Highlights of 2001 (http://www.nig.ac.jp/hot/index.html)

1.

Picky RNA polymerases

Intracellular contents and assembly states of all twelve subunits of the RNA polymerase II
in the fission yeast Schizosaccharomyces pombe.
Kimura, M, Sakurai, H, Ishihama, A.
Eur. J Biochem., 268, 612-619 (2001 )2.

Centromere assembly in vertebrate cells

CENP-H, a constitutive centromere component, is required for centromere targeting of
CENP-C in vertebrate cells.
Fukagawa, T, Mikami, Y., Nishihashi, A., Regnier, v., Haraguchi, T, Hiraoka, Y.,
Sugata, N., Tadokora, K., Brown, W., Ikemura, T
EMBO J, 20, 4603-4617 (2001).
3.

Translational repression determines neuronal potential

Translational repression determines a neuronal potential in Drosophila asymmetric cell
division.
Okabe, M., Imai, T, Kurusu, M., Hiromi, Y., Okano, H.
Nature, 4 / /, 94-98 (20011.
4.

A genome-wide survey of reproductive barriers

A genome-wide survey of reproductive barriers in an intraspecific hybrid
Harushima, Y., Nakagahra, M., Yano, M., Sasaki, T, Kurata, N.
Genetics, /59,883-892 (2001)
5.

Initiation of eukaryotic DNA replication

Sld3, which interacts with Cdc45 (Sld41, functions for chromosomal DNA replication in
Saccharomyces cerevisiae.
Kamimura, Y., Tak, Y.-S., Sugino, A., Araki, H.
EMBOJ, 20,2097-2107 (2001).
6.

Regulation of imprinted X-inactivation by Tsix

Regulation of imprinted X-chromosome inactivation in mice by Tsix.
Sado, T, Wang, Z., Sasaki, H., Li, E.
Development, 128, 1275-1286 (2001)
7.

Taming the genome by DNA methylation

Mobilization of transposons by a mutation abolishing full DNA methylation in
Arabidopsis.
Miura, A., Yonebayashi, S, Watanabe, K, Toyama, T, Shimada, H., Kakutani, T
Nature, 4//,212-214 (2001)

CONTENTS
Introduction'
STAFF
COUNCIL" ............ 1
2
6

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

A-a. DIVISION OF MOLECULAR GENETICS
Intracellular levels of RNA polymerase sigma subunits and transcription factors in E. coli
Akira ISHIHAMA, Etsuko KOSHIO, Hiroto MAEDA, Akira IWATA, Katsunori YATA and
Kaneyoshi YAMAMOTO
8Transcription activation by SdiA, an E. coli homolog of the quorum-sensing regulator
Kaneyoshi YAMAMOTO, Etsuko KOSHIO, Nobuyuki FUJITA and Akira ISHIHAMA
8
Systematic Search for Zinc-binding Proteins in Escherichia coli
Akira KATAYAMA, Takeshi NISHINO and Akira ISHIHAMA
9Discontinuous transition of E. coli phenotypes during transition from exponential growth
to stationary phase
Hideki MAKINOSHIMA and Akira ISHIHAMA
9Compilation, classification and phylogenetic analysis of bacterial transcription factors
Nobuyuki FUJITA and Akira ISHIHAMA

10Intracellular level of the RNA polymerase II in the fission yeast stays constant but the levelof CTD phosphorylation varies depending on the phase and rate of cell browth
Hitomi SAKURAI, Makoto KIMURA and Akira ISHIHAMA
10Interaction between RNA polymerase II and the carboxy-terminal domain (CTD) phosphatase
Fcp1
Makoto KIMURA, Hisako SUZUKI and Akira ISHIHAMA
10
Identification and characterization of S. pombe proteins interacting with the Rpb7 subunitof RNA polymerase II
Hiroshi MITSUZAWA, Emi KANDA and Akira ISHIHAMA
11
Identification and characterization of S. pombe TBP-associated factors (TAFsl
Hiroshi MITSUZAWA and Akira ISHIHAMA
11
An RNA effector in activation of influenza viral RNA polymerase
Ayae HONDA, Kiyohisa MIZUMOTO and Akira ISHIHAMA

12

A-b. DIVISION OF MUTAGENESIS
Ubiquitin conjugating enzyme affecting recombination at matllocus in fission yeast
Joon-Hyun PARK and Fumiaki YAMAO

13

Analysis of Rad6 postreplicative repair (PRR) pathway in fission yeast
Joon-Hyun PARK and Fumiaki YAMAO

14

Ubiquitin-conjugating enzymes involved in degradation of mitotic cyclin
Hiroaki SEINO,. 14

Characterization of SCFGRR1 that ubiquitinates G1 cyclin Cln2 in Saccharomyces cerevisiae
Tsutomu KISHI··············································
14
A-c. DIVISION OF NUCLEIC ACID CHEMISTRY
Host proteins involved in transcription of Sendai virus (SeV) genome
Division of Nucleic Acid Chemistry
Kiyohisa MIZUMOTO··························15
Research Activities
Mechanisms of transcription regulation by eubacteria-type RNA polymerase

CONTENTS

Kan TANAKA···································16

B. DEPARTMENT OF CELL GENETICS

B-a. Division of Cytogenetics
Recombinogenic activity of chimeric recA genes (Pseudomonas aeruginosa/Escherichia coli):a search for RecA protein regions responsible for this activity
Irina V. BAKHLANOVA, Tomoko OGAWA, Vladislav A. LANZOV:
17
Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA
Xiong Yu, Steven A. JACOBS, Stephen C. WEST, Tomoko OGAWA and Edward H. EGELMAN......................................................................................................... 17
Integrative study on chromosome evolution of mammals, ants and wasps based on theminimum interaction theory
Hirotami IMAI, Yoko SATTA, Naoyuki TAKAHATA······································17
B-b. Division of Microbial Genetics
Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in
Saccharomyces cerevisiae
Yoichiro KAMIMURA, Yon-Soo T AK and Hiroyuki ARAKI'
18
Phosphorylation of Sld2 by cyclin-dependent protein kinase
Hiroshi MASUMOTO, Sachiko MURAMATSU, Yoichiro KAMIMURA and Hiroyuki ARAKI....................................................... ··················································18
Functional analysis of Sld5 and Psfl
Yoichiro KAMIMURA and Hiroyuki ARAKI·······························
B-c. Division of Cytoplasmic Genetics
Linkage analysis and linkage disequilibrium mapping of functional psychoses
Takeo YOSHIKAWA

,

19

20

QTL mapping of mouse models of depressive disorder
Takeo YOSHIKAWA

20

Gene expression analysis of a model animal for depression using GeneChips
Takeo YOSHIKAWA

20

Candidate gene approaches
Takeo YOSHIKAWA

20

C. DEPARTMENT OF DEVELOPMENTAL GENETICS
C-a. Division of Developmental Genetics
Translational regulation of zygotic gene expression
Masataka OKABE, Takao IMAI, Mitsuhiko KURUSU, Hideyuki OKANO and Yasushi HIROM!
...................................................... ···················································22
A common molecular basis required for Drosophila sensory organ formation
Nao NIWA, Masataka OKABE and Yasushi HIROMI

22

Function of nuclear receptor Seven-up in the Drosophila central nervous system
Makoto KANAI, Masataka OKABE, Yasushi HIROMI

22

Identification of the down stream factors of a nuclear receptor, Seven-up
Motomi MATSUNO, Yasushi HIROMI

23

The functional analyses of TFIIH p52 subunit
Motomi MATSUNO, Yasushi HIROMI

23

Differentiation of Drosophila longitudinal glia
Yoshihiro YUASA, Masataka OKABE, Yasushi HIROMI23

FGF signal is required for glial ensheathment by redistributing Neuroglian protein
Kaz T AKIZAWA, Emiko SUZUKI, Yoshiki HOTTA and Yasushi HIROMI
Genetic dissection of Spred, a novel negative regulator of ras signaling

CONTENTS

24

Shu KONDO, Yasushi HIROMI

24

Receptors as presenters of positional information
Masaki HIRAMOTO, Yasushi HIROMI

24

GCM-motif protein function in mammalian hematopoiesis
Toshihiko HOSOYA, Yasuno IWASAKI, Motomi OSATO, Tomomasa YOKOMIZO, Chieko
HIGASHI-TAKIZAWA, Yoshiaki ITo, Kazuhiro IKENAKA, Yasushi HIROMI, Yoshiki HOTTA...................................................... ···················································25
Presentation methods that are friendly to color-blind people
Masataka OKABE and Kei ITO··:
· .. ··· 25

Hydra Peptide Project: An epitheliopeptide Hym-301 is involved in head formation
Toshio TAKAHASHI, Hiroshi SHIMIZU, Masayuki HATTA, Osamu KOIZUMI,Yoshitaka
KOBAYAKAWA and Toshitaka FUJISAWA
25
Diffuse nerve net in hydra body column represents a primitive enteric nervous system
Hiroshi SHIMIZU and Toshitaka FUJISAWA
26
C-b. Division of Gene Expression
Segmentation gene product Fushi tarazu is an LXXLL motif-dependent coactivator for orphan
receptor FTZ-Fl
Taiga SUZUKI, Haruhisa KAWASAKI, Ruth T. YU, Hitoshi UEDA and Kazuhiko UMESONO...................................................... ···················································26
Molecular cloning of human squamous cell carcinoma antigen 1 gene and characterizationof its promoter
Katsuyuki HAMADA, Hiroto SHINOMIYA, Yoshihiro ASANO, Toshimasa KIHANA, Mari
IWAMOTO, Yasushi HANAKAWA, Koji HASHIMOTO, Susumu HIROSE, Satoru KyO and
Masaharu ITO
27
Gene expression of human squamous cell carcinoma antigens 1 and 2 in human cell lines
Katsuyuki HAMADA, Yasushi HANAKAWA, Koji HASHIMOTO, Mari IWAMOTO, Toshimasa
KIHANA, Susumu HIROSE, Masahiko NAKAMURA and Masaharu ITO
27
A novel growth-related nuclear protein binds and inhibits rat aldolase B gene promoter
Tomoko YABUKI, Satoru MIYAGI, Hitoshi UEDA, Yasushi SAITOH and Ken-ichi TSUTSUMI...................................................... ···················································27
C-c. Division of Early Embryogenesis
In situ scre~n for novel genes expressed in the yolk syncytial layer
Takuya SAKAGUCHI, Atsushi KUROIWA and Hiroyuki TAKEDA

28

Role of FGF/MAPK signaling in the developing telencephalon of zebragfish embryos
Minori SHINYA, Sumito KOSHIDA, Atsushi KUROIWA and Hiroyuki TAKEDA
28
FGF/MAPK signaling and somite maturation in vertebrate segmentation
Atsushi SAWADA, Minori SHINYA, Yun-Jin Jiang, Atsushi KAWAKAMI, Atsushi KUROIWA
and Hiroyuki TAKEDA
29
Molecular analysis of zebrafish midline mutant chameleon
Atsushi KAWAKAMI, Rolf KARLSTROM, Hiroyuki TAKEDA, William S. TALBOT, Alexander
F. SCHIER····························································································· 29
EST project in medaka fish
Takanori NARITA, Tetsuaki KIMURA, Tomoko JINDO and Hiroyuki TAKEDA
29
Genetic screening for mutations affecting early embryogenesis in medaka fish
Daisuke KOBAYASHI, Tadao KITAGAWA, Shigeo TAKASHIMA, Takahiro KAGE, Hayato
YOKOI, Atsuhi SAWADA, Takanori NARITA, Tetsuaki KIMuRA, Tomoko JINDO, Kazuo ARAKI,
Yuji ISHIKAWA and Hiroyuki TAKEDA··············30C-d. Division of Physiological Genetics
Functional and structural analyses of mammalian Polycomb group complexes
Haruhiko KOSEKI

30

CONTENTSFunctional nucleosomal phases over HS2 of the human {3 -LCR
Ryoiti KIYAMA (National Institute of Advanced Industrial Science and Technology)...................................................................................... ···················31
Functional genotyping based on DNA rearrangements and its application
Ryoiti KIYAMA (National Institute of Advanced Industrial Science and Technology)...................................................... ···················································32
D. DIVISION OF POPULATION GENETICS

D-a. Division of Population Genetics
Local changes in mutation pressure in Drosophila genomes
Toshiyuki T AKANO-SHIMIZU

33

Genetic study of evolutionary changes in the genes involved in Drosophila bristle formation
Toshiyuki TAKANO-SHIMIZU
33
Genetic and molecular dissection of the within- and between-species variation in the sex
comb teeth number in Drosophila
Toshiyuki T AKANO-SHIMIZU
34
D-b. Division of Evolutionary Genetics
Functional analyses of centromere of higher vertebrate cells
Tatsuo FUKAGAWA, Atsushi OKAMURA, Ai NISHIHASHI, Yoshikazu MIKAMI, Toshimichi
IKEMURA······· .. ······························34
Chromosome-wide measurement of replication timing for human chromosomes llq and 21q:
disease-related genes and genome-synteny breakage in timing-switch regions
Yoshihisa WATANABE, Asao FUJIYAMA, Yuta ICHIBA, Yoshiyuki SAKAKI and Toshimichi
IKEMURA········································35
Analysis of codon- and oligonucleotide-composition diversity of bacterial genomes with aself-organizing map (SaM)
Shigehiko KANAYA, Takashi ABE, Makoto KINOUCHI, Yuko YAMADA, Yoshihiro KUDO
and Toshimichi IKEMURA
35
Murine equivalent of the human histo-blood group ABO gene is a cis-AB gene and encodesa glycosyltransferase with both A and B transferase activity
Miyako YAMAMOTO, Xiao-Hong LIN, Yoshihiko KOMINATO, Yukiko HATA, Reiko NODA,
Naruya SAITOU and Fumiichiro YAMAMOTO:
36
CAMUS DB for amino acid sequence data
Sadahiko MIsu, Takayasu IIZUKA, Yuichi KAWANISHI, Kaoru FUKAMI and Naruya SAITOU...................................................... ···················································36
Haplotype analysis of human alpha2-HS glycoprotein (fetuin)
Motoki aSAWA, Isao YUASA, Takashi KITANO, Jurgen HEINKE, Mika KANEKO, Naruya
SAITOU and Kazuo UMETSU...................................................... ···················································37
D-c. Division of Theoretical Genetics
Autonomous formation of spatial pattern in development
Shigeru KONDO
Development of ontology and database for .the cell signaling system
Takako TAKAI and Toshihisa TAKAGI
·.. ·
·
·
·
·
·
Knowledge representation of Signal transduction pathways and logical inferences
Ken-ichiro FUKUDA and Toshihisa TAKAGI
E. DEPARTMENT OF INTEGRATED GENETICS

E-a. Division of Human Genetics
Genome analysis of the mouse 7F4/ F5 imprinted domain

CONTENTS

38
· 38
38

Hiroyuki SASAKI, Hisao SHlROHZU, Takaaki YOKOMINE, Chikako SUDA, Wahyu
PURBOWASITO, Tsunehiro MUKAI, Atsushi TOYODA, Masahira HATTORI and Yoshiyuki
SAKAKI··········································40Regulation of imprinting' of the mouse Igf2/ H19 sub-domain
Hiroyuki SASAKI, Ko ISHIHARA, Hiroyasu FURUUMI, Yuzuru KATO and Wolf REIK ..... 40
Role of de novo DNA methyltransferases Dnmt3a/ Dnmt3b in genomic imprinting
Hiroyuki SASAKI, Naomi TSUJIMOTO, Masahiro KANEDA and Shoji TAJIMA····40DNA methylation, imprinting and human disorders
Hiroyuki SASAKI, Hisao SHIROHZU, Shin-ich MIZUNO, Takeo KUBOTA and Shoji TAJIMA...................................................... ···················································40
Studies on the possibilities of genomic imprinting and Z chromosome dosage compensationin chicken
Hiroyuki SASAKI, Takaaki YOKOMINE, Asato KUROIWA, Yoichi MATSUDA and Masaoki
TSUDUKI········································41
Development of a universal DNA chip system applicable for any organism
Hiroyuki SASAKI, Hisao SHIROHZU, Shin-ichi MIZUNO, Tadafumi IINO, Hidetoshi OZAWA,
Teruhisa OTSUKA and Kosuke TASHIRO
41
Role for Tsix in X chromosome inactivation
Takashi SADO, En LI and Hirosyuki SASAKI···· .. ········ .. 41
Role for Dnmt3a and Dnmt3b in X chromosome inactivation
Takashi SADO, Masaki OKANO, En LI and Hiroyuki SASAKI··················, 41
Human genome resources and their application to the human and primate genome analysis
Asao FUJIYAMA, Ayuko MOTOYAMA, Satoru YOSHIDA, Yoko KUROKI, Yutaka NAKAHORI,
Tatsuo NAKAYAMA, Mieko KODAlRA, Norio TAKAHASHI and Naruya SAITOU
41
Whole genome analysis of signal-transduction pathways in fission yeast
Yong-Sik BONG, Inaho DANJO, Nobuya OGAWA and Asao FUJIYAMA
42
E-b. Division of Agricultural Genetics
Developmental abnormalities induced by DNA hypomethylation mutation of Arabidopsis
Tetsuji KAKUTANI, Tetsu KINOSHITA, Asuka MIURA, Koichi WATANABE and Masaomi
KATO············································43
DNA hypomethylation mutation in rice
Tetsuji KAKUTANI, Koichi WATANABE and Asuka MIURA

43

E-c. Division of Brain Function
Short-Range and Long-Range Guidance of Olfactory Bulb Axons
Tatsumi HIRATA···················································································· 44
Mosaic development of the olfactory cortex with Pax6-dependent and -independent
components
Tatsumi HIRATA, Yoshiko TAKAGI, Yasufumi SATO
44
E-d. Division of Applied Genetics
The rice retrotransposon Tos17prefers low-copy-number sequences as integration targets
Muneo YAMAZAKI, Hidehito TSUGAWA, Akio M!YAO, Masahiro YANO, Jiang Wu, Shinichi
YAMAMOTO, Takashi MATSUMOTO, Takuji SASAKI, Hirohiko HIROCHlKA
45
Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17
insertion. Tagging of a zeaxanthin epoxidase gene and a novel OsTATC gene
Ganesh K. Agrawal, Muneo YAMAZAKI, Masatomo KOBAYASHI, Rei HIROCHIKA, Akio
MIYAO, Hirohiko HIROCHIKA················45
Isolation and characterization of rice phytochrome A mutants
Makoto TAKANO, Hiromi KANEGAE, Tomoko SHINOMURA, Akio MIYAO, Hirohiko
HIROCHlKA, Masaki fuRUYA", 46
Linear DNA intermediates of the Ttol retrotransposon in Gag particles accumulated in

CONTENTS

stressed tobacco and Arabidopsis thaliana
Shin TAKEDA, Kazuhiko SUGIMOTO, Tetsuji KAKUTANI, Hirohiko HIROCHIKA
46
Control of X-chromosome inactivationin early mouse embryogenesis
Nobuo TAKAGI··············· .. ··············47
Search for genes involved in X chromosome inactivation using murine T (X;16) 16H
translocation
Nobuo TAKAGI··········47
F. DEPARTMENT OF GENETIC STRAINS RESEARCH CENTER

F-a. Division of Mammalian Genetics Laboratory
Male-specific reproductive failure caused by X-chromosomal substitution between two mouse
subspecies
Ayako OKA, Nobuo TAKAGI, Kiyotaka TOSHIMORI, Toshiyuki TAKANO-SHIMIZU, Akihiko
MITA, Yoichi MIZUSHINA, Noriko SAKURAI, Hiromi YAMAMOTO and Toshihiko SHIROISHI...................................................... ···················································49
Genetic analysis of a mouse mutant, X-linked polydactyly (Xpn
Yukari YADA, Shigeru MAKINO, Sadao ISHIWA and Toshihiko SHIROISHI.
Analysis of a spontaneous mouse mutation, mesenchymal dysplasia (mes)
Shigeru MAKINO and Toshihiko SHIROISHI

49
49

Genetic analysis of hot taste and pain sensitivity in the Mishima battery of mouse strains
Tamio FURUSE, David A. BLIZARD, Kazuo MORIWAKI, Kazumi YAGASAKI, Toshihiko
SHIROISHI and Tsuyoshi KOIDE
50
A quantitative genetic analysis of spontaneous activity in the wild-derived mouse strains
Tamio FURUSE, Toshiyuki TAKANO-SHIMIZU, Kazuo MORIWAKI, Toshihiko SHIROISHI and
Tsuyoshi KOIDE
50
F-b. Division of Mammalian Development Laboratory
Molecular mechanism of somite segmentation
Yu TAKAHASHI, Aki ISHIKAWA, Aya KITABAYASHI and Yumiko SAGA"
Molecular clock and somitogenesis
Hiroki KOKUBO and Yumiko SAGAThe lineage analysis of Mespl-expressing cells
Satoshi KITAJIMA and Yumiko SAGA

51
52
52

Cloning and functional analysis of mouse Nanos genes
Masayuki TSUDA, Yumiko SASAOKA, Makoto KISO, Seiki HARAGUCHI and Yumiko SAGA...................................................... ···················································52
F-c. Division of Genetic Strains Resarch Center, Plant Genetics Laboratory
Genetic dissection of embryogenesis and gametogenesis of rice (Oryza sativa)
A. Regulation of expression of KNOX family class 1 homeobox genes of rice
Yukihiro ITO, Yasuo NIWA, Nori KURATA

53

B. Genetic analysis of sporogenesis using sterile mutants of rice
Ken-Ichi NONOMURA, Kazumaru MIYOSHI, Mitsugu EIGUCHI, Akio MIYAO, Hirohiko
HIROCHIKA and Nori KURATA·53

C. Structural and functional analysis of rice HAP3 family genes
Nori KURATA, Akiko SERIZAWA, Kazumaru MIYOSHI, Yukihiro ITO
D. Isolation and sequence analysis of receptor-like protein kinase genes of rice
Yukihiro ITO, Nori KURATA

54
54

Positional cloning of a heterochronic gene, PLA1, regulating the plastchron and the durationof vegetative phase in rice
Byoung-Ohg AHN, Kazumaru MIYOSHI, Yukihiro ITO, lun-khi ITOH, Yasuo NAGATO, Non
KURATA····································.·····54

CONTENTS

Construction of rice artificial chromosomes
Tadzunu SUZUKI, Ken-Ichi NONOMURA, Nori KURATA···········55
Analysis of rice genome configuration in the nucleus; isolation and characterization of rice
nuclear protein genes
Kazuki MORIGUCHI, Yukihiro ITO, Yukiko YAMAZAKI, Nori KURATA
56
Genome-wide analysis of reproductive barriers and positional cloning of a reproductive barrier
A. Quantitative Analysis of genotype segregation for reproductive barriers
Yoshiaki HARUSHIMA, Nori KURATA·······56
B. Positional Cloning of a Segregation Distortion Gene Detected in a Progeny of a Cross
between japonica and indica rice
Yoshiaki HARUSHIMA, Nori KURATA·································57

Generation and screening of enhancer trap lines of rice
Yukihiro ITO, Mitsugu EIGUCHI, Nori KURATA

57

F-d. Division of Microbial Genetics
HscA is involved in the dynamics of FtsZ-ring formation in Escherichia coli K-12
Tsuyoshi UEHARA, Hiroshi MATSUZAWA and Akiko NISHIMURA
58
kdsA mutations affect FtsZ-ring formation in Escherichia coli K-12
Hiroshi FUJISHIMA, Masaaki W ACHI and Akiko NISHIMURA'"
59
F-e. Laboratory Invertebrate Genetics
UDP-sugar transporter implicated in glycosylation and processing of Notch
Satoshi GOTO, Misako TANIGUCHI, Masatoshi MURAOKA, Hidenao TOYODA, Yukiko SADO,
Masao KAWAKITA and Shigeo HAYASHI
59
G. CENTER FOR GENETICS RESOURCE INFORMATION

G-a. Genetic Informatics Laboratory
ORYZABASE--INTEGRATED RICE SCIENCE DATABASE-Takahiro YAMAKAWA, Kazu MITSUI, Nori KURATA, Atsushi YOSHIMURA, Yasuo NAGATO
and Yukiko YAMAZAKI·······················60
PEC: Profiling of Escherichia coli Chromosome
Yukiko YAMAZAKI, ToruIKEGAMI, Takehiro YAMAKAWA, Kazu MITSUI, Takeshi
KAWABATA, Ken NISHIKAWA, Tadahiro MORI, Akiko NISHIMURA and Junichi KATO...................................................... ···················································60
G-b. Genome Biology Laboratory
NEXTDB: The nematode expression pattern database
Tadasu SHIN-I, Yuji KOHARA

61

Clustering analysis of gene expression patterns in C.elegans
Masahiro ITO, Yohei MINAKUCHI, Michiko SERIZAWA and Yuji KOHARA
Semi-automatic system for creation of cell shape model in C.elegans embryogenesis
Hideaki HIRAKI and Yuji KOHARA

61
61

Translational control of maternal glP-l mRNA by POS-1 and its interacting protein PIP-1
Ken-ichi OGURA, Norihito KISHIMOTO, Shohei MITANI, Keiko GENGYO-ANDO, Yuji KOHARA...................................................... ···················································62
An isoform of eIF4E is a component of germ granules and is required for spermatogenesis
in C. elegans
Anahita AMIRI, Brett D. KEIPER, Ichiro KAWASAKI, Yuan FAN, Yuji KOHARA, Robert E.
RHOADS and Susan STROME
62
Toward understanding of tbx-9
Yoshiki ANDACHI63

The Caenorhabditis elegans unc-32 Gene Encodes Alternative Forms of a Vacuolar ATPasea Subunit

CONTENTSNathalie PUJOL, Claire BONNEROT, Jonathan J. EWBANK, Yuji KOHARA and Danielle
THIERRY-MIEG
63
Gene expression profiles in Ciona intestinalis tailbud embryos
Yutaka SATOU, Naohito TAKATORI, Lixy YAMADA, Yasuaki MOCHIZUKI, Makoto
HAMAGUCHI, Hisayoshi ISHIKAWA, Shota CHIBA, Kaoru IMAI, Shungo KANO, Seiko D.
MURAKAMI, AIde NAKAYAMA, Atsuo NISHINO, Yasunori SASAKURA, Gohki SATOH, Taishin
SHIMOTORI, Tadasu SHIN-I, Eiichi SHOGUCHI, Miho M. SUZUKI, Norio TAKADA, Nanami
UTSUMI, Natsue YOSHIDA, Hidetoshi SAIGA, Yuji KOHARA and Nori SATOH
63
Profiles of Maternally Expressed Genes in Fertilized Eggs of Ciona intestinalis
Takahito NISHIKATA, Lixy YAMADA, Yasuaki MOCHIZUKI, Yutaka SATOU, Tadasu SHIN-I,
Yuji KOHARA, Nori SATOH
64

H. STRUCTURAL BIOLOGY CENTER
H-a. Biological Macromolecules Laboratory
Thin Layer Illumination Microscopy as a Novel Technique for Single Molecule Fluorescence
Imaging of the Inside of Cells
Makio TOKUNAGA··············
66
Single Molecule Imaging of Nucleocytoplasmic Transport in Cells and Quantitative Analysis
of Interaction with Nuclear Pores
Makio TOKUNAGA and Naoko IMAMOTO
·
·
·
·66
A novel protein pl05: recruitment into mRNA-containing complex and actin- andmicrotubule-dependent transport
Nobuyuki SHIINA, Kazumi SHINKURA and Makio TOKUNAGA······························· 66
Single molecule measurement of intermolecular and intramolecular interactions using
subpiconewton intermolecular force microscopy
Michio HIROSHIMA and Makio TOKUNAGA
67
Single molecule measurement of protein folding by intermolecular force microscopy
Isao SAKANE, Michio HIROSHIMA, Kunihiro KuwAJIMA and Makio TOKUNAGA"67
A Novel in vitro Assay System of Nucleocytoplasmic Transport
Atsuhito OKONOGI, Michio HIROSHIMA, Nobuyuki SHIINA, Naoko IMAMOTO and Makio
TOKUNAGA
67
H-b. Molecular Biomechanism Laboratory
The promoter arrest of E. coli RNA polymerase and the effect of the Gre factors
Motoki SUSA, Tomoko KUBORI, Hiroki NAGAI and Nobuo SHIMAMOTO
68
70
Inactivation of u by oligomerization and identification of the role of its spacer region
Hiroki NAGAI, Taciana KASCIUKOVIC, Richard S. HAYWARD, Yumiko SATO and Nobuo
SHIMAMOTO
68
Does RNA polymerase track a groove of DNA during sliding?
Kumiko SAKATA-SOGAWA and Nobuo SHIMAMOTO

69

H-c. Multicellular Organization Laboratory
Analysis of Synthetic Dauer-constitutive Mutations in the Nematode Caenorhabditis elegans
Kiyotaka OHKURA, Tomoko YABE, Kohji MIYAHARA, Eiko TSUCHIYA, Takeshi ISHIHARA
and Isao KATSURA
69
Analysis of Mutants That Show Abnormality in the Selection between Two Behaviors andin Behavioral Plasticity
Takeshi ISHIHARA, Yuichi IINO, Akiko MOHRI, Ikue MORI and Isao KATSURA
70
C. elegans Mutants in the Associative Learning of Odorants and Food
Ichiro TORAYAMA, Takeshi ISHIHARA and Isao KATSURA
72

CONTENTSFluoride-resistant Mutants of the Nematode Caenorhabdilis elegans
Akane OIsm, Minoru KAWAKAMI, Akiko KAMAMOTO, Takeshi ISIDHARA and Isao KATSURA...................................................... ···················································72
H-d. Biomolecular Structure Laboratory
Crystallographic Study of FI-ATPase
Yasuo SmRAKIHARA, Aya SmRAToRI and Chikako SHIRAKIHARA
Crystallographic Study of the Transcription Activator, PhoB
Kazuyasu SHINDOH, Katsumi MAENAKA and Yasuo SHIRAKIHARA
Structural and functional studies of immunoglobulin(lg)-like receptors
Katsumi MAENAKA, Kimie AMANO and Yasuo SHIRAKIHARA
Structural study of Na+-translocating ATPase
Yasuo SHIRAKIHARA

73
73
74
74

Structural analysis of glutaminase from Micrococcus luleus K-3
Yasuo SHIRAKIHARA and Aya SHIRATORI···········································75
H-e. Gene Network Laboratory
Nucleocytoplasmic exchange of macromolecules
Naoko IMAMOTO
75
Analysis of translocation process through nuclear pores using single molecule imaging
techniques
Naoko IMAMOTO and Makio TOKUNAGA
76
Analysis of nuclear migration of importin {3 through the nuclear pore complex
Shingo KOSE, Hitomi OZAWA, Yoshihiro YONEDA and Naoko IMAMOTO
Analysis of mechanism of nuclear import and export of {3 -catenin
Makiko KOIKE and Naoko IMAMOTO
Nuclear import under stress condition and normal condition
Maiko FURUTA, Shingo KOSE and Naoko IMAMOTO·· .. ··76
76
77

Molecular cloning of a novel importin Q; homologue from rice, by which constitutive
photomorphogenic 1 (COPl) nuclear localization signal (NLS)-protein is preferentiallynuclear imported
Chang-lie lIANG, Kazuhiro SHOJI, Rikyu MATSUKI, Akihiki BABA, Noritoshi INAGAKI,
Hiroshi BAN, Toshisuke IWASAKI, Naoko IMAMOTO, Yoshihiro YONEDA, Xing-Wa,ng DENG
and Naoki YAMAMOTO
77
Transforming growth factor- {3 induces nuclear import of Smad3 in an importin- {31 andRan-dependent manner
Akira KURISAKI, Shingo KOSE, Yoshihiro YONEDA, Carl-Henrik HELDIN and Aristidis
MOUSTAKAS
78
I. CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPANI-a. Laboratory for DNA Data Analysis
Expression profile of normal developing mouse eye and brain
Chi Chiu WANG, Kazuho IKEO, Takashi GOJOBORI, Michael Scott ROGERS·············· 79
The study of the genetic program for planarian brain using eDNA chip technology
Masumi NAKAZAWA and Takashi GOJOBORI
79
Comparative study for the CNS evolution using neural-related genes found in the planarian
EST project
Katsuhiko MlNETA, Masumi NAKAZAWA, Francese CEBRIA, Kazuho lKEo, Kiyokazu AGATA
and Takashi GOJOBORI
79
Identification of Neurotransmitter Receptor Genes under Significantly Relaxed Selective
Constraint by Orthologous Gene Comparisons between Humans and Rodents
Hisakazu IwAMA and Takashi GOJOBORI80

CONTENTS

Simulating interaction among multiple gene expression with GASS (Genetic Algorithm for
S-System) model
Takaho ENDO and Takashi GOJOBORI
80
Identification of apoptotic machinery in lower metazoan phylum, platyhelminthlung-Shan HWANG, Kazuho IKEO and Takashi GOJOBORI··········81
In Silico Chromosome Staining: Reconstruction of Giemsa Bands from the Whole Human
Genome Sequence
Yoshihito NIIMURA and Takashi GOJOBORI
81
I-b. Laboratory for Gene-Product Informatics
GTOP: a database of protein structures predicted from genome sequences
Takeshi KAWABATA, Satoshi FUKUCHI, Keiichi HOMMA, Motonori OTA, liro ARAKI,
Takehiko ITo, Nobuyuki ICHIYOSHI and Ken NISHIKAWA·····82
Comparison of energy components of proteins from thermophilic and mesophilic organisms
Akira R. KINJO and Ken NISHIKAWA·····································
82
Protein Surface Amino Acid Compositions Distinctively Differ Between Thermophilic andMesophilic Bacteria
Satoshi FUKUCHI and Ken NISHIKAWA·························································· 83
Knowledge-based potential defined for a rotamer library to design protein sequences
Motonori OTA, Yasuhiro ISOGAI and Ken NISHIKAWA·······························
83
X-Ray Crystalline Structures of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile,
Pyrococcus furiosus, and Its Cys-Free Mutant
Hideaki TANAKA, Masanobu CHINAMI, Tsunehiro MIZUSHIMA, Kyoko OGASAHARA,
Motonori OTA, Tomitake TSUKIHARA and Katsuhide YUTANI
84
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 and Muneo YAMAZAKI...................................................... ···················································84
Parallel evolution of ligand specificity between Lacl/GalR family repressors and periplasmic
sugar-binding proteins
Kaoru FUKAMI-KoBAYASHI, Yoshio TATENO and Ken NISHIKAWA
85
I-d. Labqratory for Research and Development of Biological Databases
Research and Development for DDBl
85
Research and Development for WFCC-MIRCEN World Data Centre for Microorganisms(WDC)···········································86
Others···············································86
J. RADIOISOTOPE CENTER(1) Radioisotope Center
Yoshiharu YAMAICHI, Katsunori YATA and Hironori NIKI

88

K. EXPERIMENTAL FARM

(1) Development of new experimental lines and reevaluation of the genetic stocks of rice
Ken-Ichi NONOMURA, Mitsugu EIGUCHI, Toshie MIYABAYASHI, Nori KURATA . ··········89
ABSTRACTS OF DIARY FOR 2001
FOREIGN VISITORS IN 2001

90
93

AUTHOR INDEX····························································································· 95

CONTENTSIntroduction
This is the 52nd annual report of the National Institute of Genetics (NIG). The planning of administrativereform, aimed at reorganizing research institutes and national universities into "agencies", has continued
from the previous year. In September, the Council on the Administrative Reforms for National Universities issued
a preliminary report, showing the future reform path for research institutes such as NIG. I believe this reform
should not be just a restructuring for the purpose of efficiency, but one that will, in the future, aid the improvementof academic research and higher education. In order to achieve this, we need to further improve the quality of
our research and create a solid justification for our existence.
I'm happy to report here that we continued to produce many excellent research achievements this year. Large
projects supported by NIG, such as the DNA Data Bank of Japan, have also progressed smoothly. Thanks to
improvements to our home page and the completion of the "Internet Museum of Genetics" , our activities topopularize knowledge and research results of genetics have also started. This year is memorable for the studyof genetics because of the elucidation of human genome draft sequence. We have already obtained full knowledgeof genome sequences about many organisms, including many bacteria, nematode and fruit fly. By adding humangenome to this list, we have really entered a new era. We would like to seriously consider about its influenceon our future research.
The following personnel changes of the faculty took place during 2001. Professor Tomoko Ogawa(Vice-Director and the head of the Department of Cell Genetics) who for many years contributed to the developmentof NIG became emeritus reaching the mandatory retirement age. Professor Akira Ishihama (Department of
Molecular Genetics) succeeded Prof. Ogawa as the Vice-Director. The Center for Information Biology was
reorganized as the Center for Information Biology and DNA Data Bank of Japan, adding a new laboratory: theLaboratory of Gene-Expression Analysis. We made two new recruitments this year: Assistant Professor Tetsu
Kinoshita has joined the Division of Agricultural Genetics and Assistant Professor Hiroki Kokubo (Genetic
Strains Research Center) transferred from Okazaki National Institute for Basic Biology, valuable additions to
the research staff. On the other hand, Professor Hiroyuki Takeda and assistant professor Atsushi Kawakami(Early Development) left NIG to form a new group at the Graduate Course in the University of Tokyo. AssistantProfessor Toshihiko Hosoya (Developmental Genetics), Assistant Professor Shigeo Tanaka (Cell Genetics) andAssistant Professor Tadashi Imanishi (Center for Information Biology) have left the institute to extend theircareer in other institutions.
The following personnel changes of the administrative staff took place: in April, Akira Kobayashi, Headof the General Mfairs Section, was transferred to Shinshu University as Head of General Affairs, and was replaced
by Yukio Tomiyama. In October, Kiyotaka Uezumi, Head of Administration, was transferred to Chiba University
as Head of Student Affairs and was replaced by Kenji Ishikawa.
As the Genetics Department of the Graduate University for Advanced Studies, 41 students studied in our
PhD program. Eleven students entered the program this year, and seven have obtained the degree of Doctorof Science. Five foreign researchers and eight graduate students were supported by the COE (Center of Excellence)
Program.

Yoshiki Hotta, Director-General

Introductioh

1

STAFF (as of December 31, 2001)
Director-General

HOTTA, Yoshiki, D. Med.
Vice-Director

ISHIHAMA, Akira, D. Sc.
Members
1. Department of Molecular Genetics

ISHIHAMA, Akira, D. Sc., Head of the Department
Department of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUJITA, Nobuyuki, D. Sc.
MITSUZAWA, Hiroshi, D. Sc.
KIMURA, Makoto, D. Sc.
Division of Mutagenesis
YAMAO, Fumiaki, D. Sc., Associate Prrofessor
KISHI, Tsutomu, D. Eng.
SEINO, Hiroaki, D. Sc.
Division of Nucleic Acid Chemistry
MIZUMOTO, Kiyohisa, D. Sc., Adjunct Professor
TANAKA, Kan, D. Ag, Adjunct Associate Professor

2. Department of Cell Genetics
ARAKI, Hiroyuki, D. Sc., Head of the Department
Division of Cytogenetics
IMAI, Hirotami T., D. Sc., Associate Professor
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., Adjunct Professor
3. Department of Developmental Genetics
HIROSE, Susumu, D. Sc., Head of the Department
Department of Developmental Genetics
HIROMI, Yasushi, D. Sc., Professor
FUJISAWA, Toshitaka, Ph. D., Associate Professor
SHIMIZU, Hiroshi, D. Eng.
OKABE, Masataka, D. Med., D. Sci.
Division of Gene Expression
HIROSE, Susumu, D. Sc., Professor
UEDA, Hitoshi, D. Ag., Associate Professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-aki, D. Ag.

2

STAFF

Division of Early Embryogensis
TAKEDA, Hiroyuki, D. Sc., Professor
KAWAKAMI, Atsushi, D. Sc.
Division of Physiological Genetics
KOSEKI, Haruhiko, D. Med., Adjunct Professor
KIYAMA, Ryoiti, D. Sc., Adjunct Professor

4. Department of Poqulation Genetics
IKEMURA, Toshimichi, D. Sc., Head of the Department
Division of Population Genetics
TAKANO, Toshiyuki, D. Sc.
Division of Evolutionary Gentics
IKEMURA, Toshimichi, D. Sc., Professor
SAITOU, Naruya, Ph. D., Associate Professor
TENZEN, Toyoaki, D. Ag.
FUKAGAWA, Tatsuo, D. Sc.
Division of Theoretical Genetics
KONDO, Shigeru, Ph. D., D. Sc., Adjunct Profesor
TAKAGI, Toshihisa, D. Eng., Adjunct Profesor
5. Department of Integrated Genetics
SASAKI, Hiroyuki, D. Med., Head of the Department
Department of Human Genetics
SASAKI, Hiroyuki, D. Med., Professor
FUJIYAMA, Asao, D. Sc., Associate Professor
SADO, Takashi, D. Sc.
Division of Agricultural Genetics
KAKUTANI, Tetsuji, D. Sc., 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., Adjunct Professor
6. Genetic Strains Research Center
SHIROISHI, Toshihiko, D. Sc., Head of the Center
Mammalian Genetics Laboratory
SHIROISHI, Toshihiko, D. Sc., Professor
KOIDE, Tsuyoshi, D. Med.
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
NISHIMURA, Akiko, D. Ag., Associate Professor

STAFF

3

Invertebrate Genetics Laboratory
HAYASHI, Shigeo, D. Sc., Professor
GOTO, Satoshi, D. Sc.

7. Center for Genetic Resource Information
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
SOGAWA, Kumiko, D. Sc.
NAGAI, Hiroki, D. Sc.
Multicelluar Organization Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.
Biomolecular Structure Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate Professor
MAENAKA, Katsumi, D. Eng.
Gene Network Laboratory
IMAMOTO, Naoko, D. Med., Associate Professor
KOSE, Shingo, D. Med.
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
OTA, Motonori, D. Sc.
Laboratory for Gene Function
TATENO, Yoshio, Ph. D., D. Sc., Professor
FUKAMI-KOBAYASHI, Kaoru, Ph. D.
Laboratory for Molecular Classification
SUGAWARA, Hideaki, D. Eng., Professor
MIYAZAKI, Satoru, D. Sc.
10. Radioisotope Center
NIKI, Hironori, D. Med., Head of the Center

4

STAFF

11. Experimental Farm
KURATA, Nori, D. Ag., Head of the FarmNONOMURA, Ken-ichi, D. Ag.
12. Technical Section
ISHII, Yuriko, Chief of the Section
13. Department of Administration
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, 2001)
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 Science, 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, 2001)
Chairman
ISHIHAMA, Akira; Professor, National Insutitute of Genetics
Vice-chairman
SEKIGUCHI, Mutsuo; Professor, Fukuoka Dental College
Outside 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: Director-General, Institute for Virus Research, Kyoto University
MATUURA, Etsuko; Professor, Faculty of Science, Ochanomizu University
OGAWA, Tomoko; Vice-President, Iwate College of Nursing
SASAZUKI, Takehiko; Professor, Medical Institute of Bioregulation, Kyusyu University
SHINOZAKI, Kazuo; Chief Scientist, RIKEN Tsukuba Institute
TAJIMA, 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
KOHARA, Yuji; Professor, National Insutitute of Genetics
SASAKI, Hiroyuki; Professor, National Insutitute of Genetics
SHIMAMOTO, Nobuo; Professor, National Insutitute of Genetics
SHIRaISHI, Toshihiko; Professor, National Insutitute of Genetics

ADVISORY COMMITTEE

7

A-a. DIVISION OF MOLECULAR GENETICS(Rob), Cra (FruR), CRP, CueR, CusR, DeoR, DnaA,
DnaK, Dps, EF-Tu, FadR, Fir, Fis, Fur, GreA, GroEL,
H-NS, HepA, Hfq, H-NS, Hu, HydG, IciA, IclR, IHF,
llvY, InfB; KdpE, Lac!, LeuO, Lrp, MerR, MntR, NusA,
OmpR, OxyR, PhoB, PhoP, PspF, RecA, Rho, RpsB,
RpbF, Rsd, SdiA, SoxR, SoxS, Ssp, StpA, ThiI, ZntRand Zur. The determination of intracellular levels ofthese factors is in progress by using quantitative
Western blotting for lysates of E. coli cells growing at
various phases and at various temperatures. For details
see refs. 1, 2, 3, 5, 6, 18, 20, 21.

