ISSN OOn-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 381987

Published by
THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken. Japan1988

Annual Repor~of theNational Ins~itute of Genetics
No. 38, 1987

Published by

The National Institute of Genetics, Japan
1988

CONTENTS
General statement
Staff .....
Council and Advisory Committee ..
Association for propagation of the knowledge of genetics '"Projects of research for 1987 .
Research activities in 1987 ..
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1. Molecular Genetics
Mapping of functional sites on subunits of Escherichia coli RNA
polymerase. ISHIHAMA, A., FUJITA, N., UESHIMA, R. and
GLASS, R. E.
Strength and regulation of Escherichia coli promoters. I. Preparation of a promoter collection and determination of two
parameters of promoter strength. ISHIHAMA, A., ENDO, So,
AlBA, H., FUJITA, N., UESHIMA, Ro and NAKAYAMA, M.
Strength and regulation of Escherichia coli promoters. II. Effectof DNA supercoiling and temperature shift on promoter activity of the rpoH gene encoding the heat-shock sigma subunitof RNA polymerase. UESHIMA, R., FUJITA, No and ISHIHAMA, A....
Structure and function of genes for RNA polymerase-binding
proteins: Regulation of expression of stringent starvation
protein (SSP) gene. SERIZAWA, H. and FUKUDA, Ro
RNA polymerase and transcriptional signals of Micrococcus luteus.
NAKAYAMA, M., FUJITA, N., OSAWA, S. and ISHIHAMA, A. ...
Processing of tRNA precursors in Escherichia coli: RNase P is
involved in both 5' and 3' termini of leuX tRNA precursor.
NOMURA, T. and ISHIHAMA, A
Molecular mechanism of influenza virus RNA transcription. I.
Role of NP on RNA chain elongation. HONDA, A., UEDA,
K., NAGATA, K. and ISHIHAMA, A. . ...
Molecular mechanism of influenza virus RNA transcription. II.
Inhibition of transcription by matrix protein. HANKINS,
R. W., NAGATA, K. and ISHIHAMA, Ao .
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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Transcription and replication of influenza virus RNA: An in vitro
system using isolated nuclei from virus-infected cells. NAGATA, K. and ISHIHAMA, A. .
.
Translational regulation of influenza virus mRNAs. YAMANAKA,
K., NAGATA, K. and ISHIHAMA, A.
Establishment of mouse cells expressing influenza viral genes.
YAMANAKA, K., NAGATA, K., HOSAKA, Y. and ISHIHAMA. A. ..
Control of influenza virus gene expression: Quantitative analysis
of each viral RNA species in infected cells. HATADA, E.,
HASEGAWA, M., MUKAIGAWA, J., SHIMIZU, K. and FUKUDA,

R.

Studies on the function(s) of influenza virus nonstructural proteins.
HATADA, E., HASEGAWA, M., SHIMIZU, K., HATANAKA, M.,
and FUKUDA, R. .
.
Analysis of a novel influenza A virus PB2 mutant, defective in theregulation of three polymerase genes. MUKAIGAWA, J., HATADA, E., FUKUDA, R. and SHIMIZU, K.
Assignment of the function of nuclear factor 1. MATSUMOTO, K,
NAGATA, K. and HANAOKA, F.
GTP induces knotting, catenation and relaxation of DNA withstoichiometric amounts of DNA topoisomerase II from
Bombyx mori and HeLa cells. HIROSE, S., TABUCHI, H. and
YOSHINAGA, K. .
.
In vitro transcription of eukaryotic genes is affected differently by
the degree of DNA supercoiling. HIROSE, S. and SUZUKI, Y.

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Changes in organization of mitochondrial and nuclear DNA in
cytoplasmic reversion of male sterile rice. NAWA, S., YAMADA, M.-A. and SANO, Y. .
.
Messenger RNA synthesis of Sendai virus (HVJ). MIZUMOTO,K.
Mechamism of mRNA capping reaction. MIZUMOTO, K. .
.
Regulation of initiation of chromosomal replication in bacteria.
YOSHIKAWA, H
.

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iii

CONTENTSII.

Microbial Genetics
A synthetic translation terminator gene: A tool for dissecting
translation direction of gene. MARUYAMA, I. N., HORIKOSHI,
K., NAGASE, Y., SOMA, M., NOBUHARA, M., YASUDA, S. and
HIROTA, Y. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determination of gene products and coding regions from the
murE-murF region of Escherichia coli. MARUYAMA, I. N.,
YAMAMOTO, A. and HIROTA, Y.
A note on the parA mutation in Escherichia coli. KATO, J.,SUZUKI, H., NISHIMURA, Y. and HIROTA, Y.
A new mutation causing a defect in chromosome partition in
Escherichia coli. NISHIMURA, Y., KATO, J., YAMADA, M.,
SUZUKI, H. and HIROTA, Y.
Organization of the parC-sufI gene region in Escherichia coli.
KATO, J., NISHIMURA, Y., YAMADA, M., SUZUKI, H. and
HIROTA, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A mutant defective in the processing of penicillin-binding protein
3 of Escherichia coli. HARA, H., NISHIMURA, Y, YASUDA, S.,SUZUKI, H. and HIROTA, Y.
Processing in the C-terminal portion of penicillin-binding protein
3 of Escherichia coli. HARA, H., KATO, J., SUZUKI, H. and
HIROTA, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
In vitro system for the processing of penicillin-binding protein 3 ofEscherichia coli. HARA, H., SUZUKI, H. and HIROTA, Y. . . . .
Purification and characterization of the dnaA protein of Escherichia
coli. YASUDA, S.
Involvement of the dnaK protein in replication of oriC DNA in
vitro. SAKAKIBARA, Y. and YASUDA, S. . . . . . . . . . . . . . . . . . . .
Inhibitor of DNA replication in Escherichia coli, a possible
regulatory protein YASUDA, S. . . . . . . . . . . . . . . . . . . . . . . . . . .
Mapping of a whole set of cell division genes in Escherichia coli
K12. NISHIMURA, A., and IROBE, R.
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Immunogenetics
Sex-specific recombinational hot spot in the mouse major histocompatibility complex. SHIROISHI, T., HANZAWA, No, SAGAI,
To and MORIWAKI, K.
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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Extensive polymorphisms of the MHC class II genes in Japanesewild populations of rat. GOTOH, H., KAwAMOTO, Y., SHIROISHI, T. and MORIWAKI, K.
Introduction of human steroid 21-hydroxylase gene into mice:
An attempt at gene therapy. GOTOH, H., SAGAI, To, SHIROISHI, T. and MORIWAKI, K .. o'o'Trans-subspecies transmission of mouse H-2 polymorphism.
SAGAI, To, SHIROISHI, T. and MORIWAKI, K
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Developmental and Somatic Cell Genetics
Preservation of frozen Drosophila embryos after irradiation bylaser microbeam. KURODA, Y, TAKADA, Y. and KASUYA,
T
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Studies on sex differentiation of embryonic cells of Drosophila
65melanogaster. KURODA, Y, SAKAGUCHI, B. and OISHI, K ..
Injury effect in hydra head regeneration. SHIMIZU, H. and SUGIYAMA, T.
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Role of interstitial cell migration in generating position-dependent
patterns of nerve cell differentiation in Hydra. FUJISAWA, T.
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Genetic studies on the lifespan of adult silkworms: Effect of re.
moval of brain. MURAKAMI, A. and SHIMADA, J
Genetic studies of parthenogenesis in the Bombyx silkworm:
Genetic factors responsible for the high frequency of spon.
taneous parthenogenesis. MURAKAMI, A
Secretory mode of fibrous cuticle (FC) in silkworm. MORI, T.,
SHIMADA, J. and MURAKAMI, A.
On the sex of artificially-induced parthenotes after treatment of
newly laid unfertilized ova with hot-water in Bombyx mori.
MURAKAMI, A.
Embryonic development of a maternally affected embryonic-lethal
mutant in Drosophila melanogaster. (II). MINATO, K. and
YAMADA, M.-A. .
.
Isolation and characterization of a maternally affected embryoniclethal mutant rescued by transplantation of normal egg cytoplasm in Drosophila melanogaster. YAMADA, M.-A. andNAWA, S
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CONTENTSv

A novel mobile DNA element consisting of tandem repeats at thewhite locus of Drosophila simulans. INOUE, Y. H. and YAMAMOTO, M.-T
. 74
Genetics of an unstable white mutant in Drosophila simulans: Reversion, suppression and somatic instability. INOUE, Y. H.,
TAIRA, T. and YAMAMOTO, M.-T
. 75
The ninja mobile element is abundant specifically in a strain of
Drosophila simulans. YAMAMOTO, M.-T. and INOUE, Y. H.

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Grandsonless strain of Drosophila simulans carries maternally inheritable sex-ratio factor which specifically dysfunctions Ybearing sperms. YAMAMOTO, M.-T. and WATANABE, T. K ... 76
Studies on gene expression in mouse cerebellum. AOTA, S. and
IKEMURA, T. 78
Myofibril assembly is linked with vinculin, a-actinin and cell-substrate contacts in cardiac myocytes in vitro. SHIMADA, Y.... 78
Myogenesis in vitro as seen with the scanning electron microscope.
SHIMADA, Y
. 79

V.

Cytogenetics
Expression of ribosomal RNA gene clusters in laboratory strainsof mice demonstrated by cytogenetic techniques. KURIHARA,
Y., SUH, D. S., SUZUKI, H. and MORIWAKI, K.
Chromosomal polymorphism in the ant Myrmecia (pilosula) n= 1.
IMAI, H. I., TAYLOR, R. W., CROZIER, R. H., CROSLAND,
M. W. J. and BROWNING, G. P. .
.
A note on trophic egg-feeding by the queen of the primitive ant
Myrmecia (pilosula) n=l. MASUKO, K.
.

VI.

Mutagenesis and Radiation Genetics
Inactivation of transforming DNA with gamma-rays at low dose
rates. SADAIE, Y. and KADA, T.
Tissue-specific radiation sensitivity and its relation to differentiation in a mouse mutant, wasted. TEZUKA, H. and INOUE, T.

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Mutagen-modifying effects of vitamin A and vitamin E in cultured.
Chinese hamster cells. KURODA, Y. .

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Mutagenicity of N'-aminocytidine and its derivatives in Chinese
hamster V79 cells. KURODA, Y., NOMURA, A., NEGISHI, K.
and HAYATSU, H.
Major genetic loci regulating susceptibility to pulmonary adenomas
in CXB recombinant inbred strains of mice. MIYASHITA, N.
and MORIWAKI, K.
Genetic analysis of the reciprocal translocation between a sex
chromosome (Y) and an autosome (5) in silkworm. MURAKAMI, A.

VII.

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Population Genetics
Molecular evolutionary clock and the neutral theory. KIMURA,
M
A stochastic model of compensatory neutral evolution. KIMURA,
M
Very slightly deleterious mutations and the molecular clock.
OHTA, T.
A model of evolution for accumulating genetic information.
OHTA, T.
Simulating evolution by gene duplication. OHTA, T.
On the overdispersed molecular clock. TAKAHATA, N.
Adult lactose absorption and milk use from the standpoint ofgene-culture theory. AOKI, K.
Toward a theory for the evolution of cultural communication:
Coevolution of signal transmission and reception. AOKI, K.
and FELDMAN, M. W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gene-culture waves of advance. AOKI, K.

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VIII. Evolutionary Genetics
Molecular evolutionary rates of oncogenes. GOJOBORI, T. and
YOKOYAMA, S.
99
Organization and nucleotide sequence of a densovirus genome
imply a host-dependent evolution of the parvoviruses. BANDO,
H., KUSUDA, J., GOJOBORI, T., MARUYAMA, T. and KAWASE,
S.
99
Evolutionary origin of pathogenic determinants in enterotoxigenic
Escherichia coli and Vibrio cholerae 01. YAMAMOTO, T.,
GOJOBORI, T. and YOKOTA, T.
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CONTENTS

vii

Molecular evolution and phylogeny of the human AIDS viruses
LAV, HTLV-III, and ARVo YOKOYAMA, S. and GOJOBORI,
To
o. o.
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o. o. o. 100
Molecular phylogeny of the human immunodeficiency and related
retroviruses. YOKOYAMA, S., MORIYAMA, E. N. and GoJOBORI, T
o. o' " o101
Six chloroplast genes (ndhA-F) homologous to human mitochondrial genes encoding components of the respiratory chain
NADH dehydrogenase are actively expressed: Determinationof the splice sites in ndhA and ndhB pre-mRNAso MATSUBAYASHI, T., WAKASUGI, T., SHINOZAKI, K., YAMAGUCHISHINOZAKI, K., ZAITA, N., HIDAKA, To, MENG, B. Y., OHTO,
C., TANAKA, M., KATO, A., MARUYAMA, T. and SUGIURA, M.
000000.

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Male-driven molecular evolution demonstrated by different ratesof silent substitutions between autosome- and sex chromosomelinked genes. MIYATA, T., HAYASHIDA, H., KUMA, K. and
YASUNAGA, T.
o.
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Nucleotide sequence of immunoglobulin epsilon genes of chimpanzee and orangutan: DNA molecular clock and hominoid
evolution. SAKOYAMA, Yo, HONG, K.-Jo, BYUN, S. M.,
HISAJIMA, H., VEDA, S., YAOITA, Y., HAYASHIDA, H., MIYATA,
To and HONJO, T.
o. o.
o. o.
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Global variation in G+C content along genome DNA sequenceswith reference to chromosome banding structures of verte104
brate genomes. IKEMURA, To and AOTA, S.
Codon usage tabulated from the GenBank genetic sequence data.
AOTA, S., GOJOBORI, T., ISHIBASHI, F., MARUYAMA, T. and
IKEMURA, To
o.
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Serological survey of mouse complement factor H in common
laboratory and wild-derived strains: A new allotype H.3.
HARADA, Y., MORIWAKI, K. and TOMITA, T.... o. o.
105Genetic characterization of the mouse strain with morphologicalphenotypes suggesting a descendant from ancient oriental
stocks of laboratory mice (Japanese waltzing mice). YONEKAWA, H., NIELSEN, J. T., MATSUSHITA, Y., WAKANA, S.,
KATOH, H., MIYASHITA, N. and MORIWAKI, K. .
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viii

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Ribosomal DNA spacer-length polymorphisms in rice.

SANO, Y.

108
Rice carries rDNA repeats at two unlinked loci. SANO, Y. 108
A chromosomal interchange induced in a near-isogenic line carrying
an alien sterility gene. SANO, Y. .
. 109

IX.

Human Genetics
Oncogene activation by Philadelphia translocations in chronic
myeloid leukemia and acute lymphoblastic leukemia. IMAMURA, T. and NAKASHIMA, H.
III
Evolutionary implications of mitochondrial DNA polymorphismin human populations: Comparison of two different populations in Japan. HORAI, S., HAYASAKA, K., GOJOBORI, T. and
MATSUNAGA, E. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 113
Phylogenetic relationships among Japanese, rhesus, Formosan, andcrab-eating monkeys inferred from restriction enzyme analysisof mitochondrial DNAs. HAYASAKA, K., HORAI, S., GoJOBORI, T., SHOTAKE, T., NOZAWA, K. and MATSUNAGA, E.
115

X.

Applied Genetics
Alien pollen primacy (APP) in the rice plant. SATO, Y.-I., TANG,
L.-H. and ISHIKAWA, R
117
The Ph-Aph association in rice, and its significance in IndicaJaponica differentiation. SATO, Y.-I. and MORISHIMA, H. .... 118
Intra-populational genetic diversity found in indigenous rice cultivars of Yunnan. MORISHIMA, H
118
Characteristics of weed rice strains collected from various Asian
countries. TANG, L.-H. and MORISHIMA, H. .
119
Isozyme genes useful for Indica-Japonica classification of common
rice. TANG, L.-H. and SATO, Y.-I.
120
Location of Pgd-l locus in rice. ISHIKAWA, R., KINOSHITA, T.
and MORISHIMA, H.
121
Genetic aspects of differentiation into annuals and perennials in
Asian wild rice. BARBIER, P. and MORISHIMA, H
122Electrophoretic analysis by immobilized pH gradient of sevenprotein fractions sequentially extracted from rice endosperm.
ENDO, T
123

CONTENTS

ix

Multiple alleles at the wx locus as a major factor for amylose content in rice grain. SANO, Y. .
124

XI. Database
Constructing a DNA database. MIYAZAWA, S
125
Publication from Genetic Resources Section, Genetic Stocks Research Center. !YAMA, S
126
Publication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 127
Abstract of diary for 1987 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 134
Foreign visitors in 1987
. . . . .. 136
Author index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 138

GENERAL STATEMENT
This report presents an outline of research activities carried out in our
institute during the year 1987. The total expenditure (not including thatfor facilities) for 1987 FY (April 1987-March 1988) amounted to 1 billion
70 million yen, a half of which was for personnel expenses. In addition,
grants-in-aid amounting to 190 million yen were rendered to selected staff
members by the Ministry of Education, Science and Culture (Mombusho).
Changes in personnel allotments included new positions for three members
approved from April: two research staff members (joshu), one for the DNA
Research Center and the other for the Genetic Stock Research Center, and an
administrative official for the Department of Administration. Thus the total
number of positions available for regular staff is 96, of which 56 are forresearch staff. Concerning facilities, a new building for the DNA ResearchCenter was completed at the end of January, and two greenhouses for riceand mulberries were also completed at the end of March.
The variety and breadth of research conducted in this institute is reflected in the honors bestowed on its members, the degree of international
participation in joint research and conferences and the contributions to thescientific community and the community at large made in the last year.
I am happy to note that three of our colleagues were honored in the past
year. Prof. Motoo Kimura, Laboratory of Population Genetics, wasawarded two prizes: the Asahi Prize for 1986 by the Asahi Shimbun Press,
and the John J. Carty Award for the Advancement of Science by the National Academy of Sciences, U.S.A., for his outstanding contributions to
the theoretical aspects of population and evolutionary genetics, most notably for his neutral theory of molecular evolution now widely accepted.
In addition, because of his distinguished services to friendship between
Japan and France through biology, he was decorated with the Chevalier del'Ordre National du Merite by the French Government. He was elected
as an honorary member of the Genetics Society of the United Kindgom.
Prof. Tomoko Ohta of the same laboratory was awarded the Weldon Memorial Prize for 1986 by Oxford University. It is given every three years
to the person who has in recent years published the most noteworthy contributions to Biometric Science. Prof. Kimura also received the same

