Highlights of 2002 (http://www.nig.ac.jp/hot/index.html)

1.

MEN-~, a secretory, proteir.l with an tDL receptc>r motif, regulates sensory

integration ar.ld learning in C. elegams.
HEN-1, a secretory proteili1 with! an LDL receptor motif, regulates sensory integrationand learning in C. elegons
Ismihara,l, lino,Y., MGhri,A., Mori,!., Gengyo-Ali1do,K., Mitani,S. Katsura,!.

Cell, 109, 639-649 (2002)

2.

Discontinuous phenotype char.l~es during bacterial differentiCltion

fractionation of c. coii Cell Populations of Different Stages (bring Growth Transition to
Statiofi1ary Phase.Makinoshima, H., Nishilillura, A. and Ishihama, A.

Mol. Microbiol. 43, 2689-280 (2002)

3.

Identification of. a substrate of S-pbase-cyclin-depen€lent protein kinase

S-Cdk-dependent pmospmorylation of Sld2 essential for ehromosomal DNA replicationin budding yemt.Maswmoto, H., MUJrmnatsw, S., KOIillimura, 1:'., and Araki, H.

Nature 4 15, 651-655 (2002)

4.

CENP-I is essential for centromere fl!lnction in ¥ertebrate cells

CENP-I is essential for centromere function in vertebrate cells.Ai Nisbimashi, lfokuko
hlaraguomi, Yosushi hiiraoka, Toshimiobi Ikemura, Vinciane Regnier, Helen Dodson,
William C. EeHfilShaw, and Tatsuo Fukagowa

De¥.Cell, 2, 463-476 (2002)

5.

A genome-wide survey of reproductive barriers

A genome-wide survey of repr@ductive barriers in an intraspecific hybrid.Yoshiaki
lrIarushima, MGsahiro Nakagahm, Masahiro Yano, Takuji Sasaki and Nori Kurata.

Gemetics, 159>, 883-892, (2001)

CONTENTS
Introduction'" . . . ... .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. .. .. . . . . . . . . 1
STAFF
2
COUNCIL
6
ADVISORY COMMITTEE·················································································· 7

A. DEPARTMENT OF MOLECULAR GENETICS
A-a. DIVISION OF MOLECULAR GENETICS
Comparative genomics of bacterial transcription factors
Nobuyuki FUJITA··················································································· 8
In vitro selection of transcription factor binding sequences from random genomic library
Nobuyuki FUJITA and Shizuko ENDO
8
Identification of proteins that interact with the Rpb7 subunit of RNA polymerase II in fission
yeast
Hiroshi MITSUZAWA
8
Identification of TBP-associated factors (TAFs) in fission yeast
Hiroshi MITSUZAWA

9Interaction between RNA polymerase II and the carboxy-terminal domain phosphatase Fcpl
in the fission yeast Schizosaccharomyces pombe
Makoto KIMURA
9
Analysis of the function of Tfg3 subunit of general transcription factor TFIIF
Makoto KIMURA··· .. ·······10A-b. DIVISION OF MUTAGENESISHyper-recombination and hyper-resistance to DNA damage in fission yeast defective inubiquitin-conjugating enzyme Ubc7
Joon-Hyun PARK, Yufuko AKAMATSU, Hiroshi IWASAKI and Fumiaki YAMAO .. · ...... ·11
Regulatory network of the yeast cell growth by the SCF ubiquitin ligase
Tsutomu KISHI············· .. ······························································ '.'11
The ubiquitin-conjugating enzymes involved in ubiquitination of mitotic cyclin
Hiroaki SEINO
12
A-c. DIVISION OF NUCLEIC ACID CHEMISTRY
Synthesis of a membrane protein with two transmembrane regions
Saburo AIMOTO

12

Synthesis of human T-cell leukemia virus type-l protease
Saburo AIMOTO
Preparation of peptide thioesters by an Fmoc solid phase method
Saburo AIMOTO

12
'

13

Molecular mechanisms of the regulatory inactivation of DnaA protein
Kazuyuki FUJIMITSU, Masayuki SU'ETSUGU, Hironori KAWAKAMI, Satoshi NISHIDA,
Tadahiro OHMURA, Tadashi UEDA and Tsutomu KATAYAMA
13
Structure-function relationship analysis of DnaA DNA-binding domain
Takafumi IWURA, Takayuki OBITA, Masayuki SU'ETSUGU, Tadashi UEDA, Taiji IMoTO
and Tsutomu KATAyAMA···············
14
B. DEPARTMENT OF CELL GENETICS

B-a. DIVISION OF CYTOGENETICS
Membrane traffic: the intracellular transport network essential for the formation andmaintenance of the multi-cellular system

CONTENTS

Tamotsu YOSHIMORI

15

Analysis of relationship between autophagy and the endosomal system
Atsuki NARA, Noboru MlZUSHIMA, Akitsugu YAMAMOTO, Yukiko KABEYA, Yoshinori
OHSUMI and Tamotsu YOSHIMORI
15
Molecular dissection of the endosomal sorting and transport by using semi-intact cells
Atsuki NARA and Tamotsu YOSHIMORI
15
Studies on role of autophagy in protection against abnormal protein aggregation within cells
Shisako SHOU]I, David H. PERLMUTTER, Akira KAKIZUKA and Tamotsu YOSHIMORI ... 16
Analysis of autophagic response to invasion of pathogenic bacteria into cells
Kayoko TSUDA, Ichiro NAKAGAWA, Atsuo AMANO and Tamotsu YOSHIMORI
16
Identification of a mammalian enzyme catalyzing the Apg12 conjugation essential for
autophagosome formation
Noboru MIZUSHIMA, Yoshinori OHSUMI and Tamotsu YOSHIMORI
16
B-b. DIVISION OF MICROBIAL GENETICS
Functional characterization of Slds which genetically interact with Dpbll in budding yeast
Yoichiro KAMIMURA and Hiroyuki ARAKI
17
Molecular mechanism of the initiation of DNA replication regulated by S-CDK
Yon-Soo TAK, Yoichiro KAMIMURA and Hiroyuki ARAKI· .. · .. · .. ··Functional analysis of the Dpbll protein
Sachiko MURAMATSU and Hiroyuki ARAKI

18
18

Counteractions of DNA polymerase ~; and yCHRAC for epigenetic inheritance of telomere
position effect variegation
Tetsushi IIDA and Hiroyuki ARAKI
19
Binding of oriC DNA protects DnaA from phospholipid-induced nucleotide releaseSeiichi YASUDA

20

B-c. DIVISION OF CYTOPLASMIC GENETICS
Linkage disequilibrium mapping of schizophrenia
Takeo YOSHIKAWA

20QTL (Quantitative Trait Loci) mappin'g of mouse prepulse inhibition
Takeo YOSHIKAWA

21

Gene expression analysis of an animal model for depression using GeneChips
Takeo YOSHIKAWA'
Candidate gene analyses
Takeo YOSHIKAWA···21······21

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

C-a. DIVISION OF DEVELOPMENTAL GENETICS
Function of the nuclear receptor Seven-up in the Drosophila central nervous system
Makoto KANAI, Masataka OKABE and Yasushi HIROMI
Regulation of prospero expression in longitudinal glial cells
Yoshihiro YUASA and Yasushi HIROMI
Role of Drosophila Spred in Ras signaling
Shu KONDO and Yasushi HIROMI

~~

23
23
23

Going through the segmental boundary: Changing the responsiveness to the segmentary
repeated guidance cue presented by Frazzled
Masaki HIRAMOTO and Yasushi HIROMI
24
Intra-axonal patterining
Takeo KATSUKI, Masaki HIRAMOTO and Yasushi HIROMI
24
Identification of New Peptides and Peptide Genes by Combining the Hydra Peptide Project
and the Hydra EST Project

CONTENTSToshitaka FUJISAWA, Chiemi FUjISAWA, Yasuharu TAKAKU, Shiho HAYAKAWA and
Takashi GOjOBORI24
The identification of peduncle of Hydra as a possible earliest heart in metazoan evolution
Hiroshi SHIMIZU and Toshitaka FUjISAWA
25
The identification of diffuse nerve net in the body column of Hydra as the enteric nervous
systemHiroshi SHIMIZU and Toshitaka FUjISAWA
25
C-b. DIVISION OF GENE EXPRESSION
Role of GAGA factor-dependent chromatin remodeling in Hox gene expression
Tsukasa SHIMOjIMA, Masahiro OKADA, Takahiro NAKAYAMA, Hitoshi UEDA, Hiroshi
HANDA, Tadashi WADA and Susumu HIROSE·············
26
Role of DNA topology in the formation of active chromatin
Kuniharu MATSUMOTO, Hirofumi FURUUAsuI, Hitoshi UEDA, Koji AKASAKA and Susumu
HIROSE··········································27
Functional analysis of transcriptional coactivator MBFI
Qin-Xin Lm, Marek JINDRA, Hitoshi UEDA, Yasushi HIROMI and Susumu HIROSE·· .. · 27
Mechanism of transcriptional regulation of the FTZ-FI geneYasuo AGAWA, Susumu HIROSE and Hitoshi UEDA
· .. ···27
Role of FTZ-FI during development
Haruhisa KAWASAKI, Yasunari KAYASHIMA, Masa-aki YAMADA, Susumu HIROSE andHitoshi UEDA
28
C-c. DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY
Genetic studies on vertebrate development using zebrafish as a model animal: Developmentof transposon-mediated insertional mutagenesis methods in zebrafish
Koichi KAWAKAMI
29
Characterization of the hagoromo gene from cichlid fishes in the East Africa
Yohey TERAI, Naoko MORIKAWA, Koichi KAWAKAMI and Norihiro OKADA
C-d. DIVISION OF PHYSIOLOGICAL GENETICS
Functional and structural analyses of mammalian Polycomb group comlexes
Haruhiko KOSEKI

30

30

Nucleosome positioning and regulation of gene expression
Ryoiti KIYAMA, Yoshiaki ONISHI and Yuko WADA-KIYAMA

32

A novel gene, Kank, is a potential growth suppressor of renal cell carcinoma
Badal C. Roy, Shubhashish SARKAR, Naoya HATANO, Kazuo GOUjI, Teiichiro AOYAGIand Ryoiti KIYAMA
32
Development and application of DNA microarrays using estrogen responsive genes
Ryoiti KIYAMA

33

D. DEPARTMENT OF POPULATION GENETICS

D-a. DIVISION OF POPULATIONGENETICS....
Adaptive evolution of the IgA hinge region in primates
Kenta SUMIYAMA, Naruya SAITOU and Shintaroh UEDA35
NJML+ P: A hybrid algorithm of the maximum likelihood and neighbor-joining methods
using parallel computing
Satoshi OOTA and Naruya SAITOU
35
Extreme mtDNA homogeneity in continental Asian populations
Hiroki OOTA, Takashi KITANO, Feng JIN, Isao YUASA, Li WANG, Shintaroh UEDA, Naruya
SAITOU and Mark STONEKING
35
A large-scale analysis of human mitochondrial DNA sequences with special reference to the
population history of East Eurasian

CONTENTSHiroki OOTA, Naruya SAITOU and Shintaroh UEDA

36

Understanding the dynamics of Spinocerebellar Ataxia 8 (SCA8) locus through a comparative
genetic approach in humans and apes
Aida M. ANDRES, M. SOLDEVILA, Naruya SAITOU, F. CALAFELL, Jaume BERTRANPETIT.,
and others
36
Helicobacter pylori in North and South America before Columbus
Yoshio YAMAOKA, Naruya SAITOU, David Y. GRAHAM and others

36

A phylogenomic study of the OCTase genes in Pseudomonas syringae pathovars: thehorizontal transfer of the argK-tox cluster and the evolutionary history of OCTase genes
on their genomes
Hiroyuki SAWADA, Shigehiko KANAYA, Masayuki TSUDA, Fumihiko SUZUKI, Kozi AzEGAMI,
and Naruya SAITOU···························36Construction and analysis of a human-chimpanzee comparative clone map
Asao FUJIYAMA, Hidemi WATANABE, Atsushi TOYODA, Todd D. TAYLOR, Takehiko ITOH,
Naruya SAITOU, Masahira HATTORI, Yoshiyuki SAKAKI and others
37
Genetic and molecular dissection of the within- and between-species variation in the sex
comb tooth number in Drosophila
Haruki TATSUTA and Toshiyuki TAKANO-SHIMIZU
38
DNA variation in the gustatory and olfactory receptor gene families of Drosophila melanogaster
Akira KAWABE and Toshiyuki TAKANO-SHIMIZU
38
D-b. DIVISION OF EVOLUTIONARY GENETICS
Informatics for unveiling hidden genome signatures
Takashi ABE, Yuta ICHIBA, Yoko KOSAKA, Nanayo ISHIHARA and Toshimichi IKEMURA........................................................ ·· .. ········ .. ······························· .. ··39
A novel bioinformatic strategy for unveiling hidden genome signatures of eukaryotes:
Self-organizing map of oligonucleotide frequency
Takashi ABE, Yuta ICHIBA, Jian-Ping SONG, Yoko KOSAKA, Kiyomi KITA and Toshimichi
IKEMURA········································39
Functional analyses of centromere of higher vertebrate cells
Tatsuo FUKAGAWA, Ai NISHIHASHI, Atsushi OKAMURA, Tetsuya HORI, Masahiro NOGAMI,
Yoshikazu MIKAMI, Yoko MIYAUCHI, Jian-Ping SONG, Kazuko SUZUKI, Mitsuko
YOSHIKAWA, Yuko FUKAGAWA and Toshimichi IKEMURA
39
Chromosome-wide assessment of replication timing for human chromosomes llq and 21q:
Genome-synteny breakpoints between mouse and human chromosomes at or near
replication-transition regions
Yoshihisa WATANABE, Yuta ICHIBA and Toshimichi IKEMURA
40

E. DEPARTMENT OF INTEGRATED GENETICS
E-a. DIVISION OF HUMAN GENETICS
Comparative analysis of an imprinted domain in the mouse and its oI1hologue in the chicken
Hiroyuki SASAKI, Takaaki YOKOMINE, Hisao SHIROHZU, Chikako SUDA, Wahyu
PURBOWASITO, Masaoki TSUZUKI, Atsushi TOYODA, Masahira HATTORI and Yoshiyuki
SAKAKI;
42
Imprinting mechanisms of the mouse Igf2/ H19 sub-domain
Hiroyuki SASAKI, Ko ISHIHARA, Yuzuru KATO, Hiroyasu FURUUMI and Wolf REIK ..... 42
Role of de novo DNA methyltransferases Dnmt3a/Dnmt3b in the establishment of genomic
imprinting
Hiroyuki SASAKI, Masahiro KANEDA, Takashi SADO, Naomi TSUJIMOTO, Kepji KUMAKI,
Shoji T AJIMA, Masaki OKANO and En LI
42
Molecular pathology of human disorders associated with DNA methylation ~nd imprinting

CONTENTSHiroyuki SASAKI, Hisao SHIROHZU, Hiroyasu FURUUMI, Takeo KUBOTA, Kenji KUROSAWA
and Shoji T AJIMA
42
Investigation on the possibility of Z chromosome dosage compensation in chicken
Hiroyuki SASAKI, Takaaki YOKOMINE, Asato KUROIWA, Yoichi MATSUDA and Masaoki
TSCDUKI········································43Development of a universal DNA chip system applicable to any organism
Hiroyuki SASAKI, Shin-ichi MIZUNO, Tadafumi IINO, Hidetoshi OZAWA, Teruhisa OTSUKA,
Kosuke TASHIRO and Takashi GOJOHBORI
43
Role of de novo methylatransferases in X chromosome inactivation
Takashi SADO, Masaki OKANOl, En LI and Hiroyuki SASAKI······························ 43
Effect of Tsix-disruption on Xis! expression
Takashi SADO and Hiroyuki SASAKI
43
Role of Dnm!3L in spermatogenesis and genomic imprinting during oogenesis
Kenichiro HATA and En LI

44

E-b. DIVISION OF AGRICULTURAL GENETICS
Developmental abnormalities induced by DNA hypomethylation mutation of Arabidopsis
Tetsuji KAKUTANI, Tetsu KINOSHITA, Asuka MIURA, Yuki KINOSHITA and Masaomi KATO...................................................... ···················································44
Charcterization of transposons activated by DNA hypomethylation mutants
Asuka MIURA, Masaomi KATO, Yuki KINOSHITA and Tetsuji KAKUTANI·······45
Inheritance of epigenetic developmental abnormality
Tetsuji KAKUTANI, Asuka MIURA and Tetsu KINOSHITA···································· 45
Epigenetic Control of FWA Gene Expression in Endosperm
Tetsu KINOSHITA, Asuka MIURA, Yuki KINOSHITA and Tetsuji KAKUTANI
45
DNA hypomethylation mutation in rice
Tetsuji KAKUTANI and Asuka MIURA·····46
Analysis of nucleosome assembly during DNA replication
Tomohiro KUBO, Akira SHIMIZU, Bruce STILLMAN and Keiichi SHIBAHARA'
46
Physiological implications of CAF-l and ASFI in Arabidopsis
Hidetaka KAYA, Takashi ARAKI and Keiichi SHIBAHARA···································· 47
E-c. DIVISION OF BRAIN FUNCTION
Mosaic development of the olfactory cortex with Pax6-dependent and -independent
components
Tatsumi HIRATA, Tadashi NOMURA, Yoshiko TAKAGI, Yasufumi SATO, Naomi TOMIOKA,
Hajime FUJISAWA and Noriko OSUMI·······47
Expression of Nogo protein by growing axons in the developing nervous system
Hirokazu TOZAKI, Takahiko KAWASAKI, Yoshiko TAKAGI and Tatsumi HIRATA······· 48
E-d. DIVISION OF APPLIED GENETICSThe slender rice mutant, with constitutively activated gibberellin signal transduction, hasenhanced capaGity for abscisic acid level
Akira IKEDA, Yu~aka SONODA, Paolo VERNIERI, Pierdomenico PERATA, Hirohiko HIROCHIKA
and Junji yAMAGUCHI·············································································· 48
Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid·biosynthesis
Masaki MORI, Takahito NOMURA, Hisako OOKA, Masumi ISHIZAKA, Takao YOKOTA,
Kazuhiko SUGIMOTO, Ken OKABE, Hideyuki KAJIWARA, Kouji SATOH, Koji YAMAMOTO,
Hirohiko HIROCHIKA and Shoshi KIKUCHI
49
Organ-spiicific alternative transcripts of KNOX family class 2 homeobox genes of rice
Yukihiro ITO, Hirohiko HIROCHIKA and Nori KURATA

49

An active Dl'fA transposon family in rice

CONTENTS

Ning JIANG, Zhirong BAO, Xiaoyu ZHANG, Hirohiko HIROCHIKA, Sean R. EDDY, Susan R.
MCCOUCH and Susan R. WESSLER
49
Non-random X Chromosome Inactivation in Mouse Embryos Carrying Searle's T(X;16)16H
Translocation Visualized using X-linked lacZ and GFP Transgenes
Nobuo TAKAGI, Michihiko SUGIMOTO, Shinpei YAMAGUCHI, Mitsuteru ITO, Seong-Seng
TAN and Masaru OKABE
50
Mapping of the breakpoint in the mouse T(X;16)l6H translocation that causes non-random
X chromosome inactivation.
Mitsuteru ITO and Nobuo TAKAGI·· 50
New XX ES cell lines carrying GFPand lacZtransgenes on the maternal or the paternal X
chromosome
Shirin FARIVAR, Seong-Seng TAN, Masaru OKABE and Nobuo TAKAGI·51
A developmental biological study on the t-complex recessive lethal mutation tclw5 in themouse
Miehihiko SUGIMOTO, Kuniya ABE, Seong-Seng TAN, Masaru OKABE and Nobuo TAKAGI................................................................. ···· .. ························· .. ·· .. ···51
F. GENETIC STRAINS RESEARCH CENTER
F-a. MAMMALIAN GENETICS LABORATORY
Developmental Geneties of Mouse Preaxial Polydacylous Mutations
Yukari YADA, Tomoko SAGAI, Sadao ISHIWA and Toshihiko SHIROISHI
53
Phylogenie conservation of a cis-acting regulator that controls polarized expression of Sonie
hedgehog in limb buds
Tomoko SAGAI, Hiroshi MASUYA, Masaru TAMURA, Kunihiko SHIMIZU, Yukari YADA,
Shigeharu WAKANA, Yoichi GONDO, Tetsuo NODA and Toshihiko SHIROISHI
53
Identification and characterization of the responsible gene of mouse skin mutations,
Recombination-induced mutation 3 (Rim3) and Rex denuded (Ret,n)Masaru TAMURA, Shigekazu TANAKA, Tomoaki FUJII, Aya AOKI and Toshihiko SHIROISHI....................................................... ······················ .. ··················· .. ·····54
Male-specific reproductive failure caused by X-{;hromosomal substitution between two mouse
subspecies
Ayako OKA, Nobuo TAKAGI, Kiyotaka TOSHIMORI, Toshiyuki TAKANO, Akihiko MITA,
Yoiehi MIZUSHINA, Noriko SAKURAI, Hiromi YAMAMOTO and Toshihiko SHIROISHI .... 55
F-b. MAMMALIAN DEVELOPMENTAL LABORATORY
ecular mechanism of somite segmentation
Yu TAKAHASHI and Yumiko SAGA"··56
Functional analyses of hesrl, 2 and 3
Hiroki KOKUBO and Yumiko SAGAThe lineage analysis of Mespl-expressing cells
Satoshi KITAJIMA and Yumiko SAGA

56
,,:,

57

Cloning and functional analysis of mouse nanos genes
Masayuki TSUDA, Yumiko SASAOKA, Makoto KISO, Seiki HARAGUCHI and Yumiko SAGA...................................................... ···················································57
F-c. MOUSE GENOMICS RESOURCE LABORATORY
Quantitative trait analyses of sensitivities to heat evoked pain and capsaicin in mouse strains
Tamio FURUSE, Kazumi YAGASAKI, Toshihiko SHIROISHI and Tsuyoshi KOIDE
58
Analysis of Locol and Loco2 involved in spontaneous locomotor activity of wild-derived
mouse strains
Juzoh UMEMORI, Tamio FURUSE, Hiromi SANO, Kazuto KOBAYASHI, Toshihiko SHIROISHI
and Tsuyoshi KOIDE
58

CONTENTS

F-d. PLANT GENETICS LABORATORY
Genetic dissection of rice development
A. Regulation of expression of KNOX family class 1 homeobox genes of rice
Yukihiro ITO and Nori KURATA

59

B. Structural and functional analysis of rice OsHAP3 genes
B-1, Identification and expression analysis of OsHAP3 genes
Nori KURATA, Kazumaru MIYOSHI, Thirumurugan THIRUVENGADAM and Yukihiro ITO...................................................... ···················································59
B-2, Functional analysis of OsHAP3A
Yukihiro ITO, Kazumaru MIYOSHI and Nori KURATA

59

B-3, Functional analysis of OsHAP3D and OsHAP3E
Thirumurugan THIRUVENGADAM, Yukihiro ITO, Kazumaru MIYOSHI and Nori KURATA..................................................................... ······ .. · .. · .. ····· .. ···············60
C. Genetic analysis of sporogenesis during sexual plant reproduction using sterile mutantsof rice
Ken-Ichi NONOMURA, Toshiyuki FUKUDA, Mutsuko NAKANO, Mitsugu EIGUCHI, Akio
MIYAO*, Hirohiko HIROCHIKA* and Nori KURATA
60
D. Molecular and genetic analysis of PLASTOCHRON genes, timekeepers for shootdevelopment in rice
D-1, Cloning and characterization of PLAI gene encoding a cytochrome P450 protein,
CYP78All
Byoung-Ohg AHN, Kazumaru MIYOSHI, Yukihiro ITO, Jun-Ichi ITOH, Yasuo NAGATO andNori KURATA···············60
D-2, Chromosomal location of PLA2 gene
Kazumaru MIYOSHI, Yukihiro ITO, Taiji KAWAKATSU, Jun-Ichi ITOH, Yasuo NAGATO andNori KURATA
61
E. Isolation and sequence analysis of receptor-like protein kinase genes of rice

Yukihiro ITO and Nori KURATA

61

Generation and screening of enhancer trap lines of rice
Yukihiro ITo, Kazumaru MIYOSHI, Thirumurugan THIRUVENGADAM, Mitsugu EIGUCHI
and Nori KURATA.'
61
Analysis of nuclear and chromosomal factors that maintain genome function
A. Finding of various plant nuclear proteins using yeast nuclear transportation trap system
Kazuki MORIGUCHI and Nori KURATA··62
B. Construction and introduction of rice artificial chromosomes
Tadzunu SUZUKI, Ken-Ichi NONOMURA and Nori KURATA· .. ···· .. ·62
Genome-wide analysis of reproductive barriers anc~ positional cloning of a reproductive barrier
A. Interactions of reproductive barriers
Yoshiaki HARUSHIMA and Nori KURATA
63
B. Positional Cloni~ of a Segregation Distortion Gene Detected in a Progeny of a Cross

between Japonica and Indica rice
Yoshiaki HARUSHIMA and Nori KURATA63

A new resource work for wild rice species involved in the activity as a center of national~ioresource project for rice
Nori KURATA, Toshie MIYABAYASHI, Yukiko YAMAZAKI*, Mitsugu EIGUCHI and Ken-Ichi
NONOMURA
64
F-e. MICROBIAL GENETICS LABORATORY
Timing of cell division
Akiko NISHIMURA'····64Global regulation of cell division: Isolation of a whole set of cell division mutants

CONTENTSKimiko SAKA, Noriko MATSUMOTO and Akiko NISHIMURA···················65
Guanosine 5'-diphosphate 37-diphosphate (ppGpp) synthetic activities on Escherichia coli
SpoT domains.
Chizuko FUJITA, Akiko NISHIMURA, Ryoko IWAMOTO and Kenji IKEHARA
65
F-f. INVERTEBRATE GENETICS LABORATORY
RNAi mutant fly bank for comprehensive analyses of gene function in Drosophila
Ryu UEDA, Misako TANIGUCHI, Kaoru SAIGO and Kuniaki TAKAHASHI·················· 66
F-g. LABORATORY FOR FRONTIER RESEARCH
Lineage Development in the Central Nervous System
Takaka Isshki

67

G. CENTER FOR GENETICS RESOURCE INFORMATION

G-a. GENETIC INFORMATICS LABORATORY
PEC
Takehiro YAMAKAWA, Junichi KATO and Yukiko yAMAZAKI
CARD R-DB

69

Takehiro YAMAKAWA, Hideki KATO, Naomi NAKAGATA, Kenichi YAMAMURA and Yukiko
YAMAZAKI69
JMSR
Takehiro YAMAKAWA and Yukiko YAMAZAKI

69

OryzabaseTakehiro YAMAKAWA, Shingo UENO, Nori KURATA, Atsushi YOSHIMURA, Hikaru SATO,
Yasuo NAGATO and Yukiko YAMAZAKI;
69
KOMUGI
Takehiro YAMAKAWA, Takashi ENDO, Yasunari OGIHARA, Hitoshi TSUJIMOTO, Taihachi
KAWAHARA, Tetsuro SASAKUMA and Yukiko YAMAZAKI
70
G-b. GENOME BIOLOGY LABORATORY
Genome Biology Laboratory, Center for Genetic Resource Information
NEXTDB: The nematode expression pattern database
Tadasu SHIN-I, Ikuko SUGIURA, Masumi OBARA, Wakako SHIMIZU, Yuko SUZUKI, JeanTHIERRY-MIEG, Danielle THIERRY-MIEG, Yutaka SUZUKI, Sumio SUGANO, Kazuko OISHI,
Masako SANO, Hisayo NOMOTO, Shinobu HAGA, Satoko NISHIZAKA, Hiroko HAYASHI,
Fumiko OHTA, Sachiko MIURA, Keiko HIRONO, Kyoko NAKATA, Hiroko UESUGI, Yasuko
SUGIYAMA and Yuji KOHARA·····71
Analysis of protein distributions of maternal genes by antibody staining in Celegans
Yumiko UETA, Masahiro ITO, Hiroko OCHI, Chihiro HI]IKATA, Keiko HIRONO, Yoshinori
UKAI, Midori SHINOHARA, Hisayoshi TORII, Emi SHIMIZU, Miki TAKENAKA, Yoshitaka IBA,
Yoshikazu KUROSAWA and Yuji KOHARA· .. ····71
Roles of POS-l and PIP-l in the translational regulation of maternal mRNA in C elegans
Norihito KISHIMOTO, Ken-ichi OGURA and Yuji KOHARA
71
Systematic identification of cis-regulatory elements of the genes that are expressed in specificnerve cells
Hiroshi KAGOSHIMA, Akiko KAMAMOTO and Yuji KOHARA··············72
Systematic RNAi Analysis of Genes Expressed in C elegans Primordial Germ Cells, Z2 andZ3Ichiro KAWASAKI, Junko KAJIWARA, Michiko SERIZAWA~ Masahiro ITO and Yuji KOHARA...................................................... ···················································72
Effect of LIM binding factor LDB-l on the function of T-box transcription factor TBX-9 in

C elegans

CONTENTSYoshiki ANDACHI:

73

Computer Simulation of the Cellular Arrangement Using Physical Model in Early Cleavageof the Nematode C. elegans
Atsushi KAJITA, Masayuki YAMAMURA and Yuji KOHARA
73
Toward a four-dimensional database of development of the nematode Ceanorhabditis elegans
Masahiro ITO, Yohei MINAKUCHI, Michiko SERIZAWA and Yuji KOHARA
73
Automatic system for creation of cell shape model in C. elegans embryogenesis
Hideaki HIRAKI and Yuji KOHARA
74
Towards comparative genomics: Genomic/EST sequencing
Kazuko OISHI, Shinobu HAGA, Hisayo NOMOTO, Masako SANO, Satoko NISIZAKA, Fumiko
OHTA, Hiroko HAYASHI, Sachiko MIURA, Tomomi MORISHITA, Tomoko ENDO, Motoyo
TAMIYA, Noriko HASEGAWA, Masumi MIZUKOSHI, Etsuko YOKOYAMA, Junko MIYAMOTO,
Shigeko IIYAMA, Tadasu SHIN-I, Shigeru SAITO, Kumiko KAWAGUCHI, Naoko SAKAMOTO,
Yasuko SUGIYAMA and Yuji KOHARA
75
The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins
Dan ROKHSAR Lab, Yuji KOHARA Lab, Michael LEVINE Lab, Nori SATOH Lab and Ascidian
research community············
75
H. STRUCTURAL BIOLOGY CENTER

H-a. BIOLOGICAL MACROMOLECULES LABORATORY
Thin Layer Illumination Microscopy as a Novel Technique for Single Molecule Fluorescence
Imaging Inside Cells
Makio TOKUNAGA···················"
78
Single Molecule Imaging of Nucleocytoplasmic Transport in Cells and Quantitative Analysis
of Interaction with Nuclear Pores
Makio TOKUNAGA and Naoko IMAMOTO
78
p105: a novel protein component of mRNA-transporting granules in neuronal dendrites
Nobuyuki SHIINA, Kazumi SHINKURA and Makio TOKUNAGA'
78
Single molecule measurement of intermolecular and intramolecular interactions using
subpiconewton intermolecular force microscopy
Michio HIROSHIMA and Makio TOKUNAGA
79
Mechanism for passive force generation of invertebrate connectin revealed by single molecule
measurement
Michio HIROSHIMA, Atushi FUKUZAWA, Koscak MARUYAMA, Sumiko KIMURA and Makio
TOKUNAGA'
79
A Novel in vitro Assay System of Nucleocytoplasmic Transport
Atsuhito OKONOGI, Michio HIROSHIMA, Nobuyuki SHIINA, Naoko IMAMOTO and Makio
TOKUNAGA'
79
Molecular Imaging of Translation Initiation Factors for Their Transport and the Local
Translation in Neuronal Dendrites
Hiraku MIYAGI, Nobuyuki SHIINA and Makio TOKUNAGA'········80
H-b. MOLECULAR BIOMECHANISM LABORATORY
The promoter arrest of E. coli RNA polymerase and the effect of the Gre factors
Shouji YAGI, Motoki SUSA, Tomoko KUBORI, Hiroki NAGAI and Nobuo SHIMAMOTO ··80
Inactivation of 0'70 by oligomerization and identification of the role of its spacer region
Usha PADMANABHAN, Hiroki NAGAI, Taciana KASCIUKOVIC, Richard S. HAYWARD, Yumiko
SATO and Nobuo SHIMAMOTO
81
Existence of tracking a groove of DNA by RNA polymerase during its sliding
Kumiko SAKATA-SOGAWA and Nobuo SHIMAMOTO

81

CONTENTS

Applicability of thermodynamics to equilibria in biology
Nobuo SHIMAMOTO and Jun-ichi TOMIZAWA······

,

···82

H-c. MULTICELLULAR ORGANIZATION LABORATORY
Analysis of synthetic dauer-constitutive mutants in the nematode Caenorhabditis elegans
Kiyotaka OHKURA, Tomoko YABE, Akiko KAMAMOTO, Takeshi ISHIHARA and Isao KATSURA.......................................................... ,·····83
Molecular genetic studies on sensory integration and behavioral plasticity in C. elegans
Takeshi ISHIHARA, Yuichi IINO, Akiko MOHRI, Ikue MORI, Keiko GENGYo-ANDO, Shohei
MITANI and Isao KATSLJRA
84

C. elegans mutants in the associative learning of odorants and foodIchiro TORAYAMA, Takeshi ISHIHARA and Isao KATSURA

85

Fluoride-resistant mutants of the nematode Caenorhabditis elegans
Akane OISHI, Minoru KAWAKAMI, Akiko KAMAMOTO, Takeshi ISHIHARA and Isao KATSURA...................................................................................................... ···85
H-d. BIOMOLECULAR STRUCTURE LABORATORY
Crystallographic Study of FI-ATPase
Yasuo SHIRAKIHARA, Aya SHIRATORI and Chikako SHIRAKIHARA

86

Comprehensive Crystallographic Study of Transcription factors and Genome-partitioning
Factors from E. coli
Yasuo SHIRAKIHARA, Aya SHIRATORI, Hisako INOUE and Megumi SEKI
87
Crystallographic Study of the Transcription Activator, PhoB, complexed with cognate DNA
Kazuyasu SHINDOH, Katsumi MAENAKA and Yasuo SHIRAKIHARA
87
Structural and functional studies of immunoglobulin(Ig)-like receptors
Katsumi MAENAKA, Tsukasa SHIMOjIMA, Kimie AMANO, Tomoko MOTOHASHI and Yasuo
SHIRAKIHARA
88
Structural analysis of glutaminase from Micrococcus luteus K-3
Yasuo SHIRAKIHARA and Aya SHIRATORI

88

1. CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPANI-a. LABORATORY FOR DNA DATA ANALYSIS
Genetic variation of Japanese gamecock and its implication to the cultural background of
cockfighting
Tomoyoshi KOMIYAMA, Kazuho IKEO and Takashi GOjOBORI89
EST analysis of a normalized library derived from 5-day regenerating planarian.
Jung Shan HWANG, Katsuhiko MINETA, Kazuho IKEO and Takashi GOjOBORI ·········89
Analysis of evolutionary relationships between interacting proteins in S. cerevisiae
Takashi MAKINO, Kazuho IKEO, Yoshiyuki SUZUKI and Takashi GOjOBORI
90
Estimation of loss of functions in metabolic pathways by comparison of 94 species
Tsuyoshi TANAKA, Kazuho IKEO and Takashi GOjOBORIThe genome sequence and structure of rice chromosome 1
Sasaki T, Matsumoto T, Yamamoto K, Sakata K, Baba T, Katayose Y, Wu J, Niimura
Y, Cheng Z, Nagamura Y, Antonio BA, Kanamori H, Hosokawa S, Masukawa M, Arikawa
K, Chiden Y, Hayashi M, Okamoto M, Ando T, Aoki H, Arita K, Hamada M, Harada
C, Hijishita S, Honda M, Ichikawa Y, Idonuma A, Iijima M, Ikeda M, Ikeno M, Ito S,
Ito T, Ito Y, Ito Y, Iwabuchi A, Kamiya K, Karasawa W, Katagiri S, Kikuta A, Kobayashi
N, Kono I, Machita K, Maehara T, Mizuno H, Mizubayashi T, Mukai Y, Nagasaki H,
Nakashima M, Nakama Y, Nakamichi Y, Nakamura M, Namiki N, Negishi M, Ohta I,
Ono N, Saji S, Sakai K, Shibata M, Shimokawa T, Shomura A, Song J, Takazaki Y,
Terasawa K, Tsuji K, Waki K, Yamilgata H, Yamane H, Yoshiki S, Yoshihara R, Yukawa
K, Zhong H, Iwama H, Endo T, Ito H, Hahn JH, Kim HI, Eun My, Yano M, Jiang J,

CONTENTS

90

Gojobori T

91

Exhaustive search for T-box genes in the whole genome shotgun sequence of Ciona intestinalis
Kohji HOTTA, Hiroki TAKAHASHI, Yasuaki MOCHIZUKI, Takashi GOJOBORI and Nori SATOH............................... ,·····91
I-b. LABORATORY FOR GENE-PRODUCT INFORMATICS
GTOP: A database of protein structures predicted from genome sequences
Takeshi KAWABATA, Satoshi UKUCHI, Keiichi HOMMA, Motonori OTA, Jiro ARAKI, Takehik.o
ITO, Nobuyuki ICHIYOSHI and Ken NISHIKAWA
93
A systematic investigation identifies a significant number of probable pseudogenes in the

Escherichia coli genome
Keiichi HOMMA, Satoshi FUKUCHI, Takeshi KAWABATA, Motonori OTA and Ken NISHIKAWA...................................................... ···················································94
Identification of amino acids involved in protein structural uniqueness: Implication for denovo protein design
Yasuhiro ISOGAI, Motonori OTA, Anna ISHII, Manabu ISHIDA and Ken NISHIKAWA .... 94
I-c. LABORATORY FOR GENE FUNCTION RESEARCH
Evolution of the MHC class I genome region in man and chimpanzee
Yoshio TATENO, Kaoru FUKAMI-KOBAYASHI, Hidetoshi INoKo, Takashi SHIINA and Muneo
YAMAZAKI
95
Parallel evolution of ligand specificity between LacI/GalR family repressors and periplasmic
sugar-binding proteins
Kaoru FUKAMI-KOBAYASHI, Yoshio TATENO and Ken NISHIKAWA···95
I-d. LABORATORY FOR RESEARCH AND DEVELOPMENT OF BIOLOGICAL DATABASES
Information systems for molecular data···················· .... · .. · .... ··· .. ··· .. · .. ··············· 96
Information systems on microbes···································································· 97
Applications of IT to the International Nucleotide Sequence Database"97
J. RADIOISOTOPE CENTER

Yoshiharu YAMAICHI, Katsunori YATA, Yasuyuki OGATA and Hironori NIKI ........ · ...... ·99

K. EXPERIMENTAL FARM
Development of new experimental strains and reevaluation of the genetic stocks of rice
Ken-Ichi NONOMURA, Mitsugu EIGUCHI, Toshie MIYABAYASHI and Nori KURATA ..... 100
A new resource work for wild rice species involved in the activity as a center of nationalbioresource project for rice
Nori KURATA, Toshie MIYABAYASHI, Yukiko YAMAZAKI, Mitsugu EIGUCHI and Ken-Ichi
NONOMURA
100

L. TECHNICAL SECTION .. ··········· .. ·······

102

ABSTRACTS OF DIARY FOR 2002

103

FOREIGN VISITORS IN 2002

105

AUTHOR INDEX··········································

106

CONTENTSIntroduction
This is the 53 rd annual report of the National Institute of Genetics (NIG). Japanese Government isdeciding a reform plan to convert National Institutes as well as National Universities into a kind of"independent agencies". It is supposed to reduce governmental control and increase independence. We
are, however, somewhat worrying about the reform, since less control may mean less public support
in the future. In order to survive through the "political turmoil", it is more important than ever to enhance
our scientific activities to receive world-wide recognition from both scientific and public communities.
The background of genetics is changing very rapidly through the increasing rate of genome
sequencings. We must realize that the accumulation of the genome-wide knowledge about many biological
species, combined with the rapid progress in computer and information science, will revolutionize genetics.
NIG has an advantage in the "scientific turmoil", since we have wide spectra of research and databasegroups, and they are in close contact in every-day activity. NIG's mottoes are, "From Molecules to
Individuals", "From Development to Evolution" and "From Experiment to Theory". We are supporting
DDBJ (DNA Data Bank of Japan) as one of the three world-wide genome database centers. Another powerof NIG is in the variety of studies with many model organisms. We have groups working with bacteria,
yeast, nematode, Drosophila, zebra fish, medaka, mouse and man. Rice and Arabidopsis groups are also
very active. Less popular but increasingly important organisms used here include planaria, hydra andchordate. The combinations of such a variety of scientific research will provide us an additional impetus
in the post-genome sequence era.
We have a weak point, however. NIG has had no affiliated hospital, and has been strong in biology,
but weak in medicine. Fortunately, Shizuoka Cancer Hospital has been open in the close location. It
will add Cancer Research Center in a few years, and we have started to discuss about our futurecollaborations.
We welcome and appreciate any comments, advice and suggestions from everyone who reads theannual report.

Yoshiki Hotta, Director-General

Introduction

1

STAFF (as of December 31, 2002)
Director-General
HOTTA, Yoshiki, D. Med.
Vice-Director

KOHARA, Yuji, D. Sc.
Members
1. Department of Molecular Genetics

SHIMAMOTO, Nobuo, D. Sc., Head of the Department

Division of Molecular Genetics
FUnTA, Nobuyuki, D. Sc.
MITSUZAWA, Hiroshi, D. Sc.
KIMURA, Makoto, D. Sc.
Division of Mutagenesis
YAMAO, Fumiaki, D. Sc., Associate Professor
KISHI, Tsutomu, D. Eng.
SEINO, Hiroaki, D. Sc.
Division of Nucleic Acid Chemistry
AIMOTO, Saburo, D. Sc., Adjunct Professor
KATAYAMA, Tsutomu, D. Sc., Adjunct Professor
2. Department of Cell Genetics

ARAKI, Hiroyuki, D. Sc" Head of the Department

Division of Cytogenetics
YOSHIMORI, Tamotsu, D. Med., Professor
IMAI, Hirotami T., D. Sc., Associate Professor
UMEBAYASHI, Kyohei, D. Ag.
Division of Microbial Genetics
ARAKI, Hiroyuki, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate Professor
KAMIMURA, Yoichiro, D. Med.
Division of Cytoplasmic Genetics
YOSHIKAWA, Takeo, D. Med., Adjunct Professor
YANAGIDA, Mitsuhiro, D. Sc., Professor (Adjunct)
3. Department of Developmental Genetics

HIROSE, Susumu, D. Sc., Head of the Department

Division of Developmental Genetics
HIROMI, Yasushi, D. Sc., Professor
FUJISAW A, Toshitaka, Ph. D., Associate Professor
SHIMIZU, Hiroshi, D. Eng.
OKABE, Masataka, M. D., Ph.D.
Division of Gene Expression
HIROSE, Susumu, D. Sc., Professor
UEDA, Hitoshi, D. Ag., Associate Professor
MINATO, Kiyoshi, M. Sc.

2

STAFF

YAMADA, Masa-aki, D. Ag.
Division of Molecular and Developmental Biology
KAWAKAMI, Koichi, D. Sc., Associate Professor
Division of Physiological Genetics
KOSEKI, Haruhiko, D. Med., Professor (Adjunct)
KIYAMA, Ryoiti, D. Sci., Adjunct Professor

4. Department of Population Genetics
IKEMURA, Toshimichi, D. Sc., Head of the Department
Division of Population Genetics
SAITOU, Naruya, Ph. D., Professor
T AKANa, Toshiyuki, D. Sc., Associate Professor
Division of Evolutionary Gentics
IKEMURA, T~shimichi, D. Sc., Professor
FUKAGAWA, Tatsuo, D. Sc., Associate Professor (Adjunct)
Division of Theoretical Genetics
KONDO, Shigeru, Ph. D., D. Sc., Professor (Adjunct)
TAKAGI, Toshihisa, D. Eng., Professor (Adjunct)
5. Department of Integrated Genetics
SASAKI, Hiroyuki, D. Med., Head of the Department
Division of Human Genetics
SASAKI, Hiroyuki, D. Med., Professor
SADO, Takashi, D. Sc.
HATA, Kenichiro, D. Med.
Division of Agricultural Genetics
KAKUTANI, Tetsuji, D. Sc., Associate Professor
SHIBAHARA, Kei-ichi, M. D., Ph. D., Associate Professor
KINOSHITA, Tetsu, D. Sc.
Division of Brain Fanction
HIRATA, Tatsumi, D. Med., Associate Professor
KAWASAKI, Takahiko, D. Sc.
Division of Applied Genetics
HIROCHIKA, Hirohiko, D. Sc., Adjunct Professor
TAKAGI, Nobuo, D. Sc., Professor (Adjunct)
6. Genetic Strains Research Center
SHIROISHI, Toshihiko, D. Sc., Head of the Center
Mammalian Genetics Laboratory
SHIRaISHI, Toshihiko, D. Sc., Professor
Mouse Genomics Resource Laboratory
KOIDE, Tsuyoshi, D. Med., Associate Professor
Mammalian Development Laboratory
SAGA, Yumiko, D. Sc., Professor
KOKUBO, Hiroki, D. Sc.
Plant Genetics Laboratory
KURATA, Nori, D. Ag., Associate Professor
ITO, Yukihiro, D. Ag.
Microbial' Genetics Laboratory

STAFF

3

NISHIMURA, Akiko, D. Ag., Associate Professor
Invertebrate Genetics Laboratory
UEDA, Ryu, D. Sc., Professor
TAKAHASHI, Kuniaki, D. Sc.
Laboratory for Frontier Research
ISSHIKI, Takako, D. Sc., Associate Professor
7. Center for Genetic Resource Inform'ation
KOHARA, Yuji, D. Sc., Head of the Center
Genetic Informatics Laboratory
YAMAZAKI, Yukiko, D. Sc., Associate Professor
FUJITA, Masaya, D. Eng.
Genome Biology Laboratory
KOHARA, Yuji, D. Sc., Professor
ANDACHI, Yoshiki, D. Sc.
8. Structural Biology Center
KATSURA, Isao, D. Sc., Head of the Center
Biological Macromolecules Laboratory
TOKUNAGA, Makio, D. Sc., Professor
SHIINA, Nobuyuki, D. Sc.
Molecular Biomechanism Laboratory
SHIMAMOTO, Nobuo, D. Sc., Professor
Multicelluar Organization Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.
Biomolecular Structure Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate Professor
Gene Network Laboratory
9. Center for Information Biology and DNA Data Bank of JapanGOJOBORI, Takashi, D. Sc., Head of the Center
Laboratory for DNA Data Analysis
GOJOBORI, Takashi, D. Sc., Professor
IKEO, Kazuho, D. Sc.
Laboratory for Gene-Product Informatics
NISHIKAWA, Ken, D. Sc., Professor
FUKUCHI, Satoshi, D. Sc.
Laboratory for Gene Function Research
TATENO, Yoshio, Ph. D., D. Sc., Professor
FUKAMI-KOBAYASHI, Kaoru, Ph. D.
Laboratory for the Research and Development of Biological Databases
SUGAWARA, Hideaki, D. Eng., Professor
MIYAZAKI, Satoru, D. Sc.
10. Radioisotope Center
NIKI, Hironori, D. Med., Head of the Center
OGATA, Yasuyuki, D. Pharm. Sci.
11. Experiment,ul Farm

4

STAFF

KURATA, Nori, D. Ag., Head of the FarmNONOMURA, Ken-ichi, D. Ag.
12. Technical Section
ISHII, Yuriko, Chief of the Section
13. Department of Administration
ISHIKAWA, Kenji, Head of the Department
TOMIYAMA, Yukio, Chief of the General Affairs Section
TAKAHASHI, Shouji, Chief of the Finance Section

STAFF

5

COUNCIL (as of December 31, 2002)
Chairman
SUGIMURA, Takashi; President Emeritus, National Cancer Center
Vice-chairman
OSAWA, Shozo; Adviser, Biohistory Research Hall
Members (Alphabetical order)
HIROBE, Masaaki; President,University of Shizuoka
ISHII, Shiro; Member, Council for Science and Technology Policy, Cabinet Office
IWATSUKI, Kunio; Professor, University of the Air
KATSUKI, Motoya; Director-General, National Institute for Basic Biology
KODAIRA, Keiichi; President, The Graduate University for Advanced Studies
KURODA, Reiko; Professor, Graduate School of Arts and Sdence, The University of Tokyo
KYOGOKU, Yoshimasa; Director-General, Biological Information Research Center, National Insttute ofAdvanced Industrial Science and Technology
MATSUBARA, Ken-ichi; President, DNA Chip Research Inc.
MATSUO, Minoru; President, Nagoya University
MIURA, Kin-ichiro; Professor, Emeritus, National Institute of Genetics
MOHRI, Hideo; President, Okazaki National Research Institutes
OKADA, Masukichi; Vice-Director, International Institute for Advanced Studies
OSAKI, Hitoshi; Director-General, Center for National University Finance
OTSUKA, Eiko; Fellow, National Institute of Advanced Industrial Science and Technology
SHINJI, Isoya; President, Tokyo University of Agiculture
TOYOSHIMA, Kumao; Director, Sumitomo Hospital
TSUNEWAKI, Koichiro; President, Fukui Prefectural University
YAMANOUCHI, Kazuya; Senior Scientific Staff, Nippon Institute for Biological Science

6

COUNCILADVISORY COMMITTEE (as of December 31, 2002)
Chairman
KOHARA, Yuji; Professor, National Insutitute of Genetics
Vice-chairman
SEKIGUCHI, Mutsuo; Director, Biomolecular Engineering Research InstituteOutside Members (Alphabetical order)GO, Michiko; Professor, Graduate School of Science, Nagoya University
HANAOKA, Fumio; Professor, Institute of Molecular and Cellular Biology, Osaka University
ISONO, Katsumi; Professor, Faculty of Science, Kobe University
ITO, Koreaki: Professor, Institute for Virus Research, Kyoto University
MATUURA, Etsuko; Professor, Faculty of Science, Ochanomizu University
OGAWA, Tomoko; Vice-President, Iwate College of Nursing
SASAZUKI, Takehiko; Director-General, International Medical Center of Japan Research Institute
SHINOZAKI, Kazuo; Chief Scientist, RIKEN Tsukuba Institute
T AJIMA, Fumio; Professor, Graduate School of Science, The University of Tokyo
Inside Menbers (Alphabetical order)
ARAKI, Hiroyuki; Professor, National Insutitute of Genetics
GOJOBORI, Takashi; Professor, National Insutitute of Genetics
HIROMI, Yasushi; Professor, National Insutitute of Genetics
HIROSE, Susumu; Professor, National Insutitute of Genetics
IKEMURA, Toshimichi; Professor, National Insutitute of Genetics
KATSURA,'Isao; Professor, National Insutitute of Genetics
NISHIKAWA, Ken; Professor, National Institute of Genetics
SASAKI, Hiroyuki; Professor, National Insutitute of Genetics
SHIMAMOTO, Nobuo; Professor, National Insutitute of Genetics
SHIROISHI, Toshihiko; Professor, National Insutitute of Genetics

ADVISORY COMMITTEE

7

A. DEPARTMENT OF
MOLECULAR GENETICS

Possible horizontal gene transfer events were inferredby several methods including massive principal
component analysis of codon usage pattern. The
results suggest that horizontal gene transfer occurs
at much higher frequency with the members of certaintranscription factor families such as Xre and CadC
families than those ·of other families. An integrated
Web-based database of bacterial transcription factors,
with identification and classification system for newly
determined sequences, is being constructed.

A-a. DIVISION OF MOLECULAR GENETICS(1) Comparative genomics of bacterial transcription(2) In vitro selection of transcription factor binding
sequences from random genomic libraryfactors
Nobuyuki FUJITA and Shizuko ENDO
Nobuyuki FUJITATranscription factor is one of the largest and themost divergent protein families in bacteria. Genomewide comparison of transcription factors among
bacterial species, therefore, will be an important stepfor understanding bacterial strategy for survival,
adaptation, and pathogenicity. Complete genomic
sequences of 44 bacterial species were searched forbacteria-type transcription factors based on sequencesimilarity. Using known transcription factors, mainly
from Escherichia coli and Bacillus subtilis, as initialqueries, a total of ~6,000 candidate proteins werecollected and classified into more than 50 protein
families primarily according to the structure of
DNA-binding domain. All bacterial species withmedium to large genomic size, irrespective of theirtaxonomic group, share most of the transcription factor
families, suggesting that these protein families haveancient phylogenetic origins. However, similarity andphylogenetic analysis of each protein family allowed
unequivocal assignment of orthologs only withinclosely related bacteria because of the high degree ofsequence diversity. Total number of transcription
factors in each bacteria showed fairly good correlation
with the genome size except for cyanobacterial andmycobacterial species in which the number oftranscription factors is significantly smaller. Bacteriawith smaller genomic size, typically less than 1.5 Mbp,
retain only a few transcription factors. Similar surveyof 11 archaeal and 2 eukaryotic genomes indicated thatarchaea share limited numbers of transcription factor
families with bacteria, and virtually no bacterialtranscription factor is conserved in eukaryotes.

