ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No.49

1998

Publ/sbed by

THE NATIONAL INSTITUTE OF GENETICS
M/sb/ma, Sb/zuoka-i{en:· Japan1999

Annual Reportof theNational Institute of Genetics
No. 49, 1998

(Center for Information Biology)

Published by

The National Institute of Genetics, Japan

1999

CONTENTS
General statement
Staff
Council and advisory committee
Research activities in 1998
A.

.
.
.
.

Department of Molecular Genetics
A-a. Division of Molecular Genetics
Mapping of Subunit-subunit Contact Surfaces on the 13 and 13'
Subunits of Escherichia coli RNA Polymerase. KATAYAMA,
A., NOMURA, T., FUJITA, N. and ISHIHAMA, A.
Search for Novel Transcription Factors Interacting with the 13 and
13' Subunits of Escherichia coli RNA Polymerase. NOMURA,
T., KATAYAMA, A., FUJITA, N., MEARES, C.F. and ISHIHAMA, A.

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3
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Structural requirements for the interdomain linker of a subunit of Escherichia coli RNA polymerase. FUJITA, N., ENDO,
S. and ISillHAMA, A.
Transcriptional Activation Mediated by the Carboxy-Terminal
Domain of RNA Polymerase a Subunit: Multipoint Monitoring with a Fluorescent Probe. OZOLINE, O.N., FuJITA, N. andISillHAMA,A.
Regulatory Mechanisms of Sigma Subunit Activities of Escherichia
coli RNA Polymerase. JISHAGE, M., MAEDA, H., DASGUPTA, D.,
FuJITA, N. and ISHIHAMA, A..
Role of the as subunit in stationary phase survival of E. coli:
Search for as-dependent promoters and analysis of rpoS
mutations. SUBBARAYAN, P.R. and ISHIHAMA, A.
.
Growth Phase-Dependent Variation in the Levels of Twelve Species of the Nucleoid Protein in Escherichia coli. TALUKDER,
A.A., IWATA, A., NISHIMURA, A., UEDA, S. and ISillHAMA, A. ..

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Growth Phase-Coupled Changes in the Ribosome Pattern of Escherichia coli and Effect of Polyamines on Ribosome
Dimerization. WADA, A., IGARASHI, K, KURLAND, C.G. and
ISHIHAMA, A.
Cloning and Identification of Subunit 4 (Rpb4) of RNA Polymerase II from the Fission Yeast Schizosaccharomyces
pombe. SAKURAI, H., MITSUZAWA, H., KIMURA, M. and
ISHIHAMA, A.
Subunit Assembly of Schizosaccharomyces pombe RNA Polymerase II. KIMURA, M. and ISHIHAMA, A.
Isolation and Characterization ofTemperature-sensitive Mutationsin the Subunit 3 Gene (rpb3) of RNA Polymerase II of the
Fission Yeast Schizosaccharomyces pombe. MITOBE, J.,
MITSUZAWA, H., YASUI, K and ISHIHAMA,A.
Isolation and Characterization ofTemperature-sensitive Mutationsin the Genes for Rpb6, Rpb7 and Rpbll of RNA Polymerase II
ofthe Fission Yeast Schizosaccharomyces pombe. ISHIGURO,
A., MITSUZAWA, H., KOMOTO, M., NOGI, H. and ISHIHAMA, A.
..................................................................................................
Identification ofthe RNA Cap 1-binding Sites on the PB2 Subunit of Influenza Virus RNA Polymerase. HONDA, A.,MIZUMOTO, K and ISHIHAMA, A.
Search for Host Factor(s) Interacting with Influenza Virus RNA
Polymerase. HONDA, A., OKAMOTO, T. and ISHIHAMA, A.....
Expression of Functional Influenza Viral RNA Polymerase in
the Methanotropic Yeast Fichia pastoris. HWANG, S.J.,
YAMADA, K, NAKADE, K and ISHIHAMA, A.
Isolation from TMV-infected Tobacco ofthe RNA-dependent RNAPolymerase with the Subunit Composition of 126K1183K
Heterodimer. WATANABE, T., HONDA, A., IWATA, A., DEDA,
S., HIBI, T. and ISHIHAMA, A.
A-b. Division of Mutagenesis
Characterization of SCFGrrl that ubiquitinates G1 cyclins in Sac-

charomyces cerevisiae. KISHI, T. and YAMAO, F.

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CONTENTS

iii

Ubiquitin pathway functioning in mitotic regulatory mechanism.
TADAKI, T., SEINO, H. and YAMAO, F.
Degradation of Cig2 protein is regulated by ubiquitin-pathway
involving UbcP4 andAPC complex. SEINO, H. and YAMAO,F
Survey and functional analysis of ubiquitin-conjugating enzymes
in S. pombe. Park, J.H., SEINO, H. and YAMAO, F.
A-c. Division of Nucleic Acid Chemistry
The Roles of Mrell of Budding Yeast in Recombination.
TOMIZAWA, J.
A new congenital muscular dystrophy with mitochondrial
structural abnormalities. NISHINO, I., GOTO, Y, HORAI, S.
and NONAKA, I.
Peopling of Japan inferred from mitochondrial DNA polymorphisms in East Asians. HORAI, S. and OMOTO, K.
Confirmation that a T-to-C mutation at 9176 in mitochondrial
DNA is an additional candidate mutation for Leigh's
syndrome. MAKINO, M., HORAI, S., GOTO, Y and NONAKA, I.
..................................................................................................
Phylogenetic analysis of mitochondrial DNA in Japanese pedigrees of sensorineural hearing loss associated with theA1555G mutation. ABE, S., USAMI, S., SHINKAWA, H.,
WESTON, M.D., OVERBECK, L.D., HOOVER, D.M., KENYON,
J.B., HORAI, S., KIMBERLING, w'J.
B. Department of Cell Genetics
B-a. Division of Cytogenetics
Mechanisms ofYeast Genetic Recombination.

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OGAWA, T., OHTA,

T., OSHIUMI, H. and MATSUDA, S.
The hepatitis B virus X protein is a co-activator of activatedtranscription that modulates the transcription machineryand distal binding activators. OHTA, T.
Analysis of the role of TFIIE in transcriptional regulation
through structure-function studies of the TFIIE {3subunit. OHTA, T.

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DNA supercoiling factor localizes to puffs on polytene chromosomes in Drosophila melanogaster. OHTA, T.
45
Biochemical basis ofhyper-recombinogenic activity of Pseudomonas
aeruginosa RecA protein in Escherichia coli cells. ALEXEEV, A.
.................................................................................................. 46
B-b. Division of Microbial Genetics
Dpbll controls association of DNA polymerases with replication
origins in Saccharomyces cerevisiae. MASUMOTO, H. and
ARAKI, H

Sld2, which interacts with Dpbll in Saccharomyces cerevisiae,
is required for chromosomal DNA replication. KAMIMURA,
Y. and ARAKI, H. ..
Function of the Sld5/Psfl complex which interacts with DNA
polymerase II( E) and Dpbll in Saccharomyces cerevisiae.
TAKAYAMA, Y., KAMIMuRA, Y. and ARAKI, H.
Is there an additional monofunctional glycosyltransferase for cellwall peptidoglycan biosynthesis in Escherichia coli? HAM,
H
B-c. Division of Cytoplasmic Genetics
Studies on behavioral disorders of Fyn tyrosine kinase knockout mice. Nna, H.
Studies on behavioral disorders of NMDAR subunit knockout
mice. Nna, H.
C.

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Department of Developmental Genetics
C-a. Division of Developmental Genetics
Regulation of ras signaling by Sprouty and EDL ensures constant outcome of induction. OKABE, M., YAMADA, T., KRAMER,
S. and HIROMI, Y.
54
A screen for proteins that interact with the ligand binding domain of a nuclear receptor, Seven-up. KOSE, H., WEST, S.,SUZUKI, E. and HIROMI, Y.
55
Relationships between organogenesis and positional information in Drosophila imaginal discs. NIWA, N., OKABE, M. and
HIROMI, Y.
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CONTENTS

v

Molecular mechanisms of Neuron-glia developmental decision.
AKIYAMA-ODA, Y., VMFSONO, Y., TAKIZAWA, K and HCYITA, Y. ..

Axon guidance mechanisms. TAKIZAWA, K, HIRAMOTO, M.,
SASAMURA, T., SHINZA, M. and HOTIA, Y.
The gcm transcriptional regulator family. HIGASHI, C., HOSOYA,
T., HOTIA, Y.
Hydra Peptide Project: A Novel Neurotransmitter that Controls
Muscle Contraction in Hydra. YUM, S., TAKAHASHI, T.,
HATTA, M., SHIMIZU, H., KOIZUMI, 0., KOBAYAKAWA, Y andFUJISAWA, T. .
Feeding and wounding response of hydra: hydra nervous system is more advanced than a diffuse system. SHIMIZU, H.
..................................................................................................
Cob. Division of Gene Expression
Chromatin remodeling and transcription. HIROSE, S.
Chromatin remodeling mediated by Drosophila GAGA factor and
ISWI activates fushi tarazu gene transcription in vitro.
OKADA, M. and HIROSE, S.
DNA supercoiling factor localizes to puffs on polytene chromosomes in Drosophila melanogaster. KOBAYASHI, M., AlTA,
N., HAYASHI, S., OKADA, K, OHTA, T. and HIROSE, S.
Yeast coactivator MBFl mediates GCN4-dependent transcriptional activation. TAKEMARU, K, HARASHlMA, S., VEDA, H.
and HIROSE, S.
Comparison of sequences of a transcriptional coactivator MBF2
from three Lepidopteran species Bombyx mori, Bombyx
mandarina and Samia cynthia. LIU, Q.-X., VEDA, H. and
HIROSE, S.
DSIF, a novel transcription elongation factor that regulates RNA
polymerase II processivity, is composed of human Spt4 andSpt5 homologs. WADA, T., TAKAGI, T., YAMAGUCHI, Y,
FERDOUS, A., lMAI, T., HIROSE, S., SUGIMOTO, S., YANO, K,
HATZOG, GA, WINSTON, F., BURATOWSKI, S. and HANDA, H...

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COco Division of Physiological Genetics
NELF, a multisubunit complex containing RD, cooperates withDSIF to repress RNA polymerase II elongation. YAMAGUCHI,
Y., TAKAGI, T., HAsEGAWA, J., WADA, T. and HANDA, H. ........ 67
Gene expression level and species-specific diversity of codon usage based on multivariate analysis. KANAYA, S., YAMADA,
Y., KUDO, Y., IKEMURA, T.
68

D.

Department of Population Genetics
D-a. Division of Population Genetics
Local recombination- and mutation-effects on molecular evolution in Drosophila. TAKANO, T.
Study of species difference as observed as interspecific hybrid
anomaly in Drosophila: genetic screens for genes involvedin the bristle loss of hybrids. TAKANO, T.
D-b. Division of Evolutionary Genetics
Non-B DNA structures spatially and sequence-specificallyassociated with individual centromeres in the human interphase nucleus. OHNO, M., TENZEN, T., WATANABE, Y.,
YAMAGATA, T., KANAYA, S. and IKEMURA, T.
Studies on codon usages and tRNA genes of 18 unicellar organisms and quantification of Bacillus subtilis tRNAs: Gene expression level and species-specific diversity of codon usage
based on multivariate analysis. KANAYA, S. and lKEMURA, T.
..................................................................................................
Triplex-forming DNAs in the Human Interphase Nucleus Visualized In Situ by PolypurinelPolypyrimidine DNA Probes andAntitriplex Antibodies. Triplex-forming DNAs Are Spatially
Associated with Centromeres. OHNO, M., TENZEN, T.,
SASAKI, H., LEE, J.S. and IKEMURA, T.
Early and late replication zone borders are present in and aroundthe human X-inactivation center (XIe): correlation with chromosome bands. WATANABE, Y., TENZEN, T. and IKEMURA, T.
..................................................................................................

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Compartmentalized DNA-replication timing and GC% levels inand around the human MHC region. TENZEN, T., YAMAGATA,
T., NOGAMI, M., WATANABE, Y., RANAYA, S., ANDO, A., INOKO,
H. and IKEMURA, T.
Conserved evolution of the Rh50 gene compared to its homologous
Rh blood group gene. KITANO, T., SUMIYAMA, K, SIllROISHI, T.
and SAITOU, N.
D-c. Division of Theoretical Genetics
Evolution by nearly-neutral mutations. OHTA, T.
On the pattern of polymorphisms at major histocompatibility
complex loci. OHTA, T...
E.

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Department of Integrated Genetics
E-a. Division of Human Genetics
Cloning and structural analysis ofthe mouse 7F4/F5 imprinted
domain. SASAKI, H., RATO, R., YOKOMINE, T., MIZUNO, S.,
SIllROHZU, H., ISHIHARA, K and MUKAI, T.
Regulation of imprinting of the mouse Igf2/H19 sub·domain.
SASAKI, H., ISHIHARA, K, RATO, R., FURUUMI, H., ORNO, M.,
ZUBAIR, M., HATANO, N. and IWAKI, T.
Cloning of a family of novel mammalian methyltransferases.
SASAKI, H., CHIJIWA, T., FURUUMI, H., PuRBowASITO, w., MIzuNo,
S., TAJIMA, S. and KONO, T.
Human genome resources and their application for the analysisof chromosome21. FUJIYAMA, A., MOTOYAMA, A. and PARK,
H.-S
Whole genome analysis of signal-transduction pathways infission yeast. BONG, Y.-S., DANJO, I. and FUJIYAMA, A. ......
E·b. Division of Applied Genetics
Expression pattern of the a-amylase gene during seed germinationin rice and barley. SUGIMOTO, N., TAKEDA, G., NAGATO, Y. and
YAMAGUClll, J.
panicle phytomer 1 mutations affect the panicle architecture of
rice. TAKAHASlll, M., NAGASAWA, N., KITANO, H. and NAGATO,Y................................................................................................

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A recessive heterochronic mutation, p1astochron 1, shortens theplastochron and elongates the vegetative phase in rice.
ITOH, J., HASEGAWA, A, KITANO, H. and NAGATO, Y.
86
Isolation and characterization of a rice homeobox gene,
OSH15. SATO, Y, SENTOKU, N., NAGATO, Y and MATSUOKA, M.
.................................................................................................. 86
Molecular mechanisms of hereditary neurodegenerative
diseases. TSUJI, S.
87

R Genetic Strains Research Center
F-a. Mammalian Genetics Laboratory
Analysis of the genetic structure of Ph hotspot in the classII of
MHC. ISOBE, T., YOSHINO, M., LINDAHAL, KF., MIZUNO, K,
KOIDE, T., MORIWAKI, K, SHIROISHI, T.
Mapping of modifier gene suggests interaction oftwo preaxial polydactylous mutant genes, Rim4 and 1st, in mouse. MASUYA,
H., SAGAI, T. and SHIROISHI, T.
Phenotype characterization and fine mapping of a preaxial polydactyly mutant, luxate (lx). YADA, Y, MASUYA, H., MITA, A
and SHIROISHI, T. ..
Physical mapping ofpolysynductylous mutation, hemimelic extra toes (Hx). SAGAI, T., MASUYA, H. and SHIROISHI, T. ......
Positional cloning of the mouse skeletal mutation, TaB-short(Ts). SHIMIZU, K, KOIDE, T., MITA,A, UCHIDA, K, WAKANA,
S., KIKKAWA, Y, YONEKAWA, H., SASAKI, H. and SHIROISHI, T.
..................................................................................................

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Polymorphism at Tail-short (Ts) locus among standard inbred
strains affects the viability of Ts heterozygotes. ISHIJIMA,
J., MITA, A, UCHIDA, K, SHIROISHI, T.
96
Cloning of a responsible gene for mouse mutant Rim3. SAEKI,
N., SATO, H., SAGAI, T., MASUYA, H., SHIROISHI, T.
97
Genetic and physical mapping of Idd4 that controls onset of
IDDM in mice. WAKANA, S., SHIROISHI, T., MORIWAKI, K,
MARUYAMA, C., WATANABE, Y, NOMURA, T.
97
Behavioral study of inbred strains established from wild mice.

CONTENTS
II. Learning and memory. KOIDE, T., MORIWAKI, K. and
SHIRaIsHI, T.
98
F -b. Mammalian Development Laboratory
Analysis of Cellular and Molecular Mechanisms in the Development of Mouse Fetal Germ Cells and Sex Differentiation of
Gonads. NAKATSUJI, N., TAMURA, M., CHUMA, S., KANNO,
Y., SAKURAI, T., HUANG, Z., SAITO, T. and TADA, T.
100
Molecular mechanism controlling neuronal differentiation in
mammals. SAITO, T., HAMA, T., SAKAMOTO, S., SABA, R. and
NAKATSUJI, N.
102
Reprogramming of parental-origin-specific epigenotype
in mouse germ-line development. TADA, T., TADA, M. and
NAKATSUJI, N.
103
F -c. Plant Genetics Laboratory
Structural and Functional Analysis of the Genes Expressed in
Early Embryogenesis and Regeneration in Rice (Oryza sativa)(l)-a. Functional Analysis of Homeobox Genes Expressed in Embryogenesis and Regeneration Process in Rice. ITO, Y.,
CHUJO, A., TAKAYA, K., NIWA, Y, KURATA, N.
106(l)-b. Cloning and StructurallFunctional Analysis of Four LEC 1
Related Genes in Rice. MIYOSHI, K., KURATA, N.
108
Study for the Nucleus and Chromosome Organization in Rice.
(2)·a. Isolation and Structural Analyses for The Centromeric
Region of Rice Chromosomes Toward Construction of Rice
Artificial Chromosome (RAC). NONOMURA, K., KURATA, N.
.................................................................................................. 108(2)-b. Production of Mini-Chromosome Rice Lines for Studying
Centromere Structure. NONOMURA, K., KURATA, N.
109
Analysis of Genetic Factors causing Segregation Distortion(3)·a. Quantitative Analysis of genotype segregation for reproductive barriers. HARUSHIMA, Y, KURATA, N.
110(3)-b. Positional Cloning of a Segregation Distortion Gene Detected in a Progeny of a Cross between japonica and indicaix

x

nee. HARUSHIMA, Y., KURATA, N.
Generation of Enhancer Trap Lines of Rice. ITO, Y., EIGUCHI,
M., KURATA, N.
Generation and Screening of Retrotransposon Insertion Linesin Rice. EIGUCHI, M., NAGATO, Y., MIYOSHI, K., ITO, Y.,
KURATA, N..
F -d. Microbial Genetics Laboratory
Timing of cell division in Escherichia coli. NISHIMURA, A. ......
Hsc66 Participates in The FtsZ Ring Formation in Escherichia
coli. UEHARA, T., MATSUZAWA, H. and NISHIMURA, A.
Post genome project: Systematic analysis of cell division genes
in Escherichia coli. HORIE, M., SAKA, K., TADENUMA, M.,
MATSUMOTO, K., ITO, H., MORl, H., SUGAWARA, H., GOTO, K.,NISHIMURA, A.
F -e. Invertebrate Genetics Laboratory
Specification and proximo-distal pattern formation of the wing
and leg in Drosophila. GOTO, S., KUBOTA, K. and HAYASHI,
S..
Cell biological study of the tubular epithelial network formationin the tracheal system. IKEYA, T., CHIHARA, T., TANAKAMATAKATSU, M. and HAYASHI, S..
Kinase-independent activity of Cdc2/Cyclin A prevents S phasein the Drosophila cell cycle. HAYASHI, S. and YAMAGUCHI,
M.
plexus, a gene required for adult wing vein patterning. MATAKATSU,
H., GAMOU, S., TADOKORO, R. and HAYASm, S..
Enhancer trap screen for genes involved in pattern formation. GoTo,
S., TAKEucm, H., TANIGUCHI, M. and HAYASm, S.
Interaction between Drosophila EGF receptor and vnddetermines
three dorsoventral domains ofthe neuroectoderm. YAGI, Y.,SUZUKI, T. and HAYASHI, S
..

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G. Center for Genetic Resource Information
G-a. Genetic Informatics Laboratory
Cross-species Database. YAMAZAKI, Y.
.
Genetic Resources Database2-1. Wheat Genetic Resources Database -KOMUG1. YAMAZAKI,Y., TSUJIMOTO, H., KAWAHARA, T. and OGIWARA, Y.
2-2. Rice Genetic Resources in Japan. YAMAZAKI, Y., KURATA,
N., YOSHIMURA, A. and NAGATa, Y.
.
2-3. Barley Germplasm Database. SATO, K., SAITO, M. and
YAMAZAKI, Y.
2-4. Mouse Microsatellite Database. SHIROISHI, T., TSUCHIYA,
R. and YAMAZAKI, Y.
2-5. Profiling ofE.coli chromosome database. KATO, J., IKEGAMI,
T., YAMAKAWA, T., MITSUI, K., YAMAZAKI, Y.
2-6. Arabidopsis genetic resource database. GoTO, S., TSUCHIYA,R., YAMAZAKI, Y.
G-b. Genome Biology
Systematic analysis of mRNA distribution by whole mount insitu hybridization. MOTOHASHI, T., OHBA, T., SUGIURA, 1.,
OBARA, M., KITAYAMA, S., SUZUKI, T., SHIN-I, T. and KOHARA,Y.

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NEXTDB:The nematode expression pattern map database.
SHIN-I, T. and KOHARA, Y.
Expressed Genes in C. elegans. THIERRY-MIEG, J., THIERRYMIEG, D., SHIN-I, T. and KOHARA, Y..
Protein Expression PatternAnalysis ofMatemal mRNAs. ONAMI,
S., NAGAOKA, T. and KOHARA, Y.
Systematic RNAi experiments with maternal genes. HIRaNo,..
K., ONAMI, S. and KOHARA, Y
Toward a four-dimensional database of gene expression in
C. elegans. ITOH, M., MOTOHASHI, T., MINAKUCHI, Y. and
KOHARA, Y..
Pos-l encodes a cytoplasmic zinc-finger protein essential for
germline specification in C. elegans.

TABARA, H., HILL, R.J.,

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MELLO, C., PRIESS, J.R. and KOHARA, Y.
128
Studies on POS-1 interacting proteins. OGURA, K. and KoHARA,
Y.
131
tbx-9 encodes a transcription activator. ANDACHI, Y.
132

H. Structural Biology center
H-a. Biological Macromolecules Laboratory
Single Molecule Imaging of Biological Functions. TOKUNAGA,
M
Single molecule capture, manipulation and force measurementof protein molecules. KITAMURA, K., IWANE, A., YANAGIDA,
T. and TOKUNAGA, M.
Sliding movements of actin filaments undergoing fluctuations
propelled by myosin subfragment-1 flexibly bound to thesurface. TOKUNAGA, M., IWANE, A., KITAMURA, K. and
YANAGIDA, T.
Molecular interactions measured by nanometer-sized probes using
subpiconewton intermolecular force microscopy. HIROSHIMA,
M. and TOKUNAGA, M.
A New Model of Molecular Motors. TOKUNAGA, M.
H-b. Molecular Biomechanism Laboratory
Branched pathway mechanism of transcription initiation by E.
coli RNA polymerase, and promoter-arrested initiation
complexes. KUBORI, T., NAGAI, H., Gaal, T. and SHIMAMOTO,

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N............................................................................................... 137
Role of the spacer region of a 70 and Inactivation by oligomerization.
NAGAI, H., KASCIUKovIC, T., Hayward, R.S., SATO, Y. and
SHIMAMOTO, N.
138
Single-Molecule Dynamics ofTranscription: Sliding ofproteins along
DNA. KINEBUCHI, T., KABATA, H., TAKEUCHI, M., SHIMAMOTO,
N., KUROSAWA, 0., ARAMAKI, H., W ASHIZU, M.
139
H-c. Multicellular Organization Laboratory
Fluoride-resistant Mutants of the Nematode Caenorhabditis
elegans. TAKE-UCHI, M., OISHI, A., ISHIHARA, T. andKATsURA,
1.
141

CONTENTS
Analysis of Synthetic Dauer-constitutive Mutations. MIYAHARA,
K, ISHIHARA, T. and KATSURA, 1.
Analysis of che-2, a gene required for the sensory cilia in
C. elegans. FUJIWARA, M., ISHIHARA, T. and KATSURA, 1. ...
Functional Analysis of Interneurons as Studied by Selection between Two Behaviors. ISHIHARA, T. and KATSURA, 1.
Reverse Genetics of Genes Containing PDZ domains. HISHIDA,
R, ISHIHARA, T. and KATSURA, 1.
H-d. Biomolecular Structure Laboratory
Crystallographic Study of F1-ATPase: Structural Analysis ofSupramolecule. SHIRAKIHARA, Y.
Crystallographic Study of the Transcription Activator,
PhoB. AKIBA, T. and SHIRAKIHARA, Y.
Crystallographic Study of the Transcription Repressor,
CamR FUKUSHI, K and SHIRAKIHARA, Y.
Crystallization of RNA Polymerase and its subunits. MURAKAMI,
K, KIMURA, M., ISHIHAMA, A. and SHIRAKIHARA, Y.
Crystallization of Mrell fragment from yeast. MURAKAMI, K,
OHTA, T., OGAWA, T. and SHIRAKIHARA, Y.
Crystallization of Na+-translocating ATPase. SHIRAKIHARA, Y.
..................................................................................................
I.

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Center for Information Biology
I-a. Laboratory for DNA Data Analysis
Different evolutionary histories in two subgenomic regions of
the Major Histocompatibility Complex. GAUDIERI, S.,KULSKI, J.K, DAWKINS, RL. and GOJOBORI, T.
Coevolution ofPERBll (MIC) and HLA Class I Genes with HERV-16
and Retroelements by Extended Genomic Duplication. KULSKI,
J.K., GAUDIERI, S., MARTIN, A. and DAWKINS, RL.
Extensive nucleotide variability within a 370 kb sequence from thecentral region of the Major Histocompatibility Complex.
GAUDIERI, S., KULSKI, J.K., DAWKINS, R.L. and GoJOBORI, T.
Elevated levels of non-coding polymorphism within the Major
Histocompatibility Complex. GAUDIERI, S., DAWKINS, R,

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HABARA, K and GOJOBORI, T.
154
Characterisation of the PERBll gene family in Primates.
CATTLEY, S.K, LONGMAN, N., GAUDIERI, S., DAWKINS, R.L.
and LEELAYUWAT, C.
155
Identification of Neural pathways in Planarian. TAZAKI, A.,GAUDIERI, S., IKEO, K, GOJOBORI, T., WATANABE, K and
AGATA, K.
156
A low rate of nucleotide changes in Escherichia coli K-12
estimated from a comparison of the genome sequences
between two different substrains. ITOH, T. and GOJOBORI, T.
.................................................................................................. 156
Slow Evolutionary Rate ofGB Virus ClHepatitis G Virus. SUZUKI,
Y., KATAYAMA, K, FUKUSHI, S., KAGEYAMA, T., OYA, A.,
OKAMURA, H., TANAKA, Y., MIZOKAMI, M., GOJOBORI, T.
158
Intragenic Recombinations in Rotaviruses. SUZUKI, Y., GoJOBORI,
T., NAKAGOMI, O.
158
I-b. Laboratory for Gene-Product Informatics
Feasibility of the inverse protein folding protocol. OTA, M. andNISHIKAWA, K.
162
Genes from nine genomes are separated into their organisms inthe dinucleotide composition space. NAKASHIMA, H., OTA,
M. and NISHIKAWA, K.
162
Constructing the Protein Mutant Database. KAWABATA, T., OTA,
M. and NISHIKAWA, K.
163
I-c. Laboratory of Gene Function Research
DNA Data Bank of Japan at Work on Genome Sequence Data.
TATENO, Y., FUKAMI-KOBAYASHI, K, MIYAZAKI, S., SUGAWARA,
H. and GOJOBORI, T.
164
Formal design and implementation of improved DDBJ DNA
database with a new schema and object oriented library.
OKAYAMA, T., TAMURA, T., GOJOBORI, T., TATENO, Y., lKEo, K,
MIYAZAKI, S., FUKAMI·KoBAYASHI, K and SUGAWARA, H.

165

CONTENTS
I-d. Laboratory for Molecular Classification
Data processing system for DDBJ. SUGAWARA, R., MIYAZAKI,S., GOJOBORI, T., TATENO, Y., IKEO, K., FUKAMI-KOBAYASHI,
K., TAMURA, T. and OKAYAMA, T.
Information system for the study of biodiversity. SUGAWARA,
R., MIYAZAKI, S. and OGAWA, R.

xv

166
166

J.

RadIoisotope Center
Functional analysis of the phoB/cotA region of the Bacillus
subtilis chromosome. SADAIE, Y., YATA, K. and OHSHIMA,
R
169
Molecular Organization of Restriction and Modification System
Genes of Bacillus subtilis. OHSHIMA, R., YATA, K. andSADAIE, Y.
170
Rapid isolation of RNA polymerase from sporulating cells ofBacillus subtilis. FUJITA, M. and SADAIE, Y.
170

K.

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

N............................................................................................... 172
Abstracts of diary for 1998
Foreign visitors in 1998
Association for propagation of the knowledge of genetics
Author index
,.

173
179
180
182

GENERAL STATEMENT

National Institute of Genetics (NIG) was established 49 years ago as acenter for genetics research. Major contributions of NIG in the research ingenetics, in particular, plant genetics, population genetics and molecular anddevelopmental genetics, have made it one of the distinguished centers withworldwide recognition. In 1984, NIG was reorganized into an Inter-university Research Institute to promote collaborative activities. Together withseven inter-university research institutes, we founded the Graduate University for Advanced Studies in 1988. We are serving as Department of Geneticsof the Graduate School of Life Science. This year, we have 32 graduate students and 10 special research students from other universities, including thosefrom abroad. Eight students obtained Ph.D. this year. In addition, we have
the Center Of Excellence (COE) program with which 5 foreign and 8 Japanese
postdoctoral fellows conducted research.
We have been carrying out several research-related services. The DNA Data
Bank of Japan (DDBJ) is one of the three central data banks in the world thatgather, annotate, store and distribute information on DNA sequences. In
recent years, it receives data input not only from Japanese institutions but
also from institutes in other Asian countries. Recent development of newtechnology using various model organisms has enhanced the importance ofgenetics as the bases of many branches of biological studies. We have established Genetic Strains Research Center and Genetic Resource Information
Center that are designed to organize and support the use of genetic strains
and resources. Our institute also stores and distributes various organisms
with genetically characterized traits. Among them, services with Escherichia coli, mice and Drosophila are particularly significant. These service activities will continue to develop in the coming years. Our institute is uniquely
suited for pursuing cooperative work with scientists of various disciplines by
sharing the genetic resources.
This year, we have started two new research groups, DIvisions of Early Embryogenesis and of Brain Functions. Such complex biological systems have2

been new targets of genetics studies. Recent development of various molecular and developmental technologies are now enabling us to genetically dissecting these biological mechanisms. We would like to exploit fundamental anduniversal nature of genetics to tackle these difficult biological problems. We
hope that such new activities will rejuvenate NIG in the 21"t century.
In the past year we also saw a number of changes in the staff members of
NIG. Prof. Takashi Imamura, Division of Human Genetics, and Prof. Hiroko
Okino-Morishima, Division of Agricultural Genetics retired at the end of
March. We would like to express our gratitude for their contribution to ourinstitute. Newly appointed as professors are Drs. Hiroyuki Araki, Division of
Microbial Genetics and Hiroyuki Sasaki, Division of Human Genetics. Dr.
Toshihiko Shiroishi was also promoted as a professor of Mammalian Genetics
Laboratory, and Prof. Yuji Kohara moved from Gene Network Laboratory to
newly established Genome Biology Laboratory. Promoted as research associates are, Dr. Toshihiko Hosoya, Division of Developmental Genetics, Dr.
Yoichiro Kamimura, Division of Microbial Genetics, and Dr. Takashi Tada,
Mammalian Development Laboratory. On the other hand, the following members left NIG to take new positions to extend their career. Among them are
Prof. Satoshi Horai (Graduate University for Advanced Studies), Drs. HongWei Cai (Japan Forage Seed Association), Hiroshi Hara (Saitama University), Yasuaki Shirayoshi (Tottori University) and Toshihiko Akiba (MinistryofAgriculture, Forestry and Fishery).

Yoshiki Hotta

3

STAFF

(as of December 31,1998)

Director-General
HO'ITA, Yoshiki, D. Med.

Vice-Director
OGAWA, Tomoko, D. Pha.

Members
1. Department 01 Molecular Geneties
ISHIHAMA, Akira, D. Sc., Head ofthe Department
Division ofMolecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUJITA, Nobuyuki, D. Sc.
MITSUZAWA, Hiroshi, D. Sc.
KIMURA, Makoto, D. Sc.
Division ofMutagenesis
YAMAO, Fumiaki, D. Sc., Associate Professor
KISHI, Tsutomu, D. Eng.
SEINO, Hiroaki, D. Sc.
Division ofNucleic Acid Chemistry
TOMIZAWA, Jun-ichi, D. Pha., Adjunct Professor
HORAI, Satoshi, D. Med., Adjunct Professor
2. Department 01 CeO Genetics

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

4

Division ofCytoplasmic Genetics
OHTSUBO, Eiichi, D. Sc., Adjunct Professor
NIKI, Hiroaki, D. Med. Sci., Ph. D. Adjunct Professor
3. Department ofDeJ'elopmentaJ 6eneUcs

HIROSE, Susumu, D. Sc., Head ofthe Department
Division ofDevelopmental Genetics
HIROMI, Yasushi, D. Sc., Professor
FUJISAWA, Toshitaka, Ph. D., Associate Professor
SHIMIZU, Hiroshi, D. Eng.
HATTA, Masayuki, D. Sc.
OKABE, Masataka, M. D., Ph. D.
HOSOYOA, Toshihiko, D. Sc.
Division ofGene Expression
HIROSE, Susumu, D. Sc., Professor
MURAKAMI, Akio, D. Ag., D. Sc., Associate Professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-aki, D. Ag.
UEDA, Hitoshi, D. Ag.
Division ofEarly Embryogenesis
Division ofPhysiological Genetics
HANDA, Hiroshi, D. Med., Adjunct Professor
KANAYA, Shigehiko, D. Ind., Adjunct Associate Professor
4. Department ofPopulatIon 6eneUcs

IKEMURA, Toshimichi, D. Sc., Head ofthe 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.
Division ofTheoretical Genetics
HARADA-OHTA, Tomoko, Ph. D. D. Sc., Adjunct Professor

STAFFKITANO, Hiroaki, D. Eng., Adjunct Professor
5. Department olIntepated 6eneUcs

OGAWA, Tomoko, D. Pha., Head of the Department

Division ofHuman Genetics
SASAKI, Hiroyuki, D. Med., Professor
FUJIYAMA, Asao, D. Sc., Associate Professor

Division ofAgricultural Genetics
Division ofBrain Function
Division ofApplied Genetics
NAGATO, Yasuo, D. Ag., Adjunct Professor
TSUJI, Shoji, D. Med., Adjunct Professor
6. 6eneUc Strains Researcb Center
NAKATSUJI, Norio, D. Sc., Head ofthe Center

Mammalian Genetics Laboratory
SHIROISHI, Toshihiko, D. Sc., Associate Professor
KOInE, Tsuyoshi, D. Med.

Mammalian Development Laboratory
NAKATSUJI, Norio, D. Sc., Professor
SHIRAYOSHI, Yasuaki, D. Sc.
SAITO, Tetsuichiro, D. Sc.

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

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

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

7. Centerlor 6eneUc Resource In/ormation
KOHARA, Yuji, D. Sc., Head ofthe Center
Genetic Informatics Laboratory

5

6

YAMAZAKI, Yukiko, D. Sc., Associate Professor
FUJITA, Masaya, D. Eng.
Genome Biology Laboratory
KOHARA, Yuji, D. Sc., Professor
ANDACHI, Yoshiki, D. Sc.

IJ. StructuralBioJolfY Center
KATSURA, Isao, D. Sc., Head of the Center

Biological Macromolecules Laboratory
TOKUNAGA, Makio, D. Sc., Associate Professor

Molecular Biomechanism Laboratory
SHIMAMOTO, Nobuo, D. Sc., Professor
NAGAI, Hiroki, D. Sc.
MulticeJIuar Organization Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.
Biomolecular Structure Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate Professor
Gene Network Laboratory
fl. Center for Information BiololfY
GOJOBORI, Takashi, D. Sc., Head ofthe Center

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

Laboratory for Gene-Product Informatics
NISHIKAWA, Ken, D. Sc., Professor
OTA, Motonori, D. Sc.
Laboratory for Gene Function
TATENO, Yoshio, Ph. D. D. Sc., Professor
FUKAMI-KOBAYASHI, Kaoru, Ph. D.

Laboratory for Molecular Classification
SUGAWARA, Hideaki, D. Eng., Professor

STAFFMIYAZAKI, Satoru, D. Sc.

16. Radioisotope Center
SADAIE, Yoshito, D. Sc., Associate Professor, Head of the Center
11. experimental Jiilrm
KURATA, Nori, D. Ag., Head of the FarmNONOMURA, Ken-ichi, D. Ag.
12.

Teclmlcal Section
MITA, Akihiko, Chief of the Section

13. Department 01 Administration
SUNADA, Hitoshi, Head of the Department
KOBAYASHI, Akira, Chief of the General Affairs Section
KOSEKI, Kenzo, Chief of the Finance Section

7

8

COUNCIL(as of December 31,1998)

Chairman
SUGIMURA, Takashi; President,Toho University
Vice-chairman
OSAWA, Shozo; Adviser, Biohistory Research Hall
Members (Alphabetical order)
HAMA, Kiyoshi; President, Okazaki National Research Institutes
HIROTA, Eiji; President, The Graduate University for Advanced Studies
ISHII, Shiro; Professor, The International Research Center for JapaneseStudies
IWATSUKI, Kunio; Professor, College of Science Rikkyo University
KURODA, Reiko; Professor, Graduate School ofArts and Sciences, The University of Tokyo
KYOGOKU, Yoshimasa; Director, Institute for Protein Research, Osaka University
MATSUBARA, Ken-ichi; Vice-Director,International Institute for Advanced
Studies
MATSUO, Minoru; President, Nagoya University
MIURA, Kin-ichiro; Director,Institute for Biomolecular Science, Gakushuin
University
MIYAMOTO, Misako; President, Nihon Women's University
MOHRI, Hideo; Director-General, National Institute for Basic Biology
OKADA, Masukichi; Professor Emeritus, University of Tsukuba
OSAKI, Hitoshi; Director, Japan Society for the Promotion of Sciences
OTSUKA, Eiko; Professor, Faculty of Pharmaceutial Sciences, Hokkaido
University
TANAKA, Ryuso; President, Hiroshima City University
TOYOSHIMA, Kumao; President, The Center for Adult Diseases, Osaka
TSUNEWAKI, Koichiro; President, Fukui Prefectural University
YAMANOUCHI, Kazuya; Senior Scientific Staff, Nippon Institute for Biological Sciense

9

ADVISORY COMMITTEE(as of December 31,1998)

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
HINATA, Kokichi; Representative Managing Director, Research Instituteof Seed Production Co., Ltd.
ISONO, Katsumi; Professor, Faculty of Science, Kobe University
ITO, Koreaki; Professor, Institute for Virus Research, Kyoto University
KATSUKI, Motoya; Professor, The Institute of Medical Science, The
University of Tokyo
MATUURA, Etsuko; Professor, Faculty of Science, Ochanomizu University
SASAZUKI, Takehiko; Professor, Medical Institute ofBioregulation, Kyusyu
University
TAJIMA, Fumio; Professor, Graduate School ofScience, 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
lKEMURA, Toshimichi; Professor, National Institute of Genetics
ISHIHAMA, Akira; Professor, National Institute of Genetics
KATSURA, Isao; Professor, National Institute of Genetics
KOHARA, Yuji; Professor, National Institute of Genetics
NAKATSUJI, Norio; Professor, National Institute of Genetics
SASAKI, Hiroyuki; Professor, National Institute of Genetics

10

RESEARCH ACTIVITIES IN 1998

A.

DEPARTMENT OF MOLECULAR GENETICS
A-a. Division of Molecular Genetics(1)

Mapping of Subunit-subunit Contact Surfaces on the {3 and {3 •Subunits of Escherichia coli RNA Polymerase

Akira KATAYAMA, Tasuku NOMURA, Nobuyuki FUJITA and Akira ISHIHAMA
The assembly of Escherichia coli RNA polymerase takes place in a stepwise fashion under the order: 2 ~a2 - 4 0; {3 - 4 0; {3{3 '(premature core) - 4 Eo
(E represents the core enzyme with the subunit structure a2{3 (3 '). The a subunit plays a key role in the assembly of core enzyme by providing the contact
surface for both {3 and {3'subunits. In the assembled core enzyme, one a subunit contacts with {3 subunit while the other a makes direct contact with
{3' subunit. The two large subunits, {3 and {3', do not form stable binary complexes under isolated states, but after the core enzyme assembly, do makedirect contact, leading to formation of the catalytic site for RNA
polymerization. Previously we performed the fine mapping of subunit-subunit contact sites on the a subunit for dimerization and binding of {3 and {3'subunits (Kimura and Ishihama, 1995; 1996). For mapping ofthe subunitsubunit contact sites on the two large subunits, we employed two approaches:(i) analysis ofproteolytic cleavage sites on both {3 and {3' subunits for unassembled
free subunits, the intermediate subassembly 0; {3 complex, the core enzyme,
and the {3'o70complex; and (ii) analysis of complex formation between {3 fragments and the a subunit, between {3' fragments and the 0; {3 complex, andbetween {3' fragments and 0 70 subunit.
Results of the {3 subunit analysis indicated that two regions of the {3 subunit are involved in the full activity of a binding, i.e., the primary contact site
between residues 737 and 904, and the secondary region with assembly con-A.

DEPARTMENT OF MOLECULAR GENETICS

IItrol activity downstream from residue 1138. The assembly activity of 13 fragments was also examined in the presence of both a and 13' subunits. Pseudocore complexes consisting of a, 13 fragment and 13' were formed for 13(1-1318),
13 (445-1342) and 13 (737-1342), which all retain nearly the full activity of abinding. The level of pseudo-core complex formation is much lower for 13(7371138) in agreement with its low level activity of a binding. Thus, we conclude
that the primary contact site of 13' subunit on 13 is located close to the a -subunit contact sites, indicating that the binding sites for a and 13 ' subunits forma single and the same structural domain (Nomura et a1., 1999).
For mapping of the subunit-subunit contact sites on the 13 ' subunit of E. coli
RNA polymerase with the 0; 13 complex or the 0 70 subunit, we employed two
approaches. First, the protease sensitive sites were determined for unassembled
free 13' subunit (1407 amino acid residues). To identify the subunit-subunit
contact sites on the 13 ' subunit, we performed the binding assays of mixturesof proteolytic fragments of 13' with either the azl3 complex or the 0 70
subunit. For detailed analysis, we then prepared a series of recombinant 13 'fragments with or without His 6 -tag by expressing the respective cloned genesegments, and tested for their activities of the binding of azl3 complex or 0 70
subunit. Taken these results together we conclude that: (i) the two regions
between residues 515-842 and 1141-1407 are involved in binding of the 0; 13
complex, and in addition, the N-terminal proximal region, residues 1-345, hasa weak activity of azl3 binding; and (ii) the N-terminal proximal region, between residues 201-345, is central for binding of the 0 70 subunit, and otherregions, between residues 515-842 and 1141-1407, have weak activity of 0 70
subunit binding (Katayama et a1., 1999). The sites involved in the catalytic
function of RNA polymerization are all located within two spacer regions sandwiched between these three subunit-subunit contact surfaces.
(!)

Search for Novel Transcription Factors Interacting with the 13 andfJ' Subunits of Escherichia coli RNA Polymerase

Tasuku NOMURA, Akira KATAYAMA, Nobuyuki FUJITA, Claude F. MEARES l andAkira ISHIHAMA (IDept. Chem., Univ. Calif., Davis, USA)RNA polymerase of Esherichia coli is composed of the core enzyme with the12subunit structure a2 {3 {3' and one of seven species of the sigma subunit. The
N-terminal domain of a subunit plays a key role in core enzyme assembly
while its C-terminal domain interacts with class-I (a -contact) transcription
factors and upstream elements (UP) of promoter DNA. Some of E. colitranscription factors, designated as class-II (a -contact) factors, interact with thea subunit and regulate its function. Recently, several observations have beenreported, which indicate that some E. coli transcription factors interact withthe {3 and/or {3' subunits. A systematic search for the class-III ({3 -contact)and class-IV ({3'-contact) factors is in progress in this laboratory.
One new approach we employed for detection of protein-protein contacts isthe phage display screening. This method is successfully used for mapping ofan epitope against an antibody and for search of a peptide ligand against a
receptor. One advantage is the large scale of screening (10 8-10 9 phage screening per experiment) and another is the easy amplification of phage clones for
DNA sequencing. Filamentous phages used as a library display 15-mer random peptides fused to coat protein pIlI (product of phage gene III) on a phagesurface. A screening was carried out by mixing the phage library and a tethered ligate on a dish or a resin and followed by washing and elution of boundphages. So far we isolated phage clones which specifically bind to the {3 subunit of RNA polymerase, which was, after biotinylation, tethered on a surfaceof polystyrene dish mediated by streptoavidine-biotin bond. Results of thesequencing of 15-mer inserts of the cloned phages indicated that the majority
are originated from non-specific binding of phages to the {3 subunit, the biotinstreptoavidin conjugate or even to plastic dishes, because many clones contained several tryptophan residues in the 15-mer region. Likewise, when
Ni 2+ -NTA agarose resin was used for immobilization of His 6-tagged {3 subunit,
most clones contained several His residues. To exclude false positive clones,
the binding assay by using ELISA will be carried out after the isolation of thephage clones bound to the {3 subunit. A screening against anti- {3 subunit
antibodies will be performed to get a positive control for ELISA. The phageclones thus isolated should contain the epitope seqeuence of the {3 subunit.
The other approach for a large scale screening of proteins which interact
with either {3 or {3' subunit is to employ an affinity chromatography using
glutathione S-transferase (GST) gene fusion system. Both {3 and {3' subunits

A.

DEPARTMENT OF MOLECULAR GENETICS

13

of E. coli RNA polymerase were expressed as GST fused proteins under thecontrol of tacpromoter in E. coli strain W3110 in the absence of inducer IPTG.
Cell lysate prepared by sonication was centrifuged to remove ribosomal fractions and the supernatant was applied onto glutathione Sepharose column.
After washing the column, bound proteins were eluted with a buffer containing
glutathione, and separated by SDS-PAGE. Each protein band was subjectedto N-terminal amino acid micro sequencing. After N-terminal amino acidsequencing, we have detected several non-RNA polymerase proteins, including
the 110 kDa RapAlHepA protein which has been identified to be associatedwith the purified RNA polymerase. Likewise, we have identified several proteins associated the His 6-tagged {3' subunit.
In collaboration with C.F. Meares and colleagues (DC Davis), search for theclass-III (8 -contact) and class-IV (8' -contact) transcription factors is also inprogress using the novel method of contact-dependent protein cleavage with achemical protease tethered to known transcription factors. Previously weidentified the 0 70 subunit-contact sites on both 8 and 8 ' subunits by mapping
the cleavage sites generated by FeBABE tethered at various positions along
0 70 (Owens et al., 1998). As an extension ofthis line studies, we have succeded
to identify the contact sites ofthe NusA, GreA and w proteins on 8 and/or 8'
subunits (Traviglia et aI., 1999).

(3)

Structural requirements for the interdomain linker of a subunitof EscherIchIa colJ RNA polymerase

Nobuyuki FUJITA, Shizuko ENDO and Akira ISHIHAMA
The a subunit of Escherichia coli RNA polymerase is comprised of two structurally independent domains. The N-terminal domain (residues 1 to 235) isrequired and sufficient for core enzyme assembly, while the C-terminal domain (residues 249 to 329) plays key roles in transcription regulation by directly interacting with either class-I (a-contact) transcription factors or promoter UP elements. The intervening sequence connecting these two domains
has high motional flexibility as revealed by the analysis of NMR dynamics(Jeon et aI., 1997). The flexible nature of interdomain linker is also suggested by the findings that the C-terminal domain can be located along a long

14

range of the template DNA in different initiation complexes (Murakami et a1.,
1997). To analyze structural requirements, if any, for the interdomain linker,
we introduced various mutations in this region and measured in vitro transcription activity after reconstitution of the mutant a subunits into respective
holoenzymes.
Deletion of 3 amino acids from the interdomain linker exhibited 50% inhibition of the CRP-dependent 1acP1 transcription. Deletion of6 or more aminoacids completely knocked out the CRP-dependent activity. Insertion of 3
amino acids did not affect the activity whereas insertion of 1, 2, or 4 aminoacids showed 40-60% inhibition. Substitution of 10 consecutive glycine residues for almost the entire region of interdomain linker showed nearly 90%
inhibition of the CRP-dependent activity. If several conservative amino acidsubstitutions were introduced so that the linker forms a strong alpha-helix
without changing the length and charge distribution, nearly 50% activity wasretained. In contrast, if the entire linker sequence was redesigned so as toform an amphiphilic helix, no activation was observed at all. These results
altogether suggest that: (i) a sufficient length of the interdomain linker isrequired for the activation function of C-terminal domain; (ii) the C-terminal
domain has a preferred orientation relative to the rest of RNA polymerase andthe interdomain linker is not totally free for rotation; and (iii) some amino acidside chains, in addition to the flexibility ofthe peptide main chain, are important probably through the formation of loose helix-like structure for theformatiion of functional C-terminal domain. The UP element-dependent
rrnBP1 transcription responded to various linker mutations almost similarly
to the CRP-dependent 1acP1 transcription, supporting our previous proposalthat the factor-dependent activation and the UP element-dependent activation share the same structural requirements.