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

prize in 1965. Dr. Takashi Gojobori of the Laboratory of Evolutionary
Genetics won an Encouragement Award from the Genetics Society ofJapan for his "Studies on Molecular Evolution by Comparing the Nucleotide Sequences of Genes."
International personnel exchanges were very active in the past year.
Members of our staff went abroad on 27 occasions for the purpose of presenting research results at various scientific meetings, exchanging information, and carrying out collaborative studies or field investigations; threemembers staying longer than 3 months. In addition, 31 scientists visited
our instltute from abroad, with whom information and views on recent
studies were actively exchanged. Some of them delivered stimulating lectures at the Biological Symposium. Those who stayed longer than threemonths and carried out collaborative studies with our staff members were:Miss Pascale Barbier, Universite des Sciences et Techniques du Languedoc,
Montpellier, France; Dr. Nguen Xuan Hong, Hanoi University, Vietnam;
Dr. Jozefa Styrna, Jagiellonian University, Poland; Dr. Dong Sang Suh,
Genetic Engineering Center, Korea Advanced Institute of Technology,
and Dr. Sam-Eun Kim, Sericultural Experiment Station, Rural Development Administration, Republic of Korea; and Dr. Ling Hua Tang, Instituteof Food Crops, Jiangsu Academy of Agricultural Sciences, People's Republicof China.
The past year also saw a number of changes in the staff of the institute.
Dr. E. Matsunaga was reappointed on October 1 to an additional two year
term as institute director. Dr. Tom Fujishima of the Laboratory of Agricultural Genetics, who had been carrying out selection experiments on thesexual maturity of Japanese quails and genetic studies on food preferencein mice, left in September for Hisamitsu Pharmaceutical Company as the
director of its Kyudo Safety Research Division; Dr. Masatoshi Yamamotoof the Laboratory of Cytogenetics, who had been engaging in developmental and molecular genetic studies of Drosophila melanogaster, waspromoted in November to Associate professor of Miyazaki Medical
College. On the other hand, the following three joined us: Mr. Hidenori
Hayashida (postgraduate student of Kyushu University) and Dr. Hitoshi
Ueda (postdoctoral fellow, National Cancer Institute, NIH, USA) wereappointed as joshu in the DNA Research Center in April and to the Genetic
Stock Research Center in October, respectively; Dr. Asao Fujiyama (research associate of the Dept. of Biochemistry and Molecular Biology,

GENERAL STATEMENT

3

University of Chicago) was appointed as Associate professor of the Laboratory of Human Genetics in December. Dr. T. K. Watanabe, Associate
professor, was transferred in April to the Genetic Stock Research Centerfrom the Laboratory of Evolutionary Genetics. In addition, Dr. Takeshi
Seno, Saitama Cancer Center Research Institute, will join us as a professor
at the Laboratory of Mutagenesis in January 1988.
We mourn the loss of two colleagues in the past year. Dr. Taro Fujii,
Associate professor of the Plant Section of the Genetic Stock ResearchCenter, died from lung cancer on May 11 at the age of 60, and Dr. Takeo
Maruyama, Professor of the Laboratory of Evolutionary Genetics andHead of the DNA Research Center, died from a heart attack on December
11 at the age of 51.
Starting his scientific career in the institute in 1950, Dr. Fujii publisheda number of papers, under the guidance of the late Dr. Seiji Matsumura(Head of the Laboratory of Induced Mutation), in the field of radiation
genetics with plants such as wheat and Arabidopsis. He then extended his
research to the development of test systems for environmental mutagens
using soybeans and maize, and more recently to a collaborative work on
nitrogen fixation in the rhizosphere of rice. After being transferred to the
Genetic Stock Research Center, he also made efforts to conserve various
strains of higher plants, and took administrative charge of the Experimental
Farm as its head.
Dr. Maruyama joined our institute in 1966 as a staff member of the
Department of Population Genetics headed by Prof. Kimura, and heengaged in the mathematical treatment of various genetic problems including the stochastic process in population genetics. He published a numberof significant papers on the rules governing genetic variability in geographically structured populations, of which two (Genet. Res. 15: 221-225,
1970; Genetics 70: 639-651, 1972) have been adopted in the Benchmark
Papers in Genetics (Vol. 7, edited by W. H. Li, 1977). After the reorganization of the institute in 1984 he devoted himself energetically to the establishment of the DNA Data Bank of Japan (DDBJ). Thanks to his
endeavor, on-line service of the data base was made available last autumn
for researchers all over the country. While confronting the challenge of
how we could contribute to international cooperation in this field, hissudden and unexpected death means a serious loss to the institute.
Continued improvement in the physical plant of the institute has allowed

4

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

for the expansion of research and the development of sophisticated information services that will benefit the entire scientific community. As mentioned above, an upgraded electronic computer was set up in March andon-line service of the DNA data base was started in autumn by connecting
telephone circuits. As in the previous year the following works were continued by the staff of the DNA Data Analysis Section: Construction anddistribution of the DNA data base including input of data published inJapanese specialty journals, the development of analytical programs, data
analysis, and the issue and distribution of the DDBJ Newsletter (No.6).
The Genetic Stock Research Center supplies, as far as possible, uponrequest of competent researchers in and outside of Japan, specific strainsof mouse, Drosophila, E. coli, B. subtilis, etc. which are being kept in theCenter. The total number of cases supplied in 1986 amounted to 247 (28sent abroad) and the total number of strains supplied was 4773 (490 sent
abroad). In cooperation with other staff, Dr. Shin-ya Iyama continued
to systematize data-base information about location, characteristics, availability of distribution, etc., of a variety of experimental organisms preservedin universities and research institutes in Japan. In the past year he compiled the "Mouse Genetic Stock List Preserved in Universities and Institutes
in Japan, 1987" and issued "Rice Genetics Newsletter, NO.3". Thesematerials were distributed to interested researchers in and outside of Japan.
In our continuing effort to serve the community of Mishima, the institute held its yearly open house for the public on April 18th. Some of
the research activities of each laboratory were exhibited using panels,
microscopes, microcomputers, and molecular models. Scientific films
were also shown. The double cherry blossoms on the campus were attheir best and some 3000 visitors in the neighborhood enjoyed them. On
November 14, public lectures were given at the National Science Museumin Tokyo; the titles were "Genetic study on the subspecies differentiationof the mouse and its implication in the biomedical research" by Prof. Kazuo
Moriwaki and "Characteristics of codon choice pattern found for individualorganisms" by Associate prof. Toshimichi Ikemura. In spite of it being a
Saturday afternoon about 140 eager people from universities and institutes
listened to the lectures, which were followed by rather specialized questions
and lively discussion.
As reported previously, our institute was reorganized into a national
center for joint use by universities in April 1984. Other inter-university

GENERAL STATEMENT

5

research institutes of the same category, all belonging to Mombusho, included the Institute of Statistical Mathematics in Tokyo, the Institute forHigh Energy Physics in Tsukuba, the Institute of Molecular Sciences, theInstitute of Basic Biology, and the Institute of Physiological Sciences in
Okazaki. During the past years directors of the national inter-university
research institutes have deliberated with Mombusho about the plan ofcreating a "Postgraduate University for Advanced Studies" (tentatively
named) by taking advantage of existing resources of their respective
institutes. As a result, a master plan was drawn last March, and an office
was set up in April in Mombusho for preparatory operation (Head: Prof.
S. Nagakura, President, Okazaki National Research Institutes). An ad hoc
committee started in June (Chairman: Prof. I. Tanaka, President of theTokyo Institute of Technology), and it is anticipated that the new
university will be open in October 1988. Our institute, while accepting up
to six postgraduate students in genetics course every year, will represent,
together with the Institute for Basic Biology and the Institute for Physsiological Sciences, the specialty of "Life Science".
As an inter-university institute we accepted, in the last year, 39 collaborative programs, 18 workshops, 9 postgraduate students, 11 research fellows
and 9 scholarships from industries, and 2 entrusted research projects fromprivate corporations. Yet, there remain a number of problems to be solvedin order to achieve the reality of reform. Among other things, constructionof the following facilities is of the highest priority; an RI Center, which isindispensable for the expansion of research activities on the molecular level;lodgings for visiting researchers with welfare facilities, and a second mainbuilding to accommodate laboratories for joint use by visiting professors.
We are eager to do our best to accomplish the missions of our institute.
We hope to have continued encouragement and support from all persons
concerned.

STAFF
Director

MATSUNAGA, Ei, D. Med., D. Sc.
Members

1. Department of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Head of the Department
Laboratory of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUKUDA, Ryuji, D. Med., Associate professor
FUJITA, Nobuyuki, D. Sc.
NAGATA, Kyosuke, D. Pharm.
Laboratory of Mutagenesis
SADAIE, Yoshito, D. Sc., Associate professor
INOEU, Tadashi, D. Ag.
TEZUKA, Hideo
Laboratory of Nucleic Acid Chemistry (Guest member)
YOSHIKAWA, Hiroshi, D. Sc., Professor
MIZUMOTO, Kiyohisa, D. Sc., Associate professor

2. Department of Cell Genetics
MORIWAKI, Kazuo, D. Sc., Head of the Department
Laboratory of Cytogenetics
MORIWAKI, Kazuo, D. Sc., Professor
IMAI, Hirotami T., D. Sc., Associate professor
SHIROISHI, Toshihiko, D. Sc.
Laboratory of Microbial Genetics
YASUDA, Seiichi, D. Sc., Associate professor
NISHIMURA, Yukinobu, D. Sc.
HARA, Hiroshi, D. Sc.
Laboratory of Cytoplasmic Genetics (Guest member)SUZUKI, Hideho, D. Sc., Associate professor

STAFFYONEKAWA, Hiromichi, D. Sc., Associate professor

3. Department of Ontogenetics
KURODA, Yukiaki, D. Sc., Head of the Department
Laboratory of Developmental Genetics
SUGIYAMA, Tsutomu, Ph. D., Professor
NAWA, Saburo, D. Sc., Professor
FUJISAWA, Toshitaka, Ph. D., Associate professor
SHIMIZU, Hiroshi, D. Eng.
Laboratory of Phenogenetics
KURODA, Yukiaki, D. Sc., Professor
MURAKAMI, Akio, D. Ag., D. Sc., Associate professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-Aki, D. Ag.
Laboratory of Physiological Genetics (Guest member)
SHIMADA, Yutaka, D. Med., Professor
IDE, Hiroyuki, D. Sc., Associate professor
4. Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department
Laboratory of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Professor
OHTA, Tomoko, Ph. D., D. Sc., Professor
TAKAHATA, Naoyuki, D. Sc., Associate professor
AOKI, Kenichi, Ph. D.
Laboratory of Evolutionary Genetics
TUTIKAWA, Kiyosi, Associate Professor
GOJOBORI, Takashi, D. Sc.
Laboratory of Theoretical Genetics (Guest member)
MUKAI, Terumi, Ph. D., D. Sc., Professor
HORI, Hiroshi, D. Sc., Associate professor
5. Department of Integrated Genetics
IMAMURA, Takashi, D. Med., Head of the Department
Laboratory of Human Genetics
IMAMURA, Takashi, D. Med., Professor
FUJIYAMA, Asao, D. Sc., Associate professor

7

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

HORAI, Satosbi, D. Med.
NAKASHIMA, Hitosbi
Laboratory of Agricultural Genetics
MORISHIMA-OKINO, Hiroko, D. Ag., Professor
ENDO, Toru, D. Ag., Associate professor
SATO-HIRAOKA, Yoichiro, D. Ag.
Laboratory of Applied Genetics (Guest member)
WATANABE, Takesbi, D. Med., Professor

6. Research Facilities
Genetic Stock Research Center
MORIWAKI, Kazuo, D. Sc., Head of the Center
MIYASHITA, Nobumoto, D. Med.
WATANABE, Takao, K., D. Sc., Associate professor
VEDA, Hitoshi, D. Ag.
SANO, Yoshio, D. Ag.
NISHIMURA, Akiko, D. Ag.
IYAMA, Shin-ya, D. Ag., Associate professor
DNA Research Center
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Center
lKEMURA, Tosbimichi, D. Sc., Associate professor
HmosE, Susumu, D. Sc., Associate professor
MIYAZAWA, Sanzo, D. Sc., Associate professor
HAYASHIDA, Hidenori
Experimental Farm
IYAMA, Sbin-ya, D. Ag., Head of the Farm
MIYAZAWA, Akira

7.

Technical Section
SUGIYAMA, Tsutomu, Ph. D., Chief of the Section

8. Department of Administration
VEMATSU, Yoshihiro, Head of the Department
VJIIE, Jun, Chief of the General Affairs Section
TANIGUCHI, Hiroshi, Chief of the Finance Section

COUNCIL

9COUNCIL
Chairman
YAMAMURA, Yuichi; Emeritus professor, Osaka University
Vice-chairman
MOROHOSHI, Seijiro; Emeritus professor, Tokyo University of Agriculture and Technology
Members (Alphabetical order)
EGAMI, Nobuo; Director, National Institute for Environmental Studies
EGAMI, Nobuo; Director, National Institute for Environmental Studies
IINO, Tetsuo; Professor, Faculty of Science, University of TokyoINOUYE, Eiji; Emeritus professor, University of TokyoNAGAKURA, Saburo; President, Okazaki National Research Institutes
NAKAI, Junnosuke; President, Hamamatsu University School of
Medicine
NATORI, Reiji; Emeritus professor, Jikei University School of Medicine
NOMURA, Tatsuji; Director, Central Institute for Experimental Animals
OKADA, Tokindo S.; Director-General, Institute for Basic Biology
ONOE, Hisao; Dean, Faculty of Economics, Osaka Industrial University
OZEKI, Haruo; Professor, Faculty of Science, Kyoto University
SAKAI, Fuminori; Director, Japan Society for the Promotion of Science
SASA, Manabu; President, Toyama Medical and Pharmaceutical University
SATO, Ryo; Emeritus professor, Osaka University
TAZIMA, Yataro; Director, Institute of Silkworm Genetics and Breeding
WATANABE, Itaru; Emeritus Professor, Keio University
YAMAGATA, Hirotada; Professor, Faculty of Agriculture, Kyoto University

ADVISORY COMMI'ITEE
Chairman
KIMURA, Motoo; Professor, Laboratory of Population Genetics
Vice-chairman
OHBA, Shigeru; Professor, Okayama University of Science
Members (Alphabetical order)
FUKUDA, Ichiro; Professor, Faculty of Liberal Arts and Sciences, Tokyo

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Women's University
MIURA, Kin-ichiro; Professor, Faculty of Engineering, University ofTokyo
MUKAI, Terumi; Professor, Faculty of Science, Kyushu University
OKADA, Masukichi; Professor, Institute of Biological Sciences, University of Tsukuba
OSAWA, Shozo; Professor, Faculty of Science, Nagoya University
SAKAGUCHI, Bungo; Professor, Faculty of Agriculture, Kyushu University
SASAKI, Motomichi; Professor, Chromosome Research Unit, Facultyof Science, Hokkaido University
TSUNEWAKI, Koichiro; Professor, Faculty of Agriculture, Kyoto University
YAMADA, Yukio; Professor, Faculty of Agriculture, Kyoto University
IMAMURA, Takashi; Professor, Laboratory of Human Genetics
ISHIHAMA, Akira; Professor, Laboratory of Molecular Genetics
KURODA, Yukiaki; Professor, Laboratory of Phenogenetics
MORISHIMA-OKINO, Hiroko; Professor, Laboratory of Agricultural
Genetics
MORIWAKI, Kazuo; Professor, Laboratory of Cytogenetics
SUGIYAMA, Tsutomu; Professor, Laboratory of Developmental Genetics
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICSKONDO, Norio; President, Emeritus professor of Tokyo University ofAgriculture
KURODA, Yukiaki; Managing Director, Professor of National Institute of
Genetics
MORIWAKI, Kazuo; Managing Director, Professor of National Institute of
Genetics
TAZIMA, Yataro, Manager, Director of Institute of Silkworm Genetics andBreeding
MIURA, Kin-ichiro; Manager, Professor of University of Tokyo
YAMAGUCHI, Hikoyuki; Manager, Professor of University of Tokyo
MATSUNAGA, Ei; Manager, Director of National Institute of Genetics
ISHIHAMA, Akira; Manager. Professor of National Institute of Genetics

PROJECTS OF RESEARCH FOR 1987
1.

DEPARTMENT OF MOLECULAR GENETICS
Laboratory of Molecular genetics

Studies on regulatory mechanisms of gene transcription (ISHIHAMA, FUKUDA,
FUJITA and NAGATA)
Studies on molecular mechanisms of transcription and replication of animalviruses (ISHIHAMA, FUKUDA and NAGATA)
Laboratory of Mutagenesis

DNA repair and radiation sensitivity in mammals (INOUE and TEZUKA)
Biochemical factors involved in cellular repair of genetic damage and induced mutagenesis (INOUE)
Radiation genetics of Caenorhabditis elegans (SADAIE)
Molecular mechanisms of unicellular differentiation in Bacillus subtilis(SADAIE)
Laboratory of Nucleic Acid Chemisry

Studies on regulatory mechanisms of chromosome replication (YOSHIKAWA)
Molecular mechanisms of RNA 5'-cap formation (MIZUMOTO)
Studies on molecular mechanisms of transcription and replication of Sendai
virus (MIZUMOTO)
2.

DEPARTMENT OF CELL GENETICS
Laboratory of Cytogenetics

Studies on species differentiation of mouse from cyto- and molecular genetic
view points (MORIWAKI)
Immunogenetical studies on the mouse MHC (SHIROISHI and MORIWAKI)
Theoretical bases for chromosomal evolution in mammals and ants (IMAI)
Cyto- and molecular mechanism of meiotic recombination in mice (SHIRO-12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Ism, lMAI and MORIWAKI)
Cytogenetical studies on Drosophila (yAMAMOTO)
Laboratory of Microbial Genetics

DNA replication in E. coli (yASUDA)
Cellular division in E. coli (NISHIMURA and HARA)
Penicillin-binding proteins in E. coli (HARA and NISHIMURA)
Laboratory of Cytoplasmic Genetics

Peptidoglycan biosynthesis in E. coli (SUZUKI)
Studies on cytoplasmic genes during subspecies differentiation of house
mouse Mus musculus (YONEKAWA)

3.

DEPARTMENT OF ONTOGENETICS
Laboratory of Developmental Genetics

Genetic analysis of developmental mechanisms in hydra (SUGIYAMA, FUJISAWA and SHIMIZU)
Studies on transformation and cell differentiation in higher organisms (NAWA
and YAMADA)
Mitochondrial DNA organization in male-sterile cytoplasms of rice (NAWA,
SANO and FUJII)
Laboratory of Phenogenetics

Genetic studies on insect cells in tissue culture (KURODA and MINATO)
Developmental genetic studies on animal cells in tissue culture (KURODA)
Genetics of somatic mammalian cells in culture (KURODA)
Genetic studies on the reproduction in Bombyx (MURAKAMI)
Ecogenetic studies on the nerve system characters in Bombyx (MURAKAMI)
Studies on the male-specific killing activity of SRO in Drosophila (YAMADA)
Laboratory of Physiological Genetics

Electron microscopic studies on differentiation of animal cells (SHIMADA
and KURODA)PROJECTS OF RESEARCH FOR 1987

13

Nerve network formation in Hydra (KuIMA)
Genetic analysis of nervous system development in hydra (KUlMA andSUGIYAMA)
4.

DEPARTMENT OF POPULATION GENETICS
Laboratory of Population Genetics

Theoretical studies of population genetics (KIMURA, OHTA, TAKAHATA andAOKI)Studies on molecular evolution from the standpoint of population genetics(KIMURA, TAKAHATA and OHTA)
Theoretical studies on the evolution of multigene family (OHTA)
Theory of gene genealogy (TAKAHATA)
Theoretical studies on the evolution of altruism (KIMURA and AOKI)
Population genetical studies on gene-culture coevolution (AOKI)Laboratory of Evolutionary Genetics

Theory of population genetics and evolution (MARUYAMA)
Studies on molecular evolution (GOJOBORI)
Radiation genetics in mice (TUTIKAWA)
Laboratory of Theoretical Genetics

Theoretical and experimental studies of transposons in population of Drosophila (MUKAI)
Computer studies on the molecular evolution (HORI)
5.

