranslation'). In the previous first three years, we
examined 65 proteins for their crystals but identified
only three proteins that gave crystals of diffraction
quality. This year, we have concentrated to refine thecrystals obtained so far.
YmcB, a protein believed to be involved in the
genome and plasmid partitioning, was found last year
to form extremely good crystals. The SeMet derivativeof YmcB was expressed, successfully purified owing
to knowledge of purification procedures accumulated
over the last year, and was crystallized under theconditions similar to those for the native protein. Mostof the crystals were trigonal (a = 69.2A, b = 69.2A, c =172.8 A) with a resolution limit of 2.6 A, but a minor
proportion was tetragonal (a = b = 68.5A, c = 99.2A)
with a resolution limit of 2.0 A. MAD data sets werecollected from a number of SeMet derivative crystals,
because initial data sets could not be analyzed
successfully possibly due to deteriorative effects ofinappropriate crystal freezing. A data set obtained at
a late stage of study was processed in a satisfactory
way. SAD analysis of the data set with SOLVE lookedpromising, and a further analysis is going on.
This work has been done in collaboration with
Akira ISHIHAMA, Emi KANDA (Nippon Institute for
Biological Science), Hironori NIKI, Rie INABA,
Katsynori YATA, Yasushi OGATA (isotope center).
(3) Structure-based functional studies on transcription

factors

Hiroshi Itou and Yasuo Shirakihara(2) Comprehensive Crystallographic Study of Transcrip-tion factors and Genome-partitioning Factors from E.coli

Yasuo Shirakihara and Aya Shiratori

In E.coli, more than 160 transcription factors
control transcription of their target gene(s) by binding
to both their specific DNA sequence and thetranscription apparatus. Sixty-five such transcription
factors had been purified in Ishihama laboratory. In
Niki laboratory, a number of novel proteins, that are
judged to play roles in the genome and plasmid

Genomic-DNA information on number oforganisms is now available. Using the information,
comprehensive structure analysis of transcription
factors for their structure-based functional
understanding is in progress. We had alreadysucceeded in structure analysis of five of crystals; twofrom an archaeon P.horikoshii and three from abacterium C.glutamicum. As a functional approach,target DNA sequences for these proteins were
determined using SELEX (Systematic Evolution of
Ligand by EXponential enrichment) method, and this

Biomolecular Structure Laboratory97led to a tentative identification of the target genes forthese transcription factors. Based on the results fromthese two approaches, further experimental strategies
toward detailed functional understanding of theproteins were set out. Through the combination of
structure and functional analysis, we successively
determined function of the CGL2612 protein, one of the
hypothetical transcription factors from Cglutamicum,
as a transcriptional repressor that responsible for the
antimicrobial resistance system in the bacterium. The
strategy used in this study is one of the mostconvenient and powerful methods to analyze
functionally unknown transcription factors newly
identified by genomic analysis.
This work has been done in collaboration with Ui
OKADA, Yonggui Gao and Isao TANAKA at Divisionof Biological Sciences, Graduate School of Science,
Hokkaido University, and Hiroaki Suzuki and Masaaki
Wachi at Department of Bioengineering, Tokyo
Institute of Technology.
(4) Expression and crystallization Study of Eukaryotic

Transcription Factors

Hiroshi Itou and Yasuo Shirakihara
Eight eukaryotic transcription factors (four from
Hsapience, two from Olapites, one from Mmusculus,
and one from Osativa) have been selected as ourresearch target. To prepare sufficient amount of
extremely purified protein samples required for protein
crystallization experiment, optimization of protein
preparation strategies for these targets have been inprogress. Despite of the complexity of protein folding
inherent in eukaryotic proteins, we've be getting goodresults in the sample preparation step for three ofproteins, under the condition of co-expressed them withseveral chaperon proteins using an E.coli
over-expression system.
This work has been done in collaboration with
Isao TANAKA at Division of Biological Sciences,
Graduate School of Science, Hokkaido University(Hsapeience and Olapites), and Sumio SUGANO at
Division of Biosciences, Graduate School of Frontier
Sciences, Tokyo University (H sapience), Minoru
TANAKA at Laboratory of molecular genetics forreproduction, National Institute for Basic Biology(Olapites), Mitsuru MORIMOTO and Yumiko SAGA
at Mammalian Development Laboratory, Genetic

98

Biomolecular Structure Laboratory

Strains Research Center, National Institute of Genetics(M. musculus), and Ken-ichi NONOMURA atExperimental Farm, National Institute of Genetics(0 sativa).
(5) Expression and crystallization Study of Transcription factors from Pseudomonas aeruginosaYasuo Shirakihara and Aya Shiratori

Pseudomonas aeruginosa is a well-known
opportunistic bacterial pathogen, and a number oftranscription factors responsible for the pathogenicity
have been identified. After structural studies of PtxR,
PtxS, PhzR, and PA3547 done over the last two years,
we have included 12 of such transcription factors thisyear. Those were AlgP, AlgQ, Anr, FleN,Dnr,
PA0275,PA2591,RhIR, Vfr, AlgR, FleQ, FleR, and FleS.
The genes for the proteins were cloned into
pET21b so that the expressed protein has a His tag at
the C-terminus, and their protein expression was
examined by a locally developed system using E.coli
hosts. Among those twelve proteins, 6 of those (AlgQ,
FleN, Dnr, PA2591, Vfr, FleQ) were obtained in large
amounts. However, annoyingly, in cases of Anr and
AlgR, proteins that had molecular weight around 32,000
and were presumably due to a cloning vector, werestrongly expressed irrespective of host cells andexpression conditions. The rest were not expressed.
The proteins were purified with aNi-NTA column and,
when possible, an ion exchange column, and have been
subjected to crystallization studies.
This work has been done in collaboration with
Hironori ARAMAKI (Daiichi Pharmaceutical College).
(6)

Crystallization of Kid, a chromosome mover

Yasuo Shirakihara and Aya Shiratori
Kid is involved in spindle formation andchromosome movements in mitosis/meiosis. It consistsof three domains: an N-terminal kinesin-like motor
domain (35-370), a C-terminal chromosome-binding
domain (594-647) and a connecting domain (371-593)
including a coiled-coil region. So far, we have tried to
crystallize fragments of the molecule: the N-terminal
motor domain, the C-terminal chromosome-binding
domains and a longer N-terminal fragment (1-462). This
year, we examined properties of the hopeful crystals

of the longer N-terminal fragment, obtained withjeffamine or isopropanol in the presence of MgADP.
However, the observed diffraction patterns suggested
that the crystals were not genuine N-terminal fragment
crystals. Then we started a crystallization experimentof the full-length Kid, expressed in E.coli as a GST
fusion protein and subsequently the GST portion was
cleaved-off. From a preliminary crystallization study,
we think that we have to increase the available amountof the protein, improve quality pf the prep, and devise
a successful strategy for the molecule with predicteddisordered regions covering a third of the molecule.
The study was done in collaboration with Noriko
TOKAI, Miho OHSUGI and lun-ichiro INOUE at theInstitute of medical science, the university of Tokyo.
(7) Structural analysis of glutaminase from Micrococcus luteus K-3

PUBLICATIONSPapers

1. Itou, H., Okada, u., Suzuki, H., Yao, M., Wachi, M.,
Watanabe, N., Tanaka, I. (2005) The CGL2612 proteinfrom Corynebacterium glutamicum is a drug
resitance-related transcriptional repressor, J. BioI.
Chem., 280 (46) 38711 - 38719
2. Maenaka, K, Fukushi, K, Aramaki, H.,
Shirakihara, y. (2005) Expression, crystallization andpreliminary diffraction studies of the Pseudomonas
putida cytochrome P-450cam operon repressor CamR,
Acta Crystallogr, F61, 796 - 798
3. Motohashi, T., Shimojima, T., Fukagawa, T.,
Maenaka, K, Park, EY (2005) Efficient large-scaleprotein production of larvae and pupae of silkworm
by Bombyx mori nuclear polyhedrosis virus bacmid
system., BioChem Biophys Res Commun., 326 (3) 564 569

Yasuo Shirakihara and Aya Shiratori
EDUCATION

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. Its maximum
activity is observed at about 2M salt, but is also active
at low salt concentration. This is in contrast to thehalophilic proteins which are active at high saltconditions but loose activity at low salt conditions.
Previously the structure of the truncated form of the
enzyme (1"-'306) had been solved in our hands. We have
tried to solve the structure of the intact enzyme with
its MAD data and its native data (2.4 A resolution).
In the initial structure analysis of the intact form,
the refinement was stuck with R-factor of 41 %. It wasthen found that the crystal was merohedrally twinned
with a twinning factor of 0.43, close to the worst. A
refinement dealing with twinning by CNS gave an
R-factor 26% and Rfree 33%, however, the resultant
maps were not good enough to trace the rest of themolecule (307"-'456).
The structural study was done in collaboration
with Kazuaki Yoshimune and Mitsuaki Moriguchi at
Oita University, and Mamoru Wakayama at
Ritsumeikan University.

Shirakihara, Y. A Crystal Structure of Thermophilic
F1-ATPase and Crystals of Thermophilic
ATPsynthase MRC Dunn Human Nutrition Unit,
Cambridge, UK

H-e. Gene Network Laboratory
Emiko Suzuki GroupRESEARCH ACTIVITIES(1) Searching for cell-surface and secreted proteins
controlling motor axon targeting and synaptogenesis
Mitsuhiko Kurusu, Kai ZinnI and Emiko Suzuki
CDivision of Biology, California Institute ofTechnology)
The aim of this study is to understand two aspects
concerning the development of neural wiring; howmotor axons recognize specific muscle fibers as targets
for synapse formation and how their synapses expand
and change as the muscles grow. Despite the advancesin characterizing molecules that regulate motor axon
pathfin~ing in both invertebrate and vertebrate
systems, we still understand little about the questions
noted above. In particular, difficulty in identifying

Biomolecular Structure Laboratory99

molecules controllingtarget recognition was due to thelack of single loss-of function (LOF) mutations thatblock recognition of a specific muscle target whereas
many mutations affect pathfinding decisions, leading
to aberrant wiring of the neuromuscular system. This
result is most easily explained by genetic redundancy
in target labeling. If each muscle fiber were defined by
a combination of several cell surface labels, removing
one of the labels might not have a major effect ontargeting ofaxons to that fiber.
To overcome the redundancy problem, we
employed gain-of-function (GOF) screen in which
candidate molecules are ectopically expressed on all
muscle fibers. If these proteins are functional labels,
their misexpression might produce alterations in target
recognition. Furthermore, this GOF screen is available
for an identification of genes involved in thesynaptogenesis. This is assessed on the aberrant
synapse shape caused by an effect of overexpressionof candidate molecules.
To conduct this GOF screen, we first created a
database of all cell-surface and secreted (CSS) proteinsin Drosophila that are likely to be involved in specificcell-cell interactions. The database was generated bydefining all fly genes encoding proteins that containdomains known to be present in CSS proteins in othereukaryotes. It currently contains 994 genes. To drive
expression of these genes in muscles, we used the HEP"
system, in which a P element containing a block of
UAS sequences that are responsive to the yeasttranscription factor GAL4 is jumped around the
genome. Like other P elements, EPs usually land
upstream of genes. If a line bearing an EP upstreamof a gene is crossed to a 'driver' line expressing GAL4
in all muscle fibers, the gene will now be expressed athigh levels in muscles in the resulting progenies. To
find EP-like elements upstream of the CSS genes, wesearched through about 40,000 different insertions,
including the original EP set generated by PernilleRorth, the EY insertions lines generated in the Bellenlab, the GS lines developed in Japan, and the GE lines
developed by GenExel, Inc. We were able to identify
insertions that can confer expression of about 300 of
the 1005 CSS genes in our database. To screen forgenes encoding potential targeting molecules, we arecrossing each of these insertions to a muscle GAL4
driver and visualizing motor axons and neuromuscularjunction synapses in the resulting Fl progeny larvae
by immunostaining. We have already identified a

100 Gene Network Laboratory

number of genes that cause specific mistargeting
phenotypes and/or aberrant synaptic boutons when
they are expressed on muscles.
(2) High-resolution in vivo analysis of synaptogenesis
in Drosophila neuromuscular system

Franklin Carrero-Martinez 1•z, Akira Chiba1 and Emiko
Suzuki CDepartment of Structural Biology and
Neuroscience, University of Illinois, zSummer studentof JSPS program)
The embryonic Drosophila body-wall neuromuscular system serves as an ideal model system forstudying the early events during synaptogenesis. In
vivo synaptogenesis can be visualized using cell-specific
GFP marker that genetically labels single muscle cells
or neurons. Our previous studies using confocal
microscopy and electron microscopy in this systemhave revealed that neuronal growth cones and target
muscle cells interact mutually with highly dynamic
actin-based filopodia when they meet each other(Ritzenthaler et al., 2000). Several other investigations
on both vertebrates and invertebrates have also
reported such filopodial interactions, suggesting thatthese processes are common at early stages ofsynaptogenesis. However, their significance has notbeen established yet. To address this problem, we
genetically inhibited the function of certain
components that is likely to be involved in thefilopodial interaction, and analysed the results carefully
to correlate this process and synaptogenesis. We
focused on a particular synapse between an RP5 motor
neuron and an M12 muscle cell, for it is the easiest
synapse to observe with high optical and time
resolution. Using whole-mount high-resolution
time-lapse in vivo confocal imaging, we could describe
the sequence of filopodial interaction processes withup to 2-minutes temporal resolution (Carrero-Martinez
et al., submitted). Once filopodia from RP5 neuron andthose from M12 muscle cell make contact, they
gradually cluster at the definite site on M12. This isstereotypic in each segment. As soon as filopodialclusters were formed, a synaptic scaffold protein DLG,
the Drosophila orthologue of PSD95, accumulated atthese sites. As PSD95 in vertebrates is known toaccumulate at the initial site of synaptogenesis, ourresults strongly suggest that the filopodial clustering
initiates synaptogenesis. To test this possibility, wedesigned an experiment to express a truncated formof human actin-interacting protein Ezrin (Ezrin DN ) in
M12 to disturb filopodial cluster formation.
Over-expression of EzrinDN did not eliminate filopodia
in muscles, but the filopodia clustering was completely
abolished. RP5 growth cone still reached M12 under
this condition. However, it failed to form a normal
terminal and, instead, extended abnormally thin andlong processes that spread diffusely over the muscle
surface. The results indicate that filopodial cluster
provides a cellular platform on which to assemble
synaptic molecules (Carrero-Martinez et al., 2006,
submitted).
Next, we asked whether post-synaptic Dig in
filopodial cluster is essential for the synapse formation.
We over-expressed DLG DN protein that lacks both
PDZI and PDZ2 domains, the domains for theinteraction with synaptic essential proteins. DLG DN
molecule dimerizes with endogenous DLG and acts
dominant negatively. When DLG DN was expressed in
M12 of wild-type, the filopodial cluster was normallyformed. However, RP5 axon subsequently retracted
and did not make a synapse with M12. Sometimes RP5
neuron extended again and exhibited a wide range of
miswiring (Carrero-Martinez et al., 2006, submitted).
These results indicate that DLG is not required forfilopodial clustering, rather it is required for thesubsequent processes. How filopodial clustering
recruits DLG remains to be uncovered in the future
project.
(3) Formation of topological axon layer in Drosophila
mushroom body