(4) Transcriptional Activation Mediated by the Carboxy-Terminal
Domain of RNA Polymerase a Subunit: Multipoint Monitoringwith a Fluorescent Probe
Olga N. OZOLINE, Nobuyuki FUJITA and Akira ISHIHAMA
The efficiency of transcription initiation by the RNA polymerase of Escheri-

A.

DEPARTMENT OF MOLECULAR GENETICS

15

chia coli depends on the nature of the two hexanucleotide sequences, located at-10 and -35 regions, and the third signal, located upstream from the -35
element. The upstream region of promoter rrn.BP1, generally designated as
UP element, contacts directly with the C-terminal domain of RNA polymerase
a subunit (aCTD). The aCTD is also responsible for interaction with a setof protein accessory factors (class-lor a-contact factors) regulating transcription efficiency. The most studied regulatory protein from this group factors is
cAMP receptor protein (CRP), regulating transcription of more than 80
genes. Molecular mechanism of transcription regulation by CRP depends on
the position of its binding site on the promoter sequence. Promoters whichhave the CRP-binding site centered between -60 and -100 usually require a
CTD for activation. Based on the mutant studies, we proposed a possibility
that one and the same surface of a CTD participating in interaction with UPDNA and class-I transcription factors overlap each other (Murakami et a1.,
1997). For precise identification ofthe contact sites on aCTD with DNA UP
elements and protein factors, some physical approaches are required besides
genetic and biochemical studies.
A novel approach employing a fluorescent reporter label positioned at various sites within the carboxy-terminal domain (CTD) of Escherichia coliRNApolymerase a subunit was used to monitor conformational changes of the protein upon transcription initiation complex formation. Previously we carriedout some spectral measurements using the RNA polymerase containing the asubunit conjugated with a fluorescent probe, fluorescein mercuric acetate(FMMA), only at Cys269 (Ozoline et a1., 1997). As an extension, we conjugated FMMA at a single Cys residue placed by the site-directed mutagenesis
at various positions of a CTD. The FMMA-conjugated a subunits were reconstituted into the respective holoenzymes. Spectral parameters of FMMA
were compared between free and promoter-bound forms of the RNA polymerase
modified at a single position. The character of local structural changes wasthen analyzed in spatial dimension. Reliable conformational changes wereobserved for binary complexes formed with UP element-dependent promoter
rrn.BP1 and ternary complexes formed with cAMP receptor protein (CRP)dependent promoter uxuABin the presence of cAMP-CRP. Our results confirm the previous data indicating that the helix I of a CTD and loop region

16

between helices III and IV are involved in these complex formation, and indicate that a similar pathway is used by the enzyme to realize the activation
signal accepted from either the UP-DNA or the activator protein CRP.
Some spectral changes were observed when the FMMA-modified RNA polymerase was mixed with cAMP-CRP in the absence of DNA, confirming thedirect protein-protein contact. Upon binding to the uxuABpromoter in theabsence of cAMP-CRP, the different changes were registered, which were similar with those observed for binary complexes with promoters T7D and mutant
rrnBP1lacking the UP element. These changes were different from thoseobserved for the ternary complex formed on the uxuABpromoter in the presence of cAMP-CRP. When the upstream region ofT7Dwas substituted by the
rrnBP1 UP element, we found the changes typical for the latter promoter,suggesting a possibility that a CTD interacts with not only the rrnBP1-type
UP element but also different types of the promoter upstream sequence using
multiple structural elements (Ozoline et aI., submitted for publication).

(5)Regulatory Mechanisms of Sigma Subunit Activities ofEscherichia co/J RNA Polymerase

Miki JISHAGE, Hiroto MAEDA l , Dipak DASGUPTA, Nobuyuki FUJITA and Akira
ISHIHAMA (lKagoshima Univ.)
The promoter recognition specificity of Escherichia coli RNA polymerase isconferred by one of seven molecular species of the 0 subunit. Replacement of
the 0 subunit on RNA polymerase is an efficient way for switching the transcription pattern. The major 0 subunit, 0 7°, is responsible for transcriptionof most, if not all, genes expressed during the exponential cell growth. Other
six species ofthe 0 subunit are required only during certain growth stages orunder specific growth conditions. The levels of these alternative 0 subunits
vary depending on the cell growth conditions (Jishage et a1., 1997; 1998). In
addition to the level control, the activity of at least some E. coli 0 subunits is
also subject to control in various ways (Kusano et aI., 1997; 1998). For instance, the unused 0 subunits are stored in inactive forms by forming complexes with another set of proteins, often designated as anti- 0 factors, withthe regulatory activity of 0 functions. Recently we discovered a novel E. coli

A. DEPARTMENT OF MOLECULAR GENETICS

17

protein, referred to Rsd (regulator of sigma D), which forms a complex with a 70
and prevents its function (Jishage et al., 1998). Purified Rsd protein formed
complexes in vitro with a 70 but not with other a subunits, and inhibited a 70 _dependent transcription in vitro to various extents depending on the promoters used. Since Rsd is induced in the stationary phase of cell growth, unusedexcess a 70 subunit, without being involved in transcription cycle, should betrapped by Rsd. Thus, the possibility has arisen that Rsd is an anti-a factor
for the major a 70 subunit for its storage in stationary phase. In order toclarify the in vivo function of Rsd, we analyzed the influence of both depletionand over-production of Rsd on a 70_ and a s-dependent transcription in vivo.
The expression of a reporter gene, lacZ, fused to either a 70 _ or as-dependent
promoter was analyzed in the absence of Rsd or presence of over-expressed
Rsd. In the rsdnull mutant, the a 70 _ and as-dependent gene expression isincreased or decreased, respectively. On the other hand, the a 70 • and as-dependent transcription was reduced or enhanced, respectively, after over-expression of Rsd. The repression of the as-dependent transcription in the rsd
mutant is overcome by increased production of the as subunit. These observations altogether support the prediction that Rsd is involved in replacementof RNA polymerase a subunit from a 70 to as during the transition from exponential growth to stationary phase (Jishage and Ishihama, 1999).
Along this line of studies, we carried out the search for a regulatory proteinof as function. By using the GST-fusion version ofthe as subunit expressedin E. coliW31l0, we have identified one specific protein associated with theas subunit. The identification ofthis a s_ associated protein is in progress.

(6) Role of the a S subunit in stationary phase survival of E. colI:
Search for a s-dependent promoters and analysis of rpoS mutations
PR. SUBBARAYAN and Akira ISHIHAMA
Seven different molecular species ofthe a subunit, the promoter recognition
subunit of RNA polymerase, are known to exist in E. coli, each recognizing a
specific set of promoters. The a 38( a S) subunit encoded by the rpoS gene issynthesized in the stationary phase of cell growth (Jishage and Ishihama,
1995; 1996), and is involved in transcription of at least some of the stationary

18phase-specific genes. Up to now, however, no consensus sequence has been
identified for as-dependent promoters, and instead we found that most of the
as-dependent promoters are recognized by the 0 70 subunit, which is involvedin the recognition of growth-related genes in the exponential phase (Tanaka etal., 1995; Kolb et aI., 1995). Moreover, some E. coli strains lack the as subunit, but they can survive in the stationary phase (Jishage and Ishihama,1997). Thus, the physiological role of as subunit remains unclear.
In order to search for promoters under the control of the as subunit, we tried
three different systems of in vivo screening: (1) search in the stationary phase
for antibiotics-resistant E. coliW3110 transformants carrying a promoter cloning vector fused to an antibiotic resistance reporter gene; (2) search for H 20 2resistant transformants carrying a promoter cloning vector fused to the geneencoding Dps (DNA binding protein from starved cells) which confers protection against the killing effect by HP2 in the stationary phase E. coli; and (3)search in the growing phase for high level B -galactosidase expressing
transformants carrying both a promoter cloning vector fused to the lacZ reporter gene and an expression vector for the as subunit. All three trials were,
however, unsuccessful for large scale screening of the a s-dependent promoters.
A group of E. coli strains have been suggested to contain a truncated form of
the as protein. Sometime ago we found that, although the original E. coli
W3110 strain contains the full-length complete copy ofthe as subunit, one
lineage W3110 lacks the as protein and another lineage contains a truncatedform of the as subunit (Jishage and Ishihama, 1997). To understand themolecular basis of the variation in as subunit composition, we analyzed thenucleotide sequences of the entire rpoSgene for all five lineage ofthe W3110
strain. In parallel, we analyzed the rpoS gene sequence ofseveral E. coli strains,and found that a group of E. coli strains different from E. coliW3110 variant also
carry the truncated form of as subunit. Taken together with reports fromother groups, it appears that a large number of mutations have accumulated
in the rpoS gene coding for the RNA polymerase as subunit.

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Growth Phase-Dependent Variation in the Levels of Twelve Speciesof the Nucleoid Protein in Escherichia colJ

Ali Azam TALUKDER, Akira IWATA!, Akiko NISHIMURA 2, Susumu UEDA I andAkira ISHIHAMA (lNippon Inst. BioI. Sci., Ohme, Tokyo; 2Genetic Strains Res.
Cntr., Microbial. Genet. Lab. )Several lines of evidence indicate that the intracellular levels of the nucleoid proteins and their localization along the genome DNA influence not only
the conformation of nucleoid but also the functions of DNA such as replication,
recombination, repair and transcription. For the switching of global patternof gene transcription among about 4,000 genes on the E. coli genome, the understanding of growth-dependent change in the conformation ofthe nucleoid. In
this study, we performed for the first time a systematic determination of theintracellular concentrations of twelve species of the nucloeid protein, CbpA
(Curved DNA-Binding Protein A), CbpB (Curved DNA-Binding Protein B; orRob, Right Origin Binding protein), DnaA (DNA binding protein A), Dps (DNAbinding Protein from Starved cells), Fis (Factor for Inversion Stimulation),
Hfq (Host Factor for phage Q# replication), H-NS (Histone-like Nucleoid Structuring protein), HU (Heat-Unstable nucleoid protein), IciA (Inhibitor of Chromosome Initiation A), IHF (Integration Host Factor protein), Lrp (LeucineResponsive regulatory Protein), and StpA (Suppressor of td- Phenotype A). For
this purpose, we over-expressed all these nucleoid proteins using the respective cloned genes. Previously, we determined the sequence specificity andDNA-binding affinity for these twelve nucleoid proteins (Talukder andIshihama, 1999). Here we produced specific antibodies in rabbits against
each of the twelve nucleoid proteins and using the quantitative Western blot
method, we measured the intracellular concentrations of all these nucleoid
proteins in E. coli strain W3110 growing at different growth phases (Talukder
et aI., 1999).
The level is maximum at the growing phase for nine proteins, CbpB, DnaA,
Fis, Hfq, H·NS, HU, IciA, Lrp and StpA, which may play regulatory roles in
DNA replication and/or transcription ofthe growth-related genes. The orderof accumulation level in growing E. coli is: Fis>Hfq>HU>StpA>HNS>IHF*>CbpB>Dps*>Lrp>DnaA>IciA>CbpA* (stars represent the station-

20
ary-phase proteins). The major protein components of nucleoid change from
Fis, Hfq and HU in the growing phase to Dps and IHF in the stationary
phase. The order of abundance in the early stationary-phase is:Dps*>IHF*>HU>Hfq>H-NS>StpA>CbpB>DnaA>Lrp>IciA>CbpA>Fis, whilethat in the late stationary phase is: Dps*>IHF*>Hfq>HU>CbpA*>StpA>HNS>CbpB>DnaA>Lrp>IciA>Fis. The curved DNA-binding protein, CbpA, appears only in the late stationary phase. This change in nucleoid protein composition in the stationary phase is accompanied by compaction ofthe genome
DNA and silencing ofthe genome functions.

(8)

Growth Phase-Coupled Changes In the Ribosome Pattern ofEscherIchIa coJl and Effect of Polyamlnes on
Ribosome Dlmerlzatlon

Akira WADAl, Kazuei IGARASHI 2, Charles G. KURLAND 3 and Akira ISHIHAMA(I0saka Med. ColI., Dept. Physics.; 2Chiba Univ., Fac. Pharm. Sci.; 3Uppsala
Univ., Dept. Mol. BioI., Sweden)
The 70S ribosomes in Escherichia coli are converted into 100S ribosome
dimers during the growth transition from exponential to stationary phase. A
55 amino acid long basic protein called ribosome modulation factor (RMF) isrequired for ribosome dimerization. RMF is a stationary phase-specific geneproduct. Since E. coli mutants lacking the rmfgene encoding RMF are unable
to survive in the stationary-phase, RMF is considered to be an essential geneproduct for survival in the stationary phase (Ishihama, 1999). The dimeric
form of ribosomes is virtually inactive in translation of at least some mRNA
templates. This suggests that ribosome dimers are inactive, stored forms ofribosomes in stationary-phase E. coli. Overproduction of spermidine results
in reduction ofthe synthesis level ofRMF, ultimately leading to cell death in
the stationary phase (Apirakaramwong et aI., 1998). The addition of Mg2+
restores partially the cell death in the absence of RMF production.
In order to further explore the role ofRMF-dependent ribosome dimerization in cell viability, we examined the relationship between the formation ofribosome dimers and the fate of E. coli cells. For this purpose, we used a setof E. coli strains from the ECOR collection, that includes a set of 72 reference E. coli

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strains isolated from a variety of hosts and geographical locations. Distributionsof ribosomes and of ribosomal subunits were studied by zone centrifugation
analysis in sucrose density gradients of extracts from overnight cultures of 19
strains from the ECOR collection. These 19 strains were classified into fourgroups on the basis of differences in their ribosomal sedimentation patterns
revealed in extracts from 24-hour cultures. Group-I strains revealed the typical 100S dimers, while ribosomes from group-IV contained as their fastest
component the 70S monomer-like peak. Group-II and -III strains yielded ribosomes with components that sedimented at velocities intermediate between
100S dimers and 70S monomers.
Culture time-dependent change in the ribosome pattern was analyzed withextracts from representatives of each group, grown in medium E supplemented
with 2% polypeptone. All the test strains showed a common growth phasecoupled change in the sedimentation pattern for ribosomes. This consists offour discrete stages: (i) formation of 100S dimers in the early stationary
phase; (ii) transient decrease in the dimer level; (iii) resumption of dimes tothe maximum level; and (iv) dissociation of 100S dimers into 70S ribosomes
which are quickly disassembled into subassemblies. The total time length
for this cycle of ribosome pattern change was, however, different among thefour group strains, leading to the apparent difference in the ribosomal distributions when observed at fixed time points. The group I strains retained thecomplete 100S ribosome dimers at least until day 4 to 5 in the stationary
phase under the culture conditions employed, while ribosomes in the group IV
strains were disassembled earlier. The number of viable cells decreases concomitantly with the disappearance of ribosome dimers. Therefore, we propose that a close correlation exists between the time of disappearance of ribosome dimers and that of cell death. In effect, it appears that ribosome dimerization is essential for the stationary phase survival of E. coli (Wada et aI.,
submitted for publication).

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(9)

Cloning and Identification of Subunit 4 (RpM) of
RNA Polymerase II from the Fission Yeast
Scblzosaccbaromyces pombe

Hitomi SAKURAI, Hiroshi MITSUZAWA, Makoto KIMURA and Akira ISHIHAMAThe RNA polymerase II in eukaryotes is composed of more than ten different
polypeptides (Ishihama et aI., 1998). The genes coding for all 12 putative
subunits of the RNA polymerase II have been isolated from the budding yeast
Saccharomyces cerevisiae and human. Previously we reported that the purified RNA polymerase II from the fission yeast Schizosaccharomyces pombecontained at least eleven polypeptides, devoid of the component corresponding
to RPB4 of S cerevisiae (Sakurai et al., 1998). Recently, the genes coding forthe subunit 4 were cloned from human and plant Arabidopsis thaliana. We
then re-examined whether the purified RNA polymerase II from S pombe contains Rpb4 or not. Mter search for a sequence containing the conserved sequences of the subunit 4 from the budding yeast, human and plant in the
PomBase (the DNA database for S. pombe), we identified the putative rpb4
gene. From the DNA sequence of a eDNA clone that was isolated by peRusing the synthetic primers with the rpb4 sequence, Rpb4 was found to consistof 135 amino acid residues with a molecular mass of 15,362.
For identification of the protein product, we made antibodies against theover-expressed and purified protein encoded by the putative rpb4 eDNA, andcarried out Western blotting using various step fractions of RNA polymerase II
purification from S pombe. The Rpb4 band was found to co-migrate on SDSPAGE with other three subunits, Rpb8, Rpb9 and Rpbll [thus, previously, we
failed to detect Rpb4] (Sakurai et aI., 1999). As in the case ofthe corresponding subunits from higher eukaryotes such as human and plant A. thaliana,
Rpb4 is smaller in size than RPB4 from the budding yeast S. cerevisiae, andlacks several segments, which are present in the S cerevisiae RPB4, including
the highly charged sequence in the central portion. The RPB4 subunit of S.
cerevisiae is not essential for normal cell growth but is required for cell viability under stress conditions. In contrast, S pombe Rpb4 was found to be essential even under normal growth conditions. The fraction of RNA polymerase
II containing RPB4 in exponentially growing cells of S cerevisiae is about 20%,A.

DEPARTMENT OF MOLECULAR GENETICS

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but the S. pombe RNA polymerase II contains the stoichiometric amount of
Rpb4 even at the growing phase. Rpb4 and Rpb7 formed a stable heterodimer when co-expressed in Escherichia coli.
(to)

Subunit Assembly of Scblzos8ccbaromyces pombe
RNA Polymerase II

Makoto KIMURA and Akira ISHIHAMARNA polymerase II from the fission yeast Schizosaccharomyces pombe consists oftwelve species of subunits, Rpb1 to Rpb12 (Sakurai et aI., 1999). We
expressed the eleven subunits, except for Rpb4, simultaneously in the same
cultured insect cells using combinations of various recombinant bacculovirus
expression vectors (Kimura et aI., 1999) [When we started this project, thegene for Rpb4 had not yet been cloned, but recently, we have succeded thecloning of rpb4 gene and eDNA (Sakurai et al., 1999)]. For this purpose, weconstructed the recombinant bacculoviruses, each carrying one or two subunit
cDNAs. Coinfection of up to six species of the recombinant virus attained thecoexpression of any combination among all eleven subunits. For isolation ofsubunit complexes formed in the infected insect cells, a glutathione-S-transferase (GST) sequence was fused to the rpb3 eDNA as to produce GST-Rpb3
fusion protein and a histidine(His1O>-tag sequence was inserted into rpbl eDNA
to produce Rpb1H protein. After glutathione affinity and succeeding Ni2+affinity chromatographies, a subunit complex consisting of seven subunits,
Rpb1H, Rpb2, GST-Rpb3, Rpb5, Rpb7, Rpb8 and Rpbll, was identified.
Removal of anyone of the six subunits besides GST-Rpb3 did not affect theassembly of other subunits, indicating two possibilities that: (i) the Rpb3
interacts directly with other six subunits; and/or (ii) the subassembly formation depends on the multiple contacts, in which the absence of any single subunit does not affect the assembly of other subunits. Direct interaction between the Rpb3 and other six subunits were detected by pairwise coexpression
experiments. Coexpression experiments of various combinations of a few
subunits revealed that the Rpbll enhances the Rpb3-Rpb8 interaction andthat Rpb8 does the Rpb1-Rpb3 interaction. Taken together we propose thatthe multile subunit-subunit contacts are involved in stabilization of the RNA

24polymersase II consisting of twelve subunits (Kimura et a1., submitted for
publication).

(11)

isolation and Characterization of Temperature-sensItive
Mutations In the Subunit 3 Gene (rpb3) of RNAPolymerase II of the Fission Yeast
Scb/zosaccbaromyces pombe

Jiro MlTOBE, Hiroshi MITSUZAWA, Kiyoshi YASUI and Akira ISHlHAMA
The RNA polymerase II of the fission yeast Schizosaccharomyces pombeconsists of twelve subunits (Sakurai et al., 1999). The subunit 3 (Rpb3) ishomologue of the prokaryotic RNA polymerase a subunit, which plays a keyrole in subunit assembly of this complex enzyme by providing the contact
surfaces for both {3 and (3' subunits. In order to get insight into the physiological role(s) of Rpb3, we have performed mutant studies for the S. pombe
rpb3 gene. A total of nine temperature-sensitive (Ts) and three cold-sensitive (Cs) S. pombe mutants have been isolated, each carrying a single
mutation(s) in the rpb3 gene in one of the four regions (A to D) conserved
among the eukaryotic subunit 3 homologues. The three Cs mutations were
all located in the region A, in agreement with the most important role of thecorresponding region in the assembly of prokaryotic RNA polymerase, while
the Ts mutations were scattered in all four regions. Among them, seven representative Ts mutants were subjected to further analysis.
Growth curve ofthese Ts mutants was characteristic for the asembly defective mutant at non-permissive temperatures. Two recessive suppressors wereindependently isolated from Ts3-154 which produces a mutant RNA polymerase with mutation. After cloning and sequencing, it was found that one
suppressor mutation codes for a mutant Pts1, a proteasome component, andthe other encodes a mutant version of a homolog of S. cervisiae PRS7 (26S
protease regulatory subunit 7). These plasmids were not exchangeable eachother. Taken these observations together with a high reversion rate we indicate that multiple pathways exist for suppression of the Ts phenotype. Western
analysis of seven Ts strains revealed the amounts of mutant Rpb3 proteins
were significantly reduced under restrictive temperature. Since the meta-

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bolic stability of most Ts mutant Rpb3 proteins were markedly reduced at a
non-permissive temperature, these mutant Rpb3 proteins seems defective in
the assembly or the mutant RNA polymerases containing the mutant Rpb3
were inactive. In addition to these observation, the Ts phenotype of all sevenmutants were suppressed to various extents by the over-expression ofRpb11,
the pairing partner subunit in the core subassembly. We conclude that the
majority of rpb3mutations affects the subunit assembly ofRpb3, even though
the extent of influence on the subunit assembly is different depending on thelocation of mutations (Mitobe et aI., 1999).

(1!)

Isolation and Characterization of Temperature-sensitive
Mutations in the Genes for Rpb6, Rpb7 and Rpbll of
RNA Polymerase II of the Fission Yeast
Schlzosaccharomyces pombe

Akira ISHIGURO, Hiroshi MITSUZAWA, Miwa KOMOTO, Hisatoshi Nom l andAkira
ISHIHAMA (lSaitama Med. ColI., Dept. Biochem.)
The RNA polymerase II of the fission yeast Schizosaccharomyces pombeconsists of twelve subunits (Sakurai et al., 1999). The two large subunits,
Rpb1 and Rpb2, are the eukaryotic homologues of the 13 and 8' subunits,
repespectively, of prokaryotic RNA polymerase, and form the catalytic center
for RNA polymerase, as observed by photo-affinity cross-linking of nascent
RNA products (Wlasoff et al., 1999). Both subunits 3 and 11 carry limited
sequence homology with the N-terminal assembly domain of prokaryotic a
subunit. Little is known about the functions of other eight subunits. For
identification of the roles of minor subunits in the assembly and function of
RNA polymerase II, mutant studies are in progress. So far we isolated temperature-sensitive (Ts) mutants of S. pombe with mutations in the rpb3, rpb6,
rpb7and rpbll genes encoding subunits 3,6,7 and 11, respectively.
Rpb6 (142 amino acid residues) is one of the common subunits among RNA
polymerases I, II and III. From the deletion analysis, the essential region forfunctions was located within the C-terminal proximal half of rpb6 (lshiguro
et al., 1999). By random mutagenesis by PCR and after replacement of thechromosomal rpb6 gene by the mutant alleles, we isolated a total of 14 Ts

26mutants with mutations in the rpb6 gene. The mutations, however, clustered along the entire rpb6gene including the non-essential N-terminal half,
but all located within the conserved sequences among the known Rpb6 homologues.
Among the multi-copy suppressors of one rpb6mutation, we identified thegene for TFIIS, the elongation factor for RNA polymerase II, indicating thedirect protein-protein contact between Rpb6 and TFIIS. In fact, the formation in vitro of binary complex was confirmed using the purified preparationsof Rpb6 and TFIIS (Ishiguro et a1., 1999).
In the case of S. cerevisiae, Rpb7 is known to be reversibly dissociated,
forming a complex with Rpb4, from the RNA polymerase II from growing
cells. In contrast, both Rpb4 (135 amino acid residues) and Rpb7 (172 aminoacid residues) were found to be the essential subunits in S. pombe and aretightly associated with the RNA polymerase II (Sakurai et a1., 1999). By
random mutagenesis of rpb7and replacement ofthe chromosomal rpb7geneby the mutant alleles, we isolated three Ts mutants, each carrying a single
mutation in the rpb7gene. One of the suppressor mutants was identified tocarry a second mutation in rpb7, indicating the intragenic suppression for theobserved phenotypic reversion (Mitsuzawa, unpublished).
Rpbll (135 amino acid residues), another homologue of prokaryotic a subunit, forms a heterodimer with Rpb3. For isolation ofthe rpbll mutants, weconstructed a rpbll disruptant of S. pombe which can survive in the presenceof plasmid carrying the rpbll gene. In the absence ofthis plasmid, the mutant S. pombe is unable to survive, indicaing that rpbll is an essential gene forcell viability. By random mutagenesis by PCR of the rpbll gene on the plasmid and after integration ofthe mutagenized rpbll gene into the chromosome,
we isolated five Ts and three Cs (cold-sensitive) mutants, each carrying asingle amino acid substitution mutation on the rpbll gene. By GST-pull
down assays, recombinant mutant Rpbll protein was found to have reduced
activity of Rpb3 binding. The isolation of multicopy suppressors is in progress(Komoto, unpublished).

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Identification of the RNA Cap I-binding Sites on the PH! Subunitof Influenza Virus RNA Polymerase

Ayae HONDA, Kiyohisa MIZUMOT0 1 andAkira ISHIHAMA (lDiv. Nucleic Acid Chem.
and Kitazato Univ.)
Influenza virus genome is composed of eight RNA segments of negative polarity, which is transcribed into plus-strand translatable RNA by the virusassociated RNA polymerase. The viral RNA polymerase is composed of threeviral P proteins, PB1, PB2 and PA. PB1 carries the catalytic site of RNApolymerization while PB2 plays a role in capped RNA recognition for generation of transcription primers. We have been concerned with the mapping offunctional sites for each P subunit polypeptide, including the subunit-subunit
contact sites. Previously, we identified two sites of photo-affinity crosslinkingof radioactive 8-azido GTP (8-N 3 GTP) on the PB1 subunit, i.e., the amino
terminal-proximal site I and the carboxy terminal-proximal site II, each being
close to that of sequence motif A and motif D, respectively, conserved among
RNA-dependent RNA polymerases.
On the other hand, capped RNA with 32p label only at its 5' cap-1 structure
was photo-affinity cross-linked to the PB2 subunit. In order to identify thecapped RNA-binding sites on PB2, we prepared capped RNA with 32p radioactivity only at the cap-1 structure, and UV-crosslinked it to the isolated RNP(ribonucleoprotein cores consisting ofvRNA, the PB1, PB2 and PA subunits of
RNA polymerase, and NP). After RNase A treatment, the radio-labeled PB2
was isolated by SDS-PAGE, and digested, together with unlabeled PB2, with
V8 protease. Cleavage fragments were separated by SDS-polyacrylamide
gel electrophoresis. Fragments with cross-linked cap-1 were blotted on PVDF
membranes and subjected to amino acid sequencing. Results indicated that
two regions of PB2 are involved in the binding of 5' cap-1 structure of capped
RNA, one at the N-terminal proximal region downstream from the PB1-contact site and the other around 500 amino acid residue region overlapping the
motif commonly found in cellular cap-binding proteins (Honda et a1., 1999).

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(14)

Search for Host Factor(s) Interacting with
Influenza Virus RNA Polymerase

Ayae HONDA, Takuto OKAMOTO and Akira ISHIHAMA
Influenza virus RNA polymerase carries two functions, one for transcriptionof vRNAs to produce vival mRNAs and the other for replicatin of vRNAs 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 1-terminal structure of virus-associated vRNAs
indicated that RNA synthesis for replication is initiated de novo without using
primers (Honda et a1., 1997). Both purified and reconstituted RNA polymerases require primers for function, while the RNA polymerase in either
virus-infected cell extracts or lysates of cells expressing three viral P proteinscan catalyze RNA synthesis in the absence of primers. We then proposedthat an as yet unidentified host factor(s) is involved in the functional conversion of the RNA polymerase from transcriptase to replicase. Attempts arebeing made to identify host proteins which interact with each of the P proteinsusing yeast two hybrid screening system. So far several positive clones havebeen isolated for each P protein, and some of these putative PBl-, PB2- andPA-interacting host factors were found to form complexes in vitro by mixing
with the respective P proteins. Functional conversion ofthe RNA polymerase
after interaction with each of these proteins is being analyzed.

(Hi)

Expression of Functional Influenza Viral RNA Polymerase In the
Methanotroplc Yeast Plebla pas/orIs

Shan-Jung HWANG, Kazunori YAMADA!, Kohji NAKADE 1 and Akira ISHIHAMA(IMitsubishi Chem. Co., Res. Dev, Div.)For the molecular anatomy ofthe multi-functional RNA polymerase ofinfluenza virus with the activities of both RNA polymerization and RNA degradation, playing in both tanscription and replication, experimental systems are
required, for both the purification of large quantities of the RNA polymerase
and the construction of mutant RNA polymerases with a specific mutation at
a defined site of a specific subunit. The efficiency of enzyme reconstitution

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we established was still low and thus the reconstitution system is costly,
time-consuming and technically inconvenient. To meet the demand for large
amounts of the influenza virus RNA polymerase for analysis the structurefunction relationship of each P protein subunit, we succeded to establish asimultaneous expression system of all three P protein subunits in the
methylotrophic yeast Pichia pastoris, and to purify the template-free functional RNA polymerase.
The methylotrophic yeast Pichia pastoris is able to catabolize methanol as
its sole carbon source and has been developed as a host for the expression ofheterologous proteins. The major advantages of this expression system includes: (i) a strong, tightly-regulated alcohol oxidase promoter, 5'AOX1,is used;(ii) a stable production can be achieved in a large volume fermentor culture;(iii) a secretory pathway allows the product secreted into the medium, separating the foreign protein from most ofthe native proteins; (iv) the expression
system can be easily constructed; and (v) the cost is equally as low as the
Escherichia coli expression system. For the expression of influenza virus RNA
polymerase, the cDNAs for RNA segments 1, 2 and 3 of influenza virus AJPR8,
each under independent control of the alcohol oxidase gene promoter, were
integrated into the P. pastoris chromosome. For the purification purpose of P
protein complexes, a hexa-histidine (His6 tag) was added at N-terminus oftheP2 protein. Expression of the P proteins in P. pastoris was optimized byvarying the concentration of methanol and the induction time after the addition of methanol. Results of the recombinant protein expression indicate
that: (i) a reasonable amount of all three P proteins was expressed and recovered in the cell lysate; (ii) all three P proteins formed a complex(es) as detectedfrom the co-elution pattern from Ni 2+ NTA agarose column; (iii) the P protein
complex carried the model RNA-dependent transcription activity in vitro; and(iv) the RNA polymerase recognizes specific sequences for transcription initiation as determined by using various model RNA templates with or without theterminal conserved sequences of both vRNA and cRNA segments. We conclude that the functional influenza virus RNA polymerase can be formed in the
methylotrophic yeast P. pastoris.

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(16)

Isolation from TMV-infeeted Tobaeeo of the RNA-dependent RNAPolymerase with the Subunit Composition of
1!6K/183K Beterodimer

Takato WATANABE, Ayae HONDA, Akira IWATA l , Susumu UEDA 1, Tadaaki HIBI 2
and Akira ISHIHAMA (lNippon Inst. BioI. Sci., Ohme, Tokyo; and 2Univ. Tokyo,
Dept. Agricui. Environ. BioI.)
The genome of tobacco mosaic virus (TMV) consists of a single-stranded
RNA molecule of about 6,400 nucleotides in length with positive polarity, which
encodes at least four polypeptides, the 126 and 183 kDa proteins required fortranscription and replication, the 30 kDa protein for cell-to-cell virus movement in infected plants, and the 18 kDa protein for virus coat formation. Threesegments of the 183K protein, each containing the sequence motifs ofmethyltransferase (M), helicase (H) or RNA-dependent RNA polymerase (P),were expressed in Escherichia coli as fusion proteins with hexa-histidine (HisJ
tag, and domain-specific antibodies were raised in rabbits against the purified Hisa-tagged M and P proteins. By immunoaffinity column chromatography, a heterodimer(s) consisting of one molecule each ofthe 126K (amino terminal-proximal portion of 183K protein) and full-length 183K viral proteins
was isolated. To check the RNA synthesis activity for the purified 183K-126K
heterodimer, we constructed model RNA templates of short chain length, but
containing the 5'- and 3'-terminal sequences ofTMV RNA. In the presence of
this model RNA template, the 183K-126K heterodimer exhibited the activity
of synthesis in vitro of template-sized RNA, but the activity was not detectedwith RNA without the TMV terminal sequences. We propose that the TMV
RNA polymerase protomer is composed of one molecule each of the 130K and
180K proteins (Watanabe et a1., 1999).

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Publications
APIRAKAWAMWONG, A, FUKUCHI, J., KAsHIWAGI, K, KAKINUMA, Y, ITO, E., ISHIHAMA, A and
IGARASHI, K: Enhancement of cell death due to decrease in Mg2+ uptake by OmpC
(cation-selective porin) deficiency in RMF (ribosome modulation factor)-deficient mutant. Biochem. Biophys. Res. Commun. %51, 482-487, 1998.
2. BALLESTEROS, M., KUSANo, S., ISHIHAMA, A and VICENTE, M.: The ftsQlp gearbox
promoter of Escherichia coli is a major sigma S-dependent promoter in the ddlB-ftsA
region. Mol. Microbial. 30, 419-430, 1998.
3. BERTONI, G., FUJITA, N., ISHIHAMA, A and DE LORENZO, V: Active recruitment of a 54 •RNA polymerase to the Pu promoter of Pseudomonas putida : role of IHF and a CTD.
EMBO J. 17, 5120-5128, 1998.
4. 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 s70 subunit by atranscription activator. J. Bacterial., in press, 1999.
5. BOWN, J.A, OWENS, J.T., MEARES, C.F., FUJITA, N., ISHIHAMA, A, BUSBY, S.J. and MINCHIN,
S.D.: Organisation of open complexes at Escherichia coli promoters: mapping thepromoter DNA sites close to region 2.5 of the a 70 subunit of RNA polymerase. J. BioI.
Chem. %74, 2263-2270, 1999.
6. BURNS, H.D., ISHIHAMA, A and MINCHIN, S.D.: Open complex formation during transcription initiatioin at the Escherichia coli galPl promoter: the role of the RNA
polymerase a subunit at promoters lacking an UP-element. Nucleic Acids Res. %7,
2051-2056, 1999.
7. CHATTERJI, D., FUJITA, N. and ISHIHAMA, A: The mediator for stringent control, ppGpp,binds to the 13 -subunit of Escherichia coli RNA polymerase. Genes to Cells, 3, 279287, 1998.
8. COLLAND, F., FUJITA, N., KOTLARZ, D., BOWN, J.A, MEARES, C.F., ISHIHAMA, A and KOLB,
A: Positioning of as, the stationary phase a factor, in Escherichia coli RNA polymerase-promoter open complexes. EMBO J., 18, 4049-4059.
9. HARADA, Y, FUNATSU, T., MURAKAMI, K, NONOYAMA, Y, ISHIHAMA, A and YANAGIDA,
T.: Single molecule imaging of RNA polymerase-DNA interactions in real time. Biophys.
J. 76, 709-715, 1999.
10. HONDA, A, MIZUMOTO, K and ISHIHAMA, A: A simple enzymatic method for analysis of
RNA 5'-terminal structures: Quantitative determination of the 5' termini of influenza virus genome RNAs. Virus Res. 55, 199-206, 1998.
11. Honda, A, Mizumoto, K and Ishihama, A: 1\vo separate sequences of PB2 subunit
consitute the RNA cap-binding site of influenza virus RNA polymerase. Genes Cells, inpress, 1999.
12. ISHIGURO, A, KIMURA, M., YASUI, K, IWATA, A, UEDA, S. and ISHIHAMA, A: 1\vo large
subunits of the fission yeast RNA polymerase II provide a platform for the assembly of1.

32
small subunits. J. Mol. Biol. %79, 703-712, 1998.
Ishiguro, A, Nogi, Y, Hisatake, K, Muramatsu, M. and Ishihama, A: Ppb6 subunitof the fission yeast RNA polymerase II is a contact target of the transcription elongation factor TFIIS. Mol. Cell. Biol., in press, 1999.
14. ISHIHAMA, A: Modulation of the nucleoid, the transcription apparatus, and the translation machinery in bacteria for stationary phase survival. Genes to Cells 3, 135-143,
1999
15. ISHIHAMA, A, KIMURA, M. and MITSUZAWA, H.: Subunits of yeast RNA polymerases instructure and function. Curro Opn. Microbiol. 1. 190-195, 1998.
16. ISHIHAMA, A, Zou. C., KOBAYASHI, M., KUMAR, A, FUJITA, N., SAKURAI, H. and HAYWARD,
R.S.: Molecular recognition in gene transcription. In: Biomolecular Interactions in
DNA and Proteins. (Buckin, V. and Funck, T, eds.), Nova Sci. Publ. 1998.
17. JISHAGE, M. and ISHIHAMA, A.: A stationary phase protein in Escherichia coli withbinding activity to the major a subunit of RNA polymerase. Proc. Natl. Acad. Sci. USA,95, 4953-4958, 1998.
18. Katayama, A, Fujita, N. and Ishihama, A.: Mapping of subunit-subunit contact
surfaces on the 13 subunit of Escherichia coli RNA polymerase. J. Biol. Chem., in press,
1999.
19. MASUNAGA, K, MIZUMOTO, K, KATO, H., ISHIHAMA, A and TOYODA, T: Molecular mapping of influenza virus RNA polymerase by site-specific antibodies. Virology %56, 130141, 1999.

13.

20. MITOBE, J., MITSUZAWA, H. and ISHIHAMA, A.: Isolation and characterization of temperature-sensitive mutations in the subunit 3 gene (rpb3) of RNA polymerase II of
the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet., in press.
21. MIYAKE, R., MURAKAMI, K, OWENS, J.T., GREINER, D.P., OZOLINE, O.N., ISHIHAMA, A and
MEARES, C.F.: The dimeric association of E. coli RNA polymerase alpha subunits,
studied by cleavage of Fe-BABE conjugated single-cysteine alpha subunits. Biochemistry 37, 1344-1349, 1998.
22. MIYAO, T., HONDA, A, Qu, Z. and ISHIHAMA, A: Mapping of Rpb3 and Rpb5 contact
sites on two large subunits, Rpb1 and Rpb2, of the fission yeast RNA polymerase II.
Mol. Gen. Genet. %59, 123-129, 1998.
23. MUKHERJEE, A, CUI, Y, MA, W.L., LIU, Y, ISHIHAMA, A, EISENSTARK, A and CHATIERJEE,
AK: RpoS (Sigma-S) controls the expression of rmsA, a global regulator of secondary
metabolites, Harpin E" and extracellular proteins in Ervinia carotovora. J. Bacteriol.
180, 3629-3634, 1998.
24. NAGAI, S., NAGASAWA, Y, YAGIHASHI, T and ISHIHAMA, A: The cyclic AMP receptor protein (CRP) gene function is required for expression of a (cell-bound)-type hemolysis inan avian pathogenic Escherichia coli strain. Veterinary Microbiol. 60, 227-238, 1998.
25. NEGRE, D., OUDOT, C., PROST, J.-F., MURAKAMI, K, ISHIHAMA, A, COZZONE, AJ. and
CORTAY, J.-C.: FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli. J. Mol. Biol. %76, 355-365, 1998.

A
26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

DEPARTMENT OF MOLECULAR GENETICS

33

NOMURA, T., FUJITA, N. and ISHIHAMA, A.: Mapping of subunit-subunit contact surfaces on the 13 subunit of Escherichia coli RNA polymerase. Biochemistry 38, 13461355, 1999.
OZOLINE, O.N., FUJITA, N., MURAKAMI, K and ISHIHAMA, A: Topology of the Escherichia coli RNA polymerase surface involved in interaction with promoter upsteam
enhancer elements. Eur. J. Biochem. !53, 371-381, 1998.
OZOLINE, O.N., MURAKAMI, K, NEGISHI, T., FUJITA, N. and ISHIHAMA, A: Specific fluorescent labeling of two functional domains in RNA polymerase alpha subunit. PROTEINS: Structure, Function, and Genetics 30, 183-192, 1998.
OWENS, J.T., MURAKAMI, K., CHMURA, A., FUJITA, N., ISHIHAMA, A. and MEARES,
C.F.: Mapping lacUV5 DNA sites proximal to conserved regions of sigma-70 in an
Escherichia coli RNA polymerase-DNA open promoter complex. Biochemistry 37, 76707675, 1998.
OWENS, J.T., MIYAKE, R, MURAKAMI, K, FUJITA, N., CHMURA, AJ., ISHIHAMA, A and
MEARES, C.F.: Mapping the sigma-70 subunit mapping the contact sites on the core
enzyme subunits of Escherichia coli RNA polymerase by site-specific cleavage withsigma-conjugated chemical protease. Proc. Natl. Acad. Sci. USA 95, 6021-6026, 1998.
PROST, J.-F., NEGRE, D., OUDOT, C., MURAKAMI, K, ISHIHAMA, A, COZZONE, AJ. and
CORTAY, J.-C.: Cra-dependent transcriptional activation of the icd gene of Escherichia
coli. J. Bacteriol. 181, 893-898, 1999.
RYu, S., FUJITA, N., ISHIHAMA, A. and ADHYA, S.: GalR-mediated repression and activation of lacUV5 promoter: Different contacts with RNA polymerase. Gene !!3, 235245, 1998.
SAKUKAI, H., KIMURA, M. and ISHIHAMA, A: Identification ofthe gene and the proteinof RNA polymerase II subunit 9 (Rpb9) from the fission yeast Schizosaccharomyces
pombe. Gene!!!, 11-16, 1998.
Sakurai, H., Mitsuzawa, H., Kimura, M. and Ishihama, A: Rpb4 subunit of thefission yeast Schizosaccharomyces pombe RNA polymerase II is essential for cell
viability and similar in structure to the corresponding subunits of higher eukaryotes.
Mol. Cell. Biol., in press, 1999.
STEVENS, AM., FUJITA, N., ISHIHAMA, A and GREENBERG, E.P.: Involvement of the RNA
polymerase a subunit C-terminal domain in LuxR-dependent activation ofthe Vibrio
fischeriluminescence genes. J. Bacteriol., 181, 4704-4707, 1999.
SUN, W., HATTMAN, S., FUJITA, N. and ISHIHAMA, A: Activation of bacteriophage Mu
mom transcription by the C protein does not require specific interaction with thecarboxyl terminal region of the a or 0 70 subunit of Escherichia coli RNA polymerase. J.
Bacteriol. 180, 3257-3259, 1998.
Talukder, AZ. and Ishihama, A: '!\velve species of DNA-binding protein from Escherichia coli: Sequence recognition specificity and DNA binding affinity. J. Biol.
Chem., in press, 1999.
Talukder, AZ., Iwata, A,Ueda, S. and Ishihama, A: Growth phase-dependent varia-

34

39.

40.
41.

42.

43.

44.

tion in the protein composition of Escherichia coli nucleoid. J. Bacteriol., in press,
1999.
THOMAS, M.S., Zou, C., ISHIHAMA, A and GLASS, R.E.: The effect of a nested set of Cterminal substituted deletions on the function of the a subunit of Escherichia coliRNApolymerase. Int. J. Biochem. Cell Biol. 29, 1475-1483, 1998.
TRAVIGLIA, S.L., DATWYLER, S.A, YAN, D., ISHIHAMA, A. and MEARES, C.F.: Proteinprotein interactions in RNA polymerase. Biochemisty, in press.
WANG, P., YANG, J., ISHIHAMA, A. and PITTARD, AJ.: Demonstration that the TyrR
protein and RNA polymerase complex formed at the divergent P3 promoter inhibits
the binding of RNA polymerase to the major promoter PI ofthe aroPgene of Escherichia coli. J. Bacteriol. 180, 5466-5472, 1998.
WATANABE, T., HONDA, A, IWATA, A, VEDA, S., HIBI, T. and ISHIHAMA, A: Isolation of
130Kl180K heterodimer with RNA-dependent RNA polymerase activity from TMVinfected tobacco. J. Virol. 73, 2633-2640, 1999.
WLASSOFF, W.A, KIMURA, M. and ISHIHAMA, A: Functional organization of two large
subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II: Location of the catalytic sites. J. BioI. Chem. 274, 5104·5113, 1999.
YASUI, K., ISHIGURO, A and ISHIHAMA, A: Molecular assembly of RNA polymerase IIfrom the fission yeast Schizosaccharomyces pombe: Subunit-subunit contact sites on
Rpb3. Biochemistry 37, 5542-5548, 1998.

A-b. Division of Mutagenesis(1)

Characterization of SCFGrrl that ublqultlnates 61 cycllns In
Saccharomyces cerevlslae

Tsutomu KISHI and Fumiaki YAMAOSCF complexes, composed of Skp 1, Cdc53 and one of F -box proteins, havebeen implicated in Cdc34-dependent ubiquitination in Saccharomyces
cerevisiae. Grrl, which is required for degradation of G1 cyclins, Clnl andCln2, as well as for regulation of glucose repression, is an F -box protein andinteracts with Skpl through the F-box motif. Grrl also interacts in vitro
with phosphorylated Clnl and Cln2. Domain analysis was performed to understand the role ofGrrl in the degradation ofCln2. In addition to the F-box,
Grrl has another motif, leucine-rich repeats (LRR). We found that the LRR
is a domain for Cln2 binding. A deletion of half of the LRR abolished theinteraction of Grrl with phosphorylated Cln2 but not with Skpl in vivo, and a

A.

DEPARTMENT OF MOLECULAR GENETICS

35

deletion of the F-box abolished the interaction ofGrrl with Skpl but not withphosphorylated Cln2 in vivo. Based on these results, we constructed grrl
mutants that are defective in association with either Skpl or Cln2. Cln2
was highly stabilized and accumulated with the phosphorylated forms in the
mutant cells. Furthermore, Skp I associated in vivo with phosphorylated Cln2
in a Grri-dependent manner. These data suggest that Grri is required for
degradation of Cln2 through linking phosphorylated Cln2 to Skp I in SCFGrrl
complex. See Ref 1 and 2.

(2)

Ubiquitln pathway functioning in mitotic regulatory mechanism

Toshimasa TADAKI, Hiroaki SEINO and Fumiaki YAMAO
A ubiquitin-conjugating enzyme, UbcP4, in fission yeast is essential for cell
growth. In vivo depletion of the UbcP4 demonstrated that it was necessary
for cell cycle progression at two phases, G2/M and metaphase/anaphasetransitions. The G2-arrest of UbcP4-depleted cells was dependent upon chkl
which mediates checkpoint pathway. Arrest at metaphase resulted in duplicated and condensed, but not separated chromosomes. Overexpression of
UbcP4 specifically rescued the growth defect of cutEI' cells at a restrictive
temperature. cutgencodes a component of the anaphase promoting complex(APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. CdcI3, a mitotic cyclin infission yeast, was accumulated in the UbcP4-depleted cells. The phenotypes
were also confirmed by using temperature-sensitive mutants of ubcP4. These
results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC. To get more genes that could be functioning in or
around the UbcP4/APC pathway, two approaches were adopted, screening of
the multicopy suppressors of ubcP4 mutations, and search of conditional mutants suppressible by UbcP4 overproduction. Two types of multicopy suppressors, a kind oftranscription factors and a tentative component ofnuc1ear
pore complex, have been identified. Multiple essential genes associated with
the UbcP4 pathway were found through the latter screening. Characterizationof these candidates is now undergoing.

36

(3)

Degradation of Cig! protein is regulated by ublquitin-pathway
involving UbcN and APC complex

Hiroaki SErNO and Fumiaki YAMAO
Ubiquitin-conjugating enzyme UbcP4 in fission yeast works together with
anaphase promoting complex (APC), mediating ubiquitin-pathway forward
destruction of Cut2 for onset of anaphase as well as of Cdc13, a mitotic cyelin
in fission yeast, for exit from mitosis. Another cyclin, Cig2, functions during
S-phase in fission yeast, and is destroyed before mitosis. Cig2 has a degradation signal (named destruction box) similar to that of Cdcl3. From analysis with temperature-sensitive mutants of ubcP4 and cutgencoding a component ofAPC, Cig2 was suggested to be another target of the UbcP4/APC pathway. Both temperature-sensitive ubcP4 and cut9mutant cells are hypersensitive to an overproduction of Cig2 protein. Null mutation of cig2 caused adecrease of restrictive temperature. Expression of the Cig2 lacking the destruction box was deleterious to the cells. These suggested that Cig2 protein
is regulated via proteolysis involving UbcP4 and APC. The cyelin boxes of
Cig2 and Cdc13 were found exchangeable each other. The phase-dependent
destruction of Cig2 and Cdcl3, was evidently determined by their structuresofN-terminus outside the cyelin box. These results indicated that ubiquitin
pathway involving UbcP4/APC was active in both G2 phase and anaphase,and that a mechanism for the phase- and substrate-specific regulation of thispathway might exist.

