ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No.48

1997

Published by

THE NATIONAL INSTITUTE OF GENETICS
Mishima, Shizuoka-Ken, Japan

1998

Annual Reportof theNational Institute of Genetics
No. 48, 1997

(Main Building)Published by

The National Institute of Genetics, Japan

1998

CONTENTS
General statement
Staff
Council and advisory committee
Research activities in 1997
A.

.
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..
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Department of Molecular Genetics
A-a. Division of Molecular Genetics
Molecular Anatomy of the a Subunit C-terminal Domain ofEscherichia coli RNA Polymerase: Positioning of Two a Subunit Carboxy-Terminal Domains on Promoters by Two Transcription Factors. MURAKAMI, K., FUJITA, N., OZOLINE,
O.N., OWENS, J.T., BELYAEVA, T.A., MEARES, C.F., BUSBY,
S.J.W. and ISHIHAMA, A.
Molecular Anatomy of the a Subunit C-terminal Domain ofEscherichia coli RNA Polymerase: Monitoring of RNA Polymerase-DNA UP Element Interaction by a Fluorescent Probe
Conjugated to a Subunit. OZOLINE, O.N., FUJITA, N.,
MURAKAMI, K. and ISHIHAMA, A.
Molecular Anatomy of the a Subunit N-terminal Domain ofEscherichia coli RNA Polymerase: Contact Sites for Dimeric
Association Studied by Cleavage with Fe-BABE Conjugatedto Single Cysteine Residues. MIYAKE, R., MURAKAMI, K.,OWENS, J.T., FUJITA, N., MEARES, C.F. and ISHIHAMA, A. ....
Molecular Anatomy of the /3 and /3 Subunits of Escherichia coli
RNA Polymerase: Mapping ofSubunit-subunit Contact Sites
Studied by Proteolytic Cleavage. NOMURA, T., KATAYAMA,
A., CHATTERJI, D., FUJITA, N. and ISHIHAMA, A.
.
Molecular Anatomy of the a Subunits of Escherichia coli RNAPolymerase: Mapping of Contact Sites with Promoter DNA
and Core Enzyme Subunits Studied by Cleavage with Fe-

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BABE Conjugated to Single Cysteine Residues. OWENS, J.T.,
BOWN, J., MURAKAMI, K, FUJITA, N., MEARES, C.F., BUSBY,
S.J.W., MINCHIN, S.D. and ISHIHAMA, A.
Regulation of the Activity of Sigma Subunits from Escherichia
coli: Functional Interaction of Escherichia coli RNA Polymerase with Inorganic Polyphosphate. KUSANO, S.,
GOWRISHANKAR, J. and ISHIHAMA, A.
A Stationary-Phase Protein in Escherichia coli with Binding Activity to the Major Sigma Subunit of RNA Polymerase.
JISHAGE, M., IWATA, A. FUJITA, N., VEDA, S. and
ISHIHAMA, A'"
Sequence Specificity and Affinity of DNA Binding for Twelve Species of Escherichia coli DNA-binding Proteins. TALKUDER,
A.A. and ISHIHAMA, A.
Gene Organization and Protein Sequence of the Subunits of the
Fission Yeast Schizosaccharomyces pombe RNA polymerase
II. SAKURAI, H. and ISHIHAMA, A.
Two Large Subunits of the Fission Yeast RNA Polymerase II Provide Platforms for the Assembly of Small Subunits.
ISHIGURO, A YASill, K, KIMuRA, M., IWATA, A, VEDA. S and
ISHIHAMA, A.
Molecular Composition of a Core Subassembly of S pombe RNAPolymerase II. KIMURA, M., ISHIGURO, A. and ISHIHAMA, A.
..................................................................................................
Mapping of the Subunit-Subunit Contact Sites on Rpbl, Rpb2
and Rpb3 of the Fission Yeast RNA Polymerase II. YASUI,K, MIYAO, T., ISHIGURO, A., HONDA, A, Qu, Z., KIMURA, M.,
MITSUZAWA, H. and ISHIHAMA, A.
Mapping of the Functional Sites on Two Large Subunits of the
RNA Polymerase II: Photo-affinity Cross-linking of 3'-Termini of Nascent RNA. WLASSOFF, W.A., KIMURA, M.
and ISHIHAMA, A.
Genetic Mapping ofthe Functional Sites on Rpb3 and Rpbll,
Homologues of Prokaryotic Alpha Subunits. MITOBE, J.,

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CONTENTS

KOMOTO, M., YASUI, K., MITSUZAWA, H. and ISHIHAMA, A...
The Molecular Anatomy of Influenza Virus RNA Polymerase:
Identification of the RNA Cap-binding Site on PB2 Subunit. HONDA, A., MIZUMOTO, K. and ISHIHAMA, A.
The Molecular Anatomy ofInfluenza Virus RNA Polymerase:
Identification of Host Factor(s) for Interconversion between
Transcriptase into Replicase. HONDA, A., OKAMOTO, T and
ISHIHAMA, A. .
Isolation of 130Kl180K Heterodimer with RNA-dependent RNAPolymerase Activity from TMV-infected Tobacco.
WATANABE, T., HONDA, A., IWATA, A., DEDA, S., HIB!, T
and ISHIHAMA, A.
A-b. Division of Mutagenesis
A Dbiquitin Pathway Essential for Onset of Anaphase in
Mitosis. SEINO, Hand YAMAO, F.
CDC34 Dependent-Ubiquitin Pathway: Role ofGRR1 in the
Ubiquitination ofG1 Cyclin Cln2. KISHI, T. and YAMAO, F.
..................................................................................................
A-c. Division of Nucleic Acid Chemistry
Enzyme mechanism of the 5'-terminal capping of eukaryotic
mRNA. MIZUMOTO, K.

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B. Department of Cell Genetics
B-a. Division of Cytogenetics
S. cerevisiae recA homologues RAll51 and DMC1 have both
distinct and overlapping roles in meiotic recombination.

OGAWA, T.
Complete Nucleotide Sequence ofthe Chloroplast Genome fromthe Green Alga Chlorella vulgaris; The Existence of Genes
Possibly Involved in Chloroplast Division. OHTA, T.
A Recombinational Defect in the C-terminal Domain of Escherichia coli RecA2278-5 Protein is Compensated by Protein
Binding to ATP. ALEXEEVE, A. and OGAWA, T.

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B-b. Division of Microbial Genetics
Characterization of the envC mutation that affects septationand leads to chain formation in E. coli. HARA, H., NARITA,
S., YAMAMOTO, Y. and NISHIMURA, Y.
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Contribution of the P mra promoter to expression of genes in the
Escherichia coli mra cluster of cell envelope biosynthesis andcell division genes. MENGIN-LECREULX, D., AYALA, J.,
BOUHSS, A., HEIJENOORT, J.v., PARQUET, C. and HARA, H .
..................................................................................................

B-c. Division of Cytoplasmic Genetics
Targeted integration of DNA using mutant lox sites in embryonicstem cells. ARAKI, K., ARAKI, M. and YAMAMURA, K. ........
Efficiency of recombination by Cre transient expression in embryonic stem cells: Comparison of various promoters.
ARAKI, K, lMAIzuMI, T., OKUYAMA, K, OIKE, Y. and YAMAMURA,
K
Nuclear organization in fission yeast meiosis. HIRAOKA, Y.....
Chromosome dynamics in living human cells. HIRAOKA, Y. ......

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C. Department of Developmental Genetics
C-a. Division of Developmental Genetics
The Inhibition ofRas Signaling Pathway by EDL is Required for
the Neural Inducing Ability of Founder Cells. YAMADA,
T., OKABE, M KOSE H. and HIROMI, Y.
Sprouty, an Extracellular Factor that Antagonizes both FGF and
EGF Signaling Pathways. KRAMER, S., OKABE, M.,
HAKOHEN, N., KRASNOW, M. and HIROMI, Y.
Functional Dissection of the Ligand Binding Domain of an Orphan Receptor, Seven-up. KosE, H., WEST, S. and HIROMI,Y.
.
Peptide Groups Which Regulate Nerve Cell Differentiation in
Hydra. TAKAHASHI, T., SHMIZU, H., HATTA, M., YUM, S., KOIZUMI,0., SUGIYAMA, T., MUNEOKA, Y. and FUJISAWA, T.
Extracellular matrix and hydra pattern formation. SHIMIZU,
H. and SARRAS, M. P.

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COb. Division of Gene Expression
MBF1 is an evolutionarily conserved transcriptional
coactivator that connects a regulatory factor and TATA
element-binding protein. TAKEMARU, K., LI, F.-Q., VEDA,
H. and HIROSE, S.
Transcriptional activation through interaction of MBF2 withTFIIA. LI, F.-Q., TAKEMARU, K., GOTO, M., VEDA, H.,
HANDA, H. and HIROSE, S.
Temporal regulation of the mid-prepupal gene FTZ-F1: DHR3
early-late gene product is one of plural positive regulators.
KAGEYAMA, Y, MASUDA, S. HIROSE, S. and VEDA, H.
Genetic Studies on the 2nd (p )-Linkage Group in Bombyx mori(L.) Are the p and S -alleles comprising of either two independent alleles or of a common allele? MURAKAMI, A.
Genetic Studies on the 2nd (p )-Linkage Group in Bombyx mori(L.) p -allelic series (2-0.0). MURAKAMI, A.
Genetic Studies on the 2nd(p )-Linkage Group in Bombyx mori(L.) S -allelic series (2-6.1). MURAKAMI, A.
Origin of Holometabolous Metamorphosis in Insects.
MINATO, K.
Coco Division of Physiological Genetics
Interplay between positive and negative elongation factors of
RNA polymerase II transcription: drawing a new view ofDRBmediated inhibition of transcription. YAMAGUCHI,Y, TAKAGI, T., WADA, T. and HANDA, H.
Species-specific codon usage diversity in eleven prokaryotes andone eukaryote whose genomes have been completely
sequenced. KANAYA, S., KUDO, Y, IKEMURA, T.
D.

Department of Population Genetics
D-a. Division of Population Genetics
Rate variation of DNA sequence evolution in the Drosophila
lineages. TAKANo, T.S.
Study of species difference as observed as interspecific hybrid
anomaly in Drosophila. TAKANO, T.S.

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D-b. Division of Evolutionary Genetics
In situ visualization of sequence-dependent high-affinity sites
for non-B structure-forming DNAs in the human interphasenucleus. Affinity sites for triplex-forming DNAs are closely
but differentially associated with individual centromeres.
OHNO, M., TENZEN, T., YAMAGATA, T., KANAYA S. andlKEMURA, T.
Triplet repeat polymorphism within the NOTCH4 gene locatednear the junction of the HLA class II and class III regions in
narcolepsy. ANDO, A, SUGAYA, K., JUJI, T., lKEMURA, T. and
INOKO,H
Replication Timing for the GC Content Transition Area.
TENZEN, T., WATANABE, Y, ANno, A, INOKO, H. and lKEMURA, T.
..................................................................................................
Structural analysis of mouse tenascin-X: evolutionary aspects ofreduplication of FNIII repeats in the tenascin genefamily. IKUTA, T., SOGAWA, N., ARIGA, H., IKEMURA, T.,
MATSUMOTO, K.
Genomic organization of human GABAB receptor identified in
MHC class I region; proposal for differentiation of two
isoform's expression via alternative usage of promoters withand without cAMP-responsive element. YAMAGATA,
T., ANDO, A, INOKO, H., and lKEMURA, T.
Nucleotide sequence analysis ofthe HLA class I region spanningthe 237-kb segment around the HLA-B and -C genes.
MIZUKI, N., KIMURA, M., ANDO, A, lKEMURA, T. and INOKO, H .
..................................................................................................
Evolution of primate ABO blood group genes and their homologous genes. SAITOU, N. and YAMAMOTO, F.
The phylogenetic relationship of the genus Oncorhynchus species
inferred from nuclear and mitochondrial markers. KITANO, T.,
MATSUOKA, N. and SAITOU, N.
Genetic origins of the Japanese: A partial support for the "dualstructure hypothesis". OMOTO, K., and SAITOU, N.

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Reconstruction of phylogenetic trees using the maximum likelihood method in parallel environment using logic programming. OOTA, S. and SAITOU, N.
D-c. Division of Theoretical Genetics
The meaning of near-neutrality at coding and non-coding
regions. OHTA, T..
Role of gene conversion in generating polymorphisms at major
histocompatibility complex loci. OHTA, T
.

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E. Department of Integrated Genetics
E-a. Division of Human Genetics
Human Genome Resources and Their Application for the Analysis ofChromosome21. FUJIYAMA,A., DANJO, 1. and PARK, H.S.
Whole Genome Analysis of Signal-Transduction Pathways in Fis.
sion Yeast. DANJO, 1., BONG, Y-S. and FuJIYAMA, A.
Severe Lactic Acidosis and Neonatal Death in Pearson
Syndrome. MURAKI, K., GoTO, Y, NISHINO, 1., HAYASHIDANI,
M., TAKEUCHI, S., HORAI, S., SAKURA, N. UEDA, K.
..
Myoclonus Epilepsy Associated with Ragged-Red Fibers: A G-toA Mutation at Nucleotide Pair 8363 in MitochondrialtRNA(Lys) in Two Families. OZAWA, M., NISHINO, 1.,
HORAI, S., NONAKA, 1., GoTO, Y.
Polymorphism ofthe HLA-DRBI Locus in Colombian, Ecuadorian, and Chilean Amerinds. BLAGITKO, N., O'HUIGIN,
C., FIGUEROA, F., HORAI, S., SONODA, S., TAJIMA, K.,
WATKINS, D., KLEIN, J..
The Geographic Distribution of Human Y Chromosome
Variation. HAMMER, M.F., SPURDLE, A.B. KARAFET, T.,
BONNER, M.R., WOOD, E.T., NOVELLETTO, A., MALASPINA, P.,
MITCHELL, R.J., HORAI, S., JENKINS, T. and ZEGURA, S.L..
E-b. Division ofAgricultural Genetics
Mapped Genomic Locations for QTLs in Rice Reflect Gene
Clusters. CAl, H. W. and MORISHIMA, H..
Phylogenetic Study of AA Genome Wild Rice Species Viewed

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from Phenotypic and Genic Levels. AKIMOTO, M.,
SHIMAMOTO, Y. and MORISHIMA, H.
New Storage Protein Genes in Rice Detected by Acidic FormatePAGE Method. CAl, H.W. and MORISHIMA, H.
E-c. Division of Applied Genetics
Composite and dinal distribution of Glycine soja in Japan revealed by RFLP analysis of mitochondrial DNA.
TozuKA, A., FUKUSHI, H., HIRATA, T., OHARA, M.,
KANAZAWA, A., MIKAMI, T., ABE, J. and SHIMAMOTO, Y.
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Genetic diversity, geographical differentiation and evolution in
the cytoplasmic genome of the wild soybean, Glycine soja,
growing in China. SHIMAMOTO, Y., FUKUSHI, H.,
ABE, J., KANAZAWA, A., GAl, J., GAO, Z. and Xu, D.
Molecular Genetic Studies of Genomic Imprinting. SASAKI, H.
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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.
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Fine Mapping of a Preaxial Polydactyly Mutant, Rim 4.
MASUYA, H., SAGAI, T., WAKANA, S. and SHIROISHI, T.
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Genetic Analysis and Physical Mapping of Polysynductylous
Mouse Hemimelic extra toes (Hx). SAGAI, T., MASUYA, H.
and SHIROISHI, T.
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Mesenchymal Dysplasia (mes), which Exhibits Preaxial
Polydactyly. MAKINO, S., MAsUYA, H., TSUGANE, M. and
SHIROISHI, T.
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Effect of Ts Mutation on Mouse Development. ISHIJIMA, J.,
MITA, A., UCHIDA, K, SHIROISHI, T.
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Positional Cloning of the Mouse Skeletal Mutation, Tail short(Ts). SHIMIZU, K, KOIDE, T., MITA, A., UCHIDA, K.,
WAKANA, S., KIKKAWA, Y, YONEKAWA, H., SASAKI, H.

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CONTENTS

and SHIRaIsHI, N.
A susceptibility gene Idd4 controls onset of IDDM : an allele
from non- diabetic MSM strain is associated with early
onset of diabetes in mice. WAKANA, S., SHIROISHI, T.,
MORIWAKI, K., NOMURA, T.
Characterization of Hyperactive Behavior in MSM Derived fromJapanese Wild Mouse, and Hypoactive Behavior in JF1 Established from Japanese Fancy Mouse. KOIDE, T.,
MORIWAKI, K. and SHIROISHI, T.
Construction of Mouse Mutant Bank by CHL Mutagenesis.
SHIROISHI, T., FUKUDA, T., UCHIDA, K., MASUYA, M. and
MITA, A.....................................................................................
F-b. Mammalian Development Laboratory
Analysis of Cellular and Molecular Mechanisms in Developmentof Mouse Fetal Germ Cells and Sex Differentiation of
Gonads. NAKATSUJI, N., SHIRAYOSHI, Y. and SAITO, T.
Differentiation and Migration of Neural Cells in Mouse Central
Nervous System Development. SAITO, T. and NAKATSUJI, N.
..................................................................................................
Molecular Analysis of Cell Differentiation and Morphogenesis
in Postimplantation Mouse Embryos. SHIRAYOSHI, Y.
and NAKATSUJI, N.
F -c. Plant Genetics Laboratory
Structural and Functional Analysis of the Genes Expressed in
Early Embryogenesis in Rice (Oryza sativa). ITO, Y.,
EIGUCHI, M., KURATA, N.
Molecular Analysis of the Developmental Program during Regeneration Process of Rice Plant from Somatic Embryo.
KURATA, N. and EIGUCHI, M.
Positional Cloning of a Segregation Distortion Gene Detected in
the Progenies of Crosses with Japonica and Indica Rices.
HARUSHIMA, Y. and KURATA, N.
Isolation and Characterization of Rice Centromeric DNA Sequences for the Construction of Rice Artificial Choromosome.

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NONOMURA, K and KURATA, N.
Generation of Enhancer Trap Lines in Rice. ITo, Y., EIGUCHI, M.
and KURATA, N.
F-d. Microbial Genetics Laboratory
Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli. NISHIMURA, A.
ftsE ts affects translocation of K+-pump proteins into the cytoplasmic membrane of Escherichia coli. UKAI, H.,
MATSUZAWA, H., ITo, K, YAMADA, M. and NISHIMURA, A.
F-e. Invertebrate Genetics Laboratory
Leg pattern formation: specification in the embryo and proximodistal pattern formation in the larva. GOTO, S.,
KUBOTA, K. and HAYASHI, S.
Cell biological study of the tubular epithelial network formationin the tracheal system. TANAKA-MATAKATSU, M., IKEYA, T.
and HAYASHI, S.
Cdc2 dependent checkpoint couples M phase completion to initiation of S phase. HAYASHI, S.
plexus, a gene required for adult wing vein pattern.
MATAKATSU, H., GAMOU, S., TADOKORO, R. and HAYASHI, S.
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An enhancer trap screen for genes involved in patternformation. GOTO, S., TAKEUCHI, H., TANIGUCHI, M. and
HAYASHI, S.
Determination of the dorsoventral domains of neurogenesis by
the Drosophila EGF receptor. YAGI, Y. and HAYASHI, S. .....
G.

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Center for Genetic Resource Information
G-a. Genetic Informatics Laboratory
Cross-species functional gene database. YAMAZAKI, Y.
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Genetic Resources Database
2·1. Wheat Genetic Resources Database-KOMUGI. YAMAZAKI,Y., TSUCHIYA, R., TSUJIMOTO, H. and KAWAHARA, T.
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2-2. Rice Genetic Resources in Japan. YAMAZAKI, Y., SAITO, M.,
KINOSHITA, T. and MORISHIMA, K
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CONTENTS
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. Experimental Animal Database. TSUCHIYA, R.,
YAMAZAKI, Y. and ICHIKAWA, T.
2-6. Cloning Vector Database. YASUDA, S., SAITO, M. and
YAMAZAKI, Y.

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H. Stmctural Biology center
H-a. Biological Macromolecules Laboratory
Single Molecule Imaging of Enzymatic Reactions Using Objective-type Evanescent Illumination. TOKUNAGA, M.,
SAITO, K., KITAMURA, K., IWANE, A. and YANAGIDA, T.
Intermolecular Interactions measured by "Subpiconewton Intermolecular Force Microscopy". AOKI, T., HIROSHIMA,
M., YANAGIDA, T. and TOKUNAGA, M.
Single molecule capture, manipulation and force measurementof protein molecules. KITAMURA, K., IWANE, A.,
YANAGIDA, T. 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. SEN, R., KUBORI, T., NAGAI, H., HERNANDEZ,
V. J. and SHIMAMOTO, N.
Detection and characterization of conformation changes of proteins by a high-resolution protein footprinting. NAGAI, H.
and SHIMAMOTO, N.
Single-Molecule Dynamics of Transcription: Sliding of proteins
along DNA. KABATA, H., TAKEUCHI, M., KINEBUCHI, T.,
SHIMAMOTO, N., KUROSAWA, 0., ARAMAKI, H., W ASHIZU, M. ..
Functional domain of Esherichia coli Single-stranded DNA binding protein. KINEBUCHI, T., SHINDO, H., NAGAI, H.,
SHIMAMOTO, N., SHIMIZU, M.

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H·c. Multicellular Organization Laboratory
Fluoride-resistant Mutants of the Nematode Caenorhabditis
eJegans. TAKEUCHI, M., MOMI,A.,ISHIHARA, T. andKATsuRA,

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A Gene That Controls Both Hatching and Cel! Migration in C.
eJegans. HISHIDA, R., ISHIHARA, T. and KATSURA, 1.
Analysis of the Head Neural Circuit of C. eJegans as Studied by
the Formation of Dauer Larvae. KATsURA, 1., SUZUKI, N. and
ISHIHARA, T.
Reverse Genetics of Neural Genes of C. eJegans and the Analysisof Their Expression Using GFP (Jellyfish Green FluorescentProtein). ISHIHARA, T., FUJIWARA, M. and KATSURA, 1.
Functional Analysis ofInterneurons as Studied by Selection between Two Behaviors. ISHIHARA, T. and KATSURA, 1.
Functional Analysis of Neural Adhesion Molecules by Introduction of Clones Producing Antisense RNA. FUJIWARA, M.,
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.
Crystallographic Study of Escherichia coli RNA Polymerase a
Subunit. MURAKAMI, K., ISHIHAMA, A. and SHIRAKIHARA, Y.
..................................................................................................
H-e. Gene Network Laboratory
Expression pattern map of the C. eJegans genome.
KOHARA, Y., SHIN-I, T., MOTOHASHI, T., OHBA, T.,
SUGIURA, 1., OBARA, M., MIYATA, A., SANO, M., UESUGI,
H., WATANABE, H., MITANI, Y. and NAGAOKA, T.
pos-l encodes a cytoplasmic zinc-finger protein essential for
germline specification in C. eJegans. TABARA, H.,

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HILL, R.J., MELLO, C., PRIESS, J.R. and KOHARA, Y.
Development of a method to mesure the size of poly(A) tails in
Caenorhabditis elegans embryos and its application to somematernal genes. ONAMI, S. and KOHARA, Y.
Functional analysis of a Caenorhabditis elegans T-box genetbx-9. ANDACHI, Y.
I. Center for Information Biology
I-a. Laboratory for DNA Data Analysis
Genome plasticity as a paradigm of eubacteria evolution.
WATANABE, H., MORl, H., ITOH, T., GoJOBORI, T.
Constrained evolution of hepatitis B virus with overlapping
genes. MIZOKAMI, M., ORITO, E., OHBA, K., IKEO, K.,
LAu, J.Y.N. GOJOBORI, T,
Evolutionary mechanisms and population dynamics ofthe third
variable envelope region of HIV within single hosts.
YAMAGUCHI, Y. and GoJOBORl, T.
Ascidian tyrosinase gene: Its unique structure and expression in
the developing brain. SATO, S., MAsUYA, H., NUMAKUNAI T.,
SATOH, N., IKEO, K., GOJOBORI, T., TAMURA, K., IDE, H.,
TAKEUCHI, T., YAMAMOTO, H....................................................
Inference of molecular phylogenetic tree based on minimum
model-based complexity method. TANAKA, H., REN, F.,
OKAYAMA, T., GoJOBORI, T.
Molecular evolution of myelin proteolipid protein. Biochemical
and Biophysical Research Communications. KURIHARA,
T., SAKUMA, M., GOJOBORl, T.
Father-to-mother-to-infant transmission ofHIV-l: clonally transmitted isolate of infant mutates more rapidly than that of
the mother and rapidly loses reactivity with neutralizing
antibody. OKAMOTO, Y, SHIOSAKI, K, EDA, Y., TOKIYOSHI, S.,
YAMAGUCHI, Y, GoJOBORI, T., HACHIMORl, T., YAMAZAKI, S.,
HONDA, M.
Bacterial features in the genome of methanococcus jannaschii interms of gene composition and biased base composition in

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ORFs and their surrounding regions. WATANABE, H.,
GOJOBORI, T., MIURA, K.
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Evolutionary significance of intra-genome duplications on humanchromosomes. ENDo, T., lMANISffi, T., GoJOBORI, T., INOKO, H.
.................................................................................................. 143
Evolution of Nicotinic Acetylcholine Receptor Subunits.
TSUNOYAMA, K and GOJOBORI, T.
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Tie Trees Generated by Distance Methods of Phylogenetic
Reconstruction. TAKEZAKI, N.
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I-b. Laboratory for Gene-Product Informatics
Structural Requirement of Highly-Conserved Residues in
Globins. OTA, M., ISOGAI, Y and NISHIKAWA, K
148
Prediction of Protein Secondary Structure Using the 3D-ID Compatibility Algorithm. ITO, M., MATSUO, Y and NISHIKAWA, K.................................................................................................. 149
Differences in Dinucleotide Frequencies of Human, Yeast, and E.
coli Genes. NAKASffiMA, H. and NISffiKAWA, K
150
I-c. Laboratory of Gene Function
Evolutionary Motif and Its Biological and Structural
Significance. TATENO, Y, lKEO, K, WATANABE, H., ENDo, T.,
YAMAGUCffi, Y, SUZUKI, Y, TAKAHAsffi, K, TSUNOYAMA, K,
KAWAI, M., KAWANISHI, Y, NAITOU, K and GOJOBORI, T.
151
DNA Data Bank of Japan in the Age of Information Biology.
TATENO, Y. and GOJOBORI, T.
152
I-d. Laboratory for Molecular Classification
Development of the data processing system for DNA Data Bankof Japan (DDBJ). SUGAWARA, H., GOJOBORI, T., MORI, H.,
TAMURA, T., OKAYAMA, T., YAMAMOTO, H. and GOTO, K
152
Development of information systems for the study of
biodiversity. SUGAWARA, H., MIYAZAKI, S.
153
Homology modeling ofthe 3D structure of immunoglobulin.
MIYAZAKI, S. and SUGAWARA, H.
154

J.

Radioisotope Center
Feedback loops controlling the initiation of sporulation in Bacil-xv

CONTENTS

Ius subtilis. FUJITA, M. and SADAIE, Y.
156
Promoter Selectivity of the Bacillus subtilis RNA Polymerasea A and a H Holoenzymes. FUJITA, M. and SADAIE, Y.
157
Functional analysis of the phoBcotA region ofthe Bacillus subtilis
chromosome containing the konjac glucomannan utilization
operon. SADAIE, Y. and YATA, K.
157
Restricted Transcription from Sigma H or Phosphorylated SpoOA
Dependent Promoters in the Temperature-sensitive secA341
Mutant of Bacillus subtilis. ASAI, K., FUJITA, M.,
KAwAMURA, F., TAKAHASHI, H., KOBAYASHI, Y. and SADAIE, Y.
.................................................................................................. 158
The secA341 Mutation Inhibits Expression of the Bacillus
subtilis Protease Gene, aprEby Blocking DegSlDegU depending activation step. SADAIE, Y.
159
K.

Experimental FarmDevelopment, Evaluation and Distribution ofthe Genetic Stocksof Rice. NONOMURA, K., MIYABAYASHI, T., EIGUCHI,
M. and KURATA, N.

Abstracts of diary for 1997
Foreign visitors in 1997
Association for propagation ofthe knowledge of genetics
Author index

161
162
166
168
170

GENERAL STATEMENT
National Institute of Genetics (NIG) was established 48 years ago as acenter for genetics research. Major contribution of NIG in the research ingenetics, in particular population genetics, plant genetics and molecular anddevelopmental genetics, has 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 serve as Department of Genetics of the
Graduate School of Life Science. We have at present about 30 graduate students and about ten special research students from other universities, including those from abroad. This year, 10 students obtained Ph.D. In addition, we
have nine post-doctoral fellows ofthe center of excellence program. The steadyflow of young scientists is important to constantly rejuvenating our research
activities.
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. Our
institute also stores and distributes various organisms with genetically characterized traits. Among them, services with Escherichia coli, mice, rice andDrosophila are particularly significant. These service activities will continue
to develop in the coming years. Recent development of new technology using
various model organisms has enhanced the importance of genetics as bases of
biology. Therefore, it is timely that we established Genetic Strains ResearchCenter and Genetic Resource Information Center. Our institute is uniquely
suited for pursuing cooperative work with scientists of various disciplines by
sharing the genetic resources.
This year, we sponsored NIG International Symposium on "Gene Functions
to Cell Differentiation" organized by Junichi Tomizawa, former DirectorGeneral. We invited 21 foreign guests and 25 Japanese speakers. We alsoutilized this occasion to present many contributions from our Institute, andasked for comments, advice and reviews as to our activities. The reviewer's

2

critical comments are being used to make our new future plan to improve ourstandard. We sincerely hope that with guidance from people in and outside
NIG and further support from governmental and private sources, we will beable to lead the institute into a more successful future.
Junichi Tomizawa who introduced radical innovations retired at the end of
September 1997, and Yoshiki Hotta was elected as a new Director-General. I
consider that the contribution of Junichi Tomizawa in modernizing the Institute cannot be overstated. He paved a road for us to maintain our prestigious
position in the genetics research. Indeed, most of the works reported here areaccomplished under the guidance of the former Director-General.
In the past year we also saw a number of changes in the staff of theinstitute. Makio Tokunaga joined as associate professor of Structural Biology Center. Dr. Kensuke Horiuchi, Division of Microbial Genetics and Dr.
Tomoko Ohta, Division of Population Genetics, retired at the end of
March. Promoted as research associates are, Masataka Okabe (Div. of Developmental Genetics) and Tetsuichiro Saito (Genetic Strain ResearchCenter). On the other hand, the following members left NIG to take newpositions to extend their career. Among them are Kenji Izuhara (Kyusyu Univ.),
Atsushi Higashitani (Tohoku Univ.), Yasuo Ina (Biomol. Eng. Res. Inst.), Hideo
Goto (Nat. Inst. Animal Health), Kengo Kanamaru (Univ. of Tokyo) and Kenichi Matsumoto (Hokkaido Univ.). In the Department of Administration,
Hitoshi Sunada succeeded to the Dept. Head, and Hideo Kuroda moved toNagasaki Univ.

Yoshiki Hotta

3

STAFF (as of December 31,1997)
Director-General
HOTTA, Yoshiki, D. Med.

Vice-Director
OGAWA, Tomoko, D. Pha.

Members
1. Department of Molecular Cenetlcs

ISHIHAMA, Akira, D. Sc., Head of the 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
MIZUMOTO, Kiyohisa, D. Sc., Adjunct Professor

2. Department ofCell Cenetlcs
OGAWA, Tomoko, D. Pha., Head ofthe Department
Division ofCytogenetics
OGAWA, Tomoko, D. Pha., Professor
IMAI, Hirotami T., D. Sc., Associate Professor
TANAKA, Shigeo, D. Med.
OHTA, Tsutomu, D. Med.
Division ofMicrobial Genetics
YASUDA, Seiichi, D. Sc., Associate Professor
HARA, Hiroshi, D. Sc.

4Division ofCytoplasmic Genetics
YAMAMURA, Ken-ichi, D. Med., Adjunct Professor
HIRAOKA, Yasushi, D. Sc., Adjunct Associate Professor
3. Department 01Developmental Genetics

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.
HA'ITA, Masayuki, D. Sc.
OKABE, Masataka, M. D., Ph. D.
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 ofPhysiological Genetics
HANDA, Hiroshi, D. Med., Adjunct Professor
KANAYA, Shigehiko, D. Ind., Adjunct Associate Professor
4. Department 01 Population Genetics

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
ISHIKAWA, Hajime, D. Sc., Adjunct Professor

STAFF
5. Department ofIntegrated Genetics

IMAMURA, Takashi, D. Med., Head of the Department

Division ofHuman Genetics
IMAMURA, Takashi, D. Med., Professor
FUJIYAMA, Asao, D. Sc., Associate Professor
HORAI, Satoshi, D. Med., Associate Professor

Division ofAgricultural Genetics
OKINO-MoRISHIMA, Hiroko, D. Ag., Professor
CAl, Hong-Wei, D.Ag.

Division ofApplied Genetics
SHIMAMOTO, Yoshiya, D. Ag., Adjunct Professor
SASAKI, Hiroyuki, D. Med., Adjunct Associate Professor

6. Genetic Strains Research Center
NAKATSUJI, Norio, D. Sc., Head of the Center

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

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

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

7. Centerfor Genetic Resource Information
NAKATSUJI, Norio, D. Sc., Head of the Center
Genetic Informatics Laboratory

5

6

YAMAZAKI, Yukiko, D. Sc., Associate Professor
FUJITA, Masaya, D. Eng.

8. StructuralBloJolfY 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.

MulticelJuar Organization Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.

Biomolecular Structure Laboratory
SHIRAKIHARA, Yasuo, D. Sc., Associate Professor
AKIBA, Toshihiko, D. Sc.

Gene Network Laboratory
KOHARA, Yuji, D. Sc., Professor
ANDACHI, Yoshiki, D. Sc.
9. Center for Information BlololfY

GOJOBORI, Takashi, D. Sc., Head of the Center

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

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.
10.

IladJoisotope Center

SADAIE, Yoshito, D. Sc., Associate Professor, Head of the Center
11.

Experimental JiIlrm

KURATA, Nori, D. Ag., Head of the Farm
NONoMuRA, Ken-ichi, D. Ag.

12.

Tecbnlcal Section
MITA, Akihiko, Chief of the Section

13.

Department 01 Administration

SUNADA, Hitoshi, Head of the Department
TAlMA, Hitoshi, Chief of the General Mfairs Section
TAMURA, Mituo, Chief of the Finance Section

7

8

COUNCIL(as of December 31, 1997)

Chairman
SUGIMURA, Takashi; President,Toho University
Vice-chairman
OSAWA, Shozo; Adviser, Biohistory Research Hall
Members (Alphabetical order)
HAMA,Kiyoshi; President, Okazaki National Research Institutes
HARA, Hiroko; Professor, Institute for Gender Studies, Ochanomizu University
HIRASA, Takao; Presidenet, Osaka Prefectural University
HIROTA, Eiji; President, The Graduate University for Advanced Studies
IWATUSKI, Kunio; Professor, Rikkyo University
KATOH, Nobuo; President, Nagoya University
KYOGOKU, Yoshimasa; Professor, Institute for Protein Research, Osaka
University
MATSUBARA, Ken-ichi; Vice-Director,International Institute for Advanced
Studies
MIURA, Kin-ichiro; Director,Institute for Biomolecular Science, Gakushuin
University
MIYAMOTO, Misako; President, Japan Women's University
MOHRI, Hideo; Director-General, National Institute for Basic Biology
NOJIMA, Shoshichi; Professor Emeritus, The University of TokyoOSAKI, Hitoshi; Director, Japan Society for the Promotion of Science
SUGANO, Haruo; Director Emeritus, Cancer Institute
TAKANAMI, Mitsuru; Director, Kazusa DNA Research Institute
TANAKA, Ryuso; President, Hiroshima City University
TOYOSHIMA, Kumao; President, The Center for Adult Diseases, Osaka
YAMANOUCHI, Kazuya; Senior Scientific Staff, Nippon Institute for Biological Sciense

9

ADVISORY COMMITTEE(as of December 31,1997)

Chairman
MORISHIMA, Hiroko; Professor, National Institute of Genetics
Vice-chairman
OKADA, Masukichi; Professor Emeritus, University of Tsukuba
Outside Members (Alphabetical order)
Go, Mitiko; Professor, Graduate School of Science, Nagoya University
HANAOKA, Fumio; Professor, Institute of Molecular and Cellular Biology,
Osaka University
HINATA, Kokichi; Representative Managing Directer, Research Institute of
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 Insutitute of Medical Science, The
University of Tokyo
SEKIGUCHI, Mutsuo; Professor, Fukuoka Dental College
TAJIMA, Fumio; Professor, Graduate School of science, The University ofTokyo
Inside Members (Alphabetical order)
GOJOBORI, Takashi; Professor, National Institute of Genetics
HIROSE, Susumu; Professor, National Institute of Genetics
IKEMURA, Toshimichi; Professor, National Institute of Genetics
IMAMURA, Takashi; Professor, National Institute of Genetics
ISHIHAMA, Akira; Professor, National Institute of Genetics
KATSURA, Isao; Professor, National Institute of Genetics
NAKATSUJI, Norio; Professor, National Institute of Genetics
OGAWA, Tomoko; Professor, National Institute of Genetics
SHIMAMOTO, Nobuo; National Institute of Genetics
TATENO, Yoshio; National Institute of Genetics

10

RESEARCH ACTIVITIES IN 1997

A.

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

Molecular Anatomy of the a Subunit C-termlnal Domain ofEscherIchIa coli RNA Polymerase: Positioning of
Two a Subunit Carboxy-Terminal Domains on
Promoters by Two Transcription Factors

Katsuhiko MURAKAMI, Nobuyuki FUJITA, Olga N. OZOLINE, Jeffrey T.
OWENS!, Tamara A. BELYAEVA2, Claude F. MEARES!, Stephen J.W. BUSBy2 andAkira ISHIHAMA (lUniv. California, Davis, USA; 2Univ. Birmingham, Birmingham, UK)
The RNA polymerase holoenzyme of Escherichia coli is composed of thecore enzyme with subunit structure a2 {3{3' and one of multiple species of a
subunit with promoter recognition activity. Promoter selectivity of the holoenzyme is modulated by direct interaction with one or two of about 100
species of transcription factors. The RNA polymerase activity is also enhanced by direct interaction with DNA UP elements. The best characterizedtarget on the RNA polymerase involved in molecular communication with transcription factors is the carboxy-terminal domain (CTD) of a subunit that contains the contact sites for class-I transcription factors and DNA UP elements.
The binding sites oftwo aCTDs within a single RNA polymerase on the UP
element DNA were determined by hydroxyl radical-based DNA cleavage mediated by Fe-BABE [(p -bromoacetamidobenzyl)-EDTA-Fe] which was bound at
Cys-269 on the UP-recognition surface of one or both a subunits. The results
clearly indicated that the two a subunits bind in tandem to two helix turns of
the rrnBPI UP element, the {3- and {3'-associated a subunit being bound to
the promoter-proximal and promoter-distal region, respectively. Using the

A.

DEPARTMENT OF MOLECULAR GENETICS

11

same DNA cleavage method by a-conjugated Fe-BABE, interactions betweencAMP receptor protein (CRP) and aCTD were also analyzed at promoterscarrying tandem DNA sites for CRP binding. Each CRP dimer was found todirect the positioning of one ofthe two CTDs, indicating that two CRP dimers
can interact independently with the two aCTDs leading to direct their positioning along the promoter DNA. If other transcription factors interact withthe a, {3 or {3' subunits independently, RNA polymerase could potentially participate in regulatory complexes involving three or more different transcription factors. For details see Refs. 7, 11, 26 and 27.
(!)

Molecular Anatomy of the a Subunit C-terminal Domain ofEscherichia coJJ RNA Polymerase: Monitoring of RNAPolymerase-DNA UP Element Interaction by a
Fluorescent Probe Conjugated to a Subunit
Olga N. OZOLINE, Nobuyuki FUJITA, Katsuhiko MURAKAMI andAkira ISHIHAMA
Single Cys mutant a subunits were used for specific labeling with amonomercury derivative offluoresceine acetate (FMMA). The FMMA-modified RNA polymerase only at Cys269 of the a subunits was used to investigate RNA polymerase interaction with different promoters either with orwithout an UP element. Interaction with the rrnBPl UP element led to substantial alterations in the spectral parameters of the reporter label. A pronounced spectral blue shift suggests that the labeled surface of aCTD closely
approaches the charged UP DNA helix. The spectral blue shift was also observed when the modified RNA polymerase interacted with trp P, supporting the prediction that this promoter carries an rrnBP1-type UP element.
A new approach employing a fluorescent reporter label positioned in themultiple sites within the aCTD was then used to compare conformationalchanges of individual structural segments of the protein upon transcription
complex formation. FMMA was conjugated to a single Cys residue placed by
the site-directed mutagenesis at 9 different positions of aCTD. Spectral
parameters ofFMMA were compared between free and promoter-bound formsof the RNA polymerases, each carrying a modified a at a single position.
Reliable conformational changes were observed for binary complexes formed

12with UP element-dependent promoter rrnBPl and ternary complexes formed
with cAMP-CRP at class I promoter uxuAB. Our results confirm the previous data indicating that the helix I of aCTD and the loop region between
helices III and IV are involved in these complex formation and indicate thatsimilar pathways may be used by the RNA polymerase to realize the activation signal accepted from either the UP-DNA or the activator protein.
Some structural changes were also observed when the FMMA-modified RNA
polymerase was mixed with cAMP-CRP in the absence of DNA, confirming thedirect protein-protein contact. These changes were different from those observed for ternary complexes formed on the uxuAB promoter. For details see
Refs. 11, 33, 34 and 35.
(3)

Molecular Anatomy of the a Subunit N-termlnal Domain ofEscherichia coJl RNA Polymerase: Contact Sites for Dimerlc
Association Studied by Cleavage with Fe-BABE
Conjugated to Single Cysteine Residues

Reiko MIYAKE', Katsuhiko MURAKAMI, Jeffrey T.OWENS 1 , Nobuyuki
FUJITA, Claude F. MEARES' and Akira ISHIHAMA (I Univ. California, Davis,
USA)
The E. coli RNA polymerase core enzyme is assembled in vivo and invitro in the sequence: 2 a--- a2 ---'a2 8--+ a2 {3{3' (premature core) ---. E (activecore). Genetic and biochemical studies indicated that the subunit-subunit
contact sites on a including the sites for a dimerization and the contact sites
with {3 and {3' subunits are all located within the amino-terminal domain(NTD) down to residue 235, whereas the carboxy-terminal domain (CTD) is
involved in transcription regulation through direct interaction with class-I
transcription factors and DNA UP elements. These two functional domains
form independent structural domains, each being connected by a long flexible
linker.
Proximity relationships between the two associated monomers of RNA polymerase a subunit were studied using a set of four mutant a subunits, eachcarrying a single Cys residue at one ofthe naturally occuring positions. Thesemutants a subunits were conjugated with the cutting reagent Fe-BABE, andA.

DEPARTMENT OF MOLECULAR GENETICS

13

the peptide backbone was cleaved at locations near the modified Cys. Analysis
ofthe cleavage sites identified segments within approximately 12Afrom theconjugation site. The results show that intra-molecular cutting was observed
only within the individual a NTD and a CTD, supporting that the two functional domains form independent structural domains. On the other hand, forinter-molecular cutting, segments of the amino-terminal subunit assembly
domain of one a subunit and the linker region between aNTD and aCTD of
the other subunit are located close to each other on the three dimensional
structure, implying that the two a NTD are aligned in anti-parallel fashion.
For details see Refs. 7, 15 and 24.

(4)

Molecular Anatomy of the 8 and 8' Subunits of Escherichia colJ
RNA Polymerase: Mapping of Subunit-subunit Contact
Sites Studied by Proteolytic Cleavage

Tasuku NOMURA, Akira KATAYAMA, Dipankar CHATTERJI, Nobuyuki FUJITA
and Akira ISHIHAMA
The a subunit of E. coli RNA polymerase plays a key role in the assembly ofcore enzyme by providing the contact surface for both 13 and 13' subunits. In theassembled core enzyme, one a subunit contacts with 13 subunit while theother a makes direct contact with 13' subunit. We have been concerned withthe mapping of subunit-subunit contact sites on the a subunit. For mappingof the subunit-subunit contact sites on the two large subunits, we employed
two approaches: (i) analysis of proteolytic cleavage sites on both 13 and 13'
subunits for unassembled free subunits, the intermediate subassembly a213
complex and the core enzyme; and (ii) analysis of complex formation between
13 fragments and intact a subunit or between 13' fragments and the a2 13
complex. Results so far obtained indicate that two regions of the 13 subunit
is 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 control
activity downstream from residue 1138. All the a subunit- 13 fragment binary complexes were found to bind 13' subunit and form pseudo-core complexes, indicating that the regions of 13 involved in a -subunit contact also
participate in interaction with the 13' subunit.