(1) Intracellular levels of RNA polymerase sigma
subunits and transcription factors in E. coli

(2) Transcription activation by SdiA, an E. coli homologof the quorum-sensing regulator

Akira ISillHAMA, Etsuko KOSillO, Hiroto MAEDA l , Akira
IWATA2, Katsunori YATA 3and Kaneyoshi YAMAMOTO(lKagoshima University, 2Nippon Institute of
Biological Science, Tokyo, and 3Radioisotope Center)

Kaneyoshi YAMAMOTO, Etsuko KOSHIO, Nobuyuki
FUJITA and Akira ISHIHAMA

A. DEPARTMENT OF
MOLECULAR GENETICS

The RNA polymerase core enzyme with thestructure ad3{3' is converted into holoenzymes bybinding one of seven molecular species of the asubunit. Each holoenzyme recognizes and transcribes
a set of E. coli genes. The major factor affecting thegene expression pattern in E. coli is the intracellularconcentrations of these seven a subunits. We thendetermined the intracellular concentrations of thesubunits at various phases of E. coli W3110 growing
in various media and at various temperatures. In
exponential growth phase, dO (the principal a) is themost abundant species, but under certain growth
conditions, the level of minor a subunits exceeds thatof the major a subunit. The levels of functional a
subunits are, however, controlled by anti-sigma factors
with inhibitory activity of the a subunits. We thenmeasured the intracellular levels of known anti-sigma
factors such as Rsd (anti- a 70 ), FlgM (anti- a F), and
DnaK (anti- a H ).
The specificity of RNA polymerase holoenzymes
are further modulated by interaction with transcription
factors. E. coli contains a total of about 260 species of
the DNA-binding protein (N. Fujita, unpublished),
whicb all seem to function as transcription factors. We
therefore extended our measurement of the intracellularconcentrations for all these transcription factors. So
far we produced specific antibodies against
over-expressed and purified transcrip~ion factors,
including AcrR, AhpC, AhpF, ArcA, CbpA, CbpB

8

DEPARTMENT OF MOLECULAR GENETICS

Transcription factors have been classified as
activators or repressors. Recent studies, however,indicate that both activators and repressors have dual
functions, showing activation and repression oftranscription depending on the site of their DNA
binding. Escherichia coli is considered to have morethan 200 transcription factors, which interact with the
RNA polymerase and modulate its activity andpromoter selectivity. Since the mode of transcription
regulation by a transcription factor is closely correlatedwith the contact site on the RNA polymerase, we
proposed to classify these transcription factors basedon their contact subunit(s) of RNA polymerase, i.e.,class-I (a contact), class- n (a contact), class- ill ( (3contact), and class-N ({3' contact). Most class-I factors
bind to upstream of the promoter, interact with the
C-terminal domain (CTD) of the a subunit, andthereby support the RNA polymerase-binding to thepromoter. On the other hand, the class-I[ factors contact
with the C-terminal region (CTD) of a subunit andstimulate the promoter opening. Many class- mand-VI
factors influence various steps of transcription frominitiation, elongation to termination.
SdiA, an E. coli homologue of the quorum-sensing
regulator, regulates the expression of ftsQAZ operon
for cell division. Transcription ftsQ is under of two
promoter, upstreamftsQP2 and downstreamftsQP1,
which are separated by 125 bp. SdiA activatestranscription from ftsQP2 in vivo. The purified SdiA
activated transcription in vitro of ftsQP2 byreconstituted RNA polymerase containing the major

o subunit (0 D). Gel shift and DNase I footprinting

assays indicated that the binding center of SdiA was
located at -34 of the ftsQP2 promoter, and that thebinding of RNA polymerase to ftsQP2 promoter was
stimulated by SdiA. In vitro transcription studies using
the mutant RNA polymerase holoenzymes containinga CTD-deleted a subunit (a235) or a CTD-deleted rP(0529), however, indicated that the 0 CTD, but not
aCTD, is required for recruitment of the RNA
polymerase and SdiA-dependent transcription
activation. Thus we proposed such a novel mode oftranscription activation as that: (i) SdiA supports the
RNA polymerase binding to ftsQP2 as in the case ofclass-I activators; but (ii) this recruitment of RNApolymerase by SdiA depends on the presence of intacto CTD as in the case of class-II factors. In addition to
the P2 activation, SdiA inhibited RNA polymerase
binding to the ftsQP1 promoter and thereby repressedtranscription from PI. Neither aCTD nor oCTD wererequired for this inhibition. Thus, the transcription
repression of PI by SdiA may result from competition
with the RNA polymerase in binding to the ftsQP1
promoter.
The measurement of SdiA protein by Western
blotting indicates that the intracellular level of SdiA
stays constant throughout the growth phase transition
from exponential to stationary phase. However, theactivity of SdiA seems to be controlled, because thetranscription activation in vitro by SdiA increased in
the presence of acetyl homoserine lactones (AHLs), thebacterial hormones which playas the ligands for
activation of most LuxR family proteins. For details see
ref. 21.
(3) Systematic Search for Zinc-binding Proteins inEscherichia coli
Akira KATAY AMA, Takeshi NISHlN0 1 and Akira
ISHIHAMA (INippon Medical School, Tokyo)
Zinc is an essential trace element, but virtually
non-toxic, in contrast to iron, copper and mercury. Over
300 enzymes or proteins have been identified to require
zinc for functions. In zinc-containing enzymes orproteins, zinc has two major functions, i.e., catalytic
and structural. Up to the present time, the structuraland functional roles of zinc have been analysed indetails with zinc-containing proteins from higher
eukaryotes, but little is known about the zinc-binding

proteins in prokaryotes. A systematic search for
Escherichia coli proteins with the zinc-binding activity
was performed using the assay of radioactive Zn(II)binding to total E. coli proteins fractionated by two
methods of two-dimensional gel electrophoresis, i.e.,
the widely used O'Farrell system and the newly
developed radical-free and highly reducing (RFHR)
method. A total of at least 30 to 40 radioactive spots
were identified, of which 14 have been assigned fromamino-terminal sequencing. Besides 5 hitherto
known zinc-binding proteins, the newly identified
zinc-binding proteins of E. coli include acetate kinase(AckA), molecular chaperone (DnaK), serinehydroxymethyltransferase (GlyA), transketolase
isozymes (TktNTktB), translation elongation factor
Ts (Tsf), ribosomal proteins L2 (RplB), L13 (RpIM) and
one of S15 (RpsO), S16 (RpsP) or S17 (RpsQ). Together
with about 20 known Zn(II)-binding proteins, the total
number of Zn(II)-binding proteins in E. coli increased
up to more than 30 species or probably more than 3%of about 1,000 proteins expressed under laboratory
culture conditions. The specificity and affinity ofzinc-binding were analysed for some of theZn(II)-binding proteins.
(4) Discontinuous transition of E. coli phenotypes duringtransition from exponential growth to stationary phase
Hideki MAKINOSHIMA and Akira ISHIHAMA
Upon sensing an impending saturation level of the
population density, Escherichia coli cells enter into thestationary phase. During the transition fromexponential growth to stationary phase, marked
changes take place in the morphology and physiologyof E. coli cells. Approximately 1,000 genes highly
expressed in the 'exponentially growing cells aremostly turned off or markedly repressed in thestationary-phase cells and instead a set of 50-100
genes that are repressed in the growing cells begins
to be expressed. To understand the molecular events
underlying the growth phase-coupled changes in gene
expression pattern, attempts have been made tophysically separate the cells of different growth pl).ases.
By using Percoll gradient centrifugation, we succeeded
to separate E. coli cultures into more than 15 cell
populations, each forming a discrete band. The cell
separation was found to result from the difference inbuoyant density but not the size difference. The cell

DIVISION OF MOLECULAR GENETICS

9density increases upon transition from exponential
growth to stationary phase. Exponential-phase cultures
formed at least 5 discrete bands with lower densities,
whereas stationary-phase cultures formed more than
10 bands with higher densities. Two molecular markers
characterizing each cell population were identified: thefunctioning promoter species, as identified bymeasuring the expression of green fluorescent protein
under the control of test promoters; and the expressed
protein species, as monitored by quantitative
immunoblotting. These findings together suggest thatthe growth phase-coupled transition of E. coli
phenotype is discontinuous. For details see ref. 10.
(5) Compilation, classification and phylogenetic analysis
of bacterial transcription factors
Nobuyuki FUJITA and Akira ISHIHAMA
Complete genomic sequences of 36 bacterial, 11archaeal and 2 eukaryotic species were searched forbacteria-type transcription factors based on sequencesimilarity to the known factors. Transcription factors
could be classified into more than 50 protein families
primarily according to the structure of DNA-binding
domain. All bacterial species with medium to large
genome size, irrespective of their taxonomic group,
share most of the transcription factor families,
suggesting that these protein families have ancientphylogenetic origins. In contrast, bacteria with small
genome size, typically less than 1.5 Mbp, retain only
a few transcription factors. This result, together withdetailed phylogenetic analysis of each protein family,suggests that the transcription factors have decayedvery rapidly after the establishment of parasitic life
cycle of these bacterial species. Archaea share limited
numbers of transcription factor families with bacteria,
and virtually no bacterial transcription factor wasconserved in eukaryotes. Possible horizontal genetransfer events were inferred by several methods
including massive principal component analysis of thecodon usage pattern. An integrated Web-based
database of bacterial transcription factors, withidentification and classification system for newly
determined sequences, is being constructed.

of cell browth
Hitomi SAKURAI, Makoto KIMURA and Akira ISHIHAMAThe RNA polymerase II (Polll) of the fission yeast
Schizosaccharomyces pombe consists of twelve Rpb
subunits, of which four (Rpb1, Rpb2, Rpb3 and Rpbll)
form the assembly and catalytic core of Polll and five
(Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) are shared
among RNA polymerases I, II and m. The intracellularlevels of three RNA polymerase forms should beinterrelated, but the control of RNA polymerase
formation remains mostly unknown. To reveal thephysiological role and the synthesis control of eachRpb subunit, the intracellular levels of Rpb proteins
were determined in S. pombe growing at various phases
under various conditions. Results indicate that the
intracellular concentrations of Rpb proteins stay
constant at levels characteristic of the rate and phaseof cell growth, and the relative level between twelve
Rpb subunits also remain constant, together implyingthat the intracellular concentration of Pol IT stays
constant as in the case of prokaryotic RNA polymerase.
As an attempt to get i.nsights into the activity
control of Polll, we also analyzed the phosphorylationlevel of the carboxyl-terminal domain (CTD) of thelargest subunit Rpb1. Phosphorylated forms of Tyrl
and Thr4 within 29 repeats of the YSPTSPS heptapeptide
were detected in both slow-migrating IIo andfast-migrating ITa forms of Rpb1 on SDS-PAGE.
However, phosphorylated Ser2 and Ser5 were identified
only in the lIo form, indicating that Ser phosphorylation
contributes to the conformational change of CTD. The
phosphorylation levels of Ser, Thr and Tyr all varydepending on the cell culture conditions. Taken
together we conclude that the intracellular level of Pol
II stays constant, but the amount of Polll engaged intranscription cycle varies depending on the culture
conditions as estimated from the sites and levels ofphosphorylation of Rpb1 CTD. For details see refs. 8,
15,16.
(7) Interaction between RNA polymerase n and thecarboxy-terminal domain (CTD) phosphatase Fcpl
Makoto KIMURA, Hisako SUZUKI and Akira ISHIHAMA(6) Intracellular level of the RNA polymerase n in thefission yeast stays constant but the level of CTD
phosphorylation varies depending on the phase and rate

10

DIVISION OF MOLECULAR GENETICS

In transcriptional regulation, RNA polymerase II(polll) interacts and forms complexes with a number

of protein factors. To isolate and identify the pol
I-associated proteins, we constructed a Schizosaccharomyces
pombe strain carrying a FLAG-tag sequence fusedto the rpb3 gene encoding the pol II subunit Rpb3.
By immuno-affinity purification with anti-FLAG
antibody-resin, we isolated a poll-complex containing
the Rpbl subunit with a non-phosphorylated form of
the carboxyl-terminal domain (CTD). In addition to thepoll subunits, this complex was found to contain threesubunits of the transcription factor TFllF (TFllFa,
TFIIFf3, and Tfg3) and the CTD-phosphatase Fep1. The
same type of poll-complex could also be purified fromanother S. pombe strain carrying a tagged Fcpl gene. The
isolated Fcpl showed the CTD-phosphatase activity invitro. The fcPl gene was found to be essential for cell
viability. Direct interaction between purified Fcpl andpoll was observed in vito. By chemical cross-linking,
GST-pull down, and affinity chromatography, theFcpl-interacting subunit of poll was identified to be
Rpb4, which plays regulatory roles in transcription.
We also constructed a thiamine dependent rpb4
shut-off system of S. pombe. On repression of the rpb4
expression, the cell produced more non-phosphorylated
form of Rpbl, but the polll-complex isolated with theanti-FLAG antibody contained less Fcpl and more
phosphorylated form of Rpbl with the concomitant
reduction of Rpb4. This result indicates the importanceof Fcpl-Rpb4 interaction for the formation of Fcpl/
TFIlF/polll complex in vivo. For details see ref. 9.
(8) Identification and characterization of S. pombe
proteins interacting with the Rpb7 subunit of RNApolymerase II

Hiroshi MITSUZA W A, Emi KANDA and Akira ISHIHAMA
Rpb7, an RNA polymerase I (Poll) subunit thatis not shared by RNA polymerase I or ill, is required
for accurate transcription initiation from the core
promoter but is unnecessary for RNA synthesis itself.
To further understand the function of Rpb7 intranscription by Poll, we screened for proteins thatinteract with S. pombe Rpb7 using the yeast two-hybrid
system. One of the isolated clones encodes the S. pombe
homolog of the S. cerevisiae Nrdl (nuclear pre-mRNA
down regulation) protein. S. cerevisiae Nrdl has recently
been shown to be involved in poly(A)+-independent3'-end formation of snRNAs and snoRNAs. We foundthat the S. cerevisiae Rpb7 interacts with Nrdl as in thecase of the S. pombe counterparts, indicating that theinteraction have been conserved throughout evolution
and thus suggesting that the interactions has
physiological relevance. To test for direct interaction
between the S. pombeRpb7 and Nrdl-related protein
in vitro, we carried out GST pull-down assays withrecombinant proteins expressed in E. coli. Rpb7 wasproduced as a His-tagged protein while the C-terminal
region of the Nrdl-related protein was produced as afusion to GST. Because both proteins were found to
be insoluble, each protein was solubilized in a
denaturing buffer containing urea and then renatured
by dialysis followed by GST pull-down assays. As
expected, Rpb7 bound to the Nrdl-related protein.
Gene disruption revealed that the Nrdl-related protein
is essential for cell viability. We also identified aminoacid residues of Rpb7 that is important in theinteraction with the Nrdl-related protein. Our results
suggest the possibility that the Rpb7 subunit isinvolved not only in transcription initiation but also
in 3'-end formation of transcripts by Pol ll.
(9) Identification and characterization of S. pombe
TBP-associated factors (TAFs)

Hiroshi MITSUZAwA and Akira ISHIHAMA
TFllD, one of general transcription factors required
for accurate transcription initiation by RNA polymerase
I (Pol ll) in vitro, comprises the TATA-binding protein(TBP) and a set of TBP-associated factors (TAFs). To
date most T AF studies have been carried out withhuman, Drosophila, and S. cerevisiae. We have been
analyzing the T AF function using the fission yeast S.
pombe as another model organism to which genetic
methods are applicable. We have reported theidentification of two distinct WD repeat-containing
TAFs, spTAF72 and spTAF73, from S. pombe. spTAF72
but not spTAF73 is shared by another transcription
complex that contains the S. pombe homolog of theGcn5 histone acetyltransferase. Interestingly,
overexpression of spTAF72 or spTAF73 suppresses thecell cycle arrest during mitosis caused by mutations ingenes involved in ubiquitin-dependent proteolysis,suggesting a role for these WD repeat-containing TAFs
in the expression of genes involved in progression
through the M phase of the cell cycle. As an attempt
to understand the role of the WD repeat domain in
spTAF72, we screened for proteins that interact with

DIVISION OF MOLECULAR GENETICS

11

the carboxyl-terminal WD repeat-containing region of
spTAF72 using the yeast two-hybrid system. We
identified another S. pombe T AF, spTAF50, a homologof histone H4-like T AFs such as human T AF80,
Drosophila TAF6O, and S. cerevisiae TAF60. Gene disruption
revealed that spTAF50 is essential for cell viability. In vitro,
spTAF50 binds to spTAF72 but less efficiently to spTAF73;
Immunoprecipitation experiments demonstrated thatspTAF50 was present in both the TFllD and SAGA-likecomplexes as in the case of spTAF72. These results indicatethat the carboxyl-terminal region of spTAF72, which largely
consists of WD repeats, interacts with spTAF50 in the
TFllD and SAGA-like complexes, suggesting a role for the
WD repeat domain in the interaction between TAFs. For
details see ref. 12.
(10) An RNA effector in activation of influenza viral
RNA polymerase
Ayae HONDA, Kiyohisa MIZUMOT0 1 and Akira
ISHIHAMA (lDivision of Nucleic Acid Chemistry andKitasato University)
The genome of influenza virus is composed ofeight negative-strand viral RNA (vRNA) segments. The
viral RNA polymerase is composed of one molecule
each of three viral proteins, PB1, PB2 and PA, and is
involved in both transcription [vRNA-directed mRNA
synthesis] and replication [vRNA-directed synthesisof complementary RNA (cRNA) and cRNA-directed
vRNA synthesis]. In transcription, the RNA polymerase
catalyzes not only viral RNA synthesis but also thecleavage of capped host cell RNA to generate capped RNA
primers for viral mRNA synthesis and polyadenylation at
the 3'-termini of mRNA For the molecular anatomy ofthis multi-functional enzyme, we have established asimultaneous expression of three P proteins in cultured
insect cells using recombinant baculoviruses. For
purification of P protein complexes, the PA protein was
expressed as a fusion with a His tag added at its
N-terminus. By using affinity chromatography, acomplex consisting of the three P proteins was isolated
from nuclear extracts of virus-infected cells. The
affinity-purified 3P complex showed the activities ofcapped RNA-binding, capped RNA cleavage, viral modelRNA-binding, model RNA-directed RNA synthesis, andpolyadenylation of newly synthesized RNA For theendonucleolytic cleavage of host cell mRNA, its
interaction with viral RNA is not required, but the12

DIVISION OF MOLECULAR GENETICS

capped RNA cleavage by the isolaed 3P complex was
found to take place in the presence of vRNA but notof cRNA Thus we concluded that the vRNA functions
as an RNA effector for activation of the assembled but
inactive viral RNA polymerase. We also analyzed thestructural elements for the RNA effector funciton using
variant forms of the model RNA templates. For details
see ref. 4.
Publications

1. Colland, F., Fujita, N., Meares, c., Ishihama, A and
Kolb, A: The interaction between as, the stationary
phase a factor, and the core enzyme of Escherichia coli
RNA polymerase. Genes Cells, in press.
2. Culham, D.E., Lu, A, Jishage, M., Krogfelt, K.,
Ishihama, A and Wood, ].M.: The osmotic stressresponse and virulence in pyelonephritis isolates ofEscherichia coli: Contribution of RpoS, ProP, ProU andosmoregulatory mechanisms not detected in
Escherichia coliK-12. Microbiology 147, 1657-1670,2001.
3. Ghosh, P., Ishihama, A and Chatterji, D.:
Escherichia coli RNA polymerase subunit Uflnd its
N-terminal domain bind full length {3' to facilitate
incorporation into the 0:2{3 subassembly. Eur. J
Biochem. 268, 4621-4627, 2001.
4. Honda, A, Endo, A, Mizumoto, K. and Ishihama,
A: Differential roles of vRNA and cRNA in functional
modulation of the influenza virus RNA Polymerase J
Bioi. Chem. 276, 31179-31185, 2001.
5. Ishihama, A, Zou, C., Kobayashi, M., Kumar, A,
Fujita, N., Sakurai, H. and Hayward, RS.: Molecular
recognition in gene transcription. Internatl. J Med. Biol
Frontiers, in press.
6. Jishage, M., Dasgupta, D. and Ishihama, A:
Mapping of the Rsd contact site on the sigma-70
subunit of Escherichia coli RNA polymerase. J Bacteriol
183, 2952-2956, 2001.
7. Katayama, A, Tsujii, A, Wada, A, Nishino, T. and
Ishihama, A: Systematic search for zinc-binding
proteins in Escherichia col. Eur. J Biochem., in press.
8. Kimura, M., Sakurai, H. and Ishihama, A:
Intracellular contents and assembly states of all twelvesubunits of the RNA polymerase II in the fission yeast

Schizosaccharomyces pombe. Eur. J Biochem. 268,
612-619, 2001.

Functional complementation between two distantpositions in E. coli RNA polymerase as revealed bysecond-site suppression. Mol. Gen. Genet. 264, 531-538,
2001.

9. Kimura, M., Suzuki, H. and Ishihama, A:
Formation of a carboxy-terminal domain phosphatase(Fcp1)/TFIIF/RNA polymerase II (Pol II) complex in

18. Shin, M, Kang, S., Hyun, S.-]., Fujita, N., Ishihama,A, Poul Valentin-Hansen, P. and Choy, H.E.:

Schizosaccharomyces pombeinvolves direct interactidn
between Fcp1 and the Rpb4 subunit of Pol II. Mol. Cell.
BioI., 22, 1577-1588, 2002.

Repression of deoP2 in Escherichia coli by CytR:
Conversion of a transcription activator into a repressor.
EMBO J 20, 5392-5399, 2001.

10. Makinoshima, H., Nishimura, A and Ishihama, A:

19. Wigneshweraraj, S.R., Chaney, M.K, Ishihama, A
and Buck, M.: Regulatory sequences in sigma 54
localise near the start of DNA melting. J Mol. Bio!. 306,
681-701, 2001.

Fractionation of Escherichia coli cell populations at
different stages during growth transition to stationary
phase. Mol. Microbiol. 43, 269-280, 2002.
11. Mitobe, J., Mitsuzawa, H. and Ishihama, A:
Functional analysis of RNA polymerase Rpb3 mutantsof the fission yeast Schizosaccharomyces pombe. Curro
Genet. 39, 210-221, 2001.
12. Mitsuzawa, H., Seino, H., Yamao, F. and Ishihama,
A: Two WD repeat-containing T AFs in fission yeastthat suppress defects in the anaphase-promoting
complex. J BioI. Chem. 276, 17117-17124, 2001.
13. Ozoline, O.N., Fujita, N. and Ishihama, A: Modeof DNA-protein interaction between the C-terminal
domain of Escherichia coli RNA polymerase a subunit
and T7D promoter UP element. Nucleic Acids Res. 29,
4909-4919,2001.
14. Raj, V.S., Tomitori, H., Yoshida, M, Apirakaramwong,
A, Kashiwagi, K, Takio, K, Ishihama, A and Igarashi,
K: Properties of a revertant of Escherichia coli viable
under spermidine accumulation: Increase in L-glycerol
3-phosphate. J Bacterol. 183, 4493-4498, 2001.
15. Sakurai, H. and Ishihama, A: Transcription
organization and mRNA levels of the genes for all
twelve subunits of the fission yeast RNA Polymerase
II. Genes Cells 6, 13-24, 2001.

20. Wigneshweraraj, S.R., Ishihama, A and Buck, M.:
In vitro roles of invariant helix-turn-helix motif residue
R383 in sigma 54 (sigma N). Nucleic Acids Res. 29,
1163-1174,2001.
21. Yamamoto, K, Yata, K, Fujita, N. and Ishihama,
A: Novel mode of transcription regulation by SdiA, an
Escherichia coli homologue of the quorum-sensing
regulator. Mol. Micro bioI. 41, 1187-1198,2001.
22. Yasuno, K, Yamazaki, T., Tanaka, Y, Kodama, T.,
Matsugami, A, Katahira, M., Ishihama, A. andKyogoku, Y: Interaction of the C-terminal domain of
E. coli RNA polymerase a subunit with the UP
element: Recognizing the backbone structure in the
major groove surface. J Mol. BioI. 306, 213-225, 2001.
23. Yoshida, M., Kashiwagi, K., Kawai, G., Ishihama,A and Igarashi, K: Polyamine enhancement of thesynthesis of adenylate cyclase at the translation level
and the consequential stimulation of the synthesis of
RNA polymerase a 28 subunit. J BioI. Chem. 276,
16289-16295, 2001.

A-b. DIVISION OF MUTAGENESIS

16. Sakurai, H. and Ishihama, A: Intracellular levelof the RNA polymerase II in the fission yeast stays
constant but the level of CTD phosphorylation varies
depending on the growth phase. Genes Cells, in press.

(1) Ubiquitin conjugating enzyme affecting
recombination at matI locus in fission yeast

17. Sujatha, S., Ishihama, A and Chatterji, D.:

The mating type of S. pombeis determined by thePark JooN-HYUN and Fumiaki YAMAODIVISION OF MOLECULAR GENETICS

13

matllocus on chromosome 2. In homothallic cells,
mating type switching occurs when the DNA cassetteat mati is replaced with cassette containing the
opposite mating type information at mat2-Por mat3-m.
The heterothallic h+N cell carries intact mat2-P and
mat3-m information together with an aberrant
structure at mat-llocus, tandem-duplicated matl-p andmat2-m sequences. The abnormal mat-l structure
might be resulted during resolution of intermediate in
the mating type switching via DNA recombination,
preventing its further conversion. We report here that
UbcP3, one of 14 ubiquitin-conjugating enzymes infission yeast, was necessary to maintain theheterothallism of h+N cells since ubcP3-disruption
resulted in efficient conversion to homothallic
phenotype. Dysfunction of UbcP3 promoted to loop out
the tandem-duplicated segments in mati of h+N cells,
converting the mati structure to that of h 90• Thesesuggested a role of ubiquitin in recombination event
during mating type switching. Together with effectsof UbcP3-dysfunction on recombination efficiencies atother loci than mati and on gene silencing in mat
locus, a possible roles of ubiquitin in regulating
chromatin structure and functions would be implicated.
Analysis of Rad6 postreplicative repair (PRR)
pathway in fission yeast(2)Park JooN-HYUN and Fumiaki YAMAO
In the budding yeast, the RAD6 postreplicative
repair (PRR) system falls into at ·least two
sub-branches, an error-prone and an error-freepathway. Recently, it has been found that aheterodimeric complex consisting of UBC13 and l\1MS2
functions in the assembly of K63 polyubiquitin chainsfor error-free repair. However, little is known about thefunctional link between the ubiquitin system and PRR
pathway in the fission yeast. We reported the evidence
that UbcP7/UBC13 functions together with anoncanonical ubiquitin-conjugating enzyme, Mms2 in
the PRR pathway defined by Rhp6/RAD6, indicatingthat ubcP7 and mms2 are epistatic to the rhp6 grouplike the budding yeast. This observation supports thefact that functionality of the PRR pathway has beenhighly conserved in the two distantly related yeast
species. We also showed through a yeast two-hybrid
library screen that Mms2 interacts with the WD protein
Cpc2/mammalian RACK1, as well as UbcP7. This

14

DIVISION OF MUTAGENESIS

finding reinforces the possibility that they might
function together in the same PRR pathway. Epistasis
analysis reveals that cpc2 also belongs to the rhp6
pathway, the functional analysis of which is now
undergoing.
(3) Ubiquitin-conjugating enzymes involved indegradation of mitotic cyelin
Hiroaki SEINO
Cell cycle events are regulated by activation andinactivation of Cdk kinases. Inactivation of Cdk
kinases is mainly achieved by degradation of cyclins.
Especially degradation of mitotic cyclin is importantfor cell cycle progression. Mitotic cyclin is degradedby ubiquitin/proteasome system. Ubiquitin ligase
involved in ubiquitylation of mitotic cyclin is APC/C(anaphase promoting complex / cyclosome).
Ubiquitin-conjugating enzymes were revealed to be two
proteins, Ubc4 and Ubcx;E2-C in biochemical analysis
of Xenopus and clam oocyte extract. However,relationship between these two ubiquitin-conjugating
enzymes were not elucidated.
I found that fission yeast homologues of these two
ubiquitin-conjugating enzymes, UbcP1/Ubc4 and
UbcP4/Ubc11 directly function for ubiquitylation of
mitotic cyclin in non-redundant fashion in vivo.
Recently I elucidated the functional differences of these
two ubiquitin-conjugating enzymes in vivo. These
enzymes clearly have distinct functions for degradationof mitotic cyclin.
(4) Characterization of SCFGRRI that ubiquitinates G1
cyelin Cln2 in Saccharomyces cerevisiae

Tsutomu KISHI
In budding yeast, G1 cyclin Cln2 is rapidly
degraded by ubiquitin/proteasome system when yeast
cells enter S phase. Upon degradation, Cln2 is first
phosphorylated by its cognate Cln2/Cdc28, and thenthe phosphorylated Cln2 is ubiquitinated by SCF
ubiquitin ligase, which is composed of Skpl, Cdc53 andGrrl. Finally, ubiquitinated Cln2 is targeted toproteasome, where Cln2 is degraded.
To isolate genes that regulate the degradation of
Cln2, I have employed genetic screen by selecting forstable Cln2-LacZ fusion protein. One of the mutant

dog75 (Degradation of Gl cyclin) was characterized.
Phosphorylation of Cln2 is not affected in dog75
mutants, indicating that Dogl in involved in either theubiquitination of Cln2 or the degradation ofubiquitinated Cln2. I next examined whether Cln2 isubiquitinated in dog75 mutants. Cln2-HA was
immunoprecipited, and then immunoblotted with
anti-poly-ubiquitin antibody. Ubiquitinated Cln2 wasdecreased in dog75 mutants. This indicates that Dog75
is involved in the ubiquitination of Cln2. Dog75 codesfor protein that has homology with protein kinase. I
am now studying whether Dog75 protein kinase
directly activates SCF ubiquitin ligase.
Publication

1. Mitsuzawa, H, Seino, H, Yamao, F. and Ishihama,A : Two WD repeat-containing TATA-binding
protein-associated factors in fission yeast that suppress
defects in the anaphase-promoting complex. J BioI.
Chem. 276, 17117-17124, 2001.

A-c. DIVISION OF NUCLEIC ACID CHEMISTRY(1) Host proteins involved in transcription of Sendai
virus (SeV) genome

Division of Nucleic Acid Chemistry
Kiyohisa MIZUMOTO (School of Pharmaceutical
Sciences, Kitasato University)
Our previous studies indicated that the SeVtranscription-stimulatory activity from bovine brain
extract was resolved into at least two complementary
fractions, one of which may be tubulin (K Mizumoto
et al.,] Biochem., 117, 527-534,1995). We have recently
purified the host factor activity complementary totubulin, and shown that it contains at least twocomponents, and one of them is phosphoglycerate
kinase (PGK), a glycolytic enzyme (Ogino et al. J BioI.
Chem. 274, 35999-36008, 1999). Neither a substrate for
PGK, 3-phosphoglycerate, nor a competitive inhibitor
against 3-phosphoglycerate, DL-glycerol-3-phosphate,affected the transcription-stimulatory activity of PGK,
suggesting that the enzymatic activity per se of PGK
does not seem to be required for its activity. Cellular
tubulin, which stimulates both mRNA synthesis andleader RNA synthesis, was found to be integrated into
the transcription initiation complex (Takagi et al., J

Biochem. 118, 390 396, 1995, Takagi et al., Arch. Virol.
141,1623-1635,1996). On the other hand, PGK stimulates
viral mRNA synthesis at the elongation step, probably
through its interaction with tubulin that has been
integrated into the initiation complex.
Here we further purified the host factor activity
from bovine brain extract and demonstrated that theactivity consists of four complementary factors, tubulin,
PGK, p52, and an unknown factor. Surprisingly, thethird host factor, p52, was also identified as a glycolylic
enzyme, enolase (Ogino et al., Biochem. Biophys. Res.
Commun. 285, 447-455, 2001). We also demonstrated that
p52 as well as a recombinant human a-enolase binds
to tubulin, as PGK does. Our findings suggest
interesting relationships between tubulin and glycolytic
enzymes that may function as coactivators for SeV
transcription through their interact-ions with tubulin
integrated in the transcription initiation complex.
Publications

1. Chiba, H, Inokoshi, ]., Okamoto, M., Asanuma, S.,
Matsuzaki, K, Iwama, M., Mizumoto, K, Tanaka, H.,
Oheda, M., Fujita, K, Nakashima, H, Shionose, M. andOmura, S.: Actinohibin, a novel anti-HIV protein froman actinomycetes that inhibits syncytium formation:
Isolation, characterization and cytological activities.
Biochem. Biophys. Res. Comm., 282, 595-610, 2001.
2. Honda, A, Endo, A., Mizumoto, K and Ishihama,
A: Differential roles of viral RNA and cRNA infunctional modulation of the influenza virus RNA
polymerase. J BioI. Chem., 276, 31179-31185, 2001.
3. Ogino, T, Yamadera, T, Nonaka, T, Imajoh-Ohmi,
S. and Mizumoto, K: Enolase, a cellular glycolytic
enzyme, is required for efficient transcription of Sendi
virus genome. Biochem. Biophys. Res. Comm., 285,
447-455, 2001.
4. Yamochi, T., Semba, K, Tsuji, K, Mizumoto, K,
Sato, H, Matsuura, Y, Nishimoto, I., Matsuoka, M.: p70
ik3-1 is phosphorylated by cyclin-dependent protein
kinase 3 (cdk3). Eur. J Biochem. , 268, 6076-6082, 2001.
5. Tsuji, K, Mizumoto, K, Yamochi, T., Nishimoto,I., Matsuoka, M.: Differential effect of ik3-l/cables on
p53- and p73-induced cell death. J BioI. Chem., 277,
2951-2957, 2002.

DIVISION OF MUTAGENESIS

15

(2)

Research Activities

Mechanisms of transcription regulation byeubacteria-type RNA polymerase: Kan TANAKA
1) Gene silencing at the stationary growth phase

Many promoters that are actively transcribed
during the exponential phase are silenced at the onsetof the stationary growth phase. These gene repression
mechanisms have been extensively analyzed for rrnBPl
promoter, where ppGpp, H-NS and intracellular NTP
concentration are deeply involved. We analyzed theactivity of the lacUV5 promoter, a consensus promoter,during the growth phase transition, and found that thispromoter transcription is shut off at the onset of thestationary phase. This shut off was dependent on the
nucleoid protein H-NS, and numerous consensus
promoters were considered to be silenced by the similar
mechanism after the growth.
2) Mechanisms of nitrogen-assimilatory gene expression
in cyanobacteria
NtcA is a generally found transcription factor incyanobacteria, and regulates nitrogen assimilatory
genes, such as nitrate/nitrite reductases, nitrate/nitratetransporter, and glutamine synthetase. Using a
recombinant NtcA protein and a cyanobacterial RNA
polymerase, we reconstituted an in vitro transcription
system for the gInA and ntcA promoters. We havefound that 2-oxoglutarate, a carbon skeleton for thenitrogen assimilation, enhances the NtcA-DNA binding
and activates the transcription initiation. Since thisactivation occurs even in the condition where NtcA
binding sites are fully occupied by NtcA, it wassuggested that 2-oxoglutarate activated the NtcA-DNA
binding as well as a later step during the transcription
initiation.
Regulation of plastid DNA transcription by
nuclear-encoded sigma factors
3)

Plastids of higher plants are organelle, which havebeen originated from a symbiosis of ancestral
cyanobacteria. Plastids still have their own genome ofc.a. 150-kb, and the transcription is performed byeubacteria-type RNA polymerase. While subunits forcore RNA polymerase are encoded by the plastid

16

DIVISION OF NUCLEIC ACID CHEMISTRY

genome, sigma subunits are encoded by the nuclear
genome, and dominate the plastid transcription under
the control of the nucleus. Six sigma factor genes were
found in Arabidosis, and we recently isolated aT-DNA
tagged sig2 mutant line. This mutant contains poorly
developed chloroplasts of low chlorophyll contents.
Further analysis revealed that the amounts of several
chloroplast tRNA species including tRNAg1U wereseverely decreased in the mutant line. Thus, the SIG2
sigma factor was suggested to activate transcriptionof a series of plastid tRNA genes. Since tRNAglu is awell-known cofactor for the tetrapyrrole biosynthesis,
SIG2 appears to regulate both protein and chlorophyll
synthesis through the tRNA transcription during thechloroplast development.
Publications

1. Kanamaru, K., Nagashima, A., Fujiwara, M.,
Shimada, H, Shirano, Y, Nakabayashi, K, Shibata, D.,
Tanaka, K and Takahashi, H.: An Arabidopsis sigma
factor (SIG2)-dependent expression of plastid-encodedtRNAs in chloroplasts. Plant Cell Physiol42, 1034-1043,
2001.
2. Tanigawa, R., Shirokane, M., Maeda, S., Ornata,T., Tanaka, K and Takahashi, H.: Transcriptional
activation of NtcA-dependent promoters of
Synechococcus sp. PCC 7942 by 2-oxoglutarate in vitro.
Proc. Nat!. A cad. Sci. USA (in press) 2002.

B. DEPARTMENT OF
CELL GENETICS

B-a. Division of Cytogenetics(1) Recombinogenic activity of chimeric recA genes(Pseudomonas aeruginosa;Escherichia coli): a search for
RecA protein regions responsible for this activity
Irina V. BAKHLANOYAl, Tomoko OGAWA, Vladislav A.
LANZOY! (!Division of Molecular and Radiation
Biophysics, Petersburg Nuclear Physics Institute)
In the background of weak, if any, constitutive
SOS function, RecA from Pseudomonas aeruginosa(RecAPa) shows a higher frequency of recombinationexchange (FRE) per DNA unit length as compared to
RecA from Escherichia coli (RecAEc). To understandthe molecular basis for this observation and to
determine which regions of the RecAPa polypeptide
are responsible for this unusual activity, we analyzed
recAX chimeras between the recAEc and recAPa genes.
We chose 31 previously described recombination- andrepair-proficient recAX hybrids and determined their
FRE calculated from linkage frequency data andconstitutive SOS function expression as measured byusing the lacZ gene under control of an SOS-regulated
promoter. Relative to recAEc, the FRE of recAPa was
6.5 times greater; the relative alterations of FRE for
recAX genes varied from approximately 0.6 to 9.0. No
quantitative correlation between the FRE increase andconstitutive SOS function was observed. Single ([L29M]
or [Il02D]), double ( [GI36N, V142I] ), and multiple
substitutions in related pairs of chimeric RecAX
proteins significantly altered their relative FRE values.
The residue content of three separate regions within
the N-terminal and central but not the C-terminal
protein domains within the RecA molecule also
influenced the FRE values. Critical amino acids in theseregions were located close to previously identified
sequences that comprise the two surfaces for subunit
interactions in the RecA polymer. We suggest that theintensity of the interactions between the subunits isa key factor in determining the FRE promoted by RecA
in vivo. For details see Ref. 1.
Domain structure and dynamics in the helicalfilaments formed by RecA and Rad51 on DNA(2)

Xiong Yu!, Steven A. JACOBS!, Stephen C. WEST!,
Tomoko OGAWA and Edward H. EGELMAN!
0Department of Biochemistry and Molecular Genetics,
University of Virginia Health Sciences Center)
Both the bacterial RecA protein and the eukaryotic
Rad51 protein form helical nucleoprotein filaments on
DNA that catalyze strand transfer between two
homologous DNA molecules. However, only the
ATP-binding cores of these proteins have beenconserved, and this same core is also found within
helicases and the FI-ATPase. The C-terminal domainof the RecA protein forms lobes within the helical RecA
filament. However, the Rad51 proteins do not have the
C-terminal domain found in RecA, but have an
N-terminal extension that is absent in the RecA protein.
Both the RecA C-terminal domain and the Rad51
N-terminal domain bind DNA. We have used electron
microscopy to show that the lobes of the yeast andhuman Rad51 filaments appear to be formed by
N-terminal domains. These lobes are conformationally
flexible in both RecA and Rad51. Within RecA
filaments, the change between the "active" and"inactive" states appears to mainly involve a large
movement of the C-terminallobe. The N-terminal
domain of Rad51 and the C-terminal domain of RecA
may have arisen from convergent evolution to play
similar roles in the filaments. For details see Ref. 2.
Publications
1. Bakhlanova, I. V., Ogawa, T. and Lanzov, V. A.:
Recombinogenic activity of chimeric recA genes (P.
aeruginosa / E. coli): RecA protein regions responsible
for this activity. Genetics, 159, 7-15, 2001.

2. Yu, X., Jacobs, S. A., West, S. C., Ogawa, T. andEgelman, E. H.: Domain Structure and Dynamics in the
Helical Filaments Formed by RecA and Rad51 on DNA.
Proc. Nat!. A cad. Sci., USA, 98, 8419-8424, 2001.
(1) Integrative study on chromosome evolution of
mammals, ants and wasps based on the minimum

DEPARTMENT OF CELL GENETICS

17

interaction theory

Hirotami IMAI, Yoko SATTA!, Naoyuki TAKAHATA 1
rSchool of Advanced Sciences, Graduate University of
Advanced Studies)
There is well-known evidence that in many
eukaryotes, different species have different karyotypes(e.g. n=I-47 in ants and n=3-51 in mammals).
Alternative (fusion and fission) hypotheses have been
proposed to interpret this chromosomal diversity.
Although the former has long been accepted,
accumulating molecular genetics evidence seems to
support the latter. We investigated this problem froma stochastic viewpoint using the Monte Carlosimulation method under the minimum interaction
theory. We found that the results of simulations
consistently interpreted the chromosomal diversity
observed in mammals, ants and wasps, and concluded
that chromosome evolution tends to evolve as a wholetoward incr~asing chromosome numbers by centricfission. Accordingly, our results support the fission
hypothesis. We discussed the process of chromosome
evolution based on the latest theory of the molecular
structure of chromosomes, and reconfirmed that thefission burst is the prime motive force in long-term
chromosome evolution, and is effective in minimizing
the genetic risks due to deleterious reciprocaltranslocations and in increasing the potential of genetic
divergence. Centric fusion plays a biological role ineliminating heterochromatin (C-bands), but is only alocal reverse flow in contrast to the previously heldviews. For details see Ref. 1.
Publication

1. Imai, H. T., Satta, Y. and Takahata, N.: Integrative
study on chromosome evolution of mammals, ants andwasps based on the minimum interaction theory. J
Theor. Bioi. 210, 475-497, 2001.

B-b. Division of Microbial Genetics(1) Sld3, which interacts with Cdc45 (Sld4), functions forchromosomal DNA replication in Saccharomyces
cerevisiae (ref. 1)

Yoichiro KAMIMuRA, Yon-Soo T AK and Hiroyuki ARAKI

18

Division of Cytogenetics

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
synthesis during the S phase. The Dpbll protein whichforms a complex with essential DNA polymerase
E (Poh)is required for association of DNApolymerases with origins in DNA replication and forthe control of the S-phase checkpoint. To identify
factors interacting with Dpbll, we have isolated 10 sid(synthetic lethality with dpbll-l) mutations which fall
into 6 complementation groups. We have cloned
SLDl"··6 and found that SLDI 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, 3, 5 genes were
new genes essential for the cell growth (Kamimura etaI., Mol. Cell. BioI., 18, 6102-6109, 1998).
The Cdc45 protein, which binds to theminichromosomal maintenance (Mcm) proteins, has apivotal role for the initiation and elongation steps ofchromosomal DNA replication in eukaryotes. We showthat throughout the cell cycle in Saccharomyces
cerevisiae, Cdc45 forms a complex with a novel factor,
Sld3. Consistently, Sld3 and Cdc45 associatesimultaneously with replication origins in thechromatin immunoprecipitation assay: both proteinsassociate with early-firing origins in Gl phase and withlate-firing origins in late S phase. Moreover, the origin
associations of Sld3 and Cdc45 are mutually dependent.
The temperature-sensitive sld3 mutation confers defectof DNA replication at the restrictive temperature andreduces an interaction not only between Sld3 andCdc45 but also between Cdc45 and Mcm2. These
results suggest that the Sld3-Cdc45 complex associateswith replication origins through Mcm proteins. At the
restrictive temperature in sld3-5 cells, RF-A, asingle-strand DNA binding protein, does not associatewith origins. Therefore, the origin association of
Sld3-Cdc45 complex is prerequisite for origin
unwinding in the initiation of DNA replication.
(2) Phosphorylation of SId2 by cyclin-dependent protein
kinase (ref.2)

Hiroshi MASUMOTO, Sachiko MURAMATSU, Yoichiro
KAMIMURA and Hiroyuki ARAKI
Cyclin-dependent protein kinases (Cdk) are key
enzymes which control the cell cycle. For initiation of
DNA replication, S-phase specific Cdk (S-Cdk) is
required while real target of S-Cdk is obscure.
Since Sld2, which form a complex with Dpbll(Kamimura et aI., Mol. Cell. BioI., 18, 6102-6109, 1998),
has six conserved amino acid sequences for
Cdk-phosphorylation, we examined whether Sld2 is
phosphorylated by S-Cdk. As expected, in
SDS-polyacrylamide gel Sld2 migrated slower in S
phase than in Gl phase. Phosphatase treatment
converted slow mobility species of Sld2 obtained from
S phase to fast mobility specie as obtained from Gl
phase, suggesting that Sld2 is phosphorylated in S
phase.
The partially purified Cdk phophorylated the Sld2
protein purified from E. coli cells expressing it. Thisin vitro phosphorylation depends on Cdkphosphorylation motif since the Cdk did not
phosphorylate the All-A protein (see below). These
results suggest that Sld2, at least in in vitro reaction,is phosphorylated by Cdk. Then, we examined whether
Sld2 is phosphorylated by S-Cdk in in vivo. When we
expressed Sid, an inhibitor of S-Cdk, phosphorylated
form of Sld2 did not appear. Further, when we
examined S-cyclins-deleted cells that are viable sinceM-phase-cyclins compensate for them while the startof DNA synthesis delays, the slow migrating form of
Sld2 appeared later than wild-type cells. Thus, in vivo
phosphorylation of Sld2 in S phase depends on S-Cdk
activity.
Next, we substituted the serine or threonine
residues in six S-Cdk phosphorylation sites by alanine.
The mutant protein containing six alanine
substitutions (All-A) did not support cell growth but
also onset of S phase while three combinations of fivealanine substitutions supported cell growth.
Furthermore, slow mobility species of the mutant
protein containing six alanine substitutions did notappear. Moreover, when we substituted three of
replaced alanine residues of All-A by an asparatate,a phosphomimeric residue, the resultant mutant allele
support cell growth. These results suggest that S-Cdk
dependent phosphorylation of Sld2 is essential for
DNA replication.
Since Sld2 and Dpbll form a complex, weinvestigated their complex formation by tagging themand using co-immunoprecipitation assay.
Co-immunoprecipitation of Sld2 and Dpbll was
detected in S phase but not in Gl phase and Dpbll
immunoprecipitated only with phosphorylated form of
Sld2. When we expressed wild-type and six-alanine
substitution mutant proteins of SldZ in the sld2mutant
cells defective in complex formation between Sld2 andDpbll, complex formation between the mutant protein
and Dpbll did not be detected while the wild typeprotein coprecipitated with Dpbl1. These results
suggest that phosphorylation of Sld2 is required forcomplex formation between Dpbll and Sld2.
Temperature-sensitive mutants of the SLD2 genedefective in formation of the Sld2-Dpbll complex issuppressed by the increased dosage of DPBll. We
found that increased dosage of DPBll also suppresses
the growth defect of the All-A mutant. This result
further suggests that inefficient formation of the
SldZ-Dpbll complex in the All-A mutant cells gives rise
to defect of the cell growth.
Since Dpbll is required for associations of DNApolymerases with replication origins (Masaumoto etal., Mol. Cell. BioI., 20, 2809-2817, 2000), it seems likelythat S-Cdk regulates complex formation between Sld2
and Dpbll and consequently loading of DNApolymerases to replication origins.
(3) Functional analysis of Sld5 and Psfl
Yoichiro KAMIMURA and Hiroyuki ARAKIThe SLDS gene isolated as a synthetic lethality
with dpbll-l (see section (1)) encodes an essential 34 kDa
protein. We also isolated the PSFI (Partner of Sid Five)and PSF2 genes, respectively as a multicopy
supperssor of sldS-12 and psfl-l mutations. In
co-immunoprecipitation experiment, Sld5 forms acomplex with Psfl and Psf2. This complex is unstablein psfl-l cells. This result suggests that complex
formation is important for the function of these
proteins. Psfl-l mutant cells arrested with a dumbbell
shape and were defective in DNA replication at the
restrictive temperature as did sldS-12 cells. To date,many replication related proteins including Dpbll associate ARS regions using chromatin immunoprecipitation assay (CHIP). Indeed, Psfl associated withreplication regions in S phase at the same time as
Dpbll and PolE. This association with origins

Division of Microbial Genetics

19depended on Sld3 and Dpbl1. Two-hybrid assay
showed the interaction between Psfl and Dpb11 of
Sld3. These data suggest that Sld5-Psfl-Psf2 complex
intermediates association between Sld3-Cdc45 andDpb11-PoIE.
Publications
1. Kamimura, Y., Tak, Y.-S., Sugino, A. and Araki,

H: Sld3, which interacts with Cdc45 (Sld4), functions
for chromosomal DNA replication in Saccharomyces
cerevisiae. EMBO J 20, 2097-2107, 2001.
2. 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.

