ISSN 0077-4995

NATIONAL INSTITUTEJAPANANNUAL REPORT
No.51

2000

Published by

THE NATIONAL INSTITUTE OF GENETICS
Mlsblma, Sblzuoka-Ken, Japan

Annual Reportof theNational Institute of Genetics
No. 51, 2000

Published by

The National Institute of Genetics, Japan

CONTENTS
General statement
Staff.......................................................................................................
Council
Advisory committee
Research activities in 2000
A.

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Department of Molecular Genetics
A-a. Division of Molecular Genetics
Monitoring of Growth'Coupled Changes in Promoter Activities
in Escherichia coliVsing Fluorescent Proteins as Reporters.
MAKINOSHIMA, H., NISHIMURA,A. and ISHIHAMA, A
Anti'sigma Factors for the a 70 and a S Subunits of Escherichia coli
RNA Polymerase. ISHIHAMA,A., DASGUPTA, D., JISHAGE, M.
andYAMAMoTO,K
Search for Class'III ({3 'contact) and,IV ({3 "contact) Transcrip'tion Factors in Escherichia coli. KATAYAMA, A, NOMURA, T.,
FUJITA, N. and ISHIHAMA,A.
Protein'protein Contact Site Mapping of E. coli Transcription
Factors on the RNA Polymerase. SITRAMAN: S., YATA, K and
ISHIHAMA, A
Determination of Intracellular Concentrations of Transcription
Factors in Escherichia coli. ISHIHAMA, A, YATA, K, KOSHIO, E.,
IWATA, A. and VEDA, S.
Novel Mode of Transcription Regulation: Simultaneous Activa'tion and Repression by a Single Transcription Factor.
YAMAMOTO, K, YATA, K and ISHIHAMA, A................................
Systematic Search for Zn 2+'binding Proteins in Escherichia coli.
KATAYAMA, A., TSUJII, A, WADA, A, NISHINO, T. and ISHIHAMA,
A

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Protein and mRNA Levels of All Twelve Subunits of RNA Poly"merase II in Schizosaccharomyces pombe. SAKURAI, H.,
KIMURA, M. and ISHIHAMA, A,.
Isolation and Characterisation of RNA Polymerase II 'AssociatedProteins in Schizosaccharomyces pombe. KIMURA, M.,
SUZUKI, H. and ISIDHAMA, A.
Search and Analysis of Transcription Factors Interacting with
Rpb7 Subunit of Schizosaccharomyces pombe RNA Poly"merase II. MITSUZAWA, H., KANDA, E. and ISHIHAMA, A.
Analysis of General Transcription Factor TFIID from Schizosac"
charomyces pombe. MITSUZAWA, H., SEINO, H., YAMAO, F.
and ISHIHAMA, A.
Differential Roles ofvRNA and cRNA in Functional Modulationof Influenza Virus RNA Polymerase. HONDA, A., ENDO, A.
and ISIDHAMA, A.
Search for Host Factors Interacting with Influenza Virus RNA
Polymerase. HONDA, A., OKAMOTO, T., KAInO, M. and
IsmHAMA, A. .
A-b. Division of Mutagenesis
Comprehensive survey and functional analysis of ubiquitin'con"
jugating enzymes in S. pombe. PARK, J.·H., SEINO, H. and
YAMAo,F
Ubiquitin'conjugating enzymes involved in mitotic cyclin
degradation. SEINO, H. and YAMAO, F.
Characterization of SCFGrrl that ubiquitinates G 1 cyclins in
Saccaharomyces cerevisiae. KISHI, T. and YAMAO, F.
A·c. Division of Nucleic Acid Chemistry
Host proteins involved in transcription of Sendai virus (SeV)
genome. MIZUMOTO, K.
Transcriptional Regulation during the stationary growth phasein escherichia coli. TANAKA, K.
B.

Department of Cell Genetics
B"a. Division of Cytogenetics

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CONTENTS
Roles ofMre11 of Saccharomyces cerevisiaein Meiotic Recombination and Mitotic Repair Reactions. TATSUDA, D., OSffiUMI,
H., OHTA, T., TOMIZAWA, J. and OGAWA, T.
Recognition of G-DNA Structure at the Yeast Telomere Ends andits Dissolution by Mre11 of S. cerevisiae. OHTA, T., TANAKA, S.,
TATsuDA, D., OSHIUMI, H. and OGAWA, T.
Domain Structure and Dynamics in the Helical Filaments Formed
by RecA and Rad51 on DNA. Yu, X., JACOBS, S. A., WEST, S.
C., OGAWA, T. andEGELMAN, E. H.
B-b. Division of Microbial Genetics
Sld3, Which Interacts with Cdc45 (Sld4), Functions for Chromosomal
DNA Replication in Saccharomyces cerevisiae. KAMIMURA, Y.
andARAKI, H.
Phosphorylation of Sld2 by cyclin-dependent protein kinase.
MAsUMOTO, H. andARA:K:I, H....................................................
Functional analysis ofSld5 and Psfl. TAKAYAMA, Y., KAMIMURA,
Y. andARA:K:I, H.
B-c. Division of Cytoplasmic Genetics
Derivation of the relationship between neutral mutation andfixation solely from the definition of selective neutrality.
TOMIZAWA, J.
Studies on Behavioral Disorders of Knockout Mice. NIKI, H.

c.

Department of Developmental Genetics
C-a. Division of Developmental Genetics
A common basis for formation of Drosophila sensory organs.
NIWA, N., OKABE, M. and HIROMI, Y.
The relationship between sensory organ identity and positional
information in Drosophila. OKABE, M. and HIROMI, Y.
Translational repression determines a neuronal potential in
Drosophila asymmetric cell division. OKABE, M. and
HIROMI, Y.
Transcriptional regulatory mechanism of a nuclear receptor,
Seven-up. MATSUNO, M., KOSE, H. and HIROMI, Y.

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Control of glial differentiation by the homeodomain protein REPO.
YUASA, Y., OKABE, M. and HIROMI, Y
46
Sprouty regulates photoreceptor neuronal number through posi'tive and negative effects on induction. IWANAMI, M. and
HIROMI, Y.
46
Systematic identification of peptide signaling molecules in Hydra.
FUJISAWA, T., HATTA, M., SHIMIZU, H., YUM, S., HARAFuJI, N.,
KOIZUMI, 0., KOBAYAKAWA, Y., MORISHITA, F. and MATSUSHITA,

047

Morphogenetic peptides, Hym-323 and Hym-346 that are involved in foot formation. HARAFUJI, N., TAKAHASHI,
T., SHIMIZU, H, HATTA, M. and FuJISAWA, T.
47
Universal occurrence ofthe vasa'related genes among metazoans
and their germline expression in Hydra. MOCHIZUKI, K.,
FUJISAWA, C. and FuJISAWA, T.

48

Digestive movements of hydra similar to peristalsis and massperistalsis in vertebrates. SHIMIZU, H. and FUJISAWA, T.... 49
Metamorphosis regulation ofreefbuilding corals by peptide hormones.
HATTA, M. and FuJISAWA, T.

49

Cob. Division of Gene Expression
MBF2 is a tissue" and stage'specific coactivator that is regu'
lated at the step of nuclear transport in the silkworm Bombyx
51mori. LIU, Q' X., UEDA, H. and HIROSE, S.
The conserved nuclear receptor Ftz-Fl is required for embryogenesis, molting and reproduction in Caenorhabditis eJegans.
AsARINA, M., ISHIHARA, T., JINDRA, M., KOHARA, Y, KATSURA, 1.
and HIROSE, S.
51
Temporally restricted expression of transcription factor BFTZF1:
significance for embryogenesis, molting and metamorphosis
in Drosophila melanogaster.

YAMADA, M., MURATA, T.,

HIROSE, S., LAVORGNA, G., SUZUKI, E. and UEDA, H.
52
Cryptosporiium parvum: Functional complementation of a parasite transcriptional coactivator CpMBFl in yeast.
LAGIER, M. J., HIROSE, S. and KEITHLY, J. S.

ZHU, G.,
53

v

CONTENTS

COco Division of Early Embryogenesis
In situ screen for novel genes expressed in the yolk syncytial
layer. SAKAGUCHI, T., KUROIWA,A. and TAKEDA, H.
55
Role ofFGFIMAPK signaling in the developing telencephalon ofzebrafish embryos. SHINYA, M., KOSHIDA, S., KUROIWA, A
and TAKEDA, H.
FGF/MAPK signaling and somite maturation in vertebrate
segmentation. SAWADA, A., SHINYA, M., JIANG, Y. -J.,
KAWAKAMI, A, KUROIWA, A and TAKEDA, H.
Molecular analysis of zebrafish midline mutant chameleon.
KAWAKAMI, A, KARLsTROM, R., TAKEDA, H., TALBOT, W. S. and
SCHIER, A. F.
EST project in medaka fish. NARITA, T., KIMURA, T. and
TAKEDA, H.
Cod. Division of Physiological Genetics
Transcriptional Modulation by Periodic Bent DNA through Chro'matin Structure. OHNISHI, Y., KATO, M., W ANAPIRAK, C. and
KIYAMA, R.

D. Department of Population Genetics
D-a. Division of Population Genetics
Genetic screens for factors involved in the notum bristle loss ofinterspecific hybrids between Drosophila melanogaster andD. simulans. TAKANO'SHIMIZU, T.
Region-dependent regulation of mutation and crossover frequencies along Drosophila chromosomes. TAKANO-SHIMIZU. T...
D-b. Division of Evolutionary Genetics
Functional analyses of centromere of higher vertebratecells. FuKAGAWA, T., OKAMURA, A, NISHIHASHI, A., MIKAMI, Y.
and IKEMURA, T.
Analysis of DNA replication timing of centromere region of mammalian artificial chromosomes. FUKAGAWA, T., NISHIHASHI,
A., IKEMURA, T., NAKANO, M. and MAsUMOTO, H.

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57
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59

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Chromosome-wide assessment of replication timing for humanchromosomes llq and 21q reveals disease-gene-rich regions.
WATANABE, Y., FUJIYAMA, A., ICHIBA, Y., SAKAKI, Y. andlKEMURA, T.
65
Codon usage and tRNA genes in eukaryotes: correlation of codon
usage diversity with translation efficiency and with CG-dinucleotide usage as assessed by multivariate analysis.
KANAYA, S., YAMADA, Y., KINOUCHI, M., KUDO, Y. andlKEMURA,
T
65
Analysis of codon usage diversity of bacterial genes with a selforganizing map (SOM) : characterization of horizontallytransferred genes with emphasis on the E. coli0157 genome.
KANAYA, S., KINOUCHI, M., YAMADA, Y., KUDO, Y. and lKEMURA,
T................................................................................................ 66
Skew and principal component analysis of prokaryotic and eukaryotjcgenomes. ICHIBA, Y., WATANABE, Y. andlKEMURA, T. 67
Programs for constructing phylogenetic trees and networks ofclosely related sequences. SAITOU, N.
67
CAMUS DB: Development of structural database for homology
search. KIKUCHI, M., MIsu, S., lMANISHI, T. and SAITOU, N. ..... 68
Gene diversity of chimpanzee ABO blood group elucidated fromintron 6 sequences. KITANO, T., NODA, R, SUMIYAMA, K.,
FERRELL, R E. and SAITOU, N.
69
Evolution of the ABO blood group gene in Japanese macaque.
NODA, R, KITANO, T., TAKENAKA, O. and SAITOU, N.
69
Evolutionary history of the Rh blood group-related genes invertebrates. KITANO, T. and SAITOU, N.
70
Genetic structure of a 2500-year-old human population in China
and its spatiotemporal changes. WANG, L., OOTA, R.,
SAITOU, N., JIN, F., MATSUSHITA, T. and UEDA, S.
70
Sequence variation in the ABO blood group gene exon 7 of chimpanzee and bonobo. SUMIYAMA, K., KITANO, T., NODA, R,
UEDA, S., FERRELL, R E. and SAITOU, N.
71

CONTENTS

vii

D-c. Division of Theoretical Genetics
Autonomous formation of spatial pattern in development.
KONDOH, S.

73

E. Department of Integrated Genetics
E-a. Division of Human Genetics
Genome analysis of the mouse 7F41F5 imprinted domain.
SASAKI, H., SUDA, C., SHIROHZU, H., PURBOWASITO, W.,
MUKAI, T., HATTORI, M. and SAKAKI, Y. .
Regulation of imprinting of the mouse Igf2/H19 sub-domain.

75

SASAKI, H., ISHIHARA, K., FURUUMI, H., OHNO, M., UEDA, T.,
KOMINAMI, R. and UMEZAWA, A.
Role for de novo DNA methyltransferases Dnmt3a/Dnmt3b ingenomic imprinting.

75

SASAKI, H., TSUJIMOTO, N., KANEDA, M.

and TAJIMA, S.
de novo DNA methyltransferases Dnmt3a/Dnmt3b and humandisorders. SASAKI, H., SHIROHZU, H., MIZUNO, S., KUBOTA, T.
and TAJIMA, S.
Studies on the possibilities of genomic imprinting and Z chromosome dosage compensation in chicken. SASAKI, H., YOKOMINE, T., KUROIWA, A., MATSUDA, Y. and TSUDUKI, M.
Are the polycomb group genes involved in genomic imprinting?
SASAKI, H., TSUJIMOTO, N. and KOSEKI, H.
Analysis of anti-sense RNA identified in the Xist region.
SADO, T., SASAKI, H. andLI, E.
Human genome resources and their application to the humanand primate genome analysis. FuJIYAMA, A., MOTOYAMA, A.,
YOSHIDA, S., KUROKI, Y., NAKAHORI, Y., NAKAYAMA, T., KODAIRA,
M., TAKAHASHI, N. and SAITOU, N.
Whole genome analysis of signal-transduction pathways in fissionyeast. BONG, Y.-S., DANJO, 1., OGAWA, N. andFuJIYAMA, A.
E-b. Division ofAgricultural Genetics
Developmental abnormalities induced by DNA hypomethylation

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mutation of Arabidopsis. KAKUTANI, T., MIURA, A. and
WATANABE, K
DNA hypomethylation mutation in rice. KAKUTANI, T., WATANABE,
K. and MIURA, A.
E-c. Division of Brain Function
Origin and Migration of Guidepost Neurons in the Lateral Olfac"
tory Tract. TOMIOKA, N., OSUMI, N., SATO, Y., FUJISAWA, H.
and HIRATA, T,
Short- Range and Long" Range Guidance of Olfactory Bulb
Axons. HIRATA, T., FuJISAWA, H., Wu, J. Y. and RAo, Y.
E-d. Division of Applied Genetics
Analysis of genomic imprinting involved in murine X chromo"
some inactivation. TAKAGI, N.
Developmental abnormalities in tetrasomy 11 mouse em"bryos. TAKAGI, N.
Molecular analysis of the NAC gene family in rice. KIKUCHI, K,
UEKUCHI"TANAKA, M., YOSHIDA, K, NAGATO, Y., MATSUOKA, M.
and HIRANO, H.
Characterization of viviparous mutants in rice (Oryza sativa LJ
MIYOSHI, K, NAKATA, E. and NAGATO, Y.
SHOOT ORGANIZATION genes regulate shoot apical meristem
organization and the pattern of leaf primordium initiation inrice. ITOH, J- I., KITANO, H., MATSUOKA, M. and NAGATO, Y. ....

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R Genetic Strains Research Center
F-a. Mammalian Genetics Laboratory
Molecular dissection of the critical region of a mouse preaxial
polydactyly mutation, Hemimelic extratoes (Hx). SAGAI, T.,
MASUYA, H., SHIMIZU, K, TAMURA, M. and SHIROISHI, T.
Analysis of a spontaneous mouse mutation, mesenchymal dyspla"sia(me~. MAKINO, S., MAsUYA, H., YADA, Y. andSHIROISHI, T.
Analysis oftwo preaxial polydactylous mouse mutations, X"linked
polydactyly(Xp.b and luxate W. YADA, Y.,MAsUYA, H., MAKINO,
S. and SHIROISHI, T.

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CONTENTS

ix

Male sterility of a consomic strain B6. MSM·Chr.X and fine map'ping of the X'linked gene responsible for the sterility. OKA, A,
TAKAGI, N., TOSHIMORI, K, MITA, A, MIZUSHINA, Y, SAKURAI, N.
and SHIROISHI. T.
Genetic modification of the phenotypes of a mouse progeria muta'tion, Klotho (k1). JINNAI, N., MITA, A and SHIROISHI, T.
Mapping of Genes Responsible for the Performance in the Passive
Avoidance Test Using Strains Derived from Wild Mice.
KOIDE, T., FURUSE, T., TAKANO, T., MORIWAKI, K., andSHIROISHI, T.
An analysis of sensitivity to capsaicin in the Mishima battery ofmouse strains. FURUSE, T., BLIZARD, D. A, MORIWAKI, K,
MIURA, Y., YAGASAKI, K, SHIROISHI, T. and KOIDE, T.
Application of cryopreservation for wild mice embryos.
SAKURAI, N., MIzuSHINA, Y., JINNAI, N., NAKAGATA, N., KOIDE, T.
and SHIROISHI, T.
F·b. Mammalian Development Laboratory
Molecular mechanism of somite segmentation. SAGA, Y.,
TAKAHASHI, Y., TAKAGI, A andKITABAYASHI, A........................
Function ofMesp1 and Mesp2 for heart formation. SAGA, Y. and
KITAJIMA, S.
Transcriptional regulation ofMesp1 and Mesp2 genes. SAGA, Y.
andHARAGUCHI, S.
F·c. Plant Genetics Laboratory
Genetic dissection of embryogenesis, regeneration and gameto'
genesis of rice (Oryza sativa)(l)·a. Regulation for expression of KNOX family class 1 homeobox
genes of rice. ITO, Y., NIWA, Y and KURATA, N.
(l)·b. Alternative splicing ofKNOX family class 2 homeobox genes

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103

ofrice. ITO, Y., HIROCHIKA, H. and KURATA. N.
(l)·c. Isolation and sequence analysis ofreceptor-like protein kinase104

genes ofrice. TAKAYA, K, ITO, Y and KURATA, N.
(I)·d. Functional analysis of OsHAP3'] using transgenic rice.
EIGUCHI, M., MIYOSHI, K andKURATA, N.

104
ITO, Y.,105x(I)-e. Analysis of genetic regulation of meiosis and gametogen'esis in rice. NONOMURA, K- I., MIYOSHI, K, HIROCHIKA, H. and
KURATA,N.
Positional cloning of a heterochronic gene, PIal, regulating
the plastochron and the duration of vegetative phase inrice. AHN, B. '0., MIYOSHI, K, ITOH, J.I., NAGATa, Y. and
KURATA,N.
Analysis of centromere structure of rice chromosome 5 toward
construction of plant artificial chromosome. NONOMURA, K·
I., SUZUKI, T. and KURATA, N.
A large scale isolation and characterization of rice nuclear pro'tein genes. MORIGUCHI, K and KURATA, N.
Genome'wide analysis of reproductive barriers in the intra'spe'cific rice hybrids and positional cloning of one of the barriers.
(5)·a. Quantitative Analysis of genotype segregation for repro'
ductive barriers. HARUSHIMA, Y. and KURATA, N.
(5)·b. Positional Cloning of a Segregation Distortion Gene De'
tected in a Progeny of a Cross between japonica and indica
rice. HARUSHIMA, Y. and KURATA, N..
Generation of enhancer trap lines of rice. ITO, Y., EIGUCHI, M.
and KURATA, N.
F·d. Microbial Genetics Laboratory
Timing of cell division in Escherichia coli. NISHIMURA, A. ........
HscA is involved in the dynamics of FtsZ'ring formation in Es'
cherichia coli K12. UEHARA, T., MATSUZAWA, H. andNISHIMURA, A.
Systematic analysis of novel cell division genes in Escherichia coli.:
Post genome project. NAKADE, S., SARA, K, MATSUMOTO, K,
KITAGAWA, M., MORI, H. and NISHIMURA, A.
.
F-e. Invertebrate Genetics Laboratory
Control of tracheal tubulogenesis by Wingless signaling.
CHIHARA, T. and HAYASHI, S.
EGF receptor attenuates Dpp signaling and helps to distinguish
the wing and leg cell fates in Drosophila. KUBOTA, K, GOTO,

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107
108

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110
112

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CONTENTS

Xl

S., ETO, K and HAYASHI, S.

116

Repression of the wing vein development in Drosophilaby the nuclear matrix protein Plexus. MATAKATSU, H.,
BRENTRUP, D.andHAYASHI, S

.

116

.

117

Enhancer trap screen for genes involved in pattern formation.
GOTO, S., TANIGUCHI, M., SADO, Y and HAYASHI, S

G. Center for Genetic Resource
G-a. Genetic Informatics Laboratory
ORYZABASE--INTEGRATED RICE SCIENCE DATABASE-YAMAKAWA, T., MITSUI, K, KURATA, N., YOSHIMURA,A, NAGATO,
Y. and YAMAZAKI, Y.

PEC : Profiling of Escherichia coli Chromosome.

118

YAMAZAKI, Y,

IKEGAMI, T., YAMAKAWA, T., MITSUI, K., KAWABATA, T.,NISHIKAWA, K, MORI, T., NISHIMURA, A and RATa, J.

119

G-b. Genome Biology Laboratory
NEXTDB : The nematode expression pattern database.
T. and KOHARA, Y

SHIN-I,.

The worm transcriptome project.

120

THIERRY-MIEG, J. and D.,

SUZUKI, Y, SUGANO, S., OISHI, K, SANO, M., NOMOTO, H., HAGA,
S., NISHIZAKA, S., HAYASHI, H., OHTA, F., MIURA, S., DESUGI,
H., POTDEVIN, M., THIERRY-MIEG, Y, SIMONYA, v., LOWE, A.,
SHIN-I, T. and KOHARA, Y.

121

Open-reading-frame sequence tags (OSTs) support the existenceof at least 17,300 genes in C. elegans.

REBOUL, J., VAGLIO,

E, RUAL, J. F., THIERRY-MIEG, N., MOORE, T., JACKSON, C., SHINI, T., KOHARA, Y, THIERRY-MIEG, D. andJ., LEE, H., HITTI, J.,
DOUCETTE-STAMM, L., HARTLEY, J. L., TEMPLE, G. F., BRASCH,
M. A, VANDENHAUTE, J., LAMESCH, E E., HILL, D. E. and VIDAL,

M

.

122

Genome-wide functional analysis by RNAi-by-soaking with a nonredundant cDNA set.

MAEDA, I., MINAMIDA, A, YAMAMOTO,

M., KOHARA, Y and SUGIMOTO, A.

Expression based RNAi reveals the function of proteasome in

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oocyte maturation and fertilization in C. elegans.

HIRONO,K, NAKATA, K, VETA, Y, IWATA, M., ITO, M. and KOHARA, Y ... 125
Translational control of maternal glp-] mRNA by POS-l and its
interacting protein PIP-1.

OGURA, K, MITANI, S., GENGYO-

ANDO, K. and KOHARA, Y.

126

Computer Simulation of Early Cleavage of C. elegans Embryo.
KAJITA, A, YAMAMURA, M. and KOHARA, Y.
PGL-l, PGL-2 and PGL-3, a family ofP-granule proteins, func-

127tion redundantly to ensure fertility in both sexes of C. elegans.
KAwASAKI, 1., AMrRI, A, FAN, Y, KARASHIMA, T., KOHARA, Yand
STROME, S.

128

Identification of inducible innate immune defences in C. elegans.
MALLO, G., KURZ, C. L., GRANJEAUD, S., KOHARA, YandEwBANK,J................................................................................................
Functional analysis of the C. elegansT-box gene tbx-9.

ANDACHI,

Y.
H.

130
130

Structural Biology Center
H -a. Biological Macromolecules Laboratory
Single Molecule Imaging in cells.

TOKUNAGA, M.

132

Single molecule imaging of nucleocytoplasmic transport.
TOKUNAGA, M. and lMAMOTO, N.

132

Characterization ofXpl05-Xp65 complex gathered around thecentrosome in Xenopus.

SHIINA, N., SHINKURA, K. and

TOKUNAGA, M.

133

Single molecule measurement of intermolecular and intramolecular interactions'using subpiconewton intermolecular force
microscopy. HIROSHIMA, M. and TOKUNAGA, M.
134
Single molecule measurement of protein folding by intermolecularforce microscopy.

SAKANE, 1., HIROSHIMA, M., KUWAJIMA, K

and TOKUNAGA, M.

134

Single molecule measurement of nucleocytoplasmic transport
at nuclear pores.

OKONOGI, A, HIROSHIMA, M., SHIINA, N.,

KOSE, S., lMAMOTO, N. and TOKUNAGA, M.

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H-b. Molecular Biomechanism Laboratory
The promoter arrest of E. coli RNA polymerase and the effect of
the Gre factors. SUSA, M., KUBORI, T., NAGAI, H., GAAL, T.,
MISKELISHIVILI, v.G. and SHIMAMOTO, N.
Role of w subunit of E. coli RNA polymerase.

136
CHATTERJI, D.,

MUKHERJEE, A., NAGAI, H. and SHIMAMOTO, N.
Inactivation of a 70 by oligomerization and identification of therole of its spacer region. NAGAI, H., KASCIUKOVIC, T., HAy·137

WARD, R.S., SATO, y. and SHIMAMOTO, N.
Single-Molecule Dynamics of Transcription : Sliding of pro'teins along DNA. KINEBUCHI, T., SOGAWA, K, KABATA, H.,

138

SHIMAMOTO, N., KUROSAWA, 0., ARAMAKI, H. and WASHIZU, M...
H-c. Multicellular Organization Laboratory

139

Fluoride-resistant Mutants ofthe Nematode Caenorhabditis elegans.
OISHI, A., ISHIHARA, T. and KATSURA, I.
Analysis of Synthetic Dauer-constitutive Mutations.

140

MIYAHARA, K, OHKURA, K, YABE, T., IsHIHARA, T. and KATsuRA, I. ...
Analysis of Mutants That Show Abnormality in the Selection
between Two Behaviors and in Behavioral Plasticity.

141

ISHIHARA, T., IINO, Y. and KATSURA, I.
H-d. Biomolecular Structure Laboratory
Crystallographic Study ofF1-ATPase.

143
SHIRAKIHARA, Y. and

SHIRATORI, A.
X-ray crystallographic analysis of repressor protein CamR.
MAENAKA, K. and SHIRAKIHARA, Y.
Crystallographic Study of the Transcription Activator, PhoB.

146

SHINDO, K, MAENAKA, K and SHIRAKIHARA, Y.
Crystallization of E.coli nucleoid proteins. SHIRAKIHARA, Y.

148

and SHIRATORI, A.
Crystallization of the Drosophila GAGA factor.

149

147

MAENAKA, K

and SHIRAKIHARA, Y.
Structural and functional studies ofimmunoglobulin(Ig)'like reo
ceptors in immune system. MAENAKA, K. and SHIRAKIHARA,

149

Y................................................................................................

150

XIV

Crystallization of Na+-translocating ATPase.

SHIRAKIHARA, Y. ...

Crystallization ofD-aminoacylase from Alcaligenes. SHIRAKIHARA,Y.
X-ray crystallographic analysis of the anti-tumor enzyme, arginine deiminase from Mycoplasma argini. MAENAKA, K and
SHIRAKIHARA, Y.
H-e. Gene Network Laboratory

151
152

152

Nucleocytoplasmic exchange of macromolecules. IMAMOTO, N...
Single molecule imaging of nucleocytoplasmic transport at153

nuclear pores. IMAMOTO, N. and TOKUNAGA, M.
Analysis of crystal structure of the uncomplexed form of importin {3 .

154

LEE, S. J., IMAMOTO, N., SAKAI, H., NAKAGAWA, A., KOSE, S.,
KOIKE, M., YAMAMOTO, M., KUMASAKA, T., YONEDA, Y. and
TSUKIHARA, T.
Mechanism of nucleocytoplasmic translocation of importin- {3 •155

OZAWA, H., KOSE, S., KATAHIRA, J., TACHIBANA, T., HIEDA, M.,
SAKAI, H., TSUKIHARA, T., IMAMOTO, N. and YONEDA, Y.
Analysis of nuclear export of importin {3 through nuclear pores.

155

KOSE, S., YONEDA, Y. and IMAMOTO, N.
Analysis of nuclear export of {3 -catenin.

156
KOIKE, M. and

IMAMOTO, N.
Stress mediated nuclear import of 70kDa heat shock protein.
FURUTA, M., KOSE, S. and IMAMOTO, N.

157
158

I. Center for Information Biology
I-a. Laboratory for DNA Data Analysis

In silica chromosome staining: Reconstruction of Giemsa bands
from the whole human genome sequence.

NIIMURA, Y. and

GOJOBORI, T.
The evolutionary origin of brain structure characterized by gene160

expression profile: A cytoarchitectonic map of the planarianbrain defined by cDNA microarray.

NAKAZAWA, M., CEBRIA,

F., MINETA, K, IKEo, K, AGATA, K and GOJOBORI, T.
SNP profiles of the human subgenomic regions. OGASAWARA, M.,

160

CONTENTSGAUDIERI, S., lMANISHI, T. and GoJOBORI, T.
Study of molecular evolution in vsxgene families. UCHIYAMA, Y,
lKEo, K. and GOJOBORI, T.
Evolutionary features of the gene expression profile of planarianbrain. MINETA, K, lKEO, K and GoJOBORI, T.
Establishment of integrated human microsatellite database.
ENDo, T. and GoJOBORI, T.
Higher evolutionary rates in the contact regions between
catecohlamine receptors and G proteins. IWAMA, H. and
GOJOBORI, T.
Does apoptosis play an active role in the regeneration ofplanarian? HWANG, J. S. and GOJOBORI, T.
The base compositional localisation of genes in the human genome sequence. T. DANIEL, ANDREWS and GoJOBORI, T.
I-b. Laboratory for Gene-Product Informatics
Structural/functional assignment of bacteriophage T4 unknown
proteins by iterative database searches. KAwABATA, T.,
ARISAKA, F. and NISHIKAWA, K
Protein structure comparison using the Markov transition matrix of evolution. KAWABATA, T. and NISHIKAWA, K
Redesign of artificial globins: Effects of residue replacements athydrophobic sites on the structural properties. ISOGAI, Y,
ISHII, A., FUJISAWA, T., OTA, M. and NISHIKAWA, K
The genomic DNA sequences of various species are distinctively
distributed in nucleotide composition space. NAKASHIMA, H.
and NISHIKAWA, K
Physico-chemical evaluation of protein folds predicted bythreading. KINJO, A. and NISHIKAWA, K
I -c. Laboratory for Gene Function Research
Evolution of the HLA class I region in human genome.
TATENO, Y, INOKO, H. and YAMAZAKI, M.
Evolution of protein structure in the periplasmic binding protein(PLBP)superfamily. FUKAMI-KOBAYASHI, K, TATENO, Y
and NISHIKAWA, K. ..

xv
161
162
162
163

164
164
165

167
168

169

170
171

172

173

xvi

Compensatory covariation in protein evolution. FUKAMIKOBAYASHI, K. and BENNER, S. A. .
DNA Data Bank of Japan (DDBJ) in collaboration with masssequencing teams. TATENO, Y, MIYAZAKI, S., OTA, M.,
SUGAWARA, H. and GOJOBORI, T.
I -d. Laboratory for Molecular Classification
Research and Development for DDBJ(I)-a. From YAMATOII to TSUNAMI(I)-b. Expansion of Genome Information Broker (GIB)(I)-c. Application ofXML to DDBJ
Research and Development for WFCC-MIRCEN World Data
Centre for Microorganisms (WDCM)(2)-a. WDCM(2)-b. Development of a Workbench for Biological Classification
and Identification (InforBIO)
Others(3)-a. Data processing system for patent sequences(3)-b. Biological Resources Centres (BRC)(3)-c. Global Biodiversity Information Facility (GBIF)

173

174

175
175
175

176
176
176
177
177

J. Radioisotope Center
Expression and Purification of Escherichia coli Transcription
Factors. YATA, K. and ISIDHAMA, A.
179
K.

Experimental FarmDevelopment and reevaluation of genetic stocks of rice.
NONOMURA, K., EIGUCHI, M., MIYABAYASHI, T., ITo, Y. and
KURATA, N

Abstracts of diary for 2000
Foreign visitors in 2000
Author index

180
181
185
187

GENERAL STATEMENT

National Institute of Genetics (NIG) was established 51 years ago as a centerfor genetics research. Major contributions of NIG in the past, especially inplant genetics, population and evolution genetics, molecular and developmen"
tal genetics, have made it one of the distinguished centers with worldwide
recognition. In 1984, NIG was reorganized into an Inter-university ResearchInstitute to promote collaborative activities. Together with seven inter-uni"
versity research institutes, we founded the Graduate University for Advanced
Studies in 1988. We are serving as Department of Genetics in the Graduate
School of Life Science. This year, we have 37 graduate students and 10 special
research students from other universities, including those from abroad. Eightstudents obtained Ph.D. this year. In addition, we have the Center Of Excellence (COE) program with which 9 foreign and 15 Japanese postdoctoral fel"lows actively participated in research.
We have been carrying out several research-related services. The DNA Data
Bank of Japan (DDBJ) is one ofthe three central data banks in the world thatgather, annotate, store and distribute information related to DNA sequences.
In recent years, it receives data input not only from Japanese institutions but
also from institutes in other Asian countries. We have also established Genetic Strains Research Center and Genetic Resource Information Center thatare designed to organize and support the use of genetic strains and resources.
Our institute also stores and distributes various organisms with geneticallycharacterized traits. Among them, services with Escherichia coli, mice andDrosophila are particularly significant. These service activities will continue
to develop in the coming years. Our institute is uniquely suited for pursuingcooperative work with scientists of various disciplines by sharing the genetic
resources.
In the past year we saw a number of changes in the NIG staff members. Prof.
Akio Murakami retired at the end of March. We would like to express ourgratitude for his contribution to our institute. Newly appointed as professors
are Drs. Yumiko Saga (Mammalian Genetics Lab.), Tetsuji Kakutani (Div. Of
Agricultural Genetics), Naoko Imamoto (Gene Network Lab.), Hironori Niki
(Radioisotope Center) and Hitoshi Ueda (Div. Of Gene Expression>. Drs. Atsushi
Kawakami, Katsumi Maenaka, Kumiko Sogawa, Nobuyuki Siina, Shingo Kose
are appointed as research associates. On the other hand, the following members left NIG to take new positions. Among them are Drs. Tetsuichiro Saito(Kyoto Univ.), Takasi Tada (Kyoto Univ.) , Tsutomu Ohta (National Cancer
Center) and Masayuki Hatta (Ochanomizu Univ.).
Japanese Government is planning to reorganize national universities andnational institutes. We are willing to be engaged in the process actively tomake the reform appropriate for better academic achievement.

Yoshiki Hotta, Director-General

3

STAFF

(as of December 31,2000)

Director-General
HOTTA, Yoshiki, D. Med.

Vice-Director
OGAWA, Tomoko, D. Pha.

Members
1. Department of Molecular Genetics

ISHIHAMA, Akira, D. Se., Head of the Department
Department ofMolecular Genetics
ISHIHAMA, Akira, D. Se., Professor
FUJITA, Nobuyuki, D. Se.
MITSUZAWA, Hiroshi, D. Se.
KIMURA, Makoto, D. Se.
Division ofMutagenesis
YAMAO, Fumiaki, D. Se.,Assoeiate Professor
KISHI, Tsutomu, D. Eng.
SEINO, Hiroaki, D. Se.
Division ofNucleicAcid Chemistry
MIZUMOTO, Kiyohisa, D. Se., Adjunct Professor
TANAKA, Kan, D. Ag, Adjunct Associate Professor

2. Department of Cell Genetics
ARAKI, Hiroyuki, D. Se., Head of the Department
Division ofCytogenetics
OGAWA, Tomoko, D. Pha., Professor
lMAI, Hirotami, T., D. Se., Associate Professor
TANAKA, Shigeo, D. Med.
OHTA, Tsutomu, D. Med.
Division ofMicrobial Genetics
ARAKI, Hiroyuki, D. Se., Professor
YASUDA, Seiiehi, D. Se., Associate Professor

4

KAMIMURA, Yoichiro, D. Med.

Division of Cytoplasmic Genetics
TOMIZAWA, Jun-ichi, D. Pha., Adjunct Professor
NIKI, Hiroaki, D. Med. Sci., Ph. D. Adjunct Professor
3. Department ofDevelopmental Genetics

HIROSE, Susumu, D. Sc., Head ofthe Department

Department ofDevelopmental Genetics
HIROMI, Yasushi, D. Sc., Professor
FUJISAWA, Toshitaka, Ph. D., Associate Professor
SHIMIZU, Hiroshi, D. Eng.
OKABE, Masataka, M. D., Ph. D.
HOSOYA, Toshihiko, D. Sc.

Division ofGene Expression
HIROSE, Susumu, D. Sc., Professor
UEDA, Hitoshi, D. Ag., Associate Professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-aki, D. Ag.

Division ofEarly Embryogenesis
TAKEDA, Hiroyuki, D. Sc., Professor
KAWAKAMI, Atsushi, D. Sc.

Division ofPhysiological Genetics
KrYAMA, Ryoichi, D. Sc., Adjunct Professor
SHIRAKAWA, Masahiro, D. Ind., Adjunct Associate Professor

i. Department ofPopulation Genetics
IKEMURA, Toshimichi, D. Sc., Head of the Department

Division ofPopulation Genetics
TAKANO, Toshiyuki, D. Sc.

Division ofEvolutionary Genetics
IKEMURA, Toshimichi, D. Sc., Professor
SAITOU, Naruya, Ph. D., Associate Professor
TENZEN, Toyoaki, D. Ag.
FUKAGAWA, Tatsuro, D. Sc.

STAFF

Division of Theoretical Genetics
KONDo, Shigeru, Ph. D. D. Sc., Adjunct Professor
KITANO, Hiroaki, D. Eng., Adjunct Professor
5. Department ofIntegrated Genetics

SASAKI, Hiroyuki, D. Med., Head ofthe Department

Department ofHuman Genetics
SASAKI, Hiroyuki, D. Med., Professor
FUJIYAMA, Asao, D. Sc., Associate Professor
SADO, Takashi, D. Sc.

Division ofAgricultural Genetics
KAKUTANI, Tetsuji, D. Sc., Associate Professor

Division ofBrain Function
HIRATA, Tatsumi, D. Med., Professor
KAWASAKI, Takahiko, D. Sc.

Division ofBrain Function
TAKAGI, Nobuo, D. Sc., Adjunct Professor
NAGATO, Yasuo, D. Ag., Adjunct Professor

6. Genetic Strains Jlesearch 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

Plant Genetics Laboratory
KURATA, Nori, D. Ag., Associate Professor
ITO, Yukihiro, D. Ag.

Microbial Genetics Laboratory
NISHIMURA, Akiko, D. Ag., Associate Professor

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

7. Center for Genetic Resource In/ormation
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. StructuralBlolog Center
KATSURA, Isao, D. Sc., Head ofthe 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., Professor
KOSE, Shingo, D. Med.
9. Center for Information Biolog
GOJOBORI, Takashi, D. Sc., Head of the Center

Laboratory for DNA Data Analysis
GOJOBORI, Takashi, D. Sc., Professor
IKEO, Kazuho, D. Sc.
IMANISHI, Tadashi, D. Sc.

STAFF

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
FUI{AMI-KOBAYASill, Kaoru, Ph. D.

Laboratory for Molecular Classification
SUGAWARA, Hideaki, D. Eng., Professor
MIYAZAKI, Satoru, D. Sc.
10.

Radioisotope Center

ISillHAMA, Akira, D. Sc., Head ofthe Center
NIKI, Hironori, D. Med.
11.

Experimental Farm

KURATA, Nori, D. Ag., Head ofthe FarmNONOMURA, Ken-ichi, D. Ag.
12.

Technical Section

ISHII, Yuriko, Chief of the Section
13.

Department 01 Administration

UEZUMI, Kiyotaka, Head of the Department
KOBAYASill, Akira, Chief of the General Affairs Section
TAKAHASHI, Shouji, Chief of the Finance Section

7

8

COUNCIL(as of December 31,2000)

Chairman
SUGIMURA, Takashi; President, Emeritus, National Canser Center

Vice-chairman
OSAWA, Shozo; Adviser, Biohistory Research Hall

Members <Alphabetical order)
HIROBE, Masaaki; President, University of Shizuoka
HIROTA, Eiji; President, The Graduate University for Advanced Studies
ISHII, Shiro; Professor, The International Research Center for JapaneseStudies
ITO, Mitsuo; President, Okazaki National Research Institutes
IWATSUKI, Kunio; Professor, University of the Air
KURODA, Reiko; Professor, Graduate School ofArts and Sciences, The University of Tokyo
KYOGOKU, Yoshimasa; Professor, Fukui University of Technology
MATSUBARA, Ken-ichi; Vice-Director, International Institute for Advanced
Studies
MATSUO, Minoru; President, Nagoya University
MIURA, Kin-ichiro; Professor, Faculty of Science, Gakusyuin University
MIYAMOTO, Misako; President, Japan Women's University
MOHRI, Hideo; Director-General, National Institute for Basic Biology
OKADA, Masukichi; Professor Emeritus, University of Tsukuba
OSAKI, Hitoshi; Director-General, Center for National University Finance
OTSUKA, Eiko; Professor Emeritus, Hokkaido University
TOYOSHIMA, Kumao; Director, Sumitomo Hospital
TSUNEWAKI, Koichiro; President, Fukui Prefectural University
YAMANOUCHI, Kazuya; Senior Scientific Staff, Nippon Institute for Biological Science9

ADVISORY COMMITTEE (as of December 31,2000)
Chairman
OGAWA, Tomoko; Professor, National Institute 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; Professor, Institute for Virus Reserch, Kyoto University
KATSUKI, Motoya; Professor, The Institute of Medical Science, The
University of Tokyo
MATSUURA, Etsuko; Professor, Faculty of science, Ochanomizu University
SASAZUKI, Takehiko; Professor, Medical Institute ofBioregulation, Kyusyu
University
SHINOZAKI, Kazuo; Chief Scientist, RIKEN Tsukuba Institute
TAJIMA, Fumio; Professor, Graduate School of Science, The University ofTokyo
Inside Members (Alphabetical order)
ARAKI, Hiroyuki; Professor, National Institute of Genetics
GOJOBORI, Takashi; Professor, National Institute of Genetics
HIROMI, Yasushi; Professor, National Institute of Genetics
HIROSE, Susumu; Professor, National Institute of Genetics
IKEMURA, Toshimichi; Professor, National Institute of Genetics
ISHIHAMA, Akira; Professor, National Institute of Genetics
KATSURA, Isao; Professor, National Institute of Genetics
KORARA, Yuji; Professor, National Institute of Genetics
SASAKI, Hiroyuki; Professor, National Institute of Genetics
SHIROISHI, Toshihiko; Professor, National Institute of Genetics

10

RESEARCH ACTIVITIES IN 2000

A.

Department of Molecular Genetics
A-a.

Division of Molecular Genetics(1) Monitoring of Growth-Coupled Changes In Promoter Activities In
Escherichia coli Using Fluorescent Proteins as Reporters
Hideki MAKINOSHIMA, Akiko NISHIMURA l and Akira ISHIHAMA(iLaboratory of Microbial Genetics, Genetic Strains Research Center)
The pattern of gene expression in Escherichia coli changes markedly during
the growth transition from exponential to stationary phase. Of about 4,000
genes on the E. coli chromosome, a total of about 1,000 genes are expressed in
the exponential growth phase as measured by two-dimensional gel electrophoresis of whole cell extracts. The expression level of these growth-related
genes decreases markedly in the stationary phase, whereas a new set of morethan 100 genes which are not expressed in the exponential phase begins to be
expressed. The growth-related genes are transcribed by the RNA polymerase
holoenzyme containing a D, while the stationary phase genes are transcribedby E a S holoenzyme. For detailed analysis of the growth phase-coupled regulation of global gene transcription, we set up a monitoring system of the in vivo
promoter activity by using fluorescent proteins as reporters. Various kinds ofthe promoter were fused to the genes coding fluorescent proteins with different
wave lengths. To measure the activity of test promoter at various growth phases,
the reference and the test promoters were fused to the coding sequences fordifferent fluorescent proteins with the emission maximum of different wavelength and both fusion genes were inserted into the same plasmid vector ofvarious copy numbers. For instance, the reference promoter, laCUV5, was fusedto the Red fluorescent protein and the test promoter was fused to the Greenfluorescent protein. The ratio of Red and Green fluorescence was measured at

A.

DEPARTMENT OF MOLECULAR GENETICS

11

various growth phases. After analysis of various promoters from the known E
coli genes, we could show the time dependent change in promoter activity for anumber of E. coli promoters, including bolA, cbpA, dps, fie, hupA, mdh,osmY
and rmf Results indicate that the pattern oftime'dependent change in pro'
moter activity is different among these promoters. For some details see Refs.
6, 8, 13, 14 and 17.

a 70 and a S Subunits ofEscherichia coil RNA Polymerase(!) Anti-sigma Factors for the

Akira ISHIHAMA, Dipank DASGUPTA 1, Miki JrSHAGE and Kaneyoshi YAMAMOTO(Ian leave of absence from Saha Institute of Nuclear Physics, Calcutta)
The Escherichia coli genes expressed during entry of cell growth into thestationary phase is transcribed by the RNA polymerase holoenzyme contain'
ing the rpoS'coded a s, a close relative of a 0, with a mutual overlap of pro'
moter recognition properties. A set of more than 100 stationary phase' specificgenes is expressed under the control of a s. The intracellular concentration ofa S subunit increases during the transition from exponential growth to sta'tionary phase. The level of a ° subunit for growth'related gene transcription,
however, stays constant, without being degraded in the stationary phase, sug'gesting that in addition to the level control, the activities of a subunits must
be controlled. So far various mechanisms have been identified, including asso'
ciation of the antisigma factors, changes in the cytoplasmic compositions, andmodulation of the nucleoid configuration. Recently we discovered a novel E.
coli protein, referred to Rsd (regulator of§igma IV, which forms a complex witha 70 and represses its function. Purified Rsd protein formed complexes in vitro
with a 70 but not with other a subunits, and inhibited a 7°'dependent tran'
scription in vitro to various extents depending on the promoters used. Bothdepletion and over-production ofRsd enhances or suppresses, respectively, a 70.
dependent transcription in vivo, with concomitant decrease or increase in as.
dependent transcription. After analysis of the a 79 cleavage sites by FeBABE
tethered on Rsd and of the binary complex formation of Ala 'substituted a 70
subunits with Rsd, the contact site of Rsd on the a 79 subunit was identified

12

to be within region 4.2, including amino acid residues L595 and L598.
As an extension ofthis line of studies, we have searched for anti-sigma factor
for the a S subunit, and found that in late stationary"phase cells, a certain
fraction of a S is associated with Dps (DNA-binding protein in starved cells).
Purified Dps forms in vitro binary complexes with a S but not with other asubunits, threby interfering with the formation of a S holoenzyme. Accordingly
transcription in vitro of a s-dependent promoters was inhibited by the addition of Dps. In agreement with these in vitro observations, the expression invivo of lacZ under the control of as-dependent fic and katE promoters increased in mutants lacking Dps. In late stationary phase of cell growth, unbound cytoplasmic form ofDps was found to exist, which may form complexes
with a s. These findings altogether raise a possibility that Dps is an anti"
sigma factor for the stationary phase-specific a S subunit. For details see
Refs. 1, 6, 7, 9 and 14.

(3) Search for Class-III ( (3 -contact) and -IV ( (3 '-contact)
Transcription Factors In Escherichia colJ
Akira KATAYAMA, Tasuku NOMURA, Nobuyuki FUJITA andAkira ISHIHAMAThe RNA polymerase holoenzyme of Escherichia coli is composed of the core
enzyme with the subunit composition of a 2 {3 B ' and one of seven different
species of the a subunit. For regulated transcription initiation of most inducible genes, additional protein or nucleotide factors are required. Most protein
factors with the regulatory activities of transcription interact directly with the
RNA polymerase. Transcription factors with enhancing or inhibiting activities of transcription were classified as activators or repressors. Several lines
of evidence indicate that these transcription factors have dual functions, transcription activation and repression, depending on the sites of DNA binding. In
E. coli, at least 100-150 transcription factors are considered to interact directly with the RNA polymerase. Since the mode of transcription regulation bytranscription factors is related to the mode of protein-protein interaction, we
proposed to classify these transcription factors on the basis of contact subunitof RNA polymerase. So far a number of class-I ( a -contact) and -II ( a-con-A.

