ISSN 0077-4995

NA TIONAl INSTITUTE OF GENETICSJAPANANNUAL REPORT1993

No. 44

Published by

THE NATIONAL INSTITUTE OF GENETICS
Mishima, Sizuoka-ken, Japan
1994

Annual Reportof theNational Institute of Genetics
No. 44, 1993

Published by

The National Institute of Genetics, Japan
1994

CONTENTS
General statement
Staff. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Council and advisory committee
Association for propagation of the knowledge of genetics
Projects of research for 1993. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Research activities in 1993. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.

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3
7
9
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16

Molecular Genetics
Molecular anatomy of Escherichia coli RNA polymerase: Functional map of the amino-terminal assembly domain of alpha subunit. KIMURA, M., FUJITA, N. and ISHIHAMA, A. . . . . . . . . 16
Molecular anatomy of Escherichia coli RNA polymerase: Functional map of the carboxy-terminal contact domain with class-I
factors. NEGISHI, T., FUJITA, N., KYOGOKU, Y. and ISHIHAMA, A............................................. 17
Molecular anatomy of Escherichia coli RNA polymerase: Mappingof cAMP receptor protein contact site on one alpha subunit.
Zou, C., FUJITA, N. and ISHIHAMA, A. . . . . . . . . . . . . . . . . . . 18
Molecular anatomy of Escherichia coli RNA polymerase: Interaction between promoter - 35 and - 10 recognition domains ofsigma-70. KUMAR, A., HAYWARD, RICHARD, S. and ISHIHAMA, A............................................. 19
Promoter selectivity control of Escherichia coli RNA polymerase:
Competition between different sigma subunits. KUSANO, S.,
JISHAGE, M., FUJITA, N. and ISHIHAMA, A. .
19
Promoter selectivity control of Escherichia coli RNA polymerase:
Differential recognition of osmoregulated promoters between
EoD and &T. DING, Q., KUSANO, S., FUJITA, N. and ISHIHAMA, A
20
Structure and function of yeast RNA polymerase II: Attempts forin vitro reconstitution. AzUMA, Y., KIMURA, M., KAwAGISHI, M., YAMAGISHI, M. and ISHIHAMA, A.
21
Molecular dissection of influenza virus RNA polymerase: RNAiisynthesis by PBl subunit. KOBAYASHI, M., TOYODA, T.,
NAKADA, S., MIZUMOTO, K. and ISHIHAMA, A.
Molecular dissection of influenza virus RNA polymerase: Photoaffinity labeling of PBl with 8-azido GTP. ASANO, Y, MARUYAMA, T., MIZUMOTO, K. and ISHIHAMA, A.
Molecular dissection of influenza virus nucleoprotein (NP): Identification of RNA-binding domain. KOBAYASHI, M., ToyoDA, T., ADYSHEV, D. and ISHIHAMA, A.
Molecular dissection of influenza virus M proteins: i~entification of
growth control activity for M1. YASUDA, J. and ISHIHAMA,A
Host factors for influenza virus growth control: Cellular factor(s)for influenza virus RNA replication. TOYODA, T. and ISHIHAMA, A
Host factors for influenza virus growth control: Antiviral activity ofmouse Mxl protein. TOYODA, T., ASANO, Y. and ISHIHAMA,A
Abnormal integrity of nucleolus associated with cell cycle arrest ina mouse cell mutant of temperature-sensitive ubiquitinactivating enzyme E1. SUDHA, T., KANEDA, S., NAGAI, Y.,
YAMAO, F., TSUJI, H., SAMESHIMA, M., MATsuDA, Y. and
SENO, T.
Cell cycle control by ubiquitin system in mammalian cells; theubiquitin-activating enzyme, El, is targeted by cdc2 kinase.
NAGAI, Y., KANEDA, S., SENO, T. and YAMAO, F.
Cell cycle control by ubiquitin system in mammalian cells; Ubiquitin pathway specific for DNA synthesis. KANEDA, S.,
NAGAI, Y, SENO, T., YAGURA, T. and YAMAO, F. . . . . . . ..
Cell cycle control by ubiquitin system in yeast Saccharomyces
cerevisiae. KISHI, T., YAMAO, F. and SENO, T.
Mechanism of mRNA capping reaction. MIZUMOTO, K.
Transcription of Sendai virus (HVJ) genome. MIZUMOTO, K. ..
Wx gene expression is enhanced in response to cool tempaeratures.
HIRANO, H.-Y., KUNI-EDA, M. and SANO, Y
Kinetic study on transcription using immobilized operons: Early
events in transcription elongation. KUBORI, T. and SHIMAMOTO, N.

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CONTENTS
Single-molecule dynamics of transcription using immobilized operons: Sliding of P. putida CamR protein on DNA. KABATA,
H. and SHIMAMOTO, N.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Functional and structural study of E. coli single-stranded DNA
binding protein. NAGAI, H. and SHIMAMOTO, N.
Diploidy of Drosophila imaginal cells is maintained by a transcriptional repressor encoded by escargot. FUSE, N., HIROSE,
S. and HAYASHI, S. ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Control of esg expression during development. YAGI, Y., HIROSE,
S. and HAYASHI, S.
. . . . . . . . . . . . . . . . . . . . . . . . . . ..
Analysis of the role of the tip cells in tracheal morphogenesis.
SHIGA, Y. and HAYASHI, S.
...
. . . . ..
The function of argos in regulating cell fate decisions during
Drosophila eye and wing vein development. SAWAMOTO, K.,
OKANO, H., KOBAYAKAWA, Y., HAYASHI, S., MIKOSHIBA, K.
and T ANIMURA, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
II.

iii

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35

36
37
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Microbial Genetics
The nicking reaction in rolling circle replication requires a singlestranded nicking region and a double-stranded binding region.
HIGASHITANI, A. and HORIUCHI, K.
SOS induction in Escherichia coli by single-stranded DNA of filamentous phages: Monitoring by cleavage of LexA repressor.
HIGASHITANI, N., HIGASHITANI, A. and HORIUCHI, K. ....
Stabilization of DnaA protein by chaperone proteins. YASUDA, S.

40

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42

A mutation that suppresses thermosensitive growth defect at low
osmolarity of the Escherichia coli !:!.prc mutation is in the genecoding for penicillin-binding protein 7. HARA, H., ABE, N.,NISHIMURA, Y. and HORIUCHI, K.
A general and fast method for mapping mutations on the Escherichia coli chromosome. HIGASHITANI, A., HIGASHITANI, N.,
YASUDA, S. and HORIUCHI, K. .
.
Molecular mechanism in determination of timing of cell division in
Escherichia coli K12. NISHIMURA, A. .
.
Correlation between cell division and the nature of cell membrane.
KANAMARU, K., TAKAGI, H. and NISHIMURA, A
.

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jv

III.

Mammalian Genetics

Establishment of new strains as a tool for analyzing tumor susceptibility genes in mice. MIYASHITA, N., FUJII M., MITA, A.,.
SHIROISHI, T. and MORIWAKI, K
Genetical study on the skeleton mouse mutant, tail short (Ts).
SHIROISHI, T., MITA, A., WAKANA, S. and MORIWAKI, K.

49

50
Genetic analysis of a new mouse mutant Rim4 affecting limb
pattern formation. MASUYA, H., SAGAI, T., MORIWAKI, K.
and SHIROISHI, T. .
.
Gene mapping toward positional cloning of the Rim3 gene.
MASUYA, H., WAKANA, S., SAGAI, T., MORIWAKI, K. and
SHIROISHI, T. .
.
Germ-line transmission of ES cell lines established from mousestrains enhancing meiotic recombination at the Lmp2 hotspot
in the MHC. SAGAI, T., NAKATSUJI, N., SHIROISHI, T. and
MORIWAKI, K. .
.
Genetical analysis of rim2 coat color mutant. SAGAI, T., WAKANA, S., MORIWAKI, K. and SHIROISHI, T.
Mutagenesis with intra-MHC recombinant strain. YOSHINO, M.,
SAGAI, T., MORIWAKI, K. and SHIROISHI, T. .
.
Chromatin structure of the recombinational hotspot in the mouse
MHC. MIZUNO, K.-I., SAGAI, T., MORIWAKI, K. and SHIROISHI, T. .
.
Polymorphism of the Lmp2 gene encoding a subunit of the mouseproteasome. MIZUNO, K.-I., SAGAI, T., SHIROISHI, T. and
MORIWAKI, K. .
.
Genetic diversity and geographic distribution of Asiatic Mus musculus subspecies based on the polymorphism of mitochondrial
DNA with special reference to the origins of Japanese mouse'M. m. molossinus'. YONEKAWA, H., TAKAHAMA, S., MIYASHITA, N. and MORIWAKI, K.
Recombination in the class III region of the mouse MHC. YoSHINO, M., SAGAI, T., LINDAHL, K. F., TOYODA, Y., SHIRAYOSHI, Y., MATSUMOTO, K.-I., SUGAYA, K., IKEMURA, T.,.
MORIWAKI, K. and SHIROISHI, T

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CONTENTSv

Linkage analysis of the js(Jackson shaker) locus on the mouse
chromosome 11. WAKANA, S., YONEKAWA, H., KANEDA,
H., OKAMOTO, M., SHIROISHI, T. and MORIWAKI, K.
The relationship between recombinational frequency and sequencehomology at the hotpot in the mouse MHC. YOSHINO, M.,
SAGAI, T., MORIWAKI, K. and SHIROISHI, T.
M-phase specific phosphorylation of mouse p84 weel kinase. YASUDA, H ...
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IV. Developmental and Somatic Cell Genetics
An exhausitve screening of peptide signal molecules affecting gene
expression in hydra. SUGIYAMA, T., FUJISAWA, To, HATTA,
M., SHIMIZU, H., KOIZUMI, 0., MUNEOKA, Y., TAKAHASHI,
T., DEHARO, M., BOSCH, T. C. and DAVID, C. N....
Screening of cell adhesion molecules in hydra. KISHIMOTO, Yo,
HATTA, M., HOBMAYER, E. and SUGIYAMA, T
Vnderwinding of DNA on binding of yeast TBP to the TATA
element. TABUCHI, H., HANDA, H. and HIROSE, S.
DNA sequence requirement of a TATA element-binding proteinfrom Arabidopsis for transcrption in vitro. MUKUMOTO, F.,
HIROSE, S., IMASEKI, H. and YAMAZAKI, K.-I. .....
BmFTZ-FI activates in vitro tanscription ofthejushi tarazu gene bybinding to the FTZ-Fl recognition site. LI, F-Q., VEDA, H.
and HIROSE, S.
o.
Molecular cloning of a cDNA for BmFTZ-FI, a silkworm counterpart of Drosophila FTZ-F1. SUN, G.-c., HIROSE, S. and·UEDA, H ...
Genetic studies on the duration of pupallifespans in Bombyx mori.
MURAKAMI, A. .
Ecogenetic studies on the duration of pupal lifespans in bombycidaeo MURAKAMI, A. and NINAKI, O. o.
Embryonic process of holometabolic insects. MINATO, K.o ....
Isolation of cDNA encoding the largest subunit of TFIIA reveals
functions important for activated transcription. MA, D.,
WATANABE, H., MERMELSTEIN, F., ADMON, A., OGURI, Ko,
SUN, X., WADA, To, IMAI, T., SHIROYA, T., REINBERG, D.
and HANDA, Ho
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vi

cDNA cloning of transcription factor E4TFI subunits with Ets andNotch motifs. WATANABE, H., SAWADA, J.-I., YANO, K.-I.,
YAMAGUCHI, K., GOTO, M. and HANDA, H. ..... . . . . . ..
Genetics of fluoride-resistance of the nematode Caenorhabditis elegans. KAWAKAMI, M., ISHIHARA, T. and KATSURA, I.
Caenorhabditis elegans larval lethal mutations that cause grossmorphological changes. HISHIDA, R., ISHIHARA, T. and
KATSURA, I.
A gene that controls both hatching and cell migration in C. elegans.
HISHIDA, R., ISHIHARA, T. and KATSURA, I.
Construction of lacZ markers for various nerve cells in the head of
C. elegans. ISHIHARA, T. and KATSURA, I. .. . . . . . . . . . . ..
Analysis of the head neural system of C. elegans as assayed byformation of dauer larvae. SUZUKI, N., URASAKI, M., ISHIHARA, T. and KATSURA, I.
Systematic analysis of C. elegans eDNA-Towards an expression
map of the genome. KOHARA, Y, MOTOHASHI, T., NISHIGAKI, A., SUGIMOTO, A. and WATANABE, H.
In situ hybridization on C. elegans embryos-Analyses of candidate
clones for differentially expressed genes. TABARA, H. and
KOHARA, Y.
Gene disruption in C. elegans-Application of transposon insertion
mutant banks. ANDACHI, Y and KOHARA, Y
Developmental mechanisms and manipulation of germ cells inmouse embryos. NAKATSUJI, N. and SHIRAYOSHI, Y. ....
Molecular analysis of cell differentiation and morphogenesis in
postimplantation mouse embryos. SHIRAYOSHI, Y and NAKATSUJI, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Analysis of migration pattern of neurons in morphogenesis of themouse central nervous system. NAKATSUJI, N.
V.

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81

Population Genetics
Pattern of nucleotide substitutions in growth hormone-prolactin
gene family: A paradigm for evolution by gene duplication.
OHTA, T.
Amino acid substitution at theAdh locus of Drosophila is facilitatedby samll population size. OHTA, T.

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CONTENTS
An examination of the generation-time effect on molecular evolution. OHTA, T.
Unbiased estimation of evolutionary distance between nucleotide
sequences. TAJIMA, F. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . ..
Simple methods for testing the molecular evolutionary clock hypothesis. TAJIMA, F.
Statistical analysis of DNA polymorphism. TAJIMA, F. . . . . . . ..
The molecular descent of the major histocompatibility complex.
KLEIN, J., SATTA, Y., O'HUIGIN, C. and TAKAHATA, N. ..
Allelic genealogy and human evolution. TAKAHATA, N.
Effective population size, genetic diversity, and coalescence time in
subdivided populations. NEI, M. and TAKAHATA, N.
The synonymous substitution rate of the major histocompatibility
complex loci in primates. SATTA, Y., O'HUIGIN, c., TAKAHATA, N. and KLEIN, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
MHC polymorphismand human evolution. KLEIN, J., TAKAHATA, N. and AYALA, F. J.
Trans-specific MhC polymorphism and the origin of species inprimates. KLEIN, J., SATTA, Y, TAKAHATA, N. andO'HUIGIN, C.
Special feature: On the occasion of the 25th anniversary of theneutral theory (I) Introductory comments on major papers byProfessor Motoo Kimura. TAKAHATA, N.
Special feature: On the occasion of the 25th aniversary of theneutral theory (II) Relaxed natural selection in human populations during the pleistocene. TAKAHATA, N.
Decay of linkage disequilibrium in a finite island model. TACHIDA, H.
Genetic diversity in partially selfing populations with the steppingstone structure. TACHIDA, H. and YOSHIMARU, H.
VI.

Evolutionary Genetics

Three human MHC class III genes near the junction with the class
II locus: gene for receptor of advanced glycosylation end
products, PBX2-homeobox gene, and a Notch-homolog,
human counterpart of mouse mammary tumor gene int-3.
SUGAYA, K., FUKAGAWA, T., MATSUMOTo, K.-I., MITA, K.,

vii

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viii

TAKAHASHI, E.-I., ANDO, A., INOKO, H. and IKEMURA, T. ..
Pseudoautosomal boundary (PABIX and PABIY)-like sequence
existing near boundary of long-range G + C% mosaic domains
in the human MHC locus. FUKAGAWA, T., SUGAYA, K.,
MATSUMOTO, K.-I., OKUMURA, K., ANDO, A., INOKO, H.
and IKEMURA, T.
The distribution of tenascin-X is distinct and often reciprocal tothat of tenascin-C. MATSUMOTO, K.-I., SAGA, Y., IKEMURA, T., SAKAKURA, T. and CHIQUET-EHRIS.MANN, R. ..
Interfamilial and inrafamilial genomic diversit of human T lymphotropic virus type I strains from Papua New guinea and theSolomon Islands. NERuRKAR, V. R., SAITOU, N. and YANAGIHARA, R.
Population genetic study in Hainan Isalnd, China. I. Distribution ofblood genetic markers. OMOTO, K., MISAWA, S., SAITOU,
N., Du, C. and Du, R.
Genetic variability of major histocompatibility complex in human. . . . . . . . ..
populations. GOJOBORI, T. and IMANISHI, T.
Cloning of human, mouse and fission yeast recombination genes
homologous to RAD51 and recA. SHIOBARA, A., OGAWA,
H., MATSUDA, Y, USHIO, N., IKEO, K. and OGAWA, T.
An evolutionary role ofkringle structures. IKEO, K., TAKAHASHI,
K. and GOJOBORI, T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Molecular evolution of the major epitopes of HIV within a single
host. YAMAGUCHI, Y. and GOJOBORI, T.
Search for candidate genes on which positives selection is operating.
ENDo, T., IKEO, K. and GOJOBORI, T.
Relative efficiencies of various methods for estimating molecularphylogenetic trees such as the maximum likelihood, maximum
parsimony, neighbor-joining methods, when rate of nucleotide
substitution varies with nucleotide site. TATENO, Y, TAKEZAKI, N. and NEI, M.
. . . . . . . . . . . . ..
VII.

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99

Human Genetics

Isolation and flourescence in situ hybridization mapping of 60
cosmid clones on human chromosome 18. NAKASHIMA, H.,
SAKAI, M., INABA, R. and IMAMURA, T.
101

CONTENTS

ix

Characterization of the supernumerary der(l8) chromosomes byfluorescence in situ hybridization with single-copy DNA
probes. NAKASHIMA, H., SAKAI, M., INABA, R., HASEGAWA, T., NAKAMURA, Y. and IMAMURA, T.
103
A dinucleotide microsatellitepolymorphism on the chromosome 18
q22 locus. NAKASHIMA, H., SAKAI, M., INABA, R. and
IMAMURA, T.
105
Peopling of the Americas, founded by four major lineages of
mitochondrial DNA. HORAI, S., KONDO, R., NAKAGAWAHATTORI, Y., HAYASHI, S., SONODA, S. and TAJIMA, K. .. 105
Evolution of hominoid mitochondrial DNA with special reference
to the silent substitution rate over the genome. KONDO, R.,
HORAI, S., SATTA, Y. and TAKAHATA, N
107
Seroepidemiological survey of human T-Iymphotropic retrovirus
among indigenous populations in Taiwan. ISHIDA, T., PAN,
I-H., HORAI, S., SAITOU, N. and SUN, C.-S.
107
Mitochondiral DNA mutations at nucleotide positions 3243 and3271 in mitochondiral myopathy, encephalomyopathy, lactic
acidosis, and stroke-like episodes: a comparative study.
SAKUTA, R., GOTO, Y., HORAI, S. and NONAKA, I.. . . . . . .. 109
An A-to-G transition at nucleotide pair 11084 in the ND4 gene may
be an mtDNA polymorphism. SAKUTA, R., GOTO, Y,
NONAKA, I. and HORAI, S.
109
A T-to-G mutation at nucleotide pair 8993 in mitochondrial DNA
in a patient with Leigh's syndrome. YOSHINAGA, H., OGINO,
T., OHTAHARA, S., SAKUTA, R., NONAKA, I. and HORAI, S...
110
Identification of HSI protein as a major substrate of proteintyrosine kinases upon B-cell antigen receptormediated signaling. YAMASHITA, Y., OKADA, M., TOYOSHIMA, K., KITAMURA, D. and WATANABE, T. 111
Regulation of the expression of human CE germline transcript.
ICHIKI, T., TAKAHASHI, W. and WATANABE, T. 112
VIII. Plant Genetics
Bacterial blight resistance in wild rice populations.
H. and MIYABAYASHI, T

MORISHIMA,113

x

Life-history of wild rice species distributed in the basin of the river
Amazon. MORISHIMA, H., SHIMAMOTO, Y., OHARA, M.,
MARTINS, P. S., ANDO, A. and OLIVEIRA, G. C. X.
114
Genetic erosion in the tropics. MORISHIMA, H., SATO, Y.-I.,
SHIMAMOTO, Y., YAMAGISHI, H. and SATO, T. ..... . . . .. 116
Gamete eliminator adjacent to the wx locus detected in an IndicaJaponica hybrid of Oryza sativa. SANO, Y., EIGUCHI, M. andHIRANO, H.-Y.
117
Procedures for rejuvenating plant genetic resource accessions withminimum loss of allelic diversity. YONEZAWA, K. and MORISHIMA, H.
. . . . . . . . . . . . . . . . . . . . . . . . .. 117

IX. Database
Toward unification of taxonomy databases in a distributed computer environment. KITAKAMI, H., TATENO, Y. and GOJOBORI,
T
119
DNA database management system. KITAKAMI, H., SHINI, T.,
IKEo, K., UGAWA, Y., SAITOU, N., GOJOBORI, T. andTATENO, Y
120
Publications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Abstracts of diary for 1993
Foreign visitors in 1993 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Author index

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GENERAL STATEMENT
Our institute was established 44 years ago as a center for comprehensive
genetics research. Outstanding accomplishments in the advancement ofresearch on genetics, in particular population genetics, plant genetics andmolecular genetics, by members of our institute have made it the center ofgenetic study in our country and a major institution with world-wide recognition. In 1984 the institute was reorganized into a National Inter-university
Research Institute to promote research activities through cooperation withother institutions. Together with seven inter-university research institutes,
we formed the Graduate University for Advanced Studies, in 1988. Our
institute admits graduate students to the Department of Genetics of the
Graduate School of Life Science. We have 32 such students at present and in
addition, about ten special research students from other universities, including those from foreign countries. I consider it vital for our research activities
to have a steady flow of young scientists.
Recent rapid progress in the field of genetics using newly developed
approaches has greatly shifted the course of study in biology. I consider itnatural that research in our institute has recently focused more on molecularstudies of various aspects of genetics. This is not to ignore the importance ofmore traditional approaches to genetics. Our institute is uniquely suitable forpursuing cooperative work by scientists of various disciplines. Throughinteractions between these lines of research, our institute will flourish.
We have been carrying out several research related services. The DNA
Data Bank of Japan (DDBJ) is one of the three banks in the world thatgather, annotate, store and distribute information on DNA sequences.
DDBJ will continue to be active as a regional center for such activities. Ollr
institute also stores and distributes various organisms with genetically characterized traits. Among them, services with mice, rice and Escherichia coli are
particularly significant. These service activities will continue to develop.
However, as the manager responsible for these activities, I honestly feel
frustration running service operations with inadequate staff numbers, salaries
and funding. Nation-wide recognition of the importance of these activities
and appropriate actions to amend these conditions are essential.
I hope that with guidance from people in and outside this institute and2further support from governmental and private sources, I could lead theinstitute into a more successful future.
In the past year, the Mammalian Development Laboratory was establishedin the Genetic Stock Research Center. Prof. N. Nakatsuji and Dr. Y.
Shirayoshi were transferred to the new laboratory from the Mammalian
Genetics Laboratory, where Dr. T. Shiroishi of the Division of Cytogenetics
was transferred, being promoted to associate professor. Dr. Y. Ugawa wastransferred to the Ministry of Agriculture and Fisheries and Dr. I. Nakamura
to Iwate Biotechnology Research Center. The following three joined us asresearch members: Dr. T. Takano in the Division of Population Genetics, Dr.
T. Kishi in the Division of Mutagenesis, and Dr. K. Kanamaru in theMicrobial Genetics Laboratory of the Genetic Stock Research Center.
It is a pleasure to note that Dr. F. Tajima was honored by the Encouragement Award of the Genetics Society of Japan for his statistical studies on
DNA polymorphism.

STAFF
Director-General

TOMIZAWA, Jun-ichi, Ph. D.
Vice-Director

MORIWAKI, Kazuo, D. Sc.
Members1.

Department of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Head of the Department
Division of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUJITA, Nobuyuki, D. Sc.
Y AMAGISHI, Masahiro, D. Sc.
TOYODA, Tetsuya, D. Med.
Division of Mutagenesis
SENO, Takeshi, D. Sc., Professor
Y AMAO, Fumiaki, D. Sc., Asssociate professor
KANEDA, Sumiko, D. Pharm.
KISHI, Tsutomu, D. Eng.
Division of Nucleic Acid Chemistry (Guest member)
MIZUMOTO, Kiyohisa, D. Sc., Professor
KITAKAMI, Hajime, D. Eng., Professor

2.

Department of Cell Genetics
MORIWAKI, Kazuo, D. Sc., Head of the Department
Division of Cytogenetics
MORIWAKI, Kazuo, D. Sc., Professor
IMAI, Hirotami T., D. Sc., Associate professor
GOTO, Hideo, D. Ag.

4Division of Microbial Genetics
HORIUCHI, Kensuke, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate professor
HARA, Hiroshi, D. Sc.
HIGASHITANI, Atsushi, D. Sc.
Division of Cytoplasmic Genetics (Guest member)
YASUDA, Hideyo, D. Pharm., Associate professor
YONEKAWA, Hiromichi, D. Sc., Associate professor
3.

Department of Ontogenetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
Division of Developmental Genetics
SUGIYAMA, Tsutomu, Ph. D., Professor
FUJISAWA, Toshitaka, Ph. D., Associate professor
SHIMIZU, Hiroshi, D. Eng.
HATTA, Masayuki, D. Sc.
Division of Phenogenetics
HIROSE, Susumu, D. Sc., Professor
MURAKAMI, Akio, D. Ag., D. Sc., Associate professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-Aki, D. Ag.
Division of Physiological Genetics (Guest member)
KOIZUMI, Osamu, D. Sc., Professor
HANDA, Hiroshi, D. Med., Professor

4.

Department of Population Genetics
OHTA, Tomoko, Ph. D., D. Sc., Head of the Department
Division of Population Genetics
OHTA, Tomoko, Ph. D., D. Sc., Professor
TAJIMA, Fumio, Ph. D., Associate professor
TAKANO, Toshiyuki, D. Sc.
Division of Evolutionary Genetics
IKEMURA, Toshimichi, D. Sc., Professor
SAITOU, Naruya, Ph. D., Associate professor
MATSUMOTO, Ken-ichi, D. Ag.
MORIYAMA, Etsuko, Ph. D.

STAFF
Division of Theoretical Genetics (Guest member)
TAKAHATA, Naoyuki, D. Sc., Professor
TACHIDA, Hidenori, D. Sc., Associate professor
5.

Department of Integrated Genetics
IMAMURA, Takashi, D. Med., Head of the Department
Division of Human Genetics
IMAMURA, Takashi, D. Med., Professor
FUJIYAMA, Asao, D. Sc., Associate professor
HORAI, Satoshi, D. Med., Associate professor
NAKASHIMA, Hitoshi, D. Med.
Division of Agricultural Genetics
MORISHIMA, Hiroko, D. Ag., Professor
SANO, Yoshio, D. Ag., Associate professor
SATO, Yoichiro, D. Ag.
HIRANO, Hiro-Yuki, D. Ag.
Division of Applied Genetics (Guest member)
WATANABE, Takeshi, D. Med., Professor
YONEZAWA, Katsuei, D. Ag., Professor

6.

Genetic Stock Research Center
MORISHIMA, Hiroko, D. Ag., Head of the Center
Mammalian Genetics Laboratory
SHIROISHI, Toshihiko, D. Sc., Associate professor
MIYASHITA, Nobumoto, D. Med.
Invertebrate Genetics Laboratory
VEDA, Hitoshi, D. Ag.
Plant Genetics Laboratory
Microbial Genetics Laboratory
NISHIMURA, Akiko, D. Ag., Associate professor
KANAMARU, Kengo, M. Ag.
Genetic Resources Laboratory
TATENO, Yoshio, Ph. D., D. Sc., Associate professor
Mammalian Development Laboratory
NAKATSUJI, Norio, D. Sc., Professor
SHIRAYOSHI, Yasuaki, D. Sc.

5

6

7.

DNA Research Center
SENO, Takeshi, D. Sc., Head of the Center
DNA-Protein Inateraction Laboratory
SHIMAMOTO, Nobuo, D. Sc., Associate professor
NAGAI, Hiroki, D. Sc.
Recombinant DNA Laboratory
KATSURA, Isao, D. Sc., Professor
ISHIHARA, Takeshi, D. Sc.
DNA Synthesis Laboratory
HAYASHI, Shigeo, D. Sc.
DNA Data analysis Laboratory
GOJOBORI, Takashi, D. Sc., Professor
IKEO, Kazuho, D. Sc.
IMANISHI,
Gene Library Laboratory
KOHARA, Yuji, D. Sc., Associate professor
ANDACHI, Yoshiki, D. Sc.

8.

Radioisotope Center
SADAIE, Yoshito, D. Sc., Associate professor, Head of the Center

9.

Experimental Farm
SANO, Yoshio, D. Ag., Head of the Farm

10.

Technical Section
MITA, Akihiko, Chief of the Section

11.

Department of Administration
EBIHARA, Akira, Head of the Department
NINOMIYA, Shigeo, Chief of the General Affairs Section
MABUCHI, Kenji, Chief of the Finance Section

COUNCIL

7COUNCIL
Chairman
NAGAKURA, Saburo; President, Graduate University for Advanced
StudiesVice-chairman
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
Members (Alphabetical order)
EBASHI, Setsuro; President, Okazaki National Research Institutes
HAMAl, Osamu; Dean, Faculty of Letters, University of TokyoICHIKAWA, Atsunobu; Deputy Director, the National Institute forEnvironmental Sciences
IINO, Tetsuo; Professor, Faculty of Human Science, Waseda University
KIMURA, Motoo; Professor emeritus, National Institute of Genetics
NOJIMA, Shoshichi; Dean, Faculty of Pharmaceutical Science, Teikyo
University
ONOE, Hisao; President, Shiga University
Om, Tatsuo; Professor, Faculty of Home Economics, Kyoto Women's
University
OSAKI, Hitoshi; Director, Japan Society for the Promotion of Science
SANO, Hirotoshi; President, Tokyo Metropolitan University
SUGANO, Haruo; Director, Cancer Institute
SUGIMURA, Takashi; President emeritus, National Cancer Center
TAKANAMI, Mitsuru; Professor, Institute for Chemical Research, Kyoto
University
TAKEUCHI, Ikuo; Director-General, Institute for Basic Biology
TANAKA, Ryuso; President, Hiroshima University
YAMAGATA, Hirotada; Professor, Faculty of Agriculture, Kyoto University
YOSHIOKA, Morimasa; President, Tokyo Women's Medical College
YURA, Takashi; Director, Institute for Virus Research, Kyoto University

8

ADVISOTY COMMITTEE
ChairmanMORIWAKI, Kazuo; Professor, Division of Cytogenetics
Vice-chairman
OSAWA, Shozo; Adviser Planning Department, Japan Tabacco Inc.
Members (Alphabetical order)
FUKUDA, Ichiro; Professor, Faculty of Liberal Arts and Sciences, Tokyo
Women's University.
HINATA, Kokichi; Professor, Faculty of Agriculture, Tohoku University
ISHIWA, Sadao; Professor, Faculty of Science, Ochanomizu Women's
University
MIURA, Kin-ichiro; Professor, Faculty of Science, Gakushuin University
OKADA, Masukichi; Professor, Institute of Biological Sciences, University of Tsukuba
TAKEBE, Hiraku; Professor, Faculty of Medicine, Kyoto University
TAKEUCHI, Takuji; Professor, Faculty of Science, Tohoku University
TSUNEWAKI, Koichiro; Professor, Faculty of Agriculture, Kyoto University
YOSHIKAWA, Hiroshi; Professor, Faculty of Medicine, Osaka University
GOJOBORI, Takashi; Professor, DNA Resarch Center
HORIUCHI, Kensuke; Professor, Division of Microbial Genetics
IKEMURA, Toshimichi; Professor, Division of Evolutionary Genetics
IMAMURA, Takashi; Professor, Division of Human Genetics
ISHIHAMA, Akira; Professor, Division of Molecular Genetics
MORISHIMA, Hiroko; Professor, Division of Agricultural Genetics
NAKATUJI, Norio; Professor, Genetic Stock Research Center
OHTA, Tomoko; Professor, Division of Population Genetics
SENO, Takeshi; Professor, Division of Mutagenesis
SUGIYAMA, Tsutomu; Professor, Division of Developmental Genetics

ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE OF GENETICS

9

ASSOCIATION FOR PROPAGATION OF THE
KNOWLEDGE OF GENETICS
President
KONDO, Norio; Professor emeritus, Tokyo University of Agriculture
Managing Director
MORIWAKI, Kazuo; Professor, National Institute of Genetics
GOJOBORI, Takashi; Professor, National Institute of Genetics
Manager
TOMIZAWA, Jun-ichi; Director-General, National Institute of Genetics
NOMURA, Tatsuji; Director, Central Institute for Experimental Animnals
YAMAGUCHI, Hikoyuki; Professor emeritus, University of Tokyo
MIURA, Kin-ichiro; Professor, Faculty of Science, Gakushuin University
KURODA, Yukiaki; Professor emeritus, National Institute of Genetics
ISHIHAMA, Akira; Professor, National Institute of Genetics
Trustee
TAZIMA, Yataro; Director, Institute of Silkworm Genetics and Breeding
OSHIMA, Chozo; Honorary member, National Institute of Genetics
SAITO, Hiuga; Professor, Faculty of Medicine, Teikyo University
SHIGETO, Gakuji; Manager, Japan Society for the Promotion of Science
MATSUNAGA, Ei; Professor emeritus, National Institute of Genetics
YOSHINO, Tatsuji; President, shokabo Publishing Co.
TAKAGAKI, Yoshio; Research Director, Chugai Pharmaceutical Co.,Ltd.
SENO, Takeshi; Professor, National Institute of Genetics
Auditor
KIMURA, Motoo; Professor emeritus, National Institute of Genetics
IMAMURA, Takashi; Professor, National Institute of Genetics
MORISHIMA, Hiroko; Professor, National Institure of Genetics
Adviser
MORIWAKI, Daigoro; Honorary member, National Institute of Genetics

PROJECTS OF RESEARCH FOR 1993
1.

DEPARTMENT OF MOLECULAR GENETICS
Division of Molecular Genetics

Regulatory mechanisms of gene transcription in prokaryotes (ISHIHAMA andFUJITA)
Molecular architecture of transcription apparatus from eukaryotes (ISHIHAMA and Y AMAGISHI)
Molecular mechanisms of transcription and replication of animal and plantviruses (ISHIHAMA and TOYODA)
Division of Mutagenesis

Multiple Role of ubiquitin system in the cell-cycle regulation (SENO, Y AMAO,KANEDA and KISHI)
Division of Nucleic Acid Chemistry

Mechanism of mRNA capping (MIZUMOTO)
Transcription of the Sendai virus genome (MIZUMOTO)
Development of DNA Database management system and of unification toolof taxonomy Databases (KITAKAMI)
2.

DEPARTMENT OF CELL GENETICS
Division of Cytogenetics

Genetic differentiation of mouse species (MORIWAKI, SHIROISHI* andMIYASHITA*)
Theoretical and experimental bases for karyotype evolution (IMAI)
Study of adrenal function in mouse development by transgene (GOTOH,* Genetic stock research center.

PROJECTS OF RESEARCH FOR 1993

11

SHIROISHI* and MORIWAKI)
Division of Microbial Genetics

DNA replication in E. coli (YASUDA, HIGASHITANI and HORIUCHI)
Cell cycle regulation in E. coli (HARA, HIGASHITANI and HORIUCHI)
Division of Cytoplasmic Genetics

Studies on cytoplasmic genes during subspecies differentiation of house
mouse Mus musculus (YONEKAWA)
Cell-cycle regulation by cyclin and CDC2 kinase (YASUDA)
3.

DEPARTMENT OF ONTOGENETICS
Division of Developmental Genetics

Genetic and molecular analysis of developmental mechanisms m hydra(SUGIYAMA, FUJISAWA, SHIMIZU and HATTA)
Division of Phenogenetics

Gene expression in eukaryotes (HIROSE, VEDA and HAYASHI)
Genetic studies on the life history characters in Bombyx (MURAKAMI)
Genetic studies on the nerve system characters in Bombyx (MURAKAMI)
Genetic studies on development and growth of insect (YAMADA andMINATO)
Division of Physiological Genetics

Nerve net formation in Hydra (KOIZUMI)

12

4.

DEPARTMENT OF POPULATION GENETICS
Division of Population Genetics

Theoretical studies of population genetics (OHTA and TAJIMA)
Theoretical studies on the evolution of multigene family (OHTA)
Theory of gene genealogy (TAJIMA)
Statistics for DNA polymorphisms (TAJIMA)
Experimental population genetics on DNA polymorphism and evolution in
Drosophila (TAKANO)
Genetic studies on interspecific variation in Drosophila (TAKANO)
Division of Evolutionary Genetics

Studies on codon usage (IKEMuRA)
Studies on chromosome band structures at the DNA sequence level (IKEMURA and MATSUMOTO)
Studies on genes in HLA locus (IKEMuRA and MATSUMOTO)
Studies on functions of extracellular matrix proteins (MATSUMOTO and
IKEMuRA)
Molecular evolutionary analysis of nucleotide sequence data (SAITOU)
Studies on the genetic affinity of human populations (SAITOU)
Studies on molecular evolution of viruses (MORIYAMA)
Studies on molecular evolution of Drosophila genes (MORIYAMA)
Division of Theoretical Genetics

Population immunogenetics (TAKAHATA)
Molecular anthropology (TAKAHATA)
Theoretical studies of population genetics (TACHIDA)
5.