14

Recently we succeded to cross-link ppGpp, the regulatory molecular of stringent control, with the {3 subunit, indicating that {3 is the target of ppGpp
action. The ppGpp-binding site seems to overlap with the primary contact
site of a subunit, while the binding sites for substrates and rifampicin arelocated upstream from the a contact site.
The assembly activity of {3 fragments was also examined in the presence ofboth a and {3' subunits. Pseudo-core complexes consisting of a, {3 fragmentand {3' were formed for {3(1-1318), {3(445-1342) and {3(737-1342), which allretain nearly the full activity of a binding. The level of pseudo-core complex
formation is much lower for {3(737-1138) in agreement with its low level activity of a binding. Thus, we conclude that the primary contact site of {3' subunit on {3 is located close to the a -subunit contact site, indicating that thebinding sites for a and {3' subunits form a single and the same structural
domain.
Tryptic cleavage of unassembled free {3' subunit indicated that the initialcleavage always takes place at sites between residues 800 and 950, splitting
this large polypeptide into two fragments. The N-terminallarge fragment isfurther cleaved around residue 350 into two subfragments. Significant difference in the {3' cleavage pattern was not observed for that assembled into
the core enzyme. Attempts to isolate {3' tryptic fragments which specificallyassociate with a 2 {3 complex, the assembly intermediate, appeares to be not
so useful. We then started to express and isolate {3' fragments using cloned
recombinant genes and test their abilities of subunit assembly. Refs. 4 and
11.

(5) Molecular Anatomy of the a Subunits of EscherIchIa colJ RNA
Polymerase: Mapping of Contact Sites with Promoter DNA andCore Enzyme Subunits Studied by Cleavage with Fe-BABE
Conjugated to Single Cysteine Residues
Jeffrey T. OWENS·, Jon BOWN2, Katsuhiko MURAKAMI, Nobuyuki FUJITA,Claude F. MEARES·, Stephen J.W. BUSBy2, Stephen D. MINCHIN 2 and Akira
ISHIHAMA (I Univ. California, Davis, USA; 2Univ. Birmingham, Birmingham,
UK)

A.

DEPARTMENT OF MOLECULAR GENETICS

15

Base-specific interactions between promoter DNA and RNA polymerase areregulated by 0 subunits during transcription initiation. To map spacial relations between the primary and major 0 subunit (0 70 ) and each DNA strand
along the lac UV5 promoter in the transcriptionally active open complex, we
used a Cys-tethered cutting reagent FeBABE to cleave DNA strands. Thischemical nuclease was conjugated to single-Cys mutants of 0 70 at sites 132C,
376C, 396C, 422C, 496C, 517C or 581C. After formation of open promoter
complexes, we observed DNA cleavage spanning at least 60 bases, between
positions -48 and + 12. The results show that 0 70 region 2.1 is proximal to
the nontemplate strand between the -10 element and positions as far downstream of the transcription start site as + 12. The conserved region 3.2 isproximal to the template strand near + 1, and region 3.1 is positioned between
the -10 and -35 elements.
This FeBABE-mediated DNA cleavage method was also employed to locate the position of region 2.5 of 0 70 in transcriptionally competent complexes.
Genetic studies indicated that the region 2.5 is involved in recognition of theextended -10 element with TGnTATAAT sequence. FeBABE was then tethered at 454C, 458C, 459C and 461C near or in region 2.5. Results of hydroxyradical based DNA cleavage indeed indicated that the region 2.5 is in close
proximity to promoter at position -14/-15 and this positioning is independentof promoter sequence.
FeBABE covalently tethered at various positions of 0 70 was also used tolocate the 0 70 -binding sites on the core enzyme. The protein-cutting probe
was covalently conjugated in or near the four conserved regions of 0 70 using theseven single Cys mutants. Each FeBABE-conjugated 070 was bound to thecore enzyme, which led to cleavage of nearby sites on the 8 and 8' subunits,
but not a subunit. The cut sites from different probe-modified 0 70 subunits
are clustered in distinct regions of the two large core subunits. On the 8subunit, cleavage is observed in two regions, one between residues 383 and354, including the conserved C and Rifregions; and the other between residues
854 and 1022, including the conserved G region, regions ofppGpp sensitivity,and one of the segments forming the catalytic center of RNA polymerase. On
the 8' subunit, the cleavage was identified within the sequence 228-461, including the 8' conserved regions C and D, which comprise part of the catalytic

16

center.

(6)

For details see Refs. 7, 31 and 32.

Regulation of the Activity of Sigma Subunits from Escherichia
colJ: Functional Interaction of Escherichia colJ RNA
Polymerase with Inorganic Polyphosphate

Shuichi KUSANO, J. GOWRISHANKAR and Akira ISHIHAMA
Upon entry into the stationary phase, transcription of a 70 -dependent genes
in E. coli is mostly turned off while that of a38 ( as)-dependent genes is switchedon, even though the intracellular level of a 70 stays at a constant level without
being degraded. In order to reveal the control mechanism(s) for preferential
utilization of a:38 in the stationary phase, we tested possible effects of theincrease in trehalose, glycine betaine, glycogen or polyphosphate concentrations and the decrease in DNA superhelicity on a 70 - and a 38 -dependent transcription in vitro. All these changes have been recognized for the stationaryphase E. coli cells. The optimum concentrations of glutamate and trehalosegiving maximum transcription by Ea 38 were higher than those by Ea 70 • The
activation of Ea 38 by trehalose was additive with the transcription enhancement by decreased superhelicity of template DNA prepared from stationaryphase cells. We thus propose that the selective activation oftranscription by
Ea 38 holoenzyme takes place in the presence of specific condition(s) and factor(s)present under stress conditions.
The ubiquitous occurrence of poly P in the stationary phase bacteria issuggestive of some important physiological role(s) for this polymer. Mutants
defective in the ppk gene encoding polyphosphate kinase (PPK) and thus
lacking long-chain poly P are defective in survival in the stationary
phase. Previously we observed the association of yet unidentified acidic
compound(s) with the RNA polymerase. Several lines of evidence indicatedthat the stationary-phase RNA polymerase is associated with poly
P. Functional assays indicate that at low salt concentrations, poly P inhibitstranscription by both a 70 and a 38 holoenzymes, but at high concentrations ofpotassium glutamate, the inhibition is relieved. In the presence of high concentrations of potassium glutamate, however, only the Ea 38 holoenzyme isallowed to function. These results altogether suggest that poly P plays a role

A.

DEPARTMENT OF MOLECULAR GENETICS

17

in the promoter selectivity control of RNA polymerase under stress conditions.
For details see Refs. 11, 12, 20, 21, 22 and 36.
(7)

A Stationary-Phase Protein in Escherichia col/ with Binding
Activity to the Major Sigma Subunit of RNA Polymerase

Miki JISHAGE, Akira IWATA', Nobuyuki FUJITA, Susumu UEDA 1 and Akira
ISHIHAMA (I Nippon Institute for Biological Science, Ohme, Tokyo)
E. coli contains seven different molecular species ofthe a subunit, the promoter recogniztion subunit of RNA polymerase. Using a quantitative Western immnoblot analysis, the intracellular levels of all seven a subunits were
determined. The concentration of a 70 (the product ofthe rpoD gene) required
for transcription of most of the genes expressed during exponential phase isabout 700 molecules per cell at both log and stationary phases, while a 38
( a s, the rpoS gene product) is detected only in the stationary phase, reaching
to about 200-300 molecules per cell. The levels of a 54 ( aN, the rpoN geneproduct) and a 28 ( a F , the rpoF gene product) are maintained at 10 and 50%
the level of a70 at both exponential and stationary phases, while other minor
a subunit, a 32 ( a H , the rpoH gene product), a 24 ( a E , the rpoE gene product) and a FecI (the fecI gene product), were hardly detected under normal growth
conditions.
In the course of these studies, we realized that heterogeneity exists among
the laboratory stocks of E. coli strains with respect to two a subunits, a F and
as. In particular, marked heterogeneity was observed in not only the intracellular level but also the structure of a 38 •Upon entry into stationary phase, the level of as increases to as much as
about 30-35% the level of a 70 , while the level of a 70 stays constant, eventhough the frequency of transcription of genes under the control of a 70 decreases by more than 10-fold. Known factors which contribute to the preferential utilization of as rather than a 70 in the stationary phase or under stressconditions include increases in intracellular concentrations of potassium
glutamate, trehalose, glycine betaine and polyphosphate and a decrease in
DNA superhelicity, all of which tend to reduce the activity of E a 70 but increases that of E as. In addition, we found that in stationary phase cells,

18

0 7°but not other 0 subunits forms a complex with a hitherto unidentified
stationary phase-specific protein. This protein begins to be synthesized during the transition from exponential growth to stationary phase. In vitro transcription studies indicated that the protein interferes with the engagement of
0 70 in the transcription cycle. After sequencing, the protein was identified asa product of one URF (unidentified reading frame) revealed by the genome
sequence analysis. We propose that this protein, designated Rsd (Regulatorof Sigma D), with the 07°-binding activity plays a role in controlling the 0 70
function in stationary-phase E. coli. Possible role of Rsd as an anti- 0 70
factor is being examined. For details see Refs. 11, 12, 16 and 17.

(8)

Sequence Specificity and Affinity of DNA Binding for Twelve
Species of Escherichia colJ DNA-binding Proteins

TALKUDER Ali Azam and Akira ISHIHAMA
The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of 4.7 Mb, to which about 10 major DNA-binding
proteins are associated, altogether forming the nucleoid. The species andamounts of these genome-associated proteins are considered to affect the
template activity of genome DNA in transcription. As an initial attempt toreveal the overall configutation of E. coli genome DNA under various growth
conditions, we compared the DNA-binding specificities of twelve species ofE. coli DNA-binding proteins: CbpA, curved DNA-binding protein A; CbpB,
curved DNA-binding protein B; DnaA, DNA-binding protein A; Dps, DNAbinding protein from starved cells; Fis, factor for inversion stimulation; Hfq,
host factor for phage Q B; H-NS, histone-like nucleoid structuring protein;
HU, heat-unstable nucleoid protein; IciA, inhibitor of chromosome initiation
A; IHF, integration host factor; Lrp, leucine-responsive regulatory protein;
StpA, suppressor of td- phenotype A. Up to the present time, our knowledge
on the specifity of DNA binding by these proteins is limited. In particular,
little is known on the specificity of DNA recognition by Dpa, Hfq, H-NS andStpA. We then purified all these DNA-binding proteins using the respective
cloned genes, and examined the sequence specificity and the affinity of DNA
binding in vitro using gel-mobility shift assays of various synthetic and natu-

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DEPARTMENT OF MOLECULAR GENETICS

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ral DNA probes. Five (CpbB, DnaA, Fis, IHF and Lrp) out of the twelve DNAbinding proteins showed sequence-specific DNA-binding, while the remaining
seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA and StpA) showed sequencenonspecific DNA-binding. From the apparent dissociation constant (Kd , app)determined using the DNA probes used, the order of DNA-binding affinity wasdetermined to be: CbpB>Fis>Lrp>IciA>IHF>StpA>Dps>HU>HNS>CbpA>Hfq.

(9)

Gene Organization and Protein Sequence of the Subunits of the
Fission Yeast Sehlzosaeeharomyees pombe RNA polymerase II

Hitomi SAKURAI and Akira ISHIHAMARNA polymerase II purified from the fission yeast Schizosaccharomyces
pombe contains more than ten different species of polypeptides, tentatively
designated as subunits. Previously, we cloned and sequenced both eDNA andthe genes encoding the four large subunits, Rpb1, Rpb2, Rpb3 andRpb5. Later, other groups isolated the genes for Rpb6 and Rpb12 and eDNA
for Rpb10. For cloning of the genes for other small-sized subunits, purified
RNA polymerase II was separated into individual subunits by SDS-PAGE andeach gel band was treated for partial proteolytic digestion and proteolytic
fragments were subjected to micro-sequencing. Using primers designatedfrom the partialamino acid sequences or the published DNA sequences, wecloned both eDNA and the genes encoding four small subunits, Rpb7, Rpb8,
Rpb10 and Rpb11. These genes were found to encode Rpb7, Rpb8, RpblOand Rpbll consisting of 172 (Mr 19,103), 125 (Mr 14,300), 71 (Mr 8,276) and123 (Mr 14,127) amino acid residues, respectively. All these four subunits
are homologous to the corresponding subunits of Saccharomyces cerevisiae
RNA polymerase II. The rpb 7 gene contains one intron while the rpbB, rpblOand rpbll genes contain two introns.
In addition, using degenarate primers from the known sequences of subunit
9 from other organisms, we also cloned both the rpb9 gene and its cDNA
encoding the subunit 9 of RNA polymerase II. From the DNA sequences, Rpb9
was predicted to consist of 113 amino acid residues with the molecular mass of
13,175. S pombe Rpb9 is 47,40 and 36% identical in amino acid sequence to20
the corresponding subunits from Saccharomyces cerevisiae, human andDrosophilla melanogaster, respectively. Previously we failed to detect Rpb9
in the purified RNA polymerase II by amino-terminal micro-sequencing ofproteolytic fragments of subunits separated by SDS-gel electrophporesis.
Mter Western blot analysis using antibodies raised against the protein product of the newly isolated rpb9 gene, we found that the purified RNA polymerase II contains Rpb9. Taken all our previous observations together thegene organization and the predicated protein sequence have been determinedfor eleven subunits of S. pombe RNA polymerase II. However, it is not established yet whether all these eleven putative subunits are necessary for RNA
polymerase functions or not. For details see Refs. 13 and 37.

(10)

Two Large Subunits of the Fission Yeast RNA Polymerase II
Provide Platforms for the Assembly of Small Subunits

Akira ISHIGURO, Kiyoshi YASUI, Makoto KIMURA, Akira IWATA', Susumu
UEDA 1 and Akira ISHIHAMA (I Nippon Institute for Biological Science, Ohme,
Tokyo)The two large subunits, Rpbl and Rpb2, of S. pombe RNA polymerase II
share high levels of sequence similarity with the {3' and {3 subunits, respectively, of prokaryotic RNA polymerases. The sequence of amino-terminal domain (NTD) of a subunit, which plays a key role in the subunit assembly byproviding the contact surface for both {3' and {3 subunitsis, is conserved inboth subunit 3 (Rpb3) and 11 (Rpb11) ofeukaryotic RNA polymerase II. Since
no prokaryotic counterparts have been identified for other putative subunits of
RNA polymerase II, little is known about the functions and the assembly
mechanism of other subunits.
The subunit-subunit contact network was then analyzed for the
Schizosaccharomyces pombe RNA polymerase II consisting of more than ten
putative subunits. Previously we carried out far-Western blot analysis ofbimolecular interaction with radio-labeled subunit 3 and 5 probes. This year
we extended the analysis using other six small-sized subunits, Rpb6, Rpb7,
Rpb8, Rpbl0, Rpb11 and Rpbl2, as probes. Taken the results together thesubunit-subunit interaction was observed for a total 18 (or 19) combinations.

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DEPARTMENT OF MOLECULAR GENETICS21

All eight small-sized subunits exhibited binding activities to the two large
subunits, Rpbl and Rpb2. In addition, bimolecular interaction was observed
for the combinations of Rpb3-Rpb5, Rpb3-Rpbll (and Rpb5-Rpb8/11). The
subunit-subunit contact within the assembled RNA polymerase was then analyzed by protein-protein cross-linking using five species of bifunctional crosslinkers with different length and specificity, i.e. dimethylsuberimidate (DMS),
2-iminothiolane hydrochloride (ITL), .N;N'-o-phenylene dimaleimide (PDM),
dimethyl 3,3'-dithio-bis propionimidate (DTBP) and diepoxybutane (DEB).
Cross-linking was observed for a total of 19 combinations, including five combinations between small subunits, Rpb3-Rpb10, Rpb3-Rpbll, Rpb5-Rpb6,
Rpb6-Rpb7 and Rpb6-Rpb8. The results altogether indicate that two large
subunits Rpbl and Rpb2 provide the platform for the assembly of small subunits and besides the small subunits interact each other for limited
combinations. Direct contact of the two large subunits, Rpb1 and Rpb2, wasalso demonstrated by cross-linking. Taken the results altogether we propose
a model ofthe subunit topology within the S. pombe RNA polymerase II. For
details see Refs. 10 and 13.

(11)

Molecular Composition of a Core Subassembly of
S. pombe RNA Polymerase II

Makoto KIMURA, Akira ISHIGURO and Akira ISHIHAMARNA polymerase II purified from the fission yeast Schizosaccharomyces
pombe consists of more than ten putative subunits. We introduced a histidine cluster tag sequence into the chromosomal rpbl and rpb3 genes which
encode subunit 1 (Rpb1) and subunit 3 (Rpb3), respectively. Using thesefission yeast strains, we could easily purified RNA polymerase II by Ni 2+ affinity chromatography. Furthermore, the RNA polymerase can be obtained
as a regin-bound form. Mter step-wise dissociation ofthe Rpb1-tagged andthe Rpb3-tagged RNA polymerases fixed on Ni2+ -resin by increasing concentrations of urea or guanidium hydrochloride, Rpb2-Rpb3-Rpbll or Rpb2-Rpb3Rpbll-Rpb10 complexes were obtained. Since the complex consisting of
Rpb2, Rpb3 and Rpbll cannot be dissociated even after treatment with 6 M
urea buffer, we propose that this complex represents a core subassembly ofthe22
RNA polymerase II, analogous to the a2 {3 complex in the assembly of Escherichia coli RNA polymerase.
Using the isolated subunits and subassemblies, we detected direct binding
ofthe isolated Rpb1 protein and of the Rpb2-Rpb3-Rpbll subassembly to
DNA in aqueous solution. The affinity for DNA was, however, weaker for both
the Rpb1 subunit and the Rpb2-Rpb3-Rpbll subassembly than the intact polII, suggesting that both the Rpb1 and Rpb2 subunits in the intact pol II participate in the high affinity binding. For details see Refs. 10 and 19.

(1!)

Mapping of the Subunit-Subunit Contact Sites on Rpbt, Rpb! andRpb3 of the Fission Yeast RNA Polymerase II

Kiyoshi YASUI, Takenori MIYAO, Akira ISHIGURO, Ayae HONDA, Zhangyi QU,
Makoto KIMURA, Hiroshi MITSUZAWA and Akira ISHIHAMA
Far-Western, GST pull-down and chemical cross-linking experiments indicated that the two large subunits of the fission yeast S. pombe RNA polymerase II provide platforms for the assembly of small subunits. Mapping of
the contact sites on two large subunits, Rpb1 and Rpb2, with two small subunits, Rpb3 and Rpb5, was carried out using the two-hybrid screening system in the budding yeast Saccharomyces cerevisiae. The Rpb5 was found tointeract with any fragment of Rpb1 containing the region H which is conserved among the subunit 1 homologues of all RNA polymerases, includingthe {3' subunit of prokaryotic RNA polymerases. In agreement with the factthat Rpb5 is shared among all three forms of eukaryotic RNA polymerases,
the region H of RNA polymerase I subunit 1 (Rpa190) was also found to interact with Rpb5.
On the other hand, the two-hybrid screening of Rpb2 fragments from theRNA polymerase II indicated the presence of Rpb3 contact site on the region
H which is conserved among the subunit 2 homologues of all RNA polymerases,
including the {3 subunit of prokaryotic RNA polymerases.
Previously we indicated that Rpb3 plays an essential role in subunit assembly because it interacts with at least five subunits, two large subunits (Rpbl
and Rpb2) and three small subunits (Rpb3, Rpb5 and Rpbll), and it constitutes a core subassembly consisting of Rpb2, Rpb3 and Rpb11. By making a

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DEPARTMENT OF MOLECULAR GENETICS

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set of Rpb3 deletion derivatives, we carried out mapping of the Rpb5- andRpb11-contact sites on Rpb3. By far-Western blot and GST pull-down assays, we found that the amino acid sequence between residues 105-263 of
Rpb3 is involved in binding Rpb5, and the sequence between residues 105-297
is required for binding Rpb11. Although the Rpb5- and Rpb11-contact sites
on Rpb3 overlap each other, both subunits are able to associate with Rpb3
simultaneously. The binding ofRpb5 stabilizes the Rpb3-Rpb11 heterodimer.
For details see Refs. 11, 25 and 41.

(13)

Mapping of the Functional Sites on Two Large Subunitsof the RNA Polymerase II: Photo-affinity
Cross-linking of 3'-Termini of Nascent RNA
Wjatschesslaw A. WLASSOFF, Makoto KIMURA and Akira ISHIHAMA
To locate the catalytic site for RNA polymerization of S. pombe RNA polymerase II, the catalytically competent transcription complex was photo-affinity labeled with photo-reactive nucleotide analogues, and the proteins wereseparated by SDS-PAGE for identification ofthe labeled subunits. Proteolytic
cleavage analysis of32P-labeled subunits allowed us to identify the exact location of the sites oflabeling on the subunits of RNA polymerase II. Using
this approach, we tried to cross-link transcriptionally competent elongation
complexes with photo-reactive nucleotides located at 3'-terminal ends of growing RNA chains.
Results altogether indicate that: (i) the RNA 3'-end binding site locates
between amino acids 934 and 994 of Rpb2; and (ii) regions 306-542 of Rpb2
and 509-917 of Rpb1 are situated in the vicinity of active center, probably
forming the catalytic site. All these regions include conserved motifs of RNApolymerases, and the active site belongs to the N-terminal part of conservative motif H. Proteolytic analysis of subunits Rpb 1 and Rpb2 has revealed
their complex multidomain organization, with the most available site of proteolysis being located in the junctions between the conservative motifs.

24

(14)

Genetic Mapping of the Functional Sites on Rpb3 and Rpbll,
Homologues of Prokaryotic Alpha Subunits

Jiro MITOBE, Miwa KOMOTO, Kiyoshi YASUI, Hiroshi MITSUZAWA and Akira
ISHIHAMA
RNA polymerase II of S. pombe consists of more than ten putative subunits.
To understand how these subunits interact with one another and with othertranscription factors, we have isolated temperature-sensitive S. pombe mutants with mutations in one of the subunit genes. As a first step, we chose
rpb3 and rpbll , whose products show homology to the E. coli RNA polymerase a subunit and form a core subassembly together with Rpb2 ( {3homologue). The rpb3 or rpbll genes were randomly mutagenized by PCR
and the amplified fragments were used to replace the chromosomal copy ofrpb3 or rpbll. Transformants were then screened for temperature-sensitive growth by replica-plating. Nine mutations for the rpb3 gene and sevenmutations for the rpbll gene, all of which result in single amino acid change,
were identified so far.
For rpb3 mutations, RNA polymerase II was affinity-purified from two
rpb3 ts strains as well as a wild-type strain. Weaker interactions of the mutant Rpb3 subunits with Rpb2, Rpb5 and Rpb7 were detected by treating theresin-bound enzymes with increasing concentrations of urea. The isolation of
extragenic suppressor mutations of rpb3 ts mutations is in progress. For rpbll
mutations, recombinant mutant proteins were produced and the interaction
with Rbp3 protein was tested by GST pull-down assays. Some mutant Rpbll
protein showed weaker interactions with Rpb3 protein. The isolation ofmulticopy suppressor genes of rpbll mutations is in progress.
(fli) The Molecular Anatomy of Influenza Virus RNA Polymerase:
Identification of the RNA Cap-binding Site on PHZ Subunit
Ayae HONDA, Kiyohisa MIZUMOTO' and Akira ISHIHAMA ('Div. 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 virus-

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DEPARTMENT OF MOLECULAR GENETICS

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associated RNA polymerase. The viral RNA polymerase is composed of threeviral P proteins, PBI, PB2 and PA. PBI carries the catalytic site of RNApolymerization while PB2 plays a role in capped RNA cleavage for generation
oftranscription 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 cross-linking
ofradioactive 8-azido GTP (8-N3 GTP) on the PBI subunit, i.e., the amino
terminal-proximal site I and the carboxy terminal-proximal site II, each being
close to that of sequence motifA and motif D, respectively, conserved among
RNA-dependent RNA polymerases.
On the other hand, capped RNA with 32p label only at its 5' cap-I structure
was photo-affinity cross-linked to the PB2 subunit. Isolated radio-labeled
PB2 was digested, after mixing with unlabeled PB2, with V8 protease andcleavage fragments were separated by SDS-polyacrylamide gelelectrophoresis. Fragments with cross-linked cap-I were eluted from gelsand subjected to amino acid sequencing. Results indicated that two regionsof PB2 are involved in the binding of 5' cap-I structure of capped RNA, one at
the N-terminal proximal region downstream from the PBI-contact site andthe other around 500 amino acid residue region overlapping the motif commonly found in cellular cap-binding proteins. For details see Refs. I and 8.

(16)

The Moleeular Anatomy of Influenza Virus RNA Polymerase:
Identlfleatlon of Host Faetor(s) for Intereonverslon
between Transerlptase Into Repllease

Ayae HONDA, Takuto OKAMOTO and Akira ISHlHAMA
Transcription ofthe genome RNA by the viral RNA polymerase is initiatedby using host cell capped RNAs as primers, while the mechanism of replication initiation remained unsolved. Analysis of the 5 ' -terminal structure ofgenome RNAs indicated that RNA synthesis for replication is initiated denovo without using primers. Both purified and reconstuted RNA polymerases
require primers for function, while the RNA polymerase in either virus-infected cell extracts or lysates of cells expressing three viral P proteins cancatalyze RNA synthesis in the absence of primers. We then proposed that an26
as yet unidentified host factor(s) is involved in the functional conversion of the
RNA polymerase from transcriptase to replicase. We have searched for theputative host factor(s) using yeast two hybrid screening system. So far morethan 10 cellular proteins have been identified, which interact with either PB1
or PB2 protein. Some of these putative host factors were found to form complexes in vitro by mixing with the PB1 or PB2 proteins. Functional conversion of the RNA polymerase after interaction with each of these proteins isbeing analyzed. For details see Refs. 8 and 9.
(t 7) Isolation of t30K/t80K Heterodimer with RNA-dependent RNAPolymerase Activity from TMV-infected Tobacco
Takato WATANABE, Ayae HONDA, Akira IWATA l , Susumu UEDA 1 , Tadaaki
HIBI 2 and Akira ISHIHAMA (I Nippon Institute for Biological Science, Ohme,
Tokyo; and 2University of Tokyo, Department of Agricultural and Environmental Biology)
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, 126 and 183 kDa proteins required
for transcription and replication (referred to as 130K and 180K proteins,respectively), 30 kDa (30K) protein for cell-to-cell virus movement in infected
plants, and 18 kDa protein for virus coat formation. The complete nucleotide
sequence was determined for the putative RNA polymerase (180K protein)gene oftobacco mosaic virus (TMV) OM strain, which differed from the related
strain, Vulgare, by 51 positions in nucleotide sequence and 5 residues in aminoacid sequence. Three segments of this 180K protein, each containing thesequence motifs of methyltransferase (M), helicase (H) or RNA-dependent
RNA polymerase (P), were expressed in Escherichia coli as fusion proteins
with hexa-histidine tag, and domain-specific antibodies were raised against
purified His-tagged M and P proteins. By immunoaffinity column chromatography, a heterodimer(s) consisting of one molecule each of the 130K (amino
terminal-proximal portion of 180K protein) and full-length 180K viral proteins was isolated, which exhibited the activity of model viral RNA templatedependent RNA synthesis in vitro. We propose that the TMV RNA poly-

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merase protomer is composed of one molecule each of the 130K and 180K
proteins.

Publ icalians
1.

2.

3.

4.

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6.

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AsANO, Y. and ISHIHAMA, A: Identification of two nucleotide·biding sites on the PB I
subunit of influenza virus RNA polymerase. J. Biochem., 122,627·634, 1997.
BOUCHER, P.E., MURAKAMI, K, ISHIHAMA, A and STlBITZ, S.: The nature of DNA·bind·ing and RNA polymerase interaction of the Bordetella pertusis BvgA transcriptional
activator at the fha promoter. J. Bacteriol., 179, 1755·1763, 1997.
BUYUKUSLU, N., TRIGWELL, S., LIN, P.P., RALPHS, N.T., FUJITA, N., ISHIHAMA, A. and
GLASS, RE.: Physical mapping of a collection of Mae! ·generating amber mutations
and the functional effect of internal deletions constructed through their manipulation.
Genes and Function, 1, 119·130, 1997.
CHA'ITERJI, 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, 279·
287, 1998.
CHUGANI, S.A, PARSEK, M.R, DOUGLAS, HERSHBERGER, C.D., MURAKAMI, K, ISHIHAMA, A
and CHAKRABARTY, AM.: Activation ofthe catBCA promoter: Probing the interactionofCatR and RNA polymerase through in vitro transcription. J. Bacteriol., 179,2221·
2227, 1997.
CHOY, H.E., HANGER, RR., MI, T., MAHONEY, M., MURAKAMI, K, ISHIHAMA, A and ADHYA,
S.: Repression and activation ofprooter·bound RNA polymerase activity by Gal reo
pressor. J. Mol. BioI., 272, 293-300, 1997.
GREINER, D.P., MIYAKE, R., MORAN, J.K, JONES, AD., NEGISHI, T., ISHIHAMA, A and
MEARES, C .F.: Synthesis of the protein cutting reagent iron (S)·I·(pbromoacetamidebenzym)ethylenediaminetetraacetate and conjucation to cysteine side
chains. Bioconjugate Chemistry, 8, 44·48, 1997.
HONDA, A and ISHIHAMA, A: The molecular anatomy of influenza virus RNA poly·merase. BioI. Chern., 378, 483·488, 1997.
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., in press.
ISHIGURO, A, KIMURA, M., YASUI, K, IWATA, A, UEDA, S. and ISHIHAMA, A: 'I\vo large
subunits ofthe fission yeast RNA polymerase II provide a platform for the assembly ofsmall subunits. J. Mol. BioI. 279, 703-712, 1998.
ISHlHAMA, A: Promoter selectivity control of Escherichia coli RNA polymerase. "Nucleic
Acids & Molecular Biology, Vol. 11, Mechanism of Transcription" (F. Eckstein and D.
Lilley, eds.), pp. 53·70, Springer·Verlag, Heidelberg, 1997.
ISHIHAMA, A: Adaptation of gene expression in stationary phase bacteria. Curro Opn.

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ISHIHAMA, A, KIMURA, M. and MITSUZAWA, H.: Subunits of yeast RNA polymerases instructure and function. Curro Opn. Microbiol. 1, 190-195, 1998.
ISHIHAMA, A, Zou, C., KOBAYASHI, M., KUMAR, A, FUJITA, N., SAKURAI, H. and HAYWARD,
R.S.: Molecular recognition in gene transcription. "Biomolecular Interactions in DNA
and Proteins" (Buckin, V. and Funck, T., eds.), Nova Sci. Publ., 1998.
JEON, YH., YAMAZAKI, T, OTOMO, T, ISHIHAMA, A and KYOGOKU, Y: Flexible linker inthe RNA polymerase a subunit facilitates the independent motion of the C-terminal
activator contact domain. J. Mol. BioI., 267, 953-962, 1997.
JISHAGE, M. and ISHIHAMA, A: Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli strain W3110. J. Bacteriol., 179, 959963, 1997.
JISHAGE, M. and ISHIHAMA, A: A stationary phase protein in Escherichia coli withbinding activity to the major 0 subunit of RNA polymerase. Proc. Nat!. Acad. Sci. USA,95, 4953-4958, 1998.
KANG, J.G., HAHN, M.Y, ISHIHAMA, A and ROE, J.H.: Identification of sigma factors for
growth phase-related promoter selectivity of RNA polymerases from Streptomyces
coelicolor A3(2). Nucleic Acids Res., 25, 2566-2573, 1997.
KIMURA, M., ISHIGURO, A and ISHIHAMA, A: RNA polymerase II subunits 2, 3 and 11
form a core subassembly with DNA binding activity. J. BioI. Chern., 272, 25851-25855,
1997.
KUNDU, TK., KUSANO, S. and ISHIHAMA, A: Promoter selectivity of Escherichia coli
RNA polymerase OF holoenzyme involved in transcription of flagellar and chemotaxis
genes. J. Bacteriol., 179, 4264-4269, 1997.
KUSANO, S. and ISHIlIAMA, A: Stimulatory effect of trehalose on the formation andactivity of Escherichia coli RNA polymerase Eo3s holoenzyme. J. Bacteriol., 179, 36493654, 1997.
KUSANO, S. and ISHIHAMA, A: Functional interaction of Escherichia coli RNA polymerase with inorganic polyphosphate. Genes to Cells, 2, 433-441, 1997.
MeHALL, S.M., KLEM, TJ., FUJITA, N., ISHIHAMA, A and CHAKRABARTY, AM.: DNase I
footprinting, DNA bending and in vitro transcription analysis of ClcR and CatR on thecJcABD promoter: Evidence of a conserved transcriptional activation mechanism.
Mol. Microbiol., 24, 965-976, 1997.
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.
MIYAO, T., HONDA, A, Qu, Z. and ISHIHAMA, A: Mapping of Rpb3 and Rpb5 contact
sites on two large subunits, Rpbl and Rpb2, of the fission yeast RNA polymerase II.
Mol. Gen. Genet., 259, 123-129, 1998.
MURAKAMI, K., KIMURA, M., OWENS, J.T., MEARES, C.F. and ISHIHAMA, A.: The two

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alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged andcontact diferent halves of the DNA UP element. Proc. Natl. Acad. Sci. USA, 94, 17091714, 1997.
MURAKAMI, K, OWENS, J.T., BELAEVA, T.A, MEARES, C.F., BUSBY, S.J.w. and ISHlHAMA,
A: Positioning of two alpha subunits carboxy-terminal domains of RNA polymerase
at promoters by two transcription factors. Proc. Natl. Acad. Sci. USA, 94, 11274-11278,
1997.
NAGAI, S., NAGASAWA, Y, YAGlHASHI, T. and ISHlHAMA, 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. Infect. Immun., 60, 227-238, 1998.
NEGRE, D., BONOD-BIDAUD, C., OUDOR, C., PROST, J.-F., KOLB, A, ISHlHAMA, A, COZZONE,
AJ. and CORTAY J.-C.: DNA flexibility of the UP element is a major determinant fortranscriptional activation at the Escherichia coli acetate promoter. Nucleic Acids
Res., %5, 713-718, 1997.
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 ofEscherichia coli. J. Mol. BioI., 276, 355-365, 1998.
OWENS, J.T., MURAKAMI, K, CHMURA, A, FUJITA, N., ISHlHAMA, A and MEARES, C.F.:
Mapping lac UV5 DNA sites proximal to conserved regions of sigma-70 in an Escherichia coli RNA polymerase-DNA open promoter complex. Biochemistry, 37, 7670-7675,
1998.
OWENS, J.T., MIYAKE, R., MURAKAMI, K, FUJITA, N., CHMURA, AJ., ISHlHAMA, 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.
OZOLlNE, O.N., MURAKAMI, K, NEGISHl, T., FUJITA, N. and ISHIHAMA, A.: Specificfluorescent labeling of two functional domains in RNA polymerase alpha subunit.
PROTEINS: Structure, Function, and Genetics, 30, 1-10, 1998.
OZOLlNE, O.N., MASULlSI, I.S., CHASOVI, v.v. and ISHlHAMA, A: Functional topology ofthe bacterial RNA polymerase. "Spectroscopy of Biological Molecules: Modern Trends"(P. Carmona, R. Navarro and A Hernanz, eds.), pp. 163-164, Kluwer Academic Publ.,
Dordrecht, 1997.
OZOLlNE,O.N., FUJITA, N., MURAKAMI, K and ISHlHAMA, A: Thpology ofthe Escherichia coli RNA polymerase surface involved in interaction with promoter upsteam
enhancer elements. Eur. J. Biochem., 253, 371-381, 1998.
RAJKUMARI, K, ISHlHAMA, A and GOWRISHANKAR, J.: Evidence for transcription attenuation rendering cryptic as-dependent promoter for the osmotically regulated proU
operon of Salmonella typhimurium. J. Bacteriol., 179,7169-7173, 1997.
SAKURAI, H. and ISHlHAMA, A: Gene organization and protein sequence of the small
subunits of Schizosaccharomyces pombe RNA polymerase II. Gene, 196, 165-174,
1997.

30
38. 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 and a 70 subunit of Escherichia coli RNA polymerase. J. Bacteriol., ISO, 3257-3259, 1998.
39. 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 coli
RNA polymerase. Int. J. Biochem. Cell BioI., %9, 1475-1483, 1997.
40 . YANG, J., MURAKAMI, K., CAMAKARIS, H., FuJITA, N., ISHIHAMA, A and PITIARD, AJ.: Aminoacid residues in the a subunit C-terminal domain of Escherichia coli RNA polymerase
involved in interactions with an UP-like sequence and the TyrR protein in the control
oftranscription from the mtr promoter. J. Bacteriol., 179, 6187-6191, 1997.
41. 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)

A Ublqultln Pathway Essential for Onset of Anaphase In Mitosis

Hiroaki SEINO and Fumiaki YAMAO
A eDNA encoding a novel ubiquitin-conjugating enzyme designated as UbcP4
in fission yeast was isolated. Disruption of its genomic gene revealed that itwas essential for cell growth. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M
and metaphase/anaphase transitions. The G2-arrest of UbcP4-depleted cells
was dependent upon chkl which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had duplicated and condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpressionof UbcP4 specifically rescued the growth defect of cut9 ts cells at a restrictive
temperature. cut9 encodes a component ofthe anaphase promoting complex(APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin infission yeast, was accumulated in the UbcP4-depleted cells. These results
strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in
conjunction with APC and mediates ubiquitin pathway for degradation of "sis-A.

DEPARTMENT OF MOLECULAR GENETICS

31

ter chromatid holding protein(s)" at the onset of anaphase and possibly of
mitotic cyclin at the exit of mitosis (2). These phenotypes were confirmed byusing temperature-sensitive mutant of ubcP4, which indicated that progression of each cell cycle step mentioned above was blocked by dysfunction of the
UbcP4 pathway.
Cig2, another B-type cyclin in fission yeast, has the same type of destruction
signal as Cdc13. We examined the possibility ofthe degradation of Cig2 via
UbcP4 pathway, providing the genetic interaction between genes encoding thetwo proteins. In order to identify genes that could be functioning in or aroundthe UbcP4/APC pathway, two approaches were adopted, screening of themulticopy suppressors of ubcP4 mutations, and search of conditional mutants suppressible by UbcP4 overproduction. Characterization ofthe candidates from these screening is now undergoing.

(2)

CDC34 Dependent-Ubiqultin Pathway: Role of 6RRI in the
Ubiqultination of 61 CyelinCln2

Tsutomu KISHI and Fumiaki YAMAO
In S. cerevisiae, Cdc34, Cdc4 and Cdc53 form a complex, and are required
for the G1-S transition. CDC34 encodes a ubiquitin-conjugating enzyme,
suggesting that Cdc34-dependent proteolysis is necessory for the cell cycle
progression. Sic1, as-phase CDK inhibitor, has been identified to be an
essential target of this pathway. Other essential target proteins could be
elucidated since a cdc34 sic1 double mutant still arrests in G2.
We have isolated extragenic suppressors of a cdc34-1 sic1 mutant. One
group of suppressors was recessive, and was found to be allelic to GRRl, agene shown to be involved in degradation of G1 cyclins (Cln 1 and Cln2) aswell as glucose repression. Conversely, overproduction ofGrr1 in cdc34 sic1
cells impaired colony formation at 30t. We isolated MGOI (Multicopy suppressor of Grr1 Overproduction defect) which suppressed this growth defectwhen introduced on a multicopy vector. Sequence analysis revealed that
MGOI is identical to SKPl, which has been shown to be required for theubiquitin-meadiated proteolysis of Cln2 and Sic1 (4).
We found that Grr1 binds Skp1 in vitro and in vivo. The interaction was

32dependent on the F-box in Grrl. We also found that Grrl binds phosphorylated Cln2 in vivo. The interaction was dependent on the leucine-rich repeats(LRR). Furthermore, we demonstrated that (1) Cln2 is stabilized in cellswith mutant Grrl (Grrl-dF), which is defective in Grrl-Skpl association, (2)Cln2 is stabilized in cells with mutant Grrl (Grrl-dL), which is defective in
Grrl-Cln2 association, and that (3) phosphorylated Cln2 associated with Skpl
dependently on Grrl in vivo. These results indicate that Grrllinks phosphorylated Cln2 with Skpl (submitted).

Publ icatians
1.

2.

3.

4.

5.

IKEIIATA, H., KANEDA, S., YAMAO, F., SENO, T., ONO, T. and HANAOKA, F.: Incubation at
the nonpermissive temperature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a temperature-sensitive ubiquitin-activating
enzyme (El). Mole. Cell. BioI., 17, 1484-1489, 1997
OSAKA, F., SEINO, H., SENO, T. and YAMAO, F.: A ubiquitin-conjugating enzyme in fission
yeast, that is essential for the onset of anaphase in mitosis. Mole. Cell. BioI., 17, 33883397, 1997
NAKAJIMA, T., KIMURA, M., KURODA, K, TANAKA, M., KIKUCHI, A, SEINO, H., YAMAO, F.
and aDA, K: Introduction of Ubiquitin-Conjuguting Enzyme Activity for Degradationof Topoisomerase II a during Adenovirus EIA Induced Apoptosis. Biophys. Biochem.
Res. Commun., 239, 823-829, 1997
KISHI, T., SENO, T. and YAMAO, F.: Grrl functions in the ubiquitin pathway in
Sacchromyces cerevisiae through association with Skp1. Mole. gen. Genet., 257, 143148, 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., 180, 100-106, 1998

A-c. Division of Nucleic Acid Chemistry(1)

Enzyme mechanism of the 5' -terminal capping of eukaryotlc mRNA

Kiyohisa MIZUMOTO (School of Pharmaceutical Sciences, Kitasato University)
Previous studies demonstrated that the mammalian mRNA capping enzyme is a bifunctional enzyme containing RNA 5 ' -triphosphatase and RNA

A

DEPARTMENT OF MOLECULAR GENETICS

33

guanylyltransferase activities in a single polypeptide. In yeast, these two
activities are separated into two different subunits, ex (RNAguanylyltransferase) and {3 (RNA 5'-triphosphatase), the genes for which wehave cloned recently. It is thus interesting to compare the structural andfunctional relationships between the mammalian and yeast capping
enzymes. We isolated two human cDNAs encoding mRNA capping enzymes
termed hCAPla and hCAPlb which code for 597 and 541 amino acids,
respectively. They are different only at the region coding for the C·terminal
portion of the enzyme. Comparison of the deduced amino acid sequenceswith other cellular and viral capping enzymes showed that all the regions
conserved among RNA guanylyltransferases including the yeast ex subunit are
observed in our clones except one conserved C-terminal region which was absent in the hCAPlb protein. The purified recombinant hCAPla gene product, hCAPla, exhibited both RNA 5 ' ·triphosphatase and RNA
guanylyltransferase activities. Deletion mutant analysis ofhCAPla showedthat the N-terminal 213 amino acid fragment containing a tyrosine specificprotein phosphatase (PTP) motif catalyzed the RNA 5 '-triphosphatase activo
ity, and the C-terminal 369 amino acid fragment had the mRNA
guanylyltransferase activity. On the other hand, hCAPlb showed RNA 5'·
triphosphatase activity, but exhibited neither the enzyme-GMP covalent complex formation nor the cap structure formation. Interestingly, comparison of
the amino acid sequences between the N-terminal RNA triphosphatase do·main of hCAPla and the yeast {3 subunit showed no similarity, and the {3subunit had no PTP motif, although they catalyze the same reaction.