8

DEPARTMENT OF MOLECULAR GENETICS

Comparative genomics analysis suggests that
Escherichia coli genome codes for nearly 270
transcription factors of which about 120 are of
unknown functions. As a means to identify the target
genes of these hypothetical factors, an in vitro selection
system based on sequence-specific DNA binding ofpurified protein was set up. A plasmid library
containing 200-300 bp random genomic DNA of
Escherichia coli (100,000 independent clones)· wasconstructed and used to generate a random populationof PCR fragrnents. After several cycles of complex
formation, copurification, and reamplification,
high-affinity binding fragments were sequenced andmapped on the genome. In a pilot experiment with Histagged CRP (cAMP receptor protein), 161 independent
clones were mapped within 31 different regions on thechromosome of which 10 regions were located upstreamof known CRP-regulated genes. All clones except 4
were intragenic, suggesting high selectivity andreliability of this method. This in vitro selection systemshould complement other screening methods such as
DNA microarray analysis.
(3) Identification of proteins that interact with the Rpb7
subunit of RNA polymerase n in fission yeast
Hiroshi MITSUZA W A
RNA polymerase II (polll) in eukaryotes consistsof twelve subunits, Rpbl through Rpbl2. Rpb7 subunit
is required for accurate transcription initiation froma promoter but is unnecessary for RNA synthesis itself.
Rpb7 has RNA binding domains and binds to RNA invitro. To understand the role of Rpb7 in transcription
initiation or other processes, we carried out a

We have identified another S. pombe TAF,
spTAF50 (spTAF6), as a protein that interacts with a

two-hybrid screen for proteins that interact with Rpb7of the fission yeast Schizosaccharomyces pombe.
The screen has identified the S. pombe homologof the Saccharomyces cerevisiae Nrdl, an RNA-binding
protein implicated in 3'-end formation of small

carboxyl-terminal WD repeat-containing region of
spTAF72. spTAF50 is the S. pombe homolog of histone
H4-like T AFs such as human T AF80 (hsTAF6),
Drosophila T AF60 (dmTAF6), and S. cerevisiae T AF60
(scTAF6). Like spTAF72, spTAF50 was present in both

nucleolar RNAs transcribed by pol II. The S. pombe
protein, named Sebl for seven binding, was shown tobe essential for cell viability. Rpb7 bound directly to

the TFIID and SAGA-like complexes.
Our results have revealed that S. pombe has
T AF-containing complexes similar to those

Sebl in vitro. S. cerevisiae Rpb7 also interacted with
Nrdl, indicating that the interaction is conserved inevolution. Glu 166 and/or Asp 167 of S. pombe Rpb7,
residues near the carboxyl terminus ofthe 172-aminoacid protein, were found to be important for its
interaction with Sebl. Based on recent structural
studies, these residues are presumed to be located inclose proximity to nascent RNA transcript extended
from the active site of pol II. Our results suggest thatRpb7 subunit may anchor a processing factor to thepol II complex, thereby coupling RNA processing totranscription.
The screen has also identified actin and an
actin-related protein as Rpb7-interacting proteins. The

characterized in other organisms. S. pombe TFIID is
unique, however, in that it contains two distinct WD
repeat T AFs, one of which (spTAF73) is specific to
TFIlD and the other (spTAF72) is shared by theactin-related protein is the S. pombe homolog of S.
cerevisiae Arp3. Interaction of Rpb7 with actin and an
actin-related protein is interesting because they are
known to be components of chromatin remodeling andhistone acetyltransferase complexes and are implicatedin transcription.

(5) Interaction between RNA polymerase II and thecarboxy-terminal domain phosphatase Fcpl in the fission
yeast Schizosaccharomyces pombe(4) Identification of TBP-associated factors (TAFs) in
fission yeast

mRNA, is a multiprotein complex consisting of twelve
subunits, Rpbl-RpbI2. The largest subunit, Rpbl,
contains an unique carboxy-terminal domain (CTD) which
consists of 29 repeats in S. pombe of heptapeptide withthe consensus of YSPTSPS. The CTD is a target ofphosphorylation, and at least two phosphoisoforms ofpol II, hypophosphorylated ITA and hyperphosphorylated
IIO froms, coexist in cells. The hypophosphorylated

SAGA-like complex. In contrast, the human, Drosophila
and S. cerevisiae TFIlD complexes contain a single
species of WD repeat T AF, which is shared by otherhistone acetyltransferase complexes. The presence of
spTAF72 and spTAF73 in a single TFIlD complex
suggests that WD repeat T AFs in other organisms arepresent in two copies in TFIlD. Support for thisprediction has come from a recent analysis of S.
cerevisiae TFIID.

Makoto KIMURA
RNA polymerase II (pol II), which synthesizes all

Hiroshi MITSUZA W A
The general transcription factor TFIID consists of
the TATA-binding protein (TBP) and multiple
TBP-associated factors(T AFs). T AFs have been
proposed to function in core promoter recognition andtranscription activation. We reported the firstbiochemical identification of T AFs in the fission yeast
S. pombe. Genetic data suggest that two WD repeat
T AFs, spTAF72 (spTAF5) and spTAF73 (spTAF5bl,
regulate genes involved in progression through the Mphase of the cell cycle. We also identified a complex
containing the S. pombe homolog of Gcn5 histone
acetyltransferase (SAGA-like complex). Interestingly,
spTAF72 but not spTAF73 was shared by this complex.

CTD interacts with a transcription mediator complex,
and the IlA from of pol II is recruited on to promotersto assemble preinitiation complexes with generaltranscription factors. On the other hand, the IIO form
elongates mRNA, and the phosphorylated CTD is
supposed to be a platform for the assembly of mRNA
processing factors. The CTD is deduced to bephosphorylated by cycline-dependent kinase (CDK) 7
in general transcription factor TFIlH, and CDK8 intranscription elongation factor P-TEFb at transcription

DIVISION OF MOLECULAR GENETICS

9

initiation and elongation, respectively. Aftertermination, the phosphorylated CTD should be
dephosphorylated for another round of transcription.
To isolate and identify protein factors whichinteraction is crucial in vivo, a thiamine dependent rpb4
shut-off system of S. pombe was constructed. On
repression of the rpb4 expression, the cell produced
more IIA from of pol II, but the pol II-complex isolated

associate and regulate the pol II, an S. pombe strain
carrying a FLAG-tag sequence fused to the rpb3 geneencoding the pol II Rpb3 subunit was constructed.
From cell extract prepared as to contain non-engaged
pol II, a complex containing IIA form of pol II was
isolated by immuno-affinity purification with
anti-FLAG antibody. In addition to the pol II subunits,
this complex was found to contain three subunits(TFIIF a, TFIIF/3 and Tfg3) of general transcription
factor TFIIF, and S. pombe homologue of TFIIFinteracting CTD-phosphatase Fcpl, which is an only
identified "CTD-phosphatase" in mammals and thebudding yeast Saccharomyces cerevisiae. Though the
Fcpl had been isolated in these organisms, this typeof complex, which must be a CTD-dephosphorylatingwith the anti-FLAG antibody contained less Fcpl andmore IIO form of pol II with the concomitant reductionof Rpb4. This result indicates the importance ofcomplex, was purified for the first time, and therefore,
Fcpl was proved to be a genuine CTD-phosphatasewhich functions after transcription termination in cells.
The intracellular contents of pol II, Fcpl and Tfg3 inan exponentially growing S. pombe cell were estimatedas 10,000, 3,000 and 20,000 molecules per cell,respectively. The same complexes could be purified
from a FLAG-Fcpl and a FLAG-Tfg3 strain, and thestoichiometry of Fcpl in these complexes revealed thatmost of Fcpl in cells exists in the pol II-complex. Cellextracts which contain only engaged pol II were also
prepared from the FLAG-Rpb3 and the FLAG-Fcpl
strains, and a complex containing pol II and Fcpl was
purified by a similar method. Analysis of the complex
revealed that though most of engaged pol II was IIO
form, a small portion of it was IIA form associated with
Fcpl. This finding supports the notion that conversion

Fcpl-Rpb4 interaction for the formation of Fcpl/TFIIF/
pol II complex and CTD-dephosphorylation in vivo.
Interaction between TFIIF a //3 complex and Fcpl was
also detected by GST-pull down assay. However, thebinding was weak in S. pombe, indicating theimportance of the Rpb4-Fcpl interaction for the complex
formation. Tfg3 subunit of TFIIF did not bind to Fcpl.
To understand the regulation mechanism of
CTD-dephosphorylation through the FcplcRpb4
interaction, Fcpl and Rpb4 were dissected. Wild typeor deletion mutants of Fcpl were expressed in S. pombefrom plasmids and their assembly into the Fcpl/TFIIF/
pol II complex was analyzed. The Fcpl with N- or
C-terminal short deletion did not assemble into thecomplex, suggesting that the formation of complex
depends on the structure of Fcpl. The crystal structureof an archaeal Rpb4 homologue and several secondary
structure prediction programs suggested that S. pombe
Rpb4, which is essential for cell viability, contains
seven a-helixes. Serial mutants of the rpb4 gene withdeletions by the helix unit were constructed andexpressed constitutively in the S. pombe rpb4 shut-off
strain. The rpb4 with N-terminal deletion supported
the cell growth, the rpb4 with C-terminal deletion
showed a dominant negative effect, and other mutants
indicated a lethal phenotype. The characters of thesemutant Rpb4 is now being analyzed biochemically.
(6) Analysis of the function of Tfg3 subunit of general

between the IIA and the IIO occurs during elongation
also by the functions of Fcpl and P-TEFb.
Recombinant·S. pombe Fcpl showed one thousand
times higher hydrolysis activity for an artificialtranscription factor TFllF

substrate p-nitrophenyl phosphate than the S. cerevisiae
enzyme, and showed CTD-phosphatase activity in vitro.
Since Fcpl was reported to require a non-CTD docking
site on pol II for CTD-dephosphorylation, I tried to
identify the Fcpl-binding subunit of pol II. By chemical crosslinking, GST-pull down and affinity
chromatography, the Fcpl-binding subunit was

Three subunits (TFIIFa, TFIIF/3 and Tfg3) ofgeneral transcription factor TFIIF were identified in
the Fcpl/TFIIF/pol II complex isolated above. The

decided as the Rpb4 subunit, which plays regulatorytranscription factors in S. pombe are more similar toroles in transcription initiation. To see if the Rpb4-Fcpl

those in higher eukaryotes than in S. cerevisiae. The

10

DIVISION OF MOLECULAR GENETICS

Makoto KIMURA

identification of S. pombe Tfg3 homologue was
unexpected, because this subunit is contained in S.
cerevisiae TFIIF but not in that from higher eukaryotes,
and generally the molecular compositions ofimportance of this subunit in transcription regulation
was analyzed. Disruption of the tfg3 gene in S. pombeindicated that the tfg3is non-essential for cell growth,
but the disruptant showed ts phenotype as wasreported in S. cerevisiae. In S. serevisiae, this protein isalso reported as a subunit of another generaltranscription factor TFIID and two more complexes
participating in transcription regulation. To see if theS. pombe Tfg3 is also a subunit of TFIID, TFITD
complex was isolated from S. pombe strains containing
FLAG-tagged taf72 or taf73 subunit gene of TFIID, andthe isolated complexes were subjected toWestern-blotting with anti-Tfg3 antibody. The isolated
complexes were proved to contain the Tfg3.
Furthermore, the TFIID complex isolated from the cells
grown at higher temperature contained more Tfg3 thanthe cells grown at lower temperature, in spite that the
intracellular content of Tfg3 in the former cells was
less, suggesting that the Tfg3 plays some role at higher
temperature. Direct interaction between Tfg3 andTATA-binding protein (TBP), which is a centralcomponent of TFIID, was also detected by GST-pull
down assay, and the binding in vitro was also stronger
at higher temperature. The function of Tfg3 is now
being investigated.
Publications
1. Colland, F., Fujita, N., Ishihama, A and Kolb, A:
The interaction between as, the stationary phase afactor, and the core enzyme of Escherichia coli RNA
polymerase. Genes Cells 7, 233-247, 2002.
2. Kimura, M., Suzuki, H. and Ishihama, A:
Formation of a carboxy-terminal domain phosphatase(Fcpl)/TFITF/RNA polymerase IT (pol IT) complex in
Schizosaccharomyces pombeinvolves direct interaction
between Fcpl and the Rpb4 subunit of pol II. Mol. Cell.
BioI. 22, 1577-1588, 2002.
3. Mitsuzawa, H. and Ishihama, A: Identification ofhistone H4-like T AF in Schizosaccharomyces pombe as
a protein that interacts with WD repeat-containing
TAF. Nucleic Acids Res. 30, 1952-1958,2002.
4. Mitsuzawa, H.: Analysis of the generaltranscription factor TFITD using the fission yeast
Schizosaccharomyces pombe. Tanpakushitsu Kakusan
Koso 47, 1931-1938,2002.

5. Yamamoto, K., Ogasawara, H., Fujita, N., Utsumi,
R. and Ishihama, A: Novel mode of transcription

regulation of divergently overlapping promoters by
PhoP, the regulator of two-component system sensing
external magnesium availability. Mol. Micro bioI. 45,
423-438, 2002.

A-b. DIVISION OF MUTAGENESIS(1) Hyper-recombination and hyper-resistance to DNA
damage in fission yeast defective in ubiquitin-conjugating
enzyme Ubc7
Joon-Hyun PARK, Yufuko AKAMATSU,t Hiroshi
IWASAKI! and Fumiaki YAMAO (!Graduate School ofIntegrated Science, Yokohama City University)
Inappropriate or unregulated homologous
recombination can have deleterious effects and leadto genomic instability. In diploid eukaryotes,
homologous recombination is essential during meiosis,
but it is suppressed during mitosis by an as yet
unknown mechanism. We identified and characterized
Ubc7, one of the 14 ubiquitin-conjugating enzymes in

SChizosaccharomyces pombe. Mutants defective in Ubc7
have enhanced mitotic recombination between
tandem-repeated segments at matl or ade6. The
increased mitotic recombination was observed only in
ubc7- cells which are competent for DSB-induced
homologous recombination. Furthermore, Ubc7
mutants are hyper-resistant to UV radiation and.MMS-induced DNA damage in cells carrying wild typeRhp51. These results suggest that mitotic homologous
recombination is a two-step reaction in eukaryotes; theinitiation step establishes competence for homologous
recombination and DNA exchange, while the second
step mediates the recombination itself. This study
demonstrates that Ubc7 negatively regulates the latterstep during mitosis in S. pombe.
(2) Regulatory network of the yeast cell growth by the
SCF ubiquitin ligase
Tsutomu KISHI
The ubiquitin pathway is involved in theregulation of many basic cellular processes. The SCF
ubiquitin ligase, one of the ubiquitin ligases in the

DIVISION OF MOLECULAR GENETICS

11

ubiquitin system, regulates cell cycle progression, anddifferenciation and development. Proteins whichorganize the SCF ubiquitin ligase have been identified
and characterized. However, only a few proteins which
are ubiquitinated by the ligase are identified. This
study aims to identify proteins to be ubiquitinated by
the SCF ubiquitin ligase in budding yeast, and toassess how their degradation contributes to cellular
regulation.
(3) The ubiquitin-conjugating enzymes involved inubiquitination of mitotic cyelin
Hiroaki SErNa
Cell cycle events are regulated by sequential
activation and inactivation of Cdk kinases. Inactivationof mitotic Cdk kinases is mainly achieved bydegradation of mitotic cyclin. Degradation of mitotic
cyclin is important for mitotic transition. Mitotic cyclin
is degraded by a ubiquitin/proteasome system. The
ubiquitin ligase that is involved in ubiquitination of
mitotic cyclin is APC/C (anaphase-promoting complex!
cyclosome). In Xenopus and clam oocytes, ubiquitinconjugating enzymes that function with APC/C havebeen identified as two proteins, UBC4 and UBCx/E2-C.
Previously we reported that the fission yeast ubiquitinconjugating enzyme UbcP4/Ubcll that was homologue
of UBCx/E2-C was required for mitotic transition.
Furthermore we show other fission yeast
ubiquitin-conjugating enzyme UbcPl/Ubc4 that ishomologue of UBC4 is also required for mitotic
transition in the same manner as UbcP4/Ubcll. Bothubiquitin-conjugating enzymes are revealed to be
directly required for the degradation of mitotic cyclin
Cdcl3 by synchronization experiments. The activity of
APC/C(Ste9) that was hyper activated by overproduction of Ste9 required both ubiquitin-conjugating
enzymes. In vivo analysis of ubiquitination of Cdcl3
shown that the ubiquitin chains on Cdc13 were shortin ubcPl mutant cells and ubiquitinated Cdcl3 wastotally reduced in. ubcP4 mutant cells. It is possible that
UbcP4/Ubcll-pathway might initiate ubiquitinationof Cdcl3 and UbcPl/Ubc4-pathway might elongate theshort ubiquitin chains on Cdcl3. We conclude thatthese two ubiquitin-conjugating enzymes have distinct
and essential function for degradation of mitotic cyclin
Cdcl3.

12

DIVISION OF MUTAGENESIS

A-c. DIVISION OF NUCLEIC ACID CHEMISTRY(1) Synthesis of a membrane protein with twotransmembrane regions
Saburo ArMaTO (Institute for Protein Research, Osaka
University)
About 30% of the human genome encodes
membrane proteins, but much of the information
concerning the structure and function of thesemembrane proteins remains to be uncovered becauseof the difficulties associated with biochemical sample
preparation. As an alternative approach to obtaining
membrane proteins, chemical synthesis represents a
viable candidate. However, peptides that have beensynthesized have only a single transmembrane domain.
Membrane proteins with multiple transmembrane
domains must be synthesized, for chemical synthesis
to be recognized as an acceptable technique formembrane protein study. The strategy that was
employed for the synthesis of a multiple transmembrane protein was the thioester method. The
purification of peptide having a transmembraneregion(s), however, represents a challenge. Difficulties
are frequently encountered during the RP-HPLC
purification of membrane peptides due to theirinsolubility in the mobile phases, and irreversible
adsorption to the column. Suitable purification
conditions must be found for preparing membrane
peptide in high purity. Overcoming the problems
involved in the synthesis of a two transmembraneprotein, we succeeded in the preparation of FIFo ATP
synthase subunit c.
(2) Synthesis of human T-cell leukemia virus type-1
protease
Saburo ArMaTO (Institute for Protein Research, Osaka
University)
Human T -cell leukemia virus type 1 (HTLV-1), a
retrovirus associated with a number of humandiseases, was the first human retrovirus isolated frompatients with adult T-cell leukemia/ lymphoma by
Gallo et al. An HTLV-l gene codes an asparatic
protease (PRJ, which processes its own polyproteins,which are transcripted owing to three reading frames.

As the result of a series of cis processing, a set ofproteins is produced, which are necessary for viral
replication. Thus, HTLV-1 PR plays a key role in the
duplication of HTLV-1 in a manner analogous to thehuman immunodeficiency virus type-1 protease inacquired immunodeficiency syndrome. In the design
potent protease inhibitors for this virus, the knowledgeof the characteristics of HTLV-1 PR itself and its
substrate specificities is critical. Searched the synthetic
conditions, especially conditions related to thesupression of epimerization during segment
condensation, we succeeded in the synthesis of
HTLV-I PR that consisted of 125 amino acids.
(3) Preparation of peptide thioesters by an Fmoc solid
phase method

Saburo NMOTO (Institute for Protein Research, Osaka
University)
In 1991, Hojo and Aimoto reported on a method
for polypeptide synthesis, which employed S-alkyl
peptide thioesters (peptide thioesters) as building
blocks. Since then, improvements have been made to
the thioester method, and it has been applied to thesynthesis of a variety of polypeptides. Peptide
thioesters are currently in use as intermediates not onlyin the thioester method, but also in the native chemical
ligation method as well as related reactions used inpolypeptide synthesis. However, a very basic problem
that still remains to be solved involves the difficultyin the preparation of peptide thioesters. In particular,
the synthetic yields of phosphopeptide thioesters are
frequently low when they are prepared by a Boc solidphase method using an MBHA resin. Furthermore, as
phosphorylated amino acid residues are often locatedin acid regions in proteins, there is the additional
problem of aspartyl residue conversion to succinimide
derivatives during peptide assembly. To overcome theproblems involved in the preparation of peptide
thioesters, we investigated the possibility to synthesize
peptide thioesters by an Fmoc solid phase method andfound excellent conditions to obtain peptide thioesters.
Publications
1. Sato, T., Kawakami, T., Akaji, K., Konishi, H.,

Mochizuki, K., Fujiwara, T., Akutsu, H. and Aimoto,S.: Synthesis of a membrane protein with twotransmembrane regions. Journal ofPeptide Science, 8,172-180, 2002.
2. Nishimura, 1., Uetsuki, T., Kuwako, K., Hara, T.,
Kawakami, T., Aimoto, S. and Yoshikawa, K.: Cell
death induced by a caspase-cleaved transmembrane
fragment of the Alzheimer amyloid precursor protein.
Cell Death. Differ., 9, 199-208, 2002.
3. Teruya, K., Kawakami, T., Akaji, K. and Aimoto,S.: Total synthesis of [L40I, C90A, C109AJ-human T-cellleukemia virus type 1 protease. Tetrahedron Letters, 43,1487-1490,2002.
4. Ohtani, M., Minakata, H. and Aimoto, S.: Potent
antagonistic action of synthetic analogues of
APGWGNamide, an antagonist of molluscan
neuropeptide APGWamide. Peptides, 5, 843-852, 2002.
5. Hasegawa, K., Sha, Y. L., Bang,]. K., Kawakami,T., Akaji, K. and Aimoto, S.: Preparation ofphosphopeptide thioesters by Fmoc- and Fmoc(2-F)solid phase synthesis. Letters in Peptide Science, 8,277-284, 2002.
6. Smith, S. 0., Eilers, M., Song, D., Crocker, E., Ying,
W., Groesbeek, M., Metz, G., Ziliox, M. and Aimoto, S.:
Implications of threonine hydrogen bonding in theglycophorin A transmembrane helix dimer. BiophysicalJournal, 82, 2476-2486, 2002.
7. Smith, S. 0., Smith, c., Shekar, S., Peersen, 0.,
Ziliox, M. and Aimoto, S.: Transmembrane interactionsin the activation of the Neu receptor tyrosine kinase.
Biochemistry, 41, 9321-9332, 2002.

(1) Molecular mechanisms of the regulatory inactivationof DnaA protein

Kazuyuki FUJIMITSU, Masayuki SU'ETSUGU, Hironori
KAWAKAMI, Satoshi NISHIDA, Tadahiro OHMURA,
Tadashi UEDA and Tsutomu KATAYAMAThe ATP-DnaA protein opens duplex DNA at the
Escherichia coli origin of replication, leading to a series
of initiation reactions in vitro. When loaded on DNA,the DNA polymerase ill sliding clamp stimulateshydrolysis of DnaA-bound ATP in the presence of the
IdaB/Hda protein, thereby yielding ADP-DnaA, which

DIVISION OF NUCLEIC ACID CHEMISTRY13

is inactive for initiation in vitro. This negative feedback
regulation of DnaA activity is proposed to playa
crucial role in the replication cycle. We found that the
mutant protein DnaA R334A is inert to hydrolysis ofbound ATP although its affinities for ATP and
ADP remain unaffected. The ATP-bound DnaA
R334A protein, but not the ADP form, initiates
minichromosomal replication in vitro at a level
similar to that seen with the wild-type DnaA. When
expressed at moderate levels in vivo, DnaA R334A is
predominantly in the ATP-bound form, unlike thewild-type and DnaA E204Q proteins, which in vitrohydrolyze ATP in a sliding clamp- and IdaB/
Hda-dependent manner. Furthermore, DnaA R334A,
but not the wild-type or the DnaA E204Q proteins,promotes overinitiation of chromosomal replication.
This in vivo data supports a crucial role for boundnucleotides in regulating the activity of DnaA during
replication. Based on a homology modeling analysis,
we suggest that the R334 residue closely interacts withbound nucleotides. (see Ref. 1)(2) Structure-function relationship analysis of DnaA
DNA-binding domain
Takafumi IWURA, Takayuki OBITA, Masayuki
SU'ETSUGU, Tadashi VEDA, Taiji IMOTO and Tsutomu
KATAYAMA
DnaA protein binds specifically to a group ofbinding sites collectively called as DnaA boxes within
the bacterial replication origin to induce local
unwinding of duplex DNA. The DNA-binding domain
of DnaA, domain IV, comprises the C-terminal 94
amino acid residues of the protein. We overproduced
and purified a protein containing only this domain plus
a methionine residue. This protein was stable as amonomer and maintained DnaA box-specific binding
activity. We then analyzed its solution structure by CDspectrum and heteronuclear multi-dimensional NMR
experiments. We established extensive assignments of
the IH, 13C, and15 N nuclei, and revealed by obtaining
combined 'analyses of chemical shift index and NOE
connectivities that DnaA domain IV contains six
achelices and no f3 -sheets, consistent with results ofCD analysis. Mutations known to reduce DnaA
box-binding activity were specifically located in or near
two of the a-helices. These fiRdings indicate that the
DNA-binding fold of DnaA domain IV is unique

14

DIVISION OF NUCLEIC ACID CHEMISTRY

among origin-binding proteins. (see Ref. 3)
Publications
1. Nishida, S., Fujimitsu, K, Sekimizu, K, Ohmura,T., Veda, T. and Katayama, T.: A nucleotide switch inE. coli DnaA protein initiates chromosomal replication:
Evidence from a mutant DnaA protein defective in
regulatory ATP hydrolysis in vitro and in vivo. J BioI.
Chem. 277 (17),14986-14995,2002.
2. Onogi, T., Ohsumi, K, Katayama, T. and Hiraga,
S.: Replication-dependent recruitment of the b subunitof DNA polymerase ill from cytsolic spaces toreplication forks in Escherichia coli. J Bacterial. 184 (3),867-870, 2002.
3. Obita, T., Iwura, T., Su'etsugu, M., Yoshida, Y.,
Tanaka, Y., Katayama, T., Veda, T. and Imoto, T.:
Determination of the secondary structure in solutionof the Escherichia coli DnaA DNA-binding domain.
Biochem. Biophys. Res. Commun. 299 (1), 42-48, 2002.

Biology, 2PRESTO, Japan Science and Technology
Corporation, 3Kansai Medical University)

B. DEPARTMENT OF
CELL GENETICS

8-a. DIVISION OF CYTOGENETICS'Membrane traffic: the intracellular transport network
essential for the formation and maintenance of themulti-cellular system

We previously showed that mouse SKDl AAA
ATPase is involved in the sorting and transport fromendosomes; cells overexpressing a dominant-negative
mutant, SKDIE235Q were defective in the endosomaltransport to both the plasma membranes andlysosomes. On the other hand, in null mutants of yeast
Vps4/Csc1, a homologue of SKD1, autophagy isimpaired, suggesting that SKDl functions not only inthe endosomal system but also in autopgay directly or
indirectly. To test the involvement of SKDl inmammalian autophagy, we examined the effects ofoverexpression of SKDlE235Q on autophagy in HeLa
cells. Overexpression of SKDlE235Q using an adenovirus

Tamotsu YOSHIMORI

delivery system caused a defect in the autophagydependent bulk protein degradation. Morphological

Eukaryotic cells are equipped inside them with atransportation system delivering molecules, so-called
membrane traffic, which is operated by theprotein-regulated membrane dynamics. By performing
secretion, endocytosis, etc., membrane traffic is
involved not only in housekeeping of each cell but also
in various functions including formation of cell polarity

observations suggested that this inhibition of autophagy
results from an impairment of autophagosome-lysosome
fusion. Emergence of the effect on endosomal transport
preceded that on autophagy and SKDIE235Q localizes

and intercellular communication, which are essential
for the multi-cellular system. We aim to unravelmolecular mechanisms of membrane traffic and theirroles in higher physiological functions and diseases
in animals, which must produce knowledge
contributing clinical medicine. Weare now focusing on
two trafficking routes; autophagy and the endosomalthat the membrane traffic from endosomes to
autophagosomes was also inhibited in the dominantnegative transfectants. These data suggest that
SKD1-dependent endosomal membrane trafficking isrequired for the late stages of the autophagic pathway
and is critical in autophagosome-lysosome fusion. The
putative component(s) required for fusion withlysosomes may be supplied by the endosome-toautophagosome membrane traffic; SKDl is necessary
for the transport. (Ref. 1)

system. Autophagy is membrane traffic delivering
cytoplasmic components to lysosomes for bulk
degradation. The process is mediated by the formationof the double membrane-bound autophagosomes.
Endosomes received macromolecules taken up by
endocytosis from outside. The cargo is then either
sorted to lysosomes or recycled back to the plasma
membrane. Each molecular basis underlying autophagy
and the endosomal sorting transport are poorly
understood.

(1) Analysis of relationship between autophagy and theendosomal system
Atsuki NARA, Noboru MIZUSHIMA1.2, Akitsugu
YAMAMOTO:', Yukiko KABEYA\ Yoshinori OHSUMI 1 andTamotsu YOSHIMORI (1National Institute for Basicexclusively to endosomes but not to autophagosomes,suggesting that the effect of the mutant protein on
autophagy is indirect. We further demonstrated(2) Molecular dissection of the endosomal sorting andtransport by using semi-intact cells
Atsuki NARA and Tamotsu YOSHIMORIIn vitro reconstitution of membrane trafficking byusing cell free systems or semi-intact cell systems isa powerful tool to resolve its molecular machinery;
application of the technique to secretory pathway has
provided many crucial information about it. To
elucidate mechanisms of the sorting and transport inendosomes, we established semi-intact cells .bytreatment of HeLa cells with a bacterial toxin,
Streptricin 0, which forms micro pores on the plasma

DEPARTMENT OF CELL GENETICS

15

membranes and allow us to access the cytoplasm
directly. We succeeded to reconstitute theendosomes-to-Iysosomes transport in these cells byadding the cytosol fraction and ATP-regenerating
system. The transport was quantified by an assay

autophagosome membrane marker. Autophagy was
induced by infection of both bacteria in cultured cells.
We are examining whether autophagy attacks thebacteria or is used by them for their survival.

system using avidin and biotin that we establishedpreviously. We are now investigating a role of SKDlin the transport. We will then reconstitute the sortingof cargo in endosomes and identify molecules required
for the event.

(5) Identification of a manunalian enzyme catalyzin~ theApg12 conjugation essential for autophagosome
formation(3) Studies on role of autophagy in protection against
abnormal protein aggregation within cells
Shisako SHoun, David H. PERLMUTTERl , Akira
KAKIZUKA2 and Tamotsu YOSHIMORI (lUniversity ofPittsburgh, 2Kyoto University)
Intracelluar accumulation of aggregated abnormal
protein are linked to many diseases, including
neurodegeneration. It has been believed that cells avoid
accumulating potentially toxic aggregates bydegradation of abnormal protein mainly via
ubiquitin-proteasome system. We hypothesized that
autophagy is also involved in degradation of suchprotein, since induction of autophagy has been often
observed in the unfolded protein diseases. To examine
the possibility, we have used disease models of cultured
cells; we overexpressed the expanded polyQ fragment,
which causes Huntington disease, or mutant ex 1
antitrypsin Z, which causes degeneration of liver cells.
By analyzing these cells, we obtained preliminary datasuggesting that autophagy reduces aggregation of theabnormal proteins by degrading part of them.
(4) Analysis of autophagic response to invasion ofpathogenic bacteria into cells
Kayoko TSUDA, Ichiro NAKAGAWAI, Atsuo AMANO l andTamotsu YOSHIMORI (lOsaka University)
Some pathogenic bacteria invade into animal cells
and survive or proliferate inside cells. Such bacteria
often internalize to the membrane-bound compartments
such as phagosomes and modulate them to avoid
digestion in lysosomes. It was recently reported that
Porphyromonas gingivalis appears in autophagosomes.
We confirm this and, moreover, found that anotherpathogenic bacteria also colocalizes with LC3, an

16

DIVISION OF CYTOGENETICS

Noboru MIZUSHIMA l • 2, Yoshinori OHSUMI l andTamotsu YOSHIMORI (lNational Institute for Basic
Biology, 2pRESTO, Japan Science and Technology
Corporation)
We previously reported that the Apgl2-Apg5
conjugate, which is essential in autophagosome
formation, is generated by a ubiquitin-like protein
conjugation system. In yeast, Apgl2, following
activation by the El-like Apg7, forms a thioester with
Apgl0 (E2-like). Apg12 is finally conjugated to Apg5
via an isopeptide bond. The Apg12 system is conserved
among eukaryotes, although a mammalian counterpartof ApglO has not yet been identified. We identified themouse Apgl0 ortholog (mApgl0) by a yeast two-hybrid
screen using the mouse Apg5 (mApg5) as bait. We
demonstrated by a modified yeast two-hybrid assaythat mApgl0 mediates the conjugation of mApg12 andmApg5. The in vivo interaction of mApg12 with
mApgl0 in HeLa cells suggests that mApgl0 is an
Apgl2-conjugating enzyme, likely serving as an
Apg5-recognition molecule in the Apg12 system. This
novel two-hybrid method, which we have named"conjugation-mediated yeast two-hybrid", proves to bea simple and useful technique with which to analyze
protein-protein conjugation. (Ref. 5)
Publications
1. Nara, A., Mizushima, N., Yamamoto, A., Kabeya,Y., Ohsumi, Y. and Yoshimori, T.: SKDl AAA
ATPase-dependent endosomal transport is involvedin autolysosome formation. Cell Struct. Funct. 27,29-37,
2002.
2. Suzuki, T., Nakagawa, M., Yoshikawa, A.,
Sasagawa, N., Yoshimori, T., Ohsumi,Y., Nishino, I.,
Ishiura, S. and Nonaka, I.: The first molecular evidence
that autophagy relates rimmed vacuole formation inchloroquine myopathy. J Biochem. 131, 647-51, 2002.

3. Ikonomov, Oc., Sbrissa, D., Yoshimori, T., Cover,TL. and Shisheva, A: PIKfyve kinase and SKD1 AAA

synthesis during the S phase. The Dpb11 protein whichforms a complex with essential DNA polymerase E

ATPase define distinct endocytic compartments: only
PIKfyve expression inhibits the cell-vacuolating(Pol E) is required for association of DNA polymerases
with origins in DNA replication (Masumoto et aI., Mol.
Cell. BioI., 20, 2809-2817, 2000) and for the control of the
S-phase checkpoint (Araki et aI., Proc. Natl. Acad. Sci.
USA, 92, 11791-11795, 1995). To identify factors
interacting with Dpb11, we have isolated 10 sld(synthetic lethality with dpbll-l) mutations which fall

activity of helicobacter pylori VacA toxin. IBiol. Chem
277, 46785-46790, 2002.
4. Fujita, H., Yamanaka, M., Imamura, K., Tanaka,
Y, Nara, A, Yoshimori, T., Yokota, S. and Himeno, M.:
A dominant negative form of the AAA ATPase SKD1/
VPS4 impairs membrane trafficking out of endosomaV
lysosomal compartments: Class E vps phenotype. I Cell
Sci. 116,401-414,2002.
5. Mizushima, N., Yoshimori, T. and Ohsumi, Y.:
Mouse Apg10 as an Apg12 conjugating enzyme:
Analysis by the conjugation-mediated yeast two-hybrid
method. FEBS let. 532, 450-454, 2002.

B-b. DIViSION OF MICROBIAL GENETICS
We have been studying on eukaryotic chromosomal
DNA replication and its regulation by the cell cycle.
For this purpose, we have employed budding yeast,
Saccharomyces cerevisiae, as a model system of
eukaryotic cells. Using strong genetic system ofbudding yeast, we have identified novel factors
involving in chromosomal DNA replication andrevealed their functions in chromosomal DNA
replication. We have also focused on the S-phasecheckpoint that monitors the status of DNA replication
and arrest the cell cycle when DNA replication is
inhibited since the Dpb11 protein we found seems to
be related to the checkpoint. Furthermore, we haveextended our study to duplication mechanism ofchromatin structure accompanying DNA replication.

(1) Functional characterization of Slds which geneticallyinteract with Dpb11 in budding yeast
Yoichiro KAMIMURA and Hiroyuki ARAKI
Chromosomal DNA replication in eukaryotic cells
initiates from multiple origins which fire sequentiallythroughout the S phase; some fires early and otherslate. The pre-replicative complex (pre-RC) starts toassemble at origins from late M phase and DNA
polymerases are recruited onto origins to initiate DNA

into 6 complementation groups. We have cloned
SLDF...., 6 and found that SLDl is identical to DPB3
encoding the third largest subunit of Pol E , SLD4 isidentical to CDC45 required for the initiation andelongation step of chromosomal DNA replication and
SLD6is identical to RAD53 which has crucial role forthe cell cycle checkpoint. The SLD2 gene encodes theessential protein which forms a complex with Dpb11
when S-CDK (Cyclin-dependent kinase) phosphorylatesit (Kamimura et aI., Mol. Cell. BioI., 18, 6102-6109, 1998;
ref. 1). Since the Sld2-Dpb11 complex formation isessential for DNA replication and probably forpolymerases' association with origins, S-CDK regulatesloading of DNA polymerases onto replication origins
through complex formation between Sld2 and Dpb11.
The Sld3 and Cdc45 proteins also form a complex
required for further loading of replication proteins
including DNA polymerases and Dpb11 (Kamimura etaI., EMBO ]., 20, 2097-2107, 2001).
The remaining SLD gene, SLD5 encodes anessential 34 kDa protein. Using sld5-12, a thermosensitive
allele of SLp5, we isolated the PSFl (Partner of Rom)as a multicopy suppressor. Then the PSF3 gene wasisolated as a multicopy suppressor of the psfl-l
thermosensitive mutation. In co-immunoprecipitation
experiment, we found that Sld5 forms a complex,
together with a novel protein, Psf2, as well as Psfl andPsf3. We named this complex GINS (Go, Ichi, Ni, San;
Five, one, two, three in Japanese). PSF1. -2 and -3 genes
are all novel genes essential for cell growth. Since theamount of GINS was reduced in sld5-12 cells andoverproduction of Psf3 in sld5-12 cells restored the cell
growth and the amount of GINS, it is suggested thatPsfl, -2 and -3 proteins function through the GINS
complex. We thus examined the psfl-l and sld5-12
mutant cells and found that they arrest with adumbbell shape and are defective in DNA replication
at the restrictive temperature. Therefore, the GINS
complex is required for DNA replication.
Many replication-related proteins including Dpbll

DIVISION OF CYTOGENETICS

17

associate with replication ongms in chromatin
immunoprecipitation assay (CHIP). Indeed, the CHIP
assay revealed that Psfl also associates with origins
in S phase at the same time as Dpbll or Pol E • This

regulates the tight association of Cdc45 with pre-RC
and the association of Dpbll-Sld2 complex with origins
and their association is in mutually dependent manner.

association with origins depends on Sld3 and Dpbll.
Conversely, Psfl is required for the association of
Dpbll with origins. Moreover, two-hybrid assay
showed the interaction between Psfl and Dpblland
Sld3. These results suggest that GINS is involved ina multi-protein complex with at least Dpbll and Pol E
in S phase and intermediates the association between
Sld3-Cdc45 on origins and Dpbll-Poh to assemble thereplisome on replication origins.

(3)(2) Molecular mechanism of the initiation of DNA
replication regulated by S-CDK

Yon-Soo T AK, Yoichiro KAMIMURA and Hiroyuki ARAKIThe regulated assembly of replication factors on
origins is crucial for the control of the initiation of
replication. At the onset of S-phase, the activated
S-CDK leads to tight association of Cdc45 with pre-RC.
Furthermore, it is believed that S-CDK activates one
or more replication factors including Sld2 that has six
preferred CDK phsophorylation motifs. Sld2 forms acomplex with Dpbll predominantly in S phase and thiscomplex is required for DNA replication. The mutant
Sld2 lacking all the CDK phosphorylation sites fails
to interact efficiently with Dpbll and is defective inchromosomal DNA replication. These results suggestthat S-CDK dependent phosphorylation of Sld2
enhance the complex formation with Dpbll to functionin the initiation of DNA replication (refl).
To understand how DNA replication is initiatedby active S-CDK, we tried to clarify the relationshipbetween the tight association of Cdc45 with pre-RC andthe association of Dpbll-Sld2 complex with origins.
Since the tight allele of thermosensitive cdc45 mutation
was not available, we first isolated thermosensitive
alleles of CDC45 defective in the initiation of DNA
replication. One of the tight alleles, the cdc45-26
mutation does not affect the Sld2-Dpbll complex
formation in S phase, but abolishes association of Sld2
and Dpbll with early-origins. Conversely, chromatinbound Cdc45 significantly reduces in drc1-1 that is atemperature-sensitive allele of SLD2 and dpbll-26
mutant cells. These results suggest that, in addition
to complex formation between Sld2 and Dpbll, S-CDK

18

DIVISION OF MICROBIAL GENETICS

Functional analysis of the Dpbll protein

Sachiko MURAMATSU and Hiroyuki ARAKI
To elucidate the function of Dpbll further, we
explored defect in dpbll-l cells. Previous studies showthat Dpbll is required for the origin association of
DNA polymerases at the initiation step of DNA
replication and moves rapidly away from origins in
CHIP assay, suggesting that Dpbll functions only forthe initiation of DNA replication. However, dpbll-l cells
that were arrested by hydroxyurea (HU), an inhibitorof DNA replication, and released at the restrictive
temperature lost their viability quickly. Moreover, thecells that were released in medium with Nocodazole,
an inhibitor of mitosis, from HU at the restrictive
temperature did not restore viability. These results
suggest that Dpbll plays roles not only before HU
block but also after HU block in S phase.
We therefore first addressed whether dPbll-l cells
complete chromosomal DNA replication at the
restrictive temperature. For this purpose, we performed
pulsed field gel electrophoresis (PFGE) of chromosomal
DNA obtained from the mutant cells. Replicating DNA
does not enter the gel of PFGE by its complicated DNA.
Then we found that chromosomal DNA of dpbll-l cells
released from HU at restrictive temperature enters thegel as efficiently as that from the wild type cells. This
result suggests that dpbll-l cells roughly complete
chromosomal DNA replication.
We also examined sister chromatids separation in
dpbll-l cells by visualizing tetR-GFP fusion proteinsbound to tandem repeats of tetO integrated at near the
cenV, because chromatid cohesion occurs in S phase.
However, we did not find any difference between
dpbll-l and the wild-type cells in the sister chromatid
separation.
Further analysis of possible defect in dpbll-l cells
is under way.

(4) Counteractions of DNA polymerase E and yCHRAC
for epigenetic inheritance of telomere position effectvariegation (TPE)
Tetsushi I IDA and Hiroyuki ARAKI
Eukaryotic chromosomal DNA is packaged into
chromatin structures: Most parts are in euchromatin
and others are in heterochromatin. The chromatin
structures are stably propagated during or soon after
DNA replication. In budding yeast, when a wild-typegene is located near telomere, it is subjected to TPE
that includes transcriptional silencing and provides
heritable silent and expressed states and reversible
switching between these epigenetic states. The
silent state of telomeric gene is attributable toheterochromatin-like structure. To address how DNA
replication couples with chromatin duplication, wemonitored TPE in mutant cells lacking Dpb3 or Dpb4,
subunits of Pole. Pole, one of replicative DNApolymerases, is composed of catalytic-subunit Po12,
Dpb2, Dpb3 and Dpb4. Although Pol2 and Dpb2 areessential for DNA replication, Dpb3 and Dpb4, whichcontain histone-fold motif related to chromatin
metabolisms, are dispensable and their function is notclear.
In the assay of silencing with telomeric URA3,
dpb3/::", dpb4/::,. and pol2-n C-terminal mutant of POL2,
cells displayed partial defect of silencing, and this. defect was most evident in dpb3/::,. cells. The silencing
defect of pol2-n cells was completely suppressed bysimultaneous introduction of high copy DPB3 andDPB4. Moreover, the silencing level in dpb3/::,. dpb4/::,.
double mutant was similar to that in dpb4/::", indicatingthat the dpb4/::,. mutation is epistatic to the dpb3/::,.
mutation. In parallel, we also observed the expressionof telomeric ADE2, which gave rise to red and white
sector colonies in the wild-type strain because ofmosaic gene silencing by TPE, and found that dpb4/::,.
mutant cells form non-sectoring light pink colonies anddpb3/::,. mutant cells form white colonies. These results
suggest that the mutations in Pol Eincrease theswitching frequency between alternative epigenetic
states in TPE.
To monitor the switching between a silent and an
expressed state in each cell division, we developed thesingle-cell telomeric silencing assay that distinguishbetween a silent state (off) and an expressed state (on)of telomeric 0;2 gene in a single cell on the 0; -factor

containing medium. Using this assay, we found thatthe switching rates for both directions increased in
dpb4/::,. cells, whereas in dpb3/::,. cells the switching ratefrom off to on specifically increased. These results
suggest that Dpb4 plays roles in stable inheritance ofboth silent and expressed states, while Dpb3 is only
involved in stable inheritance of a silent state.
The epistasis of dpb4/::,. to dpb3/::", together withdifferent switching patterns in these mutants, suggeststhat Dpb4 is shared by PolE and an unknown complex
that plays counteracting roles for TPE. We thus
purified protein complexes containing Dpb4 using
anti-Flag antibody and the 5Flag-epitope tagged Dpb4
protein, and found that two distinct protein complexes,
PolE and yCHRAC, share Dpb4. yCHRAC is aputative homologue of chromatin accessibility complex,
CHRAC in higher eukaryotes, and composed of a
WAC-motif protein Itel, an ISWI-chromatin
remodeling factor homologue Isw2, a novel histone-foldprotein Dpb31 and Dpb4.
We next addressed whether yCHRAC counteracts
PolE for TPE. In the assays with telomeric URA3 andADE2, the itc1 /: ,. and dpb31/::,. mutations enhanced
telomere silencing and restored it in dpb3/::,. cells to thelevel of dpb4/::,. cells, whereas they did not affect it in
dpb4/::,. cells. Moreover, in contrast to the dpb3/::,.
mutation, the dpb31/::,. mutation increased the switchingrate from on to off, but did not affect that from off to
on. Therefore, these results suggest that yCHRAC
regulates stable inheritance of an expressed state of
TPE, independent from PolE, while they counteract for
TPE.
Taken all together, TPE at yeast telomeres iscaused by not only simple competition between
assembly of heterochromatin and establishment ofactive-chromatin but also by specific factors such as
PolE and yCHRAC, which serve for stable inheritanceof the epigenetic states.
Publications
1. Masumoto, H., Muramatsu, S., Kamimura, Y. andAraki, H: S-Cdk-dependent phosphorylation of Sld2
essential for chromosomal DNA replication in budding
yeast. Nature 415,651-655,2002.

DIVISION OF MICROBIAL GENETICS

19

(1) Binding of otiC DNA protects DnaA from
phospholipid-induced nucleotide releaseSeiichi YASUDA
DnaA is an essential protein in the initiation of
replication that occurs at a unique locus, oriC, on thechromosome in bacteria. DnaA binds to the oriC DNA
and opens the double stranded DNA at an AT-richregion within oriC. The opened single-stranded region
serves as the site of assembly of other proteins to form
a replication complex.
Comparative studies on the dnaA genes of many
bacterial strains have established that DnaA is madeof four domains. Among them domain ill belongs to agroup of the AAA + family of ATPases which binds
and hydrolyses ATP and other nucleotides. Domain
IV at the C-terminus contains a DNA-binding motif,
and is responsible for specific binding of DnaA to aconserved 9-base pair sequence called DnaA box inoriC.
Although ATP-bound, ADP-bound andnucleotide-free DnaA can all bind oriC DNA, only
ATP-bound form is active in opening double stranded
oriC. It has been known that acidic phospholipids suchas cardiolipin release ATP and ADP from DnaA. It wassuggested that phospholipids might be involved in
rejuvenation of inactive, ADP-bound DnaA by
releasing ADP, forming nucleotide-free DnaA that isreadily activated by binding ATP.
I have been stu.dying the properties of Escherichia
coli DnaA and found that the release of ATP and ADP
from DnaA by cardiolipin is inhibited by DNA.
Covalently closed circular and linear DNA of oriC or
non-oriC plasmid was equally effective in thisinhibition. The inhibition was observed only when
DNA was added before cardiolipin. Moreover, I foundthat cardiolipin inhibits oriC binding of DnaA only
when oriC DNA is added after cardiolipin. Thus,binding of DNA to DnaA seems to protect DnaA from
nucleotide release by cardiolipin. Although initial
experiments with DNA of large size did not show any
differences in the protection efficiency between one
DNA species and another, the specificity to DNA
sequence and size became evident when smaller DNA
of defined sequence was used. Synthetic double
stranded DNA of various lengths containing a random
sequence or a DnaA box sequence was tested forbinding to DnaA and protection of nucleotide release.

20

DIVISION OF MICROBIAL GENETICS

As to DnaA box-containing DNA, 17-mer was fully
active, 13-mer was intermediate, and 9-mer wascompletely inactive in both reactions. In contrast, even
21-mer of random sequence showed only low activities,
and 13-mer was completely inactive in both reactions.
This parallelism between the binding affinity to DnaAand the protection of nucleotide release observed withdifferent DNA suggests that the nucleotide release byphospholipids is inhibited by binding of DNA per seand not by a steric effect of bound DNA.
A mutant DnaA with deletion of 14 amino acids
at the C terminus was purified. This truncated DnaA
bound ATP and oriC DNA as well as the wild type
DnaA, but was inactive in in vitro replication in a crude
protein extract. Release of bound ATP by cardiolipin
was normal in this mutant protein, but was notprotected by oriC binding. This suggests that the
C-terminal sequence of DnaA is necessary for theprotection of nucleotide release by DNA. According
to the three dimensional structure of DnaA of an
Alchae, Aquifex aeolicus, the C-terminal14 amino acidsequence that was deleted in the above mutant
constitutes a major part of the C-terminal alpha helix
called 0017 which lies on the surface of the
DNA-binding domain, separated distantly from theATP-binding domain. The function of 0017 is notknown, but it might somehow be involved in exchange
of information between domains ill and IV that isessential for the initiation function of DnaA.

B-c. DIVISION OF CYTOPLASMIC GENETICS(1) Linkage disequilibrium mapping of schizophrenia
Takeo YOSHIKAWA (Lab. for Molecular Psychiatry,
RIKEN Brain Science Institute)
We have performed a genome scan ofschizophrenia pedigrees (119 families, total of 357
individuals) using family-based association tests, at
5 cM marker density for chromosomes 6, 11 and 18, andat a 10 cM density for the remaining chromosomes.
These searches have identified several genomic lociincluding the short arm of chromosome 18, as
candidate intervals for harboring disease susceptibility
genes. In 2002, we focused on chromosome 18p in detail,by typing 16 genetic markers from an approximate 14
Mb region. This analysis revealed that two markers

derived from the Cl8ar/1 gene were significantly
associated with schizophrenia (Publication # 12). We

perturbed in the depressive state and following
antidepressant treatments. In 2002, we included ~16,000

had previously cloned and reported Cl8ar/l as a brain

new ESTs (expressed tag sequences) for examination,transcript encoding a type Ib integral membrane

and conducted extensive computational work to obtainprotein. We analyzed the gene for polymorphisms andfound a novel, single nucleotide polymorphism (SNP)

information on predictive protein characteristics ofthese ESTs that could be relevant to depression andin the 5'-untranslated region. The association of this

responsiveness to drugs.

SNP with schizophrenia was confirmed in a differently
recruited case-control panel. These results suggest that

(4) Candidate gene analyses

a true disease-causing variant may be located in thegene or an adjacent region. We are presently searching

Takeo YOSHIKAWA

for functional polymorphisms that may predispose toschizophrenia.

Target genes are selected for both positional andfunctional relevance to schizophrenia, mood disorder(2) QTL (Quantitative Trait Loci) mapping of mouse
prepulse inhibition

or epilepsy. The analyzed genes in 2002 include those
for adenylate cyclase type 9 (ADCY9) (for moodTakeo YOSHIKAWA

disorder; Publication # 5), nicotinic acetylcholine

Prepulse inhibition (PPI) of the acoustic startle
response is a form of sensory motor gating. It is defined

Publication #7), NR4A2 (for schizophrenia; Publication

as an inhibition of the startle response when a low
intensity stimulus, the prepulse, precedes the startlingor elucidated a regulatory mechanism (for the CHNRA6gene) within these genes and examined their roles instimulus. PPI is one of the few paradigms in which

the development of neuropsychiatric disorders.

disorder; Publications #1,6), cholecystokinin (for moodreceptor 06 subunit (CHNRA6 gene) (for epilepsy;#10). We have identified either genetic polymorphisms

humans and rodents are tested in a similar manner.
Deficits in PPI have been reported in schizophrenia,
making PPI a useful endophenotype of schizophrenia.
Some of the genes controlling PPI are expected to

Publications
1. Toyota, T., Hattori, E., Meerabux,]., Yamada, K.,
Saito, K., Shibuya, H., Nankai, M. and Yoshikawa, T.:overlap with disease susceptibility genes. As
preliminary experiments for mapping PPI-regulating

Molecular analysis,

mutation screening,andgenes in mice by QTL, we examined several parameters(ADCY9) in mood disorders. Am. J. Med. Genet.

including: (1) mouse strain, (2) breeders, (3) apparatus

Neuropsychiatric Genet. 114, 84-92, 2002.

association study of adenylate cyclase type 9 genefor measuring PPI, (4) effects of age and sex of mice,(5) effect of seasons on both mouse purchase date andexperiment date. In addition, inherent PPI experimental

2. Yoshikawa, T., Watanabe, A., Ishitsuka, Y.,

parameters such as the magnitude of background

Nakaya, A. and Nakatani, N.: Identification of multiple
genetic loci linked to the propensity for "behavioral

noise, prepulse and pulse were also examined. Now

despair" in mice. Genome Research 12, 357-366, 2002.

that we have determined the optimal settings, we aremaking F2 generation mice for QTL mapping.

3. Akanuma, N., Saitoh, 0., Yoshikawa, T., Matsuda, H.,
Ishikura, N., Kato, M., Adachi, N. and Onuma, T.:(3) Gene expression analysis of an animal model forInterictal schizophrenia-like psychosis in a patient withdepression using GeneChips

double cortex syndrome. The Journal of Neuropsychiatry
and Clinical Neurosciences 14, 210-213, 2002.