B-c. Division of Cytoplasmic Genetics(1) Linkage analysis and linkage disequilibrium mappingof functional psychoses
Takeo YOSHIKAWA (Lab. for Molecular Psychiatry,
RIKEN Brain Science Institute)
We have been collecting pedigrees ofschizophrenia and mood disorders. As of December
2001, these pedigrees amounted to 132 families and 411
individuals including probands and their relatives.
Among them, 119 families (357 individuals) comprisethe schizophrenia group, and 13 families (54individuals), the affective disorder group. In 2001, westarted a whole genome scan of the schizophrenia
pedigree panel, and have finished analyzing all
chromosomes at a 10 cM density, and chromosomes 6,
11 and 18 at a 5 cM density, by means of Pedigree
Disequilibrium Test. In the second stage, the markers
which showed a significant linkage/association to thedisease were analyzed by using trio families (parents
and their affected offsprings) and Extended
Transmission Disequilibrium Test statistics. The
markers which still remained to be significant after thesecond stage screening were those mapped onto
chromosomes 11 and 18. We are now testing thesegenetic loci by using the third sample panel consistingof cases (unrelated schizophrenics) and controls.
(2) QTL mapping of mouse models of depressive

20

Division of Microbial Genetics

disorderTakeo YOSHIKAWA
The forced swimming test (FST) and tailsuspension test (TST) have been widely used for thescreening of anti-depressant efficacy. Clinicallyeffective drugs reduce immobility times in both tests.
The pharmacological action is thought to prevent the
animals from experiencing despair in stressful
conditions. Thus, immobility times are considered toreflect a propensity to depression in human. We foundthat the C3H/He mouse strain showed much shorter
immobility periods than the C57BL!6 strain in bothtests, suggesting that the C3H/He mice are geneticallymore resistant to such stress conditions. In 2001, we
have prepared 560 F2 intercross mice for the QTL
mapping and performed whole genome scanning ofthese mice using 120 microsatellite markers. We havedetected FST-QTLs on mouse chromosomes 6, 8, 11 and17, and TST-QTLs on chromosomes 4, 8,11 and 14(Publicaton # 8). These QTLs controlling immobility
times in FST and TST may harbor genes whtch
correspond to susceptibility genes in humandepression.
(3) Gene expression analysis of a model animal for
depression using GeneChips
Takeo YOSHIKAWA
Learned Helplessness (LH) is a good animal modelfor the chronic depression. We have examined several
parameters to efficiently produce LH rats, andsucceeded in stably making these rats. We removed
the frontal cortex and hippocampus from the brainsof LH rats, antidepressant-treated LH rats and control
rats, and extracted RNA, and compared theirexpression levels using the GeneChips. These analyses
revealed that a number of receptor genes forneurotransmitters were down-regulated in both brain
regions of LH rats, and their expression levels wereresumed to control levels by antidepressant treatments.
(4) Candidate gene approaches
Takeo YOSHIKAWA
The target genes are selected for both positional

and functional relevance. The analyzed genes in 2001
include myo-inositol monophosphatase 2 (IMPA2) (formood disorders and schizophrenia; Publication # 1),cholecystokinin (for panic disorder; Publication # 3),cholecystokinin B receptor (for panic disorder andschizophrenia; Publication #4,5), adenosine A2areceptor (for panic disorder; Publication #4), andadenylate cyclase type 9 (ADCY9) (for mood disorders;Publication #6,7). We have identified genetic
polymorphisms of these genes, and examined theirroles in the developments of psychiatric disorders.
Publications
1. Yoshikawa, T., Kikuchi, M., Saito, K, Watanabe,A, Yamada, K, Shibuya, H., Nankai, M., Kurumaji, A,
Hattori, A, Ishiguro, H, Shimizu, H, Okubo, Y, Toru,
M. and Detera-Wadleigh, S.D.: Evidence for associationof the myo-inositol monophosphatase 2 (IMPA2) gene

6. Toyota, T, Hattori, E., Meerabux, J., Yamada, K,
Saito, K, Shibuya, H., Nankai, M., Yoshikawa, T.:
Molecular analysis, mutation screening, andassociation study of adenylate cyclase type 9 gene
(ADCY9) in mood disorders. Am. J Med. Genet.
Neuropsychiatric Genet. 114, 84-92, 2002.
7. Toyota, T., Yamada, K, Saito, K, Detera-Wadleigh,S. D., Yoshikawa, T.: Association analysis of adenylate
cyclase type 9 gene using pedigree disequilibrium testin bipolar disorder. Mol. Psychiatry (in press)
8. Yoshikawa, T, Watanabe, A., Ishitsuka, Y.,
Nakaya, A, Nakatani, N.: Identification of multiple
genetic loci linked to the propensity for "behavioral
despair" in mice. Genome Research 12, 357-366, 2002.

with schizophrenia in Japanese samples. Mol.
Psychiatry 6, 202-210, 2001.
2. Toyota, T., Shimizu, H., Yamada, K, Yoshitsugu,
K, Meerabux, J., Hattori, E., Ichimiya, T., Yoshikawa,

T: Karyotype analysis of 161 unrelated schizophrenics:
no increased rates of X chromosome mosaicism orinv(9), using ethnically matched and age-stratified
controls. Schizophr. Res. 52, 171-179, 200l.
/
3. Hattori, E., Ebihara, M., Yamada,; K, Ohba, H.,
Shibuya, H., Yoshikawa, T.: Identification of acompound short tandem repeat stretch in the
5'-upstream region of the cholecystokinin gene, andits association with panic disorder but not withschizophrenia. Mol. Psychiatry 6, 465-470, 2001.

4. Yamada, K, Hattori, E., Shimizu, M., Sugaya, A,
Shibuya, H., Yoshikawa, T: Association studies of thecholecystokinin B receptor and A2a adenosine receptor
genes in panic disorder. J Neural Trans 108, 837-848,
2001.
5. Hattori, E., Yamada, K, Toyota, T., Yoshitsugu,
K, Toru, M., Shibuya, H., Yoshikawa, T.: Association
studies of the CT repeat polymorphism in the 5
upstream region of the cholecystokinin B receptor genewith panic disorder and schizophrenia in Japanese
subjects. Am. J Moo. Genet. Neuropsychiatric Genet. 105,779-782, 2001.

Division of Cytoplasmic Genetics21(2) A common molecular basis required for Drosophila
sensory organ formationc. DEPARTMENT OF
DEVELOPMENTAL GENETICS

C-a. Division of Developmental Genetics(1) Translational regulation of zygotic gene expression
Masataka OKABE, Takao IMAI!, Mitsuhiko KURUSU2,
Hideyuki OKANO! and Yasushi HIROMI
rOsaka Univ. Graduate School of Medicine, Keio Univ.
School of Medicine, 2University of Tsukuba)
Development is controlled by expressing genes inappropriate place and at appropriate timing. Althoughmost gene expressions are regulated at thetranscriptional level, translational control has its
unique advantages; for example, in rapidly supplying
large quantities of proteins after fertilization usingmaternal mRNAs stored in the oocyte. We found thata cell-fate decision during sensory organ formation
involved a translational control of zygotic gene
expression. Each mechanosensory bristle in Drosophila
is composed of four support cells and one neuron,
which are generated through asymmetric cell divisionsof a precursor cell. In the first asymmetric division,
Notch-signaling directs the asymmetry between
neuronal and non-neuronal lineages, and a zinc finger
transcriptional repressor Tramtrack69 (TTK69) acts
downstream of Notch as a determinant of non-neuronal
identity. We found that TTK69 protein expression in
the neuronal lineage is repressed translationally rather
than transcriptionally. This translational repression
was achieved by a direct interaction between the 3'
untranslated region of ttk69 mRNA and an RNAbinding protein Musashi. In one of the daughter cells
Notch-signaling inhibits Musashi activity allowing
translation of ttk69 mRNA in the non-neuronalprogeny. We propose that the constitutive translational
repression and its release by upstream signaling
pathway enable rapid gene expression even in
post-embryonic development. For details see Ref. 1.

22

DEPARTMENT OF DEVELOPMENTAL GENETICS

Nao NIWA, Masataka OKABE and Yasushi HIROMI
Sensory organs such as eye, nose and ear havediverse morphological features that are specialized forthe particular physical stimulus that each organ has
to receive. Although three sensory organs in
Drosophila, compound eye in the head, Johnston'sorgan (ear) in the antenna, and chordotonal organ(stretch receptor) in the leg, have no apparent
morphological similarities, they share at least one
property during its formation; they all require thebHLH transcription factor encoded by the atonal genefor their formation. This suggests that these diverse
sensory organs may have a common origin and sharea molecular basis for their formation. We analyzed thespatio-temporal conditions in which diverse sensory
organs form. We found that these segment-specificsensory organs form under the same molecularenvironment that is defined by Dpp and ecdysone
signals. Both signals act through the 3' enhancer
region of the atonal gene and direct its expression ina specific region of each segment. Since Atonal
expression endows a neurogenic activity, allsegment-specific sensory organs form within aneurogenic field that is common to all segments. Thissuggests that diverse sensory organs may have evolved
from an ancestral organ that was present in all
segments. Previous models on organogenesis assumed
that a master regulator of organ formation, such as theeyeless gene, directed all aspects of the formation of aparticular sensory organ, e.g., an eye. We found thateven in an eyeless mutant, a cluster of Atonal
expressing cells are present in the eye imaginal discwhen apoptosis is blocked. Therefore, the function of
the eyeless gene is not the formation of a sensory organ
per se, but rather a modification of the proto-sensory
organ by adding segment-specific features to thecommon neurogenic field. Vertebrate homologues ofdpp and atonal gene are also involved in the formationof the eye and ear, suggesting that the molecular basis
for sensory organ formation was already establishedin a common ancestor of protostomes anddeuterostomes.
(3) Function of nuclear receptor Seven-up in the
Drosophila central nervous system

Makoto KANAI, Masataka OKABE, Yasushi HIROMI(4) Identification of the down stream factors of a nuclear
receptor, Seven-up

multi-functional protein complex that is involved not
only in transcription but also in DNA repair and cell
cycle regulation. Among its nine subunits are thosethat have enzymatic functions such as kinase andhelicase activities, which are likely to be useddifferentially depending on the state of the cell. Since
most studies concerning TFIIH have so far been done
in vitro or in unicellular organisms, it is not known how
TFIIH function is regulated in a multicellularenvironment where three functions operate
simultaneously. We are approaching this problem byfocusing on the p52 subunit, which was previously
identified as a Seven-up interacting protein in ourlaboratory. We identified nine loss-of-function allelesof Drosophila p52 gene, and showed that all alleles
show growth delay and abnormal feeding behavior,and die before reaching the mature third instar larvalstage. Furthermore, mutant clones in the eye imaginaldiscs have abnormal cell cycle progression phenotypes
and cannot be maintained to the adult stage, althoughdefect in transcription is minor. The cell cycle
phenotype of p52 mutants was different from that seen
in mutations in the Cdk activating kinase subunit of
TFIIH, and were similar to defects observed uponblocking nucleotide excision repair. We propose thatp52 has essential roles in DNA repair during
development and its loss impacts upon cell cycle
regulation indirectly.

Motomi MATSUNO, Yasushi HIROMI(6) Differentiation of Drosophila longitudinal glia

Seven-up, one of the Drosophila nuclear receptor,
functions as a genetic switch between two different
photoreceptor subtypes during compound eye
development. We are trying to understand what
Seven-up target genes are and how Seven-up regulates
them. Since Seven-up ligand binding domain has atranscriptional repression activity, we searched forgenes whose expression was up-regulated in seven-up
mutant eye imaginal disc. So far we have identified 53
candidate genes, including those encoding
transcriptional factors, cytoskeleton molecules, and celladhesion molecules.

Yoshihiro YUASA, Masataka OKABE, Yasushi HIROMI

A hallmark of the nervous system is the hugediversity in the neuronal and glial cell types that it
contains. Even in a rather simple system such as aneuromere of the Drosophila embryonic central
nervous system (CNS), each unit contains more than
250 neurons that likely consist of over a hundred cell
types. These neurons are generated through sequential
divisions of ca. 30 neuronal precursors in the CNS,
called neuroblasts. The identity of cells that eachneuroblast generates depends on their birth order fromfounder neuroblasts. A recent study revealed that thetemporal information of the birth order is provided bysequential expression pattern of a group oftranscription factors called "gap proteins". Since eachneuroblast has its unique lineage pattern, factors thatlink neuroblast identity and birth order identity must
exist. Nuclear receptor Seven-up is expressed in mostneuroblasts in diverse subsections of each lineage. In
seven-up mutant the number of neurons that express
particular identity markers is altered, accompanied bya change in the expression pattern of gap proteins.
Seven-up may be involved in coordinating neuroblast
identity and birth order identity.

(5) The functional analyses of TFIIH p52 subunit
Motomi MATSUNO, Yasushi HIROMI
Basal transcriptional factor TFIIH is a

Just as neurons comprise of diverse cell types, glia
also contain many subtypes that differ in morphology,
migration patterns and specificity in neuronalrecognition. In Drosophila, transcription factor Glial
cells missing (GCM) has been identified as the most
upstream regulator of gliogenesis, providing thedistinction between neuron and glia. Since GCM isexpressed in all glia, glial subtype determination must
depend on other regulatory proteins. We have shownthat the homeodomain protein REPO directs the glial
differentiation pathway initiated by GCM. Mutant
phenotypes of repo suggest that REPO is required forthe migration and differentiation of embryonic glial
cells. In order to further understand how REPO
functions in glial terminal differentiation, we haveanalyzed the mechanism of gene regulation by REPO.

Division of Developmental Genetics

23

In the ventral nerve cord, a specific glial subtype calledlongitudinal glia is needed for the formation andmaintenance of longitudinal axon tracts. We identified
transcription factor DRI that is expressed specificallyin longitudinal glia as a new downstream target geneof REPO. DRI cooperates with transcription factor
PointedPl, another downstream target of GCM, inregulating expression of longitudinal glia specificmarker genes. We propose that cooperation of thesetranscription factors with REPO plays a key role in thelongitudinal glial differentiation.
(7) FGF signal is required for glial ensheathment byredistributing Neuroglian protein
Kaz TAKIZAWA, Emiko SUZUKI l, Yoshiki HOTTA andYasushi HIROMI (lInstitute of Medical Science,
University of Tokyo)
In the development of Drosophila CNS,
longitudinal glia migrate medially and associate withaxon tracts to be aligned in two rows, medial andlateral, before ensheathing the longitudinal axon
bundle. Molecular mechanisms underlying theseprocesses are poorly understood. We focused our study
on the expression of a cell surface protein Neuroglianthat is expressed in glial cells and is known to mediatecell adhesion in vitro. The distribution of Neuroglian
in longitudinal glia changes dramatically during glial
morphogenesis;. while initially on the cell surface,
Neuroglian accumulates at the medial edge of themedial row and at the interface of the two rows,
concomitant with the extension of the glial process. At
stage 16, filopodia-like structure appear and eventuallyconnect with each other to form a compartment
structure that covers the dorsal surface of the medial
half of each hemisegment. Electron microscopic
analysis revealed that the compartment border has adifferent morphology of cell contacts from those within
the compartment. Mutations in FGF receptor heartless
failed to redistribute the Neuroglian protein, while
Neuroglian mutant showed a malformation of thecompartment structure. These data indicate that FGF
signal mediates glial ensheathment by directing
Neuroglian relocalization.
(8) Genetic dissection of Spred, a novel negative
regulator of ras signaling

24

Division of Developmental Genetics

Shu KONDO, Yasushi HIROMI
Ras signaling is implicated in various aspects ofcell differentiation and proliferation in developing
animal tissues. The human Spred protein is a potent
negative regulator of ras signaling in tissue culture
cells, but its physiological function is not clear. The
Spred protein has a cystein-rich domain in the N
terminus that is also present in another negative
regulator of ras signaling, Sprouty, which was
identified in our laboratory. The Drosophila genome
contains one Spred homolog, and we are analyzing its
physiological role in Drosophila. We found that
Drosophila Spred is expressed in photoreceptor cells
R2, R5 and R8, the central nervous system and theembryonic muscle attachment sites. We created a Spredmutant by imprecise excision of the P-element AE33
which is inserted in an intron of the Spred gene. To
our surprise, the Spred mutant flies are fully viable andfertile, showing no apparent visible phenotypes.
Analysis of transgenic flies overexpressing Spred in
specific tissues and a genome-wide modifier screen in
the Spred mutant background to identify functional
redundancy with other genes are underway.
(9) Receptors as presenters of positional information
Masaki HIRAMOTO, Yasushi HIROMIThe basic model that is currently employed to
explain the axon patterning by secreted guidance
molecules is the Chemotropic Hypothesis proposed by
Cajal. It proposes that positional information isexpressed by the distance from the source. We found
new way to create positional information utilizing
intra-cellular compartmentalization. Netrin is anevolutionary conserved guidance molecule, which isrecognized and captured by its receptor Frazzled. We
found Frazzled creates positional information for othercells by rearranging the distribution of Netrin(Capture/ Relocation system). In this process,intra-axonal localization of Frazzled provides positionalcue for guidance of other axons. We propose that thissystem is the basic mechanism for general axonal
pattering by secreted ligands and their receptors. To
test this idea, we are investigating the patterning
mechanism by another secreted ligand Slit and its
receptor Robo. We focused on two longitudinal pioneer
axons whose medio-Iateral positioning depends on Slit

and Robo. We found that Slit receptors also exhibit
compartmentalized localization within the axon, andcan provide medio-Iateral positional information
through a non-cell autonomous effect. These results
suggest that the presentation of positional information
by intra-axonal localization of receptors is a general
strategy in axonal guidance.
(10) GeM-motif protein function in mammalian
hematopoiesis
Toshihiko HosOYA, Yasuno IwASAKI\ Motomi OSAT0 2,
Tomomasa YOKOMIZ0 2 , Chieko HIGASHI-TAKIZAWA,
Yoshiaki IT0 2, Kazuhiro IKENAKA 1, Yasushi HIROMI,
Yoshiki HOTTA (lNational Institute for Physiological
Sciences, 2Institute for Virus Research, Kyoto
University)
In Drosophila, Gem-motif transcription factors
govern binary cell-fate decisions in both neurogenesis
and hematopoiesis. Just as GCM acts as a switch
between glia and neuron during neurogenesis,
expression of GCM and GCM2 during hematopoiesis
bisects blood cell population into plasmatocytes(macrophages) and melanin-producing crystal cells.
GCM and GCM2 are expressed and required
specifically in plasmatocytes, and their misexpression
in crystal cells converts them to the plasmatocyte fate.
We investigated whether GCM-motif proteins haveconserved functions in mammals. We found that amammalian Gem-motif protein mGCMa is expressedin the embryonic blood cells. When misexpressed in
the Drosophila blood system, mGCMa exhibited thesame cell-fate transformation activity as th~ Drosophila
GCM-motif proteins. Thus mGCMa possesses
molecular characteristics required for the function asa genetic switch in hematopoiesis. mGCMa inactivation
by gene knock-out caused defects in macrophagedevelopment, suggesting an evolutionarily conservedrole of Gem-motif proteins in hematopoiesis.
(11) Presentation methods that are friendly to color-blind
peopleMasataka OKABE and Kei ITO l (lNational Institute for
Basic Biology)
In scientific presentations and publications, "color"has become a significant vehicle for information andpresentation effect. However, color perception varies
greatly among individuals; in particular, red-green
color blindness are found in 4-9% of males in various
populations, a frequency comparable to that of the AB
blood type. Thus inappropriate color choices can cause
unexpected difficulty in understanding color figures.
We are examining how color and color combinations
are perceived by various color vision types, to develop
a method for presenting color information that canconvey maximal information to most color vision types
including color blindness. We introduced this method
in talks entitled "How to make figures andpresentations that are friendly to color-blind people"
at several academic meetings in Japan and USA, andalso on our web site: http://www.nig.ac.jp/labs/
DevGen/shikimou.html.
Publications
1. Okabe, M., Imai, T., Kurusu, M., Hiromi, Y. andOkano, H.: Translational repression determines a
neuronal potential in Drosophila asymmetric cell
division. Nature 411, 94-98, 2001.
2. Takizawa, K. and Hotta, Y.: Pathfinding analysis
in a glia-Iess gem mutant in Drosophila. Dev. Genes
Eva!. 211, 30-36, 2001.
(1) Hydra Peptide Project: An epitheliopeptide Hym-301
is involved in head formation
Toshio TAKAHASHI!, Hiroshi SHIMIZU, Masayuki
HATTA2, Osamu KOIZUMI 3,Yoshitaka KOBAYAKAWA 4
and Toshitaka FUJISAWA (lUniversity of California,
Irvine, 20 chanomizu University, 3Fukuoka Women's
University, 4Kyushu University)
During the course of systematic screening of
peptide signaling molecules from Hydra
magnipapillata, we identified a 14 amino acid peptide
with an internal disulfide bond and amidation at the
C-terminus. In situ hybridization and immunohistochemistry showed that the peptide is synthesized andlocalized in the ectodermal epithelial cells in thehypostome region. During a budding process, both thegene and the peptide are expressed in the presumptive
hypostome region before tentacle formation. The
exogenous application of the peptide at 10- 6 Menhanced the tentacle formation, whereas RNAi
inhibited it. This is the first report on the amidated

Division of Developmental Genetics

25

peptide derived from epithelial cells that is involved
in morphogenesis in Hydra.
(2) Diffuse nerve net in hydra body column represents
a primitive enteric nervous system
Hiroshi SHIMIZU and Toshitaka FUJISAWA
Digestive movements in vertebrates' intestine suchas peristalsis involve three basic structures,longitudinally and circumferentially running muscle
layers and the net-like nervous system termed
myenteric plexus between them. Interestingly, thedigestive tract of Hydra also has two muscle layers andthe net-like nervous system termed nerve net in
between. Despite the similarities, Hydra's digestive
process has been thought to be a non-dynamic event.
Here we show evidence that Hydra exhibits dynamic
digestive movements similar to peristalsis and massperistalsis (defecation) in vertebrates' intestine.
Peristalsis moves the contents back and forth in the
body cavity while mass peristalsis ejects fecal contentsfrom the mouth. Nervous system is primarily
responsible for these movements since animals withno nerve cells showed only a weak peristalsis. Body
column tissue excised from an animal showed the two
types of movement by itself indicating that nerve net
in the body column is responsible for these movements.
These observations suggest that Hydra's digestive
tract is structurally and functionally similar, althoughprimitive, to vertebrates' intestine and that nerve net
in Hydra body column represents enteric nervous
system.
Publications
1. Mochizuki, K., Nishimiya-Fujisawa, C. andFujisawa, T.: Universal occurrence of the vasa-related
genes among metazoans and their germline expressionin Hydra. Development, Genes and Evolution 211,299-308, 2001.

2. Bosch, T.C.G. and Fujisawa, T.: Polyps, peptides
and patterning. Bioessays 23, 420-427, 2001.
3. Harafuji, N., Takahashi, T., Hatta, M., Tezuka, H.,
Morishita, F., Matsushima, O. and Fujisawa, T.:
Enhancement of foot formation in Hydra by a novel
epitheliopeptide, Hym-323. Development 128,437-446.4,
2001.

26

Division of Developmental Genetics

4. Shimizu, H.: Feeding and wounding responses in
Hydra suggest functional and structural polarizationof the tentacle nervous system. Compo Biochem. andPhysiol. 131, 669-674, 2002.
5. Shimizu, H.. Zhang, X., Zhang, J., Leontovich, A.,
Fei, K., Yan, 1. and Sarras, M.P.: Head Regenerationand Wound Healing in Hydra Requires De Novo
Expression of Extracellular Matrix Components andMatrix Metalloproteinases. Development. 129, 1521-1532,
2002.

C-b. Division of Gene Expression(1) Segmentation gene product Fushi tarazu is an
LXXLL motif-dependent coactivator for orphan receptor

FTZ·FlTaiga SUZUKI!, Haruhisa KAWASAKI, Ruth T. Yu i ,Hitoshi UEDA and Kazuhiko UMESONO i (lInstitute for
Virus Research, and Graduate School for Biostudies,
Kyoto University, Kyoto, 606-8507, Japan)
Orphan receptors for whom cognate ligands have
not yet been identified form a large subclass within
the nuclear receptor superfamily. To address one
aspect of how they might regulate transcription, we
analyzed the mode of interaction between the
Drosophila orphan receptor FTZ-Fl (NR5A3) and asegmentation gene product Fushi tarazu (FTZ). Strong
interaction between these two factors was detected byuse of the mammalian one- and two-hybrid interaction
assays without addition of ligand. This interaction
required the AF-2 core and putative ligand binding
domain of FTZ-Fl and the LXXLL motif of FTZ. The
requirementofthese elements was further confirmedby examination of their target gene expression in
Drosophila embryos and observation of a cuticle
phenotype in transgenic fly lines that express mutatedfactors. In Drosophila cultured cells, FTZ is required
for FTZ-Fl activation of a FTZ-Fl reporter gene. These
results reveal a resemblance in the mode of interaction
between FTZ-Fl and FTZ and that of nuclear
receptor-coactivator and indicate that direct interaction
is required for regulation of gene expression by
FTZ-F1. Thus, we propose that FTZ may represent a
novel category of LXXLL-motif dependent coactivatorsfor nuclear receptors. For details, see Ref. 1.

(2) Molecular cloning of human squamous cell carcinoma
antigen 1 gene and characterization of its promoter
Katsuyuki HAMADA!, Hiroto SHINOMIYA2, Yoshihiro
ASAN02, Toshimasa KIHANA!, Mari IWAMOTO!, Yasushi
HANAKAWA3, Koji HASHIMOT0 3, Susumu HIROSE,
Satoru Kyo4 and Masaharu IT0 1 (IDepartment of
Obstetrics and Gynecology, 2Department ofBacteriology, and 3Department of Dermatology, Schoolof Medicine, Ehime University, Shitsukawa, Shigenobu,
Onsen-gun, Ehime 791-0295, Japan and 4Department of
Obstetrics and Gynecology, Kanazawa University,
School of Medicine, Ishikawa 920-0934, Japan)
The squamous cell carcinoma antigen (SCCA)
serves as a serological marker for squamous cell
carcinomas. Molecular cloning of the SCCA genomic
region has revealed the presence of two tandemly
arrayed genes, SCCAI and SCCA2, which are 95%
identical in nucleotide sequence. SCCAI is apapain-like cysteine proteinase inhibitor, while SCCAZ
is a chymotrypsin-like serine proteinase inhibitor. We
analyzed here the sequence and the promoter activityof the 5'-flanking region of the SCCAI gene. Deletion
analysis of SCCAI and SCCAZ promoter identified a
471-bp core promoter region upstream of thetranscription start site. The transcriptional activity of
SCCAI promoter was up-regulated in squamous cell
carcinoma cells, compared with keratinocyte andadenocarcinoma cells. The ratios of SCCAI to SCCAZ
promoter activity in squamous cell carcinoma,
keratinocyte and adenocarcinoma cells wererespectively 1.6, 5.3 and 2.8. Position -50 of SCCAI and
SCCA2 promoters played an important role indetermining the promoter activities of SCCAI and
SCCAZ. These findings suggest that the transcriptional
regulation of SCCAI and SCCA2 might differ amongsquamous cell carcinoma, keratinocyte andadenocarcinoma cells, and that SCCAI promoter might
be a potential target of gene therapy for squamous cell
carcinoma. For details, see Ref. 2.
(3) Gene expression of human squamous cell carcinoma
antigens 1 and 2 in human cell lines
Katsuyuki HAMADA!, Yasushi HANAKAWA2, Koji
HASHIMOT0 2, Marl IWAMOTO!, Toshimasa KIHANA!,
Susumu HIROSE, Masahiko NAKAMURA 3 andMasaharu ITO 1 (IDepartment of Obstetrics andGynecology, and 2Dermatology, School of Medicine,
Ehime University, shitsukawa, Shigenobu, Onsen-gun,Ehime 791-0295, and 3Division of Physiology, Institutefor Protein Research, Osaka University, Yamadaoka,
Suita, Osaka 565-0871, Japan)
The squamous cell carcinoma antigen (SCCA)
serves as a serological marker for squamous cell
carcinomas. Molecular cloning of the SCCA genomic
region has revealed the presence of two tandemly
arrayed genes, SCCAI and SCCA2. SCCAI is apapain-like cysteine proteinase inhibitor, while SCCAZ
is a chymotrypsin-like serine proteinase inhibitor. Little
is known concerning how expression of the SCCAI and
SCCA2 genes is regulated in human cell lines. The
purpose of this study was to determine whether the
SCCAI gene or SCCA2 gene is more strongly
expressed in human cell lines. Squamous cell
carcinoma cell lines secreted respectively 4 times and50 times as much SCCA proteins into medium asnormal human keratinocyte and non-squamous cell
carcinoma cell lines, as measured by enzyme-linked
immunosorbent assay. Quantitative RT-PCR ELISA
digoxigenin-Iabeling assay demonstrated that SCCAImRNA expression in squamous cell carcinoma celllines was respectively 2.8 times and 42 times that in
keratinocyte and non-squamous cell carcinoma celllines. The ratio of SCCAI to SCCAZ mRNA expression
differed distinctly among squamous cell carcinoma,
keratinocyte and non-squamous cell carcinoma celllines (2.8, squamous; 24.1, keratinocyte; 11.0,
non-squamous). These findings suggest that SCCAI
is mainly expressed in squamous cell carcinoma,
keratinocyte and non-squamous cell carcinoma celllines and that the ratio of SCCAI to SCCAZ expression
might be a novel marker for the detection of squamouscell carcinoma. For details, see Ref. 3.
(4) A novel growth-related nuclear protein binds andinhibits rat aldolase B gene promoter
Tomoko YABUKI!, Satoru MIYAGI!, Hitoshi UEDA,
Yasushi SAITOH 1 and Ken-ichi TSUTSUMI 1(ICryobiosystem Research Center, Faculty ofAgriculture, Iwate University, Ueda, Morioka, Iwate
020-8550, Japan)
The promoter of the rat aldolase B (AIdE) gene that
confers liver-specific transcription has an additional

Division of Gene Expression

27

role. It functions in vivo as an origin region of DNA
replication in the cells in which the gene is repressed(Zhao, Y., Tsutsumi, R, Yamaki, M., Nagatsuka, N.,
Ejiri, S., Tsutsumi, K, 1994. Initiation zone of DNA
replication at the rat aldolase B locus encompasses
transcription promoter region. Nucleic Acids Res. 22,5385-5390). This promoter/origin region has multiple
protein-binding sites and, thus, binding of a particular
set of protein factors in AldB-expressing or
non-expressing cells seems to correlate with functionalswitch of this promoter/ origin region. In the present
study, we characterized two closely related proteins,termed AIF-C1 and AIF-C2, which are assumed to be
involved in repression of the AldB gene. These two
proteins share an identical amino acid sequence except
for a 47-residue-insertion in AIF-C1, and are membersof a gene family including heterogeneous nuclear
ribonucleoprotein (hnRNP) and CCAAT-binding factor
subunit A (CBF-A) genes. Bacterially expressed
AIF -C1 can bind sequence-specifically to the AldB
gene promoter, whereas AIF-C2 can only weakly.
Transfection experiments using mammalian expression
vectors showed that AIF-C1 down-regulates the AldB
gene promoter in rat hepatoma cells, while AIF-C2 hadno or little effect. Expressions of mRNAs encoding
these two proteins are enriched in fetal livers and inregenerating livers. These results implied that AIF-C1
and/or C2 is involved in growth-regulated repressionof the AldB gene. For details, see Ref. 4.
Publications
1. Suzuki, T, Kawasaki, H., Yu, R T, Ueda, H. andUmesono, K: Segmentation gene product Fushi tarazu
is an LXXLL motif-dependent coactivator for orphan
receptor FTZ-Fl. Proc. Nat!. A cad. Sci. USA 98,
12403-12408, 2001.

2. Hamada, K, Shinomiya, H., Asano, Y., Kihana, T.,
Iwamoto, M., Hanakawa, Y, Hashimoto, K, Hirose, S.,
Kyo, S. and Ito, M.: Molecular cloning of humansquamous cell carcinoma antigen 1 gene andcharacterization of its promoter. Biochim. Biophys. Acta
1518, 124-131, 2001.
3. Hamada, K, Hanakawa, Y., Hashimoto, K,
Iwamoto, M., Kihana, T, Hirose, S., Nakamura, M. andIto, M.: Gene expression of human squamous cell
carcinoma antigen 1 and 2 in human cell lines. Oncology
Rep. 8, 347-354, 2001.

28

Division of Gene Expression

4. Yabuki, T, Miyagi, S., Ueda, H., Saito, Y. andTsutsumi, K: A novel growth-related nuclear protein
binds and inhibits rat aldolase B gene promoter. Gene
264, 123-129, 2001.

C-c. Division of Early Embryogenesis(1) In situ screen for novel genes expressed in the yolk
syncytial layer
Takuya SAKAGUCHI, Atsushi KUROIWA 1 and Hiroyuki
TAKEDA (lNagoya University)
Mesoderm induction and its dorsoventral
specification are important processes of vertebrate
early development. When transplanted onto theanimal-pole region of host blastula embryos, thezebrafish yolk cells induce ectopic formation of themesoderm and the organizer, indicating that the yolkcell, especially yolk syncytial layer (YSL), is responsible
for mesoderm induction and its dorsoventral
patterning. Recently, genetic studies with mice andzebrafish demonstrated that Nodal could be an
endogenous mesoderm inducer. However, we still do
not know how the expression of nordal-related genes
are regulated in the YSL and/ or it's neighboring
blastomeres. To address these questions, we carriedout a large scale screening of genes expressed in thezebrafish YSL. By combination of a subtracted cDNA
library with our in situ screening method, we
have successfully obtained more than 10
independent-positive clones out of 200 clones tested(>5%). Sequencing analyses suggest that they encode
some enzymes, amino acid transporting proteins,
plasma membrane binding protein, transcription factor,
RNA binding protein, and many novel genes. Recently,
we found that one of the isolated genes, 226D7, encodesa novel sox gene that acts downstream of Nodal
signaling to specify endoderm precursors (Sakaguchi
et aI., 2001). Functional analyses of other isolated genes
are now underway.
(2) Role of FGF/MAPK signaling in the developing
telencephalon of zebragfish embryos
Minori SHINYA, Sumito KOSHIDAl, Atsushi KUROIWA 2
and Hiroyuki TAKEDA (lKondoh Differentiation and
Signaling Project ERATO, 2Nagoya University)

The telencephalon is formed in the most anterior
part of the central nervous system (CNS) and isorganized into ventral subpallial and dorsal pallial
domains. In mice, it has been demonstrated that Fgf
signaling has an important role in induction andpatterning of the telencephalon. However, the preciserole of Fgf signaling is still unclear, due to overlapping
functions of Fgf family genes. To address this, we
examine, in zebrafish embryos, the activation of Ras/
MAPK, one of the major downstream targets of Fgf
signaling. Immunohistochemical analysis reveals thatan extracellular signal-regulated kinase (ERK), a
vertebrate MAPK, is activated in the anterior neuralboundary (ANB) of the developing CNS at earlysegmentation stages. Experiments with Fgf inhibitors
reveal that ERK activation at this stage is totallydependent on Fgf signaling. Interestingly, a substantial
amount of ERK activation is observed in ace mutantsin whichfgf8 gene is mutated. We then examined thefunction of Fgf signaling in telencephalic development
by use of several inhibitors to Fgf signaling cascade
including dominant-negative form of Ras (RasNil andFgf receptor (Fgfr), and a chemical inhibitor of Fgfr,
SU5402. In treated embryos, the induction oftelencephalic territory normally proceeds but thedevelopment of the subpallial telencephalon issuppressed, indicating that Fgf signaling is required
for the regionalisation within the telencephalon. Finally, antisense experiments with morpholino-modified
oligonucleotides suggest that zebrafishfgf3, which isalso expressed in the ANB, co-operates with fgf8 in
subpallial development (Shinya et aI., 2001).
(3) FGF/MAPK signaling and somite maturation invertebrate segmentation
Atsushi SAWADA, Minori SHINYA, Yun-Jin JIANG!,
Atsushi KAWAKAMI, Atsushi KUROIWA2 and Hiroyuki
TAKEDA (lImperial Cancer Research Fund, London,
2Nagoya University)
Somite formation, a process in which reiterated
epithelial structures are progressively demarcated fromthe mesenchymal presomitic mesoderm (PSM) in a
rostrocaudal sequence, is thought to be governed bya clock-and-wavefront mechanism. Through Notch
signalling, the segmentation clock (oscillation in the
PSM cells) is coordinated and translated into a cyclic
wave of expression of hairy-related and other genes,

sweeping caudorostrally through the PSM. Thisso-called kinematic wave is proposed to operate inconjunction with a maturation wavefront that
gradually moves posteriorly, resulting in arrest of thecyclic wave and initiation of segment furrow formationin the anterior PSM. In zebrafish these two essential
components of an evolutionalry conserved
somite-making mechanism "clock and wavefront" wereshown to be Notch- and fused somites Vss)- dependent,
respectively. By experimental manipulation andanalyses in zebrafish somitogenesis mutants, we founda novel component, Fgf/ MAPK signaling, involved insegmentation. Fgf signaling activated in the posterior
PSM is a crucial positional cue in locating thematuration wavefront in the anterior PSM and inmaintaining posterior PSM cells in immature state inan aei/ deltaD-and fss-independent manner (Sawada
et aI., 2001; Saga & Takeda, 2001).
(4) Molecular analysis of zebrafish midline mutant
chameleon
Atsushi KAWAKAMI, Rolf KARLSTROM!, Hiroyuki
TAKEDA, William S. TALBOT 2, Alexander F. SCHIER3(lUniversity of Massachusetts, 2Stanford University,
3New Yolk University)
Midline tissues, including the floor plate andnotochord, produce inducing signals and pattern theneural tube and somites. A group of zebrafish mutants(midline mutants) has common defects in the ventral
neural tube, somites and dorsal aorta.. We are now
focusing on one of the midline mutants, chamekon (con).
To identify the con gene, we mapped the mutation on
LG20. We also mapped and compared the positions ofcandidate genes that may play roles in the Shhpathway. Genomic walking is now underway.
(5) EST project in medaka fish
Takanori NARITA, Tetsuaki KIMURA, Tomoko JINDO
and Hiroyuki TAKEDA
Medaka (Oryzias latipes) has several advantages
to zebrafish. For examples, medaka has about half sizeof genome (800 Mb; zebrafish-1700 Mb) and the numberof genes are expected to be lower than that of zebrafish.
Tolerance to cold temperature makes it easy to obtaina temperature-sensitive mutant which would be a

Division of Early Embryogenesis

29

powerful tool for developmental genetics. To gain
insight into genetic system of medaka, we perform alarge scale isolation of genes expressed in developing
medaka embryos.
(6) Genetic screening for mutations affecting early
embryogenesis in medaka fish
Daisuke KOBAYASHI, Tadao KITAGAWA, Shigeo
TAKASHIMA, Takahiro KAGE, Hayato YOKOI, Atsuhi
SAWADA!, Takanori NARITA, Tetsuaki KIMuRA, Tomoko
JINDO, Kazuo ARAKI 2, Yuji ISHIKAWA3 and Hiroyuki
TAKEDA (lNagoya University, 2National ResearchInstitute of Aquaculture, 3National Institute ofRadiological Sciences)
To understand the genetic pathways that govern
vertebrate ealy embryogensis, we are conducting amutant screen in medaka fish. In our screen, male fish
mutagenized with ethylnitrosourea (ENU) were
outrcossed to wild-type female and the F1 offspring
was used to establish F2 families. F2 siblings wereintercrossed and the F3 progeny was scored 24 and 48
hours after fertilization for morphological alterations
affecting neural, mesodermal and endodermal
development.
Publications
1. Sakaguchi, T., Kuroiwa, A. and Takeda, H.:
Expression of zebrafish btg-b, an anti-proliferative
cofactor, during early embryogenesis. Mechanisms ofDevelopment 104, 113-115, 2001.

2. Sakaguchi, T., Kuroiwa, A and Takeda, H.: A
novel sox gene, 226D7, acts downstream of Nodal
signaling to specify endoderm precursors in zebrafish.
Mechanisms of Development 107, 25-38, 2001.
3. Shinya, M., Koshida, S., Sawada, A, Kuroiwa, A
and Takeda, H: Fgf signalling through MAPK cascadeis required for development of the subpallial
telencephalon in zebrafish embryos. Development 128,4153-4164, 2001.
4. Sawada, A, Shinya, M., Jiang, Y,-]., Kawakami, A,
Kuroiwa, A and Takeda, H.: Fgf/ MAPK signallingis a crucial positional cue in somite boundary
formation. Development 128, 4873-4880, 2001.

30

Division of Early Embryogenesis

5. Haraguchi, S., Kitajima, S., Takagi, A, Takeda,
H., Inoue, T. and Saga, Y.: Transcriptional regulationof Mesp1 and Mesp2 genes: differential usage ofenhancers during development. Mechanisms ofDevelopment 108, 59-69, 2001.
6. Saga, Y. and Takeda, H: The making of the somite:molecular events in vertebrate segmentation. (review)
Nature Reviews Genetics 2, 835-845, 2001.

C-d. Division of Physiological Genetics
Functional and structural analyses of mammalian
Polycomb group complexes
Haruhiko KOSEKI
In order to address the functions of mammalian
Polycomb group (PcG) complexes, we intended to
identify components of the complexes by employing
biochemical and genetic approaches. For this purpose,
we focused on following three topics, (1) identificationof additional components of mammalian PcG
complexes, (2) search for target loci of mammalian PcG
and (3) Binding of PcG complexes onto Hox cluster
genes.
(1) Involvement of 155kDa spliceosome associatedprotein (SAP155), RinglB and Mph2 in mammalian PcG
complexes
We have identified SAP155 protein as an interactor
of mammalian PcG proteins, Me118 and Ring1B byyeast two hybrid system. Immunoprecipitation
analyses revealed that SAP155 was coimmunoprecitated by anti-Me118 and anti-Ring1B
antibodies from 11.5 dpc mouse embryonic extracts.
Further evidences indicating the association of SAP155
with PcG complexes were provided through genetic
analyses using Sap155-deficient mice. Sap155-/embryos die at morula stage. Sap155+/-mice exbihited
posterior transformations of the axial skeleton. These
alterations were enhanced by Me118 mutation while
repressed by Mil mutation. In addition to SAP155, we
identified Ring1B and Mph2 as interactors of Me118.
Ring1B- and Mph2-deficient mice exhibited posteriortransformations as well as Me118-, Bmil- andSap155-deficient mice suggesting their inclusion into

PcG complexes.
(2) Generation of eDNA microarray including 15K
independent clusters
For systematic screening of downstream genes for
PcG, we intended to generate representative cDNA
microarray. From full-length cDNA libraries derived
from tail bud, fetal gut and adult thymus, we have
idolated 15K clusters. In the first preliminary screening
using 2K microarray, we have found 10 clusters, whose
expressions were affected in Me118/Bmil double
homozygotes.
(3) Mapping of binding sites for Ring1B and Mph1
within Hoxb cluster locus
To map PcG responsive elements on mouse Hox
cluster locus, we have developed chromatin
immunoprecipitation (ChIP) method by using mouse
mid-gestation embryos. By using monoclonal
antibodies against Ring1B and Mph1, we havesuccessfully immunoprecipitated chromosomal DNA
from purified chromatin and binding sites for Ring1B
and Mph1 were determined with 500 base pairs
resolution. Interestingly, Ring1B was shown to tether
transcriptionally inactive region exclusively suggesting
repressive functions of RinglE.

Publications
1. Atsuta, T., Fujimura, S., Moriya, H., Vidal, M.,
Akasaka, T., Koseki, H.: Production of Monoclonal
Antibodies against Mammalian Ring 1B Proteins.
Hybridoma 20,43-46,2001.
2. Sudo, H, Tonegawa, A., Arase, Y., Aoyama, H.,
Mizutani-Koseki, Y., Moriya, H., Wilting, J" Christ, B.
and Koseki, H.: Inductive signals from thesomatopleure mediated by bone morphogenetic
proteins are essential for the formation of the sternal
component of avian ribs. Dev. Bioi. 232, 284-300, 2001.
3. Isono, K., Abe, K., Tomaru, Y., Okazaki, Y.,
Hayashizaki, Y. and Koseki, H.: Molecular cloning,
genetic mapping, and expression of the mouse Sf3b1(SAP155) gene for the U2 snRNP component ofspliceosome. Mammalian Genome 12, 192-198, 2001.
4.