Publications
TSUKAMOTO, T" SHIBAGAKI, Y., IMAJOH-OHMI. S" MURAKOSHI, T., SUZUKI, M., NAKAMURA, A,
GOTOH, H" MlZUMOTO, K: Isolation and characterization ofthe yeast mRNA capping
enzyme 8 subunit gene encoding RNA 5'-triphosphatase, which is essential for cellbiability. Biochem. Biophys. Res. Comm., 239, 116-122, 1997.
2, SERIZAWA, H" TSUCHIHASHI, Z., MlZUMOTO, K: The RNA polymerase II preinitiation
complex formed in the presence of ATP. Nucleic Acids Res., 25, 4079-4084, 1997,
3. MURAKAMI, M, WATANABE, H., Nil KURA, Y., KAMEDA, T., SAITOH, K, YAMAMOTO, M" YOKOUCHI,1.

34y, KUROIWA, A, MlZUMOTO, K, IBA, H.:

High level expression of exogenous genes by

replication-competent retrovirus vectors with an internal ribosomal entry site. Gene,
202, 23-29, 1997.
4.

TSUKAMOTO, T., SHIBAGAKI, Y., MURAKOSHI, T., SUZUKI, M., NAKAMURA, A, GOTOH, H., andMIZUMOTO, K: Cloning and characterization oftwo human cDNAs encoding the mRNA
capping enzyme. Biochem. Biophys. Res. Comm., 243, 101-108, 1998.

5.

KUROTANI, A, KIYOTANI, K, KATO, A, SHIODA, T., SAKAI, Y, MlZUMOTO, K, YOSHIDA, T.,
and NAGAI, Y: The paramyxovirus, Sendai virus, C proteins are categorically nonessential gene products but silencing their expression critically impairs viral replicationand pathogenicity. Genes to Cells, 3, 111-124, 1998.

35

B.

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

S. cerevlslae recA. homolognes HA.D51 and DMCI have both distinct
and overlapping roles In meiotic recombination

Tomoko OGAWA
Rad51 and Dmcl are Saccharomyces cerevisae homologues of the Escherichia coli recombination proteins RecA. Mutant analysis has shown thatboth proteins are required for normal meiotic recombination, for timely andefficient formation of synaptonemal complex and for normal progression out
from meiotic prophase.
We have further characterized rad51 and dmcl single mutants. A dmcl
mutation confers more severe defects in double strand break (DSB) resolution, crossover recombination and meiotic progression than does a rad51
mutant; in contrast, during return to growth, a rad51 mutation confers more
severe defects in viability and intrachromosomal recombination than does admcl mutation. Analysis of a rad51 dmcl double mutant, in parallel withsingle mutants, shows that the double mutant is more defective with respect
to the formation of crossovers during meiosis and, especially strikingly, withrespect to interhomologue and intrachromosomal recombination during return to growth. Consistent with the observation of DMCI -dependent recombination in a rad51 mutant, subnuclear complexes ofDmcl protein were detected for the first time in this mutant. In contrast to the effects on recombination, the effect of double mutant on meiotic progression was similar to thatof the rad51 single mutant.
Rad51 and Dmcl each make unique contributions to meiotic recombination.
However, the two proteins are capable of substituting for one another undersome circumstances, implying that they most likely share at least one recombination function. Recombination and cell cycle phenotypes are all consistent with the possibility that a dmcl mutation causes an arrest of the post-

36

DSB recombination complexes at a later, more stable stage than does a rad51
mutation. see Ref. 1(Z)

Complete Nucleotide Sequence of the Chloroplast Genome fromthe Green Alga Chlorella vulgaris; The Existence ofGenes Possibly Involved in Chloroplast Division

Tsutomu OHTA
The complete nucleotide sequence of the chloroplast genome (150,613bp)
from the unicellular green alga ChIarella vulgaris C-27 has been determined.
The genome contains no large inverted repeat and has one copy of rRNA
gene cluster consisting of 16S, 23S, 5S rRNA genes. It contains 31 tRNA
genes, of which the tRNA Leu (GAG) gene has not been found in land plant
chloroplast DNAs analyzed so far. Sixty-nine protein genes and eight ORFs
conserved with those found in land plant chloroplasts have also beenfound. The most striking is the existence of two adjacent genes homologous
to bacterial genes involved in cell division, minD and minE, which are arranged in the same order in Escherichia coli. This findings suggests that themechanism of chloroplast division is similar to bacterial division. see Ref. 2(3)

A Recombinational Defect In the C-termlnal Domain ofEscherichia colJ RecAZZ78-5 Protein Is Compensatedby Protein Binding to ATP
Andrei ALEXEEVE and Tomoko OGAWA
RecA2278-5 is a mutant RecA protein (RecA mut) bearing two amino acidsubstitutions, Gly-278 to Thr and Val-275 to Phe, in the alpha-helix H of the
C-terminal subdomain of the protein. RecA2278-5 mutant cells are unusualin that they are thermosensitive for recombination but almost normal for
DNA repair of UV damage and the SOS response. Biochemical analysis of
purified RecA mutant protein revealed that its temperature sensitivity issuppressed by prior binding of this protein to its ligand. In fact, the preheating of RecA mutant protein for several minutes at a restrictive temperature(42'C) in the absence ofATP resulted in inhibition at 42'C of many activities

B.

37

DEPARTMENT OF CELL GENETICS

related to homologous recombination including ss- and dsDNA binding, highaffinity binding for ATP, ss- or dsDNA-dependent ATPase, RecA-RecA interaction, and strand transfer capability. The binary complex RecA mutant::ATPunder the same conditions showed a decrease in only two activities, i.e. dsDNA
binding and high-affinity binding for ATP. Besides ATP, sodium acetate (1.5
M) was shown to be another factor that can stabilize the RecA mutant protein at 42'C, judging by restoration of its DNA-free ATPase activity. The
similarity of influence of high salt (with its non-specific binding) and ATP(binding specifically) on the apparent protein folding stability suggests thatthe structural stability of the RecA C-terminal domain is one of the conditions
for correct interaction between RecAprotein andATP in the RecA::ATP::ssDNA
presynaptic complex formation. The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not
repair) thermosensitivity of the RecA-mutant protein. Finally, we show thatthe stability of C-terminal domain appeared to be necessary for the dsDNAbinding activity of the protein. see Ref 3.

Publ ications
1.

SHINOHARA, A, GAISOR, S., OGAWA T., KLECKNER, N. and BISHOP, D. K:

2.

WAKASUGI, T., NAGAI, T., KApOOR, M., OHTA, T., YOSHINAGA, K and SUGIURA, M.:

S. cerevisiae
recA homologues RAD51 and DMCl have both distinct and overlapping roles inmeiotic recombination. Genes to Cells, Z, 615-629 (1997).
Complete

nucleotide sequence of the chloroplast genome from the green alga ChIarella vulgaris;
The existence of genes possibly involved in chloroplast division. Proc. Natl. Acad. Sci.
3.

USA 94:5967-5972 (1997).
ALEXSEYEV, AA, BAITIN, D.M., KURAMITSU, S., OGAWA, T., OGAWA, R., LANZOV V. A.:

A

recombinational defect in the C-terminal domain of Escherichia coli RecA2278-5 protein is compensated by protein binding to ATP. Mol Microbiol. Z3, 255-265.

Oral Presentations
1.

OGAWA, T. and SHINOHARA, A:

Stimulation ofRad51-mediated Recombination by Rad52

in Saccharomyces cerevisiae. Third European Conference on "Meiosis" The Netherland,
Wageningen, April.
2.

OGAWA, T., ALEXEEV, A, WATANABE, K, OHTA, T. and NABETANI, A.:

ControiofRAD51

38

3.

4.

5.

6.
7.

gene expression in the cells with DNA damage and those in meiosis. Third European
Conference on "Meiosis" The Netherland, Wageningen, April.
SHINOHARA, A. and OGAWA, T.: Stimulation of Rad5 I-mediated Recombination by Rad52
in Saccharomyces cerevisiae. FASEB Meeting on "Genetic Recombination and generearrangement", USA, Aug.
OGAWA, T. and SHINOHARA, A.: Stimulation of Rad51-mediated Recombination by
Rad52 in Saccharomyces cerevisiae. International Symposium on "Gene Functions
and Cell Differentiation", Japan, Sep.
OGAWA, T, OHTA, T. and SHINOHARA, A.: Stimulation by Rad52 of yeast Rad51-mediated Recombination. International Symposium on "Replication, recombination andrepair" (3Rl, Japan, Oct.
OGAWA, T.: Stimulation by Rad52 of yeast Rad51-mediated recombination, International Symposium on "Germ Cell Development and Meiotic Regulation", Japan, Nov.
OHTA, T, KAWANE, K., USUI, T, OGAWA, H. and OGAWA, T.: Characterization of Mrell
Protein of S. cerevisiae. Workshop on the DNA Transaction in Recombination at The
XXth Annual Meeting of Molrcular Biology, Kyoto, Dec.

B-b. Division of Microbial Genetics(1)

Characterization of the enyC mutation that affects septation andleads to chain formation in E. colJ

Hiroshi HARA, Setsuko NARITA1.2, Yoshihiro YAMAMOT0 3, and Yukinobu
NISHIMURA 1 (IFaculty of Sciences, Toho University, 2Department of ClinicalLaboratory, Sanritsu Co. Ltd., 3Department of Genetics, Hyogo College ofMedicine)
PM61 is a non-conditional morphological mutant of E. coli that forms chainsof cells of irregular lengths. It carries the only known mutant allele of envC
that appears to disturb septum formation and cell separation. The mutation also causes hypersensitivity to crystal violet, leakage of periplasmic proteins, and thermosensitivity on salt-free L agar. We cloned the envC genethat complemented all these defects and identified the mutation site of theenvC61 allele (His 366 ~ Tyr). The gene was named yibP in the DNA databases and the map position was 81.5 min. There was a report ofthe cloningof a gene at 73.5 min as envC, but that gene was probably a multicopy suppressor of the crystal violet sensitivity and was not the true envC.

B.

DEPARTMENT OF CELL GENETICS39

The envC gene product was identified as a protein of about 45 kDa foundmainly in the periplasm after the cleavage of the signal peptide. The sequence of the N-terminal half of the mature form has features characteristic of
the coiled coil structure. The C-terminal third ofEnvC shows homology to the
C-terminal regions of E. coli NlpD and YebA, and the mature form of lysostaphin, an extracellular peptidoglycan-degrading enzyme of Staphylococcus.
As was suggested for NlpD, EnvC may also be involved in the remodeling of
the peptidoglycan sacculus during cell growth and division. Overproductionof NlpD was reported to cause bulge formation and eventually cell lysis.
Disruption of the chromosomal envC gene by insertion of an antibiotic
resistance gene resulted in the same defects that the envC61 mutant showed.
The envC gene is dispensable for growth, but important for the normal septation/separation and the maintenance of integrity of the cell surface
structure. The envC':cat allele truncates the product to about one-third. It
seems likely that the envC mutation (H366Y) also leads to the loss of function.
When the wild-type envC was overexpressed, the growth was inhibitedand morphological aberration was observed. Overexpression of the envC61
mutant allele showed an even stronger inhibitory effect, and caused similarmorphological aberration and cell lysis. The inhibition was possibly due tothe interaction of the overproduced wild-type/mutant EnvC proteins with asecond protein.
(!)

Contribution of the p.r. promoter to expression of genes
in the Escherichia colJ mra cluster of cell envelope
biosynthesis and cell division genes

Dominique MENGIN-LECREULX I , Juan AYALA 2, Ahmed BOUHSS 1, Jean VAN
HEIJENOORT 1, Claudine PARQUET l , and Hiroshi HARA (ILaboratoire desenveloppes bacteiennes, Centre National de la Recherche Scientifique,
Universite Paris-sud, France, 2Centro de Biologia Molecular Severo Ochoa,
Consejo Superior de Investigationes Cientificas, Universidad Autonoma de
Madrid, Spain)
Recently, a promoter for the essential gene its!, which encodes penicillin-40binding protein 3 of Escherichia coli, was precisely localized 1.9 bp upstream
from this gene, at the beginning of the mra cluster of cell division and cellenvelope biosynthesis genes. Disruption of this promoter (P mrs) on the chromosome and its replacement by the lac promoter (P mrs::PIs) led to isopropyl13 -D-thiogalacto pyranoside (IPTG)-dependent cells that lysed in the absenceof inducer, a defect which was complemented only when the whole region from
P mrs to ftsW, the fifth gene downstream from fts!, was provided in trans on aplasmid.
In the present work, the levels of various proteins involved in peptidoglycan
synthesis and cell division were precisely determined in cells in which P mrs::PIsc
promoter expression was repressed or fully induced. It was confirmed that
the P mrs promoter is required for expression ofthe first nine genes of the mracluster: mraZ (orfC), mraW (orfB), ftsL (mraR), Its!, murE, murF, murD,and fts W. Interestingly, three- to sixfold- decreased levels ofMurO and MurC
enzymes were observed in uninduced P mrs::PIsccells. This was correlated withaccumulation ofthe nucleotide precursors UDP-N-acetylglucosamine and UDPN-acetylmuramic acid, substrates of these enzymes, and with a depletion of
the pool of UDP-N-acetylmuramyl-pentapeptide, resulting in decreased cellwall peptidoglycan synthesis. Moreover, the expression of ftsZ, thepenultimate gene from this cluster, was significantly reduced when P mrs expression was repressed. It was concluded that the transcription of the genes
located downstream from ftsW in the mra cluster, from murG to ftsZ, is also
mainly (but not exclusively) dependent on the P mrs promoter.

Publ icatians
1.

HARA, H., YASUDA, S., HORIUCHI, K. and PARK, J. T.: A promoter for the first nine genesof the Escherichia coli mra cluster of cell division and cell envelope biosynthesis genes,
including its1 and itsW. J. Bacteriol. 179, 5802·5811, 1997.

B.

DEPARTMENT OF CELL GENETICS

41

B-c. Division of Cytoplasmic Genetics(1)

Targeted Integration of DNA using mutant lox
sites In embryonic stem cells

KimiARAKI, MasatakeARAKI and Ken-ichi YAMAMURA
Site-directed integration has been achieved by using a pair of mutant lox
sites [Albert, H., et al. (1995) Plant J. 7, 649-659.] in embryonic stem (ES)cells. We established ES cell lines carrying single copy ofloxP or mutated lox
site as a target, and examined the frequency of site-specific integration by Cre
transient expression. Since the targeting vector that we constructed contains
a complete neo gene, random integrants can also form colonies like a simple
gene targetting. In this system, the frequency of site-specific integration via
the mutant lox sites reached to maximum 17%.

On the contrary, the wild

type 10xP sites yielded site-specific integration events at quite low frequencies(under 0.5%). This mutated lox system will be useful for "knock-in" using ES
cells. For the details, see Ref. 1.

(2)

Efflelency of recombination by Cre transient expression In embryonic stem cells: Comparison of various promoters

Kimi ARAKI, Takashi IMAIZUMI, Keiji OKUYAMA, Yuichi OIKE and Ken-ichi
YAMAMURA

The Cre-IoxP recombination system of bacteriophage PI is now frequently
utilized in genetic manipulation in embryonic stem (ES) cells. The level ofCre expression is critical to induce 10xP site-specific recombination in ES
cells. In order to compare the efficiency of recombination, we have constructedfour ere expression vectors driven by different promoters, that arecytomegarovirus / chicken b-actin (CAG) promoter, human polypeptide chain
elongation factor la (hEF-la) promoter, mouse phosphoglycerate kinase-l(mPGK) promoter and polyoma enhancer / herpes simplex virus thymidine
kinase (MCl) promoter. We introduced these ere expression vectors byelectroporation into three ES cell lines carrying a single copy of CAG-IoxPchloramphenicol acetyltransferase (CAT) gene-IoxP-b-galactosidase (b-gal)

42gene construct. Since the Cre-mediated recombination leads to the excisionof CAT gene, the efficiency of recombination can be monitored by b-gal
expression. No selection system was used in the experiments. The maximum recombination frequency was obtained when the CAG promoter wasused, followed by the hEF-1a promoter, the mPGK promoter and the MC1
promoter in order. These results indicate that the efficiency of recombinationin transient expression system corelates with the promoter activity of Cre
expression vector. Thus, it is important to choose the promoter for effective
recombination by Cre. For the details, see Ref. 2.

(3)

Nuclear organization in fission yeast meiosis

Yasushi HIRAOKA (Kansai Advanced Research Center, Communications Research Laboratory)
We found that in fission yeast meiotic prophase, telomeres are clusterednear the spindle pole body (SPB; a centrosome-equivalent structure in fungi)
and take the leading position in chromosome movement while centromeres
are separated from the SPB (Chikashige et aI., Science 264, 270-273,
1994). The meiotic telomere position contrasts with mitotic nuclear organization in which centromeres remain clustered near the SPB and lead chromosome movement. Thus, nuclear reorganization switching the position of centromeres and telomeres must take place upon entering meiosis. We analyzed the nuclear location of centromeres and telomeres in genetically wellcharacterized meiotic mutant strains. An intermediate structure for the telomere-centromere switching was observed in haploid cells induced to meiosis
by synthetic mating pheromone; fluorescence in situ hybridization revealed
that in these cells, both telomeres and centromeres were clustered near the
SPB. Further analyses in a series of mutants showed that the telomerecentromere switching takes place in two steps; first, association of telomereswith the SPB and second, dissociation of centromeres from the SPB. The
first step can take place in the haploid state in response to mating pheromone,but the second step does not take place in haploid cells and probably depends
on conjugation-related events. In addition, a linear minichromosome wasalso colocalized with authentic telomeres instead of centromeres, suggesting

B.

DEPARTMENT OF CELL GENETICS

43

that telomere clustering plays a role in organizing chromosomes within a meiotic prophase nucleus. (Chikashige et aI., 1997)(4)

Chromosome dynamics in living human cells

Yasushi HIRAOKA (Kansai Advanced Research Center, Communications Research Laboratory)
To examine mitotic events, such as nuclear membrane assembly and disassembly, in living mammalian cells on a microscope stage, we designed a microscope system with a precise temperature control (Hiraoka and Haraguchi,
Chromosome Research 4, 173-176, 1996). Using the computerized fluorescence microscope system with a precise temperature control, we observed dynamics of chromosomes and microtubules during mitosis in living HeLa cells.
Chromosomes were stained for double staining of chromosomes and microtubuIes, tubulin conjugated with rhodamine was microinjected into cells that
had been stained with a DNA-specific fluorescent dye, Hoechst 33342. Microinjected rhodamine-tubulin was incorporated into microtubules and visualized microtubules in living cells. We examined their dynamics in the presence of mitotic inhibitors such as an inhibitor of type II DNA topoisomerase
and inhibitors of microtubule polymerization. Continuous observation in individualliving cells has also made it possible to visualize effects of mitotic
inhibitors on dynamics of chromosomes and microtubules. These inhibitors
discoordinated cytoplasmic events and chromosomal events.
(Haraguchi et aI., 1997)

Pub I i ca t ions
1.

2.

3.

ARAKI, K, ARAKI, M. and YAMAMURA, K: Targeted integration of DNA using mutant lox
sites in embryonic stem cells. Nucleic Acid Res. ll5: 868-872, 1997.
ARAKI, K, IMAIZUMI, T., OKUYAMA, K, OIKE, Y. and YAMAMURA, K: Efficinecy ofrecombination by Cre transient expression in embryonic stem cells: comparison of various
promoters. J. Biochem. lllll: 977-982, 1997.
KIKUCHI, S., SONOBE, K, MASHIKO, S., HIRAOKA, Y. and OHYAMA, N.: Three-dimensional
image reconstruction for biological micro-specimens using a double-axis fluorescencemicroscope. Optics Comm. 138:21-26, 1997.

44
4.

CHIKASHIGE, Y., DING, D. Q., IMAI, Y., YAMAMOTO, M., HARAGUCHI, T. and HIRAOKA,
Y: Meiotic nuclear reorganization: switching the position of centromeres and telomeres in the fission yeast Schizosaccharomyces pombe. EMBO J. 16: 193-202, 1997.

5.

WATANABE, Y., SHINOZAKI-YABANA, S., CHIKASHIGE, Y., HIRAOKA, Y. and YAMAMOTO, M.:
Phosphorylation of RNA-binding protein controls cell cycle switch from mitotic to meiotic in fission yeast. Nature 386: 187-190, 1997.
SHIMANUKI, M., MIKI, F., DING, D. Q., CHIKASHIGE, Y., HlRAOKA, Y., HORJO, T. and NIWA,

6.

7.

8.

9.

0.: A novel fission yeast gene, kms1+, is required for the formation of meiotic prophasespecific nuclear architecture. Mol. Gen. Genet %54: 238-249, 1997.
KiNOSHITA, M., KUMAR, S., MIZOGUCHI, A, IDE, C., KiNOSHITA, A, HARAGUCHI, T., HIRAOKA,
Y and NODA, M.: Nedd5, a mammalian septin, is a novel cytoskeletal component
interacting with actin-based structures. Genes & Development 11:1533-1547, 1997.
HARAGUCHI, T., KANEDA, T. and HIRAOKA, Y.: Dynamics of chromosomes and microtubuIes visualized by multiple-wavelength fluorescence imaging in living mammaliancells: effects of mitotic inhibitors on cell cycle progression. Genes to Cells %:369-380,
1997.
HIRAOKA, Y., AGARD, D. A and SEDAT, J. W: Spatial arrangement of homologous chromosomes during anaphase in early embryos of Drosophila melanogaster studied bythree-dimensional fluorescence microscopy. Bioimaging 5: 183-193, 1997.

45

C.

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

The Inhibition of Ras Signaling Pathway by EDL Is Required for
the Neural Inducing Ability of Founder Celis

Takuma YAMADA, Masataka OKABE, Hiroyuki KOSE and Yasushi HIROMIIn the developing nervous system, the determination of neurons occurs in a
stepwise fashion. For example, two classes of adult photoreceptor cells (RIR8) and of embryonic chordotonal organ precursors (COPs: CI-C8) can be discerned in Drosophila. Class I (founder cells) comprises the R8 cells in the eye
and five rhomboid (rho )-expressing COPs (CI-C5), determined by a proneuralgene atonal. Class II cells (RI-R7 and C6-C8) are induced by the foundercells through activation of the Ras signaling and its nuclear target Pointed, an
ET8 transcription factor. Although requirement of Ras signal activation inClass II cells has been well documented, its role in the founder cells is largely
unknown.
To test the function of Ras signaling in the founder cells, we overexpressed
Pointed in the region including the founder cells. In both the retina and thechordotonal system, this caused a reduction in the number of neuronal cells
surrounding the founder cells rather than production of extra neurons. This
unexpected finding suggests that the expression of Pointed in the founder cells
resulted in their decreased ability to induce Class II cells. As all the molecular components of the Ras signaling pathway are also expressed in the foundercells, there must be a mechanism to inhibit Ras signaling in these cells. Here
we describe a novel gene, edl, a candidate inhibitor ofthe Ras signaling in thefounder cells. edlproduct is present in the founder cells, can inhibit the transcriptional activity of Pointed P2 in 82 cells, and directly binds Pointed P2
protein in vitro. Loss of edl function results in the down regulation of rho
expression in founder cells and causes reduction in the number ofphotoreceptor cells and COPs, resembling the effect of overexpression of Pointed. These46

results suggest that the inhibition ofRas signaling by EDL is required in thefounder cells for their neural inducing ability.

(2)

Sprouty, an Extracellular Factor that Antagonizes both
FGF and EGF Signaling Pathways

Susanne KRAMER', Masataka OKABE, Nir HAKOHEN2, Mark KRASNOW 2 andYasushi HIROMI ('Dept. Molecular Biology, Princeton University, 2Dept.
Biochemistry, Stanford University)
Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates andvertebrates. In most systems, it is primarily the distribution of the factors,
mostly governed by their synthesis, processing and diffusion, that controls thedifferential behavior of the responding cells. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila (ref. 3). It is a novel type of protein with a cystein richregion, conserved in human Sprouty homologs. In addition to the embryonictracheal system, sprouty is also expressed in the developing eye imaginal
disc, embryonic chordotonal organ precursors, and the midline glia. In all ofthese systems the EGF receptor (DER) signaling is known to participate in thecontrol ofthe correct cell number (neurons or glia). We have found that ineach of these systems, loss of sprouty results in supernumerary neurons or
glia. Furthermore, overexpression of sprouty in other tissues where EGF signaling is required for patterning, such as wing veins or ovarian follicle cells,
results in phenotypes that resemble the loss of function phenotypes ofDEE. These results suggest that Sprouty acts as an antagonist to both the
FGF and EGF signaling pathways. These receptor tyrosine kinase-mediatedpathways may share not only intracellular signaling components but also
factors that modulate their signal magnitude in the extracellular compartment.

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

(3)

47

Functional Dissection of the Ligand Binding
Domain of an Orphan Receptor, Seven-up

Hiroyuki KOSE, Steve WEST' and Yasushi HIROMI ('Dept. Molecular Biology,
Princeton University)
Nuclear receptors, such as the estrogen and the retinoic acid receptors, areligand-dependent transcription factors that possess conserved DNA binding
and ligand binding (LBD) domains. Although many members of the nuclear
receptor family have been discovered, due to the wide structural variety of
their potential ligands (steroids, retinoids, fatty acids, amino acid derivatives), it has been technically difficult to identify specific ligands for them.
Thus the majority of nuclear receptors remain "orphan". Expression studies
performed in vertebrates and in invertebrates show that many of the orphan
receptors are expressed during development, often under exquisite transcriptional control. Their discrete expression patterns suggest that these "receptors" may convey regulatory information for the function of other molecules,
rather than acting in response to their ligands. High structural similarity
within the "ligand binding domain" among orphan and the ligand-dependent
receptors hints that this domain has an important function, even ifit does notserve as a "reception domain" for a ligand. This function is likely to be interaction with other proteins and the modulation of their activity.
We are trying to understand the mechanism of action of the LBD of anorphan receptor Seven-up. Seven-up is highly conserved throughout evolution; the human and the Drosophila homologs share more than 90% aminoacids in their LBDs. This suggests that molecules that interact with thisdomain are also conserved between human and Drosophila. The seven-up
gene plays an important role in cell fate specification in the developing eye; in
its absence photoreceptor neurons R3/R41RI1R6 are transformed towards another fate, the R7 neuron. The proper function of Seven-up depends, in part,on its tight transcriptional control; misexpression of Seven-up in cells that
normally do not express seven-up causes a variety of cell fate changes that are
specific to each cell type. We found that the expression of the Seven-up LBD,
separated from its DNA binding domain, produces various cellular phenotypes similar to those observed upon misexpression of the full length Seven-up

48

protein. These phenotypes therefore are unlikely to be caused by themisregulation of the target genes of Seven-up, but rather results from thetitration of another molecule. We propose that this molecule is a protein
partner (protein X) for Seven-up as well as for other cell type-specific transcriptional regulators that, like Seven-up, control fates of individual photoreceptor subtypes. A search for protein X is now under way.

(4)

Peptide Groups Which Regulate Nerve Cell
Differentiation In Hydra

Toshio TAKAHASHI, Hiroshi SHIMIZU, Masayuki HATTA, Seungshic YUM,
Osamu KOIZUMI 1 , Tsutomu SUGIYAMA 2, Yojiro MUNEOKA3 and Toshitaka
Fujisawa(lFukuoka Women's Univ., Fukuoka, 2Ishinomaki-Senshu Univ.,
Ishinomaki, 3Hiroshima University, Hiroshima)
In the process of systematic isolation of peptide signaling molecules from
Hydra magnipapilJata (Takahashi et aI., 1997), we identified two groups of
peptides which negatively and positively regulate nerve cell differentiation in
Hydra. The negative regulators belongs to a PW family which consists of 4
members, Hym-33H, Hym-35, Hym-37 and Hym-310. These peptides (5-8amino acids long) share a common motif of PW in their C-termini. When
hydra were treated for two days with one of these peptides, about 50% of newnerve cell differentiation was inhibited. In contrast, the treatment was prolonged to 7 days, the inhibitory effect disappeared. The results were interpreted that the PW family is a negative feed back signal for nerve cell differentiation.
The positive regulator, Hym-355, is 9 amino acid long and its C-terminus isamidated. The antibody raised against its C-terminus recognized nerve cells
mostly present in the tentacles, hypostome and basal disk. Thus, Hym-355
is a neuropeptide. Hym-355, when treated for 2 days, enhanced new nerve
cell differentiation by 50 %. The co-treatment with Hym-33H, one of the PW
peptides, and Hym-355 at the same concentration of 10- 6 M canceled out theiractivities. These results suggest that the two groups of peptides are thefeedback regulators of nerve cell differentiation in Hydra.

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

(5)

49

Extracellular matrix and hydra pattern formation

Hiroshi SHIMIZU and Michael P. SARRAS (Univ. Kansas, USA)The goal of the research project is to examine the mechanism of how extracellular matrix (ECM) affects cell activities in hydra. ECM plays a crucialrole in various cell activities such as cell proliferation, cell differentiation, cell
migration, morphogenesis etc as has been examined in vertebrate cell
cultures. However, it is not at all certain whether these pieces of information
obtained in cell culture of vertebrates is true of invertebrate systems. To
examine this issue, we examined the effect of ECM on cell activities such as
mitotic activity and morphogenetic activity. Hydra ECM termed mesoglea islocated between two epithelial mono layers, the ectoderm and the endoderm.
Mesoglea does upon incision or amputaion retract away the site of the injury
stimulus thus giving rise to an area of tissue free ofECM. We then tried to
examine if there could be changes in cell activities provoked by the retractionof ECM. So far we found two things. The first is that the mitotic activity ofthe ectodermal epithelium is not altered by the retraction of ECM. The second is that head regenerative activity is restricted to the area oftissue whereretraction occurred suggesting a relationship between morphogenesis andECM. The first piece of information is of particular interest. It has beenthought that the mitotic activity of epithelium is strongly dependent upon theattachment of the cells to ECM. Those which can divide in the absence of
ECM at the base as mentioned above have been classified as tumor cells. The
present result suggests that invertebrate cells follow different rules from vertebrates and that ECM dependence of cell activities of intertebrates should bethoroughly reexamined.

Publ ications
1.

2.

BUTLAR, S.J., RAY, S. and HIROMI, Y: Klingon, a new member of the Drosophila
immunoglobulin superfamily, is required for the development ofthe R7 photoreceptor
neuron. Development, 1%4, 781-792, 1997.
ITO, K., AWANO, w., SUZUKI, K., HIROMI, Y and YAMAMOTO, D.: The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually
identical set of neurons and glial cells. Development, 1%4, 761·771, 1997

50
3.

Sprouty

HACOHEN, N., KRAMER, S., SUTHERLAND, D., HIROMI, Y. and KRASNOW, M. A:

encodes a novel antagonist of FGF signaling that patterns apical branching of the

Drosophila airways. Cell, 92, 253-263, 1998.
4.

MURATE, M., KiSHIMOTO, Y., SUGIYAMA, T., FuJISAWA, T., TAKAHASHI-IwANAGA, H., & IWANAGA,
T.:

Hydra regeneration from recombined ectodermal and endodermal tissue. II.

Transient loss of the endodermal epithelial tissue organization. Journal of Cell Sci5.

ence Vol. 110, pp.1919-1934, 1997.
TAKAHASHI, T., OHTANI, M. , MUNEOKA, Y., ARIMOTO, S. , HATTA, M., SHIMIZU, H., FUJISAWA,
T., SUGIYAMA, T. & KOIZUMI, 0.:

Structure-activity relation of LWamide peptides syn-thesized with a multipeptide synthesizer. Peptide Chemistry Vol. 1996, pp. 193-196,
6.

1997.
TAKAHASHI, T., MUNEOKA, Y., LOHMANN, J., LoPEZ de Haro, BOSCH, T.C.G., DAVID, C.N.,
BODE, H.R., KOIZUMI, 0., SHIMIZU, H., HATTA, M., FUJISAWA, T. & SUGIYAMA, T.:

Systematic

isolation of peptide signal molecules regulating development in Hydra: I. LWamide
and PW families. Proceedings of National Academy of Science, U.S.A Vol. 94, pp.
1241-1246, 1997.

C-b. Division of Gene Expression(1)

MUF1 Is an evolutionarily conserved transcriptional
coactivator that connects a regulatory factor and
TATA element-binding protein

Ken-ichi TAKEMARU, Feng-Qian LI, Hitoshi VEDA, and Susumu HIROSE

M ultiprotein b ridging factor 1 (MBF1) is a transcriptional cofactor thatbridges between the TATA element-binding protein (TBP) and the Drosophilamelanogaster nuclear hormone receptor FTZ-F1 or its silkworm counterpart
BmFTZ-Fl. A cDNA clone encoding MBF1 was isolated from the silkworm
Bombyx mori whose sequence predicts a basic protein consisting of 146 aminoacids. Bacterially expressed recombinant MBF1 is functional in interactions
with TBP and a positive cofactor MBF2. The recombinant MBFl also makes
a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1
DNA-binding domain and stimulates the FTZ-F1 binding to its recognitionsite. The central region ofMBF1 (residues 35-113) is essential for the binding ofFTZ·F1, MBF2 and TBP. When the recombinant MBF1 was added to a
HeLa cell nuclear extract in the presence of MBF2 and FTZ622 bearing the

C.

DEPARTMENT OF DEVELOPMENTAL GENETICS

51

FTZ-Fl DNA-binding domain, it supported selective transcriptional activation of the fushi tarazu gene as natural MBFl did. Mutations disrupting
the binding ofFTZ622 to DNA or MBFl, or a MBF2 mutation disrupting thebinding to MBFl all abolished the selective activation of transcription. These
results suggest that tethering of the positive cofactor MBF2 to a FTZ-Flbinding site through FTZ-Fl and MBFl is essential for the binding site-dependent activation of transcription. A homology search in the databases revealed that the deduced amino acid sequence ofMBFl is conserved acrossspecies from yeast to human. For details, see Ref. 1.
(!)

Transcriptional activation through Interaction of
MOF! with TFIIA

Feng-Qian LI, Ken-ichi TAKEMARU, Masahide GOT0 1, Hitoshi DEDA, Hiroshi
HANDA I and Susumu HIROSE (lFaculty ofBioscience and Biotechnology, Tokyo
Institute of Technology, Yokohama)Transcriptional activation ofthe Drosophila melanogaster fushi tarazu gene
by FTZ-Fl or its silkworm counterpart BmFTZ-Fl requires two cofactors MBFl
and MBF2 which do not directly bind to DNA. MBFl is a bridging moleculethat connects FTZ-Fl (or BmFTZ- Fl), MBF2 and TATA binding protein TBP.
MBF2 is a positive cofactor that activates transcription.
To elucidate the mechanism of transcriptional activation by MBF2, we isolated a cDNA coding for the factor. Northern blot analyses showed temporally restricted expression of MBF2 mRNA similar to that of BmFTZ-Fl
mRNA. The cDNA sequence predicts a polypeptide of lOkDa whereas natural MBF2 is a glycoprotein of 22kDa. The deduced amino acid sequence ofthe factor showed no homology with proteins in the databases. Farwestern
analyses and glutathione Stransferase interaction assays demonstrated thatMBF2 makes a direct contact with the {3 -subunit of TFIIA. In a HeLa cell
nuclear extract, bacterially expressed recombinant MBF2 activated transcription from various promoters as natural MBF2 did. This activation requires
the MBF2-TFIIA interaction. When recombinant MBF2 was added to the
HeLa cell nuclear extract in the presence ofMBFl and FTZ622 bearing the
DNA-binding region of FTZ-Fl, it selectively activated transcription of the

52

fushi tarazu gene. This selective activation also requires the MBF2-TFIIA
interaction.
In sum, MBF2 activates transcription through its interaction withTFIIA. Selective transcriptional activation occurs when MBF2 is recruited
to a promoter carrying the FTZ-F1 binding site by FTZ-F1 and MBF1. For
details, see Ref. 2.
(3)

Temporal regulation of the mid-prepupal gene FTZ-Ft: DHR3
early-late gene product is one of plural positive regulators

Yuji KAGEYAMA, Shoko MASUDA, Susumu HIROSE and Hitoshi UEDA
Various ecdysteroid responsive genes play important roles in insect molting
and metamorphosis. Late FTZ-F1, a member of the nuclear receptor superfamily, is a unique transcription factor which is induced by a pulse exposure of
20-hydroxyecdysone. Elucidation of the regulation mechanism of this geneduring prepupal period will help our understanding of metamorphosis at amolecular level.
Using transgenic fly lines carrying various transcription regulatory regionsof the FTZ-F1 gene fused to the LacZ gene, we investigated cis-regulatory
elements in the late FTZ-F1 transcription unit. The region which governs thestage-specific expression during prepupal period was narrowed down to 1.2 kb,from -0.7 to +0.5 kb relative to the transcription start site. Electrophoresis
mobility shift assays using staged extracts and various probes within thestage-specific region allowed us to identify binding sites for DHR3, an earlylate gene product, around 170 and 450 bp downstream of the transcription
initiation site. Mutations disrupting these binding sites reduced the reporter
gene expression without affecting the stage specificity.
In sum, our deletion and mutation studies of the cis -regulatory element of
the FTZ-F1 gene suggest that the DHR3 binding sites located in the 5' noncoding region are involved in the prepupal expression ofthe gene. These DHR3
binding sites confer high level expression while other elements are also involved in stage-specific expression. For details, see Ref. 3.

C. DEPARTMENT OF DEVELOPMENTAL GENETICS

(4)

53

Genetic Studies on the 2nd (p )-Llnkage Group In Bombyx morl (L.)
Are the p and S -alleles comprising of either two
independent alleles or of a common IHlele?

Akio MURAKAMI
The p -linkage group in the mulberry silkworm [Bombyx (mori) mori L.] isdiscovered by Tanaka (1913) and that is remarkable in which contains 15
known genetic factors as p -alleles at 0.0 unit, 4 as S -alleles at 6.1 and 7 as
Gr-alleles at 6.9. Furthermore, four known recessive genetic factors (i-lem,
ml, wm-b, and oal) and 3 dominant factors (Y, let-A, and Re) are locating at
each locus on p -linkage group. In addition to those alleles and genes, a few
genes/(pk Rt, sme, etc.) have reported to be linked with this linkage chromosome, but their loci have yet been worked out. In Bombyx silkworms, where
cover on eleven linkage groups as to larval body-colors and/or marking patterns: bd (9-22.9), Dus (10-3.9), L (4-15.3), mIn (18-35.3), ms (12-5.5), msn (1926.9), q (7-0.0), so (26-?) U(14-40.5) and Ze (3-20.8), besides p (1-0.0) and S(1-6.1). A phenotype of gene pM (moricaude), one of the p -multiple allelicmember, is fairly analogous to that of Bombyx(mori ) manadarina M. which
is widely accepted as a closely related form as a subspecies of B. mori.
L. Analyses ofthe larval body marking pattern and/or coloration among variants of p -linkage chromosome seem to be useful clues for approach on an
original form or a progenitor of B. mori.
Although there have been accumulated a large body of data for every variant
on this linkage group, certain unresolved questions are remained. There isno difference in the phenotype between the p S (striped) and S (new striped)
mutants, for example. The dominant-recessive relationship between theboth mutants is yet established, however. Tanaka (1913, '16 '25) indicatedthat the gene pM is epistatic to the pS, while Tazima (1938) proposed a different view that the former is dominant over the latter. If the latter case isacceptable, the p S becomes an original phenotype (or genotype) in stead of thegene pM for the larval body marking pattern in Bombyx silkworms. In general, a frequency offorward mutations is higher than that of back mutations
for many species. But the latter type mutation showed a high frequency inthese p - and S -allelic series than the former, indicating that the situation is

54

unusual. Also, recombination values between p -or 8 -allelic members andthe gene & (yellow blood): the values between both alleles are small, but tosome extent fluctuated. Accordingly, the gene arrangement on p -linkage groupseem to be not an appropriate.
There is a mysterious observation that thetwo mutants, 8 2 (second new striped) and p Sw (withish-throx striped), are derived from different multiple allelic series. Furthermore, there is a complicated report that the mutant 8 2 (second striped) which was obtained from an
X-ray-induced treatment of the p S line has been explained as an inversion(and/or duplication) of the p S into 8 -allelic domain (Takasaki, 1947). On thecontrary, the p Sw (whitish-thorax striped) was obtained from a high temperature shock treatment of 8 line (Tanaka, 1951, 1953). According to the mutant list published by Chikushi (1972), however, he noted that the mutant was
obtained from the pupae after treatment of X-rays instead of the temperatureshock. Such being the context, it brings forward a question whether both p and 8 -alleles are independent each other. In other words, it gives rise to apossibility that p - and 8 -allelic series are comprising of a common allele. In
any case, to reveal the question, it will contribute partly to elucidate a genearrangement of p -linkage group in this insect as well as an original form in B.
mori L.

(5)

Genetic Studies on the Znd (p )-Linkage Group in Bombyx morl (L.)
p-allelic series (Z-O.O)

Akio MURAKAMIIn this allelic series, spontaneously-appeared four mutants, pB, pM, ps andp D are listed at present. All of them are dominant over the standard type p 3
or a conventional wild-type designated as± p. A mutant insect having eitherpM mutant gene or p S gene, shows normal viability that is no embryoniclethality, but the gene p B to some extent shows poor in health condition. Six
artificially-induced Idominant mutants are registering in the list of mutants
as follows: p L (light crescent), p Sa (sable), p Sa-2 (sable-2), p G (ventral striped),pST (pale striped) and p Sw (whitish-thorax striped).
No embryonic lethality
can be observed for the p L mutant, which was obtained from p stock line
pupae treated with a high temperature shock at 42'C (Chikushi, 1938). WhileC. DEPARTMENT OF DEVELOPMENTAL GENETICS55

the two X-ray-induced sable type mutants, p Sa and p S..2, are accompanying
with a serious embryonic lethality and indicating that they have chromosomal
deficiency (Tazima, 1938, '43). The mutant p G which was obtained from an
X-ray-treated pupa, has a translocation between p -linkage chromosome and acertain autosome, T(A:2)pS, where A indicates the autosome. This translocation is no indication of the embryonic lethality. The genetic nature of an Xray induced mutant p St (Takasaki, 1947) could not further refer to beyond theoriginal brief memoranda on this mutant. A mutant p swwas derived fromafter treatment of pupae homozygous for S with X-rays (Chikushi, 1972). It
is very difficult to understand reasons why the mutant was appeared from adifferent allelic series or the S strain. This mutant seems to be in a very
normal health condition.
All of those artificially-induced mutations are what we called a dominant
mutation, but every mutational event is directed from the dominant phenotype or genotype. p sor S, to the recessive one, p Land p Sa. Accordingly, it isvery logical to say that those mutations are appeared as some structural (offunctional) changes on the chromosome or defects of the original dominantphenotype.
The body-marking pattern of larvae heterozygous for p Sis clearly lighter incolor tone than that of the homozygous ones. Also, the color tone of a trisomic
larva for the gene locus makes deeper than a normal diploid one (Takasaki,1952). A similar situation would be applicable to other p - and S· allelicmutant members: the mutant p B seems to be comprised of either duplicated or
triplicated p S(or p M) genes on this 2nd chromosome accompanied with achromosomal aberration. Furthermore, the gene p Smight the also duplicatedthe p Mwith unequal crossing-overs in long domestication (or evolutional)
processes. Curiously enough, we cannot picture a wild-type of the Salleles. In any case, such being the situation, the present author would liketo propose and alternative view that the so-called standard type of the pallele, ± P or p 3, should be replaced with the dominant genotype of either p S orpM.