Mitsuhiko Kurusu, Kai ZinnI and Emiko Suzuki
CDivision of Biology, California Institute ofTechnology)
Topographically organized neural circuits in thebrain are essential for information processing.
Although their functional significance has been
defined, the genetic mechanisms underlying theconstruction of topologically organized circuits are stilllargely unknown. Drosophila mushroom body (MB)functioning as a learning center is a good model system
to study the molecular mechanisms that control thesequential generation of neurons and their topologicalprojections into layers. Structurally, axonal projectionsof Drosophila larval MB are topologically organized into

concentric layers. Developmental analyses have shownthat four independent small axon bundles fromnewborn neurons originating from the quadruple cellclusters intermingle with each other and sort out into
the core of peduncle, and they then migrate outwards
more peripheral region as the neurons differentiate.
To define the cellular and molecular mechanisms
underlying the construction of the topologicallyorganized concentric axonal projections, we are trying
to understand how four independent axon bundles
from newly born neurons of each cell cluster sort out
into the core within the single bundle of the peduncle.
We began these studies with the hypothesis thatcell surface molecule expressed on the axons of newlyborn neurons should represent attractive cue for theaxons of follower neurons that choose the same
pathways. To identify candidate cue molecules, we
screened for known axon guidance molecules bycareful immunostaining of the MB with antibodies
against these proteins. We identified a cell adhesion
protein, N-Cadherin (N-Cad) specifically expressed on
the axons of younger neurons that are localized within
the core fibers of the peduncle. To understand thefunctional significance of the gene for core fiber
development, we conducted systematic analyses of
their loss-of-function (LOF) and gain-of-function (GOF)
MB phenotypes. Our results demonstrate that N-Cad
is essential for the four bundles ofaxons originatingfrom the quadruple cell clusters to converge into asingle tract in the proximal part of the peduncle andcoalesce into a unified core. It has been reported that
another cell adhesion protein, Fasciclin II (Fas II), isexpressed in the outer layer ofaxons from matureneurons but is absent from the core, and that Fasll isrequired for the clonal integrity of mature neurons(Kurusu et aL, 2002). These results, combined with our
newer findings, suggest a model in which N-Cad
mediated adhesion between younger axons and Fas
II-mediated adhesion between mature axons control
the formation of concentric axonal layers. This model
implies that precise temporal regulation of expressionof these cell adhesion proteins is essential for theproper development of the topologically organizedaxonal layers of the MB.
Having identified differential temporal expressionof cell adhesion molecules as a critical factor in
distinguishing the axonal projections of younger
neurons vs. mature neurons, we then wondered whatmechanisms regulate transitions in gene expression

Gene Network Laboratory101profiles in the course of differentiation of post-mitotic
neurons. One possibility is that the acquisition ofelectrical activity by young neurons may facilitate thetransition to the 'mature' state of gene expression. To
test this idea, we examined MB development under thecondition in which electrical activity was inhibitedusing dominant expression of EKO and dORK,
modified potassium channels, which shunt
depolarizing by inducing hyperpolarization.
Preliminary tests showed contradictory effects of thetwo different electric knockout systems on the core
structure and FasII distribution in the MB axon
bundles. Whereas no defect in MB morphology oraltered expression patterns of N-Cad and Fas II in
EKO-expressing MBs, dORK expression caused severemorphological abnormality in the core structurecomposed of younger axon bundles anddown-regulation of Fas II distribution in the matureneurons. Considering that the effect of electricalsilencing is stronger in dORK than EKO expressing
cells, we conclude that the phenotype induced bydORK expression is plausible, implying the layer
development of MB depending on the electrical activity.
Molecular studies on the diacylglycerol kinase
function in Drosophila photoreceptor neurons(4)

Takuma Yamada and Emiko Suzuki
Phototransduction in Drosophila photoreceptor
neurons is a G-protein coupled phosphoinositide(PI)-signaling cascade. It is the most rapid process
among G-protein coupled cascades known to date(receptor potential is generated within "'20
milliseconds after light stimulation), and the
responsiveness is dynamically regulated. Weare
studying how such elaborated signaling cascade isaccomplished, from the viewpoint of intracellularorganization of the molecules involved in this system.
Our previous studies by combination of functional
analysis and electron-microscopic immunocytochemistry
have shown that close association of the corecomponents in phototransduction cascade inphotoreceptive cell organella, rhabdomeric microvilli,
is essential for generation of light responses(Collaboration with Prof.C.Zuker at V.C. San Diego).
We also revealed that the PI regeneration cycle islocated close to rhabdomeric microvilli. At the basesof rhabdomeric microvilli, there is a network of smooth

102 Gene Network Laboratory

endoplasmic reticulum that we call subrhabdomeric
cisternae (SRC). We found that most of the enzymes
involved in PI regeneration and a phosphatidylinositol
transfer protein are localized to SRC membranes orin the cytoplasm around it. Mutations of thesecomponents not only affect light response but also
destroy the network of SRC, leading to retinaldegeneration at the end. Among the genetic mutationsof PI regeneration cycle, retinal degeneration A (rdgA),
the mutation of eye-specific diacylglycerol kinase(DGK) that mediates phosphorylation of diacylglycerol(DG) to produce phosphatidic acid (PA), causes
severest retinal degeneration. In rdgA mutants, PA in
the photoreceptor cell layer is dramatically reduced,
indicating that RDGA is a key player for the PA
homeostasis in photoreceptors. As PA molecules arethought to be required in membrane regions with large
curvatures, as is the case for SRC, and breakdown of
SRC networks is the initial process of photoreceptor
degeneration in rdgA mutants, we hypothesize that the
homeostasis of P A content in SRC maintained by
RDGA is essential for the structural integrity of SRC.
To address this hypothesis we planned to study themolecular mechanism how DGK activity of RDGA islocalized to SRC. RDGA protein molecule has two
cysteine-rich domains (CRDs) arranged in tandem in
N-terminal region. By the analogy with CRDs in potein
kinase C, it has been suggested that CRDs in DGKfunction as binding sites for DG in plasma membranes.
However, this has not been conclusively demonstrated.
To test this possibility, we designed experiments ofexpression of modified RDGA proteins with deletionor mutation within CRD regions. Our previous
experiments showed that expression of RDGA proteinthat lacks both CRDs does not rescue the degeneration
phenotype of rdgA mutants, suggeting that these
domains are essential for its function. By thatexperiment however, we could not precisely examine
the subcellular localization of expressed protein
because of cell degeneration. This year, we tried to
study the localization of modified DGK by use of
myc-tag. Expression of myc-tagged RDGA in rdgA
mutant compound eyes rescued the degeneration
phenotype, and the expressed protein localized
normally in photoreceptors. With this construct as anentry point, we are now carrying out further
experiments with modified RDGA proteins.

PUBLICATIONSPapers

1. Orihara-Ono, M., Suzuki, E., Saito, M., Yoneda, Y,
Aigaki, T., Rama, C. (2005) The slender lobes gene,
identified by retarded mushroom body development,is required for proper nucleolar organization in
Drosophila., Developmental Biology, 281, 121 - 133
2. Tokai-Nishizumi N,. Ohsugi, M., Suzuki, E.,
Yamamoto, T. (2005) The Chromokinesin Kid Is
Required for Maintenance of Proper Metaphase
Spindle Size, Mol. BioI. Cell., 16, 5455 - 5463
EDUCATION~*~~~~~~~~~~~~~~mft*~·OO~~i!rW~$

Gene Network Laboratory103

I. CENTER FOR INFORMATION BIOLOGYAND DNA DATA BANK OF JAPANI-a. Laboratory for DNA Data Analysis
Takashi Gojobori GroupRESEARCH ACTIVITIES(1) Various adaptations for the perpetual darkness inthe cave-dwelling population of Mexican tetra,
Astyanax mexicanus, by eDNA microarrays
Nobuhiko Tanaka, Shozo Yokoyama, Kazuho Ikeo andTakashi Gojobori
Mexican tetra (Astyanax mexicanus), a single
species with eyed surface (surface fish) and eyelesscave (cavefish) dwelling forms, gives unique
opportunities for studying the genetics of speciation
processes because the direction of evolutionary
processes is known with certainty: cavefish are derived
from surface-dwelling ancestors. In this study, to findgenes that can differentiate evolutionary changes
between the surface fish and the cavefish, we
conducted cDNA micro array analyses. Each of the
microarrays has 3,070 non-redundant genes from anentire body of an adult surface fish. Target genes forthe microarrays were made from an entire body of eachsurface fish and cavefish. On the basis of the resultsof comparative microarray analysis for 716 functionallyknown genes, relatively highly expressed genes in thesurface fish and in the cavefish were listed up,
respectively. As a result, a lot of functional information on relatively highly expressed genes in the surface
fish and in the cavefish was obtained and at suggested
various adaptations for the dark cave environment.
These adaptations covered behavioral, physiological,
and morphological changes between the surface fish
and the cavefish. In conclusion, at the gene expression
level, various adaptations for the perpetual darkness
were raised in the diversification process from thesurface fish to the cavefish. This is the first report thatdifferences of gene expression between the surface fish
and the cavefish were extensively examined throughout
a whole body.
(2) Compensatory change of interacting amino acids inthe coevolution of transcriptional coactivator MBFl and
TATA·box binding protein TBP
Qing-Xin Liu, Naomi Nakashima-Kamimura, Kazuho
Ikeo, Susumu Hirose and Takashi Gojobori
To elucidate the transcriptional regulation in
eukaryotic genome network, it is important tounderstand coevolution of transcription factors,
transcriptional coactivators and T ATA-box binding
protein TBP. In this study, coevolution oftranscriptional coactivator MBFl and its interacting
target TBP was first deduced from comparison of their
phylogenetic trees. The proposed coevolution was,then, evaluated experimentally by examining if
compensatory amino acid changes took place atinteracting sites of both proteins. The experiments
were conducted by identifying interaction sites andcomparing the amino acids at these sites among
different organisms. Here we provide direct evidence
for compensatory changes of transcription coactivatorand its interacting target, presenting the first reportthat transcription coactivator has undergone
coevolution with TBP.
(3) Targets analysis of the Drosophila tdfby using
oligonucleotide microarrays
Qing-Xin Liu, Kazuho Ikeo, Yasushi Hiromi, Susumu
Hirose and Takashi Gojobori
In Drosophila, the tracheas defective (tdf) geneencodes a bZIP protein that required for thedevelopment of trachea, heart, head and neural system.
The targets of tdfresponsible for these responses havenot been identified. To identify tdfdownstream genes
in a comprehensive manner, we used genome-wide
oligonucleotide arrays and analyzed differential gene
expression in wild-type embryos versus tdfmutant
embryos. Upon knockout of tdffunction, expressionof 340 genes was decreased and 338 genes wereincreased. Many of these genes can be assigned to
specific aspects of the tracheal and neural systemdevelopment. We also discovered tdftarget genes

104 CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPANwhich are likely to play specific roles in eyemorphogenesis. Moreover, we identified lim3 to be aputative direct target of tdj, carrying a TDF binding
sequence.
(4) Functional analysis of Drosophila Midline during
the neuronal development

Qing-Xin Liu, Masaki Hiramoto, Hitoshi Veda, Takashi
Gojobori, Yasushi Hiromi and Susumu Hirose
Formation of the neural network requires
coordinated pathfinding behavior of many neurons.
Each neuron must express axon guidance receptors intheir growth cone, and also act as a secretion sourceof guidance molecules. How neurons orchestrate theexpression of multiple guidance genes is poorly
understood. Here we show that Drosophila midline
encoding a T-box protein controls expression multiple
axon guidance molecules: Frazzled, ROBO, and Slit.
In midline mutant expression of all these molecules are
reduced, resulting in severe axon guidance defects,
whereas misexpression of Midline ind \.lces theirexpression. Midline is present onthe promoter of thesegenes, indicating that it directly controls transcription.
Our results provide evidence that Midline controls theswitch to axon growth.
(5) A possible regulatory role of cis sense-antisense
mRNAs in gene expression

Naoki Osato, Yoshiyuki Suzuki, Kazuho Ikeo andTakashi Gojobori

Cis sense-antisense mRNAs (CSA) are composedof a pair of mRNAs with complementary regions, andare located on each strand at the same genomic locus.
A pair of CSA changes its expression level andregulates the activation of the mRNAs. Although thenumber of known CSA was less than 100, more than
2,000 of human and mouse CSA pairs have been
predicted from the genome and cDNA sequences. Someof the predicted CSA pairs are expected to regulate
their expression, acting as CSA. However, it is unclear
what properties of CSA affect their expression. In thisstudy, we examined whether overlapping arrangements
of predicted CSA affect their expression. From human
predicted CSA pairs, we found that when the genomic
length of their overlapping regions increased, highly

expressed CSA pairs decreased. Moreover, dependingon overlapping patterns of the predicted CSA pairs in
the human genome, the distribution of their expression
levels changed. the predicted CSA pairs of the mouseshowed the same tendency of expression as that ofhuman. These findings suggest that overlapping
arrangements of CSA pairs significantly affect theirexpreSSlOn.
(6) The Rice Annotation Project Database (RAP-DB):
hub for Oryza sativa ssp. japonica genome information

Hajime Ohyanagi, Yasumasa Shigemoto, Kazuho Ikeo
and Takashi Gojobori
Rice is considered a model cereal plant becauseof its small genome size and a high degree ofchromosomal co-linearity with other major cereal crops
such as maize, wheat, barley and sorghum. The
International Rice Genome Sequencing Project(IRGSP), a consortium of publicly-funded laboratories
from 10 countries, initiated the sequencing of Oryza
sativa ssp. japonica cultivar Nipponbare in 1998 using
the clone-by-clone sequencing strategy. In 2004, thefinished-quality sequence of the entire genome wascompleted and is now available in the public domain.
The annotation of the sequence is indispensable inunderstanding the overall structure and function of therice genome. However, most of the annotations of therice genome sequences were obtained by automatedmethods. Although this provides an overview of thecomposition of the genes that comprise the genome,
the limitations in prediction programs often result inprobable errors and artifacts among predicted genes.
Therefore, in concordance with the completion of therice genome sequence, the Rice Annotation Project(RAP) was organized in 2004 with the aim of providing
standardized and highly accurate annotations of therice genome. To facilitate efficient management of theresults of annotation and to establish a platform for
integrating the data with other rice resources, anannotation database called the Rice Annotation Project
Database (RAP-DB) was developed (Ohyanagi, et al.,2006). The RAP-DB integrates the IRGSP genome
sequence and the RAP annotations with other data on
rice researches, and makes them available to the publicthrough HTTP access. All of the resources can beaccessed through http://rapdb.lab.nig.ac.jp/.