Takeo YOSHIKAWA
4. Reyes, G.D., Esterling, L. E., Corona, W., Ferraren,
Learned helplessness (LH) is a good animal modelD., Rollins, D. Y., Padigaru, M., Yoshikawa, T., Monje,

for chronic depression. By using LH rats and DNA
microarray technology, we have performed aV. D. and Detera-Wadleigh, S. D.: Map of candidate
genes and STSs on 18pll.2, a bipolar disorder andgenome-wide search of known genes (~8,000 genes)

schizophrenia susceptibility region. Mol. Psychiatry

DIVISION OF CYTOPLASMIC GENETICS

21

7, 337-339, 2002.
5. Hattori, E., Yamada, K, Ebihara, M., Toyota, T.,
Masahiro, M., Shibuya, H. and Yoshikawa, T.: An
association study of the short tandem repeat in the 5'
upstream region of the cholecystokinin gene with mooddisorders in the Japanese population. Am. J. Med.
Genet. Neuropsychiatric Genet. 114, 523-526, 2002.
6. Toyota, T., Yamada, K, Saito, K, Detera-Wadleigh,S. D. and Yoshikawa, T.: Association analysis ofadenylate cyclase type 9 gene using pedigree
disequilibrium test in bipolar disorder. Mol. Psychiatry7, 450-452, 2002.
7. Ebihara, M., Ohba, H., Qhno, S. and Yoshikawa,
T.: Genomic organization and promoter analysis of thehuman nicotinic acetylcholine receptor 06 subunit(CHNRA6) gene: Alu and other elements directtranscriptional repression. Gene 298, 101-108, 2002.
8. Ohtsuki, H., Ishiguro, H., Detera-Wadleigh, S, D.,
Toyota, T., Shimizu, H., Yamada, K, Yoshitsugu, K,
Hattori, E., Yoshikawa, T. and Arinami, T.: Association
between serotonin 4 receptor gene polymorphisms andbipolar disorder in Japanese case-control samples andthe NIMH Genetics Initiative Bipolar Pedigrees. Mol.
Psychiatry 7, 954-961, 2002.
9. Yoshitsugu, K, Meerabux, J. M. A, Asai, K and
Yoshikawa, T.: Fine mapping of an isodicentric Y
chromosomal breakpoint from a schizophrenic patient.
Am. J. Med. Genet. Neuropsychiatric Genet. 116B, 27-31,
2003.
10. Iwayama-Shigeno, Y., Yamada, K, Toyota, T.,
Shimizu, H., Hattori, E., Yoshitsugu, K, Fujisawa, T.,
Yoshida, Y., Kobayashi, T., Toru, M., Kurumaji, A,
Detera-Wadleigh, S. and Yoshikawa, T.: (in press).
Distribution of haplotypes derived from three common
variants of the NR4A2 gene in Japanese schizophrenic
patients. Am. J. Med. Genet. Neuropsychiatric Genet.
11. Ebihara, M., Ohba, H., Hattori, E., Yamada, K and
Yoshikawa, T.: (in press). Transcriptional activities ofcholecystokinin promoter haplotypes and theirrelevance to panic disorder susceptibility. Am. J. Med.
Genet. Neuropsychiatric Genet.

22

DIVISION OF CYTOPLASMIC GENETICS

12. Kikuchi, M., Yamada, K, Toyota, T., Itokawa, M.,
Hattori, E., Yoshitsugu, K, Shimizu, H. and Yoshikawa,T.: (in press). Two-step association analyses of thechromosome 18p11.2 region in schizophrenia detect alocus encompassing CI8ar/i. Mol. Psychiatry.
Oral Presentations
1. Yoshikawa, T.: QTL analysis of mouse depression

models. XII World Congress of Psychiatry, Yokohama,Japan, August
2. Itokawa, M., Yamada, K, Yoshitsugu, K, Suga, 1.,
Toru, M. and Yoshikawa, T.: Mutation analysis of the
NMDA receptor subtype 2A gene. XII World Congressof Psychiatry, Yokohama, Japan, August
3. Yamada, K, Iwayama-Shigeno, Y, Yoshida, Y.,
Toyota, T., Itokawa, M., Hattori, E., Yoshitsugu, K and
Yoshikawa, T.: Evidence for transmission distortionin schizophrenia on llq12.1 and 16P13.3. Xth World
Congress on Psychiatric Genetics, Brussels, Belgium,
October
4. Ebihara, M., Ohba, H., Hattori, E. and Yoshikawa,T.: Transcriptional and genetic re-analyses of cck
promoter repeat suggest that the frequency of the low
activity haplotype is decreased in panic disorder.
American Society of Human Genetics 52nd Annual
Meeting, Baltimore, USA October
5. Nakatani, N., Aburatni, H., Iijima, Y., IwayamaShigeno, Y. and Yoshikawa, T.: Genome-wide
expression analysis using animal model of depression.
Society for Neuroscience 32nd Annual Meeting, Orlando,
USA, NovemberHE to KR fails to occur properly within the NB lineage.

C. DEPARTMENT OF

DEVELOPMENTAL GENETICS(2) Regulation of prospero expression in longitudinal
glial cells
Yoshihiro YUASA and Yasushi HIROMIIn Drosophila, the longitudinal glia regulates
formation of longitudinal axonal bundles and neuronal

Makoto KANAI, Masataka OKABE and Yasushi HIROMI

survival. A critical factor that enables the function of
longitudinal glial cells is Prospero (PROS), a
homeodomain transcription factor. PROS is expressedin a subset of longitudinal glial cells. In pros mutant
embryos longitudinal glial cells form, but fail todifferentiate and support axonal growth. Weareanalyzing the regulation of pros expression in the

The diversity of neurons and glial cells in thecentral nervous system is generated in two steps. First,
the identities of about 30 Neuroblasts (NB) are specified
by the spatial information provided by the segment
polarity genes and the columnar genes. Second, theunique identities of the progeny of each NB are
determined by the birth order within the lineage.
Recently, Isshiki et al. (2001) showed that NBs
sequentially express transcription factors HB, KR,
PDM, CAS, and their progeny maintain the expression
pattern of the transcription factor using it as adeterminant of birth order identity. While the order ofthe expression of these transcription factors are
identical in every NB examined, the mechanism that

longitudinal glia. We focused on three transcription
factors,homeodomain protein REPO, Etstranscriptional factor PNTP1 and Retained/Deadringer (RETN/DRI), which is a member of the ARID
(AT-rich interaction domain) family. Thesetranscription factors are all expressed in longitudinal
glial cells. We found that PROS expression was absent
or reduced in mutants of any of these threetranscription factors. While ectopic expression of eachtranscription factor alone had no effect on PROS
expression, when three genes were simultaneously
misexpressed, there was a dramatic increase in thenumber of PROS-positive cells in the epidermis. Within
the upstream regulatory region of the pros gene there
were consensus binding sites for all three

C-a. DIVISION OF DEVELOPMENTAL GENETICS(1) Function of the nuclear receptor Seven-up in the
Drosophila central nervous system. instructs when to switch the expression of these genes
is unknown.
Nuclear receptor Seven-up (SVP) is a candidate fora factor that specify the temporal information for theswitching. svp is expressed in almost all NBs, but onlyin certain subsections of the NB lineage. For example,
some NB express svp at the time of NB formation, whileothers start its expression only after several cell
divisions. This suggests that every NB has a unique
svp expression pattern. To test the possibility that svp
plays a role in regulating the expression sequence ofHE, KR, PDM, CAS, we assayed the expression patternof these transcription factors in several NB lineages.
In certain NB lineages the number of HB, KR
double-positive cells was increased in a svp null mutant
compared to the wild type embryo, in which only earlyborn cells coexpress HB and KR. This phenotypesuggests that without svp function, the switching oftranscriptional factors. Thus pros could be a directtarget gene for these three transcription factors in thelongitudinal glia.
(3) Role of Drosophila Spred in Ras signaling
Shu KONDO and Yasushi HIROMI
Negative regulation of Ras signaling is known tobe essential for proper develpment and prevention ofcancer formation. Spred is an intracellular protein
originally identified in mammals as a negative
regulator of Ras signaling. We have identified a
Drosophila homolog of Spred. We found that 1) Spredcan suppress Ras signaling when overexpressed in thedeveloping eye, wh~re the differentiation of any
particular cell type depends on the activation of Ras
signaling, 2) In S2 cells, overexpressed Spred is

DEPARTMENT OF DEVELOPMENTAL GENETICS

23

localized to the plasma membrane and suppresses theactivation of MAP kinase, 3) The antiserum raised
against the Spred protein detects the antigen at thebasal membrane of embryonic muscle attachment cells,
where ras sinaling is highly active. Although thegain-of-function experiments all points to its potential
role as a Ras antagonist, the Drosophila Spred mutant
shows no detectable abnormalities, even in sensitive
backgrounds where Ras signaling is either above or
below the physiological level, hampering ourunderstanding of its physiological role in vivo.
(4) Going through the segmental boundary: Changing
the responsiveness to the segmentary repeated guidancecue presented by Frazzled
Masaki HIRAMOTO and Yasushi HIROMI
During axonal navigation growth cones must often
alter their responsiveness to guidance cues. For
example, commissural axons switch their response to
the midline cue Netrin upon crossing the midline, fromattractive to repulsive. This is achieved by the bindingof the cytoplasmic domain of the Netrin receptor DCC
to the cytoplasmic domain of the Slit receptor Robo,
causing the desensitization of DCC to Netrin.
Longitudinal axons also encounter a similar situation
when they go thorough repeated structures, leaving
and re-entering identical structural unit in eachsegment. The longitudinal pioneer neuron dMP2
initially turns posteriorly, using the Netrin cue
positioned by the Drosophila DCC homolog Frazzled.
However, dMP2growth cones stop tracking theboundary of Frazzled/Netrin-positive region after theturning event. We showed that the entry of dMP2 into
the succeeding segment requires the change in the
responsiveness to FrazzledlNetrin by turning on Robo
expression. Thus, the midline crossing phenotype ofdMP2 in robomutant is due to a non-ceIl-autonomous
effect of Frazzled.
(5) Intra-axonal patterining
Takeo KATSUKI, Masaki HIRAMOTO and Yasushi
HIROMI
How positional information for axon guidance isprovided remains as one of the central questions inneurobiology. We have previously shown that24

DIVISION OF DEVELOPMENTAL GENETICS

intra-axonal localization of a Drosophila guidance
receptor Frazzled creates positional information by
rearranging the distribution of the guidance molecule
Netrin, and proposed the "capture/relocation model of
guidance·receptors. This raises a question on how thereceptors themselves localize inside the neuron tocreate positional information. To address this issue,
we compared the distribution of several neuronalproteins in embryonic Drosophila CNS and in primary. culture. We found that several molecules exhibit
uneven but regulated subcellular distribution evenwhen neurons grow axons in culture, isolated fromtheir normal environment; These results imply thatneurons possess an intrinsic intracellular patterning
system that can localize molecules in axonal segmentson the cell surface. Neurons might utilize theircharacteristic structures with long processes to deliverwide and specific pattern of positional information
within the nervous system. Live observations should
provide us with insight into the mechanism of
localization.
Publication
1. Umesono, Y., Hiromi, Y. and Hotta, Y.:

Context-dependent utilization of Notch activity in
Drosophila glial determination. Development 129,2391-2399, 2002.

2. Okano, H., Imai, T. and akabe, M.: Musashi: atranslational regulator of cell fates. J Cell Sci. 115,
1355-1359, 2002.
(1) Identification of New Peptides and Peptide Genesby Combining the Hydra Peptide Project and the Hydra
EST Project
Toshitaka FUJISAWA, Chiemi FUJISAWA, Yasuharu
TAKAKU, Shiho HAYAKAWA 1 and Takashi GOJOBORI 1(lDNA Data Analysis laboratory, Center for
Information Biology)
We have initiated the Hydra EST Project this year.
The EST data have proved to be extremely valuablefor the Hydra Peptide Project. First, we have identified
a number of isoforms of peptide genes that encode new
species of related as well as unrelated peptides. The
expression patterns of these genes are also interesting.
For example, neuropeptide genes clearly identified
subpopulations among neurons that will lender a newtool to examine mechanisms for neuron differentiation.
Second, the genes encoding peptides that weresuggesting that the heart-like pumping appeared in
Cnidaria far preceding the appearance of blood

identified by the Peptide Project can be easily obtained.

vascular system.

Moreover, it is possible to postulate some of the
post-translational modifications of the peptides fJ;"om(3) The identification of diffuse nerve net in the bodythe precursor sequences. This vastly saves the time toknow, for example, if the C-terminus is amidated orcolumn of Hydra as the enteric nervous systemnot. This was only possible by HPLC analyses. Thus,
the EST Project is valuable for itself, but or mass also

Hiroshi SHIMIZU and Toshitaka FUJISAWA

it will greatly speed up the ongoing Peptide Project.

How nervous system appeared in metazoan
evolution is a crucial issue in neurobiology. A widely

Publications

accepted view is that the diffuse nerve net most

1. Iwao, K., Fujisawa, T and Hatta, M.: Coral Reefs
21, 127-129, 2002.

conspicuously found in the body column of Hydra aphylum Cnidaria is the remnant of the ancestral

2. Mochizuki, K., Fine, N. A., Fujisawa, T. andnervous system and is functionally meager
contributing at most to bidirectional conduction of

Gorovsky, M A.: Cell 110(6), 689-99, 2002.

stimulus. Here we present evidence that the bodycolumn nerve net is a functionally ample enteric

3. Fabila, Y., Navarro, L., Fujisawa, T., Bode, H. R.

nervous system. We find that the body column of Hydra

and Salgado, L. M.: Mech. Dev. 119(2), 157-64, 2002.

when fed with Artemia undergoes a series of
movements. Comparison of the movements with the

4. Morishita, F., Nitagai, Y., Furukawa, Y.,digestive movements in mammalian digestive tract

Matsushima, 0., Takahashi, T, Hatta, M., Fujisawa,
T, Tsunamoto, S. and Koizumi, 0.: Peptides 6542, 1-10,

showed three similarities. First, the movement in theearly stage of digestion was similar to the movement

2003.

of mammalian esophagus termed esophageal reflex.

5. Fujisawa, T: Developmental Dynamics (in press).

to that of stomach and intestine termed peristaltic
reflex. Third, the movement in the final stage was(2) The identification of peduncle of Hydra as a possible

similar to that of rectum termed defecation reflex.

earliest heart in metazoan evolution

Isolated body column tissue showed movements similar
to those in intact Hydra demonstrating that theHiroshi SHIMIZU and Toshitaka FUJISAWA

movements are indeed reflexes which are notdominated by exogeneous nervous system. We also

The heart is assumed to have evolved as the organ
for pumping blood. Here we present evidence thatfind that polyps which have no nerve cells are capable

Hydra a phylum Cnidaria has an activity to pump

These observations suggest that Hydra's diffuse nerve

gastric fluid and the mechanism for the pumping is

net is functionally equivalent to enteric nervous system
of mammals which regulates digestive movements andSecond, the movement in the middle stage was similar

similar to that of blood pumping by the heart in higherof undergoing peristaltic movement to a limited extent.

metazoans. We find that Hydra peduncle stores thegastric fluid and upon its contraction pumps the fluidthat metazoans had obtained the capacity of theseout of it to circulate throughout the coelenteron. We

Because of the structural simplicity, the diffuse nerve
net of Hydra could be a very good experimental system

also find that Hydra RFamide ill, a homologue of
cardioexcitatory peptides, elevates the pumping

movements in the very early stage of their evolution.

to elucidate the mechanism of digestive movements.

activity. Further, the expression of CnNk-2which has
70% homology to Nkx-2.5, a marker gene of heart-celldifferentiation, is expressed in the peduncle. Theseobservations demonstrate that the pumping fluid by
the peduncle has functional, neurological and genetic
similarities to the pumping blood by the heart

DIVISION OF DEVELOPMENTAL GENETICS

25

Publications
1. Shimizu, H: Comp Biochem Physiol A. 131, 669-674,
2002.

(1) Role of GAGA factor-dependent chromatin
remodeling in Hox gene expression

2. Shimizu, H, Zhang, X., Zhang, l,Leontovich, A.,

Tsukasa SHIMOJIMA, Masahiro OKADA!, Takahiro
NAKAYAMA, Hitoshi UEDA, Hiroshi HANDA2, Tadashi

Fei, K., Yan, L. and Sarras, MP.: Development 129,
1521-1532, 2002.

C-b. DIVISION OF GENE EXPRESSION
1.

Research activities
In multicellular organisms, a single fertilized egg

divides into multiple cells that give rise to tissues andorgans. We are studying gene expression during
embryonic and postembryonic development ofDrosophila. Our current research projects are:(1) Role of GAGA factor-dependent chromatin
remodeling in Hox gene expression(2) Role of DNA topology in the formation of active
chromatin(3) Functional analysis of transcriptional coactivator
MBF1(4) Mechanism of transcriptional regulation of FTZ-F1(5) Role of FTZ-F1 during development
Following members participated in our research
activities: Susumu Hirose (Professor), Hitoshi Ueda
(Associate professor), Masa-aki Yamada and Kiyoshi
Minato (Research associates), Qin-Xin Liu (Foreign
researcher), Marek Jindra USPS Short term invited
fellow), Kunihiro Matsumoto, Takahiro Nakayama andTsukasa Shimojima (Postdoctoral fellows), Haruhisa
Kawasaki, Yasunari Kayashima, Yasuo Agawa and
Hirofumi Furuhashi (Graduate students), Yuko Takada
and Kozue Uematsu (Research assistants). Our
collaborators are; Yasushi Hiromi (Professor, Divisionof Developmental Genetics), Hiroshi Handa (Professor,
Frontier Collaborative Resarch Center, Tokyo Instituteof Technology), Tadashi Wada (Associate professor,
Faculty of Bioscince and Biotechnology, Tokyo
Institute of Technology), Koji Akasaka (Associate
professor, Graduate Department of Gene Science,
Hiroshima University), Katsuyuki Hamada (Faculty of
Medicine, Ehime University).
Research activities in 2002 are supported by
Grants-in-Aid for Scientific Research from the Ministryof Education, Science, Sports, Culture and Technologyof Japan to Hirose and Ueda, Grants for Collaborative
Research from our Institute to Hirose, and a Grant
from Naito Science Foundation to Hirose.

26

DIVISION OF DEVELOPMENTAL GENETICSW ADA 3 and Susumu HIROSE (lPresent address,
Laboratory of Molecular Genetics, RIKEN Tsukuba
Institute, Tsukuba, Ibaragi-ken 305-0074, Japan;
2Frontier Collaborative Research Center, and 3Facultyof Bioscience and Biotechnology, Tokyo Institute ofTechnology, Nagatsuda, Midori-ku, Yokohama
226-8501, Japan)
The identity of the body segment of metazoans isdetermined by the expression patterns of Hox genes.
Hox gene expression is initiated by the actions of manytranscription factors during embryogenesis. Curiously,
these expression patterns are maintained after
disappearance of the transcription factors. The active
or inactive state is memorized in the chromatin
structure and transmitted to daughter cells throughcell division. Although the mechanism of thisepigenetic gene expression is not clear, genetic studies
have identified many genes involved in the process.
These genes are classified into two groups. Pc group
genes mainly govern the maintenance of the inactive
state, while trx group genes are mainly responsible forthe maintenance of the active state. Drosophila GAGA
factor is encoded by a trx group gene Trithorax-like anddirects chromatin remodeling to its binding sites. We
found that Drosophila FACT, a heterodimer of dSPT16
and dSSRPI, is associated with GAGA factor throughits dSSRPI subunit, binds to necleosomes andfacilitates GAGA factor-directed chromatin
remodeling. Moreover, genetic interactions between
Trithorax-like and spt16 implicate the GAGA
factor-FACT complex in expression of Hox genes

Ultrabithorax, Sex combs reduced and Abdominal-B.
Chromatin immunoprecipitation experiments indicatedpresence of the GAGA factor-FACT complex in theregulatory regions of Ultra bithorax. These data
illustrate a crucial role of the GAGA factor-FACT
complex in the modulation of chromatin structure forregulation of gene expression (Oral presentations 1, 5,
6,8-10,14 and 18). We have initiated collaboration with
Dr. T. Wada to extend studies on chromatintranscription.

(2) Role of DNA topology in the formation of active
chromatin
Kuniharu MATSUMOTO, Hirofumi FURUHASHI, Hitoshi
UEDA, Koji AKASAKA! and Susumu HIROSE fGraduate
Department of Gene Science, Hiroshima University,
Kagamiyama, Higashi-Hiroshima 739-8526, Japan)
Recent studies have established that chromatin
structure is important for transcriptional regulation.
Starting from analyses of DNase I hypersensitivity in
1980s, lines of evidence have been accumulated forchanges in the chromatin structure, such as DNA
methylation, chemical modifications of histones andchromatin remodeling during regulation of gene
expression. However, besides transcription-driven
supercoiling of DNA, our knowledge on conformationof chromatin DNA was elusive due to the lack ofproper probes for analyses of DNA topology in vivo.
To circumvent the situation, we have developed amethod for visualization of negatively supercoiled DNA
within the interphase genome. Using the methods, weobserved unconstrained negative supercoils in manyloci on polytene chromosomes of Drosophilamelanogaster (Oral presentations 7 and 11).
Supercoiling factor (SCF) is a protein capable ofintroducing negative supercoils into DNA in
conjunction with DNA topoisomerase n. Partial knockout of the SCF function in Drosophila melanogaster by
RNAi indicated that SCF is involved in superactivationof transcription on male X-chromosome (Oralpresentations 4 and 12). In addition, we made studies
on a Drosophila counterpart of sea urchin insulator
binding protein GSAP in collaboration with Dr. K.
Akasaka.
(3) Functional analysis of transcriptional coactivator
MBFl
Qin-Xin Lm, Marek JINDRA, Hitoshi UEDA, Yasushi
HIROMI! and Susumu HIROSE (lDivision of Developmental Genetics)
During gene activation, the effect of binding oftranscription factors to cis-acting DNA sequences istransmitted to RNA polymerase by means ofcoactivators. Although coactivators contribute to theefficiency of transcription, their developmental roles
are poorly understood. We used Drosophila to conductmolecular and genetic dissection of an evolutionarily
conserved but unique coactivator, Multiprotein
bridging factor 1 (MBFl), in a multicellular organism.
Through immunoprecipitation MBFl was found toform a ternary complex including MBFl, TATAbinding protein (TBP) and a bZIP protein Tracheae
defective (TDF)/Apontic. We have isolated a Drosophilamutant lacking the mbfl gene. In the mbfl mutant, no
stable association between TBP and TDF is detectable,
and transcription of a TDF-dependent reporter geneis reduced five fold. Although the null mutants of mbfl
are viable, tdfbecomes haplo-insufficient in mbfl
deficient background, causing severe lesions intracheae and the central nervous system, similar to
those resulting from a complete loss of tdffunction.
These data demonstrate a critical role of MBFl in thedevelopment of tracheae and central nervous system(Article 1, and Oral presentations 2 and 15)(4) Mechanism of transcriptional regulation of the
FTZ-Fl geneYasuo AGAW A, Susumu HIROSE and Hitoshi UEDA
Insect FTZ-Fl is a unique transcription factor,
because it is induced after a pulse exposure of
ecdysteroids and is expressed just before hatching,ecdysis and pupation in a stage specific manner. The
stage specific expression of FTZ-Fl is necessary forlarval ecdysis and metamorphosis, indicating that
temporary precise regulation of the gene is important
for the development of Drosophila. Factor 1-4 has been
identified as a sequence specific binding factor thatbinds to a cis-regulatory region of the FTZ-Fl gene.
The factor is present during high ecdysteroid periods,
so that it was thought as a repressor of the FTZ-Fl
gene. However, reporter assays using FTZ-Fl
regulatory regions fused to LacZ showed that therecognition sequence of Factor 1-4 is required forhigh-level expression of FTZ-Fl. We purified Factor
1-4 from embryonic extracts based on the affinity to
its recognition sequence. Sequence analysis of thepurified polypeptide revealed that it is a zinc finger
protein carrying several other interesting motifs.
Developmental Northern blotting analysis showed thatseveral mRNA isofoms are expressed from the Factor
1-4 gene and two of them are expressed during the highecdysteroid periods around prepupa in coincidencewith the developmental expression profile of Factor 1-4

DIVISION OF GENE EXPRESSION

27

detected by gel mobility shift assays. These results
suggest that the Factor 1-4 gene belongs to an earlygene in Ashburner's model and potentiates expressionof the FTZ-F1 gene (Oral presentation 16 and 19).
(5) Role of FTZ-F1 during development (Regulation mechanism of gene expression during insect
metamorphosis)
Haruhisa KAWASAKI!, Yasunari KAYASHIMA, Masa-aki
YAMADA, Susumu HIROSE and Hitoshi UEDA (lPresent
address, Advanced Research Institute for Science andEngineering, Waseda University)
Insect metamorphosis is an important event tochange their morphology from larva to completely
different adult. During the metamorphosis, manytranscription factors are induced by ecdysteroids, butlittle is known how these factors regulate space specificexpression of their target genes. To understandmolecular mechanism underlying space specific gene
expression during metamorphosis, regulatory regionsof the EDG84A gene which is expressed in the anterior
epidermis during the mid- to late prepupal periods
depending on Drosophilatemporal regulator FTZ-F1
were analyzed. Using transgenic fly reporter genecarrying various cis-regulaory regions fused to LacZ,
we identified at least two positive and three negative
elements for the space specific expression. The results
suggested that spatial regulation is established by a
combination of several transcription factors.
Candidates of regulatory factors which bind to theregulatory regions were detected by gel mobility shift
assays. By using computer search and Yeast
two-hybrid assays, we identified a factor which binds
to one of the positive regulatory elements. On the other
hand, analysis of cis-regulatory region in the EDG78E
gene which is expressed in whole epidermis during themid- to late prepupal periods represented that FTZ-F1
is necessary for expression in the anterior epidermis
but expression in the posterior epidermis is notcompletely dependent on FTZ-F1. The results
suggested complex mechanisms that govern thetemporal and spatial regulation of gene expression
during metamorphosis (Article 2, and oral presentation
3, 17, 20 and 21).
During early metamorphosis larval salivary glands
disappear by histolysis. For the histolysis, expressionof FTZ-F1 during the mid- to late prepupal periods is

28

DIVISION OF GENE EXPRESSION

necessary as revealed by persistence of larval salivary
glands in ftzfl mutant. Forced expression of Zl isofomof BR-C or E74A during late prepupal period inftzfl
mutant recovered the histolysis. These results suggestthat BR-C and E74A work redundantly in thedownstream of FTZ-F1 in the histolysis.
2. Publications(1) Articles
1. Liu, Q.-X., Jindra, M., Ueda, H., Hiromi, Y. andHirose, S.: Drosophila MBF1 is a co-activator for
Tracheae Defective and contributes to the formationof tracheal and nervous systems. Development, inpress.
2. Kawasaki, H, Hirose, S. and Ueda, H.: {3 FTZ-F1
dependent and inependent activation of Edg78E, apupal cuticle gene, during the early metamorphic
period in Drosophila melanogaster. Develop. GrowthDiffer. 44, 419-425, 2002.
(2) Oral presentations
1. Hirose, S., Nakayama, T., Shimojima, T., Okada,M. and Ueda, H.: Epigenetic gene expression through
GAGA factor-dependent chromatin remodeling. 7th
Asian Conference on Transcription, Kuala Lumpur,
Malasia, July, 2002.
2. Liu, Q.-X., Jindra, M., Ueda, H., Hiromi, Y. andHirose, S.: MBF1 serves as a transcriptional coactivatorof Tracheae defective/Apontic during development of
the trachea and central nervous system in Drosophilamelanogaster. 43rd Annual Drosophila Research
Conference, San Diego, California, USA, April, 2002.
3. Ueda, H., Kayashima, Y. and Hirose, S.:
Mechanism of space specific gene expression in
response to ecdysteroids-Transcriptional regulationof FTZ-F1 target gene EDG84A. 43rd Annual
Drosophila Research Conference, San Diego,
California, USA, April, 2002.
4. Furuhashi, H and Hirose, S.: Functional analysis
of supercoiling factor by using RNAi. 19th Workshop
on Chromosome, Kobe, Japan, Jan, 2002.
5. Hirose, S.: GAGA factor-dependent chromatin
remodeling and epigenetics. Seminar in Waseda
University, Tokyo, Japan, March, 2002.

6. Hirose, S.: FACT is involved in epigenetic gene
expression. Seminar in BERI, Osaka, Japan, June, 2002.

16. Agawa, Y., Hirose, S., Handa, H., Wada, T.,
Shinohara, A., Iwamatsu, A. and Ueda, H: Mechanismof transcriptional regulation of the FTZ-Fl gene in

7. Matsumoto, K. and Hirose, S.: In situ analyses on
negative supercoiling of DNA and transcription. NIG
workshop on Biology of Chromatin, Mishima, Japan,
November, 2002.

Drosophila melanogaster. 25th Annual Meeting ofMolecular Biology Society of Japan, Yokohama, Japan,
December, 2002.

8. Hirose, S.: FACT is involved in epigenetic
maintenance of gene expression. Seminar in Tsukuba
University, Tsukuba, Japan, November, 2002.
9. Hirose, S.: Chromatin remodeling and maintenanceof gene expression. 12th Workshop on Radiation
Biology, Mito, Japan, November, 2002.
10. Hirose, S., Shimojima, T., Nakayama, T. and Ueda,
H: FACTis involved in epigenetic maintenance of gene
expression. Symposium on Infrastructures of
Chromatin Function, 25th Annual Meeting ofMolecular Biology Society of Japan, Yokohama, Japan,
December, 2002.
11. Matsumoto, K. and Hirose, S.: Visualization of
DNA supercoiling and transcription in situ. 25th
Annual Meeting of Molecular Biology Society of Japan,
Yokohama, Japan, December, 2002.
12. Furuhashi, H and Hirose, S.: Functional analysis
of DNA supercoiling factor in Drosophila melanogaster.
25th Annual Meeting of Molecular Biology Society ofJapan, Yokohama, Japan, December, 2002.

17. Kyashima, Y., Hirose, S. and Ueda, H: Candidates
for regulator of space~specific expression of
FTZ~F1~target gene EDG84A in Drosophila. 25th
Annual Meeting of Molecular Biology Society of Japan,
Yokohama, Japan, December, 2002.
18. Hirose, S.: Maintenance of gene expression throughchromatin structure. Seminar in Tokyo Institute ofTechnology, Yokohama, Japan, December, 2002.
19. Agawa, Y., Hirose, S. and Ueda, H.: Regulation
mechanism of the FTZ~F1 gene. Workshop on Insect
02 Hayama, Japan, March, 2002.
20. Kayashima, Y., Hirose, S. and Ueda, H.: Control
mechanism to produce tissue specific gene expression
during insect metamorphosis. Workshop on Insect 02
Hayama, Japan, March, 2002.
21. Ueda, H., Kayashima, Y. and Hirose, S.:
Mechanism of gene expression in insects. Workshop
on Insect 02 Hayama, Japan, March, 2002.

C-c. DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

and Hirose, S.: Chromosome
pairing~dependentgene regulation in Drosophila. 25th
Annual Meeting of Molecular Biology Society of Japan,
Yokohama, Japan, December, 2002.

(1) Genetic studies on vertebrate development using
zebrafish as a model animal: Development oftransposon-mediated insertional mutagenesis methods

14. Nakayama, T., Shimojima, T. and Hirose, S.:
Dynamic distribution of Drosophila GAGA factor~
dFACT complex on hsp70 gene. 25th Annual Meetingof Molecular Biology Society of Japan, Yokohama,Japan, December, 2002.

Koichi KAWAKAMI

13. Ueda,

H.

15. Liu, Q.~X., Ueda, H., Hiromi, Y. and Hirose, S.:
Functional analysis of Drosophila TDF during theneuronal development. 25th Annual Meeting ofMolecular Biology Society of Japan, Yokohama, Japan,
December, 2002.

in zebrafish

In order to understand the genetic basis fordevelopmental processes in vertebrate, we have beenusing a small tropical fish, the zebrafish, as a model
animal. Because it is practically possible to breed andmaintain very large numbers of fish in the lab, andbecause zebrafish embryos develop in water and aretransparent, forward genetic approaches (i.e., collecting
a large number of mutations affecting developmental
processes and analyzing genes responsible for themVISION OF GENE EXPRESSION

29

mutant phenotypes) are feasible in the fish. Taking

retrovirus. By performing this method, we isolated aadvantage of these merits, large-scale chemicalmutagenesis screens in zebrafish had been successfully

dominant mutation hagoromo (hag), which caused
disorganized stripe patterns on the skin of the adultcarried out and hundreds of mutants defective in every

zebrafish. In the hag mutant, the provirus was locatedaspect of early developmental processes had been

within a novel gene, which we named the hagoromo

collected. It has, however, not been easy to identify thegene, encoding a novel F-box/WD40-repeat protein.

mutated genes since most of the chemically induced

Interestingly, the mouse ortholog of the hag gene wasmutations were point mutations and time-consuming

mutated in the Dactylaplasia (Dac) mutant mouse. The

positional cloning approaches are required to clone theDac mutation is also dominant and causes defects indigit formation in fore- and hindlimbs. Thus, the hag/
Dac locus is important for pattern formation invertebrates but is involved in distinct morphogenetic
events in different animals. In order to examine
whether the hag gene is involved in divergence of
pigment patterns on the skin of diverged fish species,
we cloned homologs of the hag gene from different
species of cichlid fishes in the East Africa. By
comparing amino acid sequences of these Hag
proteins, we found that the surface amino acid residuesof the WD-repeat domain, which may interact withother proteins, changed rapidly as the fish species
diverged. Thus, our studies suggested the changes in
the amino acid sequences in the surface of the
WD-repeat domain may be related to cichlid speciation
by affecting their pigment patterns.

genes. Therefore it is important to develop insertionalmutagenesis methods in zebrafish, such as the P
transposon system in Drosophila.
The Tol2 element is a transposable element
identified from the genome of the medaka fish.
Previously we have found that the Tol2 element
encodes a fully functional transposase and have shownthat the Tol2 element can transpose into the zebrafish
genome in the germ lineage. To date, the Tol2element
is the only natural transposon in vertebrate, that has
ever been shown to encode a functional transposase,and thereby from which an autonomous element has
been identified. We have been interested in developing
novel insertional mutagenesis methods in zebrafish
using the Tol2 element. Using this Tol2 transposon
system, we successfully established a protocol togenerate transgenic zebrafish very efficiently. We thenmade an exon trap construct with a promoter-less GFP

Publications

gene using the Tol2 transposon, and generated
hundreds of insertions of the exon trap construct in1.

the zebrafish genome. 10% of the insertions yielded"specific" GFP expression, indicating that the genein the WD-repeat domain encoded by the hagoromo
gene in an explosively speciated lineage of east cichlid(exon) trap method using the Tol2transposon systemfishes. Molecular Biology and Evolution 19, 574-578, 2002.

Terai, Y., Morikawa, N., Kawakami, K. and Okada,N.: Accelerated evolution of the surface amino acids

is successfully working. In those fish, GFP wasexpressed the heart, forebrain, midbrain, midbrainhindbrain boundary, hindbrain, notochord, floorplate,C-d. DIVISION OF PHYSIOLOGICAL GENETICS

etc. Our current goal is to establish methods to identify
genes expressed in a spatially and temporally regulated
fashion and play an important role in development of. Functional and structural analyses of mammalian

the structure or the organ where the GFP expression
is detected. Efforts are in progress toward this goal.

Polycomb group comlexes
Haruhiko KOSEKI(2) Characterization of the hagoromo gene from cichlid
fishes in the East Africa
Yohey TERAI,! Naoko MORIKAWA l , Koichi KAWAKAMI
and Norihiro OKADA l (1Tokyo Institute of Technology)

Most of efforts of our group are paid tounderstand the functions of mammalian Polycomb
group
(PcG)
multimeric
comlexes
duringembryogenesis. We focused on identification andfunctional annotation of individual constituents of PcG

Previously we have developed an insertionalcomlexes.

mutagenesis method in zebrafish using a pseudotyped

30

DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

(1)RinglEWe have identified RinglB, the product of the
RinglB gene, by means of its interaction with the
Polyco'mb group protein Me118. We describe
biochemical and genetic studies directed to understandthe biological role of RinglB. Immunoprecipitation
studies indicate that RinglB form part of protein
complexes containing the products of other Polycomb
group genes, such as Rae28/Mphl and M33 and that
this complexes associate to chromosomal DNA We
have generated a mouse line bearing a hypomorphic
RinglB allele which shows posterior homeotictransformations of the axial skeleton and a mild
derepression of some Hox genes (Hoxb4, Hoxb6 andHoxb8) in cells anterior to their normal boundaries of
expression in the mesodermal compartment. In
contrast, the overexpression of RinglB in chickembryos results in the repression of cHoxbgexpression
in the neural tube. These results, together with thegenetic interactions observed in compound RinglB/
Me118 mutant mice are consistent with a role for
RinglB in the regulation of Hox gene expression by
Polycomb group complexes.
(2)

Scmhl
The product of the Scmhl gene, a mammalian
homologue of Drosophila Sex comb on midleg, interacts
with two mammalian Polycomb group (PcG) proteinsMph2/Edr2 and rae28/Mphl proteins throughrespective their SPM domains, Based on thisobservation we have generated a mutant allele ofScmhl designated as Scmhl"sPM, where the Scmhl
SPM domain has been deleted, Scmhl~sPM/:'sPM mice
exhibit posterior transformations of the axial skeleton
although the penetrance is extremely low. In addition,
Scmhl/:,sPM "SPM primary embryonic fibroblasts exhibit
premature entry into senescence and early mitotic
arrest that is associated with overexpression of Pl9"'RF
and mis-segregation of chromosomes, A partiallypenetrant phenotype is also observed during
spermatogenesis. Approximately half of theScmhl l1SPM I1SPM males are infertile which correlateswith an accelerated apoptosis of spermatocytes inmeiotic prophase I, and overexpression of p53,
P2lwAFl/CIPl and Bax.
Publications
1. Nakayama, T, Kasprowicz, D.;]., Yamashita, M.,

A, Koseki, H. and Ziegler, S. F.: The Generation of
Mature, Single-Positive Thymocytes In Vivo is
Dysregulated by CD69 Blockade or Overexpression.
J Immunol. 168, 87-94, 2002.
2. Miki, T, Suzuki, M., Shibasaki, T, Uemura,H.,
Sato, T, Yamaguchi, K, Koseki, H., Iwanaga, T.,
Nakaya, Hand Seino, S.: Mouse model of Prinzmetal
angina by disruption of the inward rectifier Kir6.1.
Nature Med. 8, 466-72, 2002.
3. Murakami, H, Okawa, A., Yoshida, H, Nishikawa,S., Moriya, H. and Koseki, H: Elbow knee synostosis(Eks): A new mutation on mouse Chromosome 14.
Mammalian Genome 13,341-344,2002.
4. O-Wang,]., Kajiwara, K, Kawamura, K, Kimura,M., Miyagishima, H., Koseki, H. and Tagawa, M. An
essential role for REV3 in mammalian cell survival:
absence ofREV3 incuces p53-independent embryonic
death. Biochem Biophys Res Commun. 293(3), 1132-1137,
2002.
5. Ohara, 0., Nagase, T., Mitsui, G., Kohga, H.,
Kikuno, R., Hiraoka, S., Takahashi, Y., Kitajima, S.,
Saga, Y. and Koseki, H.: Characterizaion ofsize-fractionated cDNA libraries generated by the invitro recombination-assisted method. DNA Research.
9,47-57,2002.
6. Yamaki, M., !sono, K, Takada, Y., Abe, K,
Akasaka, T, Tanzawa, H. and Koseki, H: The mouse
Edr2 (Mph2) gene has two forms of mRNA encoding
90- and 36-kDa polypeptides. Gene; 288,103-110, 2002.
7. Suzuki, M., Mizutani-Koseki, Y., Fujimura, Y.,
Miyagishima, H., Kaneko, T., Takada, Y., Akasaka, T,
Tanzawa, H., Takihara, Y., Nakano, M., Masumoto, H.,
Vidal, M., Isono, K and Koseki, H: Involvement of the
Polycomb-group gene RinglB in the specification of
the anterior-posterior axis in mice. Development 129,4171-4183, 2002.
8. Zhang, Y., Miki, T, Iwanaga, T., Koseki, Y., Okuno,
M., Sunaga, Y., Ozaki, N., Yano, H, Koseki, H. andSeino, S.: Otx3: identification, tissue expression andfunctional characterization of a novel member of the
Otx family. J BioI Chem. 277, 28065-28069, 2002.

Schubert, 1. A., Gillard, G., Kimura, M., Didierlaurent,

DIVISION OF PHYSIOLOGICAL GENETICS

31

9. Ishigami, A., Fujita, T., Handa, S., Shirasawa, T.,
Koseki, H., Kitamura, T., Enomoto, N., Sato, N.,
Shimosawa, T. and Maruyama, N.: Senescence marker
protein-30 knockout mouse liver are highly susceptible
to TNF- and Fas-mediated apoptosis. Am. J PathoL 161,
1273-1281, 2002.
10. Akasaka, T., Takahashi, N., Suzuki, M., Koseki,
H., Bodmer, R. and Koga, H.: MBLR, a new RING
finger protein resembling mammalian Polycomb geneproducts, is regulated by cell cycle-dependent
phosphorylation. Genes Cells. 7, 835-850, 2002.

factors to promoters and/or enhancers, and thenmodifying nucleosome structure for environmental
settings and finally initiating transcription. Therefore,
at the first step, transcription is initiated under the
repressive environment including the packed
chromatin, by binding and functioning of the very
initial components in the transcriptional apparatus in
the chromatin architecture. In the [3 -globin gene
expression, an erythroid-specific enhancer binding
protein NFcE2 is critically involved in remodeling thenucleosome structure over the DNase I hypersensitivesite 2 (HS2) of the human [3 -globin locus control region([3 - LCR). We obtained evidence of interaction of NF-E2

11. Takedam, u., Utani, A., Wu,]., Adachi, E., Koseki,
H., Taniguchi, M., Matsumoto, T., Ohashi, T., Sato, M.
and Shinkai, H.: Targeted Disruption of Dermatopontin
Causes Abnormal Collagen Fibrillogenesis. ] Invest
Dermatol. 119, 678-683, 2002.
12. Yuasa, S., Nakajima, M., Aizawa, H., Sahara, N.,
Koizumi, K., Sakai, T., Usami, M., Kobayashi, S.,
Kuroyanagi, H., Mori, H., Koseki, H. and Shirasawa,T.: Impaired cell cycle control of neuronal precursor
cells in the neocortical primordium of presenilin-ldeficient mice. ] Neurosci Res 70, 501-513, 2002.

13. Shirasawa, T., Izumizaki, M., Suzuki, YI., Ishihara,A., Shimizu, T., Tamaki, M., Huang, F., Koizumi, KI.,
Iwase, M., Sakai, H., Tsuchida, E., Ueshima, K., Inoue,
H., Koseki, H., Senda, T., Kuriyama, T. and Homma,
1.: Oxygen affinity of hemoglobin regulates 02
consumption, metabolism, and physical activity. ] Biol
Chem in press, 2002.
(1) Nucleosome positioning and regulation of gene
expression
Ryoiti KIYAMAl, Yoshiaki ONISHI! and Yuko
WADA-KIYAMA2 (IRes. Center for Glycoscience, AIST
and 2Nippon Med. Sch.)The cellular regulation of expression of a gene at
a given time under specific stimulation is a central
tissue in the study of differentiation, organogenesis,
development and phenotypes as in diseases. The
genetic information in individual cells should be
expressed at specific times in the following steps: first,
modulating a higher-order chromatin structure tochange a repressive environment, second, binding

32

DIVISION OF PHYSIOLOGICAL GENETICSto the cognate motif at HS2 in a repressive state. First,
the nucleosome containing the NF-E2 binding site
showed characteristic rotational and translational
phases. Second, the binding site had less affinity forthe histone octamers than the nearby regions while
showing higher accessibility to DNase I andmicrococcal nuclease. Finally, the motif was recognizedby the exogenous NF-E2 protein expressed in nonerythroid HeLa cells as shown by ligation-mediated
PCR and chromatin immunoprecipitation assay. Theselines of evidence indicate that NF-E2 interacts with the
cognate motif on the nucleosome before chromatin isremodeled, which may be a rate-limiting step andcritical for activation of globin gene expression. Taken
together with our previous findings that thenucleosome over HS2 was aligned by a key nucleosome
located at a distance of two nucleosomes from HS2 andDNA curvature had a significant role in thisnucleosome positioning, these results suggest that not
only transcription factor binding sites on the genome
but also the chromatin structure around HS2 arranged
by a specific DNA structure and changing its status
is critical in the early steps of [3 -globin gene
expression.
(2) A novel gene, Kank, is a potel)tial growth
suppressor of renal cell carcinoma
Badal C. ROY\ Shubhashish SARKARI, Naoya HATANO\
Kazuo GOHJI 2, Teiichiro AOYAGI 3 and Ryoiti KIYAMA!
(!Res. Center for Glycoscience, AIST, 2Dept. Urol.,
Osaka Med. College, 3Tokyo Dental College)
We identified a potential tumor suppressor gene
named Kank (for kidney ankyrin repeat-containing
protein) on chromosome 9p24 by using a genome

subtraction method followed by comprehensive
analysis of loss of heterozygosity (LOH). The genecontained an open reading frame for a protein of 1194
amino acids long with an estimated MW of 130 kD. It
also contained some interesting motifs including three
coiled-coil domains in the N-terminus and fourankyrin-repeats in the C-terminus. The protein was
localized in the cytosol as revealed by immunostaining.
The Kank gene showed LOH in about 25% RCC
patients and its expression was suppressed in tumor
tissues examined by RT-PCR, immunostaining andWestern blotting. Interestingly, the genomic DNA from
RCC tumors and several kidney cancer cell lines
showed hypermethylation at CpG sites of the gene. The
gene showed growth suppression both in vivo and invitro. Overexpression of the Kank gene in
expression-negative HEK293 and G-402 cells showed
dramatic morphological changes which seemed to be
mediated by the cytoskeletal changes. Our results
demonstrate that Kank protein may function as a
growth regulator through the cytoskeleton and may
play an important role in cell migration as well as cell
growth.

according to their functions: cancer-related genes, thegenes for growth and proliferation, structural genes
and others. Using EstrArrays, we first investigated thetime course of the responses of the genes in MCF-7
cells to an estrogen, 17 f3 -estradiol. The estrogen
responsive genes were classified according to thisresult into the early and late responding types. The
results were confirmed by Northern blot analysis. We
then performed the assay for profiling gene expressionin MCF~7 cells after chemical treatment with bisphenol
A, nonylphenol, dioxin and others. The EstrArraysystem can be used in two different major fields:
environmental monitoring, and diagnosis/drug
screening. Both systems need accurate and highthroughput analysis of cellular responses which canbe monitored by the gene response. In the first field,
gene expression profiling of endocrine disruptors will
be a useful tool for monitoring effects of industrial
chemicals, and natural and artificial biomaterials. In
the second field, the EstrArray analysis of estrogen
receptor-positive cancer cell lines from various tissues
showed similar but not identical profiles, indicating
the usefulness of this custom microarray system formonitoring estrogen responses in various tissue types,(3) Development and application of DNA microarrays
using estrogen responsive genes

which is important for cancer therapy and productionof anti-estrogen drugs.

Ryoiti KIYAMA

Publications
1. Li, X.-M., Onishi, Y., Kuwabara, K., Rho, ].-y.,Estrogen is a member of the steroid-thyroid
hormones and regulates development and maintenanceof female phenotype and behavior. The primary targetof estrogen is the estrogen receptor and the
estrogen-estrogen receptor complex binds to a numberof secondary target genes, which eventually regulate
production of growth factors as a part of hormone
actions. The chemicals that mimic estrogen activity,
so called endocrine disruptors, are becomingworldwide problems. To monitor estrogen activity inthe environment as well as in the commercial products
and foods and understand the mechanisms of theeffects of endocrine disruptors on the human body, wedeveloped a microarray-based technique by means ofprofiling estrogen responsive genes. Approximately 200
cDNAs from the genes and ESTs (expressed sequencetags) were selected after screening approximately 9,200
genes for the estrogen response and they were spottedon a custom microarray, the EstrArray. The genes and
ESTs selected were classified into several groups

WadacKiyama, Y., Sakuma, Y. and Kiyama, R.:
Ligand-dependent transcriptional enhancement by
DNA curvature between two half motifs of the estrogen
response element in the human estrogen receptor 0:
gene. Gene 294, 279-290, 2002.
2. Kiyama, R. and Trifonov, E. N.: What positions
nucleosomes? ---A model (Review). FEBS Letters 523,
7-11,2002.
3. Onishi, Y. and Kiyama, R.: Interaction of NF-E2
in the human f3-g10bin locus control region before
chromatin remodeling. J Bio!. Chern. 278, 8163-8171,
2003.
4. Wanapirak, c., Kato, M., Onishi, Y., Wada-Kiyama,
Y. and Kiyama, R.: Evolutionary conservation andfunctional synergism of curved DNA at the mouse ,and other globin-gene promoters. J Mol. Evol (in press).
5. Nadkarni, A., Sakaguchi, T., Takaku, H.,

DIVISION OF PHYSIOLOGICAL GENETICS

33

Gorakshakar, A., Phanasgaonkar, S., Colah, R.,
Mohanty, D. and Kiyama, R: A novel {30-thalassemia
mutation at codon 55 (-A) and a rare 17-bp deletion atcodons 126-131 in the Indian population. Hemoglobin
26,41-47,2002.
6. Sarkar, S., Roy, B. C., Aoyagi, T., Gohji, K. andKiyama, R: A novel ankyrin repeat-containing gene
Kank located at 9p24 is a growth suppressor of renal
cell carcinoma. J Biol. Chem. 277, 36585-36591, 2002.
7. Inoue, A., Yoshida, N., Omoto, Y., Oguchi, S.,
Yamori, T., Kiyama, R and Hayashi, S.: Developmentof cDNA microarray for expression profiling of
estrogen-responsive genes. J Mol. Endocrinol. 29,
175-192, 2002.
8. Inoue, A., Hayashi, S.-i., Aoyagi, K., Nishigaki, M.,
Sasaki, H. and Kiyama, R: Evaluation of tissue-specificresponse to estrogen activity by means of a reporter
gene assay based on the differential use of promoters
A to F of the human estrogen receptor ex gene. J

Pharmacol. Toxicol. Methods 47, 129-135,2002.

34

DIVISION OF PHYSIOLOGICAL GENETICSNJML+ P: A hybrid algorithm of the maximum
likelihood and neighbor-joining methods using parallel(2)

D. DEPARTMENT OF
POPULATION GENETICS

D-a. DIVISION OF POPULATION GENETICS(1) Adaptive evolution of the IgA hinge region inprimates

Kenta SUMIYAMA, Naruya SAITOU and Sltintaroh UEDA!
(!Univ. Tokyo, Grad. Sch. Science)
IgA is a major component that prevents thepenetration of pathogenic bacteria into mucosal
surfaces. The IgA antibody is cleaved at the IgA hinge
region with high specificity by IgA-specific proteases
produced by several pathogenic bacteria. We
conducted a genomic sequence analysis of the IgA
genes of a wide spectrum of primates, including thefirst intron and second exon, which consist of the hinge
region and the CH2 domain, to find evidence of positive
selection. Because the hinge region is quite small, we
combined the largest collection of sequences that could
be clearly aligned and evaluated the total numbers ofsynonymous and nonsynonymous substitutions on thephylogenetic tree. The nonsynonymous to synonymous
substitution ratio (dN/dS test) showed that hominoids,
Old World monkeys, and New World monkeys havedN/dS ratios of 5.4,6.3, and 4.2, respectively. Fisher's
exact probability tests showed statistical significancefor the Old World monkey. Because the substitution
rates of the flanking sequences are more or less similar
to the synonymous rates of the hinge region, these highvalues of dN/dS should be the result of positive
selection at the hinge region. Combining the highsequence variability in each population and the highly
accelerated nonsynonymous substitution rates in thehinge region, we conclude that this unusual IgA
evolution is a molecular evidence of adaptive evolution
possibly caused by the host-parasite relationship. For
details, see ref. (1).

computing
Satoshi OOTA and Naruya SAITOUThe NJML method [2,3] is a hybrid algorithmof the two well-known methods to reconstructmolecular phylogenetic trees: the neighbor-joining (NJ)
method [4] and the maximum likelihood (ML) method[l]. The NJML method is considerably efficient bothin reliability and speed comparing with the otherexisting ML-based methods. By giving appropriate
parameters, the NJML method gradually approaches
the exhaustive topology search, that is supposed to be
the most accurate way to find the true phylogenetic
tree. However, it is obvious that more exhaustive
searches require more computational time. We have
implemented the NJML method by using parallel
computing to reduce the computational time, in which
the bootstrap trials and the maximum likelihood
estimation were parallelized. For details, see ref. (2).
(3) Extreme mtDNA homogeneity in continental Asian
populations

Hiroki OOTAl, Takashi KITANO, Feng Jm2, Isao YUASA3,
Li WANG 2, Shintaroh UEDA3, Naruya SAITOU and MarkSTONEKING! (!Max Planck Institute for Evolutionary
Anthropology, 2Institute of Genetics, Beijing, 3Univ.
Tokyo, Grad. Sch. Science)
Mitochondrial DNA (mtDNA) variation incontinental Asia has not been well-studied. Here, wereport mtDNA HVI sequences for 84 Xi'an and 82
Changsha Han Chinese, 89 Honshu Japanese, and 35
Vietnamese. Comparison of these sequences with other
Asian mtDNA sequences reveals high variability
within populations, but extremely low differentiation
among Asian populations. Correlations between
genetic distance and geographic distance, based on
mtDNA and Y chromosome variation, indicate a higher
migration rate in females than in males. This may
reflect patrilocality, as suggested previously, butanother plausible hypothesis is that the demographic
expansion associated with the spread of agriculture
in Asia may be responsible for the extreme genetic
homogeneity in Asia. For details, see ref. (3).