DEPARTMENT OF INTEGRATED GENETICS
Laboratory of Human Genetics

Molecular genetics of growth and differentiation of human blood forming
cells (IMAMURA, FUJlYAMA and NAKASHIMA)
Molecular genetics of human metabolic disorders (IMAMURA, FUJlYAMA
and NAKASHIMA)
Studies on DNA polymorphisms in human populations (HORAI and MATSU-

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

NAGA)
Mitochondrial DNA restriction analysis of non-human primates (HORAI,
MATSUNAGA, HAYASAKA, SHOTAKE and NozAwA)Laboratory of Agricultural Genetics

Evolutionary studies in wild and cultivated rice species (MORISHIMA and
SATO)
Genecological studies on pollination in rice plants (SATO and MORISHIMA)
Genetic studies on rice reserve proteins (ENDO)
Laboratory of Applied Genetics

Molecular genetics of human immune mechanisms (WATANABE)
6.

RESEARCH FACILITIESGenetic Stock Research Center

Studies and conservation of germplasm resources in rice and wheat species(FuJII and SANO)
Specificity of mutagen tolerance in higher plants (FuJII)
Exploitation of genetic ability of nitrogen fixation in Gramineae (FuJII,SANO and IYAMA)
Studies on genetic differentiation in rice (SANO)
Theoretical studies on breeding techniques (IYAMA)
Genetic studies of trees in natural forest (IYAMA)
Studies on gene transfer in Gramineae (SANO and FUJII)
Documentation of genetic stocks in Japan (IYAMA)
Studies on the management system of genetic stocks information (lYAMA)
Evolutionary genetics of Drosophila (WATANABE)
Molecular genetics of insect development (VEDA)
Coordination of DNA synthesis and cell division in E. coli (NISHIMURA)
Mapping of a whole set of cell division genes in Escherichia coli Kl2 (NISHIMURA)
Development of mouse embryo freezing system (MIYASHITA and MORIWAKI)
Genetic mechanisms for regulating tumor development in the laboratory
and wild mice (MIYASHITA and MORIWAKI)

PROJECTS OF RESEARCH FOR 1987
DNA Research Center

Regulatory mechanisms of gene transcription (ISHIHAMA)DNA data analysis (MARUYAMA)
Studies on codon usage (IKEMURA)
Studies on RNA molecules of mouse brain (IKEMURA)
Database design for DNA base sequences (MIYAZAWA and HAYASHIDA)
Molecular genetics of insect development (VEDA and HIROSE)
Control of gene expression in eukaryotes (HIROSE and VEDA)

15

RESEARCH ACTIVITIES IN 1987
I.

MOLECULAR GENETICS

Mapping of Functional Sites on Subunits ofEscherichia coli RNA Polymerase
Akira IsHIHAMA, Nobuyuki FUJITA, Rei UESHIMA
and Robert E. GLASS·A major form of RNA polymerase holoenzyme in exponentially growing
cells of Escherichia coli is a big complex with a subunit composition of
a21'1"a70 • The function(s) of each subunit in transcription has been analyzed by a variefy of genetic and biochemical methods (reviewed in Ishihama, A. (1988) Trends Genet., in press). In order to get a detailed understanding of the structure-function relationship, we carried out a systematic study on the mapping of functional site(s) within each subunit
polypeptide.
To determine the sites on the I' subunit, we used a collection of E. coli
mutants with nonsense mutations in the rpoB gene encoding I' subunit.
By introducing various suppressor genes, variant RNA polymerases were
produced, each carrying a single amino acid substitution at a defined site
on I' polypeptide. Results of analyses of altered functions associated withvariant RNA polymerases (Glass, R. E. et al. (1986) Mol. Gen. Genet. 203:265-268; Glass, R. E. et al. (1986) Mol. Gen. Genet. 203: 487-491; Glass,
R. E. et al. (1986) Mol. Gen. Genet. 203: 492-495; Ishihama, A. et al. (1987)
Proteins 2: 42-53) led us to propose a tentative functional map of the I'
subunit as shown in Fig. 1. To confirm this map, attempts are being madeto affinity label the functional sites with substrates and ppGpp and todirectly determine the binding sites for these nucleotides.
Two sigma subunits, a70 and a32 , carry identical sequences of 14 aminoacids in length. These sequences might play an essential role in one of thefunctions common to the two sigma subunits. In order to identify othersigma subunits in E. coli, we made antibodies against a synthetic peptide•

Department of Biochemistry, Queen's Medical Centre, Nottingham.

17

RESEARCH ACTIVITIESINltll7
Functional Map of RNA Polymerase P Subunit~

I

rplK rplA
L11

rplJ rplL

rpoB

t...-c:::::H::J:,~~~,,~::1a."·

L1

rpoC

~ "_ _~_ _.~1;::::==:;:;:~==:=:;::::::=:=:J1-••••••••••••••,::~ polymerase tl'

RNA polymerase tl., / '." .... "
~

~.,

~

, / " , 00

200

atJlurprrt't! '"

300

400

1m' "10

500

600

700

800

900

'nrrr=:r=tnrDr-ul' '"Rifu;;;Pici~sensitivity

1000

..

"00

!"m'""",

i

".,

.

..

1200

1300

1",!!""dlll~i"lli 1342....ppGppsensitIvity

Core enzyme assemblywith this consensus sigma sequence. Using these antibodies, we searched
for E. coli proteins with sigma related proteins (Fujita, N. and Ishihama,A. (1987) Mol. Gen. Genet. 210: 5-9). The specificity of cross-reactive
proteins was tested by competition assay using synthetic peptides with
contiguous sequences of 5 or 6 amino acids. These synthetic peptides withthe consensus sigma sequence and antibodies against these peptides arealso useful for identification of the function associated with the consensus
sigma sequence.
Strength and Regulation of Escherichia coli Promoters, I.
Preparation of a Promoter Collection and Determinationof Two Parameters of Promoter Strength
Akira ISHIHAMA, Shizuko ENDo*, Hiroji AIDA*, Nobuyuki FUJITA
Rei UESHIMA and Manabu NAKAYAMA

Gene expression in bacterial cells is regulated mainly at the transcription
step. The rate of transcription is determined primarily by controlling therate of initiation. The intrinsic strength of the promoter is, therefore, theprimary determinant of the expression level of individual genes in E. coli
(reviewed in Ishihama, A. (1988) Trends Genet., in press). Up to now,
however, measurement and comparison of promoter strength for various
genes or operons in E. coli has never been attempted due to technical difficulties. Previously, we established an in vitro mixed transcription system,
in which an equimolar mixture of truncated DNA fragments, each carrying*

Department of Chemistry, Tsukuba University, Tsukuba.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38
Table 1.

Collection of Escherichia coli Promoters

Gene

Gene productMapposition

No. ofpromoters

alaS
apaH
asd
cca
crp
cya
dgk
divE
did
dnaQ
frdA
gal
ginS
gin V
gltA
gnd
gar
groE
guaB
hupA
hupB
katG
kdpABC
ksgA
lac
leuX
lpp
lysT
malB
nar
ndh
nusA
plsB
ppc
pyrF
recA
rnh
rplJ
rpoD
rpoH

Alanyl-tRNA synthetase
Diadenosine tetraphosphatase
Aspartate semialdehyde dehydrogenasetRNA nucleotidely transferase (CCA enzyme)cAMP receptor protein (CRP)
Adenylate cyclase
Diglyceride kinase
Cell division control (Serine tRNA)
Respiratory D-lactate dehydrogenase
DNA polymerase III e subunit
Fumarate reductase
Galactose fermentation
Glutaminyl-tRNA synthetase
Glutamine tRNA (a suppressor tRNA)
Citrate synthase
G luconate-6-phosphate dehydrogenase
Glutathione reductase
GroE ATPase (a heat-shock protein)
IMP dehydrogenase
HU-2 protein
HU-l protein
Catalase HPIHigh-affinity-potassium transport system
16S rRNA methyltransferase
Lactose fermentation
Leucine tRNA (a suppressor tRNA)
Murein lipoprotein
Lysine tRNA (a suppressor tRNA)
Maltose fermentation
Nitrate reductase
Respiratory NADH dehydrogenase
NusA protein
Glycerol-3-phosphate acyltransferase
Phosphoenolpyruvate carboxylase
Orotidine-5'-phosphate (OMP) decarboxylase
RecA protein (Recombination)
Ribonuclease H
Ribosomal protein LlO
RNA polymerase sigma-70 subunit
RNA polymerase sigma-32 subunit

58
1
76
67
74
85
92
22
80
5
94
17
16
16
17
44
77
94
54

1
2
2

9
89
16
1
8
97
36
17
92
27
22
69
92
89
28
58
5
90
67

2
1
2
1
1

3
1
1
2

1
1
2
1
1
1
3
1
1
1
1
1
1
1
1
4
3

RESEARCH ACTIVITIES IN 1987

19

Table 1. (Continued)
Gene
rpsA
rrnE
serB
ssb
ssp
tae
trp
trxA
tufB
une

Gene product
Ribosomal protein SI
ribosomal RNA (operon E)
Phosphoserine phosphatase
Single-strand DNA-binding protein (SSB)
Stringent starvation protein (SSP)
Synthetic promoter (trp-laeUV5)
Tryptophan synthesis
Thioredoxin
Elongation factor Tu
Protein-translocating ATP synthaseMap
No. ofposition promoters
21
90
100
92
70
28
85
90
84

5
2
2
1
1
1
1
1
1
3

a specific promoter and each producing a specific transcript, is transcribedby a known amount of purified RNA polymerase holoenzyme. DNA
truncation is designed to produce transcripts with different chain length,
which can be separated by electrophoresis on polyacrylamide gels. Using
this system, two parameters of promoter strength, the affinity to RNA polymerase and the rate of open complex formation, have been determined formore than 30 promoters (reviewed in Ishihama, A. et al. (1987) RNA Polymerase and the Regulation of Transcription).
This year, we added more than 50 promoters to our collection of E. coli
promoters, as listed in Table 1. Preparation of truncated DNA fragments
is being carried out in collaboration with Aiba and his colleagues at Tsukuba
University. Determination of the promoter strength is in progress in thislaboratory.
Strength and Regulation of Escherichia coli Promoters, II.
Effect of DNA Supercoiling and Temperature Shift on
Promoter Activity of the rpoH Gene Encoding theHeat-shock Sigma Subunit of RNA Polymerase
Rei UESHIMA, Nobuyuki FUJITA and Akira ISHIHAMA

The rpoH gene of E. coli codes for the heat-shock sigma subunit (a 32 ) of
RNA polymerase. Heat-shock genes are transcribed only by RNA polymerase holoenzyme containing a 32 (Fujita, N. and Ishihama, A. (1987)
J. Bioi. Chern. 262: 1855-1859). Upon exposure to heat-shock, the in-

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

tracellular level of a32 increases rapidly. Previously, we found that theinduction of a32 synthesis is attributed to the enhancement of rpoH genetranscription (Fujita, N. and Ishihama, A. (1987) Mol. Gen. Genet. 210:
10-15). As an attempt to elucidate the enhancing mechanism of rpoH
gene transcription, the effects of DNA supercoiling and temperature-shift
on the activity of two major promoters of the rpoH gene were examinedusing an in vitro transcription system. Upstream promoter PI was notaffected by DNA supercoiling nor temperature upshift but downstream
promoter P2 was enhanced by both of these factors. Both factors facilitated
the formation of an open complex on rpoH P2, and these two effects were
not additive. Hence we assumed that these two factors act at the same
step of transcription, probably the local unwinding of DNA double-helix
at the promoter region. Different sensitivities of the rpoH major promoters
to DNA supercoiling and temperature shift indicate that these promoters
are under differential control. Taken together with the heat-shock enhancement in vivo of rpoH P2 transcription (Fujita, N. and Ishihama, A. (1987)
Mol. Gen. Genet. 210: 10-15), we conclude that temperature up-shift itself
is a major factor controlling the activity of the rpoH promoter under heatshock stress.
Structure and Function of Genes for RNA Polymerase-Binding
Proteins: Regulation of Expression of Stringent
Starvation Protein (SSP) Gene
Hiroaki SERIZAW A and Ryuji FUKUDA

A variety of Escherichia coli proteins interact with RNA polymerase andmay participate in modulating enzyme functions. To identify such transcription factors, we isolated several RNA polymerase-binding proteins andhave been studying their effects on transcription in an in vitro system. In
parallel, we have been trying to isolate the genes coding for these proteins,
and to perform genetic analyses of their physiological functions. Stringent
starvation protein (SSP) is one of the putative transcription factors. We
isolated the gene for this protein (Fukuda, R. et al. (1985) Mol. Gen. Genet.
201: 151-157), determined the nucleotide sequence of the gene (Serizawa,
H. and Fukuda, R. (1987) Nucleic Acids Research, 15, 1153-1163), andidentified its genetic map position on the E. coli chromosome (Fukuda,
R. et al. (1988) Mol. Gen. Genet. 211: 515-519).

RESEARCH ACTIVITIES IN 1987

21

This year, we continued research on the regulation of expression of the
SSP gene. As reported previously, the consensus promoter sequences are
not present at the -10 and - 35 regions upstream from the transcription
initiation site. Deletion mapping studies of the 5' flanking region, however,indicated that the 36 bp sequence upstream from the transcription initiation site was essential for the promoter function. In addition, it was foundthat an approximately 40 bp sequence further upstream from this essential
promoter region stimulated in vivo transcription several fold. This stimulatory effect could not be observed in the in vitro transcription system using
purified RNA polymerase, and either linear or superhelical template DNA
containing this cis-acting stimulatory region. The fact suggests that atranscription factor(s) interacts with this region to enhance transcription.
DNA fragments containing this cis-acting region exhibited a bending
structure, as analyzed by gel electrophoresis. The bending center was
found to be located around 50 bp upstream from the transcription initiation site.
Analysis of the 5' flanking sequence showed the presence of two genes(rplM and rpsI) for ribosomal proteins, L13 and S9. The transcription
termination signal for these genes was located around 200 bp upstream
from the transcription initiation site of the SSP gene. Detailed SI nucleasemapping analysis of in vivo RNA indicated transcription read-throughfrom these ribosomal protein genes to the SSP gene. Furthermore, transcription in the opposite direction was observed, initiating from the 3' flanking region of the SSP gene. The meaning of these transcription is notunderstood yet.
RNA Polymerase and Transcriptional Signals ofMicrococcus luteus
Manabu NAKAYAMA, Nobuyuki FUJITA, Syozo OSAWA·
and Akira ISHIHAMA

Among bacterial species, the G+C content of genomic DNA varies ina wide range from about 25 % to 74 %. The G+C content of Micrococcus
luteus DNA is 74 %, which is much higher than that of E. coli (about 50 %).
We showed that the codon usage pattern of M. luteus is much different
from that of E. coli, and extremely biased to the use of G and C in silent•

Faculty of Science, Nagoya University, Nagoya.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

positions (Ohama, T. et al. (1987) J. BacterioI. 169: 4770-4777).
In order to understand the influence of GC pressure on the structure oftranscription signals, particularly the promoter structure, we initiatedanalyses of promoter structures of M. luteus and of the structure and function of RNA polymerase, which recognizes these promoters. By mappingwith reverse transcriptase, we determined the initiation site for both invitro and in vivo transcription of the M. luteus streptomycin (str) operon.
In an in vitro run-off transcription assay using truncated DNA templates,
RNA polymerases from both M. luteus and E. coli were able to transcribein vitro correctly from this str promoter. In contrast, the enzyme of M.
luteus is unable to transcribe correctly from E. coli promoters. Thus, wereached the conclusion that the promoter sequences of M. luteus are similar
to but distinct from those of E. coli, and that RNA polymerase of M. luteus
is stricter in its promoter selection.
Processing of tRNA precursors in Escherichia coli: RNase P is
involved in both 5' and 3' termini of leuX tRNA precursor
Teruaki NOMURA and Akira ISHIHAMA

Primary transcripts of stable RNA genes such as rRNA and tRNA genes
are biologically inactive precursor molecules. Several enzymes are involved in the processing of these precursors: processing of 5'-termini iscatalyzed by RNase P, which is composed of two components, C5 protein
and Ml RNA, while processing of 3' termini is catalyzed by a combinationof at least two enzymes other than RNase P.
The leuX gene of Escherichia coli codes for a suppressor tRNA, whichinserts leucine at the amber codon. We found that this gene is transcribed
as a monomeric precursor carrying about 20 and 34 base extra sequencesat its 5' and 3' ends, respectively (Nomura, T. and Ishihama, A. (1987) J.
Mol. Bioi. 197: 659-670). In spite of the general belief that precursors of
stable RNAs are rapidly processed and cannot be detected in wild-type
E. coli cells with respect to tRNA processing enzymes, we identified theaccumulation of primary transcripts of the leuX gene. Following measurements of both precursor and mature leuX tRNA levels under various growth
conditions, we proposed a model in which the expression of leuX gene iscontrolled not only at transcription but also at the tRNA processing step
(Nomura, T. and Ishihama, A. (1987) J. Mol. Bioi. 197: 659-670).

RESEARCH ACIIVITIES IN 1987

23

An analysis of the processing in vitro of the leuX precursor revealed thatthe processing of the 5' end takes place in a single step reaction catalyzed
by RNase P while the 3' processing involved a two step reaction. The
activity of initial endonucleolytic cleavage of the 3' precursor sequence
was found to copurify along RNase P. Heat inactivation of RNase P
from two independent RNase P mutants abolished the cleavage activity ofboth the 5' and 3' ends. These results suggest that RNase P is involved in
not only 5' processing but also in the initial step of 3' processing.
In the presence of Mg2+ alone, the leuX precursor was found to be selfcleaved at a site of about 13 nucleotides towards the inside from the 5' endof mature tRNA. Self-cleaved precursor tRNA was not addititionallyprocessed by the 3' endonuclease, suggesting that 3' endonuclease recognizes
a specific conformation of precursor tRNA for action.
Molecular Mechanism of Influenza Virus RNA Transcription, I.
Role of NP on RNA Chain Elongation
Ayae HONDA*, Kenji UEDA**, Kyosuke NAGATA
and Akira ISHIHAMA

The influenza virus genome consists of eight single-stranded RNA segments of negative polarity. Three P proteins (PB1, PB2 and PA) and one
nucleocapsid protein (NP) are associated with each RNA segment, together
forming ribonucleoprotein (RNP) cores. After virus infection, the RNP
cores are transported into cell nuclei and primary transcripts are producedby the core-associated RNA polymerase (reviewed in Ishihama, A. andNagata, K. (1988) CRC Crit. Rev. Biochem. 23: 27-76). Biochemical andgenetic studies have revealed that the catalytic site for RNA synthesis islocated on the P protein complex. The role of NP on the transcription ofviral RNA genome is, however, not understood yet.
In order to assign a role(s) to NP protein on transcription, we designed
two kinds of dissociation and reconstitution experiments. RNP cores
were dissociated into RNA polymerase-RNA genome (P-RNA) complexes
and NP protein by esC! centrifugation. The P-RNA complexes werecapable of catalyzing the endonucleolytic cleavage of capped RNA, the* Present address: Bristol-Meyers Research Institute, Tokyo.
Present address: Department of Neurosciences, School of Medicine, Universityof California, San Diego.