In this connection, it is of interest to note that a phenotype of the mutant p M(moricaude) is identical to a so-called wild-type mulberry silkworm, B. (mori)mandarina (M.) and/or a closely related subspecies of B. (mori) mori

56(L.). The dominant-recessive relationship between pM and pSis equistaticeach other (Tanaka, 1933), but the latter phenotype is regarded as epistatic to
the former (Tazima; 1938). Accordingly, the present author has to be inclined
to have a view that the p sis a fundamental body-marking pattern character
among the alleles in larvae of Bombyx silkworms. As is well known facts
that color tone of the psis more deepen that of the p,'IJ. However, we cannot
say anything certain without further investigations on this point. Because
the body marking in heterozygous for the pSis less color tone than that ofhomozygous individuals. Furthermore p Strisomic silkworms show deeper inthe color tone of body marking than the disomics (or diploid) (Takasaki,1952). A similar explanation may adopt the other p - and S allelic members:
and origin of mutant p B conceive of du- (or trio) plication ofthe pM gene or pSin
the chromosome which is accompanying with some kinds of the chromosome
aberration.
The history ofthe gene p sis also able to be supported to a multiplication of
the pM gene on account of unequal crossing-overs, but the mutant p sis freefrom any chromosomal aberrations. Although the pM like phenotype in thelarval body marking pattern has constantly been recorded in various historical documents of sericulture industry from ancient era to the present. While
the p S like mutant has not been described in old documents, so far, so thatsuch the mutant seems to be recently appeared under our eyes (e.g., Yoshitake
and Sato, 1988; Yoshitake, 1988). A similar situation will be adaptable tothe mutant pBas well as U (ursa). It seems likely to consider that thesemutants, p B, P s, S etc. have been appeared in relatively recent as products ofsome unusual biological events, mutation, unequal crossing-over and soon. Among other things, domestication procedure might be accelerated thisevent. The assumption has an advantage for the interpretation of allelicstructures in both the p-and S-loci and their genetic connection between
them. It is also feasible to suppose that both allelic series may formed by theunequal crossing-over as a large gene family.
From these context, thus, the following hypothesis can be proposed that thepM is an original phenotype (or genotype) of the larval body-making pattern of
B. mori. L. However, we can not say anything certain without further investigations on this point.

C.

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DEPARTMENT OF DEVELOPMENTAL GENETICS

57

Genetic Studies on the !nd(p )-Linkage Group in Bombyx morJ (L.)
S -allelic series (!-6.1)

Akio MURAKAMIThis allele apart to some extent from the p -allelic domain at the starting
point of p -linkage group. At present, the allele is made up of 4 members, 8,8 d (dilute new striped), 8 2 and 8 w and 8 w. Curiously enough, however, we
can not make picture a phenotype ofthe wild type (+S) and recessive types inthis allele. The first two mutant genes, 8 and 8 d, are spontaneous origin,while the others are artificially induced mutants (Tanaka, 1929, 1933). The
mutants is dominant over the mutant 8 d (Tanaka, 1933). The artificiallyinduced mutant 8 w was detected after treatment of pupae with a high temperature (42'C) shock heterozygous for pip M;818 (Chikushi, 1938). The history of this mutant was described as a recessive type mutational event from agene 8, while another explanation may be also possible as a dominant typefrom a gene 8, while another explanation may be also possible as a dominant
type mutation from either the p or pM as seen in a description of p Lmutant(Chikushi, 1938).
The mutant 8 2 is a product of the X-ray-treated of the p S pupae and thathas been regarded as a chromosomal inversion (or duplication) ofthe p Sinto
the 8 -allelic series locus (Takasaki, 1947), indicating that this mutational
event was resulted in the shift of the p S gene from its original p -allele. The
8 2 mutant history is a quite reverse case of the mutant p SWamong three mutants, 8, 8 d and 8 2 • Takasaki (1948) reports slightly different recombination values between the 8 2 and +p, 1.4% (Takasaki, 1947) and between the 8 2
and p, 1.5% (Takasaki, 1948). However, he (1952) also reported a different
recombination value between the genes 8 2 and p to be as a 6.1 % (Takasaki
1952) and he followed the view of Tanaka (1933) who has reported a recombination value of 6.1 % between the genes p and 8.
Genetics relations between the p - and 8 -multiple allelic series: As has
been discussed above, the mostly mysterious phenomena in both loci is. It
remains an unsolved question whether the gene 8 might be resulted from amultiplication ofthe pMgene through the unequal crossing-overs.
Although Bombyx silkworms with a phenotype likes the pM gene for the58

larval body marking pattern have often recorded in various historical documents for the seicultural industry from the ancient era to the present, theinsects with a phenotype likes the p Sgene did not document in old age, so far.
However, it seems to have a tendency that such the phenotypic larvae wasrecently detected and/or documented (e.g., Yoshitake, 1988; Yoshitake and Sato,1986) with the development of sericulture industry. A similar tendency isalso thought to be mutant S such as p B, U etc. It is likely to say that several
mutants, p B, P s, S, U and so forth, have been appeared as products of some
unusual biological events including mutations, unequal crossing-overs, etc.,
during a long evolutional (or domestication) process might be accelerated theevent. This assumptive view may have an advantage of the explanation forthe multiple allelic structure in the p -and S -alleles and theirconnection. Among other things, there is no denying a possibility that bothalleles are made up of what we called a large gene family. In any case, it canbe inferred from those discussion above that the pM seems to be an original
phenotype or genotype for the various gene factors as to the larval body marking pattern located on p -linkage group of the Bombyx silkworm. However,
we can not say anything without further investigations on this point.

(7)

Origin of Holometabololls Metamorphosis In Insects

Kiyoshi MINATO
The evolutional origin of hoIometabolous metamorphosis in insects, which
is one of the reasons of the large success of insects in the animal world, wasinvestigated through the available literatures and the observations of manylife styles of insects in the fields. The insects with holometabolous metamorphosis or the endopterygotes are characteristic to their larval form relatively
simple and dissimilar to the adult one, and the special instar stage of "pupae"
existing between the larval and adult one.
Though the origin and meaning of the pupal stage have been controversial
until recently, it is now considered to be reasonable that the phase with theevaginated wing anlage is necessary to making the internal space for the fullgrowth of wing muscle, and that it evolved as the last larval stage equivalent
to the last nymphal stage in exopterigotes which was retained with the exter-

C.

DEPARTMENT OF DEVELOPMENTAL GENETICS

59

nal wing anlage contrary to the progression of more or less internalization of itin the previous larval instar stages (Hinton, 1963b). However, considering
the hormonal condition controlling the ecdysis into pupae, that is, the one
under the depletion ofjuvenile hormone, the pupal stage seems to be belong torather the adult one than the larval - even if the last - one, and the external
form of the pupae the almost same as that of the adult also make the same
impression as above and seems almost not to necessitate any other ecdysis of
the pupal-adult one.
Then, the larval-pupal ecdysis in endopterygotes may originated from thenecessity for the protection from predators through the excretion of newly cuticle and its hardening during the long periods necessary for the large morphological change in the phase.
The success of endopterygotes seems to be depend upon rather their very
simple larval forms themselves with few external structures such as wing
anlage, compound eyes, and more developed legs than the merits produced
through the holometabolous metamorphosis or the independent habitats eachother in the two phases of larvae and adult. They make them possible toutilize more efficiently the three dimensional environments such as the insideof soil and water, the inside of leaves, fruits and stems of various plants andtrees, and the underside of stones. Actually, they appeared to have adapted
to utilize actively the various circumstances the parts of which appeared newlyin evolution, especially in about Permian. Furthermore, these simple larval
forms may have derived from the some utilization of embryonic forms, possibly by suppressing the further development of some external morphology, evennot by the precocious hatching of embryos as in the fascinating theory statedby Berlese (1913) .

Pub I i ca t ions
1.

2.
3.

TAKEMARU, K., LI, F.-Q., VEDA, H. and HIROSE, S.: MBFl is an evolutionarily conserved
transcriptional coactivator that connects a regulatory factor and TATA element-binding protein. Proc. Nat!. Acad. Sci. VSA, 94, 7251-7256, 1997.
LI, F.-Q., TAKEMARU, K., GOTO, M., VEDA, H., HANDA, H. and HIROSE, S.: Transcriptional
activation through interaction ofMBF2 with TFIIA. Genes to Cells, Z, 143-153. 1997.
KAGEYAMA, Y., MASUDA, S., HIROSE, S. and VEDA, H.: Temporal regulation ofthe mid-

60
prepupal gene FTZ-Fl: DHR3 early-late gene product is one of plural positive regulators. Genes to Cells, Z, 559-569. 1997.

C-c. Division of Physiological Genetics(1)

Interplay between positive and negative elongation factors of
RNA polymerase II transcription: drawing a new view of
ORB-mediated Inhibition of transcription

Yuki YAMAGUCHI, Toshiaki TAKAGI, Tadashi W ADA and Hiroshi
HANDA (Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology)

The purine nucleotide analog 5,6-dichloro-1- j3-ribofuranosylbenzimidazole(DRB) is a classic inhibitor of transcription by RNA polymerase II (polII). We
have identified a factor from HeLa cell nuclear extracts that is required toconfer DRB sensitivity in a cell·free transcription system. This DRB sensi·
tivity inducing factor (DSIF), in conjunction with DRB, inhibits PolII-mediatedtranscription at the level of elongation. DSIF has been purified and is composed of 160 kDa and 14 kDa subunits. DSIF works as a negative elongation
factor in the absence of DRB. Isolation of a corresponding eDNA reveals that
DSIF comprises human homolog of yeast SPT5 and SPT4. The convergenceof biochemical studies on DSIF and genetic analysis of SPT5 and SPT4 indicates that DSIF works generally as a negative elongation factor and regulates
PolII processivity. Although DRB inhibits PolII elongation, its mode of action
has been elusive. Two factors essential for DRB action have been
identified. They are DSIF, a negative elongation factor, and pTEFb, a positive elongation factor. pTEFb was originally identified in Drosophila extracts
by Marshall and Price (J. BioI. Chem., 270, 12335-12338, 1995). These factors positively and negatively regulate PolII-mediated transcription elongation.

C.
(~)

DEPARTMENT OF DEVELOPMENTAL GENETICS

61

Species-specific codon usage diversity in eleven prokaryotes andone eukaryote whose genomes have been completely sequenced

Shigehiko KANAYA'·2, Yoshihiro KUDO l , Toshimichi IKEMURA3 ('Department ofElectric and Information Engineering, Faculty of Engineering, Yamagata
University, 2Division of Physiological Genetics, Department of Ontogenetics,
National Institute of Genetics, 3Division ofEvolutionary Genetics, Departmentof Population Genetics, National Institute of Genetics)
We characterized codon usage diversity of genes for eleven bacteria (Mycoplasma genitalium, Mycoplasma pneumoniae, Methanococcus jannaschii,
Methanobacterium thermoautotrophicum, Borrelia burgdorferi, Helicobacter
pylori, Bacillus subtilus, Escherichia coli, Haemophilus influenzae,
Archaeoglbus fulgidus, and Synechocystis sp.) and Yeast (Saccharomyces
cerevisiae ), whose genomes have been completely sequenced. Quantificationof the cellular tRNA content of E. coli, and codons optimized for its translation
system (optimal codons) were reported previously by our group. Principal
component analysis of E. coli showed that the codons positively contributing
to the first principal component (PCI) almost always correspond to the optimal codons, suggesting that the codon usage in this bacterium is strictly constrained by translation efficiency. Comparison of the gene distribution in the
PCI projection ofthe other eleven speices with that in E. coli indicates thatcodon usage in these eleven species is also constrained by translation
efficiency. The diversity for H. influenzae is similar to that for E. coli, although the G+C% (38%) of the H. infJuenzae genome is significantly different
from that of E. coli (50%). In the case of Synechocystis sp., the genes withhomologues in the chloroplast genomes of present-day plants (e.g. genes forthe photosynthetic system and ribosomal proteins) were located in restrictedzones in the multidimensional space obtained by principal component
analysis. Furthermore, in Mjannaschii and M thermoautotrophicum, mostof genes involved in methanogenesis have postive scores for PCI, suggestingthat codon usage for methanogenesis is also highly adapted to the optimal
codon structure estimated from tRNA contents in cells. For details, see Ref. 15.

62

Pub I i ca t ions
1.

KOMAI. T. YAGI. R., SUZUKI-SUNAGAWA, H., ISHIKAWA, Y. KAsUYA. A. MIYAMOTO, S., HANDA.
H. and NISHIGAKI. T.:

Inhibition of HIV-l protease by oxim derivatives. Biochem.

Biophys. Res. Commun.. Vol. %30. pp557-561, 1997.
2.

RAWASE, H., OKUWAKI. M., MIYAJI, M., OHBA. R.. HANDA, H., ISHIMI, Y, FUJII-NAKATA, T.,
KIKUCHI. A and NAGATA, K:

NAP-l is a functional homologue ofTAF-l that is re-quired for replication and transcription of the adenovirus genome in chromatin-like3.

structure. Genes Cells. Vol. t, ppl045-1056. 1997.
KITAMURA, T., HANDA, H., HAMASAKI, N. and KITAJIMA, S.:

Characterization of thehuman thrombopoietin gene promoter. J. BioI.. Chern., Vol. %7%, pp1l361-11368, 1997.
4.

MOMOSE, F., HANDA, H. and NAGATA, K:

Identification of host factors that regulate theinfluenza virus RNA polymerase activity. Biochimie, Vol. 78. ppl103-1108, 1997.
5.

lMAI. T.• MATSUDA, K, SHIMOJIMA, T.• HASHIMOTO, T, MASUHIRO. Y. KITAMOTO, T., SUGITA.
A, SUZUKI, K. MATSUMOTO. H .• MASUSHIGE. S., NOGI, Y, MURAMATSU, M., HANDA. H. and
RATO, S.:

ERC-55, a binding protein for the papilloma virus E6 oncoprotein, specifi-

cally interacts with vitamin D receptor among nuclear receptors. Biochem. Biophys.
Res. Commun.. Vol. %33, pp765-769. 1997.
6.

MURAKAMI, M., SONOBE, M.H., VI. M., RABUYAMA. Y, WATANABE. H., WADA, T., HANDA. H.
and IBA, H.:

Phosphorylation and high level expression of Fra-2 in v-src transformed

cells: a pathway of activation of endogeneous AP-l. Oncogene, Vol. t4. pp2435-2444,
1997.
7.

NABESHIMA, R.. AIZAWA, S .• NAKANO. M .• TOYAMA. K. SUGIMOTO. K. RAmoH, A, IMAI, T
and HANDA, H.:

Effects of vesnarinone on the bone marrow stromal cell-dependent

proliferation and differentiation ofHL60 cells in vitro. Exp. Hematol.. Vol. %5. pp509515, 1997.
8.

AOKI, H., SEKI, T., SAWADA, J-I.. HANDA, H. and ADACHI, H.:

The promoter ofthe andro-

gen dependent gene in the flank organ of hamsters. J. Dermatol. Sci., Vol. t5. pp36-43,
1997.
9.

AIZAWA, S., NAKANO, M., YAGUCHI. M .. KURIYAMA. Y., IWASE, 0., TOYAMA. K, TANAKA, K,
HOSHI, H., SUGIMOTO, K. RAmoH. A, IMAI. T., NABESHIMA. R. and HANDA. H.:

Possible

involvement of bone marrow stromal cells in agranulocytosis caused by vesnarinone
treatment. Acta Hematologica.• Vol. 98, ppI40-146, 1997.
10.

LI, F.Q., TAKEMOTO. K-I., GOTO, M., VEDA, H., HANDA. H and HIROSE. S.:

Molecular

cloning of MBF2 reveals its contact with TFIIA that leads to transcriptional activation. Genes Cells. Vol. %. ppI43-153, 1997.
11.

YAMAGUCHI, Y.• SAWADA, J-I., YAMADA. M .. HANDA, Hand AzUMA, N.:

Autoregulationof

Pax6 transcriptional activation by two distinct DNA-binding subdomains of the paired
domain. Genes Cells, Vol. %, pp255-261, 1997.
12.

SOWA. Y., SHIW, Y, FUJITA. T.. MATSUMOTO, T.. OKUYAMA. Y.. KATO, D .• INOUE. J-I.,C.

DEPARTMENT OF DEVELOPMENTAL GENETICS

63

SAWADA, J.I., GOTO, M., WATANABE, H., HANDA, H. and SAKAI, T.: Retinoblastoma bind·ing factor 1 site in the core promoter region of the human RB gene is activated by
hGABP/E4TFl. Cancer Res., Vol. 57, pp3145-3148, 1997.
13. HATAKEYAMA, M., OGURA, Y, SAWADA, J.I., FUJIMOTO, K., HANDA, H. and KAWAGUCHI,
H.: Preparation of dimerized polypeptide·carrying microspheres and purification of
specific proteins bound to these microspheres. Colloids and Surfaces B: Biointerfaces,
Vol. 10, pp41-49, 1997.
14. KAWAGUCHI, H., HATAKEYAMA, M. IWATO, S., FUJIMOTO, K. and HANDA, H.: Affinity latex
for DNA diagnosis. In "Advances in Polymer Biomaterials" (Akaike T Okano T Akashi
M. Terano M. and Yui N. eds.) pp. 355-362, CMC, Tokyo, 1997.
15. KANAYA, S., KUDO, Y., NAKAMURA, Y and IKEMURA, T.: Detection of genes in Escherichia
coli sequences determined by genome projects and predicton of their protein·produc·tion levels, basing on multivariate diversity in codon usage. Computer Applied Bio·sciences, Vol. 12, pp. 213-225, 1996.
16. KANAYA, S., KUDO, Y., SUZUKI, S. and IKEMURA, T: Systematization of species·specificdiversity of genes in codon usage: Comparison of the diversity among bacteria andprediction of the protein production levels in cells. Genome Informatics Series, 7, pp.
61-71, 1996.
17. KANAYA. S., KUDO. Y and IKEMURA. T.: Systematization of species· and function·spe·cific diversity in codon usage: Special reference to photosynthetic system in Synechocystis
sp. and in chloroplasts. International Workshop on Recent Advance in Genome Biologyof Micro·organisms, p. 37, 1996.
18. KANAYA. S., OKUMURA. T., MIYAUCHI. M., FUKASAWA. H. and KUDO, Y: Assessment ofprotein coding sequences in Bacillus subtilis genome using species·specific diversity ofgenes in codon usage based on multivariate analysis: comparison of the diversity
between B. subtilis and Escherichia coli. Comnunication in Biochemistry, Cell &Molecular Biology, Vol. 1, 82-92, 1997.
19. FUKASAWA, H., KANAYA, S. and KUDO Y: Detection oflntron, Exon, and Intergenic DNA
in Human Genome on the basis of Quantification Method II. Genome Informatics
Series, 8, 262-263, 1997.

64

D.

DEPARTMENT OF POPULATION GENETICS
D-a. Division of Population Genetics(1) Rate variation of DNA sequence evolution in the
DrosopblJa lineages
Toshiyuki S. TAKANO
Rate constancy of DNA sequence evolution was examined for three species ofDrosophila, using two samples: the published sequences of eight genes fromregions ofthe normal recombination rates and new data of the four AS-C(ae,se, l'se and ase) and ei genes. The AS-C and ci genes were chosen because
these genes are located in the regions of very reduced recombination in D.
melanogaster and their locations remain unchanged throughout the entire
lineages involved, yielding less effect of ancestral polymorphism in the studyof rate constancy. The synonymous substitution pattern of the three lineages
was found to be erratic in both samples. The dispersion index for replacement substitution was relatively high for the per, G6pd and ae genes. A significant heterogeneity was found in the number of synonymous substitutionsin the three lineages between the two samples of genes with different recombination rates. This is partly due to a lack of the lineage effect in the D.
melanogaster and D. simulans lineages in the AS-C and ci genes in contrast to
AKASHI's observation of genes in regions of normal recombination. The higher
codon bias in D. yakuba as compared with D. melanogaster and D. simulans
was observed in the four AS-C genes, which suggests change(s) in action ofnatural selection involved in codon usage on these genes. Fluctuating selection intensity may also be responsible for the observed locus-lineage interaction effects in synonymous substitution. For the details, see Genetics (1998)
149: 959-970.

D.

DEPARTMENT OF POPULATION GENETICS

65(2) Study of species difference as observed as interspecific hybrid
anomaly in Drosophila
Toshiyuki S. T AKANa
With 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. We previously presented evidence that bristle loss in inter-specifichybrids is found between D. melanogaster and D. simulans, but not between
pairs of D. melanogasteron one hand, and D. mauritiana and D. sechellia on
the other. Use of cell type markers suggests that the defect does not lie in cellfate decisions during bristle development, but in the maintenance of neuralfate and/or differentiation of the descendants of sensory mother cells (Genetics, in press). To isolate genes responsible for the bristle anomaly of thehybrids, deficiency screening and QTL (quantitative trait loci) mapping basedon the within-species variation of D. simulans were performed. The latter
analysis showed that a QTL accounted for more than two-thirds of the parental difference, whereas we found two distinct candidate regions from the deficiency screening. The genetic analysis of the bristle anomaly revealed a sex
difference between comparable genotypes, indicating a certain degree of sexspecificity in genotypic effect. The sex-specificity may be one reason for theinconsistent results of the two experiments, because the female hybrids werestudied in the deficiency screening, but the males in the QTL mapping
analysis. Based on the finding that the male hybrids were more susceptible
to bristle loss than the female hybrids were, duplication screening was conducted, where the hybrid males were examined for the bristle. We consequently found one candidate region, which is close to the estimated position of
the QTL with the greatest effect.

66

Publ ication
1.

OTSUKA, Y, TAKANO, T, S, and YAMAZAKI, Y.:

Genetic variation in the expression of thesix hsp genes in the presence of heat shock in Drosophila melanogaster. Genes Genet.
Syst. 72, 19-24, 1997.

D-b. Division of Evolutionary Genetics(1)

In sltn visualization of sequence-dependent high-affinity sites for
non-U structure-forming DNAs in the human interphase nucleus.
Affinity sites for triplex-forming DNAs are closely but
differentially associated with individual centromeres

Mizuki OHNO, Toyoaki TENZEN, Tetsushi YAMAGATA, Shigehiko !{ANAYA andToshimichi lKEMURA
Triplex formation occurs readily in polypurine/polypyrimidine sequences leaving single-strand unpaired DNAs that can hybridize with other single-strand
DNAs with significant complementarity. This will enable distantly spaced
DNAs in genome sequences to associate with each other in interphase nuclei.
Distantly spaced DNAs may also form transmolecular triplexes organizingthemselves into an ordered array. To investigate mechanisms governing spatial organization ofchromosomal DNAs in human interphase nuclei, we searchedin situ the sequence-dependent high-affinity sites for DNAs with specific characteristics such as triplex formation, by two methods. The one resembled
fluorescence in situ hybridization used for detecting single-strand RNAs, thus
was able to detect single-strand DNAs in nondenatured nuclei. The otherwas a lipofection assay. Both methods showed polypurine/polypyrimidine
sequences, such as (GAlTC)n, (GAAlTTC)n and those present in the humangenome, gave clear foci-type signals with different probe sequences resulting
in different signal patterns. By combining seven chromosome-specific centromere probes with polypurinel polypyrimidine probes, differential binding
characteristics of individual centromere territories in non-denatured nuclei
were revealed, yielding a model in which supramolecular structures with differential affinities to different polypurine/polypyrimidine sequences exist

D. DEPARTMENT OF POPULATION GENETICS
within and/or near individual centromere territories.
Cell Genet, 81,101-102,1998.

67

For details, Cytogenet

Triplet repeat polymorphism within the NOTCH4 gene located near
the junction of the HLA class II and class III regions in narcolepsy(!)

Asako ANDO l , Kimihiko SUGAYA 2, Takeo JUJI 3 , Toshimichi IKEMURA and
Hidetoshi INOKO I (l School of Medicine, Tokai University, 2National Instituteof Radiological Sciences, 3Japanese Red Cross Central Blood Center)
A polymorphic (CTG)n microsatellite repeat was found in the signal peptide
domain ofthe NOTCH4 gene located near the junction ofthe class II and class
III regions of the human major histocompatibility complex. This gene belongs to a multigene family of NOTCH originally identified as a differential
factor of neuronal cells. To ascertain whether the NOTCH4 gene is involvedin the development of neurogenic disease, narcolepsy, which is known to betightly associated with HLA-DRI5, this microsatellite polymorphism of the(CTG)n repeat was analyzed in Japanese patients with narcolepsy. One allele, 9 repetitions of CTG (Leu) was significantly increased in the patientgroup. However, the significant increase of this allele in the patient group
could be explained by a strong linkage disequilibrium with the HLA class II
alleles, DRBI*1501, DQAl*0102 and DQBl*0602, which were more strongly
associated with the disease. These results suggest that the (CTG)n repeat
polymorphism in NOTCH4 dose not primarily determine the susceptibility to
narcolepsy. For details, see Tissue Antigens, 50, 646-649, 1997.

(3)

Replication Timing for the GC Content Transition Area

Toyoaki TENZEN, Yoshihisa WATANABE, Asako ANno I, Hidetoshi INOK0 1, andToshimichi IKEMURA (ISchool of Medicine, Tokai University)
Mammalian genomes are composed oflong-range G+C% mosaic structures
thought to be related to chromosome bands. We previously reported an example of a boundary of megabase (Mb)-sized GC% mosaic domains at thejunction area between major histocompatibility complex (MHC) classes II and
III, proposing it as a possible chromosome band boundary. DNA replication

68
timing during S phase is known to be correlated cytogenetically with chromosome band zones, and thus the band boundaries have been predicted to contain a switch point for DNA replication timing. In this study, to identify atthe nucleotide sequence level the replication switch point during S phase, we
determined the precise DNA replication timing for MHC classes II and III,focusing on the junction area. To do this we used PCR-based quantitation of
nascent DNA obtained from synchronized human myeloid leukemia HL60
cells. The replication timing changed precisely in the boundary region with a
2-hour difference between the two sides, supporting the prediction that thisregion may be a chromosome band boundary. We supposed that replication
fork movement terminates (pauses) or significantly slows down in the switch
region, which contains dense Alu clusters, polypurine / polypyrimidine tracts(e.g., 209-bp tracts), di-, tri- or tetranucleotide repeats, and medium reiteration frequency sequences (MERs). We found also the replication switch pointin the XIST locus in the X chromosome inactivation center (XIC). For details,
see Mol. Cell. BioI. 17, 4043-4050.

(4)Structural analysis of mouse tenascln-X: evolutionary aspects ofreduplication of FNIII repeats In the tenascln gene family

Tomoki IKUTA, Norio SOGAWA, HiroyoshiARIGA 1, Toshimichi IKEMURA, Kenichi MATSUMOTO (lGraduate School of Pharmaceutical Sciences, Hokkaido
University)
Tenascin-X (TNX) is an extracellular matrix glycoprotein involved in bothprimary structural functions and modulating cellular activities in multicellular organisms. We determined the 67,977 bp nucleotide sequence of the entire mouse tenascin-X (Tnx) gene, which also includes the last exon of Creb-rp
and Cyp21. We compared it with the orthologous human locus. Conservationof both position and orientation of the three functionally unrelated genes atthis position was found. Comparison also revealed that introns 1,4 and 6 ofTnx are highly consereved between species. The sequence showed that mouse
Tnx contains 43 exons separated by 42 introns. The deduced amino-acid sequence (4114 residues) revealed that mouse Tnx has a primary structure characteristic of tenascins, which consists of a signal peptide and four heptad

D. DEPARTMENT OF POPULATION GENETICS

69

repeats followed by 18.5 epidermal growth factor-like (EGF) repeats, 31
fibronectin type III-like (FNIII) repeats, and a region homologous to fibrinogen.
cDNA clones generated by alternative splicing of eight consecutive FNIII repeats (M15-M22) as well as a proximal FNIII repeat (M3) were also
identified. The FNIII motifs that were subject to alternative splicing wereassigned to the group of recently reduplicated FNIII repeats because they havea high level of amino-acid sequence similarity. We also analyzed the evolution ofFNIII repeats in TNX. For details, see Gene, 217, 1-13, 1998.

(5) Genomic organization of human GABAB receptor identified in
MHC class I region; proposal for differentiation of two
isoform's expression via alternative usage of promoterswith and without cAMP-responsive element
Tetsushi YAMAGATA, AsakoANDol, HidetoshiINOKO I and Toshimichi IKEMURA(lSchool of Medicine, Tokai University)
Gamma-aminobutyric acid (GABA) is the most abundant and widely distributed inhibitory neurotransmitter present in the central nervous system(CNS). Receptors activated with GABA are classified into ionotropic andmetabotropic receptors (GABAAIGABAc and GABAB, respectively). TwomRNA forms of rat's GABAB receptor were characterized and predicted to be
produced by alternative splicing. In the telomeric portion ofMHC class I, we
identified the human GABAB receptor gene which encodes the two
isoforms. The gene organization showed that the two isoforms are generated
by alternative usage of promoters rather than by alternative splicing, and acAMP responsive element (CRE) is present only in the promoter for the shorter
isoform. Based on the fact that GABAB receptor negatively regulates CRE
binding protein (CREB)-mediated transcriptions in CNS, we propose that theactivation of GABAB receptor can regulate the relative expression of its two
isofoi.-ms through alternative usage of the promoters with and without CRE
and thus can differentiate numbers and relative abundance of its isoforms via
the CRE- and CREB-mediated regulatory system.

70

(6)

Nucleotide sequence analysis of the HLA class I region spanning
the U7-kb segment around the HLA-B and -C genes

Nobuhisa MIZUKI 1 , Minoru KIMURA!, Asako ANDO I , Toshimichi
IKEMURA and Hidetoshi INOK0 1 (lSchool of Medicine, Tokai University)
To elucidate the complete gene structure and to identify new genes involvedin the development ofHLA class I antigen-associated diseases in the class I
region of the human major histocompatibility complex on chromosome 6, a
YAC clone (745D12) covering the 146-kb segment around the IkBL and MICA
loci was isolated from a YAC library constructed from the B-cell line,
BOLETH. A physical map of this region was constructed by isolation of overlapping cosmid clones derived from 745D12. Ofthese, five contiguous cosmids
were chosen for DNA sequencing by the shotgun strategy to give a single contig
of 146,601 bp from 2.8 kb telomeric of the IkBL gene to exon 60fMICA. Thisregion was confirmed to contain five known genes, IkBL, BAT1,MICB,P5-1,
and HLA-X (class I fragment), from centromere to telomere, and their exonintron organizations were determined. The 3.8-1 homologue gene (3.8-1-hom)showing 99.7% identity with the 3.8-1 cDNA clone, which was originally isolated using the 3.8-kb EcoRI fragment between the HLA-54/H and the HLA-G
genes, was detected between MICA and MICB and was suggested to represent
the cognate 3.8-1 genomic sequence from which the cDNA clone wasderived. No evidence for the presence of expressed new genes could be obtained in this region by homology and EST searches or coding and exon prediction analyses. One TA microsatellite repeat spanning 2545 bases with as
many as 913 repetitions was found on the centromeric side of the MICA gene
and was indicated to be a potential hot spot for genetic recombination. The
two segments of approximately 35 kb upstream ofthe MICA and MICB genes
showed high sequence homology (about 85%) to each other, suggesting thatsegmental genome duplication including the MICA and MICB genes musthave occurred during the evolution of the human MHC. For details, see
Genomics, 47, 372-382, 1998.

D.

(7)

DEPARTMENT OF POPULATION GENETICS

71

Evolution of primate ABO blood group genes andtheir homologous genes

Naruya SAITOU and Fumiichiro YAMAMoTO'(lThe Burnham Institute, La
Jolla, California, U.S.A.)
There are three common alleles (A, B, and 0) at the human ABO blood grouplocus. We compared nucleotide sequences ofthese alleles, and relatively large
numbers of nucleotide differences were found among them. These differences
correspond to the divergence time of at least a few million years, which isunusually large for a human allelic divergence under neutral evolution. We
constructed phylogenetic networks of human and non-human primate ABO
alleles, and at least three independent appearances of B alleles from theancestral A form were observed. These results suggest that some kind ofbalancing selection may have been operating at the ABO locus. We also constructed phylogenetic trees of ABO and their evolutionarily related a -1,3galactosyltransferase genes, and the divergence time between these two genefamilies was estimated to be roughly 400 million years ago. For details, see
Ref. 1.

(8)

The phylogenetic relationship of the genus Oncorhynchus species
Inferred from nuclear and mitochondrial markers

Takashi KITANO, Norimasa MATSUOKA!, and Naruya SAITou(lFaculty of
Science, Hirosaki University)
The phylogenetic relationship among Oncorhynchus species have been analyzed by various kind of markers for a long time. However, there are threemajor disagreement among these studies. First, phylogenetic position of the
Pacific trouts group (0. mykiss and O. clarki). Second, the relationship among
three Pacific salmons (0. keta, o. nerka, and o. gorbuscha). Third, phylogenetic position of O. masou. In this study, allozyme electrophoresis was used
to analyze the phylogeneticc salmon group and the Pacific trout group. Then,mitochondrial DNA sequences (D·loop and Hind III fragment regions citedfrom Shedlock et aI., 1992 and Thomas and Beckenbach, 1989, respectively)
were re-analyzed to clear above three problems. It was clear that all Pacific

72
salmons construct a cluster and O. masou diverged firstly in this group, then,O. keta and O. gorbuscha are sister species in three Pacific salmons by allozyme
electrophoresis and mtDNA re-analyses. However, stochastic significance forbranching pattern of phylogenetic tree constructed by D-Ioop data were notobtained. For that reason, it is considered that D-Ioop sequences are affectedby parallel substitutions. These vestiges were observed by the split decomposition graph. Nevertheless, above results were supported by the graph. For
details, see Ref. 2.

(9)Genetic origins of the Japanese: A partial support for the "dualstructure hypothesis"

Keiichi OMOT0 1, and Naruya SAITou(lInternational Research Center for Japanese Studies)
Based on the morphological characteristics of the skull and teeth, Hanihara(1991) proposed the "dual structure model" for the formation of modern Japanese populations. We examine this model by dividing it into two independent hypotheses: 1) the Upper Paleo-lithic population of Japan that gave riseto the Neolithic Jomon people was of southeast Asian origin, and 2) modern
Ainu and Ryukyuan (Okinawa) populations are direct descendants ofthe Jomon
people, while Hondo (Main Island)-Japanese are mainly derived from the migrants from the northeast Asian continent after the Aeneolithic Yayoi
period. Our aim is to examine the extent to which the model is supported bygenetic evidence from modern populations, particularly from Japan and other
Asian areas. Based on genetic distance analyses using data from up to 25"classic" genetic markers, we find first that the three Japanese populations
including Ainu and Ryukyuan clearly belong to a northeast Asian clustergroup. This negates the first hypothesis of the model. Then, we find that
Ainu and Ryukyuans share a group contrasting with Hondo-Japanese andKorean, supporting the second hypothesis of the model. Based on these results, we propose a modified version of the dual structure model which may
explain the genetic, morphological, and archaeological evidence concerning theformation of modern Japanese populations. For details, see Ref. 3.

D.

DEPARTMENT OF POPULATION GENETICS

73(to) Reconstruction of phylogenetic trees using the maximum likelihood method in parallel environment using logic programming
Satoshi OOTA, and Naruya SAITOUWith rapid increase of nucleotide and amino acid sequence data, it is required to develop reliable and flexible application programs to infer molecularphylogenetic trees. The maximum likelihood method is known to be robust
among many methods for reconstruction of molecular phylogenetic trees, however, this method requires extremely high computational cost. Although parallel computation is a good solution to realize complicated inference such as
the maximum likelihood method, generally speaking, it is not so easy to implement parallel programs. In actual data analyses, furthermore, it is often
needed to modify or expand application programs. In other words, there is noperfect application program for all data analyses. Logic programming is agood candidate to implement such data analysis programs in natural sciencefields, because programs in logic programming are easy to write, easy to modify,
and easy to implement in parallel environment. We thus have developed anexperimental system for reconstruction of phylogenetic trees in parallel environment in logic programming as a part of molecular evolutionary analysis
system {¥it DeepForest}. We propose the core algorithm for parallel execution of the maximum likelihood and show its verification according to simulation using amino acid data. For details, see Ref. 4.

Publications
1.

2.

3.

4.

SAITOU, N. and YAMAMOTO, F.: Evolution of primate ABO blood group genes and theirhomologous genes. Molecular Biology and Evolution, Vol. 14, No.4, pp. 399-411, 1997.
KITANO, T., MATSUOKA, N., and SAITOU, N.: The phylogenetic relationship ofthe genus
Oncorhynchus species inferred from nuclear and mitochondrial markers. Genes andGenetic Systems, Vol. 7Z, No. I, pp. 25-34, 1997.
OMOTO, K. and SAITOU, N.: Genetic origins ofthe Japanese: A partial support for the"dual structure hypothesis". American Journal of Physical Anthropology, Vol. 10Z, No.
4, pp. 437-446, 1997.
OOTA, S. and SAITOU, N.: Reconstruction of phylogenetic trees using the maximum
likelihood method in parallel environment using logic programming. In Miyano S. and74
Takagi T. (eds.), Genome Informatics 1997, Universal Academiy Press, Tokyo, pp.
326-327, 1997.

D-c. Division of Theoretical Genetics(1)

The meaning of near-neutrality at coding and non-coding regions

Tomoko OHTA
The nature of weak selection differs between coding and non-coding regions.
Coding regions contain genetic information, whereas most non-coding regions
do not have any information. Genetic information may be regarded as interaction systems, and the NK model of Kauffman was analysed. This model
assumes that each amino acid makes a fitness contribution that depends on
the amino acid and on K other amino acids among the N that make the protein.
It is known that the fitness landscape is very rugged for K ~ 2. Population
genetic analysis of this model suggests that protein evolution obeys the nearly
neutral theory and that random genetic drift is important. In other words,
evolution becomes rapid in small populations because the proportion of nearneutrality increases among new mutations, and proteins as interactive systems evolve by shifting through random genetic drift on the multipeaked fitness landscape. The variance ofthe evolutionary rate is not quite as large asdata indicate under the model, and additional factors, such as environmental
change or population-size fluctuation, need to be considered. Weak selection
at non-coding regions may come from chromosome organization, and may beregional in character, which differs from that at coding regions. The problem
of genetic load is thought to disappear in these circumstances. For details,
see Gene 205,261-267 and J. Mol. Evol. 44, 89-814.
(!)

Role of gene conversion in generating polymorphlsms at major
histocompatibility complex lociTomoko OHTA
Multigene families are known to be evolving under the continued occurrenceof unequal crossing-over and gene conversion, and MHC loci are not

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DEPARTMENT OF POPULATION GENETICS

75

exceptions. Population genetic models of diversifying selection, with intraand inter-locus gene conversion were studied by computer simulations. Weakselection at individual amino acid sites are shown to be quite effective inenhancing polymorphisms, if gene conversion occurs. This is caused by theappearance of coadapted segments of mutations in which recombination tends
to be suppressed. In addition to the global type of diversifying selection, an
endemic type of selection was introduced in some sets of the simulations tofind out a mechanism of the rapid allelic turnover of South American Indians.
Strong selection is assumed such that the most common two alleles become
lethal in one generation. If this endemic selection occurs periodically, allelicturnover by intralocus gene conversion is shown to become rapid. Hence, interaction of selection and gene conversion has a profound effect on MHC
polymorphisms. See for details, Hereditas 127,97-103.

Publ ication
1.

2.
3.
4
5
6

Role of random genetic drift in the evolution of interactive systems. Journal of Molecular Evolution 44(Suppll):S9-S14, 1997.
The meaning of near-neutrality at coding and non·coding regions. Gene 205:261267,1997.
Role of gene conversion in generating polymorphisms at major histocompatibility
complex loci. Hereditas 127:97-103, 1997.
The meaning of near neutrality at coding and noncoding regions. Jan. 9. 1997,
Guanacaste, Republic of Costa Rica
Role of gene conversion in generating polymorphisms at major histocompatibility
complex loci. May 25, 1997, Visby, Sweden
Evolution by nearly neutral mutations. June 16, 1997, Boulder, U. S. A.'

76

E.

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

Human Genome Resources and Their Application for the
Analysis of Chromosome!1

Asao FUJIYAMA, Inaho DANJ0 1 and Hong-Seog PARK (lHuman Genome
Center, Tokyo University)
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.

(!)

Whole Genome Analysis of Signal-Transduction
Pathways in Fission Yeast

Inaho DANJ0 1, Yong-Sik BONG and Asao FUJIYAMA (lHuman Genome Center,
Tokyo University)
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 ofthelack of appropriate analysis system. To overcome this problem, we categorized genes based on their expression levels in the presence or absence of the

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DEPARTMENT OF INTEGRATED GENETICS

77

rasl gene product under different growth conditions. We utilized arrays ofclones covering entire genome of the fission yeast. Here, we demonstrate thedetection oflow molecular weight heat shock protein gene, hsp16, and showthat it is regulated by a ras-mediated signaling pathway, not by the heat shock
response in fission yeast.
(3)

Severe Lactic Acidosis and Neonatal Death in Pearson Syndrome

Kae MURAKIl, Yuichi GoT0 2, Ichizo NISHIN0 2, M. HAYASHIDANll, S. TAKEUCHI l ,Satoshi HORAI, N. SAKURAI, K. VEDA I (lDepartment of Pediatrics, Hiroshima
University School of Medicine, 2Department of Laboratory Medicine, National
Center Hospital for Mental, Nervous and Muscular Disorders)
Pearson marrow-pancreas syndrome, a fatal disease associated with mitochondrial DNA rearrangements, is characterized by refractory sideroblastic
anaemia during infancy. Only a few neonates with Pearson syndrome havebeen reported with metabolic acidosis. A female neonate who exhibited severe metabolic acidosis and anaemia at birth is described here. Her condition progressively worsened, with pancytopenia and uncontrollable metabolic
acidosis resulting in death at the age of 14 days. A 4988-base pair deletion of
mtDNA was detected in the patient's leukocytes, liver and muscle. When aneonate exhibits severe metabolic acidosis of unknown cause, the possibilityof Pearson syndrome should be considered. For details, see Ref. 1.

(4)

Myoclonus Epilepsy Associated with Ragged-Red Fibers:
A G-to-A Mutation at Nucleotide Pair 8363 in
Mitochondrial tRNA(Lys) in Two Families

Matsuko OZAWA l , Ichizo NISHIN0 2, Satoshi HORAI, Ikuya NONAKA l ,2, Yu-ichi
GOTO l ,2 (lDepartment of Laboratory Medicine, National Center Hospital for
Mental, Nervous and Muscular Disorders, 2Department of Ultrastructural
Research, National Insitute of Neuroscience)
In addition to well-known mutations at nucleotide pair 8344 and 8356 inmitochondrial DNA in patients with myoclonus epilepsy associated with raggedred fibers (MERRF), we found a new G-to·A point mutation at nucleotide 8363

78
in two Japanese families. The probands had the typical clinical characteristics of MERRF. Since the 8363 mutation was present in a heteroplasmic
state, and seen in none of 92 patients with other mitochondrial diseases or 50
normal individuals, this mutation is thought to be disease-related and probably specific to MERRF. As seen in muscle biopsies with the previous two
mutations, focal cytochrome c oxidase (CCO) deficiency was the most characteristic finding. With single fiber analysis, the CCO-negative fibers contained
a higher percentage of mutant DNA (88.4 +/- 6.6%) than CCO-positive fibers(65.1 +/- 8.0%). These findings suggest that mutations in tRNA(Lys) coding
region are related to the MERRF phenotype and are responsible for the reduced CCO activity. For details, see Ref. 5.

(5)Polymorphism of the HLA-DRBI Locus In Colombian,
Ecuadorian, and Chilean Amerlnds

N. BLAGITK0 1, C.O'HUIGIN 1, F. FIGUEROA J , S. HORAI, S. SONODA2, K.TAJIMA3,
D. WATKINS!, J. KLEIN' (IMax-Planck-Institut fur Biologic, Abteilung
Immungenetik, Tubingen, 2Department of Virology, Kagoshima University,
3Division of Epidemiology, Aichi Cancer Research Institute)
We have characterized the DRB1 genotypes in a sample of64 South American Indians drawn from populations in Chile, Colombia, and Ecuador. No
novel DRB 1 alleles were found in the total of 17 different alleles characterized, indicating that rapid allelic generation does not occur at the DRB11oci,in contrast to HLA-B. Comparison between Chilean and Colombian/Ecuadorian samples revealed no major differences in their allelic frequencies. In
the combined Amerind sample the HLA-DRB1*0407 and HLA-DRB1*1402
alleles occurred in the highest frequencies (38% and 22%, respectively).
Genetic distance measurement showed the HLA-DRB1 frequencies reported
here to agree with findings in other Amerind groups. The high frequencies ofboth HLA-DRB1*0407 and HLA-DRB1*1402 alleles, in conjunction with theirabsence in Siberian samples, suggest that migratory groups other than Siberians may have been involved in the peopling of the Americas. For details,
see Ref. 7.