Laboratory for DNA Data Analysis

105

(7) Intracellular transporters evolve slowly

Hajime Ohyanagi and Takashi Gojobori
On the contrary to prokaryotes, eukaryotes havemembranous organelles in cells. As a result, someeukaryotic proteins might be under a different
evolutionary pressure. Therefore, the gain of themembrane system could affect the evolution ofparticular proteins, such as intracellular transporters.
With the motivation of giving insight to theevolutionary studies of the membrane system, we areconducting the following analyses. First, we computed
the evolutionary rates of proteins involved in the large
molecule transport in cells. Second, we classified
proteomes into two groups; transporters and nontransporters, and compared the evolutionary rates
between of the two groups. The results suggest that
intracellular transporters, especially transporters forlarge molecules are slowly evolving now.
(8) An analysis of gene expression for sexual
dimorphism in a mouse brain

Kazuya Yuge, Kazuho Ikeo and Takashi Gojobori
Sexually differentiated animals show distinctive
sex differences of phenotypic traits such as
reproductive organs and behavioral patterns. In
particular, sex differences of behavioral patterns may
be due to sexual dimorphism of a brain. It is generally
known that in spite of remarkable differences inbehavioral patterns between the sexes, there are only
a few differences in anatomical, biochemical, andmolecular characteristics in the brain between thesexes. In particular, little is known about sex
differences in the brain at the gene expression level.
To investigate whether there are significant
differences in a gene expression pattern of adult mousebrains between the sexes, we compared the gene
expression patterns between the sexes. In practice, we
isolated a preoptic area, a hypothalamus, and apituitary from male and female mouse brains, which
are known to show sex differences at the anatomicaland biochemical levels. we then analysed a pattern ofgene expression using a 16.3 K oligo microarray, whichcontains 93% of mouse genes. As a result, we found that
there were 14 genes in preoptic area, 16 genes inhypothalamus, 133 genes in pituitary that were

106 Laboratory for DNA Data Analysis

differentially expressed between the sexes. It is notedthat two categories of genes, peptide hormone genes
and sex chromosome genes, have a remarkably
differential pattern of gene expression between thesexes. we also validated the micro array results ofpreoptic area using real-time RT-PCR by quantifying
the expression level of genes for growth honnone andglycoprotein honnones, which are the peptide honnone
encoding genes, and Eif2s3y, which is the sex
chromosome gene. As a result, these genes wereexpressed more highly in male than in female. Thesefindings suggest that peptide honnone genes and sex
chromosome genes are important for detennining sex
differences in mouse adult brains.
(9) On the origin of mechanosensory system

J ung Shan Hwang, Shiho Hayakawa, Kazuho Ikeo,
Toshitaka Fujisawa and Takashi Gojobori
Nematocyte or cnidocyte is well being used tocharacterize all members of the phylum Cnidaria andthey act as a mechanosensory cell in response to theenvironmental stimuli. Similar structures can be
identified as extrusive organelles in many protists. Not
only the nematocyst and extrusome share themorphological similarity, they both are readily
discharged when encountered stimuli and some
extrusomes could even produce toxins. Therefore, ahypothesis of protist as the origin of nematocyst is
generally proposed. More than 60 nematocyte related
genes were isolated from hydra by using cDNA
microarray. All of them have been ensured with theirexpression patterns specific to the nematocyte lineage.
Searching the sequence homology against the protein
data set of Genbank revealed that 25 have thesignificant sequence match to the currently known
proteins, and 5 out of 25 are unique to the phylum ofProtoctista. These genes mainly express at the laterstage of nematocyte differentiation, possibly they are
essential for the assembly of mature nematocyst.
Furthennore, phylogenetic analysis revealed that oneof the protein disulfide isomerase of hydra wasclustered with the ones from protists rather thanmetazoans. The findings suggested that nematocyst
must have originated from the protist. Ultrastructuresof two heterotrophic dinoflagellates, Polykrikos andNemaiodinium, have demonstrated that the species
contain nematocysts. We plan to isolate andcharacterize genes that encode the nematocyst in either
Polykrikos or Nematodinium. Currently, a cDNA libraryof Nematodinium is constructed and nematocystrelated genes can be screened out of the library. Finally,
the approach of comparative study between hydra and
Nematodinium would give insights in the evolution of
cnidarian nematocyst.
(10) To search the origin of neural tube: Gene expression
analysis of ciliary band in sea urchin embryo
Sonoko Kinjo, Kazuho Ikeo and Takashi Gojobori
Echinoderms and chordates are thought to be amonophyletic and derived from common ancestor. It
has been thought that the neural tube of chordates hasoriginated from the ciliary band of Echinoderm larva
because the concentration of neural cells is seen in bothorgans. To know whether the ciliary band ofechinoderm larva is homologous to the neural tube ofchordates, we have been analyzing the expressed genes
in the ciliary band of sea urchin embryo. For thispurpose, we isolated the ciliary band tissue from 420
samples of sea urchin larvae, and we constructed
cDNA library of those.

implying that 14% of the NS-specific genes should havechanged differentially their original functions during
evolution. If this is the case, then the remaining 86%
of the 255 NS-specific human genes have newly
emerged during evolution. In particular, we found thatthe largest portion (24%) of the 255 NS-specific genes
had emerged after divergence of urochordata andhuman but before divergence of fishes and human.
These results suggest that the main cause of the NS
evolution was the addition of new genes which took
place most actively just before or at the evolutionary
emergence of vertebrates. We also found that there areregions where the NS-specific genes are concentrated(NSC regions) in human chromosomes 1,4,11,14,17,
19,21 and X and that most of the NS-specific genes inthese NSC regions emerged after divergence ofurochordata and human but before divergence of fishes
and human. In particular, most of functions of the geneproducts in the NSC region of chromosome X are
related to binding to other molecules such as DNAs
and proteins. It suggests that the NS-specific genes inthe eight NSC regions may have contributed to theformation of network of gene interactions to othermolecules. From these results, we concluded that theformation of gene network was crucial to the evolutionof the vertebrate NS.

(11) Evolutionary studies of the nervous system based

upon comparative genomics

PUBLICATIONS

Akiko Ogura Noda

PapersTo elucidate the evolutionary process of thenervous system (NS) in metazoa, the relationshipbetween human genes specifically expressed in the NS(NS-specific genes) and the time of their evolutionary
emergence were examined. Two hundred and fifty-fivehuman NS-specific genes were obtained from the gene
expression data of the Human Full-length cDNA
Annotation Invitational (H-invitational) database. To
determine when these genes emerged for the first time
during evolution, orthologues of the 255 NS-specificgenes in 13 species (excluding human) were searched
for by homology searches against their complete
genome sequences. we found that 14% of the NSspecific orthologous genes had already emerged before
the divergence between yeast and human. This finding
suggests that a common ancestor, which should haveno nervous system, already possessed a portion of thegenes homologous to human NS-specific genes,

1. International Rice Genome Sequencing Project:
Iwama, H., Gojobori, T., Itoh, T., Niimura, Y., et al.
(2005) The map-based sequence of the rice genome,
Nature, 436,793 - 800
2. The FANTOM Consortium and RIKEN Genome
Exploration Research Group and Genome Science
Group (Genome Network Project Core Group):
Gojobori, T., Ikeo, K., et al. (2005) The transcriptionallandscape of the mammalian genome, Science, 309(5740) 1559 - 1563
3. Chiusano, M. L., Gojobori, T. and Toraldo, G.
(2005) A C+ + computational environment forbiomolecular sequence management, eMS., 2 (3) 165 180
4. Fujii, Y., Itoh, T., Sakate, R., Koyanagi, KO.,
Matsuya, A., Habara, T., Yamaguchi, K., Kaneko, Y.,
Gojobori, T. and Imanishi, T. (2005) A web tool forcomparative genomics: G-compass, GENE, 364C, 45 52

Laboratory for DNA Data Analysis

107

5. Hanada, K, Suzuki, Y., Nakane, T., Hirose, O. andGojobori, T. (2005) The origin and evolution of porcine
reproductive and respiratory syndrome viruses, Mol.
BioI. Evol., 22 (4) 1024 - 1031
6. Kijimoto, T., Watanabe, M., Fujimura, K.,
Nakazawa, M., Murakami, Y., Kuratani, S., Kohara, Y.,
Gojobori, T. and Okada, N. (2005) cimp1, a novelastacin family metalloproteinase gene from East
African cichlids, is differentially expressed between
species during growth, Mol. BioI. Evol., 22 (8) 1649 1660
7. Koyanagi, KO., Hagiwara, M., Itoh, T., Gojobori,
T and Imanishi, T. (2005) Comparative genomics ofbidirectional gene pairs and its implications for theevolution of a transcriptional regulation system, GENE,
353 (2) 169 - 176
8. Matsumura, Y,. Shimokawa, K,. Hayashizaki, Y,..
Ikeo, K,. Tateno, Y,. Kawai,]' (2005) "Development ofa spot reliability evaluation score for DNA
micro arrays.", GENE, 350 (2) 149 - 160
9. Ogasawara, M., Imanishi, T., Moriwaki, K.,
Gaudieri, S., Tsuda, H., Hashimoto, H., Shiraishi, T.,
Gojobori, T and Koide, T. (2005) Length variation of
CAG / CAA triplet repeats in 50 genes among 16 inbred
mouse strains, GENE, 349, 107 - 119
10. Ogura, A., Ikeo, K. and Gojobori, T. (2005)
Estimation of ancestral gene set of bilaterian animals
and its implication to dynamic change of gene content
in bilaterian evolution, GENE, 345 (l) 65 - 71
11. Osada, N., Hirata, M., Tanuma, R., Kusuda, J.,
Hida, M., Suzuki, Y., Sugano, S., Gojobori, T., Shen,
CK, Wu, CI. and Hashimoto, K (2005) Substitution rateand structual divergence of 5'UTR evolution:
Comparative analysis between human and cynomolgus
monkey cDNAs, Mol. BioI. Evol., 22 (l0) 1976 - 1982
12. Shirai, T., Nakato, A., Izutani, N., Nagahisa, K,
Shioya, S., Kimura, E., Kawarabayasi, Y., Yamagishi,A., Gojobori, T. and Shimizu, H. (2005) Comparative
study of flux redistribution of metabolic pathway inglutamate production by two coryneform bacteria,
Metab. Eng., 7 (2) 59 - 69
13. Tamiya, G., Shinya, M., Imanishi, T., Ikuta, T,
Makino, S., Okamoto, K, Furugaki, K, Matsumoto, T,
Mano, S., Ando, S., Nozaki, Y., Yukawa, W., Nakashige,R., Yamaguchi, D., Ishibashi, H., Yonekura, M.,
Nakami, Y., Takayama, S., Endo, T., Saruwatari, T,
Yagura, M., Yoshikawa, Y., Fujimoto, K., Oka, A.,
Chiku, S., Linsen, S., Giphart, M., Kulski,]., Fukazawa,T., Hashimoto, H., Kimura, M., Hoshina, Y., Suzuki,

108

Laboratory for DNA Data Analysis

Y., Hotta, T., Mochida, J., Minezaki, T., Komai, K.,

Shiozawa, S., Taniguchi, A., Yamanaka, H., Kamatani,N., Gojobori, T, Bahram, S. and Inoko, H. (2005) Whole
genome association study of rheumatoid arthritis using
27, 039 microsatellites, Hum. Mol. Genet., 14 (l6) 2305 2321
14. Tanaka, T., Tateno, Y. and Gojobori, T. (2005)
Evolution of vitamin B6 (Pyridoxine) metabolism by
gain and loss of genes, Mol. BioI. Evol., 22 (2)243 - 250
15. Tanaka, Y., Hanada, K, Orito, E., Akahane, Y.,
Chayama, K., Yoshizawa, H., Sata, M., Ohta, N.,
Miyakawa, Y., Gojobori, T and Mizokami, M. (2005)
Molecular evolutionary analyses implicate injection
treatment for schistosomiasis in the initial hepatitis
C epidemics in Japan, J Hepatol., 42 (l) 47 - 53
16. Tanino, M., Debily, M.-A., Tamura, T, Hishiki, T.,Ogasawara, 0., Murakawa, K, Kawamoto, S., Itoh, K,
Watanabe, S., Souza, S. ]. d., Imbeaud, S., Graudens,E., Eveno, E., Hilton, P., Sudo, Y., Kelso, ]., Ikeo, K,
Imanishi, T., Gojobori, T, Auffray, C., Hide, W. andOkubo, K. (2005) The Human Anatomic Gene
Expression Library (H-Angel), the H-Inv integrative
display of human gene expression across disparate
technologies and platforms, Nucleic Acids Res., 33(Database Issue) 567 - 572
17. Tateno, Y, Saitou, N., Okubo, K, Sugawara, H. andGojobori, T. (2005) DDBJ in collaboration with
mass-sequencing teams on annotation, Nucleic Acids
Res., 33 (Database Issue) 25 - 28
18. Yamasaki, c., Koyanagi, KO., Fujii, Y., Itoh, T.,
Barrero, R., Tamura, T., Yamaguchi-Kabata, Y.,
Tanino, M., Takeda, ]., Fukuchi, S., Miyazaki, S.,
Nomura, N., Sugano, S., Imanishi, T. and Gojobori T(2005) Investigation of protein functions throughdata-mining on integrated human transcriptome
database, H-Invitational database (H-InvDB), GENE,
364C, 99 - 107
19. Yokomine, T,. Shirohzu, H,. Purbowasito, W,.
Toyoda, A,. Iwama, H,. Ikeo, K,. Hori, T,. Mizuno, S,.
Tsudzuki, M,. Matsuda, Y,. Hattori, M,. Sakaki, Y,.
Sasaki, H. (2005) Structural and functional analysis of
a 0.5-Mb chicken region orthologous to the imprinted
mammalian Ascl2 / Mash2-Igf2-H19 region., GenomeRes., 15 (l) 154 - 165
20. ~* 'j'!f$, iiE~ -~, Ii f~Jtffi ~ (2005) If /.b. 'Y r- ry - -7, 13 *~f!l!$~fUt, 126 (1) 55 - 59
21. Iif~Jtffi ~(2005)*f~ Jl8$4i;v~- r- B*J!

*'

8$4i;~77@]*4i;C: 0~~2.~~J*~I;::, 1:.~(J)f-I.$~8,~J!{~~~7°7 7-~ ;. -60 (l) 17

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22. ~!I!f 5G~, ~* fa, 4-jJ§
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23. Nishio, Y., Usuda, Y, Gojobori, T. and Ikeo, K.
(2005) Comparative whole genome sequence analysis
of corynebacteria, Bioinformatics of Genome Regulationand Structure II, 121 - 129
24. lL1~ifffi ~,'ifJJll: 3'§PA, ~1Ji IJXili, §'{f!Y .~,B"i/F.f tv, i~w-/N,* _, ~* '&¥, fiii!! 1ti:4it,~$ .~, ~~ $, ~~ ~~, ~~ ~~, :*7-1* 0~ (2005) lfiC~JT-?' ~~~ . ~* . M:#T, DDBJ ~fIJm~, (lL1~ifffi ~, 'ifJJll: 3'§PA) , #liteJlij, 1 - 181
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SOCIAL CONTRIBUTIONS AND OTHERS~~~±ilGojobori, T.