*'!<

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

initiation of primer-dependent RNA synthesis, and the synthesis of smallsized RNA, but were unable to synthesize full-sized RNA. By adding theisolated NP protein to the P-RNA complexes, RNP complexes were reconstituted, which supported the synthesis of full-sized transcripts as didnative RNP cores. These results suggest that NP protein is required for
efficient elongation of RNA chains but not for initiation of RNA synthesis.
RNP was then dissociated into RNA-free proteins and a protein-freegenome RNA by CsTFAjglycerol centrifugation (Kato, A. et al. (1986)
Virus Res. 3: 115-127; Honda, A. et al. (1987) J. Biochem. 102: 1241-1249).
By mixing these two fractions, primer-dependent RNA-synthesizing activity
was regained. These complexes, however, produced only small-sized
RNA, presumably due to incorrect assembly of NP on viral RNA. To
confirm these observations, we developed a transfection assay system of
RNP complexes, instead of infection of native virions. Infectivity of thereconstituted RNA-protein complexes is being tested.
Molecular Mechanism of Influenza Virus RNA Transcription, II.
Inhibition of Transcription by Matrix Protein
Raleigh W. HANKINS, Kyosuke NAGATA and Akira ISHIHAMA

Until recently, the influenza virus matrix (M)-protein had been thought
to function solely as a template for the attachment of both surface proteinsand nucleocapsid cores during the course of virus maturation. Within thepast few years, however, a number of reports have been published which
implicate M protein in the inhibition of viral RNA (vRNA) transcription.
While the involvement of M protein in such a role is well supported byexperimental evidence, its exact nature has not been characterized adequately. It has been the purpose of our research to elucidate the mechanism by which influenza virus M protein effects vRNA transcription inhibition. Specifically, we set out to clarify the following aspects of this Mprotein function: 1) the stage of vRNA transcription which M protein inhibits, i.e., initiation versus elongation, 2) the portion of the basic viraltranscription unit (RNP) to which M protein binds to effect inhibition; 3)
whether this binding occurs in a specific manner.
The results of our experiments enabled us to obtain a much better pirtureon how influenza virus M protein affects vRNA transcription inhibition.
Analyses ofvRNA transcription products in the presence of varying amounts

RESEARCH ACTIVITIES IN 1987

25

of M protein, and under differing substrate conditions led us to conclude
that M protein-effected inhibition is exerted mainly at the level of vRNAtranscription initiation and/or reinitiation. When nucleocapsid protein(NP) was removed from RNP, the addition of M protein to the remaining
RNA polymerase-RNA (P-RNA) complexes had no effect on vRNA transcription. Thus, inhibition itself probably occurs when M protein binds
to NP and physically blocks movement of the viral RNA polymerase along
vRNA. Finally, the observation that only certain monoclonal anti-M
antibodies were effective in neutralizing M protein, independent of their
Western blot titers, strongly suggests that M protein-affected vRNA transcription inhibition occurs by site-specific binding of M protein to NP.
Presently, we are in the process of utilizing a battery of anti-M monoclonal
antibodies in developing an M protein epitope map of the functional site(s)involved in vRNA transcription inhibition.
Transcription and Replication of Influenza Virus RNA:An in vitro System Using Isolated Nuclei
from Virus-Infected Cells
Kyosuke NAGATA and Akira ISHIHAMA

We developed an in vitro system for transcription and replication of influenza virus RNA using isolated nuclei from influenza virus-infected HeLa
cells (Takeuchi, K. et al. (1987) J. Biochem. 101: 837-845). In this system,
two species of positive-sense RNA tanscripts (mRNA and cRNA) and
one negative-sense RNA transcript (vRNA) are synthesized.
In the course of fractionation of this nuclear system by centrifugation,
we found that the precipitates of nuclear extracts (NEP) predominantly
produced mRNA while the supernatant (NES) alone exhibited a low levelof cRNA synthesis activity. With the addition of the NES fraction, mRNA
synthesis by NEP was switched to cRNA synthesis. This activity was notdetected in nuclear extracts prepared from unifected cells and was heatlabile. Glycerol density centrifugation of the NES fraction in the presenceof high salt, however, yielded RNA polymerase complexes that supported
mRNA synthesis. These observations suggest that a regulatory factor(s)either encoded by the virus genome or induced by virus infection is involvedin the switch from mRNA to cRNA synthesis, and that this putative factor(s)can be dissociated from RNA polymerase complexes upon exposure to

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

high ionic strength.
in progess.

Isolation and characterization of such a factor(s) are

Translational Regulation of InOuenza Virus mRNAs
Kunitoshi YAMANAKA, Kyosuke NAGATA and Akira ISHIHAMA

In influenza virus-infected cells, ten kinds of viral proteins are synthesized
and their synthesis is regulated with respect to total amount and timing.
Recent studies revealed that transcription is the key step for gene expressionof influenza virus (reviewed in Ishihama, A. and Nagata, K. (1988) CRC
Crit. Rev. Biochem. 23: 27-76). The contribution of posttranscriptional
control is, however, not yet known.
To elucidate translational regulation in influenza viral gene expression,
we inserted cDNA sequences containing ATG for the initiation codon oftranslation of influenza viral mRNAs into the cap-proximal leader regionof the CAT gene in a vector, pSV2cat. Two plasmids, designated pSV2cat
and pSV2NSlcat, were constructed using cDNAs for segments 6 and 8
conding for NA and NS proteins, respectively. Both plasmids lead to thesynthesis of complete CAT protein fused to N terminal portions of NA
and NS proteins. HeLa cells were transfected with either pSV2NAIcat
or pSV2NSIcat and, after 24 hr, superinfected with influenza virus. CAT
activity in cells transfected with pSV2NSIcat reached a maximum during
the early stage of infection, whereas maximal CAT activity in cells transfectedwith pSV2NAIcat was obtained during the late stage of infection. Thesevariations in patterns of CAT activity are consistent with the known orderof gene expression of NS and NA proteins. The primer-extension analysis
indicated that intracellular distribution and metabolic stability of mRNA
encoding fused CAT protein were similar between cells transfected withpSV2NSIcat and pSV2NAlcat. From these results, we propose that translational control operates during influenza virus infection and that the signal
involved in this control is located around the translation initiation site.

RESEARCH ACTIVITIES IN 1987

27

Establishment of Mouse Cells Expressing Influenza Viral Genes
Kunitoshi YAMANAKA, Kyosuke NAGATA, Yasuhiro HOSAKA*
and Akira ISHIHAMA

Recent studies have revealed that cellular immunity plays a crucial rolein protection against influenza virus infection. In order to identify protein(s) and epitope(s), which are recognized by cytotoxic T lymphocytes,
we attempted to establish mouse cells producing individual influenza viralproteins. Along this line, we focused on nucleocapsid protein (NP) because NP from various subtypes of influenza virus share a similar antigenicity
and NP-reactive cytotoxic T lyphocytes are therefore expected to show crossreactivity against various subtype viruses.
For efficient expression of cDNA for NP protein in mouse cells, we inserted NP cDNA under control of mouse metalothionein I promoter into abovine papiloma virus-based vector containing a neomycin resistant gene.
The recombinant plasmid was transfected into mouse L cells by the calciumphosphate coprecipitation method and Neo-resistant colonies were selectedwith G-418. Expression of the NP protein gene was tested by Western
blotting analysis using anti-NP antibody. Cells expressing the NP protein
gene were tested with respect to reaction against cytotoxic T lymphocytes.
The NP-expressing mouse cells were also were used for transfection of
RNA polymerase-viral RNA (P-RNA) complexes devoid of NP (see Honda,
A. et al. in this report).
In order to determine the roles of viral proteins in transcription andreplication, attempts have been made to establish cells producing P proteinsand non-structural (NS) proteins. These cells are presently beines used to
examine self-replication capabilities of protein-free RNA, RNA polymeraseviral RNA (P-RNA) complexes and RNA polymerase-NP-viral RNA complexes (RNP).

* Research Institute of Microbial Diseases, Osaka University, Suita.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Control of Influenza Virus Gene Expression: Quantitative
Analysis of Each Viral RNA Species in Infected Cells
Eriko HATADA*, Masakazu HASEGAWA**, Jun MUKAIGAwA***,
Kazufumi SHIMIZU*** and Ryuji FUKUDA

For studies on control mechanisms of RNA synthesis in influenza virusinfected cells, it is necessary to develop a simple system for measuring thequantity of each viral RNA species. As reported last year, we establisheda quantitative hybridization system, in which the quantities of three typesof influenza virus RNAs (vRNA, mRNA and cRNA) could be measured
separately for the 8 genome segments (24 RNA species in all). As the
hybridization probes in this system, 32P-labeled RNAs of both plus andminus polarity were synthesized employing a SP-6 transcription system.
For assay, these probes were used in a large molar excess sufficient toovercome complementary RNAs present in RNA samples from infected
cells.
Employing this hybridization system, we first analyzed the time courseof the accumulation of each viral RNA species in MDCK cells infected with
AjUdornj72 (H3N2) and compared it with the time course of the viralprotein synthesis. During the early phase of infection (1 h postinfection),
appreciable levels ofmRNA appeared for segments 1,2,3,5 and 8 (encoding
the early proteins). This pattern of mRNA synthesis was similar to thatobserved in virus-infected cells in the presence of inhibitors of protein
synthesis, such as cycloheximide. The result indicates that a differential
control operates at the transcriptional level during the primary transcription. At 2 h postinfection, the synthesis of vRNA as well as cRNA began
simultaneously for all segments. The vRNA level continued to increasebeyond 5 h postinfection, while the cRNA level reached a plateau at 2 hpostinfection. mRNAs encoding the late proteins started to be synthesizedin close coupling with the rise of vRNAs, and thereafter the level of both
early and late mRNAs, except mRNAs for polymerase proteins, continued
to increase with the rise of the respective vRNAs. The mRNA levels did
not increase but rather declined after 3 h postinfection for all segments,
being completely uncoupled with vRNA levels. We could not detect any* Present address: School of Medicine, Nihon University, Tokyo.
** Present address: Chugai Exploratory Research Labs, Gotemba.
*** School of Medicine, Nihon University, Tokyo.

RESEARCH ACfIVITIES IN 1987

29

delay in the synthesis of vRNA segments encoding the late genes. Thisobservation does not agree with the previous concept on the control mechanism of viral RNA synthesis.
The accumulation pattern of mRNAs encoding three polymerase proteins
was rather exceptional. The synthesis of mRNA for P proteins ceased
after 2 h postinfection, and the mRNA level remained at a low level thereafter. The synthesis rate of NP protein increased in proportion to thelevel of its mRNA, while that of the MI protein remained at a low level
during the early phase in the presence of a high level of mRNA. It increased abruptly thereafter during the late phase of infection. Results
thus indicates that the gene expression of influenza virus is regulated not
only at the level of transcription, but also at some posttranscriptional
step(s).
Studies on the Function(s) of Inftuenza Virus
Nonstructoral Proteins
Erika HATADA*, Masakazu HASEGAWA**, Kazufumi SHIMIzu***
Masakazu HATANAKA**** and Ryuji FUKUDA

To study the function(s) of nonstructural protein (NSI) of influenza
virus, we analyzed temperature-sensitive mutants of influenza virus A/
Udorn/72 (H3N2) having lesions in RNA segment 8. As reported lastyear, the nucleotide sequence of segment 8 was determined for three ofthese mutants. Both ICRCl629 and SPC45 have mutations in the NSlcoding region, whereas ICRC516 has a mutation in the NS2-coding region.
The synthesis of virus-specific proteins was analyzed in MDCK cells infected with these mutant viruses. With both NSI ts mutants, the synthesesof two late proteins, Ml and HA, were greatly reduced at a nonpermissive
temperature of 40°C compared to 34°C. The synthesis of NSI also decreased at the nonpermissive temperature. On the other hand, no significant alteration was observed with the NS2 ts mutant in the synthesis ofviral proteins at 40°C.
Using a quantitative hybridization method as described above, the syn*
..
***
****

Present address: School of Medicine, Nihon University, Tokyo.
Present address: Chugai Exploratory Research Labs, Gotemba.
School of Medicine, Nihon University, Tokyo.
Institute for Virus Research, Kyoto University, Kyoto.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

thesis of virus-specific RNAs was analyzed. The synthesis of viral mRNAs
was retarded, reflecting the delayed and reduced synthesis of vRNAs, withall three mutant viruses. For both NSI mutants, the levels of individualmRNAs synthesized at 40°C were almost the same as those at 34°C, although severe reduction was observed at 40°C in the synthesis of the late
proteins and NSl. These observations suggest that the NSI protein is
involved in at least two distinct steps of the virus growth process: one in thesynthesis of vRNA and/or cRNA; and another in the posttranscriptional
process(es) of late protein synthesis.
Analysis of a Novel Influenza A Virus PB2 Mutant,
Defective in the Regulation of Three
Polymerase Genes
Jun MUKAIGAWA*, Eriko HATADA**, Ryuji FUKUDA
and Kazufumi SHIMIZU*

To reveal the function(s) of the PB2 protein of influenza A virus, six
temperature-sensitive (ts) mutants of A/Udorn/72 (H3N2) virus, each having
a ts mutation in the PB2 gene, were analyzed for the synthesis of viralRNAs and proteins. One of the mutants, ICRC27, exhibited novel phenotypes, and was characterized in detail. At a nonpermissive temperatureof 40°C, the synthesis of complementary RNA (cRNA; template for genome
RNA synthesis) for each genome segment was blocked, resulting in a severe
reduction in the synthesis of genome RNA. As a result, the secondarytranscription was also blocked. Accordingly, viral late proteins (M andHA) were barely detectable and the syntheses of NP and NSI proteinsdecreased and/or was delayed.
At a permissive temperature of 34°C, the syntheses of both mRNAs andcRNAs of PB2, PBI and PA segments, which stop at 2 to 3 h postinfection
with the wild-type virus, continued at least up to 7 h post infection, yielding
several times more copies than did the wild-type virus. Elevated levels ofthese mRNAs led to increased synthesis of the polymerase proteins. The
ts+ revertants of ICRC27 exhibited none of these mutant phenotypes.
These observations indicate that this mutant is defective in the regulatory
mechanism(s) underlying the synthesis of mRNAs and/or cRNAs of the* School of Medicine, Nihon University, Tokyo.
** Present address: School of Medicine, Nihon University, Tokyo.

RESEARCH ACTIVITIES IN 1987

31

three polymerase genes, and that the PB2 protein participates in this regulation.

Assignment of the Function of Nuclear Factor I
Ken MATSUMOTO*, Kyosuke NAGATA and Fumio HANAOKA*

Nuclear factor I (NFl) has been isolated from uninfected HeLa cell
nuclei as an essential factor for the initiation of adenovirus (Ad) DNA
replication, and shown to be a sequence-specific DNA-binding protein.
NFl-binding sites, (T/G)GGNe_ 7 GCCAA, have been identified for a numberof cellular and viral genes. Although these sites are located within regulatory regions for transcription, their roles have not been determined yet.
Recently, it has been proposed that NFl is identical with one of the CCAAT
box-binding proteins (CTF), which has been characterized as a transcription factor.
To test this possibility, we employed a gel retardation assay using a 32p_
labeled Ad DNA probe containing the NFl site. Formation of NFIDNA probe complexes was completely prevented by non-labeled oligonucleotides containing a NFl-binding sequence. In contrast, oligonucleotides
containing the CCAAT box did not exhibit efficient competition. DNase
I protection analysis revealed that highly purified NFl was unable to bindsuch CCAAT boxes as those present in promoter regions of the HSV-tk
gene and mouse .a-globin gene. These results suggest that NFl is a distinct
protein from CTF or that NFl binds to CCAAT boxes with very low affinity by itself.
Nuclear extracts prepared from MDCK cells (canine) contained a highlevel of NFl DNA-binding activity compared to those from HeLa cells(human) and MF3A cells (mouse). Among mouse tissues, nuclear extracts
from liver, kidney and spleen exhibited higher NFl DNA-binding activity
than those from lung, brain and heart. Furthermore, in both MDCK andmouse kidney nuclear extracts, we detected a novel DNA-binding protein,
designated nuclear factor K (NFK), that bound to a sequence adjacent to
the NFl site in Ad DNA. Since the NFK binding site in Ad DNA contains
a sequence similar to the octamer motif, NFK may be a member of theoctamer binding proteins. NFK was capable of binding to this sequence* Faculty of Parmaceutical Sciences, University of Tokyo, Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

only in the absence of NFL These findings suggest that NFK plays a rolein kidney-specific gene expression. Studies on possible involvement of
NFK in Ad DNA replication, and of both NFl and NFK in transcription
are in progress.
GTP Induces Knotting, Catenation, and Relaxation of DNA
with Stoichiometric Amounts of DNA Topoisomerase IIfrom Bomhyx mori and HeLa Cells
Susumu HIROSE, Hisahiro TABUCHI and Koichi YOSHINAGA*

Most of the biochemical properties of DNA topoisomerase II have beencharacterized under catalytic conditions in which the molar ratio of enzyme
to DNA is far below unity. This molar ratio does not seem to reflect an
intracellular state where DNA is attached to the chromosome scaffold or
nuclear matrix at a 5-90-kilobase distance possibly through topoisomerase
II. This led us to investigate the reaction of topoisomerase II understoichiometric conditions. Type II DNA topoisomerases isolated fromthe posterior silk glands of Bombyx mori and HeLa cells utilize ATP for
unknotting of knotted DNA, relaxation of supercoiled DNA, and catenation/decatenation of circular duplex DNA under catalytic conditions. In
these reactions, ATP cannot be replaced by GTP. However, GTP inducesknotting, catenation, relaxation, but not decatenation of circular duplex
DNA with stoichiometric amounts of these enzymes. Only a limited roundof the reactions proceed with a concominant hydrolysis of GTP and thenthe reaction pauses. These GTP-dependent reactions may be employed
for maintenance of the knotted an/or catenated state they formed. For
details, see J. BioI. Chern., 263, 3805-3810.
In Vitro Transcription of Eukaryotic Genes Is Affected
Differently by the Degree of DNA Supercoiling
Susumu HIROSE and Yoshiaki SUZUKI**

We examined whether topological tension of DNA is required for the
regulation of gene expression in eukaryotes. In a posterior silk gland ex* Department of Chemistry, Faculty of Science, Shizuoka University, Shizuoka.
** Department of Developmental Biology, National Institute for Basic Biology, Okazaki.