DEPARTMENT OF INTEGRATED GENETICS
Division of Human Genetics

Genetic and physical mapping of human genome (IMAMuRA and NAKA-

PROJECTS OF RESEARCH FOR 1993

13

SHIMA)
Molecular genetics of human metabolic disorders (IMAMURA and NAKASHIMA)
Molecular biology of oncogenes (FUJIYAMA)
Studies on DNA polymorphisms in human populations (HORAI)
Division of Agricultural Genetics

Evolutionary and ecological genetics in wild and cultivated nee species(MORISHIMA and SATO)
Genetic studies of speciation in rice (SANO and HIRANO)
Studies on plant gene expression (HIRANO and SANO)
Bio-archaeological analysis of plant remains (SATO and NAKAMURA)
Division of Applied Genetics

Molecular genetics of human immune mechanisms (WATANABE)
Theoretical studies on conservation biology (YONEZAWA)
6.

GENETIC STOCK RESEARCH CENTER
Mammalian Genetics Laboratory

Recombinational hotspots in the mouse MHC (SHIROISHI and MORIWAKI)
Genetic Mechanisms for regulating tumor development in the laboratory andwild mice (MIYASHITA and MORIWAKI)
Invertebrate Genetics Laboratory

Molecular genetics of insect development (VEDA and HIROSE)

14

Plant Genetics Laboratory

Microbial Genetics Laboratory

Timing of cell division in E. coli (NISHIMURA)
Division apparatus of E. coli (KANAMARU and NISHIMURA)
Genetic Resources Laboratory

Theoretical studies in molecular phylogeny (TATENO)
Mammalian Development Laboratory

Developmental mechanisms and manipulation of germ cells in mouse embryos (NAKATSUJI and SHIRAYOSHI)
Molecular analysis of cell differentiation and morphogenesis in postimplantation mouse embryos (SHIRAYOSHI and NAKATSUJI)
Analysis of migration pattern of neurons in morphogenesis of the mouse
central nervous system (NAKATSUJI)
7.

DNA RESEARCH CENTER

DNA-Protein Inateraction Laboratory

Interaction (SHIMAMOTO and NAGAI)
Recombinant DNA Laboratory

Genetics and molecular genetics of development and behavior of Caenorhabditis elegans (KATSURA and ISHIHARA)
DNA synthesis Laboratory

Molecular genetics of Drosophila melanogaster (HAYASHI and HIROSE)PROJECTS OF RESEARCH FOR 199315

DNA Data Analysis Laboratory

Computer (GOJOBORI and UGAWA)
Construction (GOJOBORI, )Molecular (lKEO and GOJOBORI)
Gene Library Laboratory

Molecular genetics of Caenorhabditics elegans development (KOHARA andANDACHI)Genome analysis of Caenorhabditis elegans (KOHARA)

8.

RADIOISOTOPE CENTER

Molecular mechanisms of sporulation in Bacillus subtilis (SADAIE)9.

EXPERIMENTAL FARMRESEARCH ACTIVITIES IN 1993

I.

MOLECULAR GENETICS

Molecular Anatomy of Escherichia coli RNA Polymerase:
Functional Map of the Amino-Terminal Assembly
Domain of Alpha Subunit
Makoto KIMURA, Nobuyuki FUJITA and Akira ISHIHAMA

The a subunit of Escherichia coli RNA polymerase plays a major role inassembly of the core enzyme. Analysis of carboxyl-terminal deletion derivatives of a indicated that the amino terminal two-thirds of a subunit, down toamino acid 235, is involved in this assembly (Igarashi, K. et al. (1991) J. Mol.
BioI. 218, 1-6; Hayward, R.S. et al. (1991) J. Mol. BioI. 221, 23-29). To
define the site(s) for core enzyme assembly, we constructed a set of rpoA
mutants with deletion or insertion mutations in this region. The overexpressed a derivatives were purified and examined for their abilities toassemble /3 and /3' subunits in vitro. Analysis of a total of 22 a deletionmutants indicated that the minimal fragment with the full assembly activity
is a(21-235), with deletions of 20 amino-terminal and 94 carboxy-terminal
amino acids (Kimura, M. et al. (1994) J. Mol. BioI. 242, 107-115). Most of
the other assembly-defective deletion mutants appeared to lack the activity offormation of stable a dimers.
On the other hand, 11 species of insertion mutants, having two extra aminoacids, Ala-Ser, at every 20 residues, showed defects in the assembly at various
steps depending on the site of dipeptide insertion. Five mutants having theinsertions between positions 40-41, 100-101, 140-141, 180-181 and 220-221
were defective in dimerization, nevertheless two of them, having the insertion
at position 180-181 and 220-221, were able to assembe/3 subunit but unable
to assemble /3' subunit. The insertion at position 200-201 made a virtuallyinactive in/3' assembly. The insertion at position 80-81 resulted in reductionof stability of aJ] complex, but the stability increased with formation of
hybrid a dimer between the wild-type and the mutant a subunits. These

I.

MOLECULAR GENETICS

17

results altogether suggest that within the assembly domain of a, the Nterminal proximal region is involved in dimerization and contact with (3 while
the C-terminal region is involved in (3' assembly.
Molecular Anatomy of Escherichia coli RNA Polymerase:
Functional Map of the Carboxy Terminal Contact
Domain with Class-I Factors
Tomofumi NEGISHI, Nobuyuki FUJITA, Yoshimasa KYOGOKU*
and Akira ISHIHAMA

The a subunit of RNA polymerase plays an essential role not only insubunit assembly but also in transcription activation by class-I transcription
factors (reviewed in Ishihama, A. (1993) J. Bacteriol. 175,2483-2489). For
detailed mapping the structural domains of a subunit, we analyzed thepattern of proteolytic cleavage by endoproteases. First we carried out limited
proteolysis by protease V8 and trypsin under mild conditions and identified
the sites of initial cleavage after sequencing of isolated proteolytic fragments.
Results indicate that the proteolytic cleavage takes place at three regions,
between amino acid residues 236-241 (full-length a is composed of 329
amino acid residues), implying that this region are exposed on protein
surface, presumably forming interdomain linkers. Thus, we propose that two
major structural domains exist within the a subunit, each being composed ofamino acid residues 8-235 (domain 1) and 242-329 (domain 2).
The N-terminal proximal fragment down to amino acid 7 is not essential
for assembly and enzyme function (Kimura, M. et al. (1994) J. Mol. BioI.
242, 107-115). Upon prolonged proteolyis, the domain 1 was deaved into
two subdomain fragments (1a and Ib) between Arg45 and Glu68. The
amino-terminal proximal la subdomain is rapidly degrated upon further
incubation with trypsin [subdomain la contains four potential cleavage sites
by trypsin]. The subdomain 1b of 168 amins acids in length is cleaved at
Gly151 into two fragments. Domain 2 carrying the contact site I region forvarious class-I transcription factors is resistant to secondary cleavage by bothV8 and trypsin, indicating that this domain is highly structured.
In order to analyze the higher-order structure of domain 2, we cloned a
DNA fragment carrying the C-terminal region of rpoA into an expression*

Osaka Univeristy, Protein Research Institute, Suita, Osaka.

18

vector and expressed in E. coli a C-terminal fragment consisting of aminoacids 233-329. The purified C-terminal fragment [a(233-329)] was subjected to IH-NMR spectroscopic analysis, which is believed to be the best
technique for analysis of static and dynamic structure of short polypeptides insolution. Preliminary results indicate the presence of regular secondary
structure in this region. Sequential signal assignment by means of 15N-single
and 15N/13C-double labeling techniques is in progress.
Molecular Anatomy of Escherichia coli RNA Polymerase:
Mapping of cAMP Receptor Protein Contact
Site on One Alpha Subunit
Chao Zou, Nobuyuki FUJITA and Akira ISHIHAMAIn transcription activation of positively controlled genes, transcription
factors make contact with RNA polymerase at specific sites. Up to now, we
identified clusters of the contact sites on narrow regions of both a (contact
site I) and a (contact site II) subunits (reviewed in Ishihama, A. (1993) J.
Bacterial. 173,2483-2389). cAMP receptor protein (CRP) contacts with the
C-terminal region of a subunit (contact site I) in transcription activation ofthelacPl promoter (Igarashi, K. and Ishihama, A. (1991) Cell 32, 319-325;
Igarashi, K. et al. (1993) Proc. Natl. Acad. Sci. USA 88, 8958-8962). The
fine mapping indicated that the contact site is clustered within a shortsegment between amino acid residues 265-270 (Zou, C. et al. (1992) Mol.
Microbial. 6,2599-2605). However, it remained unknown whether only one
or both of the two a subunits is involved in the protein-protein contact with
CRP.
This year we constructed a hybrid RNA polymerase consisting of one
wild-type and one C-terminal truncated a subunits. Results of in vitrotranscription assay indicates that this reconstituted hybrid RNA polymerase
preparation has the activity of CRP-dependent lacPl transcription half thelevel of wild-type RNA polymerase, indicating that the reconstituted hybrid
RNA polymerase is a mixture of two forms which differ in the arrangementof mutant a and that CRP interacts with only one form enzyme (Zou, C. et
al. (1994) J. Mol. BioI. 236, 1283-1288). Thus, we conclude that CRP makes
contact with only one a subunit.

I.

MOLECULAR GENETICS

19

Molecular Anatomy of Escherichia coli RNA Polymerase:
Interaction between Promoter - 35 and -10
Recognition Domains of Sigma-70
Ashok KUMAR, Richard S. HAYWARD* and Akira ISHIHAMARNA polymerase holoenzyme of E. coli is composed of core enzyme withthe subunit structure alJ/3' and one of six different species of a subunit. RNA
synthesis is catalyzed by core enzyme but a subunit is required for promoter
recognition and transcription initiation. Each molecular species of a subunit
recognizes a specific and unique set of promoters. Prokaryotic a family
proteins carry four regions of conserved sequences, of which region 2 and 4(from N-termini) are involved in recognition of promoter -10 and - 35
sequence, respectively. Previously, we demonstrated that the region 4 (- 35
recognition domain) is not needed provided that an "extended -10 sequence" with the sequence of TGxTATAAT exists in the test promoters(Kumar, A. et al. (1994) J. Mol. BioI. 232, 406-418). These C- terminallytruncated a subunits often lose the response to a group of transcription
factors including PhoB and CRP (Kumar, A. et al. (1884) J. Mol. BioI. 235,405-413) [we designated this group as "class-II transcription factors"].
The activity of C-terminally truncated a subunits lacking the - 35 recognition domain, as measured by promoter-dependent transcription by core
enzyme, was, however, less than the intact a subunit, but was enhanced by
the addition of C-terminal fragments. Thus, it appears that the - 10
recognition domain of region 2 and the - 35 recognition domain of region 4
interacts each other to enhance the sigma activity.
Promoter Selectivity Control of Escherichia coli RNA Polymerase:
Competition between Different Sigma Subunits
Shuichi KUSANo, Miki JISHAGE, Nobuyuki FUJITA and Akira ISHIHAMARNA polymerase core enzyme with the subunit structure alJ/3' is functionally differentiated into different forms of holoenzyme by interaction with oneof multiple molecular species of the a subunit. Thus, the a subunit plays amajor role in determination of the promoter recognition property of RNApolymerase (reviewed in Ishihama, A. (1979) Trends Genet. 4, 282-286;*

University of Edinburgh, Institute of Cell and Molecular Biology, Edinburgh.

20Ishihama, A. (1993) J. Bacteriol. 175,2483-2489). Up to now, six different
species of a subunit have been identified in Escherichia coli. Changes in global
transcription pattern are believed to be mainly due to a replacement on RNA
polymerase. In order to get insight into the molecular mechanism(s) of a
subunit replacement, we first observed competition between different a subunits in an in vitro transcription system in the presence of different a subunits.
For this purpose, we purified four molecular species of a subunit, aD (a70 ; theregular a 70 encoded by rpoD), as (a38 ; stationary-specific a encoded by rpoS),aH (03 2; heat-shock gene-specific a encoded by rpoH), and a F (028; flagellagene-specific a encoded by rpoF). Using an in vitro single-round transcription
assay system, we first mearused the level of each a subunit required for
maximum level transcription in the presence of a fixed amount of core
enzyme. Results indicate that oD forms a holoenzyme at an input molar ratioof I : I for aD:core, while 5- and 3-fold molar excess was required for a H andaF, respectively. In sharp contrast, about lOO-fold molar excess of d' was
required to saturate core enzyme. The affinity of each a subunit to core
enzyme is being measured directly by glycerol gradient centrifugation. Experiments are also in progress, for measurement of competition between
different a subunits and for analysis of replacement rate between free asubunit and core-bound a.
Promoter Selectivity Control of Escherichia coli RNA Polymerase:
Differential Recognition of Osmoregulated Promotersbetween EnD and Ed'
Qingquan DING, Shuichi KUSANO, Nobuyuki FUJITA and Akira ISHIHAMA

Escherichia coli normally exists in vertebrate animals but can also survivein nature, including seawater, where nutrients are scare and the osmolarity ishigh. In such hyperosmotic media, E. coli increases its internal soluteconcentration to avoid plasmolysis. A rapid increase in K + is the primary
response of E. coli to osmotic stress. The intracellular glutamate concentration also increases as to balance the increase in K + concentration. Transcription regulation of three osmoregulated gene promoters, osmB, osm Y andproU, was analyzed in an in vitro transcription system using two species ofreconstituted RNA polymerase holoenzyme, EoD and Ed'. The proU gene isinvoled in transport of glycine betaine (the secondary response solute to

I.

MOLECULAR GENETICS

21

increase in intracellular osmalarity) while osmB and osmY encode osmotically inducible periplasmic protein and lipoprotein, respectively.
Results indicate thatproU (P2) is recognized by EaD while both osmB andosm Yare transcribed by both EaD and Eif. The maximum level transcription
was observed above 300mM K glutamate (but not KCI) for osmB and osm Y,
and above 200mM K glutamate for proU. Our studies suggest that, in
addition to the increase in glycine betaine concentration, the membranecomposition changes, as a response to osmotic stock, by assemlby of OsmB
and OsmY proteins. Furthermore, the RNA polymerase itself seems to be asensor of K glutamate concentration.
Structure and Function of Yeast RNA Polymerase II:
Attempts for in vitro Reconstitution
Yoshinao AzUMA, Makoto KIMURA, Makiko KAWAGISHI,
Masahiro YAMAGISHI and Akira ISHIHAMA

Three forms of nuclear RNA polymerase exist in eukaryotes, each being
composed of more than ten polypeptide chains. Structural and functional
complexity of eukaryotic RNA polymerases must therefore be higher thanthat of prokaryotic enzymes. Recent progress in molecular cloning of thegenes coding for eukaryotic RNA polymerase proteins from various organisms has revealed that the three large components are structurally related to
the /3', /3 and a subunits of prokaryotic RNA polymerase core enzyme. Since
the core enzyme carries the catalytic activity of RNA synthesis, it is reasonable to consider that these three components of eukaryotic RNA polymerases
form an assembly with the basic function of RNA polymerization.
To address this question and furthermore to get insight into the function ofeach polypeptide component of RNA polymerase II, we tried to express in E.
coli the genes for the putative subunits of RNA polymerase II from thebudding yeast Saccharomyces cerevisiae [a collaborative study with Dr. R.
Young, Whitehead Institute, MIT]. Although we succeded the expression ofsmall subunit genes, it seems extremely difficult to express two large subunitsin E. coli because of their instability. In parallel, we cloned the genes
encoding three large subunits of RNA polymerase II from the fission yeast
Schizosaccharomyces pombe (Azuma, Y. et al. (1991) Nucleic Acids Res. 19,
461-468; Kawagishi, M. et al. (1992) Nucleic Acids Res. 21, 469-473;

22

Azuma, Y. et al. (1993) 21, 3749-3754), and tried to express the genes in E.
Again we have so far failed to get the stable expression of two large
subunits, which is therefore the major difficulty to be overcome to proceed
this line of studies.
Attempts are also being made to set up a reversible dissociation system ofpurified S. pombe RNA polymerase II. Since the reassembly of completely
dissociated RNA polymerase may require large amounts of purified proteins
judging from the efficiency of well-established assembly system of E. coli
RNA polymerase, we started to find out reconsitution conditions of partiallydissociated RNA polymerase II with low concentrations of urea.

coli.

Molecular Dissection of Influenza Virus RNA Polymerase:
RNA Synthesis by PB1 Subunit
Makoto KOBAYASHI*, Tetsuya TOYODA, Susumu NAKADA**,
Kiyohisa MIZUMOTO*** and Akira ISHIHAMARNA polymerase of influenza virus consists of three P protiens, PB 1, PB2
and PA, and catalyzes multiple step reactions in transcription and replicationof the genome RNA. Systematic studies have been carried out in thislaboratory for identification of the function(s) of each subunit P protein. For
this purpose, we established an in vitro reconstitution system of RNApolymerase from individual P proteins expressed using recombinant baculoviruses carrying cDNA for each P protein (Kobayashi, M. et al. (1992) Virus
Res. 22, 235-245). As an extension of this line studies, we examined theactivity of viral RNA synthesis in nuclear extracts prepared from insect cells
infected with each of the recombitant baculoviruses or combinations of theseviruses. RNA synthesis activity was measured using short model RNA
templates, each carrying 5'- and 3'-terminal sequences of the genome RNA
segment 8.
Results indicate that nuclear extracts expressing only PB 1 subunit are able
to catalyze viral RNA synthesis in the presence of ApG primer. In contrast,
nuclear extracts of cells expressing all three P proteins displayed complete
replication cycle of model RNA template in the absence of primer. Thus, weconclude that PB 1 is the catalytic unit of influenza virus RNA polymerase
*
**
***

Present address: Jichi Medical School, Laboratory of Biology, Kawachi, Tochigi.
Science University of Tokyo, Noda, Chiba.
Department of Molecular Genetics, Division of Nucleic Acid Chemistry.

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and it alone is able to catalyze RNA synthesis (Kobayashi, M. et a/.,submitted for publication). Essentially the same results were obtained using
nuclear extracts infected with recombinant vaccinia viruses (Toyoda, T. eta/., submitted for publication).
Molecular Dissection of Inlfuenza Virus RNA Polymerase:
Photoaffinity Labeling of PBl with 8-Azido GTP
Yukiyasu ASANO, Tokumi MARUYAMA*, Kiyohisa MIZUMOTO**
and Akira ISHIHAMA

8-Azidopurines are used for monitoring nucleotide-binding sites on proteins at the primary sequence. Upon exposure to a low dose of UV light, theazido moiety of these nucleotide analogues is converted into a highly reactive
nitrene, which forms a covalent bond with most amino acids within the
cross-linking distance. In this study, we used 8-azido guanosine 5 ' -triphosphate (8-N 3 GTP) for identification of nucleotide-binding sites on influenza
virus RNA polymerase because GTP is the first nucleotide substrate incorporated into 3' termini of primers by virion-associated RNA polymerase(Ishihama, A. et a/. (1986) J. BiD/. Chern. 261, 10417-10421). We found that
8-N3 GTP is recognized as a substrate by influenza virus RNA polymerase
and can be polymerized into RNA chains. Upon exposure of mixtures ofradioactive 8-N3 GTP and RNP cores to UV light, PBI was selectively
photolabeled and this PBI labeling was competitively inhibited by not only
GTP but also GDP, GMP and dGTP. In the presence of ApG primer, thecross-linking of GTP analogue decreased almost half the level of its absence.
These observations altogether suggest that the substrate-binding site (S site)
is located on PBI but the GTP analogue can also binds to the primer- andproduct-binding site (P site) on the same PB I subunit.

* Tokushima Bunri University, Faculty of Pharmaceutical Sciences, Tokushima.
** Department of Molecular Genetics, Division of Nucleic Acid Chemistry.

24

Molecular Dissection of Influenza Virus Nucleoprotein (NP):
Identification of RNA-binding Domain
Makoto KOBAYASHI*, Tetsuya TOYADA, Djanybek ADYSHEVand Akira ISHIHAMA

Influenza virus nucleoprotein (NP) is associated with the RNA genome ata constant interval of 15-20 nucleotides (Yamanaka, K. et al. (1990) J. BioI.
Chem. 265, 11151-11155), together forming ribonucleoprotein (RNP) cores.
Naked RNA is active as a template for viral RNA-dependent RNA polymerase (Parvin, J. et al. (1989) J. Virol. 63, 5142-515i; Honda, A. et al.
(1990) J. Biochem. 107, 624-628) but NP is required for elongation of RNA
chains (Honda, A. et al. (1988) J. Biochem. 104, 1021-1026). In order to
identify the amino acid sequence involved in RNA binding, we performed
north-western blot analysis using a set of N- and C-terminal deletion mutantsof NP produced in Escherichia coli. The RNA-binding region was mapped
between amino acid residues 91 and 188. This region contains a sequence
highly conserved among not only NP from A-, B- and C-type influenza
viruses, but also a plant virus movement protein (Kobayashi, M. et al.
(1994) J. Viral. 68, 8433-8436).
Molecular Dissection of Influenza Virus M Proteins:
Identification of Growth Control Activity for M1
Jiro YASUDA** and Akira ISHIHAMA

The genome of influenza A virus is composed of 8 RNA segments ofnegative-polarity. Segment 7 encodes two matrix proteins. In virions, Mlprotein forms a membrane matrix between ribonucleoprotein (RNP) coresand lipid envelope, while M2 protein, produced after splicing of Ml mRNA,
forms an ion channel in infected cells, thereby controlling transport andassembly of hemagglutinin (HA). Previously, we found that the difference invirus growth rate between fast-growing WSN and slow-growing Aichi is
attributed to the M gene (Yasuda, J. et al. (1994) Arch. Viral. 133,283-294).
In this study, we tried to determine which of the two M proteins, Ml and M2,
encoded by the M gene is responsible for the rapid virus growth. For this***Present address: Jichi Medical School, Laboratory of Biology, Kawachi, Tochigi.
Present address: University of Alabama, Birmingham, USA.

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25

purpose, we produced recombinant viruses, each carrying a chimeric M gene
between Aichi and WSN after transfection of chimeric RNAs, which were
produced in vitro by transcribing hybrid cDNA (Yasuda, J. et al. (1994) J.
Virol. 68, 8141-8146. Analysis of the growth rate for these transfectant
viruses indicated that the WSN M 1 protein is responsible for rapid virus
growth. Since M1 binding to RNP interferes with RNA polymerase functions (Hankins, R.W. et al. (1989) Virus Genes 3, 111-126; Hankins, R.W. etal. (1990) Res. Virol. 141, 305-314), the rate of M1 association to and/or M1
dissociation from RNP is different between WSN and Aichi M1 molecules
and this difference may ultimately lead to changes in virus growth rate.
Host Factors for Influenza Virus Growth Control:
Cellular Factor(s) for Influenza Virus RNA Replication
Tetsuya TOYODA and Akira ISHIHAMA

Replication of the influenza virus genome involves two discrete step
ractions: vRNA-directed primer-independent (unprimed) synthesis ofcRNA; and cRNA-directed unprimed synthesis of vRNA. Nuclear extracts
from both MDCK and HeLa cells infected with influenza virus A/PR/8/34
exhibited model vRNA template-directed unprimed synthesis of not only
complementary model RNA (model cRNA) but also model vRNA (Toyoda,
T. et al. (1994) Arch. Virol. 136, 269-286). Ribonucleoprotein (RNP)
complexes with the replication activity were isolated from these nuclear
extracts by gelycerol gradient centrifugation in the presence of 0.1 M KCl.
At 0.5 M KCI, however, these complexes were dissociated into stripped RNP
and soluble protein fractions. The soluble protein fraction contained theactivity of exogenous template-dependent unprimed RNA synthesis, indicating that the RNA replicase is dissociated from RNP upon exposure to highsalt concentrations. On the other hand, the high salt-treated RNP catalyzed
only primer-dependent RNA synthesis, but regained a low level activity ofexogenous template-dependent unprimed RNA synthesis by adding nuclear
extracts from uninfected cells, suggesting that host factor(s) are involved in
the functional conversion of inlfuenza virus RNA polymerase from transcriptase to replicase.

26

Host Factors for Influenza Virus Growth Control:
Antiviral Activity of Mouse Mxl Protein
Tetsuya TOYODA, Yukiyasu ASANO and Akira ISHIHAMAThe Mx1 protein is an interferon-inducible nuclear protein of mice, which
confers resistance to influenza virus infection. Using Mxl purified from both
the Mx1 gene-expressing E. coli and interferon-treated mouse liver, wedemonstrated that mouse Mx1 is a GTP-binding protein with GTPase
activity (Nakayama, M. et al. (1991) J. Bioi. Chern. 266, 21404-21408;
Nakayama, M. et al. (1992) Virus Res. 22, 227-234) and forms selfassemblies (Nakayama, M. et al. (1993) J. BioI. Chern. 268, 15033-15038).
In order to determine whether the GTPase and self-assembly activities are
required for antiviral function of Mx1, we constructed mutant Mx1 with
mutations in the regions requied for GTPase and self-assembly and examined
their antiviral activity.
For this purpose, we established cell lines expressing wild-type and mutant
Mx1 proteins each carrying single amino acid substitution. A mutant Mx1(S501) with Ser-to-He substitution at amino acid residue 50 within the GTPbinding motif has reduced GTPase activity (Nakayama, M. et al. (1993) J.
Bioi. Chern. 268, 15033-15038). Wild-type Mx1 forms big dots of selfassembles in induced nuclei, while the S501 formed linear threads of Mx1 incell nuclei. The growth of influenza virus was not affected so much even in
the presence of S501 Mxl. Another mutant (C71S) with an amino acidsubstitution of Cys-to-Ser at position 71 within the self-assembly domain
showed diffuse distribution in the nuclei. The virus growth inhibition was notaffected by the C71S mutation, suggesting that the self-assembly domain is
not involved in antiviral activity (Toyoda, T. et al., submitted for publication).

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Abnormal Integrity of Nucleolus Associated with Cell Cycle Arrestin A Mouse Cell Mutant of The Temperature-Sensitive
Ubiquitin-Activating Enzyme El
T. SUDHA*, Sumiko KANEDA, Yukiko NAGAI, Fumiaki YAMAO,
Hideo TSUJI**, Masazumi SAMESHiMA*** ,Yoichi MATSUDA** and Takeshi SENO

The ubiquitin-activating enzyme, El, catalyzes the first step of the sequential transfer of ubiquitin to target proteins. The ubiquitin system is essential
for a wide spectrum of cellular phenomena, including cell cycle control,
induced error prone DNA repair, and the stress response.
Temperature-sensitive (ts) mutants of the ubiquitin-activating enzyme, El,
isolated from a mouse cell line FM3A including the authentic ts-El mutant,
ts85, give rise to different arrest points in the cell-cycle, although the mutants
fall into the same complementation group. The mutation site in the El
coding region was mapped to show that each mutant had a different singleamino acid change but the mutation sites were clustered somewhat at the
C-terminal half of the El protein (Kaneda et ai., submitted). However, themolecular consequences leading to different cell cycle arrests have not yet
been explained.
The long term goal is to identify the target proteins, whose ubiquitination
via a specific E2(s) is impaired due to the mutation of El, which leads to a
specific terminal mutant phenotype. We attempted to identify genetic traitsof a more qualitative nature other than the ritual flow cytometric profile.
Through cytological analysis, we observed with Azure C staining, a morphological change in the nucleolus in G2-arrested mutant ts85 cells after
exponentially growing cells were incubated for 10 h at the nonpermissive
temperature (39°C). The altered nucleolar structure consisted of a V-shapedor ring-shaped arrangement of nucleolar lobes with an unstained region in thecenter. Silver staining of the nucleolar organizer region (NOR) and fluorescence in situ hybridization analysis with rDNA, both gave signals in Azure
C-positive regions as expected. Electron microscopic examination revealed
that a cloud of unidentified electron-dense particles of homogeneous size
(diameter, approx. 70nm) was present in the Azure C-negative region
*
**
***

On leave of absence from the Down's Research Society (India), Madras, India.
National Institute of Radiological Sciences, Chiba.
The Tokyo Metropolitan Institute of Medical Science, Tokyo.

28surrounded by the nucleolar lobes (Sudha et aZ., Chromosome Res., in press).
Immunochemical and biochemical characterizations of the electron-dense
particles are in progress.
When the arrested ts85 cells were released into metaphase, we observed apeculiar pulverization-like figure consisting of several chromosomes arrayed
together. NOR-silver staining, fluorescence in situ hybridization studies with
rDNA, and centromeric satellite DNA, consistently demonstrated that thepulverized regions coincided with those of NORs. Judging from the results,
this pulverization may reflect under-condensation of the NORs associatedwith delayed dissolution of the nucleolus during G2-M transition (Sudha etaZ., Chromosome Res., in press). Attempts were made to substantiate thispossibility, by chasing the abnormal nucleolar structure following releasefrom the arrest. It appears that the ubiquitin system plays a role in the
dissociation processes of the nucleolus, possibly involving the NOR.
Cell cycle control by the ubiquitin system in mammalian cells;
the ubiquitin-activating enzyme, El, is targetedby cdc2 kinase
Yukiko NAGAI, Sumiko KANEDA, Takeshi SENO
and Fumiaki YAMAO

The ubiquitin system is essential for cell growth since the mutational blockof one of the ubiquitin-conjugation steps is lethal to cells, arresting them atspecific stages of the mitotic cell cycle. Historically, ts85, a temperaturesensitive (ts) mutant of the mouse mammary carcinoma cell line FM3A, hasbeen shown to be defective in the ubiquitin-activating enzyme and arrested at
the late S to G2 period of the cell cycle under restrictive temperatures.
Recently, other types of El mutants from the same cell line were also
reported; some are arrested predominantly at S phase while others arearrested at all phases of G 1, Sand G2fM, implying that multiple steps during
the cell cycle are differentially regulated by the ubiquitin system. In addition,a series of key proteins regulating the cell cycle were shown to be ubiquitinated. Cyclin B, a regulatory subunit of the mitosis promoting factor, hasbeen shown to be degraded by the ubiquitin-dependent proteolytic pathway.
p53 and c-Mos, have also been reported to be ubiquitinated at least in vivo.
Thus, the conjugation of ubiquitin to target molecules is associated withI.

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various functions of cell cycle progression, presumably through the proteolytic breakdown of the proteins involved in the essential function of theprocesses.
It has been well established that the transitions of the critical points in the
eukaryotic cell cycle is controlled by a series of cyclin dependent protein
kinases. A variety of mammalian cyclins and kinases associated with them
have been identified. Their functions, however, are largely unknown except
for cyclin Bjp34cdc2. Which cyclin associates with which kinases, the timingof the associations, and the in vivo substrates of the kinases are a matter ofgreat interest. To investigate the coupling of the ubiquitin system and thecyclin dependent kinases during cell cycle control, we examined the cdc2
kinase mediated phosphorylation of the ubiquitin activating enzyme El inmouse cells. In the amino acid sequence of mouse E 1 enzyme deduced fromits cDNA, at least five possible target sites of phosphorylation by cdc2 kinase
were found. The E 1 enzyme from mouse cells proved to be phosphorylated
in viVo. This phosphorylation was shown to be mediated by cdc2 kinase since
FT21O, a temperature-sensitive mutant of cdc2 kinase, failed to achieve
phosphorylation. In vitro phosphorylation of cellular and recombinant EI
proteins by immuno-purified cdc2 kinase, and their peptide mapping analysis
indicated that the serine residue at the fourth position of the EI amino acidsequence was the phosphorylation site both in vivo and in vitro. These results
strongly suggest that the E I enzyme is a substrate of cdc2 kinase in the cell.
Such a post-translational modulation of El may afford enzyme specificfunctions towards the recognition of cell cycle-dependent ubiquitin pathways.
Cell cycle control by the ubiquitin system in mammalian cells;
Ubiquitin pathways specific for DNA synthesis
Sumiko KANEDA, Yukiko NAGAI, Takeshi SENO,
Tatsuo YAGURA* and Fumiaki YAMAO

Ubiquitin is activated by the ubiquitin-activating enzyme, EI, and ATP istransferred from the El enzyme to one of a family of isoenzymes calledubiquitin conjugating enzymes (E2's) which catalyze the conjugation ofubiquitin to specific target proteins. There is increasing evidence that each E2*

Department of Biology, Facuity of Science, Kansei Gakuin University, Nishinomiya
662.

30

enzyme has a preferred target protein(s). Detailed analysis of pleiotropic
phenotypes of mutations of the ubiquitin-activating enzyme (El) showedthat the inhibition of the ubiquitin system caused cell cycle arrest not only atphase G2/M but also at Gland S in mammalian cells. To identify theubiquitin pathway associated with each phase of the cell cycle, we examinedin vitro ubiquitin transfer from El enzyme to E2 conjugating enzymes in theextracts of various ts El mutants of the mouse cell line, FM3A. Ubiquitintransfer to an E2 protein of approximately 20 kDa was found to be reduced
specifically in the lysates of mutant cell lines which were predominantly
blocked in the S phase at restrictive temperature (39°C). From such S
phase-specific El mutants, we isolated partial revertants by selecting clones
that could grow at the semi-restrictive temperature, 37°C, but not at 39°C.
The arresting points during the mitotic cell cycle of these revertants at
restrictive temperatures were Gland G2/M phases, specifically suppressing
the defect in S-phase progression of the parental mutant cell lines. In theextract of the partial revertants of El mutants, the reduced ubiquitin transfer
to the particular E2 was restored. Thus, E2 (20kDa) appears to be involved
specifically in DNA synthesis. The E2 proteins including that of 20 kDa,
were purified and subjected to partial amino acid sequences for cloning of
their genes. A 2.6kb mouse El cDNA encoding the El protein lacking thefirst 255 amino acid residues, was transfected to various El mutant cells, andfound to complement their defect in S-phase progression, but not in G2-phaseprogression. This suggested a domain for the El enzyme, which is necessary
for recognition of the S phase-specific ubiquitin pathway, in the C-terminal
two thirds of the enzyme. The S phase-specific ubiquitin pathway is possibly
mediated by the E2 enzyme described above. On the contrary, the Nterminal fragment of the El enzyme seems to be essential to the G2phase-specific ubiquitin pathway, which supports the fact that the cdc2
kinase-mediated phosphorylation of the El enzyme is at its fourth serine
residue, if the phosphorylation has any functional role at all.
Cell Cycle Control by the Ubiquitin System in Yeast
Saccharomyces cerevisiae
Tsutomu KISHI, Fumiaki Y AMAO and Takeshi SENO

Yeast cells harboring a temperature-sensitive mutation in the CDC34 are

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31

arrested at the G liS phase of the cell cycle. CDC34 has already been shownto code for the ubiquitin-conjugating enzyme E2 (UBC3). Therefore,
degradation of some specific target proteins has been suggested to be essential
for cell cycle progression.
The aim of this project is to isolate and characterize the proteins to beubiquitinated by the Cdc34. In addition, we are trying to isolate the gene(s)of the protein(s) that interacts with Cdc34.
This is a new project in this laboratory. The approaches and present results
are as follows.
(1) Isolation of extragenic suppressor of cdc34 ls : We have isolated four
suppressor strains and one of the four genes was cloned and is being analyzed.
(2) Cloning of the genes whose products interact with the Cdc34: Withthe use of the yeast two-hybrid system, several candidates were screened, andtheir characterizations are under way.
(3) Detection of target proteins of the Cdc34 by using in vitro system:
Cdc34 was expressed and purified from E. coli. First, this was mixed withlabeled ubiquitin and yeast crude extracts prepared from the G 1 phase of thecell cycle. However, the protein by CDC34 was not detected. Now, we aretrying to detect the protein using yeast nuclear extracts.
Reference
F. YAMAO, Y. NAGAI, S. KANEDA, S. YOSHIDA, and T. SENO. Conditional resistance tothymineless death predominantly selects DNA synthesis- deficient mutants of mammaliancells. Mutat. Res., 289: 83-89 (1993).

Mechanism of mRNA Capping Reaction
Kiyohisa MIZUMOTO

The yeast mRNA capping enzyme is composed of two polypeptide chainsof 52 kDa (a) and 80 kDa (/3), responsible for the activities of mRNA
guanylyltransferase and RNA 5' -triphosphatase, respectively. Recently, we
have isolated the gene encoding the a subunit (CEGl) (Shibagaki, Y. et al.
(1992) J. BioI. Chern. 267, 9521-9528). In this study, the CEGl gene wasexpressed in Eschen'chia coli under the control of a bacteriophage T7 promoter and the a subunit protein was purified to near homogeneity. The enzyme32[ P]GMP covalent reaction intermediate of the recombinant a subunit wasdigested with lysylendopeptidase and trypsin, and the resulting [ 32 p]GMP_32epeptide was purified. After the [ 32P]peptide was converted to 2p]phosphoryl-peptide through periodate oxidation followed by ,B-elimination, it washydrolyzed with alkali to give a [ 32P]phosphoamino acid. [ 32 p](N-E)phospholysine was detected as the only phosphoamino acid, indicating that GMP
in the enzyme-GMP intermediate is bound to a lysine residue via a phosphoamide linkage, as previously observed with rat liver and vaccinia virus
capping enzymes (Mizumoto, K. and Kaziro, Y. (1987) Prog. Nucl. Acid
Res. Mol. BioI. 34, 1-28). Microsequensing of the P2P]GMP-peptide revealed that the GMP binding site was localized to a region between amino acids
60 and 75, which contained an internal trypsin-resistant lysine residue at
position 70. The CEGJ gene was subjected to site-directed mutagenesis invitro and mutant polypeptides were expressed in E. coli. Substitution of Lys
70 to His or lIe abolished ability to form the enzyme-GMP complex. These
results indicate that the GMP binding site in the a subunit of yeast capping
enzyme is localized to a lysine residue at position 70.
Transcription of Sendai Virus (HV J) Genome
Kiyohisa MIZUMOTO

Sendai virus (HVJ), a member of paramyxovirus group, contains a single
non-segmented negative-strand RNA genome of approximately IS kb-long.
The genetic information of this RNA genome is expressed through at least 6
monocistronic mRNA species that are transcribed by a virion associatedRNA-dependent RNA polymerase. We are studying the mechanism oftranscription of the HVJ genome using an in vitro transcription system withpurified virus particles, in which mRNA synthesis is almost entirely dependent on host factors. In HVJ-infected cells, an abundant, short and uncappedplus-strand leader RNA encoded by the first -50 nucleotide sequence of the
3'-genomic terminus is synthesized. However the significance of leader RNA
synthesis has not been understood yet. In the present study, to investigate thecorrelation of plus-strand leader RNA synthesis with mRNA production, wecharacterized the synthesis of short RNA species in the in vitro transcription
system.
Transcription reacton was carried out in the absence of UTP to discriminate the leader transcripts from nascent short mRNA chains. Several
discrete short RNAs ranging 20-25 nucleotides in length, which cor-

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33

responded to the sizes expected from premature termination points at U
residues, were synthesized. The 5'-termini of major RNA species were foundto be (p)ppA- by post-labeling procedure with mRNA capping enzyme.
They formed duplex molecules when annealed with the minus-strand genome
RNA (vRNA). These results support that the short RNAs synthesized invitro are the products transcribed from the 3'-end leader regions of the
genome and have the positive polarity. The synthesis of these short RNAs
was almost completely dependent on the addition of host cell extracts. An
activity that stimulates the leader RNA synthesis was partially purified fromHeLa cells and was shown to be separated from the host factor activity
required for mRNA synthesis.