DEPARTMENT OF MOLECULAR GENETICS

13tact) factors have been identified and the mode of action were analyzed indetails for some of these factors. This year we searched for class'III and, IVtranscription factors which interact with the {3 and/or {3 , subunits, respec'tively. For this purpose, we employed an affinity isolation of candidate pro'teins from whole cell extracts of E. coli expressiong a low level of {3 • or {3 '.
subunits fused with glutathione S·transferase (GST). After N·terminal aminoacid micro' sequencing or mass'spectrometry, we identified 7 proteins as can'didates for the class'III ({3 'contact) and 9 proteins as candidates for the class'IV ({3 "contact) factors. Proteins identified by both screenings are considered
to be associated with the assembled RNA polymerase. For instance, the 110
kDa RapAlHepA protein, the putative helicase, is one such example. Proteinsthat were identified with only either GST· {3 or GST' {3 are the candidates forclass' III or class'IV factors, which are analyzed in details. For some details
see Refs. 6, 10, 14 and 23.
I(4) Protein-protein Contact Site Mapping of E. colJ Transcription
Factors on the RNA Polymerase
Sujatha SITRAMAN, Katsunori YATA 1 andAkira ISHIHAMA(lRadioisotope Center)
Transcription regulation is a major cellular process by which a large numberof genes in Escherichia coli are controlled. The RNA polymerase holoenzyme
consists of 2 a, {3 , {3 and one of seven molecular species of the a subunit.
The function of RNA polymerase holoenzyme is further modulated throughinterplay with one or two of about 100'150 transcription factors. Transcription
factors have been classified into four groups, class I, II, III and Iv, based on thesubunit, a, a, {3 and {3 of RNA polymerase they contact in order to execute
their functions. By a combination of genetic and biochemical approaches, anumber of class' I and'II factors have been identified and their contact sites
have been mapped on the a and a subunit, respectively. Systematic searchfor class·III and ·IV factors is in progress in this laboratory (see above). For
systematic mapping of the transcription factors on the RNA polymerase, wehave devised a novel experimental system, in which the transcription factor
I

I,

14

contact sites can be detected after analysis of contact-dependent cleavage
sites on each subunit by a chemical protease, FeBABE, conjugated to the test
factors. For detection of the cleaved subunits and mapping of the cleavage
sites, different epitope tags were added to all four RNA polymerase subunits,
thereby allowing the detection of cleavage products after Western blotting. To
test the new experimental system for mapping of the factor-contact sites, weanalyzed the binding sites of all seven a factors on the core enzyme, and thecontact sites ofDnaK, GroEL, IF2 and EF-TU, which all associates stably with
the RNA polymerase in the absence of DNA, on the holoenzyme E a 70. For
details see Refs. 2, 6, 7, 10, 18, 22, 23 and 28.

(5) Determination of Intracellular Concentrations of Transcription
Factors in Escherichia colJ
Akira ISHIHAMA, Katsunori YATA, Etsuko KOSHIO, Akira IWATA' and Susumu
UEDA' ('Nippon Institute for Biological Science, Ohme, Tokyo)
Constitutively expressed genes in Escherichia coli are transcribed by the
RNA polymerase holoenzyme alone without transcription factors, but transcription of inducible genes requires additional transcription factors. A totalof about 100-150 DNA-binding proteins are considered to function as transcription factors by directly interacting with the RNA polymerase holoenzyme
when both bind to the promoters so as to increase the local concentrations. On
this view point, the expression level of those inducible genes is considered to
be determined by the intracellular concentrations of individual transcription
factors. At present, however, little is known about the intracellular levels andcontrols of transcription factors. We then initiated a systematic study of thelevel controls of transcription factors in E. coli. For this purpose, we purified
more than 50 species of the transcription factor in His-tagged forms fromoverexpressing cells using the respective cloned genes, and raised polyclonal
antibodies in rabbits against the purified transcription factors. The concentration of transcription factors in whole lysates of E. coliW3110 growing undervarious conditions were determined by using quantitative immuno-blotting.
So far we determined the intracellular levels ofArcR, DeoR, DnaK, Fur, GreA,A.

DEPARTMENT OF MOLECULAR GENETICS

15

GroE, IclR, InfB, Lad, LeuO, MerR, NusA, OxyR, PhoP, RpoZ, SdiA, SoxR,
SoxS, ThiI and TufB. For some details see Refs. 6, 8, 23 and 24.

(6) Novel Mode of Transcription Regulation: Simultaneous Activation
and Repression by a Single Transcription Factor
Kaneyoshi YAMAMOTO, Katsunori YATA 1 and Akira ISHIHAMA(lRadioisotope Center)
SdiA, the Escherichia coli homologue of quorum' sensing regulator, controls
the expression of the ftsQAZ operon for cell division. Transcription of ftsQAZ
is under the control of two promoters, a 7°'depedent upstream ftsQP2 and a 5.
dependent downstream ftsQP1, each being separated by 125 bp. SdiA actio
vates transcription in vivo from ftsQP2. SdiA was found to stimulate tran'
scription in vitro from ftsQP2, and in addition, to repress transcription fromftsQPI. Molecular basis of the dual functions of SdiA on transcription regula'tion of ftsQP1P2 was analyzed in details using reconstituted RNA polymerases
lacking the contact surfaces for class'I or class'II transcription factors. Gel
shift and DNase I footprinting assays indicated that only a single molecule of
SdiA binds between '51 and '25 with respect to the upstream P2 promoter. In
agreement with the influence on overall transcription, SdiA stimulated theRNA ploymerase binding to P2 but inhibited its binding to PI. Activation of
ftsQP2 transcription by SdiA was observed with a mutant RNA polymerasecontaining a C·terminal domain (CTD)'deleted a subunit ( a 235) but notwith RNA polymerase holoenzymes containing a 5 (E a 5) and a CTD'deleted
a 0 (E a 0529). In good agreement with the transcription assay, DNA protec'
tion of P2 was also not observed with the RNA polymerase holoenzymes, E a 5
and E a 0529. These observations altogether indicate that: (i) SdiA supports
the RNA polymerase (E a 0) binding to ftsQP2; and (ii) unlike the well'known
mechanism of RNA polymerase recruitment by protein'protein contact be'
tween class'I factors and a CTD, the recruitment of RNA polymerase to ftsQP2
by SdiA depends on the presence of a CTD. On the other hand, transcription
inhibition of the ftsQP1 promoter by SdiA was observed with all the RNA
polymerases examined, including E a 0, E a 1J529, E a 0 containing a 235, and16

E a S (and therefore in the absence of RNA polymerase-binding to the upstream P2). We predict that the PI transcription inhibition is due to conformational change in PI promoter DNA. For some details see Refs. 2,6, 18, 19 and
28.

(7) Systematic Search for Znu-blndlng Proteins In Escherichia coli
Akira KATAYAMA, Atsuko TSUJII', Akira WADA 2, Takeshi NISHINO' and Akira
ISHIHAMA (lNihon Medical School, Department of Biochemistry, Tokyo, 20saka
Medical School, Department of Physics, Suita)
As a part of the proteome analysis of Escherichia coli, attempts have been
made to identify all Zn 2+-binding proteins. For this purpose, whole cell extractsof E. coli W3110 were fractionated by two methods of two dimensional gel
electrophoresis, i.e., the widely used O'Farrell method and the RFHR (radicalfree and highly reducing) method. After gel electrophoresis, proteins were blotted onto PVDF membranes and after renaturation, exposed to binding assay
with radioactive Zn 2+. A number of novel E. coli proteins so far unidentified as
Zn 2+-binding proteins have been found to carry the Zn 2+-binding activity as
detected by this conventional metal-binding assay. To examine the affinity
and specificity ofZn 2+-binding, the genes for some newly identified Zn 2+-binding E coli proteins were cloned into an expression vector and overexpressed in
E. coliBL2l(DE3) in His-tagged forms. The purified Zn 2+-binding proteins
were analyzed, in parallel with the known Zn 2+-binding proteins such as carbonic anhydrase and Sp1, for the Zn 2+-binding affinity. The specificity ofZn 2+binding was also tested by competition assay in the presence of other metal
ions.

(8) Protein and mRNA Levels of All Twelve Subunits of RNAPolymerase II In Schlzosaccharomyces pombe
Hitomi SAKURAI, Makoto KIMURA and Akira ISHIHAMAThe RNA polymerase II of eukaryotes is composed of twelve subunits (Rpb 1
to Rpb12), of which five are shared among Pol I, Pol II and Pol III. At present,

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DEPARTMENT OF MOLECULAR GENETICS

17

however, little is known on the regulation of synthesis and assembly of thetwelve subunits. To get insight into the regulation of synthesis of these twelve
subunits in the fission yeast Schizosaccharomyces pombe, we analyzed protein and mRNA levels for all the rpb genes. Results of Western blot and competitive RT· peR analyses indicate that: Ci) the intracellular concentrations of
twelve Rpb subunits in growing S. pombe cells are different about 15-foldbetween the least abundant Rpb3 and the most abundant Rpb12; (ii) at leastcertain fractions of the abundant RNA polymerase II subunits exist as
unassembled states as analyzed by glycerol gradient centrifugation of wholecell extracts; and (iii) mRNAs for Rpb1, Rpb3, Rpb7 and Rpb9 are among thegroup oflow abundance, while the levels ofRpb6 and Rpb10 mRNAs are about
5-fold and that of Rpb2 mRNA is about 40-fold higher than the Rpb3 mRNAlevel. The protein-to-mRNA ratio (or the translation efficiency) is low for therpbl, rpb2, rpb3 and rpbll genes, encoding the homologues of subunits 13 " 13,a, and a, respectively of the prokaryotic RNA polymerase core enzyme.
As an extension, we also analyzed transcription organization by oligo-cap'
ping method. Results indicate that transcription of one group of genes includ'ing rpb3, rpb4, rpb5, rpb6, rpb7and rpblOis initiated mainly at a single site,while that of the other group of genes for rpbl, rpb2, rpb8, rpb9, rpbll andrpb12 is initiated at multiple sites. The promoters of the first group genes
contain the TATA box sequence between -26 to -62, while the second group
genes carry TATA-less promoters. Several common sequence segments, tentatively designated "Rpb motifs", were identified in the promoter regions of therpb genes. For details see Refs. 11, 12 and 20.

(9) Isolation and Characterisation of RNA Polymerase II-AssociatedProteins in Schlzosaccharomyces pombe
Makoto KIMURA, Hisako SUZUKI and Akira ISHIHAMA
Eukaryotic RNA polymerase II is a large complex with the molecular mass ofabout 500 kDa. In the light of comparison with many other smaller nucleic acidpolymerases, we can assume that only a part within the RNA polymerase II
molecule is involved in the catalytic reaction of RNA polymerization, and other18

parts have regulatory functions. In transcription regulation, the RNA polymease
II is supposed to be controlled by protein-protein interaction with a number oftranscription factors. We tried to isolate such RNA polymerase II-interacting
protein factors from polymerase complexes, which were isolated using an
epitope-tagging method. For this purpose, an S. pombe strain carrying a FLAGtagged rpb3 gene on its chromosome was constructed by gene replacement andRNA polymerase II complexes were isolated from cell extracts using antiFLAG antibody resin. By controlling the conditions for extraction, we succeeded to separate two types ofthe RNA polymerase II complex, one containing
non-phosphorylated and the other containing phosphorylated form of Rpbl.
We assume that the non-phosphorylated and the phosphorylated forms represent the RNA polymerase II molecules non-engaged and engaged, respectively,in transcription, because the C-terminal domain (CTD) of the largest subunit(Rpb1) is not phosphorylated before promoter entry but phosphorylated during promoter clearance. The proteins that have been identified as associatedwith the non-engaged RNA polymerase II complexes include the general transcription factor TFIIF and the CTD-specific phosphatase Fcpl. This complex
fraction could be separated into RNA polymerase II complex containing both
TFIIF and Fcp1 and that containing only Fcpl. The S. pombeTFIIF herewith
identified consisted of three species of subunit as in the case of Saccharomyces
cerevisiae TFIIF, but different from that in higher eukaryotes. TFIIF fromthese two yeast strains contains an additional small subunit, Tfg3, which isalso a component of other several transcriptional complexes. See Refs. 11 and
12 for related subjects.

(10) Search and Analysis of Transcription Factors Interacting with
Rpb7 Subunit of Scblzosaccbaromyces pombe RNA Polymerase II
Hiroshi MITSUZAWA, Emi KANDA and Akira ISHIHAMA
Rpb7 is an RNA polymerase II subunit that is not shared by RNA polymerase I or III. Rpb7 associates with another RNA polymerase II-specific subunit, Rpb4, to form a subassembly dissociable from the core assembly consisting of the remaining 10 subunits. Rpb7 is required for accurate transcrip-

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DEPARTMENT OF MOLECULAR GENETICS

19

tion initiation in vitro from the core promoter but is unnecessary for RNA
synthesis. These observations suggest that Rpb7 has a role in transcription
regulation. To elucidate the function of Rpb7 in transcription by RNA polymerase II, we have used the S. pombe RNA polymerase II as a model. Using theyeast two-hybrid system, we have isolated a number of Rpb7-interacting proteins. One of the isolated clones encodes a protein that has homology to the S.
cerevisiae Nrd1 protein. Nrd1 has been implicated in pre-mRNA processing
because it interacts with the C-terminal domain (CTD) ofthe largest subunit(Rpb1) of RNA polymerase II and contains RS and RNA-binding motifs, which
are often found in proteins involved in splicing. We found that, like the S.
pombe counterparts, the S. cerevisiae Rpb7 interacts with Nrd1 in the twohybrid system, indicating that the interaction has been conserved between S.
pombe and S. cerevisiae. The result suggests that the interaction ofRpb7 with
Nrd1 is not an artefact in the two-hybrid system but has physiological relevance. Thus our results suggest a novel role of Rpb7 in pre-mRNA splicing in
addition to transcription initiation. See Refs. 5 and 15 for related subjects.
(t t) Analysis of General Transcription Factor TFIID

from Sehizosaeeharomyees pombe
Hiroshi MITSUZAWA, Hiroaki SEIN0 1, Fumiaki YAMA0 1 andAkira ISHIHAMA(IDivision of Muragenesis, Department of Molecular Genetics)
The general transcription factor TFIID plays a critical role in transcription
initiation by RNA polymerase II. TFIID is a multiprotein complex comprising
the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs).
We have characterized two related TAFs in S. pombe, TAF72 and TAF73,
which have homology to WD repeat-containing TAFs such as human hTAF100,
Drosophila dTAF80, and S. cerevisiae yTAF90. We showed that TAF72 andTAF73 are present in the same complex with TBP and other TAFs, demonstrating that TAF72 and TAF73 are indeed TAFs. yTAF90 is also present in
SAGA, a histone acetylase complex distinct from TFIID. This led us to test thepossibility that TAF72 and TAF73 are shared by TFIID and other histone
acetylase complexes. SPAC1952.05, a gene predicted by the S. pombe genome

20

sequencing project, encodes a protein that is 53% identical to S.eerevisiae
Gcn5, the histone acetylase subunit of SAGA. We refer to this gene as genS +and examined whether its product Gcn5 is associated with TAF72 and TAF73.
Co'immunoprecipitation experiments demonstrated that TAF72, but not
TAF73, is associated with Gcn5. It thus seems that TAF72 is present in both
TFIID and SAGA'like complexes while TAF73 is present only in TFIID. Interestingly, overexpression of TAF72 or TAF73 can suppress the cell cycle arrestin mitosis caused by mutations in the ubeP4 + and eut9 + genes. ubeP4 + encodes a ubiquitin'conjugating enzyme whereas eut9 + encodes a subunit of aubiquitin'protein ligase complex known as anaphase-promoting complex. These
results suggest that TAF72 and TAF73 may regulate the expression of genes
involved in ubiquitin'dependent proteolysis during mitosis. Our study thus
provides evidence for a possible role of WD repeat'containing TAFs in theexpression of genes involved in progression through the M phase of the cell
cycle. For details see Ref. 15.
(1!) Differential Roles of vRNA and cRNA in Functional
Modulation of Influenza Virus RNA Polymerase
Ayae HONDA, Atsushi END0 1 and Akira ISHIHAMA('Daiichi Pharmaceutical Co., Exploratory Res. Lab., Tokyo)
The influenza virus RNA polymerase is involved in both transcription (vRNAdirected mRNA synthesis) and replication (vRNA-directed synthesis of comple'
mentary RNA (cRNA) and cRNA-directed vRNA synthesis). In transcription,
the RNA polymerase catalyzes not only RNA synthesis but also the cleavage ofcapped host RNA to generate capped RNA primers and polyadenylation at
the 3"termini of mRNA. The RNA polymerase is composed of one molecule
each ofPB1, PB2 and PA. PB1 plays a central role in RNA polymerase assembly by providing the contact surfaces for both PB2 and PA. In the virus particles, this RNA polymerase is bound to a double'stranded region of viral RNA(vRNA) formed by base'pairing between its 5" and 3"termini. Since the RNA
polymerase is tightly associated with vRNA, no purification method yielding
large amounts of functional RNA polymerase has been established. For de'

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DEPARTMENT OF MOLECULAR GENETICS

21

tailed analysis ofthe structure-function relationship of each P protein, large
amounts of functional RNA polymerase are required in a template-free form.
We established the expression system of RNA polymerase by simultaneous
infection of three kinds of the recombinant baculovirus insect cells, each
expressinng PBl, PB2 or PA protein. For purification purpose ofthe 3P complex, the PA protein was expressed as a fusion with His 6 tag added at its Nterminus. The affinity"purified 3P complex showed the activities of capped
RNA binding, capped RNA cleavage, V" and c-sense model RNA binding, modelvRNA-directed and ApG-primed RNA synthesis, and polyadenylation of newly
synthesized RNA. We conclude that a functional form of influenza virus RNA
polymerase with the catalytic specificity of transcriptase is formed inbaculovirus-infected insect cells. The capped RNA cleavage was observed in
the presence ofvRNA but not of cRNA, indicating that vRNA functions as aregulatory factor for the specificity control of viral RNA polymerase as well asa template for transcription.
Using the same set of recombinant baculoviruses, two kinds of binary 2Pcomplex, PBl"PA and PBI-PB2, were isolated. Both 2P complexes showed
partial activities of transcription and/or replication. See Refs. 3 and 4 forrelated subjects.
(13) Search for Host Factors Interacting with Influenza Virus
RNA Polymerase
Ayae HONDA, Takuto OKAMOTO, Masako KAIDO and Akira ISHIHAMA
Influenza virus RNA polymerase consisting of three viral P proteins (PBl,
PB2 and PA) carries two functions, one for transcription ofvRNAs to produce
vival mRNAs and the other for replicatin ofvRNAs to produce progeny vRNAs
via cRNA templates. Transcription of the vRNAs by the viral RNA polymerase
is initiated by using host cell capped RNAs as primers. Analysis of the 5'terminal structure ofvirus"associated vRNAs indicated that RNA synthesis
for replication is initiated de novo without using primers. Both purified andreconstituted RNA polymerases require primers for function, while the RNA
polymerase in either virus-infected cell extracts or lysates of cells expressing

22three viral P proteins can catalyze RNA synthesis in the absence of primers.
We then proposed that an as yet unidentified host factor(s) is involved in thefunctional conversion of the RNA polymerase from transcriptase to replicase.
Attempts have been made to identify host proteins, which interact with eachof the P proteins, using yeast two hybrid screening system. Several positive
clones have been isolated for each P protein, and complate cDNAs for some ofthese putative PBI-, PB2- and PA-interacting host factors have been isolated.
One of the PB I-interacting protein with the molecular mass of 54kDa (PB Ic54)
was overproduced in E. coli, and examined for possible influence on the functions of influenza virus RNA polymerase. Using anti-PBIc54 antibodies raised
in rabbits, the intracellular localization ofPBIc54 in both virus-infected anduninfected cells was analyzed by indirect immunofluorescent microscopy. After virus infection, the PBIc54 was found to colocalize with the viral RNA
polymerase, indicating that PBIc54 interacts in vivo with the viral RNA polymerase.

Pub I i ca t ions
1.

2.

3.

4.

5.

6.
7.

BOWN, J.A, KOLB, A., MEARES, C.F., ISHIHAMA, A., MINCHIN, S.D. and BUSBY, S.J.W.:
Positioning of region 4 of the Escherichia coli RNA polymerase a 70 subunit by atranscription activator. J. Bacteriol. 18%, 2982-2984 (2000)
FUJITA, N., ENDO, S. and ISHIHAMA, A.: Structural requirements for the interdomain
linker of a subunit of Escherichia coli RNA polymerase. Biochemistry39, 6243-6249(2000)HATTA, M., ASANO, Y, MASUNAGA, K., ITo, T., OKAZAKI, T., TOYODA, T., KAWAOKA, Y.,
ISHIHAMA, A and KIDA, H.: Epitope mapping ofthe influenza A virus polymerase PA
using monoclonal anbitodies. Arch Virol. 145, 957-964 (2000)
HWANG, J.-S., YAMADA, K., HONDA, A., NAKADE, K. and ISHIHAMA, A.: Expression offunctional influenza viral RNA polymerase in the methylotrophic yeast Pichia pastoris.
J. Virol. 74, 4074-4084 (2000)
ISHIGURO, A, NOGI, Y., HISATAKE, K., MURAMATsu, M. and ISHIHAMA, A: Ppb6 subunit of
the fission yeast RNA polymerase II is a contact target of the transcription elongation
factor TFIIS. Mol. Cell. Biol. %0, 1263-1270 (2000)
ISHIHAMA, A: Functional modulation of Escherichia coli RNA polymerase. Ann. Rev.
Microbiol. 54, 499-518 (2000)
ISHIHAMA, A: Molecular anatomy of RNA polymerase using protein-conjugated metalprobes with nuclease and protease activities. Chem. Commun. %000, 1091-1094 (2000)

A
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9.
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DEPARTMENT OF MOLECULAR GENETICS

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ISHIHAMA, A, Zou, C., KOBAYASIII, M., KUMAR, A, FUJITA, N., SAKURAI, H. and HAYWARD,
RS.: Molecular recognition in gene transcription. In: Biomolecularlnteractions in DNA
and Proteins. (Buckin, V. and Funck, T., edsJ, Nova Sci. Pub!., in press.
JISHAGE, M., DASGUPTA, D. and ISHIHAMA, A: Mapping of the Rsd contact site on thesigma-70 of Escherichia coli RNA polymerase. J. Bacteriol., in press.
KATAYAMA, A, FUJITA, N. and ISHIHAMA, A: Mapping of subunit-subunit contact surfaces on the {3' subunit of Escherichia coli RNA polymerase. J. Biol. Chem. 275,35833592 (2000>

11.
12.

13.

14.

15.
16.

17.

18.

19.

20.

21.

KIMURA, M. and ISIIIIIAMA, A: Involvement of multiple subunit'subunit contacts in theassembly of RNA polymerase II. Nucleic Acids Res. 28, 952-959 (2000)
KIMURA, M., SAKURAI, R. and ISHIHAMA, A: Intracellular contents and assembly statesof all twelve subunits of the RNA polymerase II in the fission yeast Schizosaccharomyces
pombe. Eur. J. Biochem. 268, 612'619 (2001)
MAEDA, R., JISHAGE, M., NOMURA, T., FUJITA, N. and ISHIHAMA, A: Promoter selectivityof the RNA polymerase holoenzyme containing the extracytoplasmic function (ECF)
sigma subunit a E or a Fod. J. Bacteriol. 182, 1181-1184 (2000)
MAEDA, R., FUJITA, N. and ISIIIIIAMA, A: Sigma competition: Comparison of binding
affinity to the core RNA polymerase among seven E. coli sigma subunits. Nucleic Acids
Res. 28, 3497-3503 (2000)
MITOBE, J., MITSUZAWA, R. and ISHIHAMA, A.: Functional analysis of RNA polymerase
Rpb3 mutants of the fission yeast Schizosaccharomyces pombe. Curro Genet., in press.
MITSUZAWA, R., SEINO, R., YAMAO, F. and ISIIIIIAMA, A: 'l\vo WD repeat'containing TAFs
in fission yeast that suppress defects in the anaphase'promoting complex. J. Biol.
Chem., in press.
OHNUMA, M., FUJITA, N., ISHIHAMA, A, TANAKA, K. and TAKAHASHI, R.: A carboxy'
terminal 16 amino acid region of a 3. of Escherichia coli is important for transcription
under high-salt conditions and sigma activities in vivo. J. Bacteriol. 182, 4628-4631(2000)OTOMO, T., YAMAZAKI, T., MURAKAMI, K., ISIIIIIAMA, A and KYOGOKU, Y.: Structural studyof the N-terminal domain of the alpha subunit of Escherichia coli solubilized with non'denaturing deteregents. J. Biochem. 128, 337-344 (2000)OZOLINE, 0., FUJITA, N. and ISHlIIAMA, A.: Transcription activation mediated by the
carboxy-terminal domain of RNA polymerase alpha-subunit: Multipoint monitoring
ssing fluorescent probe. J. Biol. Chem.275, 1119-1127 (2000)
SAKURAI, R. and ISHIHAMA, A: Transcription organization and mRNA levels of thegenes for all twelve subunits of the fission yeast RNA Polymerase II. Genes Cells 6, 2536 (2001)SHPAKOVSKI, G.V., GADAL, 0., LABARRE-MARIOITE, S., LEBEDENKO, E.N., MIKLOS, I., SAKURAI,
R., PROSHKIN, S.A, van MULLEM, V., ISHIIIAMA, A and THURIAUX, P.: Functional conservation of RNA polymerase II in the fission and budding yeasts. J. Mol. Biol. 295, 11191127 (2000)

24
22.

SITRAMAN, S., ISHIHAMA, A. and CHATTERJI, D.: Functional complementation between
two distant positions in E. coli RNA polymerase as revealed by second-site suppres'sion. Mol. Gen. Genet. 264, 531-538 (2001)

23.
24.

TALUKDER, AA, HIRAGA, S. and ISHIHAMA, A: Two types oflocalization of the DNAbinding proteins within the Escherichia coli nucleoid. Genes Cells 5, 613'626 (2000)
WADA, A, MIKKOLA, R, KURLAND, C.G. and ISHlHAMA, A: Growth phase-coupled changesof the ribosome pattern in Escherichia coli. J. Bacterial. 182, 2893-2899 (2000)

25.

WIGNESHWERARAJ, S.R., CHANEY, M.K, ISHIHAMA, A and BUCK, M.: Regulatory sequencesin sigma 54 localise near the start of DNA melting. J. Mol. Biol. 306. 681-701 (200026.

WIGNESHWERARAJ, S.R., FUJITA, N., ISHIHAMA, A. and BUCK, M.: Conservation of sigmacore RNA polymerase proximity relationships between the enhancer independent andenhancer-dependent sigma classes. EMBO J. 19, 3038'3048 (2000)

27.

WIGNESHWERARAJ, S.R, ISHIHAMA, A and BUCK, M.: In vitro roles of invariant helix-turnhelix motif residue R383 in sigma 54 (sigma N>. Nucleic Acids Res. 29, 1163-1174
(2000

28.

YAMAMOTO, K, NAGURA, R, TANABE, H., FUJITA, N., ISHIHAMA, A and UTSUMI, R: Nega'tive regulation of the bolAlp of Escherichia coliK-12 by the transcription factor OmpR
for osmolarity response genes. FEMS Microbial. Lett. 186, 257-262 (2000)

29.

YASUNO, K, YAMAZAKI, T., TANAKA, Y., KODAMA, T., MATSUGAMI, A, KATAHIRA, M., ISHlHAMA,

A and KYOGOKu, Y.: Interaction ofthe 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. Biol. 306,213'225 (2000

A-b.

Division of Mutagenesis(1) Comprehensive survey and functional analysis of ubiquitlnconjugating enzymes in S. pombe
JoonoHyun PARK, Hiroaki SEINO and Fumiaki YAMAO
Ubiquitin'dependent proteolysis plays important roles in many biologicalprocesses such as cell cycle control, signal transduction, cell differentiation,and so on. The diversity and specificity of ubiquitination are based on themolecular heterogeneity of ubiquitin'conjugating enzymes (Ubc or E2) andubiquitinoligases (E3). Thirteen Ubc's has been identified and their functions
has been characterized in budding yeast. In fission yeast, however, very little
information about Ubc's has been accumulated. We, therefore, surveyed Ube's
to elucidate their functional diversity in fission yeast. In addition to four Ubc's

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DEPARTMENT OF MOLECULAR GENETICS

25(UbcPI-P4) previously identified by us through screening by their enzymatic
activities, other eight Ubc's (UbcP5'P12) and a non' canonical Ubc, mms2,
have been found through homology search in the fission yeast genome database. The functional analyses of Ubc's were carried out through disruption of
their genes, showing that some genes are found essential for the cell growth
while its ortholog in budding yeast are not. Some Ubc's in fission yeast have no
structural homologue in budding yeast, and vice versa. Interestingly, pheno"
typic differences between the fission yeast Ubc's and their structural homo"logues in the budding yeast have been found in many cases, suggesting theimportance of functional survey of fission yeast Ubc's.
We now focus on UbcP7 which functions together with Mms2 in repair ofdamaged DNA with the same epistasis to Rad6. Recently, by two hybrid screen"ing, we identified new protein that interacts specifically with Mms2, dysfunc"tion of which resulted in higher sensitivity to DNA damage. Functional analy"
sis of this new repair protein with respect to UbcP7 and Mms2 are now under"
going.
(!) Ubiquitin-eonjugating enzymes involved in mitotic eyelin

degradation
Hiroaki SEINO and Fumiaki YAMAO
Biochemical analysis of mitotic cyclin ubiquitination using frog or clam egg
extract showed that two ubiquitin conjugating enzymes, Ubc4 and Ubc"xIE2"
C, work with ubiquitin ligase, APC (anaphase promoting complex), toubiquitinate mitotic cyclin. However, functional differences between the two
Ubc's has not yet been elucidated. We identified their homologues, ubcP4 andubcPlIubc4, respectively, in fission yeast. We found both of them were essen"tial for cell viability. Cells depleted with either of these Ubc's exhibited mi"
totic defects with accumulation of mitotic cyclin. Even in G1 cells, mitotic
cyclin was stabilized under depletion of one of them. Over"production of theeach ubiquitin"conjugating enzyme was not able to rescue the defect oftem"
perature"sensitive mutants of other one. These results indicate that the two
Ubc's are involved in degradation of mitotic cyclin in non"redundant fashion.

26

Analysis ofthe functional relationship and differences between these two Ubc's
in the mitotic cyclin destruction are now undergoing.

(3) Characterization of SCFGrrl that ublqultlnates G1 eyellns In

Saccaharomyces cerevlslae
Tsutomu KISHI and Fumiaki YAMAOSCF complexes, composed of Skp 1, Cdc53 and one of the F-box proteins, havebeen implicated in the Cdc34-dependent ubiquitination in Saccaharomyces
cerevisiae. We have found that Grr1, which is required for degradation of G1
cyclins, Cln1 and Cln2, as well as for regulation of glucose repression, is an Fbox protein interacting with Skp 1 through the F-box motif. Furthermore, wehave found that Grr1 also interacts in vitro with phosphorylated Cln1 andCln2. From these data, we have proposed that Grrl is required for degradationof Cln2 through linking phosphorylated Cln2 to Skp 1 in SCFGrrl complex.
To isolate additional genes that are required for the degradation of Cln2, I
have employed a genetic screen to select for stable Cln2-LacZ fusion protein.
One ofthe mutants dog75 (Destabilization ofG1 cyclin) was characterized. In
dog75 mutants, Cln2 was stabilized at high temperatures. Ubiquitination ofCln2 was decreased in the mutants. However, Gic2 protein, another target
ubiquitinated by SCFGrrl complex, was not stabilized in the mutant. Sequence
analysis revealed that the gene codes for a protein that has a homology withprotein kinases.

Publ ication
1.

MITSUZAWA, H., BEINO, H., YAMAO, F. and ISHIHAMA, A.: Two WD repeat containing
TAFs in fission yeast that suppress cell cycle arrest at mitosis. J. BioI. Chern. In press.

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DEPARTMENT OF MOLECULAR GENETICS

27

Division of Nucleic Acid Chemistry(1) Host proteins involved in transcription of Sendai virus(SeV) genome
Kiyohisa MIZUMOTO (School of Pharmaceutical Sciences)
Cellular proteins (host factors) may play key roles in transcription of Sendai
virus (SeV) genome. We have previously shown that the host factor activity,
which stimulates in vitro mRNA synthesis of SeV, are separated into at least
tree complementary fractions. Two of them were identified as a cytoskeletal
protein, tubulin (Mizumoto et aI., J. Biochem. 117, 527, 1995) and a glycolytic
enzyme, phosphoglycerate kinase (PGK) (Ogino et aI., J. BioI. Chem. 274, 35999,
1999). Here, the third factor activity was further resolved into two complementary fractions, and one of them was purified to an almost single polypeptide
chain with an apparent molecular weight of 52,000 (p52) (Ogino et aI. Biochem.
Biophys. Res. Commun., 285,447,2001). From biochemical and immunologi"
cal analyses, p52 was identified as a glycolytic enzyme, enolase. Recombinanthuman a 'enolase, as did p52, acted synergistically to stimulate SeV mRNA
synthesis with other three host factors. West'western blot analysis demonstrated that tubulin specifically binds enolase as well as PGK, suggestingthat these two glycolytic enzymes stimulate SeV transcription through theirinteraction with tubulin which has been integrated into a transcription initia'
tion complex (Takagi et aI., J. Biochem. 118, 390 1995).

(2) Transcriptional Regulation during the stationary growth phasein escherichia coli
KanTANAKA(2)-a. Structure and function of a sigma factor RpoS ( a :l8, as)Escherichia coli can grow in the presence of several nutrients, including organic carbon, nitrogen, sulfur and phosphate. However in most natural envi,

28
ronments, they always face shortage of some nutrients, and are found as rest·
ing cells. Therefore, survival strategy in the starved conditions must havebeen extremely important during the evolution of E. coli. We are trying tounderstand the characteristics of transcriptional apparatus in starved bacte'ria.
RNA polymerase of E. coli is composed of a core enzyme and one of sevensigma factors, and substitution of sigma subunits modulates promoter recognition specificity of RNA polymerase. A major portion of E. coli promoters
contains 'TATAAT' type-l0 consensus sequence, and this type promoters arerecognized by two forms of RNA polymerase containing either a 70 or a 38. a 70
is the principal sigma subunit, and essential for the cell viability. The expression of a 38 is induced at the stationary growth phase, and required for the
induction of genes specific for this growth phase. Although the difference ofthepromoter specificity is obvious in vivo, both RNA polymerases could recognize
many promoters in common in vitro. Thus, the molecular basis for the recognition specificity has not been well established.
During the stationary phase, cellular physiology changes in many aspects
compared with the exponential growth phase. As for the internal condition,
solutes such as potassium and glutamate are considered to accumulate. In aprevious study, Kusano et ai. as well as us studied the effects of ionic solventson the activities of E a 70 and E a 38 in vitro, and found that E a 38 is moreresistant to (or rather activated by) potassium glutamate at 150 mM or above.
Thus, E a 38 appeared to be an RNA polymerase suitable for high ionic concen'
trations. We found a conserved 16-amino acid sequence specific for a 38 proteins at the C-terminus, and purified a a 38 variant lacking this region. An
RNA polymerase holoenzyme containing this mutant sigma was found to behighly sensitive to the high salt concentration in vivo, and therefore, we concluded that this element was esseintial for the transcription at these physiological conditions (Ohnuma et aI., J. Bacterio]. 182, 4628-4631, 2000).
(2)-b. Gene silencing at the stationary growth phase
Many promoters that are actively transcribed during the exponential phaseare silenced at the onset of the stationary growth phase. These gene repres'A

DEPARTMENT OF MOLECULAR GENETICS

29

sion mechanisms have been extensively analyzed for rrnBPl promoter, where
ppGpp, H-NS and intracellular NTP concentration are deeply involved. We
analyzed the activity of the lacUV5 promoter, a consensus promoter, during
the growth phase transition, and found that this promoter is shut off at theonset of the stationary 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.

Pub I i ca t ions
1.

YOKOSUKA, J., TSUKAMOTO, T., MIURA, K, SHIOKAWA, K and MIZUMOTO, K: Cloning andcharacterization ofmRNA capping enzyme and mRNA (guanine'7-)-methyltransferase
cDNAs from Xenopus laevis. Biochem. Biophys. Res. Commun. 268, 617'624 (2000).

2.

KAJITA, E., WAKIYAMA, M., MIURA, K, MIZUMOTO, K, OKA, T., KOMURO, I., MIYATA, T.,
YATSUKI, H., HORI, K and SHIOKAWA, K: Isolation and characterization of Xenopus

laevis aldolase B cDNA and expression pattern of aldolase A, Band C genes in adulttissues, oocytes and embryos of X laevis. Biochim. Biophys. Acta 1493, 101-118,(2000).
3.

CHIBA, H., INOKOSHI, J., OKAMOTO, M., AsANUMA, S., MATSUZAKI, K, IWAMA, M., MIZUMOTO,
K, TANAKA, H., OHEDA, H., FUJITA, K, NAKASHIMA, H., SHIONOSE, M. and OMURA, S.:
Actinohivin, a novel anti-HIV protein from an actinomycetes that inhibits syncytium
formation. Biochem. Biophys. Res. Commun. 282, 595'610 (2000.

4.

OGINO, T., YAMADERA, T., IMAJOWOHMI, S. and MIZUMOTO, K: Enolase, a cellular glycolytic enzyme, is required for efficient transcription of Sendai virus genome. Biochem.

Biophys. Res. Commun. 285, 447'455 (2000.
5.

OHNUMA, M., FUJITA, N., ISHIHAMA, A, TANAKA, K and TAKAHASHI, H. (2000) A CarboxyTerminal 16-Amino-Acid Region of a 38 of Escherichia coli Is Important for Transcrip'tion under High-Salt Conditions and Sigma Activities In Vivo. J. Bacteriol. 182:46284631

6.

OIKAWA, K, FUJIWARA, M., NAKAZATO, E., TANAKA, K and TAKAHASHI, H. (2000) Charac'terization of two plastid a factors, SigAl and SigA2, that mainly function in matured
chloroplasts in Nicotiana tabacum. Gene 261, 221-228.

7.

SHIRANO, Y, SHIMADA, H., KANAMARu, K, FUJIWARA, M., TANAKA, K, TAKAHASHI, H.,
UNNO, K, SATO, S., TABATA, S., HAYASHI, H., MIYAKE, C., YOKOTO, A and SHIBATA, D.
(2000) Chloroplast development in Arabidopsis thaliana requires the nuclear-encodedtranscription factro Sigma B. FEBS Lett. 485, 178-182.

8.

KANAMARU, K, FUJIWARA, M., KIM, M., NAGASHIMA, A., NAKAZATO, E., TANAKA, K and
TAKAHASHI, H. (2000) Chloroplast targeting, distribution and transcriptional fluctua'tion of AtMinDl, a eubacteria-type factor critical for chloroplast division. Plant Cell

30
Physiol. 41, 1119-1128.
9.

FUJIWARA, M., NAGASIIIMA, A., KANAMARU, K., TANAKA, K. and TAKAHASHI, H. (2000)
Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase sigma factors in Arabidopsis thaliana. FEBS Lett. 481, 47'52.

31

B.

Department of Cell Genetics

B-a.

Division of Cytogenetics(1) Roles of Mrel1 of Saccharomyces cerevlslae in Meiotic
Recombination and Mitotic Repair Reactions
Daisuke TATSUDA, Hiroyuki OSHIUMI, Tsutomu OHTA, Jun-ichi TOMIZAWA andTomoko OGAWA.
The genetic integrity of vegetative cells is maintained by various DNA repair
systems. Among the damage, double strand breaks (DSBs) that were inducedby treatment with ionizing radiation or radiomimetic chemicals, such as methyl-methane sulfonate (MMS), are repaired by the homologous recombination or by the non-homologous end-joining. On the other hand, genomic diversity is provided by meiotic recombination. Specific DSBs introduced in the
meiotic process are repaired by homologous recombination.
We studied the involvement ofthe MREll gene of S. cerevisiae in homologous
recombination [1]. The gene was first identified by isolation of a mutant thatis defective in meiotic recombination and repair of DSBs induced by MMS [2].
Mre 11 is involved in two temporally coupled processes in the early phase of
meiotic recombination, formation of DSBs and their processing. For the DSB
formation, Mrell acts by forming the pre-DSB complex with at least eightproteins including Rad50, Xrs2, Spoll and Mei4. For processing of the DSB
ends, the Mrell forms the post-DSB complex in which Mrell holds Rad50 and
Xrs2 tightly as a binding core. Such a tight binding among these proteins was
not present in the pre-DSB complex. The C-terminal region ofthe Mrell binds
to four meiotic proteins at least. One of the proteins, Mei4, was identified(Ohta, T. unpublished results). The N-terminal region that contains a phosphodiesterase motif specifies ssDNA endonuclease, 3' to 5' ssDNA exonucleaseand 3' to 5' dsDNA exonuclease activities that are collectively required forprocessing of the DSBs.

32

Mre 11 has two sites to bind to DNA. The central site (Site"A) is required forthe DSB processing, whereas the C"terminal site (Site-B) is involved in the
DSB formation in meiosis. Mre11 also binds to recombination hotspots on thechromosomal DNA (Ohta, T. unpublished results). The site-B is not necessary
for repair of the DNA damage caused by MMS. Mre11 has two regions to bindto Rad50, one at the N-terminal side and the other at the central region ofthe
protein. Because Rad50 has been shown to bind DNA, Mre11 could bind DNA
through Rad50. The presence of two binding sites, each for DNA and for Rad50,
permits the protein to bind to DNA differently for formation ofDSBs and theirprocessing in meiotic recombination and in repair reactions.
To elucidate further the mechanisms of joining ofDSB ends, we studied joining of blunt ended DNA, using the substrate that was made by cutting pDC 18
plasmid once with Small enzyme. Mre11 protein catalized formation of linear
oligomers, mainly dimmers, but not monomeric circular molecules. The results suggested that the formation of oligomers requires homologous sequencesat the ends of dsDNA. The reaction depends on Mn++, but not on Mg++ andATP,
and is enhanced by Rad50 protein. The joining is unstable against the heating
and the alkaline treatment, indicating binding by hydrogen bonds. These results show that this end-joining activity depends on the unwinding of the endsof DNA and on annealing with the counter molecules. The reaction is independent of the nuclease activities, because the mutant protein that is defective in
the nuclease activities can join the ends.
We then examined the unwinding activity ofMre11, using various substrates
that were made by annealing ofM13 single-stranded circular or linear viral
DNA with various nucleotide sequences of oligonucleotides in order to create
ends with different structures. Mre11 released the oligonucleotide from thesubstrates that has blunt ends with 3' overhang and also that have no overhanging at the annealed oligonucleotides. On the other hand, both overhanging
ends at the annealed oligonucleotides were not unwound. These results suggested that, by Mre11, the substrates that have blunt ends were unwound.
The substrate with 5' overhang released the fragments, but those with 3' overhang were not, suggesting that 5' overhang enhances the release ofthe fragment from the blunt ends, but 5' overhanging end could not be unwound. Thus,
the unwinding activity is dependent on the structures of the ends of the sub-

B.

DEPARTMENT OF CELL GENETICS

33

strates. The activity was dependent on Manganese, but not ATP.
Mre11 also has an activity to anneal heat denatured linear plasmid DNA.
Therefore, homologous blunt ends by Mre11 must be carried out through unwinding of the ends followed by annealing of complementary ssDNA.
The ends of linear DNA were joined covalently by T4 DNA ligase. We next
examined whether the Mre11 enhances the joining. We used the condition
where a formation of oligomers and circular molecules from blunt ended linear
dsDNA was almost undetectable due to the very small concentration of theligase. However, when the Mre11 was also present, substantial amount oflinear oligomers were formed. For the reaction, homology of the ends oflinear
dsDNA molecules was not required. The Rad50 enhanced the reaction. The
joint formed from DNA fragments that had 3' or 5' overhanging ends wascleaved by the restriction enzymes which were used for the preparation of thefragments, indicating that the joining is accurate. On the other hand, the joint
formed by blunt ended fragments by the wild-type Mre11 and Mrel1-6 was
inaccurate. Because Mrel1-6 that has no nuclease activity joins the ends inaccurately, the inaccuracy by wild-type may results from processing ends andalso unwinding the ends.
These results showed that Mre11 participates in two types of end joining
reactions, homology-dependent end-association and the enhancement of covalent end-joining of non-homologous ends by DNA ligase. Mre11 associates
homologous blunt ends through unwinding and annealing activities. It also
facilitates covalent joining of non-homologous ends by bringing the ends of two
molecules close and/or holding them together. Rad50 participates in the bindings of Mre11 to DNA in this reaction. Because Mre11 uses different DNA
binding sites for different Mre11 reactions, the difference in the mode of binding of Mre 11 to DNA is critical for different roles of the protein in different
reactions. Thus, Mre11 plays a key role in selecting repair process that issuitable to repair each particular structure at the DSB site.

34(2) Recognition of G-DNA Structure at the Yeast Telomere Ends and its
Dissolution by Mrel1 of S. cereylslae
Tsutomu OHTA, Shigeo TANAKA, Daisuke TATSUDA, Hiroyuki OSHIUMI and Tomoko
OGAWA
Telomere locates at the ends of eukaryotic chromosomes and plays important roles in cell growth. It contains G-rich sequence repeats whose synthesis
is mediated by a teromerease. In Saccaromyces cerevisiae, many genes are
known to be involved in the maintenance ofthe length and mutations in thesegenes result in the shortening of the length and cell senescence. MREll, whichis involved in homologous recombination and repair of DNA damage, is responsible for the maintenance. The protein has two each of binding sites to
DNA and to Rad50. Using various mrell mutants that are deleted either one
or both DNA binding sites, site-A and site-B, we found that Mre11 binds atsite-A to the telomere ends. The G-rich sequence of single-strand tails of telomeres is suggested to form the structure called G-DNA made by pairing of
non-canonical guanine bases. We found that the site-A ofMre11 recognizes the
DNA structure, but not its primary sequence. The binding of Rad50 to Mre 11
provably controls the way of Mre 11 binding at site-B to the G-structure at thetelomere ends. We also show that Mre11 dissolved the G-structure and either
one of DNA binding sites can serve for the reaction.
It has been reported that G-rich tails of more than 30 nucleotides at the 3'
ends oftelomeres appear in the wild-type cells only at the late S-phase of thecell-cycle. Similar tails were found in mrell- 7B- from the late S to G2 phases,
whereas in mrell b. or mrell-6A-, the tails were almost undetectable throughout the cell cycle. We then examined whether Mre11 binds to the telomere invivo using chromatin immuno-precipitation assay. In the wild-type and mrell7B- cells, the telomere DNA was found only in the S-phase, whereas in mrell6A- cells, very small amount of telomere DNA was detected throughout thecell-cycle. The ability of mutant Mre11 proteins to bind the telomere roughly
correlates to the presence of the single-strand tails in corresponding mutant
strains. Taken together with similar results of the telomere length obatinedfrom the hdfl b. (yku70b.) strain, the results imply that the binding of Mre 11

B.

DEPARTMENT OF CELL GENETICS

35

protects the tails.
In conclusion, the G"rich sequence at the end of telomere forms the G-DNA
structure. Mre 11 recognizes the structure and its binding protects the tails.
Subsequently, Mre11 dissolves the structure for further elongation oftelomere. The modes of binding ofMre11 to DNA show a selective role in the process.
Thus, Mre 11 plays critical roles in maintenance and elongation of telomere
length. Ref. [3](3) 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.
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 been conserved, and this same core is also found within helicases
and the FI-ATPase. The C-terminal domain of the RecAprotein 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 isabsent 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 thatthe lobes of the yeast and human Rad51 filaments appear to be formed by Nterminal domains. While the lobes of the RecA subunit define a unique polarity for the filament formed on single stranded DNA, human Rad51 filaments
appear to polymerize on single stranded DNA with a random polarity. In addition, 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 fromconvergent evolution to play similar roles in the filaments. Ref. [4]36Publ ications
1.

2.
3.
4.

5.
6.

7.

8.
9.
10.

USUI, T., OHTA, T., OSHIUMI, H., TOMIZAWA, J., OGAWA, H. and OGAWA, T.: Complex
formation and functional versatility of Mre 11 of budding yeast in recombination. Cell
95, 705,716 (1998).
AJIMURA, M., LEEM, S'H. and OGAWA, H.: Identification of New Genes Required for
Meiotic Recombination in Saccharomyces cerevisiae. Genetics 133: 51-66 (1993).
OHTA, T., TANAKA, S., TATSUDA, D., OSHIUMI, H. and OGAWA, T., in press Nature Genet·
ics.
Yu, X., JACOBS, S. A., WEST, S. C., OGAWA, T. and EGELMAN, E. H.: Egelman1, Domain
Structure and Dynamics in the Helical Filaments Formed by RecA and Rad51 on DNA
in press Proc. Nat!. Acard. Sci., USA.
OGAWA, T.: The Roles of Mre11 of S. cerevisiae in meiotic recombination and mitotic
repair reactions. The Gordon Research Conference on "Meiosis". June, USA.
OGAWA, T.: The Roles of Mre11 of S. cerevisiae in meiotic recombination and mitotic
repair reactions. A EMBO workshop on "The Mechanisms and Consequences of Ge'
netic Recombination". France, May.
OGAWA, T.: MRE11 and Double'strand Brake Repair, A Colloquium on "Links Between
Recombination and Replication: Vital Roles of Recombination" by the National Acad'emy of sciences. California, November.
OGAWA, T.: Roles of Mre11 on Repair of Double'strand Breaks. A workshop on "Repair
Mechanisms of DNA Damage Induced by Radiation". Tokyo, March.
OGAWA, T.: Role of Mre11 on Repair of Double'strand Breaks. A workshop on "DNARepair and Mutagenesis". Sendai, September.
TANAKA, S. and OGAWA, T.: Functional analysis of MRE 11 on the maintenance oftelom'
ere length of S. cerevisiae. A workshop on "Dynamics and Algorithms of Chromosome
Function". Hiroshima, November.

B.

B-b.