(4)

Survey and functional analysis of ubiqultin-conjugatlng
enzymes in S. pombeJoon-Hyun PARK, Hiroaki SErNO and Fumiaki YAMAO
Previous works and completion of genome sequencing revealed 13 familiesof ubiquitin-conjugating enzyme (Ubc) in S. cerevisiae. Phenotype of eachUbc differs each other. Two of them are essential for growth while others arenot. Some Ubc's are functionally redundant. To understand the functional
differences ofUbc in other species, we have surveyed Ubc's in fission yeast. In
our previous works, four Ubc's (UbcPI-P4) was identified through screening by

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DEPARTMENT OF MOLECULAR GENETICS

37

their enzymatic activities. By homology search of the fission yeast genome
data base, other five Ubc's (UbcP5-P9) have been found. Now the functional
characterization ofthe Ubc's are undergoing. Some genes were found essential for the cell growth while its ortholog in the budding yeast were not (i.e.
UBCl1/ubcP4, UBC4/ubcPl). Furthermore, there exist phenotypic differences
between the fission yeast Ubc's and its structural homologues in the budding
yeast. Thus, the whole spectrum of functional diversity in Ubc does not seems
to be identical between the two yeast lines.

Pub I i ca t ions
1.

2.
3.

4.

5.

6.

KISHI, T., SENO, T. and YAMAO, F.: Grrl functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skpl. Mol. Gen. Genet. 257, 143-148,
1998.
KISHI, T. and YAMAO, F.: An essential function of Grrl for the degradation ofCln2 is toact as a binding core that links Cln2 to Skp1. J. Cell Sci. l1t, 3655-3661, 1998.
MATSUMOTO, K., OKADA, M., HORIKOSHI, Y., MATSUZAKI, H., KISHI, T., ITAYA, M., SHIBUYA,
I.: Cloning, sequencing, and disruption of the Bacillus subtilis psd gene coding forphosphatidylserine decarboxlase. J. Bacteriol. t80, 100-106, 1998.
TANAKA, H., TAKENAKA, H., YAMAO, F. and YAGURA, T.: Aphidicolin induces alterationsin Golgi complex and disorganization of microtubules of HeLa cells upon long-term
administration. J Cell Physiol176, 602-611, 1988.
NISHIYAMA, R., IKAMI, M., YAMAO, F., TSUCHIDA, S. and YAGURA, T.: Inhibition of nuclear
envelope reconstitution in Xenopus interphase egg extract by hemin. Cell Struct Funet
23, 291·301, 1988.
YAMAO, F.: JB Review: Ubiquitin System-Selectivity and Timing of Protein Destruction. J. Biochemistry 125, 223-229, 1999.

38

A-c.
(1)

Division of Nucleic Acid Chemistry

The roles of Mrel1 of budding yeast in recombination

Jun-ichi TOMIZAWA
In yeast Saccharomyces cerevisiae, the biochemical process of meiotic recombination contains two temporally coupled processes, formation and processing of double-strand breaks (DSBs). About ten genes are involved in theseprocesses. Among them the mrell gene plays pivotal roles in both processes.
Mre11 forms a complex with Rad50 and Xrs2, acting as the binding core andparticipates in DSB processing. The C-terminal region of Mrell is required
only for DSB formation and binds to some meiotic proteins. Although theseand other proteins are involved in DSB formation, Mre 11 is not necessarily
holding them. The N-terminal half of Mre 11 specifies nuclease activities thatare collectively required for DSB processing. Mre11 has a DNA binding site
for DSB formation and another site for DSB processing. It has two regions tobind to Rad50. In addition, Mre 11 repairs methyl methanesulfonate-induced
DSBs by reactions that require the nuclease activities and those do not.
This work was performed by collaboration with Tomoko Ogawa, Tsutomu
Ohta, and Hiroyuki Oshiumi of the Department of Cell Genetics of this institute and Hideyuki Ogawa and Takehiko Usui of the Department of Biology ofOsaka University. For more detail, see the report from the Department of
Cytogenetics.
(!)

A new congenital muscular dystrophy with mitochondrial
structural abnormalities

Ichizo NISHINOl,Yuichi GOT0 1, Satoshi HORAI and Ikyuya NONAKA 1 (lDepartment of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry)
We report a new form of congenital muscular dystrophy (CMD) in 4 patientsfrom three unrelated families with probable autosomal-recessive inheritance.
All patients had the clinical characteristics of merosin-positive congenital
muscular dystrophy, but had marked mental retardation. The disease was

A.

39

DEPARTMENT OF MOLECULAR GENETICS

slowly progressive and 1 patient died from dilated cardiomyopathy at the ageof 13 years. In addition to dystrophic changes with necrosis and regenerationin muscle, the most striking finding was mitochondrial depletion in the centerof the sarcoplasm. Mitochondria at the periphery of fibers were markedly
enlarged ("megaconial" appearance) with complicated cristae, and contained
a normal amount of mitochondrial DNA by in situ hybridization. Mitochondrial
enlargement may represent functional compensation for mitochondrial depletion in the central sarcoplasm, where myofibrillar degeneration occurred. For
details, see Ref. 1.

(3)Peopling of Japan inferred from mitochondriai
DNA polymorphisms in East Asians

Satoshi HORAI and Keiichi OMOT0 1 (lInternational Research Center for Japanese Studies)
Nucleotide sequences of the major noncoding (D-loop) region of human mitochondrial DNA from five East Asian populations including mainland Japanese, Ainu, Ryukyuans, Koreans and Chinese were analyzed.

Based on acomparison of 482 base pair sequences in 293 East Asians, 207 different sequence types were observed. Of these, 189 were unique to their respective
populations, whereas 18 were shared between two or three populations. Amongthe shared types, eight were found in common between the mainland Japanese and Koreans, which is the largest number in the comparison. Phylogenetic
analysis revealed that East Asian lineages were classified into at least 18
monophyletic clusters, though lineages from the five populations were completely intermingled in the phylogenetic tree. However, we assigned 14 out of
the 18 clusters for their specificity based on the population from which the
maximum number of individuals in each cluster was derived. Of note is thefinding that 50% ofthe mainland Japanese had continental specificity in which
Chinese or Koreans were dominant, while less than 20% of either Rynkyuans
or Ainu possessed continental specificity. Phylogenetic analysis of the entire
human population revealed the closest genetic affinity between the mainland
Japanese and Koreans. Thus, the results of this study are compatible withthe hybridization model on the origin of modern Japanese. It is suggested

40that about 65% of the gene pool in mainland Japanese was derived from thecontinental gene flow after the Yayoi Age. For details, see Ref. 2.

(4)

Confirmation that a T-to-C mutation at 9176 In mitochondrial DNA
Is an additional candidate mutation for Leigh's syndrome

Michiko MAKINo 1, Satoshi HORAI, Yuichi GOT0 1 and Ikuya NONAKA 1 (IDepartment of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry)
Among 80 patients with the clinical and brain imaging characteristics ofLeigh's syndrome, 11 patients had a well-known mutation at nucleotide position (nt) 8993 in mitochondrial DNA. In addition, three patients had a T-toC mutation at nt 9176 which had been described previously in only two brothers with bilateral striatal necrosis and one patient with Leigh's syndrome. In
our three patients, one had the typical clinical characteristics of Leigh's syndrome from early infancy, and two had the later onset of neurological deficits. All had a slowly progressive course and basal ganglia abnormalities by
neuroimaging. As nt 8993 and 9176 are located in the ATPase 6 coding region,
altered ATPase function may be one of the enzyme abnormalities in Leigh's
syndrome and other similar conditions with bilateral striatal necrosis. For
details, see Ref. 3.

(5)Phylogenetic analysis of mitochondrial DNA In
Japanese pedigrees of sensorineural hearing
loss associated with the A1555G mutation

Abe S, Usami S, Shinkawa H, Weston MD, Overbeck LD, Hoover DM, Kenyon
JB, Horai S, Kimberling WJ (IDepartment of Otorhinolaryngology, Hirosaki
University School of Medicine, 2Department of Genetics, Boys Town National
Research Hospital)
Thirteen Japanese families (ten of which were from the northern part ofJapan), with sensorineural hearing loss associated with the 1555 A to G
(A1555G) mitochondrial mutation, a known cause of non-syndromic hearing
loss, were phylogenetically analysed using data obtained by restriction frag-A.

DEPARTMENT OF MOLECULAR GENETICS

41

ment length polymorphism (RFLP) and D-loop sequencing of mitochondrial
DNA (mtDNA). Various types of mtDNA polymorphism were detected by
restriction enzymes and D-loop sequence. No common polymorphic pattern
throughout the 13 families was found, though three families exhibited thesame restriction patterns and the same sequence substitution in the D-loop.
To find where each ofthe 13 families are situated in the phylogenetic tree, the482-bp of D-loop sequence were compared with those of 62 normal Japanese
subjects. Despite the three families mentioned above appearing to be clustered, the remaining 10 families were scattered along the phylogenetic tree.
This indicates that there was no common ancestor for the 13 Japanese families bearing the A1555G mutation except three families, and that the A1555G
mutation occurred sporadically and multiplied through evolution ofthe mtDNA
in Japan. The present results showed that the common pathogenicity (hearing loss associated with the A1555G mutation) can occur sporadically in families which have different genetic backgrounds, even in the Japanese population. For details, see Ref.4.

Pub I i ca t ions
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.
2. NISHINO, I., KOBAYASHI, 0., GaTO, Y, KURIHARA, M., KUMAGAI, K, FUJITA, T., HASHIMOTO,
K, HORAI, S. and NONAKA, I.: A new congenital muscular dystrophy with mitochondrial structural abnormalities. Muscle & Nerve 21: 40-47, 1998.
3. HORAI, S. and OMOTO, K: Peopling of Japan inferred from mitochondrial DNA polymorphisms in East Asians. In: The origin and past of modern humans - Towards a
reconciliation. K Omoto & P. V. Tobias (eds.), pp 54-73, World Scientific, 1998.
4. MAKINO, M., HORAI, S., GOTO, Y and NONAKA, I.: Confirmation that a T-to-C mutation
at 9176 in mitochondrial DNA is an additional candidate mutation for Leigh's syndrome. Neuromuscular Disorders 8: 149-151, 1998.
5. ABE, S., USAMI, S., SHINKAWA, H., WESTON, MD., OVERBECK, LD., HOOVER, DM., KENYON,
JB., HORAI, S. and KIMBERLING, WJ.: Phylogenetic analysis of mitochondrial DNA in
Japanese pedigrees of sensorineural hearing loss associated with the A1555G mutation. Eur. J. Hum. Genet. 6(6): 563-569, 1998
1.

42

B.

DEPARTMENT OF CELL GENETICS
B-a. Division of Cytogenetics(1)

Mechanisms of Yeast Genetic Recombination

Tomoko OGAWA, Tsutomu OHTA, Hiroyuki OSHIUMI and Shimako MATSUDA
Most recombination appears to begin by forming double-strand breaks(DSBs) in the DNA. Then, one ofthe strands at the break site is processed,
producing a tail of single-stranded extension. The tail is used to search forthe complementary sequence in a partner and to promote pairing with it. Thisoutline of the basic mechanisms of homologous recombination is conserved inprokaryotes and in eukaryotes. Studies on yeast, as one of the simplest eukaryotes, should provide important information on the chromosome mechanisms of genetic recombination. We have studied two subjects. One is theversatility of yeast Mre 11 protein that plays pivotal roles in various reactionsin the early phase of meiotic recombination and in repair of DNA damage in
vegetatively growing cells. The other is functions of Rad52 andRPAin Rad51mediated recombination reaction.

(a) Complex Formation and Functional Versatility of Mre11 of Budding Yeast In Recombination
Meiotic recombination of Saccharomyces cerevisiae consists temporally
coupled two processes, formation of double-strand breaks (DSBs) and theprocessing of their ends. Mre 11 plays pivotal roles in both processes. The
C-terminal region that is specifically required for DSB formation binds tomeiotic proteins. The N-terminal region specifies ssDNA endonuclease, 3' to
5' ssDNA exonuclease and 3' to 5' dsDNA exonuclease which are collectively
required for DSB processing. Cleavage by the endonuclease near the 5'-endof a strand may yield a 3'-overhanging structure and simultaneously eliminate SpoIl. Mre11 has two sites to bind to DNA, the central site for DSB
processing and the C-terminal site for DSB formation. It has two regions to

B.

DEPARTMENT OF CELL GENETICS

43

bind to Rad50. These functional duality would permit binding to two DNA
molecules simultaneously or to the same DNA segment differently to helptransition of the complex for DSB formation to that for DSB processing. Mrell
participates in repair of methyl methanesulfonate-induced DSBs by reactions
that require nuclease activities and those do not. See Ref. 1(b) Stimulation of Yeast Rad5t-mediated Recombination by Rad5!
Rad51 catalyzes strand exchange between circular single-stranded DNA(ssDNA) and homologous linear double-stranded DNA (dsDNA) to form joint
molecules (JM), and open circular duplex DNA molecules (Oe). However,
Rad51 is much less active than RecA protein in this reaction, even in thepresence of Replication protein-A (RPA), suggesting that Rad51 might be either inherently inefficient or weak due to a missing cofactor(s).
We found that both Rad52 and RPA proteins stimulate Rad51-mediatedreaction. In the enhancement, the bindings of the Rad52 to both Rad51 and
RPA proteins are necessary. The stimulation by RPA after formation of nucleoprotein filaments has been suggested to be due to its relaxation of the secondary structures of ssDNA. However, prior access of RPA to ssDNA inhibits
the Rad51 reactions and Rad52 alleviates this inhibition. Both stimulationof the Rad51 reaction and alleviation of the inhibition by Rad52 are specific to
the Rad51 reaction, and this specificity resides in the specificity of its interaction with Rad51. We conclude that the binding plays a crucial role in therecombination, by facilitating the formation ofRad51 nucleoprotein filaments.
Although recombination is a complex process involving other gene products,
the Rad51 reactions assisted by Rad52 are likely to be central to the molecular process of homologous recombination in eukaryotes. See Ref. 2 and 3.

44

(2)

The hepatitis 8 virus X protein is a co-activator of
activated transcription that modulates thetranscription machinery anddistal binding activators

Tsutomu OHTA
Hepatitis B virus X protein (HBx) transactivates viral and cellular genes
through a wide variety of cis-elements, but the mechanism has not been wellelucidated. Evidence for nuclear events in HBx transactivation has beenreported. Here we examine the role of HBx in modulation of transcription
with a transient transfection system and an in vitro transcription
assay. Reporters bearing Gal4-binding sites were applied to avoid the effects of endogenous transcription factors with or without signaling
processes. The GaI4-DNA binding domain fused form of HBx exhibited noeffect on Gal4-responsive reporters. However, HBx augmented activated transcription by transcriptional activators, suggesting HBx retains a co-activator
but not a transcriptional activator function. The functional domain for coactivation was the same as that for HBx transactivation, and the transcription factor IIB- and RNA polymerase II subunit 5-interacting sites of HBx,
which were critical for HBx transactivation, were shown to be crucial for the coactivation function. Importantly, HBx stimulated transcription on templates
bearing the X responsive elements in vitro with endogenous activators. These
results imply that HBx acts as a co-activator that modulates transcriptional
machinery and distal-binding activators, which may explain one ofthe mechanisms of transactivation by HBx when localized in nuclei. See Ref 4.

(3)

Analysis of the role of TFIIE In transcriptional regulation
through structure-function studies of the TFIIE IJ subunit

Tsutomu OHTA
The general transcription factor TFIIE plays important roles at two distinct
but sequential steps in transcription as follows: preinitiation complex formation and activation (open complex formation), and the transition from initiation to elongation. The large subunit of human TFIIE(TFIIEalpha) binds to

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45

and facilitates the enzymatic functions of TFIIH, but TFIIE also functions
independently from TFIIH. To determine functional roles of the small subunit of human TFIIE (TFIIEbeta), deletion mutations were systematically introduced into putative structural motifs and characteristic sequences. Here
we show that all of these structures that lie within the central 227-amino acidregion ofTFIIEbeta are necessary and sufficient for both basal and activatedtranscription. We further demonstrate that two C-terminal basic regions
are essential for physical interaction with both TFIIEalpha and single-stranded
DNA, as well as with other transcription factors including the Drosophilatranscriptional regulator Kruppel. In addition, we analyzed the effects ofthe
TFIIEbeta deletion mutations on TFIIH-dependent phosphorylation of the Cterminal domain of RNA polymerase II and on wild type TFIIEbeta-driven
basal transcription. Both responsible regions also mapped within the essential 227-amino acid region. Our results suggest that TFIIE engages in communication with both transcription factors and promoter DNA via the TFIIEbeta
subunit. See Ref. 5.

(4)

DNA supercoiling factor localizes to puffs on polytene chromosomes in DrosopbJ/a meJanogaster

Tsutomu OHTA
DNA supercoiling factor (SCF) was first identified in silkworm as a proteinthat generates negative supercoils in DNA in conjunction with eukaryotic
topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA
encoding Drosophila melanogasterSCF. Northern analysis revealed 1.6- and1.8-kb mRNAs throughout development. The longer mRNA contains an open
reading frame that shares homology with mouse reticulocalbin whereas theshorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence. An antibody against SCF detected a 45-kDa protein in
the cytoplasmic fraction and a 30-kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30-kDa protein interacts with topoisomerase II in the nucleus, and hence that it is a functional
form of SCF. Immunostaining of blastoderm embryos showed that SCF ispresent in nuclei during interphase but is excluded from mitotic chromosomes.

46
In larvae, the antibody stained the nuclei of several tissues including a posterior part of the salivary gland. This latter staining was associated with naturalor ecdysteroid-induced puffs on polytene chromosomes. Upon heat treatment
oflarvae, the staining on the endogenous puffs disappeared, and strong staining appeared on heat shock puffs. These results implicate SCF in gene
expression. See Ref.G.

(5)

Biochemical basis of hyper-recomblnogenlc activity of
Pseudomonas aeruginosa RecA protein In
Escherichia coli cells

Andrei ALEXEEVThe replacement of Escherichia colirecA gene (recA[Ec]) with the Pseudomonas aeruginosa recA(pa) gene in Escherichia coli cells results in constitutive
hyper-recombination (high frequency ofrecombination exchanges per unit lengthof DNA) in the absence of constitutive SOS response. To understand thebiochemical basis of this unusual in vivo phenotype, we compared in vitro therecombination properties ofRecA (pa) protein with those ofRecA (Ec) protein.
Consistent with hyper-recombination activity, RecA (Pa) protein appeared tobe more proficient both in joint molecule formation, producing extensive DNA
networks in strand exchange reaction, and in competition with single-stranded
DNA binding (SSB) protein for single-stranded DNA (ssDNA) binding sites.
The RecA (pa) protein showed in vitro a normal ability for cleavage of the E.
coli LexA repressor (a basic step in SOS regulon derepression) both in theabsence and in the presence (i.e. even under suboptimal conditions for RecA
(Ec) protein) of SSB protein. However, unlike other hyper-recombinogenic
proteins, such as RecA441 and RecA730, RecA (pa) protein displaced insufficient SSB protein from ssDNA at low magnesium concentration to induce the
SOS response constitutively. In searching for particular characteristics of
RecA (pa) in comparison with RecA (Ec), RecA441 and RecA803 proteins, RecA
(Pa) showed unusually high abilities: to be resistant to the displacement by
SSB protein from poly(dT); to stabilize a ternary complex RecA-ATP-ssDNA
to high salt concentrations; and to be much more rapid in both the nucleationof double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-depen-

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47

dentATP hydrolysis at pH7.5. We hypothesized that the high affinity ofRecA(Pa) protein for ssDNA, and especially dsDNA, is the factor that directs theternary complex to bind secondary DNA to initiate additional acts of recombination instead of to bind LexA repressor to induce constitutive SOS response.
See Ref. 7

Publications
1.

USUI, T., OHTA, T., OSHIUMI, H., TOMIZAWA, J., OGAWA, H. and OGAWA, T.: Complex
formation and Finctional Versatility of Mre 11 of Budding Yeast in Recombination. Cell
95, 705-716, 1998.
2. SHINOHARA, A and OGAWA, T.: Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature 391, 404-407, 1998.
3. SHINOHARA, A, SHINOHARA, M., OHTA, T., MATSUDA, S. and OGAWA, T.: Rad52 forms ring
structures and co-operates with RPA in single-strand DNA annealing. Genes to Cells,
3, 145-156, 1998.
4. LIN, Y., TANG, H., NOMURA, T., DORJSUREN, D., HAYASHI, N., WEI, w., OHTA, T., ROEDER,
R. and MURAKAMI, S.: The hepatitis B virus X protein is a co-activator of activatedtranscription that modulates the transcription machinery and distal binding activators. J BioI Chern. 273, 27097-27103, 1998.
5. OKAMOTO, T., YAMAMOTO, S., WATANABE, Y, OHTA, T.. HANAOKA, F., ROEDER, R.G.and
OHKUMA, Y.: Analysis ofthe role ofTFIIE in transcriptional regulation through structure-function studies ofthe TFIIE /3 subunit. J BioI Chern. 273, 19866-19876, 1998.
6. KOBAYASHI, M., AlTA, N., HAYASHI, S., OKADA, K., OHTA, T.and HIROSE, S.: DNA supercoiling factor localizes to puffs on polytene chromosomes in Drosophila melanogaster.
Mol Cell BioI. 18, 6737-6744, 1998.
7.

NAMSARAEV, E.A, BAITIN, D., BAKHLANOVA, LV., ALEXEEV, A A, OGAWA, H.and LANZOV,
V.A: Biochemical basis of hyper-recombinogenic activity of Pseudomonas aeruginosa
RecA protein in Escherichia coli cells. Mol Microbiol. 27, 727-738. 1998.

Oral Presentations
1.

2.
3.

OHTA, T., OSHIUMI, T., OGAWA, H. and OGAWA, T.: Roles of ssDNA Endonuclease of
Mre11 Protein of Yeast, International Symposium on Mechanisms of Recombinationand its consequence, Osaka, Japan, March, 1998.
OGAWA, T.: Functions of Mre11 Protein involved in Recombination and Repair. EMBO
workshop on Genetic Recombination, Seillac France, May, 1998
OGAWA, T.: Functions of Mre11 and Rad50 Proteins involved in Recombination Initia-

48
tion Reaction. Gordon Research Conference on MEIOSIS, New London, USA, June,
1998.
4. OGAWA, T., OHTA, T., OSHIUMI, H.: Functions ofMrell Protein in Yeast Recombination.
Eighteenth International Congress of Genetics, Bejing, China, August, 1998.
5. OGAWA, T.: Mechanisms of Yeast Genetic Recombination. The 13th Workshop on
Genetic Recombination and its Control Systems. Yokohama, Dec. 1998.
6. OHTA, T., TOMIZAWA, J., OGAWA, T.: Roles of Mrell Protein in Yeast Recombination andRepair. The 21st Annual Meeting of Molecular Biology in Japan. The Symposium on
DNA Replication and Recombination. Yokohama, Dec. 1998.

B-b. Division of Microbial Genetics(1)

Dpbl1 controls association of DNA polymerases withreplication origins in Saccharomyces cereylsJae

Hiroshi MASUMOTO and Hiroyuki ARAKI
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 toinitiate DNA synthesis during the S phase. The Dpb11 protein which genetically interacts with essential DNA polymerase E (Pol E), is required for DNA
replication and the S-phase checkpoint. The physical interaction between
Pol Eand Dpb11 had not been detected. By using a cross-linker, we detected acomplex of Pol Eand Dpb11 and this complex existed in S phase.
Dpb11 associated with replication origins at the same timing with Pol Ein
S phase by chromatin immunoprecipitation assay. Association of Dpb11 withorigins depended on functions of Mcm5, a component of the pre-RC. Conversely, Pol a - primase complex and Pol Ecould not associate with origins intemperature sensitive dphll-} mutant cells at the restrictive temperature.
Therefore, Dpb 11 functions for association of DNA polymerases with origins.
Hydroxyurea inhibits late origin firing in S cerevisiae and checkpoint genes,
and checkpoint genes RAD53 and MEC}, are involved in this control. In dphll} cells, DNA polymerases associated with early and late origins in the presence of hydroxyurea at permissive temperature whereas they associated only

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DEPARTMENT OF CELL GENETICS

49

with early origins in wild-type cells. Thus, Dpbll functions for association of
DNA polymerases with origins in DNA replication and for the control oflatefiring origins in the S-phase checkpoint.

(!)

Sid!, which Interacts with Dpbl1 In Saccharomyces cerevlslae,
Is required for chromosomal DNA replication

Yoichiro KAMlMURA and HiroyukiA.RAK:I
To identify factors interacting with Dpbll, we have isolated 10 sid (synthetic lethal with dpbll-l) mutations which fall into 6 complementationgroups (sidl - 6). So far, we have cloned SLDl, 2, 3, 4 and 5 and found that
SLDl is identical to DPB3 encoding the third largest subunit of Pol E andSLD4 is identical to CDC45 required for the initiation of chromosomal DNA
replication. The SLD2, 3 and 5 genes were new genes essential for the cell
growth. We first characterized SLD2encoding an essential 52 kDa protein.
High copy SLD2suppressed the thermosensitive growth defect of dpbll-l.
Conversely, high copy DPBll suppressed the temperature-sensitive growthdefect of sld2-6. The interaction between Sld2 and Dpbll was detected in two
hybrid assay. This interaction was evident at 25"C but not at 34"C when Sld26 or Dpbll-1 replaced its wild type protein. No interaction could be detected
between Sld2-6 and Dpbll-1 even at 25"C. Immunoprecipitation experiments
confirmed that Dpbll physically interacts with Sld2.
Sld2-6 cells were defective in DNA replication at the restrictive temperature as were dpbll-l cells. Further, in dpbll-l and sld2-6 cells, the bubble
shaped replication intermediates formed in the ARS region reduced quickly
after temperature shift. These results strongly suggest the involvement of theDpb11/Sld2 complex in a step close to the initiation of DNA replication.

50

(3)

Function of the Sld5/Psft complex which interacts with DNA
polymerase II( E) and Dpbt1 in Saccharomyces cerevlslae

Yuko TAKAYAMA, Yoichiro KAMIMURA and HiroyukiARAKIThe SLD5 gene encodes an essential 34 kDa protein. We first isolated
thermosensitive alleles of the SLD5 gene by the plasmid shuffling method toknow its function. At the restrictive temperature, a thermosensitive sld5-12
mutant cells arrested with a dumbbell shape which is the typical terminal
morphology for mutants defective in DNA replication. FACS analysis showedthat the mutant cells arrest at the S-phase. Thus, Sld5 seems to be requiredfor chromosomal DNA replication.
To elucidate the function of Sld5, we isolated the PSFI (Partner of SLD
Five) gene as a multicopy suppressor ofthe sld5-12mutation. PSFI encodesa 24 kDa protein essential for cell growth. In two-hybrid analysis and coimmunoprecipitation experiment, Sld5 interacts with Psfl in yeast cells.
Moreover, the recombinant Sld5 and Psfl proteins purified from Escherichia
coli cells made a complex, indicating that Psfl directly interacts with Sld5.
We also isolated a thermosensitive psfl-l mutation by the plasmid shuffiing
method. Psfl-l mutant cells arrested with a dumbbell shape at the restrictive temperature as did sld5-12 cells. These results suggest that the Sld51
Psfl complex is required for DNA replication.

(4) Is there an additional monofunctional glycosyltransferase for cellwall peptidoglycan biosynthesis in EscherIchIa colJ?
Hiroshi HARA (Present address: Department of Biochemistry and Molecular
Biology, Saitama University)
The last stage of cell wall peptidoglycan biosynthesis in bacteria involves
two enzymatic activities, glycosyltransferase (GTase) that polymerizes glycan
chains from lipid-linked disaccharide-peptide precursors and transpeptidase(TPase) that cross-links adjacent glycan chains via short peptide chains, thelatter being sensitive to penicillins. In Escherichia coli, penicillin-binding
proteins (PBPs) la and Ib were shown to carry not only TPase but also GTase
activity. In addition to these bifunctional enzymes, we found a monofunctional

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DEPARTMENT OF CELL GENETICS

51

GTase activity in an ion-chromatographic fraction (CM-fraction) containing nodetectable PBP, purified the enzyme, and characterized it (Hara, H. and Suzuki,
H., FEBS Lett. 168, 155-160, 1984). Recently, a gene (mtgA) whose 242-residue product's sequence showed significant homology to the well-conserved fingerprints found in the N-terminal GTase domain, but not to those in the Cterminal TPase domain, of bifunctional PBPs was identified. Overexpressionof the gene led to increased peptidoglycan-synthesizing activity (Di Bernardino, M. et aI., FEBS Lett. 392, 184-188, 1996).
In this study, the chromosomal mtgA gene was destroyed by inserting Q
interposon into a position corresponding to the 51st codon. The resultant nullmutant was viable, and the CM·fraction prepared from it was not impaired forthe glycan-polymerizing activity. These results indicate that mtgA is not responsible for the monofunctional GTase we previously identified and that thegene is dispensable for growth. MtgA protein has a molecular weight of about
27 kDa, a little smaller than that ofthe enzyme purified from the CM-fraction(34 kDa). The purified enzyme required Ca2+ or Mn 2+ but not Mg2+ for its full
activity, whereas such requirement was not confirmed for the GTase activity
detected in the solubilized membrane protein fraction of mtgA·overexpressing
cells. There seems to be two species (or more) of monofunctional GTases in E
coli, although search of the whole genome sequence did not reveal an additional gene other than mtgA that could code for a protein with homology to theGTase fingerprints.

Pub I i ca t ions
1.

2.

3.
4.

KAMIMURA, Y., MASUMOTO, H., SUGINO, A and ARAKI, H.: Sld2, which interacts with
Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication. Mol.
Cell. BioI., 18, 6102-6109, 1998.
NOSKOV, V. N., ARAKI, H. and SUGINO, A: The RFC2gene, encoding the third· largest
subunit of the replication factor C complex, is required for an S phase checkpoint in
Saccharomyces cerevisiae. Mol. Cell. BioI., 18, 4914 - 4923, 1998.
LEEM, S-H., CHUNG, C-N., SUNWOO, Y. and ARAKI, H.: Meiotic role of SWI6in Saccharomyces cerevisiae. NucI. Acids Res., 26, 3154 - 3158, 1998.
SUGINO, A, OHARA, T., SEBASTIAN, J., NAKASHIMA, N. and ARAKI, H.: DNA polymerase E
encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast
Schizosaccharomyces pombe. Genes to Cells, 3, 99·110, 1998.

52
5.

6.

MENGIN-LECREULX, D., AYALA, J., BOUHSS, A, van HEIJENOORT, J., PARQUET, C. and HARA,
H.: Contribution ofthe Pmra promoter to expression of genes in the Escherichia coli
mra cluster of cell evelope biosynthesis and cell division genes. J. Bacteriol. 180, 44064412, 1998.
KAMIMURA, Y, KAWASAKI, Y, OHARA, T. and SUGINO, A: DNA helicase III of Saccharomyces cerevisiae, encoded by YER176w (HELl), highly unwinds covalently closed,
circular DNA in the presence ofa DNA topoisomerase and yRF-A J. Biochem., 25, 236244, 1999.

B-c. Division of Cytoplasmic Genetics(1)

Studies on behavioral disorders of
Fyn tyrosine kinase knockout mice

Hiroaki NIKI (Lab. for Neurobiology of Emotion, Brain Science Institute,
RIKEN)In a series of experiments we found that our Fyn-deficient mice showed
increased fearfulness in a variety of tests for fear response and enhanced
seizure susceptibility induced by intense sound and convulsive drugs. In 1997,
we found that Fyn-deficient mice were hyper-sensitive to the hypnotic effect of
ethanol. In these Fyn-deficient mice, up-regulation of tyrosine phosphorylation of NMDAR in the hippocampus by ethanol administration (usually notedin the control mice) was not observed. Acute tolerance to ethanol inhibitionof NMDAR-mediated EPSPs in the hippocampal slices developed in control
mice but not in Fyn-deficient mice. In 1998 we examined aggressiveness of
Fyn-deficient mice, and found that they showed decreased offenssive aggression (intruder-evoked attack) and increased defensive aggression (restraintinduced target biting).
Concerning the pharmacological correlates of increased fearfulness of ourFyn-deficient mice, we conducted a receptor-binding experiment in collaboration with Dr. Yoshii at Tokyo Institute of Psychiatry. In 1998 we found that
Fyn-deficient mice have a lower density of central benzodiazepine receptor
binding sites in brain tissues.
As Fyn is strongly expressed in the olfactory bulb, we examined LTP in theolfactory bulb in collaboration with Prof. Kawai at Jichi Medical School. In

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DEPARTMENT OF CELL GENETICS

53

1998 we found an impairment in LTP of the olfactory bulb along with abnormality of GABAA function in Fyn-deficient mice (Kitazawa, H. et aI, 1998).

(2)

Studies on behavioral disorders of
NMDAR subunit knockout mice

Hiroaki NIKI (Lab. for Neurobiology of Emotion, Brain Science Institute,
RIIillN)
Concerning the studies on mice lacking NMDAR subunit epsilon 1 or epsilon
4, we obtained the following results in 1998. Mice lacking epsilon 1 were lesssusceptible to audiogenic seizure, but they did not differ from wild-type micein auditory startle response. On the other hand, mice lacking epsilon 4 showed
augmentation in startle response, but they showed no difference in audiogenic
seizure susceptibility.

Publication
1.

KITAZAWA, H.,YAGI, T., MIYAKAWA, T., NIKI, H., KAWAI, N.:

Abnormal synaptic transmis-

sion in the olfactory bulb of Fyn-kinase deficient mice. J. Neurophysiol. 79:137-142,
1998.

54

C.

DEPARTMENT OF DEVELOPMENTAL GENETICS
C-a. Division of Developmental Genetics(1)

Regulation of ras signaling by Sprouty and EDL
ensures constant outcome of induction

Masataka OKABE, Takuma YAMADA, Susanne KRAMER l and Yasushi HIROMI(lInstitut fUr Genetik, Universitat WUrzburg)
Photoreceptor neurons in the Drosophila compound eye and chordotonal organs in the embryo are both formed by induction by preexisting neurons or
their precursors. In both systems, prespecified cells induce surrounding cells
to assume fates similar to its own. The inducing molecule is a TGFa-likefactor Spitz, which activates EGF receptor and the downstream raslMAPK
signaling pathway. While there are a large number of cells that are competentto be induced, the outcome of induction is remarkably constant; each ommatidium always contain eight photoreceptor neurons, and each hemisegment
contains precisely eight chordotonal organs. This constancy is achieved by theprecise control of the inducing ability and its responsiveness.
Sprouty is a novel protein with an evolutionarily conserved Cystein richmotif. Sprouty acts as an antagonist of receptor tyrosine kinase pathways
that are triggered by the extracellular signaling molecules, such as Spitz EGF
and Branchless FGF. In the absence of sproutyfunction, supernumerary photoreceptor neurons and chordotonal organs form, due to hyperactivation ofras
signaling. We find that the expression ofSprouty is induced by the ras signal.
While cells that are close to the signaling center and receive high concentration of the signal can still respond and assume neuronal fate, those that are
further away cannot cope with the signal antagonism by Sprouty and fail to
respond. Thus the role of Sprouty is to limit the field of induction by restricting the number of responding cells to those receiving the highest concentration
ofthe inducing signal.
A nuclear target ofthe raslMAPK signaling pathway is the Ets transcription

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

55

factor Pointed P2 protein. Phosphorylation of Pointed P2 by MAPK activates
the transcriptional activation function of Pointed P2, which in turn is required
for the specification of photoreceptor neurons and chordotonal organs that areformed by induction. We found that hyperactivation of pointed function incells that normally induce other photoreceptor neurons or chordotonal organs
results in a decrease in the inducing ability. The inhibition of inducing ability
by pointedis likely to operate in the induced cells, thereby preventing unlimited spread of induction. EDL is a novel Ets protein that is specifically expressed in the cells with inducing ability. EDL contains the Ets-specific Pointed
domain, but does not contain the Ets DNA-binding domain. It binds to the
Pointed domain of Pointed P2, blocks the transcriptional activation functionof Pointed P2, thereby allowing cells to express their inducing ability. For
details, see publications 1 and 2.

(Z) A screen for proteins that interact with theligand binding domain of a nuclear receptor, Seven-up
Hiroyuki KOSE, Steve WEST l , Emiko SUZUKI and Yasushi HIROMI (I Princeton
University)
Nuclear receptors are a class of transcription factors that share two conserved domains, the DNA-binding domain and the ligand-binding domain.
While some of the nuclear receptors act as true "receptors"by responding to thesignals presented by their ligands, the vast majority of nuclear receptors haveno known ligands, even though they share a domain similar to the ligand
binding domain ofligand-responsive nuclear receptors. Seven-up is one such
nuclear receptor for which ligand has not been identified. It is one of thereceptors with the most ancient origin, and is by far the most evolutionarily
conserved nuclear receptor. During compound eye development in Drosophila,
seven-up is expressed in a subset of photoreceptor neurons and specifies theiridentity as a particular neuronal subtype. Genetic evidence suggests that thefunction of seven-up is mediated by protein(s) that act through the ligandbinding domain of Seven-up.
We have carried out a yeast two-hybrid screen for molecules that interact
with the ligand-binding domain of Seven-up. One of the proteins identified is

56

a novel protein that contains the SAM domain, a protein-protein interaction
motif found in some receptor kinases, cytoplasmic scaffold and adaptor proteins, and members of the Polycomb group chromatin proteins. We have identified strains harboring transposon insertions in the vicinity of the open reading frame, and are carrying out the genetic analysis of the gene.

(3)

Relationships between organogenesis and positional information
in Drosophila imaginal discs

Nao NIWA, Masataka OKABE and Yasushi HIROMI
Recently, Drosophila genes have been identified whose misexpression induces ectopic organs, such as eyes and wings. Such genes and has been regarded as the master control genes for development of Drosophila organs.
Ectopic organs, however, does not form anywhere in the body, but appears to
respond to the prepattern present in the ectopic location. Thus there arepositions in the body that are capable ofresponding to the expression of master control genes and initiating organogenesis, in addition to those where organogenesis normally occurs. We are investigating the relationships between
the types of induced organs and the positional information surrounding ectopic organ in the imaginal discs.
As a first step, we chose as a model the ectopic eye formed by eyeless (ey)gene expression. To identify the conditions required for the ectopic eye formation, eywas ubiquitously expressed by VAS/heat shock GAL4 system in all
imaginal discs during larval stages, and we have defined the timing of ey
expression that allows ectopic eye formation, and the positions of ectopic eyes
in imaginal discs. We found that the ectopic eye formation was most frequent
when ey was expressed by heat shock for an hour during 78-85h after egg
laying, and the positions of ectopic eyes were restricted to several domains
near the anterior-posterior compartment borders in antenna, leg and wing
imaginal discs. Thus the responsiveness to ectopic eywas clearly restricted,
both spatially and temporally.
We are currently investigating the conditions for timing and domains ofthe
master control gene expression with respect to formation of other organs, suchas ocelli and chordotonal organ, and comparing those with the conditions foundC. DEPARTMENT OF DEVELOPMENTAL GENETICS

57'

for ectopic eye formation.

(4)

Molecular mechanisms of Neuron-gila developmental decision

YasukoAKrYAMA·ODA 1, Yoshihiko UMESON0 2, Kazunaga TAKIZAWA 1 and Yoshiki
HOTTA2.3 (lGraduate School of Science, University of Tokyo, 2CREST, JST, 3Na·
tional Insitute of Genetics)
The nervous system is composed of two major types of cells, neurons andglia. Despite their distinct functions, neurons and glia are derived from acommon precursor in both vertebrate and invertebrate nervous systems. In
the Drosophila central nervous system (CNS), both neurons and glia are de·
rived from common precursor cells called neuroblasts. The gem gene is ex·pressed transiently in early glial lineage. The mutation of gem causes pre'sumptive glial cells to differentiate into cells with neuron·like characteristics,
whereas gem ectopic expression forces virtually all CNS cells to become glial
cells. Thus the gem gene governs glial vs. neuronal developmental decision byturning on the glial fate while inhibiting the default neuronal fate of theneuroblasts and their progeny.
In order to further clarify the neuron·glia decision mechanisms, the cell lin·eage and gcm expression was examined in detail. NB6·4 in the thoracic seg·ment (NB6·4T) is a neuroglioblast, although the corresponding cell in theabdominal segment (NB6·4A) produces only glia. The first cell division of
NB6·4T occurred in the medial·lateral orientation, and was found to bifurcate
the glial and neuronal lineage. After division, the medial daughter cell ex·pressed GCM protein to produce three glial cells, while the lateral daughter
cell with no GCM expression produced ganglion mother cells, secondary pre,cursors of neurons. Although gem mRNA was present evenly in the cytoplasm
of NB6·4T before the first cell division, it became detected asymmetrically inthe cell during mitosis and eventually only in the medial daughter cell. In
contrast, NB6·4A showed a symmetrical distribution of gem mRNA and GCM
protein through division. Our observations suggest that mechanisms regulat·
ing gem mRNA expression and its translation play an important role in glia
and neuron (Akiyama·Oda et aI., 1999).
The regulation of gcm expression was examined in the peripheral nervous

58

system (PNS). In the Dorsal bipolar dendritic lineage, the precursor cell divides asymmetrically to generate two progeny. The gem gene is expressed in
one of the progeny, then inhibit neuronal differentiation while promoting glial
differentiation. In this lineage, activation ofthe Notch signaling pathway was
shown to be essential for gem transcription, suggesting that Notch activationinduces gem expression, which may be the first step for glial fate induction.

(5)

Axon guidance mechanisms

Kazunaga TAKIZAWA 1, Masaki HIRAMOTOl, Takeshi SASAMURAl, Makiko SHINZA1
and Yoshiki HOTIA2,3 (lGraduate School of Science, University ofTokyo, 2CREST,
JST, 3National Insitute of Genetics)
Glial functions in axonal pathfindings was investigated by examining axonal behavior in gem mutants, which lack glial cells. In the earliest stage of
normal axon bundle formation, a small number of "pioneer" neurons extend
the first axons. These axons become associated with glial cells, on which alarge number of "follower" axons elongate. In the gem mutants, the pioneer
neurons behave normally, but the follower neurons show severe phenotypes.
Axon bundle formation is disrupted and abnormal migration of glia leads tomisrouting ofaxons. These results indicate that although glial cells appear
to be dispensable for the early pathfindings of the pioneer neurons, they haveessential supportive functions in the correct tract formation by a large numberof follower axons (Takizawa, 1999).
The role of Netrin signaling in axon guidance was also investigated. Netrin(NetA and NetB in Drosophila) is a ligand molecule that functions as a component of positional information for growth cones. Frazzled have been characterized as Netrin receptors. In the developing CNS, Netrin molecule is concentrated on the Frazzled positive region. This Netrin localization disappears in
the frazzled mutants, wherea& misexpression of manipulated Frazzled localizes Netrin ectopically, &uggesting that Frazzled have a novel function to localize Netrin. Two pioneer neurons, MP1 and dMP2, track the boundary ofNetrin positive region in the wild type. Their growth cones respond to ectopic
Netrin positive region created by misexpression of either Netrin itself or manipulated Frazzled. Thus it is proposed that Frazzled captures and translo-

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DEPARTMENT OF DEVELOPMENTAL GENETICS

59

cates Netrin molecules to form barriers for MP1 and dMP2 growth cones,
thereby controlling the direction of extension (Hiramoto, 1999).
In order to isolate novel genes that might be involved in the formation ofaxon tracts, an enhancer-trap screening was performed using the tau-IacZ
gene, a marker gene that can visualize axons. Three strains that show defects in the longitudinal axon pathway have been isolated. A candidate gene,whose mutation might be responsible for the phenotype of one strain, encodesa novel kinase expressed in the nervous system from the early stages. Because
the mutant strain shows defects in the formation of neurons and glia, thiskinase may be involved in neurogenesis (Shinza, 1999).

(6)

The gcm transcriptional regulator family

Chieko HIGASHI 1, Toshihiko HOSOYA2,3, Yoshiki HOTTA2,4 (lGraduate School of
Science, University of Tokyo, 2National Insitute of Genetics, 3pRESTO, JST,
4CREST, JST)The gem protein is a member of a novel protein family, the gem-family.
Members of this family share the gem-motif, a novel protein motif that is ofabout 150 amino acids in length. It has been shown that the gem-motif has
sequence-specific DNA-binding activity and that the gem protein has transcriptional regulation activity. The gem-binding DNA sequences are found in
tandem in the upstream of a glia-specific protein whose expression is dependent on gem. Thus it is strongly suggested that the gem protein is a noveltranscriptional regulator that controls glial vs. neuronal decision by controlling expression of cell-type specific genes. The gem-family is most likely anovel family of transcriptional regulators.
Drosophila has at least another gem family gene, gcm2, which is expressedin a subset of glial cells and early hemocytes. Since gem is also expressed inglia and hemocytes, these two gene may interact in the development ofthosecell types (Higashi, 1999). Two gem genes have been isolated from Mouse,
which show tissue specific expression. Gene disruption of these mouse genes
are currently underway.

60

(7)

Hydra Peptide Project: A Novel Neurotransmitter thatControls Muscle Contraction In Hydra

Seungshic YUM, Toshio TAKAHASHI, Masayuki HATTA, Hiroshi SHIMIZU, Osamu
KOIZUMI', Yoshitaka KOBAYAKAWA2 and Toshitaka FUJISAWA (lFukuoka Women's
University; 2Faculty of Science, Kyushu University)
During a course of systematic screening of peptide signaling molecules in
Hydra, we have identified a novel neuropeptide, Hym-176, which evokes ectodermal muscle contraction of the Hydra body colum. The peptide consists of
10 amino acids with an amidated C-terminus. Since the exogenously added
Hym-176 can induce contraction of epithelial hydra which are soley made ofepithelial cells, the peptide acts directly on epithelial cells. We have cloned acDNA encoding the peptide. Within the coding sequence, a signal sequence at
the N-terminal region and one copy ofHym-176 precursor sequence was found.
In addition, another precursor sequence of Hym-357 was found. Hym-357
had been sequenced but its function is unknown. The expression of Hym-176
gene was examined by whole mount in situ hybridization. The strong signal
was observed in the neurons in the peduncle region. A weak signal was also
observed in neurons scattered sparsely along the body column. No signal was
detected in basal disk and head including tentacles. Immunohistochemistry
using antisera which specifically recognize the C-terminal half of Hym-176
confirmed the in situ hybridization result. These results together suggestthat Hym-176 is a neurotransmitter which acts at the neuro-muscular junctions and evokes muscle contraction in the peduncle region.

(8)

Feeding and wounding response of hydra: hydra nervous system Is
more advanced than a diffuse system

Hiroshi SHIMIZU
Freshwater coelenterate hydra has been considered the best example of anorganism having diffuse nervous system. By examining physiological responseof hydra, we obtained a piece of evidence that hydra's nervous system is moreadvanced than a simple diffuse nervous system. Feeding, touching and wounding stimuli were applied to tissue in tentacles or body column, and response of

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DEPARTMENT OF DEVELOPMENTAL GENETICS

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the tissue to the stimulii was examined under a dissecting microscope. Response obtained in the tentacles was of most interest. There, proximal part oftentacle to the prey attachment site or wounding site contracted enormously
whereas distal part of it contracted only slightly or did not contract at all. To
examine if nervous system is involved in the polarized responses, a similar
analysis was carried out in epithelial tentacles which have no nerve cells. It
was found that the wounding stimulus provoked contraction of tissue only inthe vicinity of the wounding site thus suggesting that nerve cells are responsible for the polarized response in wounding in the normal tentacles. It wasalso found that polarized response in feeding occurred in distal to proximal
orientation in the normal tentacles. Despite an extensive search in the bodycolumn and peduncle regions, no polarized response was found by wounding
stimulus. From these observations, I conclude that hydra's nervous system inthe tentacles has a mechanism which enables polarized responses to external
stimulii and that this nervous system is more advanced than a simple diffuse
system. I speculate that this polarization could have been a trigger to evolveto centralized nervous system.

Publications
HACOHEN, N., KRAMER, S., SUTHERLAND, D., HIROMI, Y and KRASNOW, M.A.: sprouty
encodes a novel antagonist of FGF signaling that patterns apical branching of the
Drosophila airways. Cell, 92, 253-263, 1998.
2. KRAMER, S., OKABE, M., HACOHEN, N., KRASNOW, M. A. and HIROMI, Y: Sprouty: acommon antagonist of FGF and EGF signaling pathways in Drosophila. Development, 126, 2515-2525, 1999.
3. AKIYAMA-aDA, Y, HOSOYA,T. and HOTTA, Y.: Asymmetric cell division of thoracic neuroblast 6-4 to bifurcate glial and neuronal lineage in Drosophila. Development, 126,
1967-1974, 1999.
4. AKIYAMA-aDA, Y, HOSOYA,T. and HOTTA, Y: Alteration of cell fate by ectopic expressionof Drosophila glial cells missing in non-neuronal cells. Dev. Genes & Evo\. 208,578585, 1998.
5. HOTTA, Y: Grunion and biological clocks. Kagaku, 68(2), 101, 1998.
6. HOTTA, Y: Universities in the 21th century. Gakujyutu-geppou, 51(10), 2-3, 1998.
7. HIRAMOTO, M.: An axon guidance mechanism mediated by Frazzled dependent Netrin
translocation. Thesis, University of Tokyo, 1999.
8. TAKIZAWA, K.: Analysis of Glial Differentiation and Glial Function in Neural Circuitry
1.

62

9.
10.
11.

12.

13.