Wx gene Expression is Enhanced in Response to Cool Temperatures
Hiro-Yuki HIRANO, Motoji KUNI-EDA* and Yoshio SANO

The wx locus controls amylose synthesis in the endosperm and pollen. We
found that Wx gene expression was enhanced by exposing plants to cooltemperatures during seed development and the amylose content increased inmature seeds causing a lower quality of rice.
Rice plants were grown during seed maturation at two different temperatures, 28°C (normal) and 18°C (cool), and the amount of Wx protein andamylose content in mature seeds were estimated. The results indicated that
not only amylose content but also the amount of Wx protein in seeds matured
at 18°C were higher than those matured at 28°C.
Northern blot analysis showed that the steady state level of the Wxtranscript increased about 2- or 3-fold by exposing the plants to a temperatureof 18 °C and it decreased to the initial level after the plants were grown at
28°C for 2 days. These results suggest that the response to cool temperatures
is regulated at the transcriptional level of the Wx gene and this regulation isreversible.
Next, plants were grown at 18°C for different lengths of time, 1, 2, 4, 8, or
12 days from the 7th day after anthesis, and then grown at normal temperatures (in the field) until seed maturation. The longer plants were exposed toa temperature of 18°C, the higher the levels of amylose and Wx protein
accumulated in mature seeds. The extent of increase in amylose content was
positively correlated with that of the Wx protein level.
*

Meiji Univ.

34

These results indicated that the Wx gene was activated by cool temperatures, causing the elevation of the Wx protein level. Thus, the elevated levelsof Wx protein, the enzyme for amylose synthesis, resulted in a higher content
of amylose in matured seeds.
The Wx gene transcript increased at 10 and ISoC but not at 4 or 35°C.
These results indicated that cold or heat shocks did not affect the expression
levels of the Wx gene. Thus, the Wx gene may be activated only by cooltemperatures.
Gene regulation responding to slight differences of temperatures may bevery important in the adaptation of plants for normal growth. The Wx gene
may be one of the best clues for investigating the regulation in response toslight differences of temperatures.
Kinetic Study on Transcription Using Immobilized Operons:
Early events in transcription elongation
Tomoko KUBORI and Nobuo SHIMAMOTO

A template DNA fragment was fixed to acrylamide beads by the tightbinding between avidin and biotin. The immobilized DNA was a good
template in transcription by E. coli RNA polymerases. This techniqueenabled us to separate transcripts retained in the transcription complexes
from those released with brief centrifugation. In addition, dead-end complexes which lost elongation activity can be assayed by rapidly diluting areaction mixture with a new optimal substrate solution. We studied therelationships between active transcription complexes, dead-end complexes,
and abortive release.
In the E. coli transcription system, we found the abortive release is greatly
activated in the presence of polymerase in excess of a promoter. Whentranslocation of a polymerase on DNA is inhibited by another preceding
molecule stuck at 32nt from a promoter, a misincorporation at 6nt was foundin addition to further enhancement of abortive release. The misincorporated
transcripts were never elongated into long RNA even when the stacked
molecule was removed. Thus misincorporation was checked in the early steps
in elongation.
The immobilized DNA was preincubated with holoenzyme and packed ina 5/11 column. Reactions were started by eluting the column with a buffer

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35

containing substrates. Kinetics of releases of abortive transcripts and andsigma factor were thus studied.
Single-Molecule Dynamics of Transcription Using Immobilized
Operons: Sliding of P. putida CamR protein on DNA
Hiroyuki KABATA and Nobuo SHIMAMOTO

The sliding motion of RNA polymerase along DNA during promoter
search was proved by direct visualization of single molecules of the enzyme.
To address the question whether sliding motion is common to other DNA
binding proteins, we applied a similar single molecule dynamics to a bacterial
repressor protein, P. putida CamR. The protein was observed to slide along
DNA, and the movement is common to the proteins so far examined. In theabsence of its inducer, d-camphor, CamR molecules were trapped threepositions on a A DNA, one was its cognate operator cloned on A DNA, andthe other two were likely to be regions in A homologous to the operator. The
DNA fragments containing the homologues regions competed with the
cognate sequence in a gel-shift assay when they were added 400 and 4,000
times excess to the cognate fragments. They are, therefore, very weakbinding site of CamR by gel-shift assay, but CamR stayed on them for a longtime in the direct visualization, showing the visual detection is a very sensitive
method.
Addition of the inducer, d-camphor, significantly decreased trapped molecules, but the frequency of sliding increased. The result showed that theinducer destabilized specific interactions but not those for sliding. Theinteraction in the specific complex is different from that during sliding.
Functional and Structural Study of E. coli Single-Stranded
DNA Binding Protein
Hiroki NAGAI, and Nobuo SHIMAMOTO

E. coli Single-Stranded DNA Binding Protein (SSB) is a 19 kda protein
and essential to replication and recombination. In physiological conditions,
it exists as a tetramer and binds to both single-stranded DNA and a group ofmRNAs which have a homologous RNA element, termed SSB box. Both of
the bindings are cooperative and the protein molecules form clusters on such

36

nucleic acid molecules. These bindings suggest a coupling between replication and translation through the exchange of SSB between replication forks
and mRNAs. Because the translation initiation region of the ssb genecontains three putative SSB boxes, it is possible that the expression of ssb geneis regulated autogenously. We investigated the expression from ssb-lacZ
fusion genes in cells overproducing or depleting SSB protein. The results
showed, however, that cellular amounts of SSB protein hardly affect theexpression of ssb-lacZ fusion genes, suggesting that autogenous regulation of
the ssb gene expression through putative SSB boxes is unlikely.
Accumulation of single stranded DNA in cells induces SOS response, oneof the most intensively studied stress responses in bacteria. The ssb gene has
SOS box (LexA binding site) around - 35 position of minor promoter (P I).
In contrast it has not been clear whether the synthesis of SSB protein is SOS
inducible. We directly examined both the synthesis rate of SSB protein andthe level of SSB mRNA upon exposure to SOS inducing agents. The protein
synthesis rate increased by 2 fold upon SOS induction. Amounts of transcripts from PI promoter increased by 10 fold but that from major constitutive P2 promoter was hardly affected: total amounts of mRNA increased by
2 fold. These results indicated that SSB protein is SOS inducible and theinduction occurs primarily at transcription level likely as the other SOS
inducible genes.
Diploidy of Drosophila Imaginal Cells is Maintained by aTranscriptional Repressor Encoded by Escargot
Naoyuki FUSE, Susumu HIROSE and Shigeo HAYASHIThe Drosophila escargot (esg) gene encodes a C2-H2 type zinc finger
protein. esg is expressed in imaginal discs and histoblasts in the embryo andin the larva. In some esg mutants, abdominal histoblasts become polyploid.
In our previous work, we suggested that the role of esg is to maintain the
diploidy of the imaginal cells. We have found that esg encodes a DNA
binding protein with high affinity to G/ACAGGTG, the consensus sequence
for the bHLH family transcription factor. DNA binding activity is essential
for esg function in vivo since one of the strong embryonic lethal alleles iscaused by an amino acid change within the zinc finger which reduces affinity
to DNA. In cultured cells, esg protein strongly inhibits transcriptional

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37

activation by a heterodimer of bHLH proteins SCUTE and DAUGHTERLESS, suggesting that esg may regulate processes dependent on bHLH
proteins in development. In embryos, esg protein expressed by the heat shock
promoter can rescue the polyploid phenotype of abdominal histoblasts,
demonstrating that the phenotype is due to a loss of esg function. esg
expression is required continuously during the larval period for efficient
rescue. Ectopic expression of esg in the salivary gland inhibited endoreplication. These results suggest that esg is involved in transcriptional inhibition ofendoreplication.
Control of esg Expression During Development
Yoshimasa YAGI*, Susumu HIROSE and Shigeo HAYASHI

esg is expressed in a complex pattern during embryogenesis. In thatpattern, expression in the imaginal cell primordia is thought to be responsible
for mainteinance of diploidy. Expression in embryonic gonads and testes atlater stages has a relevance for esg's role in spermatogenesis. In addition, esg
is expressed in a subset of embryonic tracheal cells which play an importantrole in tracheal morphogenesis. To understand the role of esg in thosesystem, it is important to know how such expression patterns are established.
To analyze the regulatory mechanism of esg expression, we have started todissect the cis-control region for enhancers responsible for expression in
imaginal tissues, testes, and trachea. A lacZ fusion construct containing 3.5
kb of esg promoter was introduced into the germ line to establish transformants and lacZ expression was examined in embryo, larva and adult. This
construct did not reproduce the expression pattern in imaginal and tracheal
cells, but we found that lacZ was expressed in a subset of adult testis, similarin pattern to several esg enhancer trap lines. This expression in testes isprobably responsible for the function of esg in fertility. We are currently
making more constructs to search further upstream for other enhancers.
Once we find such enhancers, we are planning to analyze interaction withgenes such as wingless.

* On leave from Department of Biology, Faculty of Science, Nagoya University.

38

Analysis of the Role of the Tip Cells in Tracheal Morphogenesis
Yasuhiro SHIGA* and Shigeo HAYASHI

Morphogenesis of embryonic trachea involves initial determination ofprimordium from ectoderm, invagination, extention and final joining of
precursors from each hemisegment to form a tubular network. During thefinal joining process, a set of marker genes is expressed in a single cell at the
tip of each growing tracheal branch. These cells will contact each other andthe joining occurs. These cells are called the tip cells and are thought to play
important roles in pathfinding, target recognition and the fusion process. To
elucidate the role of the tip cell, we started the following experiments. i)Some esg mutants are defective in tracheal morphogenesis. We found that esg
is expressed in the tip cell and its expression is down regulated during fusionsuggesting esg plays an important role in that process. The exact role of esg
is being examined using various genetic and molecular techniques. ii) Direct
ablation of the tip cell should be an effective way to assess the role of the tip
cell. The laser microscope system recently set up in NIG will be used for thisexperiment. To identify targets for ablation, we are making constructs withvarious promoters fused to green fluorescent protein (GFP). GFP has beenused successfully as a marker in live C. elegance. The constructs are now
being injected into flies to establish transformants. From these projects wehope to solve the problem of pattern formation in this relatively simple organ.
The Function of argos in Regulating Cell Fate Decisions
during Drosophila Eye and Wing Vein Development
Kazunobu SAWAMOTO**. Hideyuki OKANO**. Yoshitaka KOBAYAKAWA***.
Shigeo HAYASHI. Katsuhiko MIKOSHIBA**
and Teiichi TANIMURA***The Drosophila argos gene, which was previously called strawberry, encodes
a secreted protein with an EGF motif. argos is involved in several developmental processes regulating cell-cell interactions such as eye morphogenesis.
Loss-of-function mutations in the argos gene cause an increase in the numberof photoreceptor cells and cone cells, impaired retinal projections to the optic
*
**
***

On leave from Faculty of Agriculture. Nagoya University.
University of Tokyo.
Kyushu University.

I.

MOLECULAR GENETICS

39

lobe, and the formation of extra veins. We found the ubiquitously expressedargos product restored all these loss-of-function phenotypes. Overexpressionof argos in the wild-type background resulted in a reduced number ofphotoreceptor cells, cone cells, and pigment cells, which are phenotypes
opposite to those of the loss-of-function mutants. The argos gene is expressedin developing wing veins. Ubiquitous argos expression caused loss of veins ina dose dependent manner. This phenotype was enhanced by the loss-offunction rhomboid mutation, implying the possibility that argos and rhomboid
play key roles in a common pathway for normal wing vein formation. We
propose that argos acts as an inhibitory signal for cellular differentiation in
the developing eye and wing. This work will be published in Developmetal
Biology (Sawamoto et al., (1994) Dev. Bio/. in press).

40

II.

MICROBIAL GENETICS

The Nicking Reaction in Rolling Circle Replication
Requires a Single-Stranded Nicking Region anda Double-Stranded Binding Region
Atsushi HIGASHITANI and Kensuke HORIUCHI

The core origin for plus strand DNA replication of filamentous bacteriophage fl binds the initiator protein (gpII), which subsequently introduces a
specific nick in the plus strand. The origin consists of a nicking region and abinding region. We previously proposed that the nicking reaction with gpII
occurs via the following steps: 1) binding of gpII forms bent DNA structuresin the origin (no requirement for either Mg2+ or negative superhelicity forthis step); 2) the DNA bending induces melting of the duplex structurearound the nicking site if the DNA is negatively superhelical; and 3) a specificnick is introduced within the melted region in the presence of Mg2+ (Higashitani, A., et a/., (1993) Ann. Rep. of NIG, 43, 36-38).
In this study, the ability of gpII to cleave DNA when the nicking site is ina single-stranded state was tested using synthetic oligodeoxyribonucleotides
as substrates. One substrate used was a 5' -terminally labeled 39-mer thatcarries the fl plus strand sequence ranging from position - 9 to + 30
(relative to the nicking site), which includes the entire gpII binding sequences{3, rand o. Upon cleavage at the nicking site, this substrate should yield a 9mer fragment from its 5' end. The results showed that an extremely weakcleavage reaction took place yielding an 8-mer and a 9-mer in approximately
equal amounts. The reaction was extremely slow, continuing for more than
120min, and only about 1 per cent of the substrate was cleaved in 120 min.
When RFI was the substrate under the same conditions, the reaction wascompleted within 1min, with 60 percent of the substrate nicked. To make thegpII binding domain double-stranded, while keeping the nicking domain
single-stranded, the 39-mer was annealed to a 25-mer oligonucleotide of the
fl minus strand sequence, ranging from position + 29 to + 5, and was
subsequently used as a substrate for the nicking reaction. This annealed
product was completely cleaved by gpII in less than 5 min to yield the 9-mer.
The results indicate that efficient nicking requires the presence of the gpIIII.

MICROBIAL GENETICS

41

binding domain in a double-stranded form. Since restnctlOn fragments(linear duplexes) carrying the complete f1 origin are very poor substrates for
gpII nicking (Higashitani, A., et al., (1992) Nucl. Acids Res. 20,2685-2691),
the results also indicate that the region immediately flanking the nicking siteneeds to be single-stranded for the cleavage reaction to occur. In fact, whenthe entire region of the 39-mer was made double-stranded by annealing witha complementary oligonucleotide, the cleavage reaction was strongly inhibited. These results indicate that efficient nicking reaction with gpII takes
place only when the substrate DNA carries a single-stranded nicking region
and a double-stranded binding region. It is thus likely that the duplex
structure around the nicking site must melt first for the nicking to occur inthe natural substrate, f1 RFI.
SOS Induction in Escherichia coli by Single-stranded DNA of
Filamentous Phages: Monitoring Through Cleavage of
LexA Repressor
Nahoko HIGASHITANI, Atsushi HIGASHITANI
and Kensuke HORIUCHI

We previously reported that the SOS response is induced in Escherichia colithrough infection with mutant filamentous phages that are defective ininitiation of the complementary (minus)-strand synthesis (Higashitani, N., etal., (1992) J. Bacterial. 174, 1612-1618). In these mutants, the rate ofconversion of parental single-stranded DNA (ssDNA) to the doublestranded replicative form (RF) was extremely slow. Conversion to RF of the
ssDNA molecules that were produced by rolling-circle replication also appeared very slow. These observations strongly suggested that it was phage
ssDNA that induced the SOS response. However, since the mutants were still
able to synthesize RF to some extent, and since we monitored the SOS
induction through the expression of SOS genes, it was not possible toexclusively conclude that the phage ssDNA alone induced the SOS response.
In the present study, we monitored the SOS induction through cleavage of the
LexA repressor, which represents the first step of SOS response, after phage
infection in the presence of chloramphenicol. The LexA levels in the infected
cells were measured by Western blotting using the anti-LexA antibody. The
results indicated that infection with the mutant phage reduced the cellular42level of LexA within a few minutes. Infection with the wild-type phage did
not affect the level of LexA. These results unambiguously show that theparental ssDNA by itself can induce the SOS response in infected cells.
Stabilization of DnaA Protein with Chaperone ProteinsSeiichi YASUDA

DnaK is a major heat shock protein of Escherichia coli and participates ina variety of cellular processes including cell division and DNA replication.
We have previously shown that DnaK protects the initiator protein DnaAfrom heat inctivation by forming a complex with it. DnaA which is normallyinactivated completely by heating at 40°C for 10min, becomes resistant toheating at 60°C for lOmin in the presence of DnaK. In these experiments,
the activity of heated DnaA was assayed through in vitro replication of oriC
DNA using a crude enzyme fraction. When DnaA was assayed by oriC
DNA-binding without using the crude enzyme, the resistance of DnaA
against heating at 60°C in the presence of DnaK was not observed. Thisimplies that the crude enzyme fraction contained activity which reactivates
DnaA that had been heated at 60°C in the presence of DnaK. An assay
system for this reactivation activity was established. None of the E. coli heatshock proteins, DnaK, DnaJ, GrpE, GroES or GroEL could substitute forthe crude enzyme fraction. The reactivating activity is being isolated fromthe crude enzyme fraction of an E. coli strain carrying a temperaturesensitive dnaA mutation.
A Mutation that Suppresses Thermosensitive Growth Defect at Low
Osmolarity of the Escherichia coli 4prc Mutation Is in theGene Coding for Penicillin-Binding Protein 7
Hiroshi HARA, Noriko ABE* Yukinobu NISHIMURA*
and Kensuke HORIUCHI

The prc gene of Escherichia coli codes for a periplasmic processing enzymethat removes the C-terminal 11 residues from the precursor form of penicillin-binding protein 3 (PBP 3), a murein-synthesizing enzyme essential for
septum formation. A prc deletion mutant shows thermosensitive (ts) growth*

Department of Biomolecular Sciences, Faculty of Science, Toho University, Funabashi.

II.

MICROBIAL GENETICS

43

at low osmolarity. However, it does not form long filaments, a typicalcellular morphology found whenever the function of PBP 3 is inhibited(Hara et al. (1991) J. Bacterial. 173,4799-4813). The processing defect of
PBP 3 seems unlikely to be directly related to the growth defect of the b.prc
mutant.
We expected that the gene(s) of the target(s) of the Pre vital function
other than PBPF3 might be found as suppressor mutation(s) of b.prc, andisolated 22 independent, spontaneous suppressor mutants that restored
growth on salt-free L agar plates at 42°C. None of them restored PBP 3
processing. As for the leakage of periplasmic proteins into the medium by theb.prc mutant, only partial suppression was observed. When a prc t -carrying
mini-F plasmid was introduced, they all showed ts growth at low osmolarity
again. All the isolates looked very much alike, and one of them was further
studied.
First b.prc was displaced with the wild-type allele that was co-transducedwith eda by PI phage. The resultant strain was ts on salt-free L agar.
Chromosomal TnlO insertions co-transducible with this ts mutation wereselected from a random TnlO insertion pool. Transductions of these TnlO
insertions from strains that also carried the ts mutation to a b.prc strain gavethermoresistant transductants at expected co-transduction frequencies, showing that this ts mutation was identical to the suppressor. It seems that thismutation and b.prc similarly affect the protection of cells against thermal andosmotic stresses and are mutually suppressors of each other. The most tightly
linked TnlO insertion (90% co-transducible) was mapped to the region alittle beyond his at 44 min in a clockwise manner by Hfr matings, and then by
PI transduction, to the vicinity of fruA at 47min (75% co-transducible).
This TnlO insertion was co-transducible with other 21 suppressor mutations
at high frequencies. All these 22 suppressors isolated independently are mostprobably allelic to one another.
Among cosmids carrying chromosomal fragments around the 47 minregion in the ordered cosmid library (Tabata et al. (1991) J. Bacterial. 171,1214-1218) some were found to complement the ts mutation, and a 3.5kilobase EcaRI-HindIII fragment carrying the complementing ability was
subcloned. The nucleotide sequence of a part of this fragment has alreadybeen reported together with the adjacent did gene (Campbell et al. (1984)
Eur. J. Biachem. 144, 367-373). It included a 3'-terminal region of an open
reading frame (ORF). Removal of this region abolished the complementing

44

ability, indicating the ORF is the wild-type allele of the suppressor, which we
named sprA (~uppressor of prc). Its precise map position was determined as
47.8 min by comparison with the revised E. coli genomic restriction map(Rudd (1992) in A Short Course in Bacterial Genetics, pp. 2.3-2.43, Cold
Spring Harbor Laboratory Press). The product of the sprA gene was identified as a protein of about 31 kilodalton (kDa) by a maxicell experiment.
Examination of the deduced amino acid sequence of the C-terminal region
revealed high homology to the C-terminal region of E. coli PBPs 5 and 6.
Through PBP assay using radioactive penicillin we found that a sprAcarrying plasmid led to an overproduction of PBP 7 of about 31 kDa in an
ompT mutant, and also of PBP 8 of about 28 kDa in a wild-type strain. The
latter was reported to be a degradation product by OmpT protease of theformer during envelope fraction preparation for PBP assay (Henderson et al.
(1994) J. Bacteriol. 176, 256-259). PBP 7 was not reproducibly detected
unless overproduced, and has not been studied in detail. Sequencing of thewhole sprA gene, cloning and sequencing of the suppressor allele, and thestudy of the effect of the /).prc mutation on PBP 7 is needed to elucidate thepossible function of PBP 7 against thermal and osmotic stresses and themechanism of the suppression.
A General and Fast Method for Mapping Mutations on the
Escherichia coli Chromosome
Atsushi HIGASHITANI, Nahoko HIGASHITANI, Seiichi YASUDA
and Kensuke HORIUCHI

One of the first steps in characterizing newly isolated mutations is todetermine their positions on the chromosome map. In Escherichia coli, themapping is often carried out using genetic methods such as Hfr crosses ortransduction with P 1 phage, which usually require construction of strains
harboring a suitable set of genetic markers. With Kohara's ordered library of
DNA clones (Kohara, Y., et al., (1987) Cell, 50, 495-508) in hand, any pieceof chromosomal DNA or its transcript can now be mapped easily throughFig. 1. Schematic illustration of TnlO and the mapping method. Horizontal arrows, lIahl
and lIah2, indicate 31-mer (5'-GAT TTT TAC CAA AAT CAT TAG GGG ATTCAT C-3') and 28-mer (5'-CAT TAA GTT AAG GTG GAT ACA CAT CTT G-3')
oligonucleotides, respectively, used as PCR primers.

II.

#a.!11iaU3A

Sau3AI

Tn10

I

45

MICROBIAL GENETICS

l!ah2

I
7"

..,..I#ah2#ah1
Random insertions of Tn10
andselection or screening for Tn10insertions
co-transducible with a specific mutation: X

Sau3AI

I

I

Sau3AI

Tn10
I

I

II
mutation: X

Chromosome DNA•

Sau3AI

Sau3AI

Sau3AI digestion.----c:::lbf----,+

Self ligation

~~

\JU
+

PCR reaction
with primers #ah1 and #ah2

#ah2

0...,.

#ah1

!ah1

<-

p

~

0

#ah2

+
Fig. 1.
Hybridization of PCR products to Kohara's clones
and lordirect sequencing

46

hybridization to membranes that retain immobilized DNA of Kohara's
clones ("the E. coli gene mapping membrane" commercially available fromTakara). Therefore, if a general method to obtain DNA fragments from theregion of mutation were available, mapping of mutations would be greatly
facilitated. We have developed a general and fast method for determining
approximate positions of mutations on the E. coli chromosome. The method
consists of the following steps: (i) random insertion of transposon TnlO into
the mutant chromosome by infection with A496(b221, ell71 ::TnlO, Oam29),(ii) selection or screening for the TnlO insertion which is co-transduciblewith the given mutation, (iii) inverse PCR amplification of the chromosomal
region flanking the TnlO insertion site, and (iv) hybridization of the PCR
products to the ordered set of Kohara's clones (Fig. 1). TnlO carries 1400
bp inverted repeats, 1810, on its ends, and each repeat carries a Sau3AI site
at a distance of about 70bp from the end (Kleckner, N. (1983) in Mobile
Genetic Elements. Academic Press, pp 261-298). Hence, the border sequences between chromosomal DNA and TnlO can be cut out by Sau3AI
digestion. The digested DNA are circularized by ligation, and are subsequently used as the template for inverse PCR with two primers #ah 1 and #ah
2, which carry base sequences found in the segment between the Sau3AI site
and the end of TnlO (see Fig. 1).
In a model experiment, two genetic markers strR and lacZ, a selectable anda non-selectable mutation, respectively, were easily mapped to their known
sites using the present method.
Molecular Mechanism in Determination of Timing of Cell
Division in Escherichia coli K12
Akiko NISHIMURA

Cell division in normally growing cells strictly takes place 20 minutes after
DNA replication is terminated. This periodic process is not disturbed under
extensive growing conditions. Cells lacking DNA are found at a remarkably
low frequency in cultures of wild type strains. This fact suggests that the cellmust have mechanisms coordinating the timing of cell division to DNA
replication. I isolated novel mutants, cfcA,B, C,D,E,F, which uncouple the
DNA replication and cell division. The cfc mutants divide 1.3 to 1.5 times
more frequently per round of DNA replication. The cfcAI mutation was

II.

MICROBIAL GENETICS

47

within the a-subunit region of glycine-tRNA synthetase. From the genetic
and physiologic analysis of the cfcAI mutant, the cfcA gene was found to be
specifically involved in the regulation of cell cycle by a pathway different
from stringent response or SOS induction. The cfcBI mutation was mapped
at 1 minute on the E. coli genetic map and was complemented by a plasmidcarrying the apaGH region. The apaH gene encodes diadenosine tetraphosphate (AppppA) hydrolase and the apaH mutants show a ~ 16-fold increase in AppppA. It has been suggested that AppppA could be a pleiotropic
signal for cell growth and DNA replication (review: Zamecnik, P. (1983)
Anal. Biochem. 134, 1-10). AppppA is formed in vitro by aminoacyl-tRNA
synthetases in a reaction in which an enzyme-bound aminoacyladenylateintermediate donates AMP to ATP (Goerlich, O. et. al. (1982) Eur. J.
Biochem. 126, 135-142). However neither aminoacyl-tRNA synthetasemutants, inhibitory treatments of aminoacylation, nor structural mutants oftRNA are known to produce AppppA in vivo. The cfcAI mutation wascomplemented by an apaHt plasmid. This suggests that the cfcAI, themutant of glycine-tRNA synthetase, could synthesize AppppA in vivo andthat the AppppA nucleotide is involved in determining the timing of cell
division in a manner which couples cell division to DNA replication. I am
analyzing other cfc mutants to search for the targets of AppppA.
Correlation between Cell Division and the Nature of the Cell Membrane
Kengo KANAMURA, Hideyuki TAKAGI and Akiko NISHIMURA

In cell division, two main characters participate in the dramatic reaction,
one is a division machinery which divides the cell into two and the other is thecell membrane structure to be divided. For a systematic understanding of cell
division, biochemical studies should focus not only on the former characterbut also the latter one. However, so far, no one has studied the problem of
the correlation between cell division and the nature of the cell membrane.
Recently, we isolated seven temperature sensitive cell division mutants (jts)of E. coli which might have defects in the correlation. Thefts mutations werecomplemented by the 1.3 kb HindIII-PvuII DNA region at 27 min, including
a previously reported kdsA gene which encodes 3-deoxy-D-mannooctulosonicacid (KDO) 8-phosphate synthetase (Woisetschlager, M., and Hogenauer,G. (1987) Mol. Gen. Genet. 207, 369-373). KDO 8-phosphate is converted

48

to KDO by KDO 8-phosphatase and supplied as a part of the lipopolysaccharides (LPS). LPS is a major component of the outer membrane. Several
researches have reported that LPS is essential for maintaining the barrier
property of the outer membrane. It has also been suggested that a mutation
in KDO 8-phosphate synthetase in Salmonella typhimurium causes a defect inthe translocation of the lipid A precursor to the outer membrane andinhibition of the growth (Rick, P. D. and Osborn, M. J. (1977) J. BioI.
Chem. 252,4895-4903). Accordingly, it is likely that thesefts mutants of E.
coli inhibit cell division by somewhat altering the membrane structure,causing instability in the membrane, or through defficient translocation of
membrane proteins, at the non-permissive temperature. Because there havebeen no reports of any E. coli mutants in which a defect in membrane
structure causes inhibition of cell division, new findings about cell division
can be gained from this research. Weare now analyzing the mutation pointsof the seven mutants to ensure that the fts mutations are actually within the
kdsA gene.

49

III.

MAMMALIAN GENETICS

Establishment of New Strains As a Tool For Analyzing
Tumor Susceptibility Genes in Mice
Nobumoto MIYASHITA, Mika FUJII*, Akihiko MITA,
Toshihiko SHIROISHI and Kazuo MORIWAKITo determine the locus in the H-2 complex, which affects susceptibility to
the development of pulmonary adenomas in mice, H-2 congenic and recombinant strains were subjected to treatment with urethane. The differences
between these strains in susceptibility are probably due to polymorphism in
the two kinds of class II genes Ef3 and Ea, which encode the heterodimericglycoprotein expressed on the cell surface: the congenic strains with Ef3k andEa k alleles show more sensitivity than strains with other alleles. Using thebackcross method, we established a new H-2 congenic strain, designated as A.
B(4R). This new strain carries the H_2 h4 haplotype with an A/Wy genetic
background. The H-2 M haplotype is a recombinant haplotype between a andb, and a combination of alleles Ef3k and Ea b• As the Ea b gene has a deletion
including all parts of the Ea promoter and the first exon, the E molecule
cannot be expressed on the cell surface. To clarify whether the Ef3 and Ea
genes themselves are responsible for adenoma susceptibility or not, we will try
to establish Eak-transgenic mice using the A.B(4R)/Ms strain as a donor.
The development of tumors is multigenic, complex trait. Strains derived
from Asian wild mice have shown dominant, resistant alleles in their pulmonary adenoma susceptibility genes. To map these tumor suppression genes,
we established a new series of recombinant congenic (RC) strains, named
AXBG. These strains are made from the parental strains A/Wy (background) and BGR (wild-derived, donor). As we backcrossed only one
generation, each RC strain contains 25% (average) of the alien genome
brought onto the genetic background and a different subset of genes from thedonor. These new tools will be useful in analyzing the multigenic quantitative
polymorphisms which affect tumor development.

* Clea Japan, Inc.

50

Genetical Study on the Skeleton Mouse Mutant, Tail short (Ts)Toshihiko SHIROISHI, Akihiko MITA, Sigeharu WAKANA
and Kazuo MORIWAKIThe mutation Tail-short (Ts) arose spontaneously in the BALB/c inbred
strain in 1946 (Morgan, 1950). Mice heterozygous for Ts have shorter
kinked tails and smaller bodies than normal mice. Other skeletal abnormalities of various kinds scattered over the whole body have been observed. It was reported that Ts homozygotes were prenatal lethal. Abnormalities in homozygous mice can be traced back to 31/2 days p.c. by several
criteria including low cell number and absence of cavitation. All these
indicate the indispensable function of the Ts gene not only in skeletal
development but in the early development of mice. The Ts gene was mapped
to the teromeric region of chromosome 11. In the past year, the gene forhuman campomelic dysplasia (CMPDl) which displays features similar to
the Ts was mapped to the homologous syntenic region on human chromosome 17q24.3-q25.1 (Tommerup et al., 1993). Morgan has already reported
that mutant mice fell short of the Mendelian expectation by 30-40% in Ts/+ X +/+ matings. This indicated that some fraction of mice heterozygous
for Ts died at the time of birth, rendering genetic analysis difficult. We havestarted the fine mapping of Ts, toward positional cloning of this gene. In thisstudy, we conducted backcross matings between the Ts/+ strain and the
MSM inbred strain, derived from Japanese wild mouse. The Ts heterozygotes from this cross were less defective and the litter-size was relatively
large, compared to the cross between the Ts/ + strain and C57BL/6 strain.
Furthermore, penetrance of the Ts phenotype was almost complete in thiscross. Thus far, one microsatellite marker, DIIMitl28 has been tightly
linked to the Ts gene.
Genetic Analysis of a New Mouse Mutant Rim4
Affecting Limb Pattern Formation
Hiroshi MASUYA, Tomoko SAGAI, Kazuo MORIWAKI
and Toshihiko SHIROISHI

The complex architecture of the vertebrate limb cartilage pattern probably
develops under the control of positional signalling provided by the zone ofpolarity activity (ZPA) and the apical ectodermal ridge (AER). Signalling

III.

MAMMALIAN GENETICS

51

from ZPA controls the development along the anteroposterior axis and thesignalling from AER, along the proximodistal axis. The signaling may beinterpreted by the Hoxd and Hoxa genes to certain positional values. Rim4
(Recombination Induced Mutant 4) is a polydactyly mutant derived from an
intra-MHC recombinant. Heterozygotes of Rim4 show preaxial polydactyly
and hyperpharangy of hindfeet. Homozygotes show preaxial polydactyly andhyperpharangy of forefeet and hindfeet, reduction of the tibia and a decreasein number of vertebrae. These phenotypes closely resemble the phenotypes of
the another polydactyly mutant, Ix. Many polydactylous mutants includinglx, show hyperpharangy and tibial hemimelia. This indicates that the affected
genes in these mutants function coordinately in a process of limb development (probably affecting production of certain positional values). We
carried out linkage analyses of the Rim4 crossing with DBA/2J and NZB. As
a result, Rim4 was mapped on chromosome 6 and was tightly linked to
D6Mit16, D6Mit17, and D6Mit94. This position does not overlap with theloci of other polydactylous mutants. Several recombinants were observed
between the Hoxa and Rim4 loci. When C57BL/lOJ-Rim4/ + mice werecrossed with other laboratory strains, the frequency of the Rim4 phenotype
varied according to the strain with which the Rim4 was crossed. Thus,
penetrance was incomplete in heterozygotes, ranging from 0% in the crosswith the MSM strain to almost 100% in the cross with the NZB strain. The
order of mouse strains with respect to penetrance was; NZB > B10 = CBA >DBA >C3H > BALB/c > MSM. The differences in penetrance indicates that
the Rim4 phenotype is controlled by allelic forms of the Rim4 locus or othergenes, which are not linked to Rim4.
Gene Mapping toward Positional Cloning of the Rim3 Gene
Hiroshi MASUYA, Shigeharu WAKANA, Tomoko SAGAI,
Kazllo MORIWAKI and Toshihiko SHIROISHI

Rim3 is a hairless mutant derived from an intra-MHC recombinant,showing hyperkeratinozation in the cornea. This gene has been mapped to
the Cola-l (collagen-I) locus on chromosome 11. In the present study, we
extended the mapping of the Rim3 gene, using a cross between the C57BL/
lOJ-Rim3 and MSM strains. As a result, the Rim3 gene was tightly linked toa microsatellite marker Dl1Mit14. We also mapped the Krt-l (keratin type52I) locus to this position. We carried out PCR-screening of the YAC libraryprovided by Research Genetics, USA, using D 11 Mit14 primers, and identified
a single positive clone. Now we are characterizing this clone.
Germ-line Transmission of ES Cell Lines Established from
Mouse Strains Enhancing Meiotic Recombination
at the LMP-2 Hotspot in the MHC
Tomoko SAGAI, Norio NAKATSUJI, Toshihiko SHIROISHI
and Kazuo MORIWAKIThe wm7 MHC haplotype derived from Japanese wild mouse enhances
meiotic recombination specifically in female at the hotspot located to the 3'end of the LMP-2 gene. Molecular characterization of this hotspot andpreviously identified hotspots located in the Eb gene revealed that these two
hotspots share a similar molecular organization, consisting of three molecularelements, consensus sequence of mouse middle repetitive MT-family, tetramer (TCTG) repeated sequence and LTR repeat of mouse retro virus.
In order to elucidate the relationship between the location of the recombinational breakpoints and these elements, we intended to carry out the genetargeting experiment in order to modify the structure of the hotspot. For this
porpose we needed the ES cell lines derived from the mouse strain which
enhances the meiotic recombination at the hotspot. Because pluripotent ES
cell lines available now were produced mostly from 129 strain which is notcapable of enhancing recombination at the hotspot, we isolated the new ES
cell lines from BlO.A(R209) strain, which exhibits high recombinationalfrequency at the hotspot. These ES cell lines were tested for the chimera
formation and germ-line tansmission through the microinjection into 8 cellstage embryos of ICR strain. As the result, we obtained several chimera micewith the various coat color contribution. Finally, three ES cell lines established from BlO.A(R209) strain and one from (R209 X CBA)Fl were transmitted to the germ-line of the chimera mice. We are now preparing thetargeting vector and are planning to use these ES cell lines for the analysis of
the molecular mechanism of meiotic recombination at the LMP-2 hotspot.

III.