Meeting of Bioinformatics/Databasegroup(~~), Jeju, Korea

Gojobori, T. JBIRC H-Invitational Disease Edition,**WiI*1R
Gojobori, T. f/<;-{T-{ ~7::t""":;-~ ~ ~A*;fltIJX~£Ii>9",,~~~Ij:?J (~~), **wrr*1R
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Gojobori, T. fJ:ntegrative Annotation of the Genome,
Transcriptome and ProteomeJ, Session 6,
Transcriptome 2005 ()~~~), Shanghai, China
Gojobori, T. fGenome Sequence Annotation andComparative GenomicsJ, Session II, 1st International
Biocurator Meeting(J*~), Asilomar, California, USA~:fI~:i:lL1~:Wi~: ~*f-j.~tJNOOttb~7 :I: 0 - (AAAS Fellow)

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29. Genome Network Platform http://genomenetwork.
nig.ac.jp/
30. BSD http://bsd.genes.nig.ac.jp/bsd_web/
31. EMBEST http://cibexsv.genes.nig.ac.jp/cibest/
embest.html
32. CIBEX http://cibex.nig.ac.jp/index.jsp
33. RAP DB http://rapdb.lab.nig.ac.jp/
34. H-ANGEN http://jbirc.jbic.or.jp/hinv/h-angel/
35. H-ZNU http://www.h-invitational.jp/
EDUCATION~~'iz~T-

1. IKEO, K.

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lL 1~ifffi ~: Associate Editor of Molecular Biology and
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lL1~ifffi ~: Editorial board of Genome ResearchlL{~ifffi ~: Editorial board of BMC Genomics
lL1~ifffi ~: Editorial
board of Briefings m
Bioinformatics
lL1~:Wi ~: Editorial board of Gene Therapy andMolecular Biology
lL{~ifffi ~: Editorial board of LNCS Trans actions on
Computational Systems Biology

Laboratory for DNA Data Analysis

109

I-b. Laboratory for Gene·Product Informatics
Ken Nishikawa GroupRESEARCH ACTIVITIES(1) Predicting absolute contact numbers of native protein
structure from amino acid sequence

Akira R. Kinjo, Katsuhisa Horimoto and Ken
Nishikawa
The contact number of an amino acid residue ina protein structure is defined by the number of Cb
atoms around the Cb atom of the given residue, aquantity similar to, but different from, solvent
accessible surface area. We present a method to predictthe contact numbers of a protein from its amino acidsequence. The method is based on a simple linear
regression scheme and predicts the absolute values ofcontact numbers. When single sequences are used forboth parameter estimation and cross-validation, thepresent method predicts the contact numbers with acorrelation coefficient of 0.555 on average. Whenmultiple sequence alignments are used, the correlation
increases to 0.627, which is a significant improvement
over previous methods. In terms of discrete states
prediction, the accuracies for 2-, 3-, and 10-statepredictions are, respectively, 71.4%, 54.1%, and 18.9%
with residue type-dependent unbiased thresholds, and76.3%,59.2%, and 21.8% with residue type-independent
unbiased thresholds. The difference between accessible
surface area and contact number from a prediction
viewpoint and the application of contact number
prediction to three-dimensional structure prediction
are discussed (Kinjo, A.R. et al., 2005).
(2) Recoverable one-dimensional encoding of threedimensional protein structures

Akira R. Kinjo and Ken Nishikawa
One-dimensional (lD) structures of proteins suchas secondary structure and contact number provide
intuitive pictures to understand how the native
three-dimensional (3D) structure of a protein is encodedin the amino acid sequence. However, it is still not clear
whether a given set of 1D structures contains sufficient
information for recovering the underlying 3D structure.

110 Laboratory for Gene-Product Informatics

Here we show that the 3D structure of a protein canbe recovered from a set of three types of 1D structures,
namely, secondary structure, contact number andresidue-wise contact order which is introduced here
for the first time. Using simulated annealing molecular
dynamics simulations, the structures satisfying the
given native 1D structural restraints were sought for
16 proteins of various structural classes and of sizes
ranging from 56 to 146 residues. By selecting thestructures best satisfying the restraints, all the proteins
showed a coordinate RMS deviation of <4 A from thenative structure, and, for most of them, the deviation
was even <2 A. The present result opens a new
possibility to protein structure prediction and ourunderstanding of the sequence-structure relationship(Kinjo, A.R. and Nishikawa, K., 2005a).
(3) Genome-wide survey of transcription factors inprokaryotes reveals many bacteria-specific families notfound in archaea

Yoshiaki Minezaki, Keiichi Homma and Ken
Nishikawa
Assignment of all transcription factors (TFs) fromgenome sequence data is not a straightforward task
due to the wide variation in TFs among different
species. A DNA binding domain (DBD) and acontiguous non-DBD with a characteristic SCOP or
Pfam domain combination are observed in mostmembers of TF families. We found that most ofexperimentally verified TFs in prokaryotes aredetectable by a combination of SCOP or Pfam domains
assigned to DBDs and non-DBDs. Based on this finding,we set up rules to detect TFs and classify them into
52 TF families. Application of the rules to 154 entirely
sequenced prokaryotic genomes detected more than
18,000 TFs classified into families, which have been
made publicly available from the "GTOP-TF"
database. Despite the rough proportionality of thenumber of TFs per genome with genome size, species
with reduced genomes, i.e., obligatory parasites andsymbionts, have only a few if any TFs, reflecting anearly complete loss. Also the number of TFs issignificantly lower in archaea than in bacteria. In
addition, all but one of the 19 TF families present inarchaea are present in bacteria, whereas 33 TF families
are found exclusively in bacteria. This observation
indicates that a number of new TF families evolved

in bacteria, making the transcription regulatory system
more divergent in bacteria than in archaea (Minezaki,
Y et al., 2005).
(4) Predicting secondary structures, contact numbers andresidue-wise contact orders of native protein structurefrom amino acid sequence using critical random networks
Akira R. Kinjo and Ken Nishikawa
Prediction of one-dimensional protein structures
such as secondary structures and contact numbers isuseful for the three-dimensional structure prediction
and important for the understanding of sequencestructure relationship. Here we present a newmachine-learning method, critical random networks(CRNs), for predicting one-dimensional structures, andapply it, with position-specific scoring matrices, to theprediction of secondary structures (SS), contact
numbers (CN), and residue-wise contact orders(RWCO). The present method achieves, on average, Q3
accuracy of 77.8% for SS, correlation coefficients of 0.726
and 0.601 for CN and RWCO, respectively. The
accuracy of the SS prediction is comparable to otherstate-of-the-art methods, and that of the CN prediction
is a significant improvement over previous methods.
We give a detailed formulation of critical random
networks-based prediction scheme, and examine thecontext-dependence of prediction accuracies. In orderto study the nonlinear and multi-body effects, we
compare the CRNs-based method with a purely linear
method based on position-specific scoring matrices.
Although not superior to the CRNs-based method, thesurprisingly good accuracy achieved by the linear
method highlights the difficulty in extracting structuralfeatures of higher order from amino acid sequence
beyond that provided by the position-specific scoring
matrices (Kinjo, A.R. and Nishikawa, K, 2005b)(5) Intrinsically disordered loops inserted into thestructural domains of human proteins
Satoshi Fukuchi, Keiichi Homma, Y oshiaki Minezaki
and Ken Nishikawa
Much attention has been paid recently to proteins
with partially or fully disordered structures, which arefound to exist mostly in eukaryotes and are involved
mainly in pivotal cellular processes such astranscriptional regulation, translation and cellularsignal transduction. Long disordered sequences are
sometimes inserted within the single structural
domains of proteins, forming loops from the molecularsurface. Such intrinsically disordered loops (IDLs)
either are invisible in X-ray crystallography, or hamper
protein crystallization itself due to great flexibility.
Perhaps because of this, such long disorderedsequences have not been characterized adequately.
Here, we propose an informational method thatstringently identifies IDLs in the structural domainsof proteins using the amino acid sequence alone. A
genome-wide survey of human proteins conducted withthe method identified 50 IDL-containing proteins,several of which have experimentally determined 3D
structures. Similar searches in other entirely sequenced
organisms revealed that IDLs are prevalent ineukaryotes, while they are much less so in prokaryotes.
As there is a statistically significant coincidence
between the boundaries of IDLs and those of exons,
we suggest that IDLs were produced mainly by exon
addition in eukaryotes. IDLs are almost always locatedat the surface of proteins and are enriched withhydrophilic residues, and IDL-containing proteins tend
to be intracellular. Some of the well-characterized
proteins with IDLs illustrate that IDLs play pivotal
roles in the switching of intracellular signaling or
regulatory functions, suggesting that IDL insertion isan effective way to create functionally different domain
variants (Fukuchi, S. et al., 2006).
PUBLICATIONSPapers

1. Kinjo, A. R., Horimoto, K and Nishikawa, K (2005)
Predicting absolute contact numbers of native protein
structure from amino acid sequence., Proteins, 58, 158 165
2. Kinjo, A. R. and Nishikawa, K (2005) Recoverable.
one-dimensional encoding of protein three-dimensional
structures., Bioinformatics, 21 (10) 2167 - 2170
3. Kinjo, A. R. and Nishikawa, K (2005) Predicting
secondary structures, contact numbers andresidue-wise contact orders of native protein structurefrom amino acid sequence using critical random
networksc, Bioinformatics, 1, 67 - 74
4. Minezaki, Y, Homma, K and Nishikawa, K (2005)
Genome-wide survey of transcription factors inprokaryotes reveals many bacteria-specific families not

Laboratory for Gene-Product Informatics

111

found in archaeac, DNA Res, 12 (5) 269 - 280
5. Saka, K, Tadenuma, M., Nakade, S., Tanaka, N.,
Sugawara, H., Nishikawa, K, Ichiyoshi, N., Kitagawa,M., Mori, H., Ogasawara, N. and Nishimura, A. (2005)
A complete set of Escherichia coli open reading frames
in mobile plasmids facilitating genetic studiesc, DNA
Res, 12, 63 - 68
6. Yamasaki C, Koyanagi KO, Fujii Y, Itoh T, Barrero
R, Tamura T, Yamaguchi-Kabata Y, Tanino M,
Takeda], Fukuchi S, Miyazaki S, Nomura N, Sugano
S, Imanishi T, Gojobori T. (2005) Investigation ofprotein functions through data-mining on integrated
human transcriptome database, H-Invitational
database (H-InvDB)., Gene, 364, 99 - 107
Books
7. ~~~,

j1§JII}t(2005)~2:$:5: ?'/,/,\7'i{q):fA'J1-foiJIil ?' /'/'\7'i{f-j.~-:fA'J1·1WJ11·~UHIH\!1,@-~5HT){t~Fi'JA#!!fi~-(1&1ii~%,87 - 100

1- c. Laboratory for Gene Function Research
Yoshio Tateno GroupThe laboratory consists of five members: Dr. Yoshio
Tateno (Professor), Dr. Roberto Antonio Barrero
Gumiel (Assistant Professor), Dr. Tokumasa Horiike(Postdoc), Dr. Lihua Jin (Postdoc) and Ms. Naoko
Murakata (Secretary). We aim at the elucidation of theorigin, evolution and function of genes and proteinsin view of molecular evolution, structural biology andinformation biology in collaboration with otherresearch groups in the institute and out. Besides theresearch activities summarized below, we take part in
the DDBJ activity in particular in the database
construction and public relations.
RESEARCH ACTIVITY(1) Genomic evolution of MHC class I region in primates
Fukami-Kobayashi, K, Shiina, T., Anzai, T., Sano, K,
Yamazaki, M., Inoko, H. and Tateno, Y
To elucidate the origins of the MHC-B-MHC-C pairand the MHC class I chain-related molecule(MIC)A-MICB pair, we sequenced an MHC class I
genomic region of humans, chimpanzees, and rhesus
monkeys and analyzed the regions from an

112 Laboratory for Gene-Product Informatics

evolutionary stand-point, focusing first on LINE
sequences that are paralogous within each of the firsttwo species and orthologous between them. Because
all the long interspersed nuclear element (LINE)
sequences were fragmented and nonfunctional, they
were suitable for conducting phylogenetic study and,in particular, for estimating evolutionary time. Our
study has revealed that MHC-B and MHC-C duplicated22.3 million years (Myr) ago, and the ape MICA andMICB duplicated 14.1 Myr ago. We then estimated thedivergence time of the rhesus monkey by using otherorthologous LINE sequences in the class I regions of
the three primate species. The result indicates thatrhesus monkeys, and possibly the Old World monkeys
in general, diverged from humans 27-30 Myr ago.
Interestingly, rhesus monkeys were found to have not
the pair of MHC-B and MHC-C but many repeatedgenes similar to MHC-B. These results support ourinference that MHC-B and MHC-C duplicated after thedivergence between apes and Old World monkeys(Fukami-Kobayashi et at., 2005).
(2) Development of a spot evaluation score for DNA
microarrays.

Matsumura, Y, Shimokawa, K, Hayashizaki, Y, Ikeo,
K, Tateno, Y, and Kawai, ].
We developed a reliability index named SRED
(Spot Reliability Evaluation Score for DNA
microarrays) that represents the probability that thecalibrated gene expression level from a DNA
microarray would be less than a factor of 2 different
from that of quantitative real-time polymerase chain
reaction assays whose dynamic quantification range
is treated statistically to be similar to that of the DNA
microarray. To define the SRED score, two parameters,
the reproducibility of measurement value and therelative expression value were selected from ninecandidate parameters. The SRED score supplies theprobability that the expression level in each spot of a
micro array is less than a certain-fold different
compared to other expression profiling data, such as
QRT-PCR~ This score was applied to approximately
1,500,000 points of the expression profile in the RIKEN
Expression Array Database (Matsumura et al., 2005).

(3) Large-scale analysis of human alternative protein
isoforms: pattern classification and correlation with
subcellular localization signals.

Nakao M, Barrero RA, Mukai Y, Motono C, Suwa M,
and Nakai K.
We investigated human alternative protein
isoforms of >2600 genes based on full-length cDNA
clones and SwissProt. We classified the isoforms andexamined their co-occurrence for each gene. Further,
we investigated potential relationships between thesechanges and differential subcellular localization. The
two most abundant patterns were the one withdifferent C-terminal regions and the one with aninternal insertion, which together account for 43% of
the total. Although changes of the N-terminal region
are less common than those of the C-terminal region,
extension of the C-terminal region is much lesscommon than that of the N-terminal region, probably
because of the difficulty of removing stop codons in
one isoform. We also found that there are somefrequently used combinations of co-occurrence inalternative isoforms. We interpret this as evidence that
there is some structural relationship which produces
a repertoire of isoformal patterns. Finally, many
terminal changes are predicted to cause differential
subcellular localization, especially in targeting either
peroxisomes or mitochondria. Our study sheds newlight on the enrichment of the human proteome
through alternative splicing and related events. Our
database of alternative protein isoforms is available
through the internet (Nakao et al., 2005).
(4) Evolution of vitamin B6 (pyridoxine) metabolism by
gain and loss of genes.