DIVISION OF POPULATION GENETICS35

(4) A large-scale analysis of human mitochondrial DNA
sequences with special reference to the population historyof East Eurasian

Hiroki OOTA!, Naruya SAITOU and Shintaroh UEDA2(!Max Planck Institute for Evolutionary Anthropology,
2Univ. Tokyo, Grad. Sch. Science)
Ancient DNA analysis is very powerful for thestudies on past human populations. However, in mostcases ancient DNA is extremely degraded into shortfragments, and the information is limited because of
the damaged state. A large-scale data analysis ofhuman mitochondrial DNA (mtDNA) was carried out
to assess validity of the short nucleotide sequence forclosely related human populations. We retrieved
mtDNA data from the DDBJ/EMBL/GenBank
nucleotide sequence database and constructed a data
set containing 414 distinct mtDNA types derived from19 populations of East Eurasia and the surroundingarea. A series of new procedures were applied and an
mtDNA phylogenetic tree was constructed. Six major
star-like clusters were observed in this tree, and thecorresponding six radiation groups (I-VI) werecharacterized. Frequency distributions of eachradiation group showed remarkable difference in eachgeographical area, suggesting that the short mtDNA
nucleotide sequences were valuable in analyzing
ancient human populations. The efficient procedurefor data analysis will enhance the usefulness of ancient
DNA data. Additionally, we discuss a possibility of two
human migration routes from Africa to East Eurasia
based on the mtDNA tree topology and the coalescencetimes in each radiation group. For details, see ref. (4).

of repeats of the short tandem repeat through allele
length sizing and sequencing methods. Human allele
distributions are very similar among populations,ruling out ethnicity as a genetic risk for allele
expansion. Primate comparison shows human-specificfeatures, with longer human alleles due to a novel
variable trinucleotide repeat, not present in non-humanprimates, which increased the disease-causing
expansion likelihood. SCA8 seems to be a humanspecific disease. For details,. see ref. (5).
(6) Helicobacter pylori in North and South America
before Columbus

Yoshio YAMAOKA!, Naruya SAITOU, David Y. GRAHAM!
and others (!Baylor College of Medicine, Houston)
Abstract We present a molecular epidemiologic
study, based on an analysis of vacA, cagA and cag
right end junction genotypes from 1042 Helicobacter
pylori isolates, suggesting that H. pylori was present
in the New World before Columbus. Eight Native
Colombian and Alaskan strains possessed novel vacA
and/or cagA gene structures and were more closely
related to East Asian than to non-Asian H. pylori. Some
Native Alaskan strains appear to have originated inCentral Asia and to have arrived after strains foundin South America suggesting that H. pylori crossed theBering Strait from Asia to the New World at different
times. For details, see ref. (6).
A phylogenomic study of the OCTase genes in
Pseudomonas syringae pathovars: the horizontal transferof the argK-tox cluster and the evolutionary history of(7)

OCTase genes on their genomes
Understanding the dynamics of Spinocerebellar
Ataxia S (SCAS) locus through a comparative genetic
approach in humans and apes
(5)

Aida M. ANDRES!, M. SOLDEVILA!, Naruya SAITOU, F.
CALAFELL!, Jaume BERTRANPETIT and others (!Univ.
Pompeu Fabra, Barcelona)
Spinocerebellar Ataxia 8 (SCA8) is a neurodegenerative
disorder caused by expansion of a trinucleotide repeat.
We undertake a comparative genetic analysis among
human populations.and primate species in the normal
variation range, where forces that shaped present
diversity can be recognised. We determinate number

36

DIVISION OF POPULATION GENETICS

Hiroyuki SAWADA!, Shigehiko KANAYA 2, Masayuki
TSUDA3, Fumihiko SUZUKI!, Kozi AZEGAMI! and Naruya
SAITOU (lNational Institute for Agro-Environmental
Sciences, Tsukuba, 2Nara Institute of Science andTechnology, 3Tohoku Univ.)
Phytopathogenic Pseudomonas syringae issubdivided into about 50 pathovars due to theirconspicuous differentiation with regard topathogenicity. Based on tpe results of a phylogenetic
analysis of four genes (gyrB, rpoD, hrpL, and hrpS),
Sawada et al. (1999) showed that the ancestor of P.
syringae had diverged into at least three monophyleticgroups during its evolution. Physical maps of the
genomes of representative strains of these three groups
were constructed, which revealed that each strain had
five rrn operons which existed on one circular genome.
The fact that the structure and size of genomes vary
greatly depending on the pathovar shows thatP. syringae genomes are quite rich in plasticity andthat they have undergone large-scale genomic
rearrangements. Analyses of the codon usage and the
GC content at the codon third position, in conjunction
with phylogenomic analyses, showed that the genecluster involved in phaseolotoxin synthesis (argK-tox
cluster) expanded its distribution by conducting
horizontal transfer onto the genomes of two P. syringae
pathovars (pv. actinidiae and pv. phaseolicola) frombacterial species distantly related to P. syringae andthat its acquisition was quite recent (i.e., after theancestor of P. syringae diverged into the respective
pathovars). Furthermore, the results of a detailed
analysis of argK [an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene] , which
is present within the argK-tox cluster, revealed theplausible process of generation of an unusualcomposition of the OCTase genes on the genomes ofthese two phaseolotoxin-producing pathovars: acatabolic OCTase gene (equivalent to the orthologue
of arcB of P. aeruginosa) and an anabolic OCTase gene(argF), which must have been formed by gene
duplication, have first been present on the genome of
the ancestor of P. syringae; the catabolic OCTase genehas been deleted; the ancestor has diverged into therespective pathovars; the foreign-originated argK-tox
cluster has horizontally transferred onto the genomesof pv. actinidiae and pv. phaseolicola; and hence twocopies of only the anabolic OCTase genes (argK andargF) came to exist on the genomes of these twopathovars. Thus, the horizontal gene transfer and the
genomic rearrangement were proven to have played
an important role in the pathogenic differentiation anddiversification of P. syringae. For details, see ref. (7).

(8) Construction and analysis of a human-chimpanzee
comparative clone map

provide us with reference data to conduct comparative
genomic research on primates, which will be importantto understand what genetic information makes us
human. Here we present a first-generation humanchimpanzee comparative genome map and its initial
analysis. The map was constructed through pairedalignment of 77,461 chimpanzee bacterial artificial
chromosome end sequences with publicly available
human genome sequences. We detected candidate
positions, including two clusters on humanchromosome 21 that suggest large, nonrandom regionsof difference between the two genomes. For details, seeref. (8).
Publications
1. Sumiyama, K., Saitou, N. and Ueda, S.: Adaptive

evolution of the IgA hinge region in primates.
Molecular Biology and Evolution, 19, 1093-1099, 2002.
2. Oota, S. and Saitou, N.: NJML+ P: A Hybrid
Algorithm of The Maximum Likelihood andNeighbor-Joining Methods Using Parallel Computing.
Genome Informatics Series, 13, 434-435, 2002.
3. Oota, H., Kitano, T., Jin, F., Yuasa, 1., Wang, 1.,
Ueda, S., Saitou, N. and Stoneking, M.: Extreme
mtDNA homogeneity in continental Asian populations.
American Journal ofPhysical Anthropology, 118, 146-153,
2002.
4. Oota, H., Saitou, N. and Ueda, S.: A large-scale
analysis of human mitochondrial DNA sequences withspecial reference to the population history of East
Eurasian. Anthropological Science, 110, 293-312, 2002.
5. Andres, A. M., Soldevila, M., Saitou, N., Volpini,
V., Calafell, F. and Bertranpetit, ].: Understanding the
dynamics of Spinocerebellar Ataxia 8 (SCA8) locus
through a comparative genetic approach in humans
and apes. Neuroscience Letters, 336, 143-146,2002.
6. Yamaoka, Y.,Orito, E., Mizokami, M., Gutierrez,
0., Saitou, N., Kodama, T., Osato, M. S., Kim, ]. G.,

Asao FUJIYAMA , Hidemi WATANABE!, Atsushi
TOYODAl, Todd D. TAYLOR!, Takehiko ITOHl, Naruya

Ramirez, F. C., Mahachai, V. and Graham, D. Y.:
Helicobacter pylori in North and South America before
Columbus. FEBS Letter, 517, 180-184,2002.

SAITOU, Masahira HATTORI l , Yoshiyuki SAKAKI l andothers (IRiken Genomic Science Center, Yokohama)
The recently released human genome sequences

7. Sawada, H., Kanaya, S., Tsuda, M., Suzuki, F.,
Azegami, K. and Saitou, N. Phylogenomic study ofl

DIVISION OF POPULATION GENETICS

37

OCTase genes in Pseudomonas syringae pathovars:
Horizontal transfer of argK-tox cluster andevolutionary history of OCTase genes on theirgenomes. Journal of Molecular Evolution, 54, 437-457,
2002.
8. Fujiyama, A., Watanabe, H., Toyoda, A., Taylor,T. D., Itoh, T., Tsai, S.-F., Park, H.-S., Yaspo, M.-L,.
Lehrach, H., Chen, Z., Fu, G., Saitou, N., Osoegawa, K.,
de Jong, P. J., Suto, Y., Hattori, M. and Sakaki, Y.:
Construction and Analysis of a Human-Chimpanzee
Comparative Clone Map. Science, 295, 131-134, 2002.

chromosome substitution lines differed between thetwo backgrounds. No QTL was found in the L
background; we found three significant QTL in the H
background, but their positions were different fromthe QTL positions estimated from the wholechromosome analysis. These results suggested that
epistasis between QTL may have been important in thephenotypic evolution of this character.
(2) DNA variation in the gustatory and olfactory
receptor gene families of Drosophila melanogaster
Akira KAWABE and Toshiyuki TAKANO-SHIMIZU

9. Umetsu, K., Iwabuchi, N., Yuasa, 1., Saitou, N.,
Clark, PF., Boxshall, G., Osawa, M. and Igarashi, K.:
Complete mitochondrial DNA sequence of a tadpole
shrimp (Triops cancriformis) and analysis of museum
samples. Electrophoresis, 23, 4080-4084, 2002.
10. Tateno, Y., Imanishi, T., Miyazaki, S.,
Fukami-Kobayashi, K., Saitou, N., Sugawara, H. andGojobori, T.: DNA Data Bank of Japan (DDBJ) for
genome scale research in life science. Nucleic Acids
Research, 30, 27-30, 2002.
(1) Genetic and molecular dissection of the within- andbetween-species variation in the sex comb tooth number
in Drosophila
Haruki TATSUTA and Toshiyuki TAKANO-SHIMIZU
Sexual dimorphic characters are often among thefirst characters to change in the evolution of new
species. Sex comb, a specific row of enlarged bristles
on the first legs of males of some species of thesubgenus Sophophora, is such an example. Indeed, thesex comb is a highly variable character between andwithin species. We first analyzed a within-species
sex-comb-tooth number variation by the composite
interval mapping for F2 samples between high (H) andlow (L) sex-comb-tooth number lines of D. simulans,
finding four significant QTL, two on each majorautosome. Interestingly, one of them showed an
overdominant effect. To study possible epistasis
between QTL, we constructed the chromosome
substitution lines and recombinant inbred lines. While
we confirmed the significant effects of the two QTL
on the third chromosome by using recombinant inbredlines, the results of QTL analysis for the second

38

DIVISION OF POPULATION GENETICS

DNA variation in 55 gustatory and 54 olfactory
receptor gene candidates were analyzed for eight

Drosophila melanogaster and one D. simulans inbred
strains. The average nucleotide diversity (n) was 0.008,
which is similar to those for other D. melanogastergenes. A significant correlation was observed between
the amount of intra-specific DNA variations insynonymous sites and those in non-coding regions, andboth of them were significantly correlated with the levelof local crossover frequency. Local mutation rate or
local crossover frequency or both might play asignificant role in shaping silent-site variation. A
significant correlation was also found between thelevels of intra- and inter-specific replacement-site DNA
variations, suggesting that the strength of selective
constraint varies among loci, but is constant between
the two species. In both the gustatory and olfactory
genes, the ratio of the numbers of replacement changes
to synonymous ones was significantly higher ininter-specific divergence than in intra-specificpolymorphism. The clustered genes showing highsimilarity within each cluster had higher level of DNApolymorphism than the single-copy genes in isolation.
Gene conversion might be responsible for the highDNA polymorphism in the clustered genes. At ten lociof 109 loci analyzed, we found 12 putative null alleles:
small indels causing flame shift, large coding-sequence
deletion, and nonsense mutations. All of them werepolymorphic in a sample. Because we analyzed only
half to one-third of entire coding sequences, a single
D. melanogasterfly may have eight or more null alleles
for the gustatory and olfactory genes.
I should add that Ms Yuriko Ishii has contributed
significantly to the above two works.

Publications

Takashi ABE, Yuta ICHIBA, Jian-Ping SONG, Yoko
KOSAKA, Kiyomi KITA and Toshimichi IKEMURA

1. Furuse, T., Takano-Shimizu, T., Moriwaki, K.,
Shiroishi, T. and Koide, T.: QTL analyses of

In addition to protein-coding information, genome

spontaneous activity by using mouse strains fromsequences contain a wealth of information of interestin many fields of biology from molecular evolution toMishima battery. Mamm. Genome 13,411-415,2002.

genome engineering and biomedicine. G+C% is used

D-b. DIVISION OF EVOLUTIONARY GENETICS

as a fundamental characteristic of individual genomes
but too simple a parameter to differentiate awide variety of genomes. Because oligonucleotide(1) Infonnatics for unveiling hidden genome signatures

frequencies vary significantly among genomes, they
can be used to distinguish genomes and genomic

Takashi ABE, Yuta ICHIBA, Yoko KOSAKA, Nanayo
ISHIHARA and Toshimichi IKEMURA
With the increasing amount of available genome
sequences, novel tools are needed for comprehensive

regions. We used an unsupervised neural network
algorithm, Kohonen's self-organizing map (SOM), to
analyze di- and trinucleotide frequencies in 9
eukaryotic genomes (a total of 1.2 Gb); S. cerevisiae, S.
pombe, C. elegans, A. thaliana, D. melanogaster, Fugu,

analysis of species-specific sequence characteristics

and rice, as well as P. falciparum chromosomes 2 andfor a wide variety of genomes. We used an unsupervised neural network algorithm, a self-organizing map

genomic sequence with different window sizes was(SOM), to analyze di-, tri-, and tetranucleotide

encoded as a 16- and 64-dimensional vector giving

frequencies in a wide variety of prokaryotic andrelative frequencies of di- and trinucleotides,
respectively. From analysis of a total of 120,000
nonoverlapping 10-kb sequences and overlapping

eukaryotic genomes. The SOM, which can clustercomplex data efficiently, was shown to be an excellent

3, and human chromosomes 14, 20, 21, and 22. Eachtool for analyzing global characteristics of genome

100-kb sequences with a moving step size of 10 kb,

sequences and for revealing key combinations ofderived from a total of the 1.2 Gb sequences, clear

oligonucleotides representing individual genomes.
From analysis of 1-kb and 10-kb genomic sequences

species-specific separations of most genomic sequenceswere obtained with the SOMs. The unsupervised

derived from 65 bacteria and from 6 eukaryotes, clear

algorithm could recognize, in most of the 10-kb

species-specific separations of major portions of thesequences were obtained with the di-, tri-, andsequences, the species-specific characteristics (keytetranucleotide SOMs. The unsupervised algorithm

signature representations of each genome. (Abe et aI.,

could recognize, in most 10-kb sequences, theGenome Informatics Series, 13, 12-20, 2002).

species-specific characteristics (key combinations ofoligonucleotide frequencies) that are signature features(3) Functional analyses of centromere of higherof each genome. We were able to classify DNA

vertebrate cells

combinations of oligonucleotide frequencies) that aresequences within one and between many species into
subgroups that corresponded generally to biologicalcategories. Because the classification power is very

Tatsuo FUKAGAWA, Ai NISHIHASHI, Atsushi OKAMURA,
Tetsuya HORI, Masahiro NOGAMI, Yoshikazu MIKAMI,

high, the SOM is an efficient and fundamental

Yoko MIYAUCHI, Jian-Ping SONG, Kazuko SUZUKI,

bioinformatic strategy for extracting a wide range ofgenomic information from a vast amount of sequences.

Mitsuko YOSHIKAWA, Yuko FUKAGAWA and Toshimichi
IKEMURA(Abe et al. Genome Research, 13, 693-702, 2003).
The centromere plays a fundamental role in(2) A novel bioinfonnatic strategy for unveiling hidden
genome signatures of eukaryotes: Self-organizing mapof oligonucleotide frequency

accurate 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

DIVISION OF POPULATION GENETICS

39

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 thefunction of the centromere, we were led to develop a
genetic analysis method that utilizes the hyperrecombinogenic chicken B lymphocyte cell line DT40.
The high level of homologous recombination in DT40
cells permit efficient targeted disruption of genes ofinterest. We also generated human artificial minichromosomes using the telomere-directed breakage
method in DT40 cells. We have combined humanartificial mini-chromosomes and DT40 cells to study
chromosome segregation. We have improved thissystem and have created several cell lines withconditional knockouts of several centromere proteins
to investigate the molecular mechanism of centromere
assembly and function. In this year we focused on thefollowing subjects to the analyses of centromeres.
I) We identified a novel constitutive centromere
protein, CENP-I, which shows sequence similarity withfission yeast Mis6 protein, and we showed that CENP-I
is a constitutive component of the centromere thatcolocalizes with CENP-A, -C and -H throughout thecell cycle in vertebrate cells. To determine the precise
function of CENP-I, we examined its role in centromerefunction and assembly by generating a conditional
loss-of-function mutant in the chicken DT40 cell line.
In the absence of CENP-I, cells accumulated BubR1
at kinetochores and arrested at prometaphase with
mis-aligned chromosomes for long periods of time.
Eventually, checkpoint function appeared to be lostand cells reentered the cell cycle without undergoingcytokinesis. Immunocytochemical analysis of
CENP-I-deficient cells demonstrated that both CENP-I
and CENP-H are necessary for CENP-C, but not
CENP-A, localization to the centromere, indicating that
CENP-I plays an essential role in centromere assemblyin vertebrate cells. These experiments define anassembly pathway for the vertebrate kinetochore inwhich binding of CENP-A is followed by mutuallyinterdependent targeting of CENP-H and CENP-I, andthen by binding of CENP-C. (Nishihashi et al. Dev. Cell,
2002).
1I) Dissection of human centromeres is difficult
because of the lack of landmarks within highly
repeated DNA. We have systematically manipulated
a single human X centromere using homologous
recombination and de novo telomere formation to40

DIVISION OF EVOLUTIONARY GENETICS

generate a large series of minichromosome deletion
derivatives. On the basis of the molecular structure of
the minichromosomes recovered, the asymmetric
distribution of CENP-A, -C and -H relative to the DXZ1
a-satellite array and the identification of a single majorsite of topoisomerase 1I activity we suggest that the
kinetochore demarcates a fixed region of limited extent
within the many megabases of centromericallylocalised repeat. Moreover, our findings suggest afundamental role for topoisomerase 1I at thecentromere. (Spence et aI., EMBO ]., 2002)(4) Chromosome-wide assessment of replication timing
for human chromosomes llq and 21q: Genome-synteny
breakpoints between mouse and human chromosomes ator near replication-transition regions

Yoshihisa WATANABE, Yuta ICHIBA and Toshimichi
IKEMURA
Completion of the human genome sequencing will
greatly accelerates the development of a new branchof evolutionary genetics. Comparison of the humangenome with other mammalian genome, particularly
identification of genome-synteny breakpoints, aids inunderstanding the evolutionary history of human andother mammalian genomes. We measured replication
timing of the entire lengths of human chromosomes
11q and 21q. Megabase-sized zones that replicate earlyor late in S phase (thus early/late transition) were
defined at the sequence level and many disease-related
genes were identified in the timing-switch regions. We
then compared human and mouse genomes on achromosome level, and found that synteny breakage
between the two genomes occurred primarily at or near
the replication-transition regions found on hu~an
chromosomes 11q and 21q. The finding that synteny
breakpoints between human and mouse genomes
coincide to replication-transition regions shows thatmechanisms of replication-timing transition may havebeen involved in evolutional processes to build up thepresent chromosome structures. (Watanabe et aI.
Human Molecular Genetics, 11, 13-21, 2002).
Publications

1. Watanabe, Y, Fujiyama, A., Ichiba, Y, Hattori, M.,

Yada, T., Sakaki, Y. and Ikemura, T.: Chromosomewide assessment of replication timing for humanchromosomes 11q and 21q: disease-related genes intiming-switch regions. Human Molecular Genetics 11,
13-21, 2002.
2. Nishihashi, A., Haraguchi, T., Hiraoka, Y.,
Ikemura, T., Regnier, V., Dodson, H., Earnshaw, W. C.
and Fukagawa, T.: CENP-I is essential for centromerefunction in vertebrate cells. Dev. Cell 2, 463-476, 2002.
3. Abe, T., Kanaya, S., Kinouchi, M., Ichiba, Y.,
Kozuki, T. and Ikemura, T.: Informatics for unveilinghidden genome signatures. Genome Research, 13,
693-702, 2003.
4. Ohno, M., Fukagawa, T., Lee, 1.S. and Ikemura, T.:
Triplex-forming DNAs in the human interphasenucleus visualized in situ by polypurine/
polypyrimidine DNA probes and antitriplex antibodies.
Chromosoma 111, 201-213, 2002.
5. Abe, T., Kanaya, S., Kinouchi, M., Ichiba, Y.,
Kozuki, T. and Ikemura, T.: A novel bioinformatic
strategy for unveiling hidden genome signatures of
eukaryotes: Self-organizing map of oligonucleotide
frequency. Genome Informatics Series 13, 12~20, 2002.
6. Fukushima, A., Ikemura, T., Kinouchi, M., Oshima,T., Mori, H. and Kanaya, S.: Detection of periodicity
in eukaryotic genomes on the basis of power spectrum
analysis. Genome Informatics Series 13, 21-29, 2002.
7. Fukushima, A., Ikemura, T., Kinouchi, M., Oshima"T., Kudo, Y., Mori, H. and Kanaya, S.: Periodicities inprokaryotic and eukaryotic genomes identified bypower spectrum analysis. Gene 300, 203-211, 2002.
8. Spence, M. ]., Critcher, R., Ebersole, A. T.,
Fukagawa, T. and Farr, ]. C.: Co-localisation ofcentromere activity, CEN proteins and a
Topoisomerase II cleavage site to a fixed region oflimited extent within the human X-satellite locus DXZl.
The EMBO Journal 21, 5269-5280, 2002.
9. Hayashi, T., Seki, M., Maeda, D., Wang, W.,
Kawabe, Y., Seki, T., Saitoh, H., Fukagawa, T., Yagi,
H. and Enomoto, T.: Ubc9 is essential for viability ofhigher eukaryotic cells. Experimental Cell Research 280,
212~221, 2002.

DIVISION OF EVOLUTIONARY GENETICS

41

E. DEPARTMENT OFINTEGRATED GENETICS

E-a. DIVISION OF HUMAN GENETICS(1) Comparative analysis of an imprinted domain in themouse and its orthologue in the chicken
Hiroyuki SASAKI, Takaaki YOKOMINE, Hisao SHIROHZU,
Chikako SUDA, Wahyu PURBOWASITO\ Masaoki
TSUZUKI 2, Atsushi TOYODA3, Masahira HATTORI3 andYoshiyuki SAKAKI3 (lKyushu Univ.; 2Hiroshima Univ.;
3GSC, RIKEN)Genomic imprinting, an epigenetic gene-marking
phenomenon in mammals, causes parent-specificmonoallelic expression of a subset of genes. Imprinted
genes tend to form clusters in the genome (imprinted
domains), which may be related to the mechanism of
imprinting or to the reason for the evolution of
imprinting. As a step to understand the structural andfunctional characteristics of imprinted domains, wehave characterized a 1-Mb imprinted domain in mouse
chromosome 7F4/F5 and its orthologous domain inchicken chromosome 5 (-0.5 Mb). We found that thegenes of the chicken domain are not imprinted and,furthermore, that the chicken domain lacks the uniquetandem repeat cluster of -0.2 Mb, the H19 gene, andthe imprinting control elements, all of which arepresent in the mouse. The results indicate that the
mammalian imprinted genes were already clustered in
the common ancestor of mammals and birds and thatthe imprinting mechanism, which can affect multiple
genes in the cluster, came in later during mammalianevolution (in preparation).
(2) Imprinting mechanisms of the mouse Igf2/H19
sub-domain
Hiroyuki SASAKI, Ko ISHIHARA, Yuzuru KATO, Hiroyasu
FURUUMI and Wolf REIK 1 (lBabraham Inst.)

42

DEPARTMENT OF INTEGRATED GENETICS

The imprinted mouse 7F4IF5 domain contains twolinked imprinted genes Igj2 and H19 near its
centromeric boundary: Igf2is paternally expressed andH19 maternally expressed. It is known that thepaternal-specific methylation of the differentiallymethylated region (DMR) located upstream of H19 is
the primary cause for the Igj2/ H19 imprinting. We
investigated the changes in Igj2/ H19 imprinting in
uniparental mouse fetuses (ref. 2). We also identified
an evolutionarily conserved CTCF-dependent insulator
element at the centromeric boundary of the Igj2/ H19
sub-domain (ref. 3). Lastly, we characterized a mousemutant called minute, which has an inversion
breakpoint at about 20-kb downstream of H19(ref. 10).
The study confirmed our previous observation that
mesodermal enhancers, which are involved in the Igf2/
H19imprinting in muscles and cartilages, are locatedin the downstream region of H19. These results provide
the basis for understanding the organization of theimprinted domains and subdomains and its
significance 'in the control of monoallelic gene
expression.
(3) Role of de novo DNA methyltransferases Dnmt3a/
Dnmt3b in the establishment of genomic imprinting
Hiroyuki SASAKI, Masahiro KANEDA, Takashi SADO,
Naomi TSUJIMOTO, Kenji KUMAKI, Shoji TAJIMA\
Masaki OKAN0 2 and En LI 3 eOsaka Univ.; 2CDB,
RIKEN; 3Harvard Med. Sch.)
DNA methylation serves as an important
gene-marking mechanism for the discrimination of theparental alleles of imprinted genes. To understand how
the primary imprints are established in the male andfemale germlines, we studied the expression andlocalization of the de novo DNA methyltransferases
Dnmt3a and Dnmt3b in the male and female gonads.
We have found that Dnmt3b and a specific form of
Dnmt3a are present in the nuclei of prospermatogonia(also called gonocytes) and growing oocytes (inpreparation), in which the primary imprints are
established. We have also started to analyze
germline-specific knockout of Dnmt3a and Dnmt3bto
know which DNA methyltransferase is responsible forthe imprint establishment.
(4) Molecular pathology of human disorders associatedwith DNA methylation and imprinting

Hiroyuki SASAKI, Hisao SHIROHZU, Hiroyasu FURUUMI,
Takeo KUBOTA!, Kenji KURosAwA2 and Shoji TAJIMA30Natl Center of Neurology and Psychiatry; 2Kanagawa
Children's Hospital; 30saka Univ.)
We studied three Japanese cases with ICF(immunodeficiency, centromeric instability, facial
anomalies) syndrome, an autosomal recessive disorderwith hypomethylation of satellite DNA, and identified
three novel mutations within the coding region of
DNMT3B (ref. 5). We also reported that paternal
disomy for chromosome 14 causes a distinctive
malformation syndrome with abdominal muscular
defects, skeletal anomalies and characteristic facies (ref.
4). We speculated that DLK1, a paternally expressedgene located on this chromosome, is associated withthe malformation of the mesodermal tissues.
(5) Investigation on the possibility of Z chromosome
dosage compensation in chicken
Hiroyuki SASAKI, Takaaki YOKOMINE, Asato KUROIWA!,
Yoichi MATSUDA! and Masaoki TSUDUKI2 (lHokkaido
Univ.; 2Hiroshima Univ.)(7) Role of de novo methylatransferases in X
chromosome inactivation
Takashi SADO, Masaki OKANO!, En LI 2 and Hiroyuki
SASAKI (!CDB, RIKEN; 2Harvard Med. Sch.)X chromosome inactivation was studied in ES cells
and embryos deficient for both Dnmt3a and Dnmt3b.
In double mutant male ES cells, Xist became ectopically
activated upon differentiation. It appeared, however,that these cells did not undergo X-inactivation despite
the association of Xist RNA with the X chromosome.
In contrast, such ectopic association of Xist RNA wasobserved only in a subset of nuclei in double mutant
embryos in both sexes. Interestingly, most of nuclei infemale embryos contained a single Xist domain despite
the lack of differential methylation at the promoter
region of Xist. Furthermore, one of the two X
chromosomes in female embryos replicated late in S
phase, suggesting that X-inactivation had properly
taken place in the absence of functional de novo
methyltransferases. These results suggest thatdifferential methylation is not necessarily theunderlying mechanism for monoallelic expression of

Xistand these de novomethyltransferases may not be
We studied whether chicken Z chromosome issubject to inactivation for gene dosage compensationin ZZ males. It was found that Z-linked genes arebiallelically expressed in ZZ males (ref. 7). The results
suggest that sex-chromosome inactivation of a
mammalian X-chromosome type does not occur in thechicken.

required for the initiation of X-inactivation andpropagation of the inactive state along thechromosome.

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

Xist and its antisense partner, Tsix, are the key

Hiroyuki SASAKI, Shin-ichi MIZUNO!, Tadafumi IINO!,
Hidetoshi OZAWA!, Teruhisa OTSUKA!, Kosuke
TASHIRO! and Takashi GOJOHBORI (lKyushu Univ.)
We carried out a collaborative research project todevelop a universal DNA chip system that can be used
to study expression of any gene in any organism. We
established the basic chip design and protocols for thisinnovative chip system. Development of the universal
DNA chip system for practical use is now underway.

(8) Effect of Tsix-disruption on Xist expression
Takashi SADO and Hiroyuki SASAKI

players in X inactivation, both of which encode a
non-coding RNA. Targeted disruption of Tsixcauses
ectopic expression of Xist in the extraembryonic tissues
upon maternal transmission, which subsequently
causes embryonic lethality due to inactivation of both
X chromosomes in females and a single X chromosome
in males. Tsix, therefore, plays a crucial role inmaintaining Xis t silencing in cis and in the regulationof imprinted X-inactivation in the extraembryonictissues. We examined the effect of Tsix-disruption on
Xist expression in the embryonic lineage using
embryonic stem (ES) cells as a model system. Upon
differentiation, Xistis ectopically activated in a subset
of nuclei of male ES cells harboring the Tsix-deficient
allele on the single X chromosome. Such ectopic

DIVISION OF HUMAN GENETICS

43

expression, however, ceased during prolonged culture.
We are tempted to speculate that surveillance by thecounting mechanism somehow shuts off .ectopic Xist
expression before that X chromosome undergoes
inactivation. Counting does not seem to be impairedin Tsix-deficient ES cells (ref. 8).
(9) Role of Dnmt3L in spennatogenesis and genomic
imprinting during oogenesis

3. Ishihara, K. and Sasaki, H.: An evolutionarily
conserved putative insulator element near the
3' boundary of the imprinted Igj2/Hl9 domain. Hum.
Mol. Genet. 11, 1627-1636,2002.
4. Kurosawa, K, Sasaki, H., Sato, Y., Yamanaka, M.,
Shimizu, M., Ito, Y., Okuyama, T, Matsuo, M.,
Imaizumi, K., Kuroki, Y. and Nishimura, G.: Paternal
UPD14 is responsible for a distinctive malformation
complex. Am. J Med. Genet. 110,268-272,2002.

Kenichiro RATA and En LI 1 (IHarvard Med. Sch.)

Dnmt3L (DNA cytosine-5-methyltransferase
3-Like) encodes a protein of 421 amino acid residues.
Dnmt3L harbors a putative zing finger domain thatshares a high degree of homology with the PHD-likedomain of DNA methyltransferases Dnmt3a andDnmt3b. The C-terminal part of Dnmt3L is related to
DNA cytosine-5-methyltransferase, but it does notpossess critical motifs for methyltransferase activity.
Dnmt3L mRNA was detected in undifferentiated ES
cells and testes by Northern blot analysis. We havegenerated Dnmt3L-deficient mice by gene targeting(ref. 6). While Dnmt3L- / - female mice grew normally, .
all embryos from pregnant Dnmt3L-/- mothers diedaround EI0.5. The maternally methylated imprinted
genes, e.g. Igj2r and Pegl, were hypomethylated inembryos derived from Dnmt3L - / - females x
Dnmt3L + / + males, but paternally methylated
imprinted genes were unaffected. The embryos derived
from a [Dnmt3a- / -, Dnmt3b+ / -] ovary showed
the same aberrant methylation of imprinted genes.
Also, Dnmt3L-/- male mice showed severe defects
in spermatogenesis, which is similar to, but severer
than, the phenotype displayed by Dnmt3a- / - mice.
We speculate that Dnmt3Lfunctions via interactions
with Dnmt3a and/or Dnmt3b to control DNA
methylation in developing germ cells (ref. 6).
Publications
1. Davies, K, Bowden, 1., Smith, P., Dean, W., Hill,
D., Furuumi, H., Sasaki, H., Cattanach, B. and Reik,

W.: Disruption of mesodermal enhancers for Igj2 in theminute mutant. Development 129, 1657-1666,2002.
2. Sotomaru, Y., Katsuzawa, Y., Hatada, I., Obata, Y.,
Sasaki, H. and Kono, T: Upregulated expression of theimprinted genes Hl9and Igj2rin mouse uniparental
fetuses. J BioI. Chem. 277, 12474-12478, 2002.

44

DIVISION OF HUMAN GENETICS

5. Shirohzu, H., Kubota, T, Kumazawa, A, Sado, T,
Chijiwa, T, Inagaki, K, Suetake, 1., Tajima, S., Wakui,
K, Miki, Y., Hayashi, M., Fukushima, Y. and Sasaki,
H.: Three novel DNMT3B mutations in Japanesepatients with ICF syndrome. Am. J Med Genet. 112,
31-37, 2002.
6. Hata, K, Okano, M., Lei, H. and Li, E.: Dnmt3L
cooperates with the Dnmt3 family of de novo DNA
methyltransferases to establish maternal imprints inmice. Development 129, 1983-93, 2002.
7. Kuroiwa, A, Yokomine, T., Sasaki, H., Tsudzuki,M., Tanaka, K., Namikawa, T and Matsuda, Y.:
Biallelic expression of Z-linked genes in male chickens.

Cytogenet. Genome Res. in press, 2003.
8. Sado, T., Li, E. and Sasaki, H.: Effect of
Tsix-disruption on Xist expression in male ES cells.

Cytogenet. Genome Res. in press, 2003.

E-b. DIVISION OF AGRICULTURAL GENETICS(1) Developmental abnormalities induced by DNA
hypomethylation mutation of Arabidopsis
Tetsuji KAKUTANI, Tetsu KINOSHITA, Asuka MIURA,
Yuki KINOSHITA and Masaomi KATO
Substantial part of large genome of flowering
plants and vertebrates are repetitive sequences, suchas transposable elements and their derivatives.
Genomic regions rich in repeated sequences tend to
be inactive in transcription and recombination andhave condensed chromatin (heterochromatin). In thoseregions, cytosine residues of genomic DNA are often
methylated at high frequency. Although function ofrepeated sequences are largely unknown, uncontrolled
activation of these sequences are presumed to bedeleterious to genome stability. On the other hand,some of repetitive sequences such as centromericrepeats and telomeres are regarded as important forthe chromosome function. Weare studying control andbiological function of repetitive sequences using DNA
methylation mutants of Arabidopsis.
In Arabidopsis, genome sequencing has been
completed for the first case in plants. In addition, manytrans mutations affecting epigenetic states have been
isolated in this plant. Arabidopsis ddml (decrease in
DNA methylation) mutation results in decrease inmethylation and transcriptional de-repression in
genomic repeat sequences. The DDMl gene encodesa protein similar to the chromatin-remodeling factor
SWI2/SNF2. The most striking feature of ddml
mutation is that it induces a variety of developmental
abnormalities by causing heritable change in other loci.
The molecular basis has been clarified in two of theloci directly causing the developmental abnormalities(see below).

methyltransferase but chromatin remodeling factor,
it has not been proved that loss of DNA methylation
is sufficient for the mobilization of CACTA elements.
In order to clarify this point, we examinedmobility of CACTA elements in mutants of DNA
methyltransferase genes. Arabidopsis MET1
(METHYLTRANSFERASE 1, ortholog of mammalian
DNA methyltransferase Dnmt1) is necessary for
maintaining genomic cytosine methylation at 5'-CG-3'
sites. Arabidopsis additionally methylates non-CG sites
using CHROMOMETHYLASE3 (CMT3). We examined
the mobility of endogenous CACTA transposons in
metl, cmt3, and cmt3-metl mutants. High frequency
transposition of CACTA elements was detected in
cmt3-metl double mutants. Single mutants in either
metl or cmt3were much less effective in mobilization,
despite significant induction of CACTA transcript
accumulation. These results lead us to conclude that
CG and non-CG methylation systems redundantly
function for immobilization of transposons (Kato et aI.,
2003). CMT3 gene and non-CG methylation in plants
may have evolved as an additional epigenetic tag
dedicated to transposon control.

(2) Charcterization of transposons activated by DNA

hypomethylation mutants(3) Inheritance of epigenetic developmental abnormality

Asuka MIURA, Masaomi KATO, Yuki KINOSHITA and
Tetsuji KAKUTANI

Tetsuji KAKUTANI, Asuka MIURA and Tetsu KINOSHITAOne of the ddml-induced developmental
abnormalities, clam, is characterized by lack ofelongation in leaves, roots and shoots. This phenotype
is heritable, but somatic sectors with normal phenotype
were occasionally observed. The size and frequencyof the sector differ from plant to plant. The phenotype
was stabilized in some of the progeny families; noreversion sector was observed in such family. Throughgenetic mapping, we identified the gene responsible
for the clam phenotype; it is the D WF4 gene, which
involved in synthesis of brassinolide, a plant growth
regulator necessary for cell elongation. The unstableclam phenotype was induced by insertion of a novel
endogenous Arabidopsis transposon, which we named
CACTAl. This transposon transposes and increases
in the copy number specifically in ddml mutant
background. These results suggest that gene silencing
associated with DNA methylation is important for
suppression of transposons.
As DDMl gene does not encode DNA

Another developmental abnormality, late flowering
trait, was induced by ectopic expression of FWA geneassociated with hypomethylation of tandem repeat
upstream of the coding region. Interesting thing is thatchange in nucleotide sequence was also not observedin fwa-l and fwa-2 alleles isolated by conventionalmutagenesis. In both cases, over-expression associatedwith the hypomethylation resulted in the phenotypes.
Combining ddml mutation and linkage analysis isuseful for identifying epigenetically regulated genes
important for plant development. The ddml-induced
late flowering trait as well as the hypomethylation andectopic expression of the FWA gene were stably
inherited over generations even in the absence of thepotentiating ddml mutation.
(4) Epigenetic Control of FWA Gene Expression in
Endosperm

Tetsu KINOSHITA, Asuka MIURA, Yuki KINOSHITA and
Tetsuji KAKUTANIDIVISION OF AGRICULTURAL GENETICS

45

Although FWA is ectopically expressed in theepigenetic alleles stated above, the role of FWA geneproduct in normal development has been completely
unknown. To understand why the FWA gene isepigenetically controlled, we further examined
expression of this gene in wild type. Results of GFP
reporter system and direct detection of the transcript
both suggest that FWA is expressed specifically in theendosperm. Endosperm is a plant tissue analogous to
mammalian placenta; it serves as nutritional support
to the embryo. Furthermore, the FWA gene wasexpressed in parent-of-origin-specific manner; only
maternal gene is expressed. We are currently dissecting
molecular mechanisms of this control in the endospermusing a series of Arabidopsis mutants affecting
epigenetic states in trans.

lysine 9 methylation and heterochromatin assembly inArabidopsis. EMBO Journal. 21, 6549-6559, 2002.

(5) DNA hypomethylation mutation in rice

A newly replicated DNA is assembled into
nucleosome soon after the passage of replication fork.
This rapid formation of nucleosome is functionallylinked to DNA replication machineries and plays acritical role for the maintenance of genome integrityand epigenetic states of chromatin in proliferating cells.
Two histone binding proteins, CAF-l (Chromatin
Assembly Factor-I) and ASFI (Anti-Silencing
Functionl), are found to be involved in some processof the nucleosome assembly.
We recently developed an in vitro reconstitution
system with human cell extract to analyze a mechanismof the DNA replication-dependent nucleosome
assembly. This nucleosome assembly reaction wasformerly analyzed in an ongoing replication reactionof SV40 in vitro system. This replication-coupledsystem has proven particularly useful in identifying
CAF-l. However, its use to identify other activities
involved in the replication-dependent nucleosome
assembly has been limited, because biochemical
fractionations of the human cellcytosolic extract (theS100 extract) used in the SV40 DNA replication (whichcontains all the necessary DNA replication factors,
histones, and other chromatin assembly factors) often
resulted in a severe loss of DNA replication activity.
In order to overcome these limitations, we successfully
used replicated DNA purified by a gel filtration spincolumn as a substrate for the nucleosome assembly
reaction. The newly replicated and separated DNA wascompetent for the replication-dependent nucleosome
assembly even after separation from free protein
fractions in the S100 extract because the DNA remains

Tetsuji KAKUTANI and Asuka MIURA
Among plant species, Arabidopsis genome has
extreme feature that it contains only small proportionof repeated sequences. We therefore extended our
research by examining effect of de-repression of
genomic repeated sequences in rice. Rice has more
repeat sequences than Arabidopsis. We found a rice
EST similar to Arabidopsis DDMl gene and generated
a transgenic rice lines expressing that EST in antisenseorientation. These transgenic lines shows reduced
genomic DNA in centromeric repeats, repeat encodingrRNA and retroelement-like sequence Tos3.
Supported by MAFF rice genome project, we
examined change in transcription pattern withmicroarray. Despite the global loss of genomic DNA
methylation, expression level did not change in the
mutant for most of the EST clones on the microarray .
We are currently examining transcript level ofendogenous rice transposons, most of which are notrepresented in the ESTs.
Publications
1. Kakutani, T.: Epi-alleles in plants: Inheritance of

epigenetic information over generations. Plant Cell
Physiol. 43, 1106-1111,2002.
2. Soppe, W., Jasencakova, Z., Hauben, A, Kakutani,T., Meister, A, Huang, M., Jacobsen, S., Schubert, 1.

and Fransz, P.: DNA methylation controls histone H3

46

DIVISION OF AGRICULTURAL GENETICS

3. Kato, M., Miura, A, Bender,]., Jacobsen, S.E. and
Kakutani, T.: Role of CG and non-CG methylation in
immobilization of transposons in Arabidopsis. Current
Biology 13, 421-426, 2003.
(1) Analysis of nucleosome assembly during DNA
replication
Tomohiro KUBOt, Akira SHIMIZU!, Bruce STILLMAN2
and Keiichi SHIBAHARA3 (1 Graduate School of
Biostudies, Kyoto University, 2Cold Spring Harbor
Laboratory, USA, 3Japan Science and Technology
Corporation)

marked, at least by PCNA (Shibahara and Stillman,
Cell 96, 575-585, 1999).
With this "two step system", we showed that
N-terminal of histones H3 and H4 were not necessarily
required for their association with CAF-1 andnucleosome assembly by CAF-1 (Shibahara et al., Proc.
Nat!. Acad. Sci. USA 97, 7766-7771, 2000). In addition,
we showed that unidentified activities in the S100
extract were required for the nucleosome assembly in
addition to CAF-1 and histones (unpublished data). We
are now on the way to identify additional activities
required for the DNA replication-dependent
nucleosome assembly.

ensures stable propagation of epigenetic states ofchromatin through facilitating nucleosome assembly
during DNA replication. Rapid re-formation of
nucleosomes onto newly replicated DNA by CAF-1
would prevent transcriptional regulators from being
targeted to the DNA in a non-regulated manner,
thereby preventing random changes in gene expression patterns in daughter cells. We also isolated asfl
mutants in Arabidopsis with similar phenotypes. We
are now analyzing those mutants and/or crossed
mutants from several angles to discuss the physiological implications of the DNA replication-dependent
nuclesome assembly more precisely.

(2) Physiological implications of CAF-I and ASF1 in
Arabidopsis

References
1. Kaya, H., Shibahara, K-i., Tasaka, K-I., Iwabuchi,

Hidetaka KAYA1,2, Takashi ARAKI3 and Keiichi
SHIBAHARA4 (lFaculty of Medicine, Kyoto University,2]apan Society for the Promotion of Science, 3Kyoto
University, 4]apan Science and Technology
Corporation)

M., Stillman, B. and Araki, T.: FASCIATA genes forchromatin assembly factor-1 in Arabidopsis maintainthe cellular organization of apical meristems. Cell. 104,131-142,2001.

E-c. DIVISION OF BRAIN FUNCTION
The genetic approach of Arabidopsis was apowerful tool to see physiological implications of
CAF-1. We had analyzed loss-of-function mutants of

caj-l (fasciata) in Arabidopsis. Those caj-l mutants
displayed severely disturbed cellular and functionalorganization of both shoot apical meristem (SAM) androot apical meristem (RAM).
They also showed a varied pattern of distorted
expression of both WUS and SCR, which play key rolesin the organization of SAM and RAM, respectively.
Asymmetrical cell division of a stem cell in RAM (Co/
En initial) gives rise to two different daughter cells (aCo/En initial and a Co/En daughter cell), which thendivides periclinally to generate a cortex and an
endodermal cell. SCR is expressed only in theendodermallineage. However, in some roots ofthe caj-lmutants, ectopic expression of SCR::GFPwas observedin single cells or small groups of cells adjacent to thecells normally expressing SCR::GFP On the other
hand, the absence of expression was observed in single
cells and small groups of adjacent cells including all
types of cells that normally express SCR::GFP The
disturbances of SCR-GFP expression were varied fromroots to roots and from plants to plants.
We proposed several models to explain thoseobservations, and the most plausible was that CAF-1(1) Mosaic development of the olfactory cortex with
Pax6-dependent and -independent components
TatsumiHIRATA, TadashiNoMURA\ YoshikoTAKAGI,
Yasufumi SATO, Naomi TOMIOKA 2, Hajime FUJISAWA2
and Noriko OSUMI 1 (lDepartment of Developmental
Neurobiology, Tohoku University Graduate School of
Medicine, 2Division of Biological Science, Nagoya
University Graduate School of Science)
One of the early events in the development of thecentral nervous system is regionalization of the neuraltube into organized domains. The telencephalicprimordium is divided into two major domains: thepallium and subpallium. In each domain, various genes
are expressed in a specific manner and assign different
developmental potentials to neural progenitors.
In the present study, we examined the developmentof the olfactory cortex, using the newly developed
monoclonal antibody (mAb) 9-4c. The olfactory cortex
is the target area of olfactory bulb axons andsuggested to be derived from neural progenitors ofvarious ventricular domains during development. The
novel regional marker, mAb 9-4c, labeled neuralprogenitors at the pallio-subpallial boundary (PSB) and

DIVISION OF AGRICULTURAL GENETICS

47

their putative descendants in the deep layers of theolfactory cortex. A search for the antigen recognized
by mAb 9-4c identified reticulon I(Rtn 1), an endoplasmic reticulum (ER) protein. In the mutant embryos of
P AX6, a pallium specific transcription factor,
expression of this antigen was completely absent at
the PSB, and the number of immunopositive cells in
the olfactory cortex was markedly reduced.
Nevertheless, the guidepost neurons of olfactory bulb
axons, lot cells, developed relatively normally in the
superficial layer of the olfactory cortex in the mutant
embryos. These guidepost neurons are shown tooriginate in the pallium and eventually guide the initialprojection of olfactory bulb axons. Subsequent
projection of olfactory bulb axons in P AX6 mutant
embryos also supported the mosaic nature of theolfactory cortex development; the axons initiallyprojected via the normal pathway and formed the LOT
bundle, but later failed to develop collateral projections
over the wide olfactory cortical areas.
(2) Expression of Nogo protein by growing axons in thedeveloping nervous system

growth of central axons. Our present finding, however,suggests another function of Nogo protein in thedeveloping nervous system.
Publications
1. Hirata, T., Nomura, T., Takagi,Y., Sato, Y.,
Tomioka, N., Fujisawa, H. and Osumi, N.: Mosaic
development of the olfactory cortex with
Pax6-dependent and -independent components. Dev.
Brain Res. 136, 17-26, 2002.

2. Tozaki, H., Kawasaki, T., Takagi, Y. and Hirata,
T.: Expression of Nogo protein by growing axons in
the developing nervous system. Mol. Brain Res. 104,111-119,2002.

E-d. DIVISION OF APPLIED GENETICS(1) The slender rice mutant, with constitutively
activated gibberellin signal transduction, has enhanced
capacity for abscisic acid level

Hirokazu TOZAKI, Takahiko KAWASAKI, Yoshiko
TAKAGI and Tatsumi HIRATA

Akira IKEDA!, Yutaka SONODA!, Paolo VERNIERI 2,
Pierdomenico PERATA3, Hirohiko HIROCHIKA4and ]unji
YAMAGUCHI! (!Hokkaido University, 2National Institute

Development of the nervous system ischaracterized by the striking capacity for axonalof Agrobiological Resources, 3University of Pisa,
University of Modena and Reggio Emilia, 4National
Institute of Agrobiological Sciences).

outgrowth. The axons, however, lose the capacity withmaturation of the central nervous system. Differencesin the growing capabilities of embryonic and adultaxons can be partly attributed to the growth-inhibitory
environment of the adult nervous system. Previous
studies in vivo and in vitro suggested the role of thecentral myelin in preventing axonal growth in the adultnervous system.
In the present study, we isolated a monoclonal
antibodies (mAb) designated as NGI that strongly
bound to growing axons in the developing nervous
system. The growth cones in cultured neurons wereparticularly strongly labeled with this mAb. Althoughthese immunostaining patterns suggested theassociation of the antigen recognized by mAb NG1with growing axons, a search for this antigen led toan unexpected finding; mAb NGI recognizes Nogo, anaxonal repulsive factor in the myelin. Previous studies
show that Nogo is produced by oligodendrocytes in
the adult nervous system and acts to prevent the

The slender rice (slrl-l) mutant, carrying a lethal
and recessive single mutation, has a constitutive
gibberellin (GA)-response phenotype and behaves as
if it were saturated with GAs [Ikeda et al. (2001) Plant
Cell 13, 999J. The SLRl gene, with sequence homology
to members of the plant-specific GRAS gene family,
is a mediator of the GA signal transduction process.
In. the slender rice, GA-inducible alpha-amylase wasproduced from the aleurone layer without applying GA.
GA-independent alpha-amylase production in the
mutant was inhibited by applying abscisic acid (ABA).
Shoot elongation in the mutant was also suppressedby ABA, indicating that the slender rice responds
normally to ABA. Interestingly, shoot ABA content
was IO-fold higher in the mutant than in the wild type,while there was no difference in root ABA content.
Expression of the Rabl6A gene, which is known to be
ABA inducible, was about lO-fold higher in shoots ofthe mutant than in those of the wild type. These results

48

DIVISION OF BRAIN FUNCTION

indicate that constitutive activation of the GA signaltransduction pathway by the slrl-l mutation promotes
the endogenous ABA level.
(2) Isolation and characterization of a rice dwarf mutant
with a defect in brassinosteroid biosynthesis
Masaki MORI l, Takahito NOMURA2, Hisako OOKA3,
Masumi ISHIZAKA4, Takao YOKOTA2, Kazuhiko
SUGIMOTO l, Ken OKABE!, Hideyuki KAJIWARA l, Kouji
SATOH!, Koji YAMAMOT0 3, Hirohiko HIROCHIKA l andShoshi KIKUCHI l rNational Institute of Agrobiological
Sciences, 2Teikyo University, 3Utsunomiya, Nagaoka
University of Technology, 4National Institute forAgro-Environmental Sciences)
We have isolated a new recessive dwarf mutantof rice (Oryz'a sativa 1. cv Nipponbare). Under normal
growth conditions, the mutant has very short leaf
sheaths; has short, curled, and frizzled leaf blades; has
few tillers; and is sterile. Longitudinal sections of theleaf sheaths revealed that the cell length along thelongitudinal axis is reduced, which explains the shortleaf sheaths. Transverse sections of the leaf blades
revealed enlargement of the motor cells along thedorsal-ventral axis, which explains the curled andfrizzled leaf blades. In addition, the number of crownroots was smaller and the growth of branch roots was
weaker than those in the wild-type plant. Because exogenously supplied brassinolide considerably restored
the normal phenotypes, we designated the mutant
brassinosteroid-dependent 1 (brdl). Further, under
darkness, brdl showed constitutive photomorphogenesis. Quantitative analyses of endogenous
sterols and brassinosteroids (BRs) indicated that
BR-6-oxidase, a BR biosynthesis enzyme, would be
defective. In fact, a O.2-kb deletion was detected in the
genomic region of OsBR6ox (a rice BR-6-oxidase gene)in the brdl mutant. These results indicate that BRs are
involved in many morphological and physiologicalprocesses in rice, including the elongation andunrolling of leaves, development of tillers,skotomorphogenesis, root differentiation, andreproductive growth, and that the defect ofBR-6-oxidase caused the brdl phenotype.

(3) Organ-specific alternative transcripts of KNOX
family class 2 homeobox genes of rice
Yukihiro ITO!, Hirohiko HIROCHIKA2and Nori KURATA l(lNationallnstitute of Genetics, 2National Institute ofAgrobiological Sciences)
We identified three genes (HOS58, HOS59 and
HOS66) of rice (Oryza sativa), which encode predicted
proteins with the KNOX family class 2 homeodomain.
These proteins contain three conserved domains, the
KNOX domain, ELK domain and homeodomain, froman N-terminus to a C-terminus. In addition to similarity
of predicted amino acid sequences, these genes showeda similar exon/intron structure. cDNA cloning andreverse transcription-polymerase chain reaction
analyses of these genes indicated tissue-specificexpression of alternative transcripts. The expressionof the longer mRNAs of HOS58 (HOS58L) and HOS59L
was detected in all organs examined such as roots, leaf
blades, leaf sheaths, flowers and calli, whereas theshorter mRNAs of HOS58 (HOS58S) and HOS59S wereexpressed in leaf blades, leaf sheaths and flowers. The
expression of HOS66L was detected in roots, leaf
blades, leaf sheaths and flowers, whereas theexpression of HOS66S was detected in roots andflowers. The alternative transcripts of HOS66 arose byuse of alternative transcription start sites. The longer
transcripts contained an exon 1which encodes analanine/glycine-rich region, whereas the shorter ones
lacked it. These results suggest that theexpression of
the alternative transcripts is organ-specific, and theirproducts have different degrees of abilities for
activation or repression of transcription.
(4) An active DNA transposon family in rice
Ning JIANG!, Zhirong BA02, Xiaoyu ZHANG!, Hirohiko
HIROCHIKA3 , Sean R. EDDy 2, Susan R. MCCOUCH4and Susan R. WESSLER l (lUniversity of Georgia,
2Washington University School of Medicine, 3National
Institute of Agrobiological Resources, 4Cornell
University)
The publication of draft sequences for the two
subspecies of Oryza sativa (rice), japonica (cv.
Nipponbare) and indica (cv. 93-11), provides a unique
opportunity to study the dynamics of transposable
elements in this important crop plant. Here we report
the use of these sequences in a computational approach

DIVISION OF APPLIED GENETICS49

to identify the first active DNA transposons from riceand the first active mini-ature inverted-repeat
transposable element (MITE) from any organism. A
sequence classified as a Tourist-like MITE of 430 basepairs, called miniature Ping (mPing), was present in
about 70 copies in Nipponbare and in about 14 copies
in 93-11. These mPingelements, which are all nearly
identical, transpose actively in an indica cell-culture
line. Database searches identified a family of relatedtransposase-encoding elements (called Pong), which
also transpose actively in the same cells. Virtually all
new insertions of mPingand Pong elements were into
low-copy regions of the rice genome. Since thedomestication of rice mPing MITEs have been
amplified preferentially in cultivars adapted toenvironmental extremes-a situation that is reminiscentof the genomic shock theory for transposon activation.
Publications
1. Ikeda, A., Sonoda, Y., Vernieri, P., Perata, P.,

Hirochika, H. and Yamaguchi, J.: The slender ricemutant, with constitutively activated gibberellin signaltransduction, has enhanced capacity for abscisic acidlevel. Plant Cell Physiol. 43, 974-979. 2002.
2. Mori, M., Nomura, T., Ooka, H., Ishizaka, M.,
Yokota, T., Sugimoto, K., Okabe, K., Kajiwara, H.,
Satoh, K., Yamamoto, K., Hirochika, H. and Kikuchi,
S.: Isolation and characterization of a rice dwarf mutant
with a defect in brassinosteroid biosynthesis. Plant
Physiol. 130, 1152-1161, 2002.