MAMMALIAN GENETICS

53

Genetical Analysis of rim2 Coat Color Mutant
Tomoko SAGAI, Shigeharu WAKANA, Kazuo MORIWAKI
and Toshihiko SHIROISHI

The rim2 is one of the RIMs (recombination induced mutants) whicharose spontaneously in the BIO.A(R201) strain. The rim2 is recessive coatcolor mutant and is characterized by the diluted pigmentation on the entire
hair, the ears and tail. The progressive depigmentation of the pelage isfrequently observed in the young individuals. The amount of pigment in theireyes at birth is smaller than the original BIO.A(R201 )strain, but the eye color
becomes darker in adult animals and cannot be distinguished from the wild
type.
Chromosomal mapping of this mutation was carried out using several
micro-satellite markers. (MSM X rim2)FI X rim2 back-cross mating resulted
that the rim2 mutant gene was tightly linked with D13MITl08 marker
located between Dl3MIT27 and 36. In this region, old coat color mutation,pe (pearl), was mapped. As the phenotype of the pearl mutant resembles thatof rim2, it is possible that the rim2 is allelic to the pe.
Mutagenesis with Intra-MHC Recombinant Strains
Masayasu YOSHINO, Tomoko SAGAI, Kazuo MORIWAKI
and Toshihiko SHIROISHI

In our laboratory, hU9dreds of intra-MHC recombinants have been established from thousands of progeny of crosses between a wild-derived MHC
congenic strain, BIO.MOL-SGR, and standard inbred strains. We found thatsome of these intra-MHC recombinants have spontaneously yielded multiple
visible mutations. The mutation rate of these intra-MHC recombinants
seems to be much higher than the standard values, 3.8 X 10- 6 for recessive
and 3.4 X 10- 7 for dominant mutations, which were estimated from laboratory mouse strains. In order to understand what mechanism causes the highrate of mutation in the intra-MHC recombinants, we have screened mutants
at specific loci which can be analyzed at the molecular level. We used the PTj
7af strain as a tester in genetic crosses with the intra-MHC recombinant, as
it carries recessive mutations at seven specific (a,b,cch,d,p,s,se) loci. In
practice, we irradiated the BIO.BR.(R228) mouse strain, from which adominant visible mutation of the hairless phenotype (Rim3) was obtained,

54

and mated it with testers of the PT strain. We have detected one mutant withthe short ear (se) locus and one visible mutant with a phenotype of dominant
white spot, in 976 offspring from 5 Gy-irradiated females of BlO.BR.(R228).
In addition to these experiments, one spontaneous visible mutation with aphenotype of dominant white spot was obtained among 480 offspring from
non-treated BlO.BR.(R228). We are planning to analyze the se mutant with
DNA markers located near the se locus and to test whether or not thedominant spot mutants are affected at the W or Sllocus.
Chromatin Structure of the Recombinational Hotspot
in the Mouse MHC
Ken-ichi MIZUNO, Tomoko SAGAI, Kazuo MORIWAKI
and Toshihiko SHIROISHI

It has been established that reciprocal recombination occurs at a highfrequency in the mouse major histocompatibility complex (MHC) whensome wild-derived mouse strains are crossed with laboratory strains. The
breakpoints of these recombinations are clustered at specific sites termed
hotspots. The wm7 MHe haplotype derived from Japanese wild mouseenhances the recombination at the hotspot located in the 3'-end of the Lmp2
gene between the Pb and Ob genes (Lmp2 hotspot). The breakpoints of therecombinants turned out to be confined to a DNA fragment of 2 kb. The
molecular mechanism that controls the site-specificity of the recombinational
breakpoints has yet to be determined. One possibility is that hotspots have an
open chromatin structure leading to enhanced accessibility of DNA to therecombination machinery. In yeast, there are reports that the chromatin
structure contributes to the initiation of meiotic recombination. We attempted to examine this hypothesis focusing on the Lmp2 hotspot. First, we
fractionated testicular germ cells from several mouse strains through centrifugal elutriation after EDTA and trypsin treatment of testes. Using thismethod, we enriched pachytene cells in which meiotic recombination isthought to occur. In order to analyze the high-order structure of chromatinaround the hotspot, we tried to analyze DNase I-hypersensitivity sites(DHSSs) in the chromosomal region surrounding the hotspot. As a result,clear DHSS was not detected in the 2 kb segment of the hotspot, but DHSSs
were detected at a distance of 1.5 kb proximal to the hotspot. On the other

III.

MAMMALIAN GENETICS

55

hand, we detected faint DHSSs in the hotspot located in the second intron of
the Eb gene in the MHC as reported previously by another group. It
appeared that the presence or absence of the DHSSs was not directly related
to recombinational frequency in the crosses. In this study, the DHSSs weresimilarly present in all mouse strains examined. In addition testis cells, weanalyzed the chromatin structure in somatic cells. We detected clear DHSSs
in the Lmp2 hotspot both in spleen and liver cells. Results showed thatrecombination at the hotspots during meiosis occurs independently on thechromatin structure. Thus, the molecular mechanism of meiotic recombination on hotspots in the mouse genome is different from that in the yeast
genome.
Polymorphism of the Lmp2 Gene Encoding a Subunitof the Mouse Proteasome
Ken-ichi MIZUNO, Tomoko SAGAI, Toshihiko SHIROISHI
and Kazuo MORIWAKI

Exogenous antigenic proteins, such as viral antigens, are digested to small
octomeric or nanomeric peptides before being presented in the context of the
MHC class I molecules to the T-cell receptors (TCR). A multisubunit
protein complex, proteasome, produces this protein degradation in an energydependent manner. In mouse, the poly A signal of the Lmp2 gene which
encodes a subunit of proteasome is mapped at a distance of only 2 kb fromtetramer repeats identified in the recombinational hotspot. Recently, it has
been shown that non-synomynous substitutions at the antigen recognition siteof class I and class II genes in the MHC is more rapid than synonymous
substitutions. It has been also reported that the Lmp2 genes in laboratory
mice are polymorphic at both amino acid and nucleotide levels. Weareinterested in whether the Lmp2 gene evolved in the trans-species mode as theantigen recognition site of the class I and II genes. To clone the Lmp2 genes
in various species and subspecies of mice and determine these sequences, we
extracted mRNA from mouse spleens and cloned the genes using the
RT-PCR method. We are presently sequencing the clones of the genes.

56

Genetic Diversity and Geographic Distribution of Asiatic Mus musculus
Subspecies Based on the Polymorphism of Mitochondrial DNA
with Special Reference to the Origins of Japnese Mouse'M. m. molossinus'
H. YONEKAWA 1), S. TAKAHAMA 1), N. MIYASHITA 2 )and K. MORIWAKI 2)

Wild mice of the species, Mus musculus, were collected from Asiatic
countries and their mitochondria DNA(mtDNA) was ~nalyzed by restriction enzymes. M. m. musculus and M m. castaneus were commonly found in
this area with clear geographical distribution patterns: M. m. musculus from
Western China, Russia, Korea to Japan, andM. m. castaneus from Southeast
Asia to Southern China. As reported previously, mice with castaneus
mtDNA could also be found in northern Japan and newly in eastern Russia.
However, it was difficult to find intrasubspecific RFLVs (Restriction Fragment Length Variations) in the musculus mice as well as in the castaneus
mice. We thus determined the DNA sequences of the D-Ioop region toclarify the fine structure of both subspecies. At least one mouse was used forthis analysis in each collection locality. The total number of mtDNA
haplotypes sequenced so far is 106. As expected, intrasubspecific DNA
variations (mainly base substitutions) were found in the region. Pair-wisecomparison was performed using their DNA sequences, their genetic diversity values were calculated, and then phylogenetic trees were constructed basedon the values. The cluster of M m. musculus is clearly separated from thatof M. m. castaneus. Furthermore, several distinct subclusters exit in themusculus cluster and the same is true in the castaneus cluster. New lines ofevidence that we found in this year are that at least two components exist
both in the Japanese musculus populations and in the castaneus ones. In themusculus population, one component is closely related to musculus micecollected in Korea and northern China. In the castaneus mice, one is related
to mice trapped in Palau and the other with those of eastern Russia.

I)

2)

The Tokyo Metropolitan Institute of Medical Science.
National Institute of Genetics, Japan.

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57

Recombination in the Class III Region of the Mouse MHC
Masayasu YOSHINO', Tomoko SAGAI" Kirsten Fischer LINDAHL', Yutaka TOYODA"
Yasuaki SHIRAYOSHI" Ken-ichi MATSUMOTO s, Kimihiko SUGAYAS,
Toshimichi IKEMURA s, Kazuo MORlwAKI 6
and Toshihiko SHIROISHI'

Sites of meiotic recombination in the class II region of the mouse MHC are
not randomly distributed, but are clustered within short segments known asrecombinational hotspots. The MHC consists of three linked gene clusters.
The class I and II regions contain genes encoding highly polymorphic cellsurface antigens which are involved in the presentation and recognition offoreign antigens during immune responses, whereas the class III region of the
MHC contains miscellaneous genes, trapped in the middle of the MHC, thatare not polymorphic and do not necessarily playa role in immune responses.
The class III region contains genes encoding steroid 21-hydroxylase (Cyp21),
members of the complement cascade (C4, Sip, C2, Bf), the heat shockproteins Hsp70 and Hsp68c, and the tumor necrosis factor cytokines (Tnfa,
Tnjb). At present, it is not known if recombinational hotspots are a generalfeature of the MHC or specific to the class II region. To answer this question,
we characterized 79 Ab: H2-D recombinants using 11 DNA markers andmapped recombinational breakpoints. In addition, we determined the geneorder in the MHC class III region. We used two DNA marker genes and one
SSLP (simple sequence length polymorphism) marker which had not previously been mapped precisely. They are Tnx, the gene for an extracellularmatrix protein, tenascin X, the Notch-related Int3 gene, and a microsatellite
marker, D17Mit13. The results gave the gene order of Eb-61.1-Int3-TnxCyp21IC4-BfHsp68c-D17Mit13-TnfaITnjb-D. The crossover sites in 40 of
the 79 recombinants were confined within the EblInt3: TnxlCyp21 interval.
The results demonstrated that unequal distribution of recombination is a
general feature of the mouse MHC, suggesting the presence of a novel, Mammalian Genetics Laboratory.
Howard Hughes Medical Institute, Departments of Microbiology and Biochemistry,
University of Texas Southwestern Medical Center, Dal1as, TX 75235-9050, USA.
3
Department of Animal Pathology, Institute of Medical Science, University of Tokyo,
Shiroganedai 4--6-1, Minato-ku 108, Japan
4
Mammalian Development Laboratory.
5
Department of Evolutionary Genetics.
6
Department of Cel1 Genetics.
2

58

recombinational hotspot within the Int3 : Tnx interval.
Linkage Analysis of thejs (Jackson shaker) Locus on
Mouse Chromosome 11
Sigeharu WAKANA', Hiromichi YONEKAWA2, Hideki KANEDA 2,
Mieko OKAMOT0 2, Toshihiko SHIROISHl J
and Kazuo MORIWAKI 4

Jackson shaker Us) is a recessive mutant in the mouse, which exhibits
circling, head-shaking and deafness. These abnormalities are caused by thedisarray of stereocilia of the outer hair cells in inner ear. Thus, js would bea useful model for human congenital deafness. Thejs gene has been mappedon the distal part of mouse chromosome 11. To search for DNA markers
closely linked to this locus, we employed a detailed linkage analysis using 300
segregants of an intersubspecific backcross between C57BL/6-jsljs and MSM,
an inbred strain established from Japanese wild mice Mus musculus molossinus. The analysis was performed by ordinary PCR and PCR-SSCP using
many microsatellite markers mapped on the the distal end of the chromosome. We identified DllMITl28 as being very closely linked to thejs locus.
We started screening the YAC clone with this microsatellte marker.
The Relationship between Recombinational Frequency and
Sequence Homology at the Hotspot in the Mouse MHC
Masayasu YOSHINO, Tomoko SAGAI, Kazuo MORIWAKI
and Toshihiko SHIROISHI

The mouse major histocompatibility complex (MHC) is one of the mostuseful genetic regions for studies of the molecular mechanism of meiotic
recombination in mammalian genomes. The class I and II genes of the MHC
encode highly polymorphic cell-surface antigens, the genotypes of which canbe readily determined serologically, facilitating the rapid identification of
intra-MHC recombinants. Furthermore, the MHC region has been wellcharacterized at the molecular level, so that we can map the recombinational
12
J
4Central Institute for Experimental Animals.
The Tokyo Metropolitan Institute of Medical Science.
Genetic Stock Research Center.
Department of Cell Genetics.

III.

MAMMALIAN GENETICS

59

breakpoints at the nucleotide level. Sites of meiotic recombination in theproximal region of the mouse MHC are not randomly distributed but are
clustered within short segments known as recombinational hotspots. The H2congenic strain of BlO.MOL-SGR whose MHC haplotype of wm7 were
derived from Japanese wild mouse, Mus musculus molossinus, enhances
meiotic recombination at the Lmp2 hotspot that are located in the H2-K to
Ab interval. Heterozygotes of crosses between BlO.MOL-SGR and standard
H2 congenic strains, BlO, BlO.A or BlO.BR have yielded high frequencies ofrecombination (2.1 %) in this region, and these frequencies are 100-foldhigher than the frequencies estimated for standard crosses. Mating experiments between BlO.MOL-SGR and BlO.CAS4(R28) or BlO.CAS3, whichwere derived from Thai wild mouse, Mus musculus castaneus, revealed thatrecombination occurred at the Lmp2 hotspot at lower frequencies (0.30.6%) than in the crosses between BlO.MOL-SGR and the standard BlO
strain (2.5%). The results indicated that high recombinational frequencies incrosses with BlO.MOL-SGR depend on the counterpart strain in the cross.
In the present study, we focused on the question of what mechanism is
responsible for differences in the rate of recombination among different
crosses. For this purpose, three test crosses were made. First, B10.
MOL-SGR was crossed with the 129 strain which has the same b haplotype
as BW. Second, BlO.MOL-SGR was crossed with another standard laboratory MHC-congenic strain of BlO.D2. Third, BlO.MOL-SGR was crossedwith BlO.M or BlO.MOL-TENl whose MHCs are characteristic of Mus
musculus castaneus. The rates of recombination in these crosses werecompared with that in the cross of BlO/BlO.MOL-SGR. Summarizing our
results, 129 enhanced recombination (1.8%) as well as BlO but the frequency
in the cross of BlO.D2/BlO.MOL-SGR was lower (0.3%) than that in the
cross of BlO/BlO.MOL-SGR. Moreover, neither BlO.M nor BlO.MOL-TEN
1 produced recombination in the K-Ab interval in crosses with BlO.MOLSGR. We sequenced the Lmp2 hotspot of these strains and the sequence data
indicated that BlO had the highest homology with BlO.MOL-SGR and that
BlO.M and B10.MOL-TEN1 had the lowest. These results suggest thatrecombination frequency is parallel with the extent of sequence homology at
the Lmp2 hotspot.

60

M-phase Specific Phosphorylation of Mouse p84wee1 Kinase
Hideyo YASUDA

I have been working on the mechanism of regulation in mammalian cell
cycle progression, especially on the mechanism of regulation through protein
phosphorylation. Phosphorylation and dephosphorylation are importantmechanisms for cell proliferation. In mammalian cells, cyclin dependent
kinases (cdks) play key roles in cell cycle regulation, whose activation andinactivation are also regulated by phosphorylation and dephosphorylation(Norbury, C. and Nurse, P. (l992) Annu. Rev. Biochem. 61,441-470). CAK(cdk activating kinase) phosphorylates Thr161 or Thr160 of cdc2 or cdk2
kinase, resulting in activation of these kinases (Poon, R. Y. C., Yamashita,K., Adamczwski, J., Hunt, T., Shuttleworth, J. W. and Shuttleworth. (1993)
EMBO J. 12,3321-3329.).
On the other hand, phosphorylations of mammalian cdc2 kinase on Thr14
and Tyr15 negatively regulate kinase activity (Norbury, C., Blow, J. andNurse, P. (1991) EMBO J. 10, 3321-3329.). Phosphorylation at Tyr15 of
cdc2 kinase is catalyzed by wee I kinase, though the kinase responsible for
Thr14 phosphorylation has not been identified so far (Honda, R., Ohba, Y.
and Yasuda, H. (1992) Biochem. Biophys. Res. Commun. 186, 1333-1338),(Parker, L. L. and Piwnica-Worms, H. (1992) Science 257, 1955-1957).
We have isolated a eDNA clone of a mouse wee 1 kinase which is about
1.5-fold larger than that of the S. pombe wee 1. Its carboxyl terminal domain,which contains catalytic sites of the kinase, resembles the entire human wee
I kinase. In a cell cycle, this mouse wee 1 kinase is phosphorylated at M
phase and this phosphorylation occurs at the N-terminal domain of the kinase
in M phase extract, not G I phase extract. These results suggest that the
N-terminal and C-terminal domains of the wee I kinase are of regulatory andcatalytic, respectively, and that the human wee 1 kinase reported previously
is not an entire molecule but only a catalytic domain.

61

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS

An Exhaustive Screening of Peptide Signal Molecules Affecting
Gene Expression in Hydra
Tsutomu SUGIYAMA, Toshitaka FUJISAWA, Masayuki HATTA, Hiroshi SHIMIZU,
Osamu KOIZUMI', Yojiro MUNEOKA 2, Toshio TAKAHASHI 2,
M. DEHARo', T. C. BOSCH' and C. N. DAVID'

Past studies have provided evidence which suggests that hydra tissue
contains morphogens which play major roles in regulating regeneration andother developmental processes in this organism. Attempts made to isolatethese morphogens have not been very successful. Head-activator (seebelow), originally isolated as a factor stimulating head formation (Schallerand Bodenmueller, 1981), has a broad spectrum of activity, and its real invivo function is uncertain. Adopting a new strategy, we started a new project
to isolate peptide signal molecules in hydra.
Hydra was mass cultured to produce about 150 g of fresh tissue. Peptides
were extracted from the tissue with 5% acetic acid, and separated into 15
crude fractions. One fraction was selected as representative, and individual
peptides present in this fraction were isolated through multiple steps ofion-exchange and reverse-phase HPLC. A total of 61 peptides were obtainedin quantities ranging from 1 to 10 nmoles, suggesting that more than 300
peptides will be present in the remaining 14 fractions. Amino acid sequencesof all 61 peptides were determined, and chemical synthesis was carried out for
4 of them based on the structures determined. Synthetic and natural peptides
were compared and judged to be identical by HPLC and MS analyses. Oneof the synthetic peptides, Hyd-23, has amino acid residues identical to
Head-activator in 5 of the 10 C-terminal positions as shown:
Hyd-23: KWVQGKPTGEVKQIKF
Head-activator: < pEPPGGSKVILF
The synthetic peptides were examined for their capacity to function as
signal molecules. Hydra were treated with the peptides (10- 7 M), RNA wasI
2,

Fukuoka Women's University.
Hiroshima University.
Zoological Institute, University of Munich.

62

extracted from the treated and untreated control animals, and subjected to
Random Primer PCR Differential Display analysis (Liang and Pardee,1992). This procedure is very powerful in displaying small differences in gene
expression (poly-A RNA synthesis) in different tissue samples. Using thisassay, Hyd-23 was found to be active in altering gene expression in thetreated polyps. This suggests that this peptide may serve as a signal moleculeby binding to a specific receptor on cells and, controlling gene expression in
the cells, via the signal transduction pathway, On the basis of these results, we
intend to carry out a larger screening program as follows; (1) Peptides
present in the remaining 14 fractions (> 3(0) will be purified by HPLC. (2)Half of the purified peptides will be used to treat hydra (> 10- 7 M), andchanges in gene expression produced by the treatment will be examined by
RPPDD. (3) The remaining half will be used for structure determination of
the peptides found to be active in RPPDD. (4) Chemical synthesis will be
carried out for the peptides whose structures are determined. (5) Synthetic
peptides will be used in biological studies to examine their effect on morphogenesis, cell proliferation, cell differentiation and other developmental andphysiological processes in hydra.
Screening of Cell Adhesion Molecules in Hydra
Yasuyuki KISHIMOTO, Masayuki HATTA, Engelbert HOBMAYER*
and Tsutomu SUGIYAMA

A project has been started to isolate cell adhesion molecules involved inregeneration and other morphogenetic processes in hydra. Hydra bodycolumn tissue consists of the ectodermal and the endodermal epithelial cell
layers. These 2 layers were separated from each other through Procaine
treatment, and cut into small pieces. When two tissue pieces thus produced
were brought into contact in 3 combinations (ectodermal-ectodermal, endodermal-endodermal and ectodermal-endodermal), firm adhesion was established within 1-2 hours in all cases. In the case of ectodermal-endodermal
contact, an "epiboly"-like process occurred following the initial adhesion.
The ectodermal epithelial cells gradually spread over the endodermal tissue,and eventually formed a continuous single ectodermal epithelial cell layer tocover the entire endodermal tissue mass.

* Presently at Department of Developmental and Cell Bioloby, University of Frankfurt.

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS

63

Cell adhesion molecules located on the cell membrane probably play amajor role in these tissue interacting systems. Several approaches were taken
to isolate these molecules. In one approach, a soluble extract was produced
from a hydra membrane fraction through trypsin digestion, and its effect was
examined. The tryptic extract « 0.1 mg/ml) was found to specifically inhibit
the epiboly-like process; it inhibited the spreading of the ectodermal cells overthe endodermal tissue, without affecting the initial ectodermal-endodermal(or ectodermal-ectodermal or endodermal-endodermal) adhesion. The inhibition was reversible; The epiboly-like process, stopped midway by adding theextract, could be resumed by remoing the extract through washing.
The effect of the tryptic extract can be explained as follows. The extractcontains a tryptic fragment of an adhesion molecule. The original intact
molecule is located on one partner of the ectodermal-endodermal interaction,and its binding to the ligand located on the other partner is a crucial step inthe epiboly-like process. The fragment containing the adhesion domain of
this molecule can bind to the ligand, thereby preventing the normal ectodermal-endodermal interaction in the epiboly-like process. Other possibilities can also be considered.
The active factor in the tryptic extract has an MW of about 3 KD. HPLC
is being used currently to purify the factor. The purified factor, whenobtained, will be used to isolate a cDNA clone for the original intact
molecule. Specific antisera also will be produced. Using them, we intend to
examine the expression and roles of the molecule in hydra development.
In another line of stupy, we are using PCR to isolate hydra homologues ofknown vertebrate cell adhesion molecules. We have obtained PCR fragments
showing homology to vertebrate cadherin (protocadherin), integrin and,B-catenin, respectively. When hybridized to hydra poly-A RNA in Northern
analysis, all fragments produced clear signals at expected sizes. Attempts toisolate cDNA clones from hydra cDNA libraries have yielded 3 overlapping,B-catenin clones (80% identity in amino acid sequences to human counterpart). cDNA clones of hydra cadherin and integrin are presently being
searched for.

64

Underwinding of DNA on Binding of Yeast TBP to the TATA Element
Hisahiro TABUCHI, Hiroshi HANDA* and Susumu HIROSEThe TATA box-binding factor TBP is an essential component in theinitiation of transcription by eukaryotic RNA polymerase II. We investigated the effect of DNA supercoiling on TBP: promoter interactions
using recombinant yeast (ry) TBP. DNase I footprinting analysis showedthat ryTBP has a higher affinity for the adenovirus major late promoter in thenegatively supercoiled state than in the relaxed state. On the contrary, its
affinity for the Drosophila hsp70 promoter is constant irrespective of DNA
topology. Binding of ryTBP to these promoters induced underwinding of
duplex DNA. The functional TATA box and active ryTBP are essential forthe underwinding. The step was facilitated by negative supercoiling of DNA
on the adenovirus major late promoter but not on the Drosophila hsp70
promoter. For details, see Biochem. Biophys. Res. Commun. 192, 1432-1438,
1993
DNA Sequence Requirement of a TATA Element-Binding Proteinfrom Arabidopsis for Transcription in vitro
Fujio MUKUMOTO**, Susumu HIROSE, Hidemasa IMASEKI**
and Ken-ichi YAMAZAKI**

We analyzed the DNA sequence requirements for the functioning of
TATA elements by examining transcriptional activities associated with 24
promoters, including representatives of each of the 21 point mutations in theconsensus sequence from plants, TATATATA, both in a HeLa in vitro
system and in a chimeric in vitro system in which human TATA-binding
protein (TBP) was replaced by purified TBP of Arabidopsis. Although therelative transcriptional activities varied among these promoters, both systems
gave virtually identical results. Among the mutant TATA elements, thosewith the sequences TAGAGATA and GAGAGAGA did not show any
detectable activity. The rest had activity that ranged from 7% to 130% of
the activity associated with the consensus element. These results suggest thefunctional conservation of TBP between plants and animals. For details, see* Division of Physiological Genetics, Department of Ontogenetics.
** Scool of Agricultural Sciences, Nagoya University, Nagoya.

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DEVELOPMENTAL AND SOMATIC CELL GENETICS

65

Plant Mol. BioI. 23, 995-1003, 1993.

BmFfZ-Fl Activates in vitro Transcription of the Jushi tarazu
Gene by Binding to the FfZ-Fl Recognition Site
Feng-Qian LI, Hitoshi VEDA and Susumu HIROSE

To study the mechanism involved in transactivation by BmFTZ-Fl, we
used an in vitro transcription assay. Because the target genes of BmFTZ-Fl
in the silkworm are unknown, we used the Drosophila Jushi tarazu (jtz) geneas a model template. A single binding site for FTZ-Fl is present at around
280 bp upstream from the initiation site. We also prepared mutant templates
carrying base substitutions within the FTZ-FI recognition site. Thesemutations abolished the binding of FTZ-Fl and BmFTZ-Fl and markedly
reduced the expression of the Jtz-IacZ fusion gene in transformed Drosophila
embryos.
When the wild-type template was incubated with the posterior silk glandextract containing BmFTZ-Fl, the Jtz gene was transcribed as efficiently as
the fibroin gene added as an internal control. The mutant templates produced
five fold fewer transcripts than did the wild-type construct. When we used
posterior silk gland extract, from which BmFTZ-FI had been depleted bytreatment with latex beads coupled with the FTZ-FI site DNA, transcriptionof the Jtz gene was severely reduced while that of the fibroin gene was
unaffected. Addition of purified BmFTZ-FI to the depleted extract restored
Jtz gene transcription to the level before depletion. These results show that
BmFTZ-Fl activates transcription of theJtz gene in the posterior silk glandextract by binding to the FTZ-FI recognition site.

Molecular Cloning of a cDNA for BmFfZ-Fl, a Silkworm
Counterpart of Drosophila FfZ-Fl
Guan-Cheng SUN, Susumu HIROSE and Hitoshi VEDA

BmFTZ-Fl is a sequence-specific DNA-binding factor in the silkworm
Bombyx mori sharing similar biochemical characteristics with Drosophila
FTZ-F 1, a member of the nuclear hormone receptor superfamily, We isolated
a cDNA encoding BmFTZ-Fl. Amino acid sequences in the zinc finger
DNA-binding region and the putative ligand-binding domain of BmFTZ-FI

66

showed a strong similarity to not only FTZ-F1 but also its mammalian
homologues, LRH-1, ELP, and Ad4BP. Northern blot analyses of RNA
isolated from the middle and posterior silk glands and fat bodies showed thata 6.1-kb BmFTZ-Fl mRNA is present in all tissues so far examined.
Genetic Studies on the Duration of Pupal Lifespans in Bombyx mori
Akio MURAKAMI

Information on pupallifespans in the domesticated silkworm, B. mori (L.)
is vague: the average pupal lifespan is said to range from eleven to thirteen
days, but some pure stocks and hybrid lines lifespans of more than 20 days.
Also, certain decapitated pharate pupae in certain F 1 hybrid lines have lived
40 days (cf., Kobayashi, 1957). It is of interest that the ellS stock itself,
which is one of parent stocks above the hybrid lines, also showed a long pupal
life or "dauer (long-lived) pupa". Similar surgical extirpations of the brain in
the many other lines did not produce the so-called the long-lived pupa.
In recent observations certain long-lived pure lines have been found among
NIG preserved stocks, the J115 stock as well as the Ascoli stock and otherwith a lifespan of 18 days. Moreover, the average duration of pupal lifespanamong temperate varieties is estimated to be 10-12 days and that of tropicalvarieties is 9 days. In any case, the duration of pupal lifespan in B. moridiffers from stock to stock and is peculiar to each stock, showing that thebiological phenomenon is a genetically based. The duration of pupal lifespanof F 1 hybrid lines obtained from a reciprocal cross between the standard life(or short) and long life stocks are much the same as the short-lived parent
stock regardless of sex. This suggests that the short-lived trait for pupal
lifespan is not dominant but recessive.
In short, there are two types of stocks of B. mori: one of them has a pupal
lifespan ranging from nine to twelve days and the majority of stocks belong
to this type, and the second type has a pupal lifespan of more than 18 days
and it rather rare.
The current concept of the humoral pathway leading to adult emergencefrom pupal form in B. mori is that the prothoracic glands are stimulated by
the neurosecretary system of the brain, PTTH (prothoracicotropic hormone)or brain hormone, and then secrete the moulting hormone or ecdysone which
finally leads to pupal ecdysis to form the adult. PTTH is released into body

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DEVELOPMENTAL AND SOMATIC CELL GENETICS

67

fluid via the corpora cardiaca. Ecdysone stimulates the tissue cells to initiate
mitosis and the epidermal cells to begin elaboration of adult cuticularmatters. If the ecdyson titer in the pupal body-fluid is low, ecdyson titres
need to accumulate above the threshold dose, required for pupal periods or
prolongation of lifespan. Surgical removal of the brain or cutting off the
supply of PTTH in the pharate pupae results in prolongation of lifespan (or"dauer-pupa"). If this is the case, some stocks, which are relatively longer
lived pupallifespans than the standard stocks, can be regarded as having a
low titer of PTTH and/or ecdyson, while the majority of standard stocks andlines, which showed a short duration of pupallifespans, have a sufficient doseof both the neurosecretory substances.
It seems likely that the duration of some pupal lifespans B. mori is undergenetic control of the secretary activities of the neurosecretory complex,
PTTH and ecdyson of copora allata.
Ecogenetic studies on the Duration of Pupal Lifespans in Bombycidae
Akio MURAKAMI and Osamu NINAKI*

The majority of Lepidopteran insects including Platysamia (Hyalophora)
Secropia, Philosamia cynthia ricini, Attacus cynthia, Caligura japonica, Chilosupressalis, etc. are of a pupal diapause type. However some species including
Bombyx mori (L.) and its putative ancestor, Bombyx mandarina (M.) are ofan egg diapause type and are said to enter the diapause state at a slightly laterstage, when the embryo is dumbbell-shaped but as yet unsegmented. As
stated in the preceding report, some long-lived (ca. 17-18 days at 25 0 C)
pupal stocks of B. mori, J115, Ascoli, and others are preserved in our
institute. Pupal durations extended ca. 60% over the average term for B.
mori. No selective effects on pupallifespans for almost 15 generations wereobserved, indicating that these biological phenomena might be stabilized or
established and are under the control of genetic mechanisms. A similar
observation was found in a pure stock, Cl15 (Kobayashi, 1959). It was also
demonstrated that when the extirpation of the brain either at a later stage ofthe last instar larvae, or at an early stage of pupae in a certain F 1 hybrid line
was done, pupal durations tended to be extended up to more than 40 days.
*

Matsumoto Branch, The National Institute of Sericulture and Entomological Science,
Matsumoto, Nagano-pref.

68

The present findings and others suggest that the domesticated silkworm, B.
mori has the potential to some extent to suspend pupal development or extend
its lifespan.
In B. mandarina, the duration of pupal lifespan varies widely. One of thepresent authors, Ninaki and his collaborators (1982) showed that the lifespanof pupae, which have been cultured in insectaria for several generations
collection from fields, should considerable variation among individuals, ranging from, 11 to 44 days regardless of rearing season. This indicates that thewild silkworm, B. mandarina, can shorten its pupal durations by means of
selective pressures. It is of interest to note that the shortest pupal lifespan in
B. mandarina, 11 days, is similar to that of B. morL However, the pupal
lifespan of B. mandarina newly collected in autumn attained almost fourmonths, from the middle of September to the middle of January of the nextyear, indicating that these pupae might be passing through not only ametamorphic phase but also a hibernating phase. Some eggs laid by themated female moths immediately resumed embryonic development without
the egg-diapause form, but others entered the diapause form. Accordingly, it
seems likely that the former type pupae, which showed a remarkably long
pupal lifespan and whose female moths laid a high frequency of nondiapausing eggs, are a kind of pupal diapause type instead of an egg diapause
type, while the latter, which also showed longer pupal lifespans but whose
female moths laid diapausing eggs, are still both an egg and pupal diapause
type. Thus, it can be said that in B. mandarina neither egg or pupal diapause
form are clearly established, while in B. mori the egg diapause form is clearly
established as the main surviving strategy against unfavorable environmental
conditions throughout a long evolution and/or domestication process. SomeB. mori stocks with a relatively long-lived pupal lifespan appear to be aremnant form of the pupal diapause form of B. mandarina. Diapause isusually confined to one stage in the lifecycle. However, both the obligatory
egg and very weak facultative pupal diapause forms seem to exist in Bombycidae, B. mori (L.) and B. mandarina (M.) an exceptional case amonglepidopteran species along, with the winter moth, Operophtera brumata [I. W.
Kozhantshikov, Ent. Oboz. 31: 178 (1950)].

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69

Embryonic Process of Holometabolous Insects
Kiyoshi MINATO

Among animals of classis insecta, there are a large number of species which
are holometabolous or completely metamorphic compared to those that arehemimetabolous and ametabolous. One of the reasons is thought to be theirlarval forms relatively simple and different from adult ones, which enable
their larvae to obtain various newly-found habitats, additionally withoutcompetition from adult.
The development of various holometabolous embryos was investigatedthrough available literature and compared to that of hemimetabolous andametabolous embryos. As a result, little evidence was found showing that themore simple larval forms of holometabolous insects was a result of thehatching of these embryos at relatively less developed stages or "neoteny", assuggested by Berlese (l913). It is more likely to be a result of the suppressed
expression of some external morphology of the embryo such as the compoundeye, the more developed legs, and the relatively complex form of the body,and also the increased expression of some internal morphology such as theabdomina motive-muscle system. These active regulation mechanisms ofexpression of the embryonic features in holometabolous insects and thegenetic background are now under investigation.
Isolation of a cDNA Encoding the Largest Subunit of TFIIA
Reveals Functions Important for Activated Transcription
Dongmin MA*, Hajime WATANABE, Fred MERMELSTEIN*, Arie ADMON**,
Kiyoshi OGURI, Xiaoqing SUN*, Tadashi WADA, Takeshi IMAI,
Toshifumi SHIROYA, Danny REINBERG* and Hiroshi HANDA

Transcription factor IIA has been shown to interact with the TATAbinding protein and to act early during preinitiation complex formation. The
human factor is composed of three subunits (a, /3, r). A human cDNA clone
encoding the largest subunit of TFIIA (a) was isolated. The recombinant a
polypeptide, together with the /3 and r subunits, was capable of reconstituting***

Department of Biochemistry, Robert Wood Johnson Medical School, University of
Medicine and Dentistry of New Jersey, Piscataway, USA.
Howard Hughes Medical Institute, Department of Molecular and Cell Biology,
University of California, Berkeley, USA.

70

TFIIA activity. Studies using antibodies raised against recombinant a
polypeptide demonstrate that FTIIA can be an integral component of thepreinitiation complex. We demonstrated that TFIIA not only interacts with
TBP but also can associate with the TFIID complex. Functional assays
established that TFIIA has no apparent role in basal transcription but plays
an important role in activation of transcription. Interestingly, amino acidsequence analyses of the ,8-subunit demonstrate these residues to be entirelycontained within the carboxyl terminus of the cDNA clone encoding thea-subunit. For details see Genes & Dev. 7,2246-2257, 1993.
cDNA Cloning of Transcription Factor E4TFI Subunits with
Ets and Notch Motifs
Hajime WATANABE, Jun-ichi SAWADA, Kei-ichi YANO*, Kazuo YAMAGUCHI*,
Masahide GOTO and Hiroshi HANDAE4TFI was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a
DNA binding protein with a molecular mass of 60 kDa and a 53-kDa
transcription-activating protein. Heterodimerization of these two subunits isessential for the protein to function as a transcription factor. In this study,
we identified a new E4TFI subunit, designated E4TFl-47, which has no
DNA binding activity but can associate with E4TFl-60. We then cloned thecDNAs for each of the E4TFI subunits. E4TFI was purified, and the partial
amino acid sequence of each subunit was determined. The predicted aminoacid sequences of each cDNA clone revealed that E4TFl-60 had an ETS
domain, which is a DNA binding domain common to ets-related transcription factors. E4TFl-53 had four tandemly repeated notch-ankyrin motifs.
The putative cDNA of E4TFl-47 coded almost the same amino acid sequences as E4TFl-53. Three hundred and thirty-two amino acids of the N
termini of E4TFl-47 and -53 were identical except for one amino acidinsertion in E4TFl-53, and they differ from each other at the C terminus.
These three recombinant cDNA clones were expressed in Escherichia coli,
and the proteins behaved in the same manner as purified proteins in a gel
retardation assay. Nucleotide and predicted amino acid sequences werehighly homologous to GABP-a and -,8, which is further supported by the*

Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan.