DEPARTMENT OF CELL GENETICS

37

Division of Microbial Genetics(1) Sld3, which Interacts with Cdc45 (Sld4), functions for chromosomal DNA Replication in Saccharomyces cereylslae
Yoichiro KAMlMURA and HiroyukiARAKI
Chromosomal DNA replication in eukaryotic cells initiates from multiple
origins which fire sequentially throughout the S phase; some fires early andothers late. The pre'replicative complex (pre'RC) starts to assemble at origins
from late M phase and DNA polymerases are recruited onto origins to initiate
DNA synthesis during the S phase. The Dpbll protein which forms a complex
with essential DNA polymerase E (Pol E), is required for association of DNApolymerases with origins in DNA replication and for the control of the S'phasecheckpoint (ref.1). To identify factors interacting with Dpbll, we have isolated
10 sid (synthetic lethality with dpbll'l) mutations which fall into 6 comple'
mentation groups. We have cloned SLDl-6 and found that SLDI is identicalto DPB3 encoding the third subunit of Pol E , SLD4 is identical to CDC45
required for the initiation and elongation step of chromosomal DNA replica'tion and SLD6is identical to RAD53which has crucial role for the cell cycle
checkpoint. The SLD2, 3, 5 genes were new genes essential for the cell growth(Kamimura et al., Mol. Cell. Biol., 18, 6102'6109, 1998). We characterized
SLD3 encoding a 77kDa protein.
In order to characterize the SLD3gene, we isolated thermosensitive alleles of
the SLD3 gene. We isolated the CDC45 gene as a multicopy suppressor of
SLD3thermosensitive mutations. Conversely, high copy SLD3suppressed thethermosensitive growth defect of cdc45 mutation. Using cross'linker reagent,
the interaction between Sld3 and Cdc45 was detected throughout the cell
cycle. Two' hybrid assay suggests that the Sld3 protein encoded by athermosensitive allele reduced the efficiency of complex formation with Cdc45.
Consistent with complex formation between Sld3 and Cdc45, Sld3 and Cdc45
associated simultaneously with replication origins in the chromatin immuno'
precipitation (CHIP) assay: both proteins associated with early·firing origins
in G1 phase and with late-firing origins in late S phase. Moreover, the origin

38

association of Sld3 and Cdc45 were mutually dependent. Since thethermosensitive sid3 mutation conferred defect of DNA replication at the restrictive temperature, these results suggest that the Sld3-Cdc45 complex associates with replication origins and its association is required for DNA replication. Furthermore, at the restrictive temperature in sid3-S cells, RF-A, asingle-strand DNA binding protein, did not associate with origins. Therefore,
the origin association ofSld3-Cdc45 complex is prerequisite for origin unwinding in the initiation of DNA replication.

(!) Phosphorylation of Sid! by eyelin-dependent protein kinase
Hiroshi MAsUMOTO and Hiroyuki ARAKI
Cyclin-dependent protein kinases (Cdk) are key enzymes which control thecell 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 Cdkphosphorylation, we examined whether Sld2 is phosphorylated by S-Cdk. As
expected, in SDS-polyacrylamide gel Sld2 migrated slower in S phase than in
G 1 phase. Phosphatase treatment converted slow mobility species of Sld2
obtained from S phase to fast mobility specie as obtained from G 1 phase,suggesting that Sld2 is phosphorylated in S phase. When we expressed Sic1,
an inhibitor of S-Cdk, phosphorylated form of Sld2 did not appear. Thus, 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 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 not appear.
It suggests that S-Cdk dependent phosphorylation of Sld2 is essential for
DNA replication.
Since Sld2 and Dpbll form a complex, we investigated their complex formation by tagging them and using co-immunoprecipitation assay. Co-immuno-B. DEPARTMENT OF CELL GENETICS

39

precipitation of Sld2 and Dpb 11 was detected in S phase but not in G 1 phaseand Dpb 11 immunoprecipitated only with phosphorylated form of Sld2. When
we expressed wild-type and six-alanine substitution mutant proteins of Sld2
in the sld2 mutant cells defective in complex formation between Sld2 andDpb11, complex formation between the mutant protein and Dpb11 did not be
detected while the wild type protein coprecipitated with Dpbll. These results
suggest that phosphorylation of Sld2 is required for complex formation between Dpb11 and Sld2.
We previously showed that complex formation between Dpb11 and Sld2 isrequired for early step of DNA replication (Kamimura et aI., Mol. Cell. BioI.,
18,6102-6109, 1998), and Dpb11 associates with replication origins in S phase(ref, 1). Using CHIP assay, we found that Dpb11 and Sld2 associate simultaneously with replication origins and their origin associations are mutuallydependent. Because Dpb11 is required for loading of DNA polymerases toorigins, this finding suggests that the Dpb11-Sld2 complex associates withorigins and this association is required for DNA polymerase loading.
Taken all together, it seems likely that S-Cdk regulates complex formation
between Sld2 and Dpb11 and consequently loading of DNA polymerases toreplication origins.

(3) Functional analysis of sld5 and Psfl
Yuko TAKAYAMA, Yoichiro KAMIMURA and Hiroyuki ARAK:r
The SLDS gene isolated as a synthetic lethality with dpbll-l (see section (1) )encodes an essential 34kDa protein. To elucidate the function of Sld5, we
isolated the PSFl (Partner of SLDS) gene as a multicopy suppressor of thethermosensitive sldS-12mutation. PSFl encodes a 24kDa protein which isessential for cell growth. The protein-level ofPsfl was roughly constant during
the cell. Co-immunoprecipitation assay revealed that the Sld5 and Psfl pro·teins coexist in the same complex throughout the cell cycle. We also isolated athermosensitive psfl-l mutation by the plasmid shuffling method. High-copy
SLDS suppresses thermosensitive growth of psfl-l, suggesting that the psfl-l
mutation is defective in the interaction between Sld5 and Psfl. Actually, in

40
psfl-l cells, co-existence of Sld5 and Psfl in the complex is hardly detected.
Moreover, when we prepared the extract from psfl-l cells harboring high copy
SLD5, a complex containing Psfl and Sld5 was detected.
Psfl-l mutant cells arrested with a dumbbell shape at the restrictive temperature as did sld5-12cells. FACS analysis showed that psfl-l cells are
defective in early step of DNA replication. Indirect immunofluorescent assay
revealed that the Psfl protein is localized in nucleus throughout cell cycle,
suggesting that Psfl plays a role in the place close to chromatin DNA. As
expected, in CHIP assay, the Psfl and Sld5 proteins associated simultaneouslywith replication origin in S phase. It strongly suggests that both proteinsfunction directly for DNA replication. Moreover, the Psfl association with alate-firing origin was significantly delayed behind the association with earlyfiring origins, suggesting that Psfl associates with replication origins immediately before firing. Furthermore, in psfl-l cells, Pol E did not associate withreplication origin. Taken all together, the complex containing Psfl and Sld5
functions directly in DNA replication, before loading of DNA polymerase.

Publications
1. MASUMOTO, H., SUGINO, A. and ARAKI, H.: Dpbll controls the association between DNA
polymerases a and E and the Autonomously Replicating Sequence region of budding
yeast. Mol. Cell. BioI., 20, 2809'2817, (2000).
2. KAMIMURA, Y., TAK, Y.·S., SUGINO, A. and ARAKI, H.: SId3, which interacts with Cdc45(Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae. EMBO
J. in press (2001).

B-c.

Division of Cytoplasmic Genetics(1) Derivation of the relationship between neutral mutation andfixation solely from the definition of selective neutrality
Jun"ichi TOMIZAWA
A mutation whose fixation is independent of natural selection is termed aneutral mutation. Therefore selective neutrality of a mutation can be defined

B.

DEPARTMENT OF CELL GENETICS41

by independence of its fixation from natural selection. By the population ge"
netic approach, Kimura [Kimura, M. (1962) Genetics 47, 713"719] predictedthat the probability of fixation of a neutral mutation (u) is equal to the fre"quency of the new allele at the start (p). The approach traced the temporalsequence of the fixation process, and the prediction was obtained by assuming
the selective equality of neutral mutant and wild"type alleles during the fixation process. However, because the definition concerns only mutation and fixa"
tion, an ideal approach should deal only with these and not with the interven"ing process of fixation. The approach begins by analysis of the state of fixationof a neutral mutation, and its relation with the initial state is deduced logically from the definition. The approach shows that the equality of the alleles
during fixation process is equivalent to the equality of probability of theirultimate fixation in a steady state. Both are manifestations ofthe definitionof selective neutrality. Then, solely from this dual nature of the definition, theequality between u and p is derived directly. Therefore, the definition of selective neutrality can be represented by the equation u = p.
The use of the assumption based on the future probability of fixation ofalleles in characterizing their behavior in the process of fixation by Kimura
caused tautology.
(~)

Studies on behavioral disorders of knockout mice

Hiroaki NIKI (Lab. for Neurobiology of Emotion, Brain Science Institute,
RIKEN)In this year, we found that the analgesic effects ofmu"opioid"receptor agonist(morphine) and kappa"opioid"receptor agonist (U50488) were reduced in weaver
mutant mice having mutant G"protein activated inwardly rectifying potas"
sium channels (GIRK channels) in the brain, suggesting that the intracellularsignal pathway via GIRK channels is important for opioid-induced analgesia.
We also investigated the molecular mechanisms underlying reduced opioid oinduced analgesia in CXBK mice that were considered to lack mu"opioid recep·
tor. We found that an abnormal non"coding region ofmu"opioidoreceptor mRNAcauses the reduced opioid"induced analgesia in CXBK mice.

42

Publ icat ions
1.

IKEDA, K., KOBAYASHI, T., KUMANISHI, T., NIKl, H. and YANO, R: Involvement ofG-proteinactivated inwardly rectifying K.(GIRK) channels in opioid-induced analgesia, Neurosci.
Res. 38, 113-116, 2000.

2.

IKEIJA,K., KOBAYASHI, T., ICHIKAWA, T., KUMANISHI, T., NIKI, H. and YANO, R: The
untranslated region ofm-opioid-receptor mRNA contributes to reduced opioid sensitiv'
ity in CXBK mice, J. Neurosci. 21, 1334-1339, 2001.

43

c.

Department of Developmental Genetics
C-a.

Division of Developmental Genetics(1) A common basis for formation of Drosophila sensory organs

Nao NIWA, Masataka OKABE and Yasushi HIROMI
Insect sensory organs such as eye and antenna are specialized for particularfunctions, and form at specific positions within each segment. Since structurally diverse segments are considered to have evolved from repeated units in aworm -like ancestor, organogenesis of segment-specific sensory organs may
share a molecular basis that is common to all segments. In Drosophila compound eye formation, the eyeless gene has been identified as a master control
gene, whose mis-expression can induce ectopic eyes in several regions of the
body. Using the ectopic eye formation by eyeless mis-expression as an experimental paradigm, we visualized spatial and temporal conditions for organ
formation in segments that normally do not produce an eye.
Competence for ectopic eye formation by eyeless was clearly restricted, andrequired Dpp and ecdysone signals. Similar constraints were found for theformation of the endogenous eye and also for other sensory organs, such as
auditory receptors (Johnston's organ) and tension receptors (chordotonal organ). These signals are integrated and result in the expression of a common
proneural gene atonalin each sensory organ precursor. Our results define amolecular pathway for a common neurogenic activity, and suggest that segment-specific sensory organs originated from an ancient prototype organ.
(!)

The relationship between sensory organ identity and positional
information in Drosophila

Masataka OKABE and Yasushi HIROMIThe embryonic peripheral nervous system has three major types of sensory

44
organs; mechanosensory organ, chemosensory organ and chordotonal organ,
each generated from respective sensory organ precursors that form at specificpositions within each segment. The identity of sensory organs is determinedat the precursor stage, by instructive transcription factors such as ahomeodomain protein Cut and a Paired-type transcription factor Pox-neuro(Pox-n). We are analyzing the regulation of cut and porn expression to identify
molecules that link positional information and the organ identity.
We have analyzed cis-regulatory elements of cut and porn using transgenic
flies carrying reporter gene constructs. We identified a 120bp genomic fragment of the cut gene at 30kb upstream from its transcription initiation site asa cis-element sufficient for expression in mechano- and chemosensory precursors. We also found that a cis-regulatory element of porn gene for expressionin chemosensory precursors is located within a 4.2kb genomic fragment upstream of its transcription initiation site. In future we plan to identify transregulatory factors that act through these cis-regulatory elements. A combination of trans-acting factors is likely to comprise a network of positional information that direct specific gene expression at particular positions.

(3) Translational repression determines a neuronal potential
in Drosophila asymmetric cell division
Masataka OKABE and Yasushi HIROMI
Asymmetric cell division is a fundamental strategy for generation of cellular
diversity during animal development. Daughter cells manifest their asymmetry in their differential gene expression. The importance of transcriptional
regulation in this process has been the focus of many studies, but cell-type
specific "translational" regulation has held a more minor role. In Drosophila
sensory organ development, Notch-signaling directs the asymmetry between
neuronal and non-neuronal lineage, and a zinc finger transcriptional repressor
Tramtrack69 (TTK69) acts downstream of Notch as a determinant of nonneuronal identity. We found that repression ofTTK69 protein expression in
the neuronal lineage occurs translationally rather than transcriptionally. Thistranslational repression was achieved by a direct interaction between cis-

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DEPARTMENT OF DEVELOPMENTAL GENETICS

45

acting sequences in the 3' untranslated region of ttk69 mRNA and its trans"
acting repressor, the RNA binding protein Musashi (MSI). Although msi canact downstream of Notch, Notch"signaling does not affect MSI expression.
Thus, Notch"signaling is likely to regulate MSI activity rather than its expres"sion. Our results define cell·type specific translational control of ttk69 by MSI
as a novel downstream event of Notch'signaling in asymmetric cell division.

(4) Transcriptional regulatory mechanism of a nuclear
recepto~ Seven-up
Motomi MATSUNO, Hiroyuki KOSEl and Yasushi HIROMI (I University ofTokushima, School of Medicine)
Neuronal subtype selection within the Drosophila eye depends on the nuclear
receptor Seven'up. Seven"up is expressed in only four of the eight photoreceptor neurons in each ommatidium, and its loss of function results in the trans'
formation of these cells into another photoreceptor subtype. Furthermore, ec"
topic expression of the Seven'up ligand binding domain causes several cellfate changes in the developing eye, depending on the cell types and their devel"
opmental stages. However, little is known about the molecular mechanisms ofSeven"up action, such as the transcriptional regulatory mechanism or its down'
stream target genes, that underlie regulatory effects ofSeven"up on cell fates.
To learn more about the role ofSeven"up in gene regulation, we have initiatedgenetic analyses of two proteins, the p52 subunit ofTFIIH and a novel SAM"
domain protein Samuel, that interact with the ligand binding domain of Seven"up in yeast cells. We have identified loss of function alleles ofp52and samuel,and have shown that they are both essential for viability. These proteins may
playa role in transcriptional regulation by Seven"up. We are also searching forthe direct target genes of Seven"up by using differential display techniques.
Since Seven"up ligand binding domain has been shown to have a transcrip"
tional repression activity in the developing eye, we are examining genes whose
expression levels are upregulated when Seven"up function is removed.

46(5) Control of glial differentiation by the homeodomain protein REPO
Yoshihiro YUASA, Masataka OKABE and Yasushi HIROMIIn Drosophila, glial determination of all neuroectodermally derived glia depends on the transcription factor Glial cells missing (GCM), which serves as abinary switch between neuronal and glial cell fates. Since the expression of
GCM is transient, other factors must be responsible for the terminal differentiation of glia. Three transcription factors, Reversed Polarity (REPO),
Tramtrack69 (TTK69), and PointedPl (PNTPl), are induced by GCM in glial
cells. REPO is a paired-like homeodomain protein, expressed exclusively in
glial cells. Mutant phenotypes of repa suggest that REPO is required for the
migration and differentiation of embryonic glial cells. In order to understandhow REPO functions in glial terminal differentiation, we have analyzed themechanism of gene regulation by REPO. We showed that REPO can act as atranscriptional activator through the CAATTA motif in glial cells, and defined
three reporter genes whose expression depends on REPO function. In different
types of glial cells, REPO can act alone, or cooperate with either TTK69 or
PNTPI to regulate different target genes. Since TTK69 and PNTPI are expressed only in a subset of cells that express REPO, their cooperation withREPO may contribute to the diversity of glial cell types. In addition, we foundthat REPO is also necessary to suppress neuronal development, cooperatingwith TTK69. We propose that REPO plays a key role in glial development bylinking glial determination and their diversification.

(6) Sprouty regulates photoreceptor neuronal number through positive
and negative effects on induction
Masaki IWANAMI and Yasushi HIROMI
Neuronal differentiation of photoreceptor cells in the Drosophila eye is triggered by a secreted inducer, Spitz EGF, that results in activation of the ras/
MAPK pathway in the induced cells. The result of induction is remarkably
constant; every ommatidia has precisely eight photoreceptor neurons locatedat stereotyped positions within the ommatidium. To achieve such constancy,

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

47

the induction process must be tightly regulated in the inducing cells and theinduced cells. Sprouty is an intracellular molecule that negatively regulates
ras signaling. Sprouty is required cell, autonomously in lens"secreting cone
cells to prevent these cells from assuming the neuronal fate. However, sproutyis also expressed in presumptive photoreceptor neurons, in particular R21R5/
R7 cells. This cell·type specificity of sprouty expression is achieved by the
repression of sproutytranscription by the nuclear receptor Seven'up, in R31R4/
Rl/R6. Loss of sproutyfunction in R2/R5 results in the up'regulation ofArgos
expression, whose transcription is induced by the ras signal that Sprouty in"hibits. Since Argos is a non-autonomous antagonist of the receptor for the
inducing signal (EGFR), Sprouty also positively regulates neuronal induction
through controlling argos expression. The dual function of Sprouty is likely tohave an important role in ensuring that the induction by a secreted signal
generates a constant outcome.

(7) Systematic identification of peptide signaling molecules in Hydra
Toshitaka FUJISAWA, Masayuki HATTA, Hiroshi SHIMIZU, Seungshic YUM, Naoe
HARAFuJI 1, Osamu KOIZUMI 2, Yoshitaka KOBAYAKAWA3, Fumihiro MORISHITA4and
Osamu MATSUSHIMA4('The Grad. Univ. for Advance Studies, 2Fukuoka Women's
Univ, 3Kyushu Univ, 4Hiroshima Univ)
We have continued our efforts to systematically screen and characterize pep'tide signallimg molecules from Hydra magnipapillata. This year we have iso"lated over 100 peptides and determined amino acid sequences of about 50 ofthem. Five genes encoding peptides were cloned and characterized. The detailed characterization was carried out on the following two peptides described
below.

(8) Morphogenetic peptides, Hym-3~3 and Hym-346 that are
involved in foot formation
Naoe HARAFUJI, Toshio TAKAHASHI, Hiroshi SHIMIZU, Masayuki HATTA andToshitaka FUJISAWA

48
It is generally thought that Hydra patterning is regulated primarily by small

diffusible substances called morphogens, We have found two morphogen candidates, Hym-323 and Hym-346 that are involved in foot formation. Bothpeptides are derived from epithelial cells and thus are termed epitheliopeptides.
They are different in amino acid sequences and coded by two independent
genes. When each of them is exogenously added to hydra, it alters the positional value and widens the foot area thus favoring foot formation. In situ
hybridization and/or immunostaining analyses in intact hydra showed that
Hym -323 was expressed both in the ectedermal and endodermal epithelial
cells throughout the body except for the basal disk, whereas Hym-346was
expressed in the foot endoderm. However, Hym-346was also expressed in theendoderm ofthe tentacle bases, suggesting its involvement in tentacle formation. During foot regeneration Hym -323 continued to be expressed until thecomplete foot formation and then ceased. This suggests that Hym-323 is required only for foot formation but not for its maintenance. In contrast, Hym346 started its expression early and continued thereafter, suggesting that
Hym-346 is required for both foot formation and its maintenance.

(9) Universal occurrence of the vasa-related genes among metazoans
and their germline expression in Hydra
Kazufumi MOCHIZUKI, Chiemi FUJISAWA and Toshitaka FUJISAWA
The vasa (vas)-related genes are members of the DEAD box protein family
and are involved in germ cell formation in higher metazoans. In the present
study, we cloned the vas-related genes as well as the PLl{}related genes, othermembers of the DEAD box protein family, from lower metazoans: sponge, Hydra and planaria. The phylogenetic analysis suggested that the vas-related
genes arose by duplication of a PLl{}related gene before the appearance ofsponges but after the diversion of fungi and plants. The vas-related genes in
Hydra, Cnvasl and Cnvas2were strongly expressed in germline cells and lessstrongly expressed in multipotent interstitial stem cells and ectodermal epithelial cells. These results suggest that the vas-related genes occur universally among metazoans and that their expression in germline cells was estab-C.

DEPARTMENT OF DEVELOPMENTAL GENETICS

49

lished at least before cnidarian evolution.
(to) Digestive movements of hydra similar to peristalsis and massperistalsis In vertebrates
Hiroshi SHIMIZU and Toshitaka FUJISAWA
Digestive movements in vertebrates' intestine such as peristalsis occur as
the concerted contraction of longitudinally and circumferentially runningmuscle processes under the control by myenteric plexus lying between them.
Although very primitive, gastrovascular cavity of hydra has structural simi'
larities to the intestine such as pattern of muscle processes running and dif·fuse nerve net between. Despite these, hydra's digestive process is thought tobe a static event. In this study we obtained evidence that behaviors similar toperistalsis and mass peristalsis occur in digestive process of hydra. The peristalsis pushed the contents forward in the cavity whereas mass peristalsis
pushed them toward the mouth resulting in defecation. Tissue excised from ananimal also underwent the two movements demonstrating that body column
tissue has endogeneous activity of these movements. Interestingly, animals
having no nerve cells showed a weak peristaltic movement suggesting a formerly unknown non' neuronal mechanism of peristalsis. These observations
suggest that hydra gastrovascular cavity is, as to dynamic movements, func'
tionally equivalent to vertebrates' intestine and that the diffuse nerve net
having no ganglia distributed in the body column controls the movements asenteric nervous system.

(11) Metamorphosis regulation of reef-building eorals
by peptide hormones
Masayuki HATTA and Toshitaka FUJISAWA
Among a number of peptides isolated from hydra, a family of peptides with
the GLWa motif in the C-ternimus have the metamorphosis-inducing activity
for marine hydrozoans. We tested biological activities of those peptides on
reef-building corals, and found that one of the GLWa family, Hym -248, can50

induce metamorphosis of coral larvae in the genus Acropora. It is suggested
that the GLWa neropeptides act as internal mediators to convert external cuesand to trigger metamorphosis hormonally, and this mechanism is conserved incnidarians. In addition, this finding provides a possible technique to turn coral
seedlings production into reality, for the first time in the long history of thereef restoration research.

Publ icatians
1.

HIRAMOTO, M., HIROMI, Y, GINIGER, E. and HOTTA, Y: A Drosophila Netrin receptor,
Frazzled, guides axons by controlling the distribution of Netrin. Nature 406, 886'889(2000).

2.

MOCHIZUKI, K., NISHIMIYA-Fu.JlSAWA, C. and FUJISAWA, T.: Universal occurrence of thevasa-related genes among metazoans and their germline expression in Hydra. Development. Genes and Evolution Gn press).
3.

BOSCH, T.C.G. and FUJISAWA, T.: Polyps, peptides and patterning. Bioessays Gn
press).

4.

TAKAHASHI, T., KOBAYAKAWA, Y, MUNEOKA, Y, FUJISAWA, Y, MOHRI, S., HATTA, M., SHIMIZU,
H., FUJISAWA, T., SUGIYAMA, T., TAKAHARA, M. and KOIZUMI, 0.: Identification of a newmember of the LWamide peptide family: Physiological activity and cellular localization in Cnidarian polyps. Biological Bulletin Gn press).

5.

HARAFUJI, N., TAKAHASHI, T., HATTA, M., TEZUKA, H., MORISIIITA, F., MATSUSHIMA, O. andFU,JlSAWA, T. (2001). Enhancement offoot formation in Hydra by a novel epitheliopeptide,Hym-323. Development t%8, 437-446.

6.

MOCHIZUKI, K., SANO, H., KOBAYASHI, S., NISHIMIYA'FUJISAWA, C. and FUJISAWA, T (2000).
Expression and evolutionary conservation of nanos-related genes in Hydra. Develop'
ment, Genes and Evolution %to, 591-602.

7.

CARDENAS, M., FABILA. Y. V., YUM, S., CERBON, J., BOEHMER, F., WETZKERR, R., FUJISAWA,
T., BOSCH TC.G. and SALGADO, L.M. (2000). Selective protein kinase inhibitors blockhead specific differentiation in hydra. Cell. Signal. t%(9-1O). 649-658.

8.

TAKAHASHI, T, KOIZUMI, 0., ARIURA, Y, ROMANOVITCH, A., BOSCH, T. C. G., KOBAYAKAWA, Y,
MOHRI, S. BODE, H., YUM, S., HATTA, M. and FU.IISAWA, T (2000). A novel neuropeptide,Hym-355, positively regulates neuron differentiation in Hydra. Development 1%7, 9971005.

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

C-b.

51

Division of Gene Expression(1) MBF2 is a tissue- and stage-specific coactivator that is regulated
at the step of nuclear transport in the silkworm Bombyx mor!

Qing-Xin LIU, Hitoshi VEDA and Susumu HIROSE
Coativators MBFl and MBF2 mediate BmFTZ-Fl-dependent transcriptional
activation in vitro by interconnecting BmFTZ-Fl, TATA binding protein TBP,
and TFIIA. Here, we analyzed temporal and spatial expression patterns of
MBF2 during embryonic and larval development ofthe silkworm Bombyx mori.
MBF2 was detected in unfertilized eggs and embryos until stage 26. In stage
22 embryos, MBFl, MBF2 and BmFTZ-Fl colocalize in neural cells. During
the larval stage, MBF2 was not expressed in the fat body and trachea. In thesilk gland, MBF2 mRNA was constitutively expressed, but MBF2 protein
appeared in the period between the second day and the molting D3 stage inboth the third and fourth instars, and then disappeared. MBF2 was also detected on the second and third days ofthe fifth instar. Immunostaining during
the fourth molt showed that MBFl, MBF2 and BmFTZ-Fllocalize in the
nucleus only at the D3 stage, while the two cofactors are present in the cytoplasm at other stages. Immunoprecipitation experiments suggested that
MBFl, MBF2 and BmFTZ-Fl form a complex at the D3 stage. Transient expression ofthese factors in Schneider cell line 2 revaled that MBFl and MBF2
localize to the nucleus and enhance BmFTZ-Fl-dependent transcription only
when all three factors are present. These data illustrate the functional regulation ofMBFl and MBF2 at the step of nuclear transport and implicate MBF2
in tissue- and stage- specific transcription. For details, see Ref. 1.
(~)

The conserved nuclear receptor Ftz-Ft is required forembryogenesis, molting and reproduction
in Caenorhabdlt!s eJegans

Masako ASAHINA I2 , Takeshi ISHIHARAJ, Marek JINDRA',4, Yuji KOHARA5 , Isao
KATSURAl and Susumu HIROSE' (IDepertment of Developmental Genetics, 2Institute of Parasitology, ;lLaboratory of Multicellular Organization, 4Institute52
of Entomology, Czech Academy of Science, Ceske Budejovice, 37005 Czech
Republic, 5Laboratory of Genome Biology, National Institute of Genetics,
Mishima, Shizuoka-ken 411-8540, Japan)
Nuclear receptors are essential players in the development of all metazoans.
The nematode Caenorhabditis elegans possesses more than 200 putative
nuclear receptor genes, several-fold the number known in any other organism.
Very few of these transcription factors are conserved with components of thesteroid response pathways in vertebrates and arthropods. Ftz-Fl, one of theevolutionarily oldest nuclear receptor types, is required for steroidogenesis
and sexual differentiation in mice and for segmentation and metamorphosis
in Drosophila.
We employ two complementary approaches, direct mutagenesis and RNAinterference, to explore the role of nhr-25, a C. elegans ortholog of Ftz-F 1.
Deletion mutants show that nhr-25 is essential for embryogenesis. RNA interference reveals additional requirements throughout the postembryonic life,
namely in molting and differentiation of the gonad and vulva. All these defects
are consistent with the nhr-25 expression pattern, determined by in situ hybridization and GFP reporter activity.
Our data link the C. elegans Ftz-Fl ortholog with a number of developmental
processes. Significantly, its role in the periodical replacement of cuticle (molting) appears to be evolutionarily shared with insects and thus supports themonophyletic origin of molting. For details, see Ref. 2.

(3) Temporally restricted expression of transcription factor 8 FTZFt:
significance for embryogenesis, molting and metamorphosis in

Drosophila meJanogaster
Masa-aki YAMADA, Takehide MURATA, Susumu HIROSE, Giovanni LAVORGNA l,
Emiko SUZUKI 2,3 and Hitoshi UEDA (lLaboratory of Morecular Cell Biology,
National Cancer Institute, National Institutes of Health, Bethesda, MD 208924255, USA, 2Department of Fine Morphology, Institute of Medical Science,
University of Tokyo, 4-6-1. Shirokanedai, Minato-ku, Tokyo 108-8639, Japan,
3CREST, Japan Science and Technology Corporation)

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DEPARTMENT OF DEVELOPMENTAL GENETICS

53

FTZ-F1, a member of the nuclear receptor superfamily, has been implicatedin the activation of the segmentation gene iushi tarazu during early
embruogenesis of Drosophila melanogaster. We found that an isoform ofFTZF1, 13 FTZ-F1, is expressed in the nuclei of almost all tissues slightly before
the first and second larval ecdysis and before pupation. Severely affected itz·
f1 mutants display an embryonic lethal phenotype, but can be rescued byectopic expression of 13 FTZ-F1 during the period of endogenous 13 FTZ-F1
expression in the wild type. The resulting larvae are not able to molt, but thisactivity is rescued again by forced expression of 13 FTZ-F1, allowing progression to the next larval instar stage. On the other hand, premature expressionof 13 FTZ-F1 in wild-type larvae at mid-first instar or mid-second instar stages
causes defects in the molting process. Sensitive periods were found to be aroundthe time of peak ecdysteroid levels and slightly before the start of endogenous
13 FTZ-F1 expression. A hypomorphic itz-f1 mutant that arrests in the prepupal stage can also be rescued by ectopic, time-specific expression of 13 FTZ-FI.
Failure of salibary gland histolysis, one ofthe phenotypes of the itz-f1 mutant,is rescued by forced expression of the itz-f1 downstream gene BR-Cduring thelate prepupal period. These results suggest that 13 FTZ-Z1 regulates genes
associated with ecdysis and metamorphosis, and that the exact timing of its
action in the ecdysone-induced gene cascade is important for proper development. For details, see Ref. 3.
(4)

CryptosporiJum paryum: Functional complementation of a parasitetranscriptional coactivator CpMBFl in yeast
Guan ZHU 1 , Michael J. LAGIER 1 , Susumu HIROSE and Janet S. KEITHLY·(IWadsworth Center, New York State Department of Health, P. O. Box 22002,
Albany, New York 12201-2002)
We report here the identification of a novel multiprotein bridging factor type1 from the apicomplexan Cryptosporidium parvum (CpMBF1), one ofthe opportunistic pathogens in AIDS patients. In slime molds, insects, and humans,
MBF1-regulated systems have been associated with cell differentiation, whichindicates that CpMBF1 could be responsible for the activation of similar sys-

54tems in C parvum during its complex life cycle. Because ofthe difficulties andhigh cost in obtaining sufficient and purified C parvum material for molecular and biochemical analyses, well-characterized yeast genetic systems may
be useful for investigating the functions of C parvum genes.
In this study, the function ofCpMBFI as an interconnecting element between
a DNA-binding regulator and TATA-box-binding protein (TBP) was confirmed
using a yeast complementation assay. Under conditions of histidine starvation, an MBF I-deficient strain of Saccharomyces cerevisiae was unableto activate the HIS3gene, which encodes imidazoleglycerol-phosphate dehydratase(IGPDH), and thus became sensitive to 3-amino triazole, an inhibitor of thisenzyme. Upon introduction of parasite CpMBFI into S. cerevisiae, 3-amino
triazole resistance of the MBFI-deficient strain was restored to wild-type
levels, and Northern blot analysis revealed that CpMBFI was able to activate
HIS3transcription in response to histidine starvation. For detail, see Ref. 4.

Publ ications
1. Lw, Q. ,X., UEDA, H. and HIROSE, S.: MBF2 is a tissue- and stage'specific coactivator
that is regulated at the step of nuclear transport in the silkworm Bombyx mori. Dev.
BioI. ZZ5: 437-446 (2000).
2. AsAHINA, M., ISHIHARA, T.. JINDRA. M., KOHARA, Y.. KATSURA, I. and HIROSE. S.: The
conserved nuclear receptor FTZ'FI is required for embryogenesis, moulting and repro'
duction in Caenorhabditis elegans. Genes Cells. 5: 711-723 (2000).
3. YAMADA, M., MURATA, T., HIROSE, S., LAVORGNA, G., SUZUKI, E. and UEDA, H.: Temporally
restricted expression of FTZ, Fl transcription factor'Significance for embryogenesis,
molting and metamorphosis in Drosohila melanogaster. Deveolpment Iln 5083-5092(2000).
4. ZHU, G., LAGIER, M.J., HIROSE, S. and KEITHLY, J. S.: Cryptosporidium parvum: Func'
tional complementation of a parasite transcriptional coactivator CpMBFl in yeast.
Exp. ParasitoI. 96: 195'201 (2000).

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DEPARTMENT OF DEVELOPMENTAL GENETICS

55

C-c. Division of Early Embryogenesis(1) In situ screen for novel genes expressed In the yolk syncytial layer
Takuya SAKAGUCHI, Atsushi KUROIWA' and Hiroyuki TAKEDA ('Nagoya University)
Mesoderm induction and its dorsoventral specification are important pro"
cess of vertebrate early development. When transplanted onto the animalpole region of host blastula embryos, the zebrafish yolk cells induce ectopic
formation of the mesoderm and the organizer, indicating that the yolk cell,
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 addressthese questions, we carried out a large scale screening of genes expressed in
the zebrafish YSL. By combination of a subtracted cDNA library with our insitu screening method, we have successfully obtained more than 10 indepen"dent"positive clones out of 200 clones tested (>5%). Sequencing analyses sug"gest that they encode some enzymes, amino acid transporting proteins, plasma
membrane binding protein, transcription factor, RNA binding protein, andmany novel genes. Functional analyses of isolated genes are now underway.
(!) Role of FGF/MAPK signaling in the developing telencephalon

of zebrafish embryos
Minori SHINYA, Sumito KOSHIDA I, Atsushi KUROIWA2 and Hiroyuki TAKEDA('Kondoh Differentiation and Signaling Project ERATO, 2Nagoya University)
The telencephalon is formed in the most anterior part of the central nervous
system (eNS) and is organized into ventral subpallial and dorsal pallial do'mains. In mice, it has been demonstrated that Fgf signaling has an importantrole in induction and patterning of the telencephalon. However, the precise role56of Fgf signaling is still unclear, due to overlapping functions of Fgf family
genes. To address this, we examine, in zebrafish embryos, the activation of
RasIMAPK, one of the major downstream targets of Fgf signaling. Immunohistochemical analysis reveals that an extracellular signal-regulated kinase(ERK), a vertebrate MAPK, is activated in the anterior neural boundary (ANB)of the developing CNS at early segmentation stages. Experiments with Fgf
inhibitors reveal that ERK activation at this stage is totally dependent on Fgf
signaling. Interestingly, a substantial amount of ERK activation is observedin ace mutants in which fgf8gene is mutated. We then examined the functionof Fgf signaling in telencephalic development by use of several inhibitors to
Fgf signaling cascade including dominant"negative form of Ras (Ras Nl7) andFgfreceptor (Fgfr), and a chemical inhibitor of Fgfr, SU5402. In treated em"
bryos, the induction oftelencephalic territory normally proceeds but the devel"
opment of the subpallial telencephalon is suppressed, indicating that Fgf signaling is required for the regionalisation within the telencephalon. Finally,
antisense experiments with morpholino"modified oligonucleotides suggest thatzebrafish fgf3, which is also expressed in the ANB, co"operates with fgf8in
subpallial development.

(3) FGF/MAPK signaling and somite maturation in vertebrate
segmentation
Atsushi SAWADA, Minori SHINYA, Yun"Jin JIANG l , Atsushi KAWAKAMI, Atsushi
KUROIWA2 and Hiroyuki TAKEDA ('Imperial Cancer Research Fund, London,
2Nagoya University)
In vertebrate, segmentation process of the somite is thought to be gov~rnedby a clock"and"wavefront mechanism. Through Notch signaling, the oscillationin each cell is coordinated and translated into the cyclic wave of gene expres"sion, such as hairyrelated genes, sweeping caudorostrally through thepresomitic mesoderm (PSM)(Sawada et aI., 2000). This cyclic wave then arrests and furrow formation initiates in very anterior PSM, the region in which
PSM cells mature and become competent to segment (a maturation wavefront).
We have examined the role ofFgflMAPK signaling, activated in the posterior

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DEPARTMENT OF DEVELOPMENTAL GENETICS

57

PSM, in zebrafish somitogenesis. Transient treatment with FGF-receptor in·hibitor promotes maturation of the PSM. In this condition, the herl (hairy
related gene) wave prematurely terminates in the intermediate PSM, leading
to formation oflarge somites. Complementary to this, transplantation ofFGF8soaked beads produces smaller somites around the bead. From these results,
we conclude that FgfIMAPK signaling activated in the posterior PSM plays acrucial role in positioning a maturation wavefront to the anterior PSM.

(4) Molecular analysis of zebrafish midline mutant chameleon
Atsushi KAWAKAMI, RolfKARLSTROM', Hiroyuki TAKEDA, William S. TALBOT2
and Alexander F. SCHIER3 ('University of Massachusetts, 2Stanford University, 3New York University)
Midline tissues, including the floor plate and notochord, produce inducing
signals and pattern the neural tube and somites. A group of zebrafish mutants(midline mutants) has common defects in the ventral neural tube, somites
and dorsal aorta. It is thought that these similar phenotypes are caused bymutations in one signaling pathway. To date, several midline mutants, sonic"
you, yOU"too, detour and smooth-musc1e"omittedhave been characterized, andtheir responsible genes are sonic hedgehog (shh) , gli2, glil and smoothened,
respectively, all of which are molecules involved in the shh signaling pathway.
As shown in these examples, the midline mutants are a useful resource tostudy a complex signaling cascade and its regulation in vivo. We are now focus"
ing on another midline mutant, chameleon (con). The rescue experiments together with the decreased expression oftarget genes suggest that the mutant
phenotypes of con are caused by reduced Shh signaling. To identify the con
gene, we mapped the mutation on LG20. We also mapped and compared thepositions of candidate genes that may play roles in the Shh pathway. To iden"
tify the con gene by positional cloning, we generated DNA markers linked tocon by AFLP. By genomic walking from the markers, we identified two YAC
clones that cover the mutated region.

58(5) EST project in medaka fish
Takanori NARITA, Tetsuaki KIMURA and Hiroyuki TAKEDA
Medaka (Oryzias latipes) has several advantages to zebrafish. For examples,
medaka has about half size of genome (800 Mb; zebrafish - 1700 Mb) and thenumber of genes are expected to be lower than that of zebrafish. Tolerance tocold temperature make it easy to obtain a temperature"sensitive mutant which
would be a powerful tool for developmental genetics. To gain insight into ge"
netic system of medaka, we perform a large scale isolation of genes expressedin developing medaka embryos.

Publ ications
I.

AMANUMA, K, TAKEDA, H., AMANUMA, H. and AOKI, Y. (2000). Transgenic zebrafish forthe detection of mutations caused by compounds in aquatic environments. Nature

Biotechnology 18: 62-65.
2.

SAWADA, A., FRITZ, A., JIANG, Y.-J., YAMAMOTO, A., YAMASU, K, KUROIWA, A., SAGA, Y. and
TAKEDA, H. (2000). Zebrafish Mesp family genes, mesp'a and mesp-b are segmentally
expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to thedeveloping somites. Development lll8: 1691-1702.

3.

KONDOH, H., VCHIKAWA, M., YODA, H., TAKEDA, H., FURUTANI-SEIKI, M. and KARLSTROM, R.
O. (2000). Zebrafish mutations in Gli mediated hedgehog signaling lead to lenstransdifferentiation from the adenohypophysis anlagen. Mechanisms ofDevelopment
96: 165-174.

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

C-d.

59

Division of Physiological Genetics(1) Transcriptional Modulation by Periodic Bent DNA through
Chromatin Structure
Yoshiaki OHNISHI, Megumi RATO, Chanane WANAPIRAK and Ryoiti KrYAMA(National Institute ofAdvanced Industrial Science and Technology)
DNA bend sites appear nearly periodically at an average interval of 680 bp,
corresponding to a length offour nucleosomes, in the human {3 -globin locus aswell as other loci (Kiyama, 1998; Wada-Kiyama et a1., 1999). These sites havea high affinity for histone core particles and their deletion results in disappearance of not only the phase at the bend site but also those in the immediate
vicinity (Onishi et a1., 1998). These lines of evidence suggest that periodic bent
DNA plays a key role in chromatin folding or nucleosome formation (Kiyama,
2000). Here we show two examples where the presence of these bend sites isclosely associated with cis-acting elements in transcription.
The first example is the enhancer of the locus, which is located 11 kb upstream ofthe E -globin gene at HS2, a DNase I -hypersensitive site (HS), andcontains the binding site for an erythroid specific transcription factor, NF-E2.
The enhancer was located between two DNA bend sites whose distance was
longer than the average and can accommodate five nucleosomes (Kiyama,
2000). The nucleosomes in this region were regularly phased except the onethat is located in the middle which corresponded to the precise location ofHS2
and included the binding site for NF-E2. Several phases were adopted in thisregion in the reconstituted chromatin and in erythroid K562 cells where theglobin genes are expressed, whereas only one phase was adopted in non-erythroid HeLa cells. Meanwhile, almost unique phases were adopted at the flanking bend sites in vitro as well as in vivo. This suggests that HS2 is placed at aregion of weak nucleosome phasing activity along with factor binding sites andcould be influenced by the nucleosomal phases determined by those located at
the bend sites. Interestingly, changing the distance between one of the bendsites and NF-E2 site affected transcription efficiency, further supporting thesignificance of this site.

60

The other example is the silencers which are present in the promoter region of
the globin genes. The most characterized silencer in this locus is located 200 to
300 bp upstream of the E 'globin and overlaps the first DNA bend site fromthe cap site (Wanapirak et al., 2000). This co'localization of the silencer or the
repressor activity is true for the {3 'globin gene, and also for the C' mye, erythro'
poietin receptor and estrogen receptor genes. Furthermore, co'localization wasobserved among all ofthe human {3 'like globin genes and the globin genes ofthe other species where the locations of the first bend site upstream from thecap site are conserved. Although these sites contain transcription factor binding sites and binding sites for less characterized proteins, the mechanism of
silencing gene expression is not clear. Therefore, co' localization of DNA bendsites with silencers could become a clue for understanding the regulation.

Publ icatians
1.

2.

3.

WADA-KiYAMA, Y, SUZUKI, K. and KIYAMA, R.: DNA Bend Sites in the Human 13 -Globin
Locus: Evidence for a Basic and Universal Structural Component of Genomic DNA.
Mol. BioI. Evol., 16, 922-930, 1999.
WANAPlRAK, C., ONISHI, Y., WADA-KIYAMA, Y, OHYAMA, T. and KIYAMA, R.: Conservationof DNA Bend Sites with Identical Superhelical Twists among the Human, Mouse,
Bovine, Rabbit and Chicken 13 -Globin Genes. DNA Res. 7, 253-259, 2000.
KIYAMA, R.: Structure and Function of Periodic Bent DNA in Higher Eukaryotes.
Seibutsubutsuri (Biophysics) 40, 173-178, 2000.

61

D.

Department of Population Genetics
D-a.

Division of Population Genetics(1) Genetic screens for factors involved in the notum bristle loss ofinterspecific hybrids between Drosopbl/a me/allogasterand D. slmu/alls
Toshiyuki TAKANO-SHIMIZUInterspecific cross is a powerful means to uncover hidden within- and between-species variation in populations. one example is a bristle loss phenotype of hybrids between Drosophila melanogaster and D. simulans, thoughboth the pure species have exactly the same pattern of bristle formation on thenotum. There exists a large amount of genetic variavility in the simulans
populations with respect to the number of missing bristles in hybrids, and thevariation is largely attributable to simulans x chromosomes. Using nine molecular markers, I screened the simu/ans x chromosome for genetic factors thatwere responsible for the differences between a pair of simulans lines with high(H) and low (L) missing bristle numbers. Together with duplication-rescue
experiments, a single major quantitative locus was mapped to a 14-15 region.
Importantly, this region accounted for most of the differences between Hand Llines in three other independent pairs, suggesting segregation of Hand L
alleles at the single locus in different populations. Moreover, a deficiency screening uncovered several regions with factors that potentially cause the hybrid
bristle loss due to epistatic interactions with the other factors. For details, see
Ref. 1.
(!) Region-dependent regulation of mutation and crossover frequencies

along Drosophila chromosomes
Toshiyuki TAKANO-SHIMIZU

62

Eukaryotic chromosomes are organized into discrete high -order domains ofseveral different levels and categories such as nucleosomes, transcription units,
and replication units. On a larger scale, cromosomes of warm-blooded verte'
brates, particularly those of humans, are divided into GC"rich and GC'poor
segments of sizes varying from 200 kb to more than 1 Mh, the so'called isochore
structure. By contrast, Drosophila genomes have been considered to be much
more homogeneous with respect to G+C content along chromosomes. However,I found evidence of irregular silent"site substitution rates and a regional het"
erogeneity in GC~AT substitution pattern in Drosophila genomes. Substitu'
tions in Drosophila are generally biased toward A and T; the estimated num"
ber of AfT ---+ GIC substitutions in restricted small x telomeric regions com"pletely outnumbered that of the reverse substitutions independently in two
Drosophila lineages. The data in noncoding regions, further, suggested muta"
tional biases as a cause of the substitution biases.
Local changes in recombination rates also contribute to the irregular synony"mous substitution patterns. This is because linkage associations between
selective loci reduce the efficacy of natural selection (the so'called Hill·
Robertson effect) and most synonymous changes in Drosophila as well as inunicellular organisms are not strictly neutral but under weak selection. I foundevidence that an about 20'fold reduction in the x-telomeric crossover frequen'cies have occurred in the lineage leading to D. melanogaster. This is a region"
wide but only local effect because the total map length ofthe x chromosome did
not differ so much among the compared Drosophila species.
In sum, the above regional variations along the Drosophila chromosomes
suggest the presence of region' dependent regulation mechanisms of mutationand crossover frequencies, which, in turn, lead to a regional variation in evolu'
tionary rate within the Drosophila genomes. For details, see Ref. 2.
I should add that Ms Yuriko Ishii has contributed significantly to the above
two works.

Publ ications
1.

TAKANO'SHIMIZU, T.:

Genetic screens for factors involved in the notum bristle loss ofinterspecific hybrids between Drosophila melanogaster and D. simulans. Genetics

D.

DEPARTMENT OF POPULATION GENETICS

63

156, 269-282, 2000.

2. TAKANO-SHIMlZU, T.: Local changes i!?- GC/AT substitution biases and in crossover
frequencies on Drosophila chromosom¢s. Molecular Biology and Evolution (in press),
2001.

D-b.

Division of Evolutionary Genetics(1) Functional analyses of centromere of higher vertebrate cells
Tatsuo FUKAGAWA, Atsushi OKAMURA, Ai NISHIHASHI, Yoshikazu MIKAMI' andToshimichi lKEMURA ('Grad. Univ. Advanced Studies)
The centromere plays a fundamental role in accurate chromosome segrega'tion during mitosis and meiosis 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 whichcentromeres interact with microtubules of the spindle apparatus during cell
division is not fully understood.
To understand the function of the centromere, we were led to develop a genetic
analysis method that utilizes the hyper-recombinogenic chicken B lymphocyte
cell line DT40. The high level of homologous recombination in DT40 cells allowed targeted disruption of genes for several centromere proteins. We at·
tempted to generate several conditional knockout cell lines of several centromere proteins, including CENP'C, CENP-H, ZWlO and Mis6. We also cre'ated several temperature' sensitive CENP-C mutants with DT40 cells. Pheno'
typic analysis of these mutants revealed several things.
I) CENp·C is necessary but not sufficient for the formation of a functional
centromere and the structure of CENp·C may be regulated during the cell
cycle.
II) CENP'C may serve further functions during G 1 of the cell cycle in addition
to its role in mitosis.
III) Centromere assembly in vertebrate cells proceeds in a hierarchical man'ner in which localization ofthe centromere-specific histone CENP·A is an early
event that occurs independently ofCENP'C and CENP-H.

64IV) ZW10 is required for a spindle checkpoint function in vertebrate cells.
V) We identified a chicken counterpart of Mis6 that is centromere protein offission yeast. We found that chicken Mis6localizes to centromere during cell
cycle and is essential for function and assembly of centromere.
For detail, Fukagawa et ai. EMBO J., (2001) and Fukagawa et aI., Nuci. Acids
Res. (2001).
(!) Analysis of DNA replication timing of centromere region of

mammalian artificial chromosomes
Tatsuo FUKAGAWA, Ai NISHIHASHI, Toshimichi IKEMURA, Megumi NAKANo l andHiroshi MASUMOT0 1 (1 Univ. Nagoya)
There are two alternative approaches to the construction of mammalian artificial chromosomes (MACs). In the first, "bottom'up" approach, homologous
recombination in the yeast is used to assemble MACs from candidate se'quences that have been cloned in YACs. In the second, "top'down" approach,assembly of a chromosome from cloned constituents is avoided and, instead, anatural mammalian chromosome is whittled down to a mini'chromosome bytelomere' directed chromosome breakage. We are using both MACs to study
centromere function.
H. Masumoto and his colleague had developed a MAC by "bottom'up" ap'proach (Ikeno et aI., Nature Biotech., 1998). In the process of experiments, theycreated two kinds of cell lines; one line contains a stable mini chromosome
with alpha satellite sequence that functions as centreomere DNA. The otherline contains a chromosome in which alpha satellite sequence is integrated on
random site and does not function as centromere. We measured DNA replica'tion timing of alpha satellite sequence in both cell line. We found that replica'tion timing of alpha satellite sequence in the centromere is late in S phase,
but it is faster when alpha satellite sequence is integrated on random site in
the chromosome. We are now studYifg the significance of DNA replication
timing for centromere assembly.