Tanaka T, Tateno Y, Gojobori T.
Vitamin B(6) (VB6) functions as a cofactor of many
diverse enzymes in amino acid metabolism. Threemetabolic pathways for pyridoxal 5'-phosphate (PLP;
the active form of VB6) are known: the de novo
pathway, the salvage pathway, and the fungal type
pathway. Most unicellular organisms and plants
biosynthesize VB6 using one or two of these threebiosynthetic pathways. However, animals such as
insects and mammals do not possess any of thepathways and, thus, need to intake VB6 in their dietto survive. It is conceivable that breakdowns of thesepathways occurred in the evolutionary lineages of
insects and mammals, and one of the major reasonsfor this would be the loss of pertinent genes. We
studied the evolution of VB6 biosynthesis from theview of the gain and loss of 10 pertinent genes in 122
species whose genome sequences were completely
determined. The results revealed that each gene in thepathways was lost more than once in the entire
evolutionary lineages of the 122 species. We also found
the following three points regarding the evolution of
PLP biosynthesis: (1) the breakdown of the PLP
biosynthetic pathways occurred independently at least
three times in animal lineages, (2) the de novo pathway
was formed by the generation of pdxB in
gamma-proteobacteria, and (3) the order of the geneloss in VB6 metabolism was conserved among different
evolutionary lineages. These results suggest that theevolution of VB6 metabolism was subject to gains andfrequent losses of related genes in the 122 species
examined. This dynamic nature of the evolutionarychanges must have been responsible for the
breakdowns of the pathways, resulting in profounddifferentiation of heterotrophy among the species(Tanaka et al., 2005).
(5) DDBJ in collaboration with mass-sequencing teams
on annotation.

Tateno Y, Saitou N, Okubo K, Sugawara H, GojoboriT.
In the past year, we at DDB] (DNA Data Bank ofJapan; http://www.ddbj.nig.ac.jp) collected and released
1,066,084 entries or 718,072,425 bases including thewhole chromosome 22 of chimpanzee, the wholegenome shotgun sequences of silkworm and various
others. On the other hand, we hosted workshops forhuman full-length cDNA annotation and participatedin jamborees of mouse full-length cDNA annotation.
The annotated data are made public at DDB]. We arealso in collaboration with a RIKEN team to accept andrelease the CAGE (Cap Analysis Gene Expression) data
under a new category, MGA (Mass Sequences for
Genome Annotation). The data will be useful forstudying gene expression control in many aspects(Tateno et al., 2005).

Laboratory for Gene Function Research113

(6) Investigation of protein functions throughdata-mining on integrated human transcriptome
database, H-Invitational database.

represents a substantial contribution to resources
required for the exploration of human biology andpathology (Yamasaki et at., 2005).

Yamasaki C, Koyanagi KO, Fujii Y, Itoh T, Barrero
RA, Tamura T, Yamaguchi-Kabata Y, Tanino M,
Takeda], Fukuchi S, Miyazaki S, Nomura N, Sugano
S, Imanishi T, and Gojobori T.

(7) Novel mammalian microRNA targets identified by
an extensive transcriptome-wide affinity screening

H-Invitational Database (H-InvDB) is a humantranscriptome database, containing integrative
annotation of 41,118 full-length cDNA clones originatedfrom 21,037 loci. H-InvDB is a product of the
H-Invitational project, an international collaboration
to systematically and functionally validate humangenes by analysis of a unique set of high quality
full-length cDNA clones using automatic annotationand human curation under unified criteria. Here, 19,574
proteins encoded by these cDNAs were classified into
11,709 function-known and 7865 function-unknown
hypothetical proteins by similarity with protein
databases and motif prediction (lnterProScan). The
proportion of "hypothetical proteins" in H-InvDB was
as high as 40.4%. In this study, we thus conducted
data-mining in H-InvDB with the aim of assigningadvanced functional annotations to those hypotheticalproteins. First, by data-mining in the H-InvDB version
of GTOP, we identified 337 SCOP domains within 7865
H-Inv hypothetical proteins. Second, by data-miningof predicted subcellular localization by sosm and
TMHMM in H-InvDB, we found 1032 transmembraneproteins within H-Inv hypothetical proteins. These
results clearly demonstrate that structural prediction
is effective for functional annotation of proteins withunknown functions. All the data in H-InvDB are shownin two main views, the cDNA view and the Locus view,and five auxiliary databases with web-based viewers;
DiseaseInfo Viewer, H-ANGEL, Clustering Viewer,
G-integra and Tapa Viewer; the data also areprovided as flat files and XML files. The data consistsof descriptions of their gene structures, novel
alternative splicing isoforms, functional RNAs,
functional domains, subcellular localizations, metabolic
pathways, predictions of protein 3D structure, mappingof SNPs and microsatellite repeat motifs in relation
with orphan diseases, gene expression profiling, andcomparisons with mouse full-length cDNAs in thecontext of molecular evolution. This unique integrative
platform for conducting in silico data-mining

114

Laboratory for Gene Function Research

Barrero RA, Tamura T, Sakurai H, Hayakawa S,
Tateno Y, Ikeo K, Imanishi T and Gojobori T.
MicroRNAs (miRNAs) act as repressors of target
mRNAs via translation inhibition or messenger
degradation. In mammals, miRNAs were shown toassociate with haematopoietic cell differentiationl,
downregulation of HOXB82 and control of insulin
secretion3. Identification of reliable miRNA targets has
become one of the key issues in the understanding of
miRNA regulatory networks. Here we report a large
scale affinity screening of miRNA targets, which
utilizes structural information of accessibility tomiRNA-recognition elements (MREs). Our method
predicted 920 non-redundant miRNA-MRE pairs,
representing 688 human and mouse genes, for 142
miRNAs. We validated "'-'71% of the predicted MREs
subjected to reporter assays, which have at least 5.5
kcal higher affinities to bind miRNAs than to makepotential internal secondary structures. Most of theidentified targets (85.5%) are new for mammals, and
195 of these are involved in multiple biologicalprocesses, which support the notion that miRNAs may
regulate complex gene networks.
(8) Development of a method for constructing aphylogenetic tree by using many different orthologs

Tokumasa Horiike, Daisuke Miyata, Kazuo Hamada,
Satoshi Saruhashi, Takao Shinozawa, Yoshio Tateno
To compare the topologies between a pair ofphylogenetic trees, we developed a method, which canaccommodate the difference in the number of OTUs
between the pair in question. Applying this method toa number of orthologous gene sets, we constructed thecorresponding number of phylogenetic trees andpair-wisely compared the constructed trees. Thismethod further enables to cluster a group of theconstructed trees among which no inconsistency of tree
topology exists irrespective of the number of OTUs.
Since the one with the largest trees among the clusters

is considered to show the closest phylogenetic
relationships of the species involved to the true ones,
we also developed a statistical method to single it out.
In reality, however, we have faced the difficulty in
refining and redefining orthologous genes in the extant
orthologous databases not mention to selecting thosefrom genome sequence data in general. We will thus
find a way of selecting orthlogous genes and compilingreliable orthologous gene sets.
(9) The analysis of the evolutionary relationshipbetween duplication and alternative splicing
Jin LH., Ikeo K., Suzuki Y. and Gojobori T.
Living things Increase their functional
diversification by variable evolutionary mechanisms.
Among these mechanisms, gene duplication andalternative splicing (AS) are known as the two of major
evolutionary ways that can bring the functional
diversification by increasing gene variations. Our
research interest is to clarify the evolutionary
relationship between these two different phenomena
by utilizing all available data resources. The resultsof this study showed that gene duplication and age ofgene both give positive influence on the occurrence ofAS, in both human and mouse genomes. Furthermore,when combine the effect of gene duplication and age
of gene by using a new parameter ADE (Accumulated
Duplication Effect), the AS isoform number tends toincrease in early stage of ADE, remain stable in the
middle stage, and decrease in the later stage. Since
large ADE generally resulted from either large size or
old age of a gene family, therefore, we can conclude
that these two factors cause increase of AS isoform
numbers only to a certain level.
(10) Evolutionary study of small-RNA-mediated genesilencing pathways by investigating the evolution of
Rnase III enzymes
Jin LH., Ikeo K., Suzuki Y. and Gojobori T.
The small-RNA-mediated gene silencing pathways
are evolutionarily conserved processes. It highlights
a fundamental role of short RNAs in eukatyotic gene
regulation and antiviral defense. Recently, 3 distinct
small-RNA-mediated silencing pathways are observed,
such as the destruction of mRNA via siRNA, inhibitionof mRNA translation via miRNA, and epigenetic genesilencing via siRNA. It was also found that in thesepathways, the members of ribonclease III family play
important roles in diverse RNA maturation and decay.
Here we investigated the evolution of Rnase III
nucleases, Dicer as representative, to further figure out
the evolutionary relationship of 3 gene silencing
pathways. With the advantage of using genomic
sequences as the subject in homologue search, in
un-annotated genomic regions, we were able to detect
possible candidates for several functional domains andgenes of Rnase III nuclease members. Moreover, wefound that 150 prokaryotes including eubacteria andarchbacteria lack completely the PAZ domain of Dicer.
These results show the taxonomic-dependent evolutionof the small-RNA-mediated gene silencing pathways.
(11) A Novel Arabidopsis Gene Causes Bax-like Lethality

in Saccharomyces cerevisiae
Kawai-Yamada M., Saito Y" Jin LH., Ogawa T., Kim
KM., Jin LH., Tone Y., Hirata A., Umeda M., andUchimiya H.
Overexpression of the mammalian proapoptotic
protein Bax induces cell death in plant and yeast cells.
The Bax inihibitor-l (BI-l) gene rescues yeast and plantfrom Bax-mediated lethality. Using the Arabidopsis BI-l
(AtEI-l) gene controlled by the GALl promoter as acell death suppressor in yeast, Cdfl (fell growth 4efectfactor-]) was isolated from Arabidopsis eDNA library.
Overexpression of Cdfl caused cell death in yeast,
whereas such an effect was suppressed byco-expression of AIBI-l. The Cdfl protein fused witha green fluorescent protein was localized in the
mitochondria and resulted in the loss of mitochondrial
membrane potential in yeast. The Bax-resistant mutant
BRMI demonstrated tolerance against Cdfl-mediatedlethality, whereas the ~ alp4 strain was sensitive toedfl. Our results suggest that Cdfl and Bax cause
mitochondria-mediated yeast lethality through partiallyoverlapped pathways.
(12) Collaboration with Prof. Tadao Saito of Tohoku
University and his laboratory on the function andevolution of glucosidase and galactosidase genes in
Lacfobcillus.
Saito, T, Yamazaki Y and Tateno Y

Laboratory for Gene Function Research115

(13) Collaboration with Prof. Shintou Eguchi of theInstitute of Mathematical Statistics and his laboratoryon the statistical analyses of SNP and gene expression
data.
Eguchi S, Ikeo K and Tateno Y(14) Collaboration with Dr. Kaoru Fukami.Kobayashi
of RIKEN on evolutionary analysis of MHC genes inprimates.

6. Yamasaki C, Koyanagi KO, Fujii Y, Itoh T, Barrera
RA, Tamura T, Yamaguchi-Kabata Y, Tanino M,
Takeda], Fukuchi S, Miyazaki S, Nomura N, Sugano
S, Imanishi T, Gojobori T (2005) Investigation ofprotein functions through data-mining on integrated
human transcriptome database, H-Invitationa1
Database (H-InvDB)., Gene, 364, 99 - 107
7. 0
v o. fI*f=~ (2005) rmiRNAO)?7-"'Iv" ./'\

Ify H~}EO)fJ!:[kJ. /{-{;t7'":7 J O~-~~-Tlv. 5(4) 403 - 405

Fukami-Kobayashi K and Tateno Y

Books
8. fI*f=~. q(15) Collaboration with Prof. Naoko Takezaki of Kagawa
Medical College on the evolution of genes expressed inbrains.

.1:::~I't-J)!~-f-O)TilIUJ

Takezaki Nand Tateno Y(16) Collaboration with AHG·Evolutional Curation
System teams on annotation of expressed human genes.
Ikeo K, Suzuki Y., Ohta S., Jin LH., Yamazaki C.,
Suzuki M., Kanno M., Saichi N., Sanbonmatsu R,
Koyanagi K, Sakate R, Itoh T., and Imanishi T.
PUBLICATIONSPapers
1. Fukami-Kobayashi, K, Shiina, T., Anzai, T., Sano,
K, Yamazaki, M., Inoko, H. and Tateno, Y (2005)
Genomic evolution of MHC class I region in primates.,
Proc Natl Acad Sci USA, 102, 9230 - 9234
2. Matsumura, Y., Shimokawa, K, Hayashizaki, Y,
Ikeo, K, Tateno, Y, and Kawai,]. (2005) Developmentof a spot evaluation score for DNA microarrays, Gene,
350, 149 - 160
3. Nakao M, Barrero RA, Mukai Y, Motono C, Suwa
M, Nakai K (2005) Large-scale analysis of humanalternative protein isoforms: pattern classification andcorrelation with subcellular localization signals.,
Nucleic Acids Res, 33,2355 - 2363
4. Tanaka, T., Tateno, Y. and Gojobori, T. (2005)
Evolution of vitamin B6 (pyridoxine) metabolism by
gain and loss of genes, Mol Bioi Evol, 22, 243 - 250
5. Tateno, Y., Saitou, N., Okubo, K., Sugawara, H. andGojobori, T. (2005) DDBJ in collaboration with
mass-sequencing teams on annotation, Nucleic Acids
Res, 33, D25 - D28

116 Laboratory for Gene Function Research"'Iv" ./'\ v

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12. DDBJ7-?7"'- A http://www.ddbj.nig.ac.jp/
Welcome-e.htm1
13. CIBEX7-?7"'- A http://cibex.nig.ac.jp/index.jsp
EDUCATION~~·-tz::T-

1.

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2. Barrero RA. Introduction to the DDBJ databaseand use of it for bioinformatic research Yang-Ming
University (Taipei, Taiwan)
3. Barrero RA. Introduction to the DDBJ databaseand use of it for bioinformatic research National
Health Research Institute (Hsin Chu, Taiwan)
4. Y Tateno The fundamental of population
genetics and molecular evolution National Yang-Ming
University, Taipei, Taiwan
5. Y Tateno The fundamental of population
genetics and molecular evolution National HealthResearch Institute, Hsin-chu, Taiwan
6. ~!I!f~~r/'\-1.:t-1/7::t~T4:7A~~lfJA*y

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SOCIAL CONTRIBUTIONS AND OTHERS~~~±:flCenter for Information Biology and DNA Data Bankof Japan, NIG, National Genome Information Center,
KRIBB, Korea The 4th Korea-Japan-China
Bioinformatics Training Course Deajon, Korea
B*~~~~ mn@B*~~~~*~ **~~*fJD2005-330761,~!l!f.~,

r::J / ' t." .::L -?' Ie. J: Q~~~~,

Yasumasa Shigemoto t, Haruka Sakai, Takshi Abe,
Satoru Miyazaki tt , Hideaki Sugawara.
'Hitachi soft, ttTokyo Univ. of Sci.