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71

observation that the GABP-specific antibody can recognize human E4TFl.
For details see Mol. Cell. BioI. 13, 1385-1391, 1993.
Genetics of Fluoride-resistance in the Nematode Caenorhabditis elegans
Minoru KAWAKAMI, Takeshi ISHIHARA and Isao KATSDRA

Sodium fluoride (NaF) is toxic to almost all organisms. Biochemical
reactions in vitro suggest that it may interfere with signal transduction
systems, because it depletes Ca2+, inhibits phosphatases, and activates Gproteins. Weare trying to elucidate such a system through the isolation andcharacterization of C. elegans mutants resistant to NaF.
We isolated 13 recessive fluoride-resistant mutants that map in 5 newgenes,ftr-l X,ftr-2 V,ftr-3 IV,ftr-4 X andftr-5 V. They are grouped into two
categories, class 1 and class 2. Class 1 mutants (jlr-l, ftr-3, and ftr-4) areresistant to lOmM NaF, but they grow twice as slowly as wild type worms
and have a small brood size even in the absence of NaF. In contrast, class 2
mutants (jlr-2 andftr-5) are not completely resistant to lOmM NaF, and they
are almost normal in growth rate and brood size in the absence of NaF.
Interestingly, class 2 mutations suppress the slow growth and small brood size
but not the strong fluoride-resistance of class 1 mutations. (Katsura, I. et al.
(1994) Genetics 136, 145-154).
To understand the biochemical reactions for fluoride-sensitivity we cloned
ftr-l andftr-3 genes from transposon-insertion mutants, using the transposon
Tel as the probe. We also cloned partial-length cDNAs of these genes froma C. elegans cDNA library using fragments of the genomic clones as theprobes and determined their nucleotide sequences. The predicted amino acidsequence of theftr-l protein showed a weak homology with that of some ionchannels, but further evidence is required to draw a decisive conclusion. In
contrast, the ftr-3 protein has almost all the domains of protein kinases(Hanks, S. K. et al. (1988) Science 241, 42-52). However, since it does nothave a close relationship to any special family of protein kinases, we think it
encodes a protein kinase belonging to a new family.

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Caenorhabditis elegans Larval Lethal Mutations That Cause Gross
Morphological Changes
Ryuichi HISHIDA, Takeshi ISHIHARA and Isao KATSURA

We have been analyzing mutants that die as larvae with abnormal shapes,
in order to study essential functions in late embryonic and post-embryonicdevelopment (Katsura, I. (1993) Genetica 88, 137-146). Among thosemutants, we concentrated our study on those in which the outer surface of
the intestine is detached from the inner surface of the J:>ody wall. We callthem clr-I-like mutants, since the phenotype resembles that of clr-I among
known mutations. Ten such mutants have been isolated and mapped either toknown genes that act in signal transduction (let-23, let-34I, lag-2 and clr-I)
or to unknown genes. We think the latter genes may act also in signaltransduction or in cellular functions regulated by signal transduction. We
cloned one of the unknown genes, let(ut40, utl02), by the transposon-tagging
method and determined the nucleotide sequence, which encodes a novel
protein. We plan to test whether the phenotypes of various signaltransduction mutants are suppressed by microinjection of the wild-type cloneof let(ut40, ut102). We will also check the time and location of gene
expression using a lacZ fusion of let (ut40, ut102).
A Gene that Controls Both Hatching and Cell Migration in C. elegans
Ryuichi HISHIDA, Takeshi ISHIHARA and Isao KATSURA

Mutants in the hch-I gene are defective both in hatching and in migrationof a neuroblast (Hedgecock, E. M. et al. (1987) Development 100, 365-382).
They cannot digest protein components of the eggshell, and a neuroblast
called QL moves anteriorly instead of posteriorly during larval development.
Since we isolated a transposon-insertion mutant in hch-I, we cloned the geneby the transposon-tagging method. No significant homology to known genes
has been found. We plan to clone the cDNA for further investigation.

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73

Construction of lacZ Markers for Various Nerve Cells
in the Head of C. elegans
Takeshi ISHIHARA and Isao KATSURA

To analyze the formation and function of the neural circuit in the head of
C. elegans, we are preparing markers for various nerve cells using the"promoter trapping" method (Hope I. (1991) Development 113, 399-408).
We first made a library consisting of about 20,000 independent clones byligating genomic DNA fragments of C. elegans to a lacZ fusion vector. Then,
we divided it into about 200 pools, injected the DNA constructs of each pool
into the ovary of C. elegans, and stained the transgenic worms with Xgal.
About 20% of the transformants expressed lacZ in some nerve cells in thehead. In these cases we isolated single positive clones from the pools andinserted them into a chromosome of C. elegans. Thus we obtained stable C.
elegans strains in which almost all neurons or only one pair of head neurons,for example, express lacZ. To prepare markers for subsets of neurons, we
also used promoters of genes that probably code for neuronal proteins assuggested by the C. elegans genome project. In many cases lacZ in the vector
was replaced by the cDNA of the green fluorescent protein of the jellyfish
Aequorea victoria (Chalfie, M. et al. (1994) Science 263, 802-805). Suchworms showed fluorescence in a defined set of neurons as live animals without
any treatment.
Analysis of the Head Neural System of C. elegans as Assayedby the Formation of Dauer Larvae
Norio SUZUKI, Misako URASAKI, Takeshi ISHIHARA and Isao KATSURA

If larvae of C. elegans are in crowded conditions just after hatching and if
only a small number of bacteria are present around them, they develop into
enduring, non-feeding larvae called dauer larvae instead of third-stage larvae(L3). The decision to form dauer or L3 seems to be regulated by the headneural circuit with pheromone and the smell of bacteria as key inputs, which
act mainly on a pair of sensory organs in the head called amphids. Since theassay of dauer formation is much less time-consuming than behavioral assays
such as that of chemotaxis, we started functional analysis of the head neuralcircuit by checking dauer formation as the output.

74

It is known that some double mutants in movement or behavior (unc-31;
unc-3, unc-31;aex-3, unc-104;osm-1, unc-104;che-ll and unc-104;che-13), but
not any of the single mutants, are constitutive in dauer formation. Namely
they form dauer larvae even if they are not crowded and are fed well withbacteria. This phenomenon can be explained if we assume parallel pathways
for a dauer-inhibiting signal in the head neural circuit, where each of themutations blocks only part of the pathways. We then looked for othercombinations of known mutations that have a synthetic dauer-constitutive
phenotype. The following double mutants were found to have such aphenotype: egl-4;unc-3, egl-32;unc-3 and double mutants between either unc31 or unc-3 and one of the following, che-3, che-11, daJ-6, daJ-10, osm-1, osm5 or osm-6. Strangely enough, it is known that the latter mutations change
the structure of amphids and have a dauer-defective phenotype by themselves. We discovered also that some other chemotaxis mutations that do notaffect the amphid structure as well as most of the fluoride-resistant (jlr)mutations (see above) have a dauer-constitutive phenotype, if they are
combined with unc-31 or unc-3. These results are especially interesting to us,
because it shows that the fir mutations probably affect neural functions.
We also isolated more than 40 new mutations that have a dauerconstitutive phenotype in combination with an unc-31 mutation but not bythemselves. For this purpose we made a strain unc-31 Ex[unc-31(+)] by
microinjection of a clone of the wild-type unc-31 gene into an unc-31 mutant.
Since the injected clone is maintained as an extrachromosomal array, it issomewhat unstable, and the transformant worms produce both Unc + andUnc-31 progeny. We mutagenized such worms with ethyl methanesulfonate
and selected among the F2 progeny for those producing Unc dauers but not
Unc+ dauers. We are now mapping those mutants using a method based onpolymorphic sequence-tagged sites (Williams, B. D. et al. (1992) Genetics
131, 609-624).

Systematic Analysis of C. elegans cDNA
-Towards an Expression Map of the GenomeYuji KOHARA, Tomoko MOTOHASHI, Akiko NISHIGAKI,
Akiko SUGIMOTO and Hisako WATANABE

Aiming to ultimately understand the network of gene expression in devel-

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DEVELOPMENTAL AND SOMATIC CELL GENETICS

75

opment of the worm, we are trying to construct an expression map of the 100
Mb genome through the identification and characterization of all of its
cDNA species, whose number is estimated to be around 15,000.
cDNA were made from the mRNA of a mixed-stage population of thehim-8 strain and were size-fractionated before library construction. Thistime, a set of some 4,400 cDNA clones was selected from a library of > 2 kb
cDNA by picking up cDNA clones at random (using ;tZAPII as a vector)
and then removing abundant cDNA clones (some 20 species which occupied
ca. 20% of the library). cDNA inserts of individual clones were PCRamplified using vector primers and then subjected to (l) tag-sequencing, (2)mapping onto the genome, and (3) the surveying of expression patterns.
(1) Tag sequencing: Single reads have been made from both 5'-ends(using vector primers) and 3'-ends (using anchored oligo-dT primers inorder to minimize the effect of the long poly-A stretch) on ABI sequencers.
Thus far, we have finished the tag-sequencing for almost all the clones. Outof them, 3,089 clones gave clean 3'-tag sequences, which were used to classify
the clones into 1,478 species (808 standing-alone clones and 670 groups of 223 clones) by comparing the tags. This search also detected pairs of clones
which appeared to be generated by alternative splicing. A database searchshowed that the set of 1,478 species hit 20% (36 genes) of the C. elegans gene
entries in GenBank and 23 % of the clones showed significant similarities withgenes of other organisms. At least 49 cDNA hit the 2 Mb genomic region
which has been sequenced by the genome sequencing consortium. Analysis of
the 5'-tags are in progress.
(2) Mapping onto the genome: Most of the clones have been mappedusing the ordered YAC filter. Recently a supplementary filter (Suppoly) was
made available by Alan Coulson at the Sanger Center, U.K. Using these,
about 700 cDNA species have been mapped onto the genome; the central
regions of chromosomes were much denser for cDNA than other regions.
(3) Surveying expression patterns: Since in situ hybridization analysis
has confirmed the validity of the membrane strips on which aliquots of the 3'end region of all cDNA species, amplified from single embryos at various
developmental stages, have been dot-blotted (see Tabara & Kohara, in thisissue), we are using the membrane strips for the surveying. Thus far, 126
cDNA species mapped on chromosome 3 have been analyzed; briefly, 52
cDNA showed ubiquitous expression throughout embryogenesis, 19 appearedmaternal since they gave signals only on the dot of the 0 hr embryo, 14 cDNA

76

appeared zygotic since they gave signals on the dots from later stage embryos
but not from the 0 hr embryo, and the remainder gave no signals at all,probably due to expression after embryogenesis or a very low level ofexpression.
Part of these data are already in an integrated C. elegans genome database,
ACEDB version 2 (constructed and managed by Richard Durbin at theSanger Center, U.K.), and the remaining data will be submitted to the
database as soon as possible. The tag-sequences were also submitted to
DDBJ.
As a next step, we have picked up altogether 30,000 cDNA clones at
random from 3 different cDNA libraries; the same library of >2kb cDNA,
a library of unfractionated cDNA, and an embryonic cDNA library. We are
now making membrane blots of high-density grids of the clones, which will
be used for probing with the already characterized cDNA to make a subset ofthe clones for unidentified cDNA species. We are going to continue theabove analyses on the subset.
In situ Hybridization on C. elegans Embryos-Analyses of
Candidate Clones for Differentially Expressed GenesHiroaki TABARA') and Yuji KOHARA

Previously, we have developed a method to amplify the 3'-end region (0.51kb) of all mRNA (cDNA) species from a single embryo or a single
blastmere quantitatively. The amplified cDNA have been used for two typesof differential screening which have given several candidate clones fordifferentially expressed genes; (1) The cDNA were used as probes in conventional screening of cDNA libraries, (2) aliquots of the amplified cDNA fromvarious stages of single embryos were dot-blotted on strips of nylon membrane and then probed by individual cDNA clones. The validity of theamplification was confirmed by the fact that consistent hybridization signals
were obtained in model experiments in which the strips of the dot-blot wereprobed with stage-specific gene probes; glp-l for very early stage, unc-54 andmyo-2 for mid-late stage and vit-2 for no expression in embryogenesis.
However, the PCR amplification involved in the method could produce
potential uncertainty with respect to quantitativeness, and information about
I)

Department of Genetics, The Graduate University for Advanced Studies.

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DEVELOPMENTAL AND SOMATIC CELL GENETICS77

the distribution of mRNA within an embryo is not available from theseanalyses. Thus, we have developed a method for in situ hybridization onembryos.
The in situ method is based on our protocol for separating blastmeres, insitu protocol for Drosophila embryos and that of G. Seydoux (Carnegie
Institute, Baltimore). The main point is that the egg shell is removed bychitinase treatment and then the vitelline membrane is partially broken by
shearing force before fixation, which takes a bit of time but allows us toregulate the extent of fixation and proteinase digestion closely, leading to highsignal-to-noise ratio for early embryos. The results of a model experimentusing the stage-specific gene probes (unc-54, myo-2 and glp-l) verified themethod. As to glp-l, both AB and PI blastmeres were positive, which wasconsistent with observations by Evans and Kimble.
The results of in situ analysis on 4 cDNA clones were as follows;(1) Clone 4-1: This clone was obtained through differential screening
between the amplified cDNA from single embryos at 1.5 hr and 9.5 hr after
the 1st cleavage. It showed positive signals the dots from the 9.5 hr embryoin the dot-blot analysis. In situ analysis showed that this gene gave signals on
the embryos from the comma to the 2-fold stages, which exactly coincidedwith the period expected from the dot-blot analysis, and more precisely, theexpression was limited in cells placed along the worm body.
(2) Clone 4-3: This was obtained from the same screening as with clone
4-1, but was specific for the 1.5 hr embryo. The results of in situ analysis werevery interesting. No expression was observed in embryos earlier than the 4cell stage or later than the end of gastrulation. The expression was firstdetectable at the 8-cell stage in all blastmeres except for P3, and culminatedat the beginning of gastrulation except that one cell, probably P4, was still
free from the expression.
(3) ant-I: This clone was obtained from differential screening using
cDNA amplified from AB and PI blastmeres. However, in situ analysis
showed no clear localization of the mRNA at the 2-cell stage and showedrather ubiquitous expression during early embryogenesis. Since this gene hasa similarity to the cell cycle related gene, CKSI (cdc28 kinase subunit), thedifferential screening might have detected the difference between the stages ofcell cycle in the AB and PI cells from which the cDNA were amplified. We
are going to continue the differential screening.
(4) skn-l: Although the skn-l gene product was reported to be localized

78

in P I and therefore a good example of a maternal gene which showed
localized expression in early embryos, skn-l probes hybridized equally to
dot-blots of the eDNA amplified from AB and PI blastmeres, suggesting
there is no localization of the mRNA. In situ analysis also showed theexistence of the mRNA in both AB and PI cells. These data suggest that theexpression of the skn-l gene is regulated post-transcriptionally.
Gene Disruption in C. elegansApplication of Transposon Insertion Mutant" Banks
Yoshiki ANDACHI and Yuji KOHARAI

An efficient method for targeted gene disruption is essential to understanding gene function. A so-called gene targeting method is not available in C.
elegans due to the lack of appropriate selection markers, but mutator strainsof high-hopper transposon Tel have been used to obtain insertion mutantsfor many genes. Once a Tel insertion mutant is obtained, deletion mutants
can be generated from it, taking advantage of illegitimate excision of Tel,and, it was reported recently that gene replacement was also possible.
Building on these strategies, we developed an efficient strategy for isolating
Tc 1 insertion mutants in a desired gene from a bank of frozen mutant worms.
The main point of our method is a pooling strategy. Pools of worms of amutator strain, each of which contains about 100 worms, are cultured to the
FI generation. Part of the FI worms of each pool is stored frozen to
construct a mutant bank. Genomic DNA are extracted from the remaining
part of the FI worms and subjected to PCR assay using primers derived fromthe target gene and Tel. When a positive pool is found, frozen worms fromthe pool are cultured individually and a portion of the progeny are subjectedto the same PCR analysis. In this fashion, a strain with the Tc I insertion canbe isolated.
We have constructed mutant banks of two mutator strains; 768 pools(about 80,000 independent worms) of RW7097 (mut-6) and 192 pools(about 20,000 independent worms) of MT3126 (mut-2). To test the qualityof the banks, insertion mutants were screened for several genes. Screening of
the RW7097 bank for 13 genes revealed 9 mutant strains for 7 genes, andscreening of the MT3126 bank for 6 revealed gave 5 mutant strains for 4
genes. The MT3126 bank appears more efficient than the RW7097 bank since

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DEVELOPMENTAL AND SOMATIC CELL GENETICS79

the number of pools in the MT3126 bank is 1/4 of that of the RW7097 bank.
These figures provide an estimation for one strain having a Tel insertion ina given 4 kb genomic region obtained from the MT3126 bank.
Seven strains out of the 14 insertion strains turned out to have the Telinsertions in exons. One strain with the insertion in an exon of the unc-22
gene showed a typical twicher phenotype. However, the others didn't show
any abnormality in viability, morphology and mobility even when they were
homozygous with respect to the Tc 1 insertion. Recently it was reported thata Tel sequence which was inserted in an exon of a gene was spliced out togenerate wild-type mRNA. Therefore, we tried to isolate deletion mutantsfrom a Tc 1 insertion mutant taking advantage of the fact that excision of a
Tel frequently accompanies deletion of the flanking region. Two worms ofa Tel insertion homozygous strain were cultured in each of 50 plates tostarvation. DNA was extracted from a part of the progeny worms of eachplate, and subjected to PCR analysis using primers flanking the Tel insertionsite with a distance of about 3 kb. A pool containing deletion mutants gives
a PCR fragment shorter than the wild-type fragment (about 3 kb). A strainof the deletion mutation can be isolated by repeating sub-dividision of theworms of the positive pool and performing PCR analysis.
We have applied the procedure to the kin-16 gene which is one of theprotein kinase homologues, in collaboration with W. Morgan (The College of
Wooster, USA), and have isolated 3 deletion mutant strains. One of themturned out to have a deletion of the entire coding region of the gene, and thehomozygous strain did not show any abnormality in viability, morphology
and mobility. This suggests that the kin-16 gene is a non-essential gene orthat there are functionally exchangeable genes. Isolation of deletion mutantsfor other genes is in progress.
Developmental Mechanisms and Manipulation ofGerm Cells in Mouse Embryos
Norio NAKATSUJI and Yasuaki SHlRAYOSHI

The postimplantation period of mammalian embryogenesis includes many
important events, such as formation of the primitive streak, central nervous
system and primordial germ cells (PGCs). PGCs migrate to fetal gonads,
become the gonia cells, and take different courses of differentiation in male80

and female embryos. Weare studying cellular and molecular mechanisms ofsuch developmental processes in germ cells of mouse embryos.
There are several growth factors, such as the stem cell factor and leukemia
inhibitory factor, which are known to affect proliferation of PGCs in culture.
We found that another factor, tumor necrosis factor (TNF)-a, stimulates
proliferation of PGCs at early migration stages when added to the culture
medium (Kawase, E. et al. (1994) Devel. BioI. 161, 91-95). We also foundthat a medium conditioned with buffalo rat liver (BRL) cells has a similareffect on PGCs, but it is effective not only on the younger PGCs but also on
those at later stages, thus indicating the presence of different factor(s).
We are currently studying differentiation of PGCs into gonia cells after
they have settled in gonads. After developing culture conditions to allow
survival and differentiation of PGCs at these stages, we are now analyzing
molecular and cellular changes of germ cells at this stage. In addition to theend of proliferation and cell shape changes, we have found differences in
response to growth factors and cell surface antigens at these critical stages.
Our aim is to develop experimental methods to study developmental processes including meiosis and gamete formation in culture, and to enable
manipulation of germ cells, such as gene transfection, at various developmental stages to facilitate molecular analysis of germ cell differentiation.

Molecular Analysis of Cell Differentiation and Morphogenesis
in Postimplantation Mouse Embryos
Yasuaki SHlRAYOSHI and Norio NAKATSUJI

Determination of cell fate and cell differentiation are crucial events inmorphogenesis and embryogenesis. Weare trying to approach such problems
from a molecular aspect. One area of experimentation involves identifying
important genes in the determination and differentiation of the central
nervous system during the postimplantation period. We have constructed acDNA library from 7.5-8.5 days old mouse embryos, in which neural plateand neural tube formation is initiated, and screened for important genes
relating to CNS development.
We are currently characterizing one such gene to find out its possiblefunction in embryogenesis. For such a purpose, we are going to analyze theexpression pattern using in situ hybridization method. Further, we will study

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81

their roles in development either by ectopically expressing the gene intransgenic mice or chimaeras with transfected embryonic stem (ES) cells, orby altering the gene through the gene targeting method using ES cells andhomologous recombination. Various ES cell lines we obtained (Kawase et al.
(1994) Int. J. Devel. BioI. 38, 385-390) may be useful for such studies.
Analysis of Migration Patterns of Neurons in Morphogenesisof the Mouse Central Nervous System
Nono NAKATSUJI

In collaboration with another laboratory (Dr. I. Nagata at Tokyo Metropolitan Institute for Neuroscience), we are studying migration patterns ofneuroblasts during histogenesis of the mammalian central nervous system(CNS). We found a new type of cell behavior, "perpendicular contact
guidance," which is exhibited by eNS neurons but not by the peripheral
nervous system neurons on aligned parallel bundles of neurites. Such aperpendicular pattern occurred even on an artificially fabricated microstructure on the quartz surface (Nagata, I. et al. (1993) Development 117, 401408). We also studied migration patterns of neurons in explants of brain
cortices using fluorescence cell labeling (Nagata, I. and Nakatsuji, N. (1994)
Devel. Growth Differ. 36, 19-27), and the ultrastructure of the neuroblasts
showing perpendicular contact guidance (Ono, K. et al. (1994) Devel. GrowthDiffer. 36, 29-38). We are now analyzing the roles of perpendicular contact
guidance in the actual morphogenesis of brain cortices, together with the highresolution optical analysis of growth cone motility to find out the molecularmechanisms of perpendicularity.

82

v.

POPULATION GENETICS

Pattern of Nucleotide Substitutions in Growth Hormone-Prolactin
Gene Family: A Paradigm for Evolution by Gene Duplication
Tomoko OHTA

The growth hormone-prolactin gene family in mammals is an interesting
example of evolution by gene duplication. Divergence among members ofduplicated gene families and among species was examined by using reported
gene sequences of growth hormone, prolactin and their receptors. Sequence
divergence among species was found to show a general tendency in which ageneration-time effect is pronounced for synonymous substitutions but not so
for nonsynonymous substitutions. Divergence among duplicated genes ischaracterized by the relatively high rate of nonsynonymous substitutions, i.e.,
the rate is close to that of synonymous ones. In view of the stage- andtissue-specific expression of duplicated genes, some of the amino acid substitutions among duplicated genes is likely to be caused by positive Darwinian
selection. For details, see Genetics 134, 1271-1276.
Amino Acid Substitution at the Adh Locus of Drosophila is
Facilitated by Small Population Size
Tomoko OHTA

The number of amino acid replacement substitutions and that of synonymous substitutions are examined by using DNA sequences of the Adh locus
of Drosophila. The ratio of replacement to synonymous substitutions ishigher in sequence comparisons between species than in polymorphisms
within species. The ratio for the between-species comparisons is highest in
the Hawaiian group and lowest in the obscura group. These observations
suggest that amino acid substitutions are facilitated by small population size.
The result is in accord with the nearly neutral theory of molecular evolution.
For details, see Pro. Nat!. Acad. Sci., U.S.A 90, 4548-4551.

V.

POPULATION GENETICS

83

An Examination of the Generation-time Effect on Molecular Evolution
Tomoko OHTA

By using DNA sequences of 17 mammalian genes, the generation-time
effect is estimated separately for synonymous substitutions and nonsynonymous substitutions. Star phylogenies composed of rodentia, artiodactyla, and primates are examined. The generation-time effect is found to
be more conspicuous for synonymous substitutions than for nonsynonymous
substitutions, by using the methods of (i) Nei and Gojobori, (ii) Li, and (iii)Ina. The proportion of accepted amino acid substitutions in evolution isestimated to be almost twice as large in the primate lineage as in the rodent
lineage. This result is in accord with the nearly neutral theory of molecular
evolution. For details, see Proc. Natl. Acad. Sci. U.S.A. 90, 10676-10680.
Unbiased Estimation of Evolutionary Distance between
Nucleotide Sequences
Fumio T AJIMA

The number of nucleotide substitutions between nucleotide sequences is
one of the fundamental quantities for the study of molecular evolution. The
proportion of different nucleotide pairs between two nucleotide sequences canbe converted into the number of nucleotide substitutions per nucleotide site
(i.e., evolutionary distance) if an appropriate method is used. Although there
are many methods for such transformation, these methods have some problems. First, these methods give an overestimate when the length of nucleotide
sequence is short. Second, they cannot be applied when the argument of alogarithm of the estimation formula becomes negative; such inapplicable
cases occur quite frequently when two distantly related nucleotide sequences
are compared. That is, when x is a random variable, the expectation of -loge( 1- x) is larger than -loge {l- E(x)} and x can be larger than unity even if
E(x) is smaller than unity, where E(x) is the expectation of x.
I have obtained a new algorithm without logarithm for estimating theevolutionary distance between two nucleotide sequences. This algorithm canbe applied to many estimation methods, such as Jukes and Cantor's method,
Kimura's transition/transversion method, and Tajima and Nei's method.
Unlike ordinary methods, this algorithm is always applicable. Numerical84

computations and computer simulations indicate that this algorithm gives analmost unbiased estimate of the evolutionary distance unless the evolutionary
distance is very large. This algorithm should be useful especially when weanalyze short nucleotide sequences. It can also be applied to amino acidsequences for estimating the number of amino acid substitutions. For details,
see Mol. BioI. Evol. 10, 677-688.
Simple Methods for Testing the Molecular
Evolutionary Clock Hypothesis
Fumio T AJIMA

Whether the molecular evolutionary clock hypothesis holds or not is one of
the most important issues in molecular evolution. This hypothesis may nothold if natural selection is operating, or if mutation rate is not constant per
year.
Although there are several methods for testing this hypothesis, all methods
have some problems: (i) Absolute rate of molecular evolution cannot be
estimated unless we know the divergence time. (ii) We must know thepattern of substitution rates to estimate the number of nucleotide (or aminoacid) substitutions per site. (iii) We must also know the variation ofsubstitution rates among different sites to estimate the number of substitutions per site. (iv) The phylogenetic relationship among nucleotide (oramino acid) sequences must be known before the test is performed.
I have developed new statistical methods for testing the molecular evolutionary clock hypothesis, which overcome above problems. These methods are
based on the chi-square test and are applicable even when the pattern of
substitution rates is unknown and/or the substitution rate varies among
different sites. Furthermore, some of the methods can be applied even when
the outgroup is unknown. Using computer simulations, these methods werecompared with the likelihood ratio test and the relative rate test. The results
indicate that the powers of the present methods are similar to those of thelikelihood ratio test and the relative rate test, in spite of the fact that the latter
two tests assume that the pattern of substitution rates follows a certain modeland that the substitution rate is the same among different sites, while suchassumptions are not necessary to apply the present methods. The present
methods are so simple that they can be easily applied to both nucleotide andV.

POPULATION GENETICS

85

amino acid sequences. For details, see Genetics 135, 599-607.
Statistical Analysis of DNA Polymorphism
Fumio T AJIMA. A large amount of genetic variation can be maintained in natural populations. In order to understand the mechanism maintaining genetic variation,
we must first estimate the amount of genetic variation. There are two
measures for estimating the amount of DNA polymorphism, i.e., the average
number of pairwise nucleotide differences and the number of segregating sites
among a sample of DNA sequences. Using these two measures, we can test
the neutral mutation-random drift hypothesis (the neutral theory). The
expectation of the amount of DNA polymorphism has been studied under
several models, including population subdivision, change in population size,
and natural selection. When a population is subdivided, a large amount of
DNA polymorphism can be maintained in the population if the migration
rates among subpopulations are small. In this case the amount of DNApolymorphism in the subpopulation with lower migration rate is expected tobe smaller than that of higher migration rate. When the population sizechanges, the number of segregating sites changes more rapidly than does theaverage number of nucleotide differences. When purifying selection is
operating, the number of segregating sites is more strongly affected by theexistence of deleterious mutants than is the average number of nucleotide
differences. On the other hand, when balancing selection is operating, theeffect of the selection on the average number of nucleotide differences islarger than that on the number of segregating sites. A mutant under natural
selection affects the amount of DNA polymorphism at linked sites (hitchhiking effect). DNA sequences are not random sequences and there may beconservative and variable regions in them. For details, see pp. 37-59 in
Mechanisms of Molecular Evolution (N. Takahata and A. G. Clark, eds,
Japan Sci. Soc. Press, TokyojSinauer Associates, Sunderland, MA.) and Jpn.
J. Genet. 68, 567-595.

86

The Molecular Descent of the Major Histocompatibility Complex
Jan KLEIN, Yoko SATTA, Colm O'HUIGIN and Naoyuki TAKAHATA

In the last few years, more than 500 primate major histocompatibility
complex (Mhc) genes have been sequenced. This extraordinary sequence
information was used to draw conclusions about the manner of Mhc evolution. Mhc genes were found to evolve at a relatively slow rate with theregularity of a clock. It took from 1 to 6 million years for a new mutation to
be incorporated into an Mhc allele, and the mutation rate is comparable tothat of most other primate genes. The nonsynonymous sites coding for the
peptide-binding region (PBR) are under relatively weak positive selection
pressure (selection coefficient of a few percent only); the nonsynonymous
non-PBR sites are under moderate negative selection pressure. The positive
pressure is probably provided by parasites and is responsible for the transspecies persistence of allelic lineages at functional Mhc loci for more than 40
million years. (For details, see Annu. Rev. Immuno/ll, 269-295, 1993.)
Allelic Genealogy and Human Evolution
Naoyuki TAKAHATA

Genetic variation at most loci examined in human populations indicatesthat the (effective) population size has been __ 104 for the past 1 million years(Myr) and that individuals have been genetically united rather tightly. Alsosuggested is that the population size has never dropped to just a few
individuals, even in a single generation. These impose important requirements for a hypotheses on the origin of modern humans: a relatively large
population size and frequent migration if populations were geographicallysubdivided. Any hypothesis that assumes a small number of founding
individuals throughout the late Pleistocene can be rejected. Extraordinary
polymorphism at some loci of the major histocompatibility complex (Mhc)
rules out the past action of severe bottlenecks, or the so-called founderprinciple, which invokes only a small number of founding individuals when a
new species emerges. This conclusion may be extended to the 35-Myr-old
history of the human lineage, because some polymorphism at Mhc loci seems
to have lasted that long. Furthermore, although the population structure
prior to the late Pleistocene is less clear, owing to the insensitivity of Mhc

V.

POPULATION GENETICS

87

alleles, even at low levels of migration, the nature of Mhc polymorphism
suggests that the effective size of populations leading to humans was as large
as 105• Hence, the effective population size of humans might have become
somewhat smaller in most of the late Pleistocene. The reduction could be due
either to the then adverse environment in the Old World and/or to theincreased migration rate. It can also be argued that population explosion
fostered by the agriculture revolution had significant effects on incorporating
new alleles into human populations. (For details, see Mol. Bio/. Eva/. 10, 222, 1993.)
Effective Population Size, Genetic Diversity, andCoalescence Time in Subdivided Populations
Masatoshi NEI and Naoyuki TAKAHATA

A formula for the effective population size for the finite island model ofsubdivided populations was derived. The formula indicates that the effective
size can be substantially greater than the actual number of individuals in theentire population when the migration rate among subpopulations is small. It
was shown that the mean nucleotide diversity, coalescence time, and heterozygosity for genes sampled from the entire population can be predicted fairly
well from the theory for randomly mating populations if the effective
population size for the finite island model is used. (For details, see J. Mo/.
Eva/. 37, 2~244, 1993.)
The Synonymous Substitution Rate of the Major Histocompatibility
Complex Loci in Primates
Yoko SATTA, Colm O'HUIGIN, Naoyuki TAKAHATA and Jan KLEIN

Because the divergence of many allelic lineages at the major histocompatibility complex (MHC) loci predates species divergence, standard methods ofcalculating synonymous substitution rates are not applicable to this system.
We used three alternative methods of rate estimation: one based on theminimum number of substitutions (D m ), another on the nucleotide difference(D xy ), and the third on the net nucleotide difference (Do). We applied thesemethods to the protein-encoding sequences of primate MHC class I (A, B,
and C) and class II (DRBl) genes. To determine the reliability of the

88different estimates, we carried out computer simulations. The distribution of
the estimates based on D xy or D n , is generally much broader than that basedonD m • More importantly, the Dm-based method nearly always has the highest
probability of recovering true rates, provided that D m is not smaller than 5.
Because of its desirable statistical properties, we used the Dm-based method toestimate the rate of synonymous substitutions. The rate is 1.37±0.61 for A,
1.84±0.40 for B, 3.87± 1.05 for C, and 1.l8±0.36 for DRBlloci, always persite per 109 years. Hence despite the extraordinary polymorphism, themutation rate at the primate MHC loci is no higher than that of other loci.
(For details, see Proc. Natl. Acad. Sci. USA 90, 7480-7484, 1993.)
MHC Polymorphism and Human Evolution
Jan KLEIN, Naoyuki TAKAHATA and Francisco J. AYALA

The diversity of the human major histocompatibility complex (Mhc or
MHC), referred to as the human leukocytoe antigens (HLA), was generated
long before Homo sapiens emerged. The diversity that now shields humans
from parasites is a heritage that has been handed down through innumerable
generations over 65 million years. HLA polymorphism helps to solve various
problems concerning the origin of humans. In this article, we focused on one
convincing message of HLA polymorphism about the founding population of
Homo sapiens. (For details, see Scientific American 269,46-51, 1993.)
Trans-specific Mhc Polymorphism and the Origin of Species in Primates
Jan KLEIN, Yoko SATTA, Naoyuki TAKAHATA and Colm O'HUIGIN

The major histocompatibility complex (Mhc) is a cluster of loci controllingthe specific immune response in vertebrates. Mhc alleles often differ by alarge number of nucleotide substitutions, some of which began to accumulate
before the emergence of extant species. We applied the theory of allelicgenealogy to the primate Mhc genes with the aim of estimating the size of thefounding populations. The calculations indicated that the long-term effective
population size of the studied species was between 104 and 105 individuals andthat it most likely never dropped below 103 individuals. (For details, see J.
Med. Prematol 22, 57-64, 1993.)

v. POPULATION GENETICS

89

SPECIAL FEATURE On the Occasion of the 25th Anniversaryof the Neutral Theory (I) Introductory Comments
on Major Papers by Professor Motoo Kimura
Naoyuki TAKAHATA

Professor Motoo Kimura is best known for his neutral, random drift
theory of molecular evolution which was proposed 25 years ago. This theory
has revolutionized the way we think about molecular evolution. Yet,
population geneticists are more impressed by the power, originality, andingenuity of his research in theoretical population genetics. The topics that
he has treated during the past 40 years are diverse. Except for a few areas
such as those in evolutionary stable strategies and applied quantitative
genetics, he has indeed covered most of the problems that have been raised inmodem evolutionary biology. Because of this diversity, however, it may not
be easy to fully appreciate his papers, particularly for students and young
researchers. Therefore it is my hope to establish the background for his
papers and point out their relationship to other works and to subsequentdevelopments.
Here, 57 papers are grouped into 18 subjects. This grouping obviously does
not follow the chronological order and is neither completely unambiguous
nor mutually exclusive. Nevertheless, I have adopted this compromise tosave space (because the same topic sometimes appears repeatedly in several
papers, with a different focus), but more importantly, to emphasize theprospective value of Professor Kimura's work. (For details, see Jpn. J.
Genet. 68, 353-394, 1993.)
SPECIAL FEATURE On the Occasion of the 25th Anniversaryof the Neutral Theory (II) Relaxed Natural Selectionin Human Populations During the Pleistocene
Naoyuki TAKAHATA

Available genetic data reveals that the human population is more variable
than the chimpanzee population at the protein level, whereas the opposite is
the case at the DNA level. The lower level of silent polymorphism in thehuman population suggests that its long-term breeding size is smaller than the
chimpanzee's. The neutral theory suggests that natural selection has been

90

relaxed in the human population under an improved environment. The
possibility that the relaxation began with the emergence of Homo sapiens was
examined, because it is known that H. habilis underwent for the first time,
dramatic changes in brain size, way of life, and culture, and that thechildhood of H. erectus was already twice as long as that of chimpanzee. The
relaxation hypothesis predicts that, relative to chimpanzee, some 20% of
deleterious mutations became harmless under the changed environment
throughout the Pleistocene. More extensive study of genetic variation in
non-human primates is necessary not only to confirm the hypothesis, but also
to better understand the human genome itself. (For details, see Jpn. J. Genet.
68, 539-547, 1993.)
Decay of Linkage Disequilibrium in a Finite Island Model
Hidenori T ACHIDA

Time-dependent behavior of linkage disequilibrium when there was initial
linkage disequilibrium was studied in a finite island model assuming neutrality. Explicit expressions for linkage disequilibrium parameters were obtained.
From these expressions, the initial and the ultimate decay rates of linkage
disequilibrium parameters were found to increase and decrease, respectively,
due to finiteness of the population when recombination rate, migration rateand inverse of subpopulation size were of comparable order. Also, differentiation of the gametic parameter of linkage disequilibrium among subpopulations was found to diminish quickly. Implications of these results for theinterpretations of linkage disequilibria in natural populations were discussed.
Genetic Diversity in Partially Selfing Populations
with the Stepping Stone Structure
Hidenori TACHIDA and Hiroshi YOSHIMARU*

Plant species are sometimes partially self-fertilizing. Thus, it is necessary
to incorporate self-fertilization in the modeling of plant genetic diversity.
Here, we developed a method to derive equilibrium identity coefficients of
two genes in partially selfing populations using those in haploid populations.
Since explicit expressions are available for the identity coefficients of two* Forestry and Forest Products Research Institute

V.

POPULATION GENETICS

91

genes in haploid stepping stone models, we can obtain those in the partiallyselting population with the same geographical structure as that of the haploid
population using this method. If the mutation rate, population size andmigration rate are small, we showed that the identity coefficients for a selting
population can be approximately obtained by puttingN(I-s/2) in place of N
in the identity coefficients of the corresponding haploid population where N
and s are subpopulation size and selting rate, respectively. Applicability ofthis approximation and its extension are also discussed.

92

VI.