D. DEPARTMENT OF POPULATION GENETICS

65(3) Chromosome-wide assessment of replication timing for humanchromosomes llq and 21q reveals disease-gene-rich regions
Yoshihisa WATANABE, Asao FUJIYAMA 1•2, Yuta ICHIBA, Yoshiyuki SAKAKI 2 andToshimichi IKEMURA (lDivision of Human Genetics, 2Human Genome ResearchGroup, RIKEN Genomic Sciences Center)
Mammalian DNA replication proceeds in a precisely regulated mannerwhereby Mb-sized clusters of replicons are activated at distinct times in S
phase. The replicons are heterogeneous in size, but most are 50 to 450 kb inlength. Several to 10 (or more) contiguous replicons with origins that fire synchronously at a specific time comprise a Mb-sized domain that can be visualized cytogenetically as a band by the replication-banding method. We measured replication timing ofthe entire lengths of human chromosomes llq and
21q. Megabase-sized zones that replicate early or late in S phase (thus early/
late transition) were defined at the sequence level. Early zones were more GCrich and gene-rich than were late zones, and early/late transitions occurred
primarily at positions identical to or near GC% transitions. In the early/latetransition regions, concentrated occurrence of cancer-related genes including
CCNDl, FGF4, TIAMI and FLIl was observed. The transition regions contained other disease-related genes including APP, SODI and PTS. We also
found the single nucleotide polymorphism (SNP) frequency was higher in thetransition regions. These findings are discussed with respect to the predictionthat increased DNA damage occurs in replication-transition regions.

(4) Codon usage and tRNA genes in eukaryotes: correlation of codon
usage diversity with translation efficiency and with CGdinucleotide usage as assessed by multivariate analysis
Shigehiko !{ANAYA l, Yuko YAMADA 2, Makoto KINOUCHI 1, Yoshihiro KUDO l andToshimichi IKEMURA (IDepartment ofBio-System Engineering, Faculty of Engineering, Yamagata Univ., 2Department of Biochemistry, Jichi Medical School)
Species·specific diversity ofcodon usage in five eukaryotes (Schizosaccharomyces

pombe, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and66

Homo sapiens) was investigated with principal component analysis. Optimal
codons for translation were predicted on the basis of tRNA-gene copy numbers. Highly expressed genes, such as those encoding ribosomal proteins andhistones in S pombe, C elegans, and D. melanogaster, have biased patterns ofcodon usage, which have been observed in a wide range of unicellular organisms. In S pombe and C elegans, codons contributing positively to the principal component with the largest variance (Zl-parameter) corresponded to the
optimal codons which were predicted on the basis oftRNA gene numbers. In D.
melanogaster, this correlation was less evident, and the codons contributing
positively to the Zl-parameter corresponded primarily to codons with a C or G
in the codon third position. In X laevis and H sapiens, codon usage in thegenes encoding ribosomal proteins and histones was not significantly biased,suggesting that the primary factor influencing codon usage diversity in these
species is not translation efficiency. Codon usage diversity in these species isknown to reflect primarily isochore structures. In the present study, the second
additional factor was explained by the level of use of codons containing CG
dinucleotides, and this is discussed with respect to transcription regulation
via methylation of CG-dinucleotides, which is observed in mammalian genomes. For details, see Kanaya et aI., J. Mol. Evol. (2001).

(5) Analysis of codon usage diversity of bacterial genes with a selforganizing map(SOM): characterization of horizontally transferredgenes with emphasis on the E. colJ 0157 genome
Shigehiko !{ANAYA!, Makoto KINOUCHI', Yuko YAMADA2, Yoshihiro KUDO' andToshimichi IKEMURA ('Department of Electrical and Information Engineering,
Yamagata Univ., 2Department of 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 of protein-coding
sequences for a wide range of genomes. In the present study, we used a novel
neural-network algorithm, a self-organizing map (SOM), to efficiently andcomprehensively analyze codon usage in approximately 60,000 genes from 29
bacterial species simultaneously. This SOM makes it possible to cluster andD.

DEPARTMENT OF POPULATION GENETICS

67

visualize genes of individual species separately at a higher resolution thancan be obtained with principal component analysis. The organization of the
SaM can be explained by the tRNA compositions and genome G+C% of theindividual species. Using SaM, we examined codon usage heterogeneity in theE. coli 0157 genome, which contains "0157-unique segments (a-islands)",
and showed that the SaM is a powerful new strategy for characterization ofhorizontally transferred genes. For details, see Kanaya et al., Gene (2001).

(6) Skew and principal component analysis of prokaryotic andeukaryotic genomes
Yuta ICHIBA, Yoshihisa WATANABE and Toshimichi IKEMURA
GC skew that has been connected with asymmetries in base composition
between the leading and the lagging strands was reported previously in manyprokaryotic genomes. We analyzed mono-nucleotide and di-nucleotide skew ofyeast and human genomes, as well as of several prokaryotic genomes in thefollowing strategy. To find the most skewed nucleotide, we conducted multivariate analysis (principal component analysis; PCA) on the 5 - 100 kb segments of individual genomes. By using the skew and PCA, we have found thata significant proportion of the skews and the factor scores of PCA can be attributed to replication orientation. The present skew and the factor score canpredict several ARSs of Saccharomyces cerevisiae chromosome VI. In addition, the correlation of the skew and factor score pattern with replicationtiming of human genomes was observed. There also seems to be correlation
between factor scores and SNPs. In the cyanobacterium Synechocystis sp, themost skewed di-nucleotide and factor score can predict an origin of replication
which could not be identified by GC and AT skew.

(7) Programs for constructing phylogenetic trees and networks ofclosely related sequences
Naruya SAITOU
Nowadays increasingly many closely related sequences are deposited to the

68
DDBJIEMBLIGenBank nucleotide sequence database. To deal with those vast
number of closely related nucleotide sequences, I recently developed a series ofprograms to process closely related mass sequence data. We start from BLAST
homology search. Program pO extracts sequences homologous to query sequenceand produce FASTA format output file. After retrieving those homologous se"quences, multiple alignment usually follows. However, when we restrict oursearch only to closely related sequences, multiple alignment is not necessary,for BLAST already extracted homologous regions and those rarely have gaps.
Therefore, we can skip multiple alignment process which often takes a very
long computer time compared to BLAST search. After format transformation
via programs pI and p2, program p3 eliminates invariant sites. When we deal
with closely related sequences, many invariant site are expected to exist, andthis procedure can reduce the data file extensively. Program p4 then examinessequence identity, and all the identical sequences are joined. Consequently,
only different sequences remain. The next step is to eliminate 'single polymorphic' sites in which only one sequence have different character and all others
have the same character. This is conducted by program p5. In this fashion, we
can rapidly extract so"called phylogenetically informative sites for maximum
parsimony methods. For details, see ref. 13.

(8) CAMUS DB: Development of structural database forhomology search
M KIKUCHI l, S MISU l, Tadashi lMANISHI l and Naruya SAITOU (lDNAData Bankof Japan, Center for Information Biology, National Institute of Genetics)
DDBJIEMBUGenBank International Nucleotide Sequence Database (INSD)
is increasing with unexpected rate (doubling time is only slightly longer than
one year), and computation time for homology search is becoming longer andlonger. Because nucleotide sequences have their own nature to make copies
through DNA replication, there are many similar, evolutionarily closely reo
lated nucleotide sequences in INSD. We therefore developed new system tocluster similar sequences and make compressed sequence database consist"ing of representative sequences ofthose clusters. Each cluster consists of highly

D. DEPARTMENT OF POPULATION GENETICS

69

homologous sequences, and they are presented as multiply-aligned form. We
DDBJ [1] call those combined database as CAMUS (Compressed database
And MUltiple aligned Sequence database). Its URL is http://wolf.genes.nig.ac.jp/
camus/. For details, see ref. 14.

(9) Gene diversity of chimpanzee ABO blood group elucidated from
Intron 6 sequences
Takashi KrTANO, Reiko NODA, Kenta SUMIYAMA, Robert E. FERRELLI and Naruya
SAITOU (IDepartment of Human Genetics, University of Pittsburgh, USA)The human and non-human primate ABO blood group gene shows relatively
large numbers of nucleotide differences among the exon 7 region. In this study,
we determined intron 6 sequences for 10 alleles of common chimpanzee andfor 3 alleles of bonobo to estimate nucleotide diversities among them. Sequence length polymorphisms are observed in this region due to differences ofrepeat numbers from 1 to 5. From a phylogenetic network of intron 6 sequencesofABO blood group genes for human, common chimpanzee, and bonobo, effectsof parallel substitutions and/or some kinds of convergent events are predictedin the chimpanzee lineage. We also estimated nucleotide diversities for common chimpanzee and bonobo ABO blood group genes, and these values were0.219 and 0.208 percent, respectively. For details, see ref. 15.

(10) Evolution of the ABO blood group gene in Japanese macaque
Reiko NODA, Takashi KrTANO, Osamu TAKENAKA 1 and Naruya SAITOU (IPrimate Research Institute, Kyoto University, Inuyama)
We determined 5 sequences of Japanese macaque ABO blood group gene exon
7 (ca. 0.5kb) and 2 sequences for exon 5 and intron 6 (ca. 1.7kb). We comparedthose data with published sequences of other Old World monkey species, andthe results suggest that alleles A and B were polymorphic in the ancestral
species of macaques, and that B type allele evolved independently in macaqueand baboon lineages. For details, see ref. 16.

70(11) Evolutionary history of the Rh blood group-related
genes in vertebrates
Takashi KrTANO and Naruya SAITOU
Rh and its homologous Rh50 gene products are considered to form
heterotetramer on erythrocyte membranes. Rh protein has Rh blood groupantigen sites, while Rh50 protein does not have Rh blood group antigen sites,
and is more conservative than Rh protein. We previously determined both Rh
and Rh50 gene cDNA coding regions from mouse and rat, and carried out
phylogenetic analyses. In this study, we determined Rh50 gene cDNA coding
regions from African clawed frog and Japanese medaka fish, and examined thelong-term evolution ofthe Rh blood group genes and their related genes. We
constructed the phylogenetic tree from amino acid sequences. Rh50 genes ofAfrican clawed frog and Japanese medaka fish formed a cluster with mammalian Rh50 genes. The gene duplication time between Rh and Rh50 genes wasestimated to be about 510 million years ago based on this tree. This periodroughly corresponds to the Cambrian, before the divergence between jawlessfish and jawed vertebrates. We also BLAST-searched amino acid sequence
database, and the Rh blood group genes and their related genes were found tohave homology with ammonium transporter genes of many organisms. Ammonium transporter genes can be classified into two major groups (amt a andamt b). Both groups contain genes from three domains (bacteria, archaea, andeukaryota). The Rh blood group genes and their related genes are separated
from both amt a and b groups. For details, see ref. 17.

(1!) Genetic structure of a !500-year-old human popuiation In China
and its spatiotemporai changes
Li WANG 1,2, Hiroki OOTA 1, Naruya SAITOU, Feng JIN2, Takayuki MATSUSHITAland Shintaroh UEDA1(lGraduate School of Science, University of Tokyo, Tokyo,
2Institute of Genetics, Chinese Academy of Sciences, Beijing, China,:3Doigahama Site Anthropological Museum, Houhoku)
To examine temporal changes in population genetic structure, we comparedD. DEPARTMENT OF POPULATION GENETICS71

the mitochondrial DNA (mtDNA) sequences of three populations that lived in
the same location, Linzi of China, in different periods: 2500 years ago (theSpring-Autumn era), 2000 years ago (the Han era), and the present day. Two
indices were used to compare the genetic differences: the frequency distribu'
tions of the radiating haplotype groups and the genetic distances among the
populations. The results indicate that the genetic background of the three
populations is distinct from each other. Inconsistent with the geographicaldistribution, the 2500'year-old Linzi population showed greater genetic simi'
larity to present-day European populations than to present-day east Asian
populations. The 2000 year-old Linzi population had features that were intermediate between the present-day European/2500-year-old Linzi populations
and the present-day east Asian populations. These relationships suggest theoccurrence of drastic spatiotemporal changes in genetic structure of Chinese
people during the past 2500 years. For details, see ref. 18.

(13) Sequence variation in the ABO blood group gene exon 7 ofchimpanzee and bonobo
Kenta SUMIYAMA, Takashi KrTANO, Reiko NODA, Shintaroh UEDA, Robert E.
FERRELL' and Naruya SAITOU (IDepartment of Human Genetics, University ofPittsburgh, USA)
Human and non-human primate ABO blood group genes show relatively large
numbers of nucleotide differences. In this study, we determined exon 7 sequences for 10 individuals of common chimpanzee and for 4 individuals ofbonobo to estimate nucleotide diversities among them. Sequence data showed
the existence of chimpanzee specific 9 base deletion in the beginning of theexon 7 coding region. From a phylogenetic network of exon 7 sequences of ABO
blood group genes for human, common chimpanzee, bonobo and gorilla, effectsof parallel substitutions and/or some kinds of convergent events are inferredin the chimpanzee lineage. We also estimated nucleotide diversities for common chimpanzee and bonobo ABO blood group genes, and these values were
0.4 % and 0.2 % percent, respectively. These values are higher than that ofmost human genes. For details, see ref. 19.

72

Pub I i ca t ions
1.

2

3.

FUKAGAWA, T., MIKAMJ, Y., NISHIHASHI, A., REGNIER, Y, HARAGUCHI, T, HIRAOKA, Y., SUGATA,
N., TODOKORO, K., BROWN, W. and IKEMuRA, T.: CENP- H, a constitutive centromere
component, is required for centromere targeting of CENP-C in vertebrate cells. EMBO
J., Vol. 20, 4603-4617 (20011.
FUKAGAWA, T., REGNIER, Y and IKEMuRA, T: Creation and characterization of tempera'
ture'sensitive CENP-C mutants in vertebrate cells. Nucl. Acids Res., Vol. 29, (2000, inpress.
KANAYA, S., KINOUCHI, M., YAMADA, Y., KUDO, Y. and IKEMuRA, T.: Analysis of codon
usage diversity of bacterial genes with a self-organizing map (SOM): characterizationof horizontally transferred genes with emphasis on the E. coli 0157 genome. Gene,(2000, in press.

4.

OKAMURA, A., PENDON, C., VALDIVIA, M. M., IKEMURA, T. and FUKAGAWA, T: Gene structure, chromosomal localization and immunolocalization of chicken centromere proteins CENP'C and ZWlO. Gene, 262, 283-290 (2001).

5.

KANAYA, S., YAMADA, Y., KINOUCHI, M., KUDO, Y. and IKEMuRA, T.: Codon usage andtRNA genes in eukaryotes: correlation of codon usage diversity with translation

6.

efficiency and with CG'dinucleotide usage as assessed by multivariate analysis. J.
Mol. Evol. (2000, in press.
NAKAMURA, Y., GOJOBORI, T. and IKEMuRA, T: Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Research, 28,292 (2000).

7.

8.

KANAYA, S., FUKAGAWA, T, MilO, A., INoKo, H., KUDO, Y. and IKEMuRA, T.: Distribution ofpolypurine/polypyrimidine tract sequences in the human MHC region and their possible functions. In: Major Histocompatibility Complex, Springer-Verlag Berlin/Heidelberg/New York/Tokyo, 131-145 (2000).
OllNO, M., TENZEN, T, WATANABE, Y., YAMAGATA, T, KANAYA, S. and IKEMuRA, T: Non-B
DNA structures spatially and sequence-specifically associated with individual centromeres in the human interphase nucleus. Chromosomes Today, 13, 57-69 (2000).

NOGAMI, M., NOGAMI, 0., KAGOTANI, K., TM;UCHI, H., IKEMURA, T and OKUMURA, K.:
Intranuclear arrangement of human chromosome 12 correlates to large-scale replication domains. Chromosoma, 108, 514-522 (2000).
10. WATANABE, Y., TENZEN, T, NM;ASAKA, Y., INIKO, H. and IKEMURA, T: Replication timing
ofthe human X-inactivation center (XIC) region: correlation with chromosome bands.
9.

Gene, 252, 163-172 (2000).
11.

UEMURA, T., KUBO, E., KANARI, Y., IKEMURA, T., TATSUMI, K. and MuTO, M.: Temporaland spatial localization of novel nuclear protein NP95 in mitotic and meiotic cells. Cell
Structure and Function, 25, 149-159 (2000).

D.
12.

13.

14.

15.

16.
17.
18.

19.

DEPARTMENT OF POPULATION GENETICS73

NOGAMI, M., KOHDA, A., TAGUCHI, R., NAKAO, M., IKEMURA, T. and OKUMURA, K: Relative
locations of the centromere and imprinted SNRPNgene within chromosome 15 territories during the cell cycle in RL60 cells. Journal of Cell Science U3, 2157-2165 (2000).
SAITOU, N.: Programs for constructing phylogenetic trees and networks of closely
related sequences. I IWATUKI, K (ed,), "lIAS International Symposium on Biodiversity",
International Institute for Advanced Studies, Kyoto, pp. 45-50, 2000.
KIKUCHI, M., MISU, S., IMANISHI, T. and SAITOU, N.: CAMUS DB: Development ofstructural database for homology search. In Miyano, S., Shamir, R. and Takagi, T.
(eds.l, "Currents in Computational Molecular Biology", Universal Academy Press,
Tokyo, pp. 80-81, 2000.
KITANO, T., NODA, R, SUMIYAMA, K, FERRELL, R. E. and SAITOU, N.: Gene diversity ofchimpanzee ABO blood group elucidated from intron 6 sequences. Journal of Reredity,
91,211-214,2000.
NODA, R., KITANO, T., TAKENAKA, O. and SAITOU, N.: Evolution of the ABO blood groupgene in Japanese macaque. Genes and Genetic Systems, 75, 141-147,2000.
KITANO, T. and SAITOU, N.: Evolutionary history of the Rh blood group'related genes invertebrates. Immunogenetics, 51, 856-862, 2000.
WANG, L., OOTA, R., SAITOU, N., JIN, F., MATSUSHITA, T. and UEDA, S.: Genetic structureof a 2500-year-old human population in China and its spatiotemporal changes. Molecular Biology and Evolution, 17, 1396-1400, 2000.
SUMIYAMA, K, KITANO, T., NODA, R, UEDA, S., FERRELL, R. and SAITOU, N.: Sequencevariation in the ABO blood group gene exon 7 of chimpanzee and bonobo. Gene, vol.
Z59, pp. 75-79, 2000.

D-c.

Division of Theoretical Genetics(1) Autonomous formation of spatial pattern In development

Shigeru KONDOH
Most of biological phenomena occur as the result of complex interaction of thegenes or molecules. As the number of the element increases, the relationships
among them become terribly complex as the exponential function. Complex
phenomena like morphogenesis or neural net formation, occurs as the chain
reaction of the local interaction of cells. Therefore, it is quite difficult to imago
ine what really happens. In such case, computer simulation may help.
In our laboratory, using the skin pattern of zebra fish as a model system, we
aim to develop a practical standard method to simulate complex phenomena

74
in biology.
From the observation with the skin pattern of Pomacanthus and other fish,
we are sure that the skin pattern offish is a "wave" generated by a putative
mechanism called reaction-diffusion. However, there is no molecular information yet.
Several mutant lines that have altered skin pattern are isolated in the large
scale screening experiment done in Tubingen. Cloning of the genes will be very
helpful. On the other hand, knowing the skin structure as the field of wave
formation is also very important. Our study is, now, concentrated on this subject.
From the electro-microscopic analysis and the transplantation experiment,
followings are revealed.
(l}the skin of zebrafish is composed of the three layers, epithelium, mesenchyme and pigmented cell layer. Melanophore and iridophore are located in
the pigment cell layer. However, xhantophore locates in the mesenchyme layer.
(2)From the transplantation experiment, the positional information exist in
the mesenchyme layer.
(3)From the chimera experiment, we conclude that melanophore does not involved in the pattern formation, that is against the former anticipation.

Publ icatian
None

75

E.

Department of Integrated Genetics
E-a.

Division of Human Genetics(1) Genome analysis of the mouse 7F4/F5 imprinted domain

Hiroyuki SASAKI, Chikako SUDA, Hisao SHIROHZU, Wahyu PURBOWASIT0 1,
Tsunehiro MUKAI 2, Masahira HATTORI;] and Yoshiyuki SAKAKI 3 (JInst. of Genetic
Information, Kyushu Univ, 2Dept. of Biochemistry, Saga Medical College, 3Genome Science Center Riken)
Genomic imprinting, an epigenetic gene marking phenomenon, causes parental-origin' specific monoallelic expression of a subset of mammalian genes.
Imprinted genes tend to form clusters in specific regions ofthe genome, which
may be related to the mechanism of imprinting or the reason for the evolutionof imprinting. As a step to understand the structural and functional characteristics ofthe imprinted genome domains, we are studying an imprinted domain
in mouse chromosome band 7F41F5, which contains at least eight imprinted
genes. This region is syntenic to human llp15.5, which contains genetic lociresponsible for Beckwith-Wiedemann syndrome and some types of childhood
and adult tumors. YAC, BAC and cosmid contigs covering the 1-Mb region
were constructed and the sequence of the entire imprinted domain has now
been determined using selected BAC clones. Part of this sequence has been
described in ref. 3. A more detailed analysis of the sequence is now ongoing to
identify new imprinted genes and their regulatory elements. We have also
started to map the nuclear matrix attachment regions (MARs) of the region,
which might play an important role in the regulation of the imprinted domain.
(!) Regulation of imprinting of the mouse IgIZ/H19 sub-domain

Hiroyuki SASAKI, Ko ISHIHARA, Hiroyasu FURUUMI, Mizuki OHN0 1, Takayuki
UEDA2, Ryo KOMINAMI 3 and Akihiro UMEZAWA 4 (IInst. of Genetic Information,

76

Kyushu Univ, 2Inst. of Molecular Genetics and 3Dept. of Biochemistry, Facultyof Medicine, Niigata Univ, 4Dept. of Pathology, Faculty of Medicine, Keio Univ.)
The imprinted mouse 7F4/F5 domain contains two linked imprinted genes
Igf2 and H19 near its centromeric boundary: Igf2 is paternally expressed andH19 maternally expressed. We have identified five evolutionarily conserved
tissue-specific enhancers in the downstream region of H19which are potentially involved in the reciprocal expression patterns of the two genes (ref. 1).
We also identified a conserved 39-bp element within the differentially methylated region (DMR) upstream of H19which is capable offorming complexes
with specific nuclear factors including CTCF. Binding of one of the novel factors was inhibited by methylation of the CpG dinucleotides within the target
sequence, just as was the case for CTCF (ref. 1). These complexes may contribute to the presumed boundary function of the unmethylated DMR on the maternal chromosome, which is proposed to insulate maternal Igf2from the enhancers (ref. 2). Paternal-specific methylation of the DMR is thought to be theprimary imprint regulating the Igf2/H19 imprinting. We found that this methylation is inherited from sperm (ref. 4 and 10) and that it is established in
the gonocyte stage during fetal testis development (ref. 4). By using a novel
allele-specific RNA-FISH technique, it was also found that the paternal-specific expression of Igf2 begins in the blastocyst stage despite the presence ofthe methylation imprint in earlier pre-implantation embryos (ref. 7). Finally,
we reported that the degree of chromatin packaging is different between theparental alleles of the imprinted genes (ref. 5).

(3) Role for de noyo DNA metbyltransferases Dnmt3a/Dnmt3b
in genomic imprinting
Hiroyuki SASAKI, Naomi TSUJIMOTO, Masahiro KANEDA and Shoji TAJIMA 1(!lnst. of Protein Biochemistry, Osaka Univ.)
DNA methylation works as an important gene marking mechanism to distinguish the parental alleles of imprinted genes. To understand how the primary
imprints are established in the male and female germ cells, we are studying

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DEPARTMENT OF INTEGRATED GENETICS77

the expression and localization of the de novo DNA methyltransferases
Dnmt3a/Dnmt3b in the male and female gonads. We have also started to
produce mice lacking Dnmt3a and/or Dnmt3b only in the germ cell lineage byusing conditional gene knockout system.

(4) de novo DNA methyltransferases Dnmt3a/Dnmt3b andhuman disorders
Hiroyuki SASAKI, Hisao SHIROHZU, Shin'ich MIZUNO l , Takeo KUBOTA2 and Shoji
TAJIMA3 (IInst. of Genetic Information, Kyushu Univ, 2Natl. Center of Neurol'ogy and Psychiatry, 3Inst. of Protein Biochemistry, Osaka Univ.)
It is known that tumor suppressor genes are often methylated and inacti'
vated in a number of cancers. We have found that acute meyelogenous leuke'
mia cases with methylated p15 tumor suppressor gene have higher levels of
all three DNA methyltransferases DNMT1, DNMT3A and DNMT3B(ref. 6).
We also studied three Japanese cases with ICF (immunodeficiency, centro'
meric instability, facial anomalies) syndrome, an autosomal recessive disorder caused by DNMT3Bmutations, and identified three novel mutations.

(5) Studies on the possibilities of genomie imprinting andZ ehromosome dosage eompensation in ehieken
Hiroyuki SASAKI, Takaaki YOKOMINE, Asato KUROIWA 1, Yoichi MATSUDA l andMasaoki TSUDUKI 2 (I Chromosome Research Unit, Hokkaido Univ, 2Facultyof
Animal Production, Hiroshima Univ.)
Although it is generally thought that genomic imprinting has evolved only inmammals, this has not been tested in other vertebrate species. We have asked
whether the homologs ofthe mammalian imprinted genes show parental'ori'
gin' specific monoallelic expression in chicken. Using DNA polymorphisms iden'tified between various breeds and strains, it was shown that both IGF2 andMPRl are expressed biallelically in chicken embryos (ref. 9). We also foundthat chicken Z chromosome is not subject to inactivation for gene dosage com'pensation in ZZ males. We are currently studying the structure and function of

78

chicken DNA methyltransferases DNMT3A1DNMT3B, whose cDNAs have beencloned in our laboratory.

(6) Are the polycomb group genes involved in genomic imprinting?
Hiroyuki SASAKI, Naomi TSUJIMOTO and Haruhiko KOSEKI l ('Chiba Univ. Graduate School)
Polycomb group proteins (PcGs) are the components oflarge chromatin complexes called polycomb complexs and involved in gene silencing and insulator
functions in Drosophila. Previous experiments using transgenic flies suggested
that mammalian PcGs may be involved in the allele-specific repression of
imprinted genes. We have examined the allele-specific expression of Igf2and
H19in mice disrupted with two PcG genes Mel-18and Bmi-l. In both single
homozygotes ([Mel-18/] and [Bmi-l/-J) and double homozygotes ([Mel-18-/-,Bmi-l-/-J), the proper imprinted expression patterns of both Igf2and H19were

maintained, suggesting that these PcG genes are not involved in the imprinting of the two genes.

(7) Analysis of anti-sense RNA Identified In the X/st region
Takashi SADO, Hiroyuki SASAKI and En LI ' ('CVRC, MGH, USA)
X-chromosome inactivation is an important gene dosage compensation mechanism in female mammals. We have found several forms of processed RNAstranscribed from the anti-sense strand of Xist, an X-linked locus essential for
X-chromosome inactivation. The anti-sense RNAs (Tsix) cover the whole transcription unit of JUst and do not code for a protein. To know whether these novel
RNAs playa role in X-inactivation, we have disrupted Tsix in mice by targetedmutagenesis. The X chromosome with disrupted Tsixinappropriatelyexpressed
Xist and became inactivated, suggesting an important regulatory function of
Tsix(ref. 8). This work was awarded with a Best Poster Prize in the 14th International Mouse Genome Conference held in Narita in November 2000.

E. DEPARTMENT OF INTEGRATED GENETICS

79(8) Human genome resources and their application to the human andprimate genome analysis
Asao FUJIYAMA, Ayuko MOTOYAMA 1, Satoru YOSHIDA 1, Yoko KUROKI 2, Yutaka
NAKAHORI 2, Tatsuo NAKAYAMA3, Mieko KODAIRA\ Norio TAKAHASHI 4 and Naruya
SAITOU5 (I Genome Science Center Riken, 2Tokushima Univ, 3Miyazaki Medical
School, 4Radiation Effect Research Foundation, 5Evolution Genetics Div., National Institute of Genetics)
The goal of human genome analysis is not only sequencing entire genome nor
cataloging protein coding regions, but to understand functions retained in thehuman genome and chromosomes. Since most of human chromosomes can be
purified by means of dual-laser cell sorting system, such isolated chromosomes are good resources for the studies to understand biological functions
retained in individual chromosome. Using purified chromosomes, we have constructed human mono-chromosomal cosmid libraries (except for CM#9-12),
CM#9-12, #21, CM-Y fosmid libraries, and BAC library. In addition, we are in
the process of constructing primate libraries including chimpanzee and gorilla. Using these resources, sequencing of the long arm of human chromosome
21 has been accomplished. A total of 33,546,361 bp of DNA, distributed overfour contigs, were sequenced with very high accuracy. In order to carry out
primate comparative genomic study, we have sequenced and mapped morethan 10,000 BAC-ends on the human genome.

(9) Whole genome analysis of signal-transduction pathways
in fission yeast
Yong-Sik BONG, Inaho DANJ0 1, Nobuya OGAWA and Asao FUJIYAMA (I Cancer Center Research Institute)
In fission yeast, Schizosaccharomyces pombe, deficiency of ras1 gene causes
abnormal cell shape and abolishes mating ability. However, the signaling pathway in the cell and its target genes are largely unknown because ofthe lack ofappropriate analysis system. To overcome this problem, we categorized genes
based on their expression levels in the presence or absence ofthe rasl gene

80

product under different growth conditions. We utilized micro"arrays of clones
covering entire genome of the fission yeast.

Publications
1.

ISHIHARA, K., HATANO, N., FURUUMI, H., RATO, R., IWAKI, T., MIURA, K., JINNO, Y. and
SASAKI, H.: Comparative genomic sequencing identifies novel tissue-specific enhancers
and sequence elements for methylation-sensitive factors implicated in Igf2/Hl9im-2.

printing. Genome Res. to, 664-671, 2000.
SASAKI, H., ISHIHARA, K. and RATO, R.: Mechanisms of Igt2!Hl9imprinting: DNA methy-

3.

715, 2000.
YATSUKI, H., WATANABE, H., HATORI, M., JOH, K., SOEJIMA, H., KOMODA, H., XIN, Z., ZHU,

lation, chromatin and long-distance gene regulation (Review). J. Biochem. t27, 711-

X., HIGASHIMOTO, K., NISHIMURA, M., KURATOMI, S., SASAKI, H., SAKAKI, Y. and MUKAI, T.:
Sequence-based structural features between Kvlqt1 and Tapal in mouse chromosome 7F4/F5 corresponding to the Beckwith-Wiedemann syndrome region in human
11pI5.5: Long-stretches of unusually well conserved intronic sequence of Kvlqtl
4.

between mouse and human. DNA Res. 7, 195-206, 2000.
VEDA, T., ABE, K., MIURA, A, YUZURIHA, M., ZUBAIR, M., NOGUCHI, M., NIWA, K., RAWASE,
Y., KONO, T., MATSUDA, Y., FUJIMOTO, H., SHIBATA, H., HAYASHIZAKI, Y. and SASAKI, H.: The
paternal methylation imprint of the mouse Hl9locus is acquired in the gonocyte stage
during fetal testis development. Genes Cells 5, 649-659, 2000.

5.

WATANABE, T., YOSHIMURA, A, MISHIMA, Y., ENDO, Y., SHIROISHI, T., KOIDE, T., SASAKI, H.,AsAKURA, H. and KOMINAMI, R.: Differential chromatin packaging of genomic imprinted
regions between expressed and non-expressed alleles. Hum. Mol. Genet. 9, 30293035, 2000.

6.

MIZUNO, S., CHIJIWA, T., OKAMURA, T., AKAsHI, K., FUKUMAKI, Y., NIHO, Y. and SASAKI, H.:
Expression of DNA methyltransferases DNMTl, 3A and 3Bin normal hematopoiesis
and in acute and chronic myelogenous leukemia. Blood (in press).

7.

OHNO, M., AOKI, N. and SASAKI, H.: Allele-specific detection of nascent transcripts byfluorescence in situ hybridization reveals temporal and culture-induced changes in
Igf2imprinting during pre-implantation mouse development. Genes Cells (in press).

8.

SADO, T., WANG, Z., SASAKI, H. and LI, E.: Regulation ofimprinted X-inactivation in mice

9.

YOKOMINE, T., KUROIWA, A., TANAKA, K., TSUDZUKI, M., MATSUDA, Y. and SASAKI, H.:

by Tsix. Development (in press).
Sequence polymorphisms, allelic expression status and chromosomal locations of thechicken IGF2and MPRl genes. Cytogenet. Cell Genet. (in press).
10. HAMATANI, T., SASAKI, H., ISHIHARA, K., HIDA, N., MARUYAMA, T., YOSHIMURA, Y., HATA, J.
and VMEZAWA, A: Epigenetic mark sequence of the Hl9gene in human sperm. Biochim.

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DEPARTMENT OF INTEGRATED GENETICS

81

11.

Biophys. Acta (in press).
HATTORI, M. and FU,JIYAMA, A. et al.: The DNA sequence of human chromosome 21.

12.

PARK, H.-S., NOGAMI, M., OKUMURA, K, HATTORI, M., SAKAKI, Y. and FU,JJYAMA, A.: Newly

Nature 405,311-319,2000.
identified repeat sequences, derived from human chromosome 21q-ter, are also localized in the subtelomeric region of particular chromosomes and 2q13 and are conservedin the chimpanzee genome. FEBS Letters 475, 167-169, 2000.
13. BRUI·;LS, T., GYAPAY, G., PETIT, J.-L., ARTI(;UENAVE, F., VICO, V., QIN, S., TIN-WOLLAM,
A.M., DasILVA, C., MUSELET, D., MAVEL, D., PELLETIER, E., LEVY, M., FU,JJYAMA, A.,
MATSUDA, F., WILSON, R, ROWEN, L., HOOD, L., WEISS EN BACH, J., SAURIN, W. and HEILIG,

R: A clone map of human chromosome 14. Nature 409, 947-948, 2001.
14. The International Human Genome Consortium including A. FUJIYAMA and 55 authors:
A physical map of the human genome. Nature 409, 934-941, 2001.
15. The International Human Genome Consortium including A. FU,JJYAMA and 72 authors
from international sequencing centers: The human genome: initial sequencing andanalysis. Nature 409, 860-921, 2001.

E-b.

Division of Agricultural Genetics(1) Developmental abnormalities induced by DNA hypomethylation
mutation of Arabidopsis
Tetsuji KAKuTANI, Asuka MIURA and Koichi WATANABE
Substantial part of large genome of flowering plants and vertebrates are
repetitive sequences, such as transposable elements and their derivatives.
Genomic regions rich in repeated sequences tend to be inactive in transcription and recombination and have condensed chromatin (heterochromatin). In
addition, cytosine residues in such regions are often methylated at high fre"quency. Although function of repeated sequences are largely unknown, uncon"
trolled activation of these sequences are presumed to be deleterious to genome
stability. On the other hand, some of repetitive sequences such as centromericrepeats and telomeres are regarded as important for the chromosome func'
tion. We are studying control and biological function of repetitive sequencesusing DNA methylation mutants of Arabidopsis.
In Arabidopsis, genome sequencing has been completed for the first case inplants. In addition, many trans mutations affecting epigenetic states have82

been isolated in this plant. Arabidopsis ddml (decrease in DNA methylation)
mutation results in decrease in methylation and transcriptional de-repression in genomic repeat sequences. The DDMl gene encodes a protein similar tothe chromatin' remodeling factor SWI2/SNF2. The most striking feature of
ddml mutation is that it induces a variety of developmental abnormalities bycausing heritable change in other loci. The molecular basis has been clarified
in two ofthe loci directly causing the developmental abnormalities.
One of them, clam, is characterized by lack of elongation in leaves, roots andshoots. This phenotype is heritable, but somatic sectors with normal pheno'
type were occasionally observed. The size and frequency of the sector differ
from plant to plant. The phenotype was stabilized in some of the progeny
families; no reversion 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 clam phenotype. It is
DWF4 gene, which involved in synthesis ofbrassinolide, a plant growth regulator necessary for cell elongation. The unstable clam phenotype was induced
by insertion of a novel endogenous Arabidopsis transposon, which we named
GAGTAl. This transposon transposes and increases in the copy number specifically in ddml mutant background. These results suggest that gene silenc'
ing associated with DNA methylation is important for suppression oftransposons Gn press).
Another developmental abnormality, late flowering trait, was induced byectopic over expression of FWA gene associated with hypomethylation of tan'dem repeat upstream of the coding region. Interesting thing is that change innucleotide sequence was also not observed in fwa-l and fwa-2alleles isolated
by conventional mutagenesis. In both cases, over-expression associated withthe hypomethylation resulted in the phenotypes. Combining ddml mutationand linkage analysis is useful for identifying epigenetically regulated genes
important for plant development.
(~)

DNA hypomethylation mutation in rice

Tetsuji KAKuTANI, Koichi WATANABE and Asuka MIURA

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DEPARTMENT OF INTEGRATED GENETICS

83

Among plant species, Arabidopsis genome has extreme feature that it contains only small proportion of repeated sequences. We therefore extended our
research by examining effect of de-repression of genomic repeated sequencesin rice. Rice has more repeat sequences than Arabidopsis. We found a rice EST
similar to Arabidopsis DDMl gene and generated a transgenic rice lines expressing that EST in antisense orientation. These transgenic lines shows reduced genomic DNA in centromeric repeats, repeat encoding rRNA andretroelement-like sequence Tos3.

Pub I i ca t ions
1. SOPPE, W., JACOBSEN, S.E., ALoNso-BLANco, C., JACKSON, J., KAKUTANI, T., KOORNNEEF, M.
and PEETERS, A.J.M.: The gain of function epi-mutant FWA causes late flowering.
Molecular Cell 6: 791-802 (2000).
2.

HIROCHIKA, H., OKAMOTO, H. and KAKUTANI, T.: Silencing of Retrotransposons in
Arabidopsis and Reactivation by the ddml mutation. The Plant Celli!: 357-368(2000).

E-c.

Division of Brain Function(t) Origin and Migration of Guidepost Neurons in the Lateral

Olfactory Tract
Naomi TOMIOKA 1, Noriko OSUMI 2,Yasufumi SATO, Hajime FUJISAWA 1 and Tatsumi
HIRATA (INagoya University Graduate School, 2Tohoku University)
The early-generated neurons designated as lot cells specifically mark the
future site ofthe lateral olfactory tract (LOT) and guide LOT axons. We investigated the mechanism of how lot cells develop and get localized in the LOT
position. Lot cells differentiated from neuroepithelial cells in all regions ofthe
neocortex but not from those in the ganglionic eminence in culture. Cell tracing
analyses demonstrated that lot cells generated from the neocortex subsequently
followed a tangential migration stream ventrally toward the LOT position.
Mutant mouse embryos lacking the function oftranscription factor Gli3 showed
disturbances of the migration stream, and translocation of lot cells in the84dorsal telencephalon. These results reveal a new type of neuronal migration in
the telencephalon and introduce an unexpected dramatic feature of the earliest regionalization of the telencephalon.
(Z) Short-Range and Long-Range Guidance of Olfactory Bulb Axons

Tatsumi HIRATA, Hajime FUJISAWA 1, Jane Y. Wu 2 and Yi RA0 2 (INagoya University Graduate School, 2Washington University School of Medicine)During development, mitral cells, the major output neurons of the olfactorybulb, project their axons caudolaterally into the telencephalon and form thelateral olfactory tract (LOT). Two types of guidance cues 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, thepathway 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 mitral cell axons inorganotypic culture upon ectopic application of Slit, and inhibition of endogenous Slit signaling. 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 cell axons. Although the septum and exogenous Slit can repel olfactory bulb axons, our results cast doubts on the physi0logical relevance of the septum and endogenous Slit in guiding the projectionof mitral cell axons.

Pub I i ca t ions
1. TOMIOKA, N., OSUMI, N., BATO, Y., INOUE, T., NAKAMURA, S., FUJISAWA. H. and HIRATA, T.:
Neocortical origin and tangential migration of guidepost neurons in the lateral olfac'
2.

tory tract. J. Neurosci. ZO, 5802-5812, 2000.
HIRATA, T., FUJISAWA, H., Wu, J. Y. and RAO, Y.: Short'range guidance of olfactory bulb
axons is independed of repulsive factor slit. J. Neurosci. 2001 <in press).

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DEPARTMENT OF INTEGRATED GENETICS

E-d.

85

Division of Applied Genetics(1) Analysis of genomic imprinting involved in murine
X chromosome inactivation
Nobuo TAKAGI (Graduate School of Environmental Earth Science, Hokkaido
University)
Mouse embryos having an additional maternally inherited X chromosome(XM) invariably die before mid 'gestation with the deficient extraembryonicectoderm ofthe polar trophectoderm lineage, whereas postnatal mice having
an additional paternally inherited X chromosome (XP) survive beyond parturi'
tion. A cytogenetic study led us to hypothesize that abnormal development ofsuch embryos disomic for XM (DSXM) is attributable to two doses of active XM
chromosome in extraembryonic tissues. To test the validity of this hypothesis,
we examined the initial X chromosome inactivation pattern in embryos at theblastocyst stage by means of replication banding method as well as RNA
FISH detecting Xisttranscripts. XP was the only asynchronously replicating X
chromosome, if any, in XMXMXP blastocysts, and no such allocyclic X chromo'
some was ever detected in XMXMy embryos. In agreement with these findings,
only one Xistpaint signal was detected in 79 % ofXMXMXP cells, whereas nosuch signal was found in XMXMy cells. Thus, two X chromosomes remaining
active in the extraembryonic cell lineages due to the maternal imprinting
explain the underdevelopment of extraembryonic structures and hence early
postimplantation death of DSXM embryos. We went on to further characterize
the nature of imprinting on XP making use of androgenetic embryos produced
by pronuclear transfer. FISH analyses consistently revealed one and two Xist
paint signals in XPY and XPXP embryos, respectively from the 4'cell to morula
stage. Furthermore, XPXP androgenones surviving to embryonic day (E) 7.5
achieved random X inactivation in all tissues including those derived from thetrophectoderm and primitive endoderm characterized by XP'inactivation in
fertilized embryos. This finding supports the above hypothesis that XM is rig'
idly imprinted to avoid inactivation, whereas XP is free of such imprint in
fertilized female embryos before implantation. It is likely that imprinted in'

86

activation was derived from random inactivation not the other way around.

(2) Developmental abnormalities in tetrasomy 11 mouse embryos
Nobuo TAKAGI (Graduate School of Environmental Earth Science, Hokkaido
University)
In mammals, an excess copy of any autosomal pair invariably induces strict
developmental abnormalities mostly resulting in spontaneous abortion or stillbirth, whereas effects of an increased copy of the X chromosome are much lesssevere. The dosage compensation mechanism silencing all X chromosomes inexcess of one seems responsible for the alleviated phenotypic manifestation of
X aneuploidy. This notion is substantiated by the observation that growth offemale embryos defective in X-inactivation is severely impaired and they areabsorbed immediately after implantation. This study was initiated to clarify
whether X chromosome is involved in embryonic development disproportionately heavily than average autosomes. In this study we tried to analyze effectsof two-fold increase in chromosome 11 (tetrasomy 11) on embryonic development taking advantage of high rate of nondisjunction in mice heterozygous for
two Robertsonian translocations Rb(1O.11)8Bnr and Rb(I1.13)6Lub. About
5% of embryos recovered from heterozygous females mated with males carry"ing same translocations at E7.5 were tetrasomic for chromosome 11. Tetrasomy
11 embryos corresponding in size to E5.5 embryos showed no clear differentia"
tion between embryonic and extraembryonic region. Furthermore, dead cells
were abundant in the proamniotic cavity without mesoderm differentiation.
These abnormalities are comparable in severity to those found in embryos
having two copies of active X chromosomes. Thus, it seems probable that effects of four copies of chromosome 11 on embryonic development roughly corre"
spond to those of two active copies of the X chromosome implying that X chromosome is not special if we assume that the level of activity is twice that of
autosomes.

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DEPARTMENT OF INTEGRATED GENETICS

87(3) Molecular analysis of the NAC gene family in rice
Kazuhiro KrKucml, Minako UEKUCm-TANAKA 2, Kaoru YOSHIDA', Yasuo NAGATO',
Makoto MATSUOKA 2 and Hiroyuki HIRANO' ('Graduate School of Agriculturaland Life Sciences, University of Tokyo, 2BioScience Center, Nagoya University)
Genes that encode products containing a NAC domain, such as NOAPICALMERISTEM(NAM) in petunia, CUP-SHAPED COTYLEDON2 (CUC:!) and
NAPin Arabidopsis thaliana, have crucial functions in plant development. We
describe here molecular aspects ofthe OsNACgenes that encode proteins with
NAC domains in rice (Oryza sativa LJ. Sequence analysis revealed that theNAC genes in plants can be divided into several subfamilies, such as theNAM, ATAF, and OsNAC3 subfamilies. In rice, OsNACl and OsNAC2 areclassified in the NAM subfamily, which includes NAM and CUC2, while
OsNAC5 and OsNAC6fall into the ATAF subfamily. In addition to the members of these subfamilies, the rice genome contains the NAC genes OsNAC3,
OsNAC4 (both in the OsNAC3 subfamily), OsNAC7, and OsNAC8. These
results and Southern analysis indicate that the OsNAC genes constitute alarge gene family in the rice genome. Each OsNAC gene is expressed in a
specific pattern in different organs, suggesting that this family has diverse
and important roles in rice development. For details, see Ref. 5.

(4) Characterization of viviparous mutants in rice (Oryza sativa L.)
Kazumaru MIYOSHI, Eijirou NAKATA and Yasuo NAGATO (Graduate School of
Agricultural and Life Sciences, University of Tokyo)
Four single recessive viviparous mutants of rice, rivl-l, rivl-2, riv2and enll,
were characterized. All rivmutants showed pre-harvest germination (vivipary)
during seed development but no defects in the other traits. Vivipary was also
observed in the en]l mutant that lacked endosperm in the seeds. Examination of the viviparous nature of the mutants under three rain conditions, norain, artificial rain and natural rain, revealed that the induction ofvivipary in

88

the riv mutants required a small amount of water. In contrast, precocious
germination in the enll mutant was caused by the absence of endosperm inthe seeds, but not by the external signal of rain. In rivmutants, the precocious
germination occurred simultaneously at late stages of seed development. The
germination test in the presence of exogenous ABA revealed that sensitivity to
ABA was gradually reduced from 15 through 35 days after pollination in thewild-type seeds, and was almost lost at 40 days after pollination. A similartendency was observed in riv1 and riv2 seeds. However, at 15-35 days after
pollination, riv1-1 and riv2 seeds had significantly lower sensitivity to ABAthan the wild-type seeds. In addition, the three rivmutants lost the sensitivity to ABA at an earlier stage of seed development than the wild type. These
results indicate that riv1 and riv2have a lower sensitivity and shorter periodof sensitivity to ABA. For details, see Ref. 6.

(5) SHOOT ORGANIZATION genes regulate shoot apical meristem
organization and the pattern of leaf primordium initiation in rice
J un-Ichi ITOH l, Hidemi KITAN0 2 , Makoto MATSUOKA:] and Yasuo NAGATO l (lGraduate School ofAgricultural and Life Sciences, University of Tokyo, 2 Faculty ofAgriculture, Nagoya University, :lBioScience Center, Nagoya University)
The mechanism regulating the pattern of leaf initiation was analyzed using
shoot organization (sho) mutants derived from three loci (SH01, SH02 and
SH03J. In the early vegetative phase, sho mutants show an increased rate ofleaf production with random phyllotaxy. Resulting leaves are malformed,
thread-like, or short and narrow. Their shoot apical meristems are relatively
low and wide, i.e., flat in shape, although their shape and size are highlyvariable among plants of the same genotype_ Statistical analysis reveals thatthe shape ofthe shoot meristem rather than its size is closely correlated withthe variation ofplastochron and phyllotaxy. Rapid and random leaf production
in sho mutants are correlated with the frequent and disorganized cell divisions in the shoot meristem and with a reduction of expression domain of a ricehomeobox gene, OSHl. These changes in the organization and behavior of the
SAM suggest that sho mutants have a reduced number of indeterminate cells

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DEPARTMENT OF INTEGRATED GENETICS

89

and increased number of determinate cells, with many cells acting as leaf
founder cells. Thus the SHOgenes have an important role in maintaining theproper organization of shoot apical meristem which is essential for the normal
initiation pattern ofleafprimordia. For details, see Ref. 7.

Pub I i ca t ions
1.
2.

3.

4.
5.

6.
7.

8.
9.

10.