RESEARCH ACTIVITIES IN 1987

33

tract, covalently closed circular (ccc) DNA is in a superhelical state that
supports more transcription of the fibroin gene than does linear DNA. A
HeLa cell extract showed neither a supercoiling activity nor preference forthe transcription of ccc DNA over linear DNA. These activities could
be added to the HeLa cell extract. Phosphocellulose fractionation of theposterior silk gland extract yielded a flow-through fraction and a 0.6 M
KCI eluate fraction which were required for the supercoiling and for efficient transcription of the ccc template in the acceptor HeLa cell extract.
The 0.6 M KCI fraction had DNA topoisomerase II activity, and the flowthrough fraction contained a supercoiling factor which, with the aid of
topoisomerase II, introduced negative supercoils into ccc DNA. Whenboth fractions were added to the posterior silk gland extract, more supercoiling occurred than with the extract alone. Several genes were optimallytranscribed under various degrees of supercoiling. The fibroin gene andadenovirus 2 major late promoter were fully transcribed as partially supercoiled templates. The sericin gene required more supercoiling for fulltranscription, whereas no preference for supercoiling was seen with thetranscription of hsp70. These results suggest that DNA topology plays arole in the regulation of gene expression. For details, see Proe. Nat!. Aead.
Sci. USA, 85, 718-722.
Changes in Organization of Mitochondrial and Nuclear DNA
in Cytoplasmic Reversion of Male Sterile Rice
Saburo NAWA, Masa-Aki YAMADA and Yoshio SANO

Strains of rice which exhibit cytoplasmic male sterility (ems) contained
the two free plasmids, B-1 and B-2, not only in the mitochondrial fraction,
but also in the nuclear fraction. These DNAs were no longer apparent in
preparations of either mitochondrial or nuclear DNA from cytoplasmicallyreverted strains which were generated by the treatment of a ems strain with
EMS. Hybridization with restriction fragments revealed the presence ofvarious sequences homologous to B-1 and B-2 in the mitochondrial genomic
DNA of high molecular weight and also in the nuclear chromosomal DNA,
both in ems strains and in the normal strain. The reversion of male sterility
to the fertile conditions was accompanied by the disappearance of the freeplasmid DNAs, B-1 and B-2, and the appearance of new patterns of restriction fragments of mitochondrial or nuclear DNA with respect to sequences

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

homologous to B-1 or B-2. However, no evidence was obtained to support the possible integration of B-1 or B-2 into mitochondrial or nuclear
DNA during the reversion. Hybridization revealed several regions ofhomology, in both mitochondrial and nuclear DNA, common to the sterilestrains and to the fertile revertants, but several homologies observed in theems strains were no longer present in the fertile revertants. Thus, ems
strains contained more homologous sequences in terms of both number
and quantity of homologous regions than the normal strain or revertants.
The presence of sequences homologous to B-1 and B-2 in the mitochondrial DNA of the normal strain may mean that plasmid DNAs could
have arisen from the mitochondrial genomes by excision, and have thenbeen autonomously replicated and subsequently integrated into various
sites on the mitochondrial genome, as transposable elements. These specific
events might be associated with the origin of cytoplasmic male sterility inrice. Recombinations or rearrangements of the mitochondrial genome
during the reversion might be the cause of the different restriction patternsof DNA from ems strains and from revertants. Hybridization data revealed the presence of common DNA sequences in nuclear and mitochondrial genomes in rice. The episomal plasmids, B-1 and B-2, may
have left the mitochondria of ems strains and entered the nucleus where
they were transposed into the genome. The B-1 and B-2 sequences innuclear DNA may perhaps be able to serve as vectors of the free plasmids
for transformation in rice, since it may be possible to integrate foreign
DNA into the genome by transposition or recombination of the homologous sequences.
The presence of the free plasmids and the characteristics of the organization of the mitochondrial and nuclear genomes did not affect the abilityof the plant to produce functional pollen when the nuclear backgroundcontained nuclear restorer genes. However, both the disappearance ofthe free plasmids and the rearrangements in mitochondrial DNA of highmolecular weight or in nuclear DNA, with respect to B-1 and B-2 sequences,were involved in the reversion to fertility, and these events are indicative ofsome correlation between the organization of these sequences and cytoplasmic male sterility in rice.

RESEARCH ACTIVITIES IN 1987

35

Messenger RNA Synthesis of Sendai Virus (HVJ)
Kiyohisa MIZUMOTO

Sendai virus (HVJ), a member of the paramyxovirus group, contains asingle nonsegmented RNA genome of approximately 15 kb long withnegative polarity encoding at least seven proteins, NP, P, C, M, F, HN
and L. The genetic information of this virus appears to be expressed
through six monocistronic mRNAs corresponding to each protein, exceptthat the P and C proteins are translated from a single mRNA species with
two overlapping reading frames. It has been suggested that these mRNAs
are synthesized by transcribing the negative strand genome RNA with thevirion associated RNA-dependent RNA polymerase, which is assumed toconsist of the P and L proteins. The precise mechanism of the transcription, however, is unclear, because no efficient in vitro transcription system
has been established. We have tried to develop a transcription system inwhich faithful initiation as well as termination takes place.
We have found that the in vitro transcription directed by purified HVJ
particles is almost entirely dependent on the addition of host (HeLa) cell
extracts. The active component (transcription factor) in the extracts washeat labile and sensitive to trypsin-treatment, while refractory to nucleasetreatment. The transcription factor activity was also detected in otheranimal cells, and even in plant cells, indicating that HVJ utilizes protein(s)ubiquitously present in the eukaryotic cells for its transcription. The
transcription factor was partially purified from bovine brain and was shown
to be separated into at least two complementary fractions, one of which
could be replaced by highly purified tubulin.
The in vitro reaction products largely consisted of 18S and 12S RNA
species which were similar in size to the mRNAs made in the infected cells.
A single cap structure, m7 GpppAm, was released from in vitro poly(A)+RNA upon digestion with nuclease PI, indicating that transcription of all
the HVJ mRNA starts with the A residues. Identical 5'-cap structures
were found on the viral mRNAs made in vivo. From RNA-RNA hybridization and protection experiments with minus strand genome RNA,the in vitro products were predominantly of message sense (plus strand),and were transcribed from correct sites on the genome.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Mechanism of mRNA Capping Reaction
Kiyohisa MIZUMOTO

Most of the cellular as well as viral mRNAs in eukaryotes contain a5'-terminal cap structure, m7 GpppN. The cap structure is required for theefficient initiation of translation and for RNA splicing. It has also beensuggested that the synthesis of the methylated cap structure and/or the
association of the capping enzyme system with RNA polymerase may play
an important role in the initiation of transcription. The cap structure issynthesized at the initial stage of mRNA synthesis and is conserved at the
5'-termini of RNAs while they are processed in the nucleus and transported
to the cytoplasm. Consequently, elucidation of the mechanism of synthesisof the cap structure is important for understanding the molecular mechanismof eukaryotic gene expression.
We characterized the reactions catalyzed by the mRNA capping enzyme(mRNA guanylyltransferase) which is a key enzyme in the cap formation,
to understand the molecular mechanism of steps involved in eukaryotic
mRNA synthesis. Using mRNA capping enzymes from various eukaryotic
cells, we demonstrated that the capping reaction (GTP+ppN-RNA-+
G(5')pppN-RNA+PPi) proceeds through a covalent enzyme-GMP intermediate in which GMP is linked to the e-amino group of a lysine residue
throug ah phosphoamide bond (Mizumoto, K. and Kaziro, Y. (1987),
Prog. Nucl. Acid. Res. Mol. Bioi., 34, 1-28).
An RNA 5'-triphosphatase activity specifically hydrolyzing the r-phosphate from the 5'-triphosphate terminated RNA was found to be tightly
associated with guanylyltransferase purified from rat liver, Arternia salina,
and yeast suggesting that this is a general feature of the capping system.
The capping enzyme from rat liver and A. salina consists of a single polypeptide chain with Mrs of 69,000 and 73,000 respectively, containing catalytic domains for both guanylyltransferase and triphosphatase. In contrast, a highly purified yeast capping enzyme is composed of two separate
chains of 52 kDa (a) and 80 kDa ((3), responsible for the activities ofguanylyltransferase and triphosphatase, respectively (Itoh, N. et al. (1987),
J. Bioi. Chern., 262, 1989-1959; Mizumoto, K. and Kaziro, Y. (1987),
Frog. Nucl. Acid Res. Mol. Bioi., 34, 1-28).
To see whether the a and (3 subunits are derived from a single polypeptide
or are independent polypeptides encoded by two separate genes, we at-

RESEARCH ACTIVITIES IN 198737

tempted to isolate the gene(s) coding for the yeast capping enzyme. A
yeast genomic DNA expression library in Agtl I was screened with an antibody against the yeast capping enzyme which recognizes both a and fi
chains. One of the positive clones, AC3, contained a 3,5OO-base pair insert
of yeast DNA. From experiments based on the affinity selection of theantibody by antigens produced with AC3 in E. coli, this clone appeared tocontain the guanylyItransferase gene. The insert contained an open reading frame encoding a polypeptide of 459 amino acids with a calculated Mr
of 52,764, corresponding to the size of the 52 kDa a subunit. The identiryof the gene was further confirmed by expressing the gene in E. coli to givecatalytically active guanylyItransferase.
From a Northern blot analysis of the yeast poly(A)+ RNA, the size ofthe guanylyItransferase mRNA was estimated to be about 1,600 bases.
Since this mRNA size fairly closely corresponds to a 52 kDa polypeptide,
the a and fi chains of the yeast capping enzyme are probably encoded by
two separate genes. The disruption of one copy of the a subunit gene ina diploid yeast creates a recessive lethal mutation, indicating that the single
guanylyItransferase gene of yeast has an essential function.
Regulation of Initiation of Chromosomal Replicationin Bacteria
Hiroshi YOSHIKAWA

Regulation of initiation of the chromosomal replication by DnaAboxes in the origin region of the Bacillus subtilis chromosome.
A gene homolgous to the Escherichia coli dnaA gene and two flanking
regulatory regions which contain nine and four DNA-boxes (the sequenceof 9 nucleotides present in oriC of E. coli and serves for the initial bindingsites of DnaA protein to activate oriC: the consensus sequence is
TTATCCACA), respectively are located in the replication origin region of
the B. subtilis chromosome (Ogasawara et aI. 1985 EMBO J. 4: 3345).
Attempts to isolate an autonomously replicating fragments from these regulatory regions in order to identify oriC have not been successful because
the DnaA-box containing regions strongly inhibited plasmid transformation particularly when inserted into a high copy number plasmid pUB11 O.
Using two plasmids differing in copy number, the two regions were subdivided into three regions, A, Band C, each containing 5, 4 and 4 DnaA(I)38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

boxes, respectively, which differed in level of inhibition of transformation.
Region C is downstream of the dnaA gene and inhibits transformation in
high-copy but not in low-copy number plasmid. When a part of the
DnaA-boxes was deleted from the incompatible plasmids, they became
transformable and produced slow-growing transformants in which the initiation frequency of chromosomal replication was selectively reduced. Fastgrowing revertants were found containing the same number of plasmids as
the parent but with single base changes in the DnaA-boxes. These mutations were in the most highly conserved bases of the DnaA-box sequence.
This indicates that a sequence-specific interaction of the DnaA-box most
likely with the B. subtilis DnaA protein is responsible for the observed
incompatibility and thus appears to be involved in control of initiation
frequency of the chromosomal replication.
(2) Structure of dnaA region of Pseudomonas putida: Conservation among
the three bacteria, Bacillus subtilis, Escherichia coli and P. putida.
We have cloned from Pseudomonas putida a gene homologous toEscherichia coli dnaA, and determined the sequence of the gene and its
neighboring region. The dnaA gene and at least 3 other genes, dnaN,
recF and gyrA are found highly homolgous with the genes in the dnaA
region of E. coli and B. subtilis. A large non-translatable region immediately upstream of the dnaA gene is also conserved in the three bacteria
and contains 3, 12 and 14 DnaA-boxes (TTATCCACA and closely relatedsequences) in E. coli, P. putida and B. subtilis, respectively. The present
results confirm our hypothesis that dnaA region is the replication origin
region of the ancestral bacteria and the essential feature of the dnaA and
DnaA-box combination is conserved in most of the eubacteria to playa
central role in initiation of chromosomal replication.

RESEARCH ACTIVITIES IN 1987

II.

39

MICROBIAL GENETICS

A Synthetic Translation Terminator Gene: a Tool for
Dissecting Translation Direction of Gene
Ichiro N. MARUYAMA*, Kazuhiro HORIKOSHI**, Yasukazu NAGASE**
Masaaki SOMA**, Masahiro NOBUHARA**, Seiichi YASUDA
and Yukinori HIROTA t

A 41-nucleotide-Iong duplex DNA fragment (TER fragment), which contains a TAA translation terminator in all six reading frames joined to thelac operator sequence of Escherichia coli (Fig. 1), was synthesized. Thisfragment may be useful not only for producing a truncated protein encoded
in a plasmid, but also for the identification of the precise coding region andtranslation direction of a bacterial gene in the cloned chromosomal segment.
By inserting the TER fragment into two sites in the coding region of agene, one can determine the precise coding region and the translation direction by molecular weight measurement of the two truncated proteins. The
lac operator system was used for the selection of the plasmid containing thesynthetic fragment. We could detect the insertion of the TER fragment
into plasmid DNA which has a copy number of more than 20 per cell. The
lac operator on the plasmid binds with the lac repressor which is present at
about ten molecules per cell. As a result, the lac operon in the host chromosome is expressed.
The TER fragment was inserted into the p-Iactamase structural gene in
pBR322 in order to test in vivo activity. The plasmid produced mutant
p-Iactamase reduced in size, as expected from the insertion site, and rendered
the host bacterium constitutive for p-galactosidase. Thus, termination
Deceased on December 23,1986.
Yukinori Hirota left many works unfinished, along with a great legacy of the"temperature-sensitive mutant stock of Escherichia coli" as an achievement of his'K-project'. This and the following seven reports describe the results achievedby collaboration of the late Professor Hirota and the joint authors who worked
with him during his lifetime and continued the researches left unfinished after his
death.
* Present address: Medical Research Council Molecular Genetics Unit, Cambridge,
England.
** Mochida Pharmaceutical Co. Ltd., Tokyo.
t

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

5' GAT CC GGGIT A AIGIT A AIG T A A T T GT GAG C GGAT A A C A A TT A GTT A GTT A GC CG 3'
3' GCCC ATT CAT T C A TT AACACTC GCCTA TTGTT AAT CIAATlclAATlc GGCCTAG

BamHI linker

Lactose Operator

5'

BamHI linker

Fig. 1. Nucleotide sequence of the TER fragment.

codons and the lactose operator in the synthetic nucleotide appear to befunctional in vivo. For details, see Gene Anal. Tech., in press.
Determination of Gene Products and Coding Regions fromthe murE-murF Region of Escherichia coli
Ichiro N. MARUYAMA*, Aki-hiko YAMAMOTO* and Yukinori HIROTA!

The murE and murF genes of Escherichia coli encode diaminopimelic acidand D-alanyl-D-alanine-adding enzymes, respectively, and both are involvedin the biosynthesis of the cytoplasmic precursor of cell wall peptidoglycan,
UDP-N-acetylmuramyl-L-Ala-D-r - Glu- meso - diaminopimelyl -D-Ala- D- Ala(UDP-MurNAc-pentapeptide). Both genes are located at 2 min in thegenetic map and are contained in a large cluster of genes for cell envelope
growth and division. To understand how peptidoglycan synthesis andseptum formation are organized during cell growth in E. coli, a structural
analysis of this gene cluster is important.
The murE and murF genes are adjacent to the fts! gene and carried by
pLC26-6 (Takeda, Y. et al., 1981, Plasmid 6: 86). We subcloned DNA
segments from pLC26-6 and determined the precise position of murE andmurF genes on the restriction map as summarized in Fig. I. The productof murE and murF genes were 56 kDa and 54 kDa proteins, respectively.
These genes were transcribed in the same direction as the fts! gene (clockwise
on the genetic map). The murE gene seems to have its own promoter. It
is not clear whether murF is organized in an operon together with murE ortranscribed independently.

! See footnote to p. 39.
* Present address: Medical Research Council Molecular Genetics Unit, Cambridge,
England.
** Present address: Department of Biology, Kohnan University, Kobe.

RESEARCH ACTIVITIES IN 1987>
01a:: -0
0

W

I

E~

.2
~

(1)0!

~

C'-

(/)

Cl.0I

::.:::Z

0-

!

!

2

ftsl

!

..
murE

>

=:l

:; a::0 :;

0-

0- W

>
I

3

41

>
!

0

i

4

i

~

:l

cr~

~
i

!

5

=> 0:
:l

0

OJ

0-

w

J:

0

a.

!

i

6

Kb

..
murF

Fig. 1. Restriction map of ftsI-murF chromosomal region. Arrows indicate thepolypeptide-coding regions and transcriptional directions of the genes.

A Note on the parA Mutation in Escherichia coli
Jun-ichi KATO*, Hideho SUZUKI, Yukinobu NISHIMURA
and Yukinori HIROTA!

Thermosensitive par mutants of Escherichia coli have been described as
mutants that grow into filamentous cells with centrally located nucleoids at
restrictive temperatures and the parA mutation responsible for such characteristic morphology was mapped around 95 min close to purA (Hirota, Y.
et al., 1971, Biomembranes 2: 13). The thermosensitive growth of the parA
mutant MFTIIO was partially corrected by pLC8-47 (Nishimura, Y. et al.,
1977, Plasmid 1: 67).
The restriction sites on pLC8-47 were determined for several nucleases asshown in Fig. 1. The KpnI fragment was recloned from the chromosomal
region of pLC8-47 and various deletions were introduced (Fig. I). The
recloned constructs were introduced into MFTIIO and examined for theability to correct thermosensitivity in growth. Results are included in Fig.
1 and indicate that the ca. 1.6 kb BamHI-PvuII fragment of the chromosome
region in pLC8-47 contained a putative gene to complement the thermosensitive mutation in MFTIIO and that the HpaI site on this fragment isessential for complementing ability. However, pJK710 carrying the
BamHI-PvuII region did not correct the Par phenotype of MFTlIO, nor
did pLC8-47K which was identical with pLC8-47 except that two small
contiguous PstI fragments of the plasmid region were replaced by a kanamycin resistance gene (Fig. 1). Clearly, the chromosome region carried
by pLC8-47 does not cover the parA gene mutated in MFTlI0. MFTlI0! See footnote to p. 39.
* Department of Bacteriology, National Institute of Health, Tokyo.

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

P PSmP

1#

Sm

K

)!

E

E SBE B

X 89' K B

S\'-\II !