Koizumi, K., Nakajima, M., Yuasa, S., Saga, Y.,

Sakai, T., Kuriyama, T., Shirasawa, T. and Koseki, H.:
The role of Presenilin1 during somite segmentation.
Development 128, 1391-1402,2001.
5. Fukamachi, H, Fukuda, K., Suzuki, M., Furumoto,T., Ichinose, M., Shimizu, S., Tsuchiya, S., Horie, S.,
Shiokawa, K., Suzuki, Y., Saito, Y., Watanabe, K.,
Taniguchi, M. and Koseki, H.: Mesenchymaltranscription factor FKH6 controls gastric epithelial
development and differentiation. Bioehem. Biophys. Res.
Commun. 280, 1069-1076,2001.
6. Akasaka, T., van Lohuizen, M., van der Lugt, N.,
Mizutani-Koseki, Y., Kanno, M., Taniguchi, M., Vidal,
M., Alkema, M., Berns, A. and Koseki, H: Mice doubly
deficient for the Polycomb-Groupe genes Me118 and
Bmil reveal synergy and requirement for maintenancebut not initiation of Hox gene expression. Development
128, 1587-1597,2001.
7. Yamamoto, K., Fujii, R., Toyofuku, Y., Saito, T.,
Koseki, H., Hsu, V.W. and Aoe, T.: The KDEL receptor
mediates a retrieval mechanism that contributes toquality control at the endoplasmic reticulum. EMBO
J 20, 3082-3091, 2001.
8. Ogita, J" Isogai, E., Sudo, H., Sakiyama, S.,
Nakagawara, A. and Koseki, H.: The expression ofchicken Dan. Meeh. Dev. 109, 363-365, 2001.
9. Kimura, M., Koseki, Y., Yamashita, M., Watanabe,N., Shimizu, C., Katsumoto, T., Kitamura, T.,
Taniguchi, M., Koseki, H. and Nakayama, T.:
Regulation of Th2 cell differentiation by mel-18, amammalian polycomb group g-ene. Immunity. 15,
275-287, 2001.
(1) Functional nucleosomal phases over HS2 of thehuman j3-LCR
Ryoiti KIY AMA (National Institute of Advanced
Industrial Science and Technology)
High-level, tissue-specific expressions of the{3-globin genes require the presence of an upstream
locus control region ({3-LCR) which coincides withchromatin segments displaying erythroid-specific
DNase I hypersensitivity. The tandem NF-E2 bindingsites within the DNase I hypersensitive site 2 (HS2) ofDivision of Physiological Genetics

31the /3-LCR are important for the strong enhancer
activity. Previously, we reported that the translational
phases of the nucleosomes over HS2 of the human/3-LCR were arranged by the nucleosome located atcurved DNA acting as a key nucleosome andinfluenced on the enhancer activity (1). Here, weinvestigated the chromatin structure of HS2 region infurther details. The results of gel shift assay andmicrococcal nuclease digestion experiments using invitro reconstituted chromatin showed that only thethirty bp fragment containing the tandem NF-E2
binding sites had less nucleosome forming activity
than those from the neighboring sequences, indicatingthat this region has a specific chromatin structure
suitable for function. The results of hydroxyradical
footprinting suggested that the nucleosomal phase of
HS2 region was rotationally unique while there were
multiple translational phases and that the tandem
NF-E2 binding sites were always located outside but
not in the histone side, facilitating interaction with
NF-E2. Finally, we examined the binding of NF-E2 tothe sites in the packed chromatin over HS2 region by
ligation-mediated PCR and a chromatin immunoprecipitation assay using non-erythroid HeLa
cells, where we provided evidence for direct NF-E2
binding on the nucleosome in vivo. These results
support a crucial role of NF-E2 in the regulation of/3-globin gene expression through a direct interaction
to the cognate motifs located on the chromatin before
chromatin remodeling.
(2) Functional genotyping based on DNA rearrangements and its application
Ryoiti KIYAMA (National Institute of Advanced
Industrial Science and Technology)
We focus upon the mechanism of tumorigenesis
by loss of heterozygosity (LOH) which results in lossof function of tumor suppressor genes. For this, we firstobtained candidate genetic loci by means of genomic
subtraction between the normal and tumor (the mixed
cell type and the clear cell type of renal cell carcinoma)cell DNA from the same individual which was followed
by cloning the DNA fragments derived from the sitesof DNA arrangements. We obtained a total of 44 suchloci, which showed 10% to 90% of frequencies of LOH
among approximately 100 patients (1). They included
VHL (von Hippel Lindau), APC and IRF-l and other

32

Division of Physiological Genetics

previously reported loci as well as new loci including
5q32-q34, 6q21-q22 and 8p12. Four such loci had
minimum distances of less than 10 mega-bases (Mb).
We obtained a candidate tumor suppressor gene fromone of these loci and functional analysis of the geneis now under way. Using the information of candidate
loci, we fabricated DNA microarrays for highthroughput genotyping of the candidate genes.
Publications
1. Onishi, Y. and Kiyama, R: Enhancer activity of

HS2 of the human /3-LCR is modulated by distancefrom the key nucleosome. Nucl. Acids Res. 15, 3448-3457,
2001.
2. Hatano, N., Nishikawa, N. S., McElgunn, C.,
Sarkar, S., Ozawa, K., Shibanaka, Y., Nakajima, M.,
Gohji, K. and Kiyama, R: A Comprehensive Analysisof LOH Caused by Hemizygous Deletions in Renal Cell
Carcinoma Using a Subtraction Library. Mol. Carcinog.
31,161-170,2001.

(1) Local changes in mutation pressure in Drosophila
genomes

substitutions in the orena lineage were significantly
biased toward G and C at the consecutive yellow, lethalof scute, and asense loci, but they were significantly
biased toward A and T at sta. The substitution bias
toward G and C was centered in different areas inyakuba (significantly biased at EG:165H7.3,
EG:171Dll.2, and suppressor ofsable). The similar silent
substitution patterns in coding and noncoding regions,further, suggested mutational biases as a cause of thesubstitution biases.
In sum, this study offered evidence ofheterogeneous and species-specific (fluctuating)mutation pressure along Drosophila chromosomes.
For details, see Ref. 1.

Toshiyuki T AKANO-SHIMIZU(2) Genetic study of evolutionary changes in the genes

D. DIVISION OF
POPULATION GENETICS

D-a. Division of Population Genetics

involved in Drosophila bristle formation

The genomes of warm-blooded vertebrates,
particularly humans, are compartmentalized into theso-called isochores that are DNA segments of sizes
varying from 200 kb to more than 1 Mb and relatively
homogeneous in their DNA content. The
genome-composition organization is assumed to be
determined and maintained by the action of mutationand selection, but the degree to which these two factors
have contributed has been the subject of muchcontroversy. Regardless of the validity of the mutationand selection hypotheses, both hypotheses are basedon the finding that local environments along
chromosomes affect DNA composition of genes.
By contrast, Drosophila genomes have been
considered to be much more homogeneous with respect
to G+C content along chromosomes. However, I foundevidence of remarkable local changes in GCIAT
substitution biases on Drosophila chromosomes. The
substitution pattern at ten loci in the telomeric regionof the X chromosome was studied for four species of
the D. melanogasterspecies subgroup. D. orena and D.
erecta are clearly the most closely related species pair(the erecta complex) among the four species studied;however, the overall data at the ten loci revealed a clear
dichotomy in the silent substitution patterns between
AT-biased-substitution melanogaster and erecta
lineages and GC-biased-substitution yakuba and orena
lineages, suggesting two or more independent changes
in GCIAT substitution biases. More importantly, theresults indicated a between-locus heterogeneity in GCI
AT substitution bias in this small region independently
in the yakuba and orena lineages. Indeed, silent

Toshiyuki T AKANO-SHIMIZUInterspecific cross is a powerful means to uncoverhidden within- and between-species variation in
populations. One example of such hybrid anomaly isa bristle loss phenotype of hybrids between Drosophilamelanogaster and D. simulans, though both the purespecies have exactly the same pattern of bristle
formation on the notum. This means that the geneticarchitecture of bristle formation can change in local
populations in the absence of any obvious phenotypicalternation. There exists a large amount of genetic
variability in the simulans populations with respect
to the number of missing bristles in hybrids, and thevariation is largely attributable to simulans X
chromosomes. I screened the simulans X chromosome
for genetic factors that were responsible for thedifferences between a pair of simulans lines with high(H) and low (L) missing bri~tle numbers. A single major
quantitative locus was mapped to the 14-16 region. This
indeed explained nearly all difference between the twoparental strains and also accounted for most of thedifferences between Hand L lines in three otherindependent pairs.
We took a further fine mapping experiment to
identify the QTL: a construction of about 2500
recombinant strains between the Hand L simulans
lines. They were studied for the number of missing
bristles in hybrids with D. melanogaster and forgenotypes at several marker loci covering the 14-16
region. This ends up identification of 15 recombinants

DIVISION OF POPULATION GENETICS

33

in an about 500 kb region studied. These enable asubsequent fine mapping of the QTL.
(3) Genetic and molecular dissection ofthe within- andbetween-species variation in the sex comb teeth number
in Drosophila
Toshiyuki T AKANO-SHIMIZU
Sexual dimorphic characters are very often among
the first characters to change in the evolution of new
species, and they provide a number of advantages forstudying the genetic and molecular basis of species
differences in morphology. Males of some species ofthe subgenus Sophophora including D. melanogaster
posses sex-specific bristles on their first legs, theso-called sex comb. The sex comb is a highly variablecharacter between species and within species.
We analyzed the within-species sex-comb-teeth
number variation by the composite interval mappingfor F2 samples between high and low sex-comb-teeth
number lines. We did this with an aim to identify
quantitative trait loci and to estimate their effects,
finding four significant QTL, two on each majorautosomes. The sign of the QTLs differed among
QTLs, and one of them showed an overdominant effect.
Because of a lack of male recombination, the F2
analysis cannot study all possible genotypes even ina very large number of samples; for instance, a double
homozygote at two loci on a single chromosome neveremerges in F2 populations. Then, the chromosome
substitution or recombinant inbred strains wereconstructed, and they can now be used for our
subsequent fine mapping and for studying epistasis
between QTLs.
I should add that Ms Yuriko Ishii has contributed
significantly to the above three works.
Publication
1. Takano-Shimizu, T.: Local changes in GC/AT
substitution biases and in crossover frequencies on
Drosophila chromosomes. Molecular Biology and
Evolution 18, 606-619, 2001.

34

Division of Population Genetics

D-b. Division of Evolutionary Genetics(1) Functional analyses of centromere of higher
vertebrate cells
Tatsuo FUKAGAWA, Atsushi OKAMURA, Ai NISHIHASHI,
Yoshikazu MIKAMI, Toshimichi IKEMURA
The centromere plays a fundamental role inaccurate chromosome segregation during mitosis andmeiosis in eukaryotes. Its functions include sister
chromatid adhesion and separation, microtubule
attachment, chromosome movement and mitotic
checkpoint control. Although chromosome segregation
errors cause genetic diseases including some cancers,
the mechanism by which centromeres interact withmicrotubules of the spindle apparatus during cell
division is not fully understood.
To understand the function of the centromere, wewere led to develop a genetic analysis method thatutilizes the hyper-recombinogenic chicken B
lymphocyte cell line DT40. The high level ofhomologous recombination in DT40 cells allowed
targeted disruption of genes for several centromere
proteins. We attempted to generate several conditional
knockout cell lines of several centromere proteins,
including CENP-C, CENP-H, ZWlO and Mis6. We also
created several temperature-sensitive CENP-C mutants
with DT40 cells. Phenotypic analysis of these mutants
revealed several things.
I) CENP-C is necessary but not sufficient for theformation of a functional centromere and the structureof CENP-C may be regulated during the cell cycle.
II) CENP-C may serve further functions during G1of the cell cycle in addition to its role in mitosis.
ill) Centromere assembly in vertebrate cells proceeds
in a hierarchical manner in which localization of thecentromere-specific histone CENP-A is an early eventthat occurs independently of CENP-C and CENP-H.
N) ZW10 is required for a spindle checkpoint function
in vertebrate cells.
V) 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 centromere

function and assembly by generating a conditional
loss-of-function mutant in the chicken DT40 cell line.
In the absence of CENP-I, cells accumulated BubRl
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, kllization 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.
For detail, Fukagawa et aI., EMBO ]., 20, (2001),
Fukagawa et aI., Nucl. Acids Res., (2001). Okamuta etal., Gene, 283, (2001), and Sonoda et al., Dev. Cell, 1, (2001)(2) Chromosome-wide measurement of replication timing
for human chromosomes llq and 21q: disease-related
genes and genome-synteny breakage in timing·. witch
regions
Yoshihisa WATANABE, Asao FUllYAMA1.2, Yuta ICHIBA,
Yoshiyuki SAKAKI2and Toshimichi IKEMURA 0Divisionof Human Genetics, 2Human Genome Research Group,
RIKEN Genomic Sciences Center)
The completion of the human genome sequence
provides fundamental knowledge to a wide field of
biology. We used the sequence information to measure
replication timing of the entire lengths of humanchromosomes llq and 21q focusing on a total of 450
STSs. Mb-sized zones that replicate early or late in S
phase (thus early/ late transition) were defined at thesequence level. Early zones were more GC-rich andgene-rich than were late zones, and early/ latetransitions occurred primarily at positions identical toor near GC% transitions. In the early/ late transition
regions, concentrated occurrence of cancer-related
genes that include CCNDl encoding cyclin Dl, FGF4,
TIAM1, and FL/1 was observed. The transition regions
contained other disease-related genes including APP
associated with familial Alzheimer's disease, SODl
associated with familial ALS, and PTS associated withphenylketonuria. We also found the single nucleotide
polymorphism (SNP) frequency was high in transition
regions. These findings are consitent with theprediction that increased DNA damage occurs in
replication-transition regions. We also found thatbreakage of synteny between the human and mouse
genomes occurred primarily at or near the
replication-transition regions.
For detail, Science, 294, 2282, 2001; and Human
Molecular Genetics, 11, 13-21, 2002.
(3) Analysis of codon- and oligonucleotide-composition
diversity of bacterial genomes with a self-organizing map(80M)
Shigehiko KANAYA\ Takashi ABE2, Makoto KINOUCHI\
Yuko YAMADA3, Yoshihiro KUDO l and Toshimichi
IKEMURA (lDepartment of Electrical and Information
Engineering, Yamagata Univ., 2Xanagen, 3Departmentof Biochemistry, Jichi Medical School)
With increases in the amounts of available DNA
sequence data, it has become increasingly important
to develop tools for comprehensive systematic analysis
and comparison of species specific characteristics ofgenome sequences for a wide range of species. In thepresent study, we used a novel neural-network
algorithm, a self-organizing map (SaM), to efficiently
and comprehensively analyze codon usage inapproximately 60,000 genes from 29 bacterial species
simultaneously. This SaM makes it possible to cluster
and visualize genes of individual species separately
at a higher resolution than can be obtained withprincipal component analysis. The organization of the
SaM can be explained by the tRNA compositions andgenome G+C% of the individual species. Using SaM,
we examined codon usage heterogeneity in the E. coli
0157 genome, which contains "0157-unique segments(a-islands)", and showed that the SaM is a powerful
new strategy for characterization of horizontallytransferred genes. For details, see Kanaya et aI., Gene
(2001). With SaM, we also analyzed di-, tri-, andtetra-nucleotide frequencies of the 65 bacterial genomes
and obtained good intra- and inter-species separation,
which could not be obtained with conventional
multivariate methods.
Publications
1. Watanabe, Y., Fujiyama, A., Ichiba, Y, Hattori, M.,

Division of Evolutionary Genetics

35Yada, T., Sakaki, Y. and Ikemura, T.:
Chromosome-wide assessment of replication timing forhuman chromosomes llq and 21q: disease-related
genes in timing-switch regions. Human Molecular
Genetics, 11, 13-21, 2002.
2. Sonoda, E., Matsusaka, T., Morrison, c.,Vagnarelli, P., Hoshi, 0., Ushiki, T., Nojima, K,
Fukagawa, T., Waizenegger, I.C., Peters, J-M.,
Earnshaw, W.C. and Takeda, S.: Scd/ Rad21/ MCDI
is required for sister chromatid cohesion andkinetochore function in vertebrate cells. Dev. Cell, 1,759-770, 2001.
3. Kanaya, S., Yamada, Y, Kinouchi, M., Kudo, Y.
and Ikemura, T.: Codon usage and tRNA genes ineukaryotes: Correlation of codon usage diversity withtranslation efficiency and with CG-Dinucleotide usage
as assessed by multivariate analysis. J Mol. Evol., 53,
290-298, 2001.
4. Kanaya, S., Kinouchi, M., Abe, T., Kudo, Y.,
Yamada, Y., Nishi, T., Mori, H. and Ikemura, T.:
Analysis of codon usage diversity of bacterial genes
with a self-organizing map (SOM): characterization ofhorizontally transferred genes with emphasis on theE. coli 0157 genome. Gene, 276, 89-99, 2001.
5. Fukagawa, T., Regnier, V. and Ikemura, T.:
Creation and characterization of temperature-sensitive
CENP-C mutants in vertebrate cellso Nucleic Acids
Research, 29, 3796-3803, 2001.
60 Fukagawa, T, Mikami, Yo, Nishihashi, A, Regnier,Vo, Haraguchi, T, Hiraoka, Y, Sugata, No, Todokoro,
K., Brown, W. and Ikemura, To: CENP-H, a constitutive
centromere component, is required for centromere
targeting of CENP-C in vertebrate cells. The EMBO
Journal, 20, 4603-4617, 2001.
7. Hamada, A, Ikemura, T. and Yoshinaga, K:
Stabilization of chi/transcript by tRNA in chloroplastof green alga chiarella vulgariso J Biochemo Malo Bioi. &Biophys., 5, 277-284, 2001.
80 Okamura, A, Pendon, c., Valdivia, M. Mo, Ikemura,T. and Fukagawa, To: Gene structure, chromosomal
localization and immunolocalization of chicken
centromere proteins CENP-C and ZWI00 Gene, 262,

36

Division of Evolutionary Genetics

283-290, 2001.
90 Shina, To, Ando, A, Suto, Y, Kasai, F., Shigenari,
A, Takishima, No, Kikkawa, K, Iwata, K., Kuwano, Yo,
Kitamura, Y, Matsuzawa, Y., Sano, K, Nogami, M.,
Kawata, H., Li, S., Fukuzumi, Y., Yamazaki, Mo,
Tashiro, H., Tamiya, G., Kohda, A, Okumura, K,
Ikemura, T, Soeda, E., Mizuki, N., Kimura, Mo, Bahram,S. and Inoko, H.: Genomic anatomy of apremier major
histocompatibility complex paralogous region on
chromosome lq21-q22. Genome Research, 11, 7879-802,
2001.
(1) Murine equivalent ofthe human histo-blood group
ABO gene is a cis-AB gene and encodes a
glycosyltransferase with both A and B transferase
activity
Miyako YAMAMOTO!, Xiao-Hong LIN l , Yoshihiko
KOMINAT02, Yukiko HATA2, Reiko NODA, Naruya
SAITOU and Fumiichiro YAMAMOTO l(lThe Burnham Institute, La Jolla, USA
2Department of Legal Medicine, Faculty of Medicine,
Toyama Medical & Pharmaceutical University,
Toyama, Japan)
We have cloned murine genomic and complementary DNA that is equivalent to the human ABO geneo
The murine gene consists of at least 6 coding exonsand spans at least 11 kilobase pairs (kbp). Exon-intron
boundaries are similar to those of the human gene.
Unlike human A and B genes that encode two distinct
glycosyltransferases with different donor
nucleotide-sugar specificities, the murine gene is a
cis-AB gene that encodes an enzyme with both A andB transferase activities, and this cis-AB gene prevails
in the mouse population. Cloning of the murine AB
gene may be helpful in establishing a mouse model
system to assess the functionality of the ABO genes
in the future. For details, see ref. 1.
(2) CAMUS DB for amino acid sequence data
Sadahiko MISU 1 , Takayasu IIZUKA 1 , Yuichi
KAWANISHI 1, Kaoru FUKAMI1 and Naruya SAITOU
rCenter for Information Biology and DNA Data Bankof Japan, National Institute of Genetics)
DDBJ/ EMBLI GenBank International Nucleotide

Sequence Database is still increasing, keeping
doubling time only slightly longer than one year forthe last these years. This situation affects the increaseof DDBJ Amino acid sequence Database (DAD), which
is made from translation of nucleotide sequences in
CDS regions annotated in DDBJ, and makes
computation time for homology search of DAD longer
as well as that of DNA database. We therefore createdcompressed sequence database, consisting of highly
homologous sequence clusters in multiple aligned form
with representative sequences, the DAD version of
CAMUS (Compressed database for homology searches
And MUltiple aligned Sequence database). Website
URL of CAMUS DB is http://hypernig.nig.ac.jpj
camus/. For details, see ref. 2.
(3) Haplotype analysis
glycoprotein (fetuin)

of

human

nonsynonymous substitutions were concentrated in the
carboxyl-terminal domain. For details, see ref. 3.

Publications
1. Yamamoto, M., Lin, X.-H., Kominato, Y, Hata, Y,
Noda, R., Saitou, N. and Yamamoto, F.: Murine
equivalent of the human histo-blood group ABO geneis a cis-AB gene and encodes a glycosyltransferasewith both A and B transferase activity. Journal of
Biological Chemmistry, 276, no. 17, 13701-13708, 2001.
2. Misu, S., Iizuka, T., Kawanishi, Y, Fukami, K and
Saitou, N.: CAMUS DB for amino acid sequence data.
Matsuda, H, Wong, L., Miyano, S. and Takagi, T. (eds.),"Genome Informatics 2001", Universal Academy Press,
Tokyo, 502-503, 2001.

alpha2-HS

Motoki OSAWA\ Isao YUASA, Takashi KITANO, Jurgen
HEINKE, Mika KANEKO!, Naruya SAITOU and Kazuo
UMETSU!
(!Department of Forensic Medicine, Yamagata
University School of Medicine, Yamagata, Japan)
Alpha2-HS glycoprotein (AHSG), which isequivalent to fetuin in other species, is a protein foundin human plasma. AHSG is polymorphic with two
common alleles and many variants. To examine theintragenic haplotypes and their diversity at this locus,
a contiguous genomic DNA sequence (10.3 kb) was
analyzed in 20 samples (40 chromosomes), andhaplotypes were determined for 309 subjects. Judging
from the aligned nucleotide sequences and theconserved amino acid residues comparing human andchimpanzee AHSG, it was concluded that the type 1
allele is probably older and has evolved into four major
suballeles. The type 2 allele was generated from one
branch of the type 1 allele. AHSG*3 and *5 variants
were each found to have a single nucleotide change
in exon 7, resulting in the change of an amino acidresidue from Arg299 to Cys and from Asp258 to Asn,
respectively. It was noted that the AHSG*3 mutation
gives rise to an additional cysteine residue, which
possibly affects the conformation of the protein. The
AHSG gene was found to have a low mutation rate andno apparent recombination events. Furthermore, the3. Osawa, M., Yuasa, 1., Kitano, T., Heinke, J.,
Kaneko, M., Saitou, N. and Umetsu, K: Haplotype
analysis of human alpha2-HS glycoprotein (fetuin).
Annals of Human Genetics, 65, no. 1,27-34,2001.
4. Yuasa, I., Umetsu, K, Ago, K, Sun, c.-S., Pan, I-H,
Ishida, T., Saitou, N. and Horai, S.: Population genetic
studies on nine aboriginal ethnic groups of Taiwan.
II. Serum protein systems. Anthropological Science, 109,
no. 4, 257-273, 2001.
5. Umetsu, K, Tanaka, M., Yuasa, I., Saitou, N., Naito,
E., Ago, K, Nakayashiki, N., Miyoshi, A, Kashimura,S., Watanabe, G. and Osaw'a, M.: Multiplex amplified
product length polymorphism analysis'to rapidly detecthuman mitochondrial DNA variations. Electrophoresis,
22, 3533-3538, 2001.
6. Sanders, AR., Cao, Q., Taylor, J., Levin, T.E.,
Badner, J.A, Cravchik, A, Comeron, J.M., Saitou, N.,
Del Rosario, A, Salvi, D.A, Walczyk, KA, Goldin, L.
R, Mowry, B.]., Levinson, D.F., Crowe, RR, Silverman,
J.M. and Gejman, P.V.: Genetic Diversity of the Human
Serotonin Receptor IB (HTRIB) Gene. Genomics, 72,
1-14, 2001.

detected substitutions were nonhomogeneously
distributed at this locus. In particular, four

Division of Evolutionary Genetics

37

Mathematical sciences. 39, 44-47, 2001.

D-c. Division of Theoretical Genetics(1) Autonomous formation of spatial pattern indevelopment
Shigeru KONDO
Most of biological phenomena occur as the resultof complex interaction of the genes or molecules. As
the number of the element increases, the relationships
among them become terribly complex as the
exponential function. Complex phenomena likemorphogenesis or neural net formation, occurs as thechain reaction of the local interaction of cells.
Therefore, it is quite difficult to imagine what really
happens. In such case, computer simulation may help.
In our laborato!'y, using the skin pattern of zebra
fish as a model system, we aim to develop a practicalstandard method to simulate complex phenomena inbiology.
The formation of periodic patterns in animal skins
has been explained by the reaction-diffusion (RD)system, a hypothetical chemical reaction proposed by
A. Turing1. However, the model alone cannot explain
the directionality of stripes. To investigate themechanism regulating the direction of stripes, we havestudied differences in the pattern formation of two
species of Genicanthus that share almost identicalmorphological properties except for their stripe
direction. In both species, stripes are formed by therearrangement of the characteristic transient patternthat suggest the underlying mechanisms.
Computational analysis shows that adding diffusion
anisotropy to the standard RD model can explain allof the features of pattern formation in Genicanthus.
As only a weak anisotropy i~ required for the effect,a small change in the skin structure may cause amarked change in stripe direction, which might have
implications for evolution and initiating species
divergence.
Publications
1. Kondo, S.: Usage of computer simulation in
studying complex biological pfenomena. Tanpakushitu

kakusan koso. 46, 2461-2467, 2001.
2. Kondo, S.: Morphogenesis and Gene-expression.

38

Division of Theoretical Genetics(1) Development of ontology and database for the cellsignaling system
Takako T AKAI l and Toshihisa TAKAGI (lHuman
Genome Center, Institute of Medical Science, Universityof Tokyo)
In the post-genome sequencing era, the mostsignificant issue is the reconstruction oflivingorganisms in computer, based on their genome
information. Reconstruction and analysis of molecularinteractions among gene products, pathways, andnetworks could be addressed as its first step. We
analyzed conceptual structure of the cell signaling
system and specified the concepts as
SIGNAL-ONTOLOGY.
The ontology consists of concepts of molecules,
molecular interactions, pathway motifs, and cellularfunctions. We also developed a database for the cellsignalling system, SPARK, based on the ontology. The
database system is constructed by XML-database,compound graph representation, and ontology. All thedata contained in the database are collected fromliteratures according to controlled vocabulary in the
ontology. SIGNAL-ONTOLOGY and SPARK will be
opened soon from http://ontology.ims.u-tokyo.ac.;p/.
(2) Knowledge representation of Signal transduction
pathways and logical inferences
Ken-ichiro FUKUDA l and Toshihisa TAKAGI(lComputational Biology Research Center, National
Institute of Advanced Industrial Science andTechnology)
Our group focuses on providing methods required
to develop Signal Transduction Pathway (STP)
databases. The problem is broken down into two
subproblems, i.e., knowledge representation design of
STPs and its utilization to infer relevant biologicalhypothesis. The knowledge representation model that
we developed is based on a Compound graph modeland can cope with knowledge fragmentation, cOI:'.plex
hierarchies and various levels of details on specificbodies of knowledge (heterogeneous knowledge
granularity). Equipped with the ontologies for STPs,
the model is able to formalize the knowledge describedwith natural language or drawings of diagrams. Then,the inference procedure that a biologist performs ismodeled as a hypothetical reasoning framework on acase-base, and a prototype knowledge base was
implemented, which infers cross-talk of pathways.
Publications

1. Fukuda, K. and Takagi, T.: Knowledge
representation of signal trunsdution pathways,
Bioinformatics, 17, 829-837, 2001.
2. Fukuda, K. and Takagi, T.: Signal transduction
pathways and logical inferences, The 2001 International
Conference on Mathematics and Engineering(METMBS2001), 297-303, 2001.
3. Gno, T., Hishigaki, H, Tanigami, A. and Takagi,
T.: Automated extraction of information onprotein-protein interactions from the biological
Literature, Bioinformatics, 17, 155-161,2001.
4. Hishigaki, H, Nakai, K., Gno, T., Tanigami, A. andTakagi, T.: Assessment of prediction accuracy ofprotein function from protein-protein interaction data,
Yeast, 18, 523-531, 2001.

Division of Theoretical Genetics

39

E. DEPARTMENT OFINTEGRATED GENETICS

E-a. Division of Human Genetics(1) Genome analysis of the mouse 7F4/ F5 imprinted
domainHiroyuki SASAKI, Hisao SHIROHZU, Takaaki YOKOMINE,
Chikako SUDA, Wahyu PURBOWASITOl, Tsunehiro
MUKAI 2, Atsushi TOYODA3, Masahira HATTORI3 andYoshiyuki SAKAKI3 (lKyushu Univ.; 2Saga Medical
College; 3Genome Science Center Riken)
Genomic imprinting, an epigenetic gene-marking
phenomenon, causes parental-origin-specificmonoallelic expression of a subset of mammaliangenes. Imprinted genes tend to form clusters in specificregions of the genome, which may be related to themechanism of imprinting or the reason for theevolution of imprinting. As a step to know thestructural and functional characteristics of theimprinted genome domains, we study the mouse
chromosome region 7F4/ F5, which contains at least
12 imprinted genes. This region is syntenic to human
llpI5.5, which contains genetic loci responsible for
Beckwith-Wiedemann syndrome and some types oftumors. We have determined the complete DNA
sequence of this mouse domain of 1.0 Mb and thecorresponding chicken region of 0.5 Mb. A moredetailed analysis of the sequence is now ongoing. Sincechicken does not appear to have imprinting,comparisons of the mouse and chicken sequences
should reveal regulatory elements specifically involved
in imprinting.
(2) Regulation of imprinting of the mouse Ig(2/ H19
sub-domain
Hiroyuki SASAKI, Ko ISHIHARA, Hiroyasu FURUUMI,
Yuzuru KATO and Wolf REIK l (lBabraham Inst., UK)

40

DEPARTMENT OF INTEGRATED GENETICS

The imprinted mouse 7F4/ F5 domain contains twolinked imprinted genes Igf2 and H19 near its
centromeric boundary: Igf2is paternally expressed andH19 maternally expressed. It is known that thepaternal-specific methylation of the differentiallymethylated region (DMR) located upstream of H19 is
the primary cause for the Igf2/ H19 imprinting. We
have investigated the temporal and spatial changes in
Igf2/ H19 imprinting during mouse development (ref.
2,4 and 9). We have also identified an evolutionarily
conserved CTCF-dependent insulator element at thecentromeric boundary of the Igf2/ H19 sub-domain(submitted). Lastly, we have characterized a mousemutant called minute, which has an inversion
breakpoint at about 20-kb downstream of H19(ref. 10).
(3) Role of de novo DNA methyltransferases Dnmt3aj
Dnmt3b in genomic imprinting
Hiroyuki SASAKI, Naomi TSUJIMOTO, Masahiro KANEDA
and Shoji TAJIMA l (lOsaka Univ.)
DNA methylation works 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 germ cells, we study 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 also characterized the enzymatic
properties of Dnmt3a and Dnmt3b in vitro (ref. 6).
(4) DNA methylation, imprinting and human disorders
Hiroyuki SASAKI, Hisao SHIROHZU, Shin-ich MIZUNO l,
Takeo KUBOTA2 and Shoji TAJIMA3 (lKyushu Univ.;
2Natl. Center of Neurology and Psychiatry;
30saka Univ.)
It is known that tumor suppressor genes are

methylated and inactivated in a number of cancers. We
have found that acute meyelogenous leukemia caseswith methylated p15 tumor suppressor gene havehigher levels of all three DNA methyltransferases
DNMTl, DNMT3A and DNMT3B (ref. 1). We have

also studied three Japanese cases with ICF(immunodeficiency, centromeric instability, facial
anomalies) syndrome, an autosomal recessive disorderwith aberrant DNA methylation, and identified threenovel mutations within the coding region of DNMT3B(submitted). Lastly, we have reported that paternal
disomy for chromosome 14 causes a distinctive
malformation syndrome with abdominal muscular
defects, skeletal anomalies and characteristic facies (ref.
8).

(5) Studies on the possibilities of genomic imprinting and
Z chromosome dosage compensation in chicken
Hiroyuki SASAKI, Takaaki YOKOMINE, Asato KUROIWA!,
Yoichi MATSUDA! and Masaoki TSUDUKI 2 PHokkaido
Univ.; 2Hiroshima Univ.)
Although it is generally thought that genomic
imprinting has evolved only in mammals, this has notbeen tested in other vertebrate species. We have asked
whether the homologues of the mammalian imprinted
genes show parental-origin-specific monoallelicexpression in chicken. Using DNA polymorphisms
identified between various breeds and strains, it was
shown that both IGF2 and MPRI are expressedbiallelically in chicken embryos (ref. 5). We also foundthat chicken Z chromosome is not subject toinactivation for gene dosage compensation in ZZ males(in preparation). We are currently studying thestructure and function of chicken DNA
methyltransferases, in order to know whether thedifferences in DNA methylation machinery can be thecause for the evolution of genomic imprinting anddosage compensation of sex chromosomes.
(6) Development of a universal DNA chip systemapplicable for any organism
Hiroyuki SASAKI, Hisao SHIROHZU, Shin-ichi MIZUNO!,
Tadafumi IINO\ Hidetoshi OZAWA!, Teruhisa OTSUKA!
and Kosuke TASHIRO! (!Kyushu Univ.)
We have started a collaborative research project
to develop a universal DNA chip system that can beused to study the expression of all genes from anyorganism.
(7) Role for Tsix in X chromosome inactivation

Takashi SADO, En LI! and Hirosyuki SASAKI fHarvard
Med. Sch.)The Xist gene is essential for the initiation of X
inactivation. It is known that antisense transcription
occurs at the Xist locus to produce Tsix RNA. We havegenerated Tsix-deficient mouse and shown that Tsix
plays an important role to regulate Xist expression.
Available evidence suggests that Tsix RNA itself iscritical rather than antisense nature of transcription.
Using ES cells deficient for either Tsixor Xist, we are
currently trying to elucidate how Tsix RNA exert its
effect on Xist expression.
(8) Role for Dnmt3a and Dnmt3b in X chromosome
inactivation
Takashi SADO, Masaki OKANO!, En LI 2 and Hiroyuki
SASAKI (!RIKEN, CDB, 2Harvard Med. Sch.)To study the role for de novo methyltransferases
in X inactivation, we examined expression of Xist in
male ES cells doubly deficient for Dnmt3a and Dnmt3b,
in which the promoter region of the Xist gene isextensively demethyated. RNA FISH revealed that Xist
becomes ectopically expressed and accumulates on the
X chromosome upon induction of differentiation. It
appeared, however, that the X chromosome coated withXist RNA did not become transcriptionally inactivated,suggesting that these de novo methyltransferases are
involved in the process of X inactivation following
accumulation of Xist on X chromosome.
(9) Human genome resources and their application to
the human and primate genome analysis
Asao FUJIYAMA, Ayuko MOTOYAMA!, Satoru YOSHIDA!,
Yoko KUROKI2, Yutaka NAKAHORI 2, Tatsuo
NAKAYAMA 3, Mieko KODAIRA4, Norio TAKAHASHI 4 andNaruya SAITOU5 (!Genome Science Center 'Riken,
2Tokushima Univ., 3Miyazaki Medical School,
4Radiation Effect Research Foundation, 5National
Institute of Genetics, Evolution Genetics Div.)The goal of human genome analysis is not only
sequencing entire genome nor cataloging protein
coding regions, but to understand functions retainedin the human genome and chromosomes. This simple
notion is often misunderstood by many people, even

Division of Human Genetics

41

including many scientists. The scientific importanceof the human genome project both for science andhumanity is well proven through many publications
in major journals. The Nature paper by theInternational Human Genome Consortium is the mostfrequently cited paper in life-science field. Since mostof human chromosomes can be purified by means ofdual-laser cell sorting system, such isolated
chromosomes are good resources for the studies tounderstand biological functions retained in individual
chromosome. Using purified chromosomes, we haveconstructed human mono-chromosomal cosmid
libraries (except for CM#9-12), CM#9-12, #21, CM-Y
fosmid libraries, and BAC libraries. In addition, we arein the process of constructing primate libraries
including chimpanzee and gorilla. Using theseresources, BAC-end sequencing of chimpanzee clones
proved that the difference between human andchimpanzee sequence is less than 1.5%.
(10) Whole genome analysis of signal-transduction
pathways in fission yeast
Yong-Sik BONG, Inaho DANIa!, Nobuya OGAWA andAsao FUJIYAMA (lCancer Center Research Institute)
In fission yeast, Schizosaccharomyces pombe,
deficiency of rasl gene causes abnormal cell shape andabolishes mating ability. However, the signaling
pathway in the cell and its target genes are largely
unknown because of the lack of appropriate analysis
system. To overcome this problem, we categorized
genes based on their expression levels in the presenceor absence of the rasl gene product under different
growth conditions. We utilized micro-arrays of clones
covering entire genome of the fission yeast. Y-S. B.
earned his Ph.D. degree through this work.
Publications
1. Mizuno, S., Chijiwa, T., Okamura, T., Akashi, K,
Fukumaki, Y., Niho, Y and Sasaki, H.: Expression of
DNA methyltransferases DNMT1, 3A and 3B in
normal hematopoiesis and in acute and chronic
myelogenous leukemia. Blood 97, 1172-1179, 2001.
2. Ohno, M., Aoki, N. and Sasaki, H: Allele-specificdetection of nascent transcripts by fluorescence in situ
hybridization reveals temporal and culture-inducedchanges in Igf2 imprinting during pre-implantation

42

Division of Human Genetics

mouse development. Genes Cells 6, 249-259, 2001.
3. Sado, T., Wong, Z., Sasaki, H. and Li, E.:
Regulation of imprinted X-chromosome inactivationin mice by Tsix. Development 128, 1275-1286, 2001.
4. Hamatani, T., Sasaki, H., Ishihara, K, Hida, N.,
Maruyama, T, Yoshimura, Y, Hata,]. and Umezawa,
A: Epigenetic mark sequence of the H19 gene inhuman sperm. Biochim. Biophys. Acta 1518, 137-144,
2001.
5. Yokomine, T, Kuroiwa, A, Tanaka, K, Tsudzuki,M., Matsuda, Y. and Sasaki, H.: Sequencepolymorphisms, allelic expression status andchromosomal locations of the chicken IGF2 and MPRl
genes. Cytogenet. Cell Genet. 93, 109-113, 2001.
6. Aoki, A, Suetake, 1., Miyagawa, J., Fujio, T.,
Chijiwa, T., Sasaki, H. and Tajima, S.: Enzymatic
properties of de novo-type mouse DNA (cytosine-5)
methyltransferases. Nucl. Acids Res. 29, 3506-3512, 2001.
7. 1. Takebayashi, S., Nakao, M., Fujita, N., Sado, T.,
Tanaka, M., Taguchi, H. and Okumura, K.:
5-Aza-2-deoxycytidine induces histone hyperacetylation of mouse centromeric heterochromatin by a mechanism independent of DNA demethylation. Biochem.
Biophys. Res. Commun. 288, 921-926, 2001.
8. 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. (in press).
9. Sotomaru, Y, Katsuzawa, Y, Hatada, L, Obata, Y.,
Sasaki, H and Kono, T: Upregulated expression of theimprinted genes H19and Igf2rin mouse uniparental
fetuses. J Bioi. Chem. (in press).
10. 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 (in press).
11. The International Human Genome Consortium
including A Fujiyama and 72 authors frominternational sequencing centers: The Human Genome:

Initial Sequencing and Analysis. Nature 409, 860-921,
2001.
12. The International Human Genome Consortium
including A Fujiyama and 55 authors: A Physical mapof the human genome. Nature 409, 934-941, 2001.
13. Bruls, T., Gyapay, G., Petit, 1.-1., Artiguenave, F.,
Vico, V., Qin , S., Tin-Wollam, A.M., Da Silva, C.,
Muselet, D., Mavel, D., Pelletier, K, Levy, M., Fujiyama,A., Matsuda, F., Wilson, R, Rowen, 1., Hood, 1.,
Weissenbach, J., Saurin, W. and Heilig, R.: A clone mapof human chromosome 14. Nature 409,947-948,2001.
14. Graf, D., Timmons, P.M., Hitchins, M., Episkopou,
V., Moore, G., Ito, T., Fujiyama, A, Fisher, AG. andMerkenschlager, M.: Evolutionary conservation,
developmental expression and genomic mapping of
mammalian Twisted gastrulation. Mammalian
Genomics 12, 554-560, 2001.
15. Fujiyama, A, Watanabe, H, Toyoda, A, Taylor,T.D., Itoh, T., Yaspo, M.-1., Lehrach, H., Chen, Z., Fu,
G., Park, H.-S., Saitou, N., Tsai, S.-F., Osoegawa, K.,de long, P,J., Suto, Y., Hattori, M. and Sakaki, Y.:
Construction and Analysis of a Human-Chimpanzee
Comparative Clone Map. Science 295, 131-134,2002.

E-b. Division of Agricultural Genetics(1) Developmental abnormalities induced by DNA
hypomethylation mutation of Arabidopsis
Tetsuji KAKUTANI, Tetsu KINOSHITA, Asuka MIURA,
Koichi WATANABE and Masaomi KATO
Genomic regions rich in repeated sequences tend
to be inactive in transcription and recombination andhave condensed chromatin (heterochromatin). In
addition, cytosine residues in such regions 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. We are 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 ddmJ (decrease in
DNA methylation) mutation results in decrease inmethylation and transcriptional de-repression in
genomic repeat sequences. The DDMJ gene encodesa protein similar to the chromatin-remodeling factor
SWI2/SNF2 (]eddeloh et al., Nat. Genet. 12, 1714-) The
most striking feature of ddmJ mutation is that it
induces a variety of developmental abnormalities bycausing heritable change in other loci (Kakutani et al.,PNAS 93, 12406-). The molecular basis has beenclarified in two of the loci directly causing thedevelopmental abnormalities.
One of them, 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. We
mapped the locus responsible for the clam phenotype
at high resolution. By genotyping 926 chromosomes,
we identified the gene responsible for the clamphenotype. It is DWF4 gene, which involved insynthesis of brassinolide, a plant growth regulator
necessary for cell elongation. The unstable clamphenotype 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 ddmJ mutant
background. These results suggest that gene silencing
associated with DNA methylation is important for
suppression of transposons (ref.1).
Another developmental abnormality, late flowering
trait, was induced by ectopic over expression of FWA
gene associated with hypomethylation of tandem
repeat upstream of the coding region. Interesting thingis that change in nucleotide sequence was also not
observed in fwa-J and fwa-2 alleles isolated byconventional mutagenesis. In both cases,
over-expression associated with the hypomethylation
resulted in the phenotypes (Soppe et al" Mol. Cell 6,791-). An interesting remaining problem is function of
FWA gene product during normal development.
(2) DNA hypomethylation mutation in rice

Division of Human Genetics

43Tetsuji KAKUTANI, Koichi WATANABE 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
transgenic rice lines expressing that EST in antisenseorientation. These transgenic lines show reduced
genomic DNA methylation in centromeric repeats,
repeat encoding rRNA and retroelement-like sequence
Tos3.
Publications
1. Miura, A., Yonebayashi, S., Watanabe, K., Toyama,T., Shimada, H. and Kakutani, T.: Mobilization oftransposons by a mutation abolishing full DNA
methylation in Arabidopsis. Nature 411, 212-214, 2001.
2. Takeda, S., Sugimoto, K., Kakutani, T., Hirochika,
H.: Linear DNA intermediates of the Ttol
retrotransposon in Gag particles accumulated instressed tobacco and Arabidopsis thaliana. PlantJ28,
307-17,2001.

caudolaterally into the telencephalon and form thelateral olfactory tract (LOT). Two types of guidancecues have been suggested for this projection. Firstly,a long-range factor Slit, which is secreted from theseptum repels mitral cell axons into a caudolateral
direction. Secondly, the pathway of mitral cell axons
contains a subset of neurons designated as lot cells,
which guide the axons through short-range
interactions. It is not clear how these two guidance cues
relate to each other and how they share thephysiological roles. We examined the behavior of mitralcell axons in organotypic culture upon ectopic
application of Slit, and inhibition of endogenous Slitsignaling. The results suggested that the short-range
guidance cue in the LOT pathway functions
independently from Slit. Furthermore, our results
showed that removal of the septum and inhibition of
Slit signaling did not affect the projection of mitral cellaxons. Although the septum and exogenous Slit canrepel olfactory bulb axons, our results cast doubts on
the physiological relevance of the septum andendogenous Slit in guiding the projection of mitral cellaxons.
(2) Mosaic development of the olfactory cortex with
Pax6-dependent and -independent components
Tatsumi HIRATA, Yoshiko TAKAGI, Yasufumi SATO

3. Kinoshita, T., Harada, JJ., Goldberg, RB., Fischer,
RL.: Polycomb repression of flowering during earlyplant development. Proc Nat! Acad Sci USA. 98,
14156-14161, 2001.
4. Habu, Y., Kakutani, T. and Paszkowski, ].:Epigenetic developmental mechanisms in plants:
molecules and targets of plant epigenetic regulation.
Current Opinion in Genetics & Development 11, 215-220,
2001.

E-c. Division of Brain Function(1) Short-Range and Long-Range Guidance of Olfactory
Bulb Axons

Tatsumi HIRATA
During development, mitral cells, the major output
neurons of the olfactory bulb, project their axons

44

Division of Agricultural Genetics

The olfactory cortex is the target area of olfactory
bulb axons and suggested to be derived fromneuroepithelial progenitors of various ventriculardomains during development. We examined thedevelopment of the olfactory cortex, using the newly
developed monoclonal antibody (mAb) 9-4c, which
recognizes reticulon I-A and -B. The mAb labeledneuroepithelial progenitors at the pallio-subpallial
boundary and their putative descendants in the deep
layers of the olfactory cortex. In the Pax6 mutant
embryo, labeling at the pallio-subpallial boundary was
specifically lacking, and the number of immunopositive
cells in the olfactory cortex was markedly reduced. In
contrast, the guidepost neurons of olfactory bulb
axons, lot cells, developed relatively normally in the
superficial layer of the olfactory cortex in the mutant
embryo. These guidepost neurons have been recently
shown to originate in the pallium and eventually guidethe initial projection of olfactory bulb axons. The
olfactory bulb projection in the Pax6 mutant embryo

also suggested the dualistic nature of the olfactorycortex development; the initial projection of olfactory
bulb axons developed relatively normally, whereas the
final projection of their collateral branches wasseverely defective.
Publications

1. Hirata, T., Fujisawa, H., Wu, ]. Y. and Rao, Y.:
Short-range guidance of olfactory bulb axons is
independed of repulsive factor slit. J Neurosci. 21,
2373-2379, 2001.
2. Kawasaki, T., Bekku, Y, Suto, F., Kitsukawa, T.,
Taniguchi, M., Nagatsu, 1., Nagatsu, T., Itho, K.,
Yagi, T. and Fujisawa, H.: Requirement ofneuropilin-l-mediated Sema3A signals in patterningof the sympathetic nervous system. Development (inpress), 2002.
3. 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. (in press), 2002.

indicating that Tos17has relatively low target-site
specificity at the nucleotide sequence level. Integration
targets were widely distributed over the chromosomes;
however, preferential integration into the sucrosesynthase 2 gene and into Tos17itself was demonstrated
by PCR screening using pooled DNA prepared fromthe mutant population. Hybridization studies indicatedthat Tos17 preferentially integrates into
low-copy-number regions of the genome. In agreementwith this result, about 30% of flanking sequences
examined showed significant homology to known
genes. Taken together, these results show that Tos17
can have a significant impact on the rice genome andcan be used as a tool for efficient insertionalmutagenesis.
(2) Screening of the rice viviparous mutants generated
by endogenous retrotransposon Tos17 insertion. Tagging
of a zeaxanthin epoxidase gene and a novel OsTATC
gene

Ganesh K. AgrawaP, Muneo YAMAZAKI!, Masatomo
KOBAYASHIz, Rei HIROCHIKA!, Akio MIYA0 1, Hirohiko
HIROCHIKA l (lDepartment of Molecular Genetics,
National Institute of Agrobiological Sciences, zRIKEN
Tsukuba Institute)

E-d. Division of Applied Genetics(1) The rice retrotransposon Tos17 prefers
low-copy-number sequences as integration targets
Muneo YAMAZAKI!, Hidehito TSUGAWAz, Akio MIYAO!,
Masahiro YANO l, Jiang Wu 3, Shinichi YAMAMOTO l,
Takashi MATSUMOTO!, Takuji SASAKI!, Hirohiko
HIROCHIKA l (lNational Institute of Agrobiological
Resources, zAomori Green BioCenter, 3Institute of theSociety for Techno-innovation of Agriculture, Forestry
and Fisheries)
The rice retrotransposon Tos17is highly activatedby tissue culture. To evaluate the impact oftransposition of Tos170n the rice genome and examine
its utility for insertional mutagenesis, more than 100
sequences flanking newly transposed Tos17 copies
were characterized. The 5-bp target-site duplications
flanking Tos17 did not show any consensus sequence,and preferred nucleotides, A/T and G/C, were only
found at the second and third nucleotides from bothends of the target site duplications, respectively,

The rice retrotransposon Tos17is one of a few
active retrotransposons in plants and its transpositionis activated by tissue culture. Here, we present thecharacterization of viviparous mutants of rice inducedby tissue culture to demonstrate the feasibility of theuse of retrotransposon Tos17 as an endogenous
insertional mutagen and cloning of the tagged genefor forward genetics in unraveling the gene function.
Two mutants were shown to be caused by the insertionof Tos17. Osabal, a strong viviparous mutant withwilty phenotype, displayed low abscisic acid level andalmost no further increase in its levels upon drought.
The mutant is shown to be impaired in the epoxidationof zeaxanthin. On the other hand, Ostatc, a mutant withweak phenotype, exhibited the pale green phenotype
and slight increase in abscisic acid levels upondrought. Deduced amino acids of the causative genesof Osabal and Ostatc manifested a significantly highhomology with zeaxanthin epoxidase isolated fromother plant species and with bacterial Sec-independenttranslocase TATC protein, respectively. This is the firstexample of transposon tagging in rice.