EVOLUTIONARY GENETICS

Three Human MHC Class III Genes Near the Junction with
the Class II Locus: Gene for the Receptor of Advanced
Glycosylation End Products, PBX2 Homeobox Geneand a Notch-homolog, Human Counterpart of
Mouse Mammary Tumor Gene int-3
Kimihiko SUGAYA*, Tatsuo FUKAGAWA*, Ken-ichi MATSUMOTO,
Kazuei MITA**, Ei-ichi TAKAHASHI**, Asako ANDO***,
Hidetoshi INoKo*** and Toshimichi IKEMURA

Cosmid walking of about 250 kb from the MHC class III gene CYP21 toclass II was conducted. The gene for the receptor of advanced glycosylation
end products of proteins (RAGE, a member of immunoglobulin superfamily
molecules), PBX2 homeobox gene designated HOX12 and human counterpart of mouse mammary tumor gene int-3 were found. The contiguous
RAGE and HOX12 genes were completely sequenced, and the human int-3
counterpart was partially sequenced and assigned to a Notch homolog. This
human Notch homolog, designated NOTCH3, showed both the intracellular
portion present in the mouse int-3 sequence and the extracellular portion
absent in the int-3. It thus corresponds to the intact form of a Notch-typetransmembrane protein. About 20 kb of dense Alu clustering was found justcentromeric to the NOTCH3. For details, see Genomics, 23, 408-419(1994).

*

Department of Evolutionary Genetics, National Institute of Genetics, and The
Graduate University for Advanced Studies.
** Division of Biology, National Institute of Radiological Sciences, Anagawa, Chibaken, Japan.
*** School of Medicine, Tokai University, Isehara, Kanagawa-ken, Japan.

VI.

EVOLUTIONARY GENETICS

93

Pseudoautosomal Boundary (PABIX and PABIY)-like Sequence
Existing Near Boundary of Long-range G + C% Mosaic
Domains in the Human MHC Locus
Tatsuo FUKAGAWA1l, Kimihiko SUGAYA'l, Ken-ichi MATSUMOTO,
Katsuzumi OKUMURA2 ), Asako ANDO J ), Hidetoshi INoKo J )and Toshimichi IKEMURA

The human genome is composed of long-range G + C% mosaic structures
related to chromosomal bands. We found the human MHC locus to be an
example of megabase-level G+C% mosaic structures. Chromosome walkingof the 450 kb region bridging MHC classes II and III and base-compositional
analysis could precisely locate the boundary of the mosaic domains, disclosing a sharp G+C% transition. Near the transition point there was asequence highly homologous with the pseudoautosomal boundary of shortarms of human sex chromosomes (PABIX and PABIY) which is theinterface between sex-specific and pseudoautosomal regions. Many PAB 1
XY-like sequences (PABLs) were detected by hybridization against genomic
DNA, and the new sequences defined the complete form of PABLs of about
650 nt. Homologous sequences in the bovine genome were detected byhybridization, suggesting their evolutionarily stable maintenance and biological significance. For details, see Genomics 1995, in press.
The Distribution of Tenascin-X is Distinct and often Reciprocal
to that of Tenascin-C
Ken-ichi MATSUMOTO, Yumiko SAGA'), Toshimichi IKEMURA,
Teruyo SAKAKURA') and Ruth CHIQUET-EHRISMANN4 )We have isolated a cDNA encoding mouse tenscin-X (TN-X), a newmember of the family of tenascin genes. The TN-X gene lies in the major
histocompatibility complex (MHC) class III region, as it is the case for its
human counterpart. On Northern blots we detected a TN-X mRNA of
I)

2)
J)
4)
5)

Department of Evolutionary Genetics, National Institute of Genetics, and The
Graduate University for Advanced Studies.
Faculty of Bioresources, Mie University, Tsu, Mie-ken, Japan.
SChool of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa-ken, Japan.
Friedrich Miescher Institute, Basel, Switzerland.
Laboratory of Cell Biology, RIKEN, Tsukuba, Japan.

94

approximately 13 kb in most tissues analyzed, whereas in various mouse celllines mRNAs of 11 and 13 kb were detected, suggesting the possibility ofalternative splicing of TN-X transcripts. We raised antibodies against mouse
TN-X fragments expressed in bacteria and used these antibodies to identify
the TN-X protein in heart cell extracts and in the conditioned medium of arenal carcinoma cell lines. The subunit molecular size of TN-X is approximately 500 kD, suggesting that the protein may contain up to 40 fibronectin
type III repeats, making it the largest tenascin family member known yet.
TN-X in conditioned medium, as well as the purified pro~ein bind to heparin,
but no binding to tenascin-C (TN-C), fibronectin, laminin or collagens could
be detected. Thus the heparin-binding activity may be a common feature of
the tenascins. The TN-X mRNA as well as the protein are predominantly
expressed in heart and skeletal muscle, but the mRNA is found in mosttissues at a low level. Immunostaining showed the protein to be associatedwith the extracellular matrix of muscle tissues and with blood vessels in all of
the tissues analyzed. Although the TN-X gene lies in the MHC class III
locus, it is not expressed in the lymphoid organs analyzed, expect for the
staining around blood vessels. In skin and tissues of the digestive tract often
a reciprocal distribution of TN-X and TN-C was observed. For details, see
J. Cell BioI. 125,483-493, 1994.
Interfamilial and Intrafamilial Genomic Diversity of Human T
Lymphotropic Virus Type I Strains from Papua New Guinea
and the Solomon Islands
V. R. NERuRKAR*, N. SAITOU and R. YANAGIHARA*

To determine the interstrain genomic diversity and molecular phylogeny of
the recently identified variants of human T-cell lymphotropic virus type I(HTLV-I) in Melanesia, we enzymatically amplified, then directly sequenced
representative regions of the gag, pol, and env genes ofHTLV-I strains from10 members of four families, including one family from Papua New Guinea
and three families from the Solomon Islands. When aligned and compared toa Japanese strain of HTLV-I (ATK), the Melanesian HTLV-I strains differedby 7.6 to 8.7% in the gag, 7.1 to 9.3% in the pol, and 7.3 to 8.2% in the env
gene regions. Based on 931 nucleotides, the overall sequence divergence of*

National Institute of Neurological Disorders and Stroke, NIH, U.S.A.

VI.

EVOLUTIONARY GENETICS95

the 10 Melanesian HTLV-I strains from HTLV-I ATK was 7.3 to 8.1% (68to 75 base substitutions). The intrafamilial genetic heterogeneity among
these virus strains was nil to 0.2%, while the interfamilial sequence variation
between HTLV-I strains from the Solomon Islands and those from Papua
New Guinea was 3.4 to 4.2%, and the genetic heterogeneity among virus
strains from the three Solomon Islands families was 0.2 to 0.9%. Using the
maximum parsimony and neighbor-joining methods, phylogenetic analysis
indicated that the HTLV-I strains from Papua New Guinea and the Solomon
Islands formed a monophyletic group and that the Melanesian and cosmopolitan strains of HTLV-I have evolved along two major geographicallydependent lineages. For details, see Virology 196, 506-513, 1993.
Population Genetic Study in "ainan Island, China. I. Distributionof Blood Genetic Markers
K. OMOTO*, S. MISAWA**, N. SAITOU, C. Du*** and R. Du****Results obtained in a joint Japanese-Chinese field study in Hainan Island
are presented. Three national minority groups, Li, Miao and Hui, together
with Han as control, were studied. A total of 23 blood genetic markers
comprising 9 blood groups, 7 red cell enzymes and 7 serum proteins, and alsoalpha and beta thalassemias were examined, and phenotype and allele frequencies were tabulated. The distributions of allele frequencies differed markedly among the four groups, although falling in general within that ofsouthern Chinese. Two sample groups of Li, i.e., Qi-Li and Benti-Li, aremore similar to each other than the two sample groups of Miao, probably
reflecting different backgrounds in migratory history of Li and Miao. Judging from the high frequencies of both alpha and beta thalassemias and G-6PD
deficiency in Li and Miao, it was considered that malaria played a major rolein determining these characteristics. For details, see Anthropological Science
101, 1-24, 1993.

* International Research Center for Japanese Studies.
** University of Tsukuba.
*** Sun Yazt-sen University of Medical Sciences, China.
**** Institute of Genetics, Academica Sinica, China.

9~Genetic Variability of Major Histocompatibility Complex
in Human Populations
Takashi GOJOBORI and Tadashi IMANISHI

Human major histocompatibility complex (MHC) molecules are called
human leukocyte antigens (HLA). A variety of knowledge about HLA
structures, functions, and genes has been obtained particularly at the molecular level. The HLA gene complex in human is 4 Mbp long and is located on
chromosome 6. HLA genes are classified essentially into two groups, class I
and class II, depending on their structure and function. Genes in each classexist as a multigene family in the MHC gene region. It is well known thatseveral HLA genes exhibit an extremely high degree of polymorphisms. In
particular, HLA-DR and HLA-DQ genes in class II are highly polymorphic
in various ethnic groups. Thus, these genes are very useful for tracing anevolutionary history of human populations. In this study, we described theoutline of our phylogenetic analysis of 80 ethnic groups by use of HLA typing
data that were collected in the 11th International MHC Workshop at
Yokohama.
For more details, see Human Population Genetics: A Centennial Tribute toJ. B. S. Haldane, Plenum Publishing Co., pp 67-74.
Cloning of Human, Mouse and Fission Yeast Recombination
Genes Homologous to RAD51 and recA
Akira SHIOBARA*, Hideaki OGAWA*, Yoichi MATSUDA**.
Noriko USHIO*, Kazuho IKEO and Tomoko OGAWA*

Rad51 of Saccharomyces cerevisiae is a homologue of recA of Eschenchia
coli and plays crucial roles in both mitotic recombination and in repair ofdouble strand breaks of DNA. We have cloned genes from human, mouseand fission yeast that are homologous to rad5!. The 339 amino acid proteinspredicted for the two mammalian genes are almost identical and are highly
homologous with the yeast proteins. The mouse gene is transcribed at a highlevel in thymus, spleen, testis and ovary and at a lower level in brain andother tissues. The rad51 homologues fail to complement the DNA repair***

Depertment of Biology, Faculty of Science, Osaka Univercity.
Division of Genetics, National Institute of Radiological Science.

VI.

EVOLUTIONARY GENETICS

97

defect of rad5l mutants of S. cerevisiae. The mouse gene is located in the F 1
region of chromosome 2 and the human gene maps to chromosome 15.
For details, see Nature Genetics 4, 239-243.
An Evolutionary Role of Kringle Structures
Kazuho IKEO, Kei TAKAHASHI* and Takashi GOJOBORI

Serine preteases involved in the system of blood coagulation and fibrinolysis consist of "kringle" and protease domains. A kringle domain represents a
conspicuous secondary structure of about 80 amino acids with three sets of
intrachain disulfide bonds. Recently, a new member of kringle family was
found in the cell surface protein (ROR) which is a kind of tyrosine kinases.
To understand the evolutionary origin and the evolutionary process of the
kringle family in the ROR gene, we constructed the phylogenetic trees by useof the nucleotide sequences of kringle structures and tyrosine kinase domains.
Our molecular evolutionary analysis shows that the kringle structure sequence of the ROR gene diverged from its ancestral gene about 400 million
years ago. Because the ancestral gene of ROR appeared about 1,500 million
years ago, the kringle structure was considered to have been inserted into the
ROR gene after the appearance of the ancestral gene of ROR-type tyrosine
kinase. Our study also shows that the kringle structures play an importantrole as one of evolutionary and functional units.
Molecular Evolution of the Major Epitopes of HIVwithin a Single Host
Yumi YAMAGUCHI and Takashi GOJOBORIThe third variable (V3) region in the envelope protein of HIV is known to
be one of major epitopes. It has been reported that some amino acid changes
in the V3 region affected the binding ability of antibodies to a viral particle.
To elucidate the evolutionary mechanisms of the V3 region within a single
host, we conducted a phylogenetic analysis of HIV clones isolated from single
hosts. In this analysis, we used two sets of the nucleotide sequence data for
the V3 regions of HIV that were reported to have been isolated from single
hosts at several time points after infection. In particular, we estimated the*Department of Physiology, Shimane Medical College.

98

rates of synonymous and nonsynonymous substitutions during each timeinterval between different time points. The results obtained showed that therates of both synonymous and nonsynonymous substitutions vary with time.
In particular, we found that there was a time interval when the rate ofnonsynonymous substitution became much higher than that of synonymous
substitution. It appears that this time interval may be related to the time of
onset of AIDS.
Search for Candidate Genes on Which Positive Selection Is Operating
Toshinori ENDO, Kazuho IKEO and Takashi GOJOBORI

It has been known that for most genes examined so far, the number ofsynonymous substitutions is much higher than that of nonsynonymous substitutions. It is due to less functional constraints against synonymous substitutions because these substitutions, by definition, do not affect the structure andfunction of a protein. If there is a gene in which the number of nonsynonymous substitutions is significantly larger than that of synonymous
substitutions, we can reasonably assume that positive selection may be
operating on the gene. Because only the MHC gene can be considered as a
candidate gene of positive selection by the studies conducted so far, we
performed the extensive search for such candidate genes in the DNA database. By translating the coding regions of the genes in each entry of the DNA
data base into the amino acid sequences, we made the amino acid sequence
data base. We then conducted homology search. by taking each entry as aquery sequence to collect all homologous gene groups. The gene groups
obtained were multiply aligned with each other. Finally, the so-called cDNA
multiple alignment data base was constructed by replacing the amino acidsequences by the cDNA sequences. Using the cDNA multiple alignment database, we estimated the numbers of synonymous and nonsynonymous substitutions for all possible pairs of cDNA sequences in all available gene groups.
Although we have not obtained the final results yet, the preliminary results
suggest that there are several gene groups in which the number of synonymous substitutions is much higher than that of nonsynonymous substitutions.

VI.

EVOLUTIONARY GENETICS

99

Relative Efficiencies of Various Methods for Estimating MolecularPhylogenetic Trees Such as the Maximum Likehood,
Maximum Parsimony, Neighbor-joining Methods,
when Rate of Nucleotide Substitution Varies
with Nucleotide Site
Yoshio TATENo, Naoko TAKEZAKI* and Masatoshi NEI*

The relative efficiencies of the maximum-likelihood (ML), neighborjoining (NJ), and maximum-parsimony (MP) methods in obtaining thecorrect topology and in estimating the branch lengths for the case of four
DNA sequences were studied by computer simulation, under the assumption
either that there is variation in substitution rate among different nucleotide
sites or that there is no variation. For the NJ method, several different
distance measures (Jukes-Cantor, Kimura two-parameter, and gamma distances) were used, whereas for the ML method three different transition/transversion ratios (R) were used. For the MP method, both thestandard unweighted parsimony and the dynamically weighted parsimony ismore efficient than unweighted parsimony in obtaining the correct topology.
(2) However, both weighted and unweighted parsimony methods are generally less efficient than the NJ and ML methods even in the case where the MP
method gives a consistent tree. (3) When all the assumptions of the ML
method are satist:.~d, this method is slightly more efficient than the NJ
method. However, when the assumptions are not satisfied, the NJ methodwith gamma distances is slightly better in obtaining the correct topology thanis the ML method. In general, the two methods show more or less the same
performance. The NJ method may give a correct topology even when the
distance measures used are not unbiased estimators of nucleotide substitutions. (4) Branch length estimates of a tree with the correct topology areaffected more easily than topology by violation of the assumptions of themathematical model used, for both the ML and the NJ methods. Undercertain conditions, branch lengths are seriously overestimated or underestimated. The MP method often gives serious underestimates for certain
branches. (5) Distance measures that generate the correct topology, withhigh probability, do not necessarily give good estimates of branch lengths.
(6) The likelihood-ratio test and the confidence-limit test, in Felsenstein's
DNAML, for examining the statistical significance of branch length estimates are quite sensitive to violation of the assumptions and are generally too

100liberal to be used for actual data. Rzhetsky and Nei's branch length test is
less sensitive to violation of the assumptions than is Felsenstein's test. (7)When the extent of sequence divergence is < 5% and when> 1,000 nucleotides are used, all three methods show essentially the same efficiency inobtaining the correct topology and in estimating branch lengths. Clearly, thesimplest method, i.e., the NJ method, is preferable in this case.

101

XII.

HUMAN GENETICS

Isolation and Fluorescence in situ Hybridization Mapping of 60
Cosmid Clones on Human Chromosome 18
Hitoshi NAKASHIMA, Masako SAKAI, Rie INABA and Takashi IMAMURA

We have mapped 60 new cosmids on the short and long arms of chromosome 18 either by R or by DAPI banding and simultaneous fluorescence insitu hybridization. These markers were isolated from a hybrid MSI26-21,
made from a human-rodent hybrid cell line that retained human chromosome
18. Twenty-two of the cosmid probes map on the short arm, and 31 probes
cluster in the distal half of the long arm between bands 18q21.1 and 18q23
while 7 other probes are mapped more proximal to the centromere aroundbands 18q11.1-q12.3. The technique of fluorescence in situ hybridization has
proven to be a very efficient methodology for gene mapping. These 60 probes
will be useful in the elucidation of genetic alterations associated with diseases
such as tetrasomy 18p syndrome, 18q- syndromes, and colorectal cancer.
Chromosome 18 is involved in tetrasomy 18p and trisomy 18. The latteris the second most common chromosomal abnormality after Down syndrome. To identify genes involved in these disorders, construction ofhigh-resolution physical map of this chromosome is a rational strategy. A
number of small DNA markers and genes have been physically mapped tochromosome 18 by the use of human-rodent somatic hybrids or humanchromosome deletion panel. Using Cot-lor total human DNA as competitor, the hybridization signals from genomic repetitive DNA sequences can beappropriately suppressed so that large DNA segments cloned in artificial
yeast chromosomes (YAC) or cosmids can now be precisely localized to thecorresponding regions of the chromosomes by fluorescence in situ hybridization (FISH). For characterization of disease-related genes, localization ofmany such large DNA fragments on chromosomes will be very useful. The
resources of human 18 chromosome YAC libraries have recently available formapping, but the localization of a large number of cosmids to this chromosome has not been reported.
Cosmid DNAs were labeled by nick translation with biotin-ll-dUTP,
hybridized to chromosome DNA as described, under conditions that suppress

102

signals from repetitive sequences. Hybridized probes were detected by meansof fluorescein isothiocyanate (FITC) conjugated to avidin and biotinylated
anti-avidin antibody. Signals on metaphase were viewed using a Zeiss
Axiophot epifluorescence microscope equipped with a Cooled Charge Coupled Device (CCD) camera. A total of 60 cosmids have been mapped tometaphase chromosome 18. Twenty-two of the cosmid clones were mapped
to the short arm, one mapped to the centromere, and 37 were mapped to thelong arm of chromosome 18. None of the clones thus far analysed wasfortuitously mapped to other chromosomes, suggesting that the library used
contains DNA fragments derived only from human chromosome 18 as
expected. However, possibility for those cosmids to contain some humanrodent chimeric DNA insert could not be excluded.
Our initial study indicated that cosmid probes derived from the Giemsa
dark bands were difficult to hybridize to the R-banded chromosome spreads.
However, they might well be localized on the DAPI-banded chromosomes.
Since the thymidine synchronization to induce R-banding efficiently blocked
the cells at the moment when the R-bands have finished their replication andbefore the G-bands have began their synthesis, the Giemsa dark bands, beinglate replicating, were BrdU substituted. Thus, the chromatin structure in
G-bands might have undergone overly extensive damage by UV-exposure
process resulting in loss of DNA. We thought that the loss of DNA fromchromatin might have reduced the efficiency of in situ hybridization of theprobe relative to G-bands. Since BrdU substituted chromosomal DNAs
became more sensitive to UV-irradiation procedure, particular attention wasgiven to the analysis of these probes by omitting UV and reducing denaturation time to the minimum for DAPI multiple banding as suggested. Probesthat appeared locating at the junctional region between the G- and R-bands
were tentatively mapped on the G-band when detected only by the DAPI
banding and simultaneous FISH method, but not by the R-banding of thechromosomes. In any event, probes derived from R-bands were to be mapped
clearly on either R- or DAPI-banded chromosomes.

XII.

HUMAN GENETICS

103

Characterization of the Supernumerary der(l8) Chromosomes by
Fluorescence in situ Hybridization with
Single-copy DNA Probes
Hitoshi NAKASHIMA, Masako SAKAI, Rie INABA, Tomoko HASEGAWA,
Yasuhiro NAKAMURA and Takashi IMAMURA

Many cases of extra marker chromosomes have been reported, including
familial and 'de novo' events. In the inherited cases the extra chromosome
may not appear to be causally related to the phenotype of the probands. The
de novo cases present a variety of clinical pictures depending on the different
origins of the markers. Constitutional supernumerary marker chromosomescannot be easily identified using standard cytogenetic banding techniques
because they lack a distinct banding pattern. Marker chromosomes may be
associated with a wide range of phenotypes, making it important to identify
the chromosomal origin of the markers so that correlations with a clinicalphenotype can be evaluated. To develop a method that can differentiate
between the i(18p) and other markers including the 18q- chromosome,cosmid probes were cloned from both arms of chromosome 18 using asomatic cell hybrids, which were then mapped to chromosome 18 by fluorescence in situ hybridization (FISH). The probe was labeled with biotin andused to identify rearrangements involving the chromosome 18. This approach was used to analyze two patients with the phenotype of tetrasomy(18p) or trisomy 18.
A set of chromosome 18-specific DNA probes were obtained from a
cosmid DNA library derived from the cell line MS126, a somatic cell hybrid
containing a normal chromosome 18 as its only human component (Nakashima et al. (1993) Genomics 19, 577-580). Cosmid SCW0204F was hybridized specifically to the short arm at 18p11.2, SCW0207A hybridized to thepericentric region of chromosome 18, and SCW0205D hybridized to the longarm at 18q11.2. FISH was performed according to the methods described
elsewhere. Cosmid DNAs were labeled by nick translation with biotin-lldUTP and were hybridized to chromosome DNA under conditions thatsuppress signals from repetitive sequences. Hybridized probes were detectedby means of fluorescein isothiocyanate (FITC) conjugated to avidin. Signals
on both metaphase and interphase nuclei were viewed using a Zeiss Axiophot
epifluorescence microscope equipped with a cooled charge coupled device(CCD) camera.

104

A 15-year-01d boy was observed to have microcepahly, epicanthal folds,
hypotelorism, hypotonia, a single simian crease, and mildly delayed development. Chromosome analyses revealed a karyotype, 47, XY, +mar, for allcells examined. The marker chromosome was metacentric and potentially ani(l8p). A probe previously localized on the short arm of chromosome 18 wasselected for FISH to determine the origin of the marker chromosome.
Fluorescent signal of the probe SCW0204F DNA was present on the short
arm at 18p11.2 of the normal copies of chromosome 18. Furthermore, theprobe was hybridized to both arms of the metacentric marker chromosome.
The results clearly indicate that the marker chromosome is the i( 18p).
The other patient had phenotypic features characteristic of trisomy 18 ather birth. Cytogenetic analysis showed a karyotype, 47, XX, + 18, t(2;18)(q11;qll), inv(9)(p11q13). The probe SCW0207A specific for the pericentric region of chromosome 18 hybridized to one of the rearranged
chromosome t(2p18q), but not to the other t(18p2q) chromosome. It isnoteworthy that the triplicated hybridization signals for the pericentric
region of chromosome 18 could be obtained also from the interphase nuclei(data not shown). The probe SCW0205D hybridizing to the long arm at 18
ql1.2 of the normal copies of chromosome 18 situated on the long arm of
rearranged t(2p18q) chromosome. Therefore, the break point on the chromosome 18 must be localized between the SCW0207A locus at pericentric
region and the SCW0205D locus at 18q11.2.
One type of marker chromosome that has been reported is the isochromosome 18p. The i(l8p) chromosome is difficult to distingush from chromosome with a large deletion of l8q. Furthermore, translocation products of 18
with another chromosome may appear cytogenetically indistinguishable fromthe i(18p). In cases associated with multiple congenital anomaly, clinicalfeatures are variable and standard banding techniques allow only a likely
diagnosis. Thus, the identification of i(18p) chromosome should use an 18p
probe with attention paid to the distribution of signal on both arms of themarker chromosome as well as on normal copies of chromosome 18. While
chromosome paints may identify the origin of markers or detect translocations, band-specific single-copy sequence probes will permit detailed analysisof chromosomal rearrangements when more precise information may beneeded. To our knowledge this is about the first occasion that chromosome
18 trisomy has been reported in association with a reciprocal translocation
between chromosome 2 and 18. Although it is important to detect the origin

XII.

HUMAN GENETICS

105

of imbalance of chromosome 18, it remains to be clear whether this case is ade novo rearrangement or a malsegregant of a previously existing balanced
karyotype in a parent.
A Dinucleotide Microsatellite Polymorphism on the
Chromosome 18q22 Locus
Hitoshi NAKASHIMA, Masako SAKAI, Rie INABA
and Takashi IMAMURA

Cosmid SCW0205G contaning the (CA)n repeat was isolated from thechromosome 18-specific cosmid library that was made from a human-rodent
hybrid cell line MS125-21 (Nakashima et al. (1993) Genomics, 19, 577-580)
with chromosome 18 as the only human chromosome materials. Colonies
were gridded on nylon membranes and were screened with radio-labeled(CA)20 oligonucleotide probe. The insert DNA was digested with Sau3AI
and subcloned into pUC18 plasmid. CAW0205G, a 0.7kb Sau3AI fragment
was sequenced using primers designed for the sequence flanking the cloningsite. The dinucleotide repeat polymorphism was typed using the polymerase
chain reaction with one of the primer fluoro-Iabeled. Primer sequences wereTAT GTA AAA ACT GAA AGA ATC CAC (CA strand) and TTT TAT
TCT 1'1'1' TGA TGT TAT TGT (GT strand). The reaction products wereanalysed on a 6% denaturing acrylamide sequencing gel by the use of an
automated laser fluorescent DNA sequencer detection system (Genesis 2000
DNA Analysis System; DuPont). Allele sizes were determined by comparison to M13mpl8 sequencing peaks. Fourteen different allele-frequencies
ranged from 0.02 to 0.23, which were calculated from the genotype of 50
unrelated Japanese. The heterozygosity was 0.87. Codominant segregation
was observed in 4 informative two- to three-generation-families.
Peopling of the Americas, Founded by Four Major Lineages ofMitochondrial DNA
Satoshi HORAI, Rumi KONDO, Yuko NAKAGAWA-HATTORI, Seiji HAYASHI,
Shunro SONODA and Kazuo T AJIMA

Mongoloid descendants are now distributed over a wide area of the
Pacific-Rim region, having adapted to a variety of environments. One of the

106

major issues for research into the prehistoric dispersal of Asiatic Mongoloid
peoples is the question of the first Americans, the "Peopling of the New
World". No authority doubts that the ancestral native Americans came from
Northeast Asia across the Bering land bridge, and then dispersed and settled
in various parts of the Americas, and finally reached the southernmost part ofSouth America. However, when they came and with what genetic backgrounds and cultures, is not yet fully resolved.
Nucleotide sequence analysis of the major noncoding region of humanmitochondrial DNA from various races was extended with 72 Native Americans from 16 different local populations (nine populations from Chile, four
from Colombia, and one each from Brazil and from Maya and Apache
Indians). The sequences were determined directly from the polymerase chain
reaction products. On the basis of a comparison of the 482-bp sequences inthe 72 Native Americans, 43 different types of mitochondrial DNA sequenceswere observed. The nucleotide diversity within the Native Americans wasestimated to be 1.29% which is slightly less than the value of 1.44% from thetotal human population including Africans, Europeans, and Asians. Phylogenetic analysis revealed that most Native American lineages are classified into
four major distinct clusters. Individuals belonging to each cluster share atleast two specific polymorphic sites that are nearly absent in other humanpopulations, indicating a unique phylogenetic position of Native Americans.
A phylogenetic tree of 193 individuals including Africans, Europeans,
Asians, and Native Americans indicated that the four Native American
clusters are distinct and dispersed in the tree. These clusters almost exclusively consist of Native Americans with only a few Asians, if any. We postulatethat four ancestral populations gave rise to different waves of migration to the
New World. From the estimated coalescence time of the Asian and Native
American lineages. We infer that the first migration across the Bering landbridge took place -14,000-21,000 years ago. Furthermore, sequence differences in all pairwise comparisons of Native Americans showed a bimodal
distribution that is significantly different from Poisson. These results suggestthat the ancestral Native American population underwent neither a severebottleneck nor rapid expansion in population size, during the migration ofpeople into the Americas. For details, see Mol. Biol. Evol. 10,23-47 (1993).

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HUMAN GENETICS

107

Evolution of Hominoid Mitochondrial DNA with Special Reference
to the Silent Substitution Rate over the Genome
Rumi KONDO, Satoshi HORAI, Yoko SATTA
and Naoyuki TAKAHATA

Focusing on the synonymous substitution rate, we carried out detailed
sequence analyses of hominoid mitochondrial(mt) DNAs of ca. 5-kb length.
Owing to the outnumbered transitions and strong biases in the base compositions, synonymous substitutions in mtDNA reach rapidly a rather low
saturation level. The extent of the compositional biases differs from gene togene. Such changes in base compositions, even if small, can bring about
considerable variation in observed synonymous differences and may result in
the region dependent estimate of the synonymous substitution rate. We
demonstrate that such a region dependency is due to a failure to take properaccount of heterogeneous compositional biases from gene to gene but that theactual synonymous substitution rate is rather uniform. The synonymous
substitution rate thus estimated is 2.37 ± 0.11 X 10- 8 per site per year andcomparable to the overall rate for the noncoding region. On the other hand,
the rate of nonsynonymous substitutions differs considerably from gene togene, as expected under the neutra theory of molecular evolution. The lowest
rate is 0.8 X 10- 9 per site per year for COl and the highest rate is 4.5 X 10- 9
for ATPase 8, the degree of functional constraints (measured by ratio of thenonsynonymous to the synonymous substitution rate) being 0.03 and 0.19,
respectively. Transfer RNA (tRNA) genes also show variability in the basecontents and thus in the nucleotide differences. The average rate for II
tRNAs contained in the 5-kb region is 3.9 X 10- 9 per site per year. The
nucleotide substitutions in the genome suggest that the transition rate is about
17 times faster than the transversion rate. For details, seeJ. Mol. Evol. 36(6),217-231 (1993).
Seroepidemiological Survey of Human T-Iymphotropic Retrovirus
Among Indigenous Plations in Taiwan
Takafumi ISHIDA, I-Hung PAN, Satoshi HORAI, Naruya SAITOU
and Chie-Shan SUN

Human T-lymphotropic retrovirus (HTLV) has been considered as thecausative agent of adult T-cell leukaemia (ATL) and HTLV-I associated108

myelopathy (HAM/TSP). HTLV showing endemic and non-endemic patterns of infection among human populations is not a common virus in man.
Taiwan is situated between HTLV-endemic Ryukyu (Southwestern part ofJapan) and non-endemic mainland China. Recent seroepidemiological studies showed several seropositive cases of HTLV infection in the Taiwanese
population. In addition patients with ATL and HAM/TSP have been
identified. The Taiwanese population consists of Han Chinese and indigenous populations. The Han Chinese migrated from Fujian and Canton in
mainland China several hundred years ago and are to be found throughout
the island. The indigenous Taiwanese who live in the mountainous areas, on
the southeastern coastal region, and on an island between Taiwan and thePhilippines, have been classified into nine ethnoculturally distinguishable
populations. Ethnological data suggest that there is a close relationshipbetween the indigenous Taiwanese and southeast Asian native groups. Data
from two nationwide seroepidemiological studies of HTLV are available andboth report positive cases in the Han Chinese with prevalence rates ranging
from 0% to 1%. However, there is controversy over the presence of virus
carriers in the indigenous populations. In one study seropositivity wasreported in the north whereas in another no seropositive cases were foundamong eight indigenous population groups.
Because previous surveys did not cover all indigenous population groups, aserological survey of HTLV infection among all nine indigenous populationsof Taiwan (Ami, Atayal, Bunun, Paiwan, Puyuma, Rukai, Saisiat, Tsuo, andYami) was carried out. In all, 797 healthy subjects in Taiwan including Han
Chinese and nine indigenous populations were examined for the presence ofantibodies to HTLV by particle agglutination, indirect immunofluorescence,and Western blot test. Two seropositive cases were found in this screening.
One Saisiat male and a Han Chinese female were seropositive for HTLV.
The Western blot profile indicated the virus was type-l HTLV. For details,
see Int. J. Epidemiol. 22, 927-930 (1993).

XII.

HUMAN GENETICS

109

Mitochondrial DNA Mutations at Nucleotide Positions 3243 and 3271
in Mitochondrial Myopathy, Encephalomyopathy, Lactic Acidosis,
and Stroke-Like Episodes: a Comparative Study
Ryoichi SAKUTA, Yu-ichi GOTO, Satoshi HORAI and Ikuya NONAKAAn A-to-G transition mutation at nucleotide position (nt) 3243 in the
mitochondrial (mt)_tRNALeU(UUR) gene (3243 mutation) is commonly foundin patients with MELAS (mitochondrial myopathy, encephalopathy, lactic
acidosis, and stroke-like episodes). In our previous study, 32 of 40 patients(80%) with MELAS had this mutation, the remaining 8 patients did not. We
subsequently found a T-to-C transition in the same mt-tRNA gene but at adifferent position, at nt 3271 (3271 mutation), in three of the 8 remaining
patients. In this communication, we report the analysis of 10 other patientswith MELAS, which allowed us to estimate the incidence of the new 3271
mutation and to clarify the phenotypic differences between patients with the3243 and 3271 mutations in a total of 50 patients.
Of 50 patients with the clinical characteristics of MELAS, 38 had a point
mutation at nucleotide position (nt) 3243 in the tRNALeu(UUR) region in
mitochondrial DNA and 6 at nt 3271 in the same tRNALeu(UUR) gene. Except
for the later onset of the disease in the patients with the 3271 mutation, there
were no clinical, biochemical and pathological differences between the twogroups. Since the nt 3271 region is not located in the binding site for
mitochondrial transcription termination (mTERM) factor, which has been
proposed to be defective in the 3243 mutation, a functional defect in tRNA
itself might be responsible for the enzyme defects in MELAS patients;
however the mechanism by which the defective tRNALeu(UUR) induces thestroke-like episodes remains to be clarified. For details, see J. Neural. Sci.
115, 158-160 (1993).
An A-to-G Transition at Nucleotide Pair 11084 in the ND4 Gene
May Be an mtDNA Polymorphism
Ryoichi SAKUTA, Yu-ichi GOTO, Ikuya NONAKA and Satoshi HORAI

Lertrit et al. (Am. J. Hum. Genet. 51, 457-468, 1992) reported a new
disease-related mutation of mtDNA in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS).

110

They reported two Caucasian patients with the classical features of MELAS
and found different mtDNA mutations in each patient: in one, an A-to-Gtransition at nucleotide pair (nt) 3243 in the tRNALeu(UUR) gene (3243mutation), commonly seen in MELAS patients, and in the other, an A-to-Gtransition at nt 11084 in the ND4 gene (11084 mutation), a mutation which
Lertrit et al. claimed is a novel disease-related mutation. In 50 MELAS
patients studied in our laboratory, we found the 3243 mutation in 34 patients,
a T-to-C transition at nt 3271 in the tRNALeu(UUR) gene (3271 mutation) in 4
patients, and the 11084 mutation in 3 patients. To determine whether the11084 mutation was a disease-related mutation, we further analyzed 90
patients with a variety of other mitochondrial myopathies, as well as 105
normal Japanese individuals. The 11084 mutation was found in 9 patients(10%) with mitochondrial myopathies, including MELAS, chronic progressive external ophthalmoplegia (CPEO), and other miscellaneous mitochondrial disorders, and in 15 (14%) normal individuals. However, none of the
109 normal and diseased Caucasians, American blacks, or several patientswith mitochondrial diseases, including 7 patients with Leber hereditary optic
neuropathy, had the 11084 mutation. On the other hand, I of 10 Japanesepatients with mitochondrial respiratory disorder showed the 11084 mutation.
The high incidence of the A-to-G substitution at nt 11084 in mtDNA in theJapanese population suggests that this substitution may be a polymorphism
prevalent in the Japanese. Although nt 11084 is an evolutionarily conservedsite, and although the amino acid residues are highly invariant in various
species, our findings suggest that an A-to-G substitution at nt 11084 my be apolymorphism and may not be responsible for inducing the clinical phenotype
of MELAS. For details, see Am. J. Hum. Genet. 53, 964-965 (1993).
A T-to-G Mutation at Nucleotide pair 8993 in Mitochondrial DNA
in a Patient with Leigh's Syndrome
Harumi YOSHINAGA, Tatsuya OGINO, Shunsuke OHTAHARA, Ryoichi SAKUTA,
Ikuya NONAKA and Satoshi HORAI

We studied a patient with Leigh's syndrome using neurophysiologic,
radiologic, enzymatic, biochemical, and molecular analysis. Her clinicalcourse had started with acute encephalopathic symptoms at 7 months of age.
With repeated remission and exacerbation, she developed hypotonia andXII.