'>\1 I

IKK

8 KSmK P\ \l I ( pLC8-47(+)
L-I

lkb

pJK700 (+)

SK

pJK707 (-)

K

8
E

-....
-

(Pv)

E

H

--T"

8
8

pJK709 (+)
pJK710(+,45K+)
pJK716 (+,45K+)(PJK717 (+ ,45K+)

8

pJK713 (-,45K-)

Fig. 1. Analysis of the DNA region that corrects the thermosenstivity of parA
mutant MFTlIO.
Solid and hollow bars denote the regions derived from the chromosome andthe plasmid, respectively. Only the cloned regions are shown except for
pLC8-47. pJK716 and pJK717 had the same BamHI-Pvull chromosome
region but with an opposite orientation: in pJK717 the lac promoter was
joined to the BamHI end and in pJK716 to the Pvull end which was converted to a BamHI cohesive end in joining. A filled triangle indicates theinsertion site of a translation terminator and an arrow shows the approximate
location and the transcriptional direction of the gene that corrects the thermosensitive defect of MFTllO and codes for a 45K protein. An open triangle
with Km indicates the deleted region and the insertion site of a kanamycin
resistance gene in pLC8-47K (cl text). Symbols in parentheses following
the plasmid numbers denote that complementation of the thermosensitive
mutation in MFTlI 0 was positive (+ ) or negative ( - ) and that the synthesisof the 45K protein was detected (45K+) or not (45K-). B, BamHI; Bg
BglII; E, EcoRI; H, HpaI; K, KpnI; P, PstI; Pv, Pvull; S, SalI; Sm, SmaI;
X, XhoI.

possibly has at least two thermosensitive mutations and the major thermosensitive defect is not due to parA. The parA mutation should also giverise to a thermosensitive defect, which might be suppressed phenotypicallyto some extent under ordinary culture conditions. The major thermosensitive defect in MFTllO may be due to psd which is also mapped close topurA, because pJK717 as well as pLC8-47 corrected the thermosensitivityof GC2418 (psd-l) and YYCl76 (psd-2).
The gene product directed by the BamHI-PvuII fragment was identified
as a 45 kDa (45K) protein by comparing the electrophoretic spectra ofin vitro products directed by pJK710 and pJK713. The insertion of a translation terminator into the HpaI site on pJK710 to construct pJK713 resulted
I

RESEARCH ACTIVITIES IN 1987

43

in the simultaneous loss of abilities to direct the synthesis of the 45K protein
and to correct the thermosensitive defect in MFTllO. The synthesis of
the 45K protein in vitro was more augmented with pJK717 than with pJK716(cf legend to Fig. I), and IPTG (isopropyl j3-D-thiogalactoside) stimulated
the 45K protein synthesis directed by pJK717 in maxicells. The transcription for the 45K protein appears to proceed in the direction from the BamHI
to the Pvull site.
The 45K protein may be a psd product. The psd product, a phosphatidylserine decarboxylase, is an essential enzyme involved in the biosynthesisof phosphatidylethanolamine, the major membrane lipid of E. coli. Although it is possible that psd may affect chromosome partition, the present
results rather indicate that the parA locus is located somewhere other thanat the 95 min position.
A New Mutation Causing a Defect in Chromosome
Partition in Escherichia coli
Yukinobu NISHIMURA, Jun-ichi KATO*, Masao YAMADA**
Hideho SUZUKI and Yukinori HIROTA!

Two temperature-sensitive (ts) mutants defective in chromosome partition have been described as parA and parB in Escherichia coli. They arecharacterized by growth into filamentous cells with centrally congregatednucleoids at restrictive temperatures. We found another kind of par
mutants, JE1l215 and JEI0572, in the ts mutant stock of E. coli K12 (established with intention of the late Professor Y. Hirota to set up an exhaustive mutant collection for essential genes). The ts growth of these twomutants was corrected by pLC4-14 which is now known to cover the metC
region at 65 min. The ts mutation in JEI1215 was mapped at 65.3 min
through the results of mating and PI-transduction experiments, being
cotransducible with metC at 59 % (29/49) frequency and with gic at 20 %(5/25) frequency.
The ts mutation was transferred by cotransduction with metC+ into ametC derivative of C600. The resultant ts transductants failed to grow on
L-agar plates at 42°C and manifested a typical Par phenotype as observed! See footnote to p. 39.
* Department of Bacteriology, National Institute of Health, Tokyo.
** Present address: National Children's Medical Research Center, Tokyo.

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38
Table 1. Complementation of temperature-sensitive par mutations by plasmids
carrying dnaG alleles.
Complementation by

Strains

pGL444: :Tn5#4(dnaG+)JE11215 (pare)
MFTlOO(parB)

PC3 (dnaG)
MFTl10 (parA)

pGL444: :Tn5lf18(dnaG-)

pBS105: :Tn5lf128(dnaG-)++

Plasmids were a generous gift from Dr. J. R. Lupski and carry the dnaG segmentwith Tn5 insertion outside (dnaG+) or within (dnaG-) the dnaG gene (Lupski, J. R.,
e! al., 1982, Mol. Gen. Genet. 185: 120).

with DAPI (4',6-diamidino-2-phenylindole)-stained cells. This par mutation is neither allelic to parA nor to parB as described below and will bereferred to as parCo
The parA mutation in MFTllO was previously reported to lie at 95 min.
However, this turned out to be incorrect as described in the accompanying
report (Kato, J. et at., in this Ann. Rep. p. 41) and the map position of parA
is unknown at present. The Par phenotype of the parA mutant MFTllO
was not corrected by plasmids carrying the 65 min chromosome region andcomplementing par in JE1l215. The par mutation found in JE1l215 isclearly distinct from parA.
The parB mutation has been mapped between thyA (61 min) and argG
(69 min). Although this mutation may be suspected to lie around the 65
min map position, the ts defect of parB mutant MFTlOO was not correctedby pLC4-14 which complemented the par mutation in JE1l215. Since
dnaG has been mapped at 67 min and some dna ts mutants can reveal rather
Par-like morphology of nucleoids at restrictive temperatures, complementation of parB by the dnaG gene was investigated by transforming four relevantmutants, MFTlOO(parR), PC3(dnaG), MFTllO(parA) and JE1l215 withplasmids carrying dnaG+ or dnaG-:: Tn5. As shown in Table 1, thednaG gene carried by the plasmid complememted parB as well as dnaG,
but neither parA nor the par mutation in JE1l215. These results indicatethat the par found in JE1l215 differs from parR and that parB is likely tobe an allele of dnaG. More recently, it has been suggested by other authorsthat parB lies in the dnaG gene and affects primase (Norris, U. et at., 1986,
J. Bacteriot. 168: 494).

RESEARCH ACTIVITIES IN 1987

45

The parC transductant, EJ812, with a genetic background of C600 grew
into filaments with centrally located nucleoids at 42°C, releasing numbersof nucleoid1ess cells which were approximately of normal cell size. The
fraction of nucleoidless cells accounted for more than 20 % after 2 hr incubation at 42°C. The morphological phenotype was essentially unchangedby the introduction of recA into EJ812. Control of cell division by theprocess of chromosome partition seems to be independent of the recAmediated division control. The production of nucleoidless cells by theparC mutant may suggest rather loose coupling between cell division andchromosome partition. The loose coupling might be specific to the parC
mutation or alternatively inherent in E. coli.

Organization of the parC-sufI Gene Region in
Escherichia coli
Jun-ichi KATO*, Yukinobu NISHIMURA, Masao YAMADA **Hideho SUZUKI and Yukinori HIROTA!

The plasmid pLC4-14 which complements parC (for chromosome partition) has been shown to correct by some degree the thermosensitive defectdue to ftsI (mapped at 2 min, pbpB for penicillin-binding protein 3). The
putative gene that can suppress the ftsI mutation is termed sufI (or sui).
The restriction map of pLC4-14 was determined as drawn in Fig. 1 andthe parC-sufI region was analyzed by subcloning the chromosome region in
it. Plasmids carrying the HpaI fragment complemented parC, correcting
both the thermosensitivity in growth and the impairment of nucleoid segregation in the hosts. Complementing ability was abolished by the insertion of a translation terminator into the PvuII-2 site (Pv2 in Fig. 1).
Thus, the parC gene lies on the HpaI fragment. The allelism of parC+on pLC4-14 to parC was confirmed by cloning parC1215 in JE1l215. The
p1asmids that included the HpaI fragment directed in vitro synthesis of the
75 kDa (75K) protein. When the translation terminator was inserted into
the PvuII-2 site, the 75K protein was not produced and a 69K protein was
synthesized instead. Agreement between the complementing ability for
parC and the synthesis of 75K protein supports the notion that the 75K! See footnote to p. 39.
* Department of Bacteriology, National Institute of Health, Tokyo.
** Present address: National Children's Medical Research Center, Tokyo.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38
p ps pH P Sa B2

I )j(

ce;;;
(-

sufI

H

H S HBl~---~(------

PP

1~.~_~.~~H~J~
--

25K

ll<b

pare

Fig. 1. The restriction map of pLC4-14.
Arrows indicate the location and the transcriptional direction of the genes
for parC, 25K protein, and sufI. B1 and B2, BamHI; H, HindUI; Hp,
HpaI; P, PstI; Pvl and Pv2, PvuU; S, SmaI; Sa, Sail.

protein is a parC product. A short deletion was introduced at the BamHI
site (BI in Fig. I) by BAL-31 digestion and subsequent insertion ofa translation terminator. The in vitro product directed by the plasmid carrying thisdeletion showed an intermediate size between 75K and 69K. The 69K
protein is most likely to be a curtailed parC and thus transcription proceedsfrom right to left in Fig. I.
Plasmids carrying the BamHI fragment suppressed fts!. The suppression
occurred only under induction by IPTG (isopropyl ,B-D-thiogalactoside)
when the BI end of the BamHI fragment was joined next to the lac promoter-operator region with a mini-F replicon. Accordingly, the transcriptional direction of the sufI gene is from right to left in Fig. I and the suppression of ftsI by the sufI gene results from an increase in the amount of
sufI gene product. The sufI gene product was identified as a 55K protein
synthesized in vitro under the direction of a plasmid carrying the BamHI
fragment. The production of the 55K protein was abolished, with thesimultaneous loss of the ability to suppress ftsI, by inserting a translation
terminator into the SmaI site within the BamHI fragment. The 55K
protein radiolabeled in maxiceIIs was chased into a fast migrating component in SDS-electrophoretic gels, suggesting that the occurrence ofposttranslational processing. The sufI gene appears to be dispensable forcell viability, since the sufI gene was inactivated without loss of viability
through recombinational substitution with the sufI gene region interrupted
at the SmaI site by the insertion of a cat gene.
However, the BamHI fragment containing the sufI gene could not bedeleted even in the presence of the same fragment supplied with a plasmid.

RESEARCH ACTIVITIYS IN 1987

47

The possibility for essential genes covering the BamHI sites was exploredby insertional interruption at these BamHI sites. A polAt. strain NI126
was transformed with a derivative of pLC4-14 in which two different drug
resistance genes were inserted into each of the BamHI sites (B1 and B2
shown in Fig. 1). After the recombination process and plasmid curing at
the polA restrictive temperature, drug resistant recombinants with insertion at the B2 site were obtained but those with insertion at the Bl site were
not. Recombinants with insertion of a drug resistance gene at the Bl site
could be isolated in the presence of a mini-F derivative carrying the SmaI
fragment (Fig. 1). Viability of the isolated recombinants was lost throughcuring of the mini-F derivative. These results indicate the existence of an
essential gene covering the Bl site. The product of this gene was identified
as a 25K protein, the production of which was abolished by insertional interruption at the Bl site.
When the largest PstI fragment of pLC4-14 was placed under the lacpromoter, production of both sufI and 25K proteins was enhanced by thelac promoter ligated to the PstI site within the parC gene. The sufI geneand the gene for 25K protein are transcribed in the same direction and thus
they are possibly organized into an operon together with the parC gene at
the proximal side. However, the sufI gene and the gene for 25K protein
were significantly expressed by the PstI fragment connected in reverse orientation to the lac promoter. There may be internal promoters for thesegenes.
The localization of the gene products was examined with maxicells by
fractionating the sonicated lysates by ultracentrifugation in the presence orabsence of MgH. The parC gene product was found in the particulate
fraction in the presence of 10 mM MgH but was distributed in the soluble
fraction in the presence of 10 mM EDTA. The 25K protein was foundalmost exclusively in the particulate fraction irrespective of MgH availability. The majority of sufI gene product was present in the soluble fraction. Although it might be suspected that the sufI gene product is subject
to processing and thereby released into the periplasm, its localization in the
periplasm is not obvious at present. For details, see J. Bacteriol., in press.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

A Mutant Defective in the Processing of Penicillin-Binding
Protein 3 of Escherichia coli
Hiroshi HARA, Yukinobu NISHIMURA, Seiichi YASUDA
Hideho SUZUKI and Yukinori HIROTA!

Penicillin-binding protein 3 (PBP-3) of Escherichia coli is a cytoplasmic
membrane protein that plays an essential role in cell division. It functionsin the formation of a septum of the murein sacculus, and is one of the lethal
targets of ,8-lactam antibiotics. PBP-3 is a 588-residue polypeptide as
deduced from the nucleotide sequence of its structural gene, Its!, and issynthesized in a precursor from to be processed into a mature form distinguished by a higher electrophoretic mobility in SDS gels (Nakamura, M.
et al., 1983, Molec. Gen. Genet. 191: 1).
We found two E. coli mutants, JEIO092 and JE7304, which produced
PBP-3 with a lower electrophoretic mobility than that of wild-type PBP-3.
These mutants were investigated for possible defects in the processing of
PBP-3. JEI0092 was found in the temperature-sensitive (ts) mutant stockof E. coli. This strain was originally reported as a mutant with an altered
RNA polymerase ,8' subunit (Sugiura et al., 1977, Biochem. Biophys. Res.
Comm. 76: 739) and then found to produce the altered PBP-3. Temperature-resistant (tr) transductants of this mutant showing tr RNA polymerase
activity still produced PBP-3 with a lower mobility, indicating that the ts
defect was associated with the defective RNA polymerase rather than due
to an alteration in PBP-3. Introduction of plasmid pMS316 carrying theIts! gene into this mutant resulted in the overproduction of PBP-3 of a normal
mobility. When an Its/730 strain having the ts defect in PBP-3 was madetr by PI-transduction from this mutant, all the transductants produced
mutant-type PBP-3. These results suggest that the lower mobility of PBP-3
in JElO092 is not due to a defect in the processing mechanism but to amutation in the Its! gene. The Its! gene was cloned from JElOO92 andanalyzed for the gene product. A pulse-chase experiment revealed thatthe product was processed from the precursor form into the mature form
and that both forms showed lower mobilities than those found in wild-type
cells. The Its!92 mutation in JEIO092 appears to result in the productionof the altered PBP-3 which is larger in size but not defective in processing
and functioning.
! See footnote to p. 39.

RESEARCH ACTIVITIES IN 1987

49

The other mutant, JE7304, producing PBP-3 with a lower mobility
was found fortuitously in constructing multiple mutant strains for PBPs.
This mutant showed ts growth at a low salt concentration. The tr transductants produced wild-type PBP-3, suggesting that the ts growth waspossibly due to a mutation affecting the mobility of PBP-3. Preliminary
experiments for mapping showed that this mutation (ts-7304) was not
mapped in the fts! gene. When pMS316 was introduced into JE7304,
PBP-3 with a lower mobility was overproduced. Pulse-chase experimentswith a ts-7304 mutant harboring pMS316 showed that the processing of
PBP-3 did not occur in this mutant. These results indicate that JE7304
has a defect in the processing mechanism of PBP-3.
The ts growth due to ts-7304 was linked with a processing defect in PBP-3.
Although this may suggest that the processing of PBP-3 is of vital importance, it is also possible that the ts growth is due to a failure in theprocessing of some essential protein(s) other than PBP-3. In either case,the present results suggest that the ts-7304 gene is involved in an important
processing system.
Processing in the C-Terminal Portion of Penicillin-Binding
Protein 3 of Escherichia coli
Hiroshi HARA, Jun-ichi KATO*, Hideho SUZUKI
and Yukinori HIROTA t

It is well known that precursor forms of proteins destined for extracytoplasmic locations are processed into mautre forms by proteolytic cleavageof N-terminal signal peptides during transfer across the cytoplasmic membrane. Penicillin-binding protein 3 (PBP-3) of Escherichia coli is a cytoplasmic membrane protein essential for cell division. Its catalytic site is
likely to protrude into the extracytoplasmic side. It is synthesized in a
precursor form and processed to yield a mature form (Nakamura, M. et al.,1983, Molec. Gen. Genet. 191: 1). Proteolytic cleavage may be involved in
producing the mature PBP-3.
Two processing enzymes, signal peptidases (SPases) I and II, have been
described in E. coli. SPase II cleaves signal peptides of prelipoproteinsand SPase I processes many other extracytoplasmic proteins. The processt•

See footnote to p. 39.
Department of Bacteriology, National Institute of Health, Tokyo.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

A
pHR66

B
!

B
:

5353 PI J=::J:: :::::::::dEV B

k:::J41

5881

8

B
:

P:
5353
: ,

EV: B

LP signal
1':::::::141

5881

!

pH5101B

p

pLC26·6
p~15316

:

M
:

M
:

fts I

pHR39
pHR49

B:

K: NRE": ,:I~ SSN
:

B

N

B

R

-sB81
5881

Ii

B

B,

I

PBP-3

B

1
1I I
pACYC184
1

NRB

4971-:,15441

BC:::=:=====::::JI.:I:-J:::I-----JI

B
1=:=======fE'g-,,-5_
_
I

11

B

C:::=!====:::JI .. , ::-:::::__ .__L --.J

Li nker

pHR43

11

ColE1

B
pHR33

I

pIN-Il-B2"I-J~5601 157611

Fig. 1. Plasmids used in this study.
A, The restriction maps of the plasmids are shown with only the relevant
restriction sites: B, BamHI; E, EcoRI; EV, EcoRV; E*, EcoRI*; H, HindIII;
M, MluI; N, NarI; P, PvuII; R, RsaI; S, SalI; S3, Sau3AI. TER, thesynthetic translation terminator. Linker, the polylinker derived from
pUC18. B, PBP-3 or its derivatives encoded in the plasmids are shownwith the numbering of amino acids from the N-terminal methionine of
PBP-3. Shadowed bars, the lipoprotein singal peptide lacking the processing
site and followed by 7 residues due to a linker sequence in the pIN-II-B2
vector. Hatched bars, C-terminally extended peptides encoded in the DNA
region fused to the Its! gene. The figure is not drawn to scale.

ing of PBP-3 was not inhibited by globomycin, a cyclic peptide antibiotic
which specifically inactivates SPase II, nor was affected in the lsp mutant
defective in SPase II. Maltose-binding protein, OmpA protein and OmpF
protein (processed by SPase I), and murein lipoprotein (processed by SPaseII) were all normally processed in a ts-7304 mutant defective in the processingof PBP-3. Furthermore, kinetic studies showed that the processing of
PBP-3 was much lower in rate than the processing of other extracytoplasmic
proteins. Thus the mechanism for maturation of PBP-3 seems to be distinct from processing mechanisms known so far.
The mode of processing for PBP-3 was investigated by manipulating thecloned Its! gene. First, we examined whether the processing of PBP-3
occurs because of the removal of an N-terminal signal peptide by analyzing
the product ofpHS101 (Fig. 1). In this product, the N-terminal40 residues

RESEARCH ACTIVITIES IN 1987

51

of PBP-3 were replaced by the murein lipoprotein signal peptide which
lacked the processing site and was followed by extra 7 residues derived froma linker sequence in the vector. The pulse-chase experiment showed that
this hybrid protein was processed. The processing was not inhibited by
globomycin but prevented in the ts-7304 mutant. It is unlikely that morethan 40 residues are removed from the N terminus of PBP-3, since PBP-3
has only two cysteines at the 28th and 30th residues in the deduced sequenceand yet the (3~S]cysteine label remained uneliminated in the mature PBP-3.
Next, we examined the C-terminally truncated PBP-3. A synthetictranslation terminator was inserted into the KpnI site within the Its! geneto construct pHR33 (Fig. 1). This should result in the removal of Cterminal 91 residues of PBP-3. The truncated product was not processedin maxicells, although it seemed to be unstable and disappeared during thechase in growing cells. The truncated products which lost various lengthsof the C-terminal part (Fig. 1) were then examined. The product directedby pHR39 was unstable not only in growing cells but in maxicells. The
product directed by pHR49 was unstable in growing cells and not processedin maxicells like that directed by pHR33. The truncated product directed by
pHR43 did not appear to be processed during the 30 min chase of growing
cells pulse-labeled with (35S]methionine, but was identified as two bands
when the cell envelope was labeled with radioactive penicillin. The fastermoving band was not detected in the envelope of the ts-7304 mutant. These
results indicate that the product directed by pHR43 is processed very slowly.
In cells harboring pHR49, a single faint band of the product was detected
in a penicillin-binding assay and the band position was the same for boththe ts-7304 mutant and the wild type. Thus, we conclude that the peptide(s)essential for processing is contained in the C-terminal region between the560th and the 576th residues.
To confirm the occurrence of proteolytic cleavage in the C-terminal portion, we constructed pHR66 in which the Sau3AI fragment was deleted
from pHSIOl in order to remove the normal termination codon (Fig. 1).
This should allow the production of a molecule containing five cysteine
residues only within the extra peptide tail added to the normal C terminus
of PBP-3. The product directed by pHR66 was slowly processed as shownin a (3~S]methionine-labeling experiment, but the (3~S]cysteine label was notfound in the mature form. This indicates that the C-terminal portion isremoved during the processing of PBP-3.