Division of Brain Function45

(3) Isolation and characterization of rice phytochrome
A mutants
Makoto TAKANO!, Hiromi KANEGAE!, Tomoko
SHINOMURA2, Akio MIYAO!, Hirohiko HIROCHIKA!,
Masaki FURUYA2rNational Institute of Agrobiological
Resources, 2Hitachi Advanced Research Laboratory)
To elucidate phytochrome A (phyAl function inrice, we screened a large population of retrotransposon(Tosl7) insertional mutants by polymerase chain
reaction and isolated three independent phyA mutant
lines. Sequencing of the Tos17insertion sites confirmedthat the Tos17s interrupted exons of PHYA genes inthese mutant lines. Moreover, the phyA polypeptides
were not immunochemically detectable in these PhyA
mutants. The seedlings of phyA mutants grown incontinuous far-red light showed essentially the same
phenotype as dark-grown seedlings, indicating theinsensitivity of phyA mutants to far-red light. The
etiolated seedlings of phyA mutants also wereinsensitive to a pulse of far-red light or very low fluence
red light. In contrast, phyA mutants weremorphologically indistinguishable from wild typeunder continuous red light. Therefore, rice phyA
controls photomorphogenesis in two distinct modes ofphotoperception--far-red light-dependent highirradiance response and very low fluence
response--and such function seems to be unique andrestricted to the deetiolation process. Interestingly,
continuous far-red light induced the expression of CAB
and RBCS genes in rice PhyA seedlings, suggesting
the existence of a photoreceptor(s) other than PhyA thatcan perceive continuous far-red light in the etiolated
seedlings.
(4) Linear DNA intermediates of the Ttol
retrotransposon in Gag particles accumulated in stressed
tobacco and Arabidopsis thaliana
Shin TAKEDA!, Kazuhiko SUGIMOTO!, Tetsuji
KAKUTANI2, Hirohiko HIROCHIKA1 (lNational Instituteof Agrobiological Sciences, 2National Institute of
Genetics)
The active transcription of some plant
retrotransposons under diverse stress conditions
suggests active transposition. However, transposition
has been demonstrated only during tissue/cell culture.

46

Division of Applied Genetics

To examine whether transposition is activated underconditions other than tissue/cell culture, DNAintermediates for retrotransposition of the tobacco
retrotransposon Ttol were analyzed. Using transgenic
Arabidopsis callus expressing high levels of Ttol RNA
in a ddml hypomethylation mutant background, theexistence of extrachromosomal Ttol linear DNA
molecules in a Gag-particle fraction was demonstrated.
By combination with ligation-mediated PCRamplification, we detected Ttollinear DNA moleculesin particle fractions from callus and methyl
jasmonate-treated leaves of tobacco, but not from
non-stressed leaves. Ttol DNA intermediates could not
be detected in the tobacco corolla where Ttol isexpressed. These results indicate that thetranscriptional activation of Ttol by defense-related
stresses leads to the synthesis of DNA intermediates,
whereas post-transcriptional suppression of Ttol
activity is suggested in the corolla.

Publications
1. Teraishi, M., Hirochika,H, Okumoto, Y., Horibata,A., Yamagata, H. and Tanisaka, T.: Identification of
YAC clones containing the mutable slender glume
locus slg in rice (Oryza sativa 1.). Genome44, 1-6,2001.
2. Agrawal, G.K., Yamazaki, M., Kobayashi, M.,
Hirochika, R., Miyao, A. and Hirochika. H.: Screeningof the rice viviparous mutants generated byendogenous retrotransposon Tos17 insertion: Tagging
of a zeaxanthin epoxidase gene and a novel OsTATC
gene. Plant Physiol. 125, 1248-1257, 2001.
3. Takano, M., Kanegae, H, Shinomura, T., Miyao,
A., Hirochika, H. and Furuya, M.: Isolation andcharacterization of rice phytochrome A mutants. Plant
Cell 13, 521-534,2001.
4. Yamazaki, M., Tsugawa, H., Miyao, A., Yano, M.,
Wu, J., Yamamoto, S., Matsumoto, T., Sasaki, T. andHirochika, H: The rice retrotransposon Tos17 prefers
low-copy number sequences as integration targets. Mol
Genet Genomics 265,336-344,2001.
5. Takeda, S., Sugimoto, K., Kakutani, T. andHirochika, H: Linear DNA intermediates of the Ttol
retrotransposons in Gag-particles accumulated instressed tobacco and Arabidopsis thaliana. Plant] 28,
307-317,2001.

6. Kumar, A. and Hirochika, H.: Application ofretrotransposons as genetic tools in plant biology.
Trends Plant Sci. 6, 127-134, 2001.
7. Hirochika, H.: Contribution of the Tos17
retrotransposon to rice functional genomics. Curr.
Opin. Plant BioI. 4, 118-122, 2001.
8. Okamoto, H. and Hirochika, H.: Silencing oftransposable elements in plants. Trends Plant Sci. 6,
527-534, 2001.
9. Hirochika, H., Miyao, A., Yamazaki, M., Takeda,
S., Abe, K, Hirochika, R., Agrawal, G.K, Watanabe,T., Sugimoto, K, Sasaki, T., Murata, K, Tanaka, K,
Onosato, K, Miyazaki, A., Yamashita, Y. and Kojima,
N.: Retrotransposons of rice as a tool for the functional
analysis of genes. In Rice Genetics IV (Khush GS, Brar
DS, Hardy B, editors), 279-292, 2001.

inactivating the only X chromosome in a cell. The
present and earlier findings suggest that the paternal
Xist allele is expressed by default at cleavage stages.
Xist expression is terminated in XpO embryos as aresult of counting the number of X chromosome in acell occurring at the morula/blastocyst stage. Most
probably, therefore, X-inactivation in fertilized XX
embryos takes place in the following manner: Aftercounting the number of X chromosome in a cell,high-level Xistexpression is continued in XX embryos.
The high level Xist expression becomes irreversible indifferentiating trophectoderm cells, whereas Xist
expression reverts to the initial low level in
undifferentiated ICM. Choice of future inactive X
becomes random since Xp and Xm somehow becomes
epigenetically equivalent during this period.
(2) Search for genes involved in X chromosome
inactivation using murine T (X;16) 16H translocation(1) Control of X-chromosome inactivationin early mouseembryogenesis

Nobuo TAKAGI (Graduate School of Environmental
Earth Science, Hokkaido University)

Nobuo TAKAGI (Graduate School of Environmental
Earth Science, Hokkaido University)

The morphologically normal X chromosome isinactivated in all cells of female mice heterozygous for
Searle's T (X;16) 16H translocation. Cytogenetic andbiochemical studies suggest that the initial choice ofthe future inactive X chromosome is non-random infavor of the normal X chromosome in heterozygous
embryos. Since this non-random inactivation could be
due to breakage or position effect of a gene (s) involved
in random choice due to Searle's translocation, we
decided to characterize the translocation breakpoint
for ultimate cloning of the candidate gene. The X
chromosomal breakpoint has been placed in the 4cM
region between G6pdx and Polal. Since the breakpoint
could not be found in a YAC clone containing Polai,
we switched our endeavor to screening of BAC library.
We successfully isolated several BAC clones
containing DNA markers known to be present between
G6pdx and Polal. By FISH using these clones asprobes, we found that the breakpoint is between twoloci M-02832 and M-00766. Analysis is now under way
to locate the breakpoint in YAC clones containing
M-02832 and M-00766, respectively. At the same time
we are trying to make a complete physical map aroundthe breakpoint including M-02832 and M-00766.

One of two X chromosomes is transcriptionallysilenced in early embryonic cells of female mammals
to compensate for the two-fold differences in X-linked
gene dosage difference occurring between XY males
and XX females. It has been shown that the paternallyinherited X chromosome (Xp) is selectively inactivatedin such extraembryonic tissues as trophectoderm andprimitive endoderm which differentiate earlier thanepiblast tissues giving rise to three definitive germlayers of the entire fetus. Only Xp is inactivated introphectoderm of normally fertilized female embryos,
since the paternal Xist allele is solely expressed due
to genomic imprinting. In spite of such imprinting, wefound random inactivation in XpXp androgenetic
embryos not only in all epiblast but extraembryonictissues. Thus, we went on to further study on thecontrol of the paternal Xist allele in mouse embryos
making use of XpO embryos produced by females
heterozygous for a large paracentric X chromosome
inversion, In (X) 1H. Xist RNA FISH study revealed thatthe paternally derived Xist allele is highly expressedin every cell of the embryo from the 4-cell stage onward,
but it is terminated by the blastocyst stage without

Division of Applied Genetics

47

Publications
1. Matsui, 1., Goto, Y. and Takagi, N.: Control of Xist
expression for imprinted and random X chromosome
inactivation. Hum. Mol. Genet. 10, 1393-1401,2001.
2. Takagi, N.: The role of X chromosome inactivationin the manifestation of Rett syndrome. Brain Dev. 23,
Suppl.1: 5182-5185, 2001.

48

Division of Applied Genetics

severe abnormality of sperm heads was observed in
the advanced backcross generations. The QTL
analyses for the reduced testis weight and theabnormal spermatozoal morphology detected distinct
QTLs for each phenotype, which is located to the distal
and the central region of X chromosome respectively.
This study provided genetic basis of the initiation
process of reproductive isolation between the two
subspecies, M m. domesticus and M m. molossinus.

F. DEPARTMENT OF GENETICSTRAINS RESEARCH CENTER

F-8. Division of Mammalian Genetics Laboratory(2) Genetic analysis of a mouse mutant, X-linked
polydactyly (Xpl)(I) Male-specific reproductive failure caused by
X-chromosomal substitution between two mouse
subspecies
Ayako OKA, Nobuo TAKAGI!, Kiyotaka TOSHIMORI2,
Toshiyuki TAKANO-SHIMIZU3, Akihiko MITA, Yoichi
MIZUSHINA, Noriko SAKURAI, Hiromi YAMAMOTO andToshihiko SHIROISHI PRes. Cent. For Mol. Genet. And
Grad. Schl. Of Env. Earth Sci. Hokkaido Univ.,2l)epartment of Anatomy and Cell Biology, Miyazaki
Medical College, 3Division of Population Genetics, NlG)
In the course of constructing a consomic mousestrain, B6.MSM-ChrX, by replacing X-chromosome ofa standard laboratory strain C57BL/6J (Mus musculus
domesticus) with that of a strain MSM (Mus musculus
molossinus), we observed declining fecundity only inthe males that carry the MSM-derived X chromosome(X-MSM/Y) as the backcross generations progressed.
It suggested that epistatic interaction of X-linked
gene(s) and autosomal and/or Y-linked genets) controls
fecundity of the male mice. The observed infertility is
attributable to disruption of the interaction between
MSM-derived X-linked genets) and autosomal and/or
Y-linked gene(s) of C57BL/6J.
In this study, we carried out in-depth phenotype
characterization of the X-MSM/Y males. we also
conducted mapping of the X-linked genes responsible
for the phenotypes by quantitative trait loci (QTL)
analysis. The results of the phenotype characterization
indicated that the X-MSM/Y males failed to fertilize,
or have some defects in very early development after
the fertilization. The X-MSM/Y males had the reduced
testis weights and various severities of degeneration
at the histological level in the testis. Furthermore,
spermatozoa of the X-MSM/Y males showedmalformation in their heads and low motility. Much

Yukari YADA!, Shigeru MAKINO, Sadao ISHIWA1 andToshihiko SHIROISHI. (lOchanomizu Univ.)
X-linked polydactyly (Xpl) is a spontaneous mouse
mutation that exhibits preaxial polydactyly only on thehindlimb. To study the molecular basis underlying Xpl
phenotype, we analyzed the expression patterns ofseveral marker genes in the limb buds of Xpl embryos
by in situ hybridization. At EIl.5, ectopic expressionof genes such as Shh, Fgf4, pte, Cre and Hoxdll wereobserved at the anterior margin of the hindlimb buds.
These markers were not detected before the initialexpression of Shh gene at the posterior margin of thelimb buds. However, at EIO.5, Cli, which is adownstream gene of Shh,was expressed at the anteriorside of the Xpl hindlimb prior to the ectopic Shh
expression. All results suggested that, in the normallimb development, Xpl gene acts in the downstream
pathway of Shh signaling cascade.
To isolate the actual Xpl gene, we carried out linkage
analyses of Xpl in crosses with wild mice-derived strains.
As a result, Xpl was mapped to a O.83cM interval
between the microsatellite markers, DXMgc39 andDXMit32. Since in the human syntenic region, Xp22,
a congenital face and limb deformity, Oral-facial-digital
syndrome type I (OFDl: OMIM311200), has been
mapped, Xpl was thought to be a mouse model for
OFDl. We mapped a murine homologue for OFDl,
cXorf5, using our cross panel, and we excluded it fromthe critical region of Xpl.
(3) Analysis of a spontaneous mouse mutation,mesenchymal dysplasia (mes)
Shigeru MAKINO and Toshihiko SHIROISHIDEPARTMENT OF GENETIC STRAINS RESEARCH CENTER49

A recessive mouse mutation, mesenchymal
dysplasia (mes), causes some morphological defects
including mild preaxial polydactyly. Genetic analysis
and molecular characterization in our previous study
indicated that mes is caused by a 32 bp-deletion in the
C-terminal cytoplasmic domain of the patched (pte)gene, which encodes a transmembrane receptor protein
for Shh.
Previously, we showed that compound heterozygotesof knockout allele of pte (pte-) and mes allele, whichsurvive up to birth and die neonatally, exhibit normal
neural tube development in contrast to pte homozygotes
which die around 10 dpc with severe neural tube
defects. This suggested that the C-terminal cytoplasmic
domain is dispensable for inhibition of Shh signalingin early embryogenesis.
In contrast, the same compound heterozygotes
show ectopic expression of Gli, Shh, Gre and Fgf4 at
the anterior mesenchyme and AER. This suggested
that the C-terminal cytoplasmic domain of Ptc has arole in the negative regulation of Shh signaling at theanterior mesenchyme of the limb buds.
(4) Genetic analysis of hot taste and pain sensitivity in
the Mishima battery of mouse strains
Tamio FURUSE 1, David A. BLIZARD 2, Kazuo
MORIW AKe, Kazumi YAGASAKI\ Toshihiko SHIROISHI
and Tsuyoshi KOIDE fTokyo University of Agriculture
and Technology, 2The Penn. State University, 3Bio
Resource Center, RIKEN)
Capsaicin is the major component of red pepper,
which causes hot taste. An experiment on knockout
mice for the receptor of capsaicin, VRl, showed thatthe sensation for both capsaicin and thermal pain
mediate the same receptor. In order to approach theunderlying genetic mechanism for diversity ofpreference for red pepper, we conducted a 12-hr I-bottle
fluid intake test of capsaicin solution using animals
from the following inbred strains: 10 wild-derived
inbred strains (PGN2, BFM/2, BLG2, NJL, CHD, HMI,
CAST/Ei, SWN, KJR and MSM), 1 strain derived fromthe so-called fancy mouse (JFl), and 3 domesticatedstrains (C57BL/6J, DBNlJ and BALB/cAnN). Relative
to baseline water intake, C57BL!6J and DBA/lJ
consumed 10 percent while KJR and MSM ingested
approximately 60 percent of the 15uM capsaicin
solution. The results of I-bottle test are similar to that

50

Division of Mammalian Genetics Laboratory

displayed by these strains in the hot plate test that we
had done previously. In the I-bottle test, Fl progeny
of KJR and C57BL/6 consumed capsaicin solution
approximately same asKJR. This result indicates thatthe genes involved in capsaicin tolerance in KJR are
dominant. In order to map the loci for capsaicin
preference and pain sensation, analyses of I-bottle test
and hot plate test on F2 progenies are currently underway.
(5) A quantitative genetic analysis of spontaneous
activity in the wild-derived mouse strains
Tamio FURUSEl, Toshiyuki T AKANO-SHIMIZU2, Kazuo
MORIW AKe, Toshihiko SHIROISHI and Tsuyoshi KOIDE(iTokyo University of Agriculture and Technology,
2Division of Population Genetics, NIG, 3RIKEN
BioResource Center)
Spontaneous locomotor activity is a behavior thatis greatly influenced by the forces of natural selectionin the process of evolution. Locomotor activity ofanimals in nature plays a crucial role in resource
acquisition, territory defense and migratory behavior.
When the resources are limited or the territory is large,
animals have to move long distances to acquire foodand to protect their territory. In the laboratoryenvironment, mice have not been selected for theirability of spontaneous locomotion. Thus, the genetic
variability for behavioral performance including
spontaneous locomotor activity is still maintained asa distinct genetic feature of each mouse strain.
We previously conducted multi-phenotype
behavioral characterizations using a series of inbred
strains derived from wild mice in addition to thecommon laboratory strains, the Mishima battery ofmouse strains. In the study of spontaneous locomotor
activity in the habituated normal home cages using
infra-red sensor, we found that mice of KJR strain was
hyperactive in contrast to mice of BLG2 strain, whichshowed a low level of spontaneous locomotion. To
unravel the genetic loci involved in this behavioral
phenotype, we conducted a QTL analysis on the
backcross population of a cross between KJR and
BLG2 strains. In the QTL analysis with 376 backcrossprogeny, the maximum LOD score, 4.8, was foundbetween the markers D3Nigl and D3Mit128 andsignificantly exceeds the threshold of LOD score 3.3
for BCI. We named this most telomeric region ofchromosome 3 as Locol. Further linkage analysis using
the selected progeny which carrying the allele fromKJR at the Locol locus suggested the presence ofanother locus, Loco2, on chromosome 17. An analysis
showed that Locol and Loco2interacted epistatically.

Publications
1. Ishii, T., Serizawa, S., Kohda, A, Nakatani, H.,
Shiroishi, T., Okumura, K., Iwakura, Y., Nagawa, F.,
Tsuboi, A and Sakano, H: Projection of three subsetsof olfactory neurons expressing the odorant receptor
transgene and the two cognate endogenous alleles.
Genes Cells, 6, 71-78, 2001.
2. Tanooka, H., Sasaki, H., Shiroishi, T. andMoriwaki.: p53 pseudogene dating: identification of theorigin laboratory mice. Gene, 270, 153-159, 2001.
3. Makino, S., Masuya, H, Ishijima, ]., Yada, Y and
Shiroishi, T.: A spontaneous mouse mutation,mesenchymal dysplasia (mes), is caused by a deletionof the most C-terminal cytoplasmic domain of patched(pte). Dev. BioI. 239, 95-106, 2001.
4. Kikkawa, Y, Miura, I., Takahama, S., Wakana, S.,
Yamazaki, Y., Moriwaki, K., Shiroishi, T. andYonekawa, H.: Microsatellite Database for MSM/Ms
and JFl/Ms, molossinusu-derived strains. Mammal.
Genome, 12, 750-752, 2001.
5. Haraguchi, R, Mo, R, Hui, c.-C., Motoyama, ].,Makino, S., Shiroishi, T., Gaffield, W. and Yamada, G.:
Unique functions of Sonic hedgehog signaling duringexternal genitalia development. Development 128,
4241-4250,2001.
6. Nadeau,]., Balling, R, Barsh, G., Beier, n, Brown,S., Bucan, M., Camper, S., Carlson, G., Copeland, N.,
Eppig, J., Fletcher, C., Frankel, W. N., Ganten, D.,
Goldowitz, D., Goodnow, C., Guenet, J-1., Hicks, G.,
Hrabe de Angelis, M., Jackson, I., Jacob, H, Jenkins,N., Johnson, D., Justice, M., Kay, S., Kingsley, D.,
Lehrach, H., Magnuson, T., Meisler, M., Poustka, A,
Rinchik, E., Rossant, ]., Russell, 1., Schimenti, J.,
Shiroishi, T., Skarnes, W., Soriano, P., Stanford, W.,
Takahashi,]., Wurst, W. and Zimmer, A: Functional
Annotation of Mouse Genome Sequences. Science, 291,
1251-1255,2001.

F-b. Division of MammalianDevelopment Laboratory(1) Molecular mechanism of somite segmentation
Yu TAKAHAsm1, Aki ISHIKAWA, Aya KITABAYASm l andYumiko SAGA (INational Institute of Health Sciences)
The somites are the first morphologically distinct
segmental units formed in vertebrate embryo and giverise to metameric structures such as vertebrae, ribs andskeletal muscles. The somites are formed bysegmentation from the anterior end of the presomitic
mesoderm (PSM), which is unsegmented and posterior
to the most recently formed somite. Each somite is
subdivided into anterior (rostral) and posterior (caudal)
compartments that differ in their properties and gene
expression. The rostro-caudal polarity of a somite isestablished within the PSM prior to segmentation.
However, molecular mechanisms underlying theformation of rostro-caudal polarity and the specificationof segmental boundaries are totally unknown. Our aim
in this study is to determine the mechanisms
underlying the generation of a segmental pattern
within the PSM. Previously, we cloned Mesp2, a genecoding a novel bHLH transcription factor, MesP2.

Mesp2 is expressed in the rostral PSM. Mesp2-null mice
exhibited defective somitogenesis due to the lack ofrostral somitic compartment. By the genetic analysis,
we have shown that MesP2 playa critical role toestablish rostro-caudal polariy of somite by regulating
Dlll expression via Notch signaling pathways.
Recently, we have established a hypomorphic MesP2
allele by an introduction of zebrafish Mesp2-homologue,mesp-b. Introduced mesp-b almost rescued the Mesp2
deficiency in the homozygous mesp-b knockin mouse,indicating that mesp-b is a functional homologue ofmouse Mesp2. Segmented somites were clearlyobserved although the partial fusion of vertebral
columns remained. Interestingly, however, the natureand dosage of the mesp-b gene affected the rescue
event. A mouse line, which has a hypomorphic Mesp2
allele generated by the introduction of neo-mesp-b, gave
rise to an epithelial somite without normal rostrocaudal(RC) polarity. RC polarity was also lacking in thepresomitic mesoderm. The defects in RC polarity were
determined based on disturbed expressions of Uncx4.1
and DIll in the segmented somites and presomitic
mesoderm, respectively. In contrast, the expression of

Division of Mammalian Genetics Laboratory

51

EphA4, lunatic fringe or protocadherin, implicated in
the segment border formation, was fairly normal in
hypomorphic mutant embryos. These results suggestthat the Mesp family of transcription factors is
involved in both segment border formation andestablishment of RC polarity through different genetic
cascades.
(2) Molecular clock and somitogenesis
Hiroki KOKUBO and Yumiko SAGA
Recently, several genes, including hairy1 in chickand lunatic fringe in chick and mouse, have been
identified and shown to be expressed in the presomitic
mesoderm in a cyclic fashion with temporal periodicity
corresponding to the formation time of one somite.
Therefore these are assumed to link to a molecular
clock which governs temporal control of somitogenesis.
We have cloned novel mouse hairy and E(spl) related
subfamily genes, called hesr -1, -2, and -3, using
homology search. Comparison of sequences shows that
presence of three functional domains, bHLH, orange
and C-terminal domain, that are conserved among
Drosophila, mouse and human, but obviously distinct
from the HES family members. hesr-1 is expressed in
the caudal part of each somites and presomitic
mesoderm. In Delta-l homozygous mutant embryo,hesr-1 expression is not observed in somites andpresomitic mesoderm. In lunatic fringe and delta-3(pudgy) homozygous mutant mice, hesr-1 is expressedin a broader domain in presomitic mesoderm. Theseobservations implicate that hesr-1 might play role(s) inregulating somitogenesis through the Notch signaling
pathway. As an approach to elucidate the function ofhesr-1 during somitogenesis, we generated a knockout
mouse. However, no difference between wild type andmutant is observed so far. We speculate that there
could be functional redundancy among the hesr
proteins. We have generated knockout mice for hesr-2
and -3. We are trying to generate the compound mice
to reveal the coordinated function of these three genes.
(3) The lineage analysis of Mespl-expressing cells
Satoshi KITAJIMA 1 and Yumiko SAGA (lNational
Institure of Health Sciences)
MesPl and MesP2 are transcription factors

52

Division of Mammalian Development Laboratory

containing an almost identical hE-ILH motif. Cells lackboth genes can not contribute to the heart formation.
A lineage study using ere-lox system revealed that
Mesp1 is the earliest molecular marker expressed in theheart precursor cells. We have conducted a detaillineage analysis using Cre-reportor line, Rosa-26. The
heart is exclusively composed of cells derived from
Mespl expressing mesoderm. However, we found somecells are deprived of the {3 -gal staining, which meansthat these are derived from Mesp1-nonexpressing cells.
Among them, neural crest cells have been shown tocontribute to the conotrucus septum prior to andduring overt septation of the outflow tract. In addition,
we found several cell clusters in between the primitive
ventricles and trabecular component of both right andleft ventricles, which distribution is reasonably similar
to the cells defined as the cardiac conduction system.
We are now trying to define the identity of the cells
using several marker specific to the cardiac conduction
system. Since the lineage of these cells has been
controversial, we might be able to demonstrate their
origin is different from other cardiac cells at the
beginning of the birth.
(4) Cloning and functional analysis of mouse Nanos
genes
Masayuki TSUDA, Yumiko SASAOKA, Makoto KISO,
Seiki HARAGUCHI 1 and Yumiko SAGA (lShiga universityof Medicine)
A Drosophila gene nanos encodes RNA binding
zinc finger protein, which are shown to be involved in
the germ cell development by suppressing somaticgene expression in the germ cells. To know the possible
involvement of Nanos-homologue protein in mousegerm cell development, we tried to clone mouse nanos
genes. The first nanos gene we cloned (mNos-1) wasmaternally expressed in an unfertilized egg and thezygotic expression was observed in the neuronal celllineage especially in adult hippocampus, but it was not
expressed in the developing germ cells. The knockout
mouse developed without any significant abnormalities
so far. The second and third genes (mNos-2 andmNos-3, respectively) were then cloned by the use ofhomology with human nanos genes predicted byhuman genome project. Interestingly, these two genes
are co-expressed in a developing male gonad but not
in a female gonad. To know the function of these genes

in germ cell development, we are generating thegene-knockout mice.
Publications
1. Haraguchi, S., Kitajima, S., Takagi, A., Takeda, H.,
Inoue, T. and Saga, Y.: Transcriptional regulation of
Mesp1 and Mesp2 genes: differential usage ofenhancers during development. Meek. Dev. 108, 59-69,
2001.

2. Saga, Y., Takeda, H.: The making of the somite:
Molecular events in vertebrate segmentation. Nature
reviews genet. 2, 835-845, 2001.

similar to the wild type cDNA but with reduced
degrees. These effects were independent of directionof the cDNA and presence or absence of the promoter.
These results indicate that an extra copy of the aSH1
exons somehow induced the ectopic expression ofendogenous aSH1 in the leaf and resulted in thealtered leaf morphology. We obtained similar resultsfor other genes such as aSH6, aSH15 and aSH71.
These results suggest that both of the promoter andexon regions are not sufficient for the restricted
expression of these genes in the shoot meristem in rice.

B. Genetic analysis of sporogenesis using sterile mutantsof rice

F-C. Division of Genetic Strains ResarchCenter, Plant Genetics Laboratory(1) Genetic dissection of embryogenesis and gametogenesisof rice (Oryza sativa)
A. Regulation of expression of KNOX family class 1
homeobox genes of rice

Yukihiro ITO, Yasuo NIWA\ Nori KURATA (lShizuoka
Pref. Univ.)
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 KNaXfamily 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 are
sufficient to maintain cells in a meristematic
undifferentiated state (Ref.2). Since specific expressionof KNaXfamily class 1 homeobox genes in a shootmeristem is essential for normal development of plants,
we started to study its regulatory mechanisms. An
aSH1 promoter was shown to be active in a leaf in
addition to the shoot meristem, indicating that thepromoter region is not sufficient to confer specificexpression in the shoot meristem and other regions arenecessary for its precise expression. Introduction of
an aSH1 cDNA into rice caused ectopic expression ofaSH1 in the leaf and resulted in altered leaf
morphology. Introduction of the cDNA with a frame
shift mutation also caused altered leaf morphology

Ken-Ichi NONOMURA, Kazumaru MIYOSHI, Mitsugu
EIGUCHI, Akio MIYAO*, Hirohiko HIROCHIKA* and Nori
KURATA (*Natl. Inst. Agrobiol. ScL)
Genetic analyses for microsporogenesis andmegasporogenesis is important to understand thesystem of sexual plant reproduction. We selected
thirty-eight of rice sterile mutants defective inreproductive organ development and meiotic
chromosome behavior. All of these mutant phenotypes
segregated in a manner of single recessive genes.
These mutations were induced somaclonally bysuspension culture of rice calli, where the endogenous
retrotransposon Tos17easily transposed into multiple
genome regions. A part of mutations were known tobe caused by the insertional mutagenesis by the Tosl7.
Therefore we examined the gene-tagging events forthese mutants. Close linkage of sterile phenotype toa transposed Tos17 insertion was detected in 22 of 38
mutant lines. We are further investigating genedisruption with Tos17to isolate genes responsible forthese mutations.
In this study, a gene disruption with Tos17
insertion was already confirmed in Msp110cus, which
affect sporogenesis in sexual reproductive organs,anther and ovule. MsP1 gene encodes receptor typeserine/ threonine protein kinase. RT-PCR analysis
revealed that the expression of this gene was foundin young flowers before meiosis. In mutant anther,pollen mother cells increased abnormally and werearrested at meiotic stages, and development of
hypodermal anther wall layer was defective, resulting
in complete male sterility. In mutant ovule, megaspore
mother cells also increased abnormally, but passed

Division of Mammalian Development Laboratory53

through meiosis successfully. Multiple megaspores
occurred growth and/or space competition among
themselves during megagametogenesis and often gave
rise to an embryo sac composed of multiple
gametocytes with irregular number and position ofcells or nuclei. Such a mutant megagamete could
participate in fertilization with wild type pollens andsometimes set aberrant seeds with twin embryos. We
concluded that MsPI gene function would basicallyrelated to signal transduction for restricting thedivision of archesporial cells in very early sporogenesis,but permitting extra division only after differentiation
into hypodermal anther wall formation (Ref.7).
In addition, to analyze phenotype of meiotic
mutants according to the precise sporoe development,
we preliminary elucidated the timing of premeiotic
DNA synthesis and the relationship of stage
progression of germ cell maturation to floret
elongation. These analyses revealed that premeiotic S
phase undergoes in floret of 2.0- to 2.5-mm length andthat sporogeneous cells quickly entered meiosis after
brief G2 interphase (Ref. 8).

c. Structural and functional analysis of rice HAP3
family genes
Nori KURATA, Akiko SERIZAWA, Kazumaru MIYOSHI,
Yukihiro ITO
One of the HAP3 family genes, LECI (LEAFY
COTY LEDON 1) in arabidopsis was reported to be oneof the central regulators of seed development. HAP3
is a submit of CAATT binding protein complex which
confer regulation on promoter activity of multiple
genes. To elucide a possibility that a certain HAP3
gene of rice shows a key function in the embryo
development, we screened HAP3 family genes of ricefrom a cDNA library constructed with 3DAP (gay f!fter
Dolllination) embryos and from available genomic
sequences (about 60% of the total genome) using
conserved domains. Finally we obtained ten HAP3subunit genes, designated OsHAP3-1 to OsHAP3-10.
RT-PCR experiment revealed that three clones,
OsHAP3-1, 3-2 and 3-3, expressed ubiquitoresly inalmost organs including callus, embryo, shoot, leaf andflower, Whereas OsHAP3-4, 3-5, 3-6 and 3-7 showedslight and specific expression in embryogenesis, and
OsHAP3-8, 3-9 and 3-10 showed no expression in any
organ examined so far.

54

Several genes showing specific expression inembryo development were selected to make promoter +GUS construct, 35S promoter + cDNA construct orRNAi construct. Transformation and regeneration ofrice plants using those constructs are now in progress.
D. Isolation and sequence analysis of receptor-likeprotein kinase genes of rice
Yukihiro ITO, NoriKuRATA
We started to isolate and characterize protein
kinase genes as candidates to transduce a positional
information which is postulated to be important forplant development. We focused on a receptor-likeprotein kinase gene such as SOMA TIC
EMBRYOGENESIS RECEPTOR KINASE (SERK! ofcarrot and isolated its homologues from rice. We havecloned two genes and their cDNAs named ORKI and
ORK6. The genome clone of ORKI showed unusual
structure and detail analysis is in progress. ORK6
encodes a receptor-like protein kinase with leucin-richrepeats in a receptor domain. The amino acid sequenceof 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. We also identified four related genes indatabases. Overexpression of ORK6 or introductionof antisense or RNAi constructs of ORK6 into rice
showed no phenotypic change under a normal growth
condition. Analysis of regeneration will be carried out
using calli derived from these transgenic plants.
(2) Positional cloning of a heterochronic gene, PLAl,
regulating the plastchron and the duration of vegetative
phase in rice
Byoung-Ohg AHN, Kazumaru MIYOSHI, Yukihiro ITO,
Jun-Ichi ITOH*, Yasuo NAGATO*, Nori KURATA(*Graduate School of Agricultural Life Science,
University of Tokyo)
Heterochronic mutations affecting the timing ofdevelopmental events may be of major significance inontogeny and evolution. In plants, several heterochronicmutations that affect stepwise development of vegetative
tissue and therefore alter shoot architecture have been
identified. The recessive mutations at the rice
PLASTCHRONI (PLAI) locus cause the short

Division of Genetic Strains Resarch Center, Plant Genetics Laboratoryplastochron and ectopic expression of vegetative
programs in the reproductive phase. To understandthe molecular aspects of PLAl function during plantdevelopment, we have started to isolate the PLAl geneby map-based cloning.
A genetic and physical map was constructed toisolate a causal gene for ptal phenotype. Small-scale
mapping was carried out to determine approximate
map position of a pial locus and then a high-resolution
genetic map was made for ptal-l, one of the pial alleles,
using an F 2 population comprising 578 plal-lhomozygous plants. A high-resolution genetic map
showed that the ptal-llocus was mapped between
RFLP markers C961 and R1738A on chromosome 10,
within a 3.6cM genetic distance.
A physical map encompassing the ptal-llocus wasconstructed by overlapping Bacterial Artificial
Chromosome (BAC) clones through chromosome
walking. PCR-based RFLP markers from BAC-end
clones were developed and mapped to place the pial
locus between the markers. Physical map construction
using BAC clones indicated that a BAC clone, B44A10(167Kb), contained the PLAllocus within 74Kbp that
corresponds to a 0.52cM genetic distance.
The B44A10 clone was almost sequenced andpublished by Cold Spring Harbor Laboratory, USA.
Gene prediction analysis of the 74kb region containing
PLAllocus with GENESCAN program (MIT, USA)predicted several candidate genes for PLAl.
Comparison of the nucleotide sequences of candidate
genes between wild type and pial mutant plants
revealed that putative cytochrome P450 gene carries
a mutation within the coding region in all three allelesof pial mutants. Expression analysis by RT-PCR
indicated that cytochrome P450 gene was expressed
only in organ bearing shoot apical meristem (SAM)
such as seedling and young panicle, but not in leaves
or root. All together, the PLAllocus was strongly
suggestted to encode cytochrome P450 protein.
Molecular complementation analysis using the
genomic fragment containing wild type cytochrome
P450 gene is in progress.

Tadzunu SUZUKI, Ken-Ichi NONOMURA, Nori KURATA

cells. Fusing this feature with molecular biotechnology
such as construction ·of stable plant artificial
chromosomes will introduce us to tremendous
progression of chromosome technology. Rice, a kindof key cereals, is important not only to breeding, butalso to basic researches on account of the smallest
genome size in monocots. In this study, the candidate
DNAs which may have a centromere function wereused to create rice artificial chromosomes (RACs). Thenmethods of transferring the RACs to rice cells havebeen examined.
The candidate DNA fragments of the artificial
chromosomes were constructed using both of the yeast
artificial chromosome (YAC) and the bacterial artificial
chromosome (BAC) harboring the centromere
sequences of rice. Approximate 300 kb insert DNA of
the YAC clone is composed of two kinds of centromericrepeats of RCS2 and RCE1, which are a tandem repeat
unit of 160 bp and a dispersed repeat of 1.9 kbp
locating on all centromeres, respectively. It wassuggested that the clone was derived from a functional
center of the rice centromere (Ref.1). For transferring
the YAC into rice cells, the vector sequences of the
YAC clone was retrofitted with rice selection marker
genes. For the creation of a BAC based construct, the
100 kb of RCS2 repeats was isolated and introduced
into a binary BAC vector.
Because the transfer methods of huge size DNAs
into plant cells have not been established, two methodsof lipofection and particle bombardment system were
examined here. Lipofection can reduce damages ofDNAs more than 100 kb length and is usually applied
to transfer huge size DNAs as well as small ones into
animal cell lines. However, this method had few beenused for plant cell transformation. To clarify theusefulness of lipofection method for plant celltransformation, a trial to introduce a 4 kb plasmid
having the GFP marker gene into protoplasts isolated
from rice cultured cell line Oc was carried out. The
efficiency of transformation achieved to 2 x 10-4 • DNAsof 20 kb or less in length also revealed to be lipofected
into rice protoplasts, though the efficiency is still low.
These results indicated that lipofection method can beapplied for plant cell transformation at least fortransient expression. Further stable transformation
using larger DNAs of BAC and YAC clones remains

One of advantages of plant cells for biotechnology
is easier induction of reproductive adults than animal

to be examined.
Particle bombardment method, which has alreadybeen used in plant transformation with small plasmids,(3) Construction of rice artificial chromosomes

Division of Genetic Strains Resarch Center. Plant Genetics Laboratory55

is unsuitable for introducing large DNAs into plant
cells. However, this method is being examined fortransformation with BAC clones, since DNAs less than
100 kb had been already shown to be transferred into
rice cells. In addition, modifications of the method arein progress for reducing the damages of DNAs. In theconventional way, DNAs are spread on gold particles
and delivered into cells with the particles. In our
modified procedure, DNAs are spread on the cellsurfaces and introduced into the cells when theparticles go into the cells. The preliminary results
indicated that usual plasmids could be sufficiently
introduced. The adaptation and optimization will makea new procedure more effective than the conventional
one.
Generation of several kinds of transgenic rices
with artificial chromosomes of different centromere
repeats combination will make us possible to elucidate
functional role of each centromere components.
(4) Analysis of rice genome configuration in the nucleus;
isolation and characterization of rice nuclear protein
genes
Kazuki MORIGUCHI, Yukihiro ITO, Yukiko YAMAZAKI,
Nori KURATA
The primary aim of this study is to isolate plant
nuclear proteins exhaustively to exhibit the wholecontents of nuclear proteins using rice, which is oneof the model plants whose genome project has
progressed. The secondary aim, is to make clear what
events and factors are necessary to maintain genome
organization and function in nucleus. For this purpose,
we isolated and classified nuclear protein genes andexamined their characteristics and localization in the
nucleus to get a link to their functions.
As the first step, we applied the yeast nuclear
transportation trap (NTT) system, which was shown
to trap NLS coding cDNAs. Three cDNA libraries
derived from two panicle stages (FP: flowers in meiotic
stages, YP: flowers before and after pollination) and4 days callus after regeneration treatment have beenused. We have isolated cDNA clones derived fromabout 430 independent genes and found that the NTT
system was much less efficient for trapping nuclear
proteins of plant than animal. By a homology searchanalysis against protein databases, 45% of predicted
proteins indicated unknown function or no homology

56

and 31 % of those indicated known nuclear proteins orsimilar ones. We next examined several clones fromeach group for their intracellular localization in plantby expressing GFP fusion proteins of them in Allium
epidermal cells. Over 70% of examined proteins, which
are classified into unknown function protein, localized
in nucleus or both in nucleus and cytoplasm. Several
characteristic distribution of expressed proteins wereobserved as revealed by GFP localization vs. chromatin(DAPI) distribution. Some seemed to exist on thechromatin and others in the nuclear matrix.
Three plans are starting for further examination.
The first one is additional isolation of nuclear proteins
by introducing more efficient gene trap system forplant. The second one is to analyze alterration of
nuclear protein content in some developmental stages.
The last one is construction of makers for observing
detailed intranuclear structure and localization. Twocolored localization analysis using two different
fusion-proteins, one with GFP and the other with
DsRed2 has been employing.
(5) Genome-wide analysis of reproductive barriers andpositional cloning of a reproductive barrier
A. Quantitative Analysis of genotype segregation forreproductive barriers
Yoshiaki HARUSHIMA, 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 developed a new method for detecting
the map location and gene action of loci that contributeto the distortions of allele frequencies from Mendelian
expectation by regression analysis of allele frequencies
of markers covering an entire genome (ref. 3).
Mathematica packages for the analysis were publishedat http://shigen.lab.nig.ac.jp/rice/seganalysis/. Asianrice cultivars, Oryza sativa, can be classified into two
main types, Japonica and Indica, based on several
characteristics. To clarify the state of Japonica-Indica
differentiation, all reproductive barriers causing allele

Division of Genetic Strains Resarch Center, Plant Genetics Laboratoryfrequency distortions from Mendelian expectation in
F 2 populations were mapped and compared among
three different Japonica-Indica crosses. The numberof reproductive barriers in the three crosses was
similar, however most of the barriers were mapped at
different loci. Therefore, these reproductive barriers
formed after Japonica-Indica differentiation. In thethree Japonica-Indica cross combinations, the genetic
variations within both Japonica and Indica are small
as shown as high similarity of the restriction fragment
length of RFLP markers. Considering the high genetic
similarity within Japonica cultivars and Indica
cultivars, the differences in the reproductive barriers
on each cross were unexpectedly numerous. The
reproductive barriers of Japonica-Indica hybrids likely
evolved more rapidly than other genetic elements(ref. 5).
B. Positional Cloning of a Segregation Distortion Gene
Detected in a Progeny of a Cross between japonica andindica rice
Yoshiaki HARUSHIMA, 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 1300 F 2 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 is also being constructedusing Nipponbare BACs that were selected by acosegregated RFLP marker, C582, with the aimed gene.
BAC contig were confirmed by PCR analyses of end
sequences of BACs. Same of amplified BAC end
fragments were mapped as RFLP markers on thegenetic map. Both flanking markers of the malegametophyte gene were mapped at 9 and 2 recombinantsin the population and these markers were on the two
BAC contig.
(6) Generation and screening of enhancer trap lines ofrice
Yukihiro ITO, Mitsugu EIGUCHI, Nori KURATA
To isolate valuable mutants defective in various
steps of rice development and to clone the corresponding
genes, we are generating enhancer trap lines of rice.
We employ an enhancer trap system used in Arabidopsis
with some modifications. This system is based on the
Agrobacterium- mediated transformation using AdDs
transposable elements of maize. The enhancer trap
construct contains, in the T-DNA region, the Ds
element named Ds-GUSthat harbours a GUS coding
region with a CaMV 35S minimal promoter and ahygromycin resistance gene. Ds-GUSwas flanked by
the 35S promoter and a coding region of a selectablechlorsulfuron resistance gene, so that excision of
Ds-GUS causes connection of the 35S promoter and thecoding region and can confer chlorsulfuron resistance.
We also use a 35S-Ac transposes (AcTPase) gene
together with a bialaphos resistance gene to supply
transposase which is essential and sufficient fortransposition of Ds-GUS.
We generated forty-two transgenic rice lines witha single copy of Ds-GUS and six lines with
35S-AcTPase. We crossed four lines of Ds-GUSwith
six lines of 35S-AcTPase and obtained 675 germinaltransposants by PCR screening and 1,564 transposantsby the chlorsulfuron and hygromycin resistance at F2
generation. To obtain more transposants we crossed
other 13 original Ds-GUS lines with the 35S-AcTPaseline and obtained 926 transposants out of 3,277 F2
plants screened. We maintain F 1 plants by growth of
ratoons and continuously obtain F 2 seeds.
We also started enhancer screening by X-glucstaining at various organs and stages of ricedevelopment including germinating seeds, 2-week
seedlings, young panicles and all organs at a flowering
stage. Out of 675 transposants screened we identified
more than 10 lines that showed specific GUS staining
in one of following organs such as a root tip, a root
hair, a wounded leaf, an inflorescence, an anther, astyle or a young tiller.

Division of Genetic Strains Resarch Center. Plant Genetics Laboratory57

We will also conduct mapping of original Ds-GUS
insertion sites on rice chromosomes to selectchromosome specific Ds-G Us.
Publications
1. Nonomura, K-1. and Kurata, N.: The centromere
composition of multiple repetitive sequences on rice
chromosome 5. Chromosoma, 110, 284-291, 2001.