HUMAN GENETICS

111

symptoms of brainstem dysfunction, such as irregular respiration and swallowing difficulty. These symptoms were followed by epileptic seizures,
including simple partial seizures and tonic spasms. Both serum lactate andserum pyruvate levels were elevated, and deficient activity was detected incytochrome c oxidase in her quadriceps femoris muscle. From the earlystages, we noted an abnormality in the auditory brainstem response andvisual evoked potential, and an abnormal symmetrical low-density area in thebasal ganglia on the computed tomographic scan. We found a mitochondrial
DNA point mutation at 8993 in blood samples from both the patient and hermother using a simple polymerase chain reaction method. The ratio of wild
and mutant mitochondrial DNA calculated densitometrically on polymerase
chain reaction products was 56.6% in the patient's blood cells and 8.4% in
her mother's. This patient's disorder was thought to be maternally inherited
Leigh's syndrome. Her brother had died of the identical clinical features at
1 year 9 months of age. For details, see J. Child. Neurol. 8, 129-133 (1993).
Identification of HSl Protein as a Major Substrate ofProtein-tyrosine Kinases Upon B-cell Antigen
Receptor-mediated Signaling
Yuji YAMASHITA, Masato OKADA, Kurnao TOYOSHIMA,
Daisuke KITAMURA and Takeshi WATANABE

Crosslinking of membrane-bound immunoglobulins, which are B-cell antigen receptors, causes proliferation and differentiation of B cells or inhibitionof their growth. The receptor-mediated signaling involves tyrosine phosphorylation of cellular proteins and rapid activation of Src-like kinases. The
amino acid sequences of five proteolytic peptides of p75, a major substrate ofprotein-tyrosine kinases in the signaling, showed that p75 is the human HSI
gene product. The HS 1 gene is expressed specifically in hemopoietic cells andencodes p75 HS1 , which carries both helix-tum-helix and Src homology 3
motifs. p75 HS1 showed rapid tyrosine phosphorylation and association with a
Src-like kinase, Lyn, after crosslinking of membrane-bound IgM. Thus, p
75 HS1 may be an important substrate of Lyn and possibly other proteintyrosine kinases upon B-cell antigen receptor-mediated signaling. (Proc.
Natl. Acad. Sci., USA, 9, 3631-3635, 1993)

112

Regulation of the Expression of Human Ce Germline Transcript
Toshihiro ICHIKI, Wataru TAKAHASHI and Takeshi WATANABE

Transcriptional regulation of Ig H chain germ-line transcripts induced bycytokines, is a topic of recent interest in the understanding of the mechanism
for class recombination. Among human B cell lines examined, we found thata human IgM-producing B cell line, DND39 (EBV negative), expressed
germ-line transcripts of E constant gene (CE) when stimulated with IL-4. In
our study, the regulatory element responsible for the expression of the IL-4
induced human CE germ line transcript was determined using DND39 cells.
To identify the IL-4 responsive promoter/enhancer element, deletion analysisof the upstream region of the germ-line exon (lE) of the CE-germ-line
transcript located 5' to the switch region, was performed using a luciferase
gene as a reporter. Deletion analysis showed that a DNA fragment which lies
between - 215 and - 154 bp upstream from the most 3' transcriptional
initiation site of the human Ie gene is fully responsible for the induction ofgerm-line transcripts by IL-4. From a mutations analysis, the DNA fragment
between -163 and -152 bp, was identified as a novel IL-4 responsive
element in a human CE gene. Electrophoretic gel mobility shift assay showed
the presence of an IL-4 induced nuclear factor that specifically bound to thisIL-4 responsive element. This novel IL-4 responsive element and an IL-4
induced DNA binding protein may play an important role in the induction of
the CE gemline transcript as well as class switching to Ig E (J. Immunol., 150,
5408-5415, 1993).

113

VIII.

PLANT GENETICS

Bacterial Blight Resistance in Wild Rice Populations
Hiroko MORISHIMA and Toshie MIYABAYASHI

Population biology studies on bacterial blight resistance in wild andcultivated rice were undertaken to gain understanding of the evolutionary
dynamics of a plant-pathogen system. We demonstrated that natural populations of wild rice as well as those of the rice landrace are polymorphic inresistance to bacterial blight disease (BB), and that wild rice is more resistant
than cultivars, and the perennial ecotype of wild rice is more resistant than
the annual ecotype. The following are experimental results recently obtained.
(I) Gene analysis of resistance in wild rice.
About 20 BB resistance genes have so far been identified through genetic
research in rice. To investigate the genetic basis of resistance in wild rice
(Oryza rujipogon), three strains showing different reaction patterns to four
pathogen races (Xanthomonas oryzae pv. oryzae) were chosen from a polymorphic population of annual-type wild rice collected in Thailand and thencrossed with each other. The segregation pattern of BB resistance observedin F 2 populations suggested that at least two dominant genes are responsible
for resistance in the strains used. Most probably many more resistance genes
are preserved in this population. Analysis of F 2's derived from the crosses
with tester lines indicated that the wild strains used, carry resistance genes
different from either of the known genes (xa-5 and Xa-21) which show
similar reaction patterns.
(2) Mechanism of resistance polymorphism within populations.
We have found a high degree of BB resistance polymorphism within wild
rice populations, especially in the annual type of wild rice. In general,
resistance polymorphism can be explained by either of the following twomodels. I) If resistant plants are less fit than susceptible ones in disease-free
conditions ("resistance cost"), then a polymorphism can be maintained. 2)
If pathogens are genetically heterogeneous with each specialized in different
host genotypes, then a polymorphism can be maintained through frequencydependent selection. We examined the relationship between degree of BB
resistance and seed productivity efficiency in the absence of pathogens using

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individuals derived from a few natural populations as well as F 2 populations
derived from resistant and susceptible plants crosses. In experimental populations derived from natural populations of wild rice, the relationships
between resistance and fitness did not show any consistency, being either
positively correlated, negatively correlated or uncorrelated. In F/s, nocorrelation was found in any population. Thus, we failed to detect a trend toresistance cost in this experiment. Weare now planning to examine genetic
heterogeneity within a pathogen population which might be another possible
mechanism of resistance polymorphism.
Life-History of Wild Rice Species Distributed in the Basinof the Amazon River
Hiroko MORISHIMA, Yoshiya SHIMAMOTO*. Masashi OHARA*.
Paulo S. MARTlNS**. Akihiko ANDO** andGiancarlo C.X. OLIVEIRA**

Under a Japan-Brazil cooperative project on Amazonian plant germplasm,
we made study-trips to the Rio Negro in 1992 and to the Rio Solimoes in
1993, focusing on the genus Oryza. Two wild rice species, O. glumaepatulaand O. grandiglumis were found. A total of 103 sites inhabited by wild rice
were studied, and seed samples were collected from 68 populations of O.
glumaepatula and from 61 populations of O. grandiglumis. The former is a
diploid species carrying the same genome as cultivated rice (AA), and thelatter is an allotetraploid species carrying a different genome (CCDD).
1. O. glumaepatula was found in the Rio Solimoes (water pH is nearly
neutral) as well as in the Rio Negro (pH 4.5-5.5). O. grandiglumis was
frequently distributed in the Rio Solimoes, but rare in the Rio Negro.
Interestingly, this species was found in a tributary of the Rio Negro (Rio
Branco) which belongs to a high pH water system. This suggests that O.
glumaepatula can be grown in water with a wide range of pH, but O.
grandiglumis can not survive in low pH water.
2. Although during the present trip we could observe only the plant lifein the aquatic phase, circumstantial evidence indicated that these wild ricespecies initiate their yearly growth in the terrestrial phase (dry season) from***

University of Hokkaido.
University of Sao Paulo, Brazil.

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seedlings in O. glumaepatula, and from ratoons (asexual propagation) in O.
grandiglumis.
3. When we traveled during the later stage of the aquatic phase, many
populations of O. glumaepatula were forming free-floating vegetation, continuing to develop reproductive shoots, while plants of O. grandiglumis werestanding erect, rooted on the river bottom. These two species seemed to havedifferent strategies for coping with the drastic rise of water level; the former
species through free-floating, and the latter through a high ability forinternode elongation. O. glumaepatula proved to have a unique characteristic of brittleness of the stem which helped them to become free-floating
plants.
4. It is not known how the flowering time of wild rice distributed in the
Amazon is regulated where daylength as well as temperature are almost
constant throughout the year. According to information obtained from local
people, wild rice plants begin to flower during the period of rising river water.
Our preliminary experiments showed that both species are photoperiod
sensitive, inducing panicles in response to short day-length. In this experiment, O. glumaepatula showed a second peak of flowering under long
day-length (0. grandiglumis did not show a second peak).
5. A preliminary study of isozyme variation indicated that 0. glumaepatula showed little genetic differentiation among populations. This may be
explained by the free-floating habit of this species. In contrast, O. grandiglumis which tends to persist at the same site seemed to develop a certain degreeof population differentiation.
6. Seeds of both species were found to lack strong dormancy which is a
general characteristic of wild rice. Further, they seemed to be able to survive
in water longer than the seeds of related Oryza species distributed in othercontinents.
The present trip provided us with important information and germplasm
collections of Oryza species in the Amazon Basin. They will surely contributeto our future ecological-genetic study of American wild rice. Further, theunique characteristics found in these wild rices are expected to be utilized inrice breeding work.

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Genetic Erosion of Rice in the Tropics
Hiroko MORISHIMA, Yo-Ichiro SATO, Yoshiya SHIMAMOTO*,
Hiroshi YAMAGISHI** and Tadashi SATO***

Loss of genetic resources is called genetic erosion. Rapid progress ofgenetic erosion occurring in crop species and their wild relatives has beenrecognized as a serious problem in the field of agronomy. To estimate therate of decline of the Asian common wild rice (Oryza rufipogon), biomass(estimated as the total area of population size X rice plant cover in percent) ofwild rice plants in 21 populations (nine annual and twelve perennial types) innorthern and northeastern Thailand in 1983 and 1991 (or 1992) werecompared. The biomass in the annual populations was reduced to 13 percentof the original value on the average during the last decade, suggesting that theannual type of wild rice will disappear by the end of this century. The
biomass in the perennial populations was reduced to 73 percent. In perennial
populations, the weedy type, which is most probably a derivative of natural
hybridization between wild rice and the neighboring cultivars is increasing.
Thus, pure perennial wild rice is now being replaced by the weedy type.
Genetic erosion occurs in cultivated rice also. The number of native
varieties grown in northeastern Thailand has been reduced approximately to
50 percent during the last decade. Genetic diversity within a field has also
been reduced along with the introduction of high-yielding varieties.
To prevent the progress of genetic erosion, gene banks at international andnational levels were established for preservation of genetic resources for
major cultivated plant species, such as rice, wheat, corn, barley, beans. In thecase of rice, a gene bank was built at the International Rice ResearchInstitute (IRRI). National sites were also built in various countries. Suchconservation programmes have played important roles in maintaining genetic
resources. The laboratory conservaton of seed stocks, however, is always
subject to unconscious selection through man's manipulation and consequently potentially useful genes preserved in natural populations are endangered by various factors. The significance of in situ conservation (conservation of natural populations in their original habitats) of genetic resources,
is now recognized. From the viewpoint of agronomy, low input sustainable*

University of Hokkaido.

** Kyoto Sangyo University.
***

Institute of Genetic Ecology, Tohoku University.

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agriculture (LISA) should be encouraged to maintain desirable agroecosystems.
Gamete Eliminator Adjacent to the wx Locus Detected inan Indica-Japonica Hybrid of Oryza sativa
Yoshio SANO, Mitsugu EIGUCHI and Hiro-Yuki HIRANO

A previous study of rice revealed that Wx a (non-waxy endosperm) could
not be transferred from PTB 10 (Indica type of Oryza sativa) to T65wx(Japonica type ofO. sativa). To clarify this phenomenon, the F I hybrid was
backcrossed with T65wx. Two semi-sterile BC3F 1 plants were heterozygous
for wx, but produced only waxy seeds on selfing. Pollen analysis revealed that
non-waxy pollen grains were present at a frequency of 14.8 X 10-4, indicatingthat most W~ pollen grains were eliminated during gametogenesis. The
results showed that PTB 10 and T65wx carried S]if(t) and S1O(t), respectively, and that S1O(t) induced abortion of gametes that carried the opposite
allele, S]if(t), which was linked to Wx a• The frequency of recombination
between wx and S1O(t) suggests that the gamete eliminator detected is very
close to the wx locus. Molecular analysis of the eliminator may thus befacilitated (J. Heredity 85: 310-312).
Procedures for Rejuvenating Plant Genetic Resource Accessions
with Minimum Loss of Allelic Diversity
Katsuei YONEZAWA and Hiroko MORISHIMA

Allelic diversity in plant genetic resource accessions is reduced throughcycles of rejuvenation. Strategies for minimizing the loss of allelic diversity
were discussed based on effective population size and the probability that theinitial allelic diversity within accessions is maintained after 10 and 50 cycles
of rejuvenation. Population size (the number of plants grown for one cycleof rejuvenation) is the most manageable and important factor in determining
the adequacy of maintenance. Population size for each accession, however,cannot be large in most maintenance programmes since many accessions
must be regenerated within a limited field area. Maintaining more accessions
with a smaller population size is more efficient than maintaining fewer with alarger population size. Optimum population size therefore is not uniquely

118

specified, but depends on the total number of accessions to be maintained andfield capacity. When the population size is determined, a means for reducing
variance in progeny number per plant is useful. The SSD (single seed
descent) method is the most effective and practical means. Harvesting seeds
from not all but only some sampled plants is not advantageous even if anequal number of seeds is taken from each of the sampled plants.
The effectiveness of the SSD method, however, is not sufficiently large inpredominantly outcrossing accessions. In outcrossing accessions, controlled
pollination such as selfing and sib-mating may be used combined with the
SSD method if the accessions are self-compatible and sufficiently tolerant toinbreeding depression. Selfing by bagging would be the most handy andeffective method. In species where controlled selfing is not practical, partitioned maintenance, in which each accession is maintained in a number of
separate sub-groups, may be applied, though it is not highly rewarding. The
biparental mating system (Gale and Lawrence 1984), which is highly effective in outcrossing accessions but resource-consuming, may be applied toaccessions of special importance.

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IX.

DATABASE

Toward Unification of Taxonomy Databases in a Distributed
Computer Environment
Hajime KITAKAMI, Yoshio TATENO and Takashi GOJOBORIAll the taxonomy databases constructed with the DNA databases of theinternational DNA data banks are powerful electronic dictionaries which aid
in biological research by computer. The taxonomy databases are, however
not consistently unified with a relational format. If we can achieve consistentunification of the taxonomy databases, it will be useful in comparing many
research results, and investigating future research directions from existentresearch results. In particular, it will be useful in comparing relationships
between phylogenetic trees inferred from molecular data and those constructed from morphological data. The goal of the present study is to unify
the existent taxonomy databases and eliminate inconsistencies (errors) thatare present in them. Inconsistencies occur particularly in the restructuring of
the existent taxonomy databases, since classification rules for constructing
the taxonomy have rapidly changed with biological advancements. A repair
system is needed to remove inconsistencies in each data bank and mismatches
among data banks. We developed a new methodology for removing bothinconsistencies and mismatches from the databases on a distributed computer
environment. The methodology is implemented in a relational databasemanagement system, SYBASE.

DNA Database Management System
Hajime KITAKAMI, Tadasu SHINI, Kazuho IKEo, Yoshihiro UGAWA,
Naruya SAITOU, Takashi GOJOBORI and Yoshio TATENO

A flat-file system is inadequate for building, integrating and searching alarge-scale database in which the number of entries is increasing in an
explosive manner. The DNA database ofDDBJ (DNA Data Bank of Japan)
has been built in AWB developed by LANL on behalf of a flat-file systemsince autumn 1991. AWB is a tool for building the DNA database with theso-called GenBank-schema based on a relational database management

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system, SYBASE. Recently, we newly developed a hierarchical relational
schema for effectively building, integrating, and searching the DNA databaseon the relational database management system, SYBASE, at DDBJ. The
schema is named the "DDBJ-schema". The schema allowed us to implementa DNA database management system with two types of window interfaces,
YAMATO and ASUKA , to easily build the DNA database. The first type with
the DDBJ-schema has an interface, YAMATO, implemented in X-window
and it can be used on UNIX based workstations over computer networks.
The second type has an interface, ASUKA, implemented in the DB-Library of
SYBASE and it can be used on character terminals over telephone networks.
Both also have a window interface similar to the flat file format. Finally, we
proposed a structured SQL-programming method to be used to implement the
DNA database management system. The method is developed through the
SQL expression and the control flow language (CFL) of SYBASE, andallows us to implement a restructuring tool and a tree search tool. The
restructuring tool is needed to convert data with the GenBank-schema into
those with the DDBJ-schema when we move AWB to the new system. The
tree search tool is needed for repairing and searching the taxonomy databaseat DDBJ. For more details, please see "Molecular Bioinformatics," IEEE
Press, London pp. 61-69.

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PUBLICATIONSAIZAWA, S., YAGUCHI, M., NAKANo, M., TOYAMA, K., INOKUCHI, S., IMAI, T., YASUDA, M.,
NABESHIMA, R. and HANDA, H.: Hemopoietic supportive function of human marrowstromal cell line established by a recombinant SV40-adenovirus vector. Exp. Hematol., inpress.
ANDO, A., KIKUTI, Y. Y., KAWATA, H., OKAMOTO, N., IMAI, T., EKI, T., YOKOYAMA, K.,
SOEDA, E., IKEMURA, T., ABE, K. and INOKO, H.: Cloning of a new kinesin-related genelocated at the centromeric end of the human MHC region. Immunogenetics, 39, 194-200,
1994.
ANDO, A., SHINA, T., KAWATA, H., KIKUTI, Y. Y., GENG, L., TSUJI, K., IKEMURA, T. and
INoKo, H.: Cloning of new expressed genes located at the centromeric end of the humanmajor histocompatibility complex (MHC) region. Cytogenet. Cell Genet., in press.
ANDO, A., SHIINA, T., KAWATA, H., KIKUTI, Y. Y., GENG, L., TSUJI, K., IKEMURA, T. and
INOKO, H.: Cloning of new expressed genes located at the centromeric end of the humanmajor histocompatibility complex (MHC) region. HGM93, in press.
ARAKI, K., OHASHI, Y., SASABE, T., KINOSHITA, S., HAYASHI, K., YANF, X. Z., HOSAKO, Y.
and HANDA, H.: Immortalization of rabbit corneal epithelial cells by a recombinant SV40adenovirus vector. Ophthalmol. Vis. Sci., 34, 2665-2671, 1993.
AZUMA, Y., YAMAGISHI, M. and ISHIHAMA, A.: Subunits of the Schizosaccharomyces pombe
RNA polymerase II: Enzyme purification and structure of the subunit 3 gene. Nucleic
Acids Res., 21, 3794-3745, 1993.
CHIQUET-EHERISMANN, R., HAGIOS, C., and MATSUMOTO, K.: The tenascin gene family.
Perspect. Dev. Nuerobiol., 2, 3-7, 1994.
DANJO, I., IHA, H., ONOZAWA, T., ZHAOJUN, X. and FUJIYAMA, A.: The posttranslational
modification of C-termional amino acids of the ras super family proteins. Amino Acids,S,147, 1993.
EHRISMANN, R. C., HAGIOS, C. and MATSUMOTO, K.: The tenascin gene family. Perspect.
Dev. Nuerobiol., in press.
EIGUCHI, M., HIRANO, H.-Y., SANO, Y. and MORISHIMA, H.: Effects of water depth oninternodal elongation and floral induction in a deepwater-tolerant rice line carrying the dW3
gene. Jpn. J. Breed., 43, 135-139, 1993.
EIGUCHI, M., SANO, R., HIRANO, H.-Y. and SANO, Y.: Genetic and developmental bases forphenotypic plasticity in deepwater rice. J. Hered., 84, 201-205, 1993.
GOJOBOTI, T. and IKEo, K.: Molecular evolution of serine protease and its inhibitor with specialreference to domain evolution. Phil. Trans.: BioI Sci., The Royal Society, in press.
GOJOBORI, T., KUCHINO, Y., NISHIMURA, S. and OSAWA, S.: Experimental methods formolecular evolutionary studies. Tokyo Kagaku Dojin, Tokyo, 1993 (in Japanese).
GOJOBORI, T. and IMANISHI, T.: Genetic variability of major histocompatibility complex inhuman populations. In: "Human Population Genetics: A centennial tribute to J. B. S.
Haldane" (Majumder, P. ed.), pp. 67-74 Plenum Publishing Corp., 1993.

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GOJOBORI, T., YAMAGUCHI, Y., IKEO, K. and MIZOKAMI, M.: Evolution of pathogenic viruses
with special reference to the rates of synonymous and non-synonymous substitutions. Jpn.
J. Genet., in press.
HAMAMATSU, C., TORIYAMA, S., TOYODA, T. and ISHIHAMA, A.: Ambisence coding strategyof the rice stripe virus genome. J. Gen. Virol., 74,1125-1131,1993.
HARA, H. and PARK, J. T.: A far upstream region is required for expression of theftsI genecoding for penicilin-binding protein 3 of Escherichia coli. In "Bacterial growth and lysis" (dePedro, M. A. et al. ed), pp. 303-308, Plenum Press, New York, 1993.
HASHIMOTO, H., ISHIHARA, T., SHIGEMOTO, R., MORI, K. and NAGATA, S.: Molecularcloning and tissue distribution of a receptor for pituitary adenylate cyclase-activating
polypeptide. Neuron, 11, 333-342, 1993.
HASHIMOTO, S., KUMAI, H., NAKAI, M., HASHIMOTO, K. and NAKATSUJI, N.: Promotion ofcell adhesion on fibronectin during adenovirus infection of KB cells. Exp. Cell Res., 205,
270-275, 1993.
HAYASHI, S., HIROSE, S., METCALFE, T. and SHiRRAS, A.: Control of imaginal cell development by the escargot gene of Drosophila. Development, 118, 105-115, 1993.
HAYASHI, T., OHTSUKA, H., KUWABRA, K., MAFUNE, Y., MIYASHlTA, N., MORIWAKI, Ki.,
TAKAHASHI, Y. and KOMINAMI, R.: A variant family of mouse minor satellite located on
the centromeric region of chromosome 2. Genomics, 17, 490-492, 1993.
HAYASHIZAKI, Y., SHIBATA, H., HIROTSUNE, S., SUGINO, H., OKAZAKI, Y, SSAKI, N.,
HIROSE, K., IMoTo, H., OKUlZUMI, H., MURAMATsu, M., KOMATSUBARA, H., SHlROISHI, T., MORIWAKI, K., KATSUKI, M., HATANO, N., SASAKI, H., UDEDA, T., MISE, N.,
TAKAGI, N., PLASS, C. and CHAPMAN, V. M.: Identification of an imprinted U2afbinding
protein related sequence on mouse chromosome II using the RLGS method. Nature
Genetics, 6, 33-40, 1994.
HIGASHITANI, A., GREENSTEIN, D., HIROKAWA, H., ASANO, S. and HORIUCHI, K.: Multiple
DNA conformational changes induced by an initiator protein precede the nicking reaction
in a rolling circle replication origin. J. Mol. BioI., in press., 1994.
HIGASHITANI, N., HIGASHITANI, A. and HORIUCHI, K.: Nucleotide sequence of the primer
RNA for DNA replication of filamentous becteriophages. J. Virol., 67, 2175-2181, 1993.
HIGASHITANI, N., HIGASHITANI, A., HOTTA, Y and TABATA, S.: Characterization ofmeiosis-specific strand transfer activities in lily microsporocytes. Bull. Inst. Chern. Res.,
Kyoto Univ., 71, 259-267, 1993.
HIGASHITANI, A., NISHIMURA, Y., HARA, H., AlBA, H., MIZUNO, T. and HORIUCHI, K.:
Osmoregulation of the fatty acid receptor genefadL in Escherichia coli. Mol. Gen. Genet.,
240, 339-347, 1993.
HIRANO, H.-Y., MOCHIZUKI, K., UMEDA, M., OHTSUBO, H., OHTSUBO, E. and SANO, Y.:
Retrotransposition of a plant SINE into the wx locus during evolution of rice. J. Mol. Evol.,38, 132-137, 1994.
HONDA, R., OHBA, Y, NAGATA, A., OKAYAMA, H. and YASUDA, H.: Dephosphorylation ofhuman p34odc2 kinase on both Thr-14 and Try-15 by human cdc25B phosphatase. FEBS
Lett., 318, 331-334, 1993.
HONDA, M., SHIOSAKI, K., EDA, Y., TOKIYOSHI, S., YAMAMOTO, S., NAKASONE, T., IKEO, K.,
YAMAGUCHI, Y., GOJOBORI, T., KORBER, B., HACHIMORI, T. and YAMAZAKI, S.:

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Father-to-mother-to-infant transmission of HIV-1: Clonally transmitted isolate of infant
mutates more rapidly than that of the mother and gradually loses reactivity with neutralizing antibody. J. Virology, in press.
HORAI, S.: Different waves of migration to the New World: implications of mitochondrial DNA
polymorphism in Native Americans. Current Research in the Pleistocene. Vol. 10. pp. 4345, Oregon State University Press, 1993.
HORAI, S., KONDO, R., NAKAGAWA-HATTORI, Y, HAYASHI, S., SONODA, S. and TAJIMA, K.:
Peopling of the Americas, founded by four major lineages of mitochondrial DNA. Mol.
BioI. Evol., 10(1),23-47, 1993.
ICHIKI, T., TAKAHASHI, W. and WATANABE, T.: Regulation of the expression of human Cc
germline transcript. Identification of a novel IL-4 responsive element. J. Immunology, 150,
5408-5417, 1993.
IKEMURA, T.: CODON USAGE. In "Plant Molecular Biology LABFAX" (Croy, R. R. D.
ed.), pp. 37-48, Black well Scientific Publications, UK, 1993.
IMAMURA, T.: Moleuclar biology of the red cells---Genetic analysis of the thalassemia
syndrome. Hematology & Oncology, 28, 49-57, 1994. (in Japanese).
IMAMURA, T.: Studies on the growth and differentiation of hematopoietic stern cells. SaishinIgaku, 43, 318-326, 1993. (in Japanese).
INA, Y. and GOJOBORI, T: Statistical analysis of nucleotide sequences of the hemagglutinin
gene of human influenza A viruses. Proc. Natl. Acad. Sci., U.S.A., in press.
INA, Y, MIZOKAMI, M., ORITO, E., MORIYAMA, E. N., YAMAMOTO, M. and GOJOBORI, T.:
Reduction of synonymous substitutions in the core protein gene of hepatitis C virus. J. Mol.
Evol., 38, 50-56, 1993.
INA, Y., MIZOKAMI, M., ORITO, E., MORIYAMA, E. N., YAMAMOTO, M. and GOJOBORI, T.:
Mutation pattern of hepatitis B viruses and its implication for the effect of the anti-viraldrugs. Mol. BioI. Evol., in press.
INA, Y., MIZOKAMI, M., ORITO, E., MORIYAMA, E. N., YAMAMOTO, M. and GOJOBORI, T.:
Reduction of synonymous substitutions in the core protein gene of hepatitis C virus. J. Mol.
Evol., in press.
INOMATA, Y., WADA, T., HANDA, H., FUJIMOTO, K. and KAWAGUCHI, H.: Preparation of
DNA carrying affinity latex and purification of transcription factors with the latex. J.
Biomater. Sci., in press.
ISHIDA, T., PAN, I.-H., HORAI, S., SAITOU, N. and SUN, C.-S.: Seroepidemiological survey ofhuman T-Iymphotropic retrovirus among indigenous populations in Taiwan. International
Journal of Epidemiology, 22, 927-930, 1993.
ISHIHAMA, A.: Protein-protein communication within the transcription apparatus. J. Bacteriol.,
175, 2483-2489, 1993 (minireview).
ISHIHAMA, A. and BARBIER, P.: Molecular anatomy of viral RNA-directed RNA polymerases.
Arch. Virol. 134, 235-258, 1994 (review).
ISHIHAMA, A., TOYODA, T., KOBAYASHI, M., YASUDA, J., ASANO, Y and NAKAYAMA, M.:
Formation and control of influenza virus RNA polymerase. In "Options for the Control of
Influenza" (Hannoun, C. et al., eds.), pp. 289-297, Elsevier Sci. Publ. B. V., 1993.
ISHIHAMA, A., YAMAGISHI, M., FUJITA, N., MATSUSHIMA, H., OZAKI, M. and SAKURAI, H.:
Starvation-induced resistant state of Escherichia coli: Transcriptional and translational

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control at the stationary phase. In "Stress Proteins" (Azuma, K. ed.), J. UOEH, Vol. 15,
Suppl., pp. 25-30, 1993.
ISHIHAMA, A., Zou, C., KOBAYASHI, M., KUMAR, A., FUJITA, N., SAKURAI, H. and HAYWARD, R. S.: Molecular recognition in gene transcription. In "Molecular Recognition in
Biological Systems" (Buckin, V. et al., eds.).
IWATA, A., TSUCHIYA, T., UEDA, S., ISHIHAMA, A., ZHU, G.-S. and HIRAI, K.: Nucleotide
sequence and transcription organization of the tumor-associated region of Marek's diseasevirus. Proc. Internatl. Symp. Marek's Disease Virus, in press.
IYAMA, S., TSUNEWAKI, K. and TATENO, Y.: Catalogue of wheat experimental strains
maintained in the universities and institutes in Japan. Genetic Resources Section, National
Institute of Genetics, Mishima, 1993.
KABATA, H., KUROSAWA, 0., ARAI, I., WASHIZU, M., GLASS, R., MARGARSON, S. A. and
SHIMAMOTO, N.: Visualization of single molecules of RNA polymerase sliding along DNA.
Science, 262, 1561-1563, 1993.
KAJlTANI, M., KATO, A., WADA, A., INOKUCHI, Y. and ISHIHAMA, A.: Regulation of the
Escherichia coli hfq gene enconding host factor-I for phage Qj3. J. Bacteriol., 176, 53-534,
1994.
KANAMARU, K., KASHIWAGI, S. and MIZUNO, T.: A copper transporting P-type ATPase foundin the thy1akoid membrane of the cyanobacterium, Synechococcus sp. PCC7942. Mol.
Microbiol. (1994) in press.
KANAMARU, K., KASHIWAGI, S. and MIZUNO, T.: The cyanobacterium, Synechococcus sp.
PCC7942, possesses two distinct genes encoding cation-transporting P-type ATPases.
FEBS Lett., 330, 99--104, 1993.
KATOH, Y., MAEKAWA, M. and SANO, Y.: Effects of 5-azacytidine on somatic mutation in asoybean test system. Mutation Research, 300, 49-55, 1993.
KATSURA, I.: In Search of new mutants in cell-signaling systems of the nematode Caenorhabditis elegans. Genetica, 88, 137-146, 1993.
KATSURA, I., KONDO, K., AMANO, T., ISHIHARA, T. and KAWAKAMI, M.: Isolation,
characterization, and epistasis of fluoride-resistant mutants of Caenorhabditis elegans.
Genetics, 136, 145-154, 1994.
KATOH, Y., MAEKAWA, M. and SANO, Y.: Effects of 5-azacytidine on somatic mutation in asoybean test system. Mutation Res., 330, 49--55, 1993.
KAWAGISHI, M., YAMAGISHI, M. and ISHIHAMA, A.: Cloning and sequence determination of
the Schizosaccaromyces pombe rpb2 gene encoding the subunit 2 of RNA polymerase II.
Nucleic Acids Res., 21, 469--473, 1993.
KAWASE, E., YAMAMOTO, H., HASHIMOTO, K. and NAKATSUJI, N.: Tumor necrosis factor(TNF)-a stimulates proliferation of mouse primordial germ cells in culture. Devel. BioI.,
161, 91-95, 1994.
KAWASE, E., SUEMORI, H., TAKAHASHI, N., OKAZAKI, K., HASHIMOTO, K. and NAKATSUJI,
N.: Strain difference in establishment of mouse embryonic stem (ES) cell lines. Int. J.
Devel. BioI., 38, 385-390, 1994.
KITAGAWA, M., OKABE, T., OGINO, H., MATSUMOTO, H., SUZUKI-TAKAHASHI, I., KOKUBO,
T., HIGASHI, H., SAITOH, S., TAYA, Y., YASUDA, H., OHBA, Y., NISHIMURA, S., TANAKA,
N. and OKUMURA, A.: Butyrolactone I, a selective inhibitor of cdk2 and cdc2 kinase.

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Oncogene, 8, 2425-2432, 1993.
KITAMI, H., HARA, H., YAMANAKA, H. and MIYAZAKI, T.: Performance Evaluation for
Parallel-Mixed Integer Programming System, Optimization Methods and software, Gordon
and Breach Science Publishers, 1993/12.
KITAKAMI, H. and GOJOBORI, T.: Challenge to Genome Informatics, DNA Database System,
Journal of Computer Science (bit), Kyoritsu Press, pp. 24-36, 1993 (in Japanese).
KITAKAMI, H., TATENO, Y and GOJOBORI, T.: Toward unification of taxonomy databases ina distributed computer environment. Proceedings of the second International Conferenceon Intelligent System for Molecular Biology, AAAI Press, Menlo Park, California (inpress).
KITAKAMI, H., TATENO, Y. and GOJOBORI, T.: Toward unification of taxonomy database in a
distributed computer environment. ISMB94, in press.
KITAKAMI, H., TATENO, Y and GOJOBORI, T.: Toward Unification of Taxonomy Databases
in a Distributed Computer Environment, Research Report (Japanese Ministry of Education, Science and Culture under Grand-in-Aid for Scientific Research (B) (Grant-No.
05352027», Kyoto University, pp. 93-104, 1993.
KITAKAMI, H., YAMAZAKI, Y, IKEO, K., UGAWA, Y, SHIN-I, T., SAITOU, N., GOJOBORI, T.
and TATENO, Y: Building and search system for a large-scale DNA database. Proceedingof Mol. Bioinformatics, lEE 94 Collaqulun: London, in press.
KITAKAMI, H., YAMAZAKI, Y., IKEO, K., UGAWA, Y., SHIN-I, T., SAITOU, N., GOJOBORI, T.
and TATENO, Y.: Building and search system for a large-scale DNA database. Proceedingof Molecular Bioinformatics, The Institution of Electrical Engineers (lEE) Press, London(in press).
KITAKAMI, H., YAMAZAKI, Y., IKEO, K., UGAWA, Y., SHINI, T., SAITOU, N., Gojobori, T. andTateno, Y: Building and search system for a large-scale DNA database. Molecular
Bioinformatics, lEE Press, London, in press.
KITAKAMI, H., YAMAZAKI, Y., IKEO, K., UGAWA, Y., SHINJ, T., SAITOU, N., GOJOBORI, T.
and TATENO, Y: Building and Search System for a Large-scale DNA Database, Digest of
One-Day Colloquim "Molecular Bioinformatics", The Institution of Electrical Engineers(lEE) Press, London, pp. 61-69, 1994.
KLEIN, J., SATTA, Y., O'HUIGIN, C. and TAKAHATA, N.: The molecular descent of the major
histocompatibility complex. Ann. Rev. Immuno., 11, 269-295, 1993.
KLEIN, J., SATTA, Y., TAKAHATA, N. and O'HUIGIN, C: Trans-specific Mhc polymorphism andthe origin of species. J. Med. Primatol., 22, 57-64, 1993.
KLEIN, J., TAKAHATA, N. and AYALA, F. J.: Mhc polymorphism and human origins. Sci. Am.,
269,46-51, 1993.
KOLB, A., IGARASHI, K., ISHIHAMA, A., LAVIGNE, M., BUCKLE, M. and Buc, H.: E. coli RNA
polymerase, deleted in the C-terminal part of its a-subunit, interacts differentially with thecAMP-CRP complex at the lacPI and thegalPI promoter. Nucleic acids Res., 21, 319-326,
1993.
KONDO, R., HORAI, S., SATTA, Y and TAKAHATA, N.: Evolution of hominoid mitochondrial
DNA with special reference to the silent substitution rate over the genome. J. Mol. Evol.,36(6),217-231, 1993.
KUMAR, A., GRIMES, B., FUJITA, N., MAKINO, K., MALLOCH, R. A., HAYWARD, R. S. and126
ISHIHAMA, A.: Role of the sigma70 subunit of Escherichia coli RNA polymerase intranscription activation. J. Mol. BioI., 235, 405-413, 1994.
KUMAR, A., MALLOCH, R. A., FUJITA, N., SMILLIE, D. A., ISHIHAMA, A. and HAYWARD, R.
S.: The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiationof transcirption at an "extended minus 10" promoter. J. Mol. BioI., 232, 406-418, 1993.
KUSUBATA, M., MATSUOKA, Y, TSUJIMURA, K., ITO, H., ANDO, M., YASUDA, H., KAMIJO,
M., OHBA, Y., OKUMURA, E., KISHIMOTO, T. and INAGAKI, M.: Cdc2 kinase phosphorylation of desmin at three serine/threonine residues in the amino-terminal had domain.
Biochem. Biophys. Res. Commun., 190,927-934, 1993.
LAWLEY, B., FUJITA, N., ISHIHAMA, A. and PITTARD, A. J.: The TyrR protein of Escherichia
coli is a class I transcription activator. J. Bacteriol., in press.
LEE, H.-S., ISHIHAMA, A. and KUSTU, S.: The C-terminus of the a-subunit of RNA polymerase
is not essential for transcription activation of d 54-holoenzyme. J. Bacteriol., 175, 2479-2482,
1993.
LI, F.-Q., VEDA, H. and HIROSE, S.: Mediators for activation offushi tarazu gene transcription
by BmFTZ-FI. Mol. Cell. BioI., in press.
Luo, J., FUJITA, N., ISHIHAMA, A. and KRAKOW, J. S.: Molecular anatomy ofthet/' subunitof E. coli RNA polymerase: Identification of regions involved in assembly. Biochemistry, inpress.
MA, D., WATANABE, H., MERMELSTEIN, F., ADMON, A., OGURA, K., SUN, X., WADA, T.,
IMAI, T., SHIROYA, T., REINBERG, D. and HANDA, H.: Isolation of a cDNA encoding thelargest subunit of TFIIA reveals functions similar to coactivators. Genes Dev., 7, 22462257, 1993.
MASUDA, Y., TSUCHIMOTO, S., NISHIMURA, A. and OHTSUBO, E.: Isolation of temperaturesensitive aminoacyl-tRNA synthetase mutants from an Escherichia coli strain harboring the
pemK plasmids. Mol. Gen. genet., 238, 169-176, 1993.
MATSUMOTO, K., SAGA, Y., IKEMURA, T., SAKAKURA, T. and CHIQUET-EHRISMANN, R.: The
distribution of tenascin-X is distinct and often reciprocal to that of tenascin-C. J. Cell BioI.,
125: 483-493, 1994.
MATSUMOTO, K., SAGA, Y., IKEMURA, T., SAKAKURA, T. and CHIQUET-EHRISMANN, R.:
Mapping of the extracellular matrix protein tenascin-like gene in the s region of the H-2
complex in comparison to its human counterpart. Cytogenet. Cell Genet., in press.
MIURA, T., FUKUNAGA, T., IGARASHI, T., YAMASHITA, M., IDO, E., FUNAHASHI, S., ISHIDA,
T., WASHIO, K., VEDA, S., HASHIMOTO, K., YOSHIDA, M., OSAME, M., SINGHAl, B. Z.,
ZANINOVIZ, Y., CARTIER, L., SONODA, S., TAJIMA, K., INA, Y., GOJOBORI, T. and
HAYAMI, M.: Phylogenetic subtypes of human T-Iymphotropic virus type I and theirrelations to the anthropological background. Proc. Natl. Acad. Sci. V.S.A., in press.
MIZOKAMI, M., GOJOBORI, T. and LAU, Y. N.: Molecular evolutionary virology: Its application to HCY. Gastroenterology, in press.
MOCHIZUKI, K., OHTSUBO, H., HIRANO, H.-Y., SANO, Y. and OHTSUBO, E.: Classification andrelationships of rice strains with AA genome by identification of transposable elements atnine loci. Jpn. J. Genet., 68, 205-217, 1993.
MUKUMOTO, F., HIROSE, S., IMASEKI, H. and YAMAZAKI, K.: DNA sequence requirement ofa TATA element-binding protein from Arabdopsis for transcription in vitro. Plant Mol.