Only the morphologically normal X chromosome
is inactivated in female mice heterozygous for Searle's
X-autosome translocation, T(X;16)16H. Here we
performed a visual study of the primary and secondary
events that culminate in the completely non-random
inactivation in female embryos having thistranslocation. The data we have obtained so far
indicate that the initial choice of the future inactive X
chromosome is biased: the degree of skewing issomewhere between 70:30% and 90:10% in favor of themorphologically normal X chromosome. The majorityof genetically unbalanced cells that inactivate atranslocated X chromosome are quickly elimin.atedfrom the embryo proper by E8.5, although the survivalof such cells is sporadically observed thereafter. The
initial non-random choice demonstrated in this study
supports the contention that the T(X;16)16H
translocation disrupts one of the loci involved in the
randomness of the choice of the future inactive X
chromosome. Although the HMG-lacZtransgene in the
H253 stock mice is an excellent marker of
X-inactivation, the present study suggests that it isinfrequently de-repressed on the inactive X
chromosome.
(2) Mapping of the breakpoint in the mouse T(X;16)16H
translocation that causes non-random X chromosome
inactivation.

Mitsuteru ITO and Nobuo TAKAGI

Embryos Carrying Searle's T(X;16)16H Translocation
Visualized using X-linked lacZ and GFP Transgenes

On the assumption that there is a novel element
involved in the random choice of the future inactive
X in the area near the T16H translocation, we
attempted to map and clone the translocation
breakpoint. Previous studies showed that the T16H
breakpoint is located in a 4 cM region spanning fromG6pdx to Pola about 8 cM proximal to the
X-chromosome inactivation center. 'Using available
maps such as the radiation hybrid map, genetic map
and physical map of YACs and BACs, we made adetailed physical map near the translocation
breakpoint. FISH assay with BAC clones as probes

Nobuo TAKAGI, Michihiko SUGIMOTO, Shinpei
YAMAGUCHI, Mitsuteru ITO, Seong-Seng TAN l and
Masaru OKABE 2 (lHoward Florey Inst., Univ.
Melbourne, and 2Genome Inform. Res. CTR, Osaka
Univ.)

showed that the breakpoint is on the region that isroughly estimated about 900 kb. This region is
physically continuous with seven BAC clones and one
assembled contig. However, the analysis was hinderedby the discouraging fact that six different BAC clones
gave rise to two FISH signals one on 16x and the other

3. Ito, Y., Hirochika, H. and Kurata, N.: Organspecific alternative transcripts of KNOX family class
2 homeobox genes of rice. Gene 17, 41-47, 2002.
4. Jiang, N., Bao, Z., Zhang, X., Hirochika, H., Eddy,
SR.,McCouch, SR. and Wessler, SR.: An active DNA
transposon family in rice. Nature 421,163-167,2003.
(1) Non-random X Chromosome Inactivation in Mouse

50

DIVISION OF APPLIED GENETICS

on X16 chromosome. Further sequence analysis showed
a possibility that the X chromosome was broken
somewhere in the BACRP23-279G2 that is less than
250kb in length.
(3) New XX ES cell lines carrying GFP and IaeZ
transgenes on the maternal or the paternal X
chromosome
Shirin FARIVAR, Seong-Seng TAN!, Masaru OKABE 2
and Nobuo TAKAGI (1Howard Florey Inst., Univ.
Melbourne, and 2Genome Inform. Res. CTR, Osaka
Univ.)
Three new ES cell lines carrying the GFP and
HMG-lacZtransgenes on the same X chromosome were
established. The first ES cell line (MC58) was derived
from an XX blastocyst obtained from a GL female
doubly homozygous for GFPand HMG-lacZtransgenes
mated with a normal male (129/Sv). Two transgenes
were, therefore, carried by the maternally derived
X-chromosome. The second and third lines (GLl andGL2) were established from blastocysts obtained from
129/Sv females mated with GL male mice. In these celllines, the two transgenes were carried by the paternallyinherited X-chromosome. Differentiation was inducedby culturing MC58 cells in the absence of LIF in thebacteriological dishes to which ES cells do not adhere.
On day 3 of differentiation, half the embryoid bodies
were transferred to tissue culture dishes to the bottomof which embryoid bodies adhere and spread toenhance differentiation further. GFP-negative cells
appeared first in the outer endoderm layer 4 days after
the initiation of differentiation suggesting theoccurrence of X-inactivation. A single late replicating
X chromosome was identified in a low frequency of
metaphase cells, but {3 -galactosidase negative cell was
not observed at this stage. Loss of {3 -gal activity
occurred about two days after the first occurrence ofdifferential GFP fluorescence. Visual examination of
X-inactivation thus becomes possible in differentiating
ES cells may reveal here-to-fore hidden aspects ofrandom and nonrandom X-inactivation.
(4) A developmental biological study on the t-complex
recessive lethal mutation tclw5 in the mouse2,

Michihiko SUGIMOTO, Kuniya ABE!, Seong-Seng TAN
Masaru OKABE 3 and Nobuo TAKAGI (1Riken BRC,

2Howard Florey Inst., Univ. Melbourne, and 3Genome
Inform. Res. CTR, Osaka Univ.)
The mouse t-complex is a naturally occurring
variant of the proximal region of chromosome 17 withfour independent inversions. The t-haplotypes usuallycontain a recessive lethal mutation. In spite ofremarkable progress in genetics from the first
discovery of t-haplotypes in 1932, the mechanism of thelethality of any t-haplotypes is not yet understood.
tclwS is a t-complex recessive lethal mutation in the
twS-haplotype. Since twS/ twS embryos die soon after
implantation, tclwS gene is thought to play animportant role in embryogenesis. To understand thefunction of tclwS gene product,we tried to determine
the primary site of tclwS gene action. Histologicalexamination demonstrated that homozygotes of twS
died at the gastrulation stage due to extensive deathof the embryonic ectoderm cells while theextraembryonic ectoderm and the visceral endoderm
were less affected in these embryos. Histologically, twS/
twS embryos were first distinguished from normallygrowing littermates at E5.5. At this stage, the visceralendoderm cells of twS/twS embryos were characterizedby vacuoles smaller in number and size than normal
littermates. By aggregation with diploid wild-type
embryos, twS/ twS cells were capable of contributing
to all three germ layers. Thus, it is likely that tclwS is
not cell autonomous lethal. Furthermore, the hypothesisthat tissues other than the embryonic ectoderm are theprimary sites of tclwS action was supported by the
rescue of twS/ twS embryos by aggregation withtetraploid embryos. Hence, it is probable that the
product of tclwS is essential for the function of
extraembryonic tissues. However, embryos thus
rescued were arrested at E9.0 with the severely
underdeveloped posterior region, indicating that tclwS
is also necessary for development at midgestation.
Publications
1. Mizuno, H., Okamoto, 1. and Takagi, N.:

Developmental abnormalities in mouse embryos
tetrasomic for chromosome 11: Apparent similarity toembryos functionally disomic for the X chromosome.
Genes and Genetic Systems, 77, 269-276, 2002.
2. Goto, Y., Matsui, J. and Takagi, N.: Developmental potential of mouse tetraploid cells in diploid
tetraploid chimeric embryos. International Journal of

DIVISION OF APPLIED GENETICS

51

Developmental Biology, 46, 741-745, 2002.

3. Takagi, N., Sugimoto, M., Yamaguchi,S., Ito, M.,
Tan, 5.5. and Okabe, M.: Non-random X chromosome
inactivation in mouse embryos carrying Searle'sT(X,16)l6H translocation visualized using X-linked lacZand GFPtransgenes. Cytogenetic and Genome Research,
in press.

52

DIVISION OF APPLIED GENETICS

embryos. Thus, in these mutants, the A-P axis
formation of limbs is disrupted from the initial stage

F. GENETIC STRAINSRESEARCH CENTER

F-8. MAMMALIAN GENETICS LABORATORY(1) Developmental Genetics of Mouse Preaxial
Polydacylous Mutations
Yukari YADA!, Tomoko SAGAI, Sadao ISHIWA I andToshihiko SHIROISHI (IOchanomizu University)of limb development. Therefore, we examined theexpression of dHAND, CIi3 and AIx4 in the Ix and

Rim4 embryos. The result revealed that theirexpression was normal in Rim4 embryos, but shifted
anteriorly in the Ix limb buds, indicating that Ix geneacts in upstream of dHAND, Cft3 and Alx4 genes, while
Rim4 gene acts in downstream of them. Thus, it is most
likely that Ix controls the initial A-P axis formation
through the Shh independent signal, and that basedon the spatiotemporal expression patterns, the Rim4
gene is involved in positioning of Shh induction inposterior margin of limb buds (For detail, see ref.1).
(2) Phylogenie conservation of a cis-acting regulator
that controls polarized expression of Sonic hedgehog(Shh) in limb buds

The anteroposterior (A-P) axis patterning ofdeveloping limbs is controlled by formation of the zoneof polarizing activity (ZPA), located at the posterior

Tomoko SAGAI, Hiroshi MASUYA!, Masaru TAMURA,
Kunihiko SHIMIZU2, Yukari YADA3, Shigeharu

margin of limb buds. Sonic hedgehog (Shh) is

W AKANA!, Yoichi GONDa!, Tetsuo NODAl andToshihiko SHIROISHI (1RIKEN GSC, 2Nihon University,
30chanomizu University)

expressed at the posterior margin of limb bud, and isthought to mediate the ZPA activity. Recently, it wasreported that the A-P axis formation of limbs is notaffected by Shh null mutation in the early stage of limb

Polarized expression of the Sonic hedgehog (Shh)development. Thus, the initial A-P axis of limb is likely
determined by Shh-independent signaling pathway.

gene in the posterior mesenchyme is common fromteleost fin to tetrapod limb buds, and essential for limb

To uncover the genetic network that controls thepattern formation. But the molecular mechanism of its

early phase of the A-P axis formation of limbs, we
carried out molecular characterization of the preaxial

regulation is poorly understood.
We have performed the positional cloning of two

polydacylous

X-linkedpreaxial polydactyl mutations, Hx and M100081,

polydactyly (Xpl) and Recombination induced

localized in the vicinity of Shh gene on chromosome

mutations,

luxate([x),

mutation -4 (Rim4), which were affected in the A-P axis

5. M100081 is a novel mutant generated from the ENU

formation. Since causative genes for these mutants
have not been identified, we examined the expression

mutagenesis in RIKEN GSC. The both mutants arecharacterized with the mirror-image digit duplication

patterns of genes known to be involved in the A-P axis

coupled with an ectopic expression of Shh in theformation of limbs with the mutant embryos. In XpIanterior limb margin. In the 84kb critical region of Hx,embryos, though the expression of Shh was normal ina candidate gene, Lmbrl, was identified. However, any

the posterior of limb buds at the initial stage, thealteration was not found in the Lmbrl coding sequence

ectopic expression of Shh was detected in the anteriorof Hx, and the expression level of the Lmbrl wasside of the hindlimb buds. No ectopic expression of thesimilar between Hx and the wild type embryos. We

other genes was detected before the ectopic Shhidentified a sequence conserved between human andexpression. Thus, XpI gene likely acts in the down-mouse in intron 5 of Lmbrl. It resides approximately

stream of the Shh signaling, and mediates the

IMbp from the Shh coding region in the mouse

Shh-dependent signaling during limb morphogenesis.
On the other hand, in Ix and Rim4, the Shh expression
domain in the posterior side of the limb buds wasshifted anteriorly compared with that in wild-type

genome. Moreover, the sequence is highly conserved
throughout the tetrapod lineage. The sequences of
500bp fragment from mammalian and reptile species
and the sequence of a 300bp fragment from amphibian

GENETIC STRAINS RESEARCH CENTER

53

species were highly conserved with the average
matching rate exceeding 80% among species.
Sequence analysis of the two mutations, Hx and
M100081, detected single base-substitution located in
the conserved sequence. In order to clarify whether thesequence is a cis-acting regulator, we carried out acis-trans test for Hx mutation using Shh knockout (KO)allele. As a result, in spite of the dominant phenotypeof the Hx mutation, mice heterozygous for therecombinant chromosome harboring the Hxmutationand Shh KO allele on the same chromosome did notshow the polydactylous phenotype. This result revealed
that Hx mutation is attributed to an alteration of thecis-acting regulator of Shh.
In some vertevrate species, limb loss is often
accompanied with trunk elongation and loss ofregional differentiation in the axial skeleton. It has
been reported that in pythons, a group evolved fromthe tetrapod lizards, and that forelimbs are absent, but
hindlimb buds are formed during embryogenesis. It
is also known that the Shh expression is not detectedin python hindlimb buds, and there is no polarizing
activity. If a function of the conserved sequence inintron 5 of Lmbrl is crucial for the polarized expressionof Shh in the posterior mesenchyme of limb buds, thissequence may be lost in limbless species. We therefore
examined whether several limbless snake species anda limbless newt retain the conserved sequence.
Southern blot analysis using probes amplified for
lizard and newt did not yielded any signal for thelimbless species. Thus, it appears that loss of theconserved sequence is coupled with the loss of Shhsignaling pathway, suggesting the conserved sequence
has played a key role in tetrapod evolution. Considering the ectopic expression in Hx mutant and thehighly conserved sequence in intron 5 of Lmbrl, thewild-type cis-element is likely to be involved inpolarized Shh expression by repressing the Shh
expression in the anterior mesenchyme of developing
limb buds.
(3) Identification and characterization of the responsible
gene of mouse skin mutations, Recombination-induced
mutation 3 (Rim3) and Rex denuded (Re den )Masaru TAMURA, Shigekazu TANAKA, Tomoaki FUJII,
Aya AOKI 1 and Toshihiko SHIROISHI. (1Department of
Neurosurgery, luntendo University, Izunagaoka
Hospital)

54

MAMMALIAN GENETICS LABORATORY

Epidermis is composed of multiple cell layers, theoutermost being the body surface. Only innermost,basal layer, contains living and multiplying cells. Whena basal cell ceases to divide and begins its journey tothe skin surface, it embarks on a program of terminal
differentiation. However, the molecular mechanism bywhich epidermal stem cells differentiate is largely
unknown.
Currently, we are interested in mechanisms of
epidermal cell growth, differentiation and hairdevelopment. To study these subjects, we are using
several mutant mice, such as Recombination-induced
mutation 3 (Rim3) and Rex denuded (RIflen ). Rim3 andRet,n arose spontaneously in the congenic strain
BlO.BR (R228) and Bl0.129, respectively. Rim3 exhibits
epidermal hyperplasia, hyperkeratosis and abnormal
hair development resembling those of RIflen . Linkage
analysis and phenotype characterization indicated that
Rim3 and Ret'" might be allelic. Rim3 was mapped tothe distal portion of mouse chromosome 11 between
Grb7 and DllMit14 (Sato et. al., 1998).
Recently, we isolated several genes in the criticalregion of Rim3. Sequence analysis of Rim3 and Rete"mutations revealed that one of these genes is thecausative gene for the two mutations. To examine
expression pattern of this gene, we carried out RT-PCR
and in situ hybridization analysis for several different
tissues of adult mouse. We found that this gene ismainly expressed in hair follicules. Histologicalanalysis of Rim3 mutant mice at 3 months of age
showed thatthe interfollicnler epidermis and permanent zone of the hair follicles are highly hyperplastic
and hyperkeratinized. At this stage, the number of
BrdU-possitive cell nuclei of Rim3was six- to sevenfold
higher in the basal and the permanent zone of the hairfollicle cells than those of control wild-type mice,indicating a higher proliferation rate of those cells in
the Rim3 skin. Immunohistochemical analysis withseveral antibodies such as anti-Keratin 14, -keratin 10
and -keratin 6 demonstrated that the hair follicle
structure was converted into epidermis in Rim3, andthat differentiation of epidermal keratinocytes was
impaired in the mutant mice. These data suggested
that function of causative gene of Rim3is to regulate
cell growth and terminal differentiation of epidermis.

(4) Male-specific reproductive failure caused by Xchromosomal substitution between two mouse subspecies

functions for chromatin condensation in the sperm
head. Comparative sequence analysis of Halap-X
showed polymorphism of nucleotide sequence between

Ayako aKA, Nobuo TAKAGI!, Kiyotaka TOSHIMORI z,

C57BL!6] and MSM strains. It is most likely that mouse
X-chromosome has crucial genes that control

Toshiyuki TAKAN0 3, Akihiko MITA, Yoichi MIZUSHINA,
Noriko SAKURAI, Hiromi YAMAMOTO and Toshihikospermatogenesis through epistatic interaction withother genes on autosome(s) and lor the Y-chromosome.

SHIROISHI (IRes. Cent. For Mol. Genet. and Grad. Schl.

The sterility of the B6.MSM-ChrX males is possiblyof Env. Earth Sci. Hokkaido Dniv., Department ofcaused by incompatibility of MSM allele(s) of X-linked

Anatomy and Developmental Biology(Gl) Grad. Schl.

gene(s) and C57BL!6] allele(s) of the second genets) inof Medicine, Chiba Dniv. 'Division of Population
Genetics, NIG)

autosome(s) and/or Y-chromosome. The disruption of
the interaction may occur during the process ofConsomic strain is defined as an inbred strain, inbackcrossing to produce the consomic strains, andresult in the sterility. The whole system uncovered inwhich a certain chromosome of a strain is replaced as

this study may represent reproductive isolation

a whole by the same chromosome of another strain,

between two mouse subspecies, Mus musculus

leaving the rest of the former strain's chromosomes

domesticus and Mus musculus molossinus.

intact. We have set out construction of a consomic
mouse strain, replacing X-chromosome of a standard

Publicationlaboratory strain, C57BL!6] (Mus musculus domesticus),by its homologue of a strain MSM (Mus musculus
molossinus). During generating the X-chromosomal

1. Yada, Y., Makino, S., Chigusa-Ishiwa, S. andShiraishi, T.: The mouse polydactylous mutation, luxate(lx), causes anterior shift of the anteroposterior borderconsomic strain, B6.MSM-ChrX, we found sterility onlyin the developing hindlimb bud. Int. J. Dev. BioI. 46,

in the males. When eggs/embryos were collected from975-982, 2002.

oviducts one day after the copulation with theB6.MSM-ChrX males, none of them developed to
two-ceIl-stage embryos. This suggests that the spermsof B6.MSM-ChrX are defective in the fertilization.
Alternatively, the embryos generated from the sperms

2. Furuse, T., Blizard, D. A., Moriwaki, K., Miura, Y.,
Yagasaki, K., Shiroishi, R. and Koide, T.: Genetic
diversity underlying capsaicin intake in the Mishima
battery of mouse strams. Brain Res. Bull. 57, 49-55, 2002.

of B6.MSM-ChrX are not able to develop beyond theanalysis revealed that the B6.MSM-ChrX males have

3. Chan, B. W. H., Chan, Kwok-S., Koide, T., Yeung,Sau-m., Leung, M. B. W., Copp, A. ]., Loeken, M. R.,

reduced testis weight with variable degree of degen-Shiroishi, T. and Shum, A. S. W.: Maternal Diabetes

eration in spermatogenesis and severe anomaly in the
sperm head.

Increases the Risk if Caudal Regression Casused by
Retinoic Acid. Diabetes, 51, 2811-2816, 2002.

first cleavage of the fertilized eggs. HistologicalIn order to map gene(s) responsible for the reduced
fecundity of B6.MSM-ChrX males, we carried out

4. Isobe, T., Yoshino, M., Mizuno, K-I., Lindahl, K.

quantitative trait loci (QTL) analysis with respect toF., Kiode, T., Gaudieri, S., Gojobori, T. and Shiroishi,

the reduced testis weight and the sperm anomaly. We

T.: Molecular characterization of the Pb recombination

mapped a major QTL, responsible for the reduced testis

hotspot in the mouse major histocompatibility complex

weight, to the distal region of the X-chromosome. The

class IT region. Genomics, 80, 229-235, 2002.

other QTLs affecting sperm anomaly was mapped tothree loci on the X chromosome. In the central region

5. Furuse, T., Takano-Shimizu, T., Moriwaki, K.,of the X-chromosome, where the highest likelihood

Shiroshi, T. and Koide, T.: QTL analyses ofratio was located, several candidate genes have been
mapped so far. One of them, Halap-X encoding

spontaneous activity by using mouse strains from
Mishima battery. Mammal. Genet. 13, 411-415, 2002.

haploid-specific alanin-rich acidic protein, is locatedto this region and expressed in the nucleiplasm ofspermatids during the spermiogenesis. It likely

MAMMALIAN GENETICS LABORATORY

55

F-b. MAMMALIAN DEVELOPMENTAL LABORATORY(1) ecular mechanism of somite segmentation
Yu TAKAHASHI (National Institute of Health Sciences)and Yumiko SAGA

Dlll-Notch signaling. Based on these findings, we
propose a new model, which is different from theprevious concept that rostro-caudal patterning, i.e.,
formation of half-a-somite stripe pattern of gene
expression, can be regarded as a result of stabilizationof oscillating expression in the posterior PSM.
(2) Functional analyses of hesrl, 2 and 3

The sOIhite is the first morphologically distinct
segmental unit formed in a vertebrate embryo and giverise to metameric structures such as vertebrae, ribs andskeletal muscles. Each somite is subdivided into
anterior (rostral) and posterior (caudal) compartmentsthat differ in their properties and gene expression. The
rostro-caudal polarity of a somite is established within
the PSM prior to segmentation. Our aim is tounderstand the mechanisms underlying generation ofa segmental pattern within the PSM. Previously, wecloned Mesp2, a gene encoding a novel bHLH
transcription factor, MesP2, which is expressed in the
rostral PSM. Mesp2-null mice exhibited defective
somitogenesis due to a lack of rostral somitic
compartment. By the genetic analysis, we have shownthat MesP2 plays a critical role in establishment ofrostro-caudal polariy of a somite by regulating Dlll
expression via Notch signaling pathways.
One feature of the Notch signaling system insomitogenesis is that it utilizes several ligands. In
mouse embryos, at least two Notch ligands, DIll and
Dll3, are co-expressed in the PSM, and their expression
domains are finally segregated into the rostral or
caudal half of formed somites. Despite a large numberof studies, the functional significance of two distinct
Notch ligands in the PSM is not clear. Furthermore,
the role of Psen1, a protein involved in nuclear
translocation of the Notch intracellular domain, is not
fully understood. Therefore, we genetically analyzed
the role in rostro-caudal patterning triggered by Notch
signaling mediated by either Dlll or D1l3, therelationship between Notch signaling and Mesp2
function, and the involvement of Psen1 in Dlll- andD1l3-mediated Notch pathways. Our analyses revealed
several novel findings: 1) Dlll, D1l3 and Mesp2
constitute a complex signaling network for rostrocaudal patterning in the anterior PSM. 2) Mesp2
function in the rostro-caudal pattering downstream ofDlll or D1l3. 3) Psenl is differently involved inDIll-Notch and Dll3-Notch pathways. 4) Dll3-Notch
signaling counteracts with the Psen1-dependent

56

MAMMALIAN DEVELOPMENTAL LABORATORY

Hiroki KOKUBO and Yumiko SAGA
Notch signaling pathway has been implicated indevelopment of the cardiovascular system. hairy andenhancer of split genes are known to be direct targetsof Notch signaling pathway in Drosophila andvertebrates. We have cloned novel mouse hairy andE(spl) related subfamily genes, called hesr -1, -2, and -3.
These genes are expressed in the developing heart,
somites, limb bud and other tissues. To investigate thefunction of these genes, we have generated single
gene-knockout mice for hesr1, hesr20r hesr3. Amongthem hesr2-knockout mouse showed abnormalities inheart morphogenesis. hesr2mutant mice are high lethal
in early postnatal period with congenital heart defects,
featured by the enlarged hearts. We successfully
performed the transthoracic echocardiography in
5-day-old mice to reveal the heart dysfunction. A time
motion (M-) mode echocardiogram of the left ventricle(LV) indicated the dilated LV chamber with markedly
diminished in fractional shortening of the LV. The
Doppler echocardiogram revealed the atrio-ventricular(AV) valve regurgitation. Morphological andhistological analyses of homozygous hearts
demonstrated that the heart dysfunction was causedby septal defects in subaortic ventricle and secundum
atrium, and the regurgitation in the dysplastic andstenotic tricuspid valve. In addition, the heart rate inhomozygous mice significantly decreased. We havenoticed a drastic increase in the expression of s-myosin
heavy chain, which could lead to a change of the heartrate. These observations strongly indicate that hesr2
plays an important role in formation of functional AV
valves and thus, hesr2 gene null mice provide a usefulmodel system with which to elucidate genetic bases of
the heart dysfunction, a common disease in humaninfants.

(3) The lineage analysis of Mespl·expressing cells

Satoshi KITA]IMA (National Institute of Health Sciences)and Yumiko SAGA
MesPI and MesP2 are transcription factors
containing an almost identical bHLH motif. Cells lackboth genes are unable to contribute to heart formation.
A lineage study using Cre-Iox system revealed that
Mespl is the earliest molecular marker expressed inheart precursor cells. We have conducted a detaillineage a~alysis using Cre-reportor line, Rosa-26. The
heart is exclusively composed of cells derived from
Mespl-expressing mesoderm. However, we have foundsome cells are deprived of the {3 -gal staining, whichmeans that these are derived from Mesplnonexpressing cells. Among them, neural crest cells
have been shown to contribute to the conotrucus
septum prior to and during overt septation of theoutflow tract. In addition, we found several cell clusters
in between primitive ventricles and trabecularcomponent of both right and left ventricles, which
distribution is reasonably similar to the cells definedas the cardiac conduction system. To define whether
these cell are really contribute cardiac conduction
system, we compared distribution of Mesplnonexpressing cells with cells in the conduction
system using CCS-LacZ transgenic mouse, in which
only cells in conduction system are stained by {3 -gal.
The result strongly suggests that Mespl-nonexpressing
cells corresponds to cells in the conduction system. In
addition, we used Connexin-40 (Cx40) as a molecularmarker for the conduction system. The Cx40 signal and
Mespl-LacZ signal are segregated, which indicates that
the Mespl-nonexpressing cells are Cx4D-positive. Theseobservations strongly suggest that the origin of the
conduction system is different from other cardiac cells.
(4) Cloning and functional analysis of mouse nanos
genes

Masayuki TSUDA, Yumiko SASAOKA, Makoto KISO,
Seiki HARAGUCHI (Shiga university of Medicine) andproteins in germ cell development, we have isolated
three mouse nanos genes (There are designated as
nanosl, nanos2and nanos3). nanosl is maternally expressed in an unfertilized egg and the zygotic expression is observed in the neuronal cell lineage, especially in the adult hippocampus, but it was not expressedin developing germ cells. The knockout mouse
developed without any significant abnormalities.
Interestingly, the maternal expression was also
observed for nanos2, and the expression pattern wasquite similar to that of nanosl. Both nanos2 and nanos3
are expressed in male germ cells between embryonicdays 13.5 and 15.5, and nanos3 expression is also
observed in the bipotential gonad of 11.5 dpc and in
12.5-dpc female gonad. In addition, nanos3 is expressedin PGCs at as early as 9.5 dpc. The functions of thesegenes are examined by generating gene-knockout micefor nanos2and nanos3. A defect of nanos2-null mouse
was observed 'only in male spermatogenesis. The adulttestes showed reduced in size and weight and no germ
cell was detected. In contrast, female gonad normallydeveloped and the homozygous female was fertile,
indicating no maternal defect due to the lack of nanos2
expression in the oocyte. As expected, apoptotic cells
were observed in the testes after 15.5 dpc and theapoptosis continued up to 18. 5 dpc, indicating nanos
2 is required for the maintenance of germ cells in theembryonic testes.
Reflecting its expression pattern, we found defects
in both ovary and testes of nanos3-null mice. In adultmale and female mutants, no germ cell was observed.
In contrast to nanos2-null mice, germ cells were alreadymissing in the genital ridge, at as early as 12.5 dpc,
indicating that PGC failed to enter the genital ridge.
The' detailed mechanism leading to a loss of PGC is
now under investigation.
Publications
1. Nomura-Kitabayashi, A., Takahashi, Y, Kitajima,S., Inoue, T., Takeda, H. and Saga, Y: Hypomorphic
Mesp allele distinguishes establishment ofrostrocaudal polarity and segment border formation
in somitogenesis. Development, 129, 2473-2481, 2002.

Yumiko SAGA
A Drosophila gene nanosencodes a RNA-binding
zinc-finger protein that is shown to be involved in germ
cell development by suppressing somatic gene
expression. To know the involvement of mouse nanos

2. Ohara, 0., Nagase, T., Mitsui, G., Kohga, H.,
Kikuno, R., Hiraoka, S., Takahashi, Y., Kitajima, S.,
Saga, Y. and Koseki, H.: Characterization ofsize-fractionated cDNA libraries generated by the invitro recombination-assisted method. DNA Res. 9, 47-57,

MAMMALIAN DEVELOPMENTAL LABORATORY

57

2002.

KOBAYASHI 3, Toshihiko SHIROISHI2 and Tsuyoshi
KOIDE (1Tokyo University of Agriculture and

3. Katoh, c., Kitajima, S., Saga, Y., Kanno, ]., Horii,

Technology, 2Mammalian Genetics Laboratory, NIG,

1. and Inoue, T.: Assessment of quantitative dual-

3Fukushima Med. Univ.)

parameter flow cytometric analysis for the evaluationof testicular toxicity usingcyclophosphamide- andIt has been previously described that spontaneous

ethinylestradiol-treated rats. ]. Toxico!. Sci. 27, 87-96,

activities of mice are dependent on a light/dark rhythm.

2002.

Measuring spontaneous activity of mice revealed thatmost of mice are active during dark period, and thespontaneous activity is quite different among common

F-C. MOUSE GENOMICS RESOURCE LABORATORY

laboratory inbred strains and wild mice-derived inbred
strains. In these strains, mice of KJR strain were

(1) Quantitative trait analyses of sensitivities to heat

especially hyperactive in contrast to mice of BLG2

evoked pain and capsaicin in mouse strains.

which were hypoactive. The Fl females from the crossbetween KJR and BLG2 performed the same levels of

1
,

1
,

Tamio FURUSE Kazumi YAGASAKI Toshihiko
SHIROISHI 2 and Tsuyoshi KOIDE (lTokyo University ofAgriculture and Technology, 2Mammalian Genetics
Laboratory, NIG)
Pain sensation is an essential alert for avoiding
environmental danger and feeling tissue damage or

spontaneous activity as KJR mice, suggesting somedominance of the hyperactive phenotype. A genetic
linkage analysis using the progeny from a backcross
KJR to BLG2 revealed that two loci, Locol and Loco2,
are involved in spontaneous activity (2). We conductedphenotypic and molecular analyses to elucidate genetic
mechanism of this hyperactive phenotype.

illness. In spite of the essential role of pain sensation,

It has been proposed that hyperactivity is related

surplus pain is stressful or unpleasant for animals.
Heat evoked pain and capsaicin (8-methyl-N-vanillyl-

to dopamine and dopaminergic system. Therefore we

6-nonenamide) sensation activate the multi functional

products, DOPAC (dihydroxyphenylacetate) and HVA

pain receptor, VRl. Capsaicin is the main chemical(Homovanilate) in midbrain and striatum. In thedetermined level of Dopamine (DA) and DA degradation

component of hot chili peppers providing its hot taste

striatum of KJR, levels of DA, DOPAC and HVA wereand causes sensation in physiological pain system. In

significantly higher than that of BLG2. These data

the previous annual report, we described about genetic
diversity of sensitivity for capsaicin and pain in thesuggested that Locol and Loco2 might be involved instock of mouse strains, Mishima battery (1). In theelevated levels of DA, DOPAC, and HVA in striatum
of KJR. We also compared the localization andpresent study, we conducted QTL analyses toexpression levels of TH (tyrosine hydorogenase), DRDI

characterize genetic loci involved in the different(Dopamine receptor DIA), and DRD2 (Dopamine

sensitivity to capsaicin and heat evoked pain between

receptor D2) between KJR and BLG2. As a result,

two mouse strains, C57BL!6 and KJR. In the study, 470

expression levels of DRD2 in striatum and midbrain

F2 mice were generated from a cross of Fl miceof BLG2 were significantly higher than that of KJR.

obtained from a cross of C57BL!6 and KJR. We applied
fluid intake test of capsaicin solution and 52°C hot plate

This result suggested that D2DR is down regulated
according to high level of dopamine in stiratum of KJR.

test for the F2 progeny. In the mapping study, fouron chr2, chr7, chr8 and two significant QTLs for heat

Publication(1) Furuse, T., Blizard, D. A., Moriwaki, K., Miura, Y.,sensitivity were detected on chr8 and 18.

Yagasaki, K., Shiroishi, T. and Koide, T.: Genetic

significant QTLs for capsaicin sensitivity were detected

diversity underlying capsaicin intake in the Mishima(2) Analysis of Locol and Loco2 involved inbattery of mouse strains. Brain Research Bulletin 57,

spontaneous locomotor activity of wild-derived mouse

49-55, 2002.

strains(2)
Juzoh UMEMORI, Tamio FURUSE\ Hiromi SAN0 3, Kazuto

58

MAMMALIAN DEVELOPMENTAL LABORATORY

Furuse, T., Takano-Shimizu, T., Moriwaki, K.,

Shiroishi, T. and Koide, T.: QTL analyses ofspontaneous locomotor activity using mouse strainsfrom Mishima battery. Mammalian Genome 13,411-415,
2002.
(3) Chan, B. W. H., Chan, K.-S., Koide, T., Yeung,
S.-M., Leung, M. B. W., Copp, A. ]., Loeken, M. R.,
Shiroishi, T. and Shum, A. S. W.: Maternal Diabetes
Increases the Risk of Caudal Regression Caused by
Retinoic Acid. Diabetes 51, 2811-2816, 2002.
(4) Isobe, T., Yoshino, M., Mizuno, K., Fisher Lindahl,K., Koide, T., Gaudiery, S., Gojobori, T. and Shiroishi,
T.: Molecular characterization of the Pb recombination
hotspot in the mouse major histocompatibility complex
class I region. Genomics 80, 229-235, 2002.

F-d. PLANT GENETICS LABORATORY(1)

Genetic dissection of rice development

I-A. Regulation of expression of KNOX family class 1
homeobox genes of rice
Yukihiro ITO and Nori KURATA
To elucidate genetic programmes that control
embryogenesis and regeneration of rice, we conductedcloning and structural and functional analyses of genes
which encode transcription factors and protein kinases.
We previously identified five KNOX family class 1
homeobox genes and analysed their expression
patterns during early embryogenesis and regeneration
by RT-PCR and in situ hybridization. We also foundthat constitutive expression of these genes is sufficient
to maintain cells in a meristematic undifferentiatedstate. Since specific expression of KNOX family class
1 homeobox genes in a shoot meristem is essential fornormal development of plants, we started to study its
regulatory mechanisms.
We showed that the coding region of OSHl isinvolved in the regulation of its expression.
Introduction of OSHl cDNA without a promoter
sequence induced expression of both endogenous andintroduced OSHl in a leaf and resulted in altered leaf
morphology. This indicates that an extra copy of OSHl
induces ectopic expression of the endogenous OSHl
and the OSHl cDNA sequence has ability to induce
its own expression in leaves (in preparation). In

addition, we found that cytokinin which is known to
induce shoot regeneration from calli induces expressionof OSHl in calli and suspension culture cells of rice.
Mechanisms involved in the induction and repressionof OSHl expression and its relation to rice development
will be examined.
I-B. Structural and functional analysis of rice OsHAP3
genes
I-B-I, Identification and expression analysis of OsHAP3
genes
Nori KURATA, Kazum~ru MIYOSHI, Thirumurugan
THIRUVENGADAM and Yukihiro ITo
We identified 11 genes that encode subunits of
CAAT-box binding complex in rice by cDNA screening
and database search. We classified these genes into
two groups based on the deduced amino acidsequences, exon-intron structure and expression
patterns. The first group consists of three genes named
OsHAP3A, OsHAP3B and OsHAP3C, which are
expressed ubiquitously. The second group consists ofeight genes designated OsHAP3D to OsHAP3K, whose
expression is restricted in specific meristem-containing
organs at specific stages depending on genes. Detailanalysis of expression of these genes by in situ
hybridization and promoter-GUS fusion is underway.
I-B-2, Functional analysis of OsHAP3A
Yukihiro ITO, Kazumaru MIYOSHI and Nori KURATA
To examine roles of HAP3s on rice development,
we generated antisense and RNAi transgenic rice
plants of OsHAP3A, a member of the first group. The
transformants showed reduced expression of
OsHAP3A and had pale green leaves. This phenotype
was inherited to the next generation, albeit low
penetrance, and detail analysis was carried out withprogenies in the next generation.
We examined the chlorophyll content in the third
leaf blades of 2-week seedlings and showed that thechlorophyll content of the antisense plant was about
half of that of the vector-transformed plant.
Transmission electron microscope analysis showedthat chloroplasts of the antisense plant were greatly
affected. Development of tyrakoid membrane was
perturbed and no accumulation of starch grain was

MOUSE GENOMICS RESOURCE LABORATORY

59

observed. Quantitative RT-PCR analysis showed that
expression of nuclear-encoded photosynthesis genes
such as RBCSand cab was reduced, while expressionof chloroplast-encoded genes such as RBCL and
RPL23 was unaffected or rather up-regulated. We also
examined the expression level of OsHAP3B and
OsHAP3C, two genes highly related to OsHAP3A insequence. In the antisense plants, expression of
OsHAP3B and OsHAP3C were also reduced comparedwith the vector-transformed plant. These results
suggest that OsHAP3genes are involved in the normal
biogenesis of chloroplast through gene regulation(submitted). Considering that LECl, a member of
NF- YB/ HAP3 genes of Arabidopsis, is a criticalregulator of embryogenesis, plant NF- YB/HAP3 genes
may have diverse functions ranging from chloroplastbiogenesis to embryogenesis depending on genes.
I-B-3, Functional analysis of OsHAP3D and OsHAP3E
Thirumurugan THIRUVENGADAM, Yukihiro ITO,
Kazumaru MIYOSHI and Nori KURATA
We have been generating transgenic rice plants
in which the second group OsH4P3 genes such as
OsHAP3D, OsHAP3E, OsHAP3F, OsHAP3G and
OsHAP3H are overexpressed or suppressed byantisense or RNAi. Among them" overexpressing plantsof OsHAP3D and OsHAP3E, showing high similarity
with each other, showed abnormal phenotypes.
Transformed calli regenerated shoots with narrowleaves. After transfer to soil, the transformants were
dwarf and had small leaves. These results suggest that
OsNF- YB6is involved in norma.! development of rice,
and its overexpression somehow modified ricedevelopment and resulted in abnormal phenotypes.
I-C. Genetic analysis of sporogenesii": during sexual plant
reproduction using sterile mutants of rice
Ken-Ichi NONOMURA, Toshiyuki FUKUDA, Mutsuko
NAKANO, Mitsugu EIGUCHI, Akio MIYAO*, Hirohiko
HIROCHIKA * and Nori KURATA (* Natl. Inst. Agrobiol.
Sci.)
Genetic analyses for micro sporogenesis andmegasporogenesis is important to understand thesystem of plant meiosis and sexual reproduction. We
identified and characterized three genes of rice, MSPl,

60

PLANT GENETICS LABORATOFi:Y

PAIRl, and PAIR2, tagged by transposed Tosl7. The
MSPl (multiple sporocytes) gene encodes aleucin-rich-repeat receptor-like protein kinase. A
loss-of-function mutation in the MSPl gives rise toexcess number of both male and female sporocytes. In
the anther, development of inner wall layer isdisordered and then tapetum cells are completely
absent. The in situ expression of the MSPl suggeststhat MSPl products play important roles in preventing
the ectopic male and female sporogenesis during flower
development (submitted).
The PAIRl gene encodes a novel protein with aputative coiled-coil domain, and the PAIR2 gene is ahomologue of Arabidopsis ASYl. A loss-of-function
mutations in both genes showed failure in homologous
chromosome pairing. Furthermore, in the pairl mutant,second meiotic cell division is also defected, resulting
in formation of polyad spores instead of tetrad. The
phenotype of pairl mutant resembles to a mutation in
Arabidopsis SYNl, a gene encoding a cohesin protein.
These facts raise a possibility that the genes identified
in this study play important roles in meiotic
chromosome pairing as cooperating with the cohesin
complex.
I-D. Molecular and genetic analysis of PLASTOCHRON
genes, timekeepers for shoot development in rice
Heterochronic mutations affecting the timing ofdevelopmental events may be of major significance inonotogeny and evolution. In plants, several
heterochronic mutations that affect stepwise
development of vegetative tissue and therefore altershoot architecture have been identified. The recessive
mutations at the rice PLASTCHRONloci (PLAl and
PLA2) cause the short plastochron and ectopic
expression of vegetative programs in the reproductive
phase, indicating that the PLA genes play importantroles in temporal regulation of shoot development. To
clarify the mechanism operating in the time-controlling
process of shoot development, we have been analyzing
PLA genes.
I-D-I, Cloning and characterization of PLAI geneencoding a cytochrome P450 protein, CYP78All.
Byoung-Ohg AHN, Kazuman.l MIYOSHI, Yukihiro ITO,
Jun-Ichi ITOH, Yasuo NAGATO and Nori KURATA(Graduate School of Agricultural and Life Science,University of Tokyo)
In the previous work, we already identified strong
candidate gene for PLAI that encodes CYP78All, amember of cytochrome P450 proteins (submitted).
Some members of the plant P450 family are known
to be involved in important biochemical pathways
such as biosynthesis of phenylpropanoids, lipids,
glucosinolates, and plant growth regulators such as
gibberellins, jasmonic acid, and brassinosteroids. The
candidate P450 gene carried mutations in all pIal alleles(pIal-I, -2, -3 and plal-4). The predicted protein contains
a hydrophobic region in the N-terminus and putativeoxygen and heme-binding domains that arecharacteristic of microsomal cytochrome P450.
Complementation experiment that the wild-type P450
gene rescued the plalphenotype, confirmed that thegene was a real PLAl gene. Examination of temporaland spatial expression patterns of PLAl by in situ
hybridization revealed that PLAl is expressed
exclusively in young leaf but was not in shoot apicalmeristem (SAM). The expression pattern, potential
enzymatic activity of the protein and the mutant
phenotype of PLAI suggest that PLAI acts directly or
indirectly in the synthesis of inhibitory substances inleaf primordia. PLAl could be a molecular clue for the
inhibitory field theory, an oldest but the most widely
accepted model for leaf initiation process.

I-D-2, Chromosomal location of PLA2 gene
Kazumaru MIYOSHI, Yukihiro ITO, Taiji KAWAKATSU,
Jun-Ichi ITOH, Yasuo NAGATa and Nori KURATA(Graduate School of Agr. and Life Sci., University ofTokyo)The pla2 mutant shows similar but more severe
phenotype to that of pIal. The double mutant (plal-2/
pIaL!) took the phenotype of pla2, indicating that PLA2
is epistatic to PLAl. Thus, PLA2 gene would function
upstream of PLAI gene in shoot development.
Elucidation of functional relationship between thesegenes would help to clarify the mechanism for thetime-controlling process of shoot development. As thefirst step to isolate PLA2 gene, we mapped the PLA2
gene on an approximate chromosomal location. F2progeny analysis using 179 pla2 homozygous plants
revealed that PLA2 is located on the long arm ofchromosome 1 within the interval of 3.6 cM between

the marker L819 and S10562. Further fine mapping andcloning are in progress.
I-E. Isolation and sequence analysis of receptor-likeprotein kinase genes of rice
Yukihiro ITo and Nori KURATA
We started to isolate and characterize protein
kinase genes as candidates to transduce positional
information which is postulated to be important forplant development. We focused on a receptorlike protein kinase gene such as SOMA TIC
EMBRYOGENESIS RECEPTOR KINASE (SERK! ofcarrot and isolated its homologues from rice. We havecloned two genes and those cDNAs were named ORKl
and ORK6. The genome clone of ORKI showed
unusual structure and ORK6 encodes a receptorlike protein kinase with leucine-rich repe~ts in a
receptor domain. The amino acid sequence of ORK6
is highly similar to plant receptor-like protein kinases
such as SERK, CLAVAT Al and ERECTA of
Arabidopsis, which are known to function in a shootmeristem. The ORK6promoter fused to GUS directed
GUS activity in a root tip, a lamina joint and a callus.
Overexpression of ORK6or introduction of antisenseor RNAi constructs of ORK6 into rice showed no
phenotypic change under normal growth condition.
Analysis of regeneration is underway using calli
derived from these transgenic plants.
(2)

Generation and screening of enhancer trap lines ofrice
Yukihiro ITO, Kazumaru MIYOSHI, Thirumurugan
THIRUVENGADAM, Mitsugu EIGUCHI and Nori KURATA
To isolate valuable mutants defective in various
steps of rice development and to clone thecorresponding genes, we are generating enhancer
trap lines of rice. We employ an enhancer trap systemused in Arabidopsis with some modifications as
described in the previous reports in 2000 and 2001. Thissystem proved to transpose well the Ds-GUS elementby Ac-transposase in rice.
We continued screening of transposants from thefour Ds-GUS lines by the chlorsulfuron andhygromycin resistance. Out of 18,020 F2 plants screenedin two years, 3,711 were judged to be transposants. We

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61

also crossed with other 22 Ds-GUS lines with the
35S-AcTPase line, and screening of transposants was
carried out. Out of 6,590 F2 plants screened by PCR,
1,874 plants (28%) were judged to be transposants.
Using these transposants, we also determined thesuitable concentration of chlorsulfuron for screening
Screening of about 800 transposant lines forembryonic mutants, we identified two lines with anabnormal embryo size. One line produced seeds witha greater embryo. Seed size and differentiation ofembryonic organs seemed normal. The other line
produced seeds with a smaller embryo. The size ofembryos was greatly different from seed to seed, andin an extreme case a seed had no embryo. However,linkage analysis indicated that these genes are nottagged by Ds-GUS.
We have also been screening enhancer trap lines
using GUS gene as a reporter. A total of 60 lines whichshowed GUS expression in any organ are now
examined to detect flanking sequences of the trappedsites. GUS staining pattern and flanking sequence dataof these enhancer trap lines are being collected tohouse in the rice development database which we
collaboratively construct as a rice functional genomics
database.
(3) Analysis of nuclear and chromosomal factors thatmaintain genome function
3-A. Finding of various plant nuclear proteins using yeast
nuclear transportation trap system.
Kazuki MORIGUCHI and Nori KURATA
Nucleus is the most important intracellularorganelle, in which replication and gene expression are
regulated by accepting signals from environment as
the terminal place and responding them as the firstplace. Although the genome projects of Arabidopsis andOryza have been finished, the large-scale functional
analyses of these genes are just at its beginning on
their assignment of functions.
We have been trying a comprehensive analysis ofplant nuclear proteins by yeast nuclear transportation
trap (NTT) system using Oryza sativa cDNA libraries.
At present, about 520 gene species were obtained.
Based on homology search results, 33% of themencodes nuclear and nuclear/cytoplasmic proteins, but
43% and 24% of them are shown to encode functionally

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PLANT GENETICS LABORATORY

unknown and generally thought as non-nuclear
proteins, respectively. By observing the localization of
a subset of those fusion proteins with GFP, we
confirmed that nuclear localizing proteins were actuallyenriched not only in those unknown function proteinsgroup but also even in those non-nuclear proteinsgroup. These results showed that it is difficult topredict nuclear proteins only by in silico analysis basedon the previous data. We also analyzed expression
profiles of the isolated nuclear protein genes in several
organs. At present, 43 and 29 genes, which are
up-regulated at meiotic stage and early embryoenriched panicles, respectively, have been found by
microarray analysis. Relating to these genes, twoapproaches will be applied. One is the analysis of
nuclear-cytoplasmic shuttling proteins, which were
found during our NTT screening. We have been
generating transgenic rice strains introduced
GFP-tagged those genes. The other is the analysis of
the plant nuclear proteins network using yeast
two-hybrid system. We are mainly focusing on plant
nuclear matrix proteins, those content has beenthought to be quite different from those in mammals.
3-B. Construction and introduction of rice artificial
chromosomes
Tadzunu SUZUKI, Ken-Ichi NONOMURA and Nori
KURATA
Generation of transgenic rice plants which harbor
artificial chromosomes will be a useful tool not only
to elucidate factors necessary for maintaining
chromosome architecture and basal function but also
in introducing large genomic fragments into rice undercontrolled condition. To create rice artificial
chromosomes, the candidate DNA construction andapplication of several transformation methods on ricecells have been carried out. The candidate DNA
fragments of 40-100 kb derived from centromere
regions of rice were integrated into bacterial artificial
chromosomes (BAC). A YAC vector carrying 300 kb
chromosome 5 centromeric region, telomere repeats andselection markers had also constructed.
The transfer method of huge size DNAs into plant
cells have not been established. For introduction of theconstructed artificial chromosome vectors, two methodsof lipofection and particle bombardment system were
examined here. Trials of lipofection method using small

plasmids or BAC vectors and rice cultured cells,
showed it worked at least for transient expression of
the small plasmid. Particle delivery system was shown
more effective. Several lines of calli, which survived ona selection media, were obtained by particle
bombardment introduction of BAC vectors, although
the growth of these calli was slower than that of thecalli transformed with control vectors. RT-PCR
analysis showed that only one line expressed themarker gene while the others did not or less. Thismight suggest that the marker gene was suppressedby the centromeric repeats inserted in the vector, or
hopefully that the construct existed unstably as an
episomal state. Further examination wil be needed.
(4)

Genome-wide analysis of reproductive barriers andpositional cloning of a reproductive barrier
4-A. Interactions of reproductive barriers

Yoshiaki HARUSHIMA and Nori KURATA
Genetic mechanisms for isolation of 'species' arecalled as reproductive barriers and these include
hybrid incompatibility, hybrid inviability, hybrid
sterility, hybrid breakdown, etc. The distortions ofallele frequencies from Mendelian expectation inprogeny of inter- or intra- species hybrid due to hybrid
sterility genes, hybrid breakdown genes andgametophytic competition genes have been often
observed. We have studied reproductive barriers
between Japonica and Indica cultivars. We previously
mapped all reproductive barriers causing allele
frequency distortions from Mendelian expectation in
Nipponbare (Japonica) and Kasalath (Indica) F z
populations. These mapped reproductive barriers
would be resulted from interactions between alleles,
between segregating different loci within agametophyte or a zygote, between genome andcytoplasmic factors, and between parent genome andprogeny genome. To detect interactive loci within asegregating gametophyte or zygote in F 2 population,a simple x 2 test for independence of allele frequencies
for all pairs of 1055 DNA markers covering the entire
genome were performed. A high value of x 2 would be
expected for linked marker pairs and for marker pairs
in the vicinity of interactive reproductive barriers. The
contour plots of the x 2 results are symmetrical about
the diagonal and high x 2 values in that region are due

to linkage between markers. There were regions
showing high x 2 values other than in the diagonal
region. The x 2 values show peaks. Some DNA markers
of interaction peaks were also the closest markers to
the reproductive barriers identified by regression
analysis, other interactive loci did not induce apparent
segregation distortion. To verify relevance between thebarriers and the interaction peaks, favorable orunfavorable genotype combinations were investigated.
Several zygotic barriers might be resulted from theinteractions detected in this study; however there wereno relevance between the gametophytic barriers andthe interaction peaks. To investigate reproducibilityof the interaction peaks, we performed the independence test of the non-linked marker combinations withthe high X Z values by another population from thesame cross. Some interactive peak that close toreproductive barrier were reproducible; however mostof the combinations of the markers that were not close
to the reproductive barriers did not show high x 2
values in the different population.
4-B. Positional Cloning of a Segregation Distortion Gene
Detected in a Progeny of a Cross between Japonica andIndica rice

Yoshiaki HARUSHIMA and Nori KURATA
The aim of this study is isolation of the mostprominent barrier on chromosome 3 detected in F 2 of
Nipponbare-Kasalath hybrid by positional cloning, andelucidation of the molecular nature of the individual
reproductive barriers. We have clarified the aimed gene
was a male gametophyte gene that interact withmaternal locus on chromosome 6. In other words, thepollen with Kasalath genotype at the gametophyte
gene preferentially fertilized by 94% probability inmaternal plant that is heterozygote or Kasalathhomozygote at the interactive locus on chromosome
6. For detailed mapping the gametophyte gene, we haveselected plants with recombination in 1.9 cM intervalfrom 1000 F; and 473 backcross plants and retrieved
their genomic DNA of the selected plants from bulked
young leaves of their selfed seedling. Considering the
genotypes of the interactive locus, the genetic map of
the male gametophyte gene is being constructed by thedosage analysis using the selected population. The
physical map of this region was constructed using
Nipponbare BACs and P ACs. A PAC that cover the

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63

candidate region was sequenced and physical lengthof the candidate region was about 50 kb and there wasno detectable insertion or deletion between Nipponbare
and Kasalath by Southern analysis. To narrow down
the candidate region, we found the flanking PCRmarkers of the candidate region by the sequence
information, selected recombinants from another 2600
F 2 plants by the flanking PCR markers, and mapped
the aimed gene precisely . We narrowed down thecandidate region to 5.3 Kb. Sequencing of thecorresponding Kasalath genome, and survey of gene
expression in pollen from the candidate region are in
progress.