2. Ito, Y., Eiguchi, M. and Kurata, N.: KNOX
homeobox genes are sufficient in maintaining cultured
cells in an undifferentiated state in rice. Genesis, 30,
231-238, 2001.
3. Harushima, Y., Nakagahra, M., Yano, M., Sasaki,
T. and Kurata, N.: A genome-wide survey of reproductive
barriers in an intraspecific hybrid. Genetics, 159,
883-892, 2001.
4. Moriguchi, K., Maeda,Y., Satou, M., Niken, S. N.
H., Kataoka, M., Tanaka, N. and Yoshida, K.: The
complete nucleotide sequence of a plant root-inducing(Ri) plasmid indicates its chimeric structure andevolutionary relationship between tumor-inducing (Ti)and symbiotic (Sym) plasmids in Rhizobiaceae. J Mol.
Bioi. 307, 771-784, 2001.
5. 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.
6. Ahn, B. 0., Miyoshi, K., Itoh, ]. 1., Nagato, Y. and
Kurata, N.: A genetic and physical mapping of theregion containing PLASTOCHRONl,a heterochronic
gene, in rice (Oryza sativa, L). Theor. Appl. Genet. (inpress).
7. Nonomura, K-1., Miyoshi, K., Eiguchi, M., Miyao,
A., Hirochika, H. and Kurata, N.: acdl mutant aberrant

for archesporial cell division in the anther and ovuleof rice. Rice Genet. Newsl. 18 (in press).
8. Miyoshi, K., Kurata, N. and Nonomura, K.1.:
Detection ofthe pre-meiotic DNA synthesis in pollen
mother cells by immunofluorescence technique in rice.
Rice Genet. Newsl 18 (in press).
9. Kurata, N., Nonomura, K-I. and Harushima, Y.:

58

Rice genome organization focusing on centromere andgenome interaction studies. Annals of Botany (in press).
10. Kurata, N. and Fukui, K.: Chromosome researchin genus Oryza. in Monograph in genus Oryza. Nanda
JS. Ed, Springer (in press).
11. Ito, Y., Hirochika, H. and Kurata, N.: Organspecific alternative transcripts of KNOX family class
2 homeobox genes of rice. Gene, 288, 41-47, 2002.

F-d. Division of Microbial Genetics(1) HscA is involved in the dynamics of FtsZ-ringformation in Escherichia coli K·12

Tsuyoshi UEHARA, Hiroshi MATSUZAWA l and Akiko
NISHIMURA (1Department of Bioscience andBiotechnology, Aomori University)
FtsZ, a homologue of eucaryotic tubulin, localizes
throughout the cytoplasm in non-dividing E. coli.
However, it assembles in cytokinetic rings, however,at the early stages of septation. Factors controlling the
dynamics of FtsZ ring formation are unknown, and themolecular mechanism governing these dynamics is yet
to be determined. At 42°C, JEI0715 mutant bacteria
formed multinucleated filaments with a highly reduced
number of FtsZ-rings at potential division sites. The
JElO715 phenotype resulted from a mis-sense mutationin the hscA gene which encodes a heat shock Hsp70
family protein, with a single alanine-to-valine substitution
at position 192 within the ATPase domain. BothJElO715 and the hscA knockout strain of JEI0715 werecompletely complemented by a plasmid-born, wild type
hscA gene, but not by a mutant-type hscA 715 gene. An
hscA conditional knockout of the wild-type strain
under non-permissive conditions exhibited longer rod
cells with an abnormal localization of FtsZ. The
over-expression of dnaK partially complemented the
JEI0715 mutation. In vitro, the ATPase activity of themutant protein, HscA715, was reduced to 63% of thewild-type HscA activity. HscA cosedimented with
FtsZ-polymers in the presence of GTP. HscA is
involved in FtsZ-ring formation, through a chaperon-likeinteraction with FtsZ. Defects in hscA, however, canpartially be compensated for by redundant genes,
including the wild-type dnaK. (For details, see Ref. 1)

Division of Genetic Strains Resarch Center, Plant Genetics Laboratory

(2)kdsA mutations affect FtsZ-ring formation in

Escherichia coli K-12

F-e. Laboratory Invertebrate Genetics

Hiroshi FUJISHIMA, Masaaki WACHI and Akiko
NISHIMURA(1) UDP-sugar transporter implicated in glycosylationand processing of Notch

No one has, as yet, addressed the relationshipbetween the nature of the outer membrane and cell
division. kdsA encodes 3-deoxY-D-manno-octulosonicacid (KDO) 8-phosphate,synthetase which catalyses thefirst step in the synthesis of KDO, the linker between
lipidA and oligosaccharide of lipopolysaccharide (LPS).
Seven temperature-sensitive mutants containing
mis-sense mutations in kdsA were affected in the
production of KDO and all mutants stopped dividing
at 4tC and formed filaments with either one or no FtsZ
ring. All observed defects were reversed by theplasmid-born wild-type kdsA gene. Western blotting
analysis, however, demonstrated that the amount of
FtsZ protein was not affected by the mutation. The
mutants were more susceptible to various hydrophorbic
materials, such as novobiocine, eosine Y and SDS at
36°C. Methylene blue, however, restored kdsA mutant
growth. Plasmid-born wild-type msbA, encoding a lipid
A transporter in the ABC transporter family, partiallysuppressed kdsA mutantation. A mutation of IpxA,
functioning at the first stage in lipid A biosynthesis,
inhibited both cell division and growth, producing
short filaments. These results indicate that the
instability of the outer membrane, caused by the defectin KDO biosynthesis, affects the FtsZ-ring formation.
(For details, see Ref. 2)

Satoshi GoTO, Misako TANIGucm, Masatoshi MURAOKA l,
Hidenao TOYODA2, Yukiko SADO, Masao KAWAKITA l andShigeo HAyASH!
(iDepartment of Physiological Chemistry, The Tokyo
Metropolitan Institute of Medical Science (Rinshoken),
Bunkyo-ku, Tokyo 113-8613, Japan 2Division of
Analytical Sciences, Faculty of Pharmaceutical Sciences,
Chiba University, Inage, Chiba 263-8522, Japan)

Publications
1. Uehara, T., Matsuzawa, H. and Nishimura, A.:
HscA is involved in the dynamics of FtsZ-ringformation in Escherichia coliK-12. Genes eel/s6, 803-814,
2001.

Glycosylation modifies protein activities in various
biological processes. We report here functions of anovel UDP-sugar transporter (UST74C) localized to theGolgi apparatus in cellular signaling of Drosophila.
Genetic analyses demonstrated that UST74C isrequired for both Wingless and Notch signaling inembryos, and the requirement becomes restricted toNotch signaling in larvae. Larval phenotypes indicatethat both Fringe-dependent and independent pathwaysof Notch are affected. Biochemical analyses demonstrated
both glycosylation and proteolytic maturation of Notch
are defective in mutant larvae. The results suggest thatchanges in nucleotide-sugar level can differentiallyaffect Wg, and two distinct aspects of Notch signaling.
Publication
1. Goto, S., Taniguchi, M., Muraoka, M., Toyoda, H.,
Sado, Y., Kawakita, M. and Hayashi, S.:UDP-sugartransporter implicated in glycosylation and processingof Notch. Nature Cell Biology, 3, 816-822, 2001.

2. Fujishima, H., Nishimura, A, Wachi, M., Takagi,
H., Hirasawa, T., Teraoka, H., Nishimori, K, Kawabata,T., Nishikawa, K and Nagai, K: kdsA mutations affect
FtsZ-ring formation in Escherichia coli. K-12.
Microbiology. 148, 103-112,2002.
3. Makinoshima, H., Nishimura, A and Ishihama, A:
Fractionation of E. coli cell populations of different
stages during growth transition to stationary phase.
Mol. Micro bioi. 43, in press, 2002.

Division of Microbial Genetics

59Oryzabase is available from http://www.
shigen.nig .ac.jp/rice/ oryzabase/.

G. CENTER FOR GENETICSRESOURCE INFORMATION(2) PEC: Profiling of Escherichia coli Chromosome
Yukiko YAMAZAKI, Toru IKEGAMI 1 , Takehiro
YAMAKAWA, Kazu MITSUI, Takeshi KAWABATA, Ken
NISHIKAWA, Tadahiro MORI 2, Akiko NISHIMURA and
Junichi KATO fUniversity of Tokyo, 2Nara Institute of
Science and Technology)

G-a. Genetic Informatics Laboratory(1) ORYZABASE··INTEGRATED RICE SCIENCEDATABASE ••Takahiro YAMAKAWA, Kazu MITSUI, Nori KURATA,
Atsushi YOSHIMURA, Yasuo NAGATO and Yukiko
YAMAZAKI The Oryzabase is a a!, Yasuo NAGAT0 2andYukiko YAMAZAKI fKyushu University, 2University ofTokyo)The Oryzabase is a comprehensive rice science
database established in the collaboration with rice
researcher's committee in Japan. The database isoriginally aimedddimededzabase consists of five parts,
which are (1) genetic resource stock information, (2)gene dictionary, (3) chromosome maps, (4) mutant
images, and (5) fundamental knowledge of rice science.
The database includes more than 10000 collectionsof germplasms collected or developed by classicalbreeding and/or new molecular biological method asa result of long history of rice breeding in Japan. The
Oryzabase map represents the integration of sevendifferent maps from classical linkage map to the latest
YAC physical map provided by Rice Genome Project.
We've completed the internal cross-linking of related
information such as common markers on different
maps, genes and the respective mutant images, strains
and their marker genes, and so on. Using Web-based
good interface, the rice gene dictionary has been
constantly updated by the specialists coupled with theactivity of Rice Gen of Rice Gene Cooperative.
Oryzabase uses an object-oriented databasemanagement software with Java2D for application.
We are planning to do more extensive
cross-referencing of Oryzabase to the major DNA
sequence database, literature database and other ricedatabase such as Ricegenes in order to provide thewealth of information to rice researchers.

60

CENTER FOR GENETICS RESOURCE INFORMATION

Profiling of Escherichia coli chromosome (PEC) isa database a database atabase s, (v) structural
information proposed by bioinformatics researchersand (vi) a tool for graphical display of different geneclasses in different colors. The database will be a very
useful tool providing a cyber space to meetbioinformaticians and experimethat compiles allnecessary information for functional analysis of E. coli
genes. The Japanese E. coli genetic resource committee
has extensively contributed to the development of thedatabase, which provides a comprehensive interface
for experimental researchers. Available from thedatabase are (i) deletion mutant information obtained
from experimental research, (ii) annotated gene
information including gene classification, (iii) genetic
stock information, (iv) similarity search results for eachgenes / orf PEC is a vaila ble at http: / / www.
shigen.nig .ac.jp / ecoli / pec /.
Since the whole genome sequence of E.coli waspublished in 1997, enormous efforts have been madeto analyze the functions of the genes/orfs. Further
innover innov. Further innovFurther innovbiologists,
however, are not always satisfied with such databases.
On the other hand, computational scientists face
problems on how to annotate their analytical results
generated and/or calculated from sequence data.
To resolve the dilemma, we'v developative researchis still needed to complete those tasks. Many different
E.coli databases are now available through theInternet, but most databases have been constructedby computer scientists with the contents derived fromsequence analysis. Experimental e developed the PEC
aiming to provide experimental scientists a chance tomeet and communicate with computer scientists andvice versa through the Internet. The database includes
original deletion mutant data and genetic stock
available through ts incorporated into PEC. The
database also provides analytical data such asnucleotide composition proposed by computerscientists with a comprehensive interface. To create
an overview, we are planning to develop off-line toolby which the user can createhe Internet. Most genes/
ods in the database are classified into three groups,
essential, non-essential, and unknown according toeither experimental results or data published in journal
articles. The basic information of genes/orfs are
acquired from resources available to the public andannotated by researchers before their incorporation
into the database. BLAST/PSI-BLAST homology
search and motif search using PROSITE and pfam
database have also been performed for all genes/ods
periodically and the result an original image file byapplying his/her own data set.
Publications
1. Yamazaki, Y., Yoshimura, A., Nagato, Y. and

Kurata, N.: Oryzabase-Integrated map and mutant
database-,Plant and Animal Genome Diego, CA,
January
2. Yamazaki, Y., Yoshimura, A., Nagato Y. and
Kurata, N.: ORYZABASE--INTEGRATED RICESCIENCE DATABASE ,Fourth International Rice
Genetics Symposium, Laguna, Philippines, October
Genome Biology Lab.

G-b. Genome Biology Laboratory(1) NEXTDB: The nematode expression patterndatabase
Tadasu SHIN-I, Yuji KOHARA
We updatea "NEXTDB" that integrates all theinformation of ESTs, gene expression patterns andgene functions of C.elegans which are being produced
and analyzed in this laboratory, and, made it open.
Images of whole mount in situ hybridization for mRNA
were taken by CCD cameras equipped on Zeiss
Axioplan microscopes, loaded to the Sun workstation
to process and arrange them properly in the database.
Then, they were annotated with respect todevelopmental stages and expression patterns on the
database. Images of immunostaining taken on Zeiss
LSM510 confocal microscope, and images anddescriptions of RNAi phenotypes, were also stored in

the database. New version of NEXTDB contains about
120,000 cDNAs that have been classified to 12,000
unique cDNA groups, in situ images of about 9,000
cDNA groups, RNAi phenotype images of 160 groups,
and immunostaining images of 120 groups. The newversion is available over the Internet. URL:<http://
nematode.lab.nig.ac.jp/>(2) Clustering analysis of gene expression patterns inC.elegans
Masahiro ITO, Yohei MINAKUCHI, Michiko SERIZAWA
and Yuji KOHARA
In this lab, whole mount in situ hybridization has
been performed using the cDNAs that have beenclassified in our EST project. Thus far, in situ analysis
for some 7600 genes was finished. Minimal annotation
has been given to the in situ images; 10 stages forembryogenesis, 4 stages for larval-adult stage, on
average 10 patterns (cell(s), tissue, region) per stage,
3 levels of relative intensity of signals per each pattern.
Using the information ((1O+4)xlOx3=420 dimensions),
we have done clustering analysis of the in situ patterns.
The gene expression patterns were roughly clusteredby the factor analysis using the Euclid distance. This
treatment classified the embryonic patterns of 5064
genes into 39 clusters that contained 5 to 3355 genes
per cluster (12 clusters had more than 50 gene
members). Then, we focused on several celli 1ineage/
stage-specific clusters; gut specific cluster (910 genes),
hypodermis specific cluster (421 genes), and body wall
specific cluster (72 genes). The three clusters were
further classified by the Ward method. This treatment
produced 7 sub-clusters along the developmental timecourse from the gut cluster and 6 sub clusters from thehypodermis and body wall muscle clusters. This meansthat we know the time of transcription initiation of themember genes in the individual cell lineages. The same
sub-cluster might be regulated by the samemechanisms. Thus, we are now trying to extractcommon sequence motifs, possible candidates fortranscriptional regulatrory signals, in the 5'- upstream
region of the genes in the same sub-cluster.
(3) Semi-automatic system for creation of cell shapemodel in C.elegans embryogenesis
Hideaki HIRAKI and Yuji KOHARA

Genetic Informatics Laboratory61

C. elegans is a model organism that allows us toobserve details of the developmental process as it istransparent and has a limited number of cells. Because
cell to cell interactions have critical roles in early
embryogenesis, it is very important to grasp the cell
shapes, the arrangement of them and the contacts
among them. A polygon model of the early
embryogenesis was built in our laboratory from a timeseries of 3-dimensional images of differential
interference contrast (DIC) microscopy. The model has
been used as the basis to model the early
embryogenesis regarding the regulation of gene
expressions or the physical process of cleavages, andas the template superimposed with the images ofantibody staining to identify the localization of various
proteins. Building such model took long time and much
effort because it is difficult to extract automatically thecell boundaries in DIC images and the outlines of thecells had to be traced by hand.
Accordingly, we have been developing a system
to build the model of the cell shapes from a time seriesof confocal microscopic images of the plasma
membranes stained with a fluorescent dye
automatically by computer. In detail, we developed
computer programs to extract the cell shapes with theseeded region growing method on 3-dimensional image
and to extract candidates of the seeds with the distancetransformation filter. We applied them to a dataset of
24-200 cell stages successfully. The system has not been
fully automatic yet as the seeds of the regions have to
be selected from the candidates by the operator to
obtain a good result. We are improving the system to
be more automated and are planning to make4-dimensional databases consisting of the cell lineages
constructed from time series datasets. And then, we
will apply the system to compare the cellulararrangements and the cell-to-cell contacts among therelative species of C. elegans and the mutants such as
created by RNAi.

(4) Translational control of maternal yip-l mRNA by
POS-! and its interacting protein PIP-!
Ken-ichi OGURA, Norihito KISIDMOTO, Shohei MITANI\
Keiko GENGYo-ANDO\ Yuji KOHARA fTokyo Women's
Medical University)
The translation of maternal glP-l mRNA is
temporally and spatially controlled in C. elegans

62

Genome Biology Laboratory

embryos. Its 3' UTR (Untranslated region) is importantin this regulation. Here we show that a CCCH zincfinger protein, paS-I, represses glP-l mRNAtranslation by binding to a spatial control region
within the 3' UTR. We identified an RNP-typeRNA-binding protein as a PaS-I-interacting protein,
PIP-I. PIP-I is present in oocytes to early embryos andoverlaps with POS-I in the cytoplasm and P granulesof posterior blastomeres. PIP-I binds to a subregionof the temporal control region in the 3' UTR and isrequired for the translation of glP-l mRNA. We proposethat POS-I cooperates with PIP-I to control translationof the maternal glP-l mRNA.
(5) An isoform of eIF4E is a component of germ
granules and is required for spermatogenesis in C.
elegans
Anahita Amiri!, Brett D. Keiper, Ichiro KAWASAKI!,
Yuan Fan\ Yuji KOHARA, Robert E. Rhoads2and Susan
Strome!, (1Indiana University, 2Louisiana State
University Health Sciences Center)
Control of gene expression at the translational
level is crucial for many developmental processes. The
mRNA cap-binding protein, eIF4E, is a key player inregulation of translation initiation; appropriate levels
of eIF4E are essential for normal cell-cycle regulationand tissue differentiation. The observation that eIF4E
levels are elevated during gametogenesis in several
organisms suggests that eIF4E might have a specificrole in gamete formation as well. We show that one of
the five isoforms of C. elegans eIF4E, IFE-I, is enrichedin the germline and is a component of germ granules(P granules). The association of IFE-I with P granules
requires the P-granule protein PGL-I. In vitro PGL-I
interacts directly with IFE-I, but not with the other four
isoforms of eIF4E. Analysis of animals depleted of
IFE-I by RNAi shows that IFE-I is required forspermatogenesis, specifically for efficient progression
through the meiotic divisions and for the productionof functional sperm, in both hermaphrodites and males.
The requirement for IFE-I is highly sensitive totemperature. IFE-I is not required for oogenesis, as
ife-l (RNAi) hermaphrodites produce viable progeny
when normal sperm are supplied. Consistent with a
primary role in spermatogenesis, ife-l mRNA levels arehighest in regions of the gonad undergoing
spermatogenesis. Our results suggest that C. elegans

spermatogenesis requires either this specific isoform
of eIF4E or an elevated level of eIF4E.
(6)Toward understanding of tbx-9

or decrease of their expression truly reflects on thechange of expression pattern of them are in progress.
The Caenorhabditis elegans unc-32 Gene Encodes
Alternative Forms of a Vacuolar ATPase a Subunit(7)

Yoshiki ANDACHI
tbx-9 is a member of the T-box family oftranscription factors. T-box genes have been found invarious metazoans, and many of the genes play animportant role in pattern formation especially inembryogenesis. tbx-9 encodes a transcription activator
since the tbx-9 product can induce the transcriptionof a reporter gene containing the tbx-9 binding
sequence upstream of the gene. The expression of tbx-9
is first observed in nucleus of the E cell at the 8-cellstage by in situ hybridization analysis. The expression
lasts up to its daughters, Ea and Ep. At this stage, Ca,
Cp and some AB-derived cells that seem to be
precursors of hypodermal cells start tbx-9 expression.
The expression of tbx-9 can also be seen in 4 body wall
muscle precursor cells of MS descendants at the
200-cell stage. The latest expression of tbx-9 is in
precursor cells of hypodermis that disappears justbefore morphogenesis. A tbx-9 deletion mutation
produced by gene disruption causes disorganizationin embryogenesis that occurs predominantly inposterior part of the body. At least one reason for the
deformity is a defect in the positioning of body wall
muscle cells at the comma stage. Penetrance of thephenotype is low, suggesting the presence of anothergene that is functionally redundant with tbx-9. Indeed,
all of the tbx-9 mutant embryos given dsRNA of tbx-8,
another T -box gene phylogenetically most related totbx-9, show abnormality in the overall length of the
body. In these embryos body wall muscle cells areapparently detached from hypodermis. Although N2
embryos given tbx-8 dsRNA also reveal
disorganization in their bodies, penetrance of thephenotype is low and the deformity is observed
predominantly in the central part of the body.
To elucidate the nature of transcription regulation
responsible for the tbx-9 mutant phenotype, I havebeen trying to identify target genes of tbx-9 by cDNA
microarray analysis. Search for genes whose
expression level is altered in embryos of the strains inwhich the amount of the tbx-9 product has been
modified, and tests of these genes by in situ
hybridization analysis to know whether the increase

Nathalie PUJOL1.2, Claire BONNEROT2, Jonathan J.
EWBANK!, Yuji KOHARA and Danielle THIERRY-MIEG2(lCNRS, INSERM, France, 2CRBM, CNRS, France)
Eukaryotes possess multiple isoforms of the asubunit of the VO complex of vacuolar-type
H +-ATPases (V -ATPases). Mutations in the
V -ATPase a3 isoform have recently been shown toresult in osteopetrosis" a fatal disease in humans, but
no function has yet been ascribed to other isoforms.
In Caenorhabditis elegans, the unc-32 mutant was
originally isolated on the basis of its movement defect.
We have isolated four new mutant alleles, the strongestof which is embryonic lethal. We show here that unc-32
corresponds to one of the four genes encoding a
V-ATPase a subunit in the nematode, and we present
their expression patterns and a molecular analysis of
the gene family. unc-32 gives rise via alternative
splicing to at least six transcripts. In the uncoordinatedalleles, the transcript unc-32 B is affected, suggestingthat it encodes an isoform that is targeted to synaptic
vesicles of cholinergic neurons, where it would control
neurotransmitter uptake or release. Other isoforms
expressed widely during embryogenesis are mutatedin the lethal alleles and would be involved in otheracidic organelles. Our results indicate that V-ATPasea subunit genes are highly regulated and havetissue-specific function.
(8) Gene expression profiles in Ciona intestinalis tailbud

embryos

Yutaka SATOU!, Naohito TAKATORI!, Lixy YAMADA l,
Yasuaki MOCHIZUKI!, Makoto HAMAGUCHI!, Hisayoshi
ISHIKAWA!, Shota CHIBA!, Kaoru IMAI!, Shungo KANO!,
Seiko D. MURAKAMI l, Akie NAKAYAMA!, Atsuo
NISHINO!, Yasunori SASAKURA!, Gohki SATOH!, Taishin
SHIMOTORI!, Tadasu SHIN-I, Eiichi SHOGUCHI!, Miho M.
SUZUKI!, Norio TAKADA!, Nanami UTSUMI 2, Natsue
YOSHIDA2, Hidetoshi SAIGA2, Yuji KOHARA and Nori
SATOHl (iKyoto University, 2Tokyo Metropolitan
University)

Genome Biology Laboratory63

A set of 3423 expressed sequence tags derived fromthe Ciona intestinalis tailbud embryos was categorized
into 1213 independent clusters. When compared with
DNA Data Bank of Japan database, 502 clusters ofthem showed significant matches to reported proteins
with distinct function, whereas 184 lacked sufficient
information to be categorized (including reported
proteins with undefined function) and 527 had no
significant similarities to known proteins. Sequencesimilarity analyses of the 502 clusters in relation to thebiosynthetic function, as well as the structure of themessage population at this stage, demonstrated that390 of them were associated with functions that manykinds of cells use, 85 with cell-cell communication and27 with transcription factors and other gene regulatory
proteins. All of the 1213 clusters were subjected towhole-mount in situ hybridization to analyze the gene
expression profiles at this stage. A total of 387 clusters
showed expression specific to a certain tissue or organ;
149 showed epidermis-specific expression; 34 were
specific to the nervous system; 29 to endoderm; 112 tomesenchyme; 32 to notochord; and 31 to muscle. Many
genes were also specifically expressed in multiple
tissues. The study also highlighted characteristic gene
expression profiles dependent on the tissues. In
addition, several genes showed intriguing expression
patterns that have not been reported previously; forexample, four genes were expressed specifically in thenerve cord cells and one gene was expressed only inthe posterior part of muscle cells.
This study provides molecular markers for eachof the tissues and! or organs that constitutes the Ciona
tailbud embryo. The sequence information will also be
used for further genome scientific approach to explore
molecular mechanisms involved in the formation of oneof the most primitive chordate body plans.
(9) Profiles of Maternally Expressed Genes in Fertilized
Eggs of Ciona intestinalis
Takahito NISHIKATA1, Lixy YAMADA!, Yasuaki
MOCHIZUKI1, Yutaka SATOU2, Tadasu SHIN-I, Yuji
KOHARA, Nori SATOH2 (IKonan University, 2Kyoto
University)
A set of 1,378 expressed sequence tags (ESTs), boththe 5'-most and 3'-most ends, derived from Ciona
intestinalis fertilized eggs was categorized into 1,003
independent clusters. When compared with sequences

64

Genome Biology Laboratory

in databases, 452 of the clusters showed significant
matches with reported proteins, while 190 showedmatches with putative proteins for which there is notenough information to categorize their function, and361 had no significant similarities to known proteins.
Sequence similarity analyses of the 452 clusters in
relation to the biological function as well as the
structure of the message population at this stage
demonstrated that 362 of them have functions thatmany kinds of cells use, 65 are associated with cell-cellcommunication, including a candidate cDNA for sonichedgehog, and 25 are transcription factors. Sequence
prevalence distribution analysis demonstrated that thegreat majority (78%) of the mRNAs are rare mRNAs or
are represented by a single clone/cluster. All of the
1,003 clusters were subjected to whole-mount in situ
hybridization to analyze the distribution of thematernal mRNAs in fertilized eggs, and a total of 329
genes showed localized distribution of the mRNAs: 16
showed cortical localization, 12 showed
mitochondrial-like distribution, 99 crescent-like
distribution, 63 partial localization, and 139 weak
localization. When the distribution pattern of all thematernally expressed mRNAs was examined in the
8-cell stage embryos, it became evident that 248 genes
which have localized mRNA patterns at the fertilized
egg stage lose their localized distribution by the 8-cellstage. In contrast, 13 genes newly gain a localized
pattern by the 8-cell stage. In addition, a total of 39
genes showed distinct in situ signals in the nucleusof blastomeres of the 8-cell stage embryo, suggesting
early zygotic expression of these genes by this stage.
These results suggest that complicated cytoplasmic
movements are associated with the characteristic
distribution of maternal mRNAs, which in tum support
proper embryonic axis formation and establishmentof the genetic network for embryonic cell specification.
Publications
1. Pujol, N., Bonnerot, C., Ewbank, J.. Kohara, Y. andThierry-Mieg, D.: The C. elegans unc-32 gene encodes
alternative forms of the100-kDa a subunit of
Vacuolar-ATPases.J BioI. Chem. 276,11913-11921, 2001.
2. Maeda, I., Kohara, Y., Yamamoto, M. and
Sugimoto, A.: Large-scale analysis of gene function in
Caenorhabditis elegans by high-throughput RNAi.
Current Biology, 11, 171-176, 2001.

3. Reboul,]., Vaglio, P., Tzellas, N., Thierry-Mieg, N.,
Moore, T., Jackson, C., Shin-I, T., Kohara, Y.,
Thierry-Mieg, D., Thierry-Mieg,]., Lee, H., Hitti, ].,Doucette-Stamm, L., Hartley,]., Temple, G., Brasch, M.,
Vandenhaute,]., Lamesch,Pl., Hill, D. and Vidal, M.:
Open-reading-frame sequence tags (OSTs) support theexistence of at least 17,300 genes in C.elegans. Nature
Genetics, 27, 332-336, 2001.

Y., Satoh, N. and Satou, Y.: EST analysis of gene
expression in testis of the ascidian Ciona intestinalis.
Molecular Reproduction and Development (in press).
11. Morino, K, Katsumi, H., Akahori, Y., Iba, Y.,
Shinohara, M., Ukai, Y., Kohara, Y., Kurosawa, Y.:
Antibody fusions with fluorescent proteins: a versatile
reagent for profiling protein expression. J Immunol
Methods. 257, 175-84, 2001.

4. Navarro, RR, Eun Yong Shim, E.-Y., Kohara, Y,
Singson, A and Blackwell, T.K: cgh-l, a Conserved
Predicted RNA Helicase Required for Gametogenesis
and Protection from Physiological Germline Apoptosis
in C. elegans. Development 128, 3221-3232, 2001.
5. Hanazawa, M., Motti, M., Ueno, N., Kohara, Y andlino, Y.: Use of cDNA subtraction and RNAinterference screens in combination reveals genes
required for germ-line development in Caenorhabditis
elegans. Proc. Natl. Acad. Sci. USA 98,8686-8691,2001.
6. Altun-Gultekin, Z., Andachi, Y., Tsalik, E.L.,
Pilgrim, D., Kohara, Y. and Hobert, 0.: A regulatorycascade of three homeobox genes, ceh-10, ttx-3 andceh-23 , controls cell fate specification of a definedinterneuron class in C.elegans. Development 128,
1951-1969, 2001.
7. Satou, Y, Takatori, N., Yamada, L., Mochizuki, Y,
Hamaguchi, M., Ishikawa, H., Chiba, S., Imai, K, Kano,
S., Murakami, S., Nakayama, A, Nishino, S., Sasakura,Y., Satoh, G., Shimotori, T., Shin-i, T., Shouguchi, E.,
Suzuki, M., Takada, N., Utsumi, N., Yoshida, N., Saiga,
H., Kohara, Y., Satoh, N.: Gene expression profiles in
Ciona intestinalis tailbud embryos. Development 128,2893-2904, 2001.
8. Amiri, A, Keiper, B.D., Kawasaki, 1., Fan, Y.,

Kohara, Y., Rhoads, RE., Strome, S.: An isoform of
eIF4E is a component of germ granules and is required
for spermatogenesis in C. elegans. Development 128,
3899-3912,2001.
9. Nishikata, T., Yamada, L., Mochizuki, Y, Satou,
Y., Shin-i, T., Kohara, Y., Satoh, N.: Profiles ofmaternally expressed genes in fertilized eggs of Ciona
intestinalis. Developmental Biology 238, 315-331, 2001.
10. Inaba, K, Padma, P., Satouh, Y, Shin-i, T., Kohara,

Genome Biology Laboratory65

H. STRUCTURAL BIOLOGYCENTER

H-a. Biological Macromolecules Laboratory(1) Thin Layer Illumination Microscopy as a Novel
Technique for Single Molecule Fluorescence Imaging of
the Inside of Cells
Makio TOKUNAGA
Objective-type total internal reflection fluorescencemicroscopy, which we have developed, enabled us tovisualize single molecules on or just below the cellsurfaces. However, the technique is applied only when
fluorescent molecules are from the glass-medium or
glass-cell surface to a depth of less than about 150 nm.
To overcome the limitation, I have further developed
a new single-molecule microscopy by devising the wayof light illumination for fluorescence excitation.
Specimens were illuminated with a thin-layered laser
beam with the thickness of about 5 to 10 micrometers.
The present microscopy has a significant
advantage of low background and high sensitivity, so
it allowed us to visualize single fluorescent moleculesin the inside of cells up to the depth of about 10 Jl mfrom the glass-medium interface. It is also useful forobservation of living cell, which is sensitive to theillumination light, and for time-lapse observation overa long period.
(2) Single Molecule Imaging of Nucleocytoplasmic
Transport in Cells and Quantitative Analysis ofInteraction with Nuclear Pores
Makio TOKUNAGA and Naoko IMAMOT0 1 (IGene
Network Laboratoy)
We examined behavior of green fluorescent protein(GFP)-tagged importin beta and GFP-tagged cargo indigitonin permeabilized cells, using the above newtechnique of thin layer illumination microscopy.

66

STRUCTURAL BIOLOGY CENTER

Although recent years have seen considerable progressin researches on nucleocytoplasmic transport,
molecular mechanisms of the transport and theinteraction between nuclear pore complex (NPC) andmolecules remain to be solved. Fluorescence imagesof importin beta or cargo molecules bound to the
nuclear rim were obtained. Images were composed ofmany fluorescent spots, which represented single
NPC's. Furthermore, imaging of single molecules of
GFP-importin beta or GFP-cargo on the NPC has been
also achieved.
Both visualization of single pores and single
molecules enabled us to analyze images quantitatively
at the molecular level and to obtain kinetic parametersin cells, i.e., retention time at the pore, number ofmolecules bound to pores, and binding constant. It was
found that there were two kinds of binding site ofimportin beta on NPC in the absence of cargo and
Ran-GTP, weak binding sites and strong ones. The
former gathers many proteins on NPC, and the latteris related with the active site of transport. Thus, single
molecule imaging has opened a new way to obtain
quantitative information on kinetics of molecularinteractions in cells.
(3) A novel protein pl05: recruitment intomRNA-containing complex and actin- andmicrotubule -dependent transport
Nobuyuki SHIINA, Kazumi SHINKURA and Makio
TOKUNAGA
p105 is a novel protein we identified originally as
an antigen of a monoclonal antibody against Xenopus
centrosomes. Here, we characterized the molecularconstituent of pl05-contaning complex and observed
the behavior of the complex.
In pl05-expressing cultured cells, pl05 was
localized in electron-dense structures, which contained
ribosome-like particles and membranous structures.
The p105-containing structures were immunostainedwith EF-l alpha (a translation elongation factor) andbeta-COP (a component of coated vesicles) antibodies,supported the existence of ribosomes and membranes
in the structures. The p105-containing structures also
contained mRNAs. Actin mRNA was not contained in
the structures, indicating that some specific mRNAs
were localized in the structures. Lines of biochemical
evidence suggested that pl05 was associated with

ribosomes via mRNA. These findings revealed thatp105 forms a complex with specific mRNAs, ribosomes,translational machinery and beta-CaP-containing
membranes.
p105 was fused with GFP and its behavior wasstudied by time-laps observation. The p105-containing
complex was transported close to the plasma
membrane in a microtubule- and actin-dependent
manner, and moved concomitantly with dynamic
membranes, e. g. ruffling membranes and processes.
These characteristics of the plG5-containing
complex are similar to those of mRNA transporters
identified in mammalian neurons and Drosophila
oocytes. We are now characterizing the complex further
in the point of view of cell polarity.
(4) Single molecule measurement of intermolecular andintramolecular interactions using subpiconewton
intermolecular force microscopy

Michio HIROSHIMA and Makio TOKUNAGAIntermolecular force microscopy (IMF) is able tomeasure forces of intermolecular and intramolecularinteractions at single molecular level. This microscopy
has achieved the force resolution of subpiconewton
with the handmade cantilever which is over lOG-foldmore sensitive than commercial ones. And
incorporating the feedback system which use laser
radiation pressure to reduce thermal fluctuation of thecantilever, the cantilever position is controlled with ananometre accuracy.
In conventional atomic force microscopy (AFM),
optical lever method has been used for sensing thecantilever position, however, there were a few problems
in applying this method to our IMF. We have devised
new method in which the cantilever inclined at 45
degrees is illuminated and its position is detected as
displacement of the image projected on thephotosensor. In this refined system, the measurable
range of fo:tces is over lOGpN which is comparable tothat of the horizontal IMF developed in last year.
With the refined IFM, we have measured thebinding forces between single bases of DNA and theunfolding process of single globular protein. Forces
curves of single-molecule or single-bond interactions
were obtained with the sub-nanometer and piconewton
resolution, which were never obtained by conventionalscanning probe microscopy.

(5) Single molecule measurement of protein folding byintermolecular force microscopy

Isao SAKANE, Michio HIROSHIMA, Kunihiro KuwAlIMA!
and Makio TOKUNAGA (!Department of Physics,
University of Tokyo)
We measured mechanical unfolding events ofsingle staphyloc<?ccal nuclease (SNase) molecules with
the intermolecular force microscope. Cysteinyl residues,
which have an activity to chemisorb to gold, wereintroduced into both the N- and C-termini of SNase.
One terminus of the protein was attached to thegold-coated glass surface and the other terminus was
attached to the probe tip of the intermolecular force
microscope. Then the protein was extended
mechanically to obtain relations between the extension
length and the force exerted on the protein. From therelations of force-extention length, a partially unfolded
structure or an intermediate structure was found forthe first time. The extension length of whole the protein
was 45 nm, while partial unfolding yielded extension
length of 15 nm. Supported with the result of theexperiment in the presence of Ca ion, this partiallyunfolded structure was attributed to a structure inwhich alpha-helices in the C-terminal region are
unfolded. Multi-pathways in the mechanical unfolding
were also found. The results suggested two ways ofthe mechanical unfolding: unfolding by way of thepartial unfolded structure, and direct unfolding
through no intermediate structure.
(6) A Novel in vitro Assay System of Nucleocytoplasmic
Transport

Atsuhito OKONOGI, Michio HIROSHIMA, Nobuyuki
SHIINA, Naoko IMAMOTO! and Makio TOKUNAGA rGene
Network Laboratoy)
We have developed a novel in vitro assay systemof nucleocytoplasmic transport. We aim at applicationof the method to new single-molecule experiments,
imaging and nano- or force-measurement.
Nuclear envelope was formed on a planar surface
of. a small agarose plate. At first, agarose plates weremodified with glutation. Then glutation-agarose plates
were coated with GST-RanGDP fusion protein. Nuclear
envelope was formed onto the RanGDP-coated surface
using extracts from Xenopus laevis frog eggs.

Biological Macromolecules Laboratory67Formation of Nuclear Pore Complexes was confirmed
by observing import of fluorescently labeled proteins.
This new cell-free system has marked advantages: 1)
Solutions in both sides, pseudo-cytoplasmic andpseudo nucleoplasmic sides, can be replaced
independently. 2) The shape of the nuclear envelope
can be changed as one likes, for example, a verticalplane and a horizontal plane. 3) The system doesn't
contain no organelle or cellular structures except thenuclear envelope. Using the in vitro system together
with single molecule techniques or fluorescenceresonance energy transfer should provide aninnovative and powerful tool to investigate molecularmechanisms of transport.
Publications
1. Kitamura, K., Ishijima, A., Tokunaga, M.,
Yanagida, T.: Single-Molecule Nanobiotechnology.
jSAP International, 4, 4-9, 2001.

2. Aoki, T., Sowa, Y., Yokota, H., Hiroshima, M.,
Tokunaga, M., Ishii, Y, Yanagida, T.: Non-Contact
Electrostatic Surface Force Imaging of Single Protein
Filaments using Intermolecular Force Microscopy.

Single Mol., 3, 183-190, 2001.

H-b. Molecular Biomechanism Laboratory(1) The promoter arrest of E. coli RNA polymerase andthe effect of the Gre factors
Motoki SUSA 1, Tomoko KUBORI!, Hiroki NAGAI! andNobuo SHIMAMOT0 1 (lStructural Biology Center,
National Institute of Genetics and and Department of
Genetics, School of Life Science, The Graduate
University for Advanced Studies)
Transcription initiation has been conventionallyassumed to be a multi-step sequential process,although additional steps could exist. The in vitro
initiation from T7Al promoter in particular apparently
behaves in a manner that can be fully explained by thesequential pathway. However, the initiation froml,PRAL 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

68

Biological Macromolecules Laboratory

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 l,PRAL 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.
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 l,PRAL
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 atp, are decreased bydisruption of greA and greB. Their transcriptions wereenhanced in a purified reconstitution system by adding
GreA or GreB. Among them, atp operon was examinedmost detail. The formation of moribund complex at the
atp 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 u70 by oligomerization andidentification of the role of its spacer region
Hiroki NAGAI!, Taciana KASCIUKOVIC2, Richard S.
HAYWARD 2, Yumiko SATO! and Nobuo SHIMAMOTO!

fNat Inst. of Genet/Grad. School of Adv. Stud., 22Inst.
Of Cell and Molec. BioI., Edinburgh Univ., Scotland)
We have found that 0 70, the major 0 factors of E.
coli, forms aggregate in vivo and in vitro at a hightemperature within physiological condition. The
oligomeric 0 70 has little transcriptional activity and theoligomerization in vitro showed a sharp temperaturedependence. We have constructed a strain with adisrupted rpoD ( 0 70 ), and plasmid born 0 70 supports its
growth. We have observed a positive correlation
between the intracellular concentration of 0 70 andupper limit of growing temperature. This raises a
possibility that 0 70 is a molecular thermometer.
The major 0 factors of proteobacteria mostly havea big spacer region between the conserved regions 1
and 2 which is not conserved in eubacteria. In E. coli
700
this region has extensive acidic patches which may
be concerned with the property of 0 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
tubeculosis sigA failed to complement the disruption,
and we are now testing B. subtilis sigA and others. The
mutant 0 70 lacking the spacer region of aa130-374 (dSR) complemented the disruption at 30 and 25°C,
proving that 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) Does RNA polymerase track a groove of DNA duringsliding?
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, RNApolymerase first binds to a nonspecific site and thenreaches a promoter by repeated nonspecific associationand dissociation and by sliding along DNA. Sliding
is one-dimensional random diffusion of proteins along
DNA, the existence of which has been proved for E.
coli RNA polymerase and several other DNA binding
proteins by single-molecule dynamics and kinetics.

There are two possible modes of movement duringsliding. 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 relative rotational motion
around the axis.
To determine the sliding mode for E. coli RNA
polymerase, we devised an assay for detecting the
tracking of a groove as rotation of DNA based on
single-molecule manipulation. A nonspecific DNA is
fixed to a 1 /l m polystyrene bead coated withstreptavidin by several biotin residues with norotational freedom. A 0.1 /l m fluorescent bead iscovalently attached to the polystyrene bead to detect
the rotational motion of the DNA-fixed beads undera fluorescent microscope. The polystyrene bead is heldwith optical tweezers at a distance from the surface
of a glass slide on which RNA polymerase has been
fixed. In this configulation, we moved RNA polymerase
relative to the DNA by shifting the slide continuously
in one direction. If RNA polymerase tracks a groove,it should rotate DNA and the rotation can be detected
as the movement of the fluorescent bead.
We have observed rotational motions of the beads
that could be caused by groove tracking of RNApolymerase in a small number of cases. Such motions
were observed only when the distances between beads
and the surface are smaller than the length of DNA.
However, the number of such observation is notenough to conclude groove tracking. A short lifetimeof sliding complex, about 1 sec in solution, complicatesthe estimation of the frequency of rotational motions.
Further quantitative determination is undergoing toprove or disprove groove tracking.
Publication
1. Sen, R., Nagai, H. and Shimamoto, N.:
Conformational switching of Escherichia coli RNA
polymerase-promoter binary complex is facilitated byelongation factors GreA and GreB. Gene. Cells 6,389-402, 2001.