Formation in Drosophila Central Nervous System. Thesis, University of Tokyo, 1999.
SHINZA, M.: Isolation of novel genes involved in the nervous system development in
Drosophila. Master's thesis, University of Tokyo, 1999.
HIGASHI, C.: Analysis of the gcm2 gene, a novel gene that has a gem-motif. Master's
thesis, University of Tokyo, 1999.
GRENS, A., SHIMIZU, H., HOFFMEISTER, S., BODE, H.R. and FUJISAWA, T.: PedibinlHym346 lowers positional value thereby enhancing foot formation in hydra. Development,
VoLl!6, 517-524. 1999.
YUM, S., TAKAHASHI, T., HATIA, M. and FUJISAWA, T.: The structure of a preprohormoneof a neuropeptide, Hym-176 in Hydra magnipapillata. FEBS Letters Vol. 439,31-34.
1998.
YUM, S., TAKAHASHI, T., KOIZUMI, 0., MIURA, Y., KOBAYAKAWA, Y., MOHRI, S. and FUJISAWA,
T.: A novel neuropeptide, Hym-176 induces contraction of the ectodermal muscle inhydra. Biochem. Biophys. Res. Comm. Vol. 248, 584-590. 1998.

C-b. Division of Gene Expression(1)

Chromatin remodeling and transcription

Susumu HIROSE
The fundamental building unit of chromatin is the nucleosome, in which 146
base pairs of DNA wind around an octamer of core histones. The chromatin
structure is essential not only for the compact packaging of the eukaryotic
genome but also for regulation of transcription. In vivo evidence for a role ofchromatin in the global repression oftranscription has been obtained by placing a histone gene under the control of an inducible promoter in the yeast
Saccharomyces cerevisiae. When expression of core histone H4 or H2B isrepressed, certain genes including PH05, CUPl and HIS3, that are normallyinactive, become activated. Subsequent studies have led to a consensus thattranscriptional activation and repression are achieved through chromatin acetylation and deacetylation, respectively.
Genetic analyses of transcriptional regulation in yeast have led to the identification of a number of SWI and SNF genes (SM for mating type switching,
SNF for sucrose non-fermenting). These genes are required for proper expression of certain inducible promoters including PH05, CUPl, and HIS3. Among
SWIISNF gene products, SWIl, SWI2/SNF2, SWI3, SNF5, and SNF6 are

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DEPARTMENT OF DEVELOPMENTAL GENETICS

63

functionally interdependent, suggesting that they exert their function as acomplex. Yeast SWI/SNF complex has been purified and found to possess achromatin remodeling activity. In Drosophila, three distinct chromatin remodeling factors, NURF, ACF and CHRAC, have been identified in addition to
the SWIISNF (BRAHMA) complex. All of these chromatin remodeling com·
plexes contain a SWI2/SNF2-related subunit harboring an ATP-dependent
DNA helicase domain. It has been postulated that the SWI2/SNF2-related
proteins may achieve ATP-driven DNA translocation to disrupt histone-DNAinteractions. Interestingly, recent data demonstrate that chromatin remodeling in the promoter region is necessary for transcription. For details, see Ref.
1.
(!)

Chromatin remodeling mediated by Drosophl/a GAGA factor and
ISWI activates lushl tarazu gene transcription In yltro

Masahiro OKADA and Susumu HIROSEGAGA factor is known to remodel the chromatin structure in concert with
the nucleosome remodeling factor NURF in an embryonic S150 extract of Drosophila. The promoter region of the DrosophIla fushi tarazu (ftz) gene carries
several binding sites for GAGA factor. Both the GAGA factor-binding sites
and GAGA factor per se are necessary for the proper expression of ftz in vivo.
We observed transcriptional activation of the ftz gene when a preassembled
chromatin template was incubated with GAGA factor and the S150 extract.
The chromatin structure within the ftzpromoter was specifically disrupted byincubation of the preassembled chromatin with GAGA factor and the S150
extract. Both transcriptional activation and chromatin disruption were
blocked by an antiserum raised against ISWI or by base substitutions in the
GAGA factor-binding sites in the ftzpromoter region. These results demonstrate that GAGA factor and ISWI-mediated disruption ofthe chromatin structure within the promoter region of ftz activates transcription on the chromatin
template. For details, see Ref. 2.

64

(3)

DNA supercoiling factor localizes to puffs on polytene
chromosomes in DrosopblJa meJanogBster

Masatomo KOBAYASHI', NorikoAITA 1, Shigeo HAYASHI, Kohichi OKADA, Tsutomu
OHTA and Susumu HIROSE (lThese authors contributed equally to this work)
DNA supercoiling factor (SCF) was first identified in silkworm as a proteinthat generates negative supercoils into DNA in conjunction with eukaryotic
topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA
encoding Drosophila SCF. Northern analysis revealed 1.6 and 1.8 kb mRNAs
throughout development. The longer mRNA contains an open reading framethat shares homology with mouse reticulocalbin whereas the shorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence.
An antibody against SCF detected a 45 kDa protein in the cytoplasmic fraction and a 30 kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30 kDa protein interacts with topoisomerase
II in the nucleus, and hence it is a functional form of SCF. Immunostaining of
blastoderm embryos showed that SCF is present in nuclei during interphasebut excluded from mitotic chromosomes. In larvae, the antibody stained nuclei of several tissues including a posterior part of the salivary gland. Thislatter staining was associated with natural or ecdysteroid-induced puffs onpolytene chromosomes. Upon heat treatment of larvae, the staining on theendogenous puffs disappeared and strong staining appeared on heat shock
puffs. These results implicate SCF role in gene expression. For details, see
Ref. 3.

(4)

Yeast coactivator MUF1 mediates GCN4-dependenttranscriptional activation

Ken-ichi TAKEMARu, Satoshi lIARASHIMA 1, Hitoshi UEDA and Susumu HIROSE(lDepartment of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka)
Transcriptional coactivators playa crucial role in gene expression by communicating between regulatory factors and the basal transcription machinery.
The coactivator MBF1 (multiprotein bridging factor 1) was originally identi-

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

65

fied as a bridging molecule that connects the Drosophila nuclear receptor FTZF1 and TATA-binding protein TBP. The MBF1 sequence is highly conserved
across species from yeast to human. Here we provide in vitro and in vivo
evidence that yeast MBF1 mediates GCN4-dependent transcriptional activation by bridging the DNA-binding region of GCN4 and TBP. These findings
indicate that the coactivator MBF1 functions by recruiting TBP to promoterswhere DNA-binding regulators are bound. For details, see Ref. 4.

(5)Comparison of sequences of a transcriptional coactivator MBF!
from three Lepidopteran species Bombyx morJ,
Bombyx mandarlna and Samla cynthia

Qing-Xin LID, Hitoshi DEDA and Susumu HIROSE
MBF2 was first isolated from the silkworm Bombyx mori as a positive
cofactor that activates transcription through its interaction with TFIIA. To
identify conserved domain(s) within the MBF2 molecule, we isolated cDNAs
encoding MBF2 homologues from other silkworms Bombyx mandarina andSamia cynthia. Bacterially expressed and purified MBF2 of B. mandarina
and S. cynthia activated transcription in vitro. The predicted amino acid sequences ofMBF2 from two Bombyx species share 97% homology. When wecompared between B. mori and S. cynthia factors, the homology reduced to50%. Four regions in MBF2 are conserved among these three species. Two ofthem are present in the middle region of MBF2 that is essential for the transcriptional activation. For details, see Ref. 5.

(6)

DSIF, a novel transcription elongation factor that regulates RNA
polymerase II processlvlty, is composed ofhuman SpU and Spt5 homologs

Tadashi WADA1,2, Toshiyuki TAKAGI1,2, Yuki YAMAGUCHI 2, Anwarul FERDOUS 2,
Takeshi IMAI 2, Susumu HIROSE, Seiji SUGIMOT03, Keiichi YAN03, Grant A.
HATZOG 4 , Fred WINSTON 4 , Stephen BURATOWSKI 5 and Hiroshi HANDA2 (lTheseauthors contributed equally to this work, 2Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226, Japan, 3Tokyo
Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Machida-shi 194, Ja-

66pan, 4Department of Genetics and 5Department of Biological Chemistry andMolecular Pharmacology, Harvard Medical School, Boston, Massachusetts
02115 USA)We report the identification of a transcription elongation factor from HeLa
cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in
conjunction with the transcription inhibitor 5,6-dichloro-1- 13 ·D·
ribofuranosylbenzimidazole (DRB). This factor, termed ORB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF
has been purified and is composed of 160-kD (P160) and 14-kD (P14) subunits.
Isolation of a eDNA encoding DSIF p160 shows it to be a homolog ofthe Saccharomyces cerevisiae transcription factor Spt5. Recombinant Spt4 protein,
the human homolog of yeast Spt4, is functionally equivalent to DSIF p 14,
indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In
addition to its negative role in elongation, DSIF is able to stimulate the rate ofelongation by RNA Pol II in a reaction containing limiting concentrations ofribonucleoside triphosphates. A role for DSIF in transcription elongation isfurther supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF
and genetic analysis of Spt4 and Spt5 in yeast indicates that DSIF associateswith RNA Pol II and regulates its processivity in vitro and in vivo. For details,
see Ref. 6.

Publ icatians
1.

2.

3.

4.

HIROSE, S.: JB Review: Chromatin remodeling and transcription. J. Biochem., 6.
1060·1064, 1998.
OKADA, M. and HIROSE, S.: Chromatin remodeling mediated by Drosophila GAGA
factor and ISWI activates fushi tarazu gene transcription in vitro. Mol. Cell. BioI., 18,2455·2461, 1998.
KOBAYASHI, M., AlTA, N., HAYASHI, S., OKADA, K., OHTA, T. and HIROSE, S.: DNA supercoiling factor localizes to puffs on polytene chromosomes in Drosophila melanogasterMol. Cell. Bioi., 18, 6737-6744, 1998.
TAKEMARU, K., HARASHIMA, S., UEDA, H. and HIROSE, S.: Yeast coactivator MBF1 mediates GCN4-dependent transcriptional activation Mol. Cell. BioI., 18, 4971-4976,
1998.

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DEPARTMENT OF DEVELOPMENTAL GENETICS

67

5.

LIU, Q.-X, UEDA, H. and HIROSE, S.: Comparison of sequences of a transcriptional
coactivator MBF2 from three Lepidopteran species Bombyx mori, Bombyx mandarina
and Samia cynthia. Gene, 220, 55-59, 1998.
6. WADA, T., TAKAGI, T., YAMAGUCHI, Y, FERDOUS, A., IMAJ, T., HIROSE, S., SUGIMOTO, S.,
YANO, K., HARTZOG, G. A., WINSTON, F., BURATOWSKI, S. and HANDA, H.: DSIF, a noveltranscription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev. 12, 343-356, 1998.
7. KOBAYASHI, M. and HIROSE, S.: Functional dissection of DNA supercoiling factor: EFhand domains and C-terminal HDEF motif are essential for its activity. Genes to
Cells, in press.

C-c. Division of Physiological Genetics(1)

NELF, a multi subunit complex containing RD, cooperates withDSIF to repress RNA polymerase II elongation

Yuki YAMAGUCHI, Toshiaki TAKAGI, Jun HAsEGAWA, Tadashi WADA and Hiroshi
HANDA (Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology)
The purine nucleotide analog 5,6-dichloro-1- B- ribofuranosylbenzimidazole(DRB) is a classic inhibitor of transcription elongation by RNA polymerase II(pol II). Since DRB generally affects class II genes, factors involved in thisprocess must play fundamental roles in pol II elongation. Two elongation
factors essential for DRB action were identified, namely DSIF (Wada et aI.,
Genes Dev., 12, 343-356, 1998) and P-TEFb (Marshall and Price, J. BioI. Chern.,
270, 12335-12338, 1995). Recently, we have identified and purified a third
protein factor required for DRB-sensitive transcription from HeLa cell nuclear
extracts. This factor, termed negative elongation factor (NELF), cooperates
with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five peptides, the smallest of which is identicalto RD, a putative RNA-binding protein of unknown function (Yamaguchi et aI.,
Cell, 97, 41-51, 1999). We have shown a molecular mechanism for DRB action and a regulatory network of positive and negative elonagtion factors.

68

(2)

Gene expression levei and species-specific diversity ofcodon usage based on multivariate analysis

Shigehiko KANAYA1. 2, Yuko YAMADA3, Yoshihiro KUDO l , Toshimichi IKEMURA4(lDepartment of Electrical and Information Engineering, Faculty of Engineering, Yamagata University, 2Division of Physiological Genetics, Department of
Ontogenetics, National Institute of Genetics, 3Department of Biochemistry,
Jichi Medical School, 4Division of Evolutionary Genetics, Department ofPopulation Genetics, National Institute of Genetics)
We examined codon usage in Bacillus subtilis genes by multivariate analysis, quantified its cellular levels of individual tRNAs, and found a clear constraint oftRNA contents on synonymous codon choice. Individual tRNA levels were proportional to the copy number ofthe respective tRNA genes. This
indicates that the tRNA gene copy number is an important factor to determine
in cellular tRNA levels, which is common with Escherichia coli and yeast Saccharomyces cerevisiae. Codon usage in 18 unicellular organisms whose genomes have been sequenced completely was analyzed and compared with thecomposition oftRNA genes. The 18 organisms are as follows: yeast S. cerevisiae,
Aquifex aeolicus, Archaeoglobus fulgidus, B. subtilis, Borrelia burgdorferi,
Chlamydia trachoma tis, E. coli, Haemophilus influenzae, Helicobacter pylori,
Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma pneumoniae,
Pyrococcus horikoshii, Rickettsia prowazekii, Synechocystis sp., and Treponema
pallidum. Codons preferred in highly expressed genes were related to thecodons optimal for the translation process, which were predicted by the composition of isoaccepting tRNA genes. Genes with specific codon usage are discussed in connection with their evolutionary origins and functions. The origin
and terminus of replication could be predicted on the basis of codon usage
when the usage was analyzed relative to the transcription direction of individual genes.

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DEPARTMENT OF DEVELOPMENTAL GENETICS

69

Publ ications
1.

HIRAMOTO, M., SHIMIZU, N., SUGIMOTO, K, TANG, J., KAWAKAMI, Y, ITO, M., AIZAWA,

2.

S.,TANAKA, H., MAKINO, I. and HANDA H.: Nuclear targetted suppression of NF-kB
activity by the novel quinone derivative E3330. J. Immunol. 160, 810-819, 1998.
HIRAMOTO, M., A!ZAWA, S., IWASE, 0., NAKANO, M., TOYAMA, K, HOQUE. M., NABESHIMA, R.,
KAIDOW, A, Imai, T., HOSHI, H. and HANDA, H.: Stimulatory effects of substance P on
CD34 positive cell proliferation and differentiation in vitro are mediated by the modu-

4.

lation of stromal cell function. Int. J. Mol. Med. 1, 347-354, 1998.
WADA, T., TAKAGI, T., YAMAGUCHI, Y., FERDOUS, A, IMAI, T., HIROSE, S., SUGIMOTO, S.,YANO,
K, HARTZOG, G. A, WINSTON, F., BURATOWSKI, S. and HANDA, H.: DSIF, a novel negative transcription elongation factor that regulates RNA polymerase II processivity, iscomposed of human Spt4 and Spt5 homologs. Genes Dev. lZ, 343-356, 1998.
HARTZOG, G.A, WADA, T., HANDA, H. and WINSTON, F.: Evidence that Spt4, Spt5, and5.

Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae.
Genes Dev. lZ, 357-369, 1998.
HIRANO, F., TANAKA, H., HIRANO, Y., HIRAMOTO, M., HANDA, H., MAKINO, I. and SCHEIDEREIT,

6.

C.: Functional interference ofsp1 and NF-kB through the same DNA binding site.
Mol. Cell. BioI. 18, 1266-1274, 1998.
YAMAGUCHI, Y., WADA, T. and HANDA, H.: Drawing a new view ofDRB: Interplay

3.

7.

8.

9.

10.

11.

between positive and negative elongation factors. Genes Cells 3, 9-15, 1998.
VASSIAS, I., HAZAN, D., MICHEL, Y, SAWA, C., HANDA, H., GOUYA, L. and MORINET,
F.: Regulation of human B19 parvovirus promoter expression by hGABP (E4TF1)
transcription factor. J. BioI. Chern. Z73, 8287-8293, 1998.
HATAKEYAMA, M., IWATO, S., FUJIMOTO, K, HANDA, H. and KAWAGUCHI, H.: DNA-carrying
latex particles for DNA diagnosis. 1. Separation of normal and point mutant DNAs
according to the difference in hybridization efficiency. Colloids and Surfaces B:
Biointerfaces 10, 161-169, 1998.
HATAKEYAMA, M., NAKAMURA, K, IWATO, S., HANDA, H., FUJIMOTO, K and KAWAGUCHI,
H.: DNA-carrying latex particles for DNA diagnosis. 2. Distinction of normal andpoint mutant DNA using Sl nuclease. Colloids and Surfaces B: Biointerfaces 10, 171178, 1998.
YAMAGUCHI, Y, WADA, T., SUZUKI, F., TAKAGI, T., HASEGAWA, J. and HANDA, H.: Casein
kinase II interacts with the bZip domains of several transcription factors. Nucleic
Acids Res. Z6, 3854-3861, 1998.
SUZUKI, F., GOTO, M., SAWA, C., ITO, S., WATANABE, H., SAWADA, J-1. and HANDA,H.:

Functional interactions of transcription factor hGABP subunits. J. BioI. Chern.

Z73, 29302-29308, 1998.

12.

WADA, T., TAKAGI, T., YAMAGUCHI, Y, WATANABE, D. and HANDA, H.:

Evidence that p-

TEFb alleviates the negative effect ofDSIF on RNA polymerase II-dependent tran-

70

13.

14.

15.

16.

17.

18.

19.

20.
21.

scription in vitro. EMBO J. 17,7395·7403, 1998.
AJ.ZAWA, S., NAKANO, M., IWASE, 0., YAGUCHI, M., HIRAMOTO, M., HOSHI, H., NABESHIMA, R.,
SHIMA, D., HANDA, H. and TOYAMA, K: Bone marrow stroma from refractory anemia of
myelodysplastic syndrome is defective in its ability to support normal CD34-positive
cell proliferation and differentiation in vitro. Leuk.Res. %3, 239-246, 1999.
YAMAGUCHI, Y, TAKAGI, T., WADA, T., YANO, K, FURUYA, A., SUGIMOTO, S., HASEGAWA,
J.and HANDA, H.: NELF, a multiple complex containing RD, cooperates with DSIF
and RNA polymerase II elongation. Cell 97, 41-51, 1999.
HANDA, H, YAMAGUCHI, Y and WADA, T: Purification of DNA binding proteins. In High
Resolution Chromatography: A Practical Approach. (ed. P.A.Miller) Oxford University
Press, 283-302, 1999.
YAMAGUCHI, Y, WADA, T., WATANABE, D., TAKAGI, T., HASEGAWA, J. and HANDA,
H.: Structure and function of the human transcription elongation factor DSIF. J. BioI.
Chern. %74, 8085-8092, 1999.
KANAYA, S., YAMADA, Y, KUDO, Y and IKEMURA, T: Studies of codon usage and tRNA
genes of 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: Gene
expression level and species-specific diversity of codon usage based on multivariate
analysis. Gene (accepted).
KANAYA, S., KUDO, Y, OKAZAKI, T, ABE, T, IKEMURA, T: Classification of E. coli genes
by self-organization mapping of codon usage in bacteria with completely sequenced
genome. International Symposium, Frontier ofthe Genome Biology of Escherichia coli,
P3, 1998.
KANAYA, S., KUDO, Y, ABE, T., OKAZAKI, T, CARLOS, Del Carpio, IKEMURA, T: Gene
classification by self-organization mapping of codon usage in bacteria with completely
sequenced genome. Genome Informatics Series, No.9, 369·371, 1998.
KUNISAWA, T, KANAYA, S. and KUTTER, E.: Comparison of synonymous codon distribution patterns of bacteriophage and host genomes. DNA Res., 5, 1-8, 1998.
NAKAYAMA, K, KANAYA, S., OHONISHI, M., TERAWAKI, Y and HAYASHI, T: The complete
nucleotide sequence of4>CTX, a cytotoxin-convering phage of Pseudomonas aeruginosa:
implications for phage evolution and horizontal gene transfer via bacteriophages.
Molecular Microbiol., 31, 399-419, 1999

71

D.

DEPARTMENT OF POPULATION GENETICS
D-a. Division of Population Genetics(1)

Local recombination- and mutation-effects onmolecular evolution in Drosophila

Toshiyuki TAKANODeleterious mutations clearly arise at a much higher rate than advantageous and compensatory mutations, indicating the importance of effective elimination of deleterious mutations from a population. Linkage associations between selective loci reduce the efficacy of natural selection (the so-called HillRobertson effect), and thus recombination rates affect accumulation rates ofdeleterious and advantageous mutations. I previously showed the significant difference in the synonymous-substitution pattern found in the achaetescute complex (AS-C) genes in two Drosophila lineages, higher codon-bias in D.
yakuba, and lower bias in D. melanogaster (for detail, see Ref. 1). Besides
these genes, the functionally unrelated yellow (y)gene showed the same substitution pattern, suggesting a phenomenon specific to the X.chromosome telomere region (the yand AS-C genes are all in the tip of the X chromosome).
Because the numbers of AlT-+G/C substitutions were not significantly different from those of G/C-+AIT in the y noncoding regions of these species, a AT/
GC mutational bias could not thoroughly account for the synonymous-substitution biases. By contrast, we did find about a 14-fold difference in recombination rates in the X.chromosome telomere regions between the two species,
suggesting that the reduction of recombination rates in this region resulted in
the reduction of the efficacy of selection in D. melanogaster.
The ylocus also revealed a very high GC-bias of the substitutions in the D.
orena branch, which increased the G+C content at silent sites by about 5%
since the divergence from D. erecta. The ratio of the number of AIT -+ G/C
substitutions to the number of the reverse in the orena branch was significantly different from those at the Adh and Amy loci. The ratio was also sig-

72
nificantly different from those at the yin the D. erecta branch. This suggeststhat the local mutation pressure changed in the orena branch specifically at
the y locus. This change, in turn, probably led to the higher substitution ratein the orena branch compared with the erecta.
In conclusion, this study provided evidence that local changes in recombination rates and mutational pressure are contributing to the irregular synonymous-substitution patterns in Drosophila.

(2) Study of species difference as observed as interspecific hybrid
anomaly in Drosophila: genetic screens for genes involved in thebristle loss of hybrids
Toshiyuki T AKANOWith the aim of revealing genetic variation accumulated among closely related species during the course of evolution, this study focuses on loss ofmacrochaetae on the notum as one of the developmental anomalies seen ininter-specific hybrids between D. melanogaster and its closely related species.
I previously presented evidence that bristle loss in inter-specific hybrids isfound between D. melanogasterand D. simulans, but not between pairs of D.
melanogaster on one hand, and D. mauritiana and D. sechellia on theother. Genetic analysis showed that the D. sim ulans X chromosome confers alarge effect on hybrid bristle loss, although X-autosome interaction is presumably involved. Use of cell-type markers suggests that the defect does not lie
in cell fate decisions during bristle development, but in the maintenance ofneural fate and/or differentiation of the descendants of sensory mothercells. For detail, see Ref. 2.
To isolate genes responsible for the bristle loss of hybrids, deficiency screening and QTL (quantitative trait loci) mapping based on the within-species
variation of D. simulanswere performed. The latter analysis revealed that asingle major effect locus (or cluster ofloci) on the X chromosome accounted forabout two-thirds of the parental difference. I have screened this region for
duplications that affect the hybrid bristle phenotypes. One duplication rescued hybrids from the bristle loss to a great degree. Subsequently, deficiencies were induced on this duplication with 4000 rad of gamma-ray

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irradiation. In a screen of 13000 mutagenized chromosomes, 31 deletions
were recovered. Physical mapping of the deletion-induced duplications andother non-rescue duplications allowed us to localize the locus to a small regionin polytene interval 14A-B. Molecular mapping at fine scale is now under way.

Publication
TAKANo, T. S.: Rate variation of DNA sequence evolution in the Drosophila lineages.
Genetics 149, 959·970, 1998.
2. TAKANo, T. S.: Loss of notum macrochaetae as an interspecific hybrid anomaly between Drosophila melanogaster and D. simulans. Genetics 149, 1435-1450, 1998.
1.

D-b. Division of Evolutionary Genetics(1)

Non-U DNA structures spatially and sequence-specificallyassociated with individual centromeres in thehuman interphase nucleus

Mizuki OHNOl, Toyoaki TENZEN, Yoshihisa WATANABE, Tetsushi YAMAGATA,
Shigehiko KANAYA and Toshimichi lKEMURA (l Inst. Genetic Information, Kyushu
Univ.)
Chromosome bands are associated with various genome characteristics such
as DNA replication timing and long-range GC% mosaic structures (i.e.
isochores), and therefore their boundaries are thought to correspond to switch·
ing regions for replication timing and isochore borders. We previously founda boundary for both the Mb-sized GC% mosaic domains and the replicationtiming zones at the junction between human MHC classes II and III. Within
this junction region, there is a 209-bp polypurine/polypyrimidine sequences(purlPyr tract) with triplex-forming potential. Triplex formation occurs readilyin PurlPyr tract leaving single-strand DNAs that can hybridize with othersingle-strand DNAs and RNAs. Distantly spaced DNAs may also form
transmolecular triplexes organizing themselves into an ordered array. Therefore, triplex formation is thought important as a molecular mechanism determining spatial organization of genome DNA in the nuclei. To investigate

74

biological significance of non-B forming DNAs, unusual DNA structures suchas triplexes were searched in the human interphase nucleus with a modified
fluorescence in situ hybridization method; "nondenaturing" hybridization. Various
PurlPyr tracts present in the human genome were found to have triplex-forming potential in the interphase nucleus. The triplexes visualized by DNA
probes and antitriplex antibodies were characteristically spatially associatedwith centromeres. We hypothesize that different triplex-forming DNAs associate, sequence-specifically, with distinct centromeres in the interphasenucleus. For details, Chromosomes Today 13, in press, 1999.

Studies on codon usages and tRNA genes of 18 unicellar organisms
and quantlflcation of Baclllos sobtllis tRNAs: Gene expression leveland speeles-speclflc diversity of codon usage based on
multivariate analysis(!)

Shigehiko KANAYA and Toshimichi lKEMURA
We examined codon usages in Bacillus subtilis genes by multivariate analysis, and also quantified cellular contents of individual tRNAs of the species.
Clear constraint of tRNA contents on synonymous codon choice was observed.
Contents of individual tRNAs were proportional to copy number of the respective tRNA genes, in common with Escherichia coli and yeast Saccharomyces
cerevisiae. This shows that the tRNA gene number is an important factor todetermine cellular tRNA content. Codon usages of eighteen unicellular organisms whose genomes have been completely sequenced, were similarily analyzed and compared with compositions of tRNA genes. The eighteen organisms are as follows; yeast S. cerevisiae, Aquifex aeolicus, Archaeoglobus
fulgidus, B. subtJiis, Borrelia burgdorferi, Chlamydia trachoma tis, E. coli,
Haemophilus influenzae, Helicobacter pylori, Methanococcus jannaschii,
Methanobacterium thermoautotrophicum, Mycobacterium tuberculosis, Mycoplasma genitarium, Mycoplasma pneumoniae, Pyrococcus horikoshii, Rickettsia prowazekli; Synechocystis sp., and Treponema pallidum. Codon usage
in highly expressed genes were related with the codons optimal for translation
predicted by composition ofisoacceptor tRNA genes. Genes with specific codon
usage were also discussed in connection with these specific functions as well

D. DEPARTMENT OF POPULATION GENETICS75

as their specific evolutionary origin; e.g., the evolutionary processes that establish chloroplast and mitochondria genomes were discussed in view of codon
usage of cyanobacteria and proteobacterium genes, respectively. The origin
and terminus of replication could be predicted for a few species using thespecies-specific codon heterogeneity. For details, Gene, in press, 1999.

(3)

Triplex-forming DNAs in the Human Interphase Nucleus Visualized
In SltD by Polypurine/Polypyrimidine DNA Probes andAntitriplex Antibodies. Triplex-forming DNAs Are
Spatially Associated with Centromeres

Mizuki ORNOl, Toyoaki TENZEN, Hiroyuki SASAKI l, Jeremy S. LEE 2 andToshimichi IKEMURA (lInst. Genetic Information, Kyushu Univ., 2Departmentof Biochemistry, University of Saskatchewan, Canada)
Triplex formation occurs readily in polypurine/polypyrimidine tracts underphysiological conditions leaving the unpaired DNAs available for hybridization with other single-stranded DNAs. Single-strand formation andtransmolecular triplex formation could enable sequences spaced distantly along
the genome to associate with each other and organize nuclear DNA into ordered structures. Multiple copies ofpolypurine/polypyrimidine sequences arepresent in the human genome, and their locations are scattered among andwithin chromosomes. Triplex-forming DNAs in the human interphase nucleus
were investigated using a modified fluorescence in situ hybridization methodthat employed polypurine/polypyrimidine probes. This technique, which isused to detect RNA, will detect single-stranded DNAs in nondenatured nuclei.
Polypurine/polypyrimidine probes such as (GAlTC)n and (GAAlTTC)n gave
sequence-specific, foci-type signals that overlapped with or were closely associated with triplexes immunolocalized by known antitriplex antibodies. The
triplexes visualized differentially with distinct polypurine/polypyrimidine
probes were associated spatially with centromeres in the interphase nucleus
in a sequence-specific manner. Several trinucleotide repeats that can form
non-B structures, including those associated with trinucleotide-expansion diseases, gave foci-type signals, suggesting that several non-B structures could
form in the nucleus.

76

(4)

Early and late replication zone borders are present In andaround the human X-Inactivation center (XIC):
correlation with chromosome bands

Yoshihisa WATANABE, Toyoaki TENZEN and Toshimichi IKEMURA
The human genome is composed of long-range G+C% mosaic structures,
which are thought to be related to chromosome bands. Replication timing
during S phase is associated with chromosome bands; thus, band boundaries
are thought to correspond to regions where replication timing switches. The
proximal limit of the human X-inactivation center (XIC) has been localized
cytologically to the junction zone between Xq13.1 and Xq13.2. Using PCRbased quantification of newly replicated DNA from cell-cycle fractionated THP1 cells, the precise replication timing in and around XIC was determined at themolecular level. We found two regions where replication timing changes fromearly to late. One is near the XIST locus, and the other is located near a large
inverted duplication proximal to XIC. This identified the 1-Mb late replication zone (from XIST to the large inverted duplication) as G-band Xq13.2. We
found several common characteristics between the XIST region and the MHC
class II-III junction which was previously defined as a chromosome bandboundary. These characteristics included differential, dense clustering of Alu
and LINE repeats, and the presence of polypurine/polypirimidine tracts,
MER41A, MER57 and MER58B.

(5)

Compartmentalized DNA-replication timing and GC'Ie levels In andaround the human MDC region

Toyoaki TENZEN, Tetsushi YAMAGATA, Masahiro NOGAMI, Yoshihisa WATANABE,
Shigehiko KANAYA, Asako AND0 1, Hidetoshi INOK0 1 and Toshimichi IKEMURA(lDept. Mol. Life Sci., Tokai Univ. School of Med.)
Genomes of warm-blooded vertebrates have long-range GC% mosaic structures (isochores), which are thought to be related to chromosome bands. DNA
replication timing, gene density, and repeat sequence density also are related
to chromosome bands. Basing on these genome characteristics connectedD. DEPARTMENT OF POPULATION GENETICS

77

with chromosome bands, we may define their boundaries at the molecularlevel. We previously reported a concordant transition of GC% level and replication timing in the junction region of the human MHC classes II and III andproposed the junction to be a band boundary at a high resolution level. In thiswork, we determined DNA replication timing in and around the MHC region
and again found the concordant transition of DNA replication timing and GC%
levels. The compartmentalized structures is the following. Non-MHC region on the centromeric-side (very GC-rich; very early replication): class II (ATrich; relatively late replication): class III (very GC-rich; very early replication):class I (intermediate GC%; early replication): telomeric non-MHC region (ATrich; late replication). Several characteristic structure including polypurine/
polypyrimidine tract were found in the transition regions. The human genome contain a large number ofpolypurine / polypyrimidine tracts. Polypurine/ polypyrimidine sequences have the potential to form triplex and are sites
where DNA polymerases pause in vitro. We have shown the 209-bp tract in
the class II and III junction to form triplex in vitro and in the human interphasenucleus.

(6)Conserved evolution of the Rh50 gene compared toits homologous Rh blood group geneTakashi KITANO, Kenta SUMIYAMA, Toshihiko SHIROISHI 1 and Naruya SAITOU(lMammalian Genetics Laboratory)
We have sequenced the complete coding region of the Rh blood group gene formouse and rat and that of Rh-related 50kD glycoprotein (Rh50) for mouse, rat,and crab-eating macaque. Phylogenetic analyses ofRh and Rh50 amino acidsequences indicate that the Rh50 gene has been evolving about two times
more slowly than the Rh blood group gene both in primates and rodents. Thisconservative nature of the Rh50 gene suggests its relative importance to the
Rh blood group gene. The time of gene duplication that produced the Rh andRh50 genes was estimated to be about 240-310 million years ago. We also
conducted window analyses of synonymous and nonsynonymous nucleotide
substitutions for those two genes. Some peaks where nonsynonymous substitutions are higher than synonymous ones were located on outer membrane

78
regions. This suggests the existence of positive Darwinian selection on Rh
and Rh50 genes through host-parasite interactions. For details, see Ref. 9.

Publ ications
1.

2.

3.

4.

5.

6.

7.
8.

9.

10.

11.

OHNO, M., TENZEN, T, WATANABE, Y., YAMAGATA, T., KANAYA, S. and IKEMURA, T.: NonB DNA structures spatially and sequence-specifically associated with individual centromeres in the human interphase nucleus. Chromosomes Today, 13, in press, 1999.
IKEMuRA, T., OHNO, M., UEMURA, T., SASAKI, H. and TENzEN, T.: Genome and subnuclear organization of non-B forming DNAs in mammalian chromosomes. Cytogenet
Cell Genet, 8t, 101-102, 1998.
SHIINA, T., TAMIYA, G., OKA, A., YAMAGATA, T., YAMAGATA, N., KIKKAWA, E., GOTO, K,
MIZUKI, N., WATANABE, K, FUKUZUMI, Y., TAGUCHl,S., SUGAWARA, C., ONO, A, CHEN, L.,
YAMAZAKI, M., TASHIRO, H., ANDO, A, IKEMuRA, T., KiMURA, M. and INoKo, H.: Nucleotide
sequencing analysis of the 146 kb segment around the IKBL and MICA genes at thecentromeric end of the HLA class I region. Genomics, 47, 372-382, 1998.
IKUTA, T., SOGAWA, N., ARIGA, H., IKEMURA, T. and MATSUMOTO, K: Structural analysis
of mouse tenascin-X: evolutionary aspects of reduplication of FNIII repeats in thetenascin gene family. Gene, %t7, 1-13, 1998.
KANAYA, S., KUDO, Y., ABE, T., OKAZAKI, T., CARPIO, C.D. and IKEMuRA, T.: Gene classification by self-organization mapping of codon usage in bacteria with completely sequenced genome. Genome Informatics Series, No.9, 369-371, 1998.
ANDO,A, KiKUTI, Y.,ABE, K., SHIGENARI,A, KAWATA, H., IKEMURA, T andINoKo, H.: cDNA
cloning, Northern hybridization and mapping analysis of a putative GDS (guanine
nucleotide dissociation stimulator of G proteins)-related protein gene at the centromeric ends ofthe human and mouse MHC regions. Immunogenetics, in press, 1999.
NAKAMURA, Y., GOJOBORI, T and IKEMuRA, T: Codon usage tabulated from the international DNA sequence databases. Nucl. Acids Res. %6, 334, 1998.
KANAYA, S., KUDO, Y. and IKEMuRA, T: Systematization of species-specific diversity ofgenes in codon usage: Comparison of the diversity among six prokaryotes and discrimination of photosynthetic genes in cyanobacteria Synechocystis sp. based on the diversity of genes in chloroplast genome codon usage. Gene, in press, 1999.
KITANO, T., SUMIYAMA, K, SHIROISHI, T. and SAITOU, N.: Conserved evolution of theRh50 gene compared to its homologous Rh blood group gene. Biochemical and Biophysical Research Communications, %49, 78-85, 1998.
OOTA, S.: ThreeTree: A new method to reconstruct phylogenetic trees. In Miyano S.
and Takagi T. eds., Genome Informatics 1998, Universal Academy Press, Tokyo, 340341, 1998.
KUDO, T., IKEHARA, Y., TOGAYACHI, A., KANEKO, M., HIRAGA, T, SASAKI, K and NARIMATSU,

D.

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H.: Expression Cloning and Characterization of a Novel Murine a1,3Fucosyltransferase, mFuc-TIX, that synthesizes the Lewis x (CD15) Epitope in Brain
and Kidney. J. BioI. Chern., 273, 26729-26738, 1998.
SUMIYAMA, K., WASHIO-WATANABE, K., aNa, T., YOSHIDA, M.C., HAYAKAWA, T. and UEDA,
S.: Human class III POU genes, POU3F1 and POU3F3, map to chromosomes 1p34.1
and 3p14.2. Mammalian Genome, 9, 180-181, 1998.

D-c. Division of Theoretical Genetics(1)

Evolution by nearly-neutral mutations

Tomoko OHTA
Genetic systems are complex and interact at various levels; among aminoacids or nucleotide sites, among gene products, and among regulatory regions
and proteins. Patterns of synonymous and nonsynonymous substitutions ofmammalian genes indicate that nonsynonymous substitutions are nearly neutral, coming from interactions among amino acids. As an interactive system, the NK model of Kauffman was analysed. This model assumes thateach amino acid makes a fitness contribution that depends upon the amino acidand upon K other amino acids among the N that make the protein. Throughsimulations, it was found that there are numerous nearly-neutral mutationsin this model, and that evolution is rapid in small populations and slow inlarge ones. The system moves on the rugged fitness landscape by mutation,
random genetic drift and selection. Small populations have more chance toattain novel genetic systems than large ones because of larger effects of random drift, but the chance of extinction becomes greater. For details, see
Genetica 102/103, 83-90.

(2)

On the pattern of polymorphisms at major
histocompatibility complex lociTomoko OHTA
The pattern of polymorphisms at major histocompatibility complex loci wasstudied by computer simulations and by DNA sequence analysis. Two typesof selection overdominance plus short-term selection and maternal-fetal in-80compatibility, were simulated for a gene family with intra- and interlocus geneconversion. Both types of selection were found to be consistent with the observed patterns of polymorphisms. It was also found that the more interlocus
conversion occurs, the higher the divergence becomes at both nonsynonymousand synonymous sites. The ratio of nonsynonymous-to-synonymous divergenceamong alleles decreases as the interlocus conversion rate increases. These
results agree with the interpretation that the rate of interlocus conversion islower in human genes than in genes of other nonprimate mammals. This isbecause, in the latter, synonymous divergence at the ARS (antigen recognition
site) is often higher than that at the non-ARS, whereas in the former, this is
not so. Also, the ratio of nonsynonymous to synonymous substitutions at the
ARS tends to be higher in human genes than in other mammalian genes. The
main difference between overdominance plus short-term selection and maternal-fetal interaction is that the number of alleles and heterozygosity per locus
are higher in the latter than in the former under the presumed selection
intensities. However, the average divergence among alleles tends to be lower
in the latter than in the former under similar conditions. For details, see
Jour. Mol. Evol., 46, 633-638.

Pub Iica t ions
1.
2.

OHTA, T.: Evolution by nearly-neutral mutations. Genetica 103, 83-90, 1998.
OHTA, T.: On the pattern of polymorphisms at major histocompatibility complex loci.
Journal of Molecular Evolution 46,633-638, 1998.

Oral Presentations
1.
2.
3.

OHTA, T.: Nearly neutral mutations and the molecular clock. Aug. 12, Beijin, China.
OHTA, T.: Nearly neutral theory of molecular evolution. Aug. 23 Fukuoka.
OHTA, T.: Nearly neutral theory. Oct. 6, Workshop, Towards a Comprehensive Dynamics of Evolution, Santa Fe, U. S. A.

81

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DEPARTMENT OF INTEGRATED GENETICS
E-a. Division of Human Genetics(1)

Cloning and structural analysis of themouse 7F4/F5 imprinted domain

Hiroyuki SASAKI, Reiko KAT0 1, Takaaki YOKOMINE 1, Shin-ichi MIZUNO', Hisao
SHIROHZU 1, Ko ISHIHARA l and Tsunehiro MUKAI 2 (lInstitute of Genetic Information, Kyushu University, 2Department of Biochemistry, Saga Medical College)
Our main research subject is genomic imprinting, which causes parentalorigin-specific monoallelic expression of a subset of mammaliangenes. Imprinted genes tend to form clusters in specific regions of the
genome. This may be related to the mechanism of imprinting or the reason
for the evolution of imprinting. As a step to understand the structural andfunctional characteristics of the imprinted genome domains, we study an imprinted domain in mouse chromosome band 7F41F5. This region is syntenic
to human llp15.5, which contains loci responsible for Beckwith-Wiedemannsyndrome and childhood and adult tumors. To date, at least eight imprinted
genes have been mapped to this mouse region. YAC, BAC, and cosmid contigs
covering the I-Mb region were constructed and a physical map of the region
was produced. Large-scale sequencing of selected BAC clones is now ongoing.
The obtained sequence information will be used to identify new imprinted
genes and their regulatory elements. Since most imprinted genes are associated with a CpG island, we also developed a quick and simple method to
identify CpG islands (Ref. 4), which can be used to isolate new imprinted
genes.

82(!)

Regulation of imprinting of themouse IgIZ/H19 sub-domain

Hiroyuki SASAKI, Ko ISHIHARA 1, Reiko RAT0 1, Hiroyasu FURUUMI·, Mizuki
OHN0 1, Mohamad ZUBAIR 1, Naoya HATAN0 1 and Tohru IWAKI 2 (llnstitute ofGenetic Information, Kyushu University, 2Department of Neuropathology, Neurological Institute, Faculty of Medicine, Kyushu University)
At the centromeric end of the above imprinted domain, there are two reciprocally imprinted genes, Igf2and H19: Igf2is paternally expressed and H19maternally expressed. It was previously shown that the parental-allele-specific expression ofthese genes occurs through a competition for a set of endoderm-specific enhancers located downstream of H19. To look for enhancers
responsible for the imprinted expression in the tissues other than endoderm,
we sequenced a 40-kb region containing H19in both mouse and human. A
comparison ofthe sequences from the two species revealed a total often evolutionarily conserved non-coding segments, two of which precisely coincided to
the known endoderm enhancers. We then tested the function of the remaining eight segments in transgenic mice and found that at least five ofthem weretissue-specific enhancers (three mesoderm-specific and two ectoderm-specificenhancers). More detailed studies on the mechanism of Igf2/H19imprinting
is now being conducted.

(3)

Cloning of a family of novel mammalian
methyltransferases

Hiroyuki SASAKI, Takahito CHIJIWA!, Hiroyasu FURUUMI 1, Wahyu PURBOWASIT0 1,
Shin-ichi MIZUNO·, Shoji TAJIMA2 and Tomohiro KON0 3 (llnstitute of Genetic
Information, Kyushu University, 21nstitute of Protein Biochemistry, Osaka
University, 3NODAI Research Institute, Tokyo University ofAgriculture)
In genomic imprinting, DNA methylation works as an important marking
mechanism to distinguish the parental alleles of imprinted genes. We performed a TBLASTN search ofthe dbEST database using the catalytic domainof the known mammalian DNA(cytosine-5) methyltransferase Dnmtl, andidentified cDNAs that potentially code for novel types of methyltransferase. We

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DEPARTMENT OF INTEGRATED GENETICS

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cloned full-length cDNAs for the mouse proteins and confirmed their
methyltransferase activity using an in vitro assay. It will be interesting toinvestigate the roles of these novel methyltransferases in genomic imprinting.

(4)

Human genome resources and their application for the
analysis of chromosome!lAsao FUJIYAMA, Ayuko MOTOYAMA 1 and Hong-Seog PARK! (lGenome Science
Center Riken)
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 haveconstructed human mono-chromosomal cosmid libraries (except for CM#9 12) and #21 fosmid library, and BAC library. Unlike other libraries, ourcosmid and fosmid libraries employed random fragmentation/cloning protocolthat enabled us to efficiently construct highly randomized libraries. Using
these resources, detailed analyses on human 21qcen and 21qter regions are in
progress.

(5)

Whole genome analysis of signal-transduction pathways In
fission yeast

Yong-Sik BONG, Inaho DANJO! and Asao FUJIYAMA (lMax Planck Institute,
Munich)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 of thelack of appropriate analysis system. To overcome this problem, we categorized genes based on their expression levels in the presence or absence of theras1 gene product under different growth conditions. We utilized arrays ofclones covering entire genome of the fission yeast. Here, we demonstrate the84

detection of low molecular weight heat shock protein gene, hsp 16, and showthat it is regulated by a ras-mediated signaling pathway, not by the heat shock
response in fission yeast.

Pub I i ca t ions
HATANO, N., EVERSOLE-CIRE, P., FERGUSON-SMITH, AC., JONES, P.A, SURANI, M.A &SASAKI, H.: Enhancer-dependent, locus-wide regulation of the imprinted mouse insulin-like growth factor II gene. J. Biochem. Ill3, 984-991, 1998.
2. SHIBATA, H., YODA, Y, RATO, R, VEDA, T., KAMIYA, M., HIRAlWA, N., YOSIKI, A, PLASS, C.,PEARSALL, RS., HELD, W.A, MURAMATSU, M., SASAKI, H., KUSAKABE, M. & HAYASHIZAKI,Y.: A methylation imprint mark in the imprinted gene Grfl/Cdc25Mm locus shares acommon feature with the U2afbp-rs gene: an association with a short tandem repeat
and a hypermethylated region. Genomics 49, 30-37, 1998.
3. ISHIHARA, K., RATO, R, FURUUMI, H., ZUBAIR, M. & SASAKI, H.: Sequence of a 42-kb
mouse region containing the imprinted H19locus: identification of a novel musclespecific transcription unit showing biallelic expression. Mamm. Genome 9, 775-777,
1998.
4. RATO, R. & SASAKI, H.: Quick identification and localization of CpG islands in large
genomic fragments by partial digestion with Hpall and Hhal DNA Res. 5, 287-295,
1.

5.

6.

1998.
YOSHIKAWA, H., FUJIYAMA, A. and MATSUBARA, K.: A novel human gene located on
Xpll.2-pl1.4 which escapes X-inactivation detected and isolated using a two-dimensional DNA mapping method. Genomics, 49, 237-246, 1998.
DANJOH, 1. and FUJIYAMA, A: Ras-mediated signaling pathway regulates the expression of a low molecular weight heat shock protein in fission yeast.(1999) Gene, in press

DEPARTMENT OF INTEGRATED GENETICS

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85

E-b. Division of Appl ied Genetics(1) Expression pattern of the a -amylase geneduring seed germination In rice and barley
Noriko SUGIMOTO l , Genkichi TAKEDA l , Yasuo NAGATO l , and Junji YAMAGUCHI 2(IGraduate School of Agricultural and Life Sciences, University of Tokyo,
2BioScience Center, Nagoya University)

In situ hybridization analysis shows some similarities in the expression
pattern between rice and barley a -amylase genes, RAmylA and Amyl: i) the
expression of these genes initiates in the scutellar epithelium and continues
in the aleurone layer, ii) the gene expression in the aleurone layer initiatesfrom the region close to the embryo. However, we also identified some differences between these species; i.e., i) expression of the rice a -amylase gene
initiates in the terminal regions of scutellar epithelium, whereas middle-basal
region is the initial expression site of the barley gene, ii) the progress of thegene expression in rice aleurone layer is faster than that in barleyone. Additionally, analyses using embryo mutants indicate that the abolishment of the scutellum results in no a -amylase gene expression. For details, see Ref. I.
(!)

pan/de pbytomer 1 mutations affect the

panicle architecture of rice
Masayuki TAKAHASHI l , Nobuhiro NAGASAWA l , Hidemi KITAN0 2 and Yasuo
NAGATO l (IGraduate School of Agricultural and Life Sciences, University ofTokyo, 2Faculty ofAgriculture, Nagoya University)
We have characterized three panicle phytomer 1 (papl) mutations from theviewpoint of phytomer. In papl mutants, rachis phytomers were strongly affected: severe reduction of rachis internode length and the increase in the number ofrachis internodes. In addition, bracts were frequently overdeveloped. On
the contrary, papl differently affected primary branch phytomers: reduction inboth the number and length of internodes. Rudimentary and empty glumes
were frequently elongated. Floral organs were mostly normal. However, a double

86mutant between papl and fonl inducates that PAPl has a distinct role in theregulation of floral organ number. Thus PAPl participates in the regulation ofpanicle phytomers. For details, see Ref.2.

(3) A recessive heterochronlc mutation, pJastochroD 1, shortens theplastochron and elongates the vegetative phase In rice
Jun-Ichi ITOH 1, Atsusi HAsEGAWA2, Hidemi KrTAN0 3and Yasuo NAGAT0 1 (lGraduate School ofAgricultural and Life Sciences, University of Tokyo, 2Aichi University of Education, 3Faculty ofAgriculture, Nagoya University)
We identified two recessive alleles of a rice heterochronic gene, pIastochronl1 (pIal-l) and pIal-2, that reduce the length ofthe plastochron to about halfthat ofthe wild type. The number ofleaves produced in the vegetative phase
was nearly twice that in the wild type. Panicle development was severely
disturbed in pIal mutants; primary rachis branch primordia were converted
into vegetative shoots. Thus, vegetative and reproductive programs are simultaneously expressed during the reproductive phase of pIal. Accordingly,
pIal is a heterochronic mutation that extends the vegetative period. The
PLAl gene is considered to regulate the duration of the vegetative phase via
controlling the rate ofleaf production in the meristem. For details, see Ref.3.