Huestis, G., Rocheford, T. and Laurie, D.A.:
Comparative mapping of the barley Ppd-Hl
photoperiod response gene region, which lies close toa junction between two rice linkage segments. Genetics,
161, 825-834, 2002.

(5) A new resource work for wild rice species involvedin the activity as a center of national bioresource project

7. Kurata, N. and Fukui, K.: Chromosome researchin genus Oryza. in Monograph in genus Oryza. Nanda .
]S. Ed, Springer (in press).

for rice.
Nori KURATA, Toshie MIYABAYASHI, Yukiko
YAMAZAKI', Mitsugu EIGUCHI and Ken-Ichi NONOMURA(* Genetic Strains Information Lab.)

5. Kurata, N., Nonomura, K-1. and Harushima, Y.:
Rice genome organization: the centromere and genome
interactions Annals of Botany 90,427-435,2002.
6. Yamazaki, Y., Yoshimura, A., Nagato, Y. and
Kurata, N.: Recent advances in Oryzabase (Integrated
Rice Database). Rice Genetics News let. 19, 7-8, 2002.

F-e. MICROBIAL GENETICS LASaRATORY(1) Timing of cell division

In 2002, the national bioresource project started
for representative genetic and biological resources
under the MEXT. The plant genetics laboratory andexperimental farm act as a center for rice genetic
resources project. In this project, we deal with about
2000 wild rice accessions including 9 genomes and over
20 species collected all over the world. Propagation,
seed storage, distribution and database construction
for wild rice species are progressed. Details are shownin the report of Experimental Farm.
Publications
1. Harushima, Y., Nakagahra, M., Yano, M., Sasaki,

T. and Kurata, N.: Diverse variation of reproductive
barriers in three intraspecific rice crosses. Genetics.
313-322, 2002.
2. Ahn, B. 0., Miyoshi, K., Itoh, ]. I., Nagato, Y. and
Kurata, N.: A genetic and physical mapping of theregion containing PLASTOCHRON1,a heterochronic
gene, in rice (Oryza sativa, L). Theor. Appl. Genet. 105,654-659, 2002.
3. Ito, Y., Hirochika, H. and Kurata, N.: Organspecific alternative transcripts of KNOX family class
2 homeobox genes of rice. Gene, 288, 41-47, 2002.
4. Dunford, RP., Yano, M., Kurata, N., Sasaki, T.,

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PLANT GENETICS LABORATORY

Akiko NISHIMURA
During the cell cycle.of E. coh several fundamental
events take place through strictly periodic processes,
although the synthetic patterns of DNA, cell mass
increase, and membrane in the cell cycle are completely
different from each other. As a result, two identicaldaughter cells are produced. The cell must havemechanisms coordinating each event. Cell division, forexample, occurs with constant period (20 min), and thecoefficient of variation in cell size at the time of cell
division is relatively small, at about 10%. These facts
suggest that the E. coli must have mechanisms to theinitiation of cell division at a specific cell length, andthat the consistency of the cell cycle process is basedon coordination mechanisms between various
intracellular reactions and cell division. DNA
replication and cell division also follow a strict
timetable before two identical daughter cells can beproduced. If cells were to divide too early, progeny
would be smaller than normal and more importantly,
replicating DNA strands could be torn apart and/or
annucleoid cells could be produced. The cell must
therefore have mechanisms for coordinating each
event. I have isolated cfc mutants, which uncouple DNA
replication and cell division. E coli cells possessing cfc

mutations divide before reaching the optimal size at
which cfc+ cells divide, and many small cells with asingle nucleoid are produced. Mutations affect thetiming of cell division, but not the initiation mass forchromosome replication. This is due to a reduction in
the period between nuclear division and cell division,and a compensatory increase in the interval between
cell division and initiation of the next round of DNA
replication. These mutations relieve cells from divisionarrest caused by the inhibition of DNA replication. As
such, cfc mutants are defective in the signalingmechanisms that couple DNA replication and cell
division. I have concluded that Ap4A forms part of thissignaling mechanism. I have also isolated the Ap4A
binding protein, which is structurally well conserved
from human to bacteria. This displays nucleotide
binding domains, and interacts with tubulin ineucaryotic cells. I demonstrated that knockout of thisgene produces longer rod cells and over-expression ofthis gene produces numerous small cells. To analyze
the molecular mechanisms of timing in cell division,
I am investigating the mode of expression of this genein a synchronized cell cycle, localization of geneproducts within cells, in vitro function of gene products,
and relationships with Ap4A.
(2) Global regulation of cell division: Isolation of awhole set of cell division mutants
Kimiko SAKA, Noriko MATSUMOTO and Akiko
NISHIMURA
The entire nucleotide sequence of E. coli has been
analyzed, and 4311 ORFs have been demonstrated, but
the functions of more than half of these ORFs remain
unknown. The greater part of these ORFs are
considered to be involved in coordinating cellproliferation. To thoroughly analyze the hierarchy andnetwork responses in expression of cell division genes,
as one of the model cases for post-genome analysis,
we constructed a whole set of mobile plasmid clones
of ORFs, facilitating genetic studies of E. coli. Using
these clones, we have identified 229 cell division genes
by the method of complementation. Of total identified,
5% were known fts genes, 27% were novel genes, 25%
were involved in the key reaction such as DNA
replication and cell division, 19% were involved incellular processes such as ion transport and signaltransduction, and 24% were involved in metabolism.

These results support our speculation that theconsistency of the cell cycle process is based on
coordination mechanisms between various intracellular
reactions and cell division. Using these mutants, weare planning to investigate the hierarchy of expression
for these genes, and the effects of their mutations on
global regulation of cells, using DNA chip techniques.
(3) Guanosine 5'-diphosphate 37-diphosphate (ppGpp)
synthetic activities on Escherichia coli SpoT domains.
Chizuko FUJITA, Akiko NISHIMURA, Ryoko IWAMOTO
and Kenji IKEHARA

Escherichia coli SpoT protein with 702 amino acidresidues is a bifunctional enzyme catalyzing both
guanosine 5'-diphosphate 3'-diphosphate (ppGpp)
degradation and its synthesis. First, we investigated
how many domains are comprised in SpoT protein, bylimited hydrolysis of the protein with serine proteases,a -chromotrypsin and elastase. Based on the results,
we deduced that SpoT protein is composed of two
major domains, an N-terminal half domain from Metlto Phe373 and a C-terminal half domain from Glu374
to Asn702 (C-terminal end). In addition, by a furthera -chymotrypsin digestion two cleaved sites were foundat Arg196 in the N-terminal half domain (D12) and at
Lys475 in the C-terminal half domain (D34), respectively,
to produce four minor domains, D1, D2, D3 and D4.
Next, plasmids expressing the major two domains (D12and D34) and four minor domains (D1, D2, D3 and D4)
were constructed. Consequently, the deduced SpoT
minor domains as well as the maj or domains wereexpressed as stable protein units except for D4. D4 may
be also folded into a stable protein in E. coli cells, sincehigh expression of D4 from a plasmid results in hostcell lethality. E. coli relA, spar double null strains
expressing D1, D2 and D12 recovered cell growth inM9 minimal medium, whereas the transformants of D3,
D4 and D34 did not grow in the minimal medium. It
indicates that ppGpp synthetic activities could be
restricted in the N-terminal half domain (D12, D1 and
D2). For details, see Ref. 3.
Pubrications
1. Fujishima, H., Nishimura, A., Wachi, M., Eirasawa,T., Teraoka, E., Nishimori, k., Kawabata, T.,
Nishikawa, K. and Nagai, K.: The kdsA mutations
affect FtsZ-ring formation in Escherichia coli K-12.

MICROBIAL GENETICS LABORATORY

65

Microbiol. 148, 103-112,2002.
2. Makinoshima, H., Nishimura, A and Ishihama, A:
Fractionation of E. coli cell populations of different
stages during growth transition to stationary phase.
Mol. Microbiol. 43, 269-280, 2003.
3. Fujita, C., Nishimura, A, Iwamoto, R. and Ikehara,
K.: Guanosine 5'-diphosphate 3'-diphosphate (ppGpp)
synthetic activities on Escherichia coli SpoT domains.
Biosci. Biotechnol. Biochem. 66, 1515-1523, 2002.
4. Makinoshima, H., Aizawa, S., Hayashi, H., Miki,
T., Nishimura, A. and Ishihama, A.: Growthphase-coupled alterations in cell structure and functionof Escherichia coli. ]. Bacteriol. 185, 1338-1345, 2002.

F-f. INVERTEBRATE GENETICS LABORATORY(1) RNAi mutant fly bank for comprehensive analysesof gene function in Drosophila
Ryu UEDA, Misako TANIGUCHI, Kaoru SAIG0 1 andKuniaki TAKAHASHI (IGraduate School of Science,
University of Tokyo).

Genome sequencing projects have revealed thenumber of genes for several model organisms forgenetics. The small worm Caenolabditis elegans, which.is composed of only 959 cells and has no complex
organs such as an alimentary canal, cardiovascularsystem, or central nervous system, has 19,000 genes
in its genome. On the other hand, Drosophilamelanogaster, which has a long and sophisticated alimentary canal, a tubular heart that circulates
hemolymph, and a large brain composed of over 10'
cells, harbors only 13,500 genes. Considering there issuch a small number of fly genes, each one of them may
have an essential function in fly development andbehavior. In other words, it may be easy to detect andanalyze gene function in the fly by reverse genetics
because the abnormal phenotype will frequently appear
when knocking down a target gene whose function is
unknown. We are planning 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 13,500

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MICROBIAL GENETICS LABORATORY

genes in the fly? We use RNA interference (RNAi) to
knock down the activity of the target gene. RNAi is
one of the emerging technologies with which toinvestigate gene function in multicellular organisms.
When introduced 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 a sequence-specific manner, and consequently
brings a loss-of-function mutation phenotype to thehost cell. The detailed mechanism of this RNAi
phenomenon has not yet been elucidated, but it works
efficiently in many multicellular organisms, including
humans.
We coupled the RNAi with the GAL4-UAS gene
expression system to induce a conditional
loss-of-function 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 yeasttranscription factor, GAL4, in a specific cell/tissue orat a specific developmental stage in favor of the GAL4transgene. The other fly line harbors a transgene on
the chromosome, 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 loss-of-function
mutation by the RNAi mechanism. Such inducible
RNAi caused by the transcription of an IR sequence
was first successfully adopted to gene function
analysis in C. elegans. It was then also found to beeffective in fly genetics. By making a UAS-transformation vector containing an IR sequence of the gene
predicted by the fly genome project, and by introducing
it into a fly line (IR fly), a mutant phenotype of the genecan be easily observed in any cell or at any developmental stage of the progeny, whenever the IR fly iscrossed to an appropriate GAL4 driver fly.
We are expanding this inducible RNAi to thewhole genome of the fly. This process involves two
major procedures.
1) in vitro construction of transformation vectors
containing an IR sequence from each of the 13,500
predicted genes.
2) Transformation of IR vectors by injecting them

into fly eggs and establishment of IR fly lines bytraditional geneti~ methods.
As of the end of 2002, over 4000 transformation
vectors had been constructed, 2500 of which have beensuccessfully introduced into the fly. Considering thepace of this work, the construction of vectors for over
11,000 genes (accounting for 80% of the entire genome)
should be accomplished during the next fiscal year.
Completion of the transformation will require about
one additional year. Clearly then, we will be able toobtain a powerful tool with which to determine thefunction of the entire fly genome in the near future.
Along with the establishment of IR fly lines, basic
characterization of the target genes is conducted using
these fly lines. All of the IR fly lines are crossed to the
Act5C-GAL4 fly. The Act5C-GAL4 induces the
UAS-transgene in all cells at all developmental stages.
Thus, if the gene targeted by RNAi has functions thatare indispensable for fly development, the progeny of
IR and GAL4 flies should die before the adult flies
emerge. Among the 1617 genes tested, 51.7% of the flylines showed lethality. This value is rather highcompared to that obtained by classical genetics (25%),
while the fact that many of the genes tested here wereconsidered to have important functions in various
aspects of fly development by our collaborators may
bring about such a high score. Detailed analyses on
known genes and greater accumulation of data arenecessary. We are currently collaborating with 30
groups. The usefulness of inducible RNAi forinvestigating gene function in Drosophila is being
revealed in many aspects. We published 3 papers in2002 using RNAi flies.
This work was supported in part by financial
assistance to Dr. Ueda from the Mitsubishi Kagaku
Institute of Life Sciences (MITILS).
Publications

1. Inaki, M., Kojima, T., Ueda, R. and Saigo, K:
Requirements of high levels of Hedgehog signaling
activity for medial-region cell fate determination in
Drosophila leg: identification of pxb, a putative
Hedgehog signaling attenuator gene repressed along
the anterior-posterior compartment boundary. Mech.
Develop. 116, 3-18, 2002.
2. Leulier. F., Vidal, S.S., Saigo, K, Ueda, R. andLemaitre, B.: Inducible expression of double strand
RNA reveals a role of dFADD in the regulation of
antibacterial response in Drosophila. Curr. BioI. 12,
996-1000, 2002.
3. Hayashi, S., Ito, K, Sado, Y., Taniguchi, M.,
Akimoto, A., Takeuchi, H., Aigaki, T., Matsuzaki, F.,
Nakagoshi, H., Tanimura, T., Ueda, R., Uemura, T.,
Yoshihara, M. and Goto, S.: GETDB, a database
compiling expression patterns and molecular locationsof a collection of Gal4 enhancer traps. Genesis, 34, 58-61,
2002.
4. Doi, N., Zenno, S., Ueda, R., Ohki-Hamazaki, H.,
Ui-Tei, K. and Saigo, K.: Requirement of Dicer andeIF2C translation initiation factors for shortinterfering-RNA-mediated gene silencing in
mammalian cells. Curr. BioI. 13,41-46,2003.

F-g. LABORATORY FOR FRONTIER RESEARCH
Lineage Development in the Central Nervous System
Takako Isshki,.,.

Generation of multiple cell types during
embryogenesis is orchestrated temporally as well as
spatially. Yet we know relatively little about how
different cell fates are generated over time. Neuralprogenitors often generate diverse neurons and glia 'in stereotyped order, and the cell fate of a neuron/glia
is tightly linked to its birth order within a parental
progenitor lineage. Our group focuses on elucidating
the molecular mechanisms of birth order-dependent
specification of neural cells within a lineage by
employing a molecular genetic approach using the
Drosophila central nervous system as a model system.
Previously, we have reported that most Drosophila
neuroblasts sequentially express the transcription
factors Hunchback - Kruppel-Pdm-Castor over time,
while differentiated daughter cells maintain theexpre"ssion profile of the transcription factor present
at their birth. Moreover, we showed that Hunchback
and Kruppel, and possibly Pdm and Castor regulate
temporal identity within a lineage. These findings haveprovided the clues to study the molecular mechanismsof temporal specification, and raised many newquestions, such as how the expressions of the temporal

INVERTEBRATE GENETICS LABORATORY

67

identity genes are regulated.
The regulatory genomic region driving thetranscription of hunchback in the CNS was identified.
This region can be divided into two parts: the part for
driving Hb expression in most neuroblasts, and thepart for maintaining Hb expression in differentiateddaughter cells. Dissection and molecular analysis of
the region should reveal what kind of enhancers arepresent, how transcriptional regulation within the CNS
is coordinated. Furthermore, to identify the new factors
responsible for driving the sequential expression of the"temporal identity" genes revealed so far, I started
performing EMS mutagenesis, screening for alterations
in several neuroblast lineages, with emphasis on theneuroblast 7-1 and Z-3 lineages.
Previous study showed that Hb and Kr could
regulate the expressions of the subsequent temporal
identity genes, suggesting that Hb and Kr themselves
are components of the molecular clock keeping timein neuroblasts. In castor mutants, neuroblasts produce
extra daughter cells with mid-born cell fates, thus
progression from the middle to late stage of neuroblast
lineage during development is severely perturbed or
prolqnged. This indicates that Castor also participatesin the molecular clock as a factor accelerating the clock.

•

•
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LABORATORY FOR FRONTIER RESEARCH

G. CENTER FOR GENETICSRESOURCE INFORMATION

G-a. GENETIC INFORMATICS LABORATORY(1) PEC

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

CARD R-DB

Takehiro YAMAKAWA, Hideki KATO, Naomi NAKAGATA,
Kenichi YAMAMURA and Yukiko YAMAZAKI
CARD R-DB is the genetically engilleered mice
database of the Center for Animal Resources andDevelopment (CARD), Kumamoto University,
established as a core center for the production,
cryopreservation and supply of mice.
The database system has been implemented witha direct online data submission and validation system.
Each entry belongs to at least one group and one entry
consists of strain-specific and/or gene specificinformation. Although the data type of most items is
free text type, strain and gene names strictly follow
internationally approved vocabulary. We havedeveloped data submission and validation tools by
which the user can easily submit their data throughthe Internet. The administrator can validate theinformation right after the submission and thenautomatically update the databC\.Se on the same web
screen. The database is managed through therelational database system (Oracle) with Java servlet
applications. The CARD R-DB is accessible at http://
cardb.cc.kumamoto-u.ac.jp/transgenic/.
(3)

JMSR

Takehiro YAMAKAWA and Yukiko YAMAZAKI
JMSR (Japan Mouse Strain Resource) database
I' d es a common gateway 0 f
prOVI
mouse '
genetIc
resources from different organizations. The current
database is compiled of information from fourorganizations including Kumamoto University,
National Institute of Genetics, Tokushima University
and the Japan Animal Strain Committee. Each dataconsists of minimal items such as (1) Strain name, (2)Strain group, (3) Stock type, (4) Genes, (5) Model diseasename and/or application field and (6) URL address from
where full information of the certain strain can be
retrieved. We use the free PostgreSQL relational
database in order to make the system freely available
under an Apache open source license. JSMR expects
new members to join in the near future. Data
distribution in XML format with the associateddocument type definition (DTD) file is also an ongoingproject. JMSR provides web access at http://
www.shigen.nig.ac.jp/mousehmsr/.
(4)

OryzabaseTakehiro YAMAKAWA, Shingo UENO, Nori KURATA,
Atsushi YOSHIMURA, Hikaru SATO, Yasuo NAGATO andYukiko YAMAZAKI

CENTER FOR GENETICS RESOURCE INFORMATION

69

Oryzabase represents an integrated rice science
database, containing(1) Rice Genetic Resources, (2) Mutant Phenotype
Collection,(3lGene Dictionary, (4) Genetic Maps, (5)Developmental stage and gene expression, (6) Basic
Information and (7)Useful site information.
Current database compiles ca. 11000 strains
maintained by 25 stock centers. Oryzabase also
provides the "Core Collection of Wild Rice" to show
representative wild rice accessions, which cover 9
different genomes (AA, BB, CC, BBCC, CCDD, EE, FF,
GG, and HHJJ) and 18 different species. All collections
have been characterized and presented with image
data. Mutant phenotype collection is currently classified into 5 groups such as Coloration, Heterochrony,
Reproductive organ, Seed, and Vegetative organ.
Oryzabase also provides a web-based.fiata submission
system by which users can upload the mutant
phenotype image files and submit them to the
database.
The gene information of Oryzabase is based on thereport of the Committee on Gene symbolization,
Nomenclature and Linkage Groups.
Ontology is one powerful idea to specify a certain
concept and useful especially to compare concepts
common between different fields. Among many
biological ontology projects recently established, theGene Ontology (GO) Project is the most popular andwide spread because major databases such as Flybase,
SGD, MGI, TAIR, WormBase, PomBase, RGD, EBI,TIGR have \llready joined. Gramene database, acomparative mapping resource for grains maintainedby Cornell University, now proposes three riceontologies, gene ontology (GO), traits ontology (TO) andplant ontology (PO) that defines the plant anatomy
and/ or plant developmental stages. Coordinating theGramene's efforts, all genes of the current dictionary
were assigned to the above ontologies (GO/TO) in the
Oryzabase. The assignment of GO for newly coming
genes obtained from genomic sequences as well as PO
assignment is an ongoing project in collaboration withrice researchers and other rice databases. To make thiscollaboration easier, a comprehensive viewer 'called"GOALL", that will give researchers an overview of thecurrent GO and help them understand how ontology
works and how it is useful, has been developed.
Oryzabase is available at http://www.
shigen.nig.ac.jp/rice/oryzabase/.

•70

GENETIC INFORMATICS LABORATORY(5) KOMUGITakehiro YAMAKAWA, Takashi ENDO, Yasunari
OGIHARA, Hitoshi TSUJIMOTO, Taihachi KAWAHARA,
Tetsuro SASAKUMA and Yukiko YAMAZAKI
KOMUGI was originally established as a wheat
genetic resource database by collecting strain stock
information from several Japanese universities andgrew to become an integrated wheat information
database. The current version of KOMUGI databaseconsists of (1) strain resource information, (2) EST andDNA resource information, (3) wheat gene catalogue,(4) composite maps, (5) wheat information service (WIS),
and (6) DNA sequence collection of wheat (Triticeae,
A vena, Secale and soon.). The gene catalogue ispublished every 5 years and only supplemental data
is annually published. To make an up-to-date genecatalogue available through the Internet, we havedeveloped a management system of a gene catalogue
called "MacGene:'. Through this, by which thecatalogue keeper can easily submit and/or update thedata automatically. The KOMUGI database is available
to the public through the Internet at http://
www.shigen.nig.ac.jp/wheat/komugi/.
Publications(1) Matsuoka, Y., Yamazaki, Y., Ogihara, Y. andTsunewaki, K.: Whole chloroplast genome comparisonof rice, maize, and wheat implications for chloroplast
gene diversification and phylogeny of cereals. Mol. BioI.
Evol. 19, 2084-2091, 2002.
(2) Kitakami, H, Kanbara, T., Mori, Y, Kuroki, S. andYamazaki, Y.: Modified PrefixSpan Method for Motif
Discovery in Sequence Databases. PRICAI2002, LNAI
2417, 482-491, 2002.
(3) Kanbara, T., Mori, Y, Kitakami, H, Kuroki, S. andYamazaki, Y.: Discovering Motifs in Amino Acid
Sequences using a Modified PrefixSpan Method.
Currents in Computational Molecular Biology 96-97,
2002.

G-b. GENOME BIOLOGY LABORATORY
Genome Biology Laboratory, Center for Genetic
Resource Information(I) NEXTDB: The nematode expression patterndatabase
Tadasu SHIN-I, Ikuko SUGIURA, Masumi OBARA,
Wakako SHIMIZU, Yuko SUZUKI, Jean THIERRY-MIEG',
Danielle THIERRY-MIEG 1, Yutaka SUZUKI2, Sumio
SUGAN02, Kazuko OISHI, Masako SANO, Hisayo
NOMOTO, Shinobu HAGA, Satoko NISHIZAKA, Hiroko
HAYASHI, Fumiko OHTA, Sachiko MIURA, Keiko
HIRONO, Kyoko NAKATA, Hiroko UESUGI, Yasuko
SUGIYAMA and Yuji KOHARA PNCBL NIH, 2Institute ofMedical Sciences, 0. Tokyo)
We continued to update 'NEXTDB' that integrated
all the information of ESTs, gene expression patterns
and gene functions of Celegans which were produced
and analyzed in this laboratory. Images of whole mountin situ hybridization for mRNA were taken by
microscopes equipped with DIC (differential
interference contrast) apparatus, loaded to the Sun
workstation to process and arranged properly in the
database. Then, they were annotated with respect todevelopmental stages and expression patterns. Current
version of NEXTDB contains EST information of
136,467 cDNA clones (5'-EST: 126,431, 3'-EST: 116,616)
that have been classified to 11,903 unique cDNA
groups, in situ images of 9,183cDNA groups and RNAi
phenotype images of 295 groups. The cDNA clones arefreely available for academic use. Thus far, the total
23,460 clones have been sent to the total 4,829 requests
from 26 countries. NEXTDB is available at URL: http://
nematode.lab.nig.ac.jp/. Many collaborations based on
NEXTDB have been conducted (See Ref. 2, 3, 6, 9, 12)(2) Amilysis of protein distributions of maternal genes
by antibody staining in C.elegans.
Yumiko UETA, Masahiro ITO, Hiroko OeHI, Chihiro
HIJIKATA, Keiko HIRONO, Yoshinori UKAIl, Midori
SHINOHARA', Hisayoshi TORII~, Emi SHIMIZU', Miki
TAKENAKA2, Yoshitaka IBA" Yoshikazu KUROSAWA 1
and Yuji KOHARA (lInstitute for Antibtlriies, 2MBL,
lFujita Health Univ.)

The accumulation of mRNA expression patternsof Celegans that are being produced in this lab
provides unique sets of genes that are expressed in
specific developmental stages, cell-lineages, tissues andso on. The gene expression data led us systematic
analyses to elucidate the molecular mechanisms
governing the biological process. Since we areparticularly interested in early embryogenesis, we havefocused on a set of genes whose mRNA are maternallysupplied and disappear by early stage of gastrulation
or localize to specific cells in early embryos. We found477 (about 8.8 %) such genes when the expression
analysis of about 5000 genes was finished. To analyze
the subset genes systematically with respect to the
distribution of the protein products, we started to raisemouse antisera against the 477 maternal gene products
using bacterially.expressed partial proteins. Until now,
we obtained antisera against 282 gene products, andimmuno-stained embryos and gonads using theantisera.
The spatial immunostaining patterns were as
follows; nucleus (32%), cytoplasm (35%), plasma
membrane (19%), nuclear membrane (10%) andP-granules (20%). The temporal staining patterns wereas follows; the staining signals were first observed
from-the mitotic region in gonad (32%), oogenesis (12%)
and from 2-cell stage (11 'Yo) . We confirmed thespecificity of some of the antibodies by testing if thestaining signal disappeared in RNAi-treated specimen.
(3) Roles of POS-I and PIP·I in the translational
regulation of maternal mRNA in C. elegans
Norihito KISHIMOTO, Ken-ichi OGURA' and Yuji
KOHARA (lYokohama City 0.)
Translatinal control of maternal mRNA is
important for cell fate specification in early embryosof C elegans. For example, GLP-l protein, a Notch
family receptor, is translated in anterior cells but notin posterior cells, although glP-l mRNA is present inall of the cells. Several regulatory regions in glP-l
3'UTR (untranslated region) have been identified as
spatial control region (SCR) and temporal control
region (TCR). We found that POS-l specificallyinteracted with SCR and negatively regulated thetranslation of glp-l mRNA. A maternal factor POS-lregulates cell fate determination of early embryos(Tabara et al., Development 126 1-111999). POS-l has

GENOME BIOLOGY LABORATORY

71

TISll-type (CCCH) zinc-finger motifs, and is present
in cytoplasm of posterior blastomeres, being atemporary component of P granules. PIP-l (POS-linteracting protein) has an RNP-type RNA-binding
motif and is present in cytoplasm of oocytes and early
embryos, also being a temporary component of P
granules. We found that PIP-l positively regulatesmaternal glp-l mRNA translation. Based on thesefindings, we proposed a combinatorial mechanism forthe translation regulation by the two proteins, POS-land PIP-I.
To examine the roles of POS-l and PIP-l intranslational control of maternal mRNA, we studiedinteractions between POS-I, PIP-l and 3'UTRs ofvarious maternal mRNAs by using the yeast tri-hybrid
system. Other RNA-binding proteins -MEX-l, MEX-3,
MEX- &- were also studied. Futhermore, we mapped
the protein binding region in POS-l, PIP-l and another
POS-l interacting protein. Based on the results, we
propose that the cell fate specification involvescascades of tranlational control in which POS-l and
PIP-l appear to control poly(A) tail length and regulatetranslation of maternal mRNA. (See Ref. 14)(4) Systematic identification of cis-regulatory elementsof the genes that are expressed in specific nerve cells
Hiroshi KAGOSHIMA, Akiko KAMAMOTO and Yuji
KOHARA. To understand the mechanism of transcriptional
regulation, we have taken a simple strategy. We havebeen trying to extract consensus regulatory sequenceof the genes which share spatial and temporal
expression pattern in a certain cell.
There are a large number of GFP reporters thatare known to show cell-specific expression in particular
set of cells in C. elegans. One can accordingly expectthat the same transcription machinery might regulate
the genes which are expressed in a certain cell. If so,their regulatory core regions should share common
sequences. To test this idea, we checked the expression
patterns of a series of deletion promoter::GFP reporters
in transgenic worms, and identified minimal enhancer'core" for the cell-specific expression.
We currently focus on head neuron-specificpromoters. Thus far, we narrowed down 7 promotersfor the expression in the thermosensing neuron AFD;
for example, 30 bp sequence of gcy-8 (guanylyl cyclase),

72

GENOME BIOLOGY LABORATORY

80 bp sequence of nhr-38 (nuclear hormone receptor).
Interestingly, within these sequences we found thebinding consensus for the transcription factor OTXl,
which has important roles for head development in fly
and vertebrate. It has been revealed that ttx-l (OTXl
homolog of C. elegansl regulates both gcy-8 and nhr-38
expression in AFD (Satterlee et a1. 2001). Systematic
promoter analysis also demonstrated that somedeletion cause ectopic expression in additional neurons.
This observation suggests negative regulatory
mechanism seems to participate in the cell
specification.
(5) Systematic RNAi Analysis of Genes Expressed in C.
elegans Primordial Germ Cells, Z2 and Z3
Ichiro KAWASAKI, Junko KAjIWARA, Michiko SERIZAWA,
Masahiro ITO and Yuji KOHARA
Germ lineage is unique and distinctive in several
fundamental aspects from the other somatic lineages.
In C. elegans, embryonic germline precursor cells (PIto P 4) are generated through 4-rounds of unequal
divisions and set"aside from somatic blastomeres earlyin embryogenesis. Although the embryonic germline
cells inherit and accumulate many maternal mRNAs,
they are transcriptionally inactive partly due to thenegative regulation by pie-l function. The germ lineage becomes first transcriptionally active inmorphogenesis-stage embryos after P 4 is divided into
the two primordial germ cells, 22 and 23, when PIE-lprotein is already gone. After hatching, the germ
lineage becomes one of the most active lineages in theorganism. 22 and 23 divide throughout larval
development, giving rise to -2,000 germ cells in an adult
hermaphrodite. Then they progressively undergo
meiosis, spermatogenesis, and oogenesis to produce
gametes that generate the next generation.
In this lab, a systematic whole mount in situ
hybridization analysis has been performed against the
non-redundant cDNA groups that had been identified
and classified in our EST project. Through theanalysis, thus far, about 9,000 cDNA groups (genes)
have been annotated for their temporal and spatial
expression patterns. By screening germline specificexpression patterns, we have identified that as manyas 700 genes are enriched or specifically expressed in
22 and 23 ot"in their immediate daughter cells either
in morphogenesis-stage embryos or in Ll, L2-stage

larvae. Assuming that some of those Z2, Z3-expressing
genes play pivotal roles in activation of post-embryonic
germline-developmental programs, we performed
systematic RNAi analysis against those 700 genes
using Ll-soaking method. Among them, 115 genes
showed strong sterile phenotypes. Furthermore, 34 ofthem (25 are novel) exhibited Glp (Germ Line
Proliferation defective) phenotype. We have started
analyzing these new Glp genes more closely tounderstand their functions and interplays at molecularlevels.
(6) Effect of LIM binding factor LDB-I on the functionof T-box transcription factor TBX-9 in C. elegans
Yoshiki ANDACHI
tbx-9 is a member of the T-box family oftranscription factors. tbx-9 is expressed in a few
precursor cells of intestine, body-wall muscle andhypodermis. A tbx-9 deletion mutation causes
disorganization in embryogenesis that predominantly
occurs in posterior part of the body, though thepenetrancy was very low.
'!"
To further elucidate the function of tbx-9, I
searched for its binding by the yeast two-hybrid
system, and found LDB-l as one of the interacting
proteins. !db-] is a orthologue of vertebrate LIM domainbinding factor CLIM-2/NLl/Ldb] and is expressed in
all the blastomeres of early stage embryo. Two Ldb]
monomers form a homodimer and each monomer binds
to LIM transcription factor or Pitx transcription factor,
so that they constitute a complex of transcription
factors. Thus fur, relation between T -box factor andLdb] factor has not been reported. Their interaction
was confirmed by the in vitro pulldown assay. RNAi
experiments with ldb-] ds-RNA caused 3-times higher
penetrancy of the tbx-9phenotype in tbx-9mutant but
no phenotype in the N2 wild type. These results
suggest that tbx-9and !db-] work for the same function.
It is speculated that tbx-9 transcription factor forms
with another transcription factor through themediation of ldb-] dimers to synergistically regulate
the expression of their target genes.

(7) Computer Simulation of the Cellular Arrangement
Using Physical Model in Early Cleavage of the NematodeC. elegans
Atsushi KAJITA, Masayuki YAMAMURA 1 and Yuji
KOHARA (1 Tokyo Institute of Technology)
The ultimate goal of bioinformatics is toreconstruct biological systems in the computer. Since
biological systems have many levels, it is important tofocus on an appropriate level. In our first applicationof computer modeling to the early development of thenematode C. elegans, we focus on the cellulararrangement in early embryos. This plays a very
important role in cell fate determination by cell-cellinteraction, and is regarded as a system, one level
higher than the system of gene regulation within cells.
It is largely restricted by physical conditions that
seemed feasible to model by computer.
We constructed a computer model of the C.
elegans embryo, currently up to the 4-cell stage, using
a deformable and dividable triangulated network. The
model is based solely on cellular-level dynamics. We
found that the optimal ranges of three parameters that
affect the elongation of dividing cells led, in computer
simulations, to almost the same cellular arrangements
as in real embryos. The nature of the model and therelationship with real embryos were examined.
In addition, we modeled new physical phenomenaof cell division, cell rounding and stiffening; we then
combined them with already modeled phenomena,
contractile ring contraction and cell elongation. We
investigated effectiveness of the new model on thecellular arrangement by computer simulations. We
found that cell rounding and stiffening only during theperiod of cell division were effective to generate almost
identic;al cellular arrangements to in real embryos.
Since cells could be soft during the period between cell
divisions, implementation of the new model resulted
in cell shapes similar to real embryos. The nature of
the model and its relationship to real embryos are
discussed. (See Ref. 10, 11)(8) Toward a four-dimensional database of developmentof the nematode Ceanorhabditis elegans
Masahiro ITO, Yohei MINAKUCHI, Michiko SERIZAWA
and Yuji KOHARAGENOME BIOLOGY LABORATORY

73

One of the most challenging targets in the post
genomics era is computer simulation of development.
C elegans is thought to be the most suitable system
for this purpose since the complete list of cellcomponents has been identified and expression
patterns and functions of all genes are being analyzed
systematically. We wish to integrate such informationand to create a four-dimensional (3D + time course)
database of gene expression and development, basedon which better computer modeling and simulation
would be achieved.
First, we need a good framework for such 4D
database. For this purpose, we made a computer
graphics (CG) of early embryogenesis up to 86-cellstage based on the image data taken on 4D microscope.
4D microscope can record DIC (differential interference
contrast) images of live embryos at different focal
planes and along time course. First, we reconstructed
3D structure from a series of images of optically sliced
section. Since it was very difficult for conventional
image processing software to extract the cell contours
from the DIC images, we adopted a manual procedure:
we traced the contours of individual cells and nuclei
by hanm on thousands of original images of the 4D
microscopic data; then, we reconstructed 3D structuresby stacking them and making relationships amorig thecontour data. Resulting 3D structures of embryos atvarious developmental stages were connected witheach other by an interpolation method to generate a-computer graphics. The CG provides various
information of early embryogenesis such as individual
cell volume, e~tent of cell-cell contact, and so on, which
are difficult to measure on the original microscopic
images. We are using the CG as a framework of our
4D database of Celegans development.
Second, onto the CG framework, we planned toincorporate expression data of many genes at
subcellular level. However, the assignment of 3D
distribution of gene products (protein, mRNA) ofembryos at various developmental stages is difficult
and tedious. We need to automate this process. For thispurpose, we developed a new system, named SPI, forsuperimposing fluorescent confocal microscopic data
onto the computer graphics framework.
The scheme of this system is as follows: (1)acquirement of serial sections of fluorescent confocal
images with triple colors, that is, DAPI for nuclei, Cy-3
for an internal marker and Cy-5 for the query geneproduct; (2) identification of several features of the74

GENOME BIOLOGY LABORATORY

stained embryos such as the contour, developmental
stage and the position of the internal marker; (3)selection of computer graphics images of thecorresponding stage for template matching; (4)superimposition of serial sections onto the computer
graphics; (5) assignment of the position of the query
gene products. The Snakes algorithm was used for theidentification of embryo contour. Detection accuracyof embryo contour was 92.1% when it was applied toembryos of 2 to 28-cell stage. The accuracy of themethod of developmental stage prediction was 74.6%
for embryos at 2-12 cell stage. Since the accuracy forembryos at later stage was unsatisfactory due toexperimental noise effect, we adopted manual judgment
by scientist only the later stage embryos. Finally, oursystem chose optimal CG and performed thesuperposition and the assignment of the gene productdistribution successfully. Initial 4D gene expression
database containing 56 maternal genes wasestablished. (Manuscript submitted)(9) Automatic system for creation of cell shape modelin C. elegans embryogenesis•

Hideaki HIRAKI and Yuji KOHARA

C elegans if? an excellent model organism for thestudies of development as its body is transparent andconsists of a small number of cells. CeIl to cellinteractions play critical roles in early embryogenesis,
therefore, it is very important to have information
about the arrangements of cells, cell shapes and thecontact among them. A polygon model of the early
embryogenesis of a wild type embryo was built in ourlaboratory based on a time series of 3-dimensional
images of differential interference contrast (DIC)
microscopy. This type of computer model is very usefulfor comparative studies among mutant embryos,
however, building of the model takes long time andmuch effort because it is difficult to extract the cellboundaries in DIC images automatically. Indeed, theoutlines of the cells were traced by hand to build thecurrent model. Therefore, in this work we aredeveloping an automated system to build such a
computer model of the cells in embryos based on a timeseries of confocal microscopic images of embryos
whose plasma membrane is stained with a fluorescentdye. The system can fully automatically construct thecomputer models of 1-24 cell stages from an assured

seed sets of 24 cell stage. Dsing the overlapping
information in the time series of the constructed cell
shape models, the system can reconstruct virtuallycorrect cell lineage. Now we are trying to process a real
dataset of 24-200 cell stages, and are planning to apply
this system to compare the cellular arrangements andthe cell-to-cell contacts among mutant embryos andthe embryos from other species closely related to C.
elegans.
(10) Towards comparative genomics: Genomic/EST
sequencing
Kazuko OISHI, Shinobu HAGA, Hisayo NOMOTO,
Masako SANO, Satoko NISIZAKA, Fumiko OHTA, Hiroko
HAYASm, Sachiko MIURA, Tomomi MORISmTA, Tomoko
ENDO, Motoyo TAMIYA, Noriko HASEGAWA, Masumi
MIZUKOSHI, Etsuko YOKOYAMA, Junko MIYAMOTO,
Shigeko IIYAMA, Tadasu SHIN-I, Shigeru SAITO, Kumiko
KAWAGUCm, Naoko SAKAMOTO, Yasuko SUGIYAMA andYuji KOHARA
As a core facility of the group grant "Genome"(supported by MEXT and consisted of about 300 labs),
we have established a DNA sequencing center.
Currently our capacity is 8 million reads per year. Thusfar, we have performed the followings in collaboration
with the members of the group grant (in theparenthesis);(a) EST: the nematode Caenorhabditis elegans, 180,864
reads (Yuji Kohara, NIG), Ascidian Ciona intestinalis,
684,096 reads (Nori Satoh, Kyoto U), Ascidian Ciona
savignyo, 7,300 reads (Nori Satoh, Kyoto U), Ascidian
Halocynthia roretzi, 73,986 reads (Kazuhiro Makabe,
Kyoto D.), Hagfish Eptatretus burgeri, 62,208 reads(Masanori Kasahara, the Graduate U for Advanced
Studies), Medaka fish Oryzias latipes , 268,320 reads(Hiroyuki Takeda, D. Tokyo and Akira Kudo, Tokyo
Inst. Tech.), ciclid fish Haplochromis chilotes, 50,112
reads (Norihiro Okada, Tokyo Insti. Tech.), frog
Xenopus laevies, 146,496 reads (Makoto Motii, NIBB),
slime mold Dictyostelium discodeum, 183,648 reads(Yoshimasa Tanaka, Tsukuba D.), read algae
Cyanidioschyzon merolae, 76,704 reads (Tsuneyoshi
Kuroiwa, U Tokyo), moss Physcomitrella patens subsp.
patens, 104,064 reads (Mitsuyasu Hasebe, NIBB), barley,
147,648 reads (Kazuhiro Satoh, Okayama U), wheat
Triticim aestivum cv. Chinese Spring, 276,096 reads(Yasunari Ogiwara, Yokohama City D.), Japanesemorning glory, 30,720 reads (Eiji Nitasaka, Kyushu 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
distributed from this laboratory.
(b) Genome (BAC, fosmid clones): nematodePristionchus pacifficus 2 BACs (Yuji Kohara, NIG and
Ralf Sommer, MPI, Germany), fly Drosophila sechelia
2 cosmids (Toshiro Aigaki, Tokyo Metoropolitan D.),
Acidian Ciona intestinalis 16 BACs (Nori Satoh, KyotoU), medaka fish Oryzias latipes, 5 BACs (HOX region)(Hiroshi Hori, Nagoya D.), Chimpanzee, 12 BACs
(Naruya Saitou, NIG), Gorilla, 9 fosmids (Naruya
Saitou , NIG), liverwort Marchantia polymorpha L 11
P ACs (Kanji Ohyama, Kyoto U).
(c) Genome (whole genome): read algae Cyanidioschyzon merolae 16Mb (Tsuneyoshi Kuroiwa, U Tokyo),
Ascidian Ciona intestinalis 160Mb (Nori Satoh, Kyoto

U)
The main target at the moment is the wholegenome shotgun sequencing of Medaka fish Oryzias
latipes (8010Mb). (See Ref. 1, 4, 5, 7, 8, 15)(11) The draft genome of Ciona intestinalis: insights
into chordate and vertebrate origins.
Dan ROKHSAR Lab!, Yuji KOHARA Lab, Michael LEVINELab 2, • Nori SATOH Lab 3 and Ascidian researchcommunity (IDOE Joint Genome Institute, DSA, 2DC
Berkeley, DSA, 3 Kyoto U)
The first chordates appear in the fossil record at
the time of the Cambrian explosion, nearly 550 million
years ago. The modern ascidian tadpole represents aplausible approximation to these ancestral chordates.
Here we present the draft genome sequence of the mostthoroughly analyzed ascidian, Ciona intestinalis. The
Ciona genome contains protein-coding genes, similar
to the number in other invertebrates, but only half thegene number found in vertebrates. Vertebrate genefamilies are typically found in simplified form in Ciona,suggesting that ascidians contain the basic ancestralcomplement of genes involved in cell signaling anddevelopment. The ascidian genome has also acquired
a number of lineage-specific innovations, including agroup of genes engaged in cellulose metabolism thatare related to those in bacteria and fungi.
http://genome.jgi-psf.org/ciona4/ciona4.home.html (SeeRef. 13)

GENOME BIOLOGY LABORATORY

75

Publications
1. Kawashima, "T., Kawashima, S., Kohara, Y.,
Kanehisa, M. and Makabe, K.W.: Update of MAGEST:
Maboya Gene Expression patterns and Sequence Tags.
Nucleic Acids Research, 30,119-120,2002.

2. Morita, K., Flemming, AJ., Sugihara, Y., Mochii,M., Suzuki, Y., Yoshida, S., Wood, W.B., Kohara, Y.,
Leroi, AM. and Ueno, N.: A C. elegansTGF-{3, DBL-l,controls the expression of LON-I, a PR-related protein,that regulates polyploidization and body length. EMBO]. 21, 1063-1073, 2002.
3. Mallo, G.V., C.L., Kurz, Couillault, C., Pujol, N.,
Granjeaud, S., Kohara, Y. and Jonathan]. Ewbank, ].].:
Inducible Antibacterial Defence System in C.elegans.
Current Biology 12,1209-1214,2002.
4. Fujiwara, S., Maeda, Y., Shin-i, T., Kohara, Y.,
Takatori, N., Satou, Y. and Satoh, N.: Gene expression
profiles in Ciona intestinalis cleavage-stage embryos.
Mechanisms of Development 112, 115-127, 2002.
5. Ogasawara, M., Sasaki, A, Metoki, H., Shin-i, T.,
Kohara, Y., Satoh, N. and Satou, Y.: Gene expression
profiles in young adult Ciona intestinalis. Dev. Genes
Evol., 212, 173-185, 2002.
6. Iwahashi, ]., Kawasaki, 1., Kohara, Y.,
Gengyo-Ando, K., Mitani, S., Ohshima, Y., Hamada,N., Hara, K., Kashiwagi, T. and Toyoda, T.:
Caenorha bditis elegans reticulon interacts with RME-lduring embryogenesis. BBRC 293, 698-704, 2002.
7. Satou, Y., Takatori, N., Fujiwara, S., Nishikata, T.,
Saiga,H., Kusakabe, T., Shin-i, T., Kohara, Y. and
Satoh, N.: Ciona in tes tinalis eDNA projects: expressedsequence tag analyses and gene expression profiles
during embryogenesis. Gene 287, 83-96, 2002.
8. Kusakabe, T., Yoshida, R., Kawakami, 1.,
Kusakabe, R., Mochizuki, Y., Yamada, L., Shin-i, T.,
Kohara, Y., Satoh, N., Tsuda, M. and Satou, Y.: Geneespression profiles in tadpole larvae of Ciona
intestinalis. Developmental Biology 242, 188-203, 2002.
9. Ichimiya, H., lIino, 0., Kohara, Y. and Ishii, N.:
VBP-l is necessary for morphogenesis in

Caenorhabditis elegans. Oncology Reports 10, 293-295,

76

GENOME BIOLOGY LABORATORY(2003).
10. Kajita, A, Yamamura, M. and Kohara, Y.:
Computer Simulation of the Cellular Arrangement in
Early Cleavage of the Nematode C. elegans.
Bioinformatics 19, 704-716, 2003.
11. Kajita, A, Yamamura, M. and Kohara, Y.: PhysicalModeling of the Cellular Arrangement in C. elegans
Early Embryo: Effect of Rounding and Stiffening of theCells. Proceedings of the 13th International Conference
on Genome Informatics (GIW 2002), 13, 224-232, 2002.
12. Matsuno, T., Ura, K., Sonoda, R., Kohara, Y.,
Uesugi, H., Arizono, K., Iguchi, T. and Tominaga, N.:
Sensing of chemical substances using gene expression
patterns in Caenorhabditis elegans. Sensors andMaterials, 14, 395-406, 2002.
13. Dehal, P., Satou, Y., Campbell, RK., Chapman,].,Degnan, B., De Tomaso, A, Davidson, B., Di Gregorio,
A, Gelpke, M., Goodstein, D., Harafuji, N., Hastings,
K., Ho, 1., Hotta, K., Huang, W., Kawashima, T.,
Lemaire, P., Martinez, D., Meinertzhagen, lA, Necula,S., Nonaka, M., Putnam, N., Rash, S., Saiga, H., Satake,M., Terry, A, Yamada, L., Wang, H., Awazu, S., Azumi,
K., Boore, ]., Branno, M., Chin-bow, S., DeSantis, R.,
Doyle, S., Francino, P., Keys, D., Haga, S., Hayashi, H.,
Hino, K., Imai, KS., Inaba, K., Kano, S., Kobayashi, K.,
Kobayashi, M., Lee, B., Makabe, KW., Manohar, c.,Matassi, G., Medina, M., Mochizuki, Y., Mount, S.,
Morishita, T., Miura, S., Nakayama, A, Nishizaka, S.,
Nomoto, H., Ohta, F., Oishi, K., Rigoutsos, 1., Sano, M.,
Sasaki, A, Sasakura, Y., Shoguchi, E., Sin-I, T.,
Spagnuolo, A, Stainier, D., Suzuki, MM., Tassy, 0.,Takatori, N., Tokuoka, M., Yagi, K., Yoshizaki, F.,
Wada, S., Zhang, c., Hyatt, PD., Larimer, F., Detter, C.,
Doggett, N., Glavina, T., Hawkins, T., Richardson, P.,
Lucas, S., Kohara, Y., Levine, M., Satoh, N. andRokhsar, D.: The draft genome of Ciona intestinalis:insights into chordate and vertebrate origins. Science
298, 2157-2167, 2002.
14. Ogura, K., Kishimoto, N., Mitani, S., Gengyo-Ando,K. and Kohara, Y.: Translational control of maternal
glP-l mRNA by POS-l and its interacting protein
SPN-4 in Caenorhabditis elegans. Development 130,2495-2503, 2003.

15. Ohta, N., Matsuzaki, M., Misurni, 0., Miyagishima,S., Nozaki, H., Tanaka, K., Shin-i, T., Kohara, Y. andKuroiwa, T.: Complete sequence and analysis of theplastid genome of the unicellular red alga
Cyanidioschyzon merolae. DNA Research 10, 67-77, 2003,

.of

.J'

~

GENOME BIOLOGY LABORATORY77

H. STRUCTURAL BIOLOGYCENTER

H-a. BIOLOGICAL MACROMOLECULES lABORATORY(1) Thin Layer Illumination Microscopy as a Novel
Technique for Single Molecule Fluorescence Imaging
Inside Cells
Makio TOKUNAGA
Thin-layer illumination fluorescence microscopy,
a novel microscopy for single molecule imaging, hasbeen further developed. Specimens were illuminatedwith a thin-layered laser beam with the thickness ofabout 3 to 5 micrometers. This microscopy has asignificant advantage of low background and highsensitivity, so it allowed us to visualize single
fluorescent molecules inside cells up to the depth ofabout 10 /lm from the glass-medium interf~ce. Despiteof imaging inside cells, clear images of single GFP
molecules were obtained. The resolving power, i.e. theresolution distinguishing two point images, of 70 nm
has been achieved using image analysis of images ofsingle nuclear pores. The present microscopy is also
useful for observation of living cell, which is sensitive
to the illumination light, and for time-lapse observation
over a long period.
(2) Single Molecule Imaging of Nucleocytoplasmic
Transport in Cells and Quantitative Analysis ofInteraction with Nuclear Pores
Makio TOKUNAGA and Naoko IMAMOT0 1. 2 (lGene
Network Laboratoy, "Riken)

'"

:..

Single molecules of green fluorescent protein(GFP)-tagged importin beta, a carrier protein, and
GFP-tagged cargo protein are clearly visualized on
the nuclear envelope. These images are almost as
clearly as those on cover glass surfaces. At higher
concentrations of GFP-tagged proteins, images of78

STRUCTURAL BIOLOGY CENTER

nuclear pores were obtained, and image analysis
resolved single nuclear pore images. Quantitative
analysis of single-molecule and single-pore images
enabled us to obtain kinetic parameters of theinteractions with the NPC, i.e., the retention time, thenumber of bound molecules and the binding constant.
It was found that there were two kinds of binding siteof importin beta on NPC in the absence of cargo and
Ran-GTP, weak binding sites and strong ones. The
former gathers many molecules up to a maximum of
approximately one hundred molecules per NPC. The
latter vanished in the presence of cargo and in theabsence of Ran-GTP, suggesting that accessibility to
the strong binding site holds the key of the transport.
Thus, single molecule imaging has opened a new way
to obtain quantitative information on kinetics ofmolecular interactions in cells.
(3) pI05: a novel protein component ofmRNA-transporting
granules in neuronal dendrites
Nobuyuki SHIINA, Kazumi SHINKURA and Makio
TOKUNAGA.,mRNA transport and subsequent local translationof the mRNAs in neuronal dendrites are widelybelieved to be essential for local regulation of synaptic
efficacy. The dendritic mRNAs are transported indensely packed granules containing the mRNAs andribosomes, called RNA granules.
p105 is a novel protein we originally identified asa centrosome- and microtubule-associated protein.
Expression of p105 protein in cultured cells induces
formation of cytoplasmic granules containing
ribosomes, translation factors (EF-1 alpha) and mRNAs.
In this year, we showed that p105 was expressed highlyin neurons. These characteristics of pI05 protein
suggested that the p105-induced granules might beequivalent to the RNA granules in neurons.
Immunostaining of rat hippocampal slices showed that
pI05 was associated with granules containing. ribosomes and mRNAs in neuronal dendrites. Staufen,
a protein for mRNA transport, and FMRP, a protein
for translational regulation, were identified as thecomponents of the granule. Microtubule- andactin-dependent motors, kinesin and myosin, were also
the components of the granule. Furthermore, mRNAs
associated with the granule were identified. Many ofthe mRNAs, such as CaM kinase IT alpha, CREB andBDNF mRNAs, were the key molecules for long-term
synaptic plasticity. These lines of evidence indicated

I-Connectin can be extended up to 3.5 ~ m upon stretch

that p105 is a novel protein associated with

regions in I-connectin: two long PEVK regions, one

mRNA-transporting granules which might beunique sequence region and Ser-, Glu- and Lys-rich

responsible for synaptic plasticity in neuronaldendrites.