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DEPARTMENT OF INTEGRATED GENETICS

79

The Geographic Distribution of Human Y Chromosome Variation

M. F. HAMMER', A. B. SPURDLE2, T. KARAFET', M.R. BONNER!, E. T. WOOD', A.
NOVELLETT0 3, P. MALASPINA3, R. J. MITCHELL4, S. HORAI, T. JENKINS 2 and S.
L. ZEGURA 5 ('Laboratory of Molecular Systematics and Evolution, and5Deaprtment ofAnthropology, University ofArizona, 2Department of Human
Genetics, South African Institute for Medical Research and the University of
Witwatersrand, 3Departimento di Biologia, Universita degli Studi, 4Department of Genetics and Human Variation, La Trobe University)
We examined variation on the nonrecombining portion ofthe human Y chromosome to investigate human evolution during the last 200,000 years. The
Y-specific polymorphic sites included the Y Alu insertional polymorphism or"YAP" element (DYS287), the poly(A) tail associated with the YAP element,
three point mutations in close association with the YAP insertion site, an A-Gpolymorphic transition (DYS271), and a tetranucleotide micro satellite(DYS19). Global variation at the five bi-allelic sites (DYS271, DYS287, andthe three point mutations) gave rise to five "YAP haplotypes" in 60 populations from Africa, Europe, Asia, Australasia, and the New World (n=1500).
Combining the multi-allelic variation at the microsatellite loci (poly(A) tailand DYS19) with the YAP haplotypes resulted in Ii total of 27 "combination
haplotypes". All five of the YAP haplotypes and 21 of the 27 combination
haplotypes were found in African populations, which had greater haplotype
diversity than did populations from other geographical locations. Only subsets of the five YAP haplotypes were found outside of Africa. Patterns ofobserved variation were compatible with a variety of hypotheses, including
multiple human migrations and range expansions. For details, see Ref. 6.

Pub I i ca t ions
1.

TENZEN,T, YAMAGATA, T., FUKAGAWA, T, SUGAYA, K, ANoo, A., INOKO, H., GoJOBORI, T,
FUJIYAMA, A., OKUMURA, K, IKEMURA, T: Precise switching of DNA replication timing
in the GC content transition area in the human major histocompatibility complex.

2.

YOSHIKAWA, H., NAGAI, H., OH, KS., TAMAI, S., FUJIYAMA, A., NAKANISHI, T, KA.JIYAMA,
G. and MATSUBARA, K: Chromosome Assignment ofAberrant Not I Restriction DNA

Mol. Cell. BioI., 17, 4043·4050, 1997.

80

3.

4.

5.

6.

7.

Fragments in Primary Hepatocellular Carcinoma. Gene, 197, 129-135, 1997.
IHA, H., TAKIMOTO, M., DANJOH, 1. and FUJIYAMA, A: Identification and character·ization of a novel trans-membrane protein gene, pdhl, from Schizosaccharomyces
pombe. DNA Research, 4, 393-396, 1997.
MURAKI, K, GOTO, Y., NISHINO, I., HAYASH lDAN I, M., TAKEUCHI, S., HORAl, S., SAKURA,
N. and UEDA, K: Severe lactic acidosis and neonatal death in Pearson syndrome. J.
Inher. Metab. Dis. 20: 43-48, 1997.
OZAWA, M., NISHI NO, 1., HORAl, S., NONAKA, 1. and GOTO, Y: Mutations in mitochondrial tRNALy. are responsible for myoclonus epilepsy associated with ragged-red fibers(MERRF) and cytochrome c oxidase defiency: an additional novel G-to-A mutation at
nucleotide pair 8363 in tRNALy. in two families. Muscle & Nerve 20: 271-278,1997.
HAMMER, M.F., JENKINS, T., BONNER, M.R., SPUDLE, AB., WOOD, E.T., MITCHELL, R.J.,NOVELLETTO, A, HORAI, S. and ZEGURA, S.L.: The geographic distribution of human Y
chromosome variation. Genetics 145: 787-805, 1997.
BLAGITKO, N., O'HUIGIN, C., FIGUEROA, F., HORAI, S., SONODA, S., TAJIMA, K, WATKINS,
D. and KLEIN, J.: Polymorphism of the HLA-DRBllocus in Colombian, Ecuadorian,and Chilean Amerinds. Human Immunology 54 (1): 74-81, 1997.

E-b. Division of Agricultural Genetics(1)

Mapped Genomic Locations for QTLs In Rice Reflect Gene Clusters

Hong-Wei CAl and Hiroko MORISHlMA
Wild and cultivated rices differ in a set of correlated characters called domestication syndromes. All those characters are quantitative traits which
could be analyzed only by statistical genetic methods in the past. Recent
advances in genome research enabled us to map quantitative trait loci (QTL)
on the genome. As preliminarily reported last year, to look into the genetic
basis of preferential character association underlying domestication syndromes, we analyzed 125 recombinant inbred lines (RILs) derived from a crossbetween wild and cultivated rice strains.
First, 12 linkage maps were constructed from segregation data of 140 RFLP
and isozume markers. Then QTL analysis was performed by examining
cosegregation between 32 quantitative traits and 140 markers. The interval
mapping method revealed one to 24 QTLs for every 25 characters that weredetected, respectively. Among those traits, diagnostic characters for wild

E. DEPARTMENT OF INTEGRATED GENETICS81

and cultivated types, such as seed dormancy, awn length, anther length, aswell as some Indica /J aponica diagnostic traits, such as KCI03 resistance,
apiculus hair length, and germination speed proved to be controlled by relatively large number of QTLs.
It was found that several QTLs each of which controls different wild / cultivated diagnostic characters tend to cluster on the specific regions ofchromosomes. Similar clusters were redundantly found over the genome.
This cluster phenomenon was understood as multifactorial linkages resulting
in domestication syndromes. Multifactorial linkages refer to partial linkages inevitably caused by more or less random distribution of multiple factors
for different characters on a limited number of chromosomes. Further, it was
found that near the chromosomal regions harboring those gene clusters cultivar alleles segregated at higher frequency than expected among
RILs. Directions of gene effect of each QTL forming clusters were coincidently the same as predicted from parental phenotypes. Most probably, geneclusters were inconsciously selected for their coadapted character association
under cultivation pressure during developing RILs. This inference was supported by the fact that at the isozyme loci located near the clusters frequenciesof cultivar alleles gradualy increased in RILs during P to P, and that locioutside of the clusters did not show such a trend. Thus, clusters of QTLs
found in the present study must be the product moulded by multifactorial
linkages and natural selection which worked on coadapted gene sets. Such alinkage system does not prevent, but does restrict recombination in the evolutionary process.

(2)

Phylogenetic Study of AA Genome Wild Rice Species Viewed from
Phenotypic and Genic Levels

Masahiro AKIMOTO, Yoshiya SHIMAMOTO and Hiroko MORISHIMA
Variability of 21 quantitative characters and 29 isozyme loci were studied
for five AA genome wild rice species; Oryza rufipogon, O. glumaepatula, 0.
meridionalis, 0. longistaminata and 0. barthii. Polymorphism at phenotype
and isozyme levels were respectively examined by principal component analySIS.

82

O. ruflpogon and 0. glumaepatula, showing higher phenotype variability
than O. meridionalis and O. barthii, were divided into two and three clusters inthe scatter diagram, respectively; namely O. rufipogon-r, II and O.
glumaepatula-r to III. 0. ruflpogon-r and 0. glumaepatula-r, which were categorized as perennial types, were plotted overlapping each other. While, 0.
rufipogon-II joined 0. meridionalis and 0. barthii, forming an annual typegroup. On the other hand, isozyme analysis showed that five species formed
respective clusters without overlapping, indicating that they are geneticallydifferentiated from each other.
Phenotypes are subjected to natural selection. The plants, even with different phylogenetic backgrounds, are likely to develop similar phenotypes when
they grow in similar environments. The previously mentioned perennial wild
species prefer stable and less disturbed environments where density-dependent mortality is high. While, annual types tend to inhabit unstable andoccasionally disturbed environments where density-independent mortality ishigh. Judging from the incongruence between inter- and intra-species variation pattern revealed by phenotype and genotype, similarities in phenotypes
were most probably homoplasy caused by evolutionary convergence.
Diversities of mitochondria DNA and chloroplast DNA were examined forthe same materials by RFLP analysis. Results of both analysis reflected
geographical distributions of five species rather than their phenotypic variation pattern. This also indicated that the phenotype similarities were
homoplasy. For details, see Ref. 1.
(3)

New Storage Protein Genes in Rice Detected by Acidic FormatePAGE MethodHong-Wei CAl and Hiroko MORISHIMA
Acidic PAGE was widely used to detect variations of salt-soluble proteins ofwheat and barley. We applied this method to analyze variation in seed storage proteins of rice using 98 cultivars and 25 wild rice strains. Protein was
extracted from the individual ground seeds using 0.05M NaCl and 20% sucrose as an extraction buffer.
Salt-soluble proteins were separated into more than ten bands showing

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DEPARTMENT OF INTEGRATED GENETICS

83

variability among strains. At least three loci (tentatively namedAPAGE 1,2
and 3) could be identified. Segregation of 1:1 ratio for APAGE 1 and 2, respectively, was confirmed in recombinant inbred lines of several crosses. Throughlinkage analysis with 140 RFLP and isozyme markers, these two protein lociwere mapped on chromosome 5 and 11, respectively.
Four alleles were found at PAGE 1 locus. Allele 1 and 2 were frequently
found in J aponica and Indica types of cultivars, respectively. Allele 3 was
frequent in the perennial type of wild rice, though annual types harbored exclusively allele 2. So far, many gene markers were found to be diagnostic inclassifying Indica and Japonica types. But, few markers are known useful todistinguish both wild and cultivated rice, as well as perennial and annualtypes of wild rice. A PAGE 1 could be a useful marker for the evolutionary
study in rice. For details, see Ref.4.

Publications
1.

2.

3.
4.
5.

6.

7.

AKIMOTO, M., SHIMAMOTO, Y. and MORISHIMA, H.: Genetic population structure of wild
rice Oryza glumaepatula distributed in the Amazon flooded area influenced by its lifehistory traits. Molecular Ecology 7,1371-1381,1998
AKIMOTO, M., SHIMAMOTO, Y. and MORISHIMA, H.: Genetic differentiation in Oryza
glumaepatula and its phylogenetic relationship with other AA genome species. Rice
Genet. Newsletter 14, 37-39, 1997
CAl, H.W. and MORISHIMA, H.: Indica-Japonica differentiation of Bangladesh rice cuItivars detected by isozyme analysis. Rice Genet. Newsletter 14, 29-30, 1997
CAl, H.W. and MORISHIMA, H.: New storage protein genes detected by Acidic Formate
PAGE method. Rice Genet. Newsletter 14, 76-78, 1997
KOHN, J.R., LEYVA, N., DOSSEY, R., PAREDES, 0., SOBRAL, B. and MORISHIMA
H.: Quantitative trait locus analysis oftrait variation among annual and perennial
ecotypes Oryza rufipogon. Int. Rice Res. Notes, 22(2), 4-5, 1997
SUH, H.S., SATO, Y. 1. and MORISHIMA, H.: Genetic charcterization of weedy rice (Oryza
sativa L.) based on morpho-physiology, isozymes and RAPD markers. Tbeor Appl
Genet, 94, 316-321, 1997
TANG, L.H., MORISHIMA, H.: Genetic characterization of weedy rices and the inference
on their origins. Breeding Science 47, 153-160, 1997

84

E-c. Division of Appl ied Genetics(1)

Composite and clinal distribution of GlycIne soja In Japan revealed by RFLP analysis of mitochondrial DNA

Azumi TOZUKA 1, Hirohumi FUKUSHI 1, Toshiyuki HIRATA 1, Masashi OHARA 1,
Akira KANAZAWAl, Tetsuo MIKAMI 1, JunABE 1 and Yoshiya SHIMAMOT0 1 (IFaculty of Agriculture, Hokkaido University, Sapporo)
Wild soybean (Glycine soja Sieb. et Zucc.), regarded as the progenitor ofcultivated soybean (G. max(L.) Merr.), is widely distributed in East Asia. We
have collected 1097 G. soja plants from all over Japan and analyzed restriction fragment length polymorphism (RFLPs) of mitochondrial DNA(mtDNA). Based on the RFLPs detected by gel blot analysis using coxII andatp6 as probes, the collected plants were divided into 18 groups. Five mtDNA
types accounted for 94% of the plants examined. The geographic distributionof mtDNA types revealed that wild soybeans grown in Japan consisted of a
mixture of plants with different types ofmtDNA in many regions, occasionallyeven within sites. Some ofthe mtDNA types showed marked geographic clines
among the regions. Some wild soybeans possessed mtDNA types that wereidentical to those widely detected in cultivated soybeans. Our results suggest that the analysis of mtDNA could solve the maternal lineage among
plants of the genus Glycine subgenus Soja. For details, see Ref. I.

(2)

Genetic diversity, geographical differentiation and evolution In
the cytoplasmic genome of the wild soybean,
Glycine soja, growing In China

Yoshiya SHIMAMOT0 1, Hirohumi FUKUSHI 1, JunABEl, Akira KANAZAWA 1, Junyi
GAI 2, Zhong GA0 2 and Donghe Xu 2 (lFaculty of Agriculture, Hokkaido
University, Sapporo, 2S oybean Research Institute, Nangjing Agriculture
University, Nangjing)
RFLPs of chloroplast and mitochondrial DNA have been used to characterize the cytoplasmic genome of wild soybean, Glycine soja, growing in China.
We have found from the RFLPs of chloroplast (cp) and mitochondria (mt) that

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DEPARTMENT OF INTEGRATED GENETICS85

the cytoplasmic genome of most Chinese wild soybeans have a combination of
cpIII with mt-a or a combination of cpII with mt-b accompanied by mtlV or
mtV. CpII was not observed in combination with mt-a and cpIII was notobserved in combination with mt-b. The regional distribution of these two
types showed clines with opposite directions. The Yangtze River Valley had
the greatest diversity in cp and two mt profiles detected in this study and thecytoplasmic genome combining these three profiles showed the highest degreeof polymorphism in this region. The Yangtze River Valley may be a center ofcytoplasmic diversity of wild soybean and may contain various genetic resources of soybean. For details, see Ref.2.

(3)

Molecular Genetic Studies of Genomic Imprinting

Hiroyuki SASAKI (Institute of Genetic Information, Kyushu University)
Genomic or parental imprinting refers to a biological process that modifies
the genome differently in the male and female germline, leading to differential
activity of the parental genomes in the offspring. In order to understand themolecular mechanisms involved in imprinting, we -study the imprinted genecluster in the distal portion of mouse chromosome 7, in which the insulin 2(Ins2), insulin-like growth factor 2 (Igf2) and H19are contained. The results
obtained last year were as follows.
(1) We cloned and characterized a biallelically expressed gene, called L23mrp,
which is located downstream of H19. Furthermore, we showed that L23mrpis
functionally insulated from the H19 enhancers, suggesting that there may be
a domain boundary between H19 and L23mrp (ref. 1). (2) We found a newfeature common to many imprinted genes, i.e., the arrest-specific up-regulation in embryonic fibroblast cells in culture, which may give us a clue to understand the evolution of imprinting (ref. 2). (3) We identified and characterized
multiple sense and antisense transcripts in the mouse Igf2region, which arealso subject to genomic imprinting (ref. 3). (4) We showed that Igf2, which
has three different promoters, is controlled mainly by a locus-wide mechanism(in press). (5) We have sequenced a 30-kb putative boundary region of theimprinted Ins2/Igf2/H19 domain located between H19 and L23mrp (inpress). (6) A convenient method to detect and localize CpG islands in a large

86

genomic region has been developed (submitted). This technique should be
useful in identifying new imprinted genes. (7) We have determined the timing of the erasure and establishment of the methylation imprint, which ispresent in the 5' flank of H19, in the parental germline (submitted). (8) To
study the structure and function of the imprinted domain on distal chromosome 7, we constructed YAC, BAC and cosmid contigs covering the region andare now trying to close the final gaps. We also plan to sequence the entire
region.

Publ icatians
TOZUKA, A, FUKUSHI, H., HIRATA, T., OHARA, M., KANAZAWA, K., MIKAMI, T., ABE, J. and
SHIMAMOTO, Y.: Composite and dinal distribution of Glycine soja in Japan revealedby RFLP analysis of mitochondrial DNA Theor. Appl. Genet., 96, 170-176, 1998.
2. SHIMAMOTO, Y, FUKUSHI, H., ABE, J., KANAZAWA, A, GAO, Z., Xu, D. and GAl, J.: Genetic
diversity, geographical differentiation and evolution in the cytoplasmic genome of thewild soybean, Glycine soja, growing in China. Genetic Resources and Crop Evolution,
45, 433-439, 1998.
3. ZUBAIR, M., HILTON, K., SAAM, J.R., SURANI, M.A., TILGHMAN, S.M. and SASAKI,
H.: Structure and expression of the mouse L23mrp gene downstream of the imprinted H19gene: biallelic expression and lack of interaction with the H1genhancers.
Genomics 45, 290-296, 1997.
4. HAYASHIDA, T., EVERSOLE-CIRE, P., JONES, P.A and SASAKI, H.: Imprinted genes are upregulated by growth arrest in embryonic fibroblasts. J. Biochem. 122, 901-903, 1997.
5. MOORE, T., CONSTANCIA, M., ZUBAIR, M., BAILLEUL, B., FElL, R., SASAKI, H. and REIK,
W.: Multiple imprinted sense and antisense transcripts, differential methylationand tandem repeats in a putative imprinting control region upstream of mouse Igf2.
Proc. Natl. Acad. Sci. USA 94, 12509-12514, 1997.
1.

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GENETIC STRAINS RESEARCH CENTER

F-a. Mammal ian Genetics Laboratory(1)

Analysis of the Genetic Structure of Pb
Hotspot in the C1assII of MHC

Taku ISOBEI, Masayasu YOSH1N0 2, Kirsten FISHER LINDAHAL 2, Kenichi
MIZUN0 3, Tsuyoshi KOIDE, Kazuo MORIWAKI 1 , Toshihiko SHIROISHI(lGrad.Univ.Advanced.Studies, 2Univ. Texas, 3Inst.Phys.Che. Res.)In mammals, the murine major histcompatibility complex (MHC) is a uniqueregion where breakpoints of meiotic recombination have been systematicallystudied at molecular level. In this region, meiotic recombinations do not occur at random but are clustered in restricted regions known as hotspot. Thusfar, four hotspots have been identified in this region. Among four, Ph hotspot
has been poorly characterized. In the present study, we constructed a finerestriction map ofthe 15kbp of DNA fragment which contains the recombinational breakpoints. Molecular mapping revealed that five recombinants wereconfined to a 2.4 kb DNA segment located proximal to the 3'end of Ph gene.
Comparing the molecular structure of the four hotspots including Ph hotspot
in the mouse MHC class II, we found that all hotspots are located in introns or
3' end of genes, but not at 5' end of genes. This contrasts to hotspots identified in budding yeast, in which most of hotspots are located at 5' end of genes
overlapping with transcription initiation sites. Furthermore, we could not
find the consensus sequenses and motifs common to all four hotspots.
We have started analysis of higher chromatin structure around the hotspots
in meiotic prophase, focusing on topological relation of the hotspots to structure of synaptonemal complex. We have established the protocol using the
FISH method and immunostaining method together.

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(2)

Fine Mapping of a Preaxial Polydactyly Mutant, Hlml

Hiroshi MASUYA, Tomoko SAGAI, Shigeharu WAKANA I and Toshihiko
SHIROISHI (lCentral Institute for Experimental Animals)
In vertebrate limb development, the anteroposterior axis formation is mediated by a signal molecule, Sonic hedgehog. The localized expression of Sonic
hedgehog at the posterior margin of developing limb (limb bud) is disrupted inseveral preaxial polydactyly mutants in mouse. Rim4 is a preaxial polydactyly mutation which induces the ectopic Sonic hedgehog expression at the anterior margin oflimb bud.
Toward positional cloning of Rim 4 gene, we have carried out the fine mapping of Rim4 gene in crosses with two laboratory strains, DBA/2J or NZB.
Because the penetrance of Rim4 was incomplete, we used only the backcrossprogeny which showed polydactyly. As a result, Rim4 was mapped to Chromosome 6 and between D6Mit279 and D6Mit320. For the assignment of
the genetic markers in this region, we conducted a linkage analysis based on
the backcross panel using the intersubspecific cross; (C57BL10 X MSM)F1 x
C57BL10. We mapped 13 microsatellite markers in the Rim4 critical region
by SSLP analysis using 1500 progeny. Analysis ofYAC clones which contains these genetic markers revealed that gene members which belong IgVk
familiy (lgVklo, 33, 23 and 28), CD8familiy (CD8a and CD8b), IgJk and IgCk
located in this region.

(3)

Genetic Analysis and Physical Mapping of Polysynductylous
Mouse Hemimelic extra toes (Dx)

Tomoko SAGAI, Hiroshi MASUYA and Toshihiko SHIROISHI
Many limb mutants are known in the mouse. Some of them have abnormal three axes (proximo-distal, antero-posterior and dorso-ventral) formation
governed by particular signaling centers. One of the mutants, hemimelicextra toes (Hx) shows preaxial polyductyly on all four feet, associated withhemimelia, shortening of the radius, tibia and talus. Hx is located on theproximal region of Chr 5 and is very closely linked to hammer-toe (Hm) mutation, which shows interdigital webbing regression probably due to an impair-

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ment of apoptosis. Because sonic hedgehog (shh) gene that mediates anteroposterior signaling has been also mapped to this region and ectopic expression of shh has been detected in the limb bud of Hx, it has been proposed thatshh is a candidate gene for Hx.
We have mated the several laboratory and wild inbred strains with Hx heterozygotes and observed the phenotypes of the progeny. The progeny fromdifferent mating crosses exhibited characteristic phenotypes. In particular,
hemimelia that characterized the original mutant phenotype reproducibly disappeared in the progeny from some crosses. The results suggested a possibility that genetic background of the strains modified the phenotype of Hx
mutation. We are trying to fix these phenotypes by continuous backcross tothe corresponding strains.
The linkage analysis of Hx mutation using 1570 backcross progeny fromintersubspecific cross have mapped the candidate gene to a 0.6 cM segment.
One of the recombinants could separate the coding region of shh from Hx gene,and excluded the coding region of shh gene as a candidate for Hx gene. We
assume that there is at least one or two genes in this region in addition to shh,which may be responsible for the antero-posterior specification and theapoptotic cell death. In human, a complex bilateral limb deformity (NicoleiHamel polysynductyly) has been mapped to the syntenic region of chromosome
7q36. For the positional cloning of Hxand Hm genes, we have established a
YAC contig covering the Hx region. The result showed that Hx and shh genes
are colocalized within a 1.5 Mb single YAC clone snd Hxgene is included in a
600 kb single YAC clone. The construction ofBAC contig covering the 1.5 Mb
DNA which includes Hx and shh genes is now under way.
(4)

Mesenchymal Dysplasia (mes), which
Exhibits Preaxial Polydactyly

Shigeru MAKIN0 1 , Hiroshi MASUYA, Mizuyo TSUGANE 2 and Toshihiko
SHIROISHI (IGrad. Univ. Adv. Stud., 2Sapporo Univ. Medic.)
Numerous preaxial polydactyly mutations in mouse are caused by duplication of zone of polarizing activity (ZPA), which is coupled with mirror-image
duplication of digits. A recessive mutation, mes, shows preaxial polydactyly.

90

In addition, mes causes multiple skeletal anomalies, including a shortened
and wider face with wide-set eyes, a domened head and kinky tail. Excess
skin and increased musculature in the shoulders and hips are seen in the
mutant.
We examined whether mes exhibits symmetrical digit duplication similar
to other preaxial polydactyly. Toluidine blue and alizarin red staining indicated that mes has thickened feet and bifurcate metacarpal bones and metatarsal bones, but not mirror-image duplications of the skeletal pattern of thedigits and truncated zeugopod like Rim4, which exhibits ectopic expression of
Shh and Fgf4 genes at the anterior margin ofthe limb. Furthermore, mes had
abnormally shaped xiphisternum and bifurcate sternum. On the basis ofthese findings, it appeared that mes mutation alters normal growth and differentiation of mesenchyme-derived cells. Preaxial polydactyly may not be
due to abnormally axial pattern formation.
To determine the chromosomal location of mes, we carried out linkage analysis by cross with MSM strain. As a result of 79 backcross progeny, mes was
mapped between D13Mit42 and D13Mit210. This region includes six potential candidate genes for meso Three candidates, Pte, Pitxl and mdac, playa
role in positional specification during the pattern formation. As mes may be
a mutation in a gene involved in early mesenchyme formation or growth, theother three, Mad3, Gas} and Fgfr4, which playa role in cell growth or differentiation, are better candidates.

(5)

Effect of Ts Mutation on Mouse Development

Junko ISHIJIMA, Akihiko MITA, Kikue UCHIDA, Toshihiko SHIROISHI
Mice with the Tail-short (Ts) mutation have a shortened, kinky tail andnumerous skeletal abnormalities, including a homeotic anteroposterior patterning problem involving the axial skeleton. The phenotype is variable ondifferent genetic backgrounds as is seen in many other mouse dysmorphogenic
mutants. The viability of Ts heterozygotes varies dramatically, dependingon the mouse strain crossed with the mutant strain. At the extremes, theheterozygotes are viable or lethal prenatally. Detailed linkage analysis indicated that a single chromosomal region, genetically inseparable from the Ts

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91

locus, is responsible for the difference. This result suggests that there exists
polymorphism at Ts locus among inbred strains, and the allelism affects theviability of the Ts heterozygotes.
The different manifestations of the phenotype of Ts heterozygotes between
these two crosses was useful to understand the function of the Ts gene indifferent stages of mouse embryogenesis. We investigated the phenotype of
the Ts mutation in crosses with the two groups of strains, which give viable
and lethal Ts embryos respectively. Morphological and histological analyse
indicated that Ts embryos from the lethal cross exhibit more severe developmental defects. In neural plate stage, the embryonic region was poorly developed compared with the extraembryonic region due to formation of excess
mesoderm in the extraembryonic region. The inappropriate allocation of themesodermal cells at the early gastrulation stage may cause shortage of progenitors of the embryonic mesodermal cell in the primitive streak of Ts
heterozygotes. In the later developmental stage, several lines of evidence forthe disturbance in establishment of the segmental identity were observed in
Ts heterozygous embryos, which is associated with alternations of expression
pattern of marker genes. Among neural tube defects, malformation of thehindbrain was common phenotype of Ts heterozygotes. The expression boundary of some genes which thought to be important for maintaining the segmental identity of hindbrain was altered. Additionally, in the viable cross Ts
heterozygous mice showed several homeotic transformations in the axial skeletal formation with high reproducibility. Most characteristic two phenotypes
were posterior transformation of the cervical vertebra to the thoracic vertebra,
and an additional vertebra in the upper thoracic region with anterior transformation of all the prevertebrae caudal to the additional vertebra. We hypothesize that Ts gene plays a crucial role in establishment of mouse axial bodyplan from early gastrulation stage through to early fetal stage. Further study
to connect the complex mutant phenotypes observed beyond different developmental stages would certainly provide a clue to clarify the functions of Ts gene.

92

(6)

Positional Cloning of the Mouse Skeletal
Mutation, TaJl short ( Ts)Kunihiko SHIMIZU 1, Tuyoshi KOIDE, Akihiko MITA, Kikue UCHIDA, Shigeharu
WAKANA2, Yoshiaki KIKKAWA 3, Hiromichi YONEKAWA 3, Hiroki SASAKI 4 andToshihiko SHIROISHI(lDepartment of Pediatric Dentistry, Nihon University School of Dentistry at
Matsudo, 2Central Institute for Experimental Animals, 3Department ofLaboratory 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 teromericregion 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 phenotype similar to Ts and has been mapped to human syntenic region,
17q21-24.
To elucidate the function of the Ts gene in mouse embryogenesis and verify
whether it is a model for MES, we are trying to clone the gene by using themethod of positional cloning. First, we employed a fine mapping of this genebased on a large scale intersubspecific backcross between the mutant stock
TsJILe-Ts/+ and Japanese wild mouse-derived MSM strains. Ts gene wasmapped to a 0.16cM region between two microsatellite markers, DllMit128
and DllMit256. We screened mouse YAC and BAC libraries with themicrosatellite markers tightly linked to the Ts locus and have obtained YAC
and BAC clones. Further chromosome walking with the isolated clones allowed us to construct a complete BAC contig covering the Ts causative gene.
This contig consists of 6 BAC clones, which spans a 250-300 kb DNA fragment.
We have isolated several cDNA clones from the critical region by directed
cDNA selection using the corresponding BAC clones to search for candidate
genes for Ts. Characterization ofthese cDNA clones is now underway to identify the Ts causative gene.

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GENETIC STRAINS RESEARCH CENTER93

A susceptibility gene Idd4 controls onset of IODM :an allele from non- diabetic MSM strain
is associated with early onsetof diabetes in mice

Shigeharu WAKANAl, Toshihiko SHIROISHI 2, Kazuo MORIWAKI 3, Tatsuji
NOMURA! (lCentral Institute for Experimental Animals, 2National Instituteof Genetics, 3The Graduate Univ. for Advanced Studies)
The NOD mouse is a widely used model of human insulin-dependent diabetes mellitus (IDDM). Because genetic loci responsible for IDDM are as many
as eightteen, the NOD mouse is also used as a model for studying genetic
complex traits. The susceptibility loci have been identified by linkage analysis in crosses between the NOD mouse and other non-diabetic mouse strains.
We have studied the inheritance ofthe susceptibility to IDDM by genetic crossbetween NOD/Shi and a Japanese wild mouse-derived MSM strain. Threeloci Iddl, Idd3and IddlOappeared to control the incidence ofinsulitis. However,the contributions of other Idd loci including Idd4 were not detected in theabove cross. To investigate the effect ofMSM allele of Idd4without influenceof the other susceptibility genes, we have established the congenic strain for
Idd4 by introducing the chromosomal segment containing Idd4locus from
MSM strain into the NOD genetic background. The incidence of diabetes andinsulitis in the NOD/Shi. Idd4 nod/m.m congenic mice was not lower than that in
NOD mice. On the other hand, the incidence of diabetes at early onset(-2Owks) in the female NOD/Shi. Idd4 m• m/m • m mice was rather higher than thatin NOD mice or the NOD/Shi. Idd4 nod/ m• m congenic mice. This result indicatedthat an allele from non-diabetic MSM strain is associated with early onset of
diabetes in mice. For fine mapping the Idd4, we have produced a series of six
recombinant strains which carry various segment from MSM chromosome inthe congenic region containing Idd4. Comparing with the incidence of early
onset diabetes among the congenic strains, we mapped Idd4 within a shortsegment approximately less than O.8cM on chromosome 11.

94

(8)

Characterization of Hyperactive Behavior in MSM Derived fromJapanese Wild Mouse, and Hypoactive Behavior in
JFl Established from Japanese Fancy Mouse

Tsuyoshi KOIDE, Kazuo MORIWAKI 1 and Toshihiko SHIROISHI (lGrad. Univ.
Adv. Stud.)
Behavior of mouse has been studied from many aspects using only small
numbers of laboratory strains. However, those laboratory mice have been
domesticated in the course of establishing them as inbred strains, and hence
their characteristic behavior has been lost by the domestication process. On
the other hand, wild mice behave differently from the laboratory mice. Thisimplies that laboratory mice that are used in many fields of brain research
have accumulated spontaneous mutations in genes which control their
behavior. In this point of view, use of inbred strains derived from wild mice
will be more important in future brain research and behavioral studies. We
have started a new project in that behavioral patterns of inbred strains established from wild mice and laboratory strains is compared in many aspects ofmouse behavior, and the difference between the strains is genetically dissected.
An inbred strain JF1 has been established from Japanese fancy mice which
was highly domesticated and obedient. On the other hand, another inbred
strain MSM which was established from Japanese wild mouse is still very
active. To quantify the general activity in these two mouse strains, locomotor
activity of each mouse strain was measured for continuous four days in thecycle of 12 hours light and 12 hours dark. Both of JF1 and MSM strains areactive in the dark, but the overall activity of MSM is extremely higher thanthat of JFl. This difference in the locomotor activity is studied by geneticalanalysis. We have inter-crossed (JF1 and MSM)F1 progeny, and resultant
F2 progeny were tested for the locomotor activity. Based on the linkage analysis of the progeny, we intend to map the loci that control the difference inlocomotor activity between JF1 and MSM strains.

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95

Construction of Mouse Mutant Bank by CRL Mutagenesis

Toshihiko SHIROISHI, Tatsuya FUKUDA), Kikue UCHIDA, Miyoko MASUYA andAkira MITA (IChugai Pharmaceutical Co.)
Chemotherapic agent chlorambucil (CHL) is highly mutagenic in male germ
cells of the mouse. Post meiotic germ cells especially early spermatids are
the most sensitive to the mutagenic effects of this agent. It is establishedthat CHL induces predominantly chromosomal deletions. The optimum rateof mutation is estimated to be 10-3 per locus per gamete, when progeny siredfrom males treated with CHL is screened. This implies that we can expect toobtain a mutation at a given locus by screening 1, 000 progeny at about 80%
probability.
We have started a project to construct mouse mutant bank by CHL
mutagenesis. In this project, males ofMSM established from Japanese wild
mouse, Mus musculus molossinus, are treated with CHL and crossed withfemales of strain C3HIHeJ. The F1 progeny generated from the above crossare expected to have random chromosomal deletions over MSM genome. Since
MSM and C3HIHeJ strains are genetically very remote, the polymorphism
between these two strains allows to detect deletions of MSM alleles at themost loci. We have confirmed that ordinary nusive agarose gel electrophoresis is useful to distingush alleles of MSM from that of C3H/HeJ by RFLP
analysis for PCR-amplified fragments of 3' non-coding region of the mostgenes. We have also tested whether it is possible to store sperms of F1 hybrids generated from cross of (C3HIHeJ x MSM) in liquid nitrogen and to use
for in vitro fertilization to produce mice with deletion mutations. It appearedthat the frozen sperms of the F1 hybrids were preserved and used for in vitro
fertilization without loss of capability of fertilization.
Currently, we are expanding the F1 progeny sired from mutagenized malesof MSM strain toward construction of the sperm bank with saturated
mutations. We also intend to develop the system to detect chromosomal
deletions by genome scanning based on two dimensional acrylamide gel electrophoresis.

96

Pub Ii ca t ions
1.

MAsUYA, H., SAGAI, T., MORIWAKI, K. and SHIROISHI, T.:

Multigenic control of the local-

ization of the zone of polarizing activity in limb morphogenesis in the mouse. Devl.
BioI., 182: 42-51, 1997.
2.

REUSS, F.U., FRANKEL, WN., MORIWAKI, K., SHIROISHI, T. and COFFIN, J.M.:

Genetics ofintracisternal-A-partcle-related envelope-encoding proviral elements in mice. J. Virol.,70: 6450-6454, 1997.
3.

OYAKE, M., ONODERA, 0., SHIROISHI, T., TAKANo, H., TAKAHASHI, Y, KOMINAMI, R.,MORIWAKIK., IKEUCHI, T., IGARASHI, S., TANAKA, H. and TSUJI, S.: Molecular cloning of murine
homologue Dentatoubral Pallindoluysian Atrophy (DRPLA) eDNA: Strong conservation of a polymorphic CAG repeat in the murine gene. Genomics, 40: 205-207, 1997.

4.

WAKANA, S., SHIROISHI, T., PUSCHEL, A.W. and IMAI, K.:

The mouse sepaphorin F(Serna!) maps to chromosome 15. Mammal. Genome, 8, 698-699, 1997.
5.

TANAKA, Y, NARUSE, I., MAEKAWA, T., MASUYA, H. and SHIROISHI, T. and ISHII, S.:

Abnormalskeletal patterning in embryos lacking a single Cbp alleleL A partial similarity with
Rubinstein-Tabybi syndrome. Proc. Natl. Acad. Sci. USA, 94, 10215-10220, 1997.
6.

KAGITANI, F., KUROIWA, Y, WAKANA, S., SHIROISHI, T., MIYOSHI, N., KOBAYASHI, S., NISHIDA,
M., KOIIDA, T., KANEKO-IsHINO, T. and ISHINO, F.:

Peg5/Neuronatin is an imprinted

gene located on sub-distal chromosome 2 in the mouse. Nucl. Acids Res., 25, 34283432, 1997.
7.

SHIROISHI, T., UEDA, Y., BALL, S., NAGAMINE, C.M., SAGAI, T. and MORIWAKI, K.:

Genetic

differentiation of mouse Y chromosome. J. Repro. Dev., 43, Suppl.: 17-18, 1997.
8.

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

SATOH, 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,
10.

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.
11.

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

Genomic structure andchromosomal localization of the mouse Oggl gene that is involved in the repair of 8hydroxyguanine in DNA damage. Mammal. Genome, 9: 32-37, 1998.
12.

HASHIMOTO, Y, SHINDO-OKADA, N., TANI, M., NAGAMACHI, Y, TAKEUCHI, K., SHIROISHI, T.,

F. GENETIC STRAINS RESEARCH CENTER

97

TOMA, H. and YOKOTA, J.: Expression of the Elml gene, a novel gene ofCCN family,
suppresses In vivo tumor growth and metastasis of K-1735 murine melanoma cells. J.
Exp. Med. 187: 289-296, 1998.
13. 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 membrane-bound CD4. Eur. J. Immunol. 28:403-412, 1998.
14. OBATA, Y., KANEKO-IsHINO, T., KOIDE, T, TAKAI, Y, VEDA, T, SHIROISHI, T, ISHINO, F. and
KONo, T.: Disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes during embryogenesis. Development, 125: 15531569, 1998.
15. 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.

F-b. Mammal ian Development Laboratory(1)

Analysis of Cellular and Molecular Mechanisms in Development of
Mouse Fetal Germ Cells and Sex Differentiation of Gonads

Norio NAKATSUJI, Yasuaki SHIRAYOSHI and Tetsuichiro SAITO
We have been studying proliferation and growth regulation of mouse primordial germ cells (pGCs) during migratory stages (7.5 - 10.5 days post coitum) byusing a culture system.

They show a temporary proliferation in culture, but

their growth is arrested at a time corresponding to 12.5 -13.5 dpc when theydifferentiate into gonia after arriving at fetal gonads.

We tried a few meth-ods for gene transfection of mouse primordial germ cells (PGCs) in culture asa prelude to the investigation of molecular mechanisms of germ cell
development.

Using the CaP04 co-precipitation method, 18 % ofPGCs trans-

fected with plasmid pSV-LTexpressed simian virus 40 large tumor antigen(SV 40 T-Ag) transiently.

However, we did not detect any effects on the pro-liferation and survival ofPGCs obtained from embryonic gonads at 11.5 dpc
during 2 days of culture after transfection.

PGCs isolated from 11.5 dpc go-nads change from spread- to round-shape and exhibit growth arrest during a
few days of culture, and these rounded PGCs quickly disappear from theculture.

We found that the transfection and expression of Bel,XL or adenovi-

rus type 2 EIB 19,000-molecular-weight protein (E1B 19K) significantly pro-98
moted the survival ofPGCs and retarded the disappearance of rounded PGCs
from the culture system. These results suggest that Bel-XL or EIB 19Kcan
prevent the apoptosis of PGCs and inhibit cell death of rounded PGCs inculture. For details, see Ref. 1.
To extend our studies on PGCs, we are carrying out two lines ofexperiments. Firstly, we are developing a co-culture system of germ cells andgonadal somatic cells to investigate cell interaction during the entry into meiosis
or mitotic arrest by germ cells. Secondly, we have been trying to identify
novel genes involved in sex-differentiation of germ cells and gonads at thesestages. Sex-differentiation of mouse fetal gonads starts by appearance oftestis cords in testis at 12.5 dpc, followed by mitotic arrest of germ cells in
testis and initiation of meiosis in ovary starting at 13.5 dpc. It has been
known that male somatic cells direct germ cells into the spermatogenic differentiation in the testis. Such initial sex-differentiation is proceeded by expression of Szygene in precursors of sertoli cells of male gonads at around 11.5
dpc. Since the identification of Sry, a transcription factor, as the testis-determining gene, vigorous attempts have been made to discover the following
cascade of gene groups which should be in play to bring about differentiation of
the male germ cells and testicular somatic tissues, but with only a limited
success. Previous studies have also identified several genes important forsex differentiation in mammals, including SF-l/Ad4BP, WT-l, Sox 9 andflaxl. However, a number of other unknown genes should be involved in sex
differentiation of gonads. We have been trying to identify such genes in miceby using the subtraction and differential hybridization method to obtain genes
whose expression is specific for the 13.5 dpc testis after subtraction by thefemale gonad cDNA.
Thirty-two clones were analyzed, and 20 were found to be known male-gonad-specific genes, including Mullerian inhibiting substance and Desert
hedgehog: Remaining 12 clones represent novel sequences. We chose a novel
gene containing typical basic helix-loop-helix domains for further
investigation. Its cDNA size was 1,237 bp and it contained a protein coding
region of 540 bp that presumably codes 179 amino acids. 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

F. GENETIC STRAINS RESEARCH CENTER

99

than 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.

(2)

Differentiation and Migration of Neural Cells in
Mouse Central Nervous System Development

Tetsuichiro SAITO and Norio NAKATSUJI
The mammalian central nervous system comprises an enormous number ofcell types that develop from the neuroepithelium. We have been trying several approaches at the both cellular and molecular levels to understand how
the different cell types are generated. Members ofthe family of basic-helixloop-helix (bHLH) transcription factors, such as Mashl and Neurogenin, havebeen established to play important roles in mammalian neurogenesis. We
have focused on the regulatory cascades, i.e. upstream and downstream genes,
ofthe neural bHLH genes. PHD1, a member ofthe Paired-like homeodomain(PHD) family is expressed downstream of Mash 1 during the differentiation ofboth dorsal spinal cord and olfactory sensory neurons. In contrast, other
PHD genes, such as Phox2 and DRG11 , are expressed downstream ofthe bHLH
genes, in different lineages of neurons, suggesting that the cascade from bHLH
to PHD transcription factors may be important for neuronal identity
determination. Moreover, unc-4, which is most closely-related gene to PHDl ,has been shown to determine the identity of a motorneuron in C.
elegans. These suggest that the cascade during neurogenesis is conserved
even among species.
Another homeobox gene, MBHl (mammalian BarH homologue), is expressedin an exactly complementary pattern to Mashl and overlapping with
Neurogenin2 in the developing nervous system. Forced expression of MBHl
down-regulates Mashl expression and up-regulates Neurogenin2 in differentiating P19 cells, suggesting that MBHl is a regulator of the neural bHLH
genes. In order to clarify the relation between MBHl and bHLH genes andbetween bHLH and PHD genes, we are trying to identify downstream target
genes of MBHl and the bHLH genes.
We also established immortalized cell lines from early neuroepithelium

100through isolation of the forebrain region at E8.5 or E9.5 from mouse embryos
harboring transgene tsA58, the temperature sensitive SV40 T-antigen gene
(T-Ag) driven by the H2-Kb promoter. NES8 lines obtained from such embryos at E8.5 expressed marker proteins of the neural precursor cells, but they
could not be induced to differentiate into neurons or glia in various culture
conditions. Half of the NES9 lines established from embryos at E9.5 weresimilar to the NES8 lines, but the remaining half expressed neuronal or glial
differentiation marker proteins dependent on culture conditions. These results suggest that the early neuroepithelium at E8.5 is made of homogeneous
cell population of the early stem cells, and there is a beginning of cell determination and differentiation at E9.5 producing various types of multipotent
stem cells and neuronal progenitor cells. Also, in collaboration with Dr. 1.
Nagata at Tokyo Metropolitan Institute for Neuroscience and Dr. K. Ono at
Shimane Medical College, we studied migration patterns of granule cells indevelopment of mouse cerebellum. For details, see Ref. 3.

(3)

Molecular Analysis of Cell Differentiation and Morphogenesis In
Postlmplantatlon Mouse Embryos

Yasuaki SHlRAYOSHI and Norio NAKATSUJI
We cloned one Notch related gene "int-3" from the 8.5 day eDNA library. The
entire structure of the int-3gene resembles that of Notch or vertebrate Notch
homologues, and the int-3 gene showed 50·60% identity to the related Notch
homologues in its amino acid sequence. We consider int-3to be one ofthemouse homologues of Notch. RT-PCR and in situ hybridization analysis revealed that int-3 was expressed in blood vessels in 9.5-10.5 day oldembryos. The expression pattern of int-3 coincided with the expression ofreceptor tyrosine kinase Flk-l , which is regarded as a major regulator ofvasculogenesis and angiogenesis. These results suggested that int-3might
be involved in the formation of blood vessels in early mouse embryos. For
details, see Ref. 2.