(4)

Isolation and characterization of a ricehomeobox gene, 08815

Yutaka SAT0 1, Naoki SENTOKU 1 , Yasuo NAGAT0 2 and Makoto MATSUOKA 1(lBioScience Center, Nagoya University, 2Graduate School ofAgricultural andLife Sciences, University of Tokyo)
We isolated a cDNA designated OSH15 that encodes a KNOTTED-type
homeodomain protein. Transgenic tobacco plants showed a dramatically altered morphology. In early embryogenesis, OSH15 is expressed at a region
where shoot apical meristem would develop later. Thereafter, OSH15 expression in the shoot apical meristem ceased but was detected in a ring-shaped
pattern at the boundary of some embryonic organs. This expression pattern
was also observed in the vegetative and reproductive shoots as if OSH15 dis-E.

DEPARTMENT OF INTEGRATED GENETICS

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tinguishes each phytomer near the shoot apex. OSH15 may play roles inmorphogenetic events around the shoot apical meristem. For details, see
Ref.4.

(5)

Molecular mechanisms of hereditaryneurodegeneratlve diseases

Shoji TSUJI (Niigata University)
The research is focused to elucidate molecular mechanisms of human neurological diseases and eventually to develop therapeutic measures. Among theneurological diseases, the research is fOlYused on diseases caused by expansions of CAG repeats encoding polyglutamine stretches, which include hereditary spinocerebellar degenerations. We have demonstrated that the prevalence rates of individual spinocerebellar degenerations substantially vary
among ethnic populations. We have recently found that the frequencies ofintermediate alleles, i.e. alleles with large CAG repeats within the normal
range, show good correlations with the prevalence rates of the corresponding
spinocerebellar degenerations, suggesting that the intermediate alleles are
the source for the mutant alleles. We have recently succeeded in creatingtransgenic mice showing instability of the CAG repeats similar to those observed in human diseases, which should be a good mouse model for investigating the molecular mechanisms of the instability of the CAG repeats. With
regard to the mechanisms ofneurodegeneration caused by CAG repeat expansions, we have demonstrated that truncated mutant proteins with expanded
polyglutamine stretches result in aggregate formation and lead to apoptosis.

88

Pub I i ca t ions
1.

SUGIMOTO, N., TAKEDA, G., NAGATO, Y and YAMAGUCHI, J.:

Temporal and spatial ex·pression of the a -amylase gene during seed germination in rice and barley. Plant Cell2.

Physiol. 39, 323-333, 1998.
TAKAHASHI, M., NAGASAWA, N., KITANO, H. and NAGATO, Y:

panicle phytomer 1 muta-

tions affect the panicle architecture of rice. Theor. Appl. Genet. 96, 1050-1056, 1998.
3.

ITOH, J.-I., HASEGAWA, A, KITANO, H. and NAGATO, Y:

A recessive heterochronic muta-tion, plastochron 1, shortens the plastochron and elongates the vegetative phase inrice. Plant Cell 10, 1511-1521, 1998.
4.

SATO, Y, SENTOKU, N., NAGATO, Y and MATSUOKA, M.:

Isolation and characterization ofa rice homeobox gene, OSH15. Plant Mol. BioI. 38, 983-998, 1998.
5.

ITOH, J.-1. and NAGATO, Y:

A mutation associated with phyllotaxy and leafbalde·sheath boundary in rice. Rice Genet. Newslett. 15:90-93, 1998.
6.

KINAE, T., SATOH, H., KITANO, H. and NAGATO, Y:

A mutiple radicle1 mutation affects

the number ofradicles and the vegetative growth in rice. Rice Genet. Newslett. 15, 937.

96, 1998.
KIKUCHI, K, YOSHIDA, KT., NAGATO, Y. and HIRANO, H.-Y:

Chromosomal location of

the OsNACgene. Rice Genet Newslett 15 :160-161, 1998.
8.

IGARASHI, S., KOIDE, R., SHIMOHATA, T., YAMADA, M., HAVASHI, Y, TAKANO, H., DATE, H.,
OVAKE, M., SATO, T, SATO, A, EGAWA, S., IKEUCHI, T, TANAKA, H., NAKANO, R., TANAKA, K,
HOZUMI, I., INUZUKA, T, TAKAHASHI, H. and TSUJI, S.:

Suppression of aggregate forma·tion and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA
protein with an expanded polyglutamine stretch. Nature Genet. 18, 111-117, 1998.
9.

ONODERA, 0., IDEZUKA, J., IGARASHI"S., TAKIVAMA, Y, ENDO, K, TAKANO, H., OVAKE, M.,
TANAKA, H., INUZUKA, T., HAVASHI, T, YUASA, T., ITo, J., MIYATAKE, T and TsUJI, S.:

Progressive

atrophy of cerebellum and brainstem as a function of age and the size of the expanded
CAG repeats in the MJD1 gene in Machado·Joseph disease. Ann. Neurol. 43, 288-296,
10.

1998.
OKUIZUMI, K and TSUJI, S.:

Alzheimer's disease as a polygenic disease. Neuropathol-ogy 18, 111-115, 1998.
11.

SAITO, M., MATSUMINE, H., TANAKA, H., ISHIKAWA, A, SHIMODA-MATSUBAVASHI, S., SCHAFFER,
AA, MIZUNO, Y and TSUJI, S.:

Refinement ofthe gene locus for autosomal recessive

juvenile parkinsonism (AR-JP) on chromosome 6q25.2-27 and identification of markers exhibiting linkage disequilibrium. J. Hum. Genet. 43, 22-31, 1998.
12.

TANNO, Y, OKUIZUMI, K and TSUJI, S.:

Mitochondrial DNA polymorphisms in Japa-

nese sporadic Alzheimer's disease. Neurobiol. Aging 19, S47-S51, 1998.
13.

IKEUCHI, T, SANPEI, K, TAKANO, H., SASAKI, H., TASHIRO, K, CANCEL, G., BRICE, A, BIRD,
T.D., SCHELLENBERG, G.D., PERICAK-VANCE, M., WELSH-BoHMER, KA, CLARK, L.N.,
WILHELMSEN, K and Tsu.JI, S.:

A novel long and unstable CAG/CTG trinucleotide

E.

89

DEPARTMENT OF INTEGRATED GENETICS

repeat on chromosome 17q. Genomics 49, 321-326, 1998.
14.

HAYASHI, Y., KAKITA, A, YAMADA, M., EGAWA, S., OYANAGI, S., NAITO, H., TSUJI, S. and
TAKAHASHI, H.:

Hereditary dentatorubraI-pallidoluysian atrophy: ubiquitinated fila-

mentous inclusions in the cerebellar dentate nucleus neurons. Acta Neuropathol. 95,
479-482, 1998.
15.

TAKIYAMA, Y, SHIMAZAKI, H., MORITA, M., SOUTOME, M., SAKOE, K, ESUMI, E., MURAMATSU,
S., YOSHIDA, M., IGARASHI, S., TANAKA, H., TSUJI, S., SASAKI, H., WAKISAKA, A, NAKANO, L
and NISHIZAWA, M.:

Maternal anticipation in Machado-Joseph disease (MJD): somematernal factors independent of the number of CAG repeat units may playa role ingenetic anticipation in a Japanese MJD family. J. Neurol. Sci. 155, 141-145, 1998.
16.

ILLARIOSHIKIN, S.N., MARKOVA, E.D., SLOMINSKY, P. A, MIKLINA, N.l., POPOVA, S.N.,
LIMBORSKA, S.A, TSUJI, S., IVANOVA-SMOLENSKAYA, LA:

The GTP cyclohydrolase I genein Russian families with dopa-responsive dystonia. Arch. Neurol. 55, 789-792, 1998.
17.

HERRMANN, T, SCHINDLER, D., TARE, H., ONODERA, 0., IGARASHI, S., POLACK,A, ZEHNPFENNING,
D. and TSUJI, S.:

Molecular cloning, structural organization, sequence, chromosomal

assignment, and expression of the mouse alpha-N-acetylgalactosaminidase gene. Gene
ll11, 205-214, 1998.
18.

ZHOU, Y.-X., WANG, G.-X., TANG, B.-S., LI, W-D., WANG, D.-A, LEE, H.-S., SAMBUUGHIN,
N., ZHOU, 1.-S., TSUJI, S., YANG, B.-X. and GOLDFARB, 1.G.:

Spinocerebellar ataxia

type 2 in China: Molecular analysis and genotype-phenotype correlation in ninefamilies. Neurology 51, 595-598, 1998.
19.

SASAKI, H., WAKISAKA, A, SANPEI, K, TAKANO, H., IGARASHI, S., IKEUCHI, T., IWABUCHI, K,
FUKAZAWA, T, HAMADA, T, YUASA, T, TSUJI, S. and TASHIRO, K:

Phenotype variation

correlates with CAG repeat length in SCA2 - A study of 28 Japanese patients. J.
Neurol. Sci. 159, 202-208, 1998.
20.

GOOD, P, YODA, A, SAKAKIBARA, S., YAMAMOTO, A, IMAI, T, SAWA, H., IKEUCHI, T, TSUJI, S.,
SATOH, H. and OKANO, H.:

The Human Musashi Homolog 1 (MSIl) gene encoding thehomologue of MusashilNrp-l, a neural RNA-binding protein putatively expressed in
21.

CNS stem cells and neural progenitor cells. Genomics 5ll, 382-384, 1998.
TAKANo, H., CANCEL, G., IKEUCHI, T, LoRENZETTI, D., MAWAD, R., STEVANIN, G., DIDIERJEAN,

0., DURR, A, OYAKE, M., SHIMOHATA, T, SASAKI, R., KOIDE, R., IGARASHI, S., HAYASHI, S.,
Close associations between prevalence rates of dominantly inherited spinocerebellar ataxias withTAKIYAMA, Y, NISHIZAWA, M., TANAKA, H., ZOGHBI, H., BRICE, A, TSUJI, S.:

CAG-repeat expansions and frequencies of large normal CAG alleles in Japanese andCaucasian populations. Am. J. Hum. Genet. 63, 1060-1066, 1998.
22.

KOIDE, T, IGARASHI, S., KIKUGAWA, K, NAKANO, R., INUZUKA, T, YAMADA, M., TAKAHASHI,
H. and TSUJI, S.:

Formation of granular cytoplasmic aggregates in cells expressing

mutant Cu/Zn superoxide dismutase associated with familial amyotrophic lateralsclerosis. Neurosci. Lett. ll57, 29-32, 1998.
23.

SIDRANSKY, E., BURGESS, C., IKEUCHI, T., LINDBLAD, K, LONG, R.T, PHILIBERT, R.A.,
RAPOPORT, J., SHALLING, M., TSUJI, S. and GINNS, E.!.:A triplet repeat on 17q accounts

90
for most expansions detected by the repeat expansion detection technique. Am. J.
Hum. Genet. 6%, 1548-1551, 1998.
24.

OKUIZUMI, K., TAKANO, H., SEKI, K., ONISHI, Y, HORIKAWA, Y. and TSUJI, S.:

Genetic

polymorphism of the tau gene and neurodegenerative diseases with tau pathology
among Japanese. Ann. Neurol. 44, 707, 1998.
25.

HAYASHI, Y, KAKITA, A., YAMADA, M., KOIDE, R., IGARASHI, S., TAKANO, H., IKEUCHI, T.,
WAKABAYASHI, K., EGAWA, S., TSUJI, S. and TAKAHASHI, H.:

Hereditary dentatorubral-pallidoluysian atrophy: detection of widespread ubiquitinated neuronal and glial
intranuclear inclusions in the brain. Acta Neuropathol. 96, 547-552, 1998.
26.

SATO, T., OYAKE, M., NAKAMURA, K., NAKAO, K., FUKUSHIMA, Y, ONODERA, 0., IGARASHI, S.,
TAKANO, H., KIKUGAWA, K., isHIDA, Y, SHIMOHATA, T., KOIDE, R., IKEUCHI, T., TANAKA, H.,
FUTAMURA, N., MATSUMURA, R., TAKAYANAGI, T., TANAKA, F., SOBUE, G., KOMURE, 0.,
TAKAHASHI, M., SANO, A., ICHIKAWA, Y, GOTO, J., KANAZAWA, I., KATSUKI, M. and TSUJI,
S.:

Transgenic mice harboring a full-length human mutant DRPLA gene exhibit age-dependent intergenerational and somatic instabilities of CAG repeats comparable tothose in DRPLA patients. Hum. Mol. Genet. 8,99-106, 1999.
27.

AIDA, H., TAKAKUWA, K., NAGATA, H., TSUNEKI, I., TAKANO, M., TSUJI, S., TAKAHASHI, T.,
SONODA, T., HATAE, M., TAKAHASHI, K., HASEGAWA, K., MIZUNUMA, H., TOYODA, N., KAMATA,
H., TORII, Y, SAITO, N., TANAKA, K., YAKUSHIJI, M., ARAKI, T. and TANAKA, K.:

Clinicalfeatures of ovarian cancer in Japanese women with germline mutations of BRCAl.
Clin. Cancer Res. (in press)
28.

TAKANO, H., KOIKE, R., ONODERA, 0., SASAKI, R. and TSUJI, S.:

Mutational analysis andgenotype-phenotype correlation of 29 unrelated Japanese patients with X-linked
adrenoleukodystrophy (ALD). Arch. Neurol. (in press)
29.

TSUJI, S.:

Molecular genetics of dentatorubral-pallidoluysian atrophy (DRPLA). In"Genetic Instabilities and Hereditary Neurological Diseases" (edited by Wells R. D.
and Warren S. T.), pp213-220, Academic Press, San Diego, 1998.
30.

SANPEI, K. and TSUJI, S.:

DIRECT technologies. In "Genetic Instabilities and Heredi-tary Neurological Diseases" (edited by Wells R. D. and Warren S. T.), pp455-462,
Academic Press, San Diego, 1998.
31.

TSUJI, S.:

Dentatorubral-pallidoluysian atrophy (DRPLA). In "Analysis of Triplet

Repeat Disorders" (edited by Rubinsztein D. C. and Hayden M. R.), pp209-218, BIOS
Scientific Publishers, Oxford, 1998.

91

F.

GENETIC STRAINS RESEARCH CENTER
F-a. Mammal ian Genetics Laboratory(1)

Analysis of the genetic structure of Ph hotspot In theclassII of MDC

Taku ISOBEI, Masayasu YOSIDN0 2, Kirsten FISCHER LINDAHL2, Kenichi MIZUN0 3,
Tsuyoshi KOIDE, Kazuo MORIWAKII, Toshihiko SHIROISHI ('Grad.Univ.Advanced
Studies, 2Univ. Texas, 3Inst. Phys. Chem. Res.)In the mouse major histocompatibility complex (MHC) class II region, thebreakpoints of meiotic recombination are not distributed at random, but are
clustered in specific sites, called hotspots. All of the hotspots are locateddownstream or inside of genes in the MHC. Thus far, four hotspots havebeen identified in this region. Among four, a hotspot in the vicinity of Ph gene
(hereafter we called Ph hotspot) has been poorly characterized.
In the present study, we constructed a fine restriction map of the 15kbp of
DNA fragment around the Ph hotspot. Molecular mapping revealed that fiverecombinants were confined to a 2.4kb DNA region located to the 3'end of Ph
gene. Comparing the molecular structure of the four hotspots including Ph
hotspot, we found that all hotspots were located in introns or 3' end of genes,
but not at 5' end of genes. We could not detect any sign of the transcription in
the 15kbp DNA fragment encompassing the Ph hotspot by Northern blot hybridization analysis using male meiotic cells. This contrasts to hotspots
identified in budding yeast, in which most of hotspots are located at 5' end ofgenes overlapping with transcription initiation sites. We sequenced the 2.4kb
DNA fragment including the Ph hotspot from recombinants and their parental strains. As the result, recombinational breakpoints of at least five reo
combinants were confined to a 341bp of DNA segment located in the intron of
the Ph gene. The other recombinant had a breakpoint in the 2.9kb region
distal to the intron. The sequence of the hotspot was searched in the database to identify known. We found that the sequence has MT-middle repeti-

92

tive family, a sequence homologous to the transmembrane domain of rat Ph
gene and the motif of rat tumor necrosis factor (TNF).
In addition, we have studied higher chromatin structure around the hotspots
in meiotic prophase by FISH analysis with DNA probes for the hotspots andthe other regions in the MHC class II, being followed by immunostaining with
anti-synaptonemal complex (SC) proteins antibody. This study indicated thatthe DNA region including the Lmp2hotspot was randomly placed in relation
to the SC.

(2)

Mapping of modifier gene suggests interaction of two preaxial
polydactylous mutant genes, Rlm4 and 1st, in mouse

Hiroshi MASUYA, Tomoko SAGAI and Toshihiko SHIROISHI
The anteroposterior axis patterning in vertebral limb morphogenesis is established by the formation ofthe zone of polarizing activity (ZPA) at the posterior margin oflimb buds. The Sonic hedgehog (Shh) gene is expressed exclusively at the posterior margin of limb buds, and it mediates the polarizing
activity. A preaxial polydactyly mutant Rim4 that arose spontaneously in
the C57BL/IO background shows ectopic expression of Shh at the anterior
margin of the limb bud and results in mirror-image duplication of thedigits. Crossing the Rim4 heterozygotes with an inbred strain, MSM, completely suppresses the Rim4phenotype, while crossing the same mice with the
NZB strain demonstrates almost a full penetrance of the phenotype. In thisstudy, we carried out mapping of the modifier gene for Rim4, based on thepolymorphism between the MSM and NZB strains. As a result, a major
modifier gene was mapped close to Aristaless-like4 (Alx4) , which is the causative gene of another preaxial polydactyly mutation, Strong's luxoid (lst) , on
Chromosome 2. Double heterozygotes of Rim4 and 1st J showed a more severe
polydactyly phenotype than that of the heterozygotes of either single
mutation. We found three amino acid substitutions in the Alx4 gene between
the MSM and NZB alleles. All these results suggest the possibility thatgenetic interaction between Rim4 and Alx4 genes plays a key role in establishing anteroposterior axis formation in limb development.

F.

(3)

GENETIC STRAINS RESEARCH CENTER

93

Phenotype characterization and fine mapping ofa preaxial polydactyly mutant, luxate (lx)

Yukari YADA 1, Hiroshi MAsUYA, Akihiko MITA and Toshihiko SHIROISHI(lOchanomizu Univ.)
luxate (Ix) is a spontaneous mouse mutant and mapped to the proximal
region of Chromosome 5. Ix heterozygotes have preaxial polydactyly including
hyperphalangy of the first digit only on the hindlimb. The homozygotes show
missing of the first digit, hemimelia with the shortened tibia and sacralization of the 26th vertebra.
In addition to several skeletal anomalies, various kidney defects such as
horseshoe kidney, polycystic kidney and hydronephrosis were often observed in
the mutant. These kidney defects seemed to be influenced by the genetic
backgrounds. Only 10 percent of Ix homozygotes on the NZB background exhibited kidney defect, while as many as 55 percent exhibited the same phenotype on the C57BU6 background. The Ix phenotypes were completely suppressed when the mutant was mated with an inbred strain MSM that wasestablished from Japanese wild mouse. In order to carry out the linkage
analysis of Ix mutation, we have mated Ix homozygotes with the consomic
strain, B6.MSM-Chr5, in which the whole Chr5 is introduced from MSM strain
into the genetic background of C57BU6 strain. By using this consomic strain,
Ix exhibited full penetrance ofthe phenotypes. Thus, we took an advantage touse high frequency of polymorphism between MSM and C57BU6 in the gene
mapping of Ix mutation. Based on 112 backcross progeny from the above
cross, Ixwas mapped to an interval between D5Mit77and D5Mitl08, and wastightly linked to the markers D5Mit267, D5Mit150 and D5Mit130.

(4)

Physical mapping of polysynductylous mutation,
hemimelic extra toes (Ux)

Tomoko SAGAI, Hiroshi MASUYA and Toshihiko SHIROISHI
Two limb deformity mutations, Hemimelic extra toes (Hx) and hammer-toe
Hx shows preaxial polydactyly on all four feet associated with hemimelia, shortening ofthe radius, tibia(Hm) are located on the proximal region of Chr 5.

94
and talus in the original C57BU10 background. On the otherhand, Hm mutation shows interdigital webbing regression, which is probably due to an
impairment of apoptosis process in limb development. Hx and Hm are tightly
linked and only one recombinant has been reported in the1,664 offspring fromthe backcross of these two mutants. In the syntenic region of human chromosome 7q36, a congenital limb deformity with polysyndactyly has been
mapped. The causative gene(s) has not been identified yet.
Toward positional cloning of the genes involved in limb morphogenesis in
this region, we have carried out the precise genetic and physical mapping of Hx
mutation. A linkage analysis using 1,570 backcross progeny with MSM strain
showed that Hx mutation was localized to a 0.6cM interval between sonichedgehog (shh) gene which mediates antero-posterior signaling in limb development and interleukin 6 (1L6) gene. Presence of one recombinant excluded
a possibility that the coding sequence of shh is altered in Hxmutation. However
a possibility that cis regulatory element of shh is affected in Hx still remain,
since Hx is very close to shh and ectopic expression of shh is observed in theanterior margin of the limb bud of Hxmutant.
We constructed YAC and BAC contigs from the shh coding region to the Hxlocus. The result showed that Hx and the shh coding region are colocalized
within a 1.5 Mb single YAC clone, and Hxis included in a 600 kb single YAC
clone. As a next step for the more precise mapping of Hx, we carried out
shotgun sequencing of a 150kb BAC clone contained in the 600 kb YAC
clone. We generated new STS markers from the BAC clone. At the sametime, in order to have new recombinants, we crossed Hx with three inbred
strains, P/J, MAL and BLG2 and obtained 730 backcross progeny. By thelinkage analysis of them using the new STS markers, we obtained five newrecombinants in 600 kb DNA fragment of Hxcritical region. No recombination has occurred in this region among previous 1,570 progeny from cross with
MSM. Thus, the result indicated that use of different crosses with plural
strains should be very useful for the isolation of recombinants in given
regions. Finally the new recombinants mapped Hx mutation to a 150 kb
single BAC clone. We have finished construction of cosmid contig covering
the 150kb DNA fragment. Now we are trying cDNA selection to isolate thetranscripts in this regions.

F.

(5)

GENETIC STRAINS RESEARCH CENTER

95

Positional cloning of the mouse skeletal mutation,
Tail-short (Ts)Kunihiko SHIMIZU!, Tsuyoshi KOIDE, Akihiko MITA, Kikue UCHIDA, Shigeharu
WAKANA 2, Yoshiaki KIKKAWA3, Hiromichi YONEKAWA3, Hiroki SASAKI 4 andToshihiko SHIROISHI (lDepartment of Pediatric Dentistry, Nihon University
School of Dentistry at Matsudo, 2Central Institute for Experimental Animals,
3Department of Laboratory Experimental Animal Science, The Tokyo Metropolitan Institute for Medical Science, 4Genetics Division, National Cancer
Reserch Insutitute)
A mouse mutation Tail-short (Ts) exhibits shortened kinky tail and numerous skeletal abnormalities including homeotic anteroposterior patterning problem along the axial skeleton. Ts gene was previously mapped to the telomericregion of chromosome 11. Ts is likely to be a mouse model for a human skeletal dysmorphology known as Meckel syndrome (MES;OMIM2400), since MES
has phenotypes similar to Ts and has been mapped to human syntenic region,
17q21-24.
To elucidate the function of the Tsgene in mouse embryogenesis and to verify
whether it is a model for MES, we are trying to clone the gene by means ofpositional cloning. First, we employed a fine genetic mapping of this genebased on a large scale intersubspecific backcross between the mutant stock
TsJ/Le-Ts/+ and Japanese wild mouse-derived MSM strain. Ts gene wasmapped to a 0.16 cM region between two microsatellite markers, DllMit128
and DllMit256. Subsequently, we screened mouse YAC and BAC libraries
with the microsatellite markers tightly linked to the Ts locus and obtained
YAC and BAC clones. The ends ofthe isolated BAC clones were sequenced todevelop new STSs for further screening of clones and for securing the overlaps
between the clones. Further chromosome walking with the isolated clones
allowed us to construct a complete BAC contig covering the Ts causative
gene. The critical Tsregion was narrowed down to a DNA fragment between
two new STSs, DllRin56and DllNigl7. This contig consists of3 BAC clones,
which spans a 250 kb DNA fragment. In order to search candidate genes for
Ts, we have isolated several cDNA clones from the critical region by directed
cDNA selection using the corresponding BAC clones. We have also obtained

96

several cDNA clones which is derived from the mouse embryonic cDNA
library. We sequenced all cDNA clones mapped to the corresponding BAC
contig, and we searched related sequences on the database using BLAST and
GRAIL. Three ESTs were isolated from the BAC clones in the critical Ts
region. The first EST is expressed in mouse myotube, the second in mouseembryo (E14.5) and the last EST is ubiquitously expressed. Now we arecharacterizing these ESTs to identify the Ts causative gene.

(6)

Polymorphism at Tall-short (Ts) locus among standard Inbred
strains affects the viability of Ts heterozygotes

Junko ISHIJlMA, Akihiko MITA, Kikue UCHIDA, Toshihiko SHIROISHI
Tail-short (Ts) heterozygotes have variable types of malformation including
kinky tail, vertebral homeotic transformations, developmental retardationand neural tube defects. Expression of the phenotype depends on the mousestrain to which the mutant is crossed. The variation in the Ts phenotypes
observed in crosses with different inbred strains ranges from viable to dominant full lethal. Detailed linkage analysis indicated that a single chromosomal region, genetically inseparable from the Ts locus, is responsible for thedifference. This result suggests that there exists functional polymorphism
at Ts locus among inbred strains, and the different manners of the interactions between the Ts mutant gene and polymorphic allele ofthe inbred strain
may affect the viability of the Ts heterozygotes. We investigated the phenotype of the Ts mutation in crosses with the two groups of strains, which give
viable and lethal Ts embryos respectively. Ts heterozygous embryos derived
from the lethal cross exhibited more severe defects than those from the viablecross. Morphological anomalies of the Ts heterozygotes in the lethal crosswere observed in as early as neural plate stage. The embryonic region was
poorly developed compared with the extraembryonic region due to formation ofexcess mesoderm in the extraembryonic region. In the later developmental
stage, the umbilical vein does not develop properly, which may be the cause of
the lethality of the Ts heterozygotes around 12.5 dpc.

F.

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GENETIC STRAINS RESEARCH CENTER

97

Cloning of a responsible gene for mouse mutant Rim3

Norihisa SAEKI l, Hajime SAT0 2, Tomoko SAGAI, Hiroshi MASUYA, Toshihiko
SHIROISHI(lUniv. of the Ryukyus, 2Tohoku Univ.)

Rim3is a mouse mutant characterized by abnormalities in ectoderm-derived epithelium. Macroscopically it shows alopecia, coarse feature of theentire skin and turbidity of the cornea. Microscopical observation shows hyperkeratosis, acanthosis and few hair follicles in the skin, hyperkeratosis,
acanthosis, hypergranulosis and flat-shaped basal cells in corneal
epithelium. The phenotype significantly resembles that ofpreviously reported
mutant Rex denuded (Re Den). Since Rim3is mapped to the distal portion ofchromosome 11, where Re Den is reported to localize, it is highly likely that both
mutations are alleles of the same gene that functions in development of ectoderm-derived epithelium.
In the process of drawing up a detail physical map of the region in which
Rim3is localized, it was disclosed that just just 3 clones of bacterial artificial
chromosome (BAC) cover the entire region. Using these BAC clones, we haveattempted to isolate the responsible gene by a method of direct cDNA
selection. We have already isolated 3 fragments of partial cDNAs that sharesequences of expression sequence tag (EST) expressed in mouse skin. An
investigation to characterize the feature of these cDNAs is in progress.
(8)

Genetic and physical mapping of Iddl that controls onset of
IODM in mice

Shigeharu WAKANA l, Toshihiko SHIROISHI, Kazuo MORIWAKI 2, Chika
MARUYAMA l , Yuka WATANABE l, Tatsuji NOMURAl(lCentral Institute for Experimental Animals, 2Grad. Univ. Adv. Stud)The development oflDDM in the NOD mouse is under the control of multiple
insulin dependent diabetes (Idd) genes. One of them, Idd4, was mapped tomouse chromosome 11 and defined as a broad peak in a linkage analysis
based on a cross between NOD and B10.H-2 g7 strains (Todd J.A.et.al,
1991,1993). For fine mapping ofthe Idd4 gene without any influence of othersusceptibility genes, we have established a congenic strain for Idd4 by intro-

98
ducing the chromosomal segment ofthe MSM strain, which spans from Acrb to
Mpo on chromosome 11, into the NOD genetic background. The incidence ofdiabetes at early onset (-20wks) in female NOD/Shi.Idd4msm/m,mmice was somewhat higher than that in NOD mice or NOD/Shi.Idd4nodlmSmcongenic mice. This
result indicated that an Idd4 allele from the non-diabetic MSM is more susceptible to early onset of diabetes than that of NOD mice. In this study, we
produced a series of eight recombinant strains that carry various segments of
the MSM-derived chromosome in the congenic region containing Idd4. Bycomparing the incidence of early onset diabetes among these recombinantstrains, we mapped Idd4 within a short segment less than approximately 0.8
cM on chromosome 11. To construct a physical map across the Idd4 region,
YAC and BAC libraries were screened with the DNA markers on theregion. Finally, a contig of the three YAC and twelve BAC clones was obtained, which completely covered the Idd4region.

(9)

Behavioral study of inbred strains established fromwild mice. II. Learning and memory

Tsuyoshi KOIDE, Kazuo MORIWAKI 1, and Toshihiko SHIROISHI (lGrad. Univ. Adv.
Stud.)
Behavior of mouse is studied from many aspects using only small numbersof laboratory strains. However, those laboratory mice have been domesticated in the course of establishing laboratory strains, and their characteristic
behavior has been lost by the domestication. Another problem we will confront during the analyses of behavior of mice is that the variation of behavior
is limited because the most of the laboratory strains has been establishedfrom relatively small mouse colony and the genetic diversity between them issmall. In this point of view, use of inbred strains originated from variety ofwild mice in different countries, which belong to different mouse subspecies,
will provide a good chance to find novel behavioral patterns. We have started
a new project in that behavioral pattern of variety of inbred strains are stud·
ied and compared. Currently, we use eight wild-derive strains, MSM, NJL,
BLG2, BFM/2, SWN, KJR, HMI and CAST/Ei, as well as JF1 establishedfrom Japanese fancy mouse and two laboratory strains, C57BLl6 and DBA/I.

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First, we studied ability for the learning and memory in mice by using a passive avoidance test. As a result, we found a wide range of difference in theperformance among the strains used in this test. It has been reported that
C57BLl6 has the good ability in the passive avoidance test, while DBA/I has
the poor ability. We obtained essentially same result for the C57BLl6 andDBA/I in our study. In addition to this, we noticed that although BFM, KJR,
SWN, and MSM have the good performance similar to that of C57BLl6 strain,
CASTlEi and BLG2 has less ability than DBA/I strain. There is significant
difference in the ability in the passive avoidance test between the good performers that include BFM/2, KJR, SWN, MSM and C57BLl6 strains, and thepoor performers that include CAST lEi and BLG2. This difference would allow us to conduct a genetic analysis for the learning and memory ability using
this system. Currently, we are conducting a genetic cross between strains of
the good and poor performers to map gene(s) responsible for the difference inthe performance in the passive avoidance test.

Pub I i ca t ions
1.

KOIDE, T., MORIWAKI, K., UCHIDA, K., MITA, A., SAGAI, T., YONEKAWA, H., KATOH, H.,MIYASHITA, N., TSUCHIYA, K., NIELSEN, T. J. and SHIROISHI, T:

A new inbred strain JF1

established from Japanese fancy mouse carrying the classical piebald allele. Mammal. Genome, 9: 15-19, 1998.
2.

SATO, H., KOIDE, T., MASUYA, H., WAKANA, S., SAGAI, T., ISHIGURO, S., TAMAI, M. and
SHIROISHI, T:

A new mutation Rim3 resembling Reden is mapped close to reinoic acidreceptor alpha (Rara) gene on mouse chromosome 11. Mammal. Genome, 9: 20-25,
3.

1998.
SAGAI, T, KOIDE, T, ENDO, M., TANOUE, K., KIKKAWA, H., YONEKAWA, H., ISHIGURO, S.,
TAMAI, S., MATSUDA, Y, WAKANA, S. and SHIROISHI, T:

rim2 (recombination induced

mutation 2) is a new allele of pearl and a mouse model of human Hermansky-Pudlak
syndrom (HPS): genetic and physical mapping. Mammal. Genome, 9: 2-7, 1998.
4.

TANI, M., SHIMURA, K., KOHNo, T., SIIIROISHI, T., WAKANA, S., KIM, S.-R., NOHMI, T.,
KASAl, H., TAKENOSHITA, S., NAGAMACHI, Y and YOKOTA, J.:

Genomic structure andchromosomal localization of the mouse Ogg 1 gene that is involved in the repair of 8hydroxyguanine in DNA damage. Mammal. Genome, 9: 32-37, 1998.
5.

HASHIMOTO, Y, SHINDO-OKADA, N., TANI, M., NAGAMACHI, Y, TAKEUCHI, K., SHIROISHI, T.,
TOMA, H. and YOKOTA, J.:

Expression ofthe Elm1 gene, a novel gene ofCCN family,

suppresses In vivo tumor growth and metastasis ofK-1735 murine melanoma cells. J.

100
Exp. Med. 187: 289-296, 1998.
6.

NAGASE, H., WANG, C-R., YOSHIMOTO, T, SUGISHITA, C., SHIROISHI, T, MATSUZAWA, A. and
NARIUCHI, H.:

Novel mutant mice secreting soluble CD4 without expression of mem-brane-bound CD4. Eur. J. Immunol. 28:403-412, 1998.
7.

OBATA, Y, KANEKO-IsHINO, T, KOIDE, T, TAKAI, Y, UEDA, T., SHIROISHI, T., ISHINO, F. and
KONO, T.:

Disruption of primary imprinting during oocyte growth leads to the modi-

fied expression of imprinted genes during embryogenesis. Development, lZ5:15531569, 1998.
8.

MIWA, T, NONAKA, M., OKADA, N., WAKANA, S., SHIROISHI, T and OKADA, H.:

Molecular

cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal
9.

chromosome 1. Immunogenet., in press.
ISHIJIMA, J., YASUI, H., MORISHIMA, M. and SHIROISHI, T.:

Dominant lethality of themouse skeletal mutation Tail-short (Ts) is determined by the Ts allele from mating
partner. Genomics, 49:341-350, 1998.
10.

KATOH-FuKUI, Y., TSUCHIYA, R., SHIROISHI, T. HASHIMOTO, N., NOGUCHI, K. and
HIGASHINAKAGAWA, T:

Male sex-reversal in M33 mutant mice. Nature, 393:688-692,

1998.
11.

VARIN-BLANK, N., DaNDI, E., TOSI, M., HERNANDEZ, C., BOUCONTET, 1., GoTOH, H., SHIROISHI,
T, MORIWAKI, K. and MEO, T.:

Male-specific transcription initiation of the C4-S1p genein mouse liver follows activation of STAT5. Proc. Natl. Acad. Sci. USA. in press.
12.

KITANO, T., SUMIYAMA, K., SHIROISHI, T. and SAITOU, N.:Conserved evolution of theRh50 gene compared to its homologous Rh blood group gene. BBRC, 249: 78-85, 1998.
13.

TAKAHASHI, M., TAMURA, K., BUSCHER, D., MAsUYA, H., YONEI-TAMURA, S., MATSUMOTO,

K., NAITOH-MATsuo, M., TAKEUCHI, J., OGURA, K., SHIROISHI, T, OGURA, T and BELMONTE,
J. C. I.: The role of Alx-4 in the establishment of anteroposterior porality duringvertebrate limb development. Development 125: 4417-4425, 1998.

F-b. Mammal ian Development Laboratory(1)

Analysis of Cellular and Molecular Mechanisms in theDevelopment of Mouse Fetal Germ Cells andSex Differentiation of Gonads

Norio NAKATSUJI, Masaru TAMURA, Shinichiro CHUMA, Yasuhiko KANNO,
Takayuki SAKURAI, Zhenyong HUANG, Tetsuichiro SAITO, and Takashi TADA
We have been studying proliferation and growth regulation of mouse primordial germ cells (PGCs) during migratory stages by using a culture

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system. They show a temporary proliferation in the culture, but their growthis arrested when they differentiate into gonia after arriving at fetal gonads. We
are developing a culture system of germ cells to investigate the regulation of
the entry into meiosis by germ cells. Also, we have been trying to identify
novel genes involved in sex-differentiation of germ cells and gonads at thesestages.
After PGCs arrive at genital ridges at around 10.5 dpc they continue todivide mitotically until about 13.5 dpc. Then, they take different paths ofdevelopment according to their surrounding gonadal sex. Germ cells in malegonads are considered to arrest at the GO stage of the cell cycle asprospermatogonia. In females, germ cells enter into meiosis and arrest at
the prophase of the first meiotic division. Surface markers of PGCs which
are common to the early totipotent stem cells start to diminish gradually after
these transitions into mitotic arrest or meiosis. Germ cells resume mitosis
as spermatogenic stem cells or meiotic progress and oocyte maturation after
birth. Previous studies have suggested that sex-differentiation of the fetal
germ cells is determined by the surrounding gonadal somatic environments,
rather than their own chromosomal constitution. However, studies on the
differentiation of PGCs has been limited to experiments using organ or reaggregate cultures. Thus, examination of meiotic transition by PGCs andidentification of its regulative factors have been impeded, due to the lack of
the dissociated culture system of PGCs in which meiotic transition could be
detected and which enables precise analysis on meiotic transition by
PGCs. We have developed a primary culture system in which PGCs isolated
and dissociated from mitotically dividing stages entered into early phase of
the first meiotic division under the two-dimensional dissociated culture
condition. We have demonstrated, with this system, that expression of meiotic proteins by PGCs occurs in the absence of embryonic gonadal somatic cells
implying that an autonomous program for transition into meiosis is alreadypresent before reaching embryonic gonads. It is also demonstrated that LIFgp 130 signal, previously known as an inhibitor of differentiation of embryonicstem cells and promoter of the PGC survival, represses meiotic transition by
PGCs in vitro. Such in vitro systems developed in our study shall provide abase for analysis on mechanisms and factors involved in meiotic transition by

102

PGCs of mammalian embryos.
Sex-differentiation of mouse fetal gonads starts by an appearance of testis
cords in testis at 12.5 dpc, followed by mitotic arrest of germ cells in testis andinitiation of meiosis in the ovary starting at 13.5 dpc. We have been trying to
identify such genes in mice by using the subtraction and differential hybridization method to obtain genes whose expression is specific for the 13.5 dpc
testis after subtraction by the female gonad eDNA. So far, we have isolated
several clones representing novel sequences. We chose a novel gene containing typical basic helix-loop-helix domains for further investigation. We named
it nephgonadin because its strong expression was observed in the kidney andgonad. At 13.5 dpc, expression of nephgonadin was stronger in the testis
than the ovary. In adults, however, the expression level was decreased in thetestis, while it was increased in the ovary. We are now examining functions ofnephgonadin in gonad development.
(!)

Molecular mechanism controlling neuronaldifferentiation in mammals

Tetsuichiro SAITO, Taro HAMA, Shuichi SAKAMOTO, Rie SABA and Norio
NAKATSUJI
The mammalian nervous system comprises an enormous number of cell
types. We have been trying several approaches to understand how the cell
types are generated at the molecular level. Members of the family of basichelix-loop-helix (bHLH) transcription factors, such as MASH 1 and Neurogenin,play important roles in mammalian neurogenesis. We have focused on theregulatory cascades ofthe neural bHLH genes.
MBHl (mammalian BarHhomologue) is expressed in an exactly complementary pattern to Mashl and overlapping with neurogenin2in the developing nervous system. Forced expression of MBHl down-regulates Mashl expression and up-regulates neurogenin2in differentiating P19 cells, suggestingthat MBHl is a regulator ofthe neural bHLH genes. Neurogenin2, however,is also expressed in some areas which do not express MBHl. Besides, Drosophila has two BarH genes. These suggest that there may be more BarH genes
in mammals. To identify another mammalian BarH gene, we have performed

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peR using degenerated primers and obtained a DNA fragment encoding ahomeodomain similar to the MBHl protein. Using the DNA fragment as aprobe, several cDNA clones were obtained which encodes the same protein,hereafter designated as MBH2. The MBH2 protein is more closely related to
the MBHl protein than any other BarH proteins, suggesting that the MBHl
and MBH2genes may have been duplicated after the divergence of insects andvertebrates. The FIL peptide motif is also detected in the N-terminal portion ofthe MBH2 protein. MBH2expression is transient during neurogenesis
as well as MBHl. MBH2mRNA was detected in the developing diencephalon and dorsal spinal cord. More detailed analysis of MBH2 expression iscurrently being processed.
MBHl expression is restricted to specific regions in the developing nervous
system, such as the dorsal diencephalon and retinal ganglion cell layer. In
order to learn the mechanisms that regulate MBHl expression, MBHl genomic DNA clones were obtained and characterized. The MBHl gene contains at least two introns, one of which splits the homeodomain-coding region,
like the Drosophila BarHl and BarH2 genes. Reporter plasmids containing
a portion of the MBHl gene have been constructed to find out regulatory sequences for MBHl expression using transgenic mice.
PHDl, a member ofthe Paired-like homeodomain (PHD) family, is expressed
downstream of Mashl during the differentiation of both dorsal spinal cord andolfactory sensory neurons. Other PHD genes, such as Phox2and DRGll, arealso expressed downstream of the bHLH genes, in different lineages of neurons, suggesting that the cascade from bHLH to PHD transcription factors
may be important for neuronal identity determination. In order to clarify the
relation between Mashl and PHDl, we are trying to identify downstream
target genes of Mashl.

(3)

Reprogramming of parental-origin-specific epigenotype
in mouse germ-line development

Takashi TADA, Masako TADA 1 and Norio NAKATSUJI (lPRESTO; Precursory
Research for Embryonic Science and Technology, JST)In mammals, maternal and paternal genomes are not functionally equiva-

104

lent and both genomes are essential to complete normal development. Thisphenomenon known as genomic imprinting is caused by accumulated effects ofimprinted genes, which are expressed exclusively from the maternal or paternal allele. Allele-specific transcription of imprinted genes is regulated by
epigenetic modification (imprint) such as DNA methylation, histone acetylation and chromatin structure, but not by modification ofthe DNA sequence initself. One of the important aspects is that parental imprints never endure
for more than one generation, indicating that both paternal and maternal
imprints must be reprogrammed to paternal imprint during spermatogenesis
and to maternal imprint during oogenesis. To address the mechanism ofreprogramming the parental imprints in the germ line, imprinting status ofembryonic germ (EG) cells established from primordial germ cells (PGCs)
soon after entering to gonads ofEll.5 and 12.5 embryos was analyzed. Obvious
overgrowth and morphological abnormalities of chimeric embryos with these
XY and XX EG cells suggest that reprogramming of the parental imprints is
initiated before the onset of meiosis. DNA methylation analysis reinforced
this idea. Although the maternal or paternal allele is differentially methylated in somatic cells, both alleles of the majority of imprinted genes examined lost DNA methylation and this hypomethylation situation was maintained before and after cell differentiation. This biallelic hypomethylation
resulted in biallelic activation in some imprinted genes and biallelic silencing
in others as revealed by mRNA in situ hybridization. These findings clearly
demonstrated that the initial key step of reprogramming of the parental imprints is the erasure ofthe existing parental imprints in the germ line (ref. 6 ).
Erasure of the parental imprints in EG nuclei led us to consider that thetrans-acting factor(s) involved in the initial step may be maintained in EG
cells. To address this question, somatic cells in which the parental imprints
have been established were cell-hybridized with EG cells. Differential DNA
methylation of imprinted genes in somatic nuclei was drastically erased andboth alleles showed hypomethylation in the hybrid cells. Maternally methylated and repressed Pegl/lv!estin somatic cells was demethylated and reactivated in chimeras with the hybrid cells. It is likely that EG cells retain dominant trans-acting factor(s) to induce erasure of parental imprints as similar togerm cells. Recent our unique approach to elucidate mechanisms of the im-

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printing erasure is that a single maternal human chromosome 7, carrying an
imprinted gene PEGIIMESTand tagged with the transgene neo, was introduced into mouse embryonic stem (ES) cells (ESIP) and EG cells (EGIP) by the
microcell-mediated chromosome transfer technique. Transcription from maternal human PEGIIMEST, which is methylated and repressed in somatic
cells was detected in E7.5 and 9.5 chimeras only with EGIP but not in chimeras
with ES/P. Reactivation of human PEGIIMESTin mouse EG/P derivatives
indicates that the mouse erasure factor(s) is capable of reprogramming thehuman maternal imprint. This trait may be unique to germ cells as seen in
EGIP cells but not in ESIP cells.
Imprinting erasure is tightly linked to the genome-wide demethylation inpremeiotic germ cells. Erasure of epigenetic modification including DNA methylation and imprints may play an important role to prevent inheritance of apredefined parental epigenetic modification to offspring. If so, this process isessential for the preparation to impose appropriate epigenetic modification
on gametic genomes. We are now focusing our attention on the identificationof the trans-acting factor(s) involved in the erasure event.

Publications
1.

MOCHIZUKI, T, SAIJOH, Y, TSUCHIYA, K, SHIRAYOSHI, Y, TAKAI, S., TAYA, C., YONEKAWA, H.,
YAMADA, K, NIHEI, H., NAKATSUJI, N., OVERBEEK, P. A., HAMADA, H. and YOKOYAMA,
T.:

Cloning of inv, a gene that controlslefJright asymmetry and kidney development.

Nature 395,177-181,1998.
2.

SUGIHARA, K, NAKATSUJI, N., NAKAMURA, K, NAKAO, K, HASHIMOTO, R., OTANI, H.,
SAKAGAMI, H., KONDO, H., NOZAWA, S., NBA, A. and KATSUKI, M.:

Rac1 is required for theformation of three germ layers during gastrulation. Oncogene 17, 3427-3433, 1998.
3.

aNa, K, NAGATA, I., HAMA, T and NAKATSUJI N.:

Perpendicular contact guidance of

CNS neurons: is it operative in brain cortex development? "Neural Development"(Eds., Uyemura, K, Kawamura, K, Yazaki, T.), Keio Univ. Symp. for Life Science andMedicine. VoU!, pp.50-56, 1999.
4.

SAITO, T., SAWAMOTO, K, OKANO, H., ANDERSON, D. J. and MIKOSHIBA, K:

Mammalian

BarH homologue is a potential regulator of neural bHLH genes. Dev. BioI. 199, 216225,1998.
5.

LIN, J. H., SAITO, T, ANDERSON, D. J., LANCE-JONES, C., JESSELL, T M. and NIBER, S.:
Functionally related motor neuron pool and muscle sensory afferent subtypes definedby coordinate ETS gene expression. Cell 95, 393-407, 1998.

106
6.

TADA, T., TADA, M., HILTON, K., BARTON, S.C. SADO, T., TAKAGI, N. and SURANI, M.A.:
Epigenetic switching of imprintable loci in embryonic germ cells. Dev. Genes Evo!., 207,
551-561, 1998.

7.

PARK,J-I., YOSHIDA, I., TADA, T., TAKAGI, N., TAKAHASHI, Y. andKANAGAwA, H.:

Differentiative

potential of a mouse parthenogenetic embryonic stem cell line revealed by embryoid
body formation in vitro. Jpn. J. Vet. Res., 46, 19·28, 1998.
8.

PARK, J.I., YOSHIDA, I., TADA, T., TAKAGI, N., TAKAHASHI, Y. and KANAGAWA, H.:

Trisomy8 does not affect differentiative potential in a murine parthenogenetic embryonic stem
cell line. Jpn. J. Vet. Res., 46, 29·35, 1998.

F-c. Plant Genetics Laboratory(1)

Structural and Functional Analysis of the Genes Expressed in
Early Embryogenesis and Regeneration in Rice (Oryza sativa)(I)-a. Functional Analysis of Homeobox Genes Expressed in Embryogenesis
and Regeneration Process in RiceYukihiro ITO, Atsushi CHUJO, Kazuhiko TAKAYA, Yasuo NIWAI, Nori KURATA(lShizuoka 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 eight KNI-like homeobox genes and analysed their expression patterns during early embryogenesis and regeneration by RT-PCR and in situ
hybridization (see ref.7). To gain insight into functions of these genes
overexpression experiments in rice were carried out. When one of the cloned
gene OSHl/HOS24 under the control of a cauliflower mosaic virus 35S RNA
promoter (P35S) were introduced into rice calli by Agrobacterium-mediatedtransformation, most of the transformed calli did not regenerate even on regeneration medium and continued to grow as mostly undifferentiated calli. As
OSHl/HOS24 is known to be expressed in a shoot meristem, we postulated
that OSHl/HOS24 functions to maintain cells in the shoot meristem in an
undifferentiated state and its overexpression causes inhibition ofthe normal
regeneration of calli. In addition we found that overexpression of OSHl/
HOS24 induced expression of other homeobox genes HOS3 and HOSl6. In

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early embryogenesis these three homeobox genes are expressed in a same
region where the shoot meristem will appear. This suggests that such a expression network may also occur in embryogenesis.
To understand relationship between regeneration and OSHIIHOS24, a promoter region of the gene was fused to a coding region of green fluorescentprotein (GFP) and resultant chimeric gene was introduced into ricecalli. Several days after the transfer of the calli to the regeneration medium,the fluorescent signal was detected from some ofthem, and generation of green
spots were later observed only from the fluorescent-positive calli except
one. This correlation of the expression of the gene and the regeneration suggests that OSH1IHOS24 may play an important roll during the regeneration
processes and this gene can be used as a suitable molecular marker to study
the regeneration process.
We also carried out structural and expression analyses of HAZI, a homeobox
gene classified into a PHD-finger type. Nucleotide sequence analysis of agenomic clone and a cDNA clone of HAZI indicates that the predicted protein
contains characteristic motives such as nuclear localization signals, Zn finger,
acidic regions and homeodomain from N-terminus to C-terminus. This domain organization was same as a maize homeodomain protein Zmhoxl. Expression of HAZI was detected in all organs examined including leaf blade,leaf sheath, root and flowers containing an embryo at some stages. In situ
hybridization analysis indicated that HAZI was expressed in an entire embryo at a globular stage, specially at outer layers with a higher level. This
indicates that in a globular embryo outer-inner differentiation was occurred,
though no phenotypic organ differentiation was observed.
We started to isolate and characterize a protein kinase gene as a candidate
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 of carrot and isolated
its homologue from rice. Sequencing and expression analyses are in progress.