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

In Vitro System for the Processing of Penicillin-Binding
Protein 3 of Escherichia coli
Hiroshi HARA, Hideho SUZUKI and Yukinori HIROTA'

The mature form of penicillin-binding protein 3 (PBP-3) of Escherichia
coli, a cytoplasmic membrane protein essential for cell division, is produced
by proteolytic cleavage of the C-terminal portion of its precursor form as
described in the accompanying report (Hara, H. et al., in this Ann. Rep.
p. 49). To investigate this unique processing reaction in detail, we tried toreconstitute the reaction in vitro.
When a plasmid carrying the structural gene for PBP-3, jts!, was introduced into a ts-7304 mutant defective in the processing of PBP-3, alarge amount of the precursor form was accumulated in the membrane
fraction. This membrane fraction served as a "substrate" of the in vitro
reaction. The membrane fraction prepared from an jts1730 mutant, inwhich the processing activity is normal but penicillin-binding ability of
PBP-3 is lost, was used as an "enzyme". Two membrane fractions,"substrate" and "enzyme", were mixed and incubated in the presence of
0.2 % Triton X-IOO and then analyzed for PBP-3 through the binding ofradioactive benzylpenicillin and subsequent electrophoretic separation on
SDS gels. The precursor form in the "substrate" fraction was convered
into the mature form. Thus the processing reaction could be reproducedin vitro. The in vitro processing was enhanced by nonionic detergents,
Triton X-IOO and n-octyl p-D-glucoside, but to a somwehat smaller extent
with the latter. The detergent might help the interaction between the"substrate" and "enzyme" membrane vesicles. The addition of ATP
showed no effect. Even under the optimal conditions and after a long
period of incubation, at most half the amount of the precursor form wasprocessed.
Proteins in the "enzyme" membrane fraction were solubilized with 2 %Triton X-IOO in the presence of 1 M NaCI, fractionated by ion-exchange
chromatography with DEAE-cellulose and then with CM-cellulose. The
fractionated proteins were assayed for processing activity by mixing withthe "substrate" membrane fraction. The activity was recovered in the fraction which adsorbed to the CM-cellulose. Not more than half the amountof the precursors was processed by the solublized enzyme as observed fort

See footnote to p. 39.

RESEARCH ACTIVITIES IN 1987

53

processing by the "enzyme" membrane fraction. The processing site of
the precursor molecules might be enclosed inside the membrane vesicles inhalf of the vesicle population. The substrate precursor form was similarly
solubilized from the "substrate" membrane fraction and concentrated byadsorption to DEAE-cellulose. The solubilized precursors were completely
inert for processing. The precursor molecules of PBP-3 might be alteredin conformation when released from the membrane structure or might besusceptible to processing when incorporated into putative division complexes.
The in vitro processing system will be useful for biochemical analysis of
the processing reaction and for purifying the enzyme for C-terminal processing.
Purification and Characterization of the dnaA Proteinof Escherichia coli
Seiichi YASUDA

The dnaA protein of E. coli plays a central role in the initiation of chromosome replication. The dnaA gene has been cloned on a runaway replication plasmid and the dnaA protein has been shown to be overproduced in
E. coli strains harboring the plasmid (Yasuda and Takagi, 1983, J. Bacteriol.
154: 1153). In order to elucidate the functions of the dnaA protein, it waspartially purified and characterized.
Cells having the dnaA-overproducing plasmid were lysed with the standard
lysozyme method. Cell debris was removed by ultracentrifugation and thesupernatant was fractionated by ammonium sulfate at 0.25 g/ml. The
precipitates were redissolved and dialysed against a buffer solution containing 25 mM HEPES (pH 7.6), 5 mM MgCI 2 , 2 mM dithiothreitol and 20%
glycerol. The dialysed fraction was applied to a column of CM Sephadex
C50 and the column was eluted with a linear gradient of 0-0.5 M KCl in thebuffer mentioned above. The dnaA protein was eluted at about 0.25 M
KCI. SDS-polyacrylamide gel electrophoresis of the dnaA-containing fraction showed that the dnaA protein (52 kdal) was the only major protein andwas more than 70 % pure.
Using this fraction the heat-stability of the dnaA protein was studied.
As shown in Fig. 1, incubation of the dnaA protein in 25 mM HEPES
pH 7.6 at 30°C for 10 min resulted in almost complete inactivation. The

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38+40% gl ycero1+2mM ATP--------L-...-.....::::.."'="--;;---.. +5mM
OTT

1 0 0 L - - - - -....

!

[]

"
>

+40% glycerol+2mM ATP

+'
u

'"

1

40"

50"

Temperature

Fig. 1. Heat stability of the dnaA protein. Purified dnaA protein was diluted with
25 mM HEPES pH 7.6 containing various additions and heated for 10 min attemperatures indicated. The dnaA activity was assayed with the oriCdependent replication in vitro.

addition of 20 or 40 % glycerol had marked effects on the stability of theprotein, but upon incubation at 40°C all of the activity was lost. A striking
stabilization was observed when the dnaA protein was incubated with 2 mM
ATP. The protein was still active even after heating at 55°C for 10 min in
the presence of 2 mM ATP and 40 % glycerol and there was no sign of inactivation by heating up to 55°C, if 2 mM ATP, 40 % glycerol and 5 mM
dithiotreitol were present in the dnaA protein solution. Stabilization by
ATP means that the dnaA protein interacts with ATP and changes its conformation resulting in a heat-resistant form. There were no detectable
DNA-dependent or independent ATPase activities in the dnaA fraction.
Recently Sekimizu and Kornberg showed that dnaA protein binds ATP and
ADP and that the ATP-bound form of the dnaA protein is active in rep-

RESEARCH ACTIVITIES IN 1987

55

lication reactions. Stabilization of the dnaA protein by ATP shown in
the present study supports their results and may give a clue to understanding
the dnaA protein function in the replication of oriC DNA.
Involvement of the dnaK Protein in Replicationof oriC DNA in vitro
Yoshimasa SAKAKffiARA and Seiichi YASUDA

The dnaK gene of Escherichia coli encodes a heat shock protein of 70 kdal
which is involved in DNA replication of chromosome and lambda phage.
However, its precise function in chromosome replication has been unknown.
Recently Sakakibara isolated a dnaK mutant (dnaKIII) which is defectivein the initiation of chromosome replication. In the present study we examined the participation of the dnaK protein in oriC-dependent replicationin vitro. A crude enzyme fraction (fraction II) prepared from dnaKIII
mutant cells showed almost the same level of replication activity as thatfrom wild type cells and there was only a little difference in heat-sensitivity
between them. If fraction II's were prepared, however, from cells which
had been heated at 42SC for 20-40 min just before harvest, the replication
activity was much lower in the dnaKIII mutant than in wild type cells.
The defect in the in vitro replication of the dnaKIII strain could be corrected by the addition of a small amount of fraction II from a strain which
overproduces the dnaK protein. These results indicate that the dnaK
protein is essential for the oriC-dependent in vitro replication reaction. A.
Korhberg's reconstitution system, which supports replication of oriC DNA
with only purified proteins, does not include the dnaK protein as a constituent, and hence, re-examination of the reconstitution system seems to benecessary.
Inhibitor of DNA replication in Escherichia coli,a Possible Regulatory Protein
Seiichi YASUDA

An in vitro replication system dependent on E. coli replication origin,
oriC, was developed and analysed extensively by A. Kornberg's group.
The reaction was first carried out with a crude protein fraction preparedby ammonium sulfate precipitation of cleared lysate of E. coli cells (fraction

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

II). Protein factors necessary for the reaction were fractionated andpurified from the crude fraction and now it is possible to perform the reaction using only purified proteins (reconstitution system).
A striking feature of these reactions, both the reaction with the crude
fraction and the reconstitution system, is their potent replication activities
which are much higher than those of other in vitro replication reactions
including bacteriophage DNA-dependent one. This is in marked contrast
with the replication reaction in vivo; the chromosome is replicated only
once in a generation (ca 20 min at most), whereas bacteriophage DNAs
are replicated in an almost uncontrolled fashion.
From these results it seems to be reasonable to postulate that the oriCdependent in vitro replication system lacks regulatory factors which arepresent in E. coli cells and which regulates replication in a negative way.
To test this hypothesis experiments were carred out to detect in E. coli celllysates a factor which specifically inhibits the oriC-dependent in vitro replication reaction.
Protein fractions were prepared from cleared lysates of E. coli cells by
precipitation with various concentrations of ammonium sulftae. No
significant inhibitory activity was found in fractions prepared by lysing
cells in the presence of a low salt concentration. However, when cells
were lysed in the presence of KCI at concentrations higher than 0.5 M, aconsiderable inhibitory activity on the oriC-dependent replication reaction
was found in a fraction prepared by precipitation with ammonium sulfate
added at 0.28 g per ml of cleared lysate. This activity was present in astrain defective in DNases and no exo- or endonuclease activity was foundin this fraction, suggesting that the inhibition is not due to DNA degradation. Furthermore, the inhibitor was not active on q>X174 single stranded
DNA-dependent replication. This implies that the inhibitor is not a
general inhibitor of DNA replication.
In order to characterize the inhibitor further, partial purification was
carried out. The inhibitor was adsorbed to phosphocellulose and waseluted at 0.5 M KCI. This fraction, although not entirely pure, was usedin the following experiments. The inhibitor was rather temperaturesensitive and lost 80% of its activity by heating at 65°C for 10 min. Thistemperature-sensitivity and the property to adsorb to phosphocellulose
suggest that the inhibitor is proteinous in nature. The inhibitor acts at
an early stage of oriC DNA replication, because addition of the inhibitor

RESEARCH ACTIVITIES IN 1987

57

after brief preincubation of the reaction mixture had no effect on the incorporation of nucleotides. The inhibitor was also found to be active on
ColEl DNA replication in vitro. It is not clear whether these inhibitory
activities are due to a single protein or different ones. Further purification and assignment of its gene are in progress.
Mapping of a Whole Set of Cell Division Genes in
Escherichia coli K12
Akiko NISHIMURA and Reiko TROBE

E. coli is supposed to have more than one hundred Its genes, which are
involved in cell division. Up to now only twenty Its mutants among different genes have been identified. Two kinds of E. coli culture banks ofthis stock center have made it possible to identify a whole set of Its genes
in a short time. One bank is Hirota's collection of 5000 temperaturesensitive (TS) mutant strains, and the other is Clark & Carbon's collection(Neidhartdt et al. (1983), Microbial. Rev., 47, 231-284) of 2000 strains
which harbor ColEl plasmids carrying small random segments of the E.
coli chromosome (pLC-plasmids). Hirota's TS collection contains 410
unknown Its mutant strains, which grow normally at 30°C but make filament cells at 42°C because of a TS defect in the process of cell division.
Each of the 2000 streptomycin-sensitive F+ strains containing pLCplasmids was transferred via a metal replicating block with 48 needles fromstock cultures maintained in Micro-Test III plates to L-agar plates. These
master plates, after overnight incubation at 37°C, were replica-plated onlawns of above 410 each of streptomycin-resistant F- TS (Its) strains on
L-agar plates supplemented with 100 ugjml of streptomycin. These plates
were incubated for 2 days at 42°C to allow selective growth of recipients
which had acquired a hybrid plasmid correcting the TS defect.
As a result, the TS defects of 350 Its mutants were corrected by 496 kindsof pLC-plasmids. Within those, 147 Its strains were complemented for
the TS defect by pLC-plasmids whose molecular species of the inserted
chromosome segments were analyzed already. Part of the results areshown in Fig. 1.
Interestingly each of the 27 Its mutants was complemented for the TS
defect by 2 different species of pLC-plasmids, and 5 (4, 1) Its strains werecomplemented by 3 (4, 5) different species of pLC-plasmids respectively.

58

ANNYAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Fig. 1. Fts mutations in E. coli K12.
The outmost numbers show the strain number of Its mutants which wascomplemented for TS defect by the pLC-plasmids connected with a dottedlines. The plasmid information is from Neidhardt et al. (Microbiol Rev.,
47: 231-284, 1983) and the gene information (in the inner part of circle) isfrom Bachmann (Microbiol Rev., 48: 18D-230, 1983), but only Irs mutants
complemented by pLC-plasmids are shown. The dark blocks indicate
chromosome segments inferred to be carried on the designated plasmids.
The Its mutants which was complemented for TS defect by plural kinds ofpLC-plasmids differed from each other in their chromosome moiety aremarked by L:o. at the shoulder of Its strain numbers.

The Its mutants which were complemented for the TS defect by plural
kinds of pLC-plasmids, differed from each other in chromosome moiety,are marked by f:, at the shoulder of the Its strain numbers in Fig. 1. Transduction analysis by PI-phage showed that TS mutation of those Its mutants
was mapped at only one of those different loci. One pLC-plasmid contains the Its gene itself and the others are supposed to contain suppressor
genes which could correct the TS mutation of Its only when the gene is

RESEARCH ACTIVITIES IN 198759

present in multiple copies in a cell (Takeda et al. (1981), Plasmid, 6, 86-98).
This kind of putative suppressor gene for Its II1utations was found in 23 %of the total analyzed.
.
The remaining 110 Its strains were complemented for the TS defect by
one species of pLC-plasmid.
158 Its mutants were complemented for the TS defect by 345 pLCplasmids, whose mapping position of chromosomal moiety are unknown.
Continued research is necessary to determine the physical mapping positionof those unknown pLC-plasmids by hybridization techniques using thegene library of Kohara's collection (Kohara et al. (1987), Cell, 50, 495-508).
This research was supported by a Grant-in-Aid for Scientific Research
on Priority Areas.

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

III.

IMMUNOGENETICS

Sex-specific Recombinational Hot Spot in the Mouse Major
Histocompatibility Complex
Toshihiko SHIROISHI, Naoto HANZAWA, Tomoko SAGAI
and Kazuo MORIWAKI

Some Asian wild mice show increased recombination frequency within
the MHC region in crosses with inbred strains. We previously reported
one of such H-2 congenic strain, BlO.MOL-SGR, whose H-2 was derived
from Japanese wild mouse (Shiroishi et at. 1982, 1987). Any crosses between BlO.MOL-SGR and inbred strains showed a 100 fold higher recombination frequency in the K-A p interval of the MHC. Surprisingly, theincrease in recombination was observed only in female meiosis.
In the present study, we mapped crossover points of fifteen independent
recombinations which were generated from crosses between BlO.MOLSGR and inbred strains. Out of fifteen, seven were produced from crossesof BIOI BlO.MOL-SGR and eight from crosses of BlO.A/BlO.MOL-SGR.
We used polymorphic restriction sites between parental strains as genetic
markers to locate the crossover points at the DNA level. Results indicatedthat all crossover points are mapped to a DNA segment of 30Kbp inlength, which is located between the A p3 and A p2 genes. Nine of them were
further confined to a short stretch of approximately lKbp DNA.
As previously reported, recombination involving BlO.MOL-SGR showsa unique directional bias in the appearance of recombinant types depending
on strain combinations of crosses. From crosses with the BlO strain,most recombinants retain K of BIO and A p of BlO.MOL-SGR, while
reciprocal types scarcely appeared. On the other hand, from crosses with
BIO.A, recombinants preferentiallY retain K of BlO.MOL-SGR and A pof BlO.A. Interestingly this directional bias reflected the fine locationsof crossover points in the hot spot of IKbp DNA. All three recombinations which retain K of the BIO strain were confined to a 700bp DNA
segment proximal to the MspI site in BlO.MOL-SGR. Five recombinations which retain the K of BIO.A strain were mapped to a 400bp DNA
segment distal to the same MspI site. These results suggest that the recombinational hot spot of 1Kbp in length is split into two parts and each

RYSEARCH ACTIVITIES IN 1987

61

combination of crosses favors a different part of the hot spot of IKbp
DNA. Thus it became clear that BlO.MOL-SGR has a female-specificrecombinational hot spot in the A pa -A p2 interval of the MHC.
In order to analyze the molecular structure of this recombinational hot
spot and to examine whether this segment has the consensus DNA sequence
for the site-specific recombination, we attempted to clone the hot spot from
BlO.MOL-SGR and its counter regions from other parental strains, BlO.A
and BlO, using a cosmid vector. So far we have succeeded in cloning
these DNA segments of approximately 40 Kb in length from all three mousestrains. The DNA sequencing analysis around the recombinational hot
spot is now proceeding.
Extensive Polymorphisms of the MHC Class II Genes inJapanese Wild Populations of Rat
Hideo GOTOH, Yoshi KAWAMOTO*, Toshihiko SHIROISHI
and Kazuo MORIWAKIThe major histocompatibility complex of the rat (RTl), which is homologous to the HLA complex in humans and the H-2 complex in mice, encodes three sets of class II molecules referred to as RT1.B, RT1.D, andRT1.H. The polymorphism of the RTl class II genes has been determined
serologically in inbred strains of rat and in wild rat in Europe and theUSA. Eight unique haplotypes have been described for RTl class II genes
in inbred strains of rat, and low polymorphism has been observed in wild
populations of rat. We surveyed polymorphisms of RTl class II genes inJapanese wild populations by restriction fragment length polymorphism
analysis. The present preliminary survey discovered extensive polymorphisms in these populations. We confirmed that most inbred strains of
rat originated from wild populations in Europe and the USA, and thatmany unknown RTl class II haplotypes exist in Asian populations.