GOTO, Y and TAKAGI, N.: Maternally inherited X chromosome is not inactivated inmouse blastocysts due to parental imprinting. Chromo Res. 8: 101-109, 2000.
SUGIMOTO, M., TAN, S.-S. and TAKAGI, N.: X chromosome inactivation revealed by the Xlinked lacZtransgene activity in periimplantation mouse embryos. Int. J. Dev. BioI.
44: 177-182, 2000.
TADA, T., OBATA, Y, TADA, M., GOTO, Y, NAKATSUJI, N., TAN, S.-S., KONO, T. and TAKAGI,
N.: Imprint switching for non-random X-chromosome inactivation during mouse oocyte
growth. Development 127: 3101-3105,2000.
OKAMOTO, 1., TAN, S. oS. and TAKAGI, N.: X chromosome inactivation in XX androgenetic
embryos surviving implantation. Development 127,4137-4145.
KIKUCHI, K, UEKUCHI-TANAKA, M., YOSHIDA, K T., NAGATO, Y, MATSUOKA, M. and HIRANO,
H.-Y. (2000) Molecular analysis of the NAC gene family in rice. Mol. Gen. Genet. 262:
1047-1051.
MIYOSHI, K, NAKATA, E. and NAGATO, Y. (2000) Characterization of viviparous mutantsin rice (Oryza sativa L.). Breed. Sci. 50: 207-213.
ITOH, J.-1., KITANO, H., MATSUOKA, M. and NAGATO, Y. (2000) SHOOT ORGANIZATION
genes regulate shoot apical meristem organization and the pattern of leaf primordium
initiation in rice. Plant Cell 12: 2161-2174.
HAYASHIDA, E., SATOH, H. and NAGATO, Y. (2000) ADL genes are required for adaxialabaxial pattern formation in rice leaves. Rice Genet. Newslett. 17: 28-31.
IKEDA, K, NAGASAWA, N. and NAGATO, Y (2000) ABERRANTPANICLE ORGANIZATION
1 gene regulates meristem identity in reproductive phase. Rice Genet. Newslett. 17:
31-34.
AsAI, K, SATOH, N., SASAKI, H., SATOH, H. and NAGATO, Y. (2000) A heterochronic geneassociated with juvenile-adult phase change in rice. Rice Genet. Newslett. 17: 34-37.

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Genetic Strains Research Center

F-a.

Mammal ian Genetics Laboratory(1) Molecular dissection of the critical region of a mouse preaxial
polydactyly mutation, Hemimellc extratoes (Hx)
Tomoko SAGAI, Hiroshi MASUYA 1, Kunihiko SHIMIZU 2 , Masaru TAMURA3 andToshihiko SHIROISHI (IMouse Funet. Genomics Res. Group, RIKEN GSC, 2Nihon
Univ. Sch. of Dent. at Matsudo,3Kyoto UnivJ
As described in the previous report, we identified a possible candidate geneof a preaxial poydactyly (PPD) mutation, Hemimelic extra toes (Hx), by the
positional cloning. This gene, Lmbrl, is a mouse homolog of human c7orf2,
which was identified in the human syntenic region 7q36. In human, a genomic
deletion including the exon4 of c7orf2was detected in the patients with bilateral congenital defeet of the upper and lower extremities. However, their phe"notypes are different from the typical PPD, and no coding sequence alterations
or genomic rearrangements of c7orf2were detected in five independent families with PPD. In the mouse, it was reported that in Hxmouse, Lmbrl expres"sion in the developing limb bud was dramatically altered. In opposition to theresults in the report, we could not detect clear difference of Lmbrl expression
between Hx and wild type by Northern blotting analysis, quantitative PCR
and whole mount in situ hybridization. Thus, the evidence that Lmbrl is acausative gene for Hxis still circumstantial. Alternatively, Hxphenotype may
be caused by alteration of a long"range cis"element ofthe sonic hedgehog gene(shh) that is localized to the down stream ofthe Hxmutation within a distanceless than 1Mb, because Hxembryos indeed show an ectopic expression of shh
in the anterior limb margin, like several other preaxial polydactyly mutants.
As another approach to search the mutation responsible for Hxphenotype,we carried out sequencing of the genome of the Hxcritical region. Lmbrl com"prises 17 exons and may span over a 150kb genomic region. Our physicalF.

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mapping narrowed down the Hxcritical region to about a 100kb region that iscovered by a single BAC clone. We have assembled the cosmid clones covering
this region and sequenced them. Thus far we have read about 90% of thisregion.
(!) Analysis of a spontaneous mouse mutation,mesenchymal dysplasia (mes)

Shigeru MAKINO, Hiroshi MASUYA 1, Yukari YADA and Toshihiko SHIROISHI(i RIKEN GSC)
A recessive mouse mutation, mesenchymal dysplasia (mes), which arose spontaneously on Chromosome 13, causes excess skin, increased body weight andmild preaxial polydactyly. Fine gene mapping indicated that mes is tightly
linked to patched (pte) that encodes a transmembrane receptor protein forShh, a key signaling molecule in vertebrate development. Molecular characterization of the pte gene of the mes mutant and an allelism test using a pte
knockout allele (ptd demonstrated that mes is caused by a deletion of themost C-terminal cytoplasmic domain of the pte gene. Since mes homozygous
embryos exhibit normal pattern formation and neural tube development ascompared with pte- homozygotes, which die around 10 dpc with severe neuraltube defects, the C-terminal cytoplasmic domain lost in mes mutation is dispensable for inhibition ofShh signaling in early embryogenesis. However, compound heterozygotes of pte- and mes alleles, which survive up to birth and dieneonatally, have increased body weight and over-proliferation of mesenchymal
cells in the dorsal trunk and the lung. These findings indicate that Ptc is anegative regulator of the proliferation of mesenchymal cells, and that the Cterminal cytoplasmic domain ofPtc is involved in its repressive action.

(3) Analysis of two preaxial polydactylous mouse mutations, X-linked
polydactyly (Xpl) and luxate (Ix)
Yukari YADA 1, Hiroshi MASUYA 2 , Shigeru MAKINO and Toshihiko SHIROISHI(lOchanomizu Univ, 2RIKEN GSC)

92

The anteroposterior axis patterning on vertebral limb morphogenesis is controlled by the formation ofthe zone of polarizing activity (ZPA), located at theposterior margin of limb buds. The Sonic hedgehog (Shh) gene that is expressedat the posterior margin of limb buds mediates ZPA activity. However, regulation of the polarized expression of Shh in developing limb buds is poorly understood. X-linked polydactyly (Xpl) is a spontaneous mouse mutation, which
exhibits preaxial polydactyly only on the hindfeet. An analysis by in situ hybridization revealed ectopic expression of Shh and Fgf4 at the anterior marginof the hindlimb bud in the Xpl embryos. The trancription gene Gli, which is adownstream of Shh, was expressed at the anterior side of the Xpl hindlimb
prior to the Shh expression. These data indicate that Xpl gene likely acts in thedownstream or independent pathway of Shh signaling cascade. To isolate theXpl gene, we performed the linkage analysis using 2,452 backcross progeny
from cross between B6-Xpll+ and wild mouse-derived strains, MSM, KJR and
BLG2. The Xpl gene was mapped to a O.89cM interval between the
microsatellite markers, DXMgc39 and DXMit32. Now, we are constructing BAC
contig covering the causative gene of Xpl.
Another mouse mutation, luxate (Ix), also exhibits preaxial polydactyly on
the hindfeet. We found that Fgf4 and Gre are expressed only in the anterior
half of limb buds of the Ix mutants. In spite of such anterior shift of Fgf4 andGre, endogenous expression of Shh was not altered. This observation indicatesthat the Fgf4 expression is not directly involved in induction and maintenanceof the endogenous Shh expression. In 11.5 day embryos ofthe Ix mutants, FgfB
was expressed in the whole AER, but its expression domain was narrowed
toward the anterior side prior to the Shh expression. These results suggestthat the Shh expression is maintained by Fgf8in Ix limb buds, and that theanterior shift of Fgf4 and Gre is induced by the Fgf8 signal. Furthermore, we
observed anterior shift ofthe hindlimb position in the Ix mutants. Thus, the Ix
gene is likely involved in positioning of hindlimb field and determing the FgfB
expression domain in normal limb development.

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GENETIC STRAINS RESEARCH CENTER

93(4) Male sterility of a eonsomie strain 86.MSM-Chr.X and finemapping of the X-linked gene responsible for the sterility
Ayako OKA, Nobuo TAKAGI I, Kiyotaka TOSHIMORI 2, Akihiko MITA, Youichi
MIZUSHINA, Noriko SAKURAI and Toshihiko SHIROISHI (IRes. Cent. For Mol. Genet.
And Grad. Schl. Of Env. Earth Sci. Hokkaido Univ, 2Department of Anatomy
and Cell Biology, Miyazaki Medical College)
Hybrid sterility is a mechanism that safeguards the integrity of species by
preventing a free flow of genetic information between related species. The
infertility of inter-specific hybrids usually affects the heterogametic sex, whichin the case of mammals is the male. In the mouse, male infertility has beenfound in hybrids between two species, Mus spretus and Mus. Musculus, fromwhich most of the laboratory strains have been established. Understanding
hybrid sterility might give us an insight into not only mechanism by which a
new species is evolved, but also genetic regulation of gametogenesis in males.
Since several years ago, we have set out to construct a series of consomic
strains that substitute each chromosomes of Japanese wild mouse-derived
MSM strain (Mus musuculus molossinus) for the chromosomes of a laboratory
strain C57BU6J (Mus musculus domesticus). These inter-subspecific consomic
strains would be of great use to study all kinds of multigenic traits. During theprocess of production of X-chromosome consomic strain, in which the X chromosome of C57BLl6J(B6) was replaced by the MSM'derived one, we noticed
the lowered fertility ofthe males. It seemed attributable to the disturbance of
coordination between gene(s) on the X chromosome ofMSM (X' MSM) and gene(s)on the B6 chromosomes. Those genes are thought to have been separated by along period of evolution, approximately one million years. Previously, the severe reproductive disadvantage was reported in B6 strain that carries the Xchromosome of MSM strain (Takagi et aI., 1994). The decline of the fertility
was emphasized as backcross generations proceeded. Unlike X' MSMY males, X.
BSY males in the same backcross generation were fully fertile. We observedthat most epididymal spermatozoa from XMSMY males had shortened distal
part ofthe head, while spermatozoa from B6 strain and X'BSY males had wellbalanced hook-like head. To estimate the fertility of X'MSMY males, in vitro

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fertilization (IVF) was performed with their capacitated spermatozoa. Significant low fertility ofIVF was observed in X.MSMY males (6.1 ± 7.2%; N=6),
though XRGY males retained high frequency (80.2 ± 23.3%). The frequency of
pregnancy and the size oflitters in natural mating were also evaluated. X' MSMY
males in N2 generation retained their fertility, i.e.4 out of 4 males were fertile,
mean litter size 5.7 ± 3.5. Their fertility decreased with the progression of the
backcross generations, and X MSM Y males in N6 generation were fully sterile,
while X. B6Y males in the same generation retained high fertility, i.e. 4 out of 4
were fertile, mean litter size 8.5 ± 1.7. In addition, the average of paired testicular weights were reduced in comparison with that of B6 males (B6; 102.7± 14.7mg, X'MSMY; 76.4 ± 13.5mg, N=8) ..
We intended to map gene(s) responsible for the morphological anomalies intheir spermatozoa. QTL (quantitative trait loci) analysis demonstrated themost intense linkage with DXMit166, and the weaker linkage with an interval
between DXMit97 and DXMit217. It is of interest to note that the locus that
controls the testis weight was mapped to an interval between DXMit217 andDXMit160, which was possibly separated from the loci that control spermmorphology.

(5) Genetic modification of the phenotypes of a mouse progeria
mutation, Kiotho (kl)Nobuyoshi JINNAI, Akihiko MITA and Toshihiko SHIROISHI
The klotho (k1) mouse was originally identified by insertional mutagenesis.
The homozygous mutant mice show various phenotypes resembling humanaging. The manifestations include short lifespan, atherosclerosis, gonadal atrophy, skin atrophy, emphysema, ataxia and ectopic calcification. They areused as model of human premature aging syndromes. Thus far, the klotho
mutation has been introduced into various genetic backgrounds of the standard laboratory mouse strains. These mice didn't show remarkable differencein their phenotype in comparison with the klotho original stock, which was
hybrid of C57BLl6J and C3H/J strains. By contrast, when the genetic background was replaced by MSM strain that was derived from Japanese wild

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mice, we found that the mutant mice exhibited unique phenotypes different
from the original stock. The klotho gene is expressed at the highest level inkidney. Nevertheless, no phenotype except for weak calcification has been observed in mice with genetic background ofthe standard laboratory strains. In
this study, we observed severe pathological phenotype in the kidney of themutant mice with the MSM background. On the other hand, abnormality inspermatogenesis, which is a representative phenotype of the original klotho
mutation, is relieved at least at histological level. Furthermore, it is of interest to note that some of mutant mice homozygous for klotho exhibited pheno'
type indistinguishable from wild,type mice in the stage of early generations of
the backcrossing to MSM, and they survived over about two years. The variation of phenotypes depending on genetic background suggests existence ofmodifier genes for the klotho phenotype and the presence of polymorphisms of
the relevant gene(s) between the original stock and MSM strains. Now, we areconducting a linkage analysis to identify modifier genes interacting with theklotho gene.
(6) Mapping of Genes Responsible for the Performance in the Passive
Avoidance Test Using Strains Derived from Wild Mice
Tsuyoshi KOIDE, Tamio FURUSE 1, Toshiyuki TAKANO, Kazuo MORIWAKI 2 andToshihiko SHIROISHI (ITokyo University ofAgriculture and Technology, 2Grad.
Univ. Adv. Stud.)
Many aspects of mouse behavior have been studied using only a relatively
small sample of available laboratory strains. An inherent problem in analyzing mouse behavior is that genetic diversity is limited among currently available strains. In this respect, the use of strains which are derived from a variety
of wild mice should provide a means to identify novel behavioral phenotypes.
We previously invested several behavioral phenotypes using females of anumber of mouse strains derived from wild mice of different subspecies, BFM/2,
NJL, BLG2, HMI, CASTlEi, KJR, SWN, MSM, a strain derived from fancy
mice, JFl, and two laboratory strains, C57BLl6 and DBA/I. We reported thatthe two strains, CASTlEi and BLG2, are the poor learners, while five strains,

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BFM/2, KJR, SWN, MSM and C57BLl6, are the good learners in the passive
avoidance test. In order to map the genes involved in the different performance
between strains, we collected the progeny from a backcross, (C57BLl6 x
BLG2)Fl x BLG2. The learning performance in the passive avoidance test was
analyzed in each mouse. The tail DNAs were prepared for the following genetic
study. The genome wide scanning using microsatellite markers was conductedusing 282 mouse progeny from the cross. The typing data of genome wide
scanning was analyzed by using the computer packages, MapManager QTX
and QTL Cartographer. Linkage was observed on chromosomes 2, 7 and 18 at
the suggestive level. Further genetic mapping is currently underway by increasing the number of mouse progeny in order to confirm the mapped data bystatistical analysis. At the same time, we are on the way of making congenic
strains by backcrossing C57BLl6 to BLG2 strain and selected the mice maintaining ability of good performance in the passive avoidance test. At the fifth
generation of backcross, genome wide scanning was conducted. We found thepersistence of the C57BLl6 alleles at the mapped loci on chromosomes 2,7,
and 18 in two of three congenic lines, which supported the idea that mappedloci are involved in performance in passive avoidance test.

(7) An analysis of sensitivity to capsaicin in the Mishima battery ofmouse strains
Tamio FURUSE, DavidA. BLIZARD', Kazuo MORIWAKI 2, Yutaka MIURA 3, Kazumi
YAGASAKI', Toshihiko SHIROISHI and Tsuyoshi KOIDE ('Tokyo University ofAgriculture and Technology, 2Center for Developmental and Health Genetics andIntercollege Graduate Program in Genetics, The Pennsylvania State University, 3Graduate University of Advanced Studies)
Taste is usually classified into five classes: sweet, salty, sour, bitter, andumami. However, another type of pungent sensation produced by red peppers
also plays an important role in the diet of different cultures. Red pepper iswidely used as condiment of food in several cultures ofthe Central American,
Asian, and African continents. Historically, red pepper has not been accepted
as condiment for diet in European and North American continent. This differ-

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ence is undoubtedly caused by both food culture and also may well be related
to different levels of perception among people for pungent sensation that iscontrolled genetically. In order to approach the underlying genetic mechanism
for diversity of preference for red pepper, we conducted a 12-hr, I-bottle intake
test of capsaicin solution using both male and female animals from the following inbred strains: 10 wild-derived inbred strains, PGN2, BFM/2, BLG2, NJL,
CHD, HMI, SWN, KJR and MSM, 1 strain derived from the so-called fancy
mouse, JFl, and 3 widely used domesticated strains, C57BU6J, DBA/IJ andBALB/cAnN. After two days of habituation, water intake was measured during the entire dark phase (20:00-8:00) for three days and the average volume ofwater intake was calculated. The concentration was then elevated successively each day as follows, 0.5, 1.0, 4.0, 7.0, 10.0 and 15.0 i1 M. Fluid intake
was calculated as percent ofthe control 3 day mean. Relative to baseline water
intake, C57BU6J and DBA/IJ consumed 10 percent while KJR and MSM
ingested approximately 60 percent of the 15 i1 M capsaicin solution. In order
to exclude the possibility that different levels of thirst affected intake of thecapsaicin solution, we conducted a short-term, 3-hr, fluid intake test withsome of the extreme strains from the first study. For measuring short-term
fluid intake, the test was initiated just before the onset of the dark phase.
Then, after three hours, the bottles were removed from the cages using a nightscope to avoid disturbing the animals in the dark phase ofthe circadian rhythm.
Essentially the same result was obtained in that KJR and MSM consumed
more capsaicin fluid than C57BU6J and DBA/IJ. In order to study whether
this was caused by preference of capsaicin over water, a 2-bottle preference
test was conducted on MSM and C57BU6J strains. The mice were individually housed. For the first two days, two bottles both containing water wereplaced on the cage and the mice habituated to the test condition. From thethird day, a bottle containing 0.5 i1 M of capsaicin fluid was substituted forone of the water bottles. The positions of the tubes were alternated every 24
hours to control for side preferences. Fluid intake was measured every 24
hours for 10 days as previously described. In the 2-bottle test, however, bothstrains consumed a strikingly low level of capsaicin fluid relative to waterwith the preference ratio approximating 10% even at 0.5 i1 M of concentration.
There was no significant difference across strains. This data show that both

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MSM and C57BLl6J strains are able to recognize the irritating sensation ofcapsaicin at low concentrations and avoid consuming capsaicin when the water is available. The pattern of strain differences of these sensitivities are
similar to that displayed by these strains in the hot plate test that we haddone previously. Because response to both heat and capsaicin are mediated by
VRI receptor, the similarity observed across hot plate and capsaicin intake
tests, raises the possibility that the physiological pathway mediated by VRI
may be polymorphic among the strains. In addition to this possibility, further
variations in the central pathways may be associated with the strain differences in capsaicin intake. In I-bottle test, Fl progenies ofKJR and C57BLl6
consumed capsaicin solution approximately same as KJR. This result indicates that the genes involved in capsaicin tolerance in KJR are dominant.

(8) Application of cryopreservatlon for wild mice embryos
Noriko SAKURAI l , Youichi MIZUSHINA, Nobuyoshi JINNAI, Naomi NAKAGATA2,
Tsuyoshi KOIDE and Toshihiko SHIROISHI (IJAC, Inc., 2Center for Animal Resources and Development, Kumamoto University)
Recently, the simple vitrification of mouse embryos was reported for preservation of the laboratory mouse strains. Although the method has been becoming popular, it is still not clear whether it is applicable to preservation ofvarious mouse strains including wild stocks. In this study, we examined efficiency of the simple vitrification of embryos of the standard inbred strains andseveral wild-derived strains, and compared the efficiency with those obtainedby the standard slow freezing methods. Embryos were obtained from in vitro
fertilization, and 2cell embryos were cryopreservated by the two different
methods. These embryos were thawed by the own methods, and morphologically normal embryos were transferred into presudopregnant females ofB6C3F 1
mice. The pregnant mice were examined on day 18 of pregnancy to count the
number of normal embryos. The result indicated that with most mouse strains
there was no difference in the efficiency of the recovery from frozen embryos
between two methods. However, efficiency by the simple vitrification with KJRJ
Ms and SWNlMs strains was lower than that by the slow freezing method.

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Contrary, with PGN2IMs strain, we observed higher efficiency by the slow
freezing method. With CHDlMs stain, we could not obtain pregnant female atall by the both methods.

Publ icatians
1.

2.

KOIDE, T., MORIWAKI, K, IKEDA, K, NIKI, H. and SHIROISHI, T.: Behavioral study on
inbred strains established from wild mice: BLGs has impaired ability for learningand memory. Mammal. Genome, 11, 664-670, 2000.
SAEKI, N., KUWAHARA, Y, SASAKI, H. and SHIROISHI, T.: Gasdermin (Gs) localizing tomouse chromosome 11 is predominantly expressed in upper gastrointestinal tract but
significantly suppressed in human gastric cancer cells. Mammal. Genome, 11, 718-

3.

724, 2000.
WATANABE, T., YOSHIMURA, A, MISHIMA, Y., ENDO, Y., SHIROISHI, T., KOIDE, T., AsAKURA, H.
and KOMINAMI, R.: Differential chromatin packaging of genomic imprinted regions

4.

between expressed and non-expressed alleles. Hum. Mol. Genet. 9, 3029-3035, 2000.
SHIMADA, Y, NISHIMURA, M., KAKINUMA, S., OKUMOTO, M., SHIROISHI, T., CLIFTON, KH.
and WAKANA, S.: Radiation-associated loss of heterozygosity at the Znfn1aI<Ikaros)
locus on chromosome11 in murine thymic lymphomas. Radiat. Res. 154, 293'300,

5.

6.

7.

2000.
SHIMIZU, K, NAGAMACHI, Y., TANI, M., KIMURA, K, SHIROISHI, T., WAKANA, S. and YOKOTA,
J.: Molecular cloning of a novel NF2/ERM/4.1 superfamily gene, ehm2, that is expressed in high-metastatic K1735 murine melanoma cells. Genomics 65, 113-120,
2000.
MIYOSHI, N., WAGATSUMA, H., WAKANA, S., SHIROISHI, T., NOMURA, M., AISAKA, K, KOHDA,
T., SURANI, M.A, KANEKO-IsHINO, T. and ISHINO, F.: Identification of an imprinted gene,Meg3/Gt12 and its human homologue MEG3, first mapped on mouse distal chromo'
some 12 and human chromosome 14q. Genes Cells 5, 211-220, 2000.
ISHII, T., SERIZAWA, S., KOHDA, A, NAKATANI, H., SHIROISHI, T., OKUMURA, K, IWAKURA, Y.,
NAGAWA, F., TSUBOI, A and SAKANO, H.: Projection of three subsets of olfactory neurons
expressing the odorant receptor transgene and the odorant receptor transgene and thetwo cognate endogenous alleles. in press, Gene to Cell, 2000.

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F-b.

Mammal ian Development Laboratory(1) Molecular mechanism of somite segmentation
Yumiko SAGA, Yu TAKAHASHI, Atsuya TAKAGI andAya KITABAYASHI (National
Institure of Health Sciences)
The somites are the first morphologically distinct segmental units formed invertebrate embryo and give rise to metameric structures such as vertebrae,ribs and skeletal muscles. The somites are formed by segmentation from theanterior end of the presomitic mesoderm (PSM), which is unsegmented andposterior to the most recently formed somite. Each somite is subdivided into
anterior (rostraD and posterior (caudaD compartments that differ in theirproperties and gene expression. The rostro"caudal polarity of a somite isestablished within the PSM prior to segmentation. However, molecular mecha"
nisms underlying the formation ofrostro'caudal polarity and the specificationof segmental boundaries are totally unknown. Our aim in this study is todetermine the mechanisms underlying the generation of a segmental pattern
within the PSM.
Previously, we cloned Mesp2, a gene coding a novel bHLH transcription fac"
tor, MesP2. Mesp2is expressed in the rostral PSM. Mesp2-null mice exhibited
defective somitogenesis due to the lack of rostral somitic compartment. Notchl
expression is greatly reduced in Mesp2-null embryos, while Mesp2expression
is downregulated in Notchl', DIlJ" or RBP-Jk-deficient embryos, indicatingthat MesP2 is in the feedback loop of the Notch signaling network. A mem"brane"bound protein presenilin"l(PSI), functions as a r 'secretase allowing
the nuclear translocation of intracellular domains of Notch in both vertebrates
and invertebrates. PSI"deficient mice exhibit defects in the segmentation as
seen in Notchl mutant mice. Based on the finding that, in mice, the lack eitherof two molecules involved in the Notch signaling pathway, MesP2 or Presenilin"
I (PSI) results in contrasting phenotypes, caudalized vs. rostralized vertebra,
we adopted a genetic approach to analyze the molecular mechanism underly"
ing the establishment ofrostro"caudal polarity in somites. By focusing on thefact that expression of a Notch ligand, DIlJ is critically important for prefigur-

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ing somite identity, we found that MesP2 plays an important role to initiate
establishment of rostro-caudal polarity by controlling two Notch signaling
pathways. Initially, MesP2 may activate a PS I-independent Notch signalingcascade to suppress DilI expression and specify the rostral half somite. While,
PSI mediated Notch signaling is required to induce DilI expression in thecaudal half somite. Therefore, MesP2- and PS I-dependent activation of Notch
signaling pathways might differentially regulate DilI expression resulting in
the establishment of the rostro-caudal polarity of somites(~)

Function of Mespt and Mesp~ for heart formation

Yumiko SAGA and Satoshi KrTAJIMA (National Institure of Health Sciences)
MesPl and MesP2 are transcription factors containing an almost identicalbHLH motif. Mespl is expressed in the early mesoderm that is destined to
become the extraembryonic and cranial-cardiac mesoderm. A lineage study
using cre-Iox system revealed that Mespl is the earliest molecular marker
expressed in the heart precursor cells. Previously, we generated MesP 1- andMesP2-single- knockout mice to define their respective functions. Disruptionof the Mespl gene resulted in a morphogenetic abnormality of the heart, cardia bifida (Saga, 1998). Mesp2'single-knockout mice revealed the critical requirement of MesP2 for the normal somitogenesis (Saga et aI., 1997). However, no notable defect was observed before somitogenesis in Mesp2'deficient
mice, which was consistent with the apparent lack of expression of Mesp2
during the early gastrulation stage. Analyses of Mespl(-j-) embryos revealed
that the defect is due to the delayed migratory activity of mesodermal cells.
However, an abnormal heart was eventually generated in association withmesodermal migration (Saga et aI., 1999). We found that this was the result of
up-regulation of the Mesp2gene. Then we generated double-knockout (dKO)mice to define the precise functions of these two genes during gastrulation.
Embryos deficient in both MesPl and MesP2lacked most ofembryonic nonaxial
mesoderm, and no posterior trunk structures developed. Furthermore, a chimera analysis revealed that dKO cells were scarcely observed in the anteriorcephalic and heart mesoderm, however, they did contribute to the formation of

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the somites, notochord and gut. These results strongly indicate that the defectin the cranial"cardiac mesoderm is cell"autonomous, whereas the defect in theparaxial mesoderm is an non·cell"autonomous secondary consequence.

(3) Transcriptional regulation of Mespl and Mesp! genes
Yumiko SAGA and Seiki HARAGUCHI (Shiga university of Medicine)

Mespl and Mesp2genes encode bHLH"type transcription factors, Mespl and
Mesp2, respectively. The expression of both genes is observed in the nascentmesoderm, and subsequently in the rostral presomitic mesoderm. Our previ"
ous studies, however, suggested that Mespl is predominantly expressed andplays a key role in gastrulation, while Mesp2 is crucial for somitogenesis. To
determine the regulatory mechanism for gene expression, we attempted to
identify the enhancer elements by transgenic analysis. At least two enhancers
which are responsible for the expression in the early mesoderm (early meso"dermal enhancer: EME) and the presomitic mesoderm (PSM enhancer: PSME)
and one suppressor, which is responsible for the rostrally restricted expres·sion in the presomitic mesoderm, were identified. Furthermore, to determine
whether each enhancer mediates the expression of Mespl and Mesp2indepen"dently, we analyzed two types of knockout mice. When the Mespl gene wasdeleted with EME (DEI), Mesp2expression was barely observed in the meso"derm of an embryo in the gastrulation stage. In contrast, in embryos that havetargeted deletion of the Mesp2 gene with PSME (p2·null), normal Mespl ex·pression was detected in the rostral presomitic mesoderm, suggesting theexistence of an additional enhancer(s) that directs Mespl expression in Mesp2null mice. Therefore, EME is probably required for the expression of both Mespl
and Mesp2in the nascent mesoderm, while PSME may represent an enhancer
specific to the Mesp2 gene and is required for the expression duringsomitogenesis.

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Pub I i ca t ions
1.

KANATANI, A., MASHIKO, S., MURAl, N., SU(;IMOTO, N., ITO, J., FUKURODA, T., FUKAMI, T.,
MORIN, N., MACNEIL, D.J., VAN DER PLOE(], L.H., SAnA, Y, NISHIMURA, S. and IHARA, M.:
Role of the Yl receptor in the regulation ofneuropeptide Y-mediated feeding: comparison of wild-type, Yl receptor-deficient, and Y5 receptor-deficient mice. Endocrinology.
141: 1011-6, 2000.

2.

SAWADA, A., FRITZ, A., JIANG, Y., YAMAMOTO, A., YAMASU, K., KUROIWA, A., SAGA, Y and
TAKEDA, H.: Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp'b confers the anterior identity to thedeveloping somites. Development. 12T 1691-702, 2000.

3.

KITAJIMA, S., TAKAGI, A., INOUE, T. and SAGA, Y: MesPI and MesP2 are essential for thedevelopment of cardiac mesoderm. Development. 127: 3215-26, 2000.

4.

TAKAHASHI, Y, KOIZUMI, K., TAKAGI, A., KITAJIMA, S., INOUE, T., KOSEKI, H. and SAGA, Y:
Mesp2 initiates somite segmentation through the Notch signalling pathway. Nat Genet.
25: 390-6, 2000.

5.

SAWADA, A., SAGA, Y and TAKEDA, H.: Zebrafish somitogenesis-roles ofmesp' and hairy'
related genes. Tanpakushitsu Kakusan Koso. 45: 2738'44, 2000.

F-c.

Plant Genetics Laboratory(1) Genetic dissection of embryogenesis, regeneration andgametogenesis of rice (Oryza satiYa)(I)-a. Regulation for expression of KNOX family class I homeobox genes of rice
Yukihiro ITO, Yasuo NIWA I and Nori KURATA (IShizuoka Pref. Univ.)
To elucidate genetic programmes that control embryogenesis and regeneration of rice, we conducted cloning and structural and functional analyses ofgenes which encode transcription factors and protein kinases. We previously
identified five KNOX family class I homeobox genes and analysed their expression patterns during early embryogenesis and regeneration process by
RT- peR and in situ hybridization. We also found that constitutive expressionof these genes are sufficient to maintain cells in a meristematic undifferentiated state. Since specific expression of KNOX family class I homeobox genes
in a shoot meristem is essential for normal development of plant, we started
to study its regulatory mechanisms. An aSH] promoter was shown to be ac-104tive in a leaf in addition to the shoot meristem, indicating that the promoter
regeion is not sufficient to confer specific expression in the shoot meristem andother regions are necessary for its precise expression. Introduction of an OSHl
cDNA into rice caused ectopic expression of endogeneous OSHl in the leaf andaltered leaf morphology. These effects were independent of direction of thecDNA and presence or absence ofthe promoter. We obtained similar results forother genes such as OSH6, OSHl5 and OSH71. These results suggest thatboth the promoter regions and the exons are responsible for the specific expression of these genes in the shoot meristem.
(I)-b. Alternative splicing of KNOX family class 2 homeobox genes of rice
Yukihiro ITO, Hirohiko HIROCHIKA' and Nori KURATA (INatl Inst. Agrobiol. Res.)
We previously identified three homeobox genes, HOS58, HOS59and HOS66,
which were classified into KNOX family class 2. We detected alternative transcripts of these genes. The difference of the alternative transcripts was existence or absence of exon 1, which encodes an amino acid sequence with shortstretches of alanin and glycin. Such characteristic sequences are suggested tomodulate transcription or repression activities ofthese transcription factors.
Because expression of each transcript of these genes was organ sepecifc, thesegene products may have different activities depending on the organs. To identify loss-of-function mutants and to elucidate functions of these genes, we
screened Tosl7-insertionallines of rice. We identified a single line in which

Tosl7 was inserted in a 3' untranslated region of HOS59 and obtained a homozygous plant for the insertion. However, no difference of morphology andexpression of HOS59 were observed between the inserted line and wild type,suggesting that the Tosl7- insertion gave no effect on HOS59 activity in thisline.
(I)-c. Isolation and sequence analysis ofreceptor-like protein kinase genes ofrice
Kazuhiko TAKAYA, Yukihiro ITO and Nori KURATA
We started to isolate and characterize a protein kinase gene as a candidate

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to transduce a positional information which is postulated to be important forplant development. We focused on a receptor-like protein kinase gene such as
SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERJ{) of carrot andisolated its homologues from rice. We have cloned two genes and their cDNAs
named ORKl and ORK6. The genome clone of ORKl showed unusual structureand detail analysis is in progress. ORK6encodes a protein with motifs characteristic to receptor-like protein kinases of plant. It contains, from N-terminus
to C-terminus, a signal peptide, a receptor domain with leucin-rich repeats, asingle transmembrane domain and a cytoplasmic protein kinase domain withserine/threonine specificity. The amino acid sequence of ORK6 is highly similar to plant receptor-like protein kinases such as SERK, CLAVATAI and
ERECTA of Arabidopsis, which are known to function in shoot meristem. Functional analysis of ORK6using transgenic rice is underway.
(I)-d. Functional analysis of OsHAP3-l using transgenic rice
Yukihiro ITO, Mitsugu EIGUCHI, Kazumaru MIYOSHI and Nori KURATA

LECl (LEAFY COTYLEDONl) gene was reported to be a central regulator ofseed development in Arabidopsis thaliana. LECl encodes HAP3 subunit protein, a component of transcriptional factor CBF (CCAAT-binding factor), which
is ubiquitously present in many eukaryotes. We have isolated one ofthe HAP3
family genes, designated OsHAP3-l from rice and introduced OsHAP3-l cDNA
driven by a CaMV 35S promoter in sense and antisense orientations into rice.
Although the sense construct gave no effect on transgenic plants, the antisense
construct caused pale green and short leaf phenotype on transgenic plants.
Similar effects were also observed in transgenic rice with an RNAi construct.
In rice there are a lot of mutants which show pale green phenotype, and mutations are mapped on chromosomes. To elucidate a possibility that OsHAP3-lcorresponds to one of such mutations, we mapped OsHAP3-l on rice chromosome. OsHAP3-l was shown to locate between 125.5 cM and 140.5 cM ofchromosome 1 in RI map and near this region virescent6is mapped (149 cM in
CL map). We are now examining that OsHAP3-l is actually virescent60r not.

106(I)-e. Analysis of genetic regulation of meiosis and gametogenesis in rice
Ken-Ichi NONOMURA, Kazumaru MIYOSHI, Hirohiko HIROCHlKA' and Non KURATA('Natl. Inst. Agr. Res'>To elucidate genetic regulation of gametogenesis including meiosis and hap'loid gamete formation in rice, we screened sterile plants from mutants induced by tissue culture, in which transposition of rice endogenous
retrotransposon Tos17 occurred to generate consequent insertional mutations.
We selected forty lines of meiotic mutants and more than 60 mutants prob'
ably defective in gamete formation or pollination process. The meiotic mutations found in this study included the aberrations such as chromosome
decondensation, asynapsis, desynapsis, meiocyte/ pollen immaturation, andso on. For cloning causal genes ofthe mutants, investigation about the linkage
between meiotic or other sterile phenotypes and Tos17 transposition is underway.
We are also planning to utilize these meiotic mutants for cytological analy'
ses of chromosome kinetics in rice meiosis.

(2) Positional cloning of a heterochronic gene, PIal, regulating theplastochron and the duration of vegetative phase In rice
Byoung-Ohg AHN, Kazumaru MIYOSHI, Jun·Ichi ITOH', Yasuo NAGATO ' and Nori
KURATA ('Graduate School ofAgricultural and Life Science, Univ. Tokyo)
Heterochronic mutations affecting the timing of developmental events may
be of major significance in ontogeny and evolution. In plants, several
heterochronic mutations that affect stepwise development of vegetative tis'sue and therefore alter shoot architecture have been identified. The recessive
mutations at the rice piastochron 1 (pial) locus cause the short plastochronand ectopic expression of vegetative programs in the reproductive phase. To
understand the molecular aspects of Pial during plant development, we aretrying to isolate the pial gene by map-based cloning.
Following rough mapping of pial-l allele on the short arm of chromosome 10
last year, we have carried out high resolution genetic and physical mapping

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using 578 F2 segregants of pial-l homozygous plants. A physical map encompassing the pial-llocus was constructed by overlapping YAC and BAC clones
through chromosome walking. The latest physical map showed that the pial1 locus was localized in an interval of about 70 kb which corresponded to 0.17
cM genetic distance. The candidate BAC clone carrying the pial gene havebeen sequenced almost entire length by the international rice sequencing
consortiam and this would facilitate positional cloning of the gene.

(3) Analysis of centromere structure of rice chromosome 5 toward
construction of plant artificial chromosome
Ken-Ichi NONOMURA, Tadzunu SUZUKI and Nori KURATA
The primary structure of the centromeric region of rice chromosome 5 wasanalyzed for long-range, as the first case in cereal species. The YAC and BAC
contigs aligned on the centromere of the rice chromosome 5 (CEN5) covered the
distance of more than 670-kbp long. The strong suppression of genetic recombination, one of the remarks for functional centromere, occurred along thecontig region. The most remarkable nature of the CEN5 is the composition of
the multiple repetitive elements. Oryza-specific short tandem repeats RCS2
clustered along less than 100-kbp long on the contig. At least fifteen copies of
the conserved domain of 1.9-kbp RCE1 centromeric repeats, which are identical to the long terminal repeats (LTRs) of gypsy type retrotransposon RIRE7,
were dispersed mainly in 320-kbp long next to RCS2 tandem clusters. Many
copies of the LTR-like sequences of RIRE3 and RIREB, another gypsy typeretrotransposon, were also found along all of the contig. On the other hand,
gag-poi regions were less conserved in the contig. These results indicated thatthe rice centromere is composed of multiple repetitive sequences and RCS2
tandem clusters probably being situated as a core of the functional centromere
in some hundreds kb to Mb length (see reference 1). Construction of rice artificial chromosomes with centromeric YAC and BAC clones selected in this study
is now on the way and after then trails to transform rice cells with artificial
chromosomes should be also carried out.

108(4) A large seale isolation and characterization of rice nuclear
protein genes
Kazuki MORIGUCHI and Nori KURATA
In many organisms, genes function in the nucleus are expected to be a 10-20%
of total genes of the organism. The proteins working in the nucleus; chromo·
somal proteins, nuclear matrix-associated proteins, transcription regulators,
proteins related to replication and transcription machineries, should havesignificant roles in the nucleus. However, little is known about biological andbiochemical status of nuclear proteins in respect to developmental stages andphysiological conditions of the plants.
The primary aim of this theme is to comprehensively isolate and structualy
analyse nuclear protein genes using rice as a monocot plant model. The second
aim is to make clear what events and factors are necessary for organizing andmaintaining genome structure and functions in the nucleus by observing the
localization, movement and expression specificity of the isolated nuclear pro·teins. For the isolation of nuclear protein genes, we use yeast nuclear transport trap (NTT) system, which can specifically trap NLS coding cDNAs (Uekiet aI., 1998. Nat. Biotechnol., 16: 1338-1342.). The two cDNA libraries frompanicles, which contain cells of early embryogenesis and meiotic stages, hasbeen used. In 2000, we started sequencing analysis oftrapped cDNA clones. At
present, we trapped more than 100 independent clones and found a tendency
that the clones encoding transcription factors are more efficiently trapped
than the results obtained in the mouse. On the other hand, the cDNA clones
encoding non-nuclear proteins have been also trapped, yet they were low efficiency. The population of non-nuclear proteins trapped by the system would beneeded to clarify.
In 2001, to inspect the statistical reliability of NTT system for the isolationof plant nuclear proteins, we start to check the nuclear localization of trapped
genes by expressing cDNA-GFP fusion proteins. Mter the inspection, we areplanning to trap and analyze their nucleotide sequences of additional 1000
independent clones.

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109(5) Genome-wide analysis of reproductive barriers in the intra-specificrice hybrids and positional cloning of one of the barriers(S)-a. Quantitative Analysis of genotype segregation for reproductive barriers
Yoshiaki HARUSHIMA and Nori KURATA
Genetic mechanisms for isolation of "species" are called as reproductive barriers and these include hybrid incompatibility, hybrid inviability, hybrid sterility, hybrid breakdown, etc. The distortions of allele frequencies from Mendelian expectation in progeny of inter- or intra - species hybrid due to hybrid
sterility genes, hybrid breakdown genes and gametophytic competition genes
have been often observed. We have developed a new method for detecting the
map location and gene action of loci that contribute to the distortions of allele
frequencies from Mendelian expectation by regression analysis of allele frequencies of markers covering an entire genome (submitted). Asian rice cultivars, Oryza sativa, can be classified into two main types, Japonica and Indica,based on several characteristics. To clarify the state of J aponica-Indica differentiation, all reproductive barriers causing allele frequency distortions from
Mendelian expectation in F 2 populations were mapped and compared among
three different J aponica -Indica crosses. The number of reproductive barriersin the three crosses was similar, however most of the barriers were mapped at
different loci. Therefore, these reproductive barriers formed after JaponicaIndica differentiation. In the three Japonica-Indica cross combinations, thegenetic variations within both Japonica and Indica are small as shown as highsimilarity of the restriction fragment length of RFLP markers. Considering
the high genetic similarity within Japonica cultivars and Indica cultivars, thedifferences 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 (submitted).

110(5)-b. Positional Cloning of a Segregation Distortion Gene Detected in a Progeny of a Cross between japonica and indica rice
Yoshiaki HARUSHIMA and Nori KURATA
The aim of this study is isolation of the most prominent barrier on chromosome 3 detected in F2 of Nipponbare-Kasalath hybrid by positional cloning,and elucidation of the molecular nature of the individual reproductive barriers. In the last year, we have clarified the aimed gene was a male gametophyte
gene that interact with maternal locus on chromosome 6. In other words, thepollen with Kasalath genotype at the gametophyte gene preferentially fertilized by 94% probability in maternal plant that is heterozygote or Kasalathhomozygote at the interactive locus on chromosome 6. For detailed mapping
the gametophyte gene, we have selected plants with recombination in 1.9cM
interval from 1000 F2 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 malegametophyte gene is being constructed by the dosage analysis using the selected population. The physical map of this region is also being constructedusing Nipponbare BACs. Nine BACs were selected by the closest RFLP marker,
C582, that was mapped 0.13cM away from the male gametophyte gene.

(6) Generation of enhancer trap lines of rice
Yukihiro ITO, Mitsugu EIGUCHI and 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 ofrice. We employ an enhancer trap system used in Arabidopsiswith some modifications. This system is based on the Agrobacterium-mediated transformation using AcIDs transposable elements of maize. The enhancer trap constructcontains, in the T-DNAregion, the Dselement named Ds-GUSthat harbours a
GUS coding region with a CaMV 35S minimal promoter and a hygromycin
resistance gene. Ds-GUSwas flanked by the 35S promoter and a coding regionof a selectable chlorsulfuron resistance gene, so that excision of Ds-GUS causes

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connection of the 35S promoter and the coding 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 for transposition of Ds·GUS. We generated forty-twotransgenic rice lines with a single copy of Ds·GUS and six lines with 35S·
AcTPase. We crossed four lines of Ds-GUSwith six lines of 35S-AcTPase andobtained F1 seeds from seventeen combinations. The exision of Ds-GUS inleaves of the F1 plants was observed in eight combinations out of seventeen.
We screened germinal transposants at F2 generation by PCR. Out of 10,524
examined in total, 675 (6%) were judged to be transposants. Since thetransposants showed the chlorsulfuron resistance as expected from the struc'ture of the vector, we further screened transposants by the chlorsulfuron andthe hygromycin resistance and 1,564 were judged to be transposants. Frequency of the germinal transposition was different from plant to plant andfurther panicle to panicle even in the same plant in both screenings. Thissuggests that the transposition of DS'GUStook place during panicle develop'
ment and that the transposants obtained from different panicles were inde'
pendent. To obtain a large number oftransposants we crossed another twenty'
two Ds·GUSlines with the 35S-AcTPase line. Screening oftransposants will
be carried out in 2001. We will also conduct mapping of Ds-GUSinsertion sites
on rice chromosomes to select chromosome specific Ds-GUSlines. Combina'
tions with the AcTPase lines and one AcTPase line could not excise the Ds in
any combinations with the Ds lines. This result indicates that this enhancer
trap system is functional in rice. Screening of germinal transposants is now inprogress.

Publications
1.

MORIGUCHI, K, MAEDA,Y., SATOU, M .. 'KATAOKA, M., TANAKA, N. and YOSHIDA, K: Analysis of unique variable region of a plant root inducing plasmid, pRi1724, by the construction of its physical map and library, DNA Research, Jun, 2000, 7, 157-163.

2.

ITO, Y., EIGUCHI, M. and KURATA, N.: Somatic and germinal transposition of a maize

3.

MORIGUCHI, K. MAEDA, Y., SATOU, M., NIKEN, S. N. H., 'KATAOKA, M., TANAKA, N. andtransposable element Ds in rice. Rice Genet. Newslet. 17: in press
YOSHIDA, K: (2001) The complete nucleotide sequence of a plant root-inducing <Ril

112

4.
5.
6.

7.

plasmid indicates its chimeric structure and evolutionary relationship between tumorinducing (Ti) and symbiotic (Sym) plasmids in Rhizobiaceae, J. Mol. BioI., in press.
NONOMURA, K.I. and KURATA, N.: The centromere composition of multiple repetitive
sequences on rice chromosome 5. Chromosoma, in press
NONOMURA, K.I. and KURATA, N.: Centromere structure of rice chromosome 5. In"RiceGenetics IV" Int.Rice Res. Inst. Press., in press.
HARUSHIMA, Y., NAKAGAHRA, M., YANO, M., SASAKI, T. and KURATA, N.: Reproductive
barriers between japonica and Indica crosses. In "RiceGenetics IV" Int.Rice Res. Inst.
Press., in press.
YAMAZAKI, Y, YOSHIMURA, A., NAGATo, Y. and KURATA, N.: Oryzabase- Integrated rice
science database-. In "RiceGenetics IV" Int.Rice Res. Inst. Press., in press.

F-d.

Microbial Genetics Laboratory(1) Timing of cell division In Escherichia coli

Akiko NISHIMURA
We found previously that cfe mutation uncouples DNA replication and cell
division, and elevates the frequency of cell division. We further analyzed thestructure and the role ofthe cfegenes. The cfc mutants divide before they reachthe size at which cfc· cells divide, and produce many small cells-each with asingle nucleoid. The mutations affect the timing of cell division, but not otherprocesses of cell cycle, such as the length of a cell cycle and the initiation massfor chromosome replication. CfcA has a missence mutation in glySa which
encodes the a -subunit of glycyl-tRNA synthetase, and cfcB 1 has an 1S2 insertion in apaH which encodes Ap4A hydrolase. The Cfc properties of both cfe
mutants were suppressed by a multicopy plasmid carrying apaH+, and theintracellular level of Ap4A in cfcA was 15-fold higher, and cfcB was lOO-foldhigher than their parent. Experiments using a wild-type cell showed that ahigh level of Ap4A caused early cell division, and a low level of Ap4A caused
delayed cell division. we have purified the GlyS-6xHis tagged proteins fromcfc· and cfc" strains and analysed the catalytic activity in vitro for Ap4A synthesis and kinetic constants of tRNA aminoacylation catalyzed by GlyS. Mutant type GlyS synthesized more Ap4A than wild type GlyS but showed lower
degradation activity ofAp4A to ADP than wild type GlyS. Catalytic activity forF.

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glycylation (KmlKcat) of GlyS from cfcA is 20~ 100 times higher than that fromwild type. Therefore, I conclude that Ap4A is a signal for induction of cell
division. High level of Ap4A is responsible for the initiation of cell division.
The glySmutation allows efficient synthesis ofAp4A.We also identified novel
Ap4A binding protein A (AbpA) and analyzed N terminal sequence of aminoacid. Conditional null mutation of abpA induced delayed cell division, andoverproduction of abpA induced Cfc phenotype.
(!) HscA is involved in the dynamics of FtsZ-ring formationin Escherichia colJ Kl!