108(1)-b. Cloning and StructurallFunctionalAnalysis of Four LEC1 Related Genesin RiceKazumaru MIYOSHI, Nori KURATA
Arabidopsis LEC 1 gene cloned recently revealed to encode a transcriptional
factor HAP3. Most striking feature of this gene is that the over expression of
LEC 1 protein causes ectopic embryogenesis on leaves of transgenic
plants. This is the first case of ectopic induction of plant embryogenesis bygene manipulation. In monocot spices, a HAP3 gene and other LEC1 homologous gene(s) are also isolated in maize and others. However, their roles andfunctions during embryogenesis in monocotyledonous plants are not known
yet. It must be very significant to analyse the signaling pathways before andafter the LEC1 homolog gene expression when embryogenesis occurs. We haveisolated several cDNA clones showing high similarity to the LEC1 gene from a
3DAP embryo cDNA library using maize HAP3 cDNA probe, which is cloned by
RT-PCR from maize developing embryo. Sequencing analysis and databasesearch revealed that several, at least three, different LEC1 homologs are expressed during embryogenesis in rice. The isolation of full length cDNAs is
now in progress. The investigation of expression pattern and the functional
analyses using loss- and/or gain-of-function mutant plants of these genes isnext subject.

(Z)

Study for the Nucleus and Chromosome Organization in Rice(2)-a. Isolation and Structural Analyses for The Centromeric Region of Rice
Chromosomes Toward Construction of Rice Artificial Chromosome (RAC)
Kenichi NONOMURA, Nori KURATA
The cereal centromeric sequence (CCS 1) conserved in some Gramineae species contains a 17-bp motif similar to the CENP-B box, which is the bindingsite for centromere specific protein CENP-B in human. To isolate centromeric units of rice (Oryza sativa L.), we performed the polymerase chain reaction (PCR) using the CENP-B box-like sequence (CBLS) as primers. A 264bp clone amplified in this method, called RCS1516, appeared to be a novel
member ofthe CCS1 family, sharing about 60% identity with the CCS1 se-F.

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quences of other cereals. Then, a 14-kb long genomic clone lRCBll carrying
RCS1516 sequence was screened and sequenced. It was found to contain 3
copies of a 1.9-kb direct repeat RCE1, with 5.1- and 1.7-kb intervening
sequences. The 300 bp of 3' end of RCE 1 were highly conserved among threecopies (>90%) and almost identical with RCS1516 sequence including a CBLS
motif. The copy number ofRCE1 was estimated ranging from 102 to 10 3 inthe haploid genome of rice. Cloned RCE 1 units were provided for fluorescentin situ hybridization (FISH) analysis, and signals were observed on almost
every primary constriction of rice chromosomes. Thus it was concluded that
RCE1 is one of the significant components of rice centromere. The RCBllclone contained at least four AfT-rich regions, which are candidate matrix attachment regions (MARs), in the intervening sequences between the RCE 1
repeats. Other elements homologous with the short centromeric repetitive
sequences, pSau3A9 and pRG5 detected in both sorghum and rice, were also
found in the clone. These results were published in Ref. 5.
(2)-b. Production of Mini-Chromosome Rice Lines for Studying Centromere
Structure
Kenichi NONOMURA, Nori KURATA
In the study of plant centromere, there is no way other than transformation
to know whether the DNA fragments derived from centromeric sequences bear
the centromeric function or not. We plan to induce mini-chromosome plant
aiming to obtain minimal functional unit of rice chromosome. Ditelocentricalien addition lines (DtAALs), which contain a pair of telocentric chromosome
from wild species (Oryza punctata, BB genome) in addition to the normal
complement of 0. sativa chromosome set (AA genome), were irradiated by
gamma-ray. DtAALs were kindly provided by Dr. H. Yasui, Kyushu Dniv. The
M 2 population with over 3000 gamma-ray irradiated DtAALs will be used forselection of mini chromosome lines carrying diminished alien chromosomes.

110

(3)

Analysis of Genetic Factors causing Segregation Distortion(3)-a. Quantitative Analysis of genotype segregation for reproductive barriers
Yoshiaki HARUSHIMA, 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. These reproductive barriers cause segregation distortion of linked marker genotypes in the population derived fromhybrid ofinter- or intra- species cross. In spite ofthe importance ofreproductive barrier in speciation, a comprehensive survey of reproductive barriers
lying inter- or intra- species have not been made because of lack of markers
covering whole genome. The interaction between the different loci ofthe parents would be expected to play important role in reproductive isolation. However,identification of interactive loci has be seemed to be difficult in generally andhas not been attempted.
In rice, a high density linkage map was constructed using an F 2 population
derived from a cross between a japonica cultivar, Nipponbare, and an indica
cultivar, Kasalath (NK) (ref. 1). In the last year, we developed a multi-response non-liner regression analysis program to explain segregation distortions of a linkage map by a few gametophytic reproductive barriers. We succeed to explain the segregation distortions on some chromosomes.
In this year, we have extended the regression analysis program considering
zygotic reproductive barriers and succeeded to explain segregation distortions
on all chromosomes in NK by 34 reproductive barriers. Moreover, we haveestimated interactive loci for segregation by applying classical X 2 independence tests for all marker combinations in the rice high density linkage map.
In order to ascertain the characteristics of the reproductive barriers between
japonica and indica rice cultivars, we analyzed reproductive barriers in twoother crosses; between Fl1087 and Dao Ren Qiao (FD) and between Fll007
and Kinandang puti (FK). The frequencies of each allele in a whole genome
were well explained by 31 and 38 reproductive barriers in FD and FK,
respectively. To examine the possibility of common barriers lying between
japonica and indica rice cultivar, the positions of the reproductive barriers

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detected in FD and FK were estimated and located on the NK map using themarkers common with those of the NK map, thus at least 92 out of 103 barriers detected in this study were at different loci. The regions of interactive
pairs for segregation distortion were completely different among the threecrosses. (in preparation for publication).
(3)-b. Positional Cloning of a Segregation Distortion Gene Detected in a Progeny of a Cross between japonica and indica rice
Yoshiaki HARUSHIMA, Nori KURATA
The molecular mechanisms of reproductive isolation have not analyzed without a few exceptions in Drosophila, etc. The aim of this study is isolation of
the most prominent barrier, gametophyte gene 2 (ga-2), on chromosome 3 in
NK by positional cloning, and elucidation of the molecular nature of the individual reproductive barriers. We specified ga-2 position between adjacentmarkers in the NK high density linkage map in last year. We have beendeveloping the near isogenic lines that have Kasalath genome at ga-210cus in
Nipponbare background by back-crossing preferable 6 progenies (BC 2F 6) thatwas selected from 276 plants with CAPS markers.

(4)

Generation of Enhancer Trap Lines of RiceYukihiro ITO, Mitsugu EIGUCHI, Nori KURATA
To isolate valuable mutants defective in various steps of rice development
and to clone the corresponding genes, we are generating enhancer trap lines ofnee. We employ an enhancer trap system used in Arabidopsis with somemodifications. This system is based on the Agrobacterium-mediated transformation using AcIDs transposable elements of maize. The enhancer trap
construct contains, in the T-DNAregion, the Dselement that harbours a GUS
coding region with a P35S minimal promoter and a hygromycin resistancegene. This Ds was flanked by a P35S and a coding region of a selectablechlorsulfuron resistance gene, so that excision of the Ds causes connection of
the P35S and the coding region and can confer chlorsulfuron resistance. We
also use a P35S-Ac transposes (AcTPase) gene together with a bialaphos re-112

sistance gene to supply transposase which is essential and sufficient for transposition of the Ds. We generated more than two hundreds transgenic ricewith Ds. Ds-flanking regions cloned only for single copy Ds carrying plants
and sequenced indicated that some Ds (before transposition) integrated in thecoding region of the genes and others in the repetitive sequences. We willconduct mapping of the integrated Ds sites on rice chromosomes to selectchromosome specific Ds lines. The examination for transposition frequencyof Ds elements when crossed by the P35S-AcTPase lines will be carried out in
1999.

(5)

Generation and Screening of Retrotransposon
Insertion Lines in Rice

Mitsugu EIGUCHI, Yasurou NAGATO l , Kazumaru MIYOSHI, Yukihiro ITO, Nori
KURATA (lGraduate School ofAgriculture and Life Sciences, University of Tokyo)
To obtain a lot of mutants affecting the steps of embryogenesis, we usedtransposon-induced mutagenesis system of rice using retrotransposon
Thsl7. Ths17is activated under tissue culture condition and transposes several copies in each regenerant. Calli of the japonica variety Nipponbare, Oryza
sativa, were induced from germinating rice seeds, transferred into N6liquidmedium for 3 months' suspension culture and then were transferred onto regeneration plates. We produced about 900 regenerated plants, each having
several transposed copies of Thsl7, and obtain M 2 seeds from all ofthe regenerated plants (NIG-TD lines). More than another insertion lines were also
propagated as a colaborative work among several laboratories (NF lines).
To isolate valuable genes taking roles for early embryogenesis, we screened
each 20 M 2 seeds of about 3000 Tosl71ines. We obtained about 100 candidate lines defective in early embryogenesis. M 3 investigation will be continued in 1999. In addition, DNAs were extracted from pooled Tos17insertionlines and will also be used for screening mutant lines with disrupted genesequences of unknown gene function (loss of function mutants).

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Publications
1.

HARUSHlMA, Y, YANO, M., SHOMURA, A, SATO, M., SHlMANO, T, KUBOKI, Y, YAMAMOTO, T,
LIN, S. Y, ANTONIO, B. A, PARCO, A., KAJIYA, H., HUANG, H., YAMAMOTO, K, NAGAMURA, Y,
KURATA, N., KHUSH, G. S. and SASAKI, T:

A high density rice genetic linkage map with

2275 markers using a single F 2 population. Genetics 148: 479-494, 1998.
2.

YOSHIMURA, S., YAMANOUCHI, U., KATAYOSE, Y, WANG, Z-X, TOKI, S., KONO, K, KURATA, N.,
YANO, M., IWATA, N. and SASAKI, T.:

Expression of Xa-l, a novel bacterial blight resis-

tance gene in rice, is induced by bacterial inoculation. Proc. Nat\. Acad. Sci. USA 95:
1663-1668, 1998.
3.

RAJYASHRI, K R., NAIR, S., OHMIDO, N., FUKUI, K, KURATA, N., SASAKI, T and MOHAN,
M.:

Isolation and FISH mapping of Yeast Artificial Chromosomes (YACs) encompass-ing an allele of the Gm2gene for gall midge resistance in rice. Theor. Appl. Genet.97:
507-514, 1998.
4.

Wu, J., KURATA, N., TANOUE, H., SHIMOKAWA, T., UMEHARA, Y, YANO, M. and SASAKI,
T:

Physical mapping of duplicated genomic regions of two chromosome ends in rice.

Genetics 150: 1595-1603,1998.
5.

NONOMURA, KI. and KURATA, N.:

Organization of 1.9-kb repeat unit RCEI in thecentromeric region of rice chromosomes. Mol. Gen. Genet. 261: 1-10, 1999.
6.

MIYOSHI, K, NAKATA, E., NAGATO, Y and HATTORI, T.:

Differential in situ expression of

three ABA-responsive genes of rice, RabJ6A, REG2 and OSBZ8, during seed development. Plant Cell Physiol.40, 443-447, 1999.
7.

ITo, Y, EIGUCHI, M. and KURATA, N.:

Expression of novel homeobox genes in early

embryogenesis in rice. Biochim. Biophys. Acta 1444: 445-450, 1999.
8.

AsHIKAWA, I., KURATA, N., SAJI, S., UMEHARA, Y and SASAKI, T:

Application of restric-tion fingerprinting with a rice microsatellite sequence to assembling rice YAC clones.
Genome 42, 330-337, 1999.

114

F-d. Microbial Genetics Laboratory(1)

Timing of cell division in Escherichia colf

Akiko NISHIMURA
We found previously that the efe mutation uncouples DNA replication andcell division, and elevates the frequency of cell division. We further analyzed
the structure and the role of the cfegenes. The cfc mutants divide before theyreach the size at which cfc+ cells divide, and produce many small cells-eachwith a single nucleoid. The mutations affect the timing of cell division, but
not other processes of cell cycle, such as the length of a cell cycle and theinitiation mass for chromosome replication. CfcA has a mutation in glySa
which encodes the a-subunit of glycyl-tRNA synthetase, and cfcBl has an IS2
insertion in apaH which encodes Ap4A hydrolase. The cfc properties of both
efemutants were suppressed by a multicopy plasmid carrying apaH+, and theintracellular level of Ap4A in cfcA was 15-fold higher, and cfcB was 100-foldhigher than their parent. Experiments using a wild-type cell showed that ahigh level ofAp4A caused early cell division, and a low level ofAp4A caused
delayed cell division. We have purified the GlyS-6xHis tagged proteins from
cfc+ and cfc· strains and analysed the catalytic activity in vitro for Ap4A synthesis and kinetic constants oftRNA 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 forglycylation (KmlKcat) ofGlyS 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. (For details, see Ref.
2)

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GENETIC STRAINS RESEARCH CENTER

115

Usc66 Participates In The FtsZ Ring Formation In
Escberlcbla coli

Takeshi UEHARA, Hiroshi MATSUZAWA', and Akiko NISHIMURA ('Department of
Biotechnology, The University of Tokyo)
We found from the analysis of a temperature-sensitive mutant of cell division, fts715, that Hsc66, the DnaKlHsp70 homolog, affected the septal ringformation. 1. The fts715 mutant stopped cell division at 42'C but did not
chromosome replication or segregation. 2. Complementation, PI-phage mediated transduction, and sequencing analysis of mutant showed that the mutation existed in the hscA gene which encodes Hsc66, and 192 alanine wassubstituted to valine. 3. Immunofluorescence microscopy revealed that inwild type cells, both Hsc66 and FtsZ ring was localized at the potential division site. However, in the fts715filaments, FtsZ ring was rarely detected at
the potential division sites except those of both ends, though Hsc66 was detected at the most potential division sites. These results indicate that Hsc66
affects the formation of FtsZ ring. The purified FtsZ is known to polymerize
in vitro in the presence of GTP at 37'C. 4. We found that FtsZ could notpolymerize under the condition at 42'C and that the addition of wild type
Hsc66 specifically alleviated the polymerization defect of FtsZ at 42'C. 5.
Moreover, the polymerized FtsZ and Hsc66 were co-sedimented. 6. To purify
the mutant type Hsc66, the maltose-binding protein-Hsc66 fusion (MBPHsc66) of wild type or mutant type Hsc66 were isolated. The mutant type
MBP·Hsc66 could not suppress the polymerization defect of FtsZ at 42'C but
the wild type MBP-Hsc66 could. We propose that Hsc66 might promote or
stabilize the polymerization of FtsZ at 42'C.

116(3) Post genome project : Systematic analysis ofcell division genes In EscherIchIa colJ
Makiko HORIE l , Kimiko SAKA, Maki TADENUMA, Kunio MATSUMOT0 1, Hiroaki
ITO, Hirosada MORI 2, Hideaki SUGAWARA, Kousuke GOTO, Akiko NISHIMURA(lKanagawa lnst. Tech., 2Nara. lnst. Sci. Tech.)
The entire nucleotide sequence of E. coli was analyzed, and 4311 ORFs havebeen demonstrated, but the functions of more than half of these genes are still
unknown. It is considered that the greater part ofthese genes are involved incoordinating cell proliferation. To analyze the hierarchy and network responses in expression of cell division genes as a model case, we have selectedmutants defective in cell division (its) from a temperature-sensitive mutantbank (kts) consisting of 5,000 strains established by Hirota et a1., and analyzed the physiological properties of the mutants and roughly mapped thesegenes. Furthermore, to assign a corresponding ORF to each its mutation by
genomic analysis, vectors were constructed and cloning has been performed for
3600RFs.

Pub I i ca t ions
I.

2.

UKAI, H., MATSUZAWA, H., ITO, K., YAMADA, M. and NISHIMURA, A: [tsE" causes defectin cell growth of Escherichia coli through affecting translocation of K+-pump proteins
into the membrane. J. Bacteriol. 180, 3663-3670, 1998.
NISHIMURA, A: Timing of cell division: Ap4A as the signal. TiBS. 23, 157-159, 1998.

F-e. Invertebrate Genetics Laboratory(1)

Specification and proximo-distal pattern formation of thewing and leg In DrosophJla

Satoshi GOTO, Kazumasa KUBOTA' and Shigeo HAYASHI('Tokyo University of
Medicine and Dentistry)
Two thoracic limbs of Drosophila, the leg and the wing, are specified from acommon cluster of cells including the sources of two secreted signaling mol-

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ecules Decapentaplegic and Wingless. We are studying how various inductive signals are combined to specify diverse sets of cell types arranged inunique spacial patterns. Differential activation of Decapentaplegic and EGF
receptor signaling appear to play key roles in this process.
The leg imaginal disc resumes cell proliferation in the second instar, maintaining the circular patterns of the proximal and distal marker expression,and elaborates its pattern by activating additional genes required for thedifferentiation of a subset of leg structures. We are investigating how theinteraction between the proximal and distal cells maintain their specific gene
expression, activate additional genes, and establish polarity along the proximodistal axis.

(2)

Cell biological study of the tubular epithelial network
formation in the tracheal system

Tomoatsu IKEYA, Takahiro CHIHARA, Miho TANAKA-MATAKATSU and Shigeo
HAYASHICoordination of cell motility and adhesion is essential for concerted movement of tissues during animal morphogenesis. The Drosophila tracheal network is formed by branching, migration and fusion of tubular ectodermal
epithelia. The tracheal tip cell is located at the end of each branch that aregoing to fuse. We are studying mechanisms of cell fate determination, patterned cell migration, and unique cell shape changes of tracheal cells by various genetic and histological techniques.

(3)

Kinase-independent activity of Cdc2/Cyclin A prevents
S phase in the Drosophila cell cycle

Shigeo HAYASHI and Masamitsu Y AMAGUCHl 1(lLaboratory of Cell Biology, Aichi
Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan)
Cdc2 dependent inhibition ofS phase is required in G2 for correct ordering of
S phase and M phase in yeast and Drosophila. This function of Cdc2 hasbeen ascribed to its activity to phosphorylate replication factors, preventing
the assembly of preinitiation complex at the origin of replication. Whether

118

this is the sole mechanism of S phase inhibition by Cdc2 in higher metazoan is
not known because pleiotropic functions of this essential cell cycle regulator
make genetic analysis difficult. We show that Cdc2 coexpressed with Cyclin
A inhibits S phase in Drosophila salivary glands and in diploid abdominal
histoblasts. A kinase defective mutant of Cdc2 failed to promote mitosis,
but still inhibited S phase with the same efficiency as the wild type protein. In
addition, Cdc2 and Cyclin A cooperatively inhibit transcriptional activation by
the essential S phase regulator E2F. Cdc2 binds to E2F in vitro, and posttranscriptionally promotes its accumulation in vivo. Furthermore, the inhibitory effect of Cdc2 on S phase is overridden by E2F. The results suggestthat inhibition of S phase by Cdc2 is achieved in part by a kinase independent
mechanism, which is likely to be mediated by inhibition of E2F. For detail,see ref. 3.
(4)plexus, a gene required for adult wing vein patterning

Hitoshi MATAKATSU 1, Sumiko GAMOU 1, Ryu-suke TADOKOR0 2and Shigeo HAYASHI(lOsaka Prefecural University, 2Kitasato University)
Veins of the adult wing are a good landmark for the anteroposterior anddorsoventral positional informations. The gene plexus is required for suppression of vein formation in specific intervein positions. We have cloned theplexus gene. The structure and biochemical properties of Plexus protein, andits genetic interaction with various vein-patterning genes suggest that plexus
is a component of a novel regulatory pathway of vein differentiation.

(5)

Enhaneer trap sereen for genes involved In pattern formation

Satoshi GOTO, Hiroko TAKEUCHI, Misako TANIGUCHI 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 Ga14-UAS system. About 4000 lines were established in collaboration with groups in Japan and were examined for the activity of enhancers
flanking the inserts in the embryo, larva and adult. The results are being
assembled into a database.

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Interaction between DrosopbJ/a EGF receptor and vnd determines
three dorsoventral domains of the neuroectoderm

Yoshimasa YAGV Toshiharu SUZUKI, I and Shigeo HAYASHI (I Laboratory of
Neurobiophysics, School of Pharmaceutical Science, The University of Tokyo)
Neurogenesis in Drosophila melanogasterstarts by an ordered appearanceof neuroblasts arranged in three columns (medial, intermediate, and lateral)in each side of the neuroectoderm. Here we show that in the intermediate
column, the receptor tyrosine kinase DER represses expression of proneuralgenes achaete and scute, and is required for the formation of neuroblasts. Mostof the early function of DER is likely to be mediated by the Ras-MAP kinasesignaling pathway which is activated in the intermediate row, since a loss of acomponent of this pathway leads to a phenotype identical to that in DER
mutants. MAP-kinase activation was also observed in the medial column
where esg and proneural gene expression is unaffected by DER. We foundthat the homeobox gene vnd is required for the expression of esgand scutein
the medial column, and we have shown that vnd acts through the negative
regulatory region ofthe esgenhancer that mediates the DER signal, suggesting the role of vndis to counteract DER dependent repression. Thus nested
expression of vnd and the DER activator rhomboid is cruicial to subdivide theneuroectoderm into the three dorsoventral domains. For detail, see ref. 1.

Pub I i ca t ionsYAGI, Y, SUZUKI, T. and HAYASHI, S.: Interaction between Drosophila EGF receptor
and vnd determines three dorsoventral domains of the neuroectoderm. Development,
125, 3625-3633, 1998.
2. KOBAYASHI, M., AlTA, N., HAYASHI, S., OKADA, K., OHTA, T. and HIROSE, S.: DNA supercoiling factor localizes to puffs on polytene chromosomes in Drosophila melanogaster.
Mol. Cel. BioI., 18, 6737-6744, 1998.
3. HAYASHI, S. and YAMAGUCHI, M.: Kinase-independent activity of Cdc2/Cyclin A prevents S phase in the Drosophila cell cycle. Genes to Cells, 4, 111-122, 1999.
1.

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CENTER FOR GENETIC RESOURCE INFORMATION
G-a. Genetic Informatics Laboratory(1)

Cross-species Database

Yukiko YAMAZAKI
The goal of this project is full cross-referencing of biological knowledge among
different species. In 1996 an integrated database of wheat and rice genetic
resource information was experimentally developed. The prototype of crossspecies functional gene database was created in 1997. In order to integrate
gene information from any different species, I've started developing the new
system (SEEGEN) this year. The data was retrieved from i) the DNA sequence database, ii) the genetic resource databases and iii) the gene dictionaries of different species. Although the current program is written in PERL
language, the SEEGEN system will be incorporated into object-oriented DB
system.
(!)

Genetic Resources Database2-1. Wheat Genetic Resources Database -KOMUGIYukiko YAMAZAKI, Hisashi TSUJIMOT0 1, Taihachi KAWAHARA 2 and Yasunari
OGIWARA 1 (lKihara Institute for Biological Research, Yokohama City University, 2Plant Germ-plasm Institute, Faculty ofAgriculture, Kyoto University)
In addition to the update of the existing database, the KOMUGI 1.1 version
offers a comprehensive interface for searching. Mutual mirroring between
Graingenes provided by USDA, U.S.A. and KOMUGI has been in agreement. As
a subgroup ofKOMUGI, the Triticeae DNA repository database compiled over
900 clones and chromosome maps of common wheat have been added to the
database. The next task of the working group will be setting up the gene dictionary tightly coupled to the KOMUGI database. The KOMUGI 1.1 version isavailable via WWW server at http://www.shigen.nig.ac.jp/wheat/wheat.html.

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2-2. Rice Genetic Resources in Japan
Yukiko YAMAZAKI, Nori KURATA, Atsushi YOSHIMURA' and Yasuo NAGAT0 2(lKyushu University, 2University of Tokyo)
The networking group of rice researchers in Japan has started to capture allavailable information about rice science, such as i) fundamental knowledge ofrice science, ii) image data of mutant rice strain, iii) gene dictionary revised
according to the new classification of gene function and iv) the integrated linkage map of phenotype and RFLP markers provided by Dr. Yoshimura. The
existing rice genetic resource database will include the above information withcross-linking each other. We decided to use object-oriented database implementation to construct the next version of Rice DB because of complicatednetwork of relationships between the contents.

2-3. Barley Germplasm Database
Kazuyoshi SATO', Mari SAITO and Yukiko YAMAZAKI ('Okayama University)
The first version of barley germplasm database has been released and is now
available through the internet at http://www.shigen.nig.ac.jp/barley/
Barley.html. The database lists 4250 germplasms and each strain consists ofagro-morphological, physiological, and pathological information as well as genetic traits. Dr. Sato is working for setting up the original database on his own
server machine at Okayama University and the data mirroring system, throughwhich the data can be updated by connecting the remote computer, is underconstruction. Our site also plans to support a project to provide CORBA(Common
Object Request Broker Architecture) access to other public databases.
2-4. Mouse Microsatellite DatabaseToshihiko SHIROISHI, Rie TSUCHIYA and Yukiko YAMAZAKI
The mouse microsatellite database in Japan (MMDBJ ver.l.O) was publicly
released with over 900 entries and is on-line available at http://www.shigen.nig.ac.jp/
mouse/mouse.html. The database provides SSLP (simple sequence length polymorphism) information among different strains especially derived from Japanese wild mouse. Although the current database has an on-line direct data

122

submission system so that researchers can deposit their own data through the
network, the revision in the structure of the database is necessary to either
combine new data with different format or to provide new service requested byresearchers. So we've started developing new database using object oriented
database management system, ObjectStore.
2-5. Profiling of E.coli chromosome database
Junichi RATO I , Tohru IKEGAMI 1, Takehiro YAMAKAWA, Kazu MITSUI, Yukiko
YAMAZAKI (IUniversity of Tokyo)
The PEC (profiling of E.coli chromosome) database has been started construction in the collaboration with Dr. Kato's group. Among many different
ways of genome analysis, the project focused on the following point of views, (1)the gene (or orf) is essential or not, (2) the codon analysis of genes, (3) the genestructural analysis such as motif, module and/or domain, and (4) the comparative analysis between different organisms. The goal of this project is to collect the relevant information as much as possible and provide reliable resources for further research. The PEC is object oriented database with javabased applications. The information, which the current PEC database uses,
is derived from E.coli strain MG 1655 and the knowledge information was
extracted from several different databases including DNA database and journal articles.
2-6. Arabidopsis genetic resource database
Shinji GOTOl, Rie TSUCHIYA, Yukiko YAMAZAKI (IMiyagi Educational University)
Arabidopsis genetic resource database has been constructed in the collaboration with Dr. Goto this year. The first release contains about 1200 strains
and the database is available at http:www.shigen.nig.ac.jp/arabidopsis. The
collection includes AIS collection (Arabidopsis Information Service, Germany)and Sendai collection developed by Dr. Goto. The database is the first genetic
resource DB ofArabidopsis in Japan and will incorporate other Brassica strainsin the near future. Data submission system, through which the strain maintainer can update his/her own data by connecting to the remote computerG.

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running the databases, has been developed.

Publ icatians
1.
2.
3.

YAMAZAKI, Y, TSUJIMOTO, H. and KAWAHARA, T.: KOMUGI Database - Wheat Genetic
Resources Database. Genes Genet. Syst. 73, 75-77, 1998.
YAMAZAKI, Y.: Wheat Genetic Resources Database in Japan. 9th International Wheat
Genetics Symposium Proceeding. !, 375-376, 1998.
YAMAZAKI, Y.: Genetic Resource Database, Protein, Nucleic Acid and Enzyme. 43,20122015, 1998.

G-b. Genome Biology Laboratory(1)

Systematic analysis of mRNA distribution bywhole mount In situ hybridization

Tomoko MOTOHASHI, Tokie OHBA, Ikuko SUGIURA, Masumi OBARA, Sayuri
KITAYAMA, Takami SUZUKI, Tadasu SHIN-I and Yuji KOHARA
In this lab, G.elegans EST project has been performed, which has identified
10,955 cDNA species based on the comparison oftag sequences of some 65,000
clones. The distribution of the cDNA species among the six chromosomes isquite even; LG1: 1856, LG2:1855, LG3:1670, LG4:1645, LG5:1713,
LGX: 1424. To clarify their expression patterns, we have established an efficient protocol for in situ hybridization on'" whole mount embryos, larvae andadults (http://watson.genes.nig.ac.jp.8080/db/method). and have applied theprocedure to the classified cDNA groups. Thus far, we have done the in situ
analysis for 3500 cDNA groups mostly from an autosome LG3 and the sex
chromosome LGX; LG1:6, LG2:522, LG3:1448, LG4:190, LG5:49 and
LGX: 1281. Analysis of the remaining 7000 cDNA groups is also in progress
and will be finished within a year. Although the experimental part ofthe insitu hybridization is very efficient, analysis of the results including taking
photos and giving annotations is a very time consuming process. Technicalinformation on our in situ methods and statistics of the results of the expression patterns will be presented. All the data will become available at (http://watson.genes.nig.ac.jp:8080/db/).

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(2)

NEXTDB: The nematode expression pattern map database

Tadasu SHIN-I and Yuji KOHARA
We have developed a WWW·based database "NEXTDB" to integrate all theinformation of ESTs, gene expression patterns and gene functions of G.elegans
which are being produced and analyzed in this laboratory.
Raw data of tag sequencing is transferred to the server machine from the
ABI sequencers and then processed such that ambiguous regions which contain a larger rate of"N" (ambiguous base), cloning vectors, and poly-A tails are
removed. The processed 3'-tags are classified to unique cDNA groups by applying a cumulative method by use of FASTA. Both 5'- and 3'-tag sequences
are mapped to cosmid sequences by use of BLASTN. BLASTX searches arealso done to confirm homologies of predicted CDSs to genes of otherorganisms. All the above processes are done automatically.
Images of whole mount in situ hybridization for detecting mRNA distribution were taken by CCD cameras equipped on Zeiss Axioplan microscopes (SeeMotohashi et al.), transferred to the server machine and then processed andarranged properly in the database. Annotations added to individual images
were also stored in the database. Images of immunostaining taken on Zeiss
LSM510 confocal microscope (See Onami et al.) and the data ofRNAi phenotypes (See Hirono et al.) were also stored and arrange properly in the
database. Finally, NEXTDB has the page for feedback information from theusers of cDNA clones including full sequences, RNAi phenotypes, anomalies,
gene functions, homologies and so on.
In order to integrate NEXTDB, we applied a hierarchical model to arrange
all the clones and cDNA groups; 1) chromosome, 2) cosmid clone, 3) CDS, 4)
cDNA group, and 5) cDNA clone. The cosmid map which connects 1) and 2)
are obtained from AceDB. The relations about cosmids and their CDS are
retrieved from the annotations of the Sanger Centre sequence data. All of
the information is linked to each other in NEXTDB and displayed visually byuse of JAVA applets. The latest version is available at the following URL.
<http://watson.genes.nig.ac.jp :8080/db/index.html>

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125

Expressed Genes In C. eJegans

Jean THIERRY-MIEG 1.2, Danielle THIERRY-MIEG 1•2, Tadasu SHIN-It and Yuji
KOHARA1(lNIG, Mishima, Japan, 2CNRS, Montpellier, France.)
A large normalized set of G.elegans cDNAs has been constructed and thetwo ends of each cDNA have been sequenced in Mishima. The resulting 118300
traces together with 6000 other cDNA sequences available from GenBank
were aligned over 95.8 megabases genomic DNA available from the G.elegans
consortium. The expressed genes were reconstructed, and the exact splicing
established. As of March 99, there are hits to 9100 genes, among which 320
are in yet unsequenced genomic holes. That is about half of the 19000 genes
predicted by the consortium, but not an exact subset, because the actual transcripts merge or split or differ in some finer way from the predictions in almost
half the cases.
To fully exploit the data, we hand edit the basecall of the traces in view of the
genomic sequence. About 4000 genes have been edited so far. Based onthat set, a minimum of one third of the genes show alternative splicing andshould generate more than one product. Alternative polyadenylation alsofrequently occurs. Transplicing to SL1 is apparent in only 35% of the probably complete genes. This may be an underestimate, but we do see SL1 in 12/
14 of the complete genes directly upstream from the rare genes transpliced to
SL2.
Because we have both the cDNA and the genomic sequence, we derive accurate data on the distribution of the genes along the genome, the exact structure of the introns, the transplicing and the 3' untranslated regions. Basedon these independent sequence data, we confirm the high quality of the genomic sequence which presents less than one error per 40 kb and very few
rearrangements. We expect that 3.2 Mb ofthe genome is still missing. The
data on our servers can be accessed at http://alpha.crbm.cnrs-mop.fr or http://
watson.genes.nig.ac.jp:8080/db/.

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(4)

Protein Expression Pattern Analysis of Maternal mRNAs

Shuichi ONAMI, Tamami NAGAOKA and Yuji KOHARA
Our eDNA/expression pattern project has identified many maternal mRNAs
whose function is unknown. To study their function, we have started protein
expression pattern analysis.
Our main interest is cell fate determination in early embryogenesis. Thus,
we focus on genes whose mRNA is maternally supplied, and 1) disappears
before 18cell-stage or 2) localizes to specific cells. We picked up 100 suchgenes out of 2000 genes based on our in situ hybridization results. We areraising rat antisera against all of these genes using bacterially expressed
partial proteins (100-150 amino acids). Until now, we have obtained themfor 54 genes and stained embryos and gonads. Fifty antisera (93%) showed
detectable staining. The sub-cellular staining patterns are classified into
perinuclear cytoplasm and plasma membrane (8), cytoplasmic granules (7),nucleus and plasma membrane (6), P-granules (6), cytoplasm (5), nucleus (5),nuclear membrane (5), plasma membrane (5), and egg shell (3). Thirty-one
antisera (57%) showed tissue-specific staining before hatching and 9 of themshowed cell-specific staining before 18cell-stage. Twelve antisera (22%)
stained specific regions of the gonad. We also analyzed RNAi phenotype forthose 100 genes (see Hirono et al.). We found 4 genes whose RNAi resulted ingerm line defects out of 11 genes whose immunostaining pattern looked like Pcell specific. We believe that this approach provides fruitful information aboutmaternal mRNAs. We are preparing to release these data on our WWW site(http://watson.genes.nig.ac.jp:8080/db/).

(5)

Systematic RNAi experiments with maternal genes

Keiko HIRONO, Shuichi ONAMI and Yuji KOHARA
The accumulation of gene expression patterns that are being produced inthis lab provides unique sets of limited numbers of genes that possibly participate in specific stages, lineages, tissues and so on. Such sets of genes
could be subjected to systematic analysis to elucidate the molecular mechanisms governing the biological processes. Since we are particularly inter-

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127

ested in early embryogenesis, we have focused on a set of genes whose mRNA
are maternally supplied and disappear before 18cell-stage or localize to specific cells in early embryos. We selected 100 such genes out of 2000 genes
based on our in situ hybridization analysis, and have analyzed them withrespect to RNAi phenotype as well as protein distribution by immunostaining.
We micro-injected double-stranded RNA into the gonads of ten N2 worms
per gene. Phenotypic analyses were performed on the injected worms themselves, F1 embryos, larvae and adults that survived (called "escapers") and F2
embryos, with respect to embryogenesis, larval growth, sterility, morphogenesis, the expression of several differentiation markers and so on. Out ofthe
100 genes, 45% showed F1 embryonic-lethality or the reduction of the number
ofF1, 34% showed various phenotype with only escapers, 18% showed sterility, and 2% did not show any phenotype.
The phenotypes of some genes were consistent with the results of protein
distribution. For example, RNAi with the cDNA group CELK02786 causedno or a very fragile egg shell, and the antibody against the gene product stained
the surface of entire embryo, probably underneath the egg shell, suggesting thegene is involved in the process of egg shell formation. RNAi of 4 out of 11
genes whose immunostaining patterns looked P-lineage specific caused germline defect. We believe that this approach of the combination of the analysisof expression patterns and RNAi phenotypes provides highly useful
information. We are preparing to release these data on our WWW site (http:/
Iwa tson. genes.nig.ac.jp: 80801db/).

(6)

Toward a four-dimensional database ofgene expression in C. eJegans

Masahiro ITOH, Tomoko MOTOHASHI, Yohei MINAKUCHI and Yuji KOHARA
One of the most challenging targets in the post genomics era is computer
simulation of development. We feel G.eJegans is the most suitable system
for this purpose, since the complete parts list of cells has been identified andthe expression patterns and functions of all genes are being analyzed
systematically. Aiming at the final goal and at integrating such information,
we are constructing a computer graphics (CG)-based four-dimensional (3D +128time course) database of gene expression in G.elegans.
Thus far, we have made a CG for early embryogenesis up to 100 cell stage
based on 4D Nomarski image data including the data kindly supplied by
Kevin O'Connel at the University of Wisconsin at Madison. Since it was very
hard for conventional image processing software to extract the contour of cells
from the Nomarski images, we adopted a 'low-tech' procedure; firstly we traced
the contour of individual cells and nuclei by hand-writing on thousands oftheoriginal images of the 4D microscopic data and then reconstructed 3D structures by piling up and making relationships among the contour data fromdifferent focal planes. The resulting 3D structures of various stage embryos
were arranged along the time course of development and connected each otherby an interpolating method to finally generate the GG of early
embryogenesis. The graphics is faithful to the real embryogenesis, thus, it
provides various information such as the volume of individual cells, the extent
of cell-cell contact and so on.
To incorporate the information of gene expression patterns into the 4D graphics database, we have developed a procedure to superimpose a fluorescentconfocal data of the distribution of mRNA and proteins onto the CG. In theprocedure, we use triple color staining, namely, DAPI and Cy-3 for nuclei and
POS-1 (p-lineage specific) as internal markers for superimposing, respectively,
and Cy-5 for a query gene product. By this fashion, we are incorporating theimmunostaining patterns of maternal gene products. We are planning to
make the 4D database accessible over the internet.

(7)

Pos-J encodes a cytoplasmic zinc-finger protein essential for
germllne specification in C. eJegans

Hiroaki TABARA, Russell J. HILL l , Craig MELL0 2, James R. PRIESS l and Yuji
KOHARA (lFred Hutchinson Cancer Research Center, Howard Hughes Medical
Institute, Seattle, WA 98109, 2University of Massachusetts Cancer Center,
Worcester, MA 01605)
In higher animals, sperm and oocytes differentiate from special cells called
germ cells. Since the germ cells must reproduce the entire organism witheach new generation, they are considered to be totipotent. Thus mechanisms

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must exist that maintain totipotency in the embryonic cells destined to produce germ cells, although very little is understood about this process for any
animal embryo.
In C. elegans embryogenesis, the germ cells arise from a stem cell-like lineage pattern. Blastomeres in the branch of the lineage that leads to germ
cells are called germline blastomeres; the sisters of the germline blastomeres
produce only somatic tissues and are called somatic blastomeres. The final
germline blastomere is called P4 and it produces only germ cell descendants.
Several differences have been described between the germline blastomeresand somatic blastomeres of the C. elegans embryo. For example, many maternally-provided mRNAs persist longer in the germline blastomeres than in
the somatic blastomeres. Cytoplasmic granules of unknown function called
P granules are partitioned asymmetrically into the germline blastomeres during each of the early divisions, ultimately becoming localized into the P4
blastomere. The relationship between the persistence of maternal mRNAs,or P granule localization, and the totipotency ofthe germline blastomeres has
not been determined.
Another difference between the germline and somatic blastomeres is in theinitiation of embryonic gene transcription; after embryonic transcription initiates at the 4-cell stage of development, new mRNA transcripts are detected
only in the somatic blastomeres. This result suggests that transcription isrepressed or blocked in the germline blastomeres. The apparent lack of transcription provides an explanation for why germline blastomeres do not respond to transcription factors such as the SKN-l and PAL-l proteins. SKNland PAL-l are translated from maternally-provided mRNAs in both germline
and somatic blastomeres. Although these transcription factors promote specific patterns of somatic differentiation in the somatic blastomeres, they appear to have no effect on the development ofthe germline blastomeres. Thus,
transcriptional repression appears to be at least part of the mechanism thatmaintains the totipotency of the early germline blastomeres.
Mutations in the genes pie-l and mex-l cause the germline blastomeres toadopt somatic fates, suggesting that they function in maintaining the totipotency of the germline blastomeres. In pie-l mutants, embryonic transcription appears to initiate simultaneously in both somatic and germline blas-

130

tomeres, such that the germline blastomeres become competent to respond to
SKN-I and PAL-I. These results suggest that pie-l(+) functions in repressing transcription in wild-type germline blastomeres. mex-l appears to playa distinct role from pie-l, because at least some transcription is properly
repressed in the germline blastomeres of mex-l mutants. PIE-I is a novel,
predominantly nuclear, protein of unknown function that is localized exclusively to a germline blastomere at each of the early embryonic cell divisions.
MEX-I is a novel, cytoplasmic protein that is localized predominantly to a
germline blastomere at each early division. PIE-I and MEX-I are both com·ponents of P granules, and both proteins contain two copies of a CCCH-type"finger" domain originally described in a vertebrate protein of unknown function called TISI1/Nup475/TTP.
We have taken two complementary approaches to identify additional genesthat establish germline/soma differences in C. elegans. As part of the C.
elegans eDNA project, we have used molecular screens to look for mRNAs thatare localized asymmetrically in the early embryo. In a second approach, wehave used genetic screens to identify maternal-effect lethal mutations thatprevent the development of germ cells. Here we describe the pos-l gene, which
was identified independently in both screens. We show that the pos-l mRNA
is distributed asymmetrically after the first cleavage of the embryo, and ispresent predominantly in germline blastomeres. Like MEX-I, POS-I is acytoplasmic protein localized predominantly to germline blastomeres. Like
both MEX-I and PIE-I, the POS-I protein is a component ofP granules andhas two copies of the CCCH finger motif. We show that pos-l mutants havedevelopmental defects that are distinct from both mex-l and pie-l mutants,suggesting that the POS-I, MEX-I and PIE-I proteins have at least somedistinct roles in the specification of germline blastomeres.

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Studies on POS-1 Interacting proteins

Ken-ich OGURA and Yuji KOHARA
The pas-l gene encodes a TIS 11 type zinc finger protein similar to PIE-! andMEX-! (Tabara et aI., Development, 126, 1-11, 1999). Pas-l mutants show
maternal effect embryonic lethality with either reduced or excess pharynx,
almost no intestine, no germ cell and extra hypodermis. POS-1 proteins localize to P-lineage, being a temporary component ofP granules. Unlike PIE1, POS-! presents in cytoplasm but not in nuclei. POS-1 is suggested tofunction in translation of maternal mRNAs such as apx-l, however, the molecular process in which POS-1 functions is largely unknown.
Aiming at understanding the molecular process, we have identified the proteins that interact with POS-1 by using the yeast two hybrid system. These
included POS-! itself, MEX-3 (a KH type RNA binding protein), a novel RNA
binding protein (named PIP-1 after POS-1 Interacting Protein), a serine/threonine protein phosphatase 2A catalytic subunit (PP2AC). We have confirmedthat POS-1 directly bound to these proteins in vitro. The results indicatedthat POS-! could form homodimer or homocomplex. MEX-3 appears to regulate the translation of maternal pa1-l mRNA, POS-1 may function in thetranslation of maternal mRNA in cooperation with MEX-3.
RNAi experiments with pip-l yielded phenotypes similar to the pas-lmutants. The pip-l mRNA was detected abundantly in early embryos, andshowed its localization to posterior blastomeres after 4 cell stage.
Immunostaining with anti-PIP-1 antibodies showed that PIP-! was present
equally in the cytoplasm of both AB and P! blastomeres at 2 cell stage, andbegan to localize to the posterior blastomeres at 4 cell stage; the staining wasstrong in P2 and EMS but weak in ABa and ABp. Interestingly, this staining
pattern is in contrast with MEX-3. PIP-! was not detected in late stageembryos. PIP-! was suggested to be a temporary component ofP granules. We
confirmed that the staining disappeared inpip-l(RNAi) embryos. Furthermore,using the two hybrid system, we found that PIP-! interacted with MEX-3 andalso MEX-l.
Finally, we revealed that the PP2AC homolog is encoded by 1et-92, a gene
required for early larval development (Clark et aI., Genetics, 119, 345-353,

1321988). Our analysis showed that LET-92 plays essential roles in the coordi·
nation of nuclear division and cytokinesis and P granule localization. It isinteresting that POS-l has putative phosphorylation sites of casein kinase 2
and PKC.

(9)

tbx-9 encodes a transcription activator

Yoshiki ANDACHI

tbx-9is one of the T-box family genes that share a DNA binding motif, T-box.
Genes of the family are found widely in metazoans, and many of them are
involved in important events for development. Three T-box genes, Brachyury(T), VegT and tbx-2, have been shown to encode transcription factors. In C.
elegans, the whole genome sequence predicts that this organism has about 20
T-box genes, and cDNA clones corresponding to the predicted genes are being
isolated by the cDNA project. As I am interested in the regulation of gene
expression, I have been studying one of the T-box genes, CELK02736 = tbx-9,
which is the first one among T-box genes whose cDNA clones have been
isolated. I previously showed that tbx-9is expressed in only a few cells ofearly-stage embryos and tbx-9deletion mutants produced by gene disruption
show aberration of morphogenesis in the posterior body region in embryogenesis, including disturbance of the row of body wall muscles.
To test whether the tbx-9 protein has a sequence-specific DNA binding
activity, the tbx-9DNA binding sequence was determined as consensus amongsequences of DNA fragments that were selected from a mixture of DNA fragments of random sequence by forming a complex with the tbx-9protein. The
determined tbx-9binding sequence was 18 bases in length and had a palindromic structure. The T product binding sequence determined by a similar procedure has been reported to be palindromic, and the binding sequences of T
and tbx-9are almost identical. To know whether the tbx-9protein is a transcription factor, transactivation of a reporter gene depending on the tbx-9binding sequence was examined. To this end, the following plasmids wereconstructed. For forced expression of tbx-9, the tbx-9gene was ligated downstream of the hsp16-41 heat shock promoter. For assay oftransactivation,
the tbx-9 binding sequence was inserted upstream of the pes-l0 minimal

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promoter and the reporter lacZ gene. Transgenic lines containing both plasmids were produced by co-injection. Ectopic expression of tbx-9by subjecting
transgenic embryos to heat shock caused activation of the lacZ gene, indicating that the tbx-9protein functions as a transcription activator. As the next
goal, I am aiming at identifying target genes of tbx-9.

Publ icatian
1.

2.

3.

The G.eJegans Sequencing Consortium:Genome sequence of the nematode C. eJegans:A platform for investigating biology. Science %8%, 2012-2018, 1998.
TABARA, H., HILL, R.J., MELLO, C., PRIESS, J. and KOHARA, Y: Pos-] encodes a cytoplasmic zinc-finger protein essential for germline specification in C. eJegans. Development t%6, 1-11, 1999.
KUWAHARA, M., ISHIBASHI, K., Gu, Y, TERADA, Y, KOHARA, Y, MARUMo, F. and SASAKI,
S.: A water channel ofthe nematode Caenorhabditis eJegans and its implications forchannel selectivity ofMIP proteins. The Am. J. Physiol.: Cell Physiol. C1459-C1464,
1998.

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H-a. Biological Macromolecules Laboratory(1)

Single Molecule Imaging of Biological Functions

Makio TOKUNAGA
In the post-genome era, novel methods are needed to elucidate functions ofmolecules found in genome. Recently, single molecule techniques have beendeveloped out of in vitro researches of motor proteins. We are trying to spreadout this new technique from in vitro to in vivo and from motor proteins to otherbiological molecules. Imaging of single fluorescent molecules has been achievedin a relatively simple manner using objective-type total internal reflection
fluorescence microscopy (TIRFM). It is found that single molecule imaging invivo is realized using the objective-type TIRFM. Furthermore, I'm developping
new microscopy to achieve followings: 1) Quantitative imaging of molecularnumbers or concentrations in vivo. It is essential to minimize the backgroundlight. 2) Imaging in vivo of interacting molecules and activated molecules.
(!)