H-c. Multicellular Organization Laboratory(1) Analysis of Synthetic Dauer-constitutive Mutationsin the Nematode Caenorhabditis elegans
Kiyotaka OHKURA, Tomoko YABE, Kohji MIYAHARA!,

Molecular Biomechanism Laboratory

69Eiko TSUCHIYA\ Takeshi ISHIHARA and Isao KATSURA(lGraduate School of Advanced Sciences of Matter,
Hiroshima University)
If newly hatched larvae of C. elegans are in a
crowded state and given a limited food supply, theysense the environmental signal of pheromone and foodwith a head sensory organ called amphid, and deviate
from the normal life cycle, ending in non-feeding larvae
called dauer larvae. 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,
Le., they develop to dauer larvae in a certain mutant
background, regardless of the environmental
conditions. The synthetic nature of the phenotype, we
think, is based on the pathway of sensory signals.
Namely, the signals are transmitted through parallel
routes, and therefore two mutations are required toblock them. We are determining the combinations ofmutations for the Sdf-c phenotype and the pattern ofsuppression of the Sdf-c phenotype by various
suppressor mutations. In this way we hope to elucidate
the pathway of sensory signals for dauer regulation.
Furthermore, to identify new genes required for thesensory signal transduction, we isolated and mapped
44 mutations that show the Sdf-c phenotype in the
unc-3l(el 69) background, where unc-3l gene encodes
CAPS protein, which acts in secretion from dense corevesicles. Eight of the mutations mapped in 4 known
genes, but most of the remaining 36 mutations, which
map in at least 13 genes, seem to be located in novel
genes, which we named sdfgenes. Some of the mutants
exhibit additional phenotypes after removal of theunc-3l(e169) mutation. A mutant in sdj-l gene avoids
volatile attractants sensed by AWC neurons and showsa thermophilic phenotype in thermotaxis. Mutants in
sdj-13 gene (2 alleles) show defects in the adaptationof chemotaxis to odorants sensed by AWC neurons,
although they show normal chemotaxis to the sameodorants. This gene encodes a homolog of mammalian
Tbx2 and Drosophila Omb, and is expressed in AWB,AWC and AS] sensory neurons and in many
pharyngeal neurons. Mutants in sdj-9 gene (5 alleles)
form dauer larvae that recover spontaneously, if thepromoter DNA fragment of daf-7gene, which encodes

70

Multicellular Organization Laboratory

a TGF-{3 protein that inhibits dauer formation, is
introduced into the mutants.
In the year 2001, the following results wereobtained.
(a) The expression of a daj-7:: GFP fusion gene
was investigated in various Sdf-c double mutants. In
wild type animals this gene is expressed only when
they adopt the normal life cycle, but not when they
form dauer larvae. Hence, the results indicate whether
the genes of the Sdf-c mutations act upstream of daj-7
in the dauer regulatory pathway. The daj-7:: GFP
fusion gene was not expressed in the double mutants
unc-3l; osmol, unc-3l; unc-3, and egl-4; unc-3, while it
was expressed in the single mutants unc-3l(CAPS),
unc-3(Olf-l/ EBF), egl-4(PKG), and osmol (required forsensory cilium formation). We speculate that theexpression of daj-7is regulated by at least two parallel
pathways: one for the activation of the UNC-3
transcription factor by an environmental signal thatenters through sensory cilia, and the other(s) containing
neurosecretion and PKG signal transduction.
(b) Using specific antibodies, we found thatSDF-13 protein is localized mainly in cytoplasm.
Although this localization does not change by thestimulus of odorants for adaptation, it is possible thatSDF-13 may be regulated somehow by nuclear
transport. We also found that the expression of sdj-13
cDNA in AWC neurons alone can restore normal
adaptation but not the synthetic dauer-constitutive
phenotype. The results indicate that SDF-13 acts in
AWC neurons for the adaptation to odorants sensed
by the same neurons.
(c) We cloned sdj-9 gene and found that it
encodes a tyrosine phosphatase-like molecule. Since
the consensus sequence HCxxGxxR is changed in thismolecule, it may lack the enzyme activity. A functional
sdj-9.: GFP fusion gene was expressed in a pair of cells
anterior to the nerve ring.
(d) The dauer larvae formed by the double
mutants unc-3l (e169); sdj-14 (5 alleles) have two uniquecharacteristics: (i) their percentage changes greatly by
the volume of the E. coli liquid culture spread on theplates, and (ii) they recover easily to L4 larvae in spiteof having the unc-3l (e169) mutation, which normally
prevents such recovery. We narrowed down theposition of sdj-14 mutations within 3 cosmid clones bymapping and rescue experiments.
(2) Analysis of Mutants That Show Abnonnality in theSelection between Two Behaviors and in Behavioral
Plasticity
Takeshi ISHIHARA, Yuichi IINO!, Akiko MOHRI2, Ikue
MORI2 and Isao KATSURA (iMolecular Genetics
Laboratory, University of Tokyo, 2Division of
Biological Science, Nagoya University)
Animals receive environmental cues, select andintegrate necessary information, and make proper
responses, while all these steps can be modified byexperience or memory. In C. elegans, many behavioral
mutants in chemotaxis and thermotaxis, for instance,
have been isolated and analyzed, and the molecularmechanisms of sensation have been elucidated. On thisbasis and as a next step, we are analyzing mutants thatshow abnormality in the learning 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 method for the interaction between
the two responses to learn a possible role ofinterneurons. Wild-type animals change theirpreference between the responses, depending on therelative concentration of copper ion and odorants. On
the basis of the C. elegans neural circuitry, the result
suggests that the two sensory signals interact witheach other in a neural circuit consisting of about 10
neurons. 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 behavioral
interaction or selection, we are isolating and analyzing
mutants that show abnormality in this assay. The
mutant ut236 has a tendency to choose avoidance ofcopper ion rather than chemotaxis to odorants,
although it shows no abnormality in each behavior incase only one of the stimuli is given. The result showsthat ut236is abnormal in the interaction between thetwo behaviors.
We identified the gene for ut236 by positional
cloning and named it hen-l. It encodes a novel secretory

protein having an LDL receptor ligand-binding
domain. Using antibodies against its recombinant
protein, we found that the gene product is localized
in the axon and cell body of each one pair of sensory
and inter-neurons. The localization in the axon was
abolished in unc-l04 (kinesin KIFIA homolog) mutants,
which show defects in the transport of synaptic
vesicles. Expression studies with various transcriptional promoters showed that this gene acts
non-ceIl-autonomously in the nervous system. Studies
using a heat-shock promoter revealed that thewild-type phenotype is recovered if hen-l gene isexpressed after the mature neural circuitry is formed,
but not during the development of the nervous system.
These results indicate that the gene product is a novel
neuromodulatory factor that acts in diverse functions
such as the processing of sensory information andvarious types of learning (See below).
We isolated another mutant, ut235, which lacks
the effect of starvation in this assay. This mutant
behaved essentially normally in many other responses
to starvation, such as the change of locomotion speed
by starvation. Interestingly, the double-mutant ut235;
ut236 showed a preference to avoidance of copper ion,
regardless of starvation. We are now trying to clone
the gene for this mutation.
The hen-l mutant also shows abnormality inlearning by paired presentation of starvation and NaCl(collaboration with Dr. !ino, University of Tokyo). Whilewild-type animals are normally attracted by NaCl, they
avoid it after the conditioning. In contrast, hen-l mutant
animals are only partially affected by the conditioning.
Since the mutant shows normal behavioral responses
to starvation alone, we think it has abnormality in the
integration of starvation and other signals.
In 2001, we investigated the role of hen-l gene inlearning by temperature and food/ starvation, in
collaboration with Dr. Mori's Laboratory in Nagoya
University. In thermotaxis, wild type animals prefer
the temperature of their growth, but avoid it after they
are starved at the same temperature. In contrast, hen-lmutant animals did not change.their behavior by
starvation, although they show normal thermotaxis
without starvation. The results indicate that the hen-l. mutant has abnormality in the behavioral plasticity
after paired presentation of temperature andstarvation, although it responds normally to each of
the two stimuli.

Multicellular Or~anization Laborato('Y71

(3) C. elegans Mutants in the Associative Learning ofOdorants and Foodlehiro TORAYAMA, Takeshi ISIDHARA 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. For instance, paired
presentation of an attractive smell and starvation, but
not the smell and food, leads to inefficient chemotaxis
to the smell. However, this phenomenon may also beinterpreted as the inhibition of olfactory adaptation
by food. To elucidate the molecular mechanism of foodand starvation signaling in such learning, we started
the isolation and characterization of mutants that show
abnormality in associative learning.
In the year 2001, we first examined the conditions
for the associative learning of smell and food/starvation and succeeded in efficient learning by using
butanone as the odorant. Butanone is an attractant for
naive worms, but chemotaxis to butanone becomesinefficient after conditioning with starvation andbutanone, while it becomes more efficient after
conditioning with food and butanone. Then, we
isolated mutants that show abnormality in these
responses. Some of them showed inefficient chemotaxis
after conditioning with food and butanone (i. e., they
adapted to butanone even in the presence of food),
while others are attracted by butanone only after
conditioning with food and butanone. Weare mapping
ut30S, one of the mutations that belong to the former
category. Interestingly, the mutant shows noadaptation to isoamyl alcohol and benzaldehyde, which
are sensed by the same sensory neurons (AWC) as
butanone.
(4) Fluoride-resistant Mutants of the Nematode
Caenorhabditis elegans
Akane OISID, Minoru KAW AKAMI\ Akiko KAMAMOTO,
Takeshi ISHIHARA and Isao KATSURA (lGothenburg
University)
Fluoride-resistant (flr, mutations of C. elegans are

72

Multicellular Organization Laboratory

recessive and grouped into two categories: class 1
mutations, which map inflr-l,flr-3andflr-4, and class
2 mutations, which map in flr-2 and flr-S (Katsura, I.
et al.: Genetics 136, 145-154, 1994). Class 1flrmutations
show many phenotypes: slow growth, short defecation
cycle periods, frequent skip of the expulsion step ofdefecation, and synthetic abnormality in dauer
formation, besides strong resistance to fluoride ion.
The flr-l gene encodes an ion channel belonging to the
DEG/ ENaC (c. elegans degenerins and mammalianamiloride-sensitive epithelial sodium channels)
superfamily, while flr-4 and flr-3 code for a novel Ser/Thr protein kinase and a kinase-like molecule,respectively, 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 1.5-foldstage, in the isthmus of the pharynx from the 3-foldstage and in a pair of head neurons called AUA from
Lllarvae to adults. Moreover, the expression of flr-3.:
lacZis detected 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-resistance of class 1 flrmutations. Hence, it
seems that the regulatory pathway bifurcates afterclass 1 genes, and that class 2 genes control only oneof 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 havemade a model in which a signal controlled by class 1
genes is transmitted from the intestine and represses
the action of class 2 gene products in the head nervous
system.
In the year 2001, we worked on the positional
cloning of flr-S gene and the expression of flr-S gene.
We mapped a flr-S mutation precisely by classicalmethods and limited the position of flr-S gene withinseveral genomic clones by the activity of rescuing theFlr-5 phenotype (suppression of the slow growthphenotype of flr-:1) in a flr-3; flr-S double mutant.
In the past study we found that a flr-3:: lacZ geneis expressed in the intestine. However, this expression
may not reflect the expression of the intrinsic flr-3 gene,because we later discovered that flr-3 gene istranscribed together with a neighboring gene as an
operon. Hence, we isolated a genomic clone covering
the operon, inserted GFP cDNA at the end of the flr-3
coding region, and introduced the GFP fusion gene into
the wild type. The transformant expressed GFP also
in the intestine. Since the transformant grows very
slowly, we are investigating whether the flr-3:: GFP
fusion gene acts as a dominant negative allele.
Publications
1. Shioi, G., Shoji, M., Nakamura, M., Ishihara, T.,

Katsura, I., Fujisawa, H. and Takagi, S.: Mutations
affecting nerve attachment of Caenorhabditis elegans.
Genetics 157, 1611-1622, 2001.
2. Katsura, 1.: Messages to the new millenniumVarious aspects of doing research. Tanpakushitsu
Kakusan Koso 46,251-254,2001.

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 a3/33yDE. The unique rotational
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
concentrated on the structural study of the a3/33YE
sub-assembly of thermophilic F1, after solution of thestructure of the a3/33 sub-assembly and committed
attempts to crystallize the a3/33y -sub-assembly.
Employing the a3/33YE sub-assembly that showcatalytic properties very similar to those of F1, we aim
to detect structural changes caused by different
nucleotide occupancy, which should provide withstructural basis for understanding the rotational
catalysis mechanism.
The Mg ion is absolutely required for catalysis,but its absence forces the sub-assembly into only
slightly inhibited state. The a3/33YE sub-assembly
crystals were grown in broad conditions that vary incontent of both nucleotide and magnesium ion.
However, the Mg-free conditions generally gave
better-diffracting crystals than the Mg-included
conditions. When experimented with Spring8 beam,
crystals grown in absence of Mg diffracted to 4 A at
lOOK. The diffraction capability of these crystals did
not depend much on nucleotide contents in the motherliquor. The better resolution of the data, compared withthat reported last year, are due to the improved
flash-cooling method that included the use of oilparatone. The diffraction data have been collected at
Spring8 from the crystals that were grown in thepresence of varying concentrations of ADP and ATP
but in absence of Mg.
Following the analysis of the data from the crystals
grown in presence of 0.5mM ADP at 4.5 A resolution
reported last year, we analyzed the data from a crystal
grown in absence of nucleotide to 4.0 A resolution.
Unexpectedly the sub-assembly molecules in the
crystal represented Mg-free one-ADP bound form,
probably because the sub-complex molecules thatbound single ADP tightly and occupied 10-20%
population in our partially nucleotide-depleted
preparations, appear to have exclusively crystallized.
In contrast to the analysis of 0.5mM ADP form
(representing two ADP bound structure), the current
analysis of one ADP bound form allowed to identify
the structures of the y-and E-subunits.
The structure of the sub-complex with Mg-free
ADP bound to only one /3 -subunit has the features
distinct from those in all the previous F1 crystal
structures with two or three nucleotide-bound/3 -subunits. The /3 -subunit with the Mg-ADP-boundcatalytic site in the previous F1-structures is replacedby the nucleotide-free /3 -subunit in this structure. The
structural change appears to be responsible for
substantial structural changes in the stalk, 30-degree
twist in major part of the y -subunit and penetrationof the C-terminal helices of the E -subunit into the a3/3 3 cavity. The new conformation of the C-terminal
helices ofthe E-subunit explains well the E-inhibition.
These structure studies were done in collaboration
with Toshiharu Suzuki and Masasuke Yoshida, atResearch Laboratory of Resource Utilization, Tokyo
Institute of technology.
(2) Crystallographic Study of the Transcription
Activator, PhoB

Multicellular Organization Laboratory73

Kazuyasu SHINDOH, Katsumi MAENAKA and Yasuo
SHIRAKIHARA
PhoB Protein is a positive transcriptional activator
for the genes in the phosphate (pho) regulon of E. coli,such as phoA and pstSthat are induced by phosphatestarvation. PhoB acts by binding to the pho box in the
promoter region, which is tpe consensus sequenceshared by the regulatory regions of the genes in theregulon. The activity of PhoB is regulated byphosphorylation by PhoR. The N-terminal domain of
PhoB is responsible for this regulatory role, whereas
the C-terminal domain (PhoBC: spanning from aresidue 125 to the C-terminus) has a DNA binding
ability. In order to resolve the detailed interaction
between PhoB and pho box DNA, we have made thecrystallographic study of PhoBC and of its complex
with the DNA.
Using the 3.0 A resolution MAD (Multiwavelength Anomalous Dispersion) data from theselenomethionyl derivative of PhoBC, a good density
map has been obtained which allows identification of
the fold of PhoBC. The observed fold, classified as avariant of the winged-helix-turn-helix, is consistent
with the previous NMR structure but includes the
DNA-binding loop that have been unclear so far.
Refinement of this model using the 2.0 A resolution
native data has come to the final stage.
We have searched for the crystallization conditionsof the complex of PhoBC and DNA and got the crystalsof the complex in the conditions using the PEG as aprecipitant. Diffraction data to 3.0 A resolution havebeen collected at PF. The data analysis has been inprogress.
This work has been done in collaboration with
Hideyasu Okamura, Yoshifumi Nishimura, Yokohama
City University and Kozo Makino, Research Institutefor Microbial Diseases, Osaka University.
(3) Structural and functional
irnrnunoglobulin(Ig)-like receptors

studiesof

Katsumi MAENAKA, Kimie AMANO and Yasuo
SHIRAKIHARA
Since the immunoglobulin (Ig) superfamily
domains are very common among the cell surface
receptors in immune system, it is important to clarify
the molecular recognition of Ig-like cell surface

74

Biomolecular Structure Laboratory

receptors towards various ligands. We are studying
two Ig-like receptors, human killer cell Ig-like receptors(KIRs) and Fcy receptors (Fcy R).
Fcy receptors (Fcy Rs) are expressed on all
immunologically active cells and bind the Fc portionof IgG to trigger a range of immunological functions.
By using surface plasmon resonance (SPR), we havestudied the kinetic and thermodynamic properties ofFc binding of human low-affinity Fcy receptors (Fcy
Rs (Ila, lIb and IIlb), whose structure have uniquetopology (intermediate between I set and C2 set)
revealed by several structural studies of KIRs and Fcy
Rs including ours. All three receptors bind Fc or IgG
with low affinities (Kn ("'" 1O~M) and fast kinetics, which
is similar to other receptors involved in cell-cellinteractions. It suggests that FcyR recognition of
IgG-coated cells is mechanistically similar to cell-cellrecognition. On the other hand, the Fc receptors show
distinct thermodynamic properties. While the bindingof the Fcy RIla and Fcy RIlb to Fc is driven by
favourable entropic and enthalpic changes, the bindingof Fcy RIll is characterised by highly unfavourable
entropic changes, suggesting that the molecular events
coupled to the binding differ amongst the low-affinity
FcyRs.
We got very small crystals for KIR2DL3 complexedwith MHC class I (HLA-Cw7) in the conditions
including PEG20000 as a precipitant, but further
refinement of crystallization conditions is needed for
determination of the complex structure.
This work has been done in' collaboration withPeter Sondermann (Max-Planck-Institut fur Biochemie,
Martinsried), Anton van der Merwe, Yvonne Jones,
David Stuart (University of Oxford), and Izumi
Kumagai, Kouhei Tsumoto, Mitsunori Shiroishi(Tohoku University).
(4) Structural study of Na+-translocating ATPase
Yasuo SHIRAKIHARA
Na+ -translocating ATPase from Enterococcus
hirae, classified as a Vacuolar-type ATPase (V-type),
is expected to have a structure similar to F1-ATPase(F-type ATPase). Na+-translocating ATPase was highly
purified and was subjected to a crystallization
experiment. However, the crystals obtained under thepreviously identified conditions (27% PEG4000, 0.2 M
LiS0 4, 10 mM Tris-HCl, pH 7.5, 10% glycerol) werefound to be from enolase that was co-purified with
ATPase, in spite of its very low content (a few percent).
Enolase was purified and again was subjected to a
crystallization experiment in order to search fordifferent crystal forms, because the original crystal
form contained as much as four molecules in theasymmetric unit. The new crystal form (14, a=b=153.5
A, c= 90.6 A) was found to be suited for the molecularreplacement analysis. Refinement, after finishing themolecular replacement calculations, has been inprogress.
The crystallization experiment with Na+translocating ATPase was carried out again for its
authentic crystals. Several crystal forms, with plate-likeand needle-like shapes, were obtained. The mosthopeful crystals diffract to 4.5 A resolution and belong
to space group P2 1 2 1 2 1with cell dimensions of
a= 122.0 A, b= 122.0A, c=240 A. Structural analysis bymolecular replacement using the F1 structure is inprogress.
This work has been done in collaboration with
Toshiaki Hosaka, Toshiyuki Meguro and Ichiro
Yamato, Science University of Tokyo.
(5) Structural analysis
Micrococcus luteus K-3

ofglutaminaseof the truncated enzyme produced good maps, allowing
us to finish the tracing the main chain. The modelbuilding is in progress. We plan to utilize the modelin determining the structure of the intact enzyme bymolecular replacement.
The structural study was done in collaboration
with Panuwan Chantawannakul, Kazuaki Yoshimuneand Mitsuaki Moriguchi at Oita University, andMamoru Wakayama at Ritsumeikan UniversIty.
Publications
1. Maenaka, K., van der Merwe, P.A., Stuart, D.l.,
Jones, E.Y. and Sondermann, P.: The human
low-affinity Fcy receptors ITa, IIb and ill bind IgG with
fast kinetics and distinct thermodynamic properties.
J BioI. Chem. 276, 44898-44904, 2001.

2. Matsui, T., Otsuka, M., Maenaka, K., Furukawa,
H., Yabe, T., Yamamoto, K., Nishioka, K. and Kato,
T.: Detection of Autoantibodies to Killer
Immunoglobulin-Like Receptors Using RecombinantFusion Proteins for Two Killer Immunoglobulin-Like
Receptors in Patients with Systemic Autoimmune
Diseases. Arthritis and Rheumatism. 44, 384-388, 2001.

from

H-e. Gene Network Laboratory
Yasuo SHIMKIHARA and Aya SHIRATORI
Nucleocytoplasmic exchange of macromolecules
Glutaminase (EC 3.5.1.2) catalyzes the hydrolytic
degradation of L-glutamine to L-glutamic acid. The
salt-tolerant glutaminase from marine Micrococcus
luteus K-3 has an unusual property that its maximun
activity is observed at about 2M salt, and also has
industrial relevance that this enzyme may be more
efficient than the conventional ones in the soy sauce
fermentation that is carried out in the high-salt
environments.
The enzyme was found to form good crystals thatdiffracted better than 3A, as reported last year.
Crystals of Se-Met derivative have been obtained in
the conditions similar to those for the native enzyme.
The MAD data for this intact enzyme was collected to
2.6A resolution However, the truncated enzyme,
loosing the C-terminal80 residues out of 480 residues
in the intact enzyme and being identified in the latest
preparation, formed much better crystals for native and
Se-Met derivative forms than the intact molecule. The
2.4 A resolution MAD data from the Se-Met derivative

Naoko IMAMOTO
Nucleocytoplasmic exchange is a very dynamic
activity, in which vast number of molecules enter andexit the nucleus in a rapid, accurate, and often
regulated manner. This exchange of molecules isimportant, in order for cells to maintain their
homeostasis, and adapt to their extracellularenvironment.
Since the identification of the first transport factor,
referred to as importin 0: and {3, which mediates
nuclear import of classical basic nuclear localization
signal (NLS)-containing substrates, significant
progress has been achieved toward our understandingof the mechanism of nucleocytoplasmic transport, as
well as the diversity of nucleocytoplasmic transport
pathways. The presence of so many transport
pathways obliges us to raise a naive but fundamental
question: What is the benefit to having such a

Biomolecular Structure Laboratory75complexity of nuclear transport pathways leading toregulatory changes of gene expression in vivo? In orderto understand basic mechanism of transport andbiological significance of diversity of transport
pathways, our present effort focuses on theunderstanding of the function of nuclear pore complex,
and identification of transport pathways and factorsthat function under different cellular conditions.
(1) Analysis of translocation process through nuclear
pores using single molecule imaging techniques
Naoko IMAMOTO and Makio TOKUNAGA l (iNational
Institute of Genetics, Biological Macromolecules
Laboratory)
In contrast to our growing knowledge of receptor
molecules for nucleocytoplasmic transport, functionof nuclear pore complex (NPC), as well as mechanismof translocation step of transport is still very poorly
understood. This is due to the complexity of NPC
structure, and difficulty in biochemical handling of
NPC as a functional unit. As one approach toinvestigate the function of NPC, we are trying tovisualize behavior of transport factors and transport
substrates at NPC, using modified objective-type !Otal!nternal reflection fluorescence microscopy (TIRFM).
We examined behavior of green fluorescent protein(GFP)-tagged importin f3 and GFP-tagged NLS-cargo
in digitonin permeabilized cells. Fluorescence imagesof importin f3 or cargo molecules bound to the nuclear
rim were obtained. Images were composed of many
fluorescent spots, which represented single NPC's.
Furthermore, imaging of single molecules of
GFP-importin f3 on the NPC has been also achieved.
Both imaging of single pores and single molecules
enabled us to analyze images quantitatively at themolecular level and to obtain kinetic parameters oftranslocation events, Le. retention time at the pore,number of molecules bound to pores, and binding
constant. Based on quantitative analysis of images, weare currently working to propose a model to explain
the molecular interactions of translocation events.
(2) Analysis of nuclear migration of importin 13 throughthe nuclear pore complex
1

Shingo KOSE, Hitomi OZAWA!, Yoshihiro YONEDA andNaoko IMAMOTO (lDepartment of Cell Biology and76

Gene Network Laboratory

Neuroscience, Graduate School of Medicine, Osaka
University)
Importin f3 is a nuclear transport factor, whichmediates the nuclear import of various nuclear
proteins. We have found that importin f3 shuttles
rapidly between the nucleus and the cytoplasm bydirect interaction with components of the nuclear porecomplex (NPC). However, it remains unclear howtranslocation directionality is determined.
The crystal structure of uncomplexed form ofimportin f3 has been solved. Its global feature is aright-handed superhelical structure formed byconsecutively stacked HEAT repeats. To understandthe mechanism of nucleocytoplasmic migration ofimportin f3, we made various importin f3 mutants.
These mutants were mutated in amino acids between
HEAT repeat 4B and 6A, which we have shown to benecessary and sufficient for the translocation activityof importin f3 through NPC. By transient transfection
and microinjection experiments, we observed that oneof the mutants showed inefficient accumulation to the
nucleoplasm, while the other localized within the
nucleus more efficiently than wild type.
To investigate the rate of nuclear accumulation ofthese mutants, we measured nuclear accumulation of
GFP-fusion importin f3 mutants in permeabilized cells
in real time. This assay revealed that the rate of nuclear
import of one mutant (inefficiently accumulating
mutant to the nucleus), but not the other (efficiently
accumulating mutant), is faster than that of wild type,while the rate of nuclear export of inefficiently
accumulating mutant, but not the efficiently
accumulating mutant, is faster than that of wild type.
These differences in the activity of translocation
through NPC between kportin f3 mutants may be dueto the change of affinities between importin f3 mutants
and components of NPC. The interaction between
importin f3 mutants and various nucleoporins are now
under investigation.
(3) Analysis of mechanism of nuclear import and exportof 13 - catenin
Makiko KOIKE and Naoko IMAMOTO

f3 -catenin plays a key role in Wnt signaling
pathway. Upon activation, f3 -catenin accumulates andenters the nucleus, where it modulates the activation

of the target gene expression. Therefore, it is importantto understand the mechanism of nucleocytoplasmictransport of {3 -catenin.
Our previous studies indicate that {3-catenin canenter the nucleus on its own, and shuttles between thecytoplasm and nucleus. The export of {3-catenin
examined in living cells showed that this protein exit
the nucleus in CRM1- independent and Ranindependent manner.
In order to determine the region of {3-cateninnecessary and sufficient for its import and export, wemade series of deletion constructs of {3-catenin,
expressed in bacteria, and purified as recombinant
proteins. Using digitonin-permeabilized cell free importassay, and recently developed our in vitro export assay,
we examined the ability of {3-catenin fragments to exitand enter the nucleus. We confirmed that a {3-catenin
fragment containing both Arm repeats 10-12 and the
C-terminus possesses most strong nuclear import andexport activity. The import and export activities ofthese fragments were also confirmed in living cells by
microinjection experiments. Further dissection of thisfragment is now under taken, in order to know whether
import and export activity of {3-catenin can beseparated.

microinjected into the cytoplasm of heat shock cells,
we found that hsc70 efficiently migrates into the
nucleus. From our results, we concluded that nuclear
import of hsc70 is strongly and specifically stimulated
under heat shock condition.
We next examined importin a/{3 mediated nuclear
import of SV40 T -antigen NLS under normal conditionand heat shock condition. When microinjected into thecell cytoplasm, we found that nuclear import efficiencyof SV40T-antigen NLS is clearly down regulated underheat-shock condition.
These results strongly indicate that different
transport pathways or transport factors function under
different cellular conditions. In order to elucidate these
phenomena at molecular level, we are now developing
an in vitro transport assay that mimics in vivotransport of heat shock condition and normal
condition.
(5) Molecular cloning of a novel importin a homologue
from rice, by which constitutive photomorphogenic 1(COPl) nuclear localization signal (NLS)-protein ispreferentially nuclear imported

Maiko FURUTA!, Shingo KOSE and Naoko IMAMOTO(1Department of Cell Biology and Neuroscience,
Graduate School of Medicine, Osaka University)

Chang-lie Jiang!, Kazuhiro SHOJI2 , Rikyu MATSUKI2,
Akihiki BABA!, Noritoshi INAGAKI!, Hiroshi BAN3,
Toshisuke IWASAKI3, Naoko IMAMOTO, Yoshihiro
YONEDA4, Xing-Wang DENGs and Naoki YAMAMOT0 6.
(INational Institute of Agrobiological Resources,
2Central Research Institute of Electric Power Industry,
3Niigata University, 40saka University, sYale
University, 60chanomizu University)

Heat-shock induces a strong stress response andalters gene expression drastically in cells. Investigationof nuclear transport activity of certain substrates underheat-shock condition and normal condition may
provide us an information concerning whether specifictransport pathway or transport factors function under
different cellular conditions.
We first examined the nuclear transport of 70kDa
heat-shock protein (hsp70/hsc70) under normal
condition and heat-shock condition. When injected into
the cell cytoplasm, only little amount of hsc70 migrated
into the nucleus. When injected into the cell nucleus,
only little amount of hsc70 migrated into the cytoplasm.
These results show that hsc70 possess very poor
nuclear import and export activity under normal
condition. On the other hand, when hsc70 is

Nuclear import of proteins that contain classicalnuclear localization signals (NLS) is initiated byimportin a, a protein that recognizes and binds to the
NLSs in the cytoplasm. We have cloned a cDNA fora novel importin a homologue from rice which is inaddition to our previously isolated rice importin ala
and a2, and we have named it rice importin alb. In
vitro binding and nuclear import assays using
recombinant importin alb protein demonstrated thatrice importin alb functions as a component of the
NLS-receptor in plant cells. Analysis of the transcriptlevels for all three rice importin a genes revealed thatgenes were not only differentially expressed but thatthey also responded to dark-adaptation in green leaves.
Furthermore, we also show that the COPI protein bears
a bipartite-type NLS and its nuclear import is mediated(4) Nuclear import under stress condition and normal
condition

Gene Network Laboratory77preferentially by the rice importin alb. These datasuggest that each of the different rice importin aproteins carry distinct groups of nuclear proteins, suchthat multiple isoforms of importin a contribute to theregulation of plant nuclear protein transport (for detail,see Refl).
(6) Transfonning growth factor- jj induces nuclear importof Smad3 in an importin- jj 1 and Ran-dependent manner

Akira KURISAKI\ Shingo KOSE, Yoshihiro YONEDA2,
Carl-Henrik HELDlN 1 and Aristidis MOUSTAKAS1•(1Ludwig Institute for Cancer Research, Sweden,
2Department of Cell Biology and Neuroscience,
Graduate School of Medicine, Osaka University)
Smad proteins are cytoplasmic signaling effectorsof transforming growth factor-/3 (TGF-/3) family
cytokines and regulate gene transcription in the
nucleus. Receptor-activated Smads (R-Smads) become
phosphorylated by the TGF-/3 type I receptor. Rapid
and precise transport of R-Smads to the nucleus is of
crucial importance for signal transduction. By focusing
on the R-Smad Smad3 we demonstrate that 1) only
activated Smad3 efficiently enters the nucleus
of permeabilized cells in an energy- andcytosol-dependent manner. 2) Smad3, via its N-terminal
domain, interacts specifically with importin-/31 and only
after activation by receptor. In contrast, the uniqueinsert of exon3 in the N-terminal domain of Smad2
prevents its association with importin-/31. 3) Nuclear
import of Smad3 in vivo requires the action of the Ran
GTPase, which mediates release of Smad3 from thecomplex with importin-/31. 4) Importin-/31, Ran, andplO!NTF2 are sufficient to mediate import of activatedSmad3. The data describe a pathway whereby Smad3
phosphorylation by the TGF-/3 receptor leads toenhanced interaction with importin-/3 1 andRan-dependent import and release into the nucleus.
The import mechanism of Smad3 shows distinct
features from that of the related Smad2 and thestructural basis for this difference maps to thedivergent sequences of their N-terminal domains (fordetail, see Ref2).
Publications
1. Jiang, C-J, Shoji, K., Matsuki, R., Baba, A, Inagaki,N., Ban, H., Iwasaki, T., Imamoto, N., Yoneda, Y., Deng,
X.-W. and Yamamoto, N.: Molecular Cloning of a Novel

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Gene Network Laboratory

Importin a Homologue from Rice, by which constitutive
photomorphogenic 1 (COP1) nuclear localization signal(NLS)-protein is preferentially nuclear imported. J BioI.
Chem. 276, 9322-9329, 2001.
2. Kurisaki, A, Kose, S., Yoneda, Y., Heldin, C.H. andMoustakas, A: Transforming growth factor-/3 induces
nuclear import of Smad3 in an importin-/31 andRan-dependent manner. Mol. BioI. Cell,.12,1079-1091,
2001.

using cDNA chip technology

I. CENTER FOR INFORMATION BIOLOGYAND DNA DATA BANK OF JAPANI-a. Laboratory for DNA Data Analysis(1) Expression profile ofnonnal d~velopingmouseeye
and brain
Chi Chiu WANG, Kazuho IKEO, Takashi GOJOBORI,
Michael Scott ROGERS! (!Department of Obstetrics andGyraecology, The ChineseUniversity of Hong Kong)
This is only the first phase to the project of"Developmental damage, embryonic molecular regulationin early embryogenesis in both normal and diabetic
environment". The application of comprehensive gene
expression profiling technologies by DNA microarray
to compare normal and diseased tissues/cells or toassess molecular alterations resulting from various
experimental interventions has the potential to provide
higWy detailed qualitative and quantitative information.
We have received a copy of 15K mouse cDNA clone set
from NIA/NIH in which there are more than 15k uniquesequence verifies clones amongst 52,374 ESTs from preand peri-implantation embryos. It has been successfully
adopted to employ to our high density in-house cDNA
chip systems for array experiments, including sufficientnumber of internal, quantitative, RNA amplification
probes and negative controls. Initially normal brain
and eye tissues from different gestational stage ofdevelopment have been speculated whilst E8.5 of
C57BL/6] strain was taken as reference for competitive
hybridization. Whereas the variances of transcript
regulations in forebrain, midbrain and hindbrain were
evaluated by using lasar captured microdissection
technology. In additional we occupied to use a newneural network system to analyse the expression
profiles. The findings have strong implications for ourcurrent understanding of the processes of brain andeye development and differentiation.
(2) The study of the genetic program for planarian brain

Masumi NAKAZAWA and Takashi GOJOBORIThe origin of the brain remains a challenging
problem in evolutionary studies. To understand thebrain on the viewpoint of the evolution, we analyzed
the central nervous system (CNS) of a lower invertebrate,planarian. We conducted a large-scale screening of thehead part-specific genes in the planarian by constructing
a cDNA microarray. We created a microarray of 3,555
non-redundant genes, randomly chosen from 9,000 EST
sequences. Competitive hybridization of cDNAs
between a head portion and the other body portion ofplanarians revealed 205 genes with head part-specificspikes, including homologues of synapsin, synaptotagmin
and arrestin genes essential in the vertebrate nervous
system. The expression patterns of the top 30 genes
showing the strongest spikes implied that the planarianbrain has undergone functional regionalization. These
data indicate that a brain character to be highlyorganized had already emerged by the time of planarian
divergence. Moreover with analysis of their function
by RNA interference (RNAi) method, we identified agene that restricts brain formation in the head region,
since brain-like structures expand into the entire regionof the body in the gene-knockout planarian. We arealso using the microarray in order to find out thedownstream genes of one of homeobox containing
gene, otx which is essential for head formation inhigher organisms. Our results show that planarian otx(DjoM) network contains the homologous genes related
to the eye development in higher organisms. These
results should give good insights to understand acomplex gene network of mammalian CNS.
(3) Comparative study for the CNS evolution usingneural-related genes found in the planarian EST project

Katsuhiko MINETA, Masumi NAKAZAWA, Francesc
CEBRIA, Kazuho IKEO, Kiyokazu AGATA and Takashi
GOJOBORI
In the bilateral animals, the centralized nervous
system was found in both clades of deuterostome andprotostome. It is thought that central nervous systems(CNSs) were derived from a common ancestor between
deuterostome and protostome. It is an essential
question to know what kinds of genes had existed in

CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPAN79

the CNS of the common ancestor. To answer thisquestion, we took a comparative approach using
different species, focusing on one of the lower bilateralanimals, planarian (Platyhelminthes, Tridadida) which
is known to possess the CNS. We determined thenucleotide sequence of expressed sequence tags (ESTs)from planarians, obtaining 3,101 non-redundant EST
clones. As a result of homology search, we found that
116 clones had significant similarity to the genes
related to the nervous system. Here, we conducted acomparative analysis using these planarian 116 EST
clones with all ORFs of the complete genome sequencesof human, D. melanogaster and C. elegans, showing
100%,97% and 95% of the evolutionary conservation ofthese nervous system-related genes, respectively.
Moreover, we found that about 30% of planarianneural-related genes had homologous sequences in A.
tkaliana and S. cerevisiae, implying that the origin ofneural-related genes was much older than the emergenceof the nervous system. Our results provide the firstevidence of an extreme extent of the conservation of the
CNS at the molecular level during a long-term evolution.
(4) Identification of Neurotransmitter Receptor Genes
under Significantly Relaxed Selective Constraint by
Orthologous Gene Comparisons between Humans andRodents
Hisakazu IWAMA and Takashi GOJOBORI
Neurotransmitter receptors (neuroreceptors) areclassified into two types, G-protein coupled receptors(GPCRs) and ligand-gated ion channels (LGICs). The
former occupies a small part of the large GPCR
superfamily, whereas the latter consists of three
superfamilies. In these superfamilies, humans androdents share almost the same set of neuroreceptor
genes. This neuroreceptor gene set is good material
to examine the degree 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 other member genes, they may have influenced thefunctional features characteristic of human neuralactivities. With the aim of identifying such neuroreceptor
genes, we collected sequence data of orthologous
neuroreceptor genes for humans, mice and rats bydatabase searches. This data set included orthologue
pairs for 141 kinds of neuroreceptor genes, which

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Laboratory for DNA Data Analysis

covered almost the whole set of neuroreceptor genes
known to be expressed in the human brain. The degreeof selective constraint was estimated by computing theratio (dNI ds) of the number of nonsynonymous
substitutions to that of synonymous substitutions. We
found that the dNI d s ratio ranged widely and its
distribution fitted a gamma distribution. In particular,
we found that four neuroreceptor genes are under thesignificantly relaxed selective constraint. They are an
ionotropic glutamate receptor subunit NMDA-2C, two
GABAA receptor subunits, i.e. GABAA- E and GABAAe, and a dopamine receptor D4• Interestingly, theseneuroreceptors ha-xe been reported to be associatedwith cognitive abilities such as memory and learning,
and responsiveness to novel stimuli. These cognitive
abilities can influence the behavioral features of anindividual. Thus, it suggests that the relaxed-constraint
neuroreceptor genes have evolved, assuring that thenervous system responds to a variety of stimuli withproper flexibility.
(5) Simulating interaction among multiple gene
expression with GASS (Genetic Algorithm for S-System)
model
Takaho ENDO and Takashi GOJOBORIDNA microarray technology and EST profilingprovide us a great amount of information about gene
expression and some of them trail expression change
along time. However, it is hardly possible to predict
expression network from the data because of theamount of data themselves. We adopted S-System todescribe gene network from observed data. S-System
is one of the model to simulate chemical reaction andrequires optimization of 2n(n + 1) parameters for nmolecules. The number of n should be more than 100
for expressed genes and it should be hard to solve thebecause of the number of parameters. Therefore we
programmed parallel processing program running onsupercomputer Supernig with 128CPUs (the program
uses 64 of them) and the program optimizes parameters
by GA(Genetic Algorithm) to save the calculation timewithin realistic period. This S-System simulating
program with GA is named GASS model. We used
ascidian larval expression data to evaluate how well
GASS model makes the profile of the genes and theresult was sufficient one. We would adopt GASS toother public DNA microarray data and would like topredict unknown interaction pathway among genes.
(6) Identification of apoptotic machinery in lower
metazoan phylum, platyhelminthlung-Shan HWANG, Kazuho IKEO and Takashi
GOJOBORI
The freshwater flatworm, planarian belongs to thephylum platyhelminth and it is phylogenetically placedat the base of the lineage of protostomes anddeuterostomes as according to the 18S ribosomal
sequences analysis. Planarian is also well known by its
regenerative ability in which piece of its body part canbe regrown into an entire body. Regeneration bears somesimilarities to embryonic development in that most basic
processes such as cell proliferation and differentiation,
axial polarity, and patterning of structures occur in bothphenomena. Our study concerns the identification ofprogrammed cell-death in the lower metazoan such asplanarian and the conservation of this process in theevolutionary development. It has been reported that two
apoptosis-related genes, caspase and Bcl-2 homologues,
were identified in two lower metazoan organisms, hydra
and sponge, respectively. Nevertheless, the involvementof apoptosis during morphogenesis has not yet been
documented in both cases and here we carried out anapproach to study how apoptosis taking part during
planarian regeneration and the conservation of thismachinery. In order to show apoptosis occurring inplanarian, planarian was cultured in the presence ofcolchicine and then dissociated cells were examined
by TUNEL assay. Signals due to DNA fragmentation
were clearly observed under the fluorescent
microscopy. Four full-length clones were isolated froma planarian cDNA library, three are caspasehomologues (Dj-casl, Dj-cas2 and Dj-cas3) and one is
Bcl-2 homologue (Dj-blg). Sequence alignment showedthat Dj-casl, 2 and 3 contain conserved amino acids
residues in the CASc domain (large subunit) andphylogenetic analysis indicated that all three clones
are clustered with caspase 3, 6 and 7. Similarly, twoconserved domains (BH2 and 3) were identified in
Dj-blg and they were shown phylogenetically closer
to the pro-apoptotic Bcl-2 family member. Whole mountin situ hybridization was carried out to the 2-hour,
I-day, 3-day, -5-day, 7-day amputated heads, tails andintact boP-yo Expressions of Dj-cas2 and 3 (expressionof Dj-casl is low) were observed at the blastema-forming

region of head piece from three to seven days after
cutting, while their expressions in tail piece were notspecific. Surprisingly, Dj-cas 2 and 3 were also
expressed in the brain region and these results were
further confirmed by the in situ hybridization onparaffin section. Whole mount in situ hybridization
detected the expression of Dj-blg in the stump region,
not in the blastema of the head piece at 5-7 days ofregeneration, while its expression in the tail piece is
specifically found at the region nearby intestine duct
at 3 days of regeneration. The present studies provide
evidence that the process of cell death plays a criticalrole during the regeneration of planarian and in fact
the whole apoptotic pathway may have well developed
and conserved back to the earliest multicellular animals.

(7) In Silico Chromosome Staining: Reconstruction of
Giemsa Bands from the Whole Human Genome Sequence
Yoshihito NIIMURA and Takashi GOJOBORI
Giemsa staining has been used for identifying
individual human chromosomes. Giemsa-dark (G)and -light (R) bands are generally thought to correspondto GC-poor and GC-rich regions; however, several
experiments showed that the correspondence is quite
poor. To elucidate the precise relationship between GC
content and Giemsa banding patterns, we developedan "in silico chromosome staining" method forreconstructing Giemsa bands computationally from thewhole human genome sequence. Here we show, 850-level
Giemsa bands are best correlated with the difference in
GC content between a local window of 2.5 Mb and aregional window of 9.3 Mb along a chromosome. The
correlations are of strong statistical significance foralmost all of 43 chromosomal arms. Our results clearlyshow that G bands are locally GC-poor regions comparedwith the flanking regions. These findings are consistent
with the model that matrix-associated regions (MARs),
which are known to be AT-rich, are present more densely
in G bands than in R bands.
Publications
1. Cebria, F., Nakazawa, M., Mineta, K., Ikeo, K.,
Gojobori, T. and Agata, K.: Dissecting planarian CNS
regeneration by the expression of neural-specific genes.
Dev. Genes Evol. (in press) (2001).
2. Gojobori, T.: DNA sequence analysis, inLaboratory for DNA Data Analysis

81Encyclopedia of Life Sciences. Macmillan Reference Ltd:(in press) (2001).
3. Sato, S., Tanaka, M., Miura, H., Ikeo, K., Gojobori,T., Takeuchi, T. and Yamamoto, H.: Functional
conservation of the promoter regions of vertebratetyrosinase genes.] Investig Dermatol Symp Proc. 6 (1),10-8,2001.
4. Bellgard, M., Schibeci, D., Trifonov, E. and Gojobori,
T: Early detection of G + C differences in bacterial
species inferred from the comparative analysis of the twocompletely sequenced Helicobacter pylori strains J Mol.
Evol. 53(4-5), 465-468, 2001.
5. Tsunoyama, T., Bellgard, M. and Gojobori, T:
Intragenic variation of synonymous substitution rates
is caused by nonrandom mutations at methylated CpG.
J Mol. Evol. 53(4-5), 456-464, 2001.
6. asawa, S. and Gojobori, T.: Preface. J Mol. Evol.
(Thomas Jukes special issue), 53(4-5), 257, 2001.
7. Suzuki, Y and Gojobori, T.: Positively selectedamino acid sites in the entire coding region of hepatitis
C virus subtypes lb. Gene. 276(1), 83-87, 2001.
8. Iwama, H. and Gojobori, T: [Evolution of molecules
involved with neural transmission: in the case ofcatecholamine receptors and G protein alpha subunits]
Tanpakushitsu Kakusan Koso. 46(11 Suppl), 1586-93,
2001.
9. Suzuki, Y., Gojobori, T. and Nei, M. ADAPTSITE:
detecting natural selection at single amino acid sites.
Bioinformatics. 17(7), 660-661, 2001.
10. Sato, T., Terabe, M., Watanabe, H., Gojobori, T,
Hori-Takemoto, C., Miura, K.: Codon and base biases
after the initiation codon of the open reading frames
in the Escherichia coli genome and their influence on
the translation efficiency. J Biochem. 129,851-860.
11. International Human Genome sequencing Consortium(DNA sequence databases: DNA Data Bank of Japan
et. al) Initial sequencing and analysis of the humangenome. Nature. 409(6822), 860-921, 2001.

Virus Evolution. Handbook of statistical genetics, eds.
DJ.Balding, M.Bishop, and C.Canning.]ohn Wiley &Sons. 377-413, 2001.
13. RIKEN Genome Exploration Research Group
Phase II Team and FANTOM Consortium (Okazaki,
Y, Gojobori, T, et a1.), Genelal organizer: YHayashizaki
Functional anotation of a full-length mouse cDNA
collection. Nature 409(6821), 685-690, 2001.

I-b. Laboratory for Gene-Product Informatics(1) GTOP: a database of protein structures predictedfrom genome sequences
Takeshi KAWABATA, Satoshi FUKUCHI, Keiichi HOMMA,
Motonori OTA, Jiro ARAKI l , Takehiko ITO l , Nobuyuki
ICHIYOSHI l and Ken NISHIKAWA (lMitsubishi 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 numberof sequences. Predictions are mainly carried out withthe homology search program PSI-BLAST, currently
the most popular among high-sensitivity profile searchmethods. GTOP also includes the results of otheranalyses, e.g. homology and motif search, detection oftransmembrane helices and repetitive sequences. We
have completed analyzing the sequences of 41 organisms,with the number of proteins exceeding 120000 in total.
GTOP uses a graphical viewer to present the analyticalresults of each ORF in one page in a "color-bar" format.
The assigned 3D structures are presented by Chimeplug-in or RasMo1. The binding sites of ligands arealso included, providing functional information. The
GTOP server is available at:
http://spock.genes.nig.ac.jp/-genome/gtop.htm1.
See Ref. 8 for details.
(2) Comparison of energy components of proteins fromthermophilic and mesophilic organisms
Akira R. KINJO and Ken NISHIKAWA

12. Suzuki, Y., Wyndham, A. and Gojobori, T.: 14.

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Laboratory for DNA Data Analysis

In order to infer the energetic determinants ofthermophilic proteins, molecular mechanics calculations
were applied to five proteins from thermophiliceubacteria and their mesophilic homologs. The energyfunction includes a hydration term as well as theelectrostatic contribution from the solvent in addition
to the usual conformational energy terms. We calculated
energy values for three different states of each protein:
the native, near-native, and unfolded structures. Theenergy difference and its components between pairsof these states were compared. The hypotheticalnear-native structures have almost the same backbone
conformation as the native structure but with largely
distorted side-chain packing, thus enabling us to
extract the energy components important for stabilizing
the native backbone topology itself, irrespective ofstructural details. It was found that the sum of theelectrostatic and hydration energies, although of large
positive values, were consistently lower for thethermophilic proteins than for their mesophilic
counterparts. This trend was observed in the energy
difference not only between the native and unfolded
structures, but also between the near-native andunfolded structures. In contrast, the energy components
regarding side-chain packing did not show any clear
tendency. These results suggest that the thermophilicproteins are stabilized so that the precise packing of
the native structure does not significantly affect thestability. Implications of this conclusion are also
discussed. See Ref. 2 for details.

terms of the amino acid composition of the protein
surface and the interior, as well as the entire aminoacid chains, by using sequence information from thegenome projects. The biased amino acid compositionof thermophilic proteins was confirmed, and thedifferences from those of mesophilic proteins were mostobvious in the compositions of the protein surface. In
contrast to the surface composition, the interior
composition was not distinctive between the thermophilic
and mesophilic proteins. The frequency of the aminoacid pairs that are closely located in the space was also
analyzed to show the same trend of the single aminoacid compositions. Interestingly, extracellular proteinsfrom mesophilic bacteria showed an inverse trend
against thermophilic proteins (i.e. a reduced numberof charged residues and rich in polar residues). Nuclear
proteins from eukaryotes, which are known to beabundant in positive charges, showed different
compositions as a whole from the thermophiles. These
results suggest that the bias of the amino acidcomposition of thermophilic proteins is due to theresidues on the protein surfaces, which may beconstrained by the extreme environment. See Ref. 3 fordetails.