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BioI., 23, 995-1003, 1993.
MURAKAMI, A.: Growth phenomena in Bombyx mori (L.)-with a special reference to genetic
factors responsible for growth acceleration and moultinism. J. Indian Sericult, in press.
MURAKAMI, A.: Ecogenetical studies on voltinism in the tropical mulberry silk worm, Bombyx
mori. Proceedings of the International congress on Tropical Sericulture Practices, pp. 1123.
NAGAI, S., YAGIHASHI, T. and ISHIHAMA, A.: DNA sequence analysis of an allelic variant of
the Actinobacillus pleuropneumoniae-RTX-toxin (ApxIA) from serotype 10. Microb.
Pathogenesis, 15, 485-495, 1993.
NAGATA, I., KAWANA, A. and NAKATSUJI, N.: Perpendicular contact guidance of CNS
neuroblasts on artificial microstructures. Development, 117,401-408, 1993.
NAGATA, I. and NAKATSUJI, N.: Migration behavior of granule cell neurons in cerebellar
cultures. I. A PKH26 labeling study in microexplant and organotypic cultures. Devel.
Growth Differ., 36, 19-27, 1994.
NAITO, M., ISHIGURO, H., FUJISAWA, T. and KUROSAWA, Y.: Presence of eight distinct
homeobox-containing genes in cnidarians. FEBS Lett., 3, 272-274, 1993.
NAKAMURA, T., IHARA, T., NUNOYA, T., KUWAHARA, H., ISHIHAMA, A. and UEDA, S.:
Immunogenicity of glycoprotein II of pseudorabies virus. Vet. Microbiol., 36, 83-90, 1993.
NAKASHIMA, K., SUGIURA, A., KANAMARU, K. and MIZUNO, T.: Signal transduction between
the two regulatory components inbolved in the regulation of the kdpABC operon in
Escherichia coli: phosphorylation-dependent functioning of the positive regulator, KdpE.
Mol. Microbiol., 7, 109-116, 1993.
NAKASHIMA, H., SAKAI, M., INABA, R. and IMAMURA, T.: Isolation and fluorescent in situ
hybridization mapping of 60 cosmid clones on human chromosome 18. Genomics, 19, 577580, 1994.
NAKASHIMA, H., SAKAI, M., INABA, R. and IMAMURA, T.: Dinucleotide repeat polymorphismat the DI8S336 locus. Hum. Mol. Gen., 3: 390, 1994.
NAKAYAMA, M., YAZAKI, K., KUSANO, A., NAGATA, K., HANAI, N. and ISHIHAMA, A.:
Structure of mouse Mxl protein: Molecular assembly and GTP-dependent conformationalchange. J. BioI. chern., 268, 15033-15038, 1993.
NAss, S. J., OLOWSON, M., MIYASHITA, N., MORIWAKI, K., BALLING, R. and IMAI, K.:
Mapping of the Mod-I locus on mouse chromosome 9. Mammalian Genome, 4, 333-337,
1993.
NEI, M. and TAKAHATA, N.: Effective population size, nucleotide diversity, and coalescence
time in subdivided populations. J. Mol. Evol., 37, 240-244, 1993.
NERURKAR, V. R., SONG, K.-J., SAITOU, N., MALLAN, R. R. and YANAGIHARA, R.: Interfamilial and intrafamilial genomic diversity of human T lymphotropic virus type I strainsfrom Papua New Guinea and the Solomon Islands. Virology, 196, 506-513, 1993.
NONAKA, M., MATSUDA, Y., SHIROISHI, T., MORIWAKI, K., NONAKA, M. and NATSUMESAKAI, S.: Molecular cloning of the b subunit of mouse coagulation factor XIII andassignment of the gene to chromosome I: Close evolutionary relationship to complement
factor H. Genomics, 15, 535-542, 1993.
OHTA, T.: Amino acid substitution at the Adh locus of Drosophila· is facilitated by small
population size. Proc. Natl. Acad. Sci. USA, 90, 4548-4551, 1993.

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OHTA, T.: Pattern of nucleotide substitutions in growth hormone-prolactin gene family: A
paradigm for evolution by gene duplication. Genetics, 134, 1271-1276, 1993.
OHTA, T.: An examination of the generation-time effect on molecular evolution. Proc. Natl.
Acad. Sci. USA, 90, 10676-10680, 1993.
OHTA, T.: Interaction of selection and drift in molecular evolution. Jpn. J. Genet., 68, 529-537,
1993.
OMOTO, K., HIRAI, M., JIN, F., MISAWA, S., WASHIO, K., TOKUNAGA, K., YAMAZAKI, K.,
SAITOU, N., Du, C., Lu, B., Luo, Z., ZENO, X., XU, J., ZHAO, H., ZHANG, Z., WEI, X.,
NIU, K., HAO, L. and Du, R.: Population genetic study in Hainan Island, China. I.
Distribution of blood genetic markers. Anthropological Science, 101,1-24,1993.
ONO, K., NAKATSUJI, N. and NAGATA, I.: Migration behavior of granule cell neurons in
cerebellar cultures. II. An electron microscopic study. Devel. Growth Differ., 36, 29-38,
1994.
Ross, W., GOSINK, K. K., SALOMON, J., IGARASHI, K., ZOU, C., ISHIHAMA, A. and GOURSE,
R. L.: A third recognition element in prokaryotic promoters: Involvement of the a subunitof RNA polymerase. Science, 262, 1407-1413, 1993.
SAKUMI, K., IGARASHI, K., SEKIGUCHI, M. and ISHIHAMA, A.: The Ada protein is a memberof class-I transcription factor of Escherichia coli. J. Bacteriol., 175, 2455-2457, 1993.
SAKURAI, M., SUGANO, M., HANDA, H., KOMAI, T., YAGI, R., NISHIGAKI, T. and YABE, Y.:
Studies of HIV-1 protease inhibitors. I. Incorporation of a reduced peptide, simple aminoalcohol, and statine aga10g at the scissile site of substrate sequences. Chern. Pharm. Bull.,41, 136l}-1377, 1993.
SAKURAI, M., HIGASHIDA, S., SUGANO, M., NISHI, T., SAITO, F, OHTA, Y., HANDA, H.,
KOMAI, T., YAGI, R., NISHIGAKI, T. and YABE, Y.: Studies of HIV-1 protease inhibitors.
II. Incorporation of four types of hydroxyethy1ene dipeptide isoesters at the scissile site of
substrate sequences. Chern. Pharm. Bull., 41, 1378-1386, 1993.
SAKUTA, R., GOTO, Y., HORAI, S. and NONAKA, I.: Mitochondrial DNA mutations at
nucleotide positions 3243 and 3271 in mitochondrial myopathy, encepha10phathy, lactic
acidosis, and stroke-like episodes: A comparative study. J. Neurol. Sci., 115, 158-160, 1993.
SAKUTA, R., GOTO, Y., NONAKA, I. and HORAI, S.: An A-to-G transition at nucleotide pair11084 in the ND4 gene may be an mtDNA polymorphism. Am. J. Hum. Genet., 53, 964965, 1993.
.
SANO, Y: A gamete eliminator reconfirmed in a Japonica-Indica hybrid. Rice. Genet. Newslett.
10,76-77, 1993.
SANO, Y.: Constraints in using wild relatives in breeding: Lack of basic knowledge on crop
genepools. In "Internat. Crop Sci. I", (Ed. D. R. Buxton), Crop Sci. Soc. Amer. Madison,
WI. 437-443, 1993.
SANO, Y.: Is an egg-killer present in rice? Theor. Appl. Genet., 86, 148-152, 1993.
SATTA, Y, O'HUIGIN, C., TAKAHATA, N. and KLEIN, J.: The synonymous substitution rate at
the major histocompatibility complex loci in primates. Proc. Natl. Acad. Sci. USA, 90,7480-7484, 1993.
SAWADA, J., GOTO, M., SAWA, C., WATANABE, H. and HANDA, H.: Transcriptional activation
through the tetrameric complex formation of E4TF1 subunits. EMBO J., in press.
SHIMIZU, H. and SUGIYAMA, T.: Suppression of head regeneration by accelerated woundPUBLICAnONS

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healing in hydra. Develop. BioI., 160, 504-511, 1993.
SHINOHARA, A., OGAWA, H., MATSUDA, Y., USHIO, N., IKEO, K. and OGAWA, T.: Cloning of
human, mouse and fission yeast recombination genes homologous to RAD51 and recA.
Nature Genetics, 4, 239~243, 1993.
SHIROISHI, T., SAGAI, T. and MORIWAKI, K.: Hotspots of meiotic recombination in the mouse
major histocompatibility complex. Genetica, 88, 187-196, 1993.
SUGAYA, K., FUKAGAWA, T., ANDO, A., INOKO, H., MATSUMOTO, K. and IKEMURA, T.:
Three genes, receptor gene for AGE, homeobox-containing gene PBX, and int-3 related
Notch-family genes found in the HLA class III region near the border to the class II on 6
p21.3. HGM93, in press.
SUGAYA, K., FUKAGAWA, T., MATSUMOTO, K., MITA, K., TAKAHASHI, E., ANDO, A., INOKO,
H. and IKEMURA, T.: Three human MHC class III genes near the junction with the class
II locus: gene for receptor of advanced glycosylation end products, PBX2-homeobox gene
and a Notch-homolog, human counterpart of mouse mammary tumor gene int-3. Genomics, 23, 408-419, 1994.
SUMI, Y., SHIROYA, T., FUJIMOTO, K., WADA, T., HANDA, H. and KAWAGUCHI, H.:
Application of cationic latex particles for protein purification. Colloids and Surfaces, inpress.
SUN, G.-c., HIROSE, S. and UUDA, H.: Intermittent expression of BmFTZ-FI, a member of thenuclear hormone receptor superfamily during development of the silkworm Bombyx marL
Dev. BioI., in press.
TABUCHI, H., HANDA, H. and HIROSE, S.: Underwinding of DNA on binding of yeast TFIID
to the TATA element. Biochem. Biophys. Res. Commun., 192, 1432-1438, 1993.
TACH IDA, H. and IIZUKA, M.: A population genetic study of the evolution of SINEs. I.
Polymorphism with regard to the presence or absence of an element. Genetics, 133, 10231030, 1993.
TACHIDA, H.: Evolution of repeated sequences in non-coding regions of the genome. Jpn. J.
Genet., 68, 549-565, 1993.
TAJIMA, F.: Unbiased estimation of evolutionary distance between nucleotide sequences. Mol.
BioI. Evol., 10, 677-688, 1993.
TAJIMA, F.: Measurement of DNA polymorphism. In "Mechanisms of Molecular Evolution"(Takahata, N. and Clark, A. G. eds.), pp. 37-59, Japan Sci. Soc. Press, TokyojSinauer
Associates, Sunderland, MA, 1993.
TAJIMA, F.: Simple methods for testing the molecular evolutionary clock hypothesis. Genetics,
135, 599-607, 1993.
TAJIMA, F: Statistical analysis of DNA polymorphism. Jpn. J. Genet., 68, 567-595, 1993.
TAKAHASHI, K., HATTORI, T., NOJI, S., NOHNO, T., SAITO, T., FUJITA, N., ISHIHAMA, A. and
TANIGUCHI, S.: Repression of in vitro transcription of the Escherichia coli fnr and narX
genes by FNR protein. FEBS Lett., 340, 59-,64, 1994.
TAKAHASHI, M., TORIYAMA, S., HAMAMATSU, C. and ISHIHAMA, A.: Nucleotide sequenceand possible ambisense coding strategy of rice virus RNA segment 2. J. Gen. Virol., 74,
769-773, 1993.
TANAKA, K., FUJITA, N., ISHIHAMA, A. and TAKAHASHI, H.: Heterogeneity of the principal
sigma factor in Escherichia coli: The rpoS gene product, d'8, is a principal sigma factor of

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RNA polymerase in stationary phase Escherichia coli. Proc. Natl. Acad. Sci. USA, 90,
3511-3515, 1993.
TAKAHATA, N.: Allelic genealogy and human evolution. Mol. BioI. Evol., 10,2-22, 1993.
TAKAHATA, N.: Haldane's contribution to the understanding of the evolution of vertebrate
immune system. In "Human Population Genetics: A centennial tribute to J. B. S. Haldane"(Partha P. Majumder, ed.), pp. 49-65, Plenum Press, 1993.
TAKAHATA, N.: Introductory comments on major papers by Professor Motoo Kimura. Jpn. J.
Genet., 68, 353-395, 1993.
TAKAHATA, N.: Relaxed natural selection in human populations during the Pleistocene. Jpn.
J. Genet., 68, 539-547, 1993.
TAKAHATA, N. and CLARK, A. G. (Edited): Mechanisms of Molecular Evolution. Japan Sci.
Soc. Press, Tokyo/Sinauer Associates, Sunderland, MA, 1993.
TAKAHATA, N., SATTA, Y. and KLEIN, J.: Trans-species polymorphism at the major histocompatibility complex loci. In "Progress in Immunology VIII". (J. Gergely, M. Benczur, Anna
Erdei, A. Falus, Gy, Fust, G. Medgyesi, Gy. Petranyi, Eva Rajnavolgyi eds.), pp. 153-158,
Springer/Verlag, 1993.
TAO, K., FUJITA, N. and ISHIHAMA, A.: Involvement of the RNA polymerase a-subunit
C-terminal region in cooperative interaction and transcription activation with OxyR protein. Mol. Microniol., 7, 859-864, 1993.
TATENO, Y, TAKEZAKI, N. and NEI, M.: Relative efficiencies of the maximum-likelihood,neighbor-joining, and maximum-parsimony methods when substitution rate varies with site.
Mol. BioI. Evol., 11,261-277, 1994.
TOMIZAWA, J.: Evolution of functional structures of RNA. In "The RNA World" (Gesteland,
R. and Atkins, J. E, eds.), pp. 419-445, Cold Spring Harbor Laboratory Press, Cold Spring
Habor, 1993.
TOYODA, T., KOBAYASHI, M. and ISHIHAMA, A.: Replication in vitro of the influenza virus
genome: Selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein
complexes. Arch. Virol., 136, 269-286, 1994.
WADA, K., WADA, Y., TANAKA, S., DOl, H., NAKAMURA, Y., SUGAYA, K., FUKAGAWA, T.
and IKEMURA, T.: A sensitive and efficient homology search method to find protein-coding
regions using protein-coding region DNA database. In "Proceedings of Genome Informatics Workshop IV" (Takagi, T. et al., eds.), pp. 347-351, Universal Academy Press, Inc.
Tokyo, 1993.
WALLIN, J., MIZUTANI, Y., IMAI, K., MIYASHITA, N., MORIWAKI, K., TANIGUCHI, M.,
KOSEKI, H. and BALLING, R.: A new pax gene, Pax-9, maps to mouse chromosome 12.
Mammalian Genome, 4, 354-358, 1993.
WATANABE, H., SAWADA, J., YANO, K., YAMAGUCHI, K., GOTO, M. and HANDA, H.: cDNA
cloning of transcription factor E4TFI subunits with ETS and NOTCH motifs. Mol. Cell.
BioI., 13, 1385-1391, 1993.
WEST, D., WILLIAMS, R., RHODIUS, V., BELL, A., SHARMA, N., ZOU, C., ISHIHAMA, A. and
BUSBY, S.: Interactions between the Escherichia coli cyclic AMP receptor protein and RNA
polymerase at class II promoters. Mol. Microbiol., 10, 789-797, 1993.
YAMAGISHI, M., MATSUSHIMA, H., WADA, A., SAKAGAMI, M., FUJITA, N. and ISHIHAMA, A.:
Regulation of the Escherichia coli rmf gene encoding ribosome modulation factor (RMF):

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Growth phase- and growth rate-dependent control. EMBO J., 12, 625-630, 1993.
YAMAMOTO, H., MIURA, H., SAITO, S., TANAKA, M., NASU, K., IKEO, K., GOJOBORI, T. and
TAKEUCHI, T.: Regulatory regions of vertebrate tyrosinase genes: Divergence functional
conservation. Proc. Natl. Acad. Sci., USA, in press.
YAMANASHI, Y., OKADA, M., SEMBA, T., TOYOSHIMA, K., WATANABE, T. and YAMAMOTO,
T.: Identification of HSI protein as a major substrate of protein-tyrosine kinases uponB-cell antigen receptor-mediated signaling. Proc. Natl. Acad. Sci. USA, 90, 3631-3635,
1993.
YAMAO, F., NAGAI, Y., KANEDA, S., YOSHIDA, S. and SENO, T.: Conditional resistance tothymineless death predominantly selects DNA synthesis-deficient mutants of mammaliancells. Mutat. Res., 289, 83-89, 1993.
YASUDA, J., TOYODA, T., NAKAYAMA, M. and ISHIHAMA, A.: Regulatory effects of M protein
variations on influenza virus growth. Arch. Virol., 133,283-294, 1993.
YASUDA, J., NAKADA, S., KATO, A., TOYODA, T. and ISHIHAMA, A.: Molecular assembly of
influenza virus: Association of the NS2 protein with virion matrix. Virology, 196, 249-255,
1993.
YOSHIKAWA, H., NAGAI, H., MATSUBARA, K. and FUJIYAMA, A.: Two-Dimensional Gel
Electrophoretograms of Human Chromosome Specific Restriction DNA Fragments. Biochern. Biophys. Res. Commun., 196, 1566--1572, 1993.
YOSHIKI, A., MORIWAKI, K., SAKAKURA, T. and KUSAKABE, M.: Histological studies on malesterility of hybrids between laboratory and wild mouse strains. Develop. Growth & Differ.,
35,271-281, 1993.
YOSHINAGA, H., OGINO, T., OHTAHARA, S., SAKUTA, R., NONAKA, I. and HORAI, S.: A
T-to-G mutation at nucleotide pair 8993 in mitochondrial DNA in a patient with Leigh's
syndrome. J. Child Neurol., 8, 129-133, 1993.
Zou, c., FUJITA, N. and ISHIHAMA, A.: Assymetric spacial arrangement of two alpha subunits
within the Escherichia coli RNA polymerase. J. Mol. BioI., 263, 1283-1288, 1994.

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ABSTRACTS OF DIARY FOR 1993

Biological Symposium
38 1st-Jan. 6
Molecular basis for the functional specificities of homeotic proteins (Katuo Tokunaga)
382nd-Jan. 11
1. Structure-function studies of RNA polymerasesigma subunits (Ashok Kumar)
2. How to tightly control transcription of a gene in
E. coli and in mammalian cells (Hermann Bujard)
3. New components of the yeast RNA polymerase IItranscription initiation complexes (Richard A.
Young)
Insights into the peopling of Melanesia and Australasia
383rd-Jan. 28
from sequence and phylogenetic analysis of HTLV-I
virus genes (Richard Yanagihara)canceled
384th-Mar. 1
The evolution of mammalian mitochondrial DNA
385th-Mar. 8(Svante PiHibo)
386th-Mar. 8
Population dynamics of DNA fingerprinting patterns
within and between populations (Ranajit Chakraborty)
387th-Mar. 10 Databases of EMBL and tools for finding protein functions (Rainer Fuchs)
388th-Mar. 18
Optimal growth in nature: The influence of bad times(G. C. Kurland)
A molecular genetic approach to protein folding: An
389th-Mar. 31
intramolecular chaperone (Masayori Inouye)
Molecular biology of rice tungro virus: Approaches to
39Oth-Mar 30
control by non-conventional protection (Roger Hull)
391st-May 13
Genetic and immunological dissection of RNA polymerase (Robert E. Glass)392nd-June 16 TyrR mediated regulation of the tyrP gene (Blair
Lawley)Rules underlying DNA recognition by a-helices of tran393rd-July 6
scription factors (Masashi Suzuki)
Population genetics issues in the forensic uses of DNA
394th-July 21

ABSTRACTS OF DIARY FOR 1993

395th-Aug. 4
396th-July 22
397th-Sep. 3
398th-Sep. 6
399th-Sep. 8
4OOth-oct. 4
401st-oct. 7
402nd-Nov. 1
403rd-Nov.9
404th-Nov. 18
405th-Nov. 18
406th-Dec. 15
407th-Dec. 20

133(Bruce S. Weir)
The response of streptomyces coelicolor upon oxidative
treatment (Jung-Hye Roe)
Molecular analysis of head formation in hydra (Thomas
Bosch)
Immortal genes: Several examples of interesting atavistic mutations (Susumu Ohno)
Streptomyces-curators of obsolete metabolic pathways?
(Charles Thompson)
Estimation of origin times and substitution rates in
multigene families (Marcy K. Uyenoyama)
The TCR /3 chain: CDR without HVR? and constancy
of its thymocyte repertoire in man (Tommaso Meo)canceled
Mx-proteins: Interferon-induced GTPases with antiviral
activity (Peter Staeheli)
Loss of a yeast telomere: arrest, recovery, and chromosome loss (V. A. Zakian)
Sry and the molecular genetics of mammalian sex determination (Robin Lovell-Badge)
Paranemic states of DNA and their role in DNA unwinding (G. Yagil)
IGD-genome information management system (Otto
Ritter)
1. Role of cell signalling and homeodomain proteins in
the life cycle of the smut fungus Ustilago maydis(Flora Banuett)
2. Control of the cell cycle and cell polarity in budding
yeast (Ira Herskowitz)

Mishima Geneticists' Club
Functional analysis of primer protein in the protein
404th-Jan. 27
primed DNA replication of Bacillus phages M2 and ¢29(Tsutomu Kishi)
The necessity and the present situation of X-ray crystal405th-Feb. 17
lography of proteins (Yasuo Shirakihara)
406th-Feb. 4
Examination of pattern formation mechanisms in Dro-

134

407th-Apr. 21
408th-Apr. 20
409th-June 4
41Oth-Ju1y 841lth-Sep. 27
412th-Nov. 19
413th-Nov. 25
414th-Dec. 13

sophila imaginal disc by enhancer trap method (Satoshi
Gotoh)
Zn-finger proteins expressed in the mouse germ line cells(Toshiaki Nose)
Molecular mechanisms of osmotic responses in E. coli
and Cyanobacteria (Kengo Kanamaru)
A functional analysis of Wnt-3A gene by gene targeting(Shinji Takada)
Molecular mechanisms of the neural network formation
in Drosophila RP neurons (Akira Chiba)
Color expression in Japanese morning glory and transposon (Shigeru !ida)
Molecular genetics of the embryonic tissue interactionin the spinal column formation (Kenji Imai)
Regulatory role of TFIIE on transcription initiation by
eukaryotic RNA polymerase II (Yoshiaki Okuma)
Folk-head gene family and molecular mechanisms ofdevelopment of the mouse body axis and central nervous
system (Hiroshi Sasaki)

FOREIGN VISITORS IN 1993

135

FOREIGN VISITORS IN 1993

Dec. 1, 1991Jan. 6, 1993
Jan. 11
Jan. 11
Jan. 11
Jan. 11
Jan. 28
Mar. 8Mar. 8Mar. 10
Mar. 18
Mar. 31
May 8-19
May 20-June 19
May 26
May 26
May 26
May 26
June 10-11
July 6
July 20-22
July 21-28
July 21-28

Thangirala Sudha, Down's Research Society, India
Katsuo Tokunaga, Zoological Institute, University ofBasel, Switzerland
Ashok Kumar, University of Edinburgh, U.K.
Sankar Adhya, National Cancer Institute, NIH, U.S.A.
Hermann Bujard, University of Heidelberg, Germany
Richard A. Young, Whitehead Institute, Nine Cambridge Center, U.S.A.
Richard Yanagihara, National Institute of Neurological
Disorders and Stroke, NIH, U.S.A.
Svante Paabo, University of Munich and ZoologicalMuseum in Munich, Germany
Ranajit Chakraborty, University of Texas, U.S.A.
Rainer Fuchs, European Molecular Biology Laboratory, Germany
G. C. Kurland, Uppsala University, Sweden
Masayori Inouye, University of Medicine and Dentistryof New Jersey, U.S.A.
Robert E. Glass, University of Nottingham, U.K.
Blair Lawley, University of Melbourne, Australia
Lin Dhung-Mao, National Chiayi Institute of Agriculture, Taiwan
Li Cheng-Chang, National Chung-Hsing University,
TaiwanHu Mao-Lin, National Chiayi Institute of Agriculture,
TaiwanLiu Ching-Yung, National Taiwan University, Taiwan
Ju-Gyeng Kang, National Seoul University, Korea
Masashi Suzuki, MRC Laboratory of Molecular Biology, U.K.
Bruce S. Weir, North Carolina State University, U.S.A.
Charles N. David, University of Munich, Germany
Thomas Bosch, University of Munich, Germany

136

July 25-30
July 26-30
July 3(}-Aug. 1
Aug. 2-5
Aug. 6-7
Aug. 15-Nov. 14
Aug. 17-Sept. 9
Aug. 25-27
Aug. 27Sept. 3
Sept. 3-8
Sept. 6
Sept. 6-8
Sept. 7-8
Sept. 7-8
Sept. 7-8
Sept. 7-8
Sept. 7-8
Sept. 7-8
Sept. 7-11
Sept. 1(}-11
Sept. 11-14
Sept. 27---Oct. 25
Oct. 4Oct. 11-17
Oct. 5Oct. 29-Nov. 1
Oct. 29-Nov. 1
Oct. 31-Nov. 3
Nov. 9Tang Ling-Hua, Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, China
Wang Xiang Kun, Beijing Agricultural University,
China
Robert A. Steen, Cornell University, U.S.A.
Jung-Hye Roe, Seoul National University, Korea
Joe Inselburg, Dartmouth Medical School, U.S.A.
G. Subramanya, Mysore University, India
Chitrakon Songkran, Pathumthani Rice ResearchCenter, Thailand
Deb Overath, University of Georgia, U.S.A.
Ashok Kumar, University of Edinburgh, U.K.
Susumu Ohno, Beckman Research Institute of the Cityof Hope, U.S.A.
Mercy K. Uyenoyama, Duke Univercity, U.S.A.
Charles Thompson, University of Bazel, Switzerland
Masatoshi Nei, Pennsylvania State University, U.S.A.
M. A. Zargar, Banaras Hindu University, India
T. K. Kundu, Indian Inst. Sci., India
P. K. Burma, Indian Inst. Sci., India
K. P. Gopinathan, Indian Inst. Sci., India
Xue Wu, Shanghai Inst. Biochem., China
Shin-Feng Tsai, Natl. Yang-Hing Med. College
D. C. Sy, University of Santo Tomas, Philippines
T. K. Kundu, Indian Inst. Sci., India
Kevin K. Murakami, University of Vermont, U.S.A.
Supan Fucharoen, Khon Kaen University, Thailand
Tommaso Meo, Institut Pasteur, France
Wang Fengshan, Tianjin Institute of Industrial Hygieneand Occupational Diseases, China
Dzhanybek M. Adyshev, Institute of Biochemistry andPhysiology, Kyrgys Academy of Sciences, Kyrgyz
Ian Tessrey, University of Freiburg, Germany
Martin Schwemmle, University of Freiburg, GermanyPeter Staeheli, University of Freiburg, Germany
V. A. Zakian, Fred Hutchinson Cancer Research Institute, U.S.A.

FOREIGN VISITORS IN 1993

Nov. 16-19
Nov. 18
Nov. 22-26
Nov. 25-26
Dec. IDee. 1Dec. 3Dec. 15
Dec. 19-21
Dec. 20-21
Dec. 20-21

137

Robin Lovell-Badge, National Institute for Medical Research, U.K.
G. Yagil, The Weizmann Institute of Science, Israel
Boriboon Somrith, Department of Agriculture, Thailand
Yoshiaki Ohkuma, The Rockefeller University, U.S.A.
Ritta H. Mikkola, Uppsala University, Sweden
Stefanie A. Margarson, University of Nottingham Medical School, u.K.
Dipankar Chatterji, Centre for Cellular and Molecular
Biology, India
Otto Ritter, German Cancer Research Institute, Germany
Mark Stoneking, University of Pennsylvania, U.S.A.
Ira Herskowitz, University of California, U.S.A.
Flora Banuett, University of California, U.S.A.

138

AUTHOR INDEX
69.42
ADYSHEV, D
24
ANDACHI, Y. .
78
ANDO, A. .
92, 93
AN DO, A.
114
ASANO, Y.
23, 26AYALA, F. J
88
AzUMA, Y
21
BOSCH, T. C.
61
CHIQUET-EHRISMANN, R.
.
93
DAVID, C. N
61
DEHARO, M
61
DING, Q
20
Du, C
95
Du, R.
.
95
EHRISMANN, R
93
EIGUCHI, M.
.
117
ENDO, T.
.
98
FISCHER-LINDAHL, K. .
57
FUJII, M.
.
49
FUJISAWA, T
61
FUJITA, N.
.
16, 17, 18, 19,20
FUKAGAWA, T.
.
92, 93
FUSE, N36
GOJOBORI, T. .
96, 97, 98, 119, 120
GOTO, M70
GOTO, Y109
HANDA, H.
.
64,69,70
HARA, H42
HASEGAWA, T.
.
103
HATTA, M. .
61,62
HAYASHI, S
36,37,38
HAYASHI, S. .
105
HAYWARD, R. S. .
19
HIGASHITANI, A
40, 41, 44
HIGASHITANI, N.
.
.41, 44HIRANO, H.-Y
33, 117
HIROSE, S. .
36, 37, 64, 65
ADMON, A.

ABE, N.

HISHIDA, R.

72
62
HORAI, S.
.
lOS, 107, 109, 110
HORIUCHI, K.
.
40, 41, 42, 44
ICHIKI, T.
112
IKEMURA, T
57, 92, 93
IKEO, K.
.
96, 97, 98, 120
IMAI, T. .
69
IMAMURA, T. .
101, 103, 105
IMANISHI, T.
96
IMASEKI, H.
.
64
INABA, R.
.
101, 103, 105
INoKo, H.
.
92, 93ISHIDA, T.
.
107
ISHIHAMA, A
16, 17, 18, 19,20,21,22,
23, 24, 25, 26
ISHIHARA, T
71, 72, 73
JISHAGE, M. .
19
KABATA, H. .
35
KANAMARU, K.
.
47
KANEDA, H. .
57
KANEDA, S. .
27,28,29
KATSURA, I.
71, 72, 73
KAWAGISHI, M.
.
21
KAWAKAMI, M.
.
71
KIMURA, M.
.
16,21
KISHI, T.
.
30
KISHIMOTO, Y.
62
KITAKAMI, H
119, 120
KITAMURA, D.
.
112
KLEIN, J.
.
86, 87, 88
KOBAYAKAWA, Y.
.
38
KOBAYASHI, M
22,23
KOHARA, Y. .
74, 76, 78
KOIZUMI, O
61
KONDO, R. .
105, 107
KUBORI, T.
.
34
KUMAR, A.
.
19
KUNI-EDA, M.
.
33
HOBMA YER, E.

.

.

AUTHOR INDEX

19,20
17
LI, F-Q
65
LINDAHL, K. F
57
MA, D.
.
69
MARTINS, P. S.
.
114
MARUYAMA, T
23
MASUYA, H.
.
50, 51
MATSUDA, Y.
27,96
MATSUMOTO, K.-1.
57, 92, 93
MERMELSTEIN, F
69
MIKOSHlBA, K. .
38
MINATO, K.
.
69
MISAW A, S.
.
95
MITA, A. .
49, 50
MITA, K.
.
92
MIYABAYASHI, T.
.
113
MIYASHITA, N.
.
.49, 56
MIZUMOTO, K.
.
22,23,31,32
MIZUNO, K-1.
54, 55MORISHIMA,H
113, 114, 116, 117
MORIWAKI, K.
. .49, 50, 51, 52, 53, 54, 55,
56, 57, 58
MOTOHASHI, T.
74
MUKUMOTO, F.
64
MUNEOKA, Y.
61
MURAKAMI, A.
.
66, 67NAGAI, H
35
NAGAI, Y. .
27, 28, 29
NAKADA, S.
.
22
NAKAGAWA-HATTORI, Y
105
NAKAMURA, Y.
.
103
NAKASHIMA, H
101, 103, 105
NAKATSUJI, N
52,79,80,81
NEGISHI, T.
.
17
NEI, M.
.
87, 99NERURKAR, V. R
94
NINAKI, O
67
NISHIGAKI, A.
.
74
NISHIMURA, A.
.
.45, 47NISHIMURA, Y.
.42
NON AKA, I.
109, 110
OGAWA, H.
.
96
OGAWA, T
96
KUSANO, S

KYOGOKU, Y.

.

139

110
69
OHARA, M. .
114
O'HUIGIN, C.
.
86, 87, 88
OHTA, T.
82, 83
OHTAHARA, S
110
OKADA, M. .
112
OKAMOTO, M.
.
58
OKANO, H
38
OKUMURA, K.
.
93
OLIVEIRA, G. C. X.
.
114
OMOTO, K
95
PAN, I-H
107
REINBERG, D.
.
69
RICHARD, S. .
19
SADAIE, Y.47
SAGA, Y93
SAGAI, T.
50, 51, 52, 53, 54, 55, 57, 58
SAITOU, N. .
94, 95, 107, 120
SAKAI, M.
.
101, 103, 105
SAKAKURA, T.
93
SAKUTA, R
109, 110
SANO, Y.
33, 117
SATO, T.
116
SATO, Y.-1.
116
SATTA, Y.
86,87,88, 107
SAMESHIMA, M.
.
27
SAWADA, J-1.
70
SAWAMOTO, K
38
SENO, T.
27, 28, 29, 30
SHIGA, Y.
.
37
SHIMAMOTO, N. .
34, 35SHIMAMOTO, Y. .
114, 116SHIMIZU, H.
.
61
SHINI, T.
120
SHIOBARA, A.
.
96
SHIRAYOSHI, Y.
57,79,80
SHIROISHI, T. ... .49, 50, 51, 52, 53, 54, 55,
57, 58SHIROYA, T.
.
69
SONODA, S. .
105
SUDHA, T
26
SUGAYA, K. .
57,92,93
SUGIMOTO, A.
.
74
OGINO, T

OGURI, K

140
SUGIYAMA, T.

61,62

SUN, c.-S. . . . . . . . . . . . . . . . . . • . . . . . . . . 107
SUN, G.-c.

• . . . . . . . . . . . . • . . . . . . . . . . . . 65

69
73
76
64
90
83, 84, 85
105

SUN, X.

.
SUZUKI, N. .
T AHARA, H.

.

T ABUCHI, H.
T ACHIDA, H.

.

T AJIMA, F.
TAJIMA, K
TAKAGI, H.

.47

56
92
TAKAHASHI, K. .
97
TAKAHASHI, T.
61
T AKAHASHl, W. .
112
TAKAliATA, N
86,87,88,89,107
T AKEZAKI, N.
.
99
TANIMURA, Y.
.
, ...• 38
TATENO, Y.
..
99,119,120
TOYODA, T.
.
22, 23, 26
TOYODA, Y.
.
57
TOYOSHIMA, K.
.
112
TSUJI, H.
27
UEDA, H.
.
65
UGAWA, Y. . . . . . . . • . . . . . . . . . , ....•. 120
URASAKI, M
73
T AKAHAMA. S.

.

TAKAHASHI, E.-I.

USHlO, N.

96
69
WAKANA, S.
. . . • . . . . . . . . . . 50,51,53,58
WATANABE, H.
.
,
69,70
WATANABE, H
74
WATANABE, T.
112
YAGI, Y . . . . , . . . . . . • . . . . . . . . . . . . . . . • . 37
YAGURA, T.
29
Y AMAGISHI, H.
.
116
Y AMAGISHl, M. .
21
Y AMAGUCHl, K.
.
, .70
YAMAGUCHI, Y.
• .•........... ,97
Y AMAO, F. . .. ,27, 28, 29, 30
YAMASHITA, Y.
..
112
YAMAZAKI, K.-I.
64
Y ANAGIHARA, R. .
94
Y ANO, K.-I.
,
70
YASUDA, H.
. .. ,60
YASUDA, J,,
24
YASUDA, S. . • . . . . . . . . . . . . . . . ,., .. 42, 44
YONEKAWA, H,
56,58
YONEZAWA, K.
.
, .117YOSHIMARU, H.
.
,, .. 90YOSHINAGA, H.
110
YOSHINO, M
53, 57, 58
Zou, C.
18
WADA, T.

.

,

,

,

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