Tsuyoshi UEHARA, Hiroshi MATSUZAWA' and Akiko NISHIMURA (IDepartment of
Bioscience and Biotechnology, Aomori University)
FtsZ, a universal and essential protein for cell division in prokaryotes, POS"
sesses GTP"binding and GTPase activity. This protein is a structural andfunctional homologue of eucaryotic tubulin, capable of dynamic assembly inthe presence of GTP. Depolymerization of FtsZ consumes GTP in vitro. FtsZ
proteins are found scattered in the cytoplasm during most ofthe cell cycle, butre"localize in the early stages of septum apparatus construction to the middleof the cell. There, these proteins assemble into a cytokinetic ring on the inner
membrane; FtsZ proteins maintain a position at the leading edge of the in"vaginating septum, followed by the assembly of other proteins, such as FtsA,
Ftsl, FtsK, FtsL, FtsN, FtsQ, FtsW, and ZipA, and constriction of the FtsZ"
ring. FtsZ"rings are therefore observed at potential division sites, even in thefilaments of these cell division mutants. After completion of cell division, theFtsZ proteins again re"localize throughout the cytoplasm of the daughter cells.
The molecular mechanism governing the dynamic localization of FtsZ proteinsremains unknown; no factors involved in the dynamics of FtsZ"ring formation
have previously been discovered.
JEI0715 mutant bacteria form multinucleated filaments at 42'C. In immu"
nofluorescence analysis of the filaments, FtsZ"ring was highly reduced at PO"
tential division sites. Mapping ofthis mutation by PI "phage mediated trans·
duction, complementation analysis, and sequencing of genomic DNA demon"

114

strated that JE10715 phenotype results from a missense mutation in the
hscA gene, corresponding to a single alanine to valine substitution at position
192 in the ATPase domain. hscA conditional knockout of wild type strain ex'
hibited an abnormal localization of FtsZ and reduced growth, although grew
producing longer rod cells, at non permissive conditions. Overexpression of
dnaKpartially complemented the JE10715 mutant. In vitro experiments demo
onstrated that the ATPase activity of HscA715 was reduced to 63% of wild,
type levels. HscA cosedimented with FtsZ'polymers in the presence of GTP. 90
angle light scattering revealed that HscA715 inhibited the polymerization of
FtsZ in the presence ofGTP at 36"C. We conclude that HscAis involved in FtsZring formation, interacting with FtsZ; defects in hscA, however, are partiallycompensated for by the redundant genes including the wild type dnaK0(3) Systematic analysis of novel cell division genes in Escherichia
coli. : Post genome project
Shinsuke NAKADE, Kimiko SAKA, Kunio MATSUMOTO, Masanari KITAGAWA,
Hirotada MORI' and Akiko NISHIMURA ('Nara. lnst. Sci. Tech,)
The entire nucleotide sequence of E. coli was analyzed, and 4311 ORFs havebeen demonstrated, but the functions of more than halfofthese genes are still
unknown. It is considered that the greater part of these genes are involved incoordinating cell proliferation to cell division. To isolate a whole set of cell
division genes and to analyze the hierarchy and network responses in expres'sion of cell division genes as a model case, we have selected 334 mutants
defective in cell division (its) from a temperature'sensitive mutant bank (kts)consisting of 5,000 strains established by Hirota et aI., and analyzed thephysiological properties of the mutants and roughly mapped these genes bycomplementation using E. coli gene bank. Furthermore, to assign a corresponding ORF to each its mutation by genetic analysis, vectors were constructed andcloning has been performed for 2,500 ORFs. Using 1,000 ORF clones, complementation test was done for 21 fts mutants; as the results, 11 fts mutants
were identified.

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Pub Ii ca t ions
1.

UEHARA. T.. MATSUZAWA. H. and NISHIMURA. A.: (200l) HscA is involved in the dynamics
of FtsZ-ring formation in Escherichia coli. (submitted).

2.

NISHIMORI, K.. TAKAGI, H .. WACHI. M., FUJISHIMA. H., KAWABATA. T., NISHIKAWA, K. andNISHIMURA, A.: (2001) The kdsA mutations affect the FtsZ-ring formation in Escheri-chia coli. (submitted).

F-e.

Invertebrate Genetics Laboratory(1) Control of tracheal tnbnlogenesis by Wingless signaling

Takahiro CHIHARA and Shigeo HAYASHIThe tubular epithelium of the Drosophila tracheal system forms a networkwith a stereotyped pattern consisting of cells and branches with distinct identity. The tracheal primordium undergoes primary branching due to an induction by the FGF homolog Branchless, differentiates cells with specialized function such as fusion cells that perform target recognition and adhesion during
branch fusion, and extends branches toward specific targets. Specification ofan unique identity for each primary branch is essential for directed migration,
as a defect in either the EGFR or Dpp pathways leads to a loss of branch
identity and the misguidance of tracheal cell migration. Here, we investigate
the role of Wingless signaling in the specification of cell and branch identity inthe tracheal system. Wingless and its intracellular signal transducer, Armadillo, have multiple functions, one specifying dorsal trunk through activationof Spalt expression and the other inducing differentiation of fusion cells in all
fusion branches. Moreover, we show that Wingless signaling regulates Notch
signaling by stimulating Delta expression at the tip of primary branches.
Those activities of Wg signaling are combined to specify the shape of dorsal
trunk and other fusion branches. For detail, see ref. 2.

116(!) EGF receptor attenuates Dpp signaling and helps to distinguish thewing and leg cell fates in Drosophila
Kazumasa KUBOTA 1, Satoshi GOTO, Kazuhiro ETO l and Shigeo HAYASHI(IDepartment of Molecular Craniofacial Embryology, Graduate School of Tokyo Medical and Dental University)
Wing and leg precursors of Drosophila are recruited from a common pool of
ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. We report herethat the receptor tyrosine kinase EGFR antagonizes the wing-promoting func'tion of Dpp and allows recruitment of leg precursor cells from uncommitted
ectodermal cells. By monitoring the spatial distribution of cells responding to
Dpp and EGFR, we show that nuclear transduction of the two signals peaks at
different position along the dorsoventral axis when the fates of wing and leg
discs are specified and that the balance of the two signals assessed within the
nucleus determines the number of cells recruited to the wing. Differential
activation of the two signals and the cross talk between them critically affectthis cell fate choice. For detail, see ref, I.

(3) Repression of the wing vein development in Drosophila by the
nuclear matrix protein Plexus
Hitashi MATAKATSU, D BRENTRup 1 and Shigeo HAYASHI (IMax Plank Institutefor Biophysics, Getchingen, Germany)
The wing of Drosophila is separated into several sectors by the wing veins.
Vein primordia are specified by the positional information provided by hedgehog and decapentaplegic in the wing imaginal disc and express the key regula'tory gene rhomboid. We have previously cloned the gene plexus involved in the
repression of rhomboid transcription. plexus was found to encode a novel nuclear
matrix protein. To understand molecular functions of plexus, we studied thefunction of net that appears to act together with plexus to repress vein differentiation. Brentrup et al have shown that net encodes a basic helix-Ioop'helix
protein expressed in the intervein territories where rho transcription is re-F.

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pressed. We have shown that plexus and net act synergistically to repress rhotranscription. Plexus affects nuclear localization of Net. Those results together
suggest a hypothesis that the function of plexus is to tether Net protein to
specific location of the nucleus to facilitate repression of specific target genes
such as rho. This idea is being tested by genetic and biochemical analyses.

(4) Enhaneer trap sereen for genes involved in pattern formation
Satoshi GOTO, Misako TANIGUCHI, Yukiko SADO and Shigeo HAYASHI
To identify novel genes and gene functions in the pattern formation of theimaginal disc and the trachea, we are conducting an enhancer trap screen
using the Gal4-UAS system. About 4500 lines were established in collabora"tion with groups in Japan and were examined for the activity of enhancers
flanking the inserts in embryos, larvae and adults. This year we sequenced
genomic DNA flanking the P-element insertion points of all the lines, andsuccessfully mapped about 3000 of them onto unique locations in the anno'tated complete genomic sequence of Drosophila. Insertions were grouped into
1722 clusters, most of which lie close to 5' ends of transcription units. Those
data should be of great value for the use of Gal4 strains in manipulating gene
expression patterns, studying P'element insertion specificity in the genome,
and the comprehensive analysis of gene functions.

Publ icatians
1.

2.

KUBOTA, K, GOTO, S., ETO, K and HAYASHI, S.: EGF receptor attenuates Dpp signaling
and helps to distinguish the wing and leg cell fates in Drosophila. Development, 127,
3769-3776, 2000.
CHIHARA, T. and HAYASHI, S.: Control of tracheal tubulogenesis by Wingless signaling.
Development, 127, 4433-4442, 2000.

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G.

Center for Genetic Resource

G-a.

Genetic Informatics Laboratory(1) ORYZABASE --INTEGRATED RICE SCIENCE DATABASE-Takahiro YAMAKAWA, Kazu MITSUI, Nori KURATA, Atsushi YOSHIMURA 1, Yasuo,
NAGAT0 2 and Yukiko YAMAZAKI (IKyushu University, 2University of Tokyo)
The Oryzabase is a comprehensive rice science database established in thecollaboration with rice researcher's committee in Japan.
The current Oryzabase consists of five parts, (1) genetic resource stock information, (2) gene dictionary, (3) chromosome maps, (4) mutant images, and (5)fundamental knowledge of rice science.
The Oryzabase map represents the integration of seven different maps fromclassical linkage map to the latest physical map provided by Rice Genome
Project.
The gene dictionary has been constantly updated by the specialists coupledwith the activity of Rice Gene Cooperative.
Oryzabase uses an object-oriented database management software with
Java2D for application.
We are planning to do more extensive cross"referencing of Oryzabase to thesequence database, literature database and other rice databases in the world.
Oryzabase is available from http://www.shigen.nig.ac.jp/rice/oryzabase/.
We are constructing the Kyushu Rice Genetic Resource DB and the NIG Rice
Genetic Resource DB separately from the Oryzabase because of the discrepancy of data sets.
These databases are enriched with rather agronomical data.
Oryzabase architecture should be updated to integrate these data.

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119(2) PEC: Profiling of Escherichia coli Chromosome
Yukiko YAMAZAKI, Toru IKEGAMIl, Takehiro YAMAKAWA, Kazu MITSUI, Takeshi
KAWABATA, Ken NISHIKAWA, Tadahiro MORI 2,Akiko NISHIMURA and Junichi KATO
(I University of Tokyo, 2Nara Institute of Science and Technology)
The Profiling of Escherichia coli chromosome (PEC) database has been con"structed to compile any relevant information that could help to characterize
the E. coli genome, especially with respect to discovering the function of eachgene. The database is intended to provide an interface comprehensible to mostexperimental researchers. The E. coli genetic resource committee of Japansupports the construction and maintenance of this database.
The database consists of 8 parts, (1) Basic information about each gene, (2)The essentiality of each gene for cell growth, classified as essential, non"es"
sential, or unknown based on information from research reports and deletion
mutation studies. The criteria for classification are described below. Refer"ences, on which the classification is based, are listed in the format "Medline(PMID)" in the section for each gene, (3) The names of the strains related toeach gene, which are stored in the Stock center ofthe Natl. Inst. Genet, Japan,(4) Results of similarity searches for each gene product (BLAST, PSI-BLAST,FASTA), (5) Structural features (domain, motif, etc.) for each gene product, (6)Results of comparative analyses of each gene (homologues in other sequenced
bacteria, etc.), (7) Information from rather long deletion mutations, and (8)Locations of Kohara clones.
PEC provides the Genomic", Linear- and Motif overviews.
In the genomic overview the E. coli genome is displayed as a circle, on which
the locations of the tRNA genes have been marked. Essential genes, non"es"
sential genes, and unknown genes are painted in different colors, allowing
easy visualization oftheir distribution on the genome. In the linear view, thegenome is linearly displayed. Each gene is displayed in a linear manner along
the genome together with its name, direction, size, location, and class (essen"tial, non "essential, unknown). Also, the regions deleted in the deletionmutant(s) for each gene are shown. This view also shows classical markers,
whose exact locations remain unknown, contig information, and the location of

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the Kohara clones. As the Kohara clones are derived from the strain W3110,
there positions were assigned by doing a homology search on the MG 1655
genome. Structural domains and motifs of a gene product are displayed graphically along with those of other genes having the same domains or motifs in the
motif overview.
PEC is available at http://www.shigen.nig.ac.jp/ecoli/pec/.

Publ ications
1. YAMAZAKI, Y., YOSHIMURA, A., NAGATa, Y. and KURATA, N.: Oryzabase-Integrated map
and mutant database', Plant and Animal Genome VIII, San Diego, CA, January.
2.

YAMAZAKI, Y., YOSHIMURA, A., NAGATa, Y. and KURATA, N.: ORYZABASE"INTEGRATEDRICE SCIENCE DATABASE, Fourth International Rice Genetics Symposium, Laguna, Philippines, October.

G-b.

Genome Bi 0 logy Labo r ato ry(1) NEXTDB: The nematode expression pattern database
Tadasu SHIN·I and Yuji KOHARA
We are updating "NEXTDB" that integrates all the information of ESTs, gene
expression patterns and gene functions of G.eleganswhich are being produced
and analyzed in this laboratory, and, are preparing to make it open by theworm meeting. Images of whole mount in situ hybridization for mRNA weretaken by CCD cameras equipped on Zeiss Axioplans, loaded to the Sun
workstation to process and arrange them properly in the database. Then, they
were annotated with respect to developmental stages and expression patternson the database. Images ofimmunostaining taken on Zeiss LSM510 confocal
microscope, and images and descriptions ofRNAi phenotypes, were also stored
in the database. New version of NEXTDB contains about 12,000 unique cDNA
groups, in situ images of about 7,600 cDNA groups, RNAi phenotype images of
160 groups, and immunostaining images of 60 groups. The new version will bedemonstrated and will be available over the Internet. URL: <http://
helix.genes.nig.ac.jp/db/index.html>

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121(2) The worm transcriptome project
Jean THIERRY-MIEG, Danielle THIERRY'MIEG, Yutaka SUZUKI2, Sumio SUGAN0 2,
Kazuko OISHI, Masako SANO, Hisayo NOMOTO, Shinobu RAGA, Satoko NISHIZAKA,
Hiroko HAYASHI, Fumiko OHTA, Sachiko MIURA, Hiroko DEsum, Michel
POTDEVlN3, Yann THIERRY-MIEG l , Vahan SIMONYA l , Adam LoWE l , Tadasu SHIN-I
and Yuji KOHARA (lNCBI, NIH, 2Institute of Medical Sciences, D. of Tokyo,
3CRBM, CNRS)As of March 2001, 16175 yk clones have been sent to researchers around theworld, in answer to 3463 requests. The main progress since the last worm
meeting has been the isolation of full length eDNA libraries, selected by thecap and stage specific. All 83,000 eDNA clones and 9000 sequences collectedfrom other sources including public databases were coaligned using the Acembly
program on the 99.4 Mb of genome sequence (many thanks to the Consortium).
We confirm the very high quality of the genome and estimate, from the unaligned clone proportion, that we are now missing only 0.7 to 1.2 Mb of genome.
The basecall of the cDNAs was edited, and cloning artifacts, such as mosaic
clones, internal deletions or inversions, internal oligo dT priming or unspliced
RNA / DNA, were flagged and treated with appropriate care: these amounted
to 1% of the clones from the first yk libraries, 5% of the capped clones and 2%of the clones in Genbank.
As of today, we have cDNAs in Acembly/AceView for just over half of theG.elegans genes: 10126 genes produce 14154 transcripts through alternative
splicing or alternative polyadenylation. We have started to annotate the proteins and to submit the results to the public databases. We will submit inpriority the genes you ask for. We use names, such as lK18 (mec-8), that
contain chromosome, megabase (letter), kilobase number and strandedness(even/odd) and allow easy distinction from the predictions.
Indeed, contrary to the outstanding quality of the genome sequence, theWormpep de novo predictions are usually incorrect: in a sample of 111 newly
hit genes, only 33% were exactly correctly predicted: 7% were not predicted, thefirst and last exons were incorrect in 35% and 28% cases respectively <in particular 9% of genes touch two predictions), and finally, in the common region,

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33% of the genes had at least one internal exon incorrectly predicted. Geneprediction remains a difficult problem.
The new capped libraries are a huge step forward for the transcriptome project.
More than 80% of the 12000 capped clones actually contain the entire mRNA,
from 5' cap to polyA. Transpliced leaders are present in up to 65% ofthe genes,
the remaining 35% genes are not transpliced (for example, collagens or surface
proteins). We identify 12 types of transpliced leaders, possibly encoded by 30
SL genes clustered in 15 loci: each SL gene is followed in the genome by astrong donor site. Among the genes transpliced, 76% are transpliced to SLI
exclusively, the remaining 24% are transpliced to SL2/SLI2. We confirm Tom
Blumenthal et ai's hypothesis relating minor SLs to operons: the 700 genes
with SL2/12 are most often located less than 300 bp downstream of an expressed gene in cis. The proportion of pure SLI genes with such a close neighbor is 20 times lower, and, for genes not transpliced, 10 times lower. The
specific transpliced leader used appears to be stage dependent: in particular,
usage of the minor leaders increases from 5% in embryos to more than 25% in
adults. In a compact genome such as C.elegans', close genes tend to be
cotranscribed. Such physical constraints for coexpression of close genes during
development could be tuned by controlled availability of minor leaders.

(3) Open-reading-frame sequence tags (08Ts) support the existence ofat least t 7,300 genes in C. eJegans
Jerome REBOUL!, Philippe VAGLI0 1, Jean FRANCOIS RUAL', Nicolas THIERRYMIEG 2, Troy MOORE\ Cindy JACKSON 3, Tadasu SHIN-I, Yuji KOHARA, Danielle
THIERRY-MIEG4, Jean THIERRY-MIEG4, Hongmei LEE 5, Joseph HITTI 5, Lynn
DOUCETTE-STAMM5, James L. HARTLEY" Gary F. TEMPLE6, MichaelA. BRASCH6,
Jean VANDENHAUTE 7, Philippe E. LAMESCH 7, David E. HILL' and Marc VIDAL'('Dana-Farber Cancer Institute and Harvard Medical School, 2Laboratoire
LSR-IMAG, :lResearch Genetics, 4NCBI, NIH, 5Genome Therapeutics Corp.,
6Life Technologies Inc., 7Universitaires Notre-Dame de la Paix)
The genome sequences of Caenorhabditis elegans, Drosophila melanogasterand Arabidopsis thaliana have been predicted to contain 19,000, 13,600 andG.

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25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, thegene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. eleganswould have 50% more genes than D. melanogaster. Second,
intron/exon predictions need to be experimentally tested. Third, complete setsof open reading frames (ORFs), or ORFeomes, need to be cloned into various
expression vectors. To simultaneously address these issues, we have designed
and applied to C. elegans the following strategy. Predicted ORFs are amplified
by peR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning, and then sequenced to generate
ORF sequence tags (OSTs), as a way to verify identity and splicing. In a sample(n=1,222) ofthe nearly 10,000 genes predicted ab initio (that is, for which no
expressed sequence tag (EST) is available so far), at least 70% were verified
by OSTs. We also observed that 27% of these experimentally confirmed genes
have a different structure from that predicted by GeneFinder. We now haveexperimental evidence that supports the existence of at least 17,300 genes in

C. elegans. Hence we suggest that gene counts based primarily upon ESTs may
underestimate the number of genes, in human and in other organisms.

(4) Genome-wide functional analysis by RNAI-by-soaking witha non-redundant eDNA set
Ikuma MAEDA', Atsuko MINAMIDA 1, Masayuki YAMAMOTO', Yuji KOHARA andAsako SUGIMOTO' (lDept. of Biophysics and Biochemistry, University of Tokyo)
The genome of C. elegans consists of approximately 19,000 genes, and thefunction(s) of many of them are still unknown. We have been performing asystematic functional analysis of the expressed genes by the RNAi-by-soaking
method 1. In this method, L4 worms are soaked in dsRNA solutions for RNA
delivery, and their F1 phenotypes as well as PO germline phenotypes are examined. As templates for in vitro dsRNA synthesis, a set of tag-sequenced
non-redundant cDNAs corresponding to -10,000 genes (representing half ofthe predicted genes) has been used. Because the size ofthe cDNA set is compa-

124rable to the number of detectable genes by a whole-genome DNA microarray,this cDNA set is likely to contain most genes expressed in the development of
C. elegans.
We examined the RNAi phenotypes for -2,800 genes (15% of the predictedgenes) to date, and 27% of them showed detectable developmental phenotypes
under a dissection microscope. The ratio of the cDNA clones that caused phenotypes was apparently higher than that ofthe predicted ORFs (13-14%)3,4,
consistent with the finding by Fraser et aL(2000) that ORFs having EST are
more likely to give RNAi phenotypes. Thus, the RNAi screen using the nonredundant cDNA set is an efficient way to comprehensively identifying essential genes for development. The general RNAi phenotypes observed were; F1
embryonic lethality (14% of the examined clones), F1 post-embryonic lethality(4.5%), PO sterility (7%), F1 sterility (1%), and morphological abnormality(0.7%). Ofthese, we analyzed the phenotypes ofFl sterility in detail with DIe
microscopy and DAPI staining, and have identified 31 genes that might play
important roles in germline development. Each cDNA clone affected a distinct
process, such as proliferation of germ cells, germline sex determination,
gonadogenesis, gametogenesis, and fertilization.
The genes corresponding to the cDNA clones we used distribute almost evenly
to all chromosomes. The incidence of the essential genes was comparable among
the autosomes (26% - 34%), however, that on the X chromosome was significantly lower (16%). Because a mutation in the essential genes on the X chromosome would directly lead to the lethality/sterility of males, they appear to
be removed from the X chromosome in the process of evolution.
We aim to complete the RNAi screen ofthe whole cDNA set. The functional
information obtained in this work will provide a starting point for further
analysis of each gene.

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125(5) Expression based RNAi reveals the function of proteasome inoocyte maturation and fertilization in C. eJegans
Keiko HIRaNO, Kyoko NAKATA, Yumiko VETA, Mina IWATA, Masahiro ITO andYujiKoHARA
Our cDNA/expression pattern project has provided unique sets of limited
numbers of genes that possibly participate in specific stages, lineages, tissues
and so on. Such sets of genes are suitable to systematic analysis to elucidate
the molecular mechanisms governing the biological processes. Since we areparticularly interested in early embryogenesis, we have focused on a set ofgenes whose mRNA are maternally supplied and disappear or localize to spe"cific cells during early stages (2'cell-early gastrulation). We found out 477(about 10%) such genes when the expression analysis of about 5,000 genes was
finished, and have done RNAi (RNA mediated interference) analysis on the
477 genes.
We microinjected double"stranded RNA into the gonads ofN2 worms. Pheno'
typic analyses were performed on the injected worms themselves, F 1 embryos,
larvae and adults that survived (called "escapers") and F2 embryos, with reo
spect to embryogenesis, larval growth, sterility, morphogenesis, and so on. Outof the 477 genes, 5% showed the reduction ofthe number ofF1, 33% showed F1embryonic lethality, 27% showed one or various phenotypes with only escapers
and 35% did not show any phenotype. Interestingly, genes in X chromosome
showed much higher ratio of no'phenotype than those in autosomes (X chromo"
some: 42/71, autosomes: 125/404).
We further analyzed early cell division using the 4D microscope for the genesthat showed more than 50% embryonic lethality (61 genes) in the RNAi screen"ing. Out ofthem, 12 genes showed the arrest at fertilized egg and no division,
29 genes showed aberrant process until 8'cell stage and 20 genes showed no
phenotype until 8"cell stage.
Interestingly, 5 out ofthe 12 genes of the severest phenotype (arrest at fertil"ized egg) related to proteasome: 2 genes are proteasome components and 3
genes are proteasome regulatory subunits. Proteasome is known to partici"
pate in the regulation of meiotic maturation and fertilization in fish and frog

126

oocytes. Thus, we examined all possible proteasome genes. In G.elegans, 14
genes for proteasome components are predicted. cDNA are available in ourcollection for 13 genes including the 2 genes in the above analysis. Their mRNA
were detected in gonads (12/13), strongly detected in extreme early stage em'bryo and disappear by early gastrulation (6113) or localize to specific cells (6113). We did RNAi experiments for these genes, and they showed high F1 embryonic lethality (12/13) and strong reduction of the number ofF1 (12113). The
affected embryos did not show any cell divisions and seemed to be arrested
during meiosis like the previous ones. Immunostaining for one of the genes
showed strong staining at nuclei of oocytes. These results indicate that
proteasome plays important roles during meiosis in G.elegans. The experi"ments on the genes of proteasome regulatory subunits are in progress. Some ofthem show similar results to that of proteasome components.
(6) Translational control of maternal tlp-J mRNA by POS-1 and Its
Interacting protein PIP-1

Ken'ichi OGURA, ShoheiMITANI 1, Keiko GENGYo-AND0 1 and Yuji KOHARA (ITokyo
Women's Medical University School of Medicine)
A maternal factor POS-1 regulates cell fate determination of early embryos(Tabara et al., Development, 126, I-II, 1999). POS'l has two TIS11 type (CCCH)
zinc finger motifs, and is present in cytoplasm of posterior blastomeres, beinga temporary component of P granules. Previously, we reported that POS-1
binds to PIp· 1 that has an RNP type RNA binding motif. PIP-I is present incytoplasm of oocytes and early embryos, also being a temporary component of
P granules. RNAi analysis showed that PIP-I is also required for cell fate
determination of early embryos.
This time, we have isolated a deletion null mutant pip·](tm29]) and foundthat the phenotypes are identical to those ofRNAi, and that POS'l and PIP'lregulate maternal glp-1 mRNA translation. Translation of the maternal glp-]
mRNA is temporally and spatially regulated by the 3' UTR (Evans et al., Cell,
77, 183-194, 1994). In wild type embryos, GLP-1 begins to be detected inanterior AB blastomeres at two'cell stage. At four-cell stage, GLP'l is onlyG.

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detected in anterior blastomeres ABa and ABp, but not in posterior EMS andP2. We found that GLP" 1 was not detected in pip'] embryos, and that GLP'l
was ectopically detected in the posterior blastomeres in POS"] embryos. At
four-cell stage embryos ofpos"], GLP" 1 was detected in posterior EMS and P2
in addition to anterior ABa and ABp. GLP-1 was not detected in pos·] oocytes
and one'cell stage embryos, suggesting that the temporal regulation is normal. We examined POS"llocalization in pip"], and PIp· 1 localization in pos·], and found that their localizations were unchanged. These results suggestthat PIp· 1 is a positive regulator and POS"l is a negative regulator of thetranslation of maternal glp-] mRNA. In pos·]; pip'] double RNAi embryos,
the GLP'l localization was identical to that of pos-] embryos, suggesting thatpos·] is genetically epistatic. Finally, by using the three hybrid system, wefound that POS"l specifically interacted with spatial control region (SCR)
identified in the 3' UTR of glp'] mRNA (Rudel and Kimble, Genetics, 157,
639"654,2001), but PIP-I did not. We will discuss possible mechanisms ofthetranslational regulation of maternal glp'] mRNA.
(7) Computer Simulation of Early Cleavage of C. eJegans Embryo
Atsushi KAJITA, Masayuki YAMAMURA' and Yuji KOHARA UTokyo Institute ofTechnology)
Cellular arrangements are important for development. In C elegans, cell,cellinteractions are essential for cell, fate determination in early embryos. The
arrangement ofthe cells in embryo is largely restricted by physical conditions
including the force and direction of cell division, the existence of hard egg shell,
the properties of cell adhesion, membrane, cytoskeleton, and so on.
Here we present a computer simulation of early cleavage of C elegans em'
bryo, which is constructed to provide a platform to examine the relationships
among various physical parameters and early cleavages by comparing theresults of simulation with real embryos of wild type and various mutants.
Currently this simulator is based solely on dynamic model of cells and, there'
fore, includes neither chemical reaction networks nor gene'regulated networks
within the cells, in order to make the model as simple as possible. For this

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purpose, we implemented cells, eggshell and cell division, and we expressed acell by a mesh bag (= membrane) containing soft balls (= cytoplasm). The unit
particles of the cell parts are connected by springs and dampers (=shock absorbers). Eggshell is approximated by a quadric function. Timing of cell division was given and cell division was carried out by contraction of a contractile
ring in the middle of the cells.
Using the simulator, we performed several simulations of early cleavages up
to 4-cell stage. The results were that the movement and arrangement of thecells were almost the same as the real embryo, except that the cell arrangement of 3-cell stage was slightly different. This means that the restriction ofcell movements by eggshell and the repulsive forces of cell division were reconstructed successfully but the lack of cytoskeleton may cause the discrepancy at
3-cell stage. In the simulation, the cleavage ofP1 is temporally a bit behindthat ofAB, which is the normal pattern of wild type. In another simulation, we
set the cleavage timing ofP1 ahead that ofAB. The results were striking; theresulting 4 cells were arranged not in the usual diamond shape but in T shapein which ABx can not contact with P2. Interestingly, this T shape, to someextent, looks like the embryo ofA. nanus in which the cleavage ofP1 is muchahead that of AB (Wiegner and Schierenberg, Dev. BioI. 204, 3-14 (1998». We
are currently improving the simulator by adding attractive forces between theparticles of cytoplasm and implementing other phenomena in early cleavages
ofthe embryo.

(8) PGL-l, PGL-2 and PGL-3, a family of P-granule proteins, function
redundantly to ensure fertlllty In both sexes of C. elegans
Ichiro KAwASAKI, Anahita AMIRI', Yuan FAN', Takeshi KARAsHIMA2, Yuji KOHARA
and Susan STROME I ('Dept. of Biology, Indiana University, 2Dept. of Biophysics
and Biochemistry, University of Tokyo)
P granules are distinctive ribonucleoprotein complexes observed specificallyin the cytoplasm of germ cells throughout development. We previously identified PGL-1 as being a constitutive protein component ofP granules. The presence of an RGG box predicts that PGL-1 is an RNA-binding component of PG.

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granules. pgl-} mutants contain defective P granules and are sterile, due todefects in proliferation and gametogenesis. Interestingly, the sterility causedby null alleles of pgl-} is highly sensitive to temperature. Our identification
and analysis of two additional pgl-}-related genes, termed pgl-2 and pgl-3,
demonstrate that the PGL proteins function redundantly, and at low temperature, PGL-2 and PGL-3 are sufficient for fertility.
PGL-2 has 34% identity with PGL-1 in its N-terminal region. PGL-3 has
62% identity with PGL-1 throughout its length and contains an RGG box.
Based on yeast two-hybrid and GST pull-down results, the three PGL proteinsinteract with each other. Furthermore, PGL-1 and PGL-3 are co-immunoprecipitated from both embryo and oocyte extracts, indicating that they are indeed in the same protein complex in vivo. Immunofluorescence analysis has
demonstrated that in wild-type worms PGL-3 is associated with P granules atmost stages of development, like PGL-1, but interestingly, PGL-2 is associated with P granules only during postembryonic development. Based on molecular epistasis results, each PGL protein associates with P granules independently of the other two.
To address whether PGL-3 functions redundantly with PGL-1, we isolated apgl-3 deletion allele. We found that pgl-}; pgl-3 double mutant hermaphrodites and males show significantly enhanced (but not 100%) sterility at low
temperature, compared to either single mutant. Double mutant hermaphrodites contain a severely underproliferated germline, indicating that the primary defect is in proliferation. Depletion of pgl-2by RNAi did not enhancesterility further.
Our findings suggest that both PGL-2 and PGL-3 are components ofP granules that interact with PGL-1 and that at least PGL-3 functions redundantlywith PGL-1 to ensure fertility in both sexes during C. elegansgermline development.

130(9) Identification of inducible innate immune defences in C. eJegans
Gustavo MALLO l , C. Leopold KURZ l , Samuel GRANJEAUD l , Yuji KOHARA andJonathan EWBANK l (lCentre d'Immunologie de Marseille-Luminy)
Until now, it has not been clear whether C. elegans possesses an inducible
system of defence or not. We have used high-density cDNA arrays to address
this issue. We have found that following infection with the Gram-negative
bacterium Serratia a number of genes are induced. We found 14 genes thatshowed a greater than 2-fold induction after both 24 hand 48 h of infection.
Among them, other than genes for which no known homologues exist, we haveidentified genes encoding lectins and lysozymes, known to be involved in immune responses in other organisms. By in situ hybridisation, we have shownthat the majority of these are expressed in the intestinal cells. Studies using
invertebrates have significantly contributed to our current understanding ofvertebrate innate immunity. Our findings thus open a new avenue for theinvestigation of evolutionary conserved mechanisms of innate immunity.

(10) Functional analysis of the C. eJegans T·box gene tbx-9
Yoshiki ANDACHIMembers of the T-box family that share a DNA binding motif play criticalroles in developmental phenomenon in metazoans. Some ofthe members havebeen shown to encode transcription factors. The complete sequence of the C.
elegans genome indicates 20 T-box genes in this organism, and nearly half ofthem have been identified by the C. elegans EST project. I have been studying
one of the C. elegansT-box genes, the cDNA group CELK02736 or tbx-9, whichis the first T-box gene found by the EST project and seems to be the mosthighly expressed T-box gene as deduced from the number of isolated cDNA
clones corresponding to each gene. I previously showed that tbx-9 encodes atranscription activator, that tbx-9is expressed in a few cells in embryogenesis,
and that the tbx-9 deletion mutant generated by gene disruption shows aberration of morphogenesis predominantly in the posterior body, including abnor-

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mality of body'wall muscle.
To further elucidate the expression pattern of tbx-9, I performed in situ hy'
bridization double'staining analysis using the pos'] and hlh'] genes as markers. The onset of the tbx-9 expression is at the B'cell stage in the E cell, theancestor cell of gut, and the expression lasts until daughters of the E cell at the
26'cell stage. At this stage, the Ca and Cp cells start to express tbx-9, which
produce body'wall muscle and hypodermis. On the other hand, at the about
200-cell stage four MS descendant cells express both tbx-9 and hlh -1. hlh'] isa homolog of vertebrate MyoD and is expressed in the cells whose clonal de'scendants give rise only to striated muscle cells. This indicates that tbx-9is
also expressed in precursors of MS-lineage bodY'wall muscle cells. Amongthese tissues whose precursor cells express tbx-9, bodY'wall muscle that showsabnormality in the tbx-9mutant was examined with respect to the formation
by immunostaining with an antibody against body'wall muscle myosin. In
wildtype embryos, the muscle cells move and form four rows along the length of
the animal by the 1.5·fold stage. In tbx-9mutant embryos, some of the muscle
cells that are different worm by worm turned out to be positioned apart fromthe rows at this stage, suggesting that tbx-9is involved in the proper arrange'
ment of the bodY'wall muscle cells.

Pub I i ca t ion
None

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H.
H-a.

Structural Biology center

Biological Macromolecules Laboratory(1) Single Molecuie Imaging in cells

Makio TOKUNAGA
Imaging of single fluorescent molecules in vitro has been achieved in a relatively simple manner using objective-type total internal reflection fluorescence microscopy (TIRFM). It is also found that single molecule imaging invivo can be realized using the objective-type TIRFM. However, the technique isapplied only when fluorescent molecules are from the glass-medium or glasscell surface to a depth ofless than about 150 nm. I have further developed thissingle-molecule technique by refining the way oflaser-beam illumination. The
new microscopy allowed visualization of single molecules in cells up to thedepth of about 10 /1 m from the glass-medium interface.

(Z) Single molecule imaging of nucleocytoplasmic transport
Makio TOKUNAGA and Naoko IMAMOT0 1 (IGene Network Laboratoy)
The above new microscopy was applied to imaging of single nuclear pores andsingle molecules involved in nucleocytoplasmic transport. Recent years haveseen considerable progress in researches of macromolecule import into the
nucleus and export from the nucleus. In contrast to detailed knowledge ofsoluble factors, molecular mechanisms of the transport and the interaction
between nuclear pore complex (NPC) and molecules have been remained to besolved.
We examined behavior of green fluorescent protein (GFP)-tagged importin
beta and GFP-tagged cargo in digitonin permeablilized cells. Fluorescence
images of importin beta or cargo molecules bound to the nuclear rim wereH.

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obtained. Images were composed of many fluorescent spots, which represented
single NPC's. Furthermore, imaging of single molecules of GFP-importin betaon the NPC has been also achieved. Both imaging of single pores and imagingof single molecules enabled us to analyze images quantitatively at the molecular level and to obtain kinetic parameters in cells, i.e., retention time at
the pore, number of molecules bound to pores, and binding constant. Thus,
single molecule imaging has opened a new way to obtain quantitative information on kinetics of molecular interactions in cells.

(3) Characterization of Xpl05-Xp65 complex gathered around thecentrosome in Xenopus
Nobuyuki SHIINA, Kazumi SHINKURA and Makio TOKUNAGA
The centrosome is nucleating center of microtubules and plays key roles toestablish and maintain the intracellular polarity. We identified a novel protein Xp 105 as one of molecules gathered around the centrosome in Xenopus.
Xpl05 was localized on granules with a diameter of 60-70 nm bordered withelectron-dense coat. We found and identified a novel protein Xp65 as a binding
protein ofXpl05. Expression ofXp65-GFP fusion protein in cultured Xenopus
A6 cells induced formation of the granules around centrosomes. The aminoacid sequence ofXp65 showed a little homology to that of a retrotransposable
element of insects and the Xp65-GFP-induced granules resembled VLP (virus·like particles) formed by retrotransposable elements, indicating that Xp65
is a retrotransposable element in Xenopus. In contrast to Xp65, Xp 105 is agene product of host cells and highly conserved from insects to mammals. So
Xp 105 should be responsible for some function(s) in/for host cells, although it
was found originally as a molecule gathered around the centrosome together
with the VLP. To get a clue to the function ofXpl05 in host cells, we observed
the behavior ofXp 105-GFP fusion protein in cultured cells. Expression ofXp 105GFP in cultured cells induced electron-dense amorphous structures in the cytoplasm, in which beta -COP was colocalized. This novel structure was totally
distinct from COP I-coated vesicles or endosomes, where beta-COP has beenreported to be localized. Dynamic movement ofXpl05-GFP at the cell periph-

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ery was found and is being investigated in further detail.

(4) Single molecule measurement of intermolecular and intramolecularInteractions using subplconewton Intermolecularforce microscopy
Michio HIROSHIMA and Makio TOKUNAGA
We have developed intermolecular force microscopy (IMF) by refining atomic
force microscopy (AFM) which is able to measure interaction forces between
biomolecules through a gap distance of nanometers and to measure intramolecular forces of single molecule. This microscopy has achieved the force resolution of subpiconewton, which is over 100-fold more sensitive than that ofconventionalAFM. A new system using a laser radiation pressure was incorporated to reduce thermal fluctuation of the cantilever and to control its position. We have further refined the IMF on the following points: 1) expansion offorce ranges up to 200 pN, 2) development of a new horizontal scanning system
in which cantilever moves horizontally and horizontal positions are controlled
by the feedback system.
Use of a new laser for optical control reduced thermal fluctuations upto 0.8nm(r.m.s,) and expanded the force range to more than 200 pN. In the horizontal
scanning system, thermal fluctuations were reduced to 0.7nm (r.m.s.) and theforce range was 90 pN. These performances are sufficient to measure inter- or
intramolecular forces at the single molecule level.

(5) Single molecule measurement of protein folding by intermolecularforce microscopy
Isao SAKANE, Michio HIROSHIMA, Kunihiro KUWAJIMA 1 and Makio TOKUNAGA(IDepartment of Physics, University of Tokyo)
Mechanical unfolding events of single protein molecules are measured with
the intermolecular force microscope. Using single molecule measurements, we
expect to obtain new information on protein folding mechanism, which would
not be obtained by conventional methods. Staphylococcal Nuclease (SNase) isH.

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a small globular protein, and is one of the proteins most generally used forfolding researches. Its folding kinetics is well characterized, and also its atomic
structure is available. Therefore, SNase is suitable for single molecule experiments. Cysteines were substituted at the both N- and C-termini of Staphylococcal Nuclease. It was attached to gold substrates by the cysteinyl residues.
Single protein molecules ofSNase were stretched mechanically with the intermolecular force microscope. Force curves, relations between force and lengthof single molecules, showed that full-stretched length of a molecule was 87 nm.
On the assumption of the molecule as a dimmer, this result corresponds withthe length of 0.29nm per amino acid, which agrees well with previously reported results. We are carrying out measurements with higher resolution to
obtain detailed information on kinetics of proteins unfolding.

(6) Single molecule measurement of nucleocytoplasmic transport
at nuclear pores
Atsuhito OKONOGI, Michio HIROSHIMA, Nobuyuki SHIINA, Shingo KOSEl, NaokolMAMOTO l and Makio TOKUNAGA (lGene Network Laboratoy)
To ravel out the molecular mechanism of nucleocytoplasmic transport, it isessential to obtain detailed knowledge ofthe interaction between nuclear porecomplex (NPC) and proteins involved in transport. We are trying to carry out
single molecule measurement of nucleocytoplasmic transport, using
subpiconewton intermolecular force microscopy. The inner radius of the nuclear
pore is thought to be approximately 20-30 nm, so we have tried to make cantilevers, probes for scanning probe microscopy, with very sharp-pointed tips.
Carbon nanotubes, whose diameter is approximately 10 nm, were used as thetip of the catilevers. In vitro assay system of nuclear transport is also essential for the single molecule measurement. Nuclear envelope were formed invitro using extracts made from Xenopus laevis frog eggs.

Pub I i ca t ions
1. YANAGIDA, T., ESAKI, S., IWANE, A.H., INOUF:, Y, ISHIJIMA, A., KITAMURA, K, TANAKA, H.
and TOKUNAGA, M.: Single-motor mechanics and models ofthe myosin motor. Philos.

136

2.

3.

Trans. R. Soc. Lond. B, 355, 441"447, 2000.
MIMORI"KIYOSUE, Y, SHIINA, N. and TSUKITA, S.: Adenomatous polyposis coli (APC)
protein moves along microtubules and concentrates at their growing ends in epithelial
cells. J. Cell BioI., 148, 505"518, 2000.
MIMORI"KIYOSUE, Y., SHIINA, N. and TSUKITA, S.: The dynamic behavior of the APC"
binding protein EBI on the distal ends of microtubules. Curro BioI., to, 865"868, 2000.

H-b.

Molecular Biomechanism Laboratory(1) The promoter arrest of E. colI RNA polymerase and theeffect of the Gre factors
Motoki SUSA 1 , Tomoko KUBORI 1 , Hiroki NAGAI 1 , T. GAAL 2, V. Georgi
MISKELISHIVILI 3 and Nobuo SHIMAMOT0 1 (1 Structural Biology Center, National
Institute of Genetics, 2Microbiology, Wisconsin-Madison, 3Institute fms Genetik
und Mikrobiologie)
The conventional model for the mechanism of transcription initiation is com"posed offour sequential steps: the binding of RNA polymerase and necessary
factors to promoter, formation of open complex that has partially melted duplex, formation of initiation ternary complex that retain short transcript, andpromoter clearance that means dissociation of RNA polymerase from a promoter. The alternative model, which we proposed in 1996, allows the promoter-RNA polymerase complex to follow two pathways, productive and arrested pathways. In productive pathway, RNA polymerase is committed tosynthesize long mature RNA, whereas in arrested pathway the enzyme forms
moribund complex that synthesize only short abortive transcripts and thenconverted into dead-end complex that has no elongation activity (Nuc. Acids
Res. 24, 1380-1381 (1997): J. Mol. BioI. 256,449-457 (1996». We observed theformation of the nonproductive complexes at several promoters, but no suchcomplexes are found at T7A1and rrnBPl promoters, indicating the existenceof two classes of promoter.
DNA and protein footprinting analyses showed that the dead-end complex isbacktracked from the promoter, lacks the strand opening and has more exposed conserved region 3 of a 70 (Sen et al. J. BioI. 275, 10899-10904 (2000).

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This inactivation was mitigated by a a 70 factor with S506F mutation in region 3 (SEN et aI., J. BioI. Chern. 273, 9872-9877(1998» or the Gre factors(GreA and GreB: Sen et aI., Gene. Cell. in press). These factors introduce
reversibility between productive and non'productive pathways so that moribund complex can be converted into productive complex at a high concentration
ofthe initiating nucleotide. This result indicates that the behavior of T7A1
and rrnBP1 promoters can be explained as reversible promoters on the basis
ofthe branched pathway mechanism. We are on the way to prove the universality of the branched mechanism.
The Gre factors that have been assigned to be elongation factors, therefore,
also work in initiation. GreB has stronger activity as an elongation factor than
GreA, but GreA turns out to be stronger than GreB as an initiation factor invitro. We have constructed single and double disruptants of peA and peB, andthe disruption of GreA shows stronger phenotype. This indicates that the Gre
factors are initiation factors also in vivo. An analysis of 2-dimensional protein
gels and an analysis using gene array indicate 300 candidates for the target of
Gre action.

(2) Role of w subunit of E. colI RNA polymerase
Dipankar CHATTERJI 2, Akakoli MUKHERJEE 2, Hiroki NAGAI' and Nobuo
SHIMAMOTO' (1 Structural Biology Center, National Institute of Genetics, 2Cen'tre of Cellular and Molecular Biology)
The function of wsubunit of E coli RNA polymerase has not been identified
for decades, and a disruption of its gene rpoZ does not have any clear phenotype. We have purified RNA polymerase core enzyme from a rpoZ disruptant
and found the enzyme is tightly bound to GroEL shaperon. The core enzyme
can bind to a 70 and active. It is inactivated, however, with a loss of binding
activity to a 70. If the associated GroEL is removed with a dye-affinity column
or denaturation by urea. Reconstitution of activity is greatly stimulated by thepresence of w (Mukherjee et aI., Eur. J. Biochem. 266, 228-235 (1999». These
results show w is structurally required for maturation of core enzyme. The
lack of any phenotype of rpoZmutant may be due to the functional suppression

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bygroEL(3) Inactivation of a 70 by oligomerization and identificationof the role of its spacer region
Hiroki NAGAI 1, Taciana KAsCIUKOVIC 2, Richard S. HAYWARD 2, Yumiko SAT0 1 andNobuo SHlMAMOT0 1 (lNat. Inst. of Genet. /Grad. School ofAdv. Stud., 2Inst. Of
Cell and Molec. BioI., Edinburgh Univ., Scotland)
We have found that a 70, the major a factors ofE. coli, forms aggregate in vivo
and in vitro at a high temperature within physiological condition. The oligo"
meric a 70 has little transcriptional activity and the oligomerization in vitro
showed a sharp temperature dependence. We have constructed a strain with adisrupted rpoD ( a 70), and plasmid born a 70 supports its growth. We haveobserved a positive correlation between the intracellular concentration of a 70
and upper limit of growing temperature. This raises a possibility that a 70 is amolecular thermometer.
The major a factors of proteobacteria mostly have a big spacer region be"
tween the conserved regions 1 and 2 which is not conserved in eubacteria. In E.
coli a 70 this region has extensive acidic patches which may be concerned withthe property of a 70 to readily interact with nonspecific and specific surfaces.
An rpoD-disrupted strain was constructed to test the viability 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 a 70 lacking the spacer regionof aa130-374 (/::" SR) complemented the disruption at 30 and 25'C, provingthat the region is not essential for growth at low temperature. At all tested
temperature this protein predominantly exists as aggregate which are in equilibrium with a small fraction of monomer. Therefore, the role of the spacer
region is the maintenance of active monomeric form.

(4) Single-Molecnle Dynamics of Transcription: Sliding ofproteins along DNA
Takashi KrNEBUCHI 1, Kumiko SOGAWA 1, Hiroyuki KABATA I2 , Nobuo SHlMAMOT0 1,

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Osamu KUROSAWA23 , HironoriARAMAKI 4 and Masao WASHIZU2 (1 Structural Biology Center, National Institute of Genetics, 2Department of Mechanical Engineering, Kyoto University, 3Advance Co., 4Department of Molecular and Life
Science, Daiichi College of Pharmaceutical Science)
We have showed the existence of a sliding motion ofprotein along DNA throughdirect visualization of single molecules of E. coli RNA polymerase (Science 262,
1561-1563 (1993». To check the generality of sliding, we applied similar single
molecule dynamics to a bacterial repressor protein, P. putida CamR, which
was observed to slide along DNA. In the absence of its inducer, d·camphor,
CamR molecules were trapped at three positions on a A. DNA, one was its
cognate operator cloned in A. DNA, and the other two are likely to be homolo·
gous to the operator. All trapping occurred at specific sites, and only sliding
complexes were observed at non-specific sites. This observation indicatesthat the non· specific complex is the sliding complex itself.
The most distinct difference between the movements of RNA polymerase and
CamR was the pathway of dissociation from their specific sites. RNA polymerase slides out of its specific site into nonspecific sites and then dissociatesfrom nonspecific sites into bulk. This two·step dissociation was not observedin the case of CamR. CamR seemed to dissociate directly into bulk and its
sliding upon dissociation from the specific site was not long enough to be
detected. CamR also slides extensively upon association to the specific site,
and thus long nonspecific DNA segment flanking the specific site accelerateassociation but not dissociation, making its affinity for the specific site on
longer DNA stronger. Thus long DNA can harvest CamR like an antenna.
There is a long-standing contradiction on E. coli TrpR that its specificity is
too small to compete binding to its operator against the predominant nonspecific sites with the copy number present in the cell. We challenged to solve thiscontradiction by introducing the concept of antenna effect by sliding. We foundthat the affinity ofTrpR for trpO strongly depends on the length of DNA and isenhanced more than 10,000 -fold. A control experiment showed that this enhancement is not due to the stabilization by an additional interaction with along DNA. Therefore, antenna effect by sliding is really present in vivo. Thiseffect open up several new ways of gene regulation and further proof of antenna

140

effect will be obtained.

Pub I i ca t ions
1.

HARADA, Y., OHARA, 0., TAKATSUKI, A., IToH, H., SIIlMAMOTO, N. and Jr., K.K. (2000).
Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase. Nature 409, 113-115.

2.

MATsuMoTo, T., MORIMOTO, Y, SHIBATA, N., KINEBUCHI, T., SHlMAMOTO, N., TSUKIHARA, T.
and YASUOKA, N. (2000). Roles of functional loops and the C-terminal segment of asingle'stranded DNA binding protein elucidated by X'ray structure. J Biochem. 1%7,
329-335.

3.

MURAMATSU, H., HOMMA, K., YAMAMOTO, N., WANG, J., SAKATA-SOGAWA, K. and SHIMAMOTO,
N. (2000). Imaging of DNA molecules by scanning near-field microscope. Mater. Sci.

Eng. C 1%, 29-32.
4.

SEN, R, NAGAI, H. and SHIMAMOTO, N. (2000). Polymerase-arrest at the A. PR promoter
during transcription initiation. J BioI. Chem. %75, 10899-10904.

5.

YAMAMOTO, T., KUROSAWA, 0., KABATA, H., SHIMAMOTO, N. and WASHlZU, M. (2000).
Molecular surgery of DNA based on electrostatic micromanipuration. IEEE Trans. on

Indst. AppI. 36, 1010-1017.

H-c.