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101

Pub I i ca t ions
1.

WATANABE, M., SHIRAYOSHI, Y., KOSHIMlZU, D., HASHIMOTO, S., YONEHARA, S., EGUCHI, Y,
TSUJIMOTO, Y and NAKATSUJI, N.:

Gene transfection of mouse primordial germ cells in

vitro and analysis of their survival and growth control. Exp. Cell Res., !SO, 76-83,

1997.

2.

SHIRAYOSHI, Y, YUASA, Y, SUZUKI, T., SUGAYA, K., KAWASE, E., IKEMURA, T. and NAKATSUJI,
N.:

Proto-oncogene int-3, a mouse Notch homologue, is expressed in endothelial cells

during early embryogenesis. Genes to Cells, !, 213-224, 1997.
3.

DNO, K., SHOKUNBI, T., NAGATA, I., TOKUNAGA, A, YASUI, Y, NAKATSUJI, N.:

Filopodia

and growth cones in the vertically migrating granule cells of the postnatal mouse
cerebellum. Exp. Brain Res., 117, 17-29, 1997.

F-c. Plant Genetics Laboratory(1)

Structural and Functional Analysis of the Genes Expressed in
Early Embryogenesis in Rice (Oryza sativa)

Yukihiro ITO, Mitsugu EIGDCHI, Nori KURATA
To elucidate the gene expression network essential for early embryogenesis
in rice, we conducted cloning and structural and functional analyses of transcription activating factor genes specifically expressed in very early
embryos. By analysing many genes having roles in the gene expression cascade essential for early embryogenesis, we aim to clarify genetic program ofembryogenesis in the monocot plant. We constructed cDNA libraries of embryos at 1 and 3 days after pollination (DAP) and selected 34 cDNA clones
probed by homeo-domain-containing PCR fragments isolated originally fromthe 3 DAP embryo. These clones were categorized into 9 kinds of clones. In
addition, we cloned another cDNA and genomic sequences carrying homeobox
using related genes of other plants. To detect the expression specificity ofthese genes, RT-PCR and in situ hybridization analyses were carried out using
fixed-cDNA libraries and embryo sections of 0 to 6 DAP embryos. Four ofthese HOS (Homeobox gene in Oryza sativa) clones out of 9 showed stage andregion specific expression patterns in the embryo. The results showed thatHOS 24 expresses on the shoot/radicle primordium at 3-4 DAP and HOS 13

102

expresses in the restricted regions of early embryo at 2-3 DAP. These genes
might have important roles in early rice embryogenesis. To know the functional properties of these genes, we constructed constitutive expression plasmid of genes and introduced into rice cells to generate transformed rice
plants. Analysis of the resulting transformants are now in progress. For
detecting the role and situation of these genes in the genetic program of embryogenesis, we cloned genomic regions of some of these genes andsequenced. To restrict the control regions for gene expression situating upstream of the genes, plasmids connecting a reporter gene under the upstream
region of the genes were constructed and transformed rice calli withthem. These experiments would reveal the cis-elements and trans-acting
nuclear factors expressed just before the genes we examine. We would like tofind out a nember of genes essentially included in the gene expression cascadein early embryogenesis of rice, through the detection of such cis- and transfactors at successive multiple steps.

(2)

Molecular Analysis of the Developmental Program during Regeneration Process of Rice Plant from Somatic Embryo

Nori KURATA and Mitsugu EIGUCHI
We have started this project to analyse genetic program in somatic embryogenesis and compare it to that of seed embryogenesis. Are the programs in
two embryogenetic process almost same or different in several steps? To see
this we are performing in situ hybridization and RT-PCR analysis also using
regenerating rice somatic callus. A part of genes specially expressed in theseed embryogenesis apeared to be transcribed in a simmilar manner in theregenerating callus.
In this project, we are also trying to bring on plantlet regeneration from thecallus ofvariousodm (organ differentiation mutant) plants for exploring possible alternative pathways used in the regenaration process of somatic embryogenesis.
The rice homologue with the very useful cell marker gene expressed specifically in regenerating cells of carrot has been also isolated and analysed whether
it will be useful for cell fate analysis in rice.

F.

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103

Positional Cioning of a Segregation Distortion Gene Detected in
the Progenies of Crosses with Japonlca and IndIca Rices

Yoshiaki HARUSHIMA and Nori KURATA
Site specific reduced gene transmission, which is called segregation distortion, has been detected in several chromosomal regions in F2 progenies ofcrosses between japonica and indica rices. Intensive analysis of three different F2 progenies using high-density DNA markers revealed many distorters
with different strength and combinational expression. Among those causative genes (or factors) for the segregation distortion, the most seviere distorter designated ga2 was mapped on chromosome 3. For positional cloningof the ga2, fine mapping of the gene was done. Flanking DNA markers very
near the locus were detected and physical mapping will be continued in 1998.

(4)

Isolation and Characterization of Rice Centromeric DNA Sequences
for the Construction of Rice Artificial Chromosome

Kenichi NONOMURA and Nori KURATA
This study focused on unravelling the mechanisms of spacial and functionalorganization of chromosomes in the nucleus. For this purpose, we decided toconstruct plant artificial chromosome with rice as an efficient tool for analysing
structural and functional components and organization of chromosome/nucleus.
The cereal centromeric sequence (CCS1) 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 humans. To isolate as many
divergent centromeric units of rice (Oryza sativa L.) as possible, we performed
the polymerase chain reaction (PCR) using the CENP-B box-like sequence(CBLS) as primers. A 264-bp clone, called RCS1516, containing this fragment appeared to be a member of the CCS 1 family, sharing about 60% identity with the nucleotide sequences of other cereals. Several genomic clones
carrying multiple copies of CBLS were isolated. A 14-kb long genomic clone
ARCBll was identified by screening with RCS1516 probe. Characterizationand nucleotide sequencing of a 14 kb clone was carried out. It was found thatthe clone contained 3 copies of a long direct repeat RCE1 with 5.1- and 1.7-kb

104intervening sequences. The 1.9-kb RCE1 repeat unit consists of three distinct parts: a variable, a semi-variable, and a conserved region. Fluorescentin situ hybridization (FISH) analysis revealed that RCE1 repeat units arelocated in the centromeric heterochromatin of rice chromosomes. Together
with further efforts for isolating a longer DNA fragments containing RCE1
repeat suitable for centromere function, several other candidate clones containing other type of centromere repeat units are successively being screened
and characterized.

(5)

Generation of Enhancer Trap Lines in RiceYukihiro ITO, Mitsugu EIGUCHI and Nori KURATA
To establish and to screen a lot of mutants affecting the steps of early embryogenesis and of chromosome organization in rice nuclei, we are generating
enhancer trap lines for most rice genes. For efficient making of trap lines, we
decided to use an AcIDs transposon system of maize, GUS or GFP genes asreporters and the T-DNAmediated gene transfer system ofAgroba.cterium. We
chose useful constructs already used in the Arabidopsis gene/enhancer trapping system and replaced some components with fragments suitable for ricetransformation system. We reconstructed the Ds vectors fitting for rice transformation and transformed rice callus with either the vector carrying Actransposase or that carrying Ds + GUS gene. We have already obtained above
100 transformants of Ds-GUS constructs in this year. Transformants are
selected for low copy number of the Ds-GUS sequence and will be located theintegrated sites by mapping of each flanking sequence of the integration. We
will conduct to cross these Ds-GUS transformed plants and Ac transformants
to obtain a lot of Ds-GUS transposed enhancer trapped progenies.

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GENETIC STRAINS RESEARCH CENTER

Publ icatians
1.

MONNA, L., MIYAO, A., ZHONG, H. S., YANO, M., IWAMOTO, M., UMEHARA, Y., KURATA, N.,
HAYASAKA, H. and SASAKI, T:

Saturation mapping with subclones ofYACs: DNA marker

production targeting the rice disease resistance gene,Pi-b. Theor. Appl. Genet.94, 170176, 1997.
2.

SHIMOKAWA, T, KURATA, N., Wu, J., UMEHARA, Y and SASAKI, T.:

Assignment ofYAC

clones spanning rice chromosomes 10 and 12. DNA Research 3, 401-406, 1997.
3.

KOIKE, K, YOSHINO, K, SUE, N., UMEHARA, Y, AsHIKAWA, I., KURATA, N., and SASAKI,
T.:

Physical mapping of rice chromosomes 4 and 7 using YAC clones. DNA Research 4,27-33, 1997.
4.

TANOUE, H., SHIMOKAWA, T, Wu, J., SUE, N., UMEHARA, Y, AsHlKAWA, I., KURATA, N. and
SASAKI, T:

Ordered YAC clone contigs assigned to rice chromosomes 3 and 11. DNA

Research 4, 133-140, 1997.
5.

UMEHARA, Y, KURATA, N., ASHIKAWA, I. and SASAKI, T.:

Yeast artificial chromosome

clones of rice chromosome 2 ordered using DNA markers. DNA Research 4, 127-131,
1997.
6.

KURATA, N., UMEHARA ,Y., TANOUE, H. and SASAKI, T.:

Physical mapping of the ricegenome with yeast artificial chromosome clones. Special issue "Oryza: from molecular
to plant." Plant Mol. Bioi 35, 101-113, 1997.
7.

FOOTE, T., ROBERTS, M., KURATA, N., SASAKI, T and MOORE, G.:

Detailed comparative

mapping of cereal chromosome regions corresponding to the PhI locus in wheat. Genetics 147, 801-807, 1997.
8.

YANO, M., HARUSHIMA, Y, NAGAMURA, Y, KURATA, N., MINOBE, Y and SASAKI,
T.:

Identification of quantitative trait loci conferring heading date of rice using ahigh-density linkage map. Theor. Appl. Genet 95, 1025-1032, 1997.
9.

HARUSHIMA, Y, YANO, M., SHOMURA, A., SATO, M., SHIMANO, T, KUBOKI, Y, YAMAMOTO, T.,
LIN, S.Y., ANTONIO, B.A., PARCO, A., KA.!IYA, 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.
10.

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-I, a novel bacterial blight resis-

tance gene in rice, is induced by bacterial inoculation. Proc.Natl.Acad.8ci.USA 95,
1663-1668, 1998

106

F-d. Microbial Genetics Laboratory(I)

Diadenosine 5',5"'-Pl,P4-tetraphosphate (Ap4A) eontrols thetiming of eell division in EscherIchIa coli

Akiko NISHIMURA
The timing of the cell division in Escherichia coli is highly regulated, but themechanism has not been identified. We found previously that cfcAl mutation uncouples DNA replication and cell division, and elevates the frequency ofcell division. We further analyzed the structure and the role ofthe cfcgenes ofcfcAll, a derivative of cfcAl, and another cfc mutant, cfcBl.
The cfc mutants divided prior to the ordinary stage of cell division, andproduced many small cells with nucleoid. However, the cells grew exponentially, and the length of a cell cycle and the initiation mass for chromosome
replication were not altered by the cfc mutations. These properties of themutants resulted from reduction ofthe period between the nucleoid divisionand the cell division in a cell cycle and compensatory elongation of the period
between the cell division and the initiation of the next round of DNA
replication. CfcAll has a mutation in glySa which encodes the a-subunit ofglycyl-tRNA synthetase, and cfcBl has an IS2 insertion in apaHwhich encodes Ap4A hydrolase. The cfc properties of both cfc mutants were suppressedby a multicopy plasmid carrying apaH+, and the intracellular level ofAp4Ain
cfcAl was fifteen times and cfcBl was one-hundred times higher than theirparent. Experiments using a wild-type cell showed that a high level ofAp4A
caused early cell division, and a low level ofAp4Acaused delayed cell division.
Ap4A is a signal for induction of cell division. High level ofAp4A is responsible for the initiation of cell division. The glyS mutation allows efficientsynthesis of Ap4A. For details, see Ref. 1 and 3.

F.

(!)

GENETIC STRAINS RESEARCH CENTER

107/tsE Is affects translocation of K+-pump proteins Into the cytoplasmic membrane of Escherichia coli

Hideki URAl, Hiroshi MATSUZAWA', Koreaki IT0 2, Mamoru YAMADA3 and Akiko
NISHIMURA (lDepartment of Biotechnology, The University of Tokyo, 2Institutefor Virus Research, Kyoto University, :3Faculty ofAgriculture, Yamaguchi Uni·
versity)
The ftsEts mutation of Escherichia coli causes defects in cell division andcell growth. We expressed alkaline phosphatase (PhoA) fusion proteins of
KdpA, Kup, and TrkH, all ofwhich proved functional in vivo as K+ ion pumps, inthe mutant cells. During growth at 4ft:, these proteins were progressively
lost from the membrane fraction. The reduction in the abundance of these
proteins inversely correlated with cell growth, but the preformed proteins in
the membrane were stable at 41'C, indicating that the molecules synthesized
at the permissive temperature were diluted in a growth-dependent manner at
a high temperature. Pulse-chase experiments showed that KdpA-PhoA wassynthesized, but the synthesized protein did not translocate into the membrane of the ftsE" cells at 41'C and degraded very rapidly. The loss of KdpAPhoA from the membrane fractions of ftsFJs cells was suppressed by a multicopy
plasmid carrying the [tsE+ gene. While cell growth stopped when the abundance ofthese proteins decreased 15-fold, the addition of high concentration of
K+ ions specifically alleviated the growth defect offtsE" cells but not cell division, and the cells elongated more than 100-fold. We conclude that one ofthecauses of growth cessation in the ftsE" mutants is a defect in the translocation
ofK+-pump proteins into the cytoplasmic membrane. For details, see Ref. 2.

Publ icatians
I.

2.

NISHIMURA, A., MORIYA, S., UKAI,H., NAGAI, K., WACHI, M. and YAMADA, Y: Diadenosine
5', 5"'·PI, P4·tetraphosphate (Ap4A) controls the timing of cell division in Escherichia
coli. Genes to Cells, Vol. Z, pp. 401.413, 1997.
UKAI, H., MATSUZAWA, H., ITO, K., YAMADA, M. and NISHIMURA, A.: [tsE" causes defectin cell growth of Escherichia coli through affecting translocation ofK+·pump proteins
into the membrane. J. Bacteriol, Vol. 180, pp3663·3670.

108
3.

NISHIMURA, A.:

Timing of cell division: Ap4A as the signal. TiES, Vol. Z3, pp. 157-159,

1998.

F-e. Invertebrate Genetics Laboratory(1)

Leg pattern formation: specification In the embryo and proxlmodistal pattern formation in the larva

Satoshi GOTO, Kazumasa KUBOTA l and Shigeo HAYASHI (lTokyo Universityof Medicine and Dentistry)
Two thoracic limbs of Drosophila, the leg and the wing, originate from acommon cluster of cells including the sources of two secreted signaling molecules Decapentaplegic and Wingless. Wingless induces Distal-less expression which marks the limb primordium. We showed that graded activity of
Decapentaplegic expressed in the dorsal side of the limb primordium is responsible for the specification of the proximal leg and wing cell
identity. Development of distal leg cells requires Wingless, but does not require detectable contribution of Decapentaplegic, and thus appears to be thedefault state. These inductive events, together with cell migration appear to
be responsible for the formation of the leg disc that is composed of the proximal and the distal domains, and the wing disc. For detail, see ref. 1.
Specification of the leg cells requires an additional signal involving the Drosophila EGF receptor DER that is transiently activated within the limb
primordium. In the absence of DER activity, proximal leg cells lose their
specific marker gene expression and the morphology of the leg disc is
disrupted. Thus a combinatorial action of DER and Decapentaplegic is important in the patterning ofthe leg.
The leg imaginal disc resumes cell proliferation in the second instar, maintaining the circular patterns of the proximal and distal marker expression,and elaborating its pattern by activating additional genes required for differentiation of a subset ofleg structures. We are investigating how the interaction between the proximal and distal cells maintain their specific gene expression, activate additional genes, and establish polarity along the proximo-distal axis.

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GENETIC STRAINS RESEARCH CENTER

109

During the course of this work, we developed a method to fluorescently detect a protein and a mRNA in whole-mount embryos. We showed that thismethod provides a single cellular resolution useful to analyze the dynamics ofgene expression and cellular movement during induction ofthe imaginal disc.
For detail, see ref. 3.

(!)

Cell biological study of the tubular epithelial network formationin the tracheal system

Miho TANAKA-MATAKATSU, Tomoatsu IKEYA and Shigeo HAYASHI
Coordination 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 have previously described the details of changes in cell
shape and cell surface properties in tip cells during branch fusion (TanakaMatakatsu et aI., Development 122: 3697-3705, 1996). To further investigate the cellular mechanism that drives these processes, we are studying thechanges in cytoskeltal structures and its correlation between distribution ofcell surface and secreted molecules. The study revealed that reorganizationof microtubular array prior to branch fusion appears to playa central role inthe dynamic change of apical-basal cell polarity in the tip cell.
Only a single tip cell differentiates in each branch ofthe trachea. To understand how cell-cell communication is involved in this singling out process, weanalyzed the function of the transmembrane receptor Notch in the trachea.
We found that Notch is required for selecting the correct number ofthe tip cell.

(3)

Cdc! dependent checkpoint couples M phase completion to initiation of S phase

Shigeo HAYASHI
Coupling of M phase completion to initiation of S phase is essential for the
maintenance of ploidy during cell proliferation. I have previously shown thatthe essential M phase regulator Cdc2 kinase is also required for inhibition of

110

S phase prior to the completion ofM phase. To further investigate the mechanism of Cdc2 dependent inhibition of S phase, I am using a genetic and biochemical approach to test a requirement for the kinase activity of Cdc2 in thisprocess.

(4)

plexus, a gene required for adult wing vein pattern

Hitoshi MATAKATSU 1, Sumiko GAMOU 1, Ryu-suke TADOKOR0 2 and Shigeo
HAYASHI (lOsaka Prefecural University, 2Kitasato Unversity)
Veins on the adult wing are a good landmark for the anteroposterior anddorsoventral positional informations. It also provides a stage where the EGF
receptor, Decapentaplegic and Notch / Delta signaling pathways interact. The
gene plexus is required for the supression of extra veins in specific intervein
positions (between vein two and three, and between vein four and five). We
have cloned the plexus gene and determined the sequence of one of its
transcript. Further molecular and genetical analysis of the gene is under
way.

(5)

An enhancer trap screen 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 ofimaginal disc and the trachea, we are conducting an enhancer trap screen using theGa14-UAS system. About 500 lines were established and were examined forthe activity of enhancers flanking the inserts in the embryo, larva and adult.
In collaboration with other groups in Japan, we will continue to screen morelines.

F.

(6)

GENETIC STRAINS RESEARCH CENTER

III

Determination of the dorsoventral domains of neurogenesis by the
DrosopbJ/a EGF receptor

Yoshimasa YAm l and Shigeo HAYASHI (IBio-oriented Technology Research Advancement Institution and School of Pharmaceutical Science, University ofTokyo)
Primary neurogenesis in the central nervous system of insects and vertebrates occurs in three dorsoventral domains in each side of the neuroectoderm.
We have previously shown that the EGF receptor homologue (DER) plays acruicial role in the dorsoventral subdivision of neuroectoderm by repressing
the expression of the zinc-finger transcription factor Escargot (ref. 2). We
further extended this study to show that DER also represses proneural genes
aehaete and Beute, and is required for the formation of the neuroblasts in theintermediate column. The activity of DER appears to be mediated by MAP
kinase.

Publications
1. GOTO, S. and HAYASHI, S.: Specification ofthe embryonic limb primordium by graded
activity of Decapentaplegic. Development, 124, 125-132, 1997.
2. YAGI, Y. and HAYASHI, S.: Role of the Drosophila EGF receptor in determination of thedorsoventral domains of escargot expression during primary neurogenesis. Genes to
Cells, %, 41-53, 1997.
3. GOTO, S. and HAYASHI, S.: Cell migration within the embryonic limb primordium ofDrosophila as revealed by a novel fluorescence method to visualize mRNA and protein.
Development Genes and Evolution, %07, 194·198, 1997.

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G.

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

Cross-species functional gene database

Yukiko YAMAZAKI
The goal of this project is full cross referencing among different species. Last
year an integrated database of wheat and rice genetic resource information
was experimentally developed. The construction of a cross-species functional
gene database started this year.
The cross-species functional gene database on trial compiled genetic information of four different organisms, that is, rice, wheat, drosophila andmouse. Three different data sources; (l)DNA sequence database, (2) genetic
resource database which we have been working on and (3) gene symbol dictionary provided by the genome group, were used for the database
construction. The genetic information was directly extracted from the DNA
sequence database and from the genetic resource database. Gathering theseinformation indexed with accession number, a Gene-Table was created. The
table consists of organism-name, strain-name, DNA-accession-number andgene-name. The gene-name of the current Gene-Table includes gene symbol,
gene name and gene product name. Redundancy was eliminated from theGene-Table manually according to the description of gene symbol
dictionary. Although the lack of controlled vocabulary in the database makes
the task difficult to accomplish, the database will be useful as the raw material for a wide range of biological sciences including comparative genomics
study in the future.

G.

CENTER FOR GENETIC RESOURCE INFORMATION

(!)

113

Genetic Resources Database2-1. Wheat Genetic Resources Database -KOMUGIYukiko YAMAZAKI, Rie TSUCHIYA, Hisashi TSUJIMOTO! and Taihachi
KAWAHARA2(lKihara Institute for Biological Research, Yokohama City University, 2Plant
Germ-plasm Institute, Faculty ofAgriculture, Kyoto University)
The KOMUGI database working group has started preparation of the second version of the database. While reducing the redundancy of current database, 1400 image files have been added to the database and the DNA repository data have also joined KOMUGI in collaboration with Dr. Ogiwara (Kihara
Institute for Biological Research). The current DNA repository database comprises 240 accessions including DNA and cDNA clones which will be commonly
usable for Triticeae groups.
2-2. Rice Genetic Resources in Japan
Yukiko YAMAZAKI, Mari SAITO, Toshiro KINOSHITA! and Keiko MORISHlMA(lHokkaido University)
A hardcopy catalogue, Rice Genetic Resources in Japan-1997, 703 pages,
has been published and distributed to over 220 sites in the world. The catalogue includes not only genetic resource information but also basic biologicalknowledge such as anatomical data, gene designations, a morphological linkage map and the citations. The information is also available through theinternet.
Dr. Nori Kurata (National Institute of Genetics) has started establishing
the networking group of rice researchers in Japan for the new approach of ricebioinformatics including rice genomic information.
2·3. Barley Germplasm Database
Kazuyoshi SATO!, Mari SAITO and Yukiko YAMAZAKI (lOkayama University)
A barley germplasm database was constructed in cooperation with Okayama
University, a major stock center for barley strains in Japan. The databaselists about 5000 strains and consists of 32 items including agro-morphologi-

114

cal, physiological, pathological and genetic traits. The database is still forinternal use only but it soon will be available through the internet.
2·4. Mouse Microsatellite DatabaseToshihiko SHIROISHI, Rie TSUCHIYA and Yukiko YAMAZAKI
The mouse microsatellite database in Japan (MMDBJ) has been constructed
and is now on·line accessible. The database provides SSLP (simple sequencelength polymorphism) information among different strains with PCR
condition. The current database has ca. 1000 entries and an on·line direct
data submission system has been developed so that researchers can deposit
their own data through the network.
2·5. Experimental Animal Database
Rie TSUCHIYA, Yukiko YAMAZAKI and Tetsuo ICHIKAWA l(lJapan Experimental Animal Cooperative)
The database comprises about 1500 experimental animal strains main·tained by 51 Japanese breeders and 1 institution. The current database only
provides the strain name and address to obtain the strain. The next plan is
to collect as much information relevant to the strain as possible.
2·6. Cloning Vector Database
Seiichi YASUDA, Mari SAITO and Yukiko YAMAZAKI
An on·line data management system ,through which the clone maintainer
can update his own data by connecting to the remote computer running thedatabases, has been developed. The system is composed of three parts, (1)data transfer mechanism, (2) data checking mechanism, and (3) tools for cre·ating the Web pages.

Publ icatians
I.

YAMAZAKI, Y., TSUJIMOTO, H. and KAWAHARA, T.:

KOMUGI Database··Wheat Genetic
Resources Database. Wheat Information Service, 85,88·91 (1997)

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H.

STRUCTURAL BIOLOGY CENTER

H-a. Biological Macromolecules Laboratory(1)

Single Molecule Imaging of Enzymatic Reactions
Using Objective-type Evanescent Illumination

Makio TOKUNAGA, Kiwamu SAITO l , Kazuo KITAMURA2, Atsuko IWANE 2 andToshio YANAGIDA2 (lDepartment of Physics, Kanazawa University, 2Department of Physiology I, Osaka University Medical School)
Imaging of single fluorescent molecules has been achieved in a relatively
simple manner using objective-type total internal reflection fluorescence microscopy (TIRFM). This method allowed visualization of single molecules
under scanning probe microscopes. Taking advantage ofthe technique of single
molecule imaging, individualATP turnovers have been visualized with a fluo·
rescentATP analogue, Cy3-ATP, using a simple experimental strategy. Clear
on/off signals were obtained that correspond to the association and dissociation of single Cy3-ATP/ADP molecules with a single myosin head molecule.
This method will allow a variety of single-molecular assays of biomolecularfunctions to be performed using fluorescently labeled substrates, ligands, messengers and so on. For details, see Ref. 4.

(2)

Intermolecular Interactions measured by "Subplconewton
Intermolecular Force Microscopy"

TakaakiAoKIl, Michio HIROSHlMA2, Toshio YANAGIDA l and Makio TOKUNAGA(lDepartment of Physiology I, Osaka University Medical School, 2Departmentof Biophysical Engineering, Osaka University)
Force sensitivity of scanning probe force microscopy was increased by incorporating a cantilever with very low stiffness, O.lpN/nm, which is over 1000fold more flexible than is typically used in conventional atomic force
microscopy. Thermal bending motions of the cantilever were reduced to less

116

than 1 nm by exerting feed-back positioning with laser radiation pressure.
The system was tested by measuring electrostatic repulsive forces or hydrophobic attractive forces in aqueous solutions. Subpiconewton intermolecular forces were resolved at controlled gaps in the nanometer range between theprobe and a material surface. Interaction forces between hydrophobic surfaces of a probe and a coverslip were measured, and a long-range attractive
force was observed out to 100 nm in pure water. This result suggests thatvery long range hydrophobic forces also operate between objects of the size of aprotein molecule. For details, see Ref. 2 and 5.

(3)

Single molecule capture, manipulation and force
measurement of protein molecules

Kazuo KITAMURA l , Atsuko IWANE l , Toshio YANAGIDA l and Makio TOKUNAGA(IDepartment of Physiology I, Osaka University Medical School)
A single myosin head molecule (myosin subfragment-1, S1) was captured
onto the tip of a scanning microprobe, using a flexibleglass microneedle, andmanipulated with subnanometer resolution. Single molecules were confirmedby examining it in the fluorescence glass microneedle at the same time using
objective-type TIRFM. Movements and forces resulting from the interactionof a captured single S1 molecule with actin filaments were measured. Mean
displacement amplitudes of individual S1 molecules ranged from 4 to 30 nm,
certainly depending upon conditions for interactions of individual molecules.
Moreover, it was found that a myosin head processively moves along the actin
filament(s) with 5.3 nm steps, and undergoes -5 steps to produce a maximum
displacement of -30 nm for each ATP hydrolysis. The results proves the loose
coupling mechanism.

(4)

A New Model of Molecular Motors

Makio TOKUNAGA
Our single-molecule researches have provided 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 single-

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molecule experiments. Because this model is based only on a simple assumption that the interaction potential between motor proteins has a asymmetric shape, the theoretical formulation is very simple. Therefore, this modelleads to a spread use of its application.

Pub I i ca t ions
1.

IWANE, AH., KITAMURA, K., TOKUNAGA, M. and YANAGIDA, T.:

Myosin subfragment-l isfully equipped with factors essential for motor function. Biochem. Biopys. Res. Comm.,

230, 76-80, 1997.
2.

TOKUNAGA, M., AOKI, T., HIROSHIMA, M., KITAMURA, K. and YANAGIDA, T.:

Subpiconewton

Intermolecular Force Microscopy. Biochem. Biopys. Res. Comm., 231, 566-569, 1997.
3.

IWANE, AH., FUNATSU, T., HARADA, Y., TOKUNAGA, M., OHARA, 0., MORIMOTO, S. and
YANAGIDA, T.:

Single molecular assay of individual ATP turnover by a myosin-GFP

fusion protein expressed in vitro. FEBS Letters, 407, 235-238, 1997.
4.

TOKUNAGA, M., KITAMURA, K., SAITO, K., IWANE, AH. and YANAGIDA, T.:

Single Molecule

Imaging of Fluorophores and Enzymatic Reactions Achieved by Objective-type Total
Internal Reflection Fluorescence Microscopy, Biochem. Biopys. Res. Comm., 235, 47-

53, 1997.
5.

AOKI, T., HIROSHIMA, M., KITAMURA, K., TOKUNAGA, M. and YANAGIDA, T:

Non-contact

scanning probe microscopy with sub-piconewton force sensitivity. Ultramicroscopy, 70,

45-55, 1997 .
6.

SAITO, K., TOKUNAGA, M., IWANE, AH. and YANAGIDA T.:

Dual-colour microscopy of single

fluorophores bound to myosin interacting with fluorescently labelled actin using antiStokes fluorescence. J. Microscopy, 188, 255-263, 1997.
7.

HARADA, Y., FUNATSU, T, TOKUNAGA, M., SAITO, K., ISHII, Y. and YANAGIDA, T.:

Singlemolecule imaging and nano-manipulation of biomolecules. Meth. Cell BioI., 55, 117-

128, 1997.
8.

FUNATSU, T., HARADA, Y., HIGUCHI, H., TOKUNAGA, M., SAITO, K., ISHII, Y., VALE, R. and
YANAGIDA, T.:

Imaging and nano-manipulation of single bio-molecules. Biophys. Chern.,

68, 63-72, 1997.
9.

ISHIJIMA, A, KOJIMA, H., FUNATSU, T., TOKUNAGA, M., HIGUCHI, H., TANAKA, H. and
YANAGIDA, T:

Simultaneous observation of individual ATPase and mechanical eventsby a single myosin molecule during interaction with actin. Cell, 92, 161-171, 1998.

118

H-b. Molecular Biomechanism Laboratory(1)

Branched pathway mechanism of transcription Initiationby E. coli RNA polymerase, and promoter-arrested
Initiation complexes

Ranjan SEN l , Tomoko KUBORI l , Hiroki NAGAI!, V. Jim HERNANDEZ 2and Nobuo
SHIMAMOTO! (lStructural Biology Center, National Institute of Genetics, 2Microbiology, SUNYlBuffalo)
Transcription initiation is conventionally supposed to be a series of steps
composed of promoter binding, isomerization, RNA synthesis, and promoter
clearance. KUBORI and SHIMAMOTO have presented that kinetics of abortiveand productive transcription at APR promoter violates this sequential mechanism and rather indicates a branched pathway mechanism (Nuc. Acids Res.
24, 1380-1381 (1996): J. Mol. BioI. 256, 449-457 (1996)). In the new mechanism one of a branch leads to dead-end inactivated complexes via complexes
which only produce abortive transcripts, moribund complex. These studies
have opened up a possibility of revision of general initiation mechanism andre-definition of promoter clearance.
To prove the existence of branched mechanism, the yields of full-length transcripts on the same amounts of templates harboring T7A1, APR, and lac
UV5 promoters were compared in a single-round transcription condition. The
results showed that at least 60% and 97% of holoenzyme bound to the APR,
and lac UV5 promoters, respectively, remained inactive in terms of the synthesis offull-Iength transcripts. This kinetically demonstrates the existenceof dead-end pathway (ref. 1).
Next we searched a switch which turns reaction pathway from productive topromoter-arrested branched pathway. We first found that misincorporation
occurs at the 5 th and 6 th positions of the APRAL transcription unit and thenon-templated transcripts are aborted. The misincorporation and other abortive synthesis are enhanced by the presence of holoenzyme only in excess to thetemplate DNA, and even more by a stall of RNA polymerase at +32 position. A
hydroxide radical footprinting showed that the stall at +32 can accommodate
one more enzyme molecule at the promoter. The enhancement is not intro-

H. STRUCTURAL BIOLOGY CENTER

119

duced by a similar stall at +73. These result suggested that an interaction,
probably physical contact, between two enzyme molecules tend to switch thetrailing molecule into promoter-arrested state (ref. 1).
Thirdly, we have investigated the mechanism of a switch which mitigate thearrest at the promoter. The S506F mutation in 0 70 , isolated by V. J.
Hernandez, is known to reduce abortive synthesis at all the promoterstested. It also enhances full-length synthesis from weak promoters, but notfrom strong ones. By kinetic and footprinting analyses we showed that the
mutant is not an up mutant, but rather shifts the equilibrium of isomerization towards closed complexes at the A PRAL promoter. The most distinctive change is the 10-100 fold stimulation of rates of over-all dissociation ofboth productive and arrested complexes with no change of binding affinity.
Moribund complexes are rapidly dissociated (backward reaction) and inactivated (forward reaction), reducing the level of its accumulation. This reduces
abortive synthesis and decreases the yield offull-Iength transcripts from thisstrong promoter. These results proposes the significance of introduction ofreversibility into otherwise irreversible separation into the two branched path·ways as a switch (SEN et aI., J. BioI. Chem. (1998».

(2) Detection and characterization of conformation changes ofproteins by a high-resolution protein footprinting
Hiroki NAGAI and Nobuo SHIMAMOTO (Structural Biology Center, National
Institute of Genetics)
The term "conformation change" of proteins has been used mostly in speculative sense rather than in description of their distinctive structuralchange. The recently developed footprinting technique, hydroxide radical protein footprinting for example, enables to identify which part of protein changes
its exposure to solvent at a level of amino-acid residues. Therefore, genetic
information can be interpreted into concrete structural changes in parts of aprotein.
We have chosen 0 70 as a test protein for establishing the correlation ofgenetics and structural biology because of abundance of its genetic information and the presence of conformational change which has been speculated

120

from several biochemical studies. The protein has 4 regions (region 1 to 4)
which are conserved among other sigma factors and other bacteria, and theregions are further divided into subdomains.
We put a oligopeptide tag which can be phosphorylated by heart muscle
kinase (HMK tag) at C-terminus of 0 70 , enabling direct visualization of
footprinting. Holoenzyme containing the phosphorylate 0 70 and binary complexes with the APRAL promoter are prepared and challenged to hydroxide
radical generated with Fenton Reaction under a single-cutting condition. The
cutting positions are referred by a series of markers which had been generated
by several chemical and enzymatic cleavage of 0 70 (Gene. Cells 2, 725-734(1998».
The major cleavage sites well corresponded the borders of functional
domains. Region 1.1 is dispensable for basic activity, but a tryptic fragment
containing 1.2 and 2 can form a complex with core enzyme which binds to the
non-template strand of a promoter DNA. The most strong cleavage was foundto the border of these distinct functional domains. The second preferential
cleavage site was found in region 3.2, which agrees with the observation that
C-terminal truncated sigma factors can promote the initiation of transcription from the extended -10 promoter but not from -35-dependent promoters.
Some genetic models was refined by this study. A N-terminal portion ofregion 2 was thought to be involved in binding to core enzyme, being supposed
to be core-interacting site. Consistently, a moderate protection was observedin this domain, but regions 1.1, and most parts of regions 2, 3, and 4 were also
protected, suggesting the presence of more extensive contact sites between
0 70 and core enzyme than have been proposed by studies using deletionmutants. Two regions, 2.4 and 4.2, have been genetically proved to interact
with promoter DNA. Consistently small segments in these regions become
protected on binding to DNA. However most parts of these regions are already protected on the stage of holoenzyme before binding to DNA. This
indicates that the major structural change occurs upon formation ofholoenzyme rather than upon binary complex formation. The acidic region between
1.1 and 1.2 has been suggested to inhibit DNA binding activity of free 0 70 , but
the footprinting clearly showed this region affected only in the presence ofDNA, denying this model. Another model claims that region 1.1 masks a

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DNA binding activity region 4.2 by intramolecular interaction, preventing free
0 70 from binding to promoter DNA but not holoenzyme. However, our results
do not provide the evidence for interaction between regions 1.1 and 3/4. Further study is required to prove (or disprove) the possible interaction between
regions 1.1 and 3/4.

(3)Single-Molecule Dynamics of Transcription:
Sliding of proteins along DNA

Hiroyuki KABATA 12 , Minoru TAKEUCHI 1, Takashi KINEBUCHI 1, Nobuo
SHIMAMOT0 1 , Osamu KUROSAWA 23 , 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 A
DNA, one was its cognate operator cloned in A DNA, and the other two arelikely to be homologous to the operator. All trapping occurred at specificsites, and only sliding complexes were observed at non-specific sites. Thisobservation indicates that the non-specific complex is the sliding complex
itself.
Based on this observation, we developed a general theory of sliding. The
theory predicts that sliding does not always cause an enhancement of specificbinding. Sliding can accelerate not only association to a specific site but also
dissociation from it. Under certain conditions sliding enhances specific binding, and the enhancement depends on the length of DNA. In this case nonspecific sites close to a specific site, work as antenna to harvest protein molecules, but distant sites are competitors for specific sites. The critical distance distinguishing these two classes of non-specific sites is also derived
from the theory. This theory solves many seemingly conflicting results previ-

122

ously obtained in kinetic assays of sliding.
We have been developing manipulating techniques for single molecules (inStructural tools for the analysis ofprotein-nucleic acid complexes D(Birkhauser Verlag AG, Basel, 1992) pp. 241-253). As a fluorescent technique todetect single molecules, we have been improving the A. victoria Green Fluorescent Protein.

(4) Functional domain of Escherichia colJ
Single-stranded DNA binding protein
Takashi KINEBUCHI 1,2, Heizaburo SHIND0 2, Hiroki NAGAI I, Nobuo SHIMAMOT0 1,
Mitsuhiro SHIMIZU 2 (lStructural Biology Center, National Institute of
Genetics, 2Department of Pharmacology, Tokyo College of Pharmaceutical Science)

E. coli Single-stranded DNA binding protein (SSB) is an essential protein
in replication and recombination. It exists as a homotetramer throughout its
physiological environment. It cooperatively binds to single-stranded DNA,and makes clusters which is recognized by DnaG primase and RecA
recombinase. The role oftetramer formation for DNA binding is studied by adeletion analysis.
A series of mutants with N-terminus or C-terminus deletion was constructed
and proteins were purified. The oligomeric state of mutant proteins were
determined by gel permeation chromatography with Superdex-75, and DNA
binding was determined by gel mobility assay.
The results showed that all the N-terminus deletions hampers DNA binding activity and induced oligomerization more than octamer. The C-terminus deletion mutants up to 115 aa also hampers DNA binding activity andtetramer formation. This correlation of DNA binding activity with tetramer
formation indicates that the DNA binding site is created only on homotetramer
molecule.

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Publ icalions
KUBORI, T. and SHIMAMOTO, N.: Physical interference between Esherichia coli RNA
polymerase molecules transcribing in tandem enhances abortive synthesis andmisincorporation. Nuc. Acids Res. 25, 2640-2647, 1997.
2. KINEBUCHI, T., SHlNDO, H., NAGAI, H., SHIMAMOTO, N. and SHIMIZU, M.: Functional
domain of Esherichia coli Single-stranded DNA binding protein as assessed by analysis of the deletion mutants. Biochemistry 36,6732-6738, 1997.
3. NAGAI, H. and SHIMAMOTO, N.: Regions of the E. coli primary sigma factor sigma70
that are involved in interaction with RNA polymerase core enzyme. Genes to Cells 2,725-734, 1997
1.

H-c. Multicellular Organization Laboratory(1)

Fluoride-resistant Mutants of the Nematode
Caenorbabdltls eleffans

Masaya TAKEUCHI, Akane MOMI, Takeshi ISHIHARA and Isao KATSURA
Fluoride-resistant mutations of C. e1egans are all recessive and grouped
into two categories: class 1 mutations (in fJr-l, fJr-3 and fJr-4) that show strong
fluoride-resistance and slow growth, and class 2 mutations (in fJr-2and fJr-5)
that show weak fluoride-resistance, normal growth and suppression of theslow growth of class 1 mutations (Katsura, 1. et a1. (1994) Genetics 136, 145154).
fJr-l encodes an ion channel belonging to the DEGIENaC superfamily, whichcontains mechano-sensory ion channels of C. e1egans and amiloride-sensitive
epithelial ion channels of mammals. fJr-4 encodes a novel SerlThr protein
kinase having a hydrophobic domain on the carboxyl-terminal side. A genomic DNA fragment that rescues fJr-3 mutations produces a polycistronicmRNA encoding a kinase-like protein and a novel protein.
A fJr-l::GFP fusion gene is expressed only in the intestinal cells, beginningfrom the comma stage of embryos to the adult stage. A fJr-4-GFP fusion geneis 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 pair of head neurons called ADA
from L11arvae stage to adults. In those fusion genes, GFP eDNA is connected124

in frame to the 3' end of the coding region of the fJr genes. Since they can
rescue the corresponding mutant phenotypes, the expression at the site andthe time mentioned above should be enough for the animal to show the wildtype phenotypes.
All the six fJr-l mutations were missense mutations at or near the aminoacid residues that are functionally important in degenerins and ENaCs, showing functional similarity between FLR-1 and those ion channels. Four fJr-4
mutations consisted of two missense mutations at conserved residues in the
kinase domain, a mutation at a splice-acceptor sequence, and a missense
mutation in the C-terminal hydrophobic region.
Class 1 mutants show pleiotropic phenotypes, including slow growth, short
defecation cycle periods, constitutive dauer larva formation in a certain mutant background (See (3)), hypersensitivity to high osmotic pressure, and abnormal gonad shape. Some of these phenotypes are suppressed by class 2
mutations, while others are not. We think that FLR-1, FLR-3 and FLR-4
probably constitute a regulatory system in the intestine. We plan to study(a) what kind of proteins are encoded by class 2 genes, (b) How the regulatory
system in the intestine can control putative neuronal functions such as theregulation of defecation cycle periods and dauer larva formation, and (c) how
the fJrgene products act each other in the regulatory system.

(2)

A Gene That Controls Both Hatching andCell Migration in C. eJeKBns

Ryuichi HISHIDA, Takeshi ISHIHARA and Isao KATSURA
Mutants in hch-l gene cannot digest protein components ofthe eggshell andhave abnormality in the migration of a post-embryonic neuroblast called QL
and its descendants (Hedgecock, E.M. et a1. (1987) Development 100, 365382). hch-l encodes a protein that has a signal peptide, a Zn protease domain,
an EGF domain and a CUB domain, and that resembles the Drosophila
TOLLOID and the mammalian BMP-l. Whole-mount in situ hybridizationof embryos showed that the mRNA is expressed in the mid-embryonic stage,
first in the dorsal and lateral sides of the middle and posterior part of embryos and then only on the lateral sides. Experiments using hch-l ::GFP fu-

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sion genes showed essentially the same results except that weak expression
continues until just after hatching (Hishida, R. et a1., 1996, EMBO J., 15:4111-4122).
We measured protease activity in the hatching fluid in vitro, using the soft
eggshells of hch-l embryos as the substrate. Activity in the hatching fluid ofa hch-l null mutant was not detectable, whereas a transformant having many
copies ofthe wild-type hch-l gene exhibited about 10 times greater proteaseactivity than the wild-type animal. Thus, hch-l gene seems to encode thehatching enzyme itself or another factor that determines the level of hatching
enzyme activity. The protease activity was inhibited by metalloprotease inhibitors but not by inhibitors of other proteases such as Ser-, Cys-, and Aspproteases.
A nonsense mutation in the N-terminal protease domain, a putative nullmutation, was recessive and had low penetrance in the cell migration
abnormality. In contrast, a missense mutation in the C-terminal Cys-richregion was semi-dominant and had high penetrance in the cell migration
abnormality. Furthermore, a transposon-insertion mutation had low penetrance in the cell migration abnormality, but showed a stronger abnormality
in hatching than the former two mutations. The poor correlation between thetwo phenotypes indicates that delayed hatching may not be the only cause of
the cell migration abnormality.