Single molecule capture, manipulation andforce measurement of protein molecules

Kazuo KITAMURA l , Atsuko IWANE 2, Toshio YANAGIDA l ,2 and Makio TOKUNAGA(ISingle Molecule Processes Project, ICORP, JST, 2Department of Physiology I,
Osaka University Medical School)
We have developed a new instrument to capture and directly manipulate
individual molecules using a scanning probe. A single myosin head molecule(myosin subfragment-l, Sl) was captured onto the tip of a scanning microprobe using a flexible glass microprobe, and manipulated with subnanometer
resolution. Single molecule was confirmed by examining in the fluorescencemicroscopy at the same time using objective-type TIRFM. Movements andforces resulting from the interaction of a captured single Sl molecule with

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actin filaments were measured. Our data are consistent with the unitarymechanical step during sliding along an actin filament of -5.3 nm, but groupsof 2 to 5 rapid steps in succession often produce displacements of -11 to -30
nm. The instances of multiple stepping are produced by single heads during
one biochemical cycle of ATP hydrolysis. The results proves that the loose
coupling mechanism is right. For details, see Ref. 2.

(3) Sliding movements of actin filaments undergoing fluctuations
propelled by myosin subfragment-l flexibly bound to the surface
Makio TOKUNAGA, Atsuko IWANE 1, Kazuo KITAMURA 2 and Toshio YANAGIDA 1•2(lDepartment of Physiology I, Osaka University Medical School, 2Single Molecule Processes Project, ICORP, JST)We have recently demonstrated that when skeletal muscle S1 is specificallyattached to a glass surface in its tail end, it can move actin as fast as intact
myosin. The specific binding was achieved by replacing a endogenous RLC by
a recombinant fusion protein of biotin-dependent transcarboxylase (BDTC, abiotinylated peptide) and chicken gizzard RLC (cgmRLC). S1 was bound to an
avidin-coated glass surface through BDTC-cgmRLC. In this work, a flexible
peptide, 5G, which is expected to have a random coil structure and -12 nm in
expanded length, was inserted between BDTC and cgmRLC (BDTC-5GcgmRLC). Thus, S1 was attached to a glass surface via the flexible chain.
Insertion of 5G did not affect the actin-activated ATPase activity. Velocity ofactin filaments caused by BDTC-5G-cgmRLC-S1 on a glass surface was similar to those by intact myosin and by BDTC-cgmRLC-Sl, respectively. It was
confirmed that the inserted chain was indeed flexible, using both electron
microscopy and video image analysis. The present results don't support thewidely accepted lever arm model.

136

(4)

Molecular Interactions measured by nanometer-sized probes using
subplconewton Intermolecular force microscopy

Michio HIROSHIMA! and Makio TOKUNAGA (lDepartment of Biophysical Engineering, Osaka University)
Interaction between biological macromolecules involves several kinds offorces
such as electrostatic forces, hydrophobic attractive forces, and so on. For adetailed understanding ofthe interactions between biological macromolecules,
it is required to obtain information about the properties ofthese forces at themolecular level. We have refined atomic force microscopy (AFM) to improve
the sensitivity of force detection and control of probe position. Force resolutionof subpiconewton has been achieved, which is over 100-fold more sensitive
than that of conventional AFM. In this study, we have measured the forces
exerted on a nanometer-sized probe using this novel microscopy. Forces between hydrophobic surfaces and between weakly charged surfaces were measured in aqueous solutions.
A long-range attractive force was observed between hydrophobic surfaces
out to 150 nm with a decay length of 30 nm in water. Both the range and the
decay length ofthe force were decreased with the increase ofKCI concentrations or with the decrease of hydrophobicity of the surfaces. A linear relation
was found between the decay length and the contact angle between the hydrophobic surfaces and drops of electrolyte solutions. This result suggests thatthe long-range attractive force results from the interaction between the hydrophobic surfaces and water.

(5) A New Model of Molecular Motors
Makio TOKUNAGA
Our single-molecule researches provided a direct evidence of the loose coupling mechanism on protein motors. I have shown that a new simple model
named "asymmetric interaction model" can explain the results of the singlemolecule experiments. Because this model is based only on a simple assumption that the interaction potential between motor proteins has a asymmetricshape, the theoretical formulation is very simple. Structural fluctuations ofH.

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protein molecules can play an important role as a fluctuation sensor, which
realize the Maxwell's demon using energies supplied by ATP hydrolysis.
Publ ications
1.

2.

ISHIJIMA, A, KOJIMA, H., FUNATSU, T., TOKUNAGA, M., HIGUCHI, H., TANAKA, H., andYANAGIDA, T.: Simultaneous observation of individual ATPase and mechanical eventsby a single myosin molecule during interaction with actin. Cell, 92, 161-171, 1998.
KITAMURA, K., TOKUNAGA, M., IWANE, A H., and YANAGIDA, T.: A single myosin headmoves along an actin filament with regular steps of 5.3 nanometres. Nature, 397, 129134, 1999.

H-b. Molecular Biomechanism Laboratory(1) Branched pathway mechanism of transcription Initiation by
E. coli RNA polymerase, and promoter-arrested
Initiation complexes
Tomoko KUBORI 1, Hiroki NAGAI l , T. Gaa12 and Nobuo SHIMAMOT0 1 (lStructural
Biology Center, National Institute of Genetics, 2Microbiology, Wisconsin-Madison)
The initiation frequency of transcription is conventionally attributed to thestability of holoenzyme-promoter binary complex, the rate/degree of strand
opening, and the rate of promoter clearance. We previously showed that a
portion of an otherwise homogeneous holoenzyme preparation following promoter binding is converted into a binary complex incapable of synthesizing
full-length RNAs and synthesizing only abortive transcripts. These non-productive complexes designated" moribund" complexes (Nuc. Acids Res. 24,
1380-1381 (1996): J. Mol. BioI. 256, 449-457 (1996».
We kinetically and biochemically isolated the nonproductive complexes at
APR' lacUV5, T5N25antiDSR promoters, and promoters with the" best" -10
or -35 box selected by SELEX. But at T7A1, rrnBP1, and the one with both ofthe" best" -10 and the -35 boxes, moribund complexes, if any, decays synchronously to the productive complexes, and are not kinetically isolated. Thus

138

promoters can be classified into two classes, but this classification depends
on reaction conditions; APR behaves like T7Al in the presence of either a mutant 0 70 or Gre factors, and T7Al becomes similar to APR at low salt. Binary
complexes with APR -type promoters are resistant to heparin while those with
T7Al type are not (SEN et al., J. Biol. Chem. (1998».
Moribund complexes formed at A PR readily convert into inactive dead-end
complexes making no RNA. DNA and protein footprinting analyses showedthat this dead-end complex is backtracked from the promoter, lacks the strand
opening and has more exposed conserved region 3 of 0 70 • This inactivation
was mitigated in the simultaneous presence of a high concentration ofinitiating nucleotide and Gre factors prior to RNA synthesis. At T5N25antiDSR
promoter moribund complex is predominant up to 2 hr, and retains the strand
opening, suggesting moribund complex is a member of open complexes.
A minimalist model consistent with all these observations is that two subspecies of open binary complex exist either in productive or moribund states,
forming a two-state switch. At promoters like APR these different forms can·not directly convert into each other. Gre factors introduce reversibility to mori·bund binary complex formation allowing rapid switching into productive bi·nary complex which is consumed up by long RNA synthesis (SEN et al., J. Biol.
Chem. (1998».

(Z) Role of the spacer region of a 71 andInactivation by oligomerization
Hiroki NAGAI l , Taciana KAsCIUKOVIC 2, Richard S. Hayward 2, Yumiko SATO l
and Nobuo SHIMAMOT0 1 (lNat. Inst. of Genet./Grad. School ofAdv. Stud., 2Inst.
of Cell and Molec. Biol., Edinburgh Univ., Scotland)
The major 0 factors of proteobacteria mostly have a big spacer region between the conserved regions 1 and 2, which is not conserved in eubacteria. In
E. coli 0 70 this region has extensive acidic patches which may be concernedwith the property of 0 70 to readily interact with nonspecific and specific sur·faces. The roles of the characteristic spacer region in the strong adhesive
nature of 0 70 were examined.

H. STRUCTURAL BIOLOGY CENTER139

An rpoD-disrupted strain was constructed to test the viability of strains
expressing plasmid-borne mutated rpoDor another sigma factor. E. coli rpoS
and M. tubeculosis sigA failed to complement the disruption, and we are now
testing B. subtilis sigA and others. The mutant 0 70 lacking the spacer regionof aa130-374 ( f:,. SR) complemented the disruption at 30 and 25°C, provingthat the region is not essential for growth allow temperature. At all tested
temperature this protein predominantly exists as oligomers which are in equilibrium with a small fraction of monomer. Both forms bind to core enzyme but
the holoenzyme formed with oligomers had 15% or less activity of the wildtype. Therefore, the role of the spacer region is the maintenance of active
monomeric form.
The protein footprinting analysis of the f:,.SR with a kinase-tag fused at the
C-terminus, which also complements the disruption, indicated that at least
region 4 and C-terminus are involved in oligomerization. According to an
atomic force microscopic observation, the oligomer forms fibers as previously
reported for wild-type 0 70 at 60°C and RpoD800 at 46°C.

(3) Single-Molecule Dynamics of Transcription: Sliding ofproteins along DNA
Takashi KINEBUCHI 1, Hiroyuki KABATA12, Minoru TAKEUCHI 1, Nobuo
SHIMAMOT0 1 , Osamu KUROSAWA 2,3, Hironori ARAMAKI4, Masao WASHIZU 2(lStructural Biology Center, National Institute of Genetics, 2Department of
Mechanical Engineering, Kyoto University, 3Advance Co., 4Department ofMolecular and Life Science, Daiichi College of Pharmaceutical Science)
We have showed the existence of a sliding motion of protein along DNA
through direct 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 ADNA, one
was its cognate operator cloned in A DNA, and the other two are likely to behomologous to the operator. All trapping occurred at specific sites, and only140

sliding complexes were observed at non-specific sites. This observation indicates that 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. RNApolymerase slides out of its specific site into nonspecific sites and then dissociate from nonspecific sites into bulk. This two-step dissociation was notobserved in 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 specificsite, and thus long nonspecific DNA segment flanking the specific site acceler·
ate association 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. coHTrpR 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 of TrpR 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
effect will be obtained.

Publications
1.

2.

3.

SHIMAMOTO. N .• KAsClUKOVlCH. T.. NAGAI, H. and HAYWARD, RS.:

Efficient solubilizationof proteins overproduced as inclusion bodies by use of an extreme concentration ofglycerol. Tech. Tips Online t01576. 1998.
SEN. R., NAGAI. R., HERNANDEZ, v.J. and SHIMAMOTO, N.: Reduction in abortive transcription from the IPR promoter by mutations in region 3 of the a 70 subunit of E. coli
RNA polymerase. J. BioI. Chern. 273, 9872-9877, 1998.
NAGAI. H. and SHIMAMOTO. N.: Most conserved regions of the E. coli primary sigma
factor are involved in interaction with RNA polymerase core enzyme. Gene. Cells 2.
725-734. 1998.

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H-c. Multicellular Organization Laboratory
Fluoride-resistant Mutants of the
Nematode Caenorhabditls eJegans(t)

Masaya TAKE.UCHI, Akane OISHI, Takeshi ISHIHARA and Isao KATSURA
Fluoride-resistant (fir) mutations of C elegans are recessive and grouped
into two categories: class 1 mutations (strongly resistant mutations in fir-I,
fir-3 and fir-4) and class 2 mutations (weakly resistant mutations in fir-2 andfir-S) (Katsura 1. et a1. (1994) Genetics 136, 145-154). From the results presented below, we think class 1 fir genes constitute an intestinal regulatory
system that responds to fluoride ion and that controls various functions, including neural functions.
Besides resistance to fluoride ion, class 1 fir mutations show many phenotypes: slow growth, short defecation cycle periods, frequent skips of the expulsion step of defecation, and synthetic abnormality in dauer formation (See (2)below). fir-I encodes an ion channel belonging to the DEGIENaC (C. elegans
degenerins and mammalian amiloride-sensitive epithelial sodium ion channels) superfamily, while fir-4 codes for a novel SerlThr protein kinase having ahydrophobic domain on the carboxyl-terminal side. A genomic DNA fragmentthat rescues fir-3mutations produces a polycistronic mRNA encoding a kinase-like protein and a novel protein. A fir-I::GFP fusion gene is expressed
only in the intestinal cells from the comma stage of embryos to the adultstage. A fir-4::GFP fusion gene is expressed in the intestinal cells from the
1.5-fold stage, in the isthmus of the pharynx from the 3-fold stage and in a pairof head neurons calledAUAfrom L1larvae to adults. In these fusion genes,
GFP cDNA is connected in frame to the 3' end of the coding region of the fir
genes. Since they can rescue the corresponding mutant phenotypes, the expression at the site and time mentioned above should be sufficient for the
animal to show wild-type phenotypes.
Class 2 fir mutations interact with class 1 mutations. Of class 1 phenotypes, class 2 mutations suppress slow growth and synthetic abnormality indauer formation, but do not suppress strong resistance to fluoride ion or abnormalities in defecation cycles.

142

In our studies last year, the following results were obtained.
(i) We introduced a nonsense mutation into each ORF in the genomic DNA
fragment that rescues the flr-3mutant phenotypes. By checking the rescue
activity of the mutant DNA fragments, we concluded that flr-3encodes a kinase-like protein.
(ii) We killed ADA neurons in wild-type and flr-4 animals. These animals
had the same defecation cycle periods and growth rate as those which suffered
no cell-killing. Hence, we concluded that ADA neurons are not required forthese phenotypes.
(iii) Fluoride ion increased the defecation cycle periods of wild-type animals
but did not change those of class 1 flr mutants. The results suggest thatfluoride ion probably enhances the activity of class 1 flrgene products in wildtype animals and thereby increases their defecation cycle periods, eventuallycausing their death.
(iv) Mutations in flr-4 gene consisted of a mutation at a splice-acceptor site,
two missense mutations in the kinase domain, and a missense mutation in
the C-terminal hydrophobic domain. All of them had the phenotypes of strong
fluoride-resistance and defecation cycle abnormalities. The two mutationsin the kinase domain, which were temperature-sensitive, had almost normal
growth rates and showed low penetrance in synthetic dauer abnormality. The
mutation in the hydrophobic domain was special in that its abnormality in the
expulsion step of defecation was suppressed by fluoride ion.
(v) The phenotypes of flr-3 mutants were suppressed by multiple copies of thewild-type flr-4 gene. The results can be explained, if FLR-3 is an activator for
FLR-4. FLR-3 has a weak but overall homology to FLR-4. This may be relevant, if FLR-3 interacts with FLR-4 directly.
(vi) We cloned flr-2 gene by the rescue method and found that it encodes a
homolog ofa TGF- /3 antagonist belonging to the gremlinlDAN/cerberus family. Twomutations in flr-2were missense mutations at conserved amino acids. Since
there are four TGF· /3 homologs in the C elegans genome, we are determining
which is the one that interacts with FLR-2.

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Analysis of Synthetic Dauer-constitutive Mutations

Kouji MIYAHARA, Takeshi ISHIHARA and Isao KATSURA
If newly hatched larvae of C. elegans are in a crowded state and given alimited food supply, they sense the environmental signal of pheromone andfood 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 of the environmental conditions. The synthetic nature ofthe phenotype, we think, is based on
the structure of the neural circuit, in which neural signals are transmitted
through parallel routes consisting of different types of neurons so that two
mutations may be required to block the signals. We are studying the combinations of mutations for the Sdf-c phenotype and the pattern of suppression of
the Sdf-c phenotype by various suppressor mutations. In this way we hope todetermine the functional neural network for dauer regulatory signals and thefunction of relevant genes in the network.
Furthermore, we isolated and mapped 44 new mutations that show the Sdfc phenotype in combinat~onwith the unc-31(e169) mutation. Many of them are
expected to cause defects in ASI neurons, since the unc-31(e169) mutant shows
dauer-constitutive phenotype, ifASI neurons are killed. Eight of the mutations mapped in 4 known genes (tax-2, osm-6, che-ll, and aex-3), but most of
the remaining 36 mutations, which map in at least 13 genes, seem to bealleles of novel genes.
Of these mutations, we investigated those in sdf-l (1 allele) and sdf-13(2alleles) in detail. It was known that the sdf-l mutant avoids benzaldehyde,
which is an attractant of wild-type animals. This year we found that it also
avoids isoamylalcohol, butanone, NaCI, and lysine, all of which are attractants ofwild-type animals. It behaved as thermophilic in thermotaxis. However,it showed normal behavior to diacetyl, pyrazine, thiazole, and high osmoticpressure, and avoided octanol more strongly than wild-type animals. In con-144

trast, the sdf-13 mutants showed normal chemotaxis to benzaldehyde, isoamyl
alcohol and diacetyl. However, they showed almost no adaptation to the former
two attractants despite showing normal adaptation to the latter. Thus, we
could obtain various behavioral mutants by isolating Sdf-c mutants. We cloned
sdf-13 gene by the rescue method and found that it encodes a homolog of
mammalian Tbx2 and Drosophila Omb, a transcription factor containing the
T-box domain.

(3)

Analysis of che-2, a gene required for thesensory cilia in C. eJegans

Manabi FUJIWARA, Takeshi ISHIHARA and Isao KATSURA
Mutants in the C. elegans che-2 gene have extremely short cilia with anabnormal posterior projection in many sensory neurons, and show defects inbehaviors that are mediated by ciliated sensory neurons, such as chemotaxis
and osmotic avoidance. We cloned che-2gene and found that it encodes a newmember ofthe WD40 protein family. Analysis of mutation sites showed thatboth the N-terminal WD40 repeats and the C-terminal non-WD40 domain arenecessary for the CHE-2 function. CHE-2-tagged green fluorescent protein,which can rescue the mutant phenotypes, is localized at the cilia of almost all
the ciliated sensory neurons (except AFD and BAG). Expression of che-2in a
subset of sensory neurons in a che-2 mutant by using a heterologous promoter
resulted in restoration of the functions and cilium morphology of only the che2-expressing neurons. Thus, che-2 acts cell-autonomously. This technique
can be used in the future for determining the function of each type of che-2expressing sensory neurons. Using a green fluorescent protein as a marker,
we found that the extension of cilia in wild type animals took place at the lateembryonic stage, whereas the cilia of che-2mutant animals remained always
short during development. Hence, the abnormal posterior projection is madedue to the inability of cilium extension, rather than the degeneration of cilia
once formed correctly. Expression of che-2 in a che-2 mutant under a heatshock promoter showed that the extension of cilia, surprisingly, can occur evenat the adult stage and that such cilia can function in behavior. However,some cilia made at the adult stage had an abnormal curved shape, which

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suggests that these cilia may not pass through the socket cell, unlike thosemade in normal development.

(4)

Functional Analysis of Interneurons as Studied by
Selection between Two Behaviors

Takeshi ISHIHARA and Isao KATSURAThe nematode C eIegans shows avoidance from copper ion and chemotaxis
to odorants by perceiving them with different sensory neurons in the head. We
devised a behavioral assay method for the interaction between the two responses to elucidate 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 present knowledge on the neural circuitry of C eIegans and theidentity of the sensory neurons that act in these behaviors.
This assay method was used for the analysis of mutants in an AMPA-typeglutamate receptor gene (gIr-1), which is expressed only in interneurons. They
showed a preference for odorants, as compared with wild-type animals. However,
if each behavior was assayed separately, the dose response curves of gIr-]
mutants were the same as those of wild-type animals. The results suggestthat GLR-l plays a role in the interaction between the two responses.
While these experiments were performed with well-fed animals, animals
starved for 5 hours showed a stronger preference for odorants, because they
responded more weakly to copper ion than well-fed animals. This starvation
effect was not detected in the presence of serotonin, which is considered toinduce a well-fed state. The behavioral change upon starvation seems reasonable, because starved worms can look for food over a wider area.
To elucidate the mechanism of selection between two behaviors and that of
the effect of starvation, we are isolating and analyzing mutants that show
abnormality in this selection. The mutant ut236 had a tendency to choose
avoidance from copper ion rather than chemotaxis to odorants, although it
showed no abnormality in each behavior. On the other hand, the mutant
ut235 lacked the effect of starvation in the above assay, but was essentially146

normal in the change oflocomotion speed by starvation and in the response to
food. Namely, this mutant has defects only in part ofthe behavioral responses
to starvation. The double-mutant ut235; ut236showed a preference to avoidance from copper ion, regardless of starvation. To identify the gene responsible for the ut236mutation, we are now attempting the rescue ofthe phenotype by microinjection of cosmid clones from the 1 Mb region where the mutation was mapped.
We also isolated new mutants from EMS (ethylmethane sulfonate)-treatedworms and worms containing movable transposons (mut-7worms). Some ofthem had a preference for chemotaxis to odorants over avoidance to copper ion,while others had little starvation effect. We are cloning the genes of the mut7mutants by the transposon-tagging method.

(5)

Reverse Genetics of Genes Containing PDZ domains

Ryuichi HISHIDA, Takeshi ISHIHARA and Isao KATSURAPDZ domains are thought to act in protein-protein interaction, often forming
clusters of membrane-bound receptors, ion channels, etc., and localizing thesemolecules to a specific site on the membrane. We searched genes that encode
proteins containing PDZ domains, in the genomic sequence of C. elegans andinvestigated the site and time oftheir expression by using GFP fusion genes. Twoofthe genes, which we are interested in, were subjected to gene disruption. Oneof them encoded a protein having a MDMl(one of intermediate filaments)-likesequence on the C-terminal side of PDZ and was expressed in many neurons
and the excretory cells as well as at the edge ofthe vulva, from larvae to adults. The
protein exists as granules in cytoplasm, often flanking nuclei on the anteroposterior axis. We isolated a mutant in which the MDMl-like sequence wasdeleted. However, it showed no clear phenotype. The other gene encoded a
MAGI-l homolog, which contained a guanylate kinase-like sequence, two WW
domains and five PDZ domains. Its GFP fusion gene was expressed in theintestine from embryos to larvae, in lateral hypodermis from embryos to adults,
in some head neurons, the anus, ventral hypodermis, the uterus, the spermatheca and the distal tip cells of the gonad from larvae to adults. Althougha mutant lacking the guanylate kinase-like sequence had no conspicuous phe-

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notype, overexpression of the gene by multicopy transformation resulted in
low-penetrance morphological abnormality in the lateral seam cells.

Publications
TAKE-ueHI, M., KAWAKAMI, M., ISHIHARA, T., AMANa, T., KONDO, K. and KATSURA, I.:1.

An

ion channel ofthe degenerin/epithelial sodium channel superfamily controls the defecation rhythm in C. elegans. Proc. Nat!. Acad. Sci. USA 95, 11775-11780, 1998.
2.

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

Characterization of a Caenorhabditis elegans recA-like gene Ce-rdh-} involved inmeiotic recombination. DNA Res. 5, 373-377, 1998.

H-d. Biomolecular Structure Laboratory(1)

Crystallographic Study of F1-ATPase: Structural Analysis ofSupramolecule

Yasuo SHIRAKIHARA
F1-ATPase, with a subunit composition of a 3 /3 3 r (] E , is a catalytic sectorof the membrane bound ATP synthase. The ATP synthase plays a central rolein energy conversion in mitochondria, chloroplasts and bacteria, generating
ATP from ADP and inorganic phosphate using energy derived from a transmembrane electro-chemical potential. We previously solved the structure of
the a 3 /3 3 sub-assembly of F1-ATPase from a thermophilic bacterium Bacillus PS3. We are extending the structural study to a nucleotide-bound form of
the a 3/13 sub-assembly and to an a 3/13 r sub-assembly.
We have examined structure of the nucleotide-soaked crystal of the a3/33
sub-assembly, hopefully assuming the structure in it to represent an nucleotide-bound form. Diffraction data from the soaked crystals were collected
before they cracked eventually, as the aJ/33 sub-assembly is destabilized bypresence of nucleotides. 12 different soaking conditions were tried. In the
ATP and MgADP soaked crystals, the a 3 /3 3 sub-assembly were placed withorientations different from that ofthe nucleotide-free sub-assembly. However
the structures themselves in these soaks were very similar to the structure of
the nucleotide-free form. Close inspection of electron density map revealed

148

that the density for the sulfate bound to the P-Ioop (phosphate binding loop) of
the {3 -subunit got significantly weaker in comparison with the equivalentdensity in the nucleotide-free crystal. In contrast, the density for the sulfate
bound to the a -subunit's P-Ioop looked very similar between nucleotide-soaked
and nucleotide-free forms. These features were commonly observed for all
12 maps except for Pi soaked one. It is likely that some transient state
between nucleotide-free and nucleotide-bound form has been captured in thisexperiment.
As the a 3/3 3 r sub-assembly exhibits kinetic properties very similar to
those of F1, and a number of interesting mutant sub-assemblies have been
engineered, the sub-assembly is thought to be a good target for structural
study. We have searched crystallization conditions for the wild type subassembly, but have experienced extreme difficulty. Both preparation method
and crystallization conditions have been examined extensively. Among various trials to improve preparations, heat treatment and ammonium sulfate
fractionation were very effective, but for example, additional columns (dyeligand columns and gel filtration column) and control in bound nucleotides
were only marginally effective. The following points have been noticed during
crystallization experiments. As a binding ligand, ATP r S was very useful,and so were CDTA,DTT,methanol as general additives. Crystals were easily
formed at 15 degree, but they were unstable when transferred to 25 degree
environment. It was possible to grow crystals at 25 degree, but only in different crystallization conditions. Crystals at the current growing conditions
seem now ready for a diffraction study, judged from their sizes.
These structure studies were done in collaboration with Masasuke Yoshida,
Toyoki Amano, Eiro Muneyuki, Yasuyuki Kato and Satoshi Tsunoda at Research Laboratory of Resource Utilization, Tokyo Institute of technology.
(2)

Crystallographic Study of the Transcription Activator, PhoB

Toshihiko AKIBA and Yasuo SHIRAKIHARA
PhoB Protein is a positive transcriptional activator for the genes in thephosphate (Pho) regulon of E. coli; such as phoA and pstB, that are induced byphosphate starvation. PhoB binds to the phD box in the promoter region,

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which is the consensus sequence shared by the regulatory regions of phoA,
phoB, phoE and Psts. PhoB is activated by phosphorylation by PhoR. The
N terminal domain ofPhoB is responsible for this regulatory role, whereas the
C terminal domain has a DNA binding ability.
From particular interest to interactive regulatory mechanisms between theN- and the C- domains, crystallization of an intact form of the protein has
been attempted. After establishing overproducing conditions and purification procedure as described previously, crystallization conditions for
unphosphorylated form were screened employing Hampton crystal screen, but
so far without success. In an attempt to crystallize a phosphorylated from,conditions to obtain the phosphorylated form were examined. A problem wasthat in the presence of acetylphosphate, which is an phosphate donor, part of
PhoB population forms a dimer. Efforts has been made to overcome this problem.
These works have been done in collaboration with Kozo Makino, ResearchInstitute for Microbial Diseases, Osaka University.

(3)

Crystallographic Study of the Transcription Repressor, CamR

Koji FUKUSHI and Yasuo SHIRAKIHARA
CamR protein is a repressor that regulates transcription of the cytochrome
P-450cam hydroxylase operon of Pseudomonas putida. Expression of the
camDCAB operon and the camR gene is regulated through interaction of the
CamR protein with the single operator located in the overlapping promoter
region between the camDCAB operon and the camR gene. D-camphor is aninducer, and binds cooperatively to CamR. CamR is a homodimer with amolecular mass of 40 kDa.
Two crystal forms have been obtained, one from polyethyleneglycol solution
in the presence or absence ofD-camphor and the other from Na-K-phosphate
solution in the presence ofD-camphor. By switching of the E.coli over-expression system from a P L promoter system to a more efficient T7 (pET system)
promoter system, and by minor modifications in purification procedure, we
eliminated a problem ofpoor reproducibility in crystallization. Although crystal forms had similar unit cell parameters and diffracted to better than 2.5A

150

resolution, they were very sensitive to temperature change, thus prohibiting
full data collection. Further efforts have been made to get rid ofthe problem.
This work has been done in collaboration with Hironori Aramaki, Daiichi
College of Pharmaceutical Sciences.

(4)

Crystallization of RNA Polymerase and Its subunits

Katsuhiko MURAKAMI, Makoto KIMURA, Akira ISHIHAMA and Yasuo
SHIRAKIHARAThe Escherichia coli RNA polymerase (RNAP) is a large assembly (a molecular weight for the core enzyme ( a 2/3/3' ) is 380 kD and that for the holo
enzyme (a2/3/3' a 7°)is 450kD). a subunit is included as a dimer into RNAP.
The amino (N)-terminal domain from residue 20 to 235 is responsible for the
enzyme assembly through the interactions with /3 and /3' subunits and thecarboxyl (C)-terminal domain of 94 residues in length is important for transcription regulation. The solution structure of the C-terminal domain is already resolved by NMR study.
For the purpose of analysis the RNAP assembly mechanism, we have tried
to crystallize a subunit. As reported previously, PEG400/CaCI2 and PEG8000/
NaAcetate gave thin-plate crystals. However, the crystals were too thin togive diffraction and further efforts have been made this year to get thicker
crystals, but so far without success.
For crystallization of RNAP, an overproducing system of the core enzyme
has been constructed(BL21(DE3)/pGEMABC), allowing 5-fold yield increase
and therefore concomitant impurity decrease. Crystallization conditions forthe core and the holo enzymes were screened employing Hampton Crystal
Screen Kit, but with no sign of crystalline materials formed. A subsequent
Dynamic light scattering study showed that both forms of the enzyme exhib·
ited a complicated pattern of aggregation depending on pH, Mg concentration,
NaCI concentration, and species of counter ion in the Tris buffer. This led us
to explore crystallization conditions around least aggregating conditions. The
experiment is under way.
Pol II from yeast is a larger and more complicated assembly, comprising 12
subunits. We tried to crystallize Rpbll, which is a homologue of the E.coli aH.

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subunit, and Rpb6. These were selected on the basis of availability of overproduction system. From dynamic light scattering and gel filtration experiments, Rpbll was found to be polydisperse with respect to aggregation state
and thus not suitable for crystallization experiment. Rpb6, with no suchapparent difficulty for crystallization, gave no crystals so far.

(5)

Crystallization of Mret t fragment from yeast

Katsuhiko MURAKAMI, Tsutomu OHTA, Tomoko OGAWA and Yasuo SHIRAKIHARA
Mrell is involved in the recombination process in yeast. We tried to crystallize a 70 amino acid fragment of Mrell, which is capable of binding DNA,
however, was overproduced as a GST fusion protein in E.coli. It was anticipated that the fusion protein may be easy to crystallize due to crystallizing
capability of the GST part, in view of the relative size of the fragment to theremaining GST part. However actual crystallization experiments failed, andwe think that it is better to deal with the fragment itself.

(6)

Crystallization of Na+-translocating ATPase

Yasuo SHIRAKIHARA
Na+-translocating ATPase from Enterococcus hirae is classified as a Vacuolar-type ATPase (V-type), and is expected to have a structure similar to FlATPase (F-type ATPase) from amino acid sequence comparison. Na+-translocating ATPase was highly purified and was subjected to crystallization
experiment. Plate shaped crystals with largest dimension of 0.2mm wasproduced from polyethylene glycol solution, and was found to diffract to 6 A
resolution. Preparation methods are being improved to get better crystals.
This work has been done in collaboration with Kakunori Tanaka and Ichiro
Yamato, Science University of Tokyo.

Publication
None

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I-a. Laboratory for DNA Data Analysis(1)

Different evolutionary histories In two subgenomlc regions of the
Major Histocompatiblllty Complex

Silvana GAUDIERI, Jerzy K. KULSKI 1, Roger L. DAWKINS I and Takashi GOJOBORI(lCentre for Molecular Immunology and Instrumentation, University of Western Australia)
The alpha and beta blocks, contain members ofthe multicopy gene families
HLA Class I, human endogenous retroviral sequence-16 (HERV-16) (previously known as P5 and PERB3), hemochromatosis candidate genes (RCG) (II,IV, VIII, IX), 3.8-1 and MIC (PERBll). In this study, we show that the two
blocks consist of imperfect duplicated segments, which contain linked members of the different gene families. The duplication and truncation sites ofthe segments are associated with retroelements. The retroelement sites
appear to generate the imperfect duplications, indels and rearrangements,
most likely via homologous recombination. Although the two blocks share
several characteristics, they differ in the number and orientation ofthe duplicated segments. On the 62.1 haplotype, the alpha block consists of at least
10 duplicated segments that predominantly contain pseudogenes and genefragments of the HLA Class I and MIC (PERBll) gene families. In contrast,
the beta block has two major duplications containing the genes HLA-B and -C,
and MICA (pERBll.1) and MICB (pERB11.2). Given the common origin between the blocks, we reconstructed the duplication history of the segments tounderstand the processes involved in producing the different organisation in
the two blocks. We then found that the beta block contains four distinct
duplications from two separate events, whereas the alpha block is characterisedby multi-segment duplications. We will discuss these results in relation to
the genetic content of the two blocks. For details, see ref. 1.

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Coevolution of PERDU (MIC) and ULA Class I Genes with UERV-16
and Retroelements by Extended Genomic Duplication

Jerzy K. KULSKI I, Silvana GAUDIERI, A. MARTINI and Roger L. DAWKINS· (ICentre for Molecular Immunology and Instrumentation, University of Western
Australia)
The recent availability of genomic sequence information for the class I region of the MHC has provided an opportunity to examine the genomic
organisation ofHLA class I (HLAcI) and PERBlllMIC genes with a view to
explaining their evolution from the perspective of extended genomic duplications rather than by simple gene duplications and/or gene conversion events.
Analysis of genomic sequence from two different regions of the MHC (thealpha- and beta-block) revealed that at least six PERBll and 14 HLAcI genes,
pseudogenes and gene fragments are contained within extended duplicatedsegments. Each segment was searched for the presence of shared (paralogous)
retroelements by Repeat Masker in order to use them as markers of evolution,
genetic rearrangements and evidence of segmental duplications. Shared Alu
elements and other retroelements allowed the duplicated segments to be classified into 5 distinct groups (A to E) that could be further distilled down to anancient preduplication segment containing a HLA and PERBll gene, an endogenous retrovirus (HERV-16) and distinctive retroelements. The
breakpoints within and between the different HLAcI segments were foundmainly within the PERBll and HLA genes, HERV-16 and other retroelementssuggesting that the latter have played a major role in duplication and indel
events leading to the present organisation ofPERBll and HLAcI genes. On
the basis of the features contained within the segments, a coevolutionary model
premised on tandem duplication of single and multipartite genomic segments
is proposed. The model is used to explain the origins and genomic organisation
ofretroelements, HERV-16, DNA transposons, PERBll and HLAcI genes as
distinct segmental combinations within the alpha- and beta-blocks of thehuman MHC. For details, see refs. 2 and 3.

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(3)

Extensive nucleotide variability within a 370 kb sequence from thecentral region of the Major Histocompatibility Complex

Silvana GAUDIERI, Jerzy K. KULSKI 1, Roger L. DAWKINS! and Takashi GOJOBORI(ICentre for Molecular Immunology and Instrumentation, University of Western Australia)
The recent availability of the genomic sequence spanning the central andtelomeric end of the major histocompatibility complex (MHC) has allowed adetailed study of its organisation, gene content and level of nucleotide
variability. Previous analyses of nucleotide variability in the MHC has focused on the coding regions of the human leukocyte antigen (HLA) Class I and
II genes. Non-coding nucleotide variability has been considered a by-productof exonic diversity. However, with the advent of genomic sequencing, the extent of non-coding nucleotide variability within the MHC has just begun to beappreciated. In this study, we compared different human haplotypes in 370
kb of sequence in the central region of the MHC to show the following: 1)
unusually high levels of non-coding nucleotide variability, up to 80 times greaterthan elsewhere in the genome; 2) non-coding nucleotide variability greaterthan 1% at nucleotide sites distant to the Class I genes; 3) nucleotide variability greater than 1% maintained over regions containing highly linked loci; and
4) distinct troughs and peaks in the level of nucleotide variability. We will
discuss these observations in relation to a possible role of nucleotide variability in the organisation of the MHC. For details, see refs. 4 and 5.

(4)

Elevated levels of non-coding polymorphism within the
Major Histocompatibility Complex

Silvana GAUDIERI, Roger DAWKINS!, Kaori HABARA and Takashi GoJOBORI (!Centre for Molecular Immunology and Instrumentation, University of Western
Australia)
The Human Major Histocompatibility Complex (MHC) is characterised bypolymorphic multicopy gene families including the Class I and II Human Leucocyte Antigens (HLA), rearrangements, insertions and deletions (indels), anduneven rates of recombination. Within the HLA Class I and II genes, positive

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selection has been shown to operate on the peptide binding region, with polymorphism at linked loci attributed to balancing selection. Recent evidence
suggests that non-coding polymorphism is unusually high within the MHC, up
to fifty times greater than elsewhere in the genome. To examine the relationship between HLA and non-coding polymorphism, we analysed continuous sequence from different human haplotypes up to 200kb distant from the HLA
genes. We report here the most extensive analysis of polymorphism within
the genome. We show that the MHC exhibits large regions of high polymorphism (> 1%) that contain linked loci. Polymorphism peaks do not necessarily coincide with the HLA genes and in some cases occur within human endogenous retroviral sequences (HERVs). Polymorphism >1% appears to be negatively correlated to recombination rate within the MHC. These results support the hypothesis that the MHC consists of polymorphic frozen blocks, where
recombination rarely, if at all, occurs within the block due to the inhibition ofrecombination by high levels of polymorphism. For details, see ref. 6.

(5)

Characterisation of the PERBll gene family in Primates

S. K. CATTLEy l , N. LONGMAN l , Silnava GAUDIERI, Roger L. DAWKINS l and C.
LEELAYUWAT l (lCentre for Molecular Immunology and Instrumentation, University of Western Australia)
PERB 11 (MIC) is a member of the Class I-like superfamily in the MHC
region. The PERB11 gene family has 5 members in the human genome, two
are transcribed and polymorphic (PERB11.1 and PERB11.2). These genes
have been implicated in autoimmune diseases (Behcet's disease, Psoriasis.
Chimpanzees appear to have fewer copies of PERB11 based on southernhybridisation analysis. Given the low prevalence of autoimmune diseases inchimpanzees, an examination of the number and position ofPERB11 members in primates (esp chimpanzee) will be used to evaluate PERB11 as adisease candidate gene in this region. PERB11.1 and PERB11.2 specific primers (designed from human copies) were used to amplify 7 chimpanzees, 1 orangutan and 6 macaques, to examine the level of inter- and intraspecies
differences. The resultant PCR products from four different PCR reactions
have been cloned. For details, see ref7.

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(6)

Identification of Neural pathways in Planarian

Akira TAZAKI 1, Silvana GAUDIERI, Kazuho IKEO, Takashi GOJOBORI, Kenji.
WATANABE l and Kiyokazu AGATA 1 (ILaboratory of Regeneration Biology, Department of Life Science, Faculty of Science, Himeji Institute of Technology)
The Planarian contains a primitive central nervous system and has beenused as a model to examine the developmental stages within the brain. We
are collaborating with Dr. Agata from the Department of Life Science, HimejiInstitute of Technology, Hyogo. His group is developing the technology of isolating cDNA libraries from single cells. The experimental plan for the brain
involves thousands of individual cell cDNA libraries. These libraries will becompared for expression levels in different developmental stages. This planrequires a great deal of computer automation from sequence output to BLAST
searches and database construction. To facilitate the construction of the automated analysis, we have started a preliminary project using the eye cell of
the planarian. The initial project will involve 1000 cDNA sequences. We
have already sequenced the 1000 cDNA inserts and are currently forming
databases ofthe completed sequences. Interesting sequences will be further
analysed for important motifs, domains. The preliminary project will provide new data on sensory developmental genes, organ specific transcripts andother interesting new sequences. For details, see ref. 8.

(7)

A low rate of nucleotide changes in Escherichia coli K-t!
estimated from a comparison of the genome sequences
between two different substrains

Takeshi ITOH and Takashi GOJOBORIThe molecular mechanisms of mutation in bacteria have been extensively
studied. However, there is a paucity of information on the evolutionary rates
of bacteria when compared with that of other organisms such as mammals
and viruses. Recently, two genomes of Escherichia coliK-12 have been independently sequenced by Japanese and American teams. These two closely
related genomes gave us a unique opportunity to investigate the evolutionary
rates of E. coliby directly comparing the genome sequences.

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In this study, we attempted to estimate the rate of nucleotide changes by adirect comparison of the DNA sequences of two mutant derivatives of E. coliK12, W3110 and MG 1655. According to laboratory history records, thesesubstrains originated from W1485 approximately 40 years ago. Of course,
W1485 is also the derivative of the K-12 wild type. Only a few differenceswere found between the two genomes in our comparative analysis. Whenusing 40 years as the divergence time between W3110 and MG1655, we estimated the rate of nucleotide changes to be at most about 10- 7 per site per year.
The rate of nucleotide changes estimated herein is relatively low compared tothose of viruses. We also compared partial genome sequences between E. coliand Shigella fJexneri. The divergence time between these two enteric bacteria has been supposed to be 25 million years ago. We then estimated the rateof nucleotide changes to be about 10-9 per site per year, which is consistent withthe rate of nucleotide changes that we have estimated between the two E. coli
substrains.
Of all the indels (insertions or deletions), except insertion sequences (ISs)and one prophage, 98.3% were indels of eight- or fewer nucleotides and 93.0%
were indels of a single nucleotide. We found 10 long indels of about one thousand or more nucleotides in length, which are mainly caused by ISs. Eight ISs
were found only in the W3110 genome, while one IS and one prophage were
found only in MG 1655. Since the sequence examined herein is approximately
45% of the entire genorp.e and nine large indels by ISs were found in that
region, the total number oflarge indels by ISs between the two substrains isestimated to be 20. Given that the divergence time is 40 years, this indicatesthat an E. coli genome has experienced IS-derived indel events at the rate of
0.25 per year. It is of particular interest to know that E. coli has acceptedsuch drastic alterations frequently, on average once in four years. As has
been reported, it is likely that ISs play an important role in genome evolution,
because they provide homologous regions large enough to mediate recombination events. In fact, recent sequencing of a 93-kb plasmid p0157, which was
extracted from E. coliOI57:H7, revealed that p0157 carried 18 IS elements. It
also revealed that p0157 has been extensively subjected to rearrangements ofthe plasmid genome. Furthermore, in the E. coliK-12 chromosomes, we foundthat ISs have been actively transposed and have been interspersed in the

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genome during short-term evolution. Therefore, the major changes ofthe genome structures may be frequently caused by IS-mediated recombination
events. For details, see ref. 28.

(8)

Slow Evolutionary Rate of GO Virus C/Hepatltis G Virus

Yoshiyuki SUZUKI, Kazuhiko KATAYAMA I , Shuetsu FUKUSHI 1, Tsutomu
KAGEYAMA 1, Akira OYA 1, Hirofumi o KAMURA2, Yasuhito TANAKA3, Masashi
MIZOKAMI 3, Takashi GOJOBORI (lBasic Research Division, BioMedical Laboratories, Inc., 2Third Department oflnternal Medicine, Kyorin University Schoolof Medicine, 3Second Department of Medicine, Nagoya City University Medical School)
With the aim of elucidating evolutionary features of GB virus C/hepatitis G
virus (GBV-C/HGV), molecular evolutionary analyses were conducted using
the entire coding region of this virus. In particular, the rate of nucleotide
substitution for this virus was estimated to be less than 9.0 x 10-6 per site per
year, which was much slower than those for other RNA viruses. The phylogenetic tree reconstructed for GBV-C/HGV, by using GB virus A (GBV-A) as
outgroup, indicated that there were three major clusters (the HG, GB, andAsian types) in GBV-CIHGY, and the divergence between the ancestor of GB
and Asian type strains and that ofHG type strains first took place more than
7,000-10,000 years ago. The slow evolutionary rate for GBV-C/HGV suggested that this virus cannot escape from the immune response ofthe host bymeans of producing escape mutants, implying that it may have evolved othersystems for persistent infection.

(9)

Intragenic Recombinations in Rotaviruses

Yoshiyuki SUZUKI, Takashi GOJOBORI, Osamu NAKAGOMI 1 (lDepartment ofMicrobiology, Akita University School of Medicine)Groups A to F rotaviruses are members of the genus Rota virus in the family
Reoviridae. Among these viral groups, groups A, B, and C are known to infecthumans, and, moreover, group Arotavirus is one ofthe most important aetiologicalagents of gastroenteritis in infants worldwide. Nucleotide substitution andI.

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gene reassortment have been reported to be the major mechanisms ofproducing antigenic variants for rotavirus strains; in particular, recombination has
been reported to occur in the "all or none" fashion. In the present study, evidence for intragenic recombination in the VP7 gene between rotavirus strains
bearing different serotypes is demonstrated for the first time. To identify theborder ofthe recombination, four sequences were used for analysis; one putative recombinant sequence, one representative sequence from each ofthe twoparental lineages considered to be involved in the recombination, and one
sequence from known outgroup. An alignment was made ofthe four sequences,and the informative sites were collected along the alignment. The informative site supported either of the three phylogenetic relationships; the clustering ofthe recombinant with one ofthe two parental sequences, with the otherparental sequence, and with the outgroup sequence. The relative frequenciesof the former two types of sites were compared between the regions before andafter each breakpoint along the alignment, using the Fisher's exact test. If
the value was statistically significant at some breakpoints, the optimum position of the recombination was determined as the point which minimize thatvalue. Intragenic recombination may be one ofthe escaping mechanisms fromthe host immune system for rotavirus. This process involves exchanging antigenic regions, and thus questioning the use of multivalent vaccines for theprevention of rotavirus infection. For details, see ref. 24.

Pub I i ca t ions
1.

2.

3.
4.
5.

GAUDIERI, S., KULSKI, J.K., DAWKINS, R.L. and GOJOBORI, T.: The generation and dispersion of multicopy gene families in the MHC by imperfect segmental duplications.
Genome Res. (in press)
KULSKI, J.K., GAUDIERI, S., MARTIN, A. and DAWKINS, R.L.: Coevolution of PERBll(MIC) and HLA class I genes with HERV-16 and retroelements by extended genomic
duplication. J. MoL EvoL (in press)
KULSK,I J.K., GAUDIERI, S., INOKO, H. and DAWKINS, R.L.: DNA analysis of a HERV-richregion within the major histocompatibility complex. J. MoL EvoL 48, 675-683, 1999.
GAUDIERI, S., KULSKI, J.K., DAWKINS, R.L. and GOJOBORI, T.: Extensive duplications
and polymorphism within the major histocompatibility complex. Gene (in press)
DAWKINS, R.L., LEELAYUWAT, C., GAUDIERI, S., TAY, G., CATTLEY, S., MARTINEZ, P. andKULSKI, J.K.: Genomics of the Major Histocompatibility Complex: Haplotypes,

160

6.
7.

8.

9.

10.

11.

12.

13.
14.
15.
16.

17.

18.

Retroviruses and Disease. Immunological Reviews 167, 275-304, 1999.
GAUDIERI, S., DAWKINS, R., HABARA, K and GOJOBORI, T.: Elevated levels of non-coding
polymorphism within the Major Histocompatibility Complex. (submitted)
CA1'rLEY, S.K, LoNGMAN, N., GAUDIERI, S., DAWKINS, R.L. and LEELAYUWAT, C.: Phylogenetic
analysis of primate PERBll(MIC) sequences suggests that the representation of thegene family differs in different primates. European Journal of Immunogenetics ll6, 233238, 1999.
TAZAKI, A., GAUDIERI, S., IKEo, K, GOJOBORI, T, WATANABE, K, AGATA, K: Neural Network in Planarian revealed by an Antibody against Planarian Synaptotagmin homologue. (submitted)
ENDO, T, IMANISHI, T, GoJOBORI, T and INOKO, H.: Corrigendum to: 'Evolutionary significance of intra-genome duplications on human chromosomes' [Gene ll05 (1997) 19-27].
Gene, ll10, 351-4, 1998.
KATSUYAMA, Y, INOKO, H., IMANISHI, T., MlzuKI, N., GOJOBORI, T and OTA, M.: Genetic
relationships among Japanese, northern Han, Hui, Uygur, Kazakh, Greek, Saudi
Arabian, and Italian populations based on allelic frequencies at four VNTR (DIS80,
D4S43, COL2Al, D17S5) and one STR (ACTBP2) loci. Hum Hered, 48, 126-37, 1998.
Lou, H., LI, H.C., KUWAYAMA, M., YASHIKI, S., FUJIYOSHI, T., SUEHARA, M., OSAME, M.,
YAMASHITA, M., HAYAMI, M., GURTSEVICH, V, BALLAS, M., IMANISHI, T and SONODA, S.: HLAclass I and class II ofthe Nivkhi, an indigenous population carrying HTLV-I in Sakhalin,
Far Eastern Russia. Tissue Antigens, 5ll, 444-51, 1998.
MlZUKI, N., OHNO, S., ANDO, H., SATO, T., IMANISHI, T, GOJOBORI, T., ISHIHARA, M., GoTO,
K, OTA, M., GENG, Z., GENG, L., LI, G. and INOKO, H.: Major histocompatibility
complex class II alleles in an Uygur population in the Silk Route of Northwest China.
Tissue Antigens, 51, 287-92, 1998.
NAKAMURA, Y., GOJOBORI, T and IKEMuRA, T: Codon usage tabulated from the international DNA sequence databases; its status 1999, Nucleic Acids Res. ll7, 292, 1999.
OPHIR, R, ITOH, T., GRAUR, D. and GOJOBORI, T.: A simple method for estimating theintensity of purifying selection in protein-coding genes. Mol. BioI. Evol. 16, 49-53, 1999.
SUGAWARA, H., MIYAZAKI, S., GOJOBORI, T. and TATENo, Y.: DNA Data Bank of Japan
dealing with large-scale data submission. Nucleic Acids Res. ll7, 25-28, 1999.
BELLGARD, M. and GOJOBORI, T: Identification of a ribonuclease H gene in both Mycoplasma genitalium and Mycoplasma pneumoniae by a new method for exhaustive
identification of ORFs in the complete genome sequences, FEBS Letters, 445, 6-8,
1999.
ITOH, T, TAKEMOTO, K, MORI, H. and GOJOBORI, T: Evolutionary instability of operon
structures disclosed by sequence comparisons of complete microbial genomes, Mol.
BioI. Evol., 16, 332-346, 1999.
HORIUCHI, M., YAMAGUCHI, Y, GOJOBORI, T, MOCHIZUKI, M., NAGASAWA, H., TOYODA, Y,
ISHIGURO, N. and SHINAGAWA, M.: Differences in the evolutionary pattern offeline Panleukopenia virus and canine Parvovirus. Virology 249, 440-452, 1998.