Multicellular Organization Laboratory(1) Fluoride-resistant Mutants of the Nematode
Caenorhabdltis eJegans

Akane OISHI, Takeshi ISHIHARA and Isao KATSURA
Fluoride"resistant (fJr) mutations of C elegans are recessive and grouped
into two categories: class 1 mutations, which map in fJrI, fJr3 and fJr4, andclass 2 mutations, which map in fJr2and fJr5(Katsura, 1. et aD Genetics 136,
145-154, 1994). Class 1 fJrmutations show many phenotypes: slow growth,short defecation cycle periods, frequent skip of the expulsion step of defeca"
tion, and synthetic abnormality in dauer formation (See (2) below), besides
strong resistance to fluoride ion. The fJrI gene encodes an ion channel belong"
ing to the DEG/ENaC (C elegansdegenerins and mammalian amiloride"sensitive epithelial sodium channels) superfamily, while fJr4 and fJr3 code for a

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novel Ser/Thr protein kinase and a kinase-like molecule, respectively, bothhaving a hydrophobic domain on the carboxyl-terminal side. A functional fJrrGFP fusion gene is expressed only in the intestinal cells from the comma
stage of embryos to the adult stage, while fJr-4::GFP is expressed in the intestinal cells from the 1.5-fold stage, in the isthmus ofthe pharynx from the 3fold stage and in a pair of head neurons called ADA from Lllarvae to adults.
Moreover, the expression of fJr-3:·'lacZis detected mainly in the intestine. We
therefore think that class 1 fJrgenes constitute a regulatory system that actsin the intestine and that controls many foodrelated functions. Class 2 fJrmutations, which confer weak resistance to fluoride ion, suppress the slow growthand dauer formation abnormality but not the defecation abnormality andstrong fluoride-resistance of class 1 fJr mutations. Hence, it seems that theregulatory pathway bifurcates after class 1 genes, and that class 2 genes control only one of the two branches. Of the class 2 genes, fJr-2 encodes a secreted
protein belonging to the gremlinlDAN/cerberous family. Although most pro'teins of this family are TGF- {3 antagonists, there is so far no evidence showing that FLR-2 interacts with any of the four TGF- {3 proteins in C. elegans.
In the year 2000, we studied the expression of fJr-2gene. Although we could notdetect the fluorescence of the functional fJr-2:GFP fusion gene that we made,we detected the staining with anti·GFP antibodies in a small number ofneurons in the pharynx, head and tail. We also detected the product ofthe endogenous fJr-2 gene with antibodies against recombinant FLR-2 protein. A small
number of neurons in the head, tail and pharynx were stained with the antibodies. The neuronal expression ofFLR-2 may be a clue to solving the mystery:
how the class 1 fJr genes, which act in the intestine, can control sensory signals.
(~)

Analysis of Synthetic Dauer-constitutive Mutations

Kouji MIYAHARA, Kiyotaka OHKURA, Tomoko YABE, Takeshi ISHIHARA and Isao
KATSURAIf newly hatched larvae of C. elegans are in a crowded state and given alimited food supply, they sense the environmental signal of pheromone and142food with head sensory organs called amphids and deviate from the normallife cycle, ending in non-feeding larvae called dauer larvae. Since the assay ofdauer formation is much less time-consuming than behavioral assays such as
chemotaxis assays, we are analyzing the head neural circuit by detecting dauer
formation as the output. We found that mutations in more than 50 known
genes show synthetic dauer-constitutive (Sdf-c) phenotypes, i.e., they develop
to dauer larvae in a certain mutant background, regardless ofthe environmental conditions. The synthetic nature of the phenotype, we think, is based on the
structure of the neural circuit, in which neural signals are transmitted throughparallel routes consisting of different types of neurons so that two mutations
may be required to block the signals. We are studying the combinations ofmutations for the Sdf-c phenotype and the pattern of suppression of the Sdf-c
phenotype by various suppressor mutations. In this way we hope to determine
the functional neural network for dauer regulatory signals and the function of
relevant genes in the network.
Furthermore, to identify genes acting in neural functions, we isolated andmapped 44 new mutations that show the Sdf-c phenotype in combination withthe unc-31(eI69)mutation. Many ofthem are expected to cause defects inASI
neurons, since the unc-31 (eI69) mutant shows dauer-constitutive phenotype,ifASI neurons are killed. Eight of the mutations mapped in 4 known genes, but
most of the remaining 36 mutations, which map in at least 13 genes, seem tobe alleles of novel genes. Of these mutants, that in sdf-l gene (1 allele) avoids
benzaldehyde, isoamyl alcohol, butanone, NaCI, and lysine, which are attractants of wild-type animals. It behaved as thermophilic in thermotaxis. Mutants in sdf-13 gene (2 alleles) showed defects in adaptation of chemotaxis toodorants sensed by AWC neurons (benzaldehyde, isoamyl alcohol andbutanone), although they showed normal chemotaxis to these odorants. This
gene encodes a homolog of mammalian Tbx2 and Drosophila Omb, a transcription factor containing the T-box domain.
The following results were obtained in 2000.
The cells in which sdf-13 gene is expressed and the candidates ofthe cells inwhich sdf-13 gene acts for olfactory adaptation were identified. We made an
sdf-I3-:GFP fusion gene in which GFP cDNA is connected to the end ofthe sdf13 coding region. This GFP fusion gene rescued the mutant phenotypes (SdfH.

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and adaptation defects) and was expressed in the pharyngeal neurons M2 and
15 from the late embryonic to the adult stage. However, since the fluorescence
was very weak, we also studied the expression of sdf-13 gene with antibodies
against the N-terminal86 amino acid residues of SDF-13, which was synthesized in E. coli cells. The antibodies stained the amphid neurons AWB, AWe,ASJ and the pharyngeal neurons 11, 13, 15, Ml, M2, M5, NSM. Furthermore,when the sdf-13 eDNA was expressed inAWB,AWe and 11 by connecting to thegcy-l0 promoter, it rescued the olfactory adaptation defects but not the Sdf
phenotypes of sdf-13 mutants. The results is consistent with the idea that sdf13acts cell-autonomously. To prove this, we plan to test whether the olfactory
adaptation defects are rescued by expression of sdf-13 in AWe neurons andthe Sdf phenotypes in ASJ neurons.
In sdf-9gene, five mutant alleles have been obtained, which complement all
the dauer-related mutations that map around the locus. We found that nearly
100% of sdf-9 mutants animals become dauer larvae and spontaneously recover from dauer to L4larvae, if the promoter of daf-7gene (a TGF- (3 genethat acts in dauer regulation) is introduced into the mutants. However, daf7.:GFP was expressed in sdf-9mutants like in wild type animals. We mapped
sdf-9mutations to the right end of chromosome V (right side of rol-!JJ by threefactor cross with known mutations and by using single nucleotide polymorphism. Presently, we are trying to clone sdf-9 gene by introducing DNA fragments of this region into the mutants and checking the rescue of the Sdfphenotype. Studies of this gene may be useful for analyzing dauer formation anddauer recovery separately.

(3) Analysis of Mutants That Show Abnormality in the Selection
between Two Behaviors and in Behavioral Plasticity
Takeshi ISHIHARA, Yuichi IINo 1 and Isao KATSURA (IMolecular Genetics Laboratory, University of Tokyo)
Animals receive environmental cues with sensory cells, select and integratemany pieces of necessary information, and make proper responses, which canbe modified by former experience or memory. In C. elegans, analyses ofbehav'

144

ioral mutants have been performed, which lead to the molecular mechanismsof sensation of odorants and temperature, for instance. On the basis on theseresults, we are analyzing mutants on learning and the selection (evaluation) of
sensory signals, to elucidate novel mechanisms of higher-order sensory signal
processing.
The nematode C. elegans shows avoidance of copper ion and chemotaxis toodorants by perceiving them with different sensory neurons in the head. We
devised a behavioral assay method for the interaction between the two responses to learn a possible role of interneurons. Wild-type animals change
their preference between the responses, depending on the relative concentration of copper ion and odorants. This suggests that the two sensory signals
interact with each other in a neural circuit consisting of about 10 neurons, on
the basis of the neural circuitry of C. elegans and the identity of the sensory
neurons that act in these behaviors.
To elucidate the mechanism of the complex behaviors mentioned above, we
are isolating and analyzing mutants that show abnormality in this assay. The
mutant ut236 has a tendency to choose avoidance of copper ion rather thanchemotaxis to odorants, although it shows no abnormality in each behavior incase only one ofthe stimuli is given. The result shows that ut236is abnormal
in the interaction between the two behaviors. Furthermore, ut236 shows abnormality in the conditioning (learning) by the paired presentation of starvation and NaCl. While wild-type animals are normally attracted by NaCI, they
avoid it after the conditioning. In contrast, the paired presentation has only
very small effect on the ut236 mutant animals. Since the mutant shows normal behavioral responses to starvation alone, we think that it has abnormality in the learning process.
We identified the gene for ut236by positional cloning. It encoded a novel
secretory protein having an LDL receptor ligand-binding domain. Using antibodies against its recombinant protein, we found that the gene product islocalized in the axon of each one pair of sensory and inter-neurons. The localization in the axon was abolished in unc-]04 (kinesin KIFIA homolog) mutants, which show defects in the transport of synaptic vesicles. Expression
studies with various transcriptional promoters showed that this gene acts
cell-non-autonomously in the nervous system. Studies using a heat-shock pro-H.

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moter showed that the wild-type phenotype is recovered if it is expressed after
the mature neural circuitry is formed,.but not if expressed during the development ofthe nervous system. These results indicate that the gene product is a
novel neuromodulatory factor that acts in diverse functions such as the modification of sensory information and various types oflearning.
In the above experiments, the assay of selection between two behaviors was
carried out with well-fed animals. When it was conducted with animals starved
for 5 hours, the animals showed a stronger preference for odorants, because
they responded more weakly to copper ion. This starvation effect was not de'tected in the presence of serotonin, which is considered to induce a well-fed
state. The behavioral change upon starvation seems appropriate, because
starved worms can look for food over a wider area.
We isolated a mutant, ut235, which lacks the effect of starvation in thisassay. This mutant behaved essentially normally in many other responses tostarvation such as the strarvation-induced increase oflocomotion speed. Interestingly, the double-mutant ut235; ut236 showed a preference to avoidanceof copper ion, regardless of starvation. To clone the gene for the ut235 mutation, we mapped it precisely by using SNP between two wild-type strains, N2
and CB4856.

Publications
1.

OKUDA, T., HAGA, T., KANAI, Y., ENDOH, E., ISHIHARA. T and KATSURA, I.: Identificationand characterization of the high' affinity choline transporter. Nature Neurosci. S, 120125 (2000).

2.

ASAHINA, M., ISHIHARA, T., JINDRA, M., KOHARA, Y, KATSURA, I. and HIROSE, S.: The
conserved nuclear receptor Ftz'F1 is required for embryogenesis, moulting and reproduction in Caenorhabditis elegans. Genes to Cells 5, 711-723 (2000).

3.

AOKI, H., SATO, S., TAKANAMI, T, ISHIHARA, T, KATSURA, I., TAKAHASHI, H. and HIGASHITANI,

A: Characterization of Ce-alt-l, an ATMlike gene from Caenorhabditis elegans. Mol.
Gen. Genet. 264, 119-126 (2000).
4.

SHIOI, G., SHOJI, M., NAKAMURA, M., ISHIHARA, T., KATSURA, I., FU.JISAWA, H. and TAKAGI,
S.: Mutations affecting nerve attachment of Caenorhabditis elegans. Genetics 157, inpress (2001)

5.

KATSURA, I.: Behavioral genetics of the nematode (in Japanese). In "Molecular biology
of behavior" (Yamamoto, D., ed,) pp29-39. Springer-Verlag Tokyo, Inc. (2000)

146

H-d.

Biomolecular Structure Laboratory(1) Crystallographic Study of F1-ATPase
Yasuo SHIRAKIHARA andAya SHIRATORIFI-ATPase, with a subunit composition of a 3 {3 3 Y a E ,is a catalytic sector of the membrane bound ATP synthase. The ATP synthase plays a central
role in energy conversion in mitochondria, chloroplasts and bacteria, generating ATP from ADP and inorganic phosphate using energy derived from a transmembrane electro-chemical potential. The rotational catalysis mechanism of
Fl is accompanied by rotation ofthe rod-like Y subunit, which is thought tocontrol the conformations ofthe three catalytic {3 subunits in a cyclic manner.
We previously solved the structure ofthe a 3 {3 3 subassembly of FI-ATPasefrom a thermophilic bacterium Bacillus PS3. We have been extending thestructural study to a 3 {3 3 Y and a 3 {3 3 Y E sub-assemblies, both of which
exhibit kinetic properties very similar to those of F 1. In this extension, we aim
to detect structural changes caused by different mode of nucleotide binding,which should provide with structural basis for understanding the rotational
catalysis mechanism.
As reported lat year, extensive studies on purification and crystallization
had been done to get crystals of a 3 (3 3 Y sub-assembly. Currently, however,crystals are not large enough for in-house diffraction study, due to their weakdiffracting capability. Usual crystals allowed us to record to resolutions of
only about 40-20A in house. After examining freezing conditions for data collection at synchrotron, we found that those crystals diffracted to resolutions of
15-10A at SPring8. Although the values were poor, they were twice better thanvalues for resolution limit obtained with laboratory x-ray source. Crystals
exhibited high mosaicity(2.5 degree). The unit cell parameters were a=190.1A,
b=194.0A, c=254.6A, a =111.7, {3 =lp.6, Y =91.0. Further efforts are being
made to extend the resolution ofthelcrystals and to find appropriate conditions for cooling crystals.
The a 3 {3 3 Y E sub-assembly crystals have been found to be much better
than the a 3 {3 3 Y sub-assembly crystals described above. The a 3 {3 3 Y E

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sub-assembly crystals, when experimented with rotating' anode source, diffracts to 4.5 A resolution at room temperature and 7 A at lOOK. With SPring8
beam, we collected a data set to resolution of 4.5 A at lOOK. After trials on
various aspects on cryo'cooling, we found the best way for preparing the cooled
crystals was to combine the rapid cooling with liquid propane and the subse'
quent annealing in the cold nitrogen gas flow. The crystals needed to be grownin the presence of 20% ethylene glycol. This cooling procedure was successful
only for medium sized crystals, like 0.2xO.2xO.1mm. The unit cell parameters
were a=225.3A, b=225.7A, c=224.6A, a =94.1, {3 =117.5, Y =117.8, and theunit cell contains 8 of the sub·assembly. The data were merged with Rmerge of
12.2% at 4.5 A resolution. Structural analysis by molecular replacement is inprogress.
These structure studies were done in collaboration with Toshiharu Suzuki
and Masasuke Yoshida, at Research Laboratory of Resource Utilization, To'
kyo Institute of technology.
(!) X-ray crystallographic analysis of repressor protein CamR

Katsumi MAENAKA and Yasuo SHIRAKIHARAThe Pseudomonas putida cam repressor (CamR) is a homodimeric proteinthat binds to the operator DNA (camO) to inhibit transcription of cytochrome
P450cam operon camDCAB. This repression is relieved by binding of the in'ducer D·camphor. The unique feature in the molecular recognition of CamR
are: (1) CamR has typical two domain structure, one of which is a DNA binding
one and the other binds the inducer D'camphor, (2) Two molecules of D'camphor can bind to one molecule of homodimeric CamR in a negative cooperative
manner, and (3) similar amino acid sequence responsible for camphor binding
exists in three proteins of cytochrome P450cam operon, CamR, cytochrome
P450 and purtidaredoxin reductase. Our structural study aims to clarify theseunique characteristics of CamR.
The selenomethionyl derivative CamR (SeMet-CamR) has been overproducedin Escherichia coli and purified by the slightly modified method for the wild,
type CamR. Preliminary crystallization trials were conducted at 297 K using

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Natrix (Hampton Research). The SeMet-CamR protein was crystallized in
the slightly modified condition of Natrix condition 15, 10% MPD, 25mM Nacacodylate, 20mM MgCI2, pH6at 15'C. Crystals reached maximum size
(0.3xO.3xO.5mm) after 1 week. MAD data using the SeMet-CamR crystal werecollected at SPring8. The collected data reveal the space group to be orthorhombic (P222) and the diffraction reached beyond 3 A resolution. A full structure determination is in progress.
This work has been done in collaboration with Hironori Aramaki, Daiichi
College of Pharmaceutical Sciences.

(3) Crystallographic Study of the Transcription Activator, PhoB
Kazuyasu SHINDO, Katsumi MAENAKA and Yasuo SHIRAKlHARA
PhoB Protein is a positive transcriptional activator for the genes in the phosphate (pho) regulon of E. coli, such as phoA and pst8, that are induced byphosphate starvation. PhoB acts by binding to the pho box in the promoter
region, which is the consensus sequence shared by the regulatory regions of
phoA, phoB, phoEand PstS The activity ofPhoB is regulated by phosphorylation by PhoR. The N-terminal domain ofPhoB is responsible for this regulatory role, whereas the C-terminal domain (spanning from a residue 125 to the
C-terminus) has a DNA binding ability.
The selenomethionyl derivative of DNA-binding C-terminal domain has been
crystallized by the micro batch method to solve its structure employing the
MAD (Multi-wavelength Anomalous Dispersion) method. Diffraction data to
2.5 A resolution were collected at SPring8 for MAD analysis. The crystals
belong to the space group P2, with unit cell dimensions a=30.63 A, b=37.54 A,
c=44.37 A, and 13 =109.42. Data analysis has been in progress.
This work has been done in collaboration with Hideyasu Okamura, Yoshifumi
Nishimura, Yokohama City University and Kozo Makino, Research Institutefor Microbial Diseases, Osaka University.

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149(4) Crystallization of E.colJ nucleoid proteins
Yasuo SHIRAKIHARA and Aya SHIRATORIThe genome ofE.coli is composed of a single molecule of circular DNA and the
10 major DNA binding proteins, together forming the nucleoid. These DNA
binding proteins play not only a structural role, but also pleiotropic regulatory
roles in global regulation of gene transcription. Three of those proteins, IciA
(Inhibitor of Chromosome Initiation A), Lrp(Leucine-Responsive regulatory
Protein) and StpA(Suppressor of td-Phenotype A) were subjected to a crystallization experiment. IciA formed needle-shaped small crystals formed fromammonium sulfate solution, and Lrp formed plate crystals from phosphate
solution. Lrp crystals diffracted to 4 A resolution.
This work has been done in collaboration with Akira Ishihama and Talukder
A. Azam in the Department of Molecular Genetics.

(5) Crystallization of the DrosophlJa GAGA factor
Katsumi MAENAKA and Yasuo SHIRAKIHARA
The Drosophila GAGA factor specifically binds the GAGA-related DNA sequences in the promoter regions of a variety of genes including fushi tarazu
and Ultrabithoraxto induce the chromatin remodeling in the GAGA binding
promoter region and the transcriptional activation. The GAGA factor is a 519
amino acid protein and consist of several functional domains including BTB/
POZ, Glutamine-rich, and DNA binding domains. Since the GAGA factor tends
to form a multimer, we also focus on each functional domain for crystallographic analysis. We established the E.coli expression system for the whole
GAGA factor and some of these domains. The purification procedure of each
recombinant protein suitable for crystallization is now being examined.
This work has been done in collaboration with Susumu Hirose and Takahiro
Nakayama, National Institute of Genetics.

150(6) Structural and functional studies of immunoglobulin(lg)-likereceptors in immune system
Katsumi MAENAKA and Yasuo SHIRAKIHARA
Among the cell surface receptors in immune system, the most common domain type, about 50% of total, is immunoglobulin(Ig) superfamily including
fibronectin type III. Therefore it is important to reveal the generality and diversity ofthe molecular recognition oflg-like cell surface receptors. By using
surface plasmon resonance (SPR), we have studied the kinetic and thermodynamic analysis of two Ig-like receptors, human killer cell Ig-like receptors(KIRs) and Fc y receptors (Fc y R), whose structures have similar uniquetopology (intermediate between I set and C2 set) revealed by several structural studies ofKIRs and FcgRs including ours.
A repertoire ofKIRs with two or three tandem Ig domains in their extracellular regions is expressed on human natural killer (NK) cells. These KIRs activate or inhibit NK cell cytotoxicity following recognition of the MHC class I
molecules on target cells. Different two domain KIRs (KIR2Ds) recognise distinct subsets of HLA-C alleles. SPR analysis showed that the ectodomain of
KIR2DL3 exhibited the allele- and peptide-specific recognition to HLA-Cw7
with fast kinetics (an affinity of -1O-6M ) and a favourable binding entropy,
similar to other cell-cell recognition_ Although the T cell receptors (TCRs) and
KIRs both show allele- and peptide-specific MHC recognition, the TCR-MHC
interactions has slow kinetics and alarge unfavourable binding entropy, whichis in contrast with those ofKIR-MHC interactions.
On the other hand, Fc y receptors (Fc y R) are expressed on immunologically
active cells and bind the Fc portion of IgG and are besides other functions
responsible for the clearance of immune complexes. SPR analysis for the interactions of the ectodomain of all human low-affinity FcyRs (Fc y RIIa, Fc y
RIlb and Fc y RIll), which consist oftwo Ig-like domains, toward Fc showed
the fast kinetics with similar low affinity (-1O-6M) as observed in other cellsurface receptors including KIRs. These observations suggest that Fc y Rs
recognize the immune complex in similar way of cell-cell interactions of cellsurface receptors in terms of kinetics.

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We further determined the crystal structure of human MHC class I molecule,
HLA-B51, which is a ligand for KIR3DL1. The HLA-B51 structure clearly
exhibited the electrostatic differences on the predicted KIR binding site fromother MHC class I molecules, suggesting that these electrostatic differences
may determine the specificity ofKIR binding.
This work has been done in collaboration with Yvonne Jones, David Stuart,
Taeko Maenaka, Anton van der Merwe (University of Oxford), Peter
Sondermann (Max' Planck, Institut fur Biochemie, Martinsried) and Izumi
Kumagai, Kouhei Tsumoto, Mitsunori Shiroishi (Tohoku University).

(7) Crystallization of Na+-transloeating ATPase
Yasuo SHlRAKIHARA
Na+-translocating ATPase from Enterococcus hirae is classified as a Vacuo
olar-type ATPase (V-type), and is expected to have a structure similar to F1ATPase (F-type ATPase) from amino acid sequence comparison. Na+-translocating ATPase was highly purified and was subjected to a crystallization experiment. The crystals were formed by the crystallization condition using 27%
PEG4000, 0.2 M LiS0 4 , 10 mM Tris'HCi, pH 7.5, 10% glycerol. In this condition, we obtained several crystal forms, plate'like, coffine-like, cubic, and needlelike forms. High resolution was obtained using the plate-like crystals. The
symmetry was C 2 , the lattice constants were 241.6, 141.1,239.1 A, and theresolution was 2.4 A. We are now analysing the diffraction pattern. This work
has been done in collaboration with Toshiaki Hosaka, Toshiyuki Meguro andIchiro Yamato, Science University of Tokyo.

(8) Crystallization of D-aminoaeylase from AJea/lgenes
Yasuo SHlRAKIHARA
D-aminoacylase catalyzes hydrolysis oh N-acylderivatives of various neutral
D-amino acids to D-amino acids and fatty acids. The enzyme is expected tohave a unique active site structure that distinguishes from abundant L-amino

152

acids, but also to have industrial relevance with a potential to produce large
amount of D-amino acids. The enzyme from Alcaligenes xylosoxydans subsp.
xylosoxydans A -6 has been crystallized from MPD or isopropanol solutions,but the crystallization was not reproducible due to varied purity of the preparations. This year, after a solid preparation procedure having been established, the crystallization condition search has been repeated with grid screen
and crystal screen from Hampton.
A related enzyme, glutaminase from a marine bacterium Micrococcus luteus
K-B, has been crystallized from a citrate solution. The crystals diffracted to atleast 3 A resolution, and had a hexagonal lattice with a=b=110.5 A, c=210.5 A.
The crystallization study was done in collaboration with Mituaki Moriguchi,
Akiko Sato and Panuwan Chantawannakul at Oita University, and Mamoru
Wakayama, at Ritsumeikan University.

(9) X-ray crystallographic analysis of the anti-tumor enzyme, argininedelmlnase from Mycoplasma arglnl
Katsumi MAENAKA and Yasuo SHlRAKIHARA
Arginie deiminase (AD) catalyzes the hydrolysis of L-arginine to L-citrulline
and ammonia, and is involved in the arginine metabolism pathway in various
microorganisms, which is the main energy source for mycoplasma. The recombinant Mycoplasma argini arginine deiminase showed a strong inhibition of
the growth of various kinds of human and mouse tumor cells by depleting Larginine. We are trying to address the structural basis of the ariginine hydrolysis mechanism ofAD, which could provide useful information to improve
the AD function as a potential anti-tumor drug.
Previously it was established that the crystals of the recombinant AD obtained from a solution containing PEG6000 and NaCI, diffracted to 3.0 A
resolution. Using the currently available preparations, crystallization conditions were again searched employing Crystal Screen I (Hampton research), tofind the condition of 25% PEG6000. 0.2M Ammonium Acetate, O.lM Tris-CI(pHS) at 297K. AD has also been crystallized in this condition but with added
20% glycerol, which is suitable for cryo-experiment. The crystals diffracted toH.

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3.oA resolution at Spring8. Preliminary characterization of the crystals indicated a PI space group. The heavy atom derivative search is being performed.
This work has been done in collaboration with Satoru Misawa, Japan Energy
Corporation.
Pub I i ca t ions
1.

SAMATEY. F., IMADA, K, VONDERVISZT, F., SHIRAKIHARA, Y. and NAMABA, K: Crystallizationof the F41 fragment of Flagellin and data collection from extremely thin crystals.
Journal of Structural Biology, 132, 106-111,2000.

2.

MAENAKA, K, MAENAKA, T., TOMIYAMA, H., TAKIGUCHI, M., STUART, D. I. and JONES. E. Y.:
Nonstandard peptide binding revealed by crystal structures ofHLA- B*5101 complexedwith HIV immunodominant epitopes. J. Immunol. 165, 3260-3267. 2000.

3.

GAO, G. F., WILLCOX, B. E., WYER, J. R., BOULTER, J. M., O'CALLAGHAN, C. A., MAENAKA,

K, STUART, D. I., JONES, E. Y, van der MERWE, P. A., BELL, J. I. and JAKOBSEN, B. K:
Classical and nonclassical class I major histocompatibility complex molecules exhibit
subtle conformational differences that affect binding to CD8 a a , J Bioi Chern. 275,
15232-15238, 2000.

H-e.

Gene Network Laboratory

Nucleocytoplasmic exchange of macromolecules
Naoko lMAMOTO
Nucleocytoplasmic exchange is a very dynamic activity, in which vast num'
ber of molecules enter and exit the nucleus in a rapid, accurate, and often
regulated manner. This exchange of molecules is important, in order for cells
to maintain their homeostasis, and adapt to their extracellular environment.
Since the identification of the first transport factor, referred to as importin aand {3, which mediates nuclear import of classical basic nuclear localization
signal (NLS)'containing substrates, significant progress has been achieved
toward our understanding of the mechanism of nucleocytoplasmic transport,
as well as the diversity of nucleocytoplasmic transport pathways. The pres'ence of so many transport pathways obliges us to raise a naive but fundamental question: What is the benefit to having such a complexity of nuclear trans'

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port pathways leading to regulatory changes of gene expression in vivo? Inorder to understand basic mechanism of transport and biological significanceof diversity of transport pathways, our present effort focuses on the understanding ofthe function of nuclear pore complex, and identification of transport pathways and factors that function under different cellular conditions.

(1) Single molecule Imaging of nucleocytoplasmic transport
at nuclear pores
Naoko IMAMOTO and Makio TOKUNAGA' (INational Institute of Genetics, Biological Macromolecules Laboratory)
A central mediator of nucleocytoplasmic traffic is the nuclear pore complex(NPC), a large supramolecular structure of protein complex consisting of about
50-100 different polypeptides with an estimated molecular mass of 125
megadaltons in vertebrates. More than one million macromolecules are estimated to pass through the nuclear envelope every minute in dividing cells. In
contrast to our growing knowledge of soluble factors involved in transport,function of nuclear pore complex (NPC), as well as mechanism of translocation
step of transport is very poorly understood.
As one approach to investigate the function of NPC, we are trying to visualize
behavior of transport factors and transport substrates at NPC, using modified objective-type total internal reflection fluorescence microscopy (TIRFM).
This method allows us single molecule imaging in vivo. In the digitonin
permeabilized cell-free transport assay, we examined a behavior of green fluorescent (GFP)-tagged importin {3. Through the imaging of single nuclear porecomplexes, and visualizing single GFP-importin {3 molecules at NPCs duringtransport, a quantitative information on kinetics of NPC-translocation ofimportin {3 can be obtained.

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155(2) Analysis of crystal structure of the uncomplexed
form of importin {3Soo Jae LEE!, Naoko IMAMOTO, Hiroaki SAKAI 1, Atsushi NAKAGAWA 1, Shingo
KOSE, Makiko KOIKE, Masaki YAMAMOTO'l, Takashi KUMASAKA 3 , Yoshihiro
YONEDA2and Tomitake TSUKIHARA' ('Institute for Protein Research, Osaka University, 2Department of Cell Biology and Neuroscience, Graduate School of
Medicine, Osaka University, :JThe Institute of Physical and Chemical Research(RIKEN»
Importin {3 is a nuclear transport factor which mediates the nuclear importof various nuclear proteins. The N-terminaI1-449 residue fragment of mouse
importin {3 (imp {3 449) possesses the ability to bidirectionally translocate
through the nuclear pore complex (NPC), and to bind RanGTP. The structure of
imp {3 449 has been solved at a 2.6 A resolution by X-ray crystallography. It
consists often copies ofthe tandemly arrayed HEAT repeat and exhibits conformational flexibility, which is involved in protein-protein interaction for
nuclear transport. The overall conformation of the HEAT repeats shows thata twisted motion produces a significantly varied superhelical architecture fromthe previously reported structure of RanGTP-bound importin {3 . These conformational changes appear to be the sum of small conformational changes
throughout the polypeptide. Such a flexibility, which resides in the stacked
HEAT repeats, is essential for interaction with RanGTP or with NPCs. Furthermore, it was found that imp {3 449 has a structural similarity with another nuclear migrating protein, namely {3 -catenin, which is composed of another type of helix-repeated structure of ARM repeats. Interestingly, the essential regions for NPC translocation for both importin {3 and {3 -catenin are
spatially well overlapped with one another. This strongly indicates the importance of helix stacking of the HEAT or ARM repeats for NPC-passage (seepublications 1 and 2).

(3) Mechanism of nucleocytoplasmic translocation of importin- {3Hitomi OZAWA 1, Shingo KOSE, Jun KATAHIRA 1, Taro TACHIBANA 1, Miki HIEDA',
Hiroaki SAKAI", Tomitake TSUKlHARA", Naoko IMAMOTO and Yoshihiro YONEDA 1

156(lDepartment of Cell Biology and Neuroscience, Graduate School of Medicine,
Osaka University, 2Division of Protein Crystallography, Institute for Protein
Research, Osaka University)
Importin t3 is a nuclear transport factor which mediates the nuclear importof various nuclear proteins. We have found that importin t3 has an intrinsic
activity to translocate through the nuclear pore complex (NPC), and this activity requires interactions with nucleoporins.
To understand the underlying mechanism of nucleocytoplasmic migration ofimportin t3 , we made various importin t3 mutants, based on crystal structureof uncomplexed form of importin t3 . These mutants harbor amino acid substitutions within the NPC binding domain, which we have shown to be necessary
and sufficient to exhibit the translocational activity of importin t3 through
NPC. By transient transfection experiments, we observed that a mutant showedinefficient accumulation to the nuclear envelope and the nucleoplasm, while
the other localized at the nucleus more efficiently than wild type.
The activities of these mutants to translocate through NPC, as well as theirabilities to support nuclear import of NLS-bearing substrates and the interaction with various nucleoporins are now under investigation.

(4) Analysis of nuclear export of importin 8 through nuclear pores
Shingo KOSE, Yoshihiro YONEDA 1 and Naoko IMAMOTO. (IDepartment of Cell
Biology and Neuroscience, Graduate School of Medicine, Osaka University)
Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC).
Small molecules «20-40 kDa) move passively through the NPC, but larger
molecules are selectively transported by nucleocytoplasmic shuttling receptors of the importin t3 family. We have previously reported that importin t3 ,which functions as a carrier of classical nuclear localization signal (NLS)mediated nuclear import, shuttles rapidly between the nucleus and the cytoplasm by direct interaction between importin t3 and components of the NPC(nucleoporins). However, the mechanism for a directional movement ofimportin
t3 at the NPC remains unclear.

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In chilled or ATP"depleted cells, importin 13 was transported into the nucleus,
while the nuclear export of importin 13 was inhibited. Further, it was foundthat the nuclear export inhibition of importin 13 is not due to nuclear retention
via binding to nucleoporins or nuclear importin a:. These data show that thenuclear export of importin 13 involves energy"requiring step(s) in living cells.
In vitro transport assay revealed that an energy"dependent nuclear export ofimportin 13 requires soluble factors. To address the issue of how the energy isutilized during the NPC-passage of importin 13 , we have now attempted topurify an energy"dependent nuclear export factor of importin 13 from mouse
Ehrlich cytosolic extract.

(5) Analysis of nuclear export of (3 -catenin
Makiko KOIKE and Naoko IMAMOTO

13 "catenin plays a key role in Wnt signaling pathway. Upon activation, 13"catenin accumulates and enters the nucleus, where it modulates the activa"tion of the target gene expression. Therefore, it is important to understand themechanism of nucleocytoplasmic transport of 13 "catenin. Our previous stud"
ies indicate that 13 "catenin can enter the nucleus on its own, and shuttles
between the cytoplasm and nucleus. The export of 13 "catenin examined inliving cells showed that this protein exit the nucleus in CRM1"independent
and Ran"independent manner. Moreover, we found that deletion mutant con"taining Arm repeats 10"12 and C"terminus of 13 "catenin efficiently entered
the nucleus on its own like full length 13 "catenin, and that this region is also
necessary and sufficient for 13 -catenin to exit the nucleus. In order to investi"
gate export of 13 "catenin, we examined the export of 13 "catenin using digitonin"permeabilized cell-free transport assay. We found that 13 "catenin arecapable to exit the nucleus on its own, but export of 13 "catenin are often regu"
lated by factor(s) present in cytosol in the presence ofATP. A nature of factor
that affect 13 "catenin export is now under investigation.

158(6) Stress mediated nuclear import of 70kDa heat shock protein
Maiko FURUTA), Shingo KOSE and Naoko IMAMOTO ('Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University)
Heat-shock induces a strong stress response that drastically alter gene expression in cells. Heat-shock response may provide us an ideal system to
examine whether specific transport pathway or transport factors function under different cellular conditions. To start with, we are examining heat-shockstress-mediated nuclear import of 70kDa heat-shock protein (hsp70/hsc70).
Both hsp70 and hsc70 efficiently migrates into the nucleus under heat"shockcondition, but this protein is largely cytoplasmic under normal condition.
In order to elucidate a mechanism of stress-mediated nuclear import ofhsp701
hsc70, we have developed transport assay to examine stress"mediated nuclear
import of hsp70/hsc70 both in living mammalian cells and in the in vitro
system using digitonin"permeabilized cells. Our present results indicate that
nuclear accumulation ofhsc70 requires soluble factors andATP, and its nuclear
accumulation is importin 0: I {3 or transportin independent. Crude cytosolic
extracts, for example, prepared from Ehrlich ascites tumor cells or Xenopus
oocytes that efficiently support nuclear import of classical NLS-containing
substrates does not support nuclear accumulation ofhsc70 efficiently, indicating that some unique mechanism is involved in stress-mediated nuclear import hsc70.

Publications
1.

IMAMOTo, N: Diversity in nucleocytoplasmic transport pathways (Review) Cell Structure and Function, l!5, 207-216 (2000)

2.

LEE, S.J., IMAMOTO, N., SAKAI, R., NAKAGAWA, A, KOSE, S., KOIKE, M., YAMAMOTO, M.,
KUMASAKA, 1., YONEIJA, Y and TSUKIHARA, 1.: *equally contributed authors: The adoption of a twisted structure of importin -{3 is essential for the protein-protein interaction
required for nuclear transport. J. Mol. BioI., 30l!, 251-264 (2000).

3.

TACHIBANA, T., RIEIJA, M., MIYAMOTO, Y, KOSE, S., IMAMoTo, N. and YONEDA, Y.: Recyclingof importin a from the nucleus is suppressed by loss of RCC1 function in living
mammalian cells. Cell Struct. Funct. l!5: 115-123 (2000).

4.

MATSUIJAE, M., KURIHARA, 1., TACHIBANA, 1., IMAMOTO, N. and YONEDA, Y: Characteriza-

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tion of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein.
FEBS Lett., 468, 171-175 (2000).
HARAGUCIII, T., KOUJIN, T, KANEDA, T, TSUTUMI, C., IMAMoTo, N., AKAZAWA, C., SUKEf;AWA,J., YONEDA, Y. and HIRAOKA, Y: Reformation of the functional nuclear envelope during
telophase: Temporal sequences determined by fluorescence imaging in living HeLa
cells. J. Cell Sci., 113, 779-794 (2000).
6. 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 ofa Novel Importina Homologue from Rice, by which constitutive photo morphogenic 1 (COP1) nuclear
localization signal (NLS)-protein is preferentially nuclear imported. J. BioI. Chern. 276,9322-9329 (2001).
7. KURISAKI, A., KOSE, S., YONEDA, Y., HELDIN, C.H. and MOUSTAKAS, A.: Transforming
growth factor- 13 induces nuclear import of Smad3 in an importin- 13 1 and Ran"
dependent manner. Mol. BioI. Cell,. in press.

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Center for Information Biology

I-a.

Laboratory for DNA Data Analysis(1) In sllJeo chromosome staining: Reconstruction of Glemsa bands
from the whole human genome sequence
Yoshihito NIIMURA and Takashi GOJOBORI
Giemsa staining has been used for identifying individual human chromosomes for over 30 years, though the molecular basis of these banding patterns
has remained elusive. The banding patterns of Giemsa-dark (G) and Giemsalight (R) bands are known to reflect the regional differences in chromatin structures and functions. In this study, we developed an "in silica staining" method
and reconstructed Giemsa bands computationally from the draft genome sequence of human. We found that G bands are the regions where the GC content
is lower than that ofthe flanking regions. By a two-window analysis, we demonstrate that Giemsa bands are strongly correlated with the difference in GC
content between windows of 2.5 Mb and 9.3 Mb for almost all of 43 chromosomal arms. Our findings suggest that isochore structures, the large-scalevariation in GC content, has evolved by a selective pressure towards AT-richness in G-band regions to maintain compact chromatin structures.
(~)

The evolutionary origin of brain structure characterized by gene
expression profile: A cytoarchitectonic map of the planarlan
brain defined by cDNA mlcroarray

Masumi NAKAZAWA, Francesc CEBRIA 1,2, Katsuhiko MINETA, Kazuho IKEO,
Kiyokazu AGATA 1,2 and Takashi GOJOBORI (lDepartment of Biology, Faculty of
Science, Okayama University, 2RIKEN Center for Developmental Biology)
The origin of the brain remains a challenging problem in evolutionary studies. To understand when and how the structural and functional brain emerged,

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we analyzed the CNS of a lower invertebrate, planarian. We conducted alarge-scale screening ofthe head part-specific genes in the planarian by constructing a eDNA microarray. We created a microarray of 1,640 nonredundantgenes, randomly chosen from 9,000 EST sequences. Competitive hybridization of cDNAs between a head portion and the other body portion of planarians
revealed 205 genes with head part-specific spikes, including homologues ofsynapsin, 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 planarian brain has undergone functional
regionalization. Here, we demonstrate the complex cytoarchitecture of the pIa·
narian brain, despite its simple superficiality of the morphology. These data
indicate that a highly organized brain had already emerged by the time ofplanarian divergence.

(3) SNP profiles of the human subgenomie regions
Michihiro OGASAWARA, Silvana GAUDIERI l , Tadashi IMANISHI and Takashi
GOJOBORI (IThe University of Western Australia)
To investigate frequencies and distributions of single-nucleotide polymorphisms (SNPs) in the human genome, we have previously constructed a SNP
profile ofthe MHC region, showing that nucleotide diversities between different haplotypes at non-HLA sites were unexpectedly high (>10%) (Gaudieri, S.
et al. (2000) Genome Research 10: 1579·1586), up to 80 times greater thanelsewhere in the genome (0.08-0.2%). In the present study, we extended ouranalysis of SNP profiles to other regions of the human genome. And we reported characteristic features of SNP profiles in several subgenomic regions,such as the beta-globin region and others in the chromosomes 7, X and Y, bycomparing between two different haplotype sequences chosen from the public
databases. In particular, we found a total of 523 SNPs in 596.62kb surveyed(one SNP in 1141 nucleotides on average, namely 0.088%)when the MHC region was excluded. The mean frequency ofSNPs obtained in our study is lower
than those of other previous reports (Wang, D.G. et a1. (1998) Science 280:
1077-1082; Yamada, R. et al. (2000) Hum Genet 106: 293-297). And our obser-

162

vations indicate that the skewness and complexity of SNP profiles depend
heavily upon subgenomic regions.

(4) Study of molecular evolution in vsx gene families
Yoshiko UCHIYAMA, Kazuho IKEO and Takashi GOJOBORI

Vsx genes are involved in the differentiation of retina, spinal cord, and nervous system of vertebrates. It is known that vertebrate vsxgenes are classified
into two subtypes, vsx 1 and vsx 2. Vsxgenes have two domains, a paired-like
homeodomain and a typical CVC domain. The relationship between these
domains and the restricted expression patterns is unclear from the viewpointof molecular evolution. Thus, we focus on the evolution of vsx genes having
pairedlike homeodomains. In this study, we performed evolutionary phyloge"
netic analysis by using 45 paired-like genes including vsxgenes. It revealed
that the duplication of vsxgenes into two subtypes had occurred specifically inthe lineage of vertebrates. It also showed that the duplication occurred about
600 million years ago, the appearance time of vertebrates. This result suggests that vsx genes would have played an important role for the formation of
spinal cord. More detailed studies on the molecular evolution of vsx genes isconducted using experimental approaches.

(5) Evolutionary features of the gene expression profile ofplanarian brain
Katsuhiko MINETA, Kazuho IKEO and Takashi GOJOBORI
Planarian is considered to possess the primitive central nervous system(CNS). Therefore, planarian can be a useful model for understanding the evo·lutionary process of the CNS. Our purpose of the present study is to identify
the planarian genes related to CNS, to observe their expression profiles, andto compare them with those of other species. We sequenced over 8,000 redundant expressed sequence tag (EST) clones from the eDNA library derived fromthe head portions ofplanarians (Dugesiajaponica). As a result, we obtained atotal of3,101 non-redundant EST clones. According to a frequency analysis,

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77% of 3, 10 1 clones appeared only once. This means that most of genes wererarely expressed. Conducting the homology search, we found that 45% of 3, 101
clones had significant similarities with proteins whose functions were known.
Among these genes, we found that at least 71 genes were involved in CNS.
Comparing these 71 planarian genes with the genes of human, mouse, fruit fry
and nematode, we found that 68 genes were well"conserved and 3 genes were
not conserved very much. This feature of gene conservation can be considered
as a reflection of the diversification of CNS during evolution, supporting ouridea that a comparative approach based on the planarian ESTs is useful for
elucidating the evolutionary process of CNS.

(6) Establishment of Integrated human microsatellite database
Takaho ENDO and Takashi GOJOBORI
We have established microsatellite database of human genomic sequences.
We cooperate with medical laboratory ofTokai University (Dr. Inoko) and madecomputer programs which find and analyze microsatellite regions in humanchromosomes.
Microsatellites are expected as genetic markers to detect disease candidate
genes because of their high polymorphism. In isolating disease genes, wewould conduct association analysis with many polymorphic markers such as
single nucleotide polymorphisms (SNPs), variable number tandem repeat(VNTR), and microsatellites. Microsatellites are known as highly polymorphic region in the repeat number and show higher linkage (about 100kb) than
SNPs (SNPs is thought to be associated in the range of 10kb).
We have acquired microsatellite markers by some experimental methods Gn
wet laboratory) so far. Today we can utilize the database from human genome
project and we have scanned all microsatellites in it. Our database integrates
the primer designing program and it provides specific markers (avoiding reo
petitive sequences) suit for various experimental conditions. We have con"structed more than 10,000 polymorphic markers through my database and webelieve they should become useful tool for isolation of disease genes in the
genome-era.

164(7) Higher evolutionary rates in the contact regions between
catecohlamine receptors and G proteins
Hisakazu IWAMA and Takashi GOJOBORIThe catecholamine (CA) neurotransmission was recently reported to playa
central role of the emotional control in humans. The CAs are the bioaminesthat are constituted of dopamine and adrenalin. The receptors (Rcs) of CAs
are G protein (GP)-coupled Rcs (GPCRs). In particular, the function of GP_
subunit (G-l works as a connecter between an outer ligand-Rc system and asystem of inner second messenger pathways (SMPs).
In order to understand how functionally important those receptors are, we
examined the evolutionary conservation of the receptor genes involved with
the CA neurotransmission system. First, we made alignments of amino acidsequences of CA Rc and G-O separately. For the alignment of CA Rc, we divided
it into two parts; ligand-binding and GP-contact regions. For the alignment of
G_, we also divided it into two parts; the Rc-contact region and the switch
region in which the SMP is activated. The rate U of amino acid substitutions
was computed for each of the four regions.
As a result, we found that each of the four regions showed different values of_; for the ligand-binding region, 1.01 _ 0.01 substitutions/year/site, for GPcontact region 1.26 _ 0.09, for Rc-contact region 1.18 _ 0.03, and for the switch
region 0.68 _ 0.02. We also found that the difference in those substitution
rates was statistically significant based on Welch's test. Thus, we concluded
that the GP-contact and Rc-contact regions have substitution rates higher
than those of both ligand- Rc and the switch regions, implying that the connecting regions of the two systems have evolved under less functional constraints.

(8) Does apoptosls play an active role In the regenerationof planarian?
Jung Shan HWANG and Takashi GOJOBORIRegeneration, a mechanism for repairing or replacing damaged or lost bodyparts. It is found in two primitive organisms, hydra and planarian, but re-1.

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stricted in higher organisms. Vertebrate regeneration is merely found in amphibian limbs and lens, mammalian liver and muscle tissue. This raises thequestions of why the regenerative ability is limited only to some primitive,
selected organisms and further that in what extent the evolution lead to theloss of regeneration capability? We used the freshwater planarian, which be"longs to the platyhelminthes family and possess three tissue layers, bilateralsymmetrical body plan as a model to study molecular mechanisms and evolu"tion significance underlying its remarkable regenerative ability. One of thephysiological mechanisms that likely takes part in the regeneration of planar"
ian is the apoptosis since one of its central role is to regulate the precisedevelopment of cell function and cell population.
Five caspase homologs have been isolated from the cDNA library of planarian and the homology search of their ORFs has been carried out using the
BLAST programs. Among the caspase family, caspase 3 has the highest ho·mology to all the isolated clones, followed by caspases 7 and 8. Sequencealignment of each amino acid sequence was generated using the CLUSTAL X
multiple alignment program. Alignments indicated that all clones containcaspase conserved sequence and a very short amino"terminal domain. Fur"thermore, Northern Blotting has been performed to one of the clone and theresult showed that its mRNA level decreases during the regeneration. It seems
that apoptosis probably plays a role in the regenerative process of planarian,however further evidence is required to ensure the Northern blot result and to
determine the role of apoptotic cascade in the processes of reproducing thewhole body, injury and wound healing.

(9) The base compositional localisation of genes in thehuman genome sequence
T. Daniel Andrews and Takashi GOJOBORI
We have analysed the distribution of genes within the finished portion of thehuman genome sequence to look for correlations between the presence of genes
and the base composition of the isochore in which they reside. A window analysis was employed to divide the approximately 650Mb of finished non-redun"

166

dant human genome sequence into contiguous 100Kb regions in which basecomposition was calculated. A new partial annotation method that uses non'redundant EST or cDNA sequence information was then applied to gain rela'tive measures of the density of coding sequences within each window. Hence,
the relationship between gene density and isochore base composition was
directly appraised. Previous studies have found high concentrations of genes
in the highest G+C regions ofthe human genome, but we have found that thegene distribution is actually far more uniform. While genes are rare in themost A+T rich regions of the genome, there is comparatively less than a fourfold enrichment of genes in the G+C richest isochores. Our findings predictthat only around 26% of all genes are to be found in the approximately 12% of
the human genome that represents the highest G+C isochore families. These
results are supported by the recently published full draft ofthe human genome
sequence obtained by the public genome sequencing effort, and by the sequence
analysis conducted by Celera Genomics. These findings have strong implications for our current understanding of the processes of isochore formation in
warm-blooded vertebrates.

Publ ications
1. TOYODA, R, SATO, S., IKEO, K, GOJOBORI, T., NUMAKUNAI, T., GODING, C.R and YAMAMOTO,
H. (2000) Pigment cell-specific expression of the tyrosinase gene in ascidians has adifferent regulatory mechanism from vertebrates. GENEZ59(1-2): 159-170. (2000)
2.

SHIMIZU, N. and GOJOBORI, T. (2000) How can human and simian immunodeficiency
viruses utilize chemokine receptors as their coreceptors? GENE 259(1 '2): 199-205.
(2000)

3.

OTA, K, KOBAYASHI, T., UENO, K and GOJOBORI, T. (2000) Evolution of heteromorphic
sex chromosomes in the order Aulopiformes. GENE 259(1 -2): 25-30. (2000)

4.

IWATA, A, KOBAYASHI, T., IKEO, K, IMANISIII, T., ONO, H., UMEHARA, Y., HAMAMATSU, C.,
SUGIYAMA, K, IKEDA, Y, SAKAMOTO, K, FUMIHITO, A, OHNO, S. and GOJOBORI, T.: Evolu'
tionary aspects of gobioid fishes based upon a phylogenetic analysis of mitochondrial
cytochrome b genes. GENEZ59(1-2): 5-15. (2000)

5.
6.