{t~· j;:filJf-t~f-t

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1) From Web services to a Bioportal

gffi*~,

1?tlmOOflM5I1;w7!J,~~~~~5lS!I.J~Jr.~ Dr. Y. Tateno served the Genetics Societyof Japan as Representative member of the Union of
Academic Societies for Natural History~Jr.~ Dr. Y. Tateno served the Microarray Gene
Expression Data Society as an Advisory Board Member~Jr.~ Dr. Y. Tateno served the Society of
Evolutionary Studies as a Member of the Committeeof Genetics under the Science Council of Japan~Jr.~ Dr. Y. Tateno served the Genetics Societyof Japan as a Member of Editorial Board

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

The publicly available bioinformatics resources,
comprising databases and analytical tools, 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 haveoverlapping functions. We need an information
environment that is conducive to efficient andappropriate bioinformatics resource utilization for awide range of users. Therefore, the Center for
Information Biology and DNA Data Bank of Japan, inalliance with the National Institute of Informatics (NIl)and the Mitsubisi Research Institute, Inc. (MRl) havestarted a three years long project since 2003, "Researchand Development of the New Generation of Bio-portal",
to enhance the information environment for the
relevant user communities. In this project, theLaboratory for Research and Development 'of Biological
Databases is responsible for the development of
biological Web services. The project site is open athttp)/www.bioportal.jp/in 2004. From there, a Web
page for links to sites complete genome sequence andannotation are also prepared and accessible, in
addition to the biological Web services. The former isnamed "Genome Menu".
2) Expansion of Genome Information Broker (GIB)

Masaki Hirahata, Naoto Tanaka, Takashi Abe, Satoru
Miyazaki t, Hideaki Sugawa.
'Tokyo Univ. of Sci.
GIB was originally created for the retrieval andanalysis of E. coli genomic information in a set. We
implemented microbial genome data into GIB
whenever genome sequencing was completed and thedata is made open to the public. At the GIB Web page(http://gib.genes.nig.ac.jp/), key word search, homology
search, links to DBGET, KEGG and GTOP andLaboratory for Gene Function Research

117

visualization of the data are available for more than
300 strains as of December 2005. We have utilized XML,CORBA and a distributed database in order to copewith the explosion of microbial genome information.
3) Development of Genome Information Broker for
Viruses genome (GIB-V)
Masaki Hirahata, Takashi Abe, Naoto Tanaka,
Hideaki Sugawa.
The importance of analyses of the viral genomes
is increasing. Comparative genomic analysis is being
advocated as the new analysis to discover knowledge
discovery.
We developed a database of viral genomes that wecall, the Genome Information Broker for Viral genomes(GIB-V). GIB-V contains data for 1487 viral and viroid
genomes and can indicate, compare, and analyze thedata contained therein. For the development of thedatabase, we applied the architecture of the Genome
Information Broker for Microbial genomes (GIB) thathave powerful comparative genomics function. GIB-V
is possible to execute the analysis for the selected viralgenomes. We equipped a function to visualize theresult of arraignment to compare the genome structureof each viral genomes. GIB-V is an effective andpowerful tool for extracting a viral genome sequences.
GIB-V is freely available at http://gib-v.genes.nig.ac.jp.
4) Development of Genome Information Broker for
Insertion sequence (GIB-IS)
Takehide Kosuge, Takashi Abe, Yoshio Tateno,
Hideaki Sugawara
The prediction of insertion sequence (IS) is one of
the important processes for bacterial genomes
annotation, and has great influence on the judgmentof the gene prediction. Although huge number of IS
data exist in the international nucleotide database(DDBJ/EMBLIGenBank), it is difficult to obtainaccurate annotations and sequences of IS, becausesome the data have erroneous annotations.
Additionally, there has been no public service whereusers can retrieve whole set of IS data. To solve theproblems of current situation, it is important to provide
users with exact IS data. We developed a IS database(named as GIB-IS) which includes whole bacterial IS

data, such as, IS name, synonym, family name, andsequences. GIB-IS is freely available at http://
bioportal.ddbj.nig.ac.jp/is/index.html.
(2) Information systems on microbes and biodiversity
1) WFCC-MIRCEN World Data Centre for Microorganisms (WDCM)
Yasumasa Shigemoto t, J unko N agaya, Hideaki
Sugawara.
tFujitsu.
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 W orId Directory of
microbial resource centers. The on-line World
Directory contains the detailed information of 469
centers in 65 countries and also the list of theirholdings. Any culture collection is able to register,
update and delete the information at http://
www.wdcm.org/. WDCM could promote the update ofthe data by culture collections funded by the American
Society for Microbiology and UNESCO.
2) Development of an e-Workbench for BiologicalClassification and Identification (InforBIO)
N aoto Tanaka, Kouji Koorikawa t, Takashi Abe,
Satoru Miyazaki tt, Hideaki Sugawara.
tHitachi soft, ttTokyo Univ. of Sci.
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 (http://lilium.genes.nig.ac.j pi index_e.html).
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 (http://
www.wdcm.org/byogen/).

118 Laboratory for Research and Development of Biological Databases

(*) The information system on pathogenic microor-ganisms has been supported by Special Coordination
Funds for Promoting Science and Technology.
4) Global Biodiversity Information Facility (GBIF)
Hideaki Sugawara, Motomi Itoh 1 and Keiichi
Matsuura 11'Univ. of Tokyo, '1 National Science Museum,Tokyo
GBIF is an international project to establish amechanism to aggregate data on diverse domains
including abiotic ones. The GBIF data system iscomposed of national nodes and we now host a Japan
GBIF node. This laboratory has implemented awrapping system based on one of GBIF standards,
namely, DarwinCore and DiGIR protocol and somebiodiversity data in Japan are now accessible in the
GBIF portal site.
(3) Applications of IT to the International Nucleotide
Sequence Database1) Development of Open Annotation System
Satoru Miyazaki 1, Takashi Abe, Hideaki Sugawara.
lTokyo Univ. of Sci.
A number of the complete genome sequences havebeen submitted to INSD since 1995. The annotation
information, however, is not consistent among genome
sequencing teams. In addition, researchers outside of
the team might have more information and knowledge
on some genes and biological molecules. Therefore, it
is quite important to develop the system which allows
any expert to evaluate the annotation given by the teamto attach more valuable information. As a new featureof INSD, we develop so-called "Open Annotation
System (OASYS)" as an annotation editor in the
distributed environment on the Internet.
(*) OASYS project has been supported by BIRD ofJapan Science and Technology Corporation OST)
2) Development of an e-Workbench for comparative
genome analysis (G-InforBIO)
Naoto Tanaka, Kouji Koorikawa 1, Takashi Abe,
Satoru Miyazaki", Hideaki Sugawara.
lHitachi soft, "Tokyo Univ. of Sci.

We have developed the G-InforBIO system forseamless integration of browse and analysis of
genomic data published in the International Nucleotide
Sequence Database (INSD: DDBJiEMBLIGenBank) andalso local data in research laboratories. Major functionsof the system are database management, data retrieval,sequence data analysis and the visualization for
genomics. The Suite is written in Java so that it isexecutable in Macintosh OSX, Windows XP, Linux andUnix machines, and will be downloadable from http://
wdcm.org/inforbio/.
The database accommodates multiple flat files(FFs) of entries registered in the INSD and alsotab-delimited files of local data. The SOAP interface
on the G-InforBIO system is used to download FFs of
microbial genome data published from DDBJ. Genome
information on the imported database can be readily
retrievable by keyword searches. A subset of thedatabase can be also retrieved for the further analysis
and graphical representation. The G-InforBIO system
includes sequence analysis programs for cluster
analysis (Blastclust, ClustalW and SOM) andhomology analysis (MegaBlast, Blast, Blat and Sim4).
The analysis can be applied to the retrieved subset anddata imported from outside. The system provides abroad-ranging utilization of genome information.
3) Exhaustive evaluation of the annotation dataobtained by simultaneous annotations of whole bacterial
genomes.
Takehide Kosuge, Toshihisa Okido, Yasumasa
Shigemoto 1, Masaki Hirahata, Naoto Tanaka, TakashiAbe, Satoru Miyazaki' 1, Hideaki Sugawara.
1 Fujitsu, llTokyo Univ. of Sci.
The number of complete genomes of
microorganism is increasing year by year. Currently,
complete genome sequence of "'300 strains areavailable from INSD (International Nucleotide
Sequence Database, http://www.insdc.org/) and they
are summarized in our web-site, GIB (http://
gib.genes.nig.ac.jp/). Users expect that they can obtain
exact and up-to-date annotations from the genomes.
In fact, users must take care for using the geneannotations of the publicized data because the qualitiesof the annotations are not uniform, e.g. difference of
blast and motif database version, prediction programs,
and parameters for using prediction program. To solve

Laboratory for Research and Development of Biological Databases

119the problems of current situation, it is important to
supply ORF data predicted by the same protocol anddatabases as a third party annotations.
We have already developed an annotation
protocols (named as GTPS) which aimed at bacterial
genomes. GTPS protocol can classify the all predicted
ORFs into several grades based on a degree of
reliability. The GTPS protocol was applied to all
chromosomes and plasmids of 123 microbial strainsat the same time. The 123 indicates the number ofstrains whose complete genome sequence waspublicized as of July, 2003. Our protocol could foundmiss-annotations which were not annotated inpublicized data because some of the ORFs areregistered in the current Swiss-Prot or RefSeq
databases. The simultaneous annotations were
updated in 2004, the number of analyzed strains
increased to 182 strains. The update data will beavailable from our web site (URL).
(4) Genomics
1) A large-scale Self-Organizing Map (SOM) unveilssequence characteristics of a wide range of eukaryote
genomes
Takashi Abe, Toshimichi Ikemura t, Hideaki
Sugawara.
tSOKEN-DAI
Novel tools are needed for comprehensive
comparisons of interspecies characteristics of massive
amounts of genomic sequences currently available. An
unsupervised neural network algorithm, Kohonen's
Self-Organizing Map (SOM), is an effective tool forclustering and visualizing high-dimensional complex
data on a single map. We modified the conventional
SOM, on the basis of batch-learning SOM, for genome
informatics making the learning process and resulting
map independent of the order of data input. We
generated the SOMs for tetranucleotide frequencies in
10- and 100-kb sequence fragments from 38 eukaryotes
for which almost complete genome sequences areavailable. SOM recognized species-specificcharacteristics (key combinations of oligonucleotide
frequencies) in the genomic sequences, permitting
species-specific classification of the sequences without
any information regarding the species. We also
generated the SOM for tetranucleotide frequencies inl-kb sequence fragments from the human genome andfound sequences for four functional categories (5' and3' UTRs, CDSs and introns) were classified primarily
according to the categories. Because the classification
power is very high, SOM is an efficient and powerfultool for extracting a wide range of genome information.
2) Phylogenetics Aualyses of Environmental Samples
on the Basis of Self-Organizing Map (SOM)
Takashi Abe,
Sugawara.
tSOKEN-DAI.

Toshimichi

Ikemura t

HideakiMetagenomic approach, which is the genome
analysis on a mixture of uncultured microorganisms,
has been recently developed to search for novel andindustrially useful genes and to study microbial
diversity in a wide variety of environments. We
previously modified the conventional SOM for genome
informatics to make the learning process and resulting,
map independent of the order of data inpue ·6). In thepresent study, we developed the SOM as a novel
bioinformatics strategy to capture and visualize
microbial diversity and relative abundance of
microorganisms within an environmental sample. First
we constructed SOMs of tri- and tetranucleotide
frequencies in 1- and 5-kb sequence fragments fromprokaryotic genomes for which complete sequence isavailable. The sequences could be classified primarily
according to species and to 11 major phylogenetic
groups without information regarding the species. For
example, 88% of 5-kb sequences were classified into thecorrect phylogenetic group. Importantly, theclassification could be done without orthologous
sequence sets, and. therefore, SOM was especially
useful to analyze novel sequences from poorly
characterized species for industrial applications andscientific studies. With the SOM method, all non-rRNAsequences in the Database that were from unidentified
or uncultured bacteria and longer than 1 kb wereclassified into major phylogenetic groups. The present
method can also be developed as a tool for surveys ofpathogenic microorganisms in environmental andclinical samples that can not be cultured easily andin sterilized samples.

120 Laboratory for Research and Development of Biological Databases

11.

PUBLICATIONS

1fJJl(~aA, ~#i(2005) ~ /' 7 Papers
1. Abe, To, Sugawara, Ho, Kanaya, So, Kinouchi, Mo,
Matsuura, Y, Tokutaka, Ho, and Ikemura, T (2005) A
large-scale Self-Organizing Map (SOM) constructedwith the Earth Simulator unveils sequencecharacteristics of a wide range of eukaryotic genomes,
Proceedings of Workshop (2005) on Self-Organizing
Maps, 187 - 194
20 Abe, T, Ikemura, To, Kanaya, So, Kinouchi, Mo, andSugawara, R (2005)A novel bioinformatics strategy for
phylogenetic study of genomic sequence fragments:
self-organizing map (SOM) of oligonucleotide
frequencies, Proceedings of Workshop (2005) on
Self-Organizing Maps, 669 - 676
30 Abe, To, Sugawara, Ho, Kinouchi, Mo, Kanaya, So,
and Ikemura, To (2005) Novel Phylogenetic Studies ofGenomic Sequence Fragments Derived from
Uncultured Microbe Mixtures in Environmental andClinical Samples, DNA Research, 12, 281 - 290
40 Hayashi, Ho, Abe, To, Sakamoto, Mo, Ohara, R,
Ikemura, T, Sakka, K, and Benno, Y (2005) Directcloning of genes encoding novel xylanases from humangut, Canadian Journal of Microbiology, 51, 251 - 259
50 Saka, Koo, Tadenuma, Mo, Nakade, So, Tanaka, No,
Sugawara, Ho, Nishikawa, K, Ichiyoshyi, No, Kitagawa,Mori, Ho, Ogasawara, No and Nishimura, A. (2005) A
Complete Set of Escherichia coli Open Reading Frames
ion Mobile Plasmids Facilitating Genetic Studies, DNA
Research, 12, 63 - 68
60 Sugawara, Ho, Miyazaki, So, Abe, To, andShigemoto, Y (2005) Biological Data Anelysis using
DDBJ Web services, Proceedings of BIOINFO 2005, 379 382
70 Uchiyama, To, Abe, T, Ikemura, To, Watanabe, K(2005) Substrate-induced gene-expression screening ofenvironmental metagenome libraries for isolation of
catabolic genes, Nature Biotechnology, 1, 88 - 93
80 IWJ-$Jti5, ~5cJ*~, 'BAA-tv, 1fJJl(~aJH2005) Web~- to 7, I;: J: Q /'\~ ::t11f¥IH~~O)*ftftfto) PJf3~,t!, ~ 1@)/,,~ ::t11f.~iJf~~, 21 - 28
Books
90 IWJ-$Jti5,~1'H!~,;;j<V J*JIDiX,

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Database
120 Gene Trek in Procaryote Space (GTPS) http://
gtps.ddbjonigoacojpl
130 Genome Information Broker for Virus (GIB-V)
http://gib-vogenesonigoacojpl
14. GIB-IS http://bioportal.ddbj.nig.ac.jp I is I
index.html
150 Genome Information Broker for Environmental
sequence (GIB-ENV) http://bioportal.ddbj.nigoacojp/
wgsl indexohtml
160 DDBJ Open Annotation System (OASYS) http://altheaoddbjonigoacojp/indexojsp
170 GBIF http://bioportal.ddbjonigoacojp/gbiC
txl ,http://gbifoddbjonig.acoj pigbiCsearchlindexohtml
EDUCATION

1. 1fJJl(~~ 1:.~M~~~ .ilJ*~
20 1fJJl(~aA Robust & advanced technologies for the
information sharing & diffusion Useful tools for thedata analysis from phenome to genome Workshop
on Bioresource Information Management (BIM) /'\/'::I