I.
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NAKAMURA, Y., GOJOBORI, T and IKEMURA, T.:

161

Codon usage tabulated from the interna-

tional DNA sequence databases. Nucleic Acids Res. 26, 334, 1998.
20.

OKAYAMA, T., TAMURA, T., GOJOBORI, T., TATENO, Y., IKEO, K., MIYAZAKI, S., FUKAMIo
KOBAYASHI, K. and SUGAWARA, H.:

Formal design and implementation of improved

DDBJ DNA database with a new schema and object oriented library. Bioinformatics
14, 472-478, 1998.
21.

PERIERE, G., GOUY, M. and GOJOBORI, T.:

The non-redundant Bacillus subtilis (NRSub)database: update 1998. Nucl. Acids Res. 26, 60-62, 1998.
22.

ROBERTSON, B., MYERS, G., HOWARD, C., BRETTIN, T., BUKH, J., GASCHEN, B., GOJOBORI, T.,
MAERTENS, G., MIZOKAMI, M., NAINAN, 0., NETEsov, S., NISHIKAWA, K., SHlN.I, T., SIMMONDS,
P., SMITH, D., STUYVER, 1. and WEINER, A.:

Classification, nomenclature, and data-base development for hepatitis C virus (HCV) and related viruses: Proposals forstandardization. Arch. Virol. 143, 2493-2503, 1998.
23.

SUZUKI, Y. and GOJOBORI, T:

The origin and evolution of human T-Celllymphotrophic

virus types I and II. Virus Genes 16, 69-84, 1998.
24.

SUZUKI, Y., GOJOBORI, T and NAKAGOMI, 0.:

Intragenic recombinations in rotaviruses.

FEBS Lett. 427, 183-187, 1998.
25.

TANAKA, Y., MIZOKAMI, M., ORITO, E., OHBA, K.-I., KATO, T., KONDO, Y., MBOUDJEKA, I.,
ZEKENG, 1., KAPTUE, L., RIKANDOU, B., M'PELE, P, TAKEHISA, J., HAYAMI, M., SUZUKI, Y.
and GOJOBORI, T.:

African origin ofGB virus Clhepatitis G virus. FEBS Lett. 423, 143-

148, 1998.
26.

TATENO, Y., FUKAMI-KoBAYASHI, K., MIYAZAKI, S., SUAGAWARA, H. and GOJOBORI, T.:DNA

Data Bank of Japan at work on genome sequence data. Nucleic Acids Res. 26, 16-20.
1998.
27.

TSUNOYAMA, K. and GOJOBORI, T.:

Evolution of nicotinic acetylcholine receptor sub-units. Mol. BioI. Evol. Hi, 518-527, 1998.
28.

ITOH, T, OKAYAMA, T., HASHIMOTO, H., TAKEDA, J.-I., DAVIS, R.w., MORI, H. and GoJOBORI,
T:

A low rate of nucleotide changes in Escherichia coli K-12 estimated from a com-

parison of the genome sequences between two different substrains. FEBS Lett. 450,
72-76, 1999.

162

I-b. Laboratory for Gene-Product Informatics(1)

Feasibility of the Inverse protein folding protocol

Motonori OTA and Ken NISHIKAWA
There are two protocols available in the protein structure (3D) - sequence(1D) compatibility method. One of them to recognize the protein fold, called
forward-folding protocol, has been widely used in the structure prediction of
new proteins. However, the other one in which a structure recognizes its homologous sequences among a sequence database, called the inverse-folding
protocol, is a more difficult application. We have investigated the feasibilityof the latter approach. A structural library, composed of about four hundred
well resolved structures with mutually dissimilar sequences, wasprepared. We examined whether each one of proteins in the library could
correctly seek their homologs by both forward- and inverse-folding
protocols. The results showed that the inverse-folding protocol is more effective than the forward-folding protocol, once the reference states ofthe compatibility functions are appropriately adjusted. This adjustment only slightly
affects the ability ofthe forward-folding search. We noticed that the scoring,in which a given sequence is remounted onto a structure according to the 3D1D alignment determined by the dynamic programming method, is only effective in the forward- folding protocol, and not in the inverse-folding
protocol. Namely, the inverse-folding search works significantly better withthe score given by the 3D-1D alignment per se, rather than that obtained by
the remounting. See Ref. 4 for the details.

(!)Genes from nine genomes are separated into their organisms In the
dinucleotide composition space
Hiroshi NAKASHIMA I, Motonori OTA and Ken NISHIKAWA (lThe School of Health
Sciences, Faculty of Medicine, Kanazawa University)
The dinucleotide composition composed of 16 components was used to analyze the protein-encoding DNA sequences in nine complete genomes: Escheri-

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chia coli, Haemophilus influenzae, Helicobacterpylori, Mycoplasma genitalium,
Mycoplasma pneumoniae, Synechocystis sp., Methanococcus Jannaschii,Archaeoglobus fulgidus and Saccharomyces cerevisiae. The dinucleotide composition was significantly different between the organisms. The distributionof genes from an organism was clustered around its center in the dinucleotide
composition space. The genes from closely related organisms such as Gramnegative bacteria, mycoplasma species and eukaryotes showed some overlap
in the space. The genes from nine complete genomes together with thosefrom human were discriminated into respective clusters with 80% accuracy
using the dinucleotide composition alone. The composition data estimatedfrom a whole genome was close to that obtained from genes, indicating thatthe characteristic feature of dinucleotides holds not only for protein coding
regions but also noncoding regions. When a dendrogram was constructedfrom the disposition of the clusters in the dinucleotide space, it resembled thereal phylogenetic tree. Thus, the distinct feature observed in the dinucleotide composition may reflect the phylogenetic relationship of organisms. See
Ref. 1 for the details.

(3)

Constructing the Protein Mutant Database

Takeshi KAWABATA, Motonori OTA and Ken NISHIKAWA
Currently the protein mutant database (PMD) contains over 81,000 mutants, including artificial as well as natural mutants of various proteins extracted from about 10,000 articles. We recently developed a powerful viewing and retrieving system, integrated with the sequence and tertiary structure
databases. The system has the following features: (i) mutated sequences are
displayed after being automatically generated from the information describedin the entry together with the sequence data of wild-type proteinsintegrated. This may be convenient for user because it allows one to see theposition of altered amino acids ( shown in a different color) in the entire se·quence of a wild-type protein; (ii) for those proteins whose 3D structures havebeen experimentally determined, the 3D structure is displayed to show mutation sites; (iii) a sequence homology search against PMD can be carried out
with any query sequence; (iv) a summary of mutations ofhomologous sequences

164

can be displayed, which shows all the mutations at a certain site of a protein,recorded throughout the PMD. See Ref. 2 for the details.

Publ icatians
1.

2.
3.

4.

NAKASHIMA, H., OTA, M., NISHIKAWA, K and 001, T.: Genes from nine genomes are
separated into their organisms in the dinucleotide composition space. DNA Res. 5,
251·259, 1998.
KAwABATA, T., OTA, M. and NISIIIKAWA, K: The Protein Mutant Database. Nuc. Acids
Res., 27, 355·357, 1999.
FUKAMI·KoBAYASHI, K, TATENo, Y. and NISHIKAWA, K: Domain dislocation: a change ofcore structure in periplasmic binding proteins in their evolutionary history. J. Mol.
BioI., 286, 279·290, 1999.
OTA, M. and NISHIKAWA, K: Feasibility in the inverse protein folding protocol. Protein
Sci. 8, 1001·1009, 1999.

I-c. Laboratory of Gene Function Research(1)

DNA Data Bank of Japan at Work on Genome Sequence Data

Yoshio TATENO, Kaoru FUKAMI·KoBAYASHI, Satoru MIYAZAKI, Hideaki SUGAWARA
and Takashi GOJOBORIWe at the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp) have recently
begun receiving, processing and releasing EST and genome sequence data submitted by various Japanese genome projects. The data include those for human, Arabidopsis thaliana, rice, nematode, Synechocystis sp., and Escherichia coli. Since the quantity of data is very large, we organized teams to
conduct preliminary discussions with project teams about data submission
and handling for release to the public. We also developed a mass submission
tool to cope with a large quantity of data. In addition, to provide genome dataon WWW.wedevelopedagenomeinformationsystemusingJava.This system (http://mol.genes.nig.ac.jp/ecolil) can in theory be used for any genome
sequence data. These activities will facilitate processing of large quantities
of EST and genome data. (See Pub.1 for details)

I.

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CENTER FOR INFORMATION BIOLOGY165

Formal design and implementation of improved DDBJ DNA
database with a new sehema and objeet oriented library

Toshitsugu OKAYAMA, Takurou TAMURA, Takashi GOJOBORI, Yoshio TATENO,
Kazuho IKEO, Satoru MIYIAZAKI, Kaoru FUKAMI·KoBAYASHI and Hideaki
SUGAWARA
New schema and systems ofthe database management for DDBJ were constructed using an object-oriented design approach. The design was accomplished in accordance with ANSI/SPARC three-level schemaarchitecture. First, the conceptual schema was designed using a functionalmodel named AIS (associative information structure) and was visualised inextended diagram format. The model is a natural extension of an ER (entity
relationship) model and describes real-world objects in binary associations
between entities with the concept of order.

Second, the schema was mappedon a relational database as a physical schema. All details are concentrated
in this schema and the layer lying above enjoys physical independence. Finally,
as another layer, external modelling was introduced for the database application interface. It provides set-at-a-time basis operations and was implemented as a C++ object-oriented library. On this common framework of a
new schema, a new annotator's workbench named Yamato II and a World Wide
Web (WWW) submission system named Sakura have been successfully developed to improve drastically daily transactions in the DDBJ.

Publ icationsI.

TATENo, Y, FUKAMI·KoBAYASHI, K, MIYAZAKI, S., SUGAWARA, H. and GOJOBORI, T: DNA
Data Bank of Japan at work on genome sequence data. Nucleic Acids Res, 26, 16-20,
1998.

2. OKAYAMA, T., TAMURA, T, GoJOBORI, T, TATENO, Y, IKEO, K, MIYAZAKI, S., FuKAMI·KoBAYASHI,
K and SUGAWARA, H.: Formal design and implementation of improved DDBJ DNA
database with a new schema and object oriented library. Bioinformatics, 14, 472·478,
1998.

166

I-d. Laboratory for Molecular Classification
The laboratory for molecular classification is composed of Professor Hideaki
Sugawara and Professor Assistant Satoru Miyazaki and carries out researchand development in the field of bioinformatics. They especially concentratein the development of information system for the capture, accumulation, evaluation and analysis of data aiming at squeeze information and knowledge from
data. By utilizing the accomplishment of the research and development, thelaboratory also carries out such important international tasks as DNA Data
Bank of Japan (DDBJ) and WFCC-MIRCEN World Data Centre for Microorganisms (WDCM). They carefully design modular systems which will be flexible, scalable and effective in daily use. Last but not least, contribution of Dr.
Y. Ogawa, Ms R. Tanabe and Ms. Y. Fujisawa to the activities of the laboratory
should be mentioned.

(1)

Data processing system for DDBJ

Hideaki SUGAWARA, Satoru MIYAZAKI,Takashi GOJOBORI,Yoshio TATENO,
Kazuho IKEO, Kaoru FUKAMI-KoBAYASHI, Taku TAMURA l and Toshitugu
OKAYAMA2 (lIdengaku-fukyukai, 2Hitachi SK)
Sequence data have increased tremendously and also become complex more
and more these years. It is not an easy task to develop a robust data management system to cope with this change in quantity and quality. We challenged
this task by utilizing object-oriented technology (ref (1)-1). We also developed a dedicated system to microbial genomes data (ref (2)-3).

(2)

Information system for the study of biodiversity

Hideaki SUGAWARA, Satoru MIYAZAKI and Hiroyuki OGAWA
The laboratory rans the World Data Center for Microorganisms (WDCM) of
theWorld Federation for Culture Collections (WFCC) and Microbial Resources
Centers Network (MIRCEN). The WDCM maintains the on-line World Directory of Collections of Cultures of Microorganisms, which include 498 orga-

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167

nizations from 60 countries. WDCM also provides a distributed and parallel
search engine of the Agent for Hunting Microbial Information on the Internet(AHMII).
In addition to the on-line directory and the agent, the laboratory has developed a polyphasic analysis system by integrating conventional taxonomy basedon phenotypes and phylogenetic analysis based on molecular data. A database system for the integration was developed by use of object-oriented technology (ref (3)-1). The laboratory did also sequence of microbial rRNA in
order to accumulate the know-how of experimentalists. For the improvementof phylogenetic analysis, information theory was applied to the analysis of
HIV evolution (ref(1)-4).
The laboratory was invited to a study group that aimed at establishingalternative methods to animal testing and contributed to the group by thedevelopment of a database and statistical analysis tool (ref (1)-5: 12).

Publ icalians
1.

2.

3.

4.
5.

6.
7.

SUGAWARA, H., MIYAZAKI, S., GOJOBORI, T. and TATENo, Y: DNA Data Bank of Japan
dealing with large-scale data submission, Nucleic Acids Research,VoIJl7, No.1, 2528,1999.
OKAYAMA, T., TAMURA, T., GOJOBORI, T., TATENo, Y, IKEO, K, MIYAZAKI, S., FUKAMIKOBAYASHI, K and SUGAWARA, H.: Formal design and implementation of an improved
DDBJ DNA database with a new scheme and object-oriented library, Bioinformatics, 14,472-478, 1998.
SUGAWARA, H. and MIYAZAKI, S.: Towards the Asia-Pacific Bioinformatics Network,
Pacific Symposium on Biocomputing '98,(eds.) Altman R. B., Dunker A. K, Hunter L.
and Klein T. E., 759-764, Hawaii, 1998.
SATOU, K, MIYAZAKI, S. and OHYA, M.: Analysis of HIV by Entropy evolution rate,
Amino Acids, 14, 343-352, 1998.
OHNO, T., AsAKURA, M., AWOGI, T. and et al.: Validation Study and Analyses of Variations of ED50 Values I. Overview of the Study and Analysis of Variations of ED50
Values, Altern. Animal Test.Experiments, 5, 1-38,1998.
OMORI, T., SAIJO, K, KATO, M. and et a1.: Validation Study on Five Cytotoxicity Assays
by JSAAE II. Statistical Analysis, Altern. Animal Test.Experiment, 5, 39-58,1998.
OMORI, T., SAIJO, K, KATO, M. and et al.: Validation Study on Five Cytotoxicity Assays
by JSAAE III Quality of Collected Data Files, Altern. Animal Test. Experiment, 5, 5973, 1998.

168
8.

TANAKA, N., AsAKURA, M., HATTORI, C. and et al.: Validation Study on Five Cytotoxicity
Assays by JSAAE IV. Details of the Colony Formation Assay, Altern. Animal Test.
Experiment, 5, 74-86, 1998.
9. ITAGAKI, H., OHNO, T., HATAO, M. and et al.: Validation Study on Five Cytotoxicity
Assays by JSAAE V. Details of the Crystal Violet Staining Assays, Altern. Animal Test.
Experiment, 5, 87-98, 1998.
10. OHNO, T., FUTAMuRA, Y., HARIHARA, A. and et al.: Validation Study on Five Cytotoxicity Assyas by JSAAE - VI. Details of the LDH Release Assay, Altern. Animal Test.
Experiment, 5, 99-118, 1998.
11. ITAGAKI, H., OHNO, T., HATAO, M. and et al.: Validation Study in Five Cytotoxicity
Assays by JSAAE - VII., Altern. Animal Test. Experiment, 5, 119-130, 1998.
12. OHNO, T., FUTAMuRA, Y, HARIHARA, A. and et al.: Validation Study on Five Cytotoxicity Assays by JSAAE - VIII. Details of the Neutral Red Uptake Assay, Altern. Animal
Test. Experiment, 5, 131·145, 1998.

Others1.
2.
3.

SUGAWARA, H.: International Nucelotide Sequence Database, Experimental Medicine,
VoU6, No.1, 30·37, 1998.
SUGAWARA, H.: Culture collections reflect the culture of countries, Bioscience andbioindustry, 56, 272-275, 1998.
MORI, H., SUGAWARA, H. and UCHIYAMA, I.: E. coli genome, Tanpakushitu-kakusankoso, 43, 1381-1387, 1998.

Oral presentation
1.

2.

MIYAZAKI, S. and SUGAWARA, H.: Prototype of a workbench for sharing information by
use if object technology, Japan Society for Culture Collections, Hiroshima, June.
SUGAWARA, H.: Evolution of sequence database, Denshi-joho-tuushin-gakkai,

Yamanashi, September.
SUGAWARA, H.: Role of a public database in the study of microbial genomes, Frontierof the Genome Biology of Escherichia coli, Okazaki, Japan, November.
4. SUGAWARA, H.: Information highway and its application to biological informatics WDCM experiences and global biodiversity information facility, 16th International
CODATA Conference, New Delhi, India, November.
5. SUGAWARA, H.: Future of in silico biology The Ninth Parallel Computing Workshop,
Kawasaki, November.

3.

169

J.
(1)

RADIOISOTOPE CENTER

Functional analysis of the phoB/cotA region of the
Bacillus subtIlls chromosome

Yoshito SADAIE, Katsunori YATA and Hideyuki OHSHlMA
The phoB-rrnE-groESL-gutR-cotA region (70kb) of the Bacillus subtilis genome contains several operons of unknown or not well known function. We
disrupted 57 orfs of unknown function and determined their transcriptional
activity with lacZ and Northern analysis. Some features of the operons foundare as follows.
A large operon involved in degradation and incorpolation of konjac
glucomannan consists of eight orfs whose predicted products include PTS system proteins as well as mannan digesting extracellular enzyme. The expression of this operon was induced by konjac glucomannan, especially by
dissaccharides and repressed by glucose or mannose.
Inherent restriction/modification system genes hsdMRlhsdMM consist of
tondemly located modification genes ydiO and ydip, and separately locatedrestriction gene ydiSwhich make an operon with two other genes, ydiR andydjA. ydiO and ydiPmake an operon and they were co-transcribed with upstream groESL gene at TO and Tl stage.
Five orfs, ydiA to ydiE, make an operon down stream of the rrnE operon.
YdiA and YdiD are not essential for growth but YdiB, Ydie, and YdiE areessential. The last gene codes for a protein similar to P.haemolytica 0sialoglycoprotein endopeptidase. This operon is transcribed during growth.
The expression of ydjF,ydjG,ydjH, and ydjI showed a peak at T 1 stage. LacZ
activity and the signal of Northern analysis coincided. They constitute an
operon down stream of the glucitol utilization operon. Some genes such as
ydgK, ydhJ, and ydhL are transcribed during growth and other genes and operons such as ydhD, ydhF, ydhU and yd;PIQ are transcribed after T4 stage.

170

(2)

Molecular Organization of Restriction and Modification System
Genes of BacIllus subtIlls

Hideyuki OHSHIMA, Katsunori YATA and Yoshito SADAIE

Bacillus subtilis has one set of inherent restriction/modification systemgenes consists of hsdMM and hsdMR on a prophage-like region at around 55
degree, a desert area of 12 kb long which has lower GC content and few orfs
except for these genes. DNA target is a six base sequence PylTCGAPu where
C is methylated. hsdMM consists of an operon composed of tandemly located genes, ydiO and ydip, for cytosin methylases, and hsdMR is anotheroperon made ofthree genes involving ydiB, coding for a restriction endonuclease. A
classical RM mutant strain lacks this region.
Disruption of ydlSresulted in high transformability and disruption of ydiOor ydiPwas successful only in the background of disrupted ydi8.
Expression of ydiO or ydip, and ydiS was examined using bgaB or lacZ asreporter genes or by Northern analysis. Transcription ofhsdMM and hsdMR
operons was observed during growth and early sporulation phase. In addition to these transcriptions, hsdMM operon was found to be co-transcribedwith upstream groESL operon at TO and T1 stage during sporulation.
The above prophage-like region carries pseudogenes reminiscent of an integrase
or a terminase of bacteriophages and more clearly of phosphomannomutase(yhxB).

(3)

Rapid Isolation of RNA polymerase fromsporulating cells of BacIllus subtIlls

Masaya FUJITA and Yoshito SADAIE
A highly ordered program of temporal and spacial gene activation during
sporulation in Bacillus subtilis is governed by the principal RNA polymerase,and RNA polymerases containing at least five developmental sigma factorsthat appear successively during sporulation. We have developed a rapid
procedure for extracting RNA polymerase from sporulating B. subtilis cells,
which involves the construction of hexahistidine tagged {3' subunit of RNApolymerase and the isolation of RNA polymerase holoenzyme with Ni2+-NTA

J. RADIOISOTOPE CENTER

171

resID. In in vitro transcription of various promoters with the RNA polymerase thus purified, we observed the temporal change of each RNA polymerase activity during sporulation. This procedure enables an isolation of
RNA polymerase within 4hours with cell pellets. Our results indicated thata principal sigma factor, a A , could be detected in a holoenzyme form during all
the stages of growth and sporulation while the other sigma factors, a H, a E, a F
a G, and a K involved in sporulation could be detected sequentially during
sporulation. Moreover, SpoOA, the central transcription factor of commitment to sporulation, was also co-purified with RNA polymerase at the earlystages of sporulation.

Publ ications
1.

2.

3.
4.

5.
6.

7.

FUJITA, M. and SADAIE, Y: Rapid isolation of RNA polymerase from sporulating cellsof Bacillus subtili. Gene 221, 185-190, 1998.
ASAI, K., FUJITA, M., KAWAMURA, F., TAKAHASHI, H., KOBAYASHI, Y and SADAIE,
Y.: Restricted transcription from Sigma H or phosphorylated SpoOA dependent promoters in the temperature-sensitive secA341 mutant of Bacillus subtilis. Biosci. Biotech.
Biochem. 62,1707-1713, 1998.
SADAIE, Y: secA341 mutation inhibition of expression of the Bacillus subtilis protease gene, aprE; Biosci. Biotechnol. Biochem. 62, 1784-1787, 1998.
HAN, W-D., KAWAMOTO, S., HOSOYA, Y, FUJITA, M., SADAIE, Y., SUZUKI, K., OHASHI, Y,
KAWAMURA, F. and OeHI, K.: A novel sporulation-control gene (spoOM) of Bacillus
subtiliswith a a H-regulated promoter. Gene 217,31-40, 1998.
FUJITA, M. and SADAIE, Y: Promoter selectivity of the Bacillus subtilis RNA polymerase a A and a H holoenzymes. J. Biochem. (Tokyo) 124: 89-97, 1998.
FUJITA, M. and SADAIE, Y: Feedback loops involving SpoOA and AbrB in in vitrotranscription of the genes involved in the initiation of sporulation in Bacillus subtilis.
J. Biochem. (Tokyo) 124, 98-104, 1998.
NANAMIYA, H., OHASHI, Y, AsAI, K., MORIYA, S., OGASAWARA, N., FUJITA, M., SADAIE, Y andKAWAMURA, F.: ClpC regulates the fate of a sporulation initiation sigma factor, a H
protein in Bacillus subtilis. Mol. Microbiol. 29,505-513,1998.

172

K.
(1)

EXPERIMENTAL FARM

Development and reevaluation of the genetic stocks of rice

Ken-ichi NONOMURA, Mitsugu EIGUCHI, Toshie MIYABAYASHI and Nori KURATA
We have conducted the reproduction and distribution of genetic stocks ofwild and cultivated rice. From October 1, 1997, we try to include new systemof rice genetic stock generation and application. Additional resources we areconducting to produce, utilize and distribute are enhancer trap and gene trap
rice line. Another trial to analyze the composition of the centromeric heterochromatin in rice chromosomes is also progressed. These projects are cooperated with the plant genetics laboratory. For details, see the reports of plant
genetics lab.

Publ icatian
1.

NONOMURA, K.I. and KURATA, N.: Organization of 1.9-kb repeat unit ReEl in thecentromeric region of rice chromosomes. Mol. Gen. Genet %61, 1·10, 1999.

173

ABSTRACTS OF DIARY FOR 1998
Biological SymposiumJan. 14
A manifold of codes in genetic sequences (Edward
N.Trifonov)
The 2nd Decade of Human Genome Informatics
Feb. 2
Support:Reflections and Future Directions relevant toother Genome efforts. (Tom Slezak)
CREB-mediated gene expression:a mechanism underlyFeb. 17
ing hippocampal Synaptic activity-induced formation of
long-term memory? (Haruhiko Bito)
From growth cone to synapse (Motojiro Yoshihara)
Feb. 18
Roles ofIslet-1 transcription factors in the regional speciFeb. 26
fication and neuronal differentiation in the brain (Hitoshi
Okamoto)Oligonucleotide arrays and the discovery of efficientMar. 5antisense reagents (E.M.Southern)
Development and evolution ofthe skull (Shigeru Kuratani)
Mar. 12
Genetically engineered foodcrops:problems and prospects
Mar. 17(peter Day)
Genetic dissection of Drosophia sexual behavior (DaisukeMar. 17
Yamamoto)
The St.Louis,Sanger,Mishima worm sequencing projectMar. 19(Jean Thierry-Mieg)
V(D)J RECOMBINATION AND ITS BIOLOGICAL RELAApr. 13
TIVES (Martin Gellert)
The role of Deltex in Notch signaling (Kenji Matsuno)
Apr. 15
Generation and positioning of cortical neurons:a cellular
Apr. 22and histological study (Masaharu Ogawa)Inactivation at promoters and control of transcription
Apr. 22
initiation(Ranjan sen)

174

Apr. 23
Apr. 27
May 8
May 13
May 13
May 14
May 18May 18June 3June 16
June 19

June 22
July 6

July 8July 15
July 23

Molecular mechanisms of synaptic connection:analysis of
the neuromuscular junction in Drosophia (Toshinao Nose)
Molecular and neural mechanisms of thermotaxis in thenematode G.elegans (Ikue Mori)
Supramolecular Structure ofthe Salmonella typhimurium
Type ill Protein Secretion system (Tomoko Kubori)
Genetic information controlling animal behavior -theanalysis of Fyn-deficient mouse (Takeshi Yagi)
Reductive evolution of endocellular parasites and the origin of mitochondria (Charles G.Kurland)
Molecular mechanism of pattern formation revealed byanalysis of the Cadherin superfamily (Tadashi Uemura)
Detection and analysis of mutation in human and mouse(Youichi Gondoh)
Genomic imprinting:gene regulation at the level of chromosome domain based upon genome modification(Hiroyuki Sasaki)
Dorsalization mechanisms in vertebrates:Genetic analysis by using zebrafish mutants (Yasuyuki Kishimoto)
Maintaining and integrating morphogen gradients. Cross
regulation ofWg and Dpp. (J. Lawrence Marsh)
Analysis of functional sequences in human centromere
and construction of artificial chromosomes (Hiroshi
Masumoto)
Mechanism of Mouse Central Olfactory Projection(Tatsumi Hirata)
Mechanisms of Zebrafish central nervous systemdevelopment:Embryo logical and genetic approach(Hiroyuki Takeda)
Regulation of neural plasticity by Ras (Atsu Aiba)
The initiation of meiotic recombination in Saccharomyces
cerevisiae (Bernard Massy)
The role ofPax-6gene in neuronal differentiation and guidance (Noriko Ohsumi)

ABSTRACTS OF DIARY FOR 1998

July 23
July 24

July 27
July 29
July 30
July 30
July 31

Aug. 3

Aug. 7

Aug. 28
Sep. 1
Sep. 14
Sep. 17

Sep. 30

175

Higher order regulation of expression of HoxD gene cluster (Takashi Kondoh)
Developmental genetics of quaking mutation affecting neurological function and embryonic development (Kuniya
Abe)
Programmed cell death in the nematode C.eJegans :asearch for new genes (Asako Sugimoto)
Chromatin structure and transcriptional regulatory
mechanisms in the yeast genome (Mitsuhiro Shimizu)
ProteinX-New player in prion propagation (Kiyotoshi
Kaneko)Constructing trees from subtrees(with applications tomammalian evolution) (Dan Graur)Relative Contributions of Mutation and Selection to the
Evolution of Bacterial DNA Base Composition (Noboru
Sueoka)Roles ofthe paraxial protocadherin(pAPC)gene in morphogenetic movement during gastrulation of the zebrafish, Xenopus and mouse embryos. (Akihito Yamamoto)Histo-blood type ABO system:from glycosyl chain antigens to genes and glycosyltransferases (Fumi-ichiro
Yamamoto)
Hedgehog protein biogenesis and signaling (Philip
A.Beachy)
Genetic control of programmed cell death in the nematodeG.eJegans. (Michael Hengartner)
Genome imprinting and germ cell development (Takashi
Tada)
Functional analysis of Dpbll which is involved in chromosomal DNA replication and cell cycle checkpoint in
Saccharomyces cerevisiae (Youichiro Uemura)
A common regulatory mechanism controls expression of
Drosophia master control genes and establishment of organ identities (Syoichiro Kurata)

176

Oct. 5Oct. 5Oct. 6
Oct. 8Oct. 13
Oct. 13
Oct. 13
Oct. 14
Oct. 15
Oct. 16
Oct. 19
Oct. 19
Oct. 19
Oct. 20
Oct. 22
Oct. 28
Nov. 2
Nov. 2Analysis of nuclear proteins which control the plant photomorphogenesis (Minami Matsui)
Structural and functional genomics in Arabidopsis
thaliana (Hiroyuki Tabata)Interaction between enhancer and promoter in Drosophia
embryos (Sumio Ohtsuki)
The Telomeres of Drosophila and the Mosquito Anpheles
gambiae (Harald Biessmann)
Signaling by wntGenes in Drosophila:Common and Pathways (Roel Nusse)
FGF signaling in the vertebrate embryo (Gail Martin)
The G.elegans Genome Project (Stephanie L.Chissoe)
Genetic and molecular mechanisms that pattern and or·
ganize in Drosophia (Thomas B.Kornberg)
Control of meiotic recombination in Saccharomyces
cerevisiae (Michael J.Lichten)
Establishing polarity in the G.elegans embryo (Kenneth
J.Kemphues)
Control of developmental pathways by Hox proteins andtheir cofactors (Richard S.Mann)
Understanding plant zygotic embryogenesis throughanalysis of somatic embryogenesis (Hiroshi Kamata)
Expression and function of homeobox genes in rice (ShinMatsuoka)Specification of the dorsal axis of Xenopus by the Wnt
signaling pathway (Randall T.Moon)
The role of signaling factors in the development of kidney(Tomoko Ishihara-Obara)
Drosophila ELAV protein is a major posttranscriptional
regular in neurons (Kalpana White)
Analysis of structure,function and development of the
Drosophia brain,using GAL4-UAS technique (Kei Itoh)
The End of Mitosis in Budding Yeast(Leland H.Johnston)ABSTRACTS OF DIARY FOR 1998

Nov. 9Nov. 9Nov. 10
Nov. 11
Nov. 19
Dec. 1

Dec. 1
Dec. 1
Dec. 7

Dec. 10
Dec. 15
Dec. 22

Dec. 24

177"Genome fusion" of two plant species beyond one hundred
million year's evolution.-Does it waste money and time?
Or,is it informative for genome biology?- (Daisuke Shibata)
Genome DNA methylation, plant development, andtransposons:an approach using Arabidopsis mutants(Tetsuhito Kakutani)
Signaling at the synapse:identification of HIG and SIF
through low-activity mutans (Chihiro Hama)
Evolution of Rh genes and antigenes in nonhuman primates (Antonie Blancher)Computer simulation of development processes (Hiroaki
Kitano)Investigation of a set of genes essential for cell growth inEscherichia coli :Identification and characterization of thenovel hda gene encoding a Dna-related protein,which isessential for chromosome replication (Jun-ichi Katoh)
Mechanism of signal transduction of the phosphate
regulon in Escherichia coli (Kouzou Makino)
Estimation of past demographic parameters when themutation rates vary among sites (Laurent Excoffier)
FAST-1 is an essential component of activin/TGF i3 -mediated mesoderm induction in the early Xenopus embryo(Minoru Watanabe)
Evaluation of Threading Specificity and Accuracy (Stephen
H.Bryant)
Isochores,selection and rendom drift. (Girogio Bernardi)
The roles ofPGL-1,a component of the germ granules,inthe germline development of the nematode C.elegans(Ichiro Kawasaki)
The roles of a organaizer specific homeo-box gene,Xlim1,in the nerve induction and the anterior-posterior pattern formation (Masanori Taira)178

Dec. 28

Dec. 28

Yeast as a model for functional interpretation of the genomes :Approach from gene-gene and protein-protein interactions (Takashi Hoh)Molecular mechanisms of homologous recombination in
Escherichia coli :Structure and functions of Holliday junction processing enzymes,RvuA, RvuB,and RvuC proteins(Hiroshi Iwasaki)

Mishima Geneticists' Club
Jan. 16
Post-translational regulation of nitrate reductase(NR) in
higher plant (Kengo Kanamaru)
Feb. 25
Transcriptional regulation of Breathless, the FGF receptor required for the tracheal development in Drosophia(Tomokazu Ohshiro)
Mar. 11
Wnt3a and Wnt7a regulate chicken limb patterning
through two distinct signaling pathways (Mineko
Kengaku)Mar. 12
tmRNA,a small RNA functioning as both tRNA and mRNA
(Akira Mutoh)

179

FOREIGN VISITORS IN 1998
Jan. 11-14
Jan. 20-21
Jan. 19-Mar. 20
Feb. 2Mar. 5-6
Mar. 17-18
Mar. 17-18
Apr. 12·14
May 26-Jun. 13
Jun. 3-4
Jun. 15-16
Jun. 16
Jul. 17·Aug. 23
Jul. 23
Jul. 30-31
Aug. 3-4
Aug. 28-29
Aug. 31-Sep. 1
Sep.28·30

Yuan Yi-da, Institute of Genetics, Chinese Academy of Sciences, China
Zhang Zhi-xin, Beijing Blood Center, China
Jean Thierry-Mieg, CNRS, Laboratoire de Physique
Mathematique, Universite Montpellier II, France
Tom Slezak, Human Genome Center, Lawrence Livemore
National Laboratory, U.S.A.
E.M.Southern, University of Oxford, U.K.
Peter Day, The State University of New Jersy, U.S.A.
Helmut Knuepffer, IPK, Gatersleben, Germany
Martin F.Gellert, Laboratory of Molecular Biology, NIH,
U.S.A.
Dipankar Chatterji, Centre for Cellular and Molecular
Biology, India
Yasuyuki Kishimoto, Max Plank Institute of Tuebingen,Germany
Bernard de Massy, Institut Curie, France
J.Lawrence Marsh, University of California at Irvine,
U.S.A.
Shozo Yokoyama, Syracuse University, U.S.A.
Takashi Kondoh, University of Geneva, Switzerland
Kiyotoshi Kaneko, University of California San
Francisco,U.S.A.
Akihito Yamamoto, University of California Los
Angeles,U.S.A.
Philip A.Beachy, Johns Hopkins University, U.S.A.
Michael Hengartner, Cold Spring Harbor Laboratory,
U.S.A.
Jin Feng, Institute of Genetics, Chinese Academy of Sciences, China

180

Sep.28-30
Oct. 6-7
Oct. 8-9
Oct. 12-13
Oct. 13
Oct. 13-14
Oct. 14
Oct. 14-15
Oct. 15-16
Oct. 19-20
Oct. 20-21
Oct. 28-29
Oct. 31-Nov.2
Nov. 10-13
Nov. 30-Dec. 3
Dec. 7-8
Dec. 10-11
Dec. 10
Dec. 21-22

Cao Jing-Long, Institute of Genetics, Chinese Academy of
Sciences, China
Sumio Ohtsuki, University of California Berkeley, U.S.A.
Harald Biessmann, University of California at Irvine,
U.S.A.
Roel Nusse, Stanford University Medical Center, U.S.A.
Stephanie L.Chissoe, Washington University School of
Medicine, U.S.A.
Gail Martin, University of California San Francisco, U.S.A.
Thomas B.Kornberg, University of California San Francisco, U.S.A.
Michael Lichren, NIH, U.S.A.
Kenneth J.Kemphues, Cornell University, U.S.A.
Richard S.Mann, Columbia University, U.S.A.
Randall Moon, University of Washington School of Medicine, U.S.A.
Kalpana White, Brandeis University, U.S.A.
Leland H.Johnston, National Institute for Medical Research, U.K.
Antoine Blancher, Hospital Purpan, France
Laurent Excoffier, University of Geneva, Switzerland
Minoru Watanabe, Harvard Medical School, U.S.A.
Steven H.Bryant, NCBI, NIH, U.S.A.
Vijaya Gopal, Centre for Celler and Molecular Biology,
India
Ichiro Kawasaki, Indiana University, U.S.A.

ASSOCIATION FOR PROPAGATION OF THE
KNOWLEDGE OF GENETICS
President
MORIWAKI, Kazuo; Vice-President, The Graduate University for Advanced
StudiesFOREIGN VISITORS IN 1998

181

Managing Director
GOJOBORI, Takashi; Professor, National Institute of Genetics
NAKATSUJI, Norio; Professor, National Institute of Genetics
Manager
HOTTA Yoshiki; Director-General, National Institute of Genetics
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
YAMAGUCHI, Hikoyuki; Professor emeritus, University of Tokyo
MIURA, Kin-ichiro; Director, Institute for Biomolecular Science, Gakushuin
University
KURODA, Yukiaki; Professor, College of Environmental Health, Azabu University
ISffiHAMA, Akira; Professor, National Institute of Genetics
SHIGETO, Gakuji; Managing Director, Inoue Foundation for Science
Trustee
TAZIMA, Yataro; Director Emeritus, Institute of Silkworm Genetics andBreeding
OSHIMA, Chozo; Honorary member, National Institute of Genetics
SAITO, Hiuga; Director and Professor, Institute ofMedical Mycology, Teikyo
University
MATSUNAGA, Ei; Professor emeritus, National Institute of Genetics
YOSHINO, Tatsuji; President, Shokabo Publishing Co.
TAKAGAKI, Yoshio; Full-time Adviser, Chugai Pharmaceutical Co.
SENO, Takeshi; Professor, National Institute of Genetics
TATENO, Yoshio; Professor, National Institute of Genetics
Auditor
IMAMURA, Takashi; Professor Emeritus, National Institute of Genetics
MORISHIMA, Hiroko; Professor Emeritus, National Institute of Genetics
KATSURA, Isao; Professor, National Institute of Genetics
Adviser
MORIWAKI, Daigoro; Professor emeritus, Tokyo Metropolitan University

182

AUTHOR INDEX
ABE, S

40
156
AlTA, N64
AKIBA, T.
148
AKIYAMA-ODA, Y.
57
ALEXEEV, A.
46
ANDACHI, Y.
132
ANDO.A.
76
ARAKI, H
48,49.50
ARAMAKI, H
139
BONG. Y.-S
83
BURATOWSKI. S
65
CATTLEY, S.K.
155
CHIHARA, T
117
CHIJIWA, T.
82
CHUJO. A.
106
CHUMA, S
100
DANJO. I.
83
DASGUPTA, D
16
DAWKINS. R.
152,153.154.155
EIGUCHI, M
111.112,172
ENDO, S
13
F'ERDOUS. A.
65
FuJISAWA. T
60
FuJITA. M
170
FuJITA, N
10.11,13.14.16
FuJIWARA. M, 144
FuJIYAMA. A.
83
FuKAMI-KoBAYASHI. K.
164,165,166
FuKUSHI. K.
149
FUKUSHI. S
158
FURUUMI, H
82
GAAL. T137
GAMOU. S
118
GAUDIERI. S
152,153.154.155.156

GaJOBORI. T

AGATA, K.

GOTO. K.

GaTO. S

GoTO. S
GOTO. YHABARA. K

HAMA. THANDA, H
HARA. HHARASHlMA. S
HARUSHlMA. Y.
HASEGAWA, A.
HASEGAWA. J
HATANO. N
HATTA, M
HATZOG, G.A.
HAYASHI. S
HAYWARD. R.S
HIBI. T.
HIGASHI, C
HILL. R.J
HlRAMOTO. M
HIROMI. Y
HIRONO. K.
HIROSE. S
HIROSHIMA, M
HISHIDA. R.
HONDA. A.
HOOVER. D.M
HORAI. S
HORIE. M
HOSOYA. T
HOTTA. Y.
HUANG. Z

152,154,156.158.
164.165.166
116
116.118
122
38.40
154
102
65.67
50
64
110.111
86
67
82
60
65
64.116,117,118,119
138
30
59
128
58
54.55.56
126
62.63,64.65
136
146
27,28.30
40
38,39,40
116
59
57.58,59
100

AUTHOR INDEXHWANG, S.J

KINEBUCHI, T

IGARASHI, K

KISHI, T

28
20
IKEGAMI, T
122
IKEMURA, T
68,74,76
IKEO, K
156,165,166
IKEYA, T.
117
IMAl, T
65
INOKO, H
76
ISHIGURO, A
25
ISHIHAMA, A.
10,11,13,14,16,17,19,20,22,
23,24,25,27,28,30,150
ISHIHARA, K
81,82
ISHIHARA, T
141,143,144,145,146
ISHIJIMA, J
96
ISOBE, T
91
ITO, H
116
ITO, Y.
106, Ill, 112
ITOH, J86
ITOH, M
127
ITOH, T.
156
IWAKI, T
82
IWANE, A
134,135
IWATA, A.
19,30
JISHAGE, M
16
KABATA, H
139
KAGEYAMA, T
158
KAMIMuRA, Y.
49,50
KANAYA, S
68,74,76
KANNO, Y.
100
KAsCIUKOVIC, T
138
KATAYAMA, A
10,11
KATAYAMA, K
158
KATO, J
122
KATO, R.
81,82
KATSURA, I.
141,143,144,145,146
KAWABATA, T
163
KAWAHARA, T
120
KENYON, J.B
40
KIKKAWA, Y
95
KIMBERLING, w'J
40
KIMURA, M
22,23,150

KITAMURA, K
KITANO, H
KITANO, T
KITAYAMA, S
KOBAYAKAWA, Y
KOBAYASHI, M
KOHARA, Y.
KOIDE, T
KOIZUMI, O
KOMOTO, M
KONO, T.
KOSE, HKRAMER, S
KUBORI, T
KUBOTA, K
KUDO, YKULSKI, J.K
KURATA, N
KURLAND, e.G
KUROSAWA, O
LEE, J.S
LEELAYUWAT, e
LINDAHAL, KF

LIU, Q-X
LoNGMAN, N
MAEDA, H
MAKINO, M
MARTIN, A.
MARUYAMA, e
MASUMOTO, H
MASUYA, H
MATAKATSU, H
MATSUDA, S
MATSUMOTO, K
MATSUOKA, M
MATSUZAWA, H
MEARES, e.F

183
139
34
134,135
85,86
77
123
60
64
123,124,125,126,
127,128,131
91,95,98
60
25
82
55
54
137
116
68
152,153,154
106,108,109,110,
111,112,121,172
20
139
75
155
91
65
155
16
40
153
97
48
92,93,97
118
42
116
86
115
II

184
MELLO, CMINAKUCHI, Y.
MITA, A.
MITOBE, J
MITSUI, K.
MITSUZAWA, H
MIYABAYASHI, T.
MIYAHARA, K.
MIYAZAKI, S
MrYOSHI, K.
MIZOKAMI, M
MIZUMOTO, K.
MIZUNO, K.
MIZUNO, S
MORI, HMORIWAKI, K.
MOTOHASHI, T
MOTOYAMA, A.
MUKAI, T
MURAKAMI, K.
NAGAI, H
NAGAOKA, T.
NAGASAWA, N
NAGATO, Y.
NAGATO, Y.
NAKADE, K.
NAKAGOMI, O
NAKASHIMA, H
NAKATSUJI, N
NnG, HNISHIKAWA, K.
NISHIMURA, A.
NISHINO, I.
NIWA, N
NIWA, Y.
NOGAMI, M
Nom, HNOMURA, T.
NOMURA, T.
NONAKA, 1.
NONOMURA, K.

128
127
93,95,96
24
122
22,24,25
172
143
164,165,166
108,112
158
27
91
81,82
116
91,97,98
123,12783
81
150,151
137,138126
85
85,86,121
112
28
158
162
100,102,103
52,53
162,163
19,114,115,116
38
56
106
76
25
10,11
97
38,40
108,109,172

OBARA, M
OGAWA, H
OGAWA, T.
OmwARA, Y
OGURA, K.
OHBA, T
OHNO, MOHSHIMA, H
OHTA, T
OHTA, TOISHI, A.
OKABE, M
OKADA, K.
OKADA, M
OKAMOTO, T.
OKAMURA, H
OKAYAMA, T.
OMOTO, K.
ONAMI, SOSHIUMI, H

OTA, MOVERBECK, L.D
OYA, A.
OZOLINE, O.N

PARK, H.-S
PARK, J.H
PRIESS, J.R.
PURBOWASITO, W.
SABA, R.
SADAIE, Y.

SAEKI, N
SAGAI, T
SAITO, M
SAITO, T
SAITOU, N
SAKA, K.
SAKAMOTO, S
SAKURAI, H
SAKURAI, T.
SASAKI, H
SASAMURA, T.

123
166
42,151120
131
123
73,75,82
169,170
42,44,45,64,151
79
141
54,56
64
63
28
158
165,166
39
126
42
162,163
40
158
14
83
36
128
82
102
169,17097
92,93,97
121
100,102
77
116
102
22
100
75,81,82,95
58

185

AUTHOR INDEX
SATO, H97

TAKEMARU, K.

64

SATO, K.

121

TAKEUCHI, H

118
139

SATO, Y86

TAKEUCHI, M

SATO, Y138

TAKE-UCHI, M

141

SEINO, H

35,36

TAKIZAWA, K.

57,58

TALUKDER, AA

19

137,138,139

TAMURA, M

100

SHIMIZU, H

60

TAMURA, T

165

SHIMIZU, K.

95

TAMURA, T

166

123,124,125

TANAKA, Y.

158
117

SENTOKU, N
SHIMAMOTO, N

SHIN-I, T.

86

SHINKAWA, H

40

TANAKA-MATAKATSU, M

SHINZA, M

58

TANIGUCHI, M

SHIRAKIHARA, y.

147,148,149,150,151

118

TATENO, Y.

164,165,166

SHIROHZU, H

81

TAZAKI, A

156

SHIROISHI, T

77,91,92,93,95,

TENZEN, T.

73,75,76

SUBBARAYAN, PR.

96,97,98,121

THIERRY-MIEG, D

125

17

THIERRY-MIEG, J

125

SUGAWARA, H

116,164,165,166

SUGIMOTO, N

85

TOMIZAWA, J

38

SUGIMOTO, S

65

TSUCHIYA, R.

121,122SUGIURA, I.

123

TSUJI, S

87

SUMIYAMA, K.

77

TsUJIMOTO, H

120

SUZUKI, E

55

UCHIDA, K.

95,96

SUZUKI, T.

119

UEDA, H

64,65
19,30

TOKUNAGA, M

134,135,136

SUZUKI, T.

123

UEDA, SSUZUKI, Y.

158

UEHARA, T

115

TABARA, H

128

UMESONO, Y.

57

TADA, M

103

USAMI, S

40

TADA, T

100,103

WADA, A20

TADAKI, T.

35

WADA, T.

65,67

TADENUMA, M

116

WAKANA, S

95,97

TADOKORO, R.

118

WASHIZU, M

139

TAJIMA, S

82

WATANABE, K.

156

TAKAGI, T.

65

WATANABE, T

30

TAKAGI, T

67

WATANABE, Y.

73,76

TAKAHASHI, M

85

WATANABE, Y.

97

TAKAHASHI, T

60

WEST, S

55

TAKANO, T

71,72

WESTON, M.D

TAKAYA, K.

106

WINSTON, F

40
,

65

TAKAYAMA, Y.

50

YADA, Y.

93

TAKEDA, G

85

YAGI, Y.

119

186
YAMADA, K.

28

YAMADA, T.

54

YAMADA, Y.
YAMAGATA, T

68
73,76

YAMAGUCHI, J

85

YAMAGUCHI, M

117

YAMAGUCHI, Y.

65,67

YAMAKAWA, T.
YAMAO, F

122
34,35,36

YAMAZAKI, Y.

120,121,122

YANAGIDA, T

134,135

YANO, K.

65

YASUI, K.

24

YATA, K.

169,170

YOKOMINE, T

81

YONEKAWA, H

95

YOSHIMURA, A

121

YOSHINO, M

91

YUM, S

60

ZUBAIR, M

82

Annual Report of the National Institute of Genetics. No.49
Issued on October 1999
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 Yasushi Hiromi & Akiko Nishimura
Published by National Institute of Genetics
Printed by Hobundo Insatsu Co., Ltd.

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