GOJOBORI, T. and OHNO, S.: 1928'2000. GENEZ59(1-2): 2-3. (2000)
NAKANE, S., SHIRABE, S., MORIUCIII, R, MIZOKAMI, A, FURUYA, T., NISIIIURA, Y, OKAZAKI,S., NAKAMURA, N., SUZUKI, Y, NAKAMURA, T., KATAMINE, S. and GOJOBORI, T.: Comparative molecular analysis of HTLV- I proviral DNA in HTLV- I infected members of a familywith a discordant HTLV·I associated myelopathy in monozygotic twins. Journal of1.

CENTER FOR INFORMATION BIOLOGY167

Neuro Virology. 6: 275-283. (2000)
7. GAUDIERI, S., DAWKINS, R L., HABARA, K, KULSKI, J.K and GOJOBORI, T.: SNP profile
within the human major histocompatibility complex reveals an extreme and inter'
rupted level of nucleotide diversity. Genome Research. 10(10): 1579-1586. (2000)
8. SEKI, M., HI<iASHIYAMA, Y, MlZOKAMI, A., KADOTA, J.-1., MORIUCHI, R, KOIINO, S., SUZIKI,
Y, TAKAHASHI, K, GO.IOBORI, T. and KATAMINE, S.: Up-regulation of human T lymphotropic
virus type 1 (HTLV- 1) tax/rex mRNA in infected lung tissues. Clin Exp Immunol.
120: 488-498. (2000)
9. SUZUKI, Y, YAMAGUCIIl'KABATA, Y and GO.IOBORI, T.: Nucleotide substitution rates of
mV-I. AIDS Reviews. 2(l): 39-47. (2000)
10. YAMAGUCHI- KABATA, Y and GOJOBORI, T. (2000) Reevaluation of amino acid variabilityof the human immunodeficiency virus type 1 gpl20 envelope glycoprotein and prediction of new discontinuous epitopes. J Virology. 74(9): 4335-4350 (2000)
11. PINEDA, D., GONZALEZ, J., CALLAERTS, P, IKEO, K, GEHRING, WJ. and SALO, E.: Searchingfor the prototypic eye genetic network: Sine oculis is essential for eye regeneration inplanarians. Proc Natl Acad Sci USA. 97(9): 4525-9. (2000)

I-b.

Laboratory for Gene-Product Informatics(1) Structural/functional assignment of bacteriophage T4 unknown
proteins by iterative database searches
Takeshi KAWABATA, FumioARISAKA' and Ken NISHIKAWA ('Department of Mo'
lecular and Cellular Assembly, Tokyo Institute of Technology)
Among the total of 274 orfs within bacteriophage T4, only half have beenreasonably well characterized, and the functions of the rest have remained
obscure. In order to predict the molecular functions of the orfs, a position'
specific iterated (PSI)· BLAST search of bacteriophage T4 against the sequence
database of known 3D structures was carried out. PSI·BLAST is one of themost powerful iterative sequence search methods using multiple sequencealignment, with the ability to detect many more proteins with distant homol'
ogy than standard pairwise methods. The 3D structures of proteins are considered to be better preserved than the sequences, and the detected distantly
homologous proteins are likely to possess highly similar 3D structures. Thirteen orfs of phage T4, whose homologues were not detected by standard pairwise
methods, were found to have significantly homologous counterparts by this168

method. The plausibility of the results was confirmed by checking whether
important residues at substrate/ligand-binding sites were conserved. Amongthem, two orfs, vs.1 and e.1, which are similar to Escherichia colilytic enzyme
and Mut Tprotein, respectively, had not been studied previously. Also, gp rIIA,
a rapid lysis protein, whose gene structure had been intensively studied during the development of molecular biology in the 1950s and yet whose molecular function remains unknown, has an N-terminal domain that is significantly
similar to the N-terminal region of the heat shock protein Hsp90. See Ref. 1
for the details.
(!) Protein structure comparison using the Markov transition

matrix of evolution
Takeshi KAWABATA and Ken NISHIKAWA
A number of automatic protein structure comparison methods have been
proposed; however, their similarity score functions are often decided by the
researchers intuition and trial-and-error, and not by theoretical background.
We propose a novel theory to evaluate protein structure similarity, which isbased on the Markov transition model of evolution. Our similarity score between structures i and j is defined as log P{j ...... I}/P(j), where P(j ...... i) is theprobability that structure j changes to structure i during the evolutionary
process, and P(j)is the probability that structure i appears by chance, this is areasonable definition of structure similarity, especially for finding evolutionarily related (homologous) similarity. The probability P(j...... iJis estimated by
the Markov transition model, which is similar to the Dayhoff's substitution
model between amino acids. To estimate the parameters of the model, homologous protein structure pairs are collected using sequence similarity, andthe numbers of structure transitions within the pairs are counted. Next thosenumbers are transformed to a transition probability matrix of the Markov
transition. Transition probabilities for longer time are obtained by multiply·
ing the probability matrix by itself several times. In this study, we generated
three types of structure similarity scores: an environment score, a residueresidue distance score, and a secondary structure elements (SSE) score. UsingI.

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these score, we developed the structure comparison program, MATRAS(MArkovian TRAnsition of protein Structure). It employs a hierarchical alignment algorithm, in which a rough alignment is first obtained by SSEs, andthen is improved with more detailed functions. We attempted an all-versusall comparison of the SCOP database, and evaluated its ability to recognize asuperfamily relationship, which was manually assigned to be homologous in
the SCOP database. A comparison with the FSSP database shows that ourprogram can recognize more homologous similarity than FSSP. We also discuss the reliability of our method, by studying the disagreement between structural classifications by MATRAS and SCOP. See Ref. 2 for the details.

(3) Redesign of artificial globlns: Effects of residue replacements athydrophobic sites on the structural properties
Yasuhiro ISOGAI 1, Anna ISHII2, Tetsuo FuJISAWA3, Motonori OTA and Ken NISHIKAWA(IRIKEN at Wako, 2Gakushuin Univ., 3RIKEN Harima InstJ
Artifical sequences of the 153 amino acids have been designed to fit themain-chain framework of the sperm whale myoglobin (Mb) structure based ona knowledge-based 3D-1D compatibility method. The previously designed artificial globin (DG 1) folded into a monomeric, compact, highly helical and globular from with overall dimensions similar to those ofthe target structure, but itlacked structural uniqueness at the side-chain level [Isogai et aI, (1999) Bio·chemistry 38, 7431-7443]. In this study, we redesigned hydrophobic sites of
DG1 to improve the structural specificity. Several Leu and Met residues in
DG1 were replaced with 13 -branches amino acids, He and Val, referring to the3D profile of DG 1 to produce three redesigned globins, DG2-4. These residue
replacements resulted in no significant changes of their compactness and ahelical contents in the absence of denaturant, whereas they significantly affected the dependence ofthe secondary structure on the concentration of guanidine hydrochloride. The analyses of the denaturation curves revealed higher
global stabilities of the designed globins than that of natural apoMb. Among
DG 1-4, DG3, in which 11 Leu residues ofDG1 and replaced with seven He andfour Val residues, and one Met residue is replaced with Val, displayed the170lowest stability but the most cooperative folding-unfolding transition and themost dispersed NMR spectrum with the smallest line width. The present
results indicate that the replacements of Leu (Met) with the {3 -branchedamino acids at appropriate sites reduce the freedom of side-chain conformation and improve the structural specificity at the expense of stability. See
Refs. 3 and 4 for the details.

(4) The genomic DNA sequences of various species are distinctively
distributed in nucleotide composition space
Hiroshi NAKASHIMA l and Ken NISHIKAWA (lKanazawa University)
The composition of di-, tri- through to hexa-nucleotides was analyzed in genomic sequence data from 18 organisms, including the complete genomes of 13
microorganisms, two complete chromosomes from protozoa and partial genome sequences from humans, Arabidopsis thaliana and Caenorhabditis
elegans. Double-stranded DNA from each genome was chopped into fragmentsof a fixed length, and the oligonucleotide composition of these fragments was
examined in multi-dimensional composition space, defined by axes corresponding to individual oligonucleotide components. Each DNA fragment was plottedat one point n this composition space. When DNA fragments of several different organisms were plotted at once, they clustered separately as clearly shownby a projection on a two dimensional plane. We examined the extent to which
DNA fragments from the 18 organisms could be correctly assigned to a species
purely in terms of nucleotide composition. The species discrimination accu"racy increased not only as the length of oligonucleotide was increased from dito hexa "nucleotide, but also as the length of fragmented DNA was increased
from 0.1 to 10 kbp. Using the symmetrical trinucleotide frequencies, 82% of
43,045 DNA fragments of 1 kbp long from 18 organisms could be correctly
identified. This accuracy rose to 96% when DNA length was increased to 10
kbp. These results indicated that the characteristic nucleotide compositions
specific to individual organisms exist throughout the genome. In addition, thecodon"usage frequencies of protein-encoding sequences (i.e, genes) were com"pared with the trinucleotide frequencies of non "coding part of a genome. The

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results strongly suggest that the well-known, species-specific codon usage bias
might be caused by the same factor that maintains a characteristic nucleotide
composition for the entire genome of each organism. See Ref. 5 for the details.

(5) Physico-chemical evaluation of protein folds predictedby threading
Akira KINJO and Ken NISHIKAWA
Protein structure prediction remains an unsolved problem. Since predictionof the native structure seems very difficult, one usually tries to predict thecorrect fold of a protein. Here the fold is defined by the approximate backbone
structure of the protein. However, physicochemical factors that determine thecorrect fold are not well understood. It has recently been reported that molecu"
lar mechanics energy functions combined with effective solvent terms can discriminate the native structures from misfolded ones. Using such a physico"chemical energy function, we studied the factors necessary for discriminationof correct and incorrect folds. We first correct selected and incorrect folds by aconventional threading method. Then, all-atom models of those folds wereconstructed by simply minimizing the atomic overlaps. The constructed correct model representing the native fold has almost the same backbone structure as the native structure but differs in side"chain packing. Finally, theenergy values of the constructed models were compared with that ofthe experi"mentally determined native structure. The correct model as well as the native
structure showed lower energy than misfolded models. However, a large energy
gap was found between the native structure and the correct model. By decomposing the energy values into their components, it was found that solvent
effects such as the hydrophobic interaction or solvent shielding and the Bornenergy stabilized the correct model rather than the native structure. The large
energetic stabilization of the native structure was attained by specific sidechain packing. The stabilization by solvent effects is small compared to thatby side"chain packing. Therefore, it is suggested that in order to confidently
predict the correct fold of a protein, it is also necessary to predict side"chain
packing. See Ref. 7 for the details.

172

Publ icatians
1.

2.
3.

4.

5.

6.

KAWABATA, T., ARISAKA, F. and NISHIKAWA, K: Structurallfunctiona1 assignment ofbacteriophage T4 unknown proteins by iterative database searches. GENE, Z59, 223-233,
2000.
KAWABATA, T. and NISHIKAWA, K: Protein structure comparison using the Markov transition matrix of evolution. PROTEINS, 41,108-122,2000.
ISOGAI, Y., ISHII, A., FUJISAWA, T., OTA, M. and NISHIKAWA, K: Redesign of artificial
globins: Effects ofresidue replacements at hydrophobic sites on the structural properties. Biochemistry, 39, 5683-5690, 2000.
ISOGAI, Y., ISHII, A., ISHIDA, M., MUKAI, M., OTA, M., NISHIKAWA, K and IIZUKA, T.:
Structural and functional properties of designed globins. Proc. Indian Acad. Sci. (Chern.
Sci.), HZ, 215-221, 2000.
NAKASHIMA, H. and NISHIKAWA, K: The genomic DNA sequences of various species are
distinctively distributed in nucleotide composition space. Res. Comm. In Biochem.,
Cell & Mol. BioI., 4, 25-45, 2000.
YUSUF ALI, M., KHAN, M. L. A., SHAKIR, M. A., FUKAMI-KOBAYASHI, K, NISHIKAWA, K and
SIDDIQUI, S.8.: Expression and cDNA cloning of klp-12 gene encoding an ortholog of thechicken chromokinesin, mediating chromosome segregation in C. elegans. J. Biochem.
Mol. BioI., 33, 138-146, 2000.

7.

KINJO, A.R., KIDERA, A., NAKAMURA, H. and NISIIIKAWA, K: Physico-chemical evaluationof protein folds predicted by threading. Eur. Biophys. J., in press.

I-c.

Laboratory for Gene Function Research(1) Evolution of the ULA class I region in human genome
Yoshio TATENO, Hidetoshi INOK0 1 and Masaaki YAMAZAKI 2 (ITokai University,
2Fujiya Bioscience Institute)
HLA class I region is occupied mainly by A, Band C loci, where Band C loci
are located in duplicated genome fragments respectively. We found several
defective LINE sequences in both fragments, and found that they were homolo'
gous between the fragments. We estimated the evolutionary origin ofB and C
loci by analyzing the LINE sequences. It is known that these loci have a great
number of alleles. Investigation into their origin led us to the suggestion thatsome of them originated from a common ancestor of human and chimpanzee,

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supporting the trans-species polymorphism that J. Klein has proposed. This
would be an interesting case where neutral LINE sequences and a gene being
subject to typical positive selection co-exist and co-evolve in a unit genome
fragment.

(2) Evolution of protein strueture in the periplasmie binding protein(PLBP) superfamily
Kaoru FUKAMI-KOBAYASHI, Yoshio TATENO and Ken NISHIKAWA
In the previous study (Fukami-Kobayashi et aI, 1999), we have shown that
one ofthe type I PLBPs is most likely to resemble a primordial PLBP, and thata type II PLBP arose from the type I PLBPs to evolve into various type II
PLBPs. Recently, the crystal structures of another type of PLBPs have beenreported. The topological arrangements of the type III PLBPs are different
from either type. To find a clue to the evolutionary origin of the type III, weconstructed phylogenetic trees for PLBPs and their partner ABC proteins, andobtained a preliminary result that the type III was closer to the type II.
Some of bacterial transcription factors such as lactose repressor (Lac!) andpurine repressor (PurR) are homologous to periplasmic binding proteins(PLBPs) in their C-terminalligand binding domains. Phylogenetic analysis ofrepressors and their PLBP homologues revealed that the attachment of the
DNA-binding domain was originated first, and substrate specificity was gainedin evolution thereafter. The tree also shows that the emergence of the attachment predates the divergence of major lineages of eubacteria.

(3) Compensatory eovariation in protein evolution
Kaoru FUKAMI-KOBAYASHI and StevenA. BENNER! ('Florida University)
Compensatory covariation arises in divergent evolution of protein sequences,when two amino acid sites have particular mutations that work together tomaintain a global property of a protein (net charge, for example), while eachmutation alone does not. We are now finding such covariations by examining
branches of an evolutionary tree of a specified group of proteins and recon-

174

structing ancestral sequences in it.

(4) DNA Data Bank of Japan (DDBJ) in collaboration with masssequencing teams
Yoshio TATENO, Satoru MIYAZAKI, Motonori OTA, Hideaki SUGAWARA and Takashi
GOJOBORI
We at DDBJ (http://www.ddbj.nig.ac.jp )process and publicise the massive
amounts of data submitted mainly by Japanese genome projects and sequenc'
ing teams. It is emphasised that the collaboration between data producing
teams and the data bank is crucial in carrying out these processes smoothly.
The amount of data submitted in 1999 is so large that it alone exceeds thetotal amount submitted in the preceding 10 years. To cope with this situation,
we have developed tools not only for processing such massive amounts of data
but also for efficiently retrieving data on demand.

Publ ications
1. TATENO, Y., MIYAZAKI, S., OTA, M., SUGAWARA, H. and GOJOBORI, T. (2000) DNA Data
Bank of Japan in collaboration with mass sequencing teams. Nucleic Acids Res Z8: 2426.
2.

MIYAZAKI, S., HASHIMOTO, H., SHIMADA, A, TATENO, Y. and SUGAWARA, H. (2000) A new fileformat and tools for the large-scale data submission to DNA Data Bank of Japan(DDBJ). In Currents in Computational Molecular Biology, pp 60 . 61, S. Miyano, R.
Shamir and T. Takagi ed., Universal Academy Press, Tokyo.

3.

AHOLA, E.E., BAIROCH, A, BAKER, WC., BECK, S., BENSON, D.A, BERMAN, H., CAMERON, G.,
CANTOR, C., DOUBET, S., HUBBHARD, T.J.P., JONES, T.A, KU;YWEGT, G.J. KOLASKAR, AS.,KUIK, Van A., LESK, A.M., MEWS, H.-W., NEUHAUS, D., PFEIFFER, F., TENEYCK, L.F.,SIMPSON, R.J., STOSSER, G., SUSSMAN, J.L., TATENO, Y., TSUGITA, H., ULRICH, E.L. and
VIEGENTHART, J.F.G. (2000) Quality control in data banks for molecular biology.
BioEssays ZZ: 1024-1034.

4.

YUSUF ALI, M., KHAN, M. L. A, SHAKIR, M. A, FUKAMI-KoBAYASHI, K., NISHIKAWA, K. and
SIDDUQUI, S. S. (2000) Expression and eDNA cloning ofklp-12 gene encoding an orthologof the chichen chromokinesin, mediatingchromosome segregation in Caenorhabditis
elegans. J. Biochem. Mol. BioI. 33: 138-146.

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Laboratory for Molecular Classification(1) Research and Development for DDBJ

(Ih. From YAMATOII to TSUNAMI
The laboratory is in charge of the development and management of databases for DDBJ. Since 1998, DDBJ has developed a data submission systemwith parser in order to process massive data originated from genome projects
and a study on biodiversity. The development was in time for the completion of
the draft sequencing of human genome in June 2000. In the meantime, thesystem could also cope with a number of cDNA sequences of human, mice andother organisms.
DDBJ expects that the draft sequences will be updated, especially by improvement of annotation. It requires streamline of the system for review andrelease of data. Therefore, DDBJ looked through the existing review system(YAMATOII) and the mass submission system resulting in planning a newdata processing system. Development of the new system is named TSUNAMI
project and is underway.
(I)-b. Expansion of Genome Information Broker (GIB)
GIB was originally created for E. coli genome for the retrieval and analysis of
genomic information in a set. We implemented microbial genome data into
GIB whenever genome sequencing were completed and the data 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 and visualization of thedata are available for 42 organisms as of February 200l.
We have utilized XML and CORBA to integrate distributed computational
resources in order to cope with the explosion of microbial genome information.
(I)-c. Application ofXML to DDBJ
Interoperability of databases is still an issue in bioinformatics. XML is a

176

powerful yet simple tool that may improve the interoperability. We defined adata type definition (DTD) for DDBJ entries. The XML will firstly implanted
into the DDBJ retrieval system "getentry". It will make it possible to integrate automatically a private database and a subset ofDDBJ based on DTD.
(2) Research and Development for WFCC-MIRCEN World Data Centre
for Microorganisms (WDCM)(2)-a. WDCM
WFCC and MIRCEN stand for World Federation for Culture Collections andMicrobial Resource Centers network respectively. The laboratory is a host of
WDCM that is the data center ofWFCC and MIRCEN. We maintain the world
directory of 500 culture collections in 60 countries in databases of "CCINFO"
and "STRAIN" that are accessible at http://wdcm.nig.ac.jp/.
(2)-b. Development of a Workbench for Biological Classification and Identification (InforBIO)
We have been interested in introduction and development new Internet technologies. We introduced the World Wide Web (WWW) as one of the first ten
WWW servers. We also developed a simultaneous search system of distributed system named Agent for Hunting Microbial Information in Internet(AHMMI). We now develop the digital workbench named InforBIO.
In InforBIO, users are able to integrate databases and analytical tools thatare distributed in the Internet including their own resources. We aim at anopen system by use of JAVA, XML, and tools of CORBA and a relational
database management system in the public domain. We will distribute theprototype ofInforBIO in CD-ROM this year.
(3) Others(3)-a. Data processing system for patent sequences
DDBJ has received patent sequences from Japan Patent Office (JPO) and1.

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disseminate them to the public. Therefore DDBJ and JPO have discussed
about a system for data transfer from JPO to DDBJ.
This year, we cooperated a survey by Japan Bioindustry Association (JBN on
data processing system in European Patent Office (EPO) and United States
Trademark and Patent Office (USTPO). It is to be noted that EPO has alreadyuses the EMBL data system that is outside EPO. USTPO also look into thepossibility to use computer resources outside. The primary reason of theoutsourcing is to realize efficient aata processing while sequence data increase
exponentially.
(3)-b. Biological Resources Centres (BRC)
The Working Party for Biotechnology (WPB) of OECD set up a task force on
BRC in February1999 to develop a policy guidance to support BRCs. Professor
Hideaki Sugawara is the chair of the task force and compiled 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.
(3)·c. Global Biodiversity Information Facility (GBIF)
GBIF is a scheme that was created by a Biological Informatics working groupin Megascience Forum of OECD. GBIF will improve the accessibility of all
kinds of data of all kinds of organisms on the globe. One of the staffs (HS)participated in the discussion of the working group for three years and it isexpected that the laboratory will contribute GBIF when it becomes reality. The
GBIF HomePage is at ..http://www.gbif.orgf..

Publ ications
1. GOTO, Y., MIYAZAKI, S. and SUGAWARA, H.: Journal ofInformation and Knowledge Vol. to.
No.4 (2000
2. MIYAZAKI, S. and SU(iAWAI{A, H.: Development of DDBJ-XML and Its Application to a
Database of cDNA,Genome Informatics 2000,Universal Academy Press, Inc (Tokyo),380-381, 2000.

178
3.

SUGAWARA, H.: Microbial Resource Centers and Data Activities (In Japanese), Biological
Resources Centers and Data Banks for Life Science (ed. Nakatsuji, N,), KyoritsuShuppan (Tokyo), 130-135, 2000.

MORI, H., SUGAWARA, H. and UCHIYAMA, 1.: Escherichia coli genome (In Japanese), Biological Resources Centers and Data Banks for Life Science (ed. Nakatsuji, N,), KyoritsuShuppan (Tokyo), 118-124, 2000.
5. MIYAZAKI, S., SUGAWARA, H. and GOJOBORI, T.: Genome Informatics (In Japanese), SaishinIgaku, 56(1), 64-71, 2000.
4.

Oral presentation
1. AMANO, M., EZAKI, T., KAWAMURA, Y. and SUGAWARA, H.: Utilization of DNA chip for the
Identification of Bacteria (In Japanese), 7th Annual Coneference of Japan Society for
Culture Collections (Sendail, 8 June 2000.
2. SUGAWARA, H. and MIYAZAKI, S.: Issues of Microbial Information Network (In Japanese),
7th Annual Coneference of Japan Society for Culture Collections (Sendai), 8 June 2000.
3. SUGAWARA, H.: Biological Resources Centers In the New Age - a report by OECD-(InJapanese), Workshop on Biological Resources Centers (Tokyo). 14 June 2000.
4. MIYAZAKI, S. and SUGAWARA, H.: WFCC World Data Centre for Microorganisms (WDCM):
Its activities and the utilization of information technology for culture collections andmicrobiology, The 9th International Congress for Culture Collections, 22-27 July 2000,
Brisbane.
5. SUGAWARA, H. and MIYAZAKI, S.: Linking microbial genetic resources in culture collections
with nucleic acid sequences, The 9th International Congress for Culture Collections,22-27 July 2000, Brisbane.
6. SALOMON, W., DOYLE, A. and SUGAWARA, H.: OECD Task Force on strategy for the longterm development and sustainability of Biological Resource Centres, The 9th International Congress for Culture Collections, 22-27 July 2000, Brisbane.
7. SUGAWARA, H.: Bioinformatics, Management and Operation of Culture Collections Training Course of the 9th International Congress for Culture Collections, 29-30 July 2000,
Briabane.
8. SUGAWARA, H.: Roles and Value of Public Databases in the time of Genomics and its
Industrialization, Proceedings of BIO JAPAN 2000 (Tokyo), 83-86, 27 Sep 2000.

179

J.

Radioisotope Center(1) Expresion and Purification of Escherichia colJ
Transcription Factors
Katsunori YATA andAkira ISHIHAMATranscription regulation is a major cellular process by which the exression ofa large number of genes on the genome are controlled. The RNA polymerase
holoenilyme of Escherichia coli is composed of the core enzyme with the subunit coimposition a 2 13 13 ' and one of seven molecular species of the a subunit. The function of RNA polymerase holoenzyme is further modulated throughinterplay with one or two of about 100-150 transcription factors. The intracellular concentrations of transcription factors are therefore considered to be amajor determinant of the expression level of most inducible genes. In order todetermine the intracellular levels of various transcription factors in E. coli, we
expressed as many transcription factors as possible in His-tagged forms us~ng
the respective cloned genes. So far more than 25 molecular species of the E.
coli transcription factor have been purified to apparent homogeneity by affinity chromatography on Ni2+- NTA columns, including AcrR, AhpC, AhpF, AreA,
DeoR, DnaK, Fur, GroEL, KdpE, IclR, IlvY, InfB, Lac!, LeoO, MerR, NusA, OxyR,
PhoP, RpsB, RpsF, PspF, SdiA, SoxR, SoxS, ThiI, and TufB. Polyclonal antibodies were raised in rabbits against the purified transcription factors. The
antibodies are being used, in Division of Molecular Genetics, for detemination
ofthe transcription factor level in celllysates by quantitative Western blot.
The purified transcription factors will also be used for identification of theircontact sites on RNA polymerase subunits by mapping of the protein-protein
contact-dependent clevaga sites by FeBABE Gron-p-bromoacetamidovenzyl
EDTA) tethered to each transcription factor.

180

Publications
1. ISHIHAMA, A.: Functional modulation of Escherichia cailRNA polymerase. Ann. Rev.
Microbial. 54, 499-518 (2000)
2. ISHIHAMA, A.: Molecular anatomy of RNA polymerase using protein-conjugated metalprobes with nuclease and protease activities. Chem. Cammun. 2000, 1091-1094 (2000)

K.

Experimenntal Farm

(1) Development and reevaluation of the genetic stocks of rice
Ken"Ichi NONOMURA, Mitsugu EIGUCHl, Toshie MIYABAYASHl, Yukihiro ITO andNoriKURATA
We have conducted the reproduction and distribution of genetic stocks of wild
and cultivated rice. From October 1, 1997, we try to include new system of ricegenetic stock generation and application. Additional resources we are conduct"
ing to produce, utilize and distribute are enhancer trap rice. Another trial toanalyze the composition of the centromeric heterochromatin in rice chromo"
somes is also progressed. These projects are cooperated with the plant genet"
ics laboratory. For details, see the reports of plant genetics lab.

Publ ication
1.

NONOMURA, K. I. and KURATA, N.: The centromere composition of multiple repetitive
sequences on rice chromosome 5. Chromosoma, 2001Gn press).

181

ABSTRACTS OF DIARY FOR 2000
Biological SymposiumJan. 7Detection of DNA-looping by single-molecule manipulation(Kumiko Sogawa)Jan. 13
Partitioning of DNA in bacteria - Intracellular localization of
DNA depending on cell division cycle (Hironori Niki)
Jan. 19
Molecular mechanism for determining dorso'ventral axis in zebra fish (Masahiko Hibi)
Jan. 20
Schrodinger's dream over 50 years - Complex electron microscopy and gene direct-reading (Kuniaki Nagayama)Jan. 25
Control mechanism of cell cycle in higher organism cells using
warts and fzr/cdhl (Hideyuki Saya)Jan. 28
Transcription of yeast rDNA, RNA polymerase specificity, rDNA
repeats and nucleolar structures (Masayasu Nomura)Feb. 4
Function and regulation of the transcription factor Oct-4 in the
mammalian germline (Hans R. Schoeler)
Feb. 15
Research papers in an age of electronic media and problems inJapan (Hideaki Tanaka)Feb. 17
A 4-dimensional analysis of the embryogenesis of C. elegans :migrations, regions, linesge and binary specification (Ralf
Schnabel)E.coli FIS controls DNA architecture and promoter activity(Georgi Muskhelishvili)
Feb. 18
Control of morphogenesis and cell proliferation by mammalianpolycomb group (Haruhiko Koseki)Mar. 1
Molecular mechanisms ofWnt signal and TGF-beta signal (Koji
Shibuya)Mar. 2
Role of Ras-MAP kinase signal transduction system in olfactory
sense of C. elegans (Yuichi lino)Mar. 7
Gene Regulation by Peptide Nucleic Acid (PNA) (Peter E. Nielsen)
Mar. 8
Protein transport in secretory organella . Protein transport to182

Mar. 13Mar. 14Mar. 15
Apr. 10

Apr. 12Apr. 17

Apr. 24
Apr. 26

May 1

May 16
May 29vacuolar of higher plants and transport apparatus of budding
yeast - (Ken Matsuoka)
Activity dependent plasticity: new insights into functional
andmorphological changes on the synaptic level (Tobias
Bonhoeffer)
Mechanism for transporting functional molecules between
nucleus and cytoplasm (Naoko Imamoto)Mechanisms of activity-dependent plasticity in the mammalianvisual cortex (Michael P. Stryker)
Development of new bacterial chromosome partitioning technique(Hironori Niki)
Function of LAP2 on nuclear envelope breakdown andreconsitution (Kazuhiro Furukawa)
Cerebral function of speech - from physics to linguistics (Kuniyoshi Sakai)Connections among transcription, chromatin, and mRNA pro·cessing (Stephen Buratowski)
Mechanisms underlying induction and progression of a neurogenic wave in the developing zebrafish retina (Ichiro Masai)
A multiprotein mediator complex is required for the stimulation
oftranscription by acitvators in human cells <Arnold J. Berk)
Development of selective techniques for visualizing synaptic neural pathways using WGA transgene and application thereof(Yoshihiro Yoshiwara)
Loose coupling of molecular mechanism (Fumio Ohsawa)
Isolation and analysis of DL gene that regulates the flower development of rice - Is ABC mode applicable to monocotyledon? .
(Hiroyuki Hirao)
Post-transcriscriptional and post-transnational control in bacteriophage lambda lysis-lysogeny decision (Prof. Amos
Oppenheim)
Signal transduction and formation of neural cells: polarity andmechanism offorming axon (Naoyuki Inagaki)
Functions of transcription factors Mespl and Mesp2 : Early me-

ABSTRACTS OF DIARY FOR 2000

183

soderm embryogenesis and segmentation (Yumiko Saga)
Role of transcription factor Gli in controlling the expression thesonic hedgehog target gene (Hiroshi Sasaki)
July 6
July 10
July 27
Aug. 1

Search for molecular mechanism for the developmental determination of plant cells (Kiyotaka Okada)
Check-point control by Chk1 and chk2 (Shuhei Matsuoka)
Gene group for controlling neuroaxonal induction and cell migration : approach using C. elegans (Takehiro Kawano)
1. Reverse transport of protein found in membrane protein degradation, 2. Identification and analysis of different PI3KVps34p
complexes that are effective for autophagy and protein transport(Akio Kihara)

Sep. 8

Attenuation ofInfluenza Viruses via Genetic Engineering: Modi-

Sep. 20

fication of the Interferon Antagonist (Prof. Peter Palese)
Molecular Dissection of Central Spindle Assembly and Cytoki-

Sep. 27

nesis (Dr. Michael Glotzer)
Intracellular signal transfer mechanism of neuronal growth cone:approach of cell-localized selective molecular targeting with CALI
method (Kotaro Takei)

Sep. 29

Predicting the future course of human influenza virus evolution(Dr. Walter Fitch)
What determines the risk of inflammatory disease in HTLV-I
infection? (Dr. Charles Bangham)

Oct. 3Oct. 12Oct. 17

The impact of Whole Genome Sequencing on Bioinformatics,
Genomics and Proteomics (Mr. Neil J. Campbell)Sensing and responding to DNA damage (Noel Lowndes)
Mechanisms of cell fate determination during embryogenesis of
ascidian by maternal localized mRNA and cellular interaction(Hiroki Nishida)

Oct. 23

How do animal DNA viruses get to the cell nucleus? (Harumi
Kasamatsu)

Nov. 1

Regulation of chromosomal DNA methylation patterns and functions thereof (Masaki Okano)Nov. 2

Gene identification from genome sequence (Tetsushi Yada)

184

Nov. 6
Nov. 10
Nov. 13

Nov. 14

Nov. 15
Nov. 30
Dec. 19

Dec. 20

Function analysis of a new bHLH family, hesr-l, '2 and -3 : po'tential regulators of vertebrate simitogenesis (Hiroki Kokubo)Germ Line, Stem Cella and Genomic Imprinting <M. Azim Surani)
DNA Methylation in Cancer Tissue (Peter A. Jones)
Novel homeobox gene isolated by EST to be dominantly expressedin female germ cell during sex determination of mouse (Nobuka
Takasaki)
DNA computing (Masayuki Yamamura)
Kinesin I is required for organelle transport in axons and foranterior-posterior axis determination (William M. Saxton)
MES PROTEINS AND MATERNAL COTROL OF GERMLINEDEVELOPMENT IN C. ELEGANS (Susan Strome)
Rat Genome Database: A Platform for Rat, Mouse and Human
Comparative Genomics (Jian Lu)
Molecular genetics of murine dorsl'vemtrallimb and mid'hind'brain development (Dr. Randy L. Johnson)
The Dynamic End: Silencing and Replocation of Telomeres in
Yeast (Daniel Gottschling)
Ubiquitin'like protein SUMO· 1 and control of chromosomal DNA
homologous recombination and cell growth (Hisahito Saito)
Codon bias evolution in Drosophila: Detecting the "footprint" of
weak selection at silent sites in DNA (Dr. Hiroshi Akashi)
X'chromosome specific localization mechanism of drosophilamale' specific lethal complex (Yuji Kageyama)
The role of the corepressor Groucho in the early embryonic function of Even'skipped (Masatomo Kobayashi)

185

FOREIGN VISITORS IN 2000
Jan. 7·8Jan. 27-28
Feb. 14-15

Feb. 17-18

Mar. 7-8

Mar. 13-14

Apr. 10
Apr. 11-12
Apr. 12-13
May 1-2

July 10
July 27-28
Sep.7-8
Sep.20-21

Dr. Alisa S W shum, Department ofAnatomy The Chinese
University of Hong kong, Hong kong
Dr. Masayasu Nomura, Department of Biological Chemistry University of California, Irvine
Hans R. Scholer, University of Pennsylvania New Bolton
Center, Center for Animal Transgenesis and Germ Cell
ReseachRalf Schnabel, Institut fur Genetik, TU Braunschweig Pat
Nolan, MRC Mouse Genome Center and Mammalian
Genetics Unit, Harwell
Peter E. Nielsen, University of Copenhagen, IMBG, Labo·ratory B, The Panum Institute, Blegdamsvej3, DK-2200
Copenhagen N, Denmark
Michael P. Stryker, Department of Physiology and W. M.
Keck Foundation Center for Integrative Neuroscience,
UCSF Tobias Bonhoeffer, Max-Planck-Institut fur Neurobiology, Munchen-Martinaried
Stephen Buratowski, Biological Chemistry and Molecular Pharmacology, harvard Medical School
Dr. Avante Paabo, Director, Max-Planck-Institute ofEvolutionary Anthropology Leipzig, Germany
Arnold J. Berk, Molecular Biology Institute, University ofcalifornia, Los Angeles
Amos Oppenheim, The Hebrew University-Hadassah
Medical School Department of Molecular Genetics andBiotechnology Jerusalem, Israel
Matsuoka Shuhei, Baylar College of Medicine, U.S.A.
Dr. Tekehiro Kawano, Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Toront
Prof. Peter Palese, Mount Sinai School of Medicine Department of Microbiology NEW York, NY 10029, U.S.A.
Dr. Michael Glotzer, Research Institute of Moleculer Pa·

186

Sep.28-29
Oct. 3-4
Oct. 12-13
Oct. 23-24
Nov. 1-2
Nov. 6-7

Nov. 10'11

Nov. 13-14

Nov. 15-16
Nov. 30-Dec. 1
Dec. 18'19
Dec. 19

Dec. 19-20
Dec. 20-21

thology Vienna, Austria
Dr. Charles Bangham, Department ofImmunology Imperial college School of Medicine St Mary's Hospital, U.K.
Mr. Neil J. Campbell, Senior Director Celera Genomics,
US.A.
Noel Lowndes, ICRF, Clare Hall Laboratories, UK.
Harumi Kasamatsu, Moleculer Biology Institute, University of California, Los Angeles, US.A.
Masaki Okano, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School
Hiroki Kokubo, University of Texas, MD. Anderson Cancer Center Department of Biochemistry and Moleculer
Biology
M. Azim Surani, Wellcome/CRC Institute of Cancer andDevelopmental Biology, University of Cambridge
Peter A. Jones, Norris Comprehensive Cancer Center,
University of Southern California
Dr. Nobuyosi Takasaki, Laboratory of Cellular and Development Biology, NIDDK, NIH
Willian M. Saxton, Department of Biology, indiana University
Mr. Jian Lu, Bioinformatics Research Center Medical
College of Wisconsin, US.A.
Randy L. Johnson, MD Anderson Cancer Center, Houston
Dr. Yoshiyuki Imai, Department of Development Biology
Stanford University School of Medicine
Dr. Hiroshi Akasi, Dept. of Biology, Institute ofMoleculer
Evolutionary Genetics Pennsylvania State University,
U.S.A.
Dr. Hisato Saitoh, Picower Institute, N.Y., U.S.A.
Yuji Kageyama, Howard Hughes Medical Institute, Baylor
College of Medicine
Dr. Masatomo Kobayasi, kimmel Cancer Institute, Tho'
mas Jefferson University

187

AUTHOR INDEXAGATA, K.
AHN, B.O
AMIRI, A.
ANDACHI, Y.

ARAKI, H
ARAMAKI, H
ARISAKA, F
AsAHINA, M
BENNER, S.A.
BLIZARD, D.A.

160
106
128
130
37,38,39
139
167
51
173
96

FURUSE, T.
FURUTA, M
FURUUMI, H
GAAL, TGAUDIERI, S
GENGYO-ANDO, K.
GOJOBORI, T.
GoTO, SGRANJEAUD, S

79
122
116
160

HAGA, S137
115

HARUSHIMA, Y.
HATTORI, M

DOUCETTE-STAMM, L.

79
11
122

EGELMAN, E.H

35

BONG, Y.S
BRASCH, M.A.
BRENTRUP, D
CEBRIA, F
CHATTERJI, D
CHIHARA, T.
DANJO, 1.
DASGUPTA, D

EIGUCHI, M
ENDO, A.
ENDO, T.
ETO, K.
EWBANK, J
FAN, YFERRELL, R.E
FuJISAWA, C
FuJISAWA, H
FUJISAWA, T
FUJISAWA, T
FuJITA, N
FUJIYAMA, A.
FUKAGAWA, T.
FUKAMI-KOBAYASHI, K.

HARAFUJI, N
HARAGUCHI, S
HARTLEY, J.L

HATTA, M

HAYASHI, H
HAYASHI, S
HAYWARD, R.S

105,110,180
20
163
116
130
128
69, 71

HIEDA, M

48
83,84
47,48,49

HIROSE, S

169
12

HONDA, A.

65,79
63,64
173

lCHIBA, Y

HILL, D.E.
HIRANO, H
HIRATA, T
HIROCHIKA, H
HIROMI, Y.
HIRONO, K.
HIROSHIMA, M
HITTI, J

HWANG, J.S
lINO, Y.
lKEGAMI, T.

95,96
158
75
136
161
126
160,161,162,
163,164,165,174
116,117
130
121
47
102
122
109,110
47,49
75
121
115,116,117
138
155
122
87
83,84
104,106
43,44,45,46
125
51,52,53
134,135
122
20,21
164
65, 67
143
119

188
IKEMURA, T.
IKEO, K.
IMAMOTO, N
IMANISHI, T
INOKO, H
ISHIHAMA, A.

63,64,65,66,67

KATSURA, 1.

51,140,141,143

160, 162

KAWABATA, T

119,167,168

132,135,153,154,

KAWAKAMI, A.

56,57

155,156,157,158

KAwASAKI, 1.

128

68,161

KEITHLY, J.S

53

172

KIKUCHI, K.

87

10, 11, 12, 13, 14,15,

KIKUCHI, M

68
16, 17

16,17,18,19,20,21,179

KIMURA, M

ISHIHARA, K.

75

KIMURA, T

58

ISHIHARA, T

51,140,141,143

KINEBUCHI, T.

139

ISHII, A.

169

KINJO, A

ISOGAI, Y.

169

KINOUCHI, M

ITO, M

125

KISHI, T

26

ITO, Y.

103,104,105,110,180

KITABAYASHI, A

100

17 1
65,66

ITOH, J."1.

88,106KITAGAWA, M

114

IWAMA, H

164

KITAJIMA, S

101

IWANAMI, M

46

KITANO, H

88

IWATA, A.

14

KITANO, T.

69,70,71

IWATA, M

125

KIYAMA, R

59

JACKSON, C

122

KOBAYAKAWA, Y.

47

JACOBS, S.A.

35

KODAIRA, M

79

JIANG, Y. "J

56

KOHARA, Y.

51,120,121,122,123,

JIN, F70

JINDRA, M

51

KOIDE, T

95,96,98

JINNAI, N

94,98

KOIKE, M

155,157

125,126,127,128,130

JISHAGE, M

11

KOIZUMI, O

47

KABATA, H

139

KOMINAMI, R

75

KAIDO, M

21

KONDOH, S

73

KAJITA, A.

127

KOSE, H45
135,155,156,158

KAKUTANI, T

81,82

KOSE, SKAMIMURA, Y

37,39

KOSEKI, H

78

KANAYA, S

65,66

KOSHIDA, S

55

KANDA, E

18

KOSHIO, E.

14

KANEDA, M

76

KUBORI, T

136

KARASHIMA, T.

128

KUBOTA, K

116

KARLSTROM, R.

57

KUBOTA, T.

KAsCIUKOVIC, T

138

KUDO, YKATAHIRA, J

155

KUMASAKA, T.

KATAYAMA, A.

12, 16

KATO, J

119

KATO, M

59

KURATA, N

77
65,66
155
103,104,105,106,107,
108,109,110,118,180

KUROIWA, A

55,56

AUTHOR INDEX189

KUROIWA, A

77

MIYAHARA, K.

KUROKI, Y.

79
139

MIYAZAKI, S

141
174

MIYOSHI, K.

87,105,106

130
134
53
122

MIZUMOTO, K.

KUROSAWA, O
KURZ, C.L.
KUWAJIMA, K.
LAGIER, M.J
LAMESCH, P.E
LAVORGNA, G
LEE, H
LEE, S.J
LI, E
LIU, Q.X
LoWE, A.
MAEDA, I.
MAENAKA, K.
MAKINO, S
MAKINOSHIMA, H

MALLO, G

52
122
155
78
51
121
123
147,148,149,150,152
91
10
130

MIZUNO, S
MIZUSHINA, Y.
MOCHIZUKI, K.
MOORE, T.
MORI, H
MORI, T.
MORIGUCHI, K.
MORISHITA, F
MORIWAKI, K.
MOTOYAMA, A.
MUKAI, T.
MUKHERJEE, A.
MURATA, T
NAGAI, H

38,64
90,91

NAGATO, Y.

MATAKATSU, H

116

NAKAGATA, N

MATSUDA, Y

77
114
45
87,88
47

NAKAGAWA, A.

70
113

NAKATA, K.

MAsUMOTO, H
MASUYA, H

MATSUMOTO, K.
MATSUNO, M
MATSUOKA, M
MATSUSHIMA, O
MATSUSHITA, T
MATSUZAWA, H
MIKAMI, Y.
MINAMIDA, A.
MINETA, K.
MISKELISHIVILI, VG
MISU, S
MITA, A.
MITANI, S
MITSUI, K.
MITSUZAWA, H
MIURA, A.
MIURA, S
MIURA, Y.
MIYABAYASHI, T.

NAKADE, S

NAKAHORI, Y.

NAKANo, M
NAKASHIMA, H
NAKATA, E.
NAKAYAMA, T

63
123
160,162
136

NAKAZAWA, M

68
93,94
126

NISHIHASHI, A.

118,119
18, 19NISHIMURA, A.

81,82
121

NISHIZAKA, S

96
180

NARITA, T.
NIIMURA, Y
NIKI, HNISHIKAWA, K.

NISHINO, T.

NIWA, N
NIWA, Y.
NODA, R.

27
77
93,98
48
122
114
119
108
47
95,96
79
75
137
52
136,137,138
87,88, 106, 118
114
98
155
79
64
170
87
125
79
160
58
160
41
63,64
119,167,168,169,
170,171,173
10,112,113,114,119
16
121
43
103
69,71

190
NOMOTO, H

121
12
106,107,180

NOMURA, T.
NONOMURA, K-1.
OGASAWARA, M

161
79
31,34,35

OGAWA, N
OGAWA, T.
OGURA, K
OHKURA, K
OHNISHI, Y.
OHNO, M
OHTA, F
OHTA, T.
OISHI, AOISHI, K

OKA, A
OKABE, M
OKAMOTO, T.
OKAMURA, A
OOTA, HOSHIUMI, H

OTA, MOZAWA, H
PARK, J.-H
POTDEVIN, M
PURBOWASITO, W.
RAO, Y.
REBOUL, J
RUAL, J.F
SADO, T
SADO, y.
SAGA, y.
SAGAI, T.
SAITOU, N
SAKA, K
SAKAGUCHI, T.

SAKAI, H
SAKAKl, Y.

""

SATO, Y.

SHIINA, N

155
24
121
75
84
122
122
78
117
100,101,102

90
67,68,69,70,71,79
114
55
155

SEINO, H

83
138
56
57
19,24,25
133,135
136,137,138,139
47,49
90
148
120,121,122
,
133

"

SAWADA, A

59
75
121
31,34
140
121
93

83
169,174OSUMI, N

SASAKI, H
SATO, Y.

SCHIER, AF

63
135
70
31,34

OKONOGI, A

SANO, M

126
141

43,44,46
21

93,98
121
75,76,77,78

SAKURAI, N"

SHIMAMOTO, N
SHIMIZU, HSHIMIZU, K
SHINDO, K
SHIN-!, T
SHINKURA, K
SHINYA, M

55,56
146,147,148,149,
150,151,152

SHIRAKIHARA, Y.
SHIRATORI, A
SHIROHZU, HSHIROISHI, T.

146,14975, 77
90,91,93,94,95,96,98"

SIMONYA, V
SITRAMAN, S"

SOGAWA, K
STROME, S"

SUDA, C"SUGANO, S"

SUGAWARA, H,

SUGIMOTO, A
SUMIYAMA, K
SUSA, MSUZUKI, E,

SUZUKI, H
SUZUKI, T.

T. DANIEL ANDREWSTACHIBANA, T.

TAKAGI, N

SAKURAI, H" .. 16

TAKAHASHI, N

TAKAGI, A

52
17

165
155
76,77

TAJIMA, S

SAKANE, 1.

75
121
174
123
69,71
136

107
121

SUZUKI, Y

65,75
134

121
13
139
128

,

100
85,93
79

AUTHOR INDEXTAKAHASHI, T.
TAKAHASHI, Y.
TAKANO, T.
TAKANO-SHIMIZU, T
TAKAYA, K.
TAKAYAMA, Y.
TAKEDA, H
TAKENAKA, O
TALBOT, W.S
TAMURA, M
TANAKA, K.
TANAKA, S
TANIGUCHI. M
TATENO. Y.
TATSUDA, D
TEMPLE, G.F,
THIERRY-MIEG, D
THIERRY-MIEG, J
THIERRY-MIEG, N
THIERRY-MIEG, Y.
TOKUNAGA, M

47
100
95
61
104
39
55,56,57,58
69
57
90
27
34
117
172,173,174
31,34
122
121,122
121,122122
121
132,133,134,135,154

TOMIOKA, N

83

TOMIZAWA, J

31,40
93
77
16
76, 78TOSHIMORI, K.
TSUDUKI, M
TSUJII, A.
TSUJIMOTO, N
TSUKIHARA, T.
UCHIYAMA, Y.
UEDA, H
UEDA, S
UEDA, S
UEDA, T
UEHARA, T
UEKUCHI-TANAKA, M
UESUGI, H
UETA, Y.
UMEZAWA, A.
VAGLIO, P.

155
162
51,52
14
70, 71
75
113
87
121
125
75

VANDENHAUTE, J

122
122

VIDAL, M

122

WADA, A.
WANAPIRAK, C
WANG, L.
WASHIZU, M
WATANABE, K.
WATANABE, Y.
WEST, S.C

Wu, J.Y.
YABE, T.
YADA, Y.
YAGASAKI, K.
YAMADA, M
YAMADA, Y.
YAMAKAWA, T
YAMAMOTO, K.
YAMAMOTO, M
YAMAMOTO, M
YAMAMURA, M
YAMAO, F
YAMAZAKI, M
YAMAZAKI, Y.
YATA, K.
YOKOMINE, T
YONEDA, Y.
YOSHIDA, K.
YOSHIDA, S
YOSHIMURA, A.

Yu, X
YUASA, Y.
YUM, SZHU, G

191
16
59
70
139
81,82
65,67
35
84
141
91
96
52
65,66
118,119
11,15
123
155
127
19,24,25,26
172
118,119
13,14,15,179
77
155, 156
87
79
118
35
46
47
53

/'

Annual Report of the National Institute of Genetics. No.51

Issued by Director-General: Yoshiki Hotta, D.Med.
National Institute of Genetics
Yata 1,111 Mishima, Shizuoka-Ken, 411-8540 Japan
Tel. 0559-81-6718
Fax. 0559-81-6715
Edited by Hiroyuki Araki & Tetsuji Kakutani
Published by National Institute of Genetics

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