7

30 IWJ-$Jti5/'\~ ::t-r-7"'- 7,~mO)£wn/'\~::t~/'7~~T~77,~~ m~~~~$77~

SOCIAL CONTRIBUTIONS AND OTHERS~~~±ft
1fJJl(~IlA~~

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I-e. Laboratory for Gene·Expression Analysis
Kosaku Okubo Group::I F/'~.FIHjf{l~U~1:.~i1:~O)~Jt 1:.~I~/'/' F7'y

7 131 - 132
100 1i.{~:tJIij~, 1fJJl(~aJH2005)DDBJO)fIJffl~
flJffl~~.lI.iliJt&

DDBJO)The laboratory for Gene-Expression Analysis consistsof the following members:
Kousaku Okubo (Professor), Osamu Ogasawara

Laboratory for Research and Development of Biological Databases

121

(Posdoc), Makiko Otsuji (Graduate student), Isao
Kubota (Graduate student), Miya Shiojima (technicalassistant),Takayasu Iizuka (systems engineer), Koji
Watanabe (systems engineer), Hiroaki Imamura(systems engineer), Sumiyo Sugimoto (Secretary).
(1) BodyMap-Xs: Anatomical breakdown of 17 million
animal ESTs for cross-species comparison of gene
expression
Ogasawara 0, Watanabe K, Imamura H, Iizuka T,
Okubo K
BodyMap-Xs (http://bodymap.jp) is a database forcross-species gene expression comparison (Ogasawara,0., et.al., 2006) ; it was created by the anatomical
breakdown of 17 million animal EST records in DDBJ
by using a sorting program tailored for this purpose.
In BodyMap-Xs, users are allowed to compare theexpression patterns of orthologous and paralogous
genes in a coherent manner; this will provide valuable
insights for the evolutionary study of gene expression
and identification of a responsive motif for a particular
expression pattern. In addition, starting from a concise
overview of the taxonomical and anatomical
breakdown of all animal ESTs, users can navigate toobtain gene expression ranking of a particular tissue
in a particular animal; this method may lead to theunderstanding of the similarities and differences
between the homologous tissues across animal species.
BodyMap-Xs will be automatically updated insynchronization with the major update in DDBJ, which
occurs periodically.
(2) Identification of differentially expressed genes inpsoriasis using expression profiling approaches.
To identify differentially expressed genes whichplay causal roles in pathogenesis and maintenance forpsoriasis, we used BodyMapping and introduced
amplified fragment length polymorphism approaches(Itho, K., et.al., 2005). From the BodyMap database, we
selected 2007 genes which specifically expressed inepithelial tissues. Among 2007 genes, we surveyed
genes which differentially expressed in involved or
uninvolved psoriatic lesional skin samples comparedwith atopic dermatitis, mycosis fungoides, and normalskin samples. Using the 2007 gene set, we examinedgene expression levels in five serial lesions from distal

122 Laboratory for Gene-Expression Analysis

uninvolved psoriatic skin to involved psoriatic plaque.
We identified seven genes such as alpha-lmicroglobulin/bikunin precursor, calnexin, claudin 1,
leucine zipper down-regulated in cancer 1, tyrosinaserelated protein 1, Yes-associated protein 1, andunc-13-like protein (Coleonyx elegans) which show
high-expression levels only in uninvolved psoriaticlesions. These seven genes, which were reported to berelated to apoptosis or antiproliferation, might havecausal roles in pathophysiology in psoriasis.
(3) Comparison of Gene Expression Patterns between
human and mice brain.
Otsuji M, Okubo K
To elucidate gene expression patterns between
human and mice brain, we construct 3'-directed cDNA
libraries from several portions from human and micebrain. From now on, we measure gene expression levels
using iAFLP method.
(4) A Theoretical Model for the Accumulation of Gene
Expression Divergence
Ogasawara 0, Okubo K
We have constructed an explicit theoretical modelthat describes the evolutionary changes caused by theaccumulation of gene expression divergence; this model
was constructed based on the assertion that aphenomenon concerning the distribution of transcriptlevels, called Zipf's law of transcriptome, would
originate from the accumulation of gene expression
divergence. Surprisingly, our model predicted that thetranscript level variation within and between species
would not be correlated; instead, the scatter plot ofsuch a transcript level variation would exhibit astriking L-shaped distribution that is very similar tothe observed distribution. It should be pointed out that
this distribution had previously been considered as
evidence" against" the effect of the accumulation ofgene expression divergence. These results indicate thatthe process of the accumulation of gene expression
divergence has a considerable effect on gene expression
evolution.

PUBLICATIONSPapers

1. Itoh K, Kawasaki S, Kawamoto S, Seishima M,
Chiba H, Michibata H, Wakimoto K, Imai Y, Minesaki
Y, Otsuji M, Okubo K. (2005) I dentification ofdifferentially expressed genes in psoriasis using
expression profiling approaches., Exp Dennatol, 14 (9)667 - 674
2. Kaimori ]Y, Takenaka M, Okubo K (2005)
Quantification of gene expression in mouse and humanrenal proximal tubules., Methods Mol Biol, 293, 209 219
3. Tanino, M., Debily, MA., Tamura, T., Hishiki, T.,Ogasawara, 0., Murakawa, K., Kawamoto, S., Itoh, K.,
Watanabe, S., de Souza, S]., Imbeaud, S., Graudens,E., Eveno, E., Hilton, P., Sudo, Y., Kelso, ]., Ikeo, K,
Imanishi, T., Gojobori, T., Auffray, c., Hide, W., andOkubo, K (2005) The Human Anatomic Gene
Expression Library (H-ANGEL), the H-Inv integrative
display of human gene expression across disparate
technologies and platforms., Nucleic Acids Res., 33, 567 572
4. Tateno, Y., Saitou, N., Okubo, K., Sugawara, H.,
and Gojobori, T. (2005) DDB] in collaboration with
mass-sequencing teams on annotation., Nucleic Acids
Res., 33, 25 - 28
5. *~*0~, B~1B'HH2005) r~.A 1-7/ Afli'ff\:;v;:iWi*Q/'\-1;t§rti§~9lbf!l!.A..O)M*f:

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46 (2480) 137 - 142
Books

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Laboratory for Gene-Expression Analysis

123

J. RADIOISOTOPE CENTERRESEARCH ACTIVITIES(1) Environmental genetics: Stationary phase-induced
genome rearrangements
Yasuyuki Ogata
In nature bacterial cells are usually in stationary
stages, in which the cells alter their genetic inheritance.
We found that illegitimate recombination is induced
when growth phase is changed from log to stationary
phase; illegitimate recombination occurred more
frequently in early-stationary phase than in mid-log
phase during formation of 1bio-transducing phage.
When we did a quantitative analysis of deletion
formation using a miniF-based plasmid, we found thatthe stationary phase-induced illegitimate recombination can be also detected in this assay system. In
addition, we indicated that the frequency of deletionof the tag atkAl double mutant was higher than thatof its isogenic wild-type strain in early-stationary
phase, thereby implying that the alkylated lesion may
be responsible for the stationary phase-induced
illegitimate recombination. Since illegitimate recombination results in genome rearrangements including
deletion, duplication, insertion, or translocation, it
seems likely that stationary phase may induce genome
rearrangements. Thus, when bacterial cells confront
unfavorable conditions for their growth, the cells seemto increase diverse mutations as if they struggle foradaptation to the environment. If the situation takes
a favorable turn, a suitable mutant clone is likely togrow preferentially, which may lead to evolution.
(2) Archaeological genetics: Isolation of ancient bacteria
from Antarctic iceberg
Yasuyuki Ogata
In Antarctica ice sheet that snow accumulates for

124 RADIOISOTOPE CENTER

about a million years and is transformed into ice exists.
We aim to isolate ancient bacteria from a piece of
iceberg and surmise the evolution of bacterial genome.
We built a clean booth in our laboratory and set up
a clean bench inside of it for the purpose of avoiding
contamination and the iceberg piece was melted,
filtrated, and particles were collected. By microscopic
surveillance we obtained bacteria-like particles and
DAPI staining revealed that these particles contained
nuclei. Moreover, we amplified genome DNA fromthese particles in vitro and sequenced them. Selforganizing map (SOM) analysis which was developed
as a novel bioinformatics strategy for phylogeneticclassification of sequence fragments obtained frompooled genome samples showed that these sequence
fragments originated from Firmicutes, Euryarchaeota,
or Cyanobacteria.
(3) Field genetics: Obtaining of Antarctic gene resources
without contamination
Yasuyuki Ogata
In Antarctica it is extremely cold and ultraviolet rays
are much irradiated. I am trying to isolate the bacteria
from Antarctica and obtain the genes responsible forsurvival under these severe environments. Noveldefense systems against DNA damage or repair
mechanism are expected. For this purpose, I had madean Antarctic exploring by accompanying the 47th
Japanese Antarctic Research Expedition (JARE47) sinceNovember 28,2005 to March 28, 2006 and collected
deep-sea water, soil, or snow and ice without
contamination; I built a clean booth on the snowfieldat S17 and dug the ice core with a sterilized ice auger
and I also set up a clean bench on the ground at
Rundvagskollane and Ongul Islands and scooped up
the soil with a sterilized spoon.

K. EXPERIMENTAL FARM

genome, species and accessions and to examine thesuspicious accessions in the core collection, we havemeasured DNA content by flow-cytometry analysis as
well as chromosome numbers by microscopeobservation. DNA content and chromosome numberof each species and accession were well characterized
as to reveal that a few accessions have been categorized
to wrong species.

RESEARCH ACTIVITIESPUBLICATIONS

Experimental Fann is responsible for preparationand cultivation of all kinds of rice strains necessary
for the studies of genetic resources and of functional
genomics. The facilities for plant growth in theExperimental Farm are also available to the collaborators outside the NIG. All works in theExperimental Farm have been carried out ascollaborative works with the Plant Genetics Lab. One
specific activity related to the wild rice collection is
introduced below. For other activities, see the reportsof Plant Genetics laboratory.

Papers(1) Examination of genetic characters for accessions ofwild rice core collection
Ken-Ichi Nonomura, Yukie Sano, 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 maintained in the NIG, several
accessions from each species were selected to prepare
core collection. About 300 selected lines had already
sub-grouped to Rankl, Rank2 and Rank3 categories,
and a part of them were characterized for theirphenotypes 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 (WI514) and CC (WOOO2) genome species with thecultivated AA (Nipponbare) species have been carriedout by eDNA clone sequencing. Some results ofcomparative analysis are presented in the report ofPlant Genet. Lab.
In addition, to confirm genome sizes of each

1. Ito, Y., Takaya, K and Kurata, N. (2005) Expressionof SERK family receptor-like protein kinase genes inrice, Biochim. Biophys. Acta, 1730, 253 - 258
2. Itoh, J. I., Nonomura, K I., Ikeda, K, Yamaki, So,
Inukai, Yo, Yamagishi, H. and Nagato, Y (2005) Rice
plant development: from zygote to spikelet, Plant Cell
Physiol., 46, 23 - 47
3. Ito, Y. and Kurata, N.(2005) Identification ofenhancer trap lines with tissue-specific GUS activities
in rice, Rice Genet. Newslet., 22,32 - 33
4. Kurata, No, Miyoshi, K., Nonomura, K. I.,
Yamazaki, Y and Ito, Y (2005) Rice mutants and genes
related to organ development, morphogenesis andphysiological traits, Plant Cell Physiol., 46, 48 - 62
5. Moriguchi, K., Suzuki, To, Ito, Yo, Yamazaki, Y,
Niwa, Y and Kurata, N. (2005) Functional isolation ofnovel nuclear proteins showing a variety of subnuclear
localizations, Plant Cell, 17, 389 - 403
6. Xu,J-Ho, Kurata, N., Akimoto, M., Ohtsubo, H. and
Ohtsubo, E. (2005) Identification and characterizationof Australian wild rice strains of Oryza meridionalis
and Oryza rufipogon by SINE insertion polymorphism,
Genes Genet. Syst., 80, 129 - 134
7. Suzuki, T., Eiguchi, M., Satoh, H., Kumamaru, T.
and Kurata, No (2005) A modified TILLING system forrice mutant screening, Rice Genet. Newsleto, 22,89 - 90
EDUCATION

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EXPERIMENTAL FARM

125

SOCIAL CONTRIBUTIONS

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126 EXPERIMENTAL FARM

L. INTELLECTUAL PROPERTY UNIT

Munehiro Tomikawa
RESEARCH ACTIVITIESThis unit makes various efforts to return theoutcome of research generated by NIG to the industrial
world and the public while respecting the scientists'priorities in terms of scientific ideas, partnership andpublications.
To be more precise, what we have done is toestablish scientific results as intellectual property,
explain the property rights to the industrial world
intelligibly, seek collaboration with commercial
industries for actualizing those results and establish
partnerships. This shows the importance of carryingout both public relations and the acquisition of
intellectual property.
This unit was set up in March 2003. Since April
2003, a secretary, Ms. Nozomi Yokoyama has devoted
all her energy to this work from starting up ,and ha{1assisted by creating the MTA( Material Transfer
Agreement) form ,and getting it off the ground. She has
also been supporting various works such as planningand promotion of publicity activities and proceduresof application for intellectual property rights. The
results of our work in 2005 are shown as follows.