(3)

Analysis of the Head Neural Circuit of C. eJegans as
Studied by the Formation of Dauer Larvae

Isao KATSURA, Norio SUZUKI and Takeshi ISHIHARA
If newly hatched larvae of C. e1egans are in a crowded state and given alimited food supply, they deviate from the normal life cycle and develop toenduring, non-feeding larvae called dauer larvae. The developmental decision to become dauer larvae is regulated by the neural circuit in a pair ofsensory organs called amphids, with the dauer pheromone and food as the
input. Since the assay of dauer 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 muta-

126

tions in more than 50 known genes show synthetic dauer-constitutive (Sdf-c)
phenotypes, i.e., they develop to dauer larvae in a certain mutant background,regardless ofthe environmental conditions. The synthetic nature ofthe phenotype, we think, is based on the structure of the neural circuit, in which two
different neurons are often positioned in parallel so that two mutations may
be required to block signals in both neurons.
We found that the pattern of combinations for the Sdf-c phenotype is specificand can be explained by a model assuming three parallel pathways. We also
found suppressor mutations of the Sdf-c phenotype of various double-mutantsand estimated the positions where the genes of the suppressor mutations act
in the parallel pathways.
We isolated and mapped 44 new mutations that show the Sdf-c phenotype
in combination with the unc-31 (e169) mutation. Many of them are expected
to cause defects inASI neurons, since the unc-31 (e169) mutant becomes dauerconstitutive by the killing ofASI neurons. Eight of the mutants were allelesof 4 known genes (tax-2, osm-6, che-ll, and aex-3) , but most ofthe remaining
36 mutations, which map in at least 13 genes, seem to be alleles of novel
genes. We continue to map them, and plan to characterize them further toelucidate their roles in sensory reception, transduction, or processing.

(4)

Reverse Genetics of Neural Genes of C. eJe~aBs and theAnalysis of Their Expression Using GFP(Jellyfish Green Fluorescent Protein)Takeshi ISHIHARA, Manabi FUJIWARA and Isao KATSURA
To study the formation and function of the neural circuit in C. elegans, we
identified about 20 promoters giving expression in specific sets of neurons byanalyzing their GFP fusion gene. Three of the promoters were selected by thepromoter trapping method, and others by looking at the genome DNA sequence.
Most of the cells that express GFP were identified for each promoter. Of thepromoters obtained by promoter trapping, H20 gave expression in all neurons,
H13 (promoter for a Zn finger transcriptional factor) only in AFD neurons, and
185 (promoter for a collagen gene) in the post-embryonic blast cell G2 and its
descendants, including RMF neurons.

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Of the genes selected by the DNA sequence, we investigated three glycine/GABAA receptor homologs and two metabotropic glutamate receptor homologs
in more detail. We determined their cDNA sequence by RT-PCR, 5'-RACE,
and 3'-RACE and isolated their deletion mutants, whose behavioral phenotypes are now under study (See (5». We also isolated a deletion mutant of
the gene encoding a Zn finger protein expressed in AFD neurons(H13). Although AFD neurons are known to playa role in thermotaxis, themutant showed normal thermotaxis.

(5)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 combined behavioral assay method for the two responses to learn
the role of interneurons. Wild-type animals change their preference between
the responses, depending on the relative concentration of the substances. Thissuggests that the two sets of sensory information interact with each other in aneural circuit consisting of about 10 neurons, based on the present knowledge
on the neural circuitry of C. eIegans and the identity of the sensory neurons forodorants.
This assay method was used for the analysis of mutants in an AMPA-typeglutamate receptor gene (gIr-l), which is expressed only in interneurons. They
had a preference for odorants, as compared with wild-type animals. However,
if each behavior was assayed separately, the dose response curves were thesame as those of wild-type animals. The results suggest that GLR-1 plays arole in the interaction between the two responses.
While the measurements were performed usually with well-fed animals, wefound that animals starved for 5 hours had a preference for odorants, because
starved animals had weaker response to copper ion. This behavioral change
seems to make sense for starved worms, because they can look for food over awide area.
The genes for metabotropic glutamate receptors (mgI) are expressed in part

128

ofthe interneurons that are considered to act in this interaction. We isolateddeletion mutants in these genes and analyzed them. A mutant in mgl-l (Gicoupled type) had preference for chemotaxis to odorants over avoidance tocopper ion, although it showed no abnormality in each of the single
behaviors. On the other hand, a mutant in mgl-2 (Gq-coupled type) lacked
the effect of starvation. Furthermore, mgl-l; mgl-2 double-mutant worms
hardly cross the copper ion barrier even when they are starved. We are now
testing if those phenotypes are caused by the mutations in the mgl genes or byside mutations.
We also isolated new mutants from worms treated with EMS and worms
containing movable transposons (mut-7worms). Some ofthem had preference for chemotaxis to odorants over avoidance to copper ion, while others had
only a small effect of starvation, We are trying to clone the mutated genes ofthe mut-7mutants by the transposon-tagging method.

(6)

Functional Analysis of Neural Adhesion Molecules byIntroduction of Clones Producing Antisense RNA

Manabi FUJIWARA, Takeshi ISHIHARA and Isao KATSURA
In C. elegans, gene disruption requires a lot of work, and the mutants obtained in this way lack the function of the gene completely in all the cells that
express it. Hence, if we can reduce, with much less labor, the function of agene specifically in certain cells, it would be useful for many studies. As theexpression of maternal genes can be repressed by the introduction of antisense
RNA into the gonad, we tried to establish a method in which the gene function
is reduced by the expression of the antisense RNA in somatic cells.
Looking at the DNA sequence of the C. elegans genome project, we selected
10 genes encoding a protein that has the FNIII domain and/or Ig domain andthat is expected to be a neural adhesion molecule. We made DNA constructs
which, under the H20 promoter (See (4», produce antisense RNA corresponding to part of the mRNA of those genes. Worms were transformed by these
DNA constructs and tested for their effect. Abnormality in locomotion was
detected for three ofthe genes, which encode homologs ofLl, TAG1 and NCAM,
respectively. We studied the gene for the Ll (NgCAM, Neuroglian) homolog

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in more detail. Antisense RNA corresponding to different parts of the genecaused abnormality in locomotion, while sense RNA had little effect. The
animals in which the antisense RNA was expressed under a neuronal promoter showed misdirection of the motor neuron processes that normally extend from the ventral to the dorsal side. The mechanism of the action of thisantisense RNA remains to be elucidated, since we could not detect the reduction of gene expression by the antisense RNA, as monitored by a GFP fusion
gene.

Pub I i ca t ions1.

Take-uchi, M., Kawakami, M., Ishihara, T., Amano, T., Kondo, K., and Katsura,
I.: An ion channel of the degenerin/epithelial sodium channel superfamily controls
the defecation rhthm in G.elegans. Proc. Nat!. Acad. Sci. USA, 95, 11775-11780, 1998.

H-d. Bi 0 mol ecui ar St rue t ur e Lab 0 rat 0 r y(1)

Crystallographic Study of Fl-ATPase:Structural
Analysis of Supramolecule

Yasuo SHIRAKIHARA
FI-ATPase, with a subunit composition of aa 8a yo E, is a catalytic sectorof the membrane bound ATP synthase which plays a central role in energy
conversion in mitochondria, chloroplasts and bacteria, generating ATP from
ADP and inorganic phosphate using energy derived from a trans-membraneelectro-chemical potential. Both the aa 8a sub-assembly and FI-ATPaseand have been challenging targets for crystallographic study because oftheirlarge sizes e.g. 320kDa for the aa 8a sub-assembly and 380 kDa for Fl. We
have solved the structure of the aa (3 a sub-assembly of FI-ATPase from athermophilic bacterium Bacillus PS3. We have extended the structural study
to another crystal form of the aa 8a sub-assembly, mutant aa 8a sub-assembly and aa 8ag sub-assembly.
The aa 8 a sub-assembly is an active complex with 20-25% of the ATPase
activity of intact FI, and has similar catalytic properties to those ofFl. The
refined structure of the nucleotide-free form of aa 8a sub-assembly from Ba-

130

cillus PS3 F1has been published this year (Ref. 1), and deposited in theProtein Data Bank with accession code 1SKY. The final model had R factor of
22.2% (free R factor 29.9%) and had a reasonable stereochemistry. In theabsence of nucleotides and the single copy subunits, the 0:3 (13 sub-assembly
adopts a strictly symmetrical structure: the three 0: and three {3 subunits
have identical conformations. The {3 subunits adopt a conformation essentially identical to that of the nucleotide-free {3 subunit in asymmetrical mitochondrial F1 structure. The 0: subunits have a conformation similar to threemutually similar conformations of 0: subunits in mitochondrial Fl. When
compared to the mitochondrial enzyme, the major differences in structure are
localised in membrane-proximal half of the enzyme, while the remainder ofthe structure is highly conserved.
As an extension of the structural study of the 0:3 {33 sub-assembly, we haveexamined structure of the nucleotide-soaked crystal of the 0:3133 sub-assembly, hopefully assuming the structure in it to represent an nucleotide-boundform. Diffraction data from the crystals were taken before they cracked
eventually, as the 0:3 {33 sub-assembly is destabilized by presence of
nucleotides. From a preliminary analysis of the data sets, ATP and MgADP
soaked crystals, among 12 crystals soaked in different conditions, are found tobe candidates in which large structural difference from the nucleotide-freeform may be observed.
As another extension, we also examined structure of a mutant 0:3 {33 subassembly where three copies of a Glu190Gln mutant {3 subunit join instead of
the wild type {3 subunit. Mutational studies indicate that Glu190 is a catalytic residue and that an 0:3 {33 Y sub-assembly containing Glu190Gln mutant {3 subunit is inactive. The structure of the a mutant 0:3 {33 sub-assemblyis nearly identical to that of the wild-type 0:3 {33 sub-assembly, except the Glnside chain pointing to the same direction as that of the Glu side chain. The
structure suggests that loss of the activity is entirely due to the change of theside chain from carboxyl to amide. The analysis opens up a way to examine
structure of mutant molecules of the 0: and {3 subunits that are of interest,
because subunits alone can not be crystallized.
The 0:3 {33 Y sub-assembly exhibits kinetic properties very similar to thoseof F1, and a number of interesting mutant sub-assemblies have been

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engineered. We have searched crystallization conditions for the sub-assembly starting from those for nucleotide-free Cl3 /33 sub-assembly. Althoughsmall crystals were obtained in the initial stages, it was extremely difficult to
get large well-ordered crystals in spite of extensive search for the crystallization conditions and of efforts to improve purity of the sub-assembly
preparation and to eliminate in homogeneous binding of endogenous
nucleotides. Further efforts are being made to lead to successful crystallization.
The structure solution of the Cl3 /33 sub-assembly was done in collaboration with Andrew Leslie, Jan Pieter Abrahams and John Walker at MRC Laboratory of Molecular Biology, Cambridge,UK. Rest ofthe works described here
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 pstS, that are induced byphosphate starvation. PhoB binds to the phD box in the promoter region,
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.
The DNA-binding C-terminal domain, spanning from a residue 125 to the
C-terminus(220), of the protein forms good crystals allowing to collect diffraction data to 2.1 A resolution. We have attempted to solve its structure by themolecular replacement method using a structural model of C terminal fragment of OmpR with 32 % sequence identity. We found that this molecularreplacement calculation was problematic due to 1) a low signal to noise ratio
generally encountered on dealing with small-sized molecule, 2) two molecules
in an asymmetric unit and 3) a low sequence identity. In spite of many efforts, current solutions are not yet convincing enough. We are trying to com-132

bine the information from a heavy atom derivative to derive a correct molecular replacement solution.
From particular interest to interactive regulatory mechanisms between theN- and the C- domains, crystallization of an intact form of the protein has
been attempted. While the protein was overproduced in the cells containing
a T7 expression vector, the appreciable expression was achieved rather in theabsence of the inducer IPTG. This can be ascribed to the toxicity of the expressed protein. By heparin-affinity chromatography and following cationexchange chromatography used in a pH gradient mode, the protein was purified to a homogeneity level thought to be sufficient for crystallization.
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 of D-camphor. Even after establishing these conditions, we had some time when no good crystals were obtained. Therefore weswitched the E.coli over-expression system from a P L promoter system to amore efficient T7 (pET system) promoter one. Combined with minor modifications in purification procedure, this resulted in more reproducible crystal
formation.
This work has been done in collaboration with Hironori Aramaki, Daiichi
College of Pharmaceutical Sciences.

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Crystallographic Study of Escherichia colJ RNA
Polymerase a Subunit

Katsuhiko MURAKAMI, Akira ISHIHAMA and Yasuo SHIRAKIHARAThe Escherichia coli RNA polymerase (RNAP) a subunit, consisting of 329
amino acid residues, is composed of the two independent structural domains
and is included as a dimer into RNAP. The amino (N)-terminal domain fromresidue 20 to 235 is responsible for the enzyme assembly through the interactions with /3 and /3' subunits and the carboxyl (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. Firstly, we examined the purification method of thea subunit from over-expressing E.coli cells and found the conditions for preparing highly purified proteins reproducibly. Screening of crystallization conditions of the a subunit was performed by several methods, including
microbatch and vapor-diffusion techniques, using numerous kinds of crystallization solutions containing different precipitants, metal ions and different
buffers with different pH. Crystallization of a subunit was also carried out
using Crystal Screening Kit solutions (Hampton Research). We found that
PEG400/CaCI2 and PEG8000/NaAcetate gave thin-plate crystals
reproducibly. We are searching for better crystallization conditions producing thicker and larger crystals.

Publications
1.

SHIRAKIHARA,Y., LESLIE, A. G.w., ABRAHAMS, J.P., WALKER, J.E., UEDA, T., SEKIMOTO,Y.,
KAMBARA, M. K., SAIKA, KAGAWA, Y. and YOSHIDA, M.:°

The crystal structure of thenucleated free 3 /3 3 sub-complex ofF1-ATPase from the thermophilic Bacillus PS3 isa symmetric trimer. Structure 5, 825-836, 1997.
2.

SHIRAKIHARA,Y., LESLIE, A. G.w., ABRAHAMS, J.P., WALKER, J. E., UEDA,T., SEKIMOTO,Y.,
KAMBARA, M. K., SAIKA, KACAWA, Y. and YOSHIDA, M.:

Crystal structure ofthe 03 /3 3

sub-complex ofF1-ATPase from thermophilic Bacillus PS3. In "Structure and Function of Macromolecular Assembly (Proceeding of The 22nd International Symposium
Division of Biophysics The Taniguchi Foundation)" (Namba, K. eds.) pp 113-120.,
1997.

134

H-e. Gene Network Laboratory(1)

Expression pattern map of the C. eJeffans genome

Yuji KOHARA, Tadasu SHIN-I, Tomoko MOTOHASHI, Tokie OHBA, Ikuko
SUGIURA, Masumi OBARA, Akiko MIYATA, Masako SANO, Hiroko UESUGI,
Hisako WATANABE, Yuko MITANI and Tamami NAGAOKA
Aiming to ultimately understand the network of gene expression in the development of the nematode C. elegans, we are constructing an expression pattern map of the 100Mb genome by identifying and characterizing its cDNA
species, whose total number is estimated to be around 15,000.
More than 100,000 cDNA clones were picked up randomly from different
cDNA libraries, stored and gridded. cDNA clones derived from already analyzed abundant cDNA species were identified by probing the high density clone
grids and removed to avoid unnecessary redundancy of analysis. The remaining clones were subjected to analyses of (1) tag-sequencing from both 5'-ends(with vector primers) and 3'-ends (with anchored oligo-dT primers), (2) mapping onto the genome, and (3) analysis of expression patterns. Thus far, 50,000
clones have been processed, and 36,500 clean 3'-tag sequences were obtained,
which were classified into 7,680 unique cDNA species (nearly half of the total
genes) by comparing the 3'-tags. About 70% of them were located on the
genomic sequence determined by the Sanger CtrIWASH-U sequencing consortium.
II
chromosome
I
III
IV
V
X unassigned
cDNAclones
5' tags
7,439 7,242 6,765 6,836 6,119 6,103 7,318
3' tags
5,734 5,535 5,132 5,144 4,747 4,765 5,408
both tags* 5,230 5,029 4,686 4,651 4,292 4,399 3,773

47,822
36,46532,060clusters

7,681

914

1,062

897

896

923

969 2,020

total*: cDNA clones whose 5' and 3'-tags are available.
We are analyzing the expression patterns during development of the classi-

H. STRUCTURAL BIOLOGY CENTER

135

fied cDNA species using the newly developed method of in situ hybridization
on whole mount specimens of embryos, larvae and adults in the standard
multi-well format. Thus far, more than 2,000 cDNA groups mostly from chromosomes 3 and X have been analyzed. The cDNA sequence and mapping
information has been made available at NEXTDB (Nematode EXpression
paTtern DataBase) http://watson.genes.nig.ac.jp:8080/db/index.html(!)

pos-J encodes a cytoplasmic zinc-finger protein essential for
germline specification in C. eJegans

Hiroaki TABARA, Russell J. HILL 1, Craig MELL0 2 , James R. PRIESS I and Yuji
KOHARA (IFred Hutchinson Cancer Research Center, Seattle, WA and2 University of Massachusetts Cancer Center, Worcester, MA, USA)
In C. elegans, germ cells arise during early embryogenesis from an invariant
sequence of asymmetric divisions that separate germ cell precursors fromsomatic precursors. We show that maternal-effect lethal mutations in thegene pos-l cause germ cell precursors to inappropriately adopt somatic cellfates. During early embryogenesis, pos-l mRNA and POS-l protein arepresent predominantly in the germ precursors. POS-l is a novel protein withtwo copies of a CCCH finger motif previously described in the germline proteins PIE-l and MEX-l in C. elegans, and in the mammalian TISll/Nup475/
TTP protein. Mutations in pos-l cause several defects in the development of
the germline blastomeres that are distinct from those caused by mutations in
pie-lor mex-l. An early defect in pos-l mutants is a failure to express APX1 protein from maternally-provided apx-l mRNA, suggesting that POS-l may
have an important role in regulating the expression of maternal mRNAs in
germline blastomeres.

136

(3)

Development of a method to mesure the size of poly(A)tails In Caenorbabdltls ele~ans embryos and
Its application to some maternal genes

Shuichi ONAMI 1 and Yuji KOHARA (IDepartment of Genetics, The Graduate
University for Advanced Studies)
Translational regulation is expected to play an important role in early embryogenesis of the nematode G.elegans, which are mostly dependent on thematernally supplied proteins and mRNAs. Although the mechanisms oftranslational regulation are largely unknown, the size ofpoly(A) is a possible
factor in the regulation, considering the findings in other organisms such as
Xenopus lavies. Thus, we developed a PCR-mediated method to measure thepoly(A) tail length of given mRNA in C.elegnas embryos at a desired
stage. Nucleic acid is extracted from 5-15 embryos and an RNA-oligonucleotide-tag is added to its 3'-end. The 3'-end region oftarget mRNA is examined by RT-PCR using gene-specific and tag-specific primers. Using thismethod, we measured the poly(A) tail length of fem-3 mRNA as a positive
control to verify the method. We also measured the poly(A) Ienth of glp-]
mRNA, whose translation is controlled temporally and blastomere-specifically during early G. elegans embryogenesis. GLP-1 protein is not detectedin oocyte, while 2cell-stage embryos express the protein only in the anterior
blastomere. We found a correlation between the poly(A) tail length and its
protein expression.
(4)

Functional analysis of a Caenorbabdltls elegans
T-box gene tbx-fl

Yoshiki ANDACHI
tbx-9is one of the genes identified by the eDNA project and encodes a proteinwith the DNA binding motif T-box. The T-box gene family consists of morethan 30 genes in various multicellular organisms, including the mouse transcription factor Brachyury. Many of the genes are known to be expressed inembryogenesis and to be involved in morphogenesis. As it is conceivable that
the T-box genes play important roles in the development of this organism, I

H. STRUCTURAL BIOLOGY CENTER

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have been studying one ofthe T-box genes, tbx-9.
Sequence analyses of the eDNA clone yk97a6 and 5'RACE PCR products
indicated that the tbx-9gene is transcribed to a product of 1.0 kb. The transcript that was detected in embryos by developmental northern blot analysis,
and in situ hybridization analysis on whole mount embryos revealed that tbx9is expressed in a few cells of early-stage embryos.
To know the phenotype of worms with mutation in the tbx-9 gene, I performed a gene disruption method using the transposon Tel, and obtained anull allele losing more than half of the gene. Worms homozygous for theallele show a phenotype of aberration in shape in the posterior half of theirbodies. The aberration occurs during embryogenesis. Immunostaining ofmutant embryos with an antibody against body-wall muscle myosin revealed
abnormality in body-wall muscle cells, indicating that tbx-9is required forproper formation of body-wall muscle in embryogenesis.
To show sequence-specific DNA binding activity of tbx-9, I determined the
DNA binding sequence of tbx-9by a strategy for the enrichment of target DNA
fragments. The binding sequence of tbx-9turned out to be nearly identical tothat of Brachyury.

Publ icatian
None

138

I.

CENTER FOR INFORMATION BIOLOGY
I-a. Laboratory for DNA Data Analysis(1)

Genome plasticity as a paradigm of eubacteria evolution

Hidemi WATANABE, Hirotada MORI 1, Takeshi ITOH 1, Takashi GoJOBORI (lNara
Institute of Science and Technology)
To test the hypotheses that eubacterial genomes leave evolutionarily stablestructures and that the variety of genome size is brought about through genome doubling during evolution, the genome structures of Haemophilus
influenzae, Mycoplasma genitalium, Escherichia coli, and Bacillus subtilis
were compared using the DNA sequences of the entire genome or substantial
portions of genome. In these comparisons, the locations of orthologous genes
were examined among different genomes. Using orthologous genes for thecomparisons guaranteed that differences revealed in physical location would
reflect changes in genome structure after speciation. We found that dynamic
rearrangements have so frequently occurred in eubacterial genomes as tobreak operon structures during evolution, even after the relatively recent
divergence between E. coli and H influenzae. Interestingly, in such eubacterial
genomes of high plasticity, we could find several highly conservative regionswith the longest conserved region comprising the S10, spc, and a operons. Thissuggest that such exceptional conservative regions have undergone strong structural constraints during evolution. See Ref. 6 for details.
(!)

Constrained evolution of hepatitis B virus with overlapping genes

Masashi MIZOKAMI 1, Etsuro ORIT0 1, Ken-ichi OHBA 1, Kazuho IKEO, Johnson
Y.N. LAU 2, Takashi GOJOBORI (lNagoya City University Medical
School, 2University of Florida)
With the aim of elucidating the evolution of a hepadnavirus family, we constructed molecular phylogenetic trees for 27 strains of hepatitis B virus (HBV)I.

CENTER FOR INFORMATION BIOLOGY

139

using both the unweighted pair-grouping and neighbor-joining methods. Allfive gene regions, P, C, S, X, and preS, were used to construct the phylogenetic trees. Using the phylogenetic trees obtained, we classified these strains
into five major groups in which the strains were closely related to each other.
Our classification reinforced our previous view that genetic classification is
not always compatible with conventional classification determined by serological subtypes. Moreover, constraints on the evolutionary process of HBV
were analyzed for amino-acid-altering (nonsynonymous) and silent (synonymous) substitutions, because two-thirds of the open reading frame (ORF), P,
contains alternating overlapping ORFs. In our unique analysis of this interesting gene structure of HBV, the most frequent synonymous substitutions
were observed in the nonoverlapped parts of the P and C genes. On the other
hand, the number of synonymous substitutions per nucleotide site for the S
gene was quite low and appeared a strongly constrained evolution. Because
the P gene overlaps the S gene in a different frame, the low rate of synonymous
substitution for the S gene can be explained by the evolutionary constraints
which are imposed on the overlapping gene region. In other words, synonymous substitutions in the S gene can cause amino acid changes in its overlapping region in a different frame. Thus, the evolution of HBV is constrained
evolutionarily by the overlapping genes. We propose calling this mode of viral evolution "constrained evolution". The evolution of HBV represents a
typical constrained evolution. See Ref. 7 for details.

(3)

Evolutionary mechanisms and population dynamics of the third
variable envelope region of DIV within single hosts

Yumi YAMAGUCHI and Takashi GOJOBORIClonal diversifications of HIV virus were monitored by periodic samplings
on each of the six patients with regard to 183- to 335-bp segments of the env
gene, which invariably included the functionally critical V3 region. Subsequently, six individual phylogenetic trees ofviral variants were constructed. It
was found that at one time or another during the course of disease progression,
viral variants were inexplicably released from a strong negative selection
against nonsynonymous base substitutions, possibly indicating positive

140

selection. This resulted in concentrated amino acid substitutions at five specific sites within the V3 region. It was noted that these sites were often
involved as antigenic determinants that provoked the host immune response
and that these sites were also involved in the determination of viral phenotypes as to their cell tropism, syncytium formation capability, and replication
rates. See Ref. 8 for details.

(4)

As~idian tyrosinase gene: Its unique structureand expression in the developing brain
Shigeru SAT0 1, Hiroshi MASUYA, Takaharu NUMAKUNAI 2, Noriyuki
SATOH3, Kazuho IKEO, Takashi GoJOBORI, Koji TAMURA 1, Hiroyuki IDE l , Takuji
TAKEUCHI4, Hiroaki YAMAMOTO (IBiological Institute, Tohoku Univ., 2The Marine Biological Station of Asamitsu, Tohoku Univ., 3Dept. of Zoology, Kyoto
Univ., 4Dept.ofBiotechnology, Ishinomaki Senshu Univ.)
Tadpole larvae of ascidians have two sensory pigment cells in the brain.
One is the otolith cell that functions as a gravity receptor, the other pigment
cell is part of a primitive photosensory structure termed the ocellus. Thesesensory cells, like vertebrate pigment cells, contain membrane-bounded melanin granules and are considered to reflect a crucial position in the evolutionary process of this cell type. To investigate the molecular changes accompanying the evolution of pigment cells, we have isolated from Halocynthia roretzi
a gene encoding tyrosinase, a key enzyme in melanin biosynthesis. The cDNA
has an open reading frame (ORF) of 596 amino acids, which is 36-39% identical in amino acid sequence to vertebrate tyrosinases. In addition, the sequence analysis of both cDNA and genomic clones reveals an unusual organization of the tyrosinase gene, an extraordinary 3' untranslated region of thetranscripts with significant homology to the coding sequence, and a single
short intron in the sequence encoding a cytoplasmic domain. Expression of
the gene is detected first in two pigment precursor cells positioned in theneural plate of early neurulae, and later in two melanin-containing pigment
cells within the brain of late tailbud embryos. Its expression pattern correlates well with the appearance oftyrosinase enzyme activity in the developing
brain. These results provide the first descrition of pigment cell differentia-

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141

tion at the molecular level in the ascidian embryo, and also will contribute toa better understanding ofthe evolution of chordate pigment cells. See Ref. 9
for details.

(5)

Inference of molecular phylogenetic tree basedon minimum model-based complexity method

Hiroshi TANAKA l , Fengrong REN 1, Toshitsugu OKAYAMA, Takashi
GOJOBORI (lMedical Research Institute, Tokyo Medical and Dental University)
In this study, starting with a newly introduced concept of data complexity("empirical data complexity"), we specify the concept of complexity more concretely in relation to mathematical modeling and introduce "model-based complexity (MBC)". Inductive inference based on the minimum model-based complexity method is then applied to the reconstruction of molecular evolutionary tree from DNA sequences. We find that minimum MBC method has good
asymptotic property when DNA sequence lengths approach to infinite andcompensates the bias of maximum likelihood method due to the difference oftree topology complexity. The efficiency of minimum MBC method for reconstruction of molecular tree is studied by computer simulation, and results
suggest that this method is superior to the traditional maximum likelihood
method or its modification by Akaike's AIC. See Ref. 10 for details.

(6)

Molecular evolution of myelin proteolipid protein. Biochemical andBiophysical Research Communications

Tadashi KURIHARA l , Mitsuhiro SAKUMA l , Takashi GOJOBORI (lInstitute of Life
Science, Soka University)
We show that the major membrane protein of central nervous system myelin,
proteolipid protein, evolved much more rapidly than it does now more than
300 million years ago. We reason that myelin proteolipid protein evolved
rapidly just after its appearance in vertebrates and that its evolutionary ratethen gradually decreased. Comparison ofthe rates between the synonymousand nonsynonymous nucleotide substitutions for the cDNA suggests the possi-

142bility that positive selection operated on myelin proteolipid protein at least
when it appeared in vertebrates. See Ref. 13 for details.

(7) Father-to-mother-to-Infant transmission of DIV-t:
clonally transmitted Isolate of Infant mutates more
rapidly than that of the mother and rapidly
loses reactivity with neutralizing antibody
Yukari OKAMOTO I , Koichi SHIOSAKI2, Yasuyuki EDA2, Sachio TOKIYOSHI 2, Yumi
YAMAGUCHI, Takashi GOJOBORI, Takashi HACHIMORI 3, Sh udo
YAMAZAKI l , Mitsuo HONDA I (lAIDS Research Center, National Institute of
Health, 2Chemo-Sero-Therapeutic Research Institute, 3Clinical Dept. of Infectious Diseases, Komagome Tokyo Metropolitan Hospital)
The sequences of the V3 loop and surrounding regions of human immunodeficiency virus type-l from a father-to-mother-to-infant trimmer were studied and the horizontal and vertical transmissions compared. The father'svirus was variable for reactivity with neutralizing antibody and sequences of
the V3 loop central core sequence. In contrast, the mother's viral sequenceswere much less diverse than those of the father, and N-glycosylation sites
were conserved. By phylogenetic analysis, the major clone, of which V3-peptide reacted with the neutralizing antibody, was found to be transmitted fromthe mother to her infant; however, the mutated minor clones did not bind to
the antibody. These finding suggest that both horizontal and vertical virustransmission were selective, and that the clonally transmitted virus in infants mutates more rapidly than viruses in the mother, to whom the virus
was horizontally transmitted. See Ref. 16 for details.

(8)

Bacterial features In the genome of methanococcus jannaschll In
terms of gene composition and biased base composition In
ORFs and their surrounding regions

Hidemi WATANABE, Takashi GOJOBORI, Kin-ichiro MIURA I (lInstitute for
Biomelocular Science, Gakushuin University)
As a result of genome projects, the complete nucleotide sequence of the1.

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143

entire genome of an archaeon, Methanococcus jannaschii, was recently determined as well as other complete sequences of bacterial and eucaryal genomes.
When all the 1680 predicted protein-cloning genes of M jannaschiiwere classified on the basis of sequence similarity, it was found that this archaeon hada chimeric set of 1016 bacterial-type, 471 eucaryal-type and 193 species- orarchaebacteria-specific genes. However, most of the genes predicted to be
involved in translation and transcription pathways including RNA genes wereof the eucaryal-type with only a few exceptions such as 16S ribosomal RNA
and some translation factor-like genes. This appeared curious since previous studies indicated that methanogens have bacterial features in gene organization and expression. To understand the apparent inconsistency between
physiological observations and the result of the classification of genes for transcription and translation, we examined the structural relatedness of the genome of M jannaschii to those of other species. In practice, we comparedbased compositional patterns in ORFs and their surrounding regions. This
made it possible to reveal the relationships among the translation- and transcription-related structures in genomes. In this study, we conducted a statistical test called 'G-test' to evaluate the base biases around the boundaries of
ORFs. We then found that M jannaschij possesses more bacterial featuresin base biases than eucaryal ones, e.g. strong G biases at the positions corresponding to the Shine-Dalgarno site. This indicates that the few exceptional
bacterial genes for translation, such as 16S ribosomal RNA and translation
factor-like genes, play crucial roles in the translation pathway in M jannaschii.
The possibility that the genome structure in the last common ancestor of allpresent species was bacterial is discussed. See Ref. 17 for details.

(9)

Evolutionary significance of intra-genome
duplications on human chromosomes

Toshinori ENDO, Tadashi IMANISHI, Takashi GoJOBORI, Hidetoshi INOK0 1 (lTokai
University School of Medicine)
Phylogenetic analyses indicated that a series of paralogous gene pairs,
found in two extensive regions on human chromosomal bands 6p21.3 and 9q3334, were created by at least two independent duplications. The duplicated144

genes on chromosomal band 6p21.3 include the genes for type 11 collagen a2
subunit (COLllA2), NOTCH4 (mouse int-3 homologue), 70 kDa heat shockprotein (HSPA1A, HSPA1B, and HSPA1L), valyl-tRNA synthetase 2 (VARS2),
complement components (C2 and C4), pre-B cell leukemia transcription factor 2 (PBX2), retinoid X receptor b (RXRB), NATIRING3, and four other proteins. Their paralogous genes on chromosomal band 9q33-34 are genes for
type 5 collagen a1 subunit (COL5Al), NOTCH1, 78 kDa glucose-regulated
protein (HSPA5), valyl-tRNA synthetase 1 (VARS1), complement component C (C5), PBX3, retinoid X receptor a (RXRA) , ORFXIRING3L, andothers. Among these, the genes for collagem complement components, NATI
RING3, PBX, and RXR appear to have been duplicated around the time ofvertebrate emergence, supporting the idea that they were duplicated simultaneously at that time. Another group of genes that includes NOTCH and HSP
appear to have diverged long before that time. A comparison ofthe physicalmaps of these two regions revealed that the genes which duplicated in thesame period were arranged in almost the same order in the two regions, withthe assumption of a few chromosomal rearrangements. We propose a possible model for the evolution ofthese regions, taking into account the molecular mechanisms of regional duplication, gene duplication, translocation, andinversion. We also propose that a comparative mapping of paralogous genes
within the human genome would be useful for identifying new genes. See Ref.
18 for details.

(10)

Evolution of Nicotinic Acetylcholine Receptor Subunits

Kazuhisa TSUNOYAMA and Takashi GOJOBORI
Acetylcholine (ACh) has long been recognized as a neurotransmitter active in nervous and muscle systems of Bilateria. The nicotinic acetylcholine
receptors (nAChR) belong to the superfamily ofreceptors involved in ligandgated ion channels in both nervous and muscle systems. Because the nAChR
in the muscle system was the first receptor to be isolated, the nAChR ofstriated muscles is the best-characterized member ofthe super family. It is
known that the nAChR in the muscle system is a hetero-oligomer composedof five subunits ( 0: 1, 13 1, Y, (j, and E). As for the nAChR in the nervous

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system, there are eight a-type (named a2 - a9) and three /3-type (classified
as /3 2 - /3 4) subunits.
It is of particular interest to understand how all 16 subunits are evolutionarily related to each other. In spite of the intensive previous works, the controversy over the evolutionary history of these subunits has not been settled
yet. In order to resolve the controversy and to elucidate the evolutionary mechanisms of these subunits, we constructed a phylogenetic tree using 84 nucleotide sequences of receptor subunits from 18 different species. The tree constructed showed that the common ancestor of all subunits may have appeared
first in the nervous system. Moreover, we suggested that the a1 subunits inthe muscle system originated from the common ancestor of a2, a3, a4, a5,
a6, and /33 in the nervous system, whereas the /31, y, 0, and E subunits inthe muscle system shared the common ancestor with the /32 and /34 subunits
in the nervous system.
Using the ratio (f) of the number of nonsynonymous substitutions to that ofsynonymous substitutions, we predicted the functional importance ofsubunits. We found that the a1 and a7 subunits had the lowest f values in
the muscle and nervous system, respectively, indicating that very strong functional constraints work on these subunits. It is consistent with the fact thatthe a 1 subunit has binding sites to the ligand and the a7 -containing receptor regulates the release of the transmitter. Moreover, the window analysisof the f values showed that strong functional constraints work on the so-called
M2 region in all 5 types ofthe muscle subunits.
For more details, see Ref.
Reference
TSUNOYAMA, K., GoJOBORI, T.,: Evolution of nicotinic acetylcholine receptor subunits. Mol. BioI. Evol., 15, 518-527, 1998.

146

(11)

Tie Trees Generated by Distance Methods of
Phylogenetic Reconstruction

Naoko TAKEZAKI (National Institute of Genetics, The Pennsylvania State
University)
Examining genetic data in recent publications, Backeljau et al. (1996) showedcases in which two or more different trees (tie trees) were constructed from asingle data set for the neighbor-joining (NJ) method and the
UPGMA. However, it is still unclear how often and in what conditions tie
trees are generated. Therefore, I examined these problems by computer
simulation. Examination of cases in which tie trees occur shows that tie
trees can appear when no substitutions occur along some interior branch(es)on a tree. However, even when some substitutions occur along interior
branches, tie trees can appear by chance if parallel or backward substitutions
occur at some sites. The simulation results showed that tie trees occur relatively frequently for sequences with a low divergence level or with a small
number of sites. For such data, UPGMA sometimes produced tie trees quite
frequently, whereas tie trees for the NJ method were generally rare. In thesimulation, bootstrap values for clusters (tie clusters) that differed among tie
trees were mostly low «60%). With a small probability, relatively high bootstrap values (at most 70% - 80%) appeared for tie clusters. The bias of thebootstrap values caused by an input order of sequences can be avoided if oneof different paths in cycles of making an NJ or UPGMA tree is chosen at
random in each bootstrap replication.
This study was published in the following paper.
TAKEZAKI N: Tie Trees Generated by Distance Methods of Phylogenetic Reconstruction. Molecular Biology and Evolution, Volt2, pp. 727-737, 1998.

Publ icatians
1.

2.

SUZUKI, Y. and GOJOBORI, T.: The origin and evolution of human T-Celllymphotrophic
virus type I and II. Virus Genes 16, 69-84, 1998.
TATENo, Y. and GOJOBORI, T.: DNA data bank of Japan in the age of information

I.

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biology. Nucl. Acids Res. %5,14-17,1997.
3.

PERRIERE, G., MoszER, I., and GOJOBORl, T.:

The NRSub database: update 1997. Nucl.

Acids Res. %5, 53-56, 1997.
4.

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

Codon usage tabulated from the interna-

tional DNA sequence databases. Nucl. Acids Res. %5, 244-245, 1997.
5.

TATENO, Y., IKEo, K., IMANISHI, T., WATANABE, H., ENOO, T., YAMAGUCHI, Y., SUZUKI, Y.,
TAKAHASHI, K., TSUNOYAMA, K., KAWAI, M., KAWANISHI, Y., NAITOU, K. and GOJOBORI
T.:

6.

Evolutionary motif and its biological and structural significance. J. Mol. Evol. 44,

38-43, 1997.
WATANABE, H., MORl, H., IToH, T. and GOJOBORI, T.:

Genome plasticity as a paradigmof eubacteria evolution. J. Mol. Evol. 44, 57-64, 1997.
7.

MIZOKAMI, M., ORITO, E., OHBA, K., IKEo, K., LAu, J.Y.N and GoJOBORI, T.:

Constrained

evolution of hepatitis B virus with overlapping genes. J. Mol. Evol. 44, 83-90, 1997.
8.

YAMAGUCHI, Y. and GOJOBORl, T.:

Evolutionary mechanisms and population dynamics

of the third variable envelope region of HIV within single hosts. Proc. Natl. Acad. Sci.
U.S.A. 94, 1264-1269, 1997.
9.

SATO, S., MASUYA, H., NUMAKUNAI, T., SATOH, N., IKEO, K., GOJOBORI, T., TAMURA, K., IDE,
H., TAKEUCHI, T. and YAMAMOTO, H.:

Ascidian tyrosinase gene: Its unique structureand expression in the developing brain. Developmental Dynamics %08, 363-374,
10.

1997.
TANAKA, H., REN, F., OKAYAMA, T. and GOJOBORI, T.:

Inference of molecular phylogenetic

tree based on minimum model-based complexity method. Proceedings of Fifth international conference on intelligent systems for molecular biology. Halkidiki, Greece I
American Association for Artificial Intelligence. Pp.319-328, 1997.
11.

TENzEN, T., YAMAGATA, T., FUKAGAWA, T., SUGAYA, K., ANoo, A., INoKo, H., GOJOBORI, T.,
FUJIYAMA, A., OKUMURA, K. and IKEMURA, T.:

Precise switching of DNA replication

timing in the GC content transition area in the human Major Histocompatibility
Complex. Mol. and Celular BiolQgy 17, 4043-4050, 1997.
12.
13.

SUZUKI, Y. and GOJOBORI, T.: The origin and evolution of ebola and marburg viruses.
Mol. BioI. Evol. 14, 800-806, 1997.
KURIHARA, T., SAKUMA, M. and GOJOBORI, T.:

Molecular evolution of myelin proteolipid

protein. Biochemical and Biophysical Research Communications. %37, 559-561, 1997.
14.

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 Research %6, 1620, 1997.
15.

MIZUKI, N., OHNO, S., ANoo, H., SATO, T., IMANISHI, T., GOJOBORI, T., ISHIHARA, M., OTA,
M., GENG, Z., GENG, L., LI, G., KIMURA, M., INoKo, H.:

Major histocompatibility com-plex class II allelles in Kazak and Han populations in the Silk Route of northwestern
China. Tissue Antigens 50, 527-534, 1997.
16.

OKAMOTO, Y., SHIOSAKI, K., EOA, Y., TOKIYOSHI, S., YAMAGUCHI, Y., GOJOBORI, T., HACHIMORI,

148

17.

18.

c10nally transmitted isolate of infant mutates more rapidly than that of the motherand rapidly loses reactivity with neutralizing antibody. MicrobiolImmunol 41, 131138, 1997.
WATANABE, H., GOJOBORI, T. and MIURA, K.: Bacterial features in the genome of
methanococcus jannaschii in terms of gene composition and biased base composition
in ORFs and their surrounding regions. GENE 205, 7-18, 1997.
ENDO, T., IMANISHI, T., GOJOBORI, T. and INOKO, H.: Evolutionary significance of intragenome duplications on human chromosomes. GENE 205, 9-27, 1997.

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

Structural Requirement of Highly-Conserved Residues in Globins

Motonori OTA, Yasuhiro ISOGAI 1 and Ken NISHIKAWA (IThe Institute ofPhysical and Chemical Research (RIKEN), Hirosawa, Wako, Saitama)
Glogins have remarkable sequence diversity, and yet maintain a common
fold. In spite of the diversity, there are highly-conserved residues at several
sites. The conserved residues were examined in terms of the structural stability, by employing the pseudo-energy functions of the structure/sequencecompatibility method. The fitness of each residue type to the structural environment was evaluated at seven highly-conserved sites: the Leu ( at the BIOsite), Phe (CD I), and Leu (F4) residues were found to fit their respective
sites due to hydrophobic interactions; and Pro (C2) stabilizes the N-terminaledge of an a -helical structure; and Phe (CD4) is stabilized by backbone hydrogen-bonding to Phe (CD I). On the other hand, the other two residues, His(E7) and His (F8), are poorly suited to the sites from a structural viewpoint,suggesting that their conservation clearly results from a heme-related functional requirement. The invariant Phe residue (CDI) has been suggested to
be important for supporting the heme. The present analysis revealed that
this residue is also well suited to the site in terms of energy. See Ref. 3 fordetails.

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Prediction of Protein Secondary Structure Using the
3D-to Compatibility Algorithm

Masahiro ITO l , Yo MATSU0 2 and Ken NISHIKAWA (IGene Library Laboratory,
2National Center for Biotechnology Information, National Library of Medicine, NIH, Bethesda, MD 20894, USA)A new method for the prediction of protein secondary structure is proposed, which relies totally on the global aspect of a protein. The prediction
scheme is as follows. A structural library is first scanned with a query sequence by the 3D-ID compatibility method developed before. All the structures examined are sorted with the compatibility score and the top 50 in thelist are picked out. Then, all the known secondary structures of the 50 proteins are globally aligned against the query sequence, according to the 3D-ID
alignments. Prediction of either a helix, {3 strand or coil is made by taking
the majority among the observations at each residue site. Besides 325 proteins in the structural library, 77 proteins were selected from the latest release ofthe Brookhaven Protein Data Bank, and they were divided into three
data sets. Data set 1 was used as a training set for which several adjustableparameters in the method were optimized. Then, the final form ofthe method
was applied to a testing set (data set 2) which contained proteins of chain
length <400 residues. The average prediction accuracy was as high as 69% in
the three-state assessment of a, {3 and coil. On the other hand, data set 3
contains only those proteins of length >400 residues, for which the present
method would not work properly because of the size effect inherent in the 3DID compatibility method. The proteins in data set 3 were, therefore, subdivided into constituent domains (data set 4) before being fed into the prediction
program. The prediction accuracy for data set 4 was 66% on average, a few
percent lower than that for data set 2. Possible causes for this discrepancy
are discussed. See Ref. 4 for details.