68 residue-repeats called SEK repeats. In the present
study, the force measurement of the single recombinantof giant sarcomeres. There are several extensible

SEK polypeptide containing biotinylated BDTC and(4) Single molecule measurement of intermolecular and

GST tags at the Nand C termini, respectively, wereintramolecular interactions using subpiconewton

performed by intermolecular force microscopy (IFM),

intermolecular force microscopya refined AFM system. The force versus extension
curves were well fit to the wormlike chain (WLC) modelMichio HIROSHIMA and Makio TOKUNAGA

and the obtained persistence length of 0.37±0.01 nm

Intermolecular force microscopy (IFM) is able

(n=ll) indicates that the SEK region is a random coilfor full length. This is the first observation of an

to measure forces of inter- and intra-molecularinteractions at single molecular level. This microscopy

entropic elasticity of fully random coil region
contributing to the physiological function ofhas achieved the force resolution of subpiconewton

invertebrate connectin.

with ultrasensitive cantilevers. The cantilever position
is controlled with nanometer accuracy by the feedback
system using laser radiation pressure, which also

(6) A Novel in vitro Assay System of Nucleocytoplasmic
Transport

reduces thermal fluctuation of the cantilever.
Using IFM, we have measured unzipping forces
between single complementary base pairs of a

Atsuhito OKONOGI, Michio HIROSHIMA, Nobuyuki
SHIINA, Naoko. IMAMOTO l ,2 and Makio TOKUNAGA

synthesized DNA oligomer. To resolve unzipping(1Gene Network Laboratoy, 2Riken)

forces of single base pairs, both the force sensitivityof piconewton and the position control with nanometer

We have developed a novel in vitro assay systemaccuracy are required. Any current method of singleof nucleocytoplasmic transport. We aim at applicationof the method to new single-molecule experiments,

molecule detection does not fulfill this requirement,
whereas IFM does. The observed force vs. extension
curves showed a series of peaks. Each peak is 10-15

imaging and nano- or force-measurement.

pN in amplitude and approximately 1 nm in width. The

Nuclear envelope was formed on a planar surface
of a small agarose plate. At first, agarose plates werepeak amplitude varied periodically, and the periodic

modified with glutation. Then glutation-agarose plates

pattern was consistent with the periodic sequence of
the DNA oligomer. These results suggested that thewere coated with GST-RanGDP fusion protein. Nuclear

observed peaks corresponded to unzipping forces ofsingle base pairs.

using extracts from Xenopus laevis frog eggs.
Formation of Nuclear Pore Complexes was confirmed(5) Mechanism for passive force generation ofThis new cell-free ~ystem has marked advantages: 1)

envelope was formed onto the RanGDP-coated surface

by observing import of fluorescently labeled proteins.
invertebrate connectin revealed by single moleculeSol1.i.tions in both sides, pseudo-cytoplasmic andmeasurement

pseudo-nucleoplasmic sides, can be replaced
independently, 2) The shape of the nuclear envelope

Michio HIROSHIMA, Atushi FUKUZAWA\ Koscakll

can be changed as one likes, for example, a verticalplane and a horizontal plane. 3) The system doesn't

MARUYAMA , Sumiko KIMURA and Makio TOKUNAGA(1Department of Biology, Chiba University)

contain organelle or cellular structures except theInvertebrate connectin (I-connectin) is a 1960 kDa

nUdear envelope. Using the in vitro system together
with single molecule techniques or fluorescenceelastic protein linking the Z line to the tip of the myosin
filament in the giant sarcomere of crayfish claw closer

resonance energy transfer should provide aninnovative and powerful tool to investigate molecular

muscle (Fukuzawa et aI., EMBO ]. 20: 4826-4835 (2001).

mechanisms of transport.

BIOLOGICAL MACROMOLECULES LABORATORY79

(7) Molecular Imaging of Translation Initiation Factors
for Their Transport and the Local Translation in
Neuronal Dendrites
Hiraku MIYAGI, Nobuyuki SHIINA and Makio
TOKUNAGA
The learning and memory is widely believed to be
associated with local plastic changes of synapses,
which requires the translation of new proteins. Someof the proteins are believed to be translated locally at
the activated dendritic synapses. We are going tovisualize translation initiation factors to investigate
how they are transported to synaptic sites in dendrites
and when and where the translation is initiated locallyin the dendritic synapses.
Translation initiation is triggered by binding ofeukaryotic translation initiation factor (elF) 4G withelF4E. Imaging of the binding should reveal the timing
and location of the protein translation after thesynaptic activation. We constructed vectors forexpression of eIF4E-GFP, CFP, YFP and eIF4G-GFP,
CFP, YFP fusion proteins. The elF4E and elF4G fusion
proteins were bound to endogenous elF4G and eIF4E,
respectively, in CHO cells, suggesting that the fusion
proteins function in vivo. These constructs will betransfected into cultured neurons. We are ~anning toobserve the dynamics of the fusion proteins uSing
single molecule microscopy, and the binding of elF4E
and elF4G fusion proteins by some methods such as
FRET (Fluorescence Resonance Energy Transfer).
Publications
1. Hirakawa, Y., Suzutoh, M., Ohnishi, H., Shingaki,T., Erying, E.M., Tokunaga, M. and Masujima, T.:
Analysis of the Nano-Kinetic Movement of a Single
DNA by a Pin-Fiber Video Scope. Analytical Sciences18, 1293-1294, 2002.

2. Fukuzawa, A., Hiroshima, M., Maruyama, K.,
Yonezawa, N., Tokunaga, M. and Kimura, S.: Single
Molecule Measurement of Elasticity of Serine-,
Glutamate- and Lysine-Rich Repeats of Invertebrate
Connectin Reveals That Its Elasticity Is Caused
Entropically by Random Coil Structure. ]. Mus. Res.
Cell Mot., 23, 449-453, 2002.

80

BIOLOGICAL MACROMOLECULES LABORATORY

H-b. MOLECULAR BIOMECHANISM LABORATORY(1) The promoter arrest of E. coli RNA polymerase andthe effect of the Gre factors
Shouji YAGI 1, Motoki SUSA 1, Tomoko KUHORI l , Hiroki
NAGAI l and Nobuo SHIMAMOT0 1 (lStructural Biology
Center, National Institute of Genetics and andDepartment of Genetics, School of Life Science, The
Graduate University for Advanced Studies)
Transcription initiation has been conventionallyassumed to be a sequential process that is composedof several steps, although additional steps can exist
in parallel. The in vitro initiation from T7Al promoter
in particular apparently behaves in a manner that canbe fully explained by the sequential pathway. However,
the initiation from APRAL promoter has been shown
to follow the branched pathway, where a part of the
enzyme-promoter complex is arrested at the promoter,raising the question as to which mechanism is general.
We found that a moribund complex, which is generated
in the arrested branch, is formed at the T7Al promoter
especially in low salt condition, indicating that theinitiation mechanism for this promoter is alsobranched. The results of DNA footprinting suggested
that holoenzyme in moribund complex is dislocated on
DNA from its position of productive complex. However,
only a small fraction of the binary complex becomesarrested at the promoter and the interconversion
between subspecies of binary complex is apparently
more reversible than at the APRAL promoter,explaining the reason why the reaction pathway looks
sequential. These findings suggest a generality of the
branched pathway mechanism, which would resolve
contradictory observations that have been reported forvarious promoters (Susa et aI., 2002).
We next addressed the question whether the
branched pathway has physiological significance inE. coli, because the both promoters we have examined
are bacteriophage promoters. The clue is the effect of
GreA and GreB on promoter arrest, which waspreviously found in the initiation at the APRAL
promoter. These two proteins relieve the arrest at thepromoter by increasing the conversion of moribund
subspecies of binary complex into productive one. By
using genomic DNA array we selected candidate genes
whose expressions are decreased in a strain with

dusrupted greA and greB. By confirmation with
Northerm blotting, I discovered that mRNA levels ofat least 3 genes, cspA, rpsA and alp, are decreased bydisruption of greA and greB. Their transcriptions wereenhanced in a purified reconstitution system by adding
GreA or GreB. Among them, alp operon was examinedmost detail. The formation of moribund complex at thealp promoter, the major promoter of alp operon, wasconfirmed by the most sensitive kinetic assay. The
transcription showed littele pause and GreA and GreB
were shown to enhance transcription through initiation
efficiency by mitigating the promoter arrest. Thesefindings suggest the pleiotropic existence of the
branched pathway in E. coli cell. They also provide a
new concept that the Gre factors are bonafide initiation
factors, although they were discovered as elongation
factors that mitigate elongation arrest in vitro.
(2) Inactivation of(770

by oligomerization andidentification of the role of its spacer region
Usha PADMANABHAN 1, Hiroki NAGAI!, Taciana
KASCIUKOVIC 2, Richard S. HAYWARD 2, Yumiko SAT0 1
and Nobuo SHIMAMOT0 1 (lNat. Inst. bf Genet./Grad.
School of Adv. Stud., 22lnst. Of Cell and Molec. BioI.,
Edinburgh Univ., Scotland)
We have found that (J70, the major (J factors of E.

coli, forms aggregate in vivo and in vitro at a hightemperature within physiological condition. The
oligomeric (J 70 has little transcriptional activity and theoligomerization in vitro showed a sharp temperaturedependence. We have constructed a strain with adisrupted rpoD ( (J 70), and plasmid born (J 70 supports
its growth. We have observed a positive correlation
between the intracellular concentration of (J70 andupper limit of growing temperature. This raises a
possibility that (J 70 is a molecular thermometer.
The major (J factors of proteobacteria mostly havea big spacer region between the conserved regions 1
and 2 which is not conserved in eubacteria. In E. coli
(J 70 this region has extensive acidic patches which may
be concerned with the property of (J 70 to readily interact
with nonspecific and specific surfaces. An
rpoD-disrupted strain was constructed to test theviability of strains expressing plasmid-borne mutated
rpoD or another sigma factor. E. coli rpoS and M
lubeculosis sigA failed to complement the disruption,
and we are now testing B. sublilis sigA and others. The

mutant (J70 lacking the spacer region of aa130-374
complemented the disruption at 30 and 25° C, provingthat the region is not essential for growth at low
temperature. At all tested temperature this protein
predominantly exists as aggregate which are in
equilibrium with a small fraction of monomer.
Therefore, the role of the spacer region is themaintenance of active monomeric form.
(3) Existence of tracking-. a groove of DNA by RNA
polymerase during its sliding
Kumiko SAKATA-SOGAWA and Nobuo SHIMAMOTO
Structural Biology Center, National Institute of
Genetics, Yata 1111 Mishima, Shizuoka 411-8540, Japan.
As the start of transcription of a gene, most RNA
polymerase molecules first bind to sites other thanpromoters where the enzyme initiates transcription.
They then reach promoters by sliding along DNA.
Sliding of proteins along DNA is one-dimensional
Brownian motion, the existence of which has been
proved for E. coli RNA polymerase and several otherDNA binding proteins by single-molecule dynamics
as well as kinetics. There are two possible modes ofmoyemen~uringsliding. In the first mode, a protein
molecule slides along the axis of DNA helix and thus
contacts to one side of DNA. In the second, a protein
molecule tracks a groove of DNA and makes a helicalmotion around the axis. We addressed the question
whether or not RNA polymerase molecule tra<;k agroove of DNA during sliding.
We first developed a devise for detecting therotational movement of a DNA molecule around its
helical axis (Shimamoto, 2002). A nonspecific DNA is
fixed to a 900 nm polystyrene bead coated withstreptavidin by several biotin residues with norotational freedom. A 90 nm fluorescent particle iscovalently attached to the polystyrene bead to visualize
the rotational motion of the DNA-fixed beads undera fluorescent microscope. The polystyrene bead is heldwith 0v.tical tweezers at a distance from the surface
so that it can rotate almost freely. We can examine theexistence of the groove-tracking with this devise. Whena DNA molecule fixed to a bead binds to an RNA
polymerase molecules moving in a direction, its sliding
with a groove-tracking should generate a rotation in
one direction of DNA molecule that can be detectedwith the devise. In this configuration, we moved RNA

MOLECULAR BIOMECHANISM LABORATORY

81

continuously in one direction. If RNA polymeraseof the Branched Pathway in Transcription Initiationby E. coli RNA Polymerase., J BioI Chern 277,

tracks a groove, it should rotate DNA and the rotation

15407-15412, 2002.

polymerase relative to the DNA by shifting the slide

can be detected as the movement of the fluorescentbead.
We have observed rotational motions of the beads

Meetings
1. Shimamoto, N.: "What are necessary for simulatingthat could be caused by groove tracking of RNA

biological events?" Temporal order of biological events:

polymerase in a small number of cases. Such motions

Orders and disorders." Kitazato University, January

were observed only when the distances between beads
and the surface are smaller than the length of DNA.

17,2002.

However, the number of such observation is notenough to conclude groove tracking. A short lifetime

2. Shimamoto, N.: "The principles of nano-world: A
pitfall in nanobiology and nano-technology."of sliding complex, about 1 sec in solution, complicates

Nano-technology: A wonder, KAST Science Seminar,

the estimation of the frequency of rotational motions.

Kawasaki, February 28, 2002.

Further quantitative determination proved theexistence of groove tracking.

3. Shimamoto, N.: "The principles of biological(4) Applicability of thermodynamics to equilibria inmolecular machines that are different from those of theconventional machines: A pitfall in nanobiology."

biology

Towards the boundary of Material Science andBiological Science, Okazaki, March 4, 2002.

Nobuo SHIMAMOTO and Jun-ichi TOMIZAWA
4. Sakata-Sogawa,

K.

and

Shimamoto,N.:

Most DNA-binding proteins are biologically"Single-molecule detection of groove-tracking of DNA

functional as a specific complex, one containing a

by RNA polymerase during sliding." The 7th Asian

special short DNA segment. Such a complex is usually

Conference on Transcription, Kuala Lunpur, Malaysia,

assumed as a state tenable for thermodynalfic analysisof binding equilibrium. Thus, forward and backward

July 23-27, 2002.

reactions should balance at equilibrium in every

5. Ampaabeng, K. G., Padmanabhan, U. andpathway, and the affinity should be independent of thelength of DNA. However, we have found that the

Shimamoto, N.: "Monitoring the state of sigma-70 invivo." The 7'1. Asian Conference on Transcription,balance at equilibrium is broken for some proteins by

Kuala Lunpur, Malaysia, July 23-27, 2002.

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

6. Shimamoto, N.: "Effect of being molecules
uncovered form single-molecule based observations."
The summer school for young biophysicists, Kobe, July
29,2002.

does not satisfy the second law (the ergodic condition)

7. Shimamoto,and thus is disqualified for thermodynamic analysis.
A general treatment of binding equilibrium, while

by RNA polymerase during sliding." The annual

maintaining the specific complex as a distinct state,is proposed on the base of the master equqtion or
chemical kinetics.
.

N.

and

Sakata-Sogawa,

K.:"Single-molecule detection of groove-tracking of DNA
meeting of Bio-imaging Society of Japan, Nagoya,November 1, 2002.
8. Sakata-Sogawa, K. and Shimamoto, N.:"Single-molecule detection of groove-tracking of DNA

Publications
1. Shimamoto, N.: Movements of RNA polymerase

by RNA polymerase during sliding." The annual

along DNA. In Methods Enzymology (San Diego,
Elsevier Science), pp. in press, 2002.

meeting of Biophysical Society of Japan, Nagoya,November 3, 2002.

..

2. Susa, M., Sen, R. and Shimamoto, N.: Generality

82

MOLECULAR BIOMECHANISM LABORATORY9. Shimamoto, N. and Miyawaki, A.: Workshop "A
new biology with new probe techniques." The annual
meeting of Molecular Biology Society of Japan,
Yokohama, December 13, 2003.
10. Sakata-Sogawa, K. and Shimamoto, N.: "Detectionof tracking a DNA groove by RNA polymerase duringsliding using DNA single molecule manipulation." Theannual meeting of Molecular Biology Society of Japan,
Yokohama, December 13, 2003.

H-c. MULTICELLULAR ORGANIZATION LABORATORY(1) Analysis of synthetic dauer-constitutive mutants inthe nematode Caenorhabditis elegans
Kiyotaka OHKURA, Tomoko YABE, Akiko KAMAMOTO,
Takeshi ISHIHARA and Isao KATSURA
If newly hatched larvae of C. elegans are in a
crowded state and given a limited food supply, theysense the environmental signal of pheromone and foodwith a head sensory organ called amphid, and deviate
from the normal life cycle, ending in non-feeding larvae
called dauer larvae. Since the assay of dauer formation
is much less time-consuming than behavioral assays
such as chemotaxis assays, we are analyzing thepathway of sensory signals in the amphid neuralcircuit by detecting dauer formation as the output. We
found that mutations in more than 50 known genes
show synthetic dauer-constitutive (Sdf-c) phenotypes,
i.e., they induce dauer larva formation in certain mutant
backgrounds, regardless of the environmental
conditions. The synthetic nature of the phenotype, we
think, is based on the pathway of sensory signals.
Namely, the signals are transmitted through parallel
routes, and therefore two mutations are required toblock them. Weare determining the combinations ofmutations for the Sdf-c phenotype and the pattern ofsuppression of the Sdf-c phenotype by various
suppressor mutations. In this way we hope to elucidate
the pathway of sensory signals for dauer regulation.
Furthermore, to identify new genes required forthe sensory signal transduction, we isolated andmapped 44 mutations that show the Sdf-c phenotype
in the unc-31(e169j background, where unc-31 geneencodes CAPS protein, which acts in secretion fromdense core vesicles. Eight of the mutations mapped in

4 known genes, but most of the remammg 36
mutations, which map at least in 13 genes, seem to belocated in novel genes, which we named sdf genes.
Some of the mutant genes have been cloned. sdj-13 andsdj-9 genes encode a homolog of the mammalian Tbx2
transcription factor and a tyrosine phosphatase-likemolecule, respectively.
In the year 2002, the following results wereobtained.
(a) The dauer larva of sd/9 mutants was different fromthe normal dauer larva but resembled the dauer-likelarva of daj-9 and daj-12 dauer-constitutive mutants.
Like these mutants, the dauer-constitutive phenotypes
of sdj-9mutants were greatly enhanced by cholesterol
deprivation. Epistasis analyses, together with therelationship between sdj-9 mutations and daj-9
expression, suggested that SDF-9 increases the activityof DAF-9 or helps the execution of the DAF-9 function.
SDF-9 was expressed in two head cells in which DAF-9
is expressed. By their position and by genetic mosaic
experiments, we identified these cells as XXXL!R cells,
which were known as embryonic hypodermal cells andwhose function at later stages was unknown. Killingof the sdj-9-expressing cells in the wild type first-stage
larva induced formation of the dauer-like larva. Since
this study on SDF-9 and former studies on DAF-9'f
showed that the functions of these proteins are related
to Steroids, XXXL/R cells seem to playa key role inthe metabolism or function of a steroid hormone(s) thatacts in dauer regulation.
(b) We cloned sdj-14 gene and found that it was thesame as mrp-l gene, which was formerly identified byits homology to multidrug resistance protein genes inother organisms. Thus, we found a function of mrp-l
concerning dauer larva formation. An sdj-14::GFP
fusion gene that can rescue sdj-14 mJltant phenotypes
was expressed in neurons, pharynx and intestine. The
expression of sdj-14 gene in each of them by extrinsic
promoters resulted in partial suppression of the Sdf
phenotype, while expression in two of the three tissues/organs resulted in more efficient suppression. It seems
that expression in all the three parts contributes to thewild type phenotype. Since there are three isoforms of
SDF-14 made by alternative splicing, we are now
investigating the difference between these isoforms infunction and expression.
"

MOLECULAR BIOMECHANISM LABORATORY

83

(2) Molecular genetic studies on sensory integration andbehavioral plasticity in C, elegans
Takeshi ISHIHARA, Yuichi IINO l , Akiko MOHRI 2, Ikue
MORI 2, Keiko GENGYO-AND0 3, Shohei MITANP and Isao
KATSURA rMolecular Genetics Laboratory, Universityof Tokyo, 2Division of Biological Science, Nagoya'
University, 3Department of Physiology, Tokyo
Women's Medical University School of Medicine)
Animals receive environmental cues, select andintegrate necessary informaTion, and make proper
responses, while aU these steps can be modified byexperience or memory. In C.elegans, many behavioral
mutants defective in chemotaxis and thermotaxis, forinstance, have been isolated and analyzed, and themolecular mechanisms of sensation have been
elucidated. On this basis and as a next step, we are
analyzing mutants that show abnormality in thelearning and selection (evaluation) of sensory signals,
to elucidate novel mechanisms of higher order sensory
signal processing.
C. elegans shows avoidance of copper ion andchemotaxis to odorants by receiving these stimuli withdifferent sensory neurons in the head. We developed
a behavioral assay for the interaction of two sensory
signals: aversive copper ion and attractive odorant,
diacetyl. Wild-type animals change their preference
between the responses, depending on the relative
concentration of copper ion and odorants. On the basisof the C. elegans neural circuitry, the result suggeststhat the two sensory signals interact with each otherin a neural <:ircuit consisting of about 10 pairs ofneurons. While well-fed animals are usually used forthis assay, we found that animals starved for 5 hours
tend to prefer chemotaxis to odorants. The change isdue to the desensitization of copper ion avoidance by
starvation, and' can be suppressed by serotonin, whichmimics the effect of food. This desensitization isadvantageous in natural environment, because starved
animals can search for food over a wider area.
To elucidate the mechanism of sensory integrationin the neuronal circuit, we are isolating and analyzing
mutants that show abnormality in this assay. The
hen-l(ut236) mutant showed much weaker tendency
to cross the Cu2+ barrier when migrating toward
attractive odorants than the wild type, although thatthe hen-l(ut236) mutant has defects neither in the
chemotaxis toward the attractive odorant nor in the
avoidance of Cu'+ ion per se.

•

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MULTICELLULAR ORGANIZATION LABORATORY

To elucidate molecular mechanisms for the sensory
integration, we cloned the hen-l gene and found thatit encodes a secretory protein with an LDL receptor
ligand binding domain. Immunostaining by using
antibody against recombinant HEN-l protein revealed
that the gene product is localized in the axon and cellbody of each one pair of sensory and inter-neurons.
The localization in the axon was abolished in unc-104(kinesin KIFIA homolog) mutants, which show defects
in the transport of synaptic vesicles. Expression studies
with various promoters showed that this gene acts
non-ceIl-autonomously in the mature nervous system.
The hen-l(ut236) mutant also shows abnormality inlearning by paired presentation of starvation and NaCI
(collaboration with Dr. lino, University of Tokyo) andby paired presentation of starvation and temperature(collaboration with Ms. Mohri and Dr. Mori, Nagoya
University). Wild type animals show chemotaxis to
NaCI under a well-fed condition, although they avoid
NaCI after conditioned with starvation and NaC!. The
hen-l(ut236) mutant showed a weaker behavioral
change than the wild type after the conditioning,although they show normal chemotaxis to NaCI undera well-fed condition. Wild type animals prefer thecultivation temp~rature under a well-fed condition,
while they avoid that temperature after conditioned in
the absence of food at the same cultivation
temperature. Although the hen-l(ut236) mutant shows
normal thermotaxis under a well-fed condition, it does
not avoid the cultivation temperature after conditioned
in the absence of food. Since starvation was used toinduce plasticity in both learning assays, we analyzed
whether hen-l(ut236) animals can sense starvation, but
we could not find any abnormality in the behavior after
simple starvation. These results indicate that thehen-l(ut236) shows defects in the behavioral plasticity
after paired presentation of starvation and NaCI or
starvation and temperature, although it responds
normally to each of these stimuli.
We also studied on another mutant ut235. Thismutant behaved essentially normal in many other
responses to starvation, such as the change oflocomotion speed on food by starvation. Interestingly,
the double mutant ut235;hen-l showed a preference to
avoidance of copper ion, regardless of starvation. We
are now trying to clone the gene for this mutation bypositional cloning.
In 2002, we isolated and analyzed the deletion alleleof hen-l, tm501. The hen-l(tm501) was isolated byscreening pools of TMP!UV mutagenized worms using
PCR (collaboration with Dr. Gengyo-Ando and Dr.
Mitani, Tokyo Women's University of Medicine). It has
a small deletion within the hen-l gene and seems a nullallele. The behavioral analyses of the hen-l(tm501)
revealed that its phenotypes are almost indistinguishable
from those of hen-l(ut236) in the sensory integrationand in the behavioral plasticity.
Molecular genetic analyses of the HEN-1 suggestthat the HEN-l functions as a neuronal modulator for
sensory integration and learning. To elucidate themolecular mechanisms of this neuromodulation, we
start investigating the protein interacting with the
HEN-l protein. First, we developed a binding assay for
identification of receptors for HEN-I. By using a
HEN-I-alkaline phosphatase fusion protein as a ligand,which was expressed by HEK293 cells, we found thatthe HEN-l specifically binds a subpopulation of the
primary culture cells in C. elegans. By using thissystem, we are trying to expression cloning of the
HEN-l receptor from an expression cDNA library of
the primary culture cells of C.elegans.
C. elegans mutants in the associative learning ofodorants and food(3)

Ichiro TORAYAMA, Takeshi ISHlliARA and Isao KATSURAThe nematode C. elegans provides a good system
for the study of associative learning. However, themechanism of this learning looks different from thatof classical conditioning, because (a) the unconditional
stimulus is usually limited to food or starvation, andbecause (b) the learning is efficient, if the conditional
stimulus is presented at the same time as but not
before the unconditional stimulus. To elucidate themolecular mechanism of associative learning in·C.
elegans and food/starvation signaling in such learning,
we are isolating and characterizing mutants that show
abnormality in the associative learning of butanone
and food/stravation. It is known that butanone attracts
wild type worms without conditioning. Conditioning
with butanone and starvation decreased the efficiency
of chemotaxis to butanone, while conditioning withbutanone and food increased it. We isolated mutantsin these behaviors, some of which decreased theefficiency of chemotaxis after conditioning with foodand butanone, while others are attracted efficiently bybutanone only after conditioning with food andbutanone. We are mapping ut305 and ut306, which
belong to the former category. Interestingly, ut305, but
not ut306, showed abnormality in adaptation toisoamyl alcohol and benzaldehyde, which are sensed
by the same type of sensory neurons (AWC) as
butanone. ut305was mapped in the righthand part ofthe linkage group X, while ut306 near the center of thelinkage group V. We are narrowing down the regionsof the mutations and trying to rescue the mutant
phenotypes by microinjection of genomic DNA
fragments from these ~egions.

Fluoride-resistant mutants of the nematode
Caenorhabditis elegans(4)

Akane OISHI, Minoru KA W AKAMI 1, Akiko KAMAMOTO,
Takeshi ISHIHARA and Isao KATSURA (IGothenburg
University)
Fluoride-resistant (flr) mutations of C. elegans arerecessive and grouped into two categories: class 1
mutations, which map in flr-l, flr-3 and flr-4, and class
2 mutations, which map in flr-2 and flr-5 (Katsura, I.
et at.: Genetics 136,145-154,1994). Class 1 flrmutations
show diverse phenotypes: slow growth, short
defecation cycle periods, frequent skip of the expulsion
step of defecation, and synthetic abnormality in dauer
formation, besides strong resistance to fluoride ion.
The flr-l gene encodes an ion channel belonging to the
DEG/ENaC (C. elegans degenerins and mammalianamiloride-sensitive epithelial sodium channels")superfamily, while flr-4 and flr-3 code fo! a novel Serf
Thr protein kinase and a kinase-like molecule,respectively, both having a hydrophobic domain on the
carboxyl-terminal side. A functionalflr-l::GFP fusion
gene is expressed only in the intestinal cells from thecomma stage of embryos to the adult stage, while
flr-4::GFP is expressed in the intestinal cells from the
I.5-fold stage, in the isthmus of the pharynx from the
3-fold stage and in a pair of head neurons called AUA
from Lllarvae to adults. Moreover, the expression ofvarious Jlr-3::lacZ and Jlr-3::GFP fusion genes is
detected only in the intestine. We therefore think thatclass 1 flr genes constitute a regulatory system thatacts in the intestine and that controls many
food-related functions. Class 2 flr mutations, which
confer weak resistance to fluoride ion, suppress theslow growth and dauer formation abnormality but not
the defecation abnormality and strong fluoride-•

MULTICELLULAR ORGANIZATION LABORATORY

85

resistance of class 1 flrmutations. Hence, it seems thatthe regulatory pathway bifurcates after class 1 genes,
and that class 2 genes control only one of the two
branches. Of the class 2 genes, flr-2 encodes a secretory
protein belonging to the gremlin/DAN/cerberus family.
On the basis of these results, we have made a modelin which a signal controlled by class 1 genes istransmitted from the intestine and represses the actionof class 2 gene products in the head nervous system.
In the year 2002, we studied expression of afunctional flr-2::GFP fusion g~e, using an improved
GFP. The expression was eIetected in many pharyngeal
neurons and some head and tail neurons. This method
was more sensitive than the antibody staining, which
was carried out a few years ago. We started
investigating whether FLR-2 binds to a subpopulationof C. elegans primary culture cells.
Publications
1. Ishihara, T., lino, Y., Mohri, A., Mori, I.,

Gengyo-Ando, K., Mitani, S. and Katsura, I.: HEN-I,
a novel secretory protein with an LDL receptor motif,
regulates sensory integration and learning in C.
elegans. Cell 109, 639-649, 2002.
2. Kuhara, A., Inada, H., Katsura, I. and Mori, I.:
Negative regulation and gain control of sensory
neurons by the C. elegans calcineurin TAX-6. Neuron
33, 751-763, 2002.

H-d. BIOMOLECULAR STRUCTURE LABORATORY(1) Crystallographic Study of F1-ATPase

Yasuo SHIRAKIHARA, Aya SHIRATORI and Chikako
SHIRAKIHARA

F1-ATPase is the catalytic sector in ATP synthasethat is responsible for ATP production in living cells.
F1 has molecular mass of 380,000 dalton and a subunit
composition of 0:3/33yOE. The unique rot~tional
catalysis mechanism of F1 includes rotation of therod-like y subunit, which is thought to control theconformations of the three catalytic /3 subunits in acyclic manner by its rotation. We have recently moved
to the structural study of the 0:3/33yOf sub-assemblyof thermophilic F1, after solution of the structure of
the 0:3/33 sub-assembly and committed attempts to
4

86

MULTICELLULAR ORGANIZATION LABORATORY

crystallize the 0:3/33y sub-assembly. Employing the
0:3/33y f sub-assembly that show catalytic properties
very similar to those of F1, we aim to detect structuralchanges caused by different nucleotide occupancy,
which should provide with structural basis forunderstanding the rotational catalysis mechanism.
The most recent structure we have obtained is thatof the 0:3/33y f sub-assembly with Mg-free ADP boundto only one /3 -subunit. The structure is unique, as thisis the first F1 structure that has one nucleotide-filledcatalytic site; all the previous structures have two or
more nucleotide-filled catalytic sites. Interpretation of
the structure was not straightforward due to lack ofbound Mg ion; there has not been much biochemical
evidence on the Mg-free state. However, based on suchsparse evidence collected together, we now believe thatthe structure represents a weakly inhibited state of F1
with the 0:3/33y portion in a state not far from an active
state but with the E -subunit in an inhibitory state. Oneof the features of the structure is that this structure
lacks the most active /3 -subunit conformation that ischaracterized as having the tightly closed catalyticinterface and has been observed in all the previous F1
crystal structures with two or three nucleotide-bound/3 -subunits. The feature may explain the very slow
catalysis in the uni-site mode (defined as having one
nucleotide-bound catalytic site). This structuraldifference in the 0:3/33 part appears to be coupled tothe substantial structural differences in the stalk,including penetration of the C-terminal helices of theE -subunit into the 0:3/33 cavity. The new conformationof the C-terminal helices of the E -subunit explains well
the weak f -inhibition in the thermophilic F1.
We have further examined the preparation method
for the 0:3/33YE sub-assembly, because the recent
preparations (over 1 year period) had refused to
crystallize, especially in presence of Mg ion.
Combination of a controlled heat treatment withchromatography using hydrophobic and ion-exchange
columns produced a preparation that restored theoriginal crystallization capability. Neither an additional
second heat treatment nor gel-filtration chromatography
gave significant benefits. Using crystals from our new
preparation, further cooling experiments were
performed. The previous difficulty in cooling the
crystals grown in presence of Mg has gone, partly
because of benefits of using the better preparation andpartly because of our new cryo conditions. Further
structural study is in progress.

These structure studies were done in collaboration
with Toshiharu Suzuki and Masasuke Yoshida, atResearch Laboratory of Resource Utilization, Tokyo
Institute of technology.
(2) Comprehensive Crystallographic Study of Transcription factors and Genome-partitioning Factors from E.
coli
Yasuo SHIRAKIHARA, Aya SHIRATORI, Hisako INOUE and
Megumi SEKI
More than 160 transcription factors have been
identified in E.coli, and they control transcription of
their target gene(s) in either positive or negative
manners, by binding to both their specific DNA
sequence and the transcription apparatus. Sixty-fivesuch transcription factors have been purified in one
step Ni-NTA column chromatography using carefully
constructed over-expression systems in Ishihama
laboratory. In Niki laboratory, a number of novel
proteins, that are judged to play roles in the genome
and plasmid partitioning from their localization
patterns, have been prepared using similarly
constructed over-expression systems. Setting thesetranscription and genome partitioning factors astargets for structural study, we have started
comprehensive structure determination in the Protein
3000 project (the sub-field of 'transcription andtranslation').
We have developed an efficient and reliable
method for screening crystallization conditions against
the large number of the provided proteins, based on
our long-time experiences in protein crystallization.
Using the system, 34 proteins have been screened sofar (30 transcription factors: AhpC, ArcA, AcrR, CueR,
DeoR, IlvY, KdpE, SdiA, SoxR, SoxS, EmrR, InfB,
LeuO, Mlc, MntR, RscB, RstA,Thil,TufB, AtoC,BaeR,
CheB, QseB, YehT, YfhA, CpxR, DcuR, NarP, EvgA,
RcsB and 4 genome & plasmid partitioning factors,
RecR,PspA,YcbW,YgfE). In spite of an initial successof finding small crystals of ArcA and SdiA, most of
the proteins did not crystallize possibly due to theconformational flexibility of the transcription factorsthat commonly have the two-domain architecture (DNAbinding and 'receiver' domains). Crystals ofselenomethionyl derivative of ArcA and heavy atom
derivative (Hg,Pt,Ir,Os) of SdiA were prepared for datacollection at PF for MAD (Multi-wavelength

Anomalous Dispersion) analysis. However all the
crystals gave poor diffraction patterns with the best
result of 8 A resolution data for the native SdiA
crystals. We plan to crystallize the transcription factors
complexed with the cognate DNA, and their constituent
domains.
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) Crystallographic Stud"Y of the Transcription
Activator, PhoB, complexed with cognate DNA
Kazuyasu SHINDOH, Katsumi MAENAKA and Yasuo
SHIRAKIHARA
PhoB Protein is a positive transcriptional activator
to regulate the genes in the phosphate (pho) regulonof E. coli that are induced by phosphate starvation.
PhoB acts by binding to the pho box in the promoter
region, which is the consensus sequence shared by theregulatory regions of the genes in the regulon. Th~
N-terminal region of PhoB is a regulatory domain,
whereas the C-terminal region (PhoBC) has a DNA
binding activity to the pho box specifically. In orderto resolve the detailed interaction between PhoB andpho box DNA, we have made the crystallographic studyof PhoBC complexed with the pho box DNA.
We have got the crystals of PhoBC complexed with
16mer pho box DNA oligomer (ACAGCTGTCAlAAATC) .
in the condition of 0.1 M Ammonium Acetate, 0.01 M
Mg Acetate, 0.025 M Sodium Cacodylate pH 6.5, 15%
w/v Polyethylene Glycol 8000, at 2S'C. The crystals havethe PI space group with the cell dimension, a=37.99
A, b= 48.98A, c=70.30A, ex =108.57', {3 =120°, y=I04.37'(2 molecule per asymmetric unit). The diffraction data
was collected at Photon Factory and Spring8 (around
2.8 A resolution). We have already solved the crystal
structure of free PhoBC by the MAD data from theselenomethionyl derivative of PhoBC. Based on themodel C?t free PhoBC, a full structure determination bymolecular replacement is in progress.
This work has been done in collaboration with
Hideyasu Okamura, Yoshifumi Nishimura, Yokohama
City University and Kozo Makino, Research Institutefor Microbial Diseases, Osaka University.

BIOMOLECULAR STRUCTURE LABORATORY87

(4) Structural and functional studies of immunoglobulin(Ig)-like receptors
Katsumi MAENAKA, Tsukasa SHIMO]IMA, Kirnie AMANo,
Tomoko MOTOHASHI and Yasuo SHIRAKIHARA
The immunoglobulin(Ig) superfamily domains arevery common among the cell surface receptors inimmune system, and so it is important to clarify themolecular recognition of Ig-like cell surface receptors
towards various ligands. We are studying Ig-like
transcript (ILTs) receptor family.
ILTs are expressed mainly on myelomonocytic cells
and some of these can recognize MHCs on target cells
to mediate an inhibitory or activating signals. ILT2 andILT4 studied here can bind to various MHC class I
molecules, shown by cellular based assays, but thedetail interactions in protein level are not clear. We
produced all recombinant proteins (ILT2, ILT4 andMHC ligands) by E.coli expression and refolding
system. Surface Plasmon resonance (SPR) assays
clearly showed that all receptors bind a wide range of
MHC class I (e.g. HLA-A, -B, -C, and G) with low
affinities (K D (10 4 -10 6M) and fast kinetics, which issimilar to other receptors involved in cell-cellinteractions. Unexpectedly ILT2 and ILT4 can bind to
nonclassical MHC, HLA-G most preferentially incomparison to other MHC ligands. Since HLA-G isexpressed on unique tissue, placenta, thymus and sometumours, this result may suggest a predominant roleof ILTs on regulation of immune cells in these special
areas. We have also done the thermodynamics for theseinteractions.
This work has been done in collaboration with
Anton van der Merwe, Yvonne Jones, (University of
Oxford), and Izumi Kumagai, Kouhei Tsumoto,
Mitsunori Shiroishi (Tohoku University).
(5) Structural analysis of glutaminase from Micrococcus luteus K-3
Yasuo SHIRAKIHARA and Aya SHIRATORIGlutaminase (EC 3.5.1.2) catalyzes the hydrolytic
degradation of L-glutamine to L-glutamic acid. The
salt-tolerant glutaminase from marine Micrococcus
luteus K-3 has an unusual property that its maximum
activity is observed at about 2M salt, and also has
industrial relevance that this enzyme may be more
efficient than the conventional ones in the soy sauce

88

BIOMOLECULAR STRUCTURE LABORATORY

fermentation that is carried out in the high-salt
environments.
After analysis of MAD data using the Se-Met
derivatives for the intact form (2.6A. resolution) and thetruncated form (2.4 A. resolution) of the enzyme done
last year, model building of the truncated form wasdone and now refinement has started. We plan toutilize the model in determining the structure of theintact enzyme by molecular replacement.
The structural study was done in collaboration
with Kazuaki Yoshimune and Mitsuaki Moriguchi at
Oita University, and Mamoru Wakayama at
Ritsumeikan University.
Publications
Shindoh, K., Maenaka, K., Akiba, T., Okamura, H.,
Nisimiura, Y., Makino, K. and Shirakihara, Y.:
Crystallization and preliminary X-ray diffraction
studies on the DNA-binding domain of thetransciptional activator protein PhoB from Escherichia
coli:, Acta Crystallogr D 58, 1862-1864, 2002.

mainland Japan. We collected the three morphologicaltraits (height, weight, shank length) and the molecular

I. CENTER FOR INFORMATION BIOLOGYAND DNA DATA BANK OF JAPANsamples of these various Shamo. The results of themorphological studies were consistent with our
phylogenetic analysis. In particular, the Okinawagroup showed bigger values of the height, weight andshank length than the other Shamo groups. Thisobservation suggests that Japanese Shamos might beof two independent origins. It is also known that

I-a. LABORATORY FOR DNA DATA ANALYSIS(1) Genetic variation of Japanese gamecock and its
implication to the cultural background of cockfighting.
Tomoyoshi KOMIYAMA, Kazuho IKEO and Takashi
GOJOBORI
The tradition of cockfighting is widespreadthroughout the world. Chickens, including gamecocks
were domesticated from the Red Jungle fowl (Gallus
gallus gallus) in Southeast Asia at an early stage ofhuman prehistory. Originally cockfighting may havebeen held for the purpose of religious ceremony orentertainment. There is no doubt that the gamecockhas evolved together with the human culture of
cockfighting a long time ago. In Japan, there is a groupof gamecock called the "Shamo" which are used
specifically for cockfighting. From archive study, we
found that gamecocks were already known at leastfrom approximately 1,100 AD. However, the geographic
distribution of cockfighting and the influx route ofgamecocks into Japan are totally unclear. The
molecular evolutionary study of gamecocks isobviously to gain useful understanding of the processof distribution of the culture itself. Through the studyof gamecocks, we aim to throw new light on theevolution of human culture. For the purpose of thisstudy, we collected blood samples of gamecocks from
11 different prefectures in Japan. A phylogenetic tree
was constructed using a total of 46 mt DNA (D-Ioop,
1l00bp) sequences including the data from theInternational Nuclear DNA database (DDBJ/EMBL!
GenBank). The phylogenetic analysis showed that theJapanese Shamos were separated into two different
groups with high bootstrap value: one group contained
only individuals from the island of Okinawa and theother group is composed of samples from Kyushu anddifferent names are used for Shamo in Japan. In
Okinawa Island, for example, "taucii" is used as a namepronouciation of which is quite similar to Chinese. In
the other part of Japan, other word is used for Shamo,
and it is very similar to the old name meaning"Thailand." These observations also supports thetheory of separate origins of Japanese Shamos; theorigins of China and Southeast Asia.
(2) EST analysis of a normalized library derived from
5-day regenerating planarian.
Jung Shan HWANG, Katsuhiko MINETA, Kazuho IKEO
and Takashi GOJOBORI
Planarian has been widely known for its
regenerative power. An adult planarian can be cut into
200-300 pieces and each piece would eventually
regenerate into an individual. Approximately 10-14
days are required for the whole regenerating process.
Although planarian regeneration had been studied formore than 200 years, no researcher has a clue of what
determines the developmental plasticity in planarianand how did it arise during the evolution. At thecurrent stage, one possible approach to be used is todisclose all candidate molecules that involve in theprocess of regeneration and then make a comparison
to the developmental/regenerative process of otherorganisms.
A normalized cDNA library was constructed from
5-day regenerating body parts which were cut
non-specifically at different body levels. Approximately
5000 clones were isolated and the number of
non-redundant clones was reduced to its one third. By
using the non-redundant ESTs, we identified genesthat participate in the regenerative process bycomparing them with homologous genes from thedatabank. From the studies, we (1) categorized clones
that are related to planarian regeneration into several
classes, (2) identified clones which show significant

CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPAN89

similarity with those related to the development ofhigher organisms, and (3) examined the expression
patterns of clones by whole mount in situ hybridizationand some clones potentially can be used as molecularmarkers. A 5-day regeneration database of planarian
will be set up in the near future.
(3) Analysis of evolutionary relationships between
interacting proteins in S. cerevisiae
Takashi MAKINO, Kazuho IKEO, Yoshiyuki SUZUKI and
Takashi GOJOBORI (National Institute of Genetics,
Laboratory for DNA Data Analysis)
Recently, large scale approaches have predicted
many new protein interactions in S. cerevisiae. However,there are not protein interactions datasets in the otherorganisms. Therefore we analyzed evolutionary
relationships between interaction of proteins bycomparing the proteins of S. pombe with those of S.
cerevisiae. Core, S. cerevisiae interactions in PPI
database DIP that are assessed of their accuracy, isused for this analysis. Each protein included in Core,
has been subdivided into functional categ;ories using. a catalogue of known and predicted protein functions
at database MIPS.. To investigate the distribution ofinteractions, we compared S. pombe with S. cerevisiae
in amino acid sequences and estimated theevolutionary distances between each homologues.
The results indicate that the proteins of related
functions interact with each other that tend to be
exposed to similar selection.
~.
(4) Estimation of loss of functions in metabolic pathways
by comparison of 94 species
Tsuyoshi TANAKA, Kazuho IKEO and Takashi GOJOBORI
There are two different mechanisms, which are thegain of functions and the loss of functions, for theevolution of metabolic pathways. However, the reports
related to the loss of functions are few compared withthe gain of functions, the gain of function is not enoughto explain the evolution of metabolic pathways. Then,
to evaluate more detail effect on the loss of functions
in metabolic pathways, we estimated the number of theloss of functions during the evolution. We used 94
species and 1507 types of Enzymatic Reactions (ERs)for estimating the number of the loss of functions in

90

LABORATORY FOR DNA DATA ANALYSIS

metabolic pathways. Each ER is corresponding to aparticular EC number. To evaluate the loss of functions
by comparing the existence of ERs among species, we
examined how many ERs existed in each species andhow many ERs species shared. As a result, the number
of ERs in each species was ranging from 101 to 744.
Moreover, only 5 out of total ERs were commonly
observed in all 94 species and about 80% of total ERs
existed only in a half of them. We also estimated thenumber of ERs which existed in ancestral species byusing phylogenetic trees and one assumption. We
assumed that an emergence of functions of an ER was
only once during evolution of 94 species. As a result,the number of ERs in ancestral species was speculated
as more than 377. Moreover, when we summed up thenumber of loss of functions of ERs during the evolutionof 94 species, there were 10792 times in 1042 kinds ofERs. These results suggested that the ERs in metabolic
pathways changed among species and the loss of. functions in ERs has occurred many times during theevolution of metabolic pathways.
Hisakazu IwAMA and Takashi GOJOBORI
Center for Information Biology and DNA Data Bankof Japan, National Institute of Genetics, Mishima-shi
Neurotransmitter receptors (neuroreceptors) areclassified into two types, G protein-coupled receptors(GPCRs) and ligand-gated ion channels. The former
occupies a small part of the large GPCR superfamily,
whereas the latter consists of three superfamilies.
In these superfamilies, humans and rodents sharealmost the same set of neuroreceptor genes. This
neuroreceptor gene set is good material to examine thedegree of selective constraint exerted on each member
gene of a given superfamily. If there are any
neuroreceptor genes under the degree of selective
constraint that is very different from that of the othermember genes, they may have influenced the functionalfeatures characteristic of human neural activities. With
the aim of identifying such neuroreceptor genes, wecollected sequence data of orthologous neuroreceptor
genes for humans, mice, and rats by database searches.
This data set included ortholog pairs for 141 kinds ofneuroreceptor genes, which covered almost the whole
set of neuroreceptor genes known to be expressed in
the human brain. The degree of selective constraint
was estimated by computing the ratio (dNI d,) of thenumber of nonsynonymous substitutions to that ofsynonymous substitutions. We found that the liN/ ds
ratio ranged widely and its distribution fitted a gamma
distribution. In particular, we found that fourneuroreceptor genes are under the significantly relaxed
selective constraint. They are an ionotropic glutamate
receptor subunit NMDA-2C, two GABAA receptor
subunits, i.e., GABAA - E and GABAA - e, and a dopamine
receptor D 4• Interestingly, these neuroreceptors havebeen reported to be associated with cognitive abilities
such as memory and learning, and responsiveness tonovel stimuli. These cognitive abilities can influence
the behavioral features of an individual. Thus, itsuggests that the relaxed-constraint neuroreceptor
genes have evolved, assuring that the nervous system
responds to a variety of stimuli with proper flexibility.
(5) The genome sequence and structure of rice
chromosome 1.
Sasaki T, Matsumoto T, Yamamoto K, Sakata K, Baba
T, Katayose Y, Wu J, Niimura Y, Cheng Z, Nagamura
Y, Antonio BA, Kanamori H, Hosokawa S, Masukawa
M, Arikawa K, Chiden Y, Hayashi M, Okamoto M,
Ando T, Aoki H, Arita K, Hamada M, Harada C,
Hijishita S, Honda M, Ichikawa Y, Idonuma A, Iijima
M, Ikeda M, Ikeno M, Ito S, Ito T, Ito Y, Ito Y, Iwabuchi
A, Kamiya K, Karasawa W, Katagiri S, Kikuta A,
Kobayashi N, Kono I, Machita K, Maehara T, Mizuno
H, Mizubayashi T, Mukai Y, Nagasaki H, Nakashima
M, Nakama Y, Nakamichi Y, Nakamura M, Namiki N,
Negishi M, Ohta I, Ono N, Saji S, Sakai K, Shibata M,
Shimokawa T, Shomura A, Song J, Takazaki Y,
Terasawa K, Tsuji K, Waki K, Yamagata H, Yamane
H, Yoshiki S, Yoshihara R, Yukawa K, Zhong H,
Iwama H, Endo T, Ito H, Hahn JH, Kim HI, Eun MY,
Yano M, Jiang J, Gojobori T.
Rice Genome Research Program, National Institute ofAgrobiological Sciences, 1-2, Kannondai 2-chome,
Tsukuba, Ibaraki 305-8602, Japan.
tsasaki@nias.affrc.go.jp
The rice species Oryza sativa is considered to bea model plant because of its small genome size,
extensive genetic map, relative ease of transformationand synteny with other cereal crops. Here we report
the essentially complete sequence of chromosome 1, thelongest chromosome in the rice genome. We summarize

characteristics of the chromosome structure and thebiological insight gained from the sequence. The
analysis of 43.3 megabases (Mb) of non-overlapping
sequence reveals 6,756 protein coding genes, of which
3,161 show homology to proteins of Arabidopsis
thaliana, another model plant. About 30% (2,073) of thegenes have been functionally categorized. Rice
chromosome 1 is (G + C)-rich, especially in its coding
regions, and is characterized by several gene families
that are dispersed or arranged in tandem repeats.
Comparison with a draft sequence indicates theimportance of a high-quality finished sequence.
(6) Exhaustive search for T-box genes in the wholegenome shotgun sequence of Ciona intestinalis
Kohji HOTTA, Hiroki TAKAHAsm1, Yasuaki MOCHIZUKI2,
Takashi GoJOBORI and Nori SATOH2
1. Department of Developmental Biology, National
Institute for Basic Biology, 38 Nishigonaka, Myodaiji,
Okazaki 444-8585, JAPAN2. Department ofZoology, Graduate School ofScience,
Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

T-box genes encode a family of transcription
factors that share an evolutionally,conserved T DNA
binding domain. T-box genes were found among
various animals, nematode, fly, sea-urchin, ascidians,
amphioxus, zebrafish, Xenopus, newt, chick andtpammals, and they make multiple T-box gene family.
Several T-box subfamilies were known (ex. Brachyury,
TBX4/5$"TBX2/3, T-brain, TBX6). Members of the
T-box gene family play important roles in thedevelopment of both vertebrate and invertebrate
embryos, including specification of the mesoderm as
well as heart and limb mo~phogenesis.To know whatkind of T-box genes exist in primitive chordate
ascidian is important for understanding theevolutionary process of the T -box genes and functional
divergence in vertebrates. In this study, we performed
exhaustive search against the Ciona intestinalis wholegenome shotgun sequence data (total 900Mbp; National
Institute of Genetics, Japan and total 450Mbp; Joint
Genome Institute, USA). As a result, seven different
types of 10 T-box genes were found in the Ciona
in testinalis genome. Among them, six predicted genes
have sequence homology with the known ciona T -box
genes (i.e. Ci-Bra, Ci- TBX6, Ci- VegTR, Ci- Tbx2/ 3,
Ci-Tbxl, Ci-mT). The other four T-box genes were

LABORATORY FOR DNA DATA ANALYSIS

91

found as novel T-box genes (Ci-Tbx20, Ci-TBX6a,
Ci-TBX6c, Ci-TBX6d) in the Ciona intestinalis genome.
A Phylogenetic analysis of these genes suggested thatthe evolutionary origin of some of these genes is quite
old, probably before the divergence between
deuterostomes and protostomes. However, there is no
gene which belongs to Tbx4/5 subfamily in Ciona
genome as well as in the other protostomes genomes.
On April 810, some of us took part in the Ciona Genome
Annotation Jamboree (Department of Energy, Joint
Genome Institute, USA). In this Jamboree, we could
confirm above-mentioned data. In addition, we foundthat TBX6b,TBX6c and TBX6d genes were tandemlyaligned on a single scaffold.
This study was reported at 'Molecular Evolutiori'
a Meeting on Evoluion, Genomics, and Bioinformatics(13-16 June, Sorrento Italy). See Dehal et al., (Science
298:2157-67, 2002) for details.
Publications
1. Katayama, K., Shirato-Horikoshi, H., Kojima; S.,

Kageyama, T., Oka, T., Hoshino, F. B., Fukushi, S.,
Shinohara, M., Uchida, K., Suzuki, Y., Gojobori, T. and
Takeda, N.: Phylogenetic .analysis of the complete
genome of 18 norwalk-like viruses, Virology, 299,
225-239, 2002.
2. Iwama, H. and Gojobori, T.: Identification of
Neurotransmitter Receptor Genes under Significantly
Relaxed Selective Constraint by Orthologous Gene
Comparisons between Humans and Rodents,
Molecular Biology and Evolution, 19 N0.11, 1891-1901,
2002.
3. Tanaka, Y., Hanada, K., Mizokami, M., Yeo, A E.
T., Shih, J. W.-K., Gojobori, T. and Alter, H. J.: A
Comparison of the molecular clock of hepatitis C virus
in the United States and Japan predicts that
hepatocellular carcinoma incidence in the United States
will increase over the next two decades, Proc. Natul.
Acad. Sci., 99, 15584-15589, 2002.
4. Fantom Consortium (Okazaki, Y., Gojobori, T., etat.), Riken Genome Exploration Research Group Phase
I & II Team, Mouse Genome Sequencing Consortium,
Scientific management: Hayashizaki, Y.: Analysis of
the Mouse Transcriptome based upon Functional
Annotation of 60,770 full length cDNAs. Nature 420,563-573, 2002.

92

LABORATORY FOR DNA DATA ANALYSIS

5. Elmerot, c., Amason, u., Gojobori, T. and Janke,
A: The mitochondrial genome of the pufferfish, Fugu
rubripes, and ordinal teleostean relationships, Gene,
295, 163-172, 2002.
6. Sasaki, T., Yamamoto, K., Sakata, K., Baba, T.,
Katayose, Y., Wu, J., Niimura, Y., Cheng, Z., Nagamura,Y., Kanamori, H., Hosokawa, S., Masukawa, M.,
Arikawa, K. and Antonio, B. A: The genome sequenceand structure of rice chromosome 1, Nature, 420,
312-316, 2002.
7. Cebria, F., Kobayashi, C,. Umesono, Y., Nakazawa,M., Mineta, K., Ikeo, K., Gojobori, T., Itoh, M., Taira,M., Alvarado, A and Agata, K.: FGFR-related genenou-darake restricts brain tissues to the head regionof planarians., Nature, 419, 620-624, 2002.
8. Cebria, F., Kudome, T., Nakazawa, M., Mineta, K.,
Ikeo, K., Gojobori, T. and Agata, K.: The expressionof neural-specific genes reveals the structural andmolecular complexity of the planarian central nervous
system, Elsevier, Mechanisms of Development 116,
199-204, 2002.
9. K., Gojobori, T. and Agata, K.: The expression ofneural-specific genes reveals the structural andmolecular complexity of the planarian central nervous
system, Elsevier, Mechanisms of Development 116,
199-204, 2002.
10. Isobe, T., Yoshino, M., Mizuno, K.-I., Lindahl, K.
F., Koide, T., Gaudieri, S., Gojobori, T. and Shiroishi,
T.: Molecular Characterization of the Pb
Recoombination Hotspot in the Mouse Major
Histocompatibility Complex Class II Region, Genomics,
80, 229-235, 2002.
11. Cebria, F., Nakazawa, M., Mineta, K., Ikeo, K.,
Gojobori, T. and Agata, K.: Dissecting planarian
central nervous system regeneration by the expressionof neural-specific genes, Develop. Growth Deffer, 44,
135-146,2002.
12. Kunihiro, S., Kawanishi, Y., Sano, M., Naito, K.,
Matsuura, Y., Tateno, Y., Gojobori, T., Yamagata, Y.,
Abe, K. and Machida, M.: A polymerase chain
reaction-based method for cloning novel members ofa gene family using a combination of degenerate andinhibitory primers, Gene, 289, 177-184, 2002.