(1) Acquirement and utilization of Intellectual Property
RightsWe have continued to promote a sense ofentitlement in the scientists belonging to NIG andDivision of Life Science in the Graduate University for
Advanced Studies (Sokendai) by giving them speecheson why entitlement is necessary to the outcome of basic
research. Also, in visiting laboratories, monitoring thescientific progress and thinking of each scientist, wehave explained how intellectual property rights can begained from their results. We have also established anefficient patenting procedure. We have prepared aninvention exposition format to minimize the scientists'burden in informing our unit of their scientific results.
Our unit helps the scientists avoid the troublesometask of acquiring of patent rights to the practicalapplications of their inventions.
In terms of patent applications, we have tried topromote acquirement of the patent rights so the NIG
scientist does not need to bear the costs necessary forthe preparation of patent documents. At this time, 9domestic patents have been newly applied for and 1
patents are under preparation, of which 12 as a wholewill be applied for as NIG patents (including 3 joint
applications and three international patents) in the
Research Organization of Information and Systems(ROIS). One patent has already come into marketing
as of November 2003. We got the royalty with a small
amount from the second year of the business sinceApril 2005.
Secondly, we have been involved in making
guidelines for the Material Transfer Agreement which
stipulates ownership, ethical codes and compensation
for biological resources based on research results. At
present, three kinds of agreements, depending on thepositioning of biological resources as intellectual
property, have been created and are in use. Each has
a simple version corresponding to specific materials
in the research field such as fly, yeast, (Form l)and anintermediate version without provision for sharing ofindustrial property rights (Form 2) and a complete
version with provision for sharing of industrial
property rights (Form 3).They have been authorized
and, a total of 370 agreements (approximately 80% ofthem are international) have been signed by the endof year.
In addition, we have participated in the
Shizuoka-TLO-Yaramaika (STLO) campaign, whichfacilitates putting patents owned by the NIG into
practical use.
(2) Acceleration of public relations activities
Ideally, this kind of work should be developed overa broad spectrum, however, we have focused our

INTELLECTUAL PROPERTY UNIT

127

attention on the following main points. We have given
priority to our response to visitors, promotion atacademic conferences, introduction of research results
to the public, and the search for potential collaboration.
Firstly, we had 12 groups of visitors (total 229
persons) in 2005: prefectural university students, legal
apprentices, high school students, elderly community
college students and others. An overview of thisinstitute, genetic science and current laboratory
experiments was given to the visitors.
Secondly, promotion at academic conferences has
been carried out in order to obtain more excellent
students and scientists. We introduced the current
research results obtained from NIG scientists at theJapanese Cell Biology Meeting (in Sendai), the JapaneseSociety for Developmental Biology meeting (in Oomiya),
and the Japanese Molecular-Biology meeting (inHakata).
Thirdly, we presented eight specifically excellent
research results in the Hot News column on the website
as an introduction of research results and academic
awards for life science to the public. We have also hadeight research results inserted in main-stream
newspapers through cooperation with the media.
Furthermore, we introduced two scientists tocompanies which were interested in their work, andfortunately, this effort led to further business-academia
collaboration.
(3) Collaboration Research with academia, industry andgovernment, and Social Action WorkI have been conducting research of Fuji Pharma
Valley Cancer Diagnostics Study Group consisting ofacademia, industry and government. The aim of thisproject is to increase Research and Development
potential in the Mt. Fuji area and its cities of Numazu,
Mishima, Fujinomia, Fuji and Nagaizumi" and to study
together with companies operating in the field of
medicine as well as the pharmaceutical industry on thedevelopment of cancer diagnostics through genomics
and proteomics approaches. Fortunately, we could
apply two patent regarding cancer diagnostics basedon regulation of chromatin DNA this December.
Recently, the participation of public facilities, such

128 Publicity and Intellectual Property Unit

as universIties and NIG, is increasing in the
Government-Industry-University-Research Cooperation
Conference. In June, We attended the Fourth
Government-Industry-University Cooperation Conference as a panelist on behalf of NIG, which was heldat Kyoto. In July, We got the matching fund with
Shizuko-Sangyou Co., Ltd. from NEDO for researchdevelopment of the thinner Multi-well Plate whichfacilitated to transport and to reserve the biologicalresources such as bacteria, plasmids and DNA andsucceeded in preparing one-well plate and multi-wellplate with 16,96 and 384 wells. It was quite meaningful
to be able to develop a relationship with industrial
people who was interested in our patent and products.
In this March, we got approval of the establishmentof venture business referred as BioROIS from ROIS.
In conclusion, acquiring intellectual property andengaging in returning excellent technology fromscientific results to the industrial world through public
relations will be important factors in gaining wide
public recognition of NIG. These efforts are expected
to facilitate the financial management of NIG, after
turning into independent administrative entities.

M. TECHNICAL SECTIONRESEARCH ACTIVITIESThe Technical section supports the research
activity of NIG in many fields. For example, we support
the Radioisotope center and Experimental farm, and
the Genetic strain research center where genetic andtransgenic strains of mice, rice, flies, and fishes areproduced, maintained, and distributed to otherinstitutions. Members of the staff additionally support
many types of experiments in various laboratories,
such as developmental, cell, and molecular genetics.
We continually update our technical services throughattending the training and annual meetings for thetechnological staffs.
PUBLICATIONSPapers
1. Ohya, Y, Sese,]" Yukawa, Mo, Sano, F., Nakatani,
Y, Saito, T. L., Saka, A, Fukuda, T., Ishihara, S., Oka,S., Suzuki, G., Watanabe, M., Hirata, A, Ohtani, M.,
Sawai, H., Fraysse, No, Latge,]. P., Francois, ]. M., Aebi,
Mo, Tanaka, S., Muramatsu, S., Araki, Ho, Sonoike, K.,
Nogami, S. and Morishita, S. (2005) High-dimensional
and large-scale phenotyping of yeast mutants., Proc.
Natl. Acad. Sci. USA., 102 (52) 19015 - 19020
2. Saka, K., Tadenuma, M., Nakade, S., Tanaka, N.,
Sugawara, H., Nishikawa, K., Ichiyoshi, N., Kitagawa,M., Mori, H., Ogasawara, N., Nishimura, A (2005) A
Complete Set of Escherichia coli Open Reading Frames
in Mobile Plasmids Facilitating Genetic Studies, DNA
RESEARCH, 63 - 68

TECHNICAL SECTION

129

BIOLOGICAL SYMPOSIUM 2005
Jan.12
Feb.16
Mar.2
Mar.7
Mar.14Mar.22
Mar.22
Mar.23Mar.24Mar.25
Apr.7
Apr.19Apr.22
Apr.27May.24May.26
Jun.8
Jun.20Jun.23
Jun.28
JuLl5
Ju1.20Ju1.25
Aug.16Aug.24
Sep.8
Sep.27
Sep.30
Oct.13
Oct.20
Oct.24
Oct.25
Nov.2
Nov.7
Nov.11
Nov.16
Nov.21
Nov.24
Nov.24
Nov.25
Nov.28

DNA ;( T /Ht:a:- r:p It\ c LJr.!f / A ? -{ F.::r.. \:::0:/ :r.

*' T -1 'Y 7 A (Kunio Shiota)

ft~A I- vAJ;t~·{H~:toft:OJllfiUtEf~*ffl.Jc'i¥f~$fJm#;IU.

(Toshihumi Inada)
Rett ~lH~$O)~ffEam : MeCP2 f~ J::o 7 t1'"'<T /' /v-7°:a:-fl-l., tcJ!~-f-~:m$fJmamO)MaA(Shinichi Horike);( 1J Jf / A 7 -( 7'7 !J -:a:-fIJffl l.,tc*9;Um~-f-A 7 !J -.:::./, Y7*<l)~~ (Taku Uchiyama)
GENETICS OF HUMAN CANCER SUSCEPTIBILITY (Kari Hemminki)
NHR-25/ Ftz-fl / SF-l: Role during epidermal differentiation in C. elegans (Masako Asahina)
The steroid deficiency gene ecdysoneless (Marek Jindra)*DIJmO)~~ C 1Jjl*H~Htr~*DIJmO)il1fIt:a:-$fJmT:O.y Y-r /v~Ji (Yukiko Goto)
Age and sex effects on human mutation rates: an old problem with new complexities (James F. Crow)
J}fJll~~:a:-JlifJmT:07t-f-amO).:::.? I- !J Jlf:a:-ffl1t\tcMaA (Yuji Yokouchi)
Jll.mO)~iiHJ:a:-tJEkf~:toT""*DI_0).3«.8"Jrn-{:}:a:-*DIJmI*J6~~~~T:OJll1J /'/"\7"" (Yasushi Okamura)-e'7'7 7 -1 'Y.y .::z.:a:-ffllt\tcmM~~:a:-JlifJmT:OI2Sl-f-O)a~MfJT : iJII:.Ifn.~~cft;:j1n~Jjj(;O)7t-f-;( 7J .:::.:J:,A(Atsuo Kawahara)
Control of Polycomb Group gene expression by Imprinting and by feed-back regulations.
(Frederic Berger)
1ftt~~<l)mMft;:fIIJ . 1JjliHf~Jjj(;O)7t-f-am (Masahiko Hibi)~~f~:to~t:o 13 f$8"Jm~11f¥IH1~Jjj(;0);( 7J .:::.:J:,A (Shigeru Kondo):ffi*5HJ:tJ-{ 1J'/'Af~:toft:O*DIJmM:J ~ .::z..:::.'i"-.y 3 /' (Tetsuya Higashiyama)
Defining a developmental path to neural fate by global expression profiling of mouse embryonic stem
cells and adult neural stem / progenitor cells (Kazuhiro Aiba)
Molecular Dissection of the Metazoan Kinetochore (lain M. Cheeseman)
How hypoxia-induced suspended animation led us to a nitric oxide mediated pathway of innate immune
induction (Pat O'Farrell)
Cyclophilin D f~*ff l.,tc ~ I-:J /' F!J ;Jll~.;&rf1i1f$O)*DIJm~f~:to~t :O~IJj (Takashi Nakagawa).y 3 7:/ 37 /,\.::r..0)':::'.::z.- t1 -* 'Y I- ?-7amMfJT (Akira Chiba)'"'< 7 A~-f-<l)~~ c iI~tE :a:-JlifJmT:O RNA ~~1J /' /"\ 7 f{ (Yasuyuki Kurihara)*!!k C.elegans 0).y-r7°A~Jjj(;f~:to~t:o.:z.~'::\'-T/'!J :tJ--e RPM-l ~~J::O DLK-l-p38 MAP ::\'--r--e~j§<l)$fJmam (Katsunori Nakata)
Ran / RCCl .y AT AO)1fT:O~llI8"Jf~~~a~ (Hitoshi Nishijima)
7JT:J-/v; ~ /~JicfT~iIiljmO);(7J.:::.:J:,A (Kazuto Kobayashi)
J1im*'BJm<l)flitEN~cjf~· i'j:$5c:a:-!J /,7T:07t-f-;(7J.:::.:J:,A (Tatsushi Igaki)\::: I- DNA m~kM~ Dmcl ;6~fTf~ 5 DNA m~kocJ;tO)7t-f-;( 7J':::':J:, A (Takashi Kinebuchi)
Germ cell development in Drosophila (Ruth Lehmann).y 37:/ 3 7/'\.::r..~~:to~t:o mRNA-like non-coding RNA O)a~ (Yuji Kageyama)
Study of Molecular Evolution: an Informal and Personal Account. (Masatoshi Nei)
Innate and Adaptive Immunity in the fruit fly <David Schneider)
Adhesion remodelling underlying tissue morphogenesis (Thomsds Lecuit)Mechanism of DNA Methylation in Development (Guoliang Xu)
Detection and processing of color information in Drosophila (Claude Desplan)
Regulation of pre-RC assembly in yeast and human cells Uohn F.X. Diffley)
Asymmetric division and self-renewal of Drosophila neural stem cells (Chris Q. Doe)
Genomic Imprinting -Causes and Consequences (Anne C. Ferguson-Smith)m./,\-{ /"\Am! DNA polymerase t;; <l)~ 7 V:;tT F~"*f~:flafj\U~:to~t:O~l.,1t\a~(Tomoo Ogi)
*l~T:r. 'Y 7;f--{ /' l-am~j:*/i:J~ DNA *l~~~1t\;6~~~ V(j'i'MR Vn\:O;6~ (Kanji Furuya)
DNA damage checkpoint signaling in budding yeast (Katsunori Sugimoto)
Cell fate specification and cell shape control in the Drosophila retina (Takashi Hayashi)

130 BIOLOGICAL SYMPOSIUM 2005

Nov.3D
Nov.3D
Dec.l
Dec.12
Dec.13
Dec.14
Dec.211t(1)1~¥:5t*JT;>j~ G.;1j.td'G.'Elli1ftAO)j::?%(Minoru Yoneda)
1~;P-;>j~ G~Q:; -y ry IJRA(1)~WJ( ~ J1t1~ (Yosuke Kaifu)
Arabidopsis f[.:jO ft Q trans-acting siRNA (1)j::P.lG*Ilm (Manabu Yoshikawa)
The Sleeping Beauty Transposon System: A Tool for Discovering Genes Involved in Cancer andDevelopment (David A. Largaespada)
Enhancer detection in zebrafish: Search for developmentally important genes and regulatory sequences(Thomas S. Becker)
Roles of transcription-coupled histone variant deposition in maintaining chromatin structure andepigenetic states (Kami Ahmad)±tHUIllJl§:l*~mlt\tc~~l't-JtJ:IlFIl~L~.m
BIli1H;/-'.A 'T A (1) !J 7' Iv?, -1 AfW*JT (Kazuhiro Yagita)

BIOLOGICAL SYMPOSIUM 2005

131

FOREIGN VISITORS IN 2005
Mar.2 Shinichi Horike
Mar.14 Kari Hemminki

Mar.22 Masako Asahina
Mar.22 Marek Jindra

Mar.24 James F. Crow
Apr.22 Frederic Berger
Jun.8 Aiba Kazuhiro
Jun.20 lain M. Cheeseman
Jun.23 Pat O'Farrell
Ju1.15 Akira Chiba
Ju1.25 Katsunori Nakata
Sep.8 Tatsushi Igaki
Oct.20 Masatoshi Nei
Oct.24 David Schneider
Oct.25 Thomas Lecuit
Nov.2 Guoliang Xu
Nov.7 Claude Desplan
Nov.ll John F.x. Diffley
Nov.16 Chris Q. Doe
Nov.21 Anne C. Ferguson-Smith
Nov.24 Ogi TomooNov.24 Kanji Furuya
Nov.25 Katsunori SugimotoNov.28 Takashi Hayashi
Dec.12 David A. Largaespada
Dec.13 Thomas S. Becker
Dec.14 Kami Ahmad

132 FOREIGN VISITORS IN 2005

The Hospital for Sick Children Toronto, Canada
Division of Molecular Genetic Epidemiology, German Cancer ResearchCenter (DKFZ), Heidelberg, Germany, Department of Biosciences at
Novum, Karolinska Institute, 141 57 Huddinge, Sweden,
Institute of Parasitology, Academy of Sciences of the Czech Republic
Institute of Entomology, Academy of Sciences of the Czech Republic,
and Department of Molecular Biology, South Bohemica University,
Czech Republic
Department of Genetics University of Wisconsin-Madison
Temasek Lifescience Laboratory, The National University of Singapore
Laboratory of Genetics, National Institute on Aging, National Institutesof HealthLudwig Institute for Cancer Research
Department of Biochemistry and Biophysics, UCSF
Department of Cell and Structural Biology, and Neuroscience University
of Illinois at Urbana-Champaign
Department of MCD Biology, University of California, Santa Cruz
Dept. Genetics, Yale Univ. Sch. Med.
Institute of Molecular Evolutionary Genetics Pennsylvania State
University
Stanford University, Dept. Microbiology and Immunology
IBDM/LGPD
Institute of Biochemistry and Cell Biology Shanghai Institutes for
Biological Sciences Chinese Academy of Sciences
NYU, Dept. Biology
Cancer Research UK London Research Institute, Clare Hall Laboratories
University of Oregon, Institutes of Neuroscience and Molecular Biology
Department of Anatomy University of Cambridge, u.K.
Genome Damage and Stability Centre, University of Sussex
Genome Damage and Stability Centre, University of Sussex
Department of Cell Biology and Molecular Medicine University of
Medicine and Dentistry of New Jersey, USA
Department of Biochemistry, Molecular Biology and Cell Biology
Northwestern University
Department of Genetics, Cell Biology and Development University of
Minnesota Cancer Center
Sars Centre for Marine Molecular Biology Bergen, Norway
Department of Biological Chemistry and Molecular Pharmacology
Harvard Medical School

Annual Report of the National Institute of Genetics. No.56
Issued by Director-General: Yuji Kohara, D.Sc.
Research Organization of Information and Systems
National Insti tu te of Genetics
Yata 1111 Mishima, Shizuoka-Ken, 411-8540 Japan
Tel. 055-981-6718
Fax. 055-981-6715
Edited by Ryu Veda
Published by National Institute of Genetics