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Differences in Dinucleotide Frequencies of Human,
Yeast, and E. colI Genes

Hiroshi NAKASHlMA l and Ken NISHIKAWA (IThe School of Health Sciences,
Faculty of Medicine, Kanazawa University, Kodatsuno, Kanazawa)
Nucleotide sequences coding proteins in human, yeast and E. coli genes
were analyzed in terms of dinucleotide occurrences. Every gene is plotted asa point in the dinucleotide space, which is spanned by 16 axes corresponding to
the 16 components of the dinucleotide. The metric unit in the space is defined using the log-odds ratio of dinucleotide occurrences in a gene. The distribution of points showed that genes from the same organism are clustered in
the space. The clusters of human and E. coli are completely separated, andthe yeast cluster sits in-between, implying that individual genes are classified into the three sources from their location. In fact, they could be identified
with accuracy of 90%, using the DNA data alone. Even genes encoding homologous proteins belonging to the same protein superfamily were discriminated by the DNA data, and were correctly identified into their sources withthe same accuracy as above. DNA sequences of non-coding regions, including
human introns, as well as human genes of GC-rich and GC-poor types, werealso analyzed in the same manner. The most significant finding is that human genomic DNA sequences, including genes and introns together, exhibit
the largest deviation of dinucleotide occurrence from the random expectation.
Possible origins for this phenomenon are discussed. See Ref. 5 for details.

Publications
I.

2.
3.
4.
5.

OTA, M. and NISHIKAWA, K: Assessment of pseudo-energy potentials by the best-five
test: a new use of the three-dimensional profiles of proteins. Protein Engineer., 10,339-351, 1997.
SUYAMA, M., MATSUO, Y. and NISHIKAWA, K: Comparison of protein structures using
3D profile alignment. J. Mol. EvoL, 44 (Suppll), S163-Sl73, 1997.
OTA, M., ISOGAI, Y. and NISHIKAWA, K: Structural requirement of highly-conserved
residues in globins. FEBS Lett., 415, 129-133, 1997.
ITO, M., MATSUO, Y. and NISHIKAWA, K: Prediction of protein secondary structure using
the 3D-lD compatibility algorithm. CABIOS, 13, 415-423, 1997.
NAKASHIMA, R., NISHIKAWA, K and 001, T.: Differences in dinucleotide frequencies of

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human, yeast, and E. coli genes. DNA Res., 4, 185-192, 1997.
GREADY, J.E., RANCANATHAN, S., SCHOFIELD, P.R., MATSUO, Y. and NISHIKAWA, K: Predictedstructure of the extracellular region ofligand-gated ion-channel receptors shows SH2like and SH3-like domains forming the ligand binding site. Protein Sci., 6, 983-998,
1997.
7. SUMIKAWA, H., FUKUHARA, K, SUZUKI, E., MATSUO, Y. and NISHIKAWA, K: Tertiary structural models for human interleukin-6 and evaluation by a sequence-structure compatibility method and NMR experimental information. FEBS Lett., 404, 234-240, 1997.

6.

I-c. Laboratory of Gene Function(1)

Evolutionary Motif and Its Biological and Structural SignificanceYoshio TATENO, Kazuho IKEO, Hidemi WATANABE, Toshinori ENDO, Yumi
YAMAGUCHI, Yoshiyuki SUZUKI, Kazunari TAKAHASHI, Kazuhisa TSUNOYAMA,
Masato KAWAI l , Yuichi KAWANISHI I , Kimitoshi NAITOU I and Takashi
GOJOBORI (I Fujitsu Limited, Makuhari System Laboratory, Mihamaku, Chiba)
We developed a method for multiple alignment of protein sequences. The
main feature of this method is that it takes the evolutionary relationships ofthe proteins in question into account repeatedly for execution, until the relationships and alignment results are in agreement. We then applied thismethod to the data of the international DNA sequence databases, which are
the most comprehensive and updated DNA databases in the world, in orderto estimate the" evolutionary motif" by extensive use of a supercomputer. Through a few problems needed to be solved, we could estimate
the length of the motifs in the range of 20 to 200 amino acids, with about 60
the most frequent length. We then discussed their biological and structural
significance. We believe that we are now in a position to analyze DNA andprotein not only in vivo and in vitro but also in silico. For details, see Ref. 1.

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DNA Data Bank of Japan in the Age of Information Biology

Yoshio TATENO and Takashi GOJOBORIDNA Data Bank of Japan (DDBJ) began its activities in 1986 in collaboration with EMBL in Europe and GenBank in the United States. DDBJ developed a data submission tool called Sakura, by which researchers can submit
their newly sequenced data on WWW from every corner of the world. The
data bank also built a database management system (Yamato II), incorporating the techniques and functions of the object-oriented database, in order toefficiently process the data it has collected. A number of research activities
in information biology are also going on at DDBJ. For details, see Ref. 2.

Publ icatians
1.

2.

TATENO, Y, IKEO, K, IMANISHI, T., WATANBE, H., ENDo, T., YAMAGUCHI, Y., 8UZUKI, Y.,
TAKAHASHI, K, TSUNOYAMA, K, KAWAI, M., KAWANISHI, Y., NAITOU, K, and GOJOBORI, T.
(1997) Evolutionary motif and its biological and structural significance. J Mol Evol44(8uppl 1) 838-843
TATENO, Y and GOJOBORI, T. (1997) DNA Data Bank of Japan in the age of information
biology. Nucleic Acids Res %5: 14-17

I-d. Laboratory for Molecular Classification(1)

Development of the data processing system for
DNA Data Bank of Japan (DDBJ)

Hideaki SUGAWARA, Takashi GOJOBORI, Hirotada MORI], Takurou TAMURA,
Toshitsugu OKAYAMA, Hikaru YAMAMOTO and Kousuke GOTO (I NARA Institute of Science and Technology)
The sequence data stored in DDBJ has increased to be more than 1G basepairs in 1997. To cope with this data explosion, we improved systems for
capturing, evaluation, storage and dissemination of sequence data and theirannotation. The submission system of SAKURA is now equipped with visualization of annotations along sequences and resulted in supporting the sub-1.

CENTER FOR INFORMATION BIOLOGY

153

mission oflong sequences. The Genome Information Broker was newly developed for the comprehensive retrieval of microbial genome data. For details,
see Ref.5 and 8, 9, 10, 14.
(!)

Development of information systems for the study of biodiversity

Hideaki SUGAWARA, Satoru MIYAZAKI
2·1. WFCC·MIRCEN World Data Centre for Microorganisms (WDCM)
The WDCM is a data center of World Federation for Culture Collections(WFCC) and Microbial Resources Centers Network(MIRCEN). The Centerhas provided WWW server since April 1, 1997. This server was highly evaluated in the field of microbiology by the American Society of Microbiology andaccessed 30,000 times a month on average. The WDCM is and will be an
important facility for the study of microbial diversity which is introduced inRef. 3 and 15.
2-2. Utilization of a broad-band network
It is prerequisite for the study of biodiversity that a number of centers
distributed on the globe are able to share information. We did real·time
experiments of virtual laboratory and distant learning system between Japanand US by use of a dedicated line of 45Mbps. For the details, see Ref. 1 and9. We also proposed a concept ofAPBioNet for the promotion ofbioinformartics
in Asia Pacific region as introduced in Ref. 13. We especially studied an
information environment composed of high-performance computers, softwareand networks. For details, see Ref. 7.

2-3. Classification and identification of microbes
The systematics is the base of the study of biodiversity and requires
polyphasic analysis of data. It is at the stage of integration of conventionaltaxonomy based on phenotypic data and phylogenetic analysis based on molecular data. We designed an information system and also evaluated several
methods of phylogenetic analysis for a large scale data. For details, see Ref.
6 and 11.

154

6 and 11.

(3)

Homology modeling of the 3D structure of Immunoglobulin

Satoru MIYAZAKI and Hideaki SUGAWARA
The one of the important aims of bioinformatics is to elucidate the relationships of sequences, 3D structure and function of biological macromolecules.

We investigated the effects of somatic mutations in immunoglo-

bulin variable region genes on the affinity maturation of antibodies using
single precursor B cell-derived anti-DNA monoclonal antibodies generated
from an autoimmune desease-prone NZB x NZW Fl mouse.

Based on analy-

ses of DNA sequences, homology modeling on a graphic workstation and molecular dynamics simulation of antigen-binding sites, we proposed a theory
that the flexibility of antigen-binding Fv loops is associated with affinitymaturation ant-DNA antibodies.

For details, see Ref. 2.

Pub I i ca t ions
1.

2.

3.
4.
5.

6.

AATAGO, T., KIKUCHI, S., TATEYAMA, J., FUKITA, M., HARADA, H., YOKOKAWA, T SUGAWARA,
H. and MIYAZAKI, S.: An, experiment of research collaboration between Japan and
U.S.A using broadband research network, 40(5), 404-413, 199704-413, 1997.
MIYAZAKI, S., SHIMURA, J., HIROSE, S., SANOKAWA, R., TSURUI, H., WAKIYA, M., SUGAWARA,
H. and SHIRAI, T.: Is structural flexibility of antigen-binding loops involved in theaffinity maturation of anti-DNA antibodies? International Immunology, VoL 9, No.5,
771-777, 1997.
SUGAWARA, H. and MIYAZAKI, S.: Introduction to Biodiversity. Japan Society for Information and Knowledge Newsletter (in Japanese), No. 43, 3-12, 1997.
SUGAWARA, H.: Utilization of High-Performance Computers for Sequence Analysis,
Scientific System Meeting Newsletter (in Japanese), No. 86, 295-314, 1997.
GOTO, K., YAMAMOTO, H., TAMURA, T., OKAYAMA, T., KOIKE, T, MIYAZAKI, S., MORI, H.,
GOJOBORI, T and SUGAWARA, H.: Visualization of Sequence and Biological Data in DNA
Data Bank of Japan: Genome Information Broker and the Enhancement of SAKURA.
Genome Informatics 1997, Universal Academy Press(Tokyo), 236-237, 1997.
FUTATSUKI, T, KAwANISHI, Y, NAITO, K., MIYAZAKI, S. and SUGAWARA, H.: Efficient Computation of Sequence Analysis in a Vector-Parallel Computer for the Study of Molecular
Evolution. Genome Informatics 1997, Universal Academy Press (Tokyo), 264-265,
1997.

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7.

SUGAWARA, H. and MIYAZAKI, S.: Information environment for microbiology in the newage. The 5th JST International Symposium New Frontier in Microbiology, Tokyo,
January.
8. SUGAWARA, H., GOTO, K, TAMAMOTO, H., KOIKE, T., OKAYAMA, T., ISHII, J., MIZUNUMA, T.,
TAMURA, T., MIYAZAKI, S., OTA, M., FUKAMI-KoBAYASHI, K, IMANISHI, T., IKEO, T., SAITOH,
N., NISHIKAWA, K, TATENO, Y. and GOJOBORI, T.: DNA Data Bank of Japan (DDBJ) in
the age of mass submission of sequences, HGM'97, Toronto, March.
9. SUGAWARA, H.: Utilization of broad-band networks for bioinformatics, Lecture Series
on Network and Evolution of Molecular Information, Mishima, May.
10. TAMURA, T., OKAYAMA, T., GOTO, K, KOIKE, T., MIYAZAKI, S., GOJOBORI, T. and SUGAWARA,
H.: Genome Information Broker: The JAVA Applet for the Processing and Presentation of Genome Data and the CGI Program for the Retrieval of the Data from the
DDBJ Database, Objects in Bioinformatics, Hinxton Cambridge UK, June.
11. MIYAZAKI, S., FUTASUKI, T., KAWANISHI, Y. and SUGAWARA, H.: Phylogenetic Analysis of
Large Scale Datasets. V-th Annual Meeting of Japan Society for Culture Collections(Wako), June, 1997.
12. SATOU, K, MIYAZAKI, S. and OHYA, M.: Analysis of HIV by Entropy evolution rate. 5thInternational congress on amino acids, Chalkidiki, Greece, August.
13. SUGAWARA, H. and MIYAZAKI, S.: A proposal of Asia-Pacific Bioinformatics Network(APBioNet). The 8th CODATAIDSAO - New Perspectives on S&T Information Process14.

15.

ing, Taejon Korea, November.
SUGAWARA, H.: Genome Information Broker for Microbes: International Workshop on
Genome Project 1997 The Frontier of Genome Research on Microorganisms. ganisms,
Tokyo, December.
SUGAWARA, H.: Information on Biological Research Resources as an Infrastructure of
the study of Biodiversity. CBI meeting (Tokyo), December, 1997.

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J.
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RADIOISOTOPE CENTER

Feedback loops controlling the initiation of
sporulation in Bacillus subtills

Masaya FUJITA and Yoshito SADAIE
The early gene expression required for the development of spores in Bacillus
subtilis is regulated by the SpoOA transcription factor. SpoOA activity isdependent on phosphorylation. The immediate source for the phosphorylation of SpoOA is the phosphorelay signal transduction system, a more complicated variation oftwo-component signal transduction systems. The first stepis the activation of protein kinases, predominantly the products of the kinAand kinBgenes, leading to ATP-dependent autophosphorylation. A third kinase, the product of the kinG gene, is required under some growth
conditions. The phosphate group is then transferred to the SpoOF protein.
SpoOF-P in turn becomes a phosphodonor for SpoOB, a phosphoproteinphosphotransferase catalysing the transfer of phosphate to the SpoOA
protein. RNA polymerase containing aA(E a A) or aH(E a H) transcribes thegenes of the phosphorelay components. Phosphorylated SpoOA is also involved in their expression and is required for the induction of a H by repressing
its repressor gene abrB. We have examined the effects of phosphorylated
SpoOA (SpoOA-P) and AbrB on in vitro transcription of the genes involved in
the SpoOA phosphorylation and initiation of sporulation. SpoOA-P repressed
E a A-dependent transcription of the kinG and E a H-dependent transcriptionof spoOA and kinA. E aH-dependent transcription of spoOFwas stimulatedby SpoOA-P at low concentrations but was repressed by higher amounts of
SpoOA-P. On the other hand, AbrB repressed E a A-dependent transcriptionof spoOH( a Hgene), kinG and abrB, although its effect was not strong. The
results obtained support the notion of control circuits in which the SpoOA and
AbrB proteins activate or repress the initial sporulation pathway. (to be published in J.Biochem.)

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157

Promoter Selectivity of the BaclJJus subtHis
RNA Polymerase aJ. and a R Holoenzymes

Masaya FUJITA and Yoshito SADAIE
The a Hof Bacillus subtilis directs transcription of a large number of earlysporulation genes, whereas the principal a factor, a A , is essential for the transcription of the genes for vegetative growth and early sporulation. We havepurified a Aand a Hproteins, and characterized their properties. The genes
encoding aAor a Hwere separately cloned into an expression vector under thecontrol of T7 promoter. Both proteins were overproduced in Escherichia coli
BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride. Antigenicities and N-terminal amino acid sequences ofthe overproduced proteins were used to identify both proteins. Unlike a A
protein, a Hprotein showed a DNA-binding ability. To compare the promoter
selectivity of the aAprotein with that ofthe aHprotein, transcription in vitroof 16 promoters was performed using RNA polymerase holoenzymes reconstituted from a purified core enzyme with either a Hor aA. These holoenzymes
correctly recognized each of the cognate promoters; aH-RNA polymerase recognized aHpromoters but not aApromoters, and vice versa. A competition experiment for core RNA polymerase using a A and a Hrevealed that a A had astronger affinity. We propose that the predicted replacement of a a subunit
in a holoenzyme from a A to a Hin vivo at late logarithmic growth phase may
require an additional factor, or the modification of a core enzyme or a factor.
( to be published in J.Biochem.)(3)

Functional analysis of the phoB/ cotA region of
the BaclJJus subtills chromosome containing
the konjac glucomannan utilization operon

Yoshito SADAIE and Katsunori YATA
The phoB-rrnE-groESL-gutR-cotA region (70kb) of the Bacillus subtJ1is genome contains several operons of unknown or not well known function. They
include mannan utilization operon, glycoprotein endopeptidase operon, glucitol
utilization operon, and an operon for inherent restriction/modification system

158

genes. A large operon involved in degradation and incorpolation of mannanconsists of eight ORFs whose predicted products includs PTS system proteins
as well as mannan digesting enzyme. The expression of this operon was
induced by konjac glucomannan and repressed by glucose or mannose. Yeast
mannan did not induce the expression of the operon. Disruption of an ORF
having a RTR domain resulted in a high expression of the operon suggestingthat this ORF is an internal repressor gene of the operon. Bacillus subtilis
chromosome harbors an operon for utilizing glucomannan, a plant-derived
carbon source.

(4)

Restricted Transcription from Sigma B or Phosphorylated SpoOA
Dependent Promoters in the Temperature-sensitive secA34J
Mutant of BacJl/us sub/Jlis

Kei ASAI 1, Masaya FUJITA2 , Fujio KAWAMURA3 , Rideo TAKAHASHI4, Yasuo
KOBAYASHI 5 and Yoshito SADAIE 2 (1,3,4 Institute of Molecular and Cellular Biosciences, The University of Tokyo, 2 Radioisotope Center, National Institute of
Genetics, 5 Laboratory of Molecular Biology and Microbial Chemistry, Tokyo
University ofAgriculture and Technology, I Present address: Department of
Cell Biology, The graduate School of Biological Sciences, Nara Institute of
Science and Thechnology, 3 Present address: Laboratory of Molecular Biology,
College of Science, Rikkyo University.)
The temperature-sensitive secA341 mutation of Bacillus subtilis affects
sporulation and sporulation-associated events as well as protein secretionand cell septation. With lacZ or bgaBfusion genes, we examined the expression of the early sporulation genes in the mutant strain. Transcriptional
expression of oR dependent kinA, spoOA (ps), phrG, spo VG and citG (P2) genes
was blocked by the secA341 mutation at 37C. On the other hand, neither
repression of the abrBgene nor induction of spoOH( oR) gene was affected.
Active RNA polymerase containing oR was, however, found to be produced in
the mutant cell. Expression of the phosphorylated SpoOA dependent spoIIG
operon was also blocked. Thus the secA341 mutation blocks some step(s) or
factor(s) required for oR·dependent transcription in vivo. (To be published in
Biosci.Biotechol.Biochem.)

J.

RADIOISOTOPE CENTER

159(5) The secA3iJ Mutation Inhibits Expression of the
Bacillus subtllis Protease Gene, aprE by Blocking
DegS/DegU depending activation step
Yoshito SADAIE
The production of extracellular protease(s) of Bacillus subtilis is induced in
the post-exponential growth phase, and is severely reduced in a strain carrying
a temperature sensitive mutation (secA341ts) in the secA gene, the product ofwhich is required for protein secretion. The expression of the extracellularserine protease gene, aprE, as monitored by the l3-galactosidase activity of anaprE-lacZtranslational fusion, was inhibited in the secA341 mutant cell andrestored by the introduction of the degU32(Hy) mutation which makes thephosphorylated DegU response regulator of a two-component system refractory to dephosphorylation and renders the cell to become a strong producer of
AprE. The secA341 mutation appears to block some activation step required
for aprEexpression. In fact, in the secA341 mutant at 37C, the expression of
the degRgene, whose product is a stabilizer of phosphorylated DegU, is completely repressed. This repression was reliefed by the deletion mutation in
the anti-sigma D gene, flgM As degR gene is transcribed by RNA polymerasecontaining sigma D, repressed expression of the degRin the secA mutant cellis due to the undiluted anti-sigma D and results in destabilization of DegU-P
leading to repressed expression of aprE. (To be published in
Biosci.Biotechol.Biochem.)

160

Publication
1.

AsAI, K, KAWAMURA, F., SADAIE, Y. and TAKAHASHI, H.:

Isolation and characterizationof a sporulation initiation mutation in the Bacillus subtiJis secA gene. J.Bacteriol. 179,
544-547.
2.

SADAIE, Y., YATA, K, FUJITA, M., SAGAI, H., ITAYA, M., KASAHARA, Y. and OGASAWARA,

Nucleotide sequence and analysis of the phoB-rrnEgroESL region of the Bacillus
subtiJis chromosome. Microbiol. 143,1861-1866.
3. KAsAIIARA, Y, NAKAI, S., OGASAWARA, N., YATA, K and SADAlE, Y.: Sequence analysis of
the groESL-cotA region of the Bacillus subtiJis genome, containing the restrictionlN.:

modification system genes. DNA Res. 4,335-339.
4.

KUNST, F.:et al. The complete genome sequence of the Gram-positive bacterium

Bacillus subtilis. Nature. 390:249-256.

161

K.
(1)

EXPERIMENTAL FARM

Development, Evaluation and Distribution of the Genetic Stocks of
Rice

Ken-ichi NONOMURA, Toshie MIYABAYASHI, Mitsugu EIGUCHI and Nori KURATA
We have conducted the reproduction, evaluation and distribution of genetic stocks of wild and cultivated rice. Especially, plenty ofthe collection ofwild species is useful for practical breeding and evolutional studies. It contains Oryza sativa complex, which is classfied into AA genome species, O.
oDicinalis complex ofBB, BBCC, CC, CCDD, EE and FF genome species, andso on. These are also interesting sources for basic science, because rice has
the smallest genome size in all cereal crops, corn, barley, wheat, rye, millet
and sorghum. These stocks have been distributed as the requests for basic
researches. Information about these stocks were collected and made up in therice genetic stock database (by Drs. Morishima, Kinoshita and Yamazaki) andis accessible at the site ofhttp://www.shigen.nig.ac.jp/NIG_rice/rice.html.
Another trial to develop genetically tagged strains by enhancer trapping are
now in progress in cooperation with the plant genetics laboratory. Care ofseeding, crossing, screening and distribution ofthese newly developing genetic
materials will be taken by the experimental farm in a few years.

Publication
None

162

ABSTRACTS OF DIARY FOR 1997
Biological Symposium
488th-Jan. 29
Anaerobic and Nirate Control of Respitory Pathway Gene
Expression in Escherichia coli (Robert P.Gunsalus)
Correct kinetic measurement by surface plasmon
489th-Feb. 17
resonance:principle and limitation of Biacore (Peter
Schuck)490th-Feb. 21"Dissecting Hydra Development Using Molecular Biology"(Robert E.Steele)
Molecular Changes in the Eye Lens during Aging and in
491st-Feb. 24
Cataract (D.Balasbramanian)492nd-Mar. 18"Organization and function of MHC class Ib genes"(Kirsten Fischer Lindahl)"Molecular mechanisms of axon guidance and cell migra493rd-Mar. 24
tions inan invertebrate spinal cord- the nematodeC.elegans" (Joseph G.Culotti)
494th-Apr. 4"Drosophila Wing Morphogenesis" (Antonio GarciaBellido)"CellJExtracellular Matrix Interactions in Hydra vulgaris"
495th-Apr. 9
Genetic Control of Morphogenesis and Evolution of the496th-Apr. 10
Eye (Walter J.Gehring)
497th-Mar.21
The structure and function of bovine mitochondrial F 1ATPase;An example of rotational catalysis (Andrew
G.W.Leslie)
498th-June 2
Role of General and Gene-Specific Co-activators in Transcription Regulation (Robert G.Regulation)
499th-June 27"Molecular Insight into Origin and Composition of Microbial Communities" (James M.Tiedje)
500th-July 9
The Role of Apoptosis in the Pathogenesis of Sacral
Agenesis (Alisa S W Shum)
501st-June 22
Chlorophyll and Bacteriochlorophyll:Theory and Experi-

ABSTRACTS OF DIARY FOR 1997

502nd-Aug. 11503rd-Sep. 12504th-Oct.8
505th-Oct. 15

506th-Nov. 4
507th-Nov. 5508th-Nov. 12509th-Nov. 19
51Oth-Nov. 21
511st-Nov. 27
512nd-Nov. 27

513rd-Dec.3
514th-Dec.8
515th-Dec. 9516th-Dec. 10
517th-Dec.15

163

ment in Unison (Ian Gould)"Mammalian Molecular Phylogeny:Everything you know
is wrong" (Dan Graur)
Coupling of Local DNA Topolpgy to tyrR Promoter
Activity (Andrew Travers)
Molecular mechanisms involved in specific recongnitionof pathogens in plants (Fumiaki Katagiri)
Molecular mechanisms of synaptic initiation:how
postsynaptic cells talk back to presynaptic cells (Akira
Chiba)"Maternal Factors That Guide Early Development Of The
Germline In C.elegans." (Susan Strome, Ichiro Kawasaki)"Translation termination in higher eukaryotes:news andviews" (Lev Kisselev)"Germ Line Establishiment in Drosophila melanogaster"(paul Lasko)
Beyond Genomes:Emerging Role of Mass Spectrometry inProteome Studies (Hiroyuki Matsumoto)"Role of Bone Morphogenetic Proteins (BMPs) in Forebrain and Eye Development" (Yasuhide Furuta)
Neurohormone Cascades:A Mechanism for Controlling
Behavioral Sequences in Insects (James W.Truman)
Orchestration of Insect Molting and Metamorphosis:
Hormonal Regulation and Molecular Switches (Lynn M.
Riddiford)"The Myosin VIIA Deafness Gene-Genetic and Functional
Analysis in Mouse and Human" (Steve D.M.Brown)"Electrostatic Properties of Biological Macromolecules"(Barry Honig)"Structure-based design" (Tom Blundell)"Biological Database Searching and Statistical
Significance" (Michael S.Waterman)
Transcriptional Behavior of Neutrophil White BloodCells(Sherman M.Weissman)

164

Mishima Geneticists' Club
50 1st-Jan. 8Characterization ofthe ADA transcriptional co-activator
complex (Junjiro Horiuchi)
502nd-Jan. 16
New methods for molecular manipulation of DNA (Masao
Washizu)
503rd-Feb. 12
Genetic variability and Cell Tropism of HIV (Nobuaki
Shimizu)
505th-Apr. 16
Characterization of RuvB like mammalian TIP49 protein
whichi has a DNA dependent ATPase/helicase activity(Takaaki Tamura)
506th-May 14
Cascades of transeviptional control factors producing
variabilities of mammalian neuroral cells (Tetsuichiro
Saito)
507th-May 21
Molecular Evolution of Rotaviruses -Toward New Paradigms- (Osamu Nakagome)
508th-June 6
Small teleost species as valuable genetic resource (Hiroshi
Hori)
509th·June 10
Bioinformatics: analyses of Escherichia coli and
Sccharomyces cerevisiae complete genomes (Katsumi
Isono)
510th-June 11
DNA replication machinery and mechanism of Sphasecheckpoint (Hiroyuki Araki)
511st-June 12
Cellular function of AAA protease FtsH in Escherichia
coli (Hikaru Ogura)512nd-June 13 Coupling of gene expression and morphogenesis-Bacterial flagella serve as a transcriptional regulator (KazuhikoKutsukake)
513rd-June 17
Transcriptional regulation of the yeast rRNA gene-Molecular genetic approach (Yasuhiro Nogi)
514th-June 18
Molecular genetics of clock genes in cyanobacteria(Masahiro Ishimura)
515th-June 23
Role of MATI in phosphorylation of Oct-1 transcription
factor by CDK activating kinase (Susumu Inamoto)516th-July 4
Centromere-specific nucleosome structure in human

ABSTRACTS OF DIARY FOR 1997

517th-Aug. 18
518th-Oct.27
519th-Nov. 13

520th-Nov. 19

521st-Nov. 13
522nd-Nov. 27
523rd-Dec. 22524th-Dec. 22

165(Kinya Yota)
Interspecific chromosome transfer as a useful too for the
study of speciation in Drosophila (Kyoichi Sawamura)
Role of SUMO/UBC9 modifier system in nuclear transport and cell cycle control (Sumihito Saito)
Cloning of the genes essential for morphogenesis of
Zebrafish using the retro virus-mediated insertionalmutagenesis (Koichi Kawakami)
Promoter-transcription activation protein interaction invivo: Roles of cooperative DNA binding and activationdomain function (Masashi Tanaka)Hepatitis virus and liver cancer
Postembryonic Development of the Drosophila Brain
Circuit (Kei Ito)
Induction gene-trapping: Identification of genes active
down stream of BDNF (Mika Karasawa)
Glial cells missing, gene for the regulation of differenciation
between neuron and glia in Drosophila (Toshihiko Hosoya)

166

FOREIGN VISITORS IN 1997
Jan. 26-Feb. 13
Feb. 14-22
Jan. 27-29
Jan. 28-29
Feb. 12-Mar. 7
Feb. 12-Mar. 31
Feb. 23-25
Mar. 10-Apr. 27
Mar. 18-19
Mar. 19-June. 1
Mar. 24
Mar. 30-Apr. 11
Apr. 1Apr. 4-5
Apr. 7
Apr. 7
Apr. 10-11
Apr. 14-15

J.Gowrishankar,Center for Cellular Molecular Biology,
India
Robert Steele, University of California Irvine,U.S.A
Robert P.Gunsalus,University of California,LosAngeles,U.S.A
AM.Chakrabarty,University of Illinois, Chicago, Chicago
Illinois,U.S.A.
Dipankar Chatterji,Centre for cellular and Molecular
Biology,India
Jeffrey T.Owens,University of California, Davis, U.S.A.
D.Balasubramanian,Centre for Cellular and Molecular
Biology,India
Talat Malik,University of Nottingham, Nottingham,
U.S.A.
Kirsten Fischer Lindahl,Howard Hughes Medical Institute U.T.Southwestern Medicine Center,U.S.A
Masatoshi Nei,Pennsylvania State University,U.S.A
Joseph G.Culotti,Samuel Lunenfeld Research Institute
Mt.Sinai Hospital,Canada
Michael P.Sarras Jr, University of Kansas Medical
Center,U.S.A
Zhangyi Qu, Harbin Medical University, China
Antonio Gercia-Bellido, Universidad Autonoma de
Madrid,Spain
Gerald Selzer, National Science Foundation,Arlington,
U.S.A.
Machi F Dilworth,National Science Foundation,Arlington,
Virginia,U.S.A.
Walter J.Gehring,University of Basel,Switzerland
Delill S.Nasser,National Science Foundation Program,

FOREIGN VISITORS IN 1997

Apr. 24-May. 21
May 18-June. 13
May 20-21
May 22-June. 15
June 18-Aug. 14
June 21-25
July 9-10
July 28-Aug. 3
July 28-Sep. 7
Aug. 4-26
Aug. 18-20
Sep. 9-Dec. 7
Sep. 9-0ct. 3
Sep. 24-0ct. 7
Oct. 4-Nov. 3
Oct. 14-15
Oct. 27
Nov. 4-5
Nov. 5-6
Nov. 12-14
Nov. 19-20
Nov. 21-22

167

U.S.A.
Goonnapa Fucharoen,Khon Kaen University,Thailand
J.Bown,University of Birmingham,School of Biochemistry,UK
Andrew G.W.Leslie,MRC Laboratory of Biology,UK
Kan Ohno,Beckman Research Institute of the City ofHope,U.S.A.
Dan Graur,TelAviv University,Israel
Matthew Bellgard,Murdoch University,Australia
Alisa S.W.Shum,The Chinese University of Hong Kong,
Hong Kong
Dipankar Chatterji,Center of Cellular and Molecular
Biology,India
Kakoli Mukherjee,Center of Cellular and Molecular
Biology,India
Giorgio Bernardi,Laboratoire de Genetique Molecularie
Institut Jacques Monod, France
Alberto Bernardi,Laboratoire de Genetique Moleculaire
Institut Jacques Monod, France
Taciana Kostionkovitch,Edinburg University,UK
Richard Hayward,Edinburg University, UKJ uncai Ma, Chinese Academy of Sciences,The Institute ofMicrobiology, China
Noboru Sueoka,University of Colorado at Boulder, U.S.A.
Akira Chiba,University of Illinois,U.S.A.
Holling Worth,R., University of Wisconsin Madison,
Madison,Wisconsin,U B.A.
Susan Strome,Indiana University,U.S.A.
Lev Kisselev,Laboratory of Molecular Bases of Oncogenesis Engelhardt Institute of Molecular Biology, Moscow
Paul Lasko,Mcgill University,Canada
Hiroyuki Matsumoto,University of Oklahoma Health
Sciences Center, Oklahoma,U.S.A.
Yasuhide Furuta,Vanderbilt University School of

168

Nov. 25-28
Nov. 25-28
Dec. 3-4
Dec. 8Dec. 8-9
Dec. 10-11
Dec. 15

Medicine,Nashville,TN,U.S.A.
Lynn M.Riddiford,University of Washington, Seattle, WA,
U.S.A.
James W.Truman,University of Washington, Seattle, WA,
U.S.A.
Steve D.M.Brown,MRC Mouse Genome Centre, Harwell,
OX,ORD,UK
Barry Honig,Columbia University,U.S.A.
Tom Blundell,University of Cambridge,U.K
Michael Waterman, University of Southern California,
U.S.A.
Sherman M.Weissman,Yale University school of medicine,
CT,U.S.A.

ASSOCIATION FOR PROPAGATION OF THE
KNOWLEDGE OF GENETICS
President
MORIWAKl, Kazuo; Vice-President, The Graduate University for Advanced
StudiesManaging Director
GOJOBORI, Takashi; Professor, National Institute of Genetics
NAKATSUJI, Norio; Professor, National Institute of Genetics
Manager
TOMIZAWA Jun-ichi; Professor Emeritus, 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
ISHIHAMA, Akira; Professor, National Institute of Genetics
SHIGETO, Gakuji; Managing Director, Inoue Foundation for ScienceASSOC. FOR PROPAG. OF THE KNOWL. OF GENET.

169

TrusteeTAZlMA, Yataro; Director Emeritus, Institute of Silkworm Genetics andBreeding
OSHIMA, Chozo; Honorary member, National Institute of Genetics
SAITO, Hiuga; Director and Professor, Institute of Medical 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, National Institute of Genetics
MORISHlMA, Hiroko; Professor, National Institute of Genetics
KATSURA, Isao; Professor, National Institute of Genetics
Adviser
KONDO, Norio; Director-General, The Research Institute of Evolutionary
Biology
MORIWAKI, Daigoro; Professor emeritus, Tokyo Metropolitan University

170

AUTHOR INDEX
ABE. J
AlaBA. T.

AKIMoTO. M
ALEXEEVE. A.
ANDACHI. Y.
ANnO. A.
AOKI. T
ARAKi. K.
ARAKI. M
ARAMAKi. H
ARIGA, H

AsAi. K.
AYALA, J
BELYAEVA, T. A.
BLAGITKO, N
BoNG. Y.-S

BONNER. M.R.
BOUHSS, A.
BOWN, JBUSBY. S. J. W.

CAl, H.W.
CHATTERJI. D
DANJO, 1.
EDA, Y.
EIGUCHI, M
ENDO. TFIGUEROA, F
FuJiSAWA, T.
FuJITA. M
FuJITA, N
FuJIWARA. M
FUJIYAMA, A.
FuKUDA, T
FUKUSHI. H
FUKUSHI, K.

GAl. J

84
131
81
36
136
67.69.70
115
41
41
121
68
158
39
10
78
76
79
39
14
10,14
80.82
13
76
142
101,102,104,161
143,15178
48
156,157.158
10,11,12,13,14.17
126,128
76
95
84
132
84

GAMOU, S

110
84
GoJOBORI,T.
138,139.140,141.
142.143,144.151,152
GOTO, K.
152
GoTO. M
51
GoTO. S108,110
GOTO, Y.
77
GOWRISHANKAR, J
16
HACHIMORI. T.
142
HAKOHEN, N
46
HAMMER, M.F
79
HANDA, H
51,60
HARA. H
38,39
HARUSHlMA, Y.
103
HATTA. M
48
HAYASHI, S
108,109.110, III
HAYASHIDANI. M
77
HElJENOORT. J.Y
39
HERNANDEZ, YJ
118
HIBI, T
26
HILL. R.J
135
HIRAOKA, Y
42,43
HIRATA, T
84
HIROMI, Y.
45,46.47
HIROSE. S
50,51,52
HIROSHIMA, M
115
HISHIDA, R.
124
HONDA. A.
22,24.25,26
HONDA, M
142
HORAI, S
77.78.79
ICHIKAWA, T
114
IDE. H140
IKEMURA, T
61.66,67.68,69.70
IKEO. K.
138.140.151
IKEYA. T...................................................... 109
GAO, ZAUTHOR INDEX171

IKUTA, T

68

KLEIN, J

78

IMAIZUMI, T

41

KOBAYASHI, Y.

158

143

KOHARA, Y.

IMANISHI, T
INOKO, H

67,69,70,143

KOIDE, T.

134,135,136
87,92,94

ISHIGURO, A.

20,21,22

KOIZUMI, O

ISHIHAMA, A.

10,11,12,13,14,16,17,18,19,

KOMOTO, M

20,21,22,23,24,25,26,133

KOSE, H

ISHIHARA,T

123,124,125,126,127,128

KRAMER, S

46

ISHIJIMA, J

90

KRAsNOW, M

46

48
24
45,47

ISOBE, T

87

KUBORI, T.

118

ISOGAI, Y

148

KUBOTA, K

108

ITO, K107

KUDO, Y.

ITO, M

149

KURATA, N

ITO, Y.

101,104

KURIHARA, T

141

ITOH, T

138

KUROSAWA, O

121

61
101,102,103,104,161

IWANE, A.

115,116

KUSANO, S

16

IWATA, A.

17,20,26

LAu, J.Y.N

138

JENKINS, T

79

LI, F.-Q

JISHAGE, M.

17

LINDAHAL, KF

87

JUJI, T

67

MAKINO, S

89

KABATA, H

121

MALAsPINA, P

79

KAGEYAMA, Y.

52

MASUDA, S

52

KANAYA, S

50,51

61,66

MASUYA, H

140

KANAZAWA, A.

84

MASUYA, H

88,89

KARAFET, T

79

MASUYA, M

95

KATAYAMA, A

13

MATAKATSU, H

110

KATSURA, 1.

123,124,125,126,127,128

KAWAHARA, T

MATSUMOTO, K

68

113

MATSUO, Y.

149

KAWAI, M

151

MATSUOKA, N

71

KAWAMURA, F

158

MATSUZAWA, H

107

KAWANISHI, Y.

151

MEARES, C. F

10,12,14

KiKKAWA, Y

92

MELLO, C

KiMURA, M

20,21,22,23

KiMURA, M

7

°

39

MIKAMI, T

84

MINATO, K

58

KiNEBUCHI, T

121,122KiNOSHITA, T

113

MINCHIN, S.D

KiSHI, T.

31

MITA, A.

KiTAMURA, K
KiTANO, T.

115,116
71

135

MENGIN-LECREULX, D

1490,92,95

MITANI, Y.

134

MITCHELL, R.J

79

172

MITOBE. J
MITSUZAWA. H
MIURA. K.
MIYABAYASHI. T

MiYAKE. R

24
22.24
142
161

OGAWA. T
OHARA. M

35.36
84

OHBA. K.
OHBA. T

138
134

OHNO. M
OHTA. T
OHTA. T

66
36
74

MIzOKAMI. M
MIzUIU. N
MIZUMOTO, K.

12
22
134
153.154
138
70
24.32

MIZUNO. K.
MOM!. A.
MORi. HMORISHIMA. H
MORISHIMA. K.

87
123
138.15280.81.82
113

MORIWAKI. K.
MOTOHASHI. T.
MUNEOKA. Y

87.93.94
134
48

MURAKAMI, A.
MURAKAMI. K.
MURAKI. K.

53.54.57
10,11.12,14.133
77

NAGAI. H
NAGAOKA, T
NAITOU, K.
NAKASHIMA. H

118.119.122
134
151
150

PARK. H.-S

NAKATSUJI. N
NARITA. S

97,99.100
38

NISHIKAWA. K.
NISHIMURA. A.
NISHIMURA. Y.
NISHINO. I.
NOMURA. T
NOMURA. T.

148.149,150
106.107
38
77
13
93

REN, FSADAIE, Y.
SAGAI, T.
SAITO, K.
SAITO, M
SAITO, T.
SAITOU. N
SAKUMA, M

NONAKA. I.
NONOMURA. K.
NOVELLETTO. A.
NUMAKUNAI. T.
OBARA. M

77
103,161
79
140
134

MIYAO. T
MIYATA. A.

MiYAZAKI. S

O'HUIGIN. C
OIKE. Y.
OKABE. M

7841.45.46

OKAMOTO. T
OKAMOTO, Y.
OKAYAMA, T.
OKUYAMA. K.
OMOTO. K.

25
142
141.152
41
72

ONAMI. S
OOTA, S

136
73

ORiTO. E.
OTA. MOWENS, J. T.
OZAWA, M

138
148
10.12.14
77

OZOLINE, O.N

10,11
76
39

PARQUET, C
PRIESS. J.R.

QU, Z

SAKURA, N
SAKURAI, H
SANO, MSARRAS. M. P
SASAKI, H

135
22
141
156.157,158.159
88
115
113,11497.99
71,72.73
141

77
19
134
49
85

AUTHOR INDEXSASAKI, H
SATO, K

92
113
SATO, S
140
SATOH, N
140
SEINO, H
30
SEN, R.
118
SHIMAMOTO, N
118,119,121,122
SHIMAMOTO, Y.
81,84
SHIMIZU, H
48,49
SHIMIZU, K.
92
SHIMIZU, M
122
SHINDO, H
122
SHIN-I, T
134
SHIOSAKI, K.
142
SHIRAKIHARA, Y.
129,131,132,133
SHIRAYOSHI, Y.
97,100SHIROISHI,T
87,88,89,90,92,93,94,95, 114
SOGAWA, N
68
SONODA, S
78
SPURDLE, AB
79
SUGAWARA, H
152,153,154
SUGAYA, K.
67
SUGIURA, I.
134
SUGIYAMA, T.
48
SUZUKI, N
125
SUZUKI, Y.
151
TABARA,H
135
TADOKORO, R.
110
TAJIMA, K.
78
TAKAGI, T
60
TAKAHASHI, H
158
TAKAHASHI, K.
151
TAKAHASHI, T
48
TAKANO, T.S
64,65
TAKEMARU, K.
50,51
TAKEUCHI, H
110
TAKEUCHI, M
121
TAKEUCHI, M
123
TAKEUCHI, S
7'7

TAKEUCHI, T

TAKEZAKI, N
TALKUDER, A A
TAMURA, K.
TAMURA, T
TANAKA, H
TANAKA-MATAKATSU, M
TANIGUCHI, M
TATENO, Y.
TENZEN, T
TOKIYOSHI, S
TOKUNAGA, M
TOZUKA, A
TSUCHIYA, R.
TSUGANE, M
TsUJIMOTO, H
TSUNOYAMA, K.
UCHIDA, K.
UEDA, H
UEDA, K.
UEDA, SUESUGI, H

URAl, H
WADA, T.
WAKANA, S
WASHIZU, M
WATANABE, H
WATANABE, H
WATANABE, T.
WATANABE, Y.
WATKINS, D
WEST, SWLASSOFF, W. A
WOOD, E.T.

Xu, D
YAGI, Y.
YAMADA, M
YAMADA, T
YAMAGATA, T

173

140
146
18
140
152
141
109
110
151,152
66,67
142
115,116
84
113,11489
113
144,151
90,92,95
50,51,52
77
17,20,26
134
107
60
88,92,93
121
134
138,142,151
26
67
78
47
23
79
84
111
107
45
66,69

174

YAMAGUCHI, Y.

139,142,151

YAMAGUCHI, Y.

60

YAMAMOTO, F

71

YAMAMOTO, H

140,152

YAMAMOTO, Y

38

YAMAMURA, K
YAMAO, F

4130,31

YAMAZAKI, S

142

YAMAZAKI, YYANAGIDA, T
YASUDA, SYASUI, K.
YATA, K.
YONEKAWA, H

112,113,114

YOSHINO, M

115,116114
20,22,24
157
92
87

YUM, S

48

ZEGURA, S.L79

Annual Report of the National Institute of Genetics. N0.48
Printed on November 5th 1998
Issued on November 12th 1998
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 Ken Nishikawa & Yasuo Shirakihara
Published by National Institute of Genetics
Printed by Hobundo Insatsu Co., Ltd.

to Hakone/'

Official
ResidencesNt
1:25,000