I-b. LABORATORY FOR GENE·PRODUCT INFORMATICS
13. Blair,]. E., Ikeo, K, Gojobori, T. and Hedges, B.:
The Evolutionary Position of Nematodes, BMC
Evolutionary Biology, 2:7,
http://www.biomedcentraI.com/1471-2148/2/7. 2002.
14. Akashi, H. and Gojobori, T.: Metabolic efficiency
and amino acid composition in the proteomes ofEscherichia coli and Bacillus subtilis, Proc. Natl. Acad.
Sci., 99, 3695-3700, 2002.
15. Ogihara, Y., Isono, K, Kojima, T., Endo, A.,
Hanaoka, M., Shiina, T., Terachi, T., Utsugi, S., Murata,M., Mori, N., Takumi, S., Ikeo, K, Gojobori, T., Murai,
R, Murai, K, Matsuoka, Y., Ohnishi, Y., Tajiri, H. andTsunewaki, K: Structural features of a wheat plastome
as revealed by complete sequencing of chloroplast
DNA, Mol Genet Genomics, 266, 740-746, 2002.
16. Tateno, Y., Imanishi, T., Miyazaki, S.,
Fukami-Kobayashi, K, Saitou, N., Sugawara, H. andGojobori, T.: DNA Data Bank of Japan (DDBJ) for
genome scale research in life science, Nucl. Acids. Res.,30, 27-30, 2002.
17. Niimura, Y. and Gojobori, T.: In silica chromosome
staining: reconstrustion of Giemsa bands from thewhole human genome sequence, Proc. Natl. Acad. Sci.,
99, 797-802, 2002.
18. Ogawa, K, Ishihara,S., Saito, Y., Mineta, K,
Nakazawa, M., Ikeo, K, Gojobori, T., Watanabe, K and
Agata, K: Induction of a noggin-like gene by ectopic
DV interaction during planarian regeneration. Dev
BioI. 2002 Oct 1; 250(1): 59-70. PMID: 12297096[PubMed-indexed for MEDLINE]
19. Callaerts, P., Lee, PN., Hartmann, B., Farfan, C.,
Choy, DW., Ikeo, K, Fischbach, KF. and Gehring, W].:de Couet HG. HOX genes in the sepiolid squid
Euprymna scolopes: implications for the evolution ofcomplex body plans. Proc Natl Acad Sci USA. 2002
Feb 19; 99(4): 2088-93. PMlD: 11842209 [PubMed-indexedfor MEDLINE]

Prof. Ken NISHIKAWA
Assist. Prof. Satoshi FUKUCHI
At present the Nishikawa laboratory consist of 14
members: Professor Ken Nishikawa, AssistantProfessor Satoshi Fukuchi, two post-doctral research
fellows (Drs. Homma and Nagashima), 8 technicalassistants (Mses. Mimura, Yamamoto, Kuromaru, Abe,
Kubota, Hongo, Ito and Suzuki), and a secretary (Ms.
Sugiyama). We aim at theoretical work on protein
molecules, such as prediction of three-dimensional
structures from amino acid sequences, theoretical basisof protein stability, and molecular dynamics ofproteins. Recently we have extended our research fields
to genome analysis based on protein three-dimensional
structures. We have developed two databases, Protein
Mutant Database (PMD) and Genomes to Protein
Structures and Functions (GTOP), and three computer tools for analysis of amino acid sequences andstructures of proteins, LIBRA (Protein structuresequence compatibility analysis), MATRAS (Protein
tertiary structure comparison), and SSThread(Prediction of protein secondary structure using
threading).
(1) GTOP: A database of protein structures predictedfrom genome sequences
Takeshi KAWABATAI, Satoshi FUKUCHI, Keiichi HOMMA,
Motonori OTA2, Jiro ARAKI 3, Takehiko IT0 3, Nobuyuki
ICHIYOSHI 3 and Ken NISHIKAWA (1 Graduate School ofInformation Science, Nara Instiute of Science andTechnology 2Global Scientific Information andComputing Center, Tokyo Institute of Technology,
3Mitsubishi Res. Ins.)
Large-scale genome projects generate an
unprecedented number of protein sequences, most ofthem are experimentally uncharacterized. Predicting
the 3D structures of sequences provides important
clues as to their functions. We constructed the
Genomes TO Protein structures and functions (GTOP)
database, containing protein fold predictions of a huge
number of sequences. Predictions are mainly carriedout with the homology search program PSI-BLAST,
currently the most popular among hig~-sensitivity

LABORATORY FOR DNA DATA ANALYSIS

93

profile search methods. GTOP also includes the resultsof other analyses, e.g. homology and motif search,detection of transmembrane helices and repetitive
sequences. We have completed analyzing the sequences of 101 organisms, with the number of proteinsexceeding 430,000 in total. GTOP uses a graphicalviewer to present the analytical results of each ORF
in one page in a 'color-bar' format. The assigned 3D
structures are presented by Chime plug-in or RasMol.
The binding sites of ligands are also included,
providing functional information. The GTOP serveris available at http://spock.genes.nig.ac.jp/~genome/
gtop.html. See Ref. 1 for details.
(2) A systematic investigation identifies a significant
number of probable pseudogenes in the Escherichia coligenomeKeiichi HOMMA, Satoshi FUKUCHI, Takeshi KAw ABATAl,
Motonori OTA2and Ken NISHIKAWA (I Graduate Schoolof Information Science, Nara Instiute of Science andTechnology 2Global Scientific Information andComputing Center, Tokyo Institute of Technology)
Pseudogenes are dysfunctional open reading
frames (ORFs) with a high homology to known genes.
Although many pseudogenes have been reported toexist in the genomes of eukaryotes and obligate
parasites, they have not received detailed analysis in
free-living bacteria. In particular, pseudogenes havenever been identified in Eschericia coli. Comparison of
the genomes of E. coli strains K-12 and 0157 identified
a number of ORFs significantly shorter than theirorthologs in the other strain. To select pseudogenes
from these ORFs, we eliminated those possibly arisingfrom of sequence errors, splitting gene candidates andthose of prophage origin. The process yielded 101 and116 ORFs in K-12 and 0157 strains, respectively.
Examinations of predicted three-dimensional
structures of the protein products revealed that morethan 90% of them are unable to fold properly and thus
dysfunctional. We therefore conclude that a significant
number of pseudogenes exist in Eschericia coli. As the
genomes of free-hving bacteria are turned over at a(3) Identification of amino acids involved in protein
structural uniqueness: Implication for de novo protein
designYasuhiro ISOGAI I, Motonori OTA2, Anna ISHII3,Manabu
ISHIDA 4 and Ken NISHIKAWA (1RIKEN Wako, 2Global
Scientific Information and Computing Center, Tokyo
Institute of Technology, 3Department of Physics,
Faculty of Science, Gakushuin University, 4Departmentof Life Sciences, Faculty of Science, Himeji Instituteof Technology)
Structural uniqueness is characteristic of native
proteins and is essential to express their biological
functions. The major factors that bring about theuniqueness are specific interactions betweenhydrophobic residues and their unique packing in theprotein core. To find the origin of the uniqueness intheir amino acid sequences, we analyzed the
distribution of the side chain rotational isomers(rotamers) of hydrophobic amino acids in protein
tertiary structures and derived deltaS(contact), theconformational-entropy changes of side chains byresidue-residue contacts in each secondary structure.
The deltaS(contact) values indicate distinct tendenciesof the residue pairs to restrict side chain conformation
by inter-residue contacts. Ofthe hydrQphobic residues
in alpha-helices, aliphatic residues (Leu, Val, lIe)
strongly restrict the side chain conformations of eachother. In beta-sheet~, Met is most strongly restrictedby contact with Ile, whereas Leu, Val and Ile are lessaffected by other residues in contact than those inalpha-helices. In designed and native protein variants,
deltaS(contact) was found to correlate with thefolding-unfolding cooperativity. Thus, it can be used
as a specificity parameter for designing artificial
proteins with a unique structure. See Ref. 4 for details.
Publications
1. Kawabata, T., Fukuchi, S., Homma, K, Ota, M.,

Araki, J., Ito, T., Ichiyoshi, N. and Nishikawa, K:
GTOP: A database of protein structures predicted fromgenome sequences. Nucl. Acids Res., 30, 294-298, 2002.

high rate, with a constant inflow of horizontal transfer
genes, we propose that superfluous genes often become
pseudogenes before eventual elimination. See Ref 2 for2. Homma, K, Fukuchi, S., Kawabata, T., Ota, M. andNishikawa, K: A systematic investigation identifies asignificant number of probable pseudogenes in thedetails.

Escherichia coli genome. Gene, 294, 25-33, 2002.

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LABORATORY FOR GENE-PRODUCT INFORMATICS

3. Nishikawa, K: Editorial: Information concept inbiology. Bioinformatics, 18, 649-651, 2002.

remarkably dynamic in view of evolution. We canobserve almost every evolutionary event in this region;
mutation, selection, transposition, duplication, deletion

4. Isogai, Y., Ota, M., Ishii, A., Ishida, M. andNishikawa, K: Identification of amino acids involvedin protein structural uniqueness: Implication for deand insertion. We have paid special attention to
evolutionary aspects of the Band C loci and MICA andMICB loci. Analysis of the human MHC class I genome

novo protein design. Prot. Engineer. 15, 555-560, 2002.

region has shown that the two pairs of the MHC and
MIC genes were each produced by genome fragment

5. Fujishima, H., Nishimura, A., Wachi, M., Takagi,

duplication. We have been estimating the evolutionary

H., Hirasawa, T., Teraoka, H., Nishimori, K, Kawabata,time of these duplications by comparing genomeT., Nishikawa, K and Nagai, K: kdsA mutations affect
FtsZ-ring formation in Escherichia coli K-12.
Microbiology, 148, 103-112, 2002.

regions around HLA-B, HLA-C, MICA and MICB lociwith the orthologous regions in the chimpanzee

6. Fukami-Kobayashi, K, Tateno, Y. and Nishikawa,

K.: Parallel evolution of ligand specificity between

repetitive sequences can be used as a molecular clock,because all of them are fragmentary without theirgenome. We calculated the duplication time by using
LINEs in the duplicated genome fragments. Those

Lad/GalR family repressors and periplasmic

original function and thus considered to have evolved

sugar--binding proteins. Molecular Biology and
Evolution, in press.

by neutral mutation. In the case of Band C loci, we
confirmed that chimpanzee has essentially orthologous

7. Kashiwagi, K, Shiba, K, Fukami-Kobayashi, K,

genome structures around these loci to those of man,indicating that the duplication of Band C loci predates

Noda, T., Noguchi, H. and Nishikawa, K.: Characterization of folding pathways of the type-1 and type-2

the divergence of human and chimpanzee lineages.

periplasmic binding proteins MglB and ArgT. J

duplication had occurred after the divergence of apes

Biochem, in press.

and old world monkey lineages. On the other hand, we
found that chimpanzee has only one MIC locus.

8. Fukuchi, S., Yoshimune, K, Wakayama, M_,
Moriguchi, M. and Nishikawa, K.: Unique amino acid

Genome structure around the gene indicated that the
chimpanzee has a fused MIC gene by deletion of acomposition of proteins in halophilic bacteria. J Mol.

region between MICA and MICB loci. We confirmed

Bio!., in press.

this evolutionary scenario by analyzing the LINE
sequences around the MIC loci of man and9. Nakashima, H., Fukuchi, S. and Nishikawa, K:
Compositional changes in RNA, DNA and proteins for

chimpanzee. We are now analyzing the MHC class I

bacterial adaptation to higher and lower temperatures.

Sequence analysis of LINEs revealed that the

genome region in rhesus monkey, to extend our study
and trace the evolution of this region further back.

J Biochem., in press.
(2) Parallel evolution of ligand specificity between Lad/
GalR family repressors and periplasmic sugar-binding

I-c. LABORATORY FOR GENE FUNCTION RESEARCH

proteins(1) Evolution ofthe MHC class I genome region in man

Kaoru FUKAMI-KoBAYASHI, Yoshio TATENO and Ken

and chimpanzee

NISHIKAWA

Yoshio TATENO, Kaoru FUKAMI-KOBAYASHI, Hidetoshi
1

INOK0 , Takashi SHIINA! and Muneo YAMAZAKI(ITokai University, 2Fujiya Bioscience Institute)2

The bacterial Lad/GalR family repressors suchas lactose operon repressor (Lad), purine nucleotide
synthesis repressor (PurR), and trehalose operon

MHC class I genome region contains not only

repressor (TreR) consist of not only the N-terminal

MHC genes, A, B, C and others, but also MHC related

helix-tum-helix DNA-binding domain but also the
C-terminalligand-binding domain that is structurally
homologous to periplasmic sugar-binding proteins_

genes, MICA and MICB and repetitive sequences,LINEs and SINEs. As well known, this region is

LABORATORY FOR GENE-PRODUCT INFORMATICS

95

These structural features imply that the repressorfamily evolved by acquiring the DNA-binding domain
in the N-terminal of an ancestral periplasmic binding
protein (PBP). Phylogenetic analysis of the LacI/GalR
family repressors and their PBP homologues revealed
that the acquisition of the DNA-binding domain
occurred first in the family, and ligand specificity thenevolved. The phylogenetic tree also indicates that theacquisition occurred only once before the divergenceof the major lineages of eubacteria, and that the Lad/
GalR and the PBP families have since undergone
extensive gene duplication/loss independently along
the evolutionary lineages. Multiple alignments of therepressors and PBPs furthermore revealed that
repressors and PBPs with the same ligand specificity
have the same or similar residues in their binding sites.
This result, together with the phylogenetic relationship,demonstrates that the repressors and the PBPs
individually acquired the same ligand specificity byhomoplasious replacement, even though their genes
are encoded in the same operon.
Publications
1. Tateno, Y., Imanishi, T., Miyazaki, S., FukamiKobayashi, K, Saitou, N., Sugawara, H. and Gojobori,T.: DNA Data Bank of Japan (DDBJ) for genome scale
research in life science. Nucleic Acids Res 30: 27-30,
2002.
2. Kunihiro, S., Kawanishi, Y.,Sano, M., Naitoh, K,
Matsu-ura, S., Tateno, Y., Gojobori, T., Yamagata, Y.,
Abe, K and Machida, M.: A PCR-based method forcloning novel members of a gene family by a
combination of degenerate and inhibitory primers.
Gene 289: 177-184, 2002.
3. Fukami-Kobayashi, K, Schrieber, D. R. andBenner, S. A.: Detecting compensatory covariation
signals in protein evolution using reconstructed
ancestral sequences., J. Mol. BioI. 319: 729-743, 2002.

6. Fukami-Kobayashi, K, Tateno, Y. and Nishikawa,
K: Parallel evolution of ligand specificity between
LacI/GalR family repressors and periplasmic
sugar-binding proteins. Mol BioI Evol (in press), 2003.

7. Miyazaki, S. and Tateno, Y.: DNA Data Bank ofJapan as an Indispensable Public Database, In:
Population and Community, B. M. Knoppers ed,
Kluwer International Press (in press), 2003.
8. Brazma, A., Ikeo, K and Tateno, Y.: Standardization of microarray experiment data, Protein, Nucleic
Acid and Enzyme (in Japanese) (in press), 2003.

I-d. LABORATORY FOR RESEARCH AND DEVELOPMENT
OF BIOLOGICAL DATABASES
1. Information systems for molecular data1)

Web services as the programmatic interfaces

It is very easy now for biologists to search, browse
and utilize a number of data resources, i.e., databases
and data analysis tools one by one thanks to theInternet and the World Wide Web. However, it is still
difficult to use the data resource in an integrated way
specific to a work flow for the research anddevelopment. In order to improve the situation, we
introduced the Web services in addition to XML
technology that we had implemented into the
nucleotide sequence database in 2001.
The Web browser such as Internet Explorer andNetscape provides human-friendly interfaces. By
contrast, the Web services provide the programmaticinterfaces, i.e., program-friendly interfaces. A use
program written in Java and Perl is able to search Web
services that provide functions in need and bind to the
Web services. In other words, the user program canintegrate various Web services according to the work
flow. The Web services that we developed are available
at http://xml.nig.ac.jp/

4. Fukami-Kobayashi, K: How to make good use of
CLUSTALW. Protein, Nucleic Acid and Enzyme 47:
1237-1239, 2002.

2)

5. Miyazaki, S., Sugawara, H., Gojobori, T. andTateno, Y.: DNA Data Bank of Japan (DDBJ) in XML,
Nucleic Acids Res 31: 13-16, 2003.

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 the data
is made open to the public. At the GIB Web page96

LABORATORY FOR GENE FUNCTION RESEARCH

Expansion of Genome Information Broker (GIB)

(http://gib.genes.nig.ac.jp/), key word search, homology
search, links to DBGET, KEGG and GTOP andvisualization of the data are available for 100 organisms
as of December 2002. We have utilized XML, CORBA
and a distributed database in order to cope with the
explosion of microbial genome information.

annotation information, however, is not consistentamong genome sequencing teams. In addition,

2. Information systems on microbes

more valuable information. As a new feature of INSD,
we develop so-called "Open Annotation System

1) WFCC-MIRCEN World Data Centre for Microorganisms (WDCM)(OASYS)" as an annotation editor in the distributed
environment on the Internet.

WFCC and MIRCEN stand for World Federation
for Culture Collections and Microbial Resource Centers
network respectively. The laboratory is the host of
WDCM and maintains the World Directory of
microbial resource centers. The on-line World Directory
contains the detailed information of 469 centers in 62
countries and also the list of their holdings. Any
culture collection is able to register, update and delete
the information at http://www.wdcm.org/' We also used
XML technology to organize the Web page.

researchers outside of the team might have more
information and knowledge on some genes andbiological molecules. Therefore, it is quite important
to develop the system which allows any expert toevaluate the annotation given by the team to attach

The information system on pathogenic
microorganisms has been supported by Special
Coordination Funds for Promoting Science andTechnology
OASYS project has been supported by BIRD ofJapan Science and Technology Corporation (JST)2)

Improvement of functions of INSD

We continued the development of an e-Workbench
named InforBIO by use of JAVA, XML and a relational

We are in charge of developing tools for theconstruction, maintenance and dissemination of INSD.
The following tools are studied and improved this year:- the automatic scheduler to diffuse entries on the datespecified by submitters- a tool to compress the whole data set of INSD
- non-redundant daily update data set of INSD
- standalone application to check the translation of
CDS in JAVA

database management system in the public domain.
We have distributed InforBIO to several laboratories
that study microbes and improved the utility andPublication
1. Sugawara, H. (ed): Bioinformatics (in japanese),robustness of InforBIO based on the feedback.

Kyoritsu Shuppan Co., Ltd. (Tokyo), 2002.

3) An information system for pathogenic microorganisms!

2. Miyazaki, S. and Sugawara, H.: Networking of
biological resource centers: WDCM experiences, Data
Science journal, 1 (2), 102-107, 2002.

2) Development of an e- Workbench for BiologicalClassification and Identification (InforBIO)

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.

3. Sugawara, H.: What can the pubic database of
DDBJ provide? (injapanese), Genome-igaku, 2 (3), 61-69,
2002.

3. Applications of IT to the International Nucleotide
Sequence Database

4. Sugawara, H: Agglomeration of Life Sciences andIT -Bioinformaitcs (in japanese), Biological resources
access (Watanabe M. and Nimura, S (ed)),Toyo-keizai-shinpou-sha (Tokyo), 205-238, 2002.

1)

Development of Open Annotation System2

More than 100 of the complete genome sequences
have been submitted to INSD since 1995. The

5. Sugawara, H: Concept and progress of biologicalresource centers in OECD (in Japanese), Microbiol.
Cult. Coll, 17 (2), 81-87, 2002.

LABORATORY FOR RESEARCH AND DEVELOPMENT OF BIOLOGICAL DATABASES

97

6. Fumoto, M., Miyazaki, S. and Sugawara, H.:
Genome Information Broker (GIB): data retrieval andcomparative analysis system for completed microbial
genomes and more, Nucleic Acid Research, 30 (1), 66-68,
2002.
7. Tateno, Y., Imanishi, T., Fukami-Kobayashi, K.,
Saitou, N., Sugawara, H. and Gojobori, T.: DNA Data
Bank of Japan (DDB]l for genome scale research in life
science, Nucleic Acid Research, 30 (1), 27-30, 2002.

98

LABORATORY FOR RESEARCH AND DEVELOPMENT OF BIOLOGICAL DATABASES

J. RADIOISOTOPE CENTER

Yoshiharu YAMAICHI, Katsunori YATA, Yasuyuki
OGATA and Hironori NIKI
We focus on chromosome dynamics of theprokaryote for its genome integrity. We are studying
the proteins and the DNA sites responsible for the'regulation of prokaryotic DNA segregation using a
combination of genetics, molecular, biochemical,
cell-biological, and genomic approaches in Escherichia
coli. Prokaryotes are not known to have a eukaryoticlike mitotic apparatus, and little is known about themechanisms controlling chromosome partitioning. We
visualized bacterial chromosomal DNA and plasmid
DNA in cells by using fluorescence in situ hybridization (FISH).
We have already reveled subcellular localizationof different DNA segments on the E. coli chromosome
during the cell division cycle by using fluorescence insitu hybridization. These chromosome segments arelocated within the cell in the same order as a genetic
map. A large chromosomal region including replication
origin, onC shows bipolar localization after replication,
which we call the Ori domain. Then, we guessed thatprobably a chromosomal partitioning site was locatedwithin the Ori domain.
Now we know complete genome sequences ofmany kinds of bacteria. It is not impossible to identify

segment including one. These results suggest that a
cis-acting site for bipolar migration of the Ori domain
is located on this chromosomal region. Furthermore,
we have analyzed that nucleoid localization and onC
migration in a series of chromosome-split mutants, anddeletion mutants. Finally, we identified a site that was
responsible for bipolar migration of the oriC segment
as a 25 bp sequence on 89.1 min of the E. coli map.
Publication

1. Kodama, K., Kobayashi, T., Niki, H., Hiraga, S.,

Oshima, T., Mori, H. and Horiuchi, T.: Amplificationof Hot DNA segments in Escherichia coli, Mol
Microbiol 45, 1575-1588, 2002.

replication origin and terminus based on only thesequence data. In contrast, we have not succeeded infinding a cis-acting site for chromosome partitioning,or a bacterial centromere. To identify a cis-acting site
for chromosome partitioning, we have constructed a
new mutant, in which a circular chromosome was split
into two circular chromosomes. In this mutant, we wereable to study an effect of a cis-acting segment onchromosome segregation. A chromosome-split mutant,in which 740 kb of the chromosome region [84.7-89.4
min] in the Ori domain is separated from the original
chromosome, showed irregular localization of nucleoids
and defective in bipolar migration of the chromosomal

RADIOISOTOPE CENTER99

of these strains for distribution to rice researchcommunity till the end of 2006. In 2002,160 accessionsof wild- and cultivated- rice seeds were renewed and559 lines were distributed to 24 institutes. Out of 2000

K. EXPERIMENTAL FARM

accessions, we chose 289 lines as core collections
ranking from one to three, according to their typicalcharacters, habitat and distribution in genome/species.
Characterization of these strains for phenotypes and(1) Development of new experimental strains andmolecular features, are processed to house in the ricedatabase "Oryzabase" with their photograps. The rice

reevaluation of the genetic stocks of ricenational bio-resource project consists of five university
members including our laboratory. As a center of rice

Ken-Ichi NONOMURA, Mitsugu EIGUCHI, Toshie

resource project, we are responsible for the

MIYABAYASHI and Nori KURATA

manegement to make rice genetic resources powerfultools for further genomic researches. Weare

Making rice genetic stocks good resources andcollaboratively progress the refinement of our rice

research works employing those resources carried out
in the experimental farm are cooperative works

resources. In this, we extract useful characters fromall genetic stocks involved in the project as electric data

between the experimental farm and the plant genetics

and stored them in the "Oryzabase". Information aboutlaboratory. From 1998, Yukihiro ITO (Plant Genetics

these strains can be accessed at the web site ofLab.) and we have been developing a new rice genetic"Oryzabase", which is a comprehensive rice genome

stock of enhancer trap lines aiming to generate overresources database at http://www.shigen.nig.ac.jp/rice/ten thousands lines, then to characterize, propagate

oryzabase/index.html. Construction of the Oryzabaseis undertaken by Yukiko Yamazaki, a manager of all

and distribute them as rice resources for functional
genomics. Other works are more or less depending on
the experiments using plant resources; e.g. many

data from organisms being dealt in the national
bio-resource project. Addition of new information andnumbers of transgenic rice, chemically induced mutant

revision of the "Oryzabase" will be performed

strains, strains of insertional mutagenesis and so on.
These materials grown in the greenhouses and theoccasionally to include more useful information for ricebasic research under the guidance of the rice genetic

exprimental farm are indispensable for plant genetics

resources committee in Japan (chair :N. Kurata).

and genomics.
For details, see the reports of plant genetics lab.

Publications
1. Harushima, Y., Nakagahra, M., Yano, M., Sasaki,(2) A new resource work for wild rice species involvedin the activity as a center of national bioresource projectfor rice

T. and Kurata, N.: Diverse variation of reproductive

Nori KURATA, Toshie MIYABAYASHI, Yukiko
YAMAZAKI *, Mitsugu EIGUCHI and Ken-Ichi
NONOMURA, (*Genetic Strains Information Lab.)

2. Ahn, B. 0., Miyoshi, K., Itoh, J. 1., Nagato, Y. and
Kurata, N.: A genetic and physical mapping of the

In 2002, the national bio-resource project was

barriers in three intraspecific rice crosses. Genetics.
313-322, 2002.

region containing PLASTOCHRON1,a heterochronic
gene, in rice (Oryza sativa, L). Theor. Appl. Genet. 105:654-659, 2002.

started for representative genetic and biologicalresources under the MEXT. The plant genetics

3. Ito, Y., Hirochika, H. and Kurata, N.: Organlaboratory and experimental farm act as a center forrice genetic resources project. In this project, we deal

specific alternative transcripts of KNOX family class
2 homeobox genes of rice. Gene, 2002.

with about 2000 wild rice accessions including 9
genomes and over 20 species collected all over the

4. Dunford, R.P., Yano, M., Kurata, N., Sasaki, T.,

world. We planned to propagate and store all seeds

Huestis, G., Rocheford, T. and Laurie, D.A.: Compar-

100 EXPERIMENTAL FARM

ative mapping of the barley Ppd-H1 photoperiod
response gene region, which lies close to a junction
between two rice linkage segments. Genetics, 161,
825-834, 2002.
5. Kurata, N., Nonomura, K-I. and Harushima, Y.:
Rice genome organization focusing on centromere andgenome interaction studies. Annals of Botany 90,
427-435, 2002.
6. Yamazaki, Y., Yoshimura, A., Nagato, Y. and
Kurata, N.: Recent advances in Oryzabase (Integrated
Rice Database). Rice Genetics Newslet. 19, 7-8, 2002.
7. Kurata, N. and Fukui, K.: Chromosome research.
in genus Oryza. in Monograph in genus Oryza. Nanda
1S. Ed, Springer (in press).

•

EXPERIMENTAL FARM 101

elements in Arabidopsis. The 74th Annual Meeting of
the Genetics Society of Japan, Fukuoka, October, 2002.

L. TECHNICAL SECTION

The Technical section supports the research
activity of NIG in many fields. For example, we support
the Radioisotope center and Experimental farm, and
the Genetic strain research center where genetic andtransgenic strains of mice, rice, flies, and E. coli areproduced, maintained, and distributed to otherinstitutions. Members of the staff additionallY support
many types of experiments in various laboratories,
such as developmental, cell, and molecular genetics.
We continually update our technical services throughattending the training and annual meetings for thetechnical officials.
Publications
1. Davies, K., Bowden, 1., Smith, P., Dean, W., Hill,
D., Furuumi, H., Sasaki, H., Cattanach, B. and Reik,

W.: Disruption of mesodermal enhancers for Igf2in theminute mutant. Development 129, 1657-1668, 2002.
2. Hayashi, S., Ito, K., Sado, Y., Taniguchi, M.,
Akimoto, A, Takeuchi, H., Aigaki, T., Matsuzaki, F.,
Nakagoshi, H., Tanimura, T., Ueda, R., Uemura, T.,
Yoshihara, M. and Goto, S.: GETDB, a database
compiling expression patterns and molecular locationsof a collection of Gal4 enhancer traps. Genesis 34, 58-61,
2002.
3. Masumoto, H., Muramatsu, S., Kamimura, Y. andAraki, H.: S-Cdk-dependent phosphorylation of Sld2
essential for chromosomal DNA replication in budding
yeast. Nature 415,651-655,2002.
Oral presentations
1. Furuumi, H.: Detection of methylated DNA bybisulfite genomic sequencing. The 13th Annual
Meeting for Technical Engineers, Okazaki, February,
2002.
2. Miura, A, Watanabe, K., Kato, M., Kotani, H.,
Kakutani, T.: Mobile endogenous CACTA family

102

TECHNICAL SECTION

Poster presentations
1. Kiso, M.: Mouse ES cell culture and Generation ofGene targeted mouse. The 13th Annual Meeting forTechnical Engineers, Okazaki, February, 2002.
2. Muramatsu, S. and Araki, H.: Functional analysisof the DPBll gene in Saccharomyces cerevisiae. The 25th
Annual Meeting of the Molecular Biology Society ofJapan, Yokohama, December, 2002.
3. Saka, K., Matsumoto, N. and Nishimura, A.:
Isolation of a whole set of cell division genes in
Escherichia coli. The 13th Annual Meeting for TechnicalEngineers, Okazaki, February, 2002.
4. Saka, K., Matsumoto, N. and Nishimura, A: Global
regulation of cell division in Escherichia coli. The 25th
Annual Meeting of the Molecular Biology Society ofJapan, Yokohama, December, 2002.

ABSTRACTS OF DIARY FOR 2002
Biological SymposiumJan.llJan.21

Cell Cycle Control and Chromosome Partition (Mitsuhiro YANAGIDA)
The Vitamin D Receptor: Molecular Variation and Functional Outcomes(Alexander P KOUZMENKO)

Jan.25The ultimate altruistic individuals: phylogeny, evolution, ecology, physiology and molecular biology

Feb.l4Evolutionary Distance Estimation under Heterogeneous Substitution Pattern

Feb.20Synchronization as a survival tool of the cellular slime mold and its generality(Seido NAGANO)

Feb.25

Molecular functions of two TERMINAL FLOWER genes of Arabidopsis (Koji GOTO)

MarA
MarA

Protein scaffolds and cell polarity in Drosophila (Elisabeth KNUST)

MaLI3

Molecular dissection of S allele which controlls self-imcompatibility in Brassica (Masao WTANABE)

MaLI8Regulatory mechanisms and function of the MAP kinase signal transduction pathways (Eisuke
NISHIDA)of aphid soldiers (Kazuma FUKATSU)(Koichiro TAMURA)

Cell type specification in the developingcentral nervous system of the zebrafish(Jose CAMPOS-ORTEGA)

MaL22

MEC3, MECI and DDC2 functions as telomere checkpoint

APL3
May.7

Designing Artificial Transcription Factors (Aseem Z.ANSAR)

June.I7
June.20
July.3(Shinichiro ENOMOTO)

Stress and developmental regulation of RNA polymerase IT C-terminal domain phosphorylation(Oliver BENSAUDE)
Rice Functional Genomics and Bioinformatics at IRR (Richard M.BRUSKIEWICH)
Functional analysis of the canoe gene in Drosophila morphogenesis (Kuniaki TAKAHASHI)

JulyA

Cell cycle regulation by myb oncogene (Masahiro OKADA)
The establishment of the stem cell fate in Drosophila germline (Miho ASAOKA)

JulyA

Dnmt3L is required for genomic imprinting during oogenesis (Kenichiro HATA)

July.9

Molecular Genetic Analysis of Plant Sex Chromosome and Application of the Microscopic
Manipulation to Genetics (Sachihiro MATSUNAGA)
Fundamental Mechanism of Axon Growth of Neuron Cells (Junichi YUASA)

July.9July.I2
Aug.I6Chromatin Inheritance during cellular proliferation (Keiichi SHIBAHARA)
Molecular mechanism of programmed cell death and its role in the determination of cell fate (Hirataka

Aug.26KANUKA)
Partition of replicated sister chromosomes in Escherichia coli (Sota HIRAGA)

Aug.27Aug.28Aug.28

Membrane traffic and sterol-roles in post-Golgi protein sorting- (Kyohei UMEBAYASHI)
Structure and dynamics of self-organization: Examples for self-driving structures composed ofmacromolecules (Shin-ichiro, M. NOMURA)
1. Activation machanism of protease complexes 2. Observation of plant organelle by atomic-force
microscope (Takafumi YAMADA)

Aug.29Aug.29Cell Cycle Control by Ubiquitin-dependent Protein Degradation (Hiroyuki YAMANO)
Maternal-effect mutations affecting the early development in zebrafish (Yasuyuki KISHIMOTO)

Aug.29Part I: T-box gene interactions mediate regional specification of the zebrafish mesoderm; Part
IT: a new midline gene required to establish all left-right asymmetri es (Kazuyuki HOSHIJIMA)

Sep.2

Interplay of a vacuolar pathogen Legionella pneumophila and its phagocytic host cells (Hiraki NAGAI)

Sep.l8Mechanism and evolution of genomic regulation of the Dlx3-Dlx7 cluster (Kenta SUMIYAMA)
The Pttern of Polymorphism on Human Chromosome 21 (Hideki INNAN)

Sep.I8Sep.24The structure of single-nucleotide variation in overlapping regions of human genome sequence

ABSTRACTS OF DIARY FOR 2002

103

Sep.30
Sep.30
Oct.7
Oct.7
Oct.9
Oct.9
Oct.10
Oct.15
Oct.17
Nov.18
Nov.19Nov.28Nov.28
Dec.5
Dec.9
Dec.l6
Dec.18(Stephen T. SHERRY)
Small RNAs in genome rearragnement in Tetrahymena (Kazufumi MOCHIZUKI)
Hydra and the evolution of the bilateral body plan (Hans MEINHARDT)
Structural determination of biomolecules by solid NMR (Kiyonori T AKEKOSHI)
Poetry and prose of genetic sequences (Edward N. TRIFONOV)
Understanding the persistence of a long linkage disequilibrium block in the human genome: lessons
fromthe GBA region of the human genome (Jaume BERTRANPETIT)
The new tide of the chemical synthesis of proteins: Its fusion with biology (Sabro AIMOTO)
Inducing and overcoming heterochromatin mediated silencing in mammals (Richard FESTENTEIN)
Functional analysis of the isthmin gene encoding a novel secretion protein (Isato ARAKI)
Distinct histone methylation patterns define active and silent chromatin states (Ken-ithi NOMA)
Mitosis and Endoreduplication: Alternative Cell Cycle Strategies in Plant Development (Yukiko
MIZUKAMI)
Coactivator MBFI supports AP-I activity and extends lifetime under oxidative stress (Marek
JINDRA)
Chromatin regulation at imprinting-control regions in the mouse (Robert FElL)
Looking back at my research life (Tomoko OHTA)
Fishing for Novel Genes (John E. DOWLING)
Man? Mouse? Mole? Some lessons on imprinting from rare clinical disorders (David T. BONTHRON)
Mechanisms Preventing Re-replication in Saccharomyces cerevisiae. (Joachim LI)
Characterization of PR-Set7, a nucleosomal histone H4lysine 20-specific methyltransfease(Kenichi
NISHIOKA)

104 ABSTRACTS OF DIARY FOR 2002

FOREIGN VISITORS IN 2002
Jan.21

Alexander P. Kouzmenko, Bone and Mineral Research Program, Garvan Institute of Medical Research,
Sydney, Australia

MarA
MarA

Elisabeth Knust, Institut fuer Genetik, Heinrich-Heine Universitaet Duesseldorf
Jose Campos-Ortega, University of Cologne, Institute for Developmental Biology

Mar.22
Apr.3

Shinichiro Enomoto, University of Minnesota
Aseem Z. Ansari, Department of Biochemistry & Genome Center University of Wisconsin-Madison

May.7

Olivier Bensaude, Regulation de l'Expression Genetique, CNRS, Ecole Normale Superieure

June.17
JulyA

Richard M. Bruskiewich, International Rice Research Institute (IRRI)
Miho ASAOKA, Department of Cell Biology Duke University Medical Center

JulyA

Kenichiro HATA, Department of Medicine Harvard Medical School Cardiovascular Research Center

Aug.29Massachusetts General Hospital
Hiroyuki Yamano, Cancer Research UK London Research Institute Clare Hall Laboratories

Aug.29Kazuyuki Hoshijima, Eccles Institute of Human genetics, Department of Human Genetics, University

USAof Utah
Sep.2
Sep.18
Sep.18Sep.24

Hiroki Nagai, Section of Microbial Pathogenesis, Yale School of Medicine
Kenta Sumiyama, Frank Ruddle laboratory, Yale University
Hideki Innan, Human Genetics Center, University of Texas Health Science Center at Houston

Sep.30

Stephen T. Sherry, National Center for Biotechnology Information, National Institute of HealthKazufumi Mochizuki, Department of Biology, University of Rochester

Sep.30Hans Meinhardt, Max Plank Institute, Tuebingen, Germany.

Oct.7

Edward N. Trifonov, Genome Diversity Center, Institute of Evolution, University of Haifa, Haifa,
IsraelOct.9
Oct.I0Jaume Bertranpetit, Unitat de Biologia Evolutiva. Universitat Pompeu Fabra. Spain
Richard Festenstein, MRC Clinical Sciences Center Hammersmith Hospital London, UK

Oct.15

Isato Araki, Max-Planck-Institute of Molecular Cell Biology and Genetics (MPI-CBG)

Oct.17
Nov.18Ken-ithi Noma, Cold Spring Harbor Laboratory
Yukiko Mizukami, University of California, Berkeley, Department of Plant and Microbial Biology

Nov.19Nov.28

Marek JINDRA, University of South Bohemia, Czech Republic

Dec.5
Dec.9

Robert Feil, Institute of Molecular Genetics, CNRSJohn E. Dowling, Harvard University
David T. Bonthron, University of Leeds Molecular Medicine Unit St. James's University Hospital,Dec.16

UK
Joachim Li, Department of Microbiology & Immunology University of California, San Francisco

Dec.18

Kenichi Nishioka, University of Medicine and Dentistry of New Jersey

FOREIGN VISITORS IN 2002 105

AUTHOR INDEX
51
39
27
60
12,13
11
27
16

ABE, K
ABE, T
AGAWA, Y
AHN, Byoung-Ohg
AIMOTO, S
AKAMATSU, Y.
AKASAKA, K.
AMANO, A,

AMANO, K. ···············································88
ANDACHI, Y
73

91
36
91
54

ANDO, T
ANDRES, Aida M
ANTONIO, BA.
AOKI, AFURUUMI, H

79
27
58
42,43

GENGYO-ANDO, K.

· .. ·84

FUKUZAWA, A
FURUHASHI, H
FURUSE, T

GOHJI, K
GOJOBORI, T.

GONDO, Y. ················································53
GRAHAM, David Y.
HAGA, SHANDA, H
HARADA, C
HARAGUCHI, S

17,18,19·············93

HASEGAWA, N

ARAKI, J.

ARAKI, T. ·················································47
ARIKAWA, K. ·············································91
ARITA, K
91

HARUSHIMA, Y

HATTORI, M
HAYAKAWA, S
HAYASHI, H

BABA, T. ··················································91
BAO, Z49

HAYASHI, M

BERTRANPETIT, J.

HUIKATA, C.

CALAFELL, F;

CHENG, Z
CHIDEN, Y.
EDDY, Sean R.
EIGUCHI, M
ENDO, S
ENDO, T.
EUN, MFARIVAR, S
FUJII, T
FUJIMITSU, K.
FUJISAWA, C
FUJISAWA, H. .. ··FUJISAWA, T.
FUJITA, C.
FUJITA, N
FUJIYAMA, A.
FUKAGAWA, T.
FUKAGAWA, Y.
FUKAMI-KOBAYASHI, K.
FUKUCHI, S
FUKUDA, T.

106 AUTHOR INDEX

·

36
36
91
91
49
60,61,64,100
870,75,91
91
51····54
13
24
·47
24,25
65
8
37
39
39
95
93,94
60

26
91
57
···········63·············75

HATA, K. ·····································,···········44
HATANO, N
32

36

AZEGAMI, K

36····71,75

HAHN, ]H. ················································91
HAMADA, M
91

AOKI, H. ··················································91
AOYAGI, T····32ARAKI, H.

32
24,43,89,90,91

HAYWARD, Richard S

HUISHITA, S
HIRAKI, H
HIRAMOTO, M
HIRATA, T
HIROCHIKA, H
HIROMI, Y.
HIRONO, K
HIROSE, S
HIROSHIMA, M
HOMMA, K.

37,42····2471,75······91
81
71
91
74
24
47,48
48,49,60
23,24,27
71
26,27,28
79
93,94

HONDA, M. ···············································91
HORI, T
39
HOSOKAWA, S

91

HOTTA, K. ················································91
HWANG, lung Shan
IBA, YICHlBA, Y.
ICHIKAWA, Y.
ICHIYOSHI, N
IDONUMA, A.
IIDA, T.
II]IMA, M

89
71
39,40
91
93
91
19
91

IINO, T
IINO,YIIYAMA, S

43····················8475

KANAYA, S
KARASAWA, W

IKEDA, A. ·················································48
IKEDA, M
91

KASCIUKOVIC, T

65
IKEMURA, T
39,40
IKENO, M
91
IKEO, K
89,90
IMAMOTO, N
78,79
IMOTO, T
14
INOKO, H
95
INOUE, H
87
ISHIDA, M
94
ISHIHARA, K
42
ISHIHARA, N
39
ISHIHARA, T.
83,84,85
ISHII, A···················94
ISHIWA, S
53
ISHIZAKA, M
49
ISOGAI, Y
94
ISSHIKI, T.
67
ITO, H········91
ITo, M
50,71,72,73
ITO, S···91
ITO, T
91,93
ITO, Y. ........................•.............. 49,59,60,61,91
ITOH, 1.
60,61
ITOH, T
37
IWABUCHI, A····91IwAMA, H:
90,91
IWAMOTO, R
65
IWASAKI, H .. ,11
IWURA, T
14
JIANG, 1.
91
JIANGl, N
49
JIN, F
35
JINDRA, M
27
KABEYA, Y
15
KAGOSHIMA, H
72
KAJITA, A
73
KAJIWARA, H
49
KAJIWARA,1.
72
KAKIZUKA, A········16
KAKUTANI, T.
44,45,46
KAMAMOTO, A.
72,83,85
KAMIMURA, Y.
17,18
KAMIYA, K
91

KATAYAMA, T

IKEHARA, K

KANAI, M. ················································23
KANAMORI, H
91

36
42

KANEDA, M

KATAGIRI, S

KATAYOSE, Y········918191········13,14
91

KATO, H. ·················································69
KATO, 1. ···························;······················69
KATO, M
44,45
KATO, Y. ··················································42
KATSUKI, T
KATSURA, 1.
KAWABATA, T.
KAWABE, A
KAWAGUCHI, K

24
83,84,85····93,94
38
75

KAWAHARA, T. ···········································70
KAWAKAMI, H
13
KAWAKAMI, K.
KAWAKAMI, M
KAWAKATSU, T.
KAWASAKI, H
KAWASAKI, 1.
KAWASAKI, T

29,30
85
······6128
72 .
·············48

KAYA, H. ·················································47
KAYASHIMA, Y
KIKUCHI, S
KIKUTA, A.. "················2849········91

KIM, H. ···················································91
KIMURA, M
9,10
KIMURA, S
KINOSHITA, T.
KINOSHITA, Y
KISHI, T
KISHIMOTO, N
KISO, M
KITA, KKITAJIMA, S
KITANO, T
KIYAMA, R
KOBAYASHI, K
KOBAYASHI, N
KOHARA, Y.
KOIDE, T
KOKUBO, H
KOMIYAMA, T.

79
44,45
44,45
············11
71
57
39
57
35
32,33
58
········91
71,72,73,74,75
58
56
89

KONDO, S. ················································23
KONO,1.
KOSAKA, Y.
KOSEKI, H························913930

AUTHOR INDEX

107

KUBO, T. ··················································46
KUBORI, T
KUBOTA, T
KUMAKI, K
KURATA, N
KUROIWA, A
KUROSAW A, K
KUROSAWA, Y
LEVINE, M
LI, E
LIU, Qin-Xin
MACHITA, K
MAEHARA, T.
MAENAKA, K.
MAKINO, T.
MARUYAMA, K
MASUKAW A, M
MASUYA, H
MATSUDA, Y.
MATSUMOTO, K.
MATSUMOTO, N
MATSUMOTO, T.
MCCOUCH, Susan R.
MIKAMI, Y
MINAKUCHI, Y
MINETA, K.
MITA, A
MITANI, S
MITSUZAWA, H.
MIURA, A.
MIURA, SMIYABAYASHI, T.
MIYAGI, H
MIYAMOTO, J.

····················80························434249,59,60,61,62,63,64,69,100
43
43
71
75
42,43,4427
91······ .. ··9187,88····90
79
91
53
43
27
65
·91
49
39
73
89
55
·84
8,9
44,45,46
71,75
64,1008075

MIYAO, A. ············································· . ··60
MIYAUCHI, Y.
39
MIYOSHI, K.
MlZUBAYASHI, T.
MlZUKOSHI, M
MIZUNO, H
MIZUNO, S
MlZUSHIMA, N
MIZUSHINA, Y
MOCHIZUKI, Y.

59,60,61
91
75
91
43
15,16
55
91

MOHRI, A. ················································84
MORI, I.

·······84

MORI, M. ·················································49
MORIGUCHI, K.
62

MOTOHASHI, T
MURAMATSU, S
NAGAI, H
NAGAMURA, Y
NAGASAKI, H
NAGATO, Y.
NAKAGATA, N

NAKAGAWA, I. ···········································16
NAKAMA, Y
91

91

NAKAMICHI, Y.

NAKAMURA, M. ··········································91
NAKANO, M
NAKASHIMA, M
NAKATA, K
NAKAYAMA, T
NAMIKI, N
NARA, ANEGISHI,M:

NIIMURA, Y
NIKI, HNISHIDA, S
N ISHIHASHI, A
NISHIKAWA, K.
NISHIMURA, A.
NISHIZAKA, S
NISIZAKA, S

'

60
···91
71
26
91
15
91
···91
99
13
39
93,94,95
64,65
71
75

NODA, T. ··················································53
NOGAMI, 'M
39
NOMOTO, H. ··········································71,75
NOMURA, T.
NONOMURA, K.
OBARA, M
aBITA, T.,

47,49
60,62,64,100
71····14

OCHI, H. ··················································71
OGATA, Y
99
OGIHARA, Y·······70

OGURA, K. ················································71
OHKURA, K
OHMURA, T
OHSUMI, Y
OHTA, F
OHTA, I.
OISHI, AOISHI, K,
aKA, A

83
13
15,16
71,75
··········91······85
71,75
55

OKABE, K. ················································49
OKABE, M

MORIKAWA, N· .. ·30

OKADA, M

MORISHITA, T.

75

OKADA, N

108 AUTHOR INDEX·······88
91
18
80,81
91
91
60,61,69············69

MUKAI, Y

23,50,51
26
30

OKAMOTO, M
OKAMURA, A
OKANO, M
OKONOGI, A
ONISHI, Y

91
39
42,43
79
32

46,47
91
78,79,80
95
80,81,82

SHIBAHARA, K.
SHIBATA, M
SHIINA, N
SHIINA, T
SHIMAMOTO, N

ONO, N. ···················································91

SHIMIZU, A. ···············································46

OOKA, H. ·················································49

SHIMIZU, E

35,36

SHIMIZU, H

OOTA, S. ··················································35

SHIMIZU, K

OSUMI, N. ·················································47

SHIMIZU, W

OOTA, H

OTA, MOTSUKA, T

93,94
43

OZAWA, H. ···············································43
P ADMANABHAN, U.
PARK, Joan-Hyun
PERATA, P
PERLMUTTER, David H
PURBOWASITO, W

81
11····················481642

SHIMOJIMA, T
SHIMOKAW A, T
SHINDOH, K
SHIN-I, T
SHINKURA, K
SHINOHARA, M
SHIRAKIHARA, CSHIRAKIHARA, Y.

REIK, W. ··················································42

SHIRATORI, A.

·····75
Roy, Badal C····32
SADO, T:
42,43
SAGA, Y .......................•................... ·····56,57
SAGAI, T················53

SHIROHZU, H

SOLDEVILA, M

SAIGO, K. ·················································66

SONG,].

ROKHSAR, D

SAITO, S
SAITOU, N
SAJI, S·····75
35,36,37······91

SHIROISHI, T.
SHOMURA, A
SHOUJI, S

SONG, Jian-Ping
SONODA, Y
STILLMAN, B

SAKA, K. ··················································65

STONEKING, M

SAKAI, K. ·················································91

SU'ETSUGU, M

SAKAKI, Y.
SAKAMOTO, N
SAKATA, K
SAKATA-SOGAWA, K.
SAKURAI, N
SANO, H
SANO, M
SARKAR, S
SASAKI, H
SASAKI, T
SASAKUMA, T
SASAOKA, Y

37,42
75
91
·······················81···········55
58
71,75
32
42,43
91
,
·····70
57

SATO, H. ··················································69
SATO, YSATOH, K
SATOH, N
SAWADA, H
SEINO, H

47,81
49
75,91
36
12

SEKI, M .. ·················································87
SERIZAWA, M

72,73

71
25
53
71
26,88
91
87
71,75
78
71
·····86
86,87,88
86,87,88
42,43
53,54,55,58
91
16············36
···91

~

39
48
46
35
13,14

SUDA, C. ··················· .. ·····························42
SUGANO, S
SUGIMOTO, K
SUGIMOTO, M
SUGIURA, I.
SUGIYAMA, Y.
SUMIYAMA, K
SUSA, MSUZUKI, FSUZUKI, K
SUZUKI, T
SUZUKI, Y
T AJIMA, S
T AK, Yon-Sao
TAKAGI, N
TAKAGI, Y.
TAKAHASHI, H.
TAKAHASHI, K
TAKAHASHI, Y
TAKAKU, Y

;

71
49
50,51
71
71,75
35
80
36
39
··········62
71,90
42,43
18
50,51,55
47,48
91
66
56
24

AUTHOR INDEX 109

TAKANO, T
TAKANO-SHIMIZU, T.
TAKAZAKI, Y········5538················91

TAKENAKA, M. :··········································71·······················75
TAMURA, M
53,54
TAN, Seong-Seng
50,51
TANAKA, S .......•............................... ·········54
TANAKA, T··········90
TANIGUCHI, M
66
TASHIRO, K
43
T ATENO, Y.
95
TATSUTA, H·····38TAYLOR, Todd D······37
TAMIYA, M

TERAI, Y. ·················································30
TERASAWA, K
THIERRY-MIEG, D
THIERRY-MIEG, Jean
THIRUVENGADAM, T.
TOKUNAGA, M
TOMIOKA, N
TOMIZAWA, J.
TORAYAMA,1.

91
71
71
59,60;61
78,79,8047
82·······85

TORII, H. ··················································71
TOSHIMORI, K
55
TOYODA, A
TOZAKI, H
TSUDA, K.
TSUDA, M
TSUDUKI, M
TSUJI, K
TSUJIMOTO, H
TSUJIMOTO, N
TSUZUKI, M
UEDA, H.

····37,42
48
16
36,57
43
······9170
42
42
26,27,28

UEDA, R. ··················································66
UEDA, S
35,36
UEDA, T.
UENO, SUESUGI, H
UETA, Y
UKAI, YUMEMORI, J.

13,14
69
71
71
71
58

VERNIERI, P. ·············································48
WADA, T. ·················································26
WADA-KIYAMA, Y.
W AKANA, S

32
53

WAKI, K. ··················································91
WANG, L. ·················································35
WATANABE, H

110 AUTHOR INDEX37

WATANABE, Y.··········································· 40
WESSLER, Susan R.

Wu, J.

49
91

YABE, T. ··················································83
YADA, Y53
Y AGASAKI, K

58

YAGI, S. ···················································80
YAMADA, M
28

···91
48
YAMAGUCHI, S
50
Y AMAICHI, Y
99
YAMAKAWA, T.
69,70
YAMAMOTO, A
15
YAMAMOTO, H.
55
YAMAMOTO, K.
49,91
Y AMAMURA, K ..................................•.......... 69
YAMAMURA, M
73
YAMANE, H
91
YAMAGATA, H

YAMAGUCHI, J.

YAMAO, F
YAMAOKA, Y.
YAMAZAKI, M
YAMAZAKI, Y.

11

36
95
64,69,70,100

YANO, M. ·················································91
YASUDA, S
20
YATA, K
YOKOMINE, T.
YOKOTA, T

99'42,4349

YOKOYAMA, E. ···········································75
YOSHIHARA, R.
91
YOSHIKAWA, M
YOSHIKAWA, T.
YOSHIKI, S
YOSHIMORI, T.
YOSHIMURA, A.
YUASA,1.

39"20,21
91
15,16
69
35

YUASA, Y. ················································23
YUKAWA, K·····91

ZHANG, X. ················································49
ZHONG, H
91

Annual Report of the National Institute of Genetics. No.53
Issued by Director-General: Yoshiki Hotta. D.Med.
National Institute of Genetics
Yata 1111 Mishima, Shizuoka-Ken, 411-8540 Japan
Tel. 055-981-6718
Fax. 055-981-6715
Edited by Hironori Niki & Yasushi Hiromi
Published by National Institute of Genetics