Protein Surface Amino Acid Compositions Distinctively

A knowledge-based potential for a rotamer library
was developed to design protein sequences. Proteinside-chain conformations are represented by 56
templates. Each of their fitness to a given structuralsite-environment is evaluated by a combined functionof the three knowledge-based terms, Le. two-bodyside-chain packing, one-body hydration and local
conformation. The number of matches between thenative sequence and the structural site-environment
in the database and that of the virtually settled
mismatches, counted in advance, were transformed into
the energy scores. In the best-14 test (assessment for
the reproduction ability of the native rotamer on its
structural site within a quarter of 56 fitness rank
positions), the structural stability analysis on mutantsof human and T4 lysozymes and the inverse-folding
search by a structure profile against the sequence
database, this function performs better than the(3)

Differ Between Thermophilic and Mesophilic Bacteria

Satoshi FUKUCHI and Ken NISHIKAWA
One of the well-known observations of proteinsfrom thermophilic bacteria is the bias of the amino acidcomposition in which charged residues are present inlarge numbers, and polar residues are scarce. On theother hand, it has been reported that the molecularsurfaces of proteins are adapted to their subcellularlocations, in terms of the amino acid composition. Thus,
it would be reasonable to expect that the differencesin the amino acid compositions between proteins ofthermophilic and mesophilic bacteria would be muchgreater on the protein surface than in the interior. We
performed systematic comparisons between proteinsfrom thermophilic bacteria and mesophilic bacteria, in(4) Knowledge-based potential defined for a rotamer
library to design protein sequences

Motonori OTA, Yasuhiro ISOGAI1 and Ken NISHIKAWA(IRIKEN at Wakol

Laboratory for Gene-Product Informatics

83

function deduced with the conventional normalizationand our previously developed function. Targeting
various structural motifs, de novo sequence design was
conducted with the function. The sequences thus
obtained exhibit reasonable molecular masses andhydrophobic/ hydrophilic patterns similar to the native
sequences of the target and act as if they were thehomologs to the target proteins in BLASTP search.
This significant improvement is discussed in terms of
the reference state for normalization and the crucialrole of short-range repulsion to prohibit residue
bumps. See Ref. 4 for details.
(5) X·Ray Crystalline Structures of Pyrrolidone Carboxyl
Peptidase from a Hyperthermophile, Pyrococcus
furiosus, and Its Cys·Free Mutant
Hideaki TANAKA!, Masanobu CHINAMI 2, Tsunehiro
MIZUSHIMA1, Kyoko OGASAHARA!, Motonori OTA,
Tomitake TSUKIHARA! and Katsuhide YUTANI! (!Ins.
Protein Res., Osaka Univ., 2Kyushu Women's Univ.)
In order to elucidate the mechanism of thethermostability of proteins from hyperthermophiles,
X-ray crystalline structures of pyrrolidone carboxyl
peptidase from a hyperthermophile, Pyrococcus furiosus(PfPCP), and its mutant protein with Ser substituted
at Cys142 and Cys188 were determined at 2.2 and 2.7
A resolution, respectively. The obtained structures
were compared with those previously reported for
pyrrolidone carboxyl peptidases from a hyperthermophilie,

Thermococcus litoralis (TlPCP), and from a mesophile,
Bacillus amyloliquefaciens (BaPCP). The PfPCP
structure is a tetramer of four identical subunits similar
to that of the TIPCP and BaPCP. The largest structuralchanges among the three PCPs were detected in the
C-terminal protrusion, which interacts with that ofanother subunit. A comparison of the three structuresindicated that the high stability of PfPCP is caused
by increases in hydrophobic interactions and hydrogenbonds. the formation of an intersubunit ion-pair
network, and improvement to an ideal conformation.
On the basis of the structures of the three proteins, it
can be concluded that PfPCP does not have any
special factors responsible for its extremely highstability and that the conformational structure of
PfPCP is superior in its combination of positive andnegative stabilizing factors compared with BaPCP. See
Ref. 5 for details.

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Publications
1. Kinjo, A., Kidera, A., Nakamura, H. and Nishikawa,
K: Physicochemical evaluation of protein folds predictedby threading. Eur. Biophys. J, 30,1-10, 2001.
2. Kinjo, A. and Nishikawa, K.: Comparison of energy
components of proteins from thermophilic and mesophilicorganisms. Eur. Biophys. J, 30, 378-384, 2001.
3. Fukuchi, S. and Nishikawa, K: Protein surface
amino-acid compositions distinctively differ between
thermophilic and mesophilic bacteria. J Mol. Biol, 309,
835-843, 2001.
4. Ota, M., Isogai, Y. and Nishikawa, K.:
Knowledge-based potential defined for a rotamer
library to design protein sequences. Protein Eng., 14,557-564, 2001.
5. Tanaka, H., Chinami, M., Mizushima, T., Ogasawara,
K, Ota, M., Tsukihara, T. and Yutani, K: X-ray
crystalline structures of pyrrolidone carboxyl peptidasefrom a hyperthermophile, Pyrococcus furiosus and its
cys-free mutant. J of Biochem. (Tokyo), 130, 107-118,
2001.
6. Nakashima, H., Yamashita, S. and Nishikawa, K.:
Directional gene distributions of two mycoplasma
species exhibited in the nucleotide composition space.
Res. Comm. In Biochem., Cell & Mol Biol, 5, 27-35, 2001.
7. Noguchi, T., Ito, M., Matsuda, H., Akiyama, Y. andNishikawa, K: Prediction of protein secondary
structure using the threading algorithm and local
sequence similarity. Res. Comm. In Biochem., Cell &Mol. Bioi., 5, 115-131, 2001.
8. 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.

I-c. Laboratory for Gene Function Research(1) Evolution of the MHC class I genome region in man
and chimpanzee
Y. TATENO, K FUKAMI-KOBAYASHI, H. INOKO! and M.

YAMAZAKI 2 (lTokai University, 2Fujiya Bioscience
Institute)
MHC class I genome region contains not only
MHC genes such as A, B, C and others but also MHC
related genes such as MICA and MICB and repetitive
sequences such as LINEs and SINEs. As well known,
this region is remarkably dynamic in view of evolution.
We can observe almost every evolutionary event in thisregion; mutation, selection, transposition, duplication,
deletion and insertion. We have paid special attention
to evolutionary aspects of the Band C loci and MICA
and MICB loci. Our first attempt is to elucidate theevolutionary origin of the Band C loci and that of
MICA and MICE. The two pairs were each produced
by genome fragment duplication. To implement ourattempt we first sequenced the MHC class I regionsof man and chimpanzee. We have then confirmed thatchimpanzee has similar genome structures of the B andC loci to those of man. Interestingly, however,chimpanzee lacks the pair of MICA and MICB. Instead,it has fused one that was reformed by deletion of aregion between the MICA and MICB loci. We are now
analyzing the two MHC class I genome sequences
paying attention to LINEs included together in the
duplicate genome fragments. Those repetitive sequences
are fragmentary and thus considered to have evolved
by neutral mutation.
(2) Parallel evolution of ligand specificity between Lad/
GalR family repressors and periplasmic sugar-binding
proteins
K FUKAMI-KoBAYASHI, Y. TATENO and K NISHIKAWA

The bacterial Lad/GalR family repressors suchas lactose operon repressor (Lad), purine nucleotide
synthesis repressor (PurR) and trehalose operon
repressor (TreR) consist of not only the N-terminal
helix-tum-helix DNA-binding domain but also the
C-terminalligand binding domain that is structurally
homologous to periplasmic sugar-binding proteins.
These structural features imply that the repressorfamily evolved by acquiring the DNA-binding domain
in the N-terminal of an ancestral periplasmic binding
protein (PBP). Phylogenetic analysis of the Lacl/GalR
family repressors and their PBP homologues revealed
that the acquirement of the DNA-binding domain
occurred first in the family, and then ligand specificity

evolved. The phylogenetic tree also indicates that theacquirement has occurred only once before the divergenceof the major lineages of eubacteria, and that the Lacl/
GalR and the PBP families have undergone extensive
gene duplicationlloss along the evolutionary lineages.
Multiple alignment of the repressors and PBPs
furthermore revealed that repressors and PBPs withthe same ligand specificity have the same or similar
residues in their binding sites. The result, together withthe phylogenetic relationship, elucidated that the
repressors and the PBPs independently acquired thesame ligand specificity by homoplasious replacement,even though their genes are encoded in the same
operon.
Publications
1. International Human Genome Sequencing
Consortium. Initial sequencing and analysis of thehuman genome. Nature 409, 860-921, 2001.

2. Tateno, Y. and Gojobori, T.: The International
DNA Data Bank and bioinformatics. Estrela88, 11-17,
2001.

3. Tateno, Y. and Gojobori, T.: The DNA Data Bankand genome research. In: Genome Medical Science, S.
Sugano ed., pp 86-92, 2001, Yodo-Sha, Tokyo.
4. Fukami-Kobayashi, K and Nishikawa, K: Parallel
evolution at molecular level: Phylogenetic analysis of
the LacI/GalR family repressors and their PBP
homologues. Genes and Genetic Systems 76, 439, 2001.
5. Fukami-Kobayashi, K.: Getting functional
information from your sequence by the use of protein
signature databases. Protein, Nucleic Acid and Enzyme
46, 2098-2103, 2001.

6. Misu, S., Iizuka, T., Kawanishi, Y., FukamiKobayashi, K, Saitou, N.: Camus DB for amino acidsequence data. Genome Informatics 12, 502-503, 2001.

I-d. Laboratory for Research andDevelopment of Biological Databases(1) Research and Development for DDBJ
1) Introduction ofXML and enhancement of processing
massive data

Laboratory for Gene Function Research

85

Satoru MIYAZAKI and Hideaki SUGAWARA
It is necessary for bioinformatics to integrate

multiple information resources such as databases andanalytical tools. Thus the interoperability is one of key
issues in bioinformatics. We have applied XML
technology to DDBJ entries and find that the technology
is useful to improve the interoperability. We name the
XML DDBJ-XML. Each DDBJ entry and the whole
DDBJ entries are now available in the XML format
Last year, we could process human draft sequencesby use of the mass submission system (MSS) and
TSUNAMI system. Even after the completion of humangenome draft sequencing, the size of the International
Nucleotide Sequence Database (INSD) expanded at therate of 1.7 times a year. It exceeded 13 millions entries
and 14 Giga base-pairs in October, 2001. Thus we keep
upgrading the system for capturing, reviewing anddistributing massive data.
2) Expansion of Genome Information Broker (GIB)
Satoru MIYAZAKI, Masaki HIRAHATA and Hideaki
SUGAWARA
GIB was originally created for E. coli genome forthe retrieval and analysis of genomic information in
a set. We implemented microbial genome data into GIB
whenever genome sequencing was completed and thedata is made open to the public. At the GIB Web page(http://www.ddbj.nig.ac.jp/). key word search, homology
search, links to DBGET, KEGG and GTOP andvisualization of the data are available for 70 organisms
as of February 2002. We have utilized XML and CORBA
to integrate distributed computational resources in order
to cope with the explosion of microbial genome
information.
3) Survey on a backbone system for genomics

sponsored by Japan Science and Technology Corporation.
(2) Research and Development for WFCC-MIRCENWorld Data Centre for Microorganisms (WDC)
1) WDCM
Yumi FUJISAWA, Satoru MIYAZAKI and Hideaki
SUGAWARA
WFCC and MIRCEN stand for World Federation
for Culture Collections and Microbial Resource Centers
network respectively. The laboratory is a host of
WDCM that is the data center of WFCC and MIRCEN.
We maintain the world directory of 500 culture collectionsin 60 countries in databases of "CCINFO" and "STRAIN"
that are accessible at http://wdcm.nig.ac.jp/. We
introduced XML technology into the database and the
WDCM Web pages. The Web site now includes on-line
system for registration and updating of the database.
We also published the World Directory of Collectionsof Cultures of Microorganisms, and Technical Information
Sheet and Guidelines for culture collections in CD-ROM.
This activity was sponsored by UNESCO.
2) Development of an e-Workbench for BiologicalClassification and Identification (InforBIO)
Naoto TANAKA, Satoru MIYAZAKI and Hideaki
SUGAWARA
We have developed an e-Workbench named
InforBIO. In InforBIO, users are able to integrate
databases and analytical tools that are distributed in
the Internet including their own resources. We aim at
an open system by use of JAVA, XML, and tools of
CORBA and a relational database management systemin the public domain. We now distribute InforBIO in
CD-ROM to get feedback from microbiologists andstaffs in culture collections.

Hideaki SUGAWARA(3) Others
DDBJ is a member of the International Nucleotide
Sequence Database. Therefore, it is a mandatory task
for DDBJ to archive all the nucleotide sequence datathat are in public domain. However, it is expected that
DDBJ will contribute more to genomics. Thus wecarried out survey on a backbone system for genomics
and drew up a schema of the system. This task was

861) Biological Resources Centers (BRC)
Hideaki SUGAWARA
The Working Party for Biotechnology (WPB) of
OECD set up a task force on BRC in February1999toLaboratory for Research and Development of Biological Databases

develop a policy guidance to support BRCs. Professor
Hideaki Sugawara is the chair of the task force andcompiled a report entitled "Biological Resource
Centres: Underpinning the future of life sciences andbiotechnology". The report calls for actions in OECD
countries and beyond. The report and a follow-up task
force were approved by WPB on 16 February 2001. He
joins the new task force and now chairs a focus groupon international linkage.

Japanese), Protein, Nucleic Acid and Enzyme, 46(6),761-763, 2001.

2) Global Biodiversity Infonnation Facility (GBIF)

Hideaki SUGA W ARA
GBIF (http://www.gbif.org/) is a scheme that was
created by a Biological Informatics working group in
Mega-science Forum of OECD and the GBIF secretariat
was established in Copenhagen in 2001. GBIF will
improve the accessibility of all kinds of data of all
kinds of organisms on the globe and we will makescientific and technical contribution to GBIF.
Publications

1. Goto, K., Miyazaki, S. and Sugawara, H.: Genome
Information Broker for Data Retrieval and Comparative
Analysis of Microbial Genomes. Journal ofJapan Societyof Information and Knowledge 10, 4-13, 2001.
2. Miyazaki, S. and Sugawara, H: Visualization offeatures in the Flat File by use of DDBJ-XML. Currents
in computational molecular biology, 249-250, 2001.
3. Sugawara, H.: Information from Genomics to
Biodiversity (in Japanese), Journal ofJapan Society ofInformation and Knowledge 10(4), 1-3,2001.
4. Sugawara, H. and Miyazaki, S.: Effects of humangenome data to DNA Data Bank of Japan (DDBJ) (inJapanese), Genome Science News, 5(2), 2-7, 2001.
5. Sugawara, H: Bioinformatics-from tools to discipline(in Japanese), Seitai-no-kagaku, 52(4), 347-353, 2001.
6. Sugawara, H: Numerical Taxonomy (in Japanese),Classification and Identification of Microorganisms(eds. Suguzki, K., Hiraishi, A. and Yokota, A), 231-24,
Springer-Verlag Tokyo, 2001.
7. Sugawara, H.: Promising Bioinformatics (inLaboratory for Research and Development of Biological Databases

87the mechanism of position recognition by the Par
proteins provide key insight into how to determine
middle site in bacterial cells.

J. RADIOISOTOPE CENTER(1)

Radioisotope Center

Yoshiharu YAMAICHI, Katsunori YATA and Hironori
NIKI
Mechanisms regulating the partitioning of thepokaryotic genome. Weare studying the proteins andthe DNA sites responsible for the regulation of
pokaryotic DNA segregation using a combination ofgenetic, molecular, biochemical, cell-biological, andgenomic approaches in Escherichia coli. Prokaryotes
are not known to have a eukaryotic-like mitotic
apparatus, and little is known about the mechanisms
controlling chromosome partitioning. We visualizedbacterial chromosome DNA and plasmid DNA in cells
using fluorescence in situ hybridization (FISH) during
the cell-division cycle. We have revealed the dynamic
migration patterns of replication origin and terminuson the chromosome during active partitioning ofdaughter chromosomes.
Current work focuses on identifying thechromosomal segments involved in positioning andmigration of the chromosomal domains. To identifyfunctional region for bipolar migration of the Ori
domain, we constructed a series of new mutants
including a circular chromosome was split into two
circular chromosomes. The chromosome-split mutantsin which 220 kb of the chromosomal region [84.7-89.4
min] in the Ori domain is separated from the original
chromosome showed irregular localization of nuceloids
and were defective in bipolar migration of oriC
segments. The results suggest that a cis-acting DNA
site for chromosome positioning! migration is locatedon this chromosomal region.
Furthermore, we are investigating the segregationof plasmid with the ParABC partitioning system. We
have revealed that altered membrane structure affects
the localization or activity of a putative plasmid
partitioning apparatus located at positions equivalent
to 1/4 and 3/4 ofthe cell length. We hope elucidating

88

RADIOISOTOPE CENTER

Publication
1. Inagawa, T., Kato, ]., Niki, H., Karata, K., Ogura,T.: Defective plasmid partition in ftsH mutants ofEscherichia coli. Mol Genet Genomics. 265, 755-762, 2001.

chromosome 5. Chromosoma, 110, 284-291, 2001.

K. EXPERIMENTAL FARM

(1) Development of new experimental lines andreevaluation of the genetic stocks of rice
Ken-Ichi NONOMURA, Mitsugu EIGUCHI, Toshie
MIYABAYASHI, Nori KURATA
Production, evaluation and maintenance of ricegenetic stocks and research works employing such
resources carried out here are cooperative works
between the experimental farm and the plant genetics
laboratory. For details, see the reports of plant geneticslab.
In brief, we conducted reproduction anddistribution of rice genetic stocks of over 5,000 wild andcultivated strains. Information about these strains canbe accessed at the web site of "Oryzabase" , which
is a comprehensive rice genome resources database at
http://www.shigen.nig.ac.jp/rice/oryzabase/index.html.
From 1998, Yukihiro ITO (Plant Genetics Lab.) and wehave been developing a new rice genetic stock ofenhancer trap lines aiming to generate over ten
thousands lines, then to characterize, propagate anddistribute them as rice resources for functional
genomics.
Addition of new information and revision of the"Oryzabase" will be performed occasionally to include
more useful information for rice basic research under
the guidance of the rice genetic resources committee
in Japan (chair :N. Kurata).
Other works are more or less depending on theexperiments using plant resources; e.g. many numbersof transgenic rice, chemically induced mutant strains,
strains of insertional mutagenesis and so on. Thesematerials grown in the greenhouses and theexprimental farm are indispensable for plant genetics
and genomics.

2. Ito, Y., Eiguchi, M. and Kurata, N.: KNOX
homeobox genes are sufficient in maintaining cultured
cells in an undifferentiated state in rice. Genesis, 30,
231-238, 2001.
3. Harushima, Y., Nakagahra, M., Yano, M., Sasaki,
T. and Kurata, N.: A genome-wide survey ofreproductive barriers in an intraspecific hybrid.
Genetics, 159, 883-892, 2001.
4. 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.
5. Ahn, B. a., Miyoshi, K, Itoh, ]. 1., 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. (inpress).
6. Nonomura, K-1., Miyoshi, K, Eiguchi, M., Miyao,
A., Hirochika, H. and Kurata, N.: acdl mutant aberrant
for archesporial cell division in the anther and ovuleof rice. Rice Genet. Newsl. 18 (in press).
7. Miyoshi, K, Kurata, N. and Nonomura, K1.:
Detection of the pre-meiotic DNA synthesis in pollen
mother cells by immunofluorescence technique in rice.
Rice Genet. Newsl. 18 (in press).
8. Kurata, N., Nonomura, K-1. and Harushima, Y.:
Rice genome organization focusing on centromere andgenome interaction studies. Annals ofBotany (in press).
9. Kurata, N. and Fukui, K: Chromosome researchin genus Oryza. in Monograph in genus Oryza. Nanda
js. Ed, Springer (in press).
10. Ito, Y., Hirochika, H. and Kurata, N.: Organspecific alternative transcripts of KNOX family class
2 homeobox genes of rice. Gene (in press).

Publications
1. Nonomura, K-1. and Kurata, N.: The centromere
composition of multiple repetitive sequences on rice

EXPERIMENTAL FARM

89

ABSTRACTS OF DIARY FOR 2001
Biological SymposiumJan. 19
Using threading in protein classification and genome analysis (Anna R. PANCHENKO)
Jan. 25
Signal transduction and developmental strategies in Drosophila hematopoiesis and neuraldevelopment (Utpal BANERJEE)The evolution of gene structure (Walter GILBERT)
Jan. 29
DNA damage checkpoints and replication controls in budding yeast (Marco FOlAN!)
Feb. 1
Gene silencing by RNA in Drosophila: mechanism and applications (Richard CARTHEW)
Feb. 9Epigenetics and the plant genome (Robert MARTIENSSEN)
Feb. 13
Machine learning with Splines (Young TRUONG)
Feb. 21
Recent advance in DNA polymerases for translesion synthesis. (Haruo OHMORI)
Feb. 27
Mechanisms and consequences of genomic imprinting (Wolf REIK)
Mar. 5DNA supercoiling and transcription in Escherichia coli-the FIS connection (Andrew TRAVERS)
Mar. 8
Molecular polymorphism of 0 alleles in five populations of different ethnic origins (Antoine
Mar. 8BLANCHER)
Evolution and dynamics of centromeric histones (Steven HENIKOFF)
Mar. 9Mar. 12 Formation of new genes and new species (Chung-I WU)
Mar. 12 How was the metazoa threshold crossed: the urmetazoa (Werner E. G. MULER)
Mar. 26 The three-dimensional structure of bacteriorhodopsin (light-driven proton pump) and aquaporin-l(membrane water channel): mechanism of proton transprot. (Kaoru MITSUOKA)
Mar. 26 Knowledge discovery by use of transcriptome analysis (Kousaku OKUBO)
Using bioinformatics to find restriction enzymes (Richard ROBERTS)
Apr. 5
Apr. 10 Regulation of DNA replication in budding yeast (Seiji TANAKA)
Apr. 13 Site specific proteolysis and its regulation in cell death and Alzheimers disease: insights andapproaches using Drosophila (Ming GUO)
Apr. 25 Molecular genetic analysis using Arabidopsis thaliana and its related species-pollen tube guidance,
reproductive isolation, and morphological evolution-(Kentaro SHIMIZU)
May. 14 Development of genome ontologies and information extraction from biological literature (Toshihisa
TAKAGI)
May. 17 Glycobiology in the post-genome era (Tatsuro IRIMURA)
May. 28 Dynamics of core histones in living human cells: relationship of nucleosome stability with chromatin
activity (Hiroshi KIMURA)
May. 29 Signaling pathways that generate olfactory diversity and odor discrimination in C. elegans.(CoriBARGMANN)
June. 6 Using yeast to analyze interactions of proteins, nucleic acids and small molecules (Stanley FIELDS)
June. 8Gene expression measurement using DNA arrays: present results, future trends (Bertrand R.
JORDAN)
June. 11 The hippocampal serotonin Type lA receptor and its interaction with the membrane (Amitabha
CHATTOPADYAY)
June. 14 SOCS-l, a negative regulator of the JAK/STAT pathway, inhibits cell growth and is silenced inhepatocellular carcinoma by DNA methylation (Hirohide YOSHIKAWA)
June. 14 DNA distortion mechanism for transcription activation by a Zinc-responsive gene-regulatory protein
ZntR in E.coli (Thomas V. O'ALLORAN)
June. 19 Mechanisms underlying vertebrate endoderm development: analyses with zebrafish mutants. (Yutaka
KIKUCHI)
June. 27 The moving landscape of comparative genomics for mammals (Stephen]. O'BRIEN)
July. 2WNT signals control FGF-dependent limb initiation and AER formation in the chick embryo

90

ABSTRACTS OF DIARY FOR 2001

July. 3
July. 4
July. 5
July. 5
July. 6
July. 13
July. 16
July. 17
July. 24
July. 26
July. 30
Aug. 31
Aug. 31
Sep. 3
Sep.26
Oct. 11
Oct. 11
Oct. 18
Oct. 24
Oct. 29
Oct. 30
Nov. 1
Nov. 8
Nov. 12
Nov. 12
Nov. 15
Nov. 15
Nov. 16
Nov. 19
Nov. 22
Nov. 26
Nov. 26
Nov. 26
Nov. 28
Nov. 28(Yasuhiko KAWAKAMI)
Regulatory mechanism of the heat shock response: search for the the thermosensors (Takashi YURA)
Flies and drugs: studying drug abuse in Drosophila (Ulrike HEBERLEIN)
Molecular signaling mechanisms guiding cell migration (Yi RAO)
Comparison of the morphological segmentation along the embryonic anterposterior axis and leg
proximodistal axis of Drosophila melanogaster (Vincent STEPHANE)
The evolution of segmentation and axis formation (Nipam PATEL)
A perfect vulva every time: changes in signaling and gene function during nemetode evolution (Ralf
SOMMER)
Associative information access using DualNAVI--Is it applicable to the navigation of biologicalinformation?- (Akihiko TAKANO)
Zebrafish mutants reveal a link between pronephric cyst formation and epithelial cell polarity (Tomoko
OBARA-ISHIHARA)
Computation and annotation of human genome sequence variations (Stephen T. SHERRY)
Analysis of genomic regulation of the Dlx3-Dlx7 cluster of mouse (Kenta SUMIYAMA)
Chromosome versus genic mechanisms of hybrid sterility in mammals (Pavel M. BORODIN)
Disease centric rat gene initiative (Peter]. TONELLATO)
Molecular genetic and evolution of color vision in vertebrates (Shozo YOKOYAMA)
A molecular mechanism for cells to find their own positions (Hisao HONDA)
Spectral imaging for single cell biochemistry (Tokuko HARAGUCHI)
Signaling from the endoplasmic reticulum-toward understanding of the molecular mechanism of
the quality control of proteins (Kazutoshi MORI)
Membrane traffic as a bearer of cell structure and function: Molecular machinery of endosomal
system and autophagy (Tamotsu YOSHIMORI)
Cloning of the human telomerase gene: complete genomic sequence and analysis of tandem repeat
polymorphism in intronic regions (Leem SUN-HEE)
Vertebrate segmentation: the mechanisms underlying boundary formation and intercellular signaling(Yoshiko TAKAHASHI)
Ran tells cells where their chromosomes are (lain MATT AJ)
Epigenetic regulation of redundant genes in polyploid genomes (Jeffrey Z. CHEN)
Initiation mechanism of DNA replication in hyperthermophilic archae, Pyrococcus (Fujihiko
MATSUNAGA)
Information extraction from molecular biology journal articles (Nigel Collier)
Genetic complexity (Sydney BRENNER)
Evolutionary relationships among the yeasts from multigene phylogenetic analyses (Cletus P.
KURTZMAN)
A novel action of axon guidance molecules: semaphorin induces axoplasmic transport and its
intracellular signal transduction mechanisms in neuronal cells (Yoshio GOSHIMA)
Coupling gene expression to flagellar assembly by secretion of anti-sigma factor (Kelly HUGHES)
Roles of DNA dynamics in adaptation and variation: Changes in DNA supercoiling and DNA
double-strand breaks induced by environmental stress (Yasuyuki OGATA)
Arabidopsis quanitative genomics (Thomas MITCHELL-OLDS)
Optical and genetic approaches toward understanding spinal circuits in zebrafish (Shin-ichi
HIGASHUIMA)
Development and its application of a Drosophila gene search system (Toshiro AIGAKI)
Towards original and exciting imagings (Atsushi MIYAWAKI)
Evolution of genome size and diversity in microorganisms (Charles G. KURLAND)
Construction of a genome-wide mutant bank of Drosophila using induced type of RNAi (Ryu UEDA)
Molecular determinants for birth order identity of nerve cells-a study using Drosophila (Takako

ABSTRACTS OF DIARY FOR 2001

91

Dec. 3
Dec. 5
Dec. 7
Dec. 7
Dec. 14
Dec. 17
Dec. 25
Dec. 25
Dec. 26
Dec. 26

92

ISSHIKI)
Genome-wide screening for mutations affecting morphogenesis in small fishes (Makoto SEIKI)
Molecular basis of cold adaptation-the role of membrane lipids (S. SHIVAll)
Regulation of cell migration in Drosophila (Penille RORTH)
Boundary formation in Drosophila wing development (Stephen COHEN)
Genetic pathway of eye primordium formation and optic stalk specification in zebrafish (Masaya
TAKE-UCHI)
Role of a cGMP-dependent protein kinase and sensory inputs in the regulation of body size andexploratory behavior of the nematode C. elegans (Manabi FUJIWARA)
Towards understanding of the regulatory mechanisms of perception and processing of environmental
signals (Makoto KOBAYASHI)
Toward understanding transcriptional gene silencing in Arabidopsis (Yoshiki HABU)
Introduction of a medaka transposon system into zebrafish: development of a new gene transfer/insertional mutagenesis technology (Koichi KAWAKAMI)
Fish as a model system to study early development, cell differentiation, morphogenesis and formationof fine brain structure of vertebrates (Kohei HATTA)

ABSTRACTS OF DIARY FOR 2001

FOREIGN VISITORS IN 2001
Jan. 19
Jan. 25
Jan. 29
Feb. 1Feb. 7
Feb. 9Feb. 13
Feb. 21Mar. 5Mar. 8Mar. 8Mar. 9Mar. 12Mar. 12Apr. 5
Apr. 13May. 29
June. 6
June. 8June. 11
June. 11
June. 27
July. 4
July. 5
July. 5
July. 6
July. 6
July. 16
July. 17
July. 24
July. 26
July. 30
Aug. 31
Aug. 31
Oct. 18
Oct. 29

Anna R. Panchenko, National Center for Biotechnology Information, NIH.
Utpal Banerjee, Department of Molecular Cell & Developmental Biology, University of California,
Los Angeles
Walter Gilbert, Dept. of Molecular and Cellular Biology, Harvard University
Marco Foiani, F.I.R.C. Institute of Molecular Oncology, Milan
Masazumi Tada, University College London
Richard Carthew, Department of Biological Sciences, University of Pittsburgh
Robert Martienssen, Cold Spring Harbor Laboratory
Young K. Truong, Department of Statistics and Applied Probability, National University of Singapore
Wolf Reik, Laboratory of Developmental Genetics and Imprinting, The Babraham Institute,Cambridge
Andrew Travers, MRC Laboratory of Molecular Biology
Antoine Blancher, Laboratory of Immunogenetics, University of Paul Sabatier, Toulouse
Steven Henikoff, Division of Basic Sciences, Fred Hutchinson Cancer Research Center
Chung-I Wu, Department of Ecology and Evolution, University of Chicago
Werner E.G. Muler, Institut fur Physiologische Chemie, Abteilung Angewandte Molekularbiologie,
Johannes Gutenberg-Universitat
Richard Roberts, New England Biolabs
Ming Guo, Department of Neurology, School of Medicine, University of California at Los Angeles
Cori Bargmann, HHMI, Departments of Anatomy and Biochemistry and Biophysics, Universityof California, San Francisco
Stanley Fields, Howard Hughes Medical Institute, Departments of Genetics and Medicine, Universityof Washington
Bertrand R. Jordan, CNRS Research Director (emeritus), Coordinator of Marseille-Genopole, Marseille,
FranceAmitabha Chattopadhyay, Centre for Cellular and Molecular Biology, Bangalore, India
Thomas V. O'Halloran, Dept. Chem. and Dept Biochem., Mol. BioI., Northwestern Univ.
Stephen]. O'Brien, Director, Laboratory of Genomic Diversity, National Cancer Institute-Frederick,
MD, USA
Ulrike Heberlein, Department of Anatomy, University of California San Francisco
Yi Rao, Anatomy of Neurobiology, Washington University School of Medicine
Vincent Stephane, Perrimon Lab, Department of Genetics, Harvard Medical School, Howard Hughes
Medical InstituteNipam Patel, University of Chicago, Dept. of Organismal Biology and Anatomy, Howard Hughes
Medical InstituteRalf Sommer, Department of Evolutionary Biology, Max-Planck Institute for Developmental Biology
David]. Miller, Biochemistry and Molecular Biology, James Cook University
Tomoko Obara-Ishihara, MGH Renal Unit, Harvard Medical School
Stephen T. Sherry, National Center for Biotechnology Information
Kenta Sumiyama, Department of Molecular, Cellular and Developmental Biology, Yale University
Pavel M. Borodin, Institute of Cytology and Genetics
Peter]. Tonellato, Bioinformatics Research Center and Rat Genome Database, Medical College of
Wisconsin
Shozo Yokoyama, Syracuse University
Sun-Hee, Leem, Department of Biology, Dong-A University
lain Mattaj, EMBL, Gene Expression Programme

FOREIGN VISITORS IN 200193

Oct. 30
Nov. 12
Nov. 12
Nov. 15
Nov. 12
Nov. 22
Nov. 26
Dec. 5
Dec. 7
Dec. 794

Jeffrey Z. Chen, Texas A&M University
Sydney Brenner, Molecular Sciences InstituteCletus P. Kurtzman, Microbial Genomics and Bioprocessing Research Unit, National Center for
Agricultural Utilization Research
Kelly Hughes, Department of Microbiology, University of Washington Seattle
Thomas Mitchell-aIds, Max-Planck Institute for Chemical Ecology
Shin-ichi Higashijima, State University of New York at Stony Brook
Charles G. Kurland, Department of Molecular Evolution, Evolutionary Biology Center, Uppsala
University
S. Shivaji, Centre for Cellular and Molecular Biology, Hyderabad
Pernille Rorth, Developmental Biology Programme, EMBL, Heidelberg
Stephen Cohen, Developmental Biology Programme, EMBL, Heidelberg

FOREIGN VISITORS IN 2001

AUTHOR INDEX
ABE, T

35
79

AGATA, K

AGRAWAL, G.K. ··········································4554

AHN, B.-O

AMANO, K. ···············································74
ANAHITA, A
62

63
18,19
82

ANDACHI, Y
ARAKI, H

ARAKI, J.

HASHIMOTO, K
HATA, YHATTA, M
HATTORI, M
HAYASHI, S
HAYWARD, Richard. S
HEINKE, J.
HELDIN, C. H
HIRAKI, H

ARAKI, K. ·················································30
ASANO, Y
27

HIRAMOTO, M

BABA, A. ··················································77

HIRATA, T

BAKHLANOVA, LV

17

HIROCHIKA, H.

BAN, H. ···················································77
BLIZARD, D.A
50

HIROCHIKA, R
HIROMI, Y.

BONG, Y.-SHIROSE, S

42

BONNEROT, C. ············································63
CEBRIA, F
79
CHIBA, S. ·················································63
CHINAMI, M:
84

HIROSHIMA, M
HOMMA, K
HONDA, A
HOSOYA, T

DANjO, 1. ··················································42
DENG, X.-W
77

HOTTA, Y

17
53,57,89
80
63

ICHIBA, Y.·

FAN, Yuan. ···············································62

IIZUKA, T

EGELMAN, E.H
EIGUCHI, M
ENDO, TEWBANK, J.J.

25,26
59
··········8,10
35,41,42
34
36
82,83
38
50
77
40
46

FUjISAWA, T
FUjISHIMA, H
FUJITA, N
FUjIYAMA, A.
FuKAGAWA, T
FUKAMI, K
FUKUCHI, S
FUKUDA, K
FURUSE, T
FURUTA, M
FURUUMI, H
FURUYA, M

GENGyo-ANDO, K. ······································62
GOjOBORI, T

79,80,81
59
27
63

GOTO, S
HAMADA, K
HAMAGUCHI, M
HANAKAWA, Y. ······· .. ·HARAGUCHI, S

·

···

···

·27
52

56,57
27
36
25
40
59
68
37
78
62
24
44
45,46,53
45
22,23,24,25
27
67
82
12
25
··· .. ···24,25
81
35
82
41

HARUSHIMA, Y.

HWANG, lung-Shan.;

ICHIYOSHI, N
IINO, T
IINO, Y. ·····IKEGAMI, T
IKEMURA, T
IKENAKA, K
IKEO, K······· .. ······· .. ···

···71
36
60
34,35
25
79,81

lMAI, H. ···················································1763IMAI, K

IMAI, T. ···················································22
IMAMOTO, N
INAGAKI, N

66,67,75,76,77
77

INOKO, H. ·················································85
ISHIHAMA, A.

8,9,10,11,12
ISillHARA, K
40
ISillHARA, T
69,71,72
ISHIKAWA, A
51
ISHIKAWA, H ..................................•.......... 63
ISillKAWA, Y
30
ISHIWA, S;. 49
ISOGAI, Y:
83
ITO, K
25

AUTHOR INDEX

95

ITO, T

27
61
82

ITO, Y.

25,53,54,56,57

ITO, M
ITO, MKITABAYASHI, A
KITAGAWA, T
KITAlIMA, S. ········ .. ····KITANO, T

ITOH,]. ···················································54

KIYAMA, R

IWAMA, H. ················································80

KOBAYAKAWA, Y. ····IWAMOTO, M
IWASAKI, T
IWASAKI, Y
IWATA, A
JACOBS, S.A
JIANG, C.-].
JIANG, Y.-].
JINDO, T
KAGE, T
KAKUTANI, T··

KAMAMOTO, A
KAMIMURA, Y

27
77
25
8
17
77
·
·29
29,30
30

KOBAYASHI, D·43,44,46
72
18,19

KOMINATO, Y

KOBAYASHI, M

KOIDE, T

KOIKE, M. ················································76
KOIZUMI, O
25

KONDO, S
KOSE, SKOSHIDA, S

KANO, S. ··············;,···································63
KARLSTROM, R
29

KURATA, N

KASCIUKOVIC, T

KUROIWA, A.

68
9

KATAYAMA, A
KATO,]. ·······
KATO, M·· .. ·· .. ······ .. · .. ····60······

43
40
69,71,72

KATO, YKATSURA, I.
KAWABATA, T·······KAWAKAMI, A
KAWAKAMI, M
KAWAKITA, M
KAWANISHI, Y
KAWASAKI, H
KAWASAKI, I.
KEIPER, B.D
KIHANA, T
KIMURA, M······60,82
29
72
59
36
26
62
62
27
10

KIMURA, T. ············································29,30
KINJO, A.R
82
KINOSHITA, T
KINOUCHI, M
KISHI, T
KISHIMOTO, N
KIso, M

96

AUTHOR INDEX

43
35
14
62
52

52
36
24,38
76,77,78
30
28
8
68
40
35

KOKUBO, H

35
11
40
46
37

KANEKO, M

85
41
61,62,63,64
50

KOHARA, Y

KANAYA, S

KANEGAE, H················· .. · .. · .. ······253045KODAIRA, M

KOSEKI, H

KANEDA, M····· .. ········· .. ······ .. ·52
37
31,32

KOBAYASHI-FuKAMI, K.

KANAI, M. ················································23
KANDA, E

51
30

KOSHIO, E
KUBORI, T
KUBOTA, T
KUDO, Y53,54,55,56,57,60,89
78
28,29,41
41
22
67

KURISAKI, A
KUROKI, Y
KURUSU, M
KuwAlIMA, K

KyO, S. ···················································27
LANZOV, V.A
LI, E. · .. ··LIN, X.-H

17··· .. ·········· .. ········ .. ········41
36

MAEDA, H. ·················································8
MAENAKA, K
73,74
MAKINO, S
MAKINOSHIMA, H
MASUMOTO, H
MATSUDA, Y
MATSUKI, R

49,49
9
18
41
77

MATSUMOTO, T. ·········································45
MATSUNO, M
23
MATSUZAWA, H
MIKAMI, Y
MINAKUCHI, Y
MINETA, K
MISU, S
MITA, A

58
34
61
79
36
49

MITANI, S

62

MITSUI, K

60
11
43,44
89
27
69
45,46,53
53,54
12,15
40,41

MITSUZAWA, H
MIURA, AMIYABAYASHI, T
MIYAGI, S
MIYAHARA, K
MIYAO, A
MIYOSHI, K
MIzUMOTO, K
MIZUNO, S

OGAWA, T

17

OGURA, K. ················································62
OHKURA, K
69
OISHI, A. ··················································72
OKA, A. ···················································49
OKABE, M

22,23,25
34

OKAMURA, A

OKANO, H. ················································22
OKANO, M
41

67
25

OKONOGI, A
OSATO, M

MIZUSHIMA, T. ···········································84

OSAWA, M. ···············································37

MIZUSHINA, Y. ···········································49

OTA, M

MOCHIZUKI, YOTSUKA, T

MOCHIZUKI, Y
MOHRI, A
MORI, 1.
MORI, TMORIGUCHI, K
MORIWAKI, K

63
64
71
71
60
56
50

MOTOYAMA, A. ··· .. ······················ .. ···· .. ·······41
MOUSTAKAS, A
78

MUKAI, T. ················································40
MURAKAMI, S.D
MURAMATSU, S
MURAOKA, M
NAGAI, H
NAGATO, Y
NAKAHORI, Y
NAKAMURA, M
NAKAYAMA, A
NAKAYAMA, T
NAKAZAWA, M

63
18
59
68
54,60
41
27
63
41
79

NARITA, T. ······························ .. ············29,30
NIIMURA, Y
81
NIKI, HNISHIHASHI, A
NISHIKATA, T
NISHIKAWA, K.
NISlliMURA, A
NISlliMURA, A

88
34
64
60,82,83,85
58
60

NISHINO, A. ··············································63
NISHINO, T
9
NIWA, N

22

NIWA, Y. ··································· .. ·············53
NODA, It36
NONOMURA, K.
OGASAHARA, K
OGAWA, N

53,55,89
84
42

82,83,84
41

OZAWA, H. ···:································ .. ······41,76

PARK, rH

13,14

PUJOL, N. ·················································63
PURBOWASITO, W

40

REIK, W. ··················································40
RHOADS, R.E

62

ROGERS, M.S

79

SADO, T. ··················································41
SADO, Y59
SAGA, Y

51,52
SAIGA, H
63
SAITOH, Y...............................•.................. 27
SAITOU, N
36,37,41
SAKAGUCHI, T
28
SAKAKI, Y
35,40
SAKANE, 1.
67
SAKATA-SOGAWA, K. ··· .. ·· .. ·SAKURAI, H
SAKURAI, N
SASAKI, H··· .. ·······: .. ··69
10
49
40,41

SASAKI, T. ················································45
SASAKURA, Y
63
SASAOKA, Y

52

SATO, Y. ···············································44,68
SATOH, G. ················································63
SATOH, N
63,64
SATOU, Y······ .. ·································63,64;
17
SAWADA, A
29,30
SCHIER, A.F
29
SEINO, H
14
SERIZAWA, A
54
SERIZAWA, M
61
SHIINA, N
66,67
SHIMAMOTO, N
68,69

SATTA, Y

AUTHOR INDEX

97

25,26··········63
73
61,63,64··········66
27
46
28,29
73
73,74,75
73,75
40,41
49,50
63
77
62

SHIMIZU, H
SHIMOTORI, T
SHINDOH, K
SHIN-I, T
SHINKURA, K
SHINOMIYA, H
SHINOMURA, T
SHINYA, M
SHIRAKIHARA, CSHIRAKIHARA, YSHIRATORI, A.
SHIROHZU, HSHIROISHI, T
SHOGUCHI, E
SHOJI, K
STROME, S

SUDA, C. ··················································40

46
68
24
10
63
26,55
40
18
63
47,49
38

SUGIMOTO, K
SUSA, MSUZUKI, E
SUZUKI, H
SUZUKI, M.M
SUZUKI, T
T AlIMA, S
TAK, Y.-S
TAKADA, N
TAKAGI, N
TAKAGI, T

TAKAGI, Y. ····· .. ············ .. ········ .. ·········· .. ····44
TAKAHASHI ,T
25

41
51
17
38
46

TAKAHASHI, N
TAKAHASHI, Y
TAKAHATA, N
TAKAI, T
T AKANO, M
TAKANO-SHIMIZU, T
TAKASHIMA, S

33,34,49,50
30

TAKATORI, N. ············································63
TAKEDA H
30

28,29
46
24

TAKEDA, H
TAKEDA, S
T AKIlAWA, K
TAKIZAWA-HIGASHI, C. ······· .. ·TALBOT, W.S
TANAKA, H
TANAKA, K
TANIGUCHI, M
TASHIRO, K
TATENO, Y. ··98···················25
29
84
16
59
41

················· .. ······················85

AUTHOR INDEXTHIERRY-MIEG, D

63
66,67,76
72
49
40
59
69
52
41
45

TOKUNAGA, M
TORAYAMA, I.
TOSHIMORI, K
TOYODA, A
TOYODA, H
TSUCHIYA, E
TSUDA, M
TSUDUKI, M. .
TSUGAWA, H

TSUJIMOTO, N. ···········································40

W ATANABE, K.

84
27
26,27
58
26
37
···63
59
79
43,44

WATANABE, Y········ .. ·· .. · .. ··· .. ·· .. ·············35

TSUKIHARA, T
TSUTSUMI, K
UEDA, HUEHARA, T
UMESONO, K
UMETSU, K
UTSUMI, N
WACHI, M
WANG, C.C

17
45

WEST, S.C

Wu,].

YABE, T. ················· .. ········· .. ··········· .. ·······69
YABUKI, T· .. ········ .. ·······························27

YADA, Y·········· .. · .. ·· .. ·········· .. ···· .. ·· .. ·······49
Y AGASAKI, K
50
YAMADA, L
YAMADA, Y

63,64
35

YAMAICHI, Y. ·············································88
YAMAKAWA, T ..... ···················· .. ···· ........ ····60
YAMAMOTO, F. ···········································36
YAMAMOTO, H:. 49
YAMAMOTO, K
YAMAMOTO, M

8
36

YAMAMOTO, N. ··························· .. ·············77
YAMAMOTO, S
45
YAMAO, F
YAMAZAKI, M
YAMAZAKI, Y

13,14
45,85
56,60

YANO, M. ························ .. ······ .. ··· .. ··········45
YATA, K. ···························· .. ·YOKOI, H
YOKOMINE, T
YOKOMIZO, T
YONEDA, Y.
YOSHIDA, N
YOSHIDA, S·············8,88
30
40,41
25
76,77,78
63
41

YOSHIKAWA, T. ··········································20
YOSHIMURA, A
60
YU, R.T. ··················································26

Yu, X
YUASA, 1.
YUASA, Y
YUTANI, K

1737··············································2384

AUTHOR INDEX

99

Annual Report of the National Institute of Genetics. No.52
Issued by Director-General: Yoshiki Hotta, D. Med.
National Institute of Genetics
Yata 1,111 Mishima, Shizuoka-Ken, 411-8540 Japan
Tel. 055-981-6718
Fax. 055-981-6715
Edited by Yasushi Hiromi, Hiroyuki Araki & Tetsuji Kakutani
Published by National Institute of Genetics