•

Department of Animal Genetics, Faculty of Agriculture, Nagoya University,

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Introduction of Human Steroid 21-hydroxylase Gene
into Mice: An Attempt at Gene Therapy
Hideo GOTOH, Tomoko SAGAI, Toshihiko SHIROISHI
and Kazuo MORIWAKIWe reported on an intra H-2 recombinant haplotype awl8 which truncatesat about 80 kb in the H-2 S region. The functional steroid 21-hydroxylase(21-0Hase) gene and the C4 gene are encoded in the deleted fragment.
Steroid 21-hydroxylase acts in the cascade of adrenocortical steroidogenesis,
and C4 protein plays a role in one of two pathways of the complement
activation system. Homozygocity for the awl8 haplotype in mice is lethal
in the early neonatal period. We assumed that the lethality of the awl8
homozygote is due to the inability to synthesize adrenocortical steroids inthe mice.
So far, we have obtained 6 transgenic mice containing the human genomic
21-0Hase gene with an H-2 genotype of either +/+ or +lawI8. If theintroduced gene is expressed in the proper tissue, awl8 homozygotes would
be rescued, and the mice should be a new and unique animal model for C4
deficiency.

Trans-subspecies Transmission of Mouse H-2 Polymorphism
Tomoko SAGAI, Toshihiko SHIROISHI and Kazuo MORIWAKI

Japanese wild mice, M.m. molossinus, have several genetic characteristics
clearly distinguishable from European wild mice, M.m. domesticus, which
are considered to be the ancestos of laboratory mice. Japanese wild mice
were estimated to have a genetic distance of approximately one million
years from European mice. Considering this unique feature of Japanesewild mice, we attempted to verify the hypothesis that H-2 polymorphism
is older than at least the subspeciation of mice. Our early research focusedon finding the H-2 antigens peculiar to Japanese wild mice and, in addition,
the antigens common to European wild mice. Serological surveys of wild
mouse populations using aIlo-antibodies indicated that three H-2K antigens,
H-2Kd, H-2Kf and H-2KU were predominant in Japanese wild populations. Their phenotypic frequencies were; d: 38 %, f: 33 % and u: 17 %,respectively. Two of them, the d and f antigens, have been reported tobe distributed widely in European and North American populations at

RESEARCH ACTIVITIES IN 1987

63

substantially high frequencies (Klein, J.). Therefore they seem to becommon antigens preserved in both subspecies. On the other hand the
H-2KU antigen was found only in Asian populations including Japan,
Taiwan and main land China. To investigate these common and population specific antigens in detail, we produced 10 monoclonal antibodies for
the H-2Kf antigen and 4 for the H-2KU antigen and made H-2 homozygouslines carrying wild-derived H-2 genes. Serological analysis using newly
established monoclonal antibodies revealed several variations in antiganic
determinants even in the same H-2K antigen which was identified by alloantibodies. It is, therefore, conceivable that each of the three H-2K
antigens discussed above is not a single antigen but is part of a group including variants distinguishable by monoclonal antibodies. In addition toprecise serological studies, we also carried out RFLP analysis for the wild
mouse populations with the aid of a DNA probe to hybridize with the 3'
non-coding region of the H-2K gene. This study indicated that each groupof H-2K antigens such as d, f and u types, identified by serology, displayed
representatively unique RFLP patterns. Of greater interest, the reactivity
for only one of monoclonal antibody was consistent with the appearance ofspecific RFLP patterns in several cases. For example, the presence ofdeterminant MS54, which is carried by the H-2Kf antigen group, always
displayed the specific band irrespective of the presence of other determinants,
while the H-2K gene identified as a member of the f group by alloantibodies
did not show the specific RFLP pattern mentioned above if it did not havethe MS54 determinant. Even out of the H-2Kf antigen group, if the MS54
determinant was present, the same specific RFLP pattern was obtained.
These results may suggest that intra-genic recombination occurred in the
middle of the H-2K genes to split the antigenic determinants into twoparts, MS54 and f-specific determinants on the external domain of theH-2K molecule.
In conclusion the present studies demonstrated that common types of
H-2K antigens beyond the subspecies barrier exist, although minor variations have accumulated in each of the subspecies. Thus it became clearthat the ancestral f allele was present before subspeciation between Japaneseand European wild mice.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS

Preservation of Frozen Drosophila Embryos after
Irradiation by Laser Microbeam
Yukiaki KURODA, Yuko TAKADA and Takahiro KASUYA*

Most stock cultures of Drosophila are being maintained in laboratories
of universities and institutes at a temperature of 25°C or slightly lower bymating male and female flies. The day-to-day operation for maintaining
these stocks entails enormous cost, labor, and time. In the present study,
the possibility of using the laser microbeam to preserve early embryos of D.
melanogaster at -196°C was examined.
The most difficult problem encountered in freezing Drosophila embryos
has involved a method in which a protective agent is introduced into theeggs through the vitelline membrane which is very tolerant to chemical
enzymes. In the present study, a UV laser microbeam was applied toinflict breaks or cuts on the micropile of the eggs in order to allow protective glycerol to enter.
Eggs were dechorionated by treatment with 3 % sodium hypochloride
solution. Eggs of the appropriate developmental stage were transferredto thick plastic slide plate, and then the plate was placed on the stage of a
microscope, which had been incorporated into a laser microinjector system.
A laser beam with a spot size 0.5 pm was focused through an incident-lightmicroscope objective onto the micropile of each egg. Exact aiming was
accomplished by pointing the target image on a TV monitor screen with alight pen.
The irradiated eggs were incubated at 25°C in Medium K-17 supplemented with fetal bovine serum and 15% glycerol. Following a 1-24 hour
incubation, the eggs were frozen rapidly at -196°C in small glass vials in
liquid nitrogen. After at least another 24 hours, the eggs were thawed andincubated in a salt solution at 25°C. A few of the eggs hatched, and thelarvae grew to adult flies. Glycerol incorporation was confirmed by staining the eggs with neutral red. The survival rate of frozen, irradiated eggs
* Microbeam Physics Laboratory, The Institute of Physical and Chemical Research,
Wako, Saitama.

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65

increasing upon incubation with glycerol for at least 16 hours. The stage
and site of eggs to be laser irradiated, the conditions of incubation, and therate of freezing and thawing are now under investigation so as to yield alarge number of viable embryos.
Studies on Sex Differentiation of Embryonic Cells
of Drosophila melanogasterYukiaki KURODA, Bungo SAKAGUcm* and Kugao OISHI"In Drosophila melanogaster, a cluster of several cells which develops togerm cells in adult flies appears as the polar cells outside of the blastoderm
at the posterior end of blastoderm embryos. Other morphological differences between male and female individuals can be distinguished only after
pupation in normal development. No practical procedures have beenfound to detect the differences between somatic male and female cells during
embryogenesis.
Sex-ratio organisms (Spiroplasma SRO) of Drosophila have male-specificlethal effects and kill only male embryos in early development. We established an experimental system in which undifferetiated cells dissociatedfrom post-gastrula embryos differentiated into larval-type cells or adulttype structures in the absence or presence of ecdysterone in culture medium.
In the present study we examined when SROs infect undifferentiated cells
obtained from post-gastrula of D. melanogaster, how they recognize somatic
male and female cells in culture, and what types of cells or tissues are affectedby SROs.
A haemolymph of 0.2 ml containing SROs was collected by a glass capillary from abdomens of 300-600 adult flies 7 to 10 days after eclosion. The
haemolymph was diluted with the same volume of salt solution, filteredthrough a Millipore filter and added to culture flasks in which embryoniccells had previously been cultured for 24 hours. After cultivation for one
or two days at 25°C, SRO-infected cells were observed under an inverted
phase microscope.
As a result, necrosis was found in some cells, suggesting that SROs hove
lethal effects on infected cells. The necrotic cells appeared as brownclusters. The tissue specificity of affected cells are under investigation.
* Department of Sericulture, Faculty of Agriculture, Kyushu University, Fukuoka.
** Department of Biology, Faculty of Science, Kobe Univeristy, Kobe.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Injury Effect in Hydra Head Regeneration
Hiroshi SHIMIZU and Tsutomu SUGIYAMA

Roles of "injury effect" in hydra regeneration was examined. Strain
reg-16 is a mutant strain which has a reduced head regenerative capacity.
Kobatake and Sugiyama (1988) have shown that head regeneration by thisstrain can be significantly improved by the reopening of wounded tissue
located at the apical regenerating end of the decapitated polyps. Thissuggests that injury inflicted upon the regenerating tissue has a positiveeffect on head regeneration in strain reg-16.
The present study was carried out to examine whether or not a similareffect of injury can be also demonstrated in head regeneration of the wild
type strain of Hydra magnipapillata (strain 105). Head was removed bysurgical amputation at midgastric region of an animal bearing the first bud
protrusion. The decapitated polyps were then devided into three groups.
In the first group (control), wound opening produced by amputation wasallowed to close and heal normally. In the second group, wound opening
was prevented from closing and healing for 24 hr by placing a nylon fishingline through the gastric cavity of the decapitated polyp. In the third group,
wound opening was covered immediately after amputation. This wasachived by grafting two decapitated polyps together in a mirror image symmetry in such a way that the wound opening of the two polyps covers eachother.
It was found that the rate of head regeneration was significantly lower inthe third group (mirror image grafting) than in the other two groups. Approximately 40 %of the animals in this group did not regenerate a hypostomeor tentacles, whereas nearly 100 % of animals regenerated normally in theother groups. Furthermore, decapitated polyps whose wound closing andhealing was delayed (second group) regenerated a significantly higher number of tentacles than in the control polyps.
These observations suggest that surgical removal of head alone does notnecessarily lead to head regeneration, and that the state of injured tissue inthe period immediately following head removal has a significant effect on
determining regeneration. The underlying mechanism responsible forthis effect is not well understood at present, but probably identical to the"injury effect" observed in hydra tissue transplantation phenomena by
MacWilliams (Deve!. Bioi., 96, 239-257 (1983)).

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67

Role of Interstitial Cell Migration in Generating
Position-Dependent Patterns of Nerve Cell
Differentiation in Hydra
Toshitaka FUJISAWA

Nerve cell differentiation in Hydra occurs predominantly in the head andfoot regions. The role of interstitial cell migration in the formation of thisposition-dependent pattern was investigated in the regenerating head.
When distal tissue was removed from the body of wild type strain of
Hydra magnipapillata, 105, nerve cell differentiation occurred at a rapid rateduring the first 48 hr of regeneration, slowing after this point. The rapid
nerve cell differentiation was due primarily to the migration of interstitialcells, some of which were nerve cell precursors. The migration, however,
did not continue but decreased considerably after the first 48 hr ofregeneration. In a mutant strain, reg-16, deficient in head regeneration, no migration of interstitial cells and hence no new nerve cell differentiation was observed in the regenerating tip. However, the interstitialcells of reg-16 were found to migrate if they found a proper environment.
These observations suggest that the migration of nerve cell precursors plays
an important role when the new nerve net is being rapidly established, e.g.
head regenevation.
Genetic Studies on the Lifespan of Adult Silkworms
Effect of Removal of Brain
Akio MURAKAMI and Jun SHIMADA*

The mean lifespan of adult silkworms was found to differ markedly withthe strain under experimental conditions (25°C), and the lifespan of females
was 1.5 times longer than that of males. The mean lifespan of females was
about 10 days, but the lifespan of the Daizo strain females was 1.5-2 days,
the shortest lifespan among silkworm strains. The hybrid females weregenerally longlived, and this characteristic was found to be dominant overshort lifespan. The relationship between lifespan and brain function has
been discussed for a long time. The brain-central nervous system plays
an important role in basic or fundamental processes. Operations on the* Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tsukui,
Kanagawa-ken.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

brain-central nerve system can be performed easily in silkworms. We
examined the effect of brain removal on the mean lifespan of Daizo,
the strain with the shortest lifespan, that of the J106 strain showing a mean
lifespan and the hybrid between the two strains.
The experiment was done on 100 silkworms each in the untreated control
and brain removal groups for each strain and both sexes. The operation
was conducted on pupae of both sexes just after pupal ecdysis up to I hour
after pupation, in a simple clean bench. As a result, the operation was
successful in about 75 % of the silkworms which grew into adults. The
removal of the brain shortened the lifespan in all strains and both sexes.
A difference between the sexes was seen in Daizo, the short lifespan strain,
but the effect of brain removal on the reduction of lifespan was small. The
lifespan of females was reduced significantly by brain removal to a lengthof lifespan equal to that of untreated males. Males usually lived for 11.5 days, so the reduction was slight. However, in the F 1 hybrid obtained
from a cross with JI06, the effect of brain removal was marked in both
sexes. The lifespan of the F 1 hybrid which was classified into the longest
lifespan group, was reduced by about 2-3 days on the average. The males
in both strains had a lifespan of about 2 days, and the removal of the brain
had less effect on the lifespan of males than on that of females.
In conclusion, brain removal was found to reduce lifespan by about 2days irrespective of the adult duration specific to the strain or sex. Thatis, the lifespan specific to the strain and the sex of adults depends on brain
function in each strain and sex.
Genetic Studies of Parthenogenesis in the Bombyx Silkworm:
Genetic Factors Responsible for the High Frequencyof Spontaneous Parthenogenesis
Akio MURAKAMIIn the domesticated silkworm (Bombyx mori), naturally-occurring parthenogenesis, a form of asexual reproduction, is a popular phenomenon
with a marked difference among strains or races ranging from a few percent
to 70% or more. Particularly, an inbred line of the Cambodge strain, oneof the tropical multivoltine races, showed a high potency (70 % or more) for
spontaneously-occurring parthenogenesis among two dozen silkworm
strains investigated so far. A similar observation with an Indian com-

RESEARCH ACTIVITIES IN 1987

69

mercial multivoltine strain, Nistari, had been reported by Chowdhury(1953).

Most eggs laid by virgin Cambodge female moths developed spontaneously into, at least, the completion stage of organogenesis, while only 10 %of the eggs from virgin females in various other strains, Daizo, Aojuku,
Cl08, J106 etc., attained the same embryonic stage as the Cambodge.
However, the rest of the parthenogenetically developed eggs generally died
before the completion stage of organogenesis regardless of strain or race.
Many eggs laid by virgin moths showed no external indications parthenogenetic development (Murakami et al., 1988). In Bombyx mori, the developing eggs are easily discernible from non-developing egg at the stage
coloration with pigment granules in the serosa membrane cells. Developing eggs become dark gray, while non-developing ones remain light yellowish-white. Accordingly, it has been suggested that the multivoltine race
may have a higher parthenogenetic potency than uni- or bivoltine races.
Eggs laid by F I female moths of a cross between the Cambodge femaleswith a high frequency and JI06 males with a low frequency did not showa high incidence of spontaneous parthenogenesis. However, eggs laid by
the F I moths of the reciprocal cross, Jl06r;;. X Cambodge 5', showed a frequency similar to that of Cambodge females. This clearly indicated thatthe genetic factor(s) responsible for a high potency for spontaneous parthenogenesis in the Cambodge strain is inherited autosomally and is not
sex-linked. Vrigin female moths of the backcross F I , (Cambodge r;;. X
JI065') r;;. X Cambodge 5', produced different egg batches with high andlow frequencies for spontaneously-occurring parthenotes. The frequenciesof parthenotes detected in the high line roughly corresponded to those of
inbred Cambodge strain moths, while those in the low lines corresponded
to those of the JI06 strain or about one fifth or one tenth of the Cambodge
strain. A similar situation was also observed in egg batches laid by thereciprocal cross mentioned above.
It is of further interest to note that a ratio of low to high lines was nearly
one to one, suggesting that a hereditary factor for the high parthenogenetic
trait in the Cambodge strain may monogenic and may be located on acertain autosome. Virgin female moths of the BF I produced only one typeof egg batch with a somewhat higher frequency in the event than that of
the JI06 strain. Egg batches laid by F 2 moths of a cross between the JI06
females and Cambodge males were two types with high and low frequencies

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

of spontaneously-occurring parthenotes having a ratio of one to three.
The present observations and others suggest that in the Bombyx silkworm,
the tropical multivoltine race possesses a high potency for parthenogenesis
compared with the uni- and/or bivoltine races.
Secretory Mode of Fibrous Cuticle (FC) in Silkworm
Tadasu MORI"', Jun SHIMADA"'''' and Akio MURAKAMI

In insect integument, new cuticle is formed at every occurrence of ecdysis.
In such a cuticularization in the fifth instar larvae of silkworm, the numberof fibrous cuticle (FC) layers (about 15 just after the fifth ecdysis) increases
to about 70 when the larvae grow for 7 days with an increase in thickness
from 10 to 40,um. In addition, an S-shaped profile can periodically beobserved in such a thickening process. However, nothing has been reported so far on detailed electron microscopic observations of the FC
secretion in insects, including silkworm, except for an assumption basedon a study using electron microscope autoradiograph, that raw materials of
FC are secreted (supplied) from epidermis (EP) in a desert locust.
In the present study, the FC formation process was observed very clearly
under an electron microscope on a boundary section between normal
and transparent integuments on the ventral side of the fifth segment ofa threeday-old fifth instar mosaic larva obtained from a cross of mo/mo:ok/ok ~ and +/+: ok/+ 6. Compared with a normal individual, no differences were found in mosaic larvae in cell adhesion and structure of
cuticle in the boundary section between normal and transparent EPs. In
these two EPs at this stage of mosaic larvae, however, the following phenomena were observed: 1) Golgi bodies were found everywhere and wereconstructed by electron-dense spheres (EOSs) covered with a membrane;2) these EOSs were transferred toward peripheral sites in cytoplasm, where
the EP adjoins the cuticle layer, and are located densely in the site; 3) someof the these EDSs were released from the cells (into a space between EP
and FC) by exocytosis; 4) at the released space, some or entire EOSs weremetamorphosed into a fibrous mass; and 5) the metamorphosed fibrousmass moved into the FC which adjoined EP and was combined with FC.
'" Kinjyo University, Nagoya.
"'''' Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tsukui,
Kanagawa-ken.

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71

These phenomena observed in the EPs clearly differ from the secretorymode of ecdysial droplets whose secretion starts at the beginning of spinneret pigmentation. On the basis of the above results, it was found thatthe secretory mode of FC consisted of two processes: a secretory process(construction of EDSs as a raw material for cuticle-formation by Golgi
bodies and release of EDSs outside the cells by means of exocytosis) and aformation process (metamorphosis of released EDSs into fibrous material,and incorporation of the material into FC and formation of fresh FC).
On the Sex of Artificially-Induced Parthenotes after Treatmentof Newly Laid Unfertilized Ova with Hot-Water
in Bombyx mori
Akio MURAKAMI

Meiosis is an important event in the course of gametogenesis and is composed of the two successive divisions. The majority of biological organisms
are the pre-reductional type: the first division is a reductional (heterotypic)
process from tetrad to dyad chromosomes and the second division is anequational (homeotypic) process from dyad chromosomes to a monad
chromosome. The other is the post-reductional type: the first division isof an equational and the second is of a reductional. The Bombyx female
is a heterogametic sex, designated in terms of the sex chromosome consitution ZW, and the male is ZZ. In the case of the pre-reductional division
type, all artificially-induced parthenogenetic individuals produced by suppression of the second division would become exclusively ZZ males, since
WW females are theoretically nonviable, whereas in the case of post-reductional type, parthenotes would result in only ZW females. Several attempts have been made in spite of serious difficulties to cytologically analyze
the maturation divisions of spontaneous parthenogenetic eggs and haveindicated that in naturally occurring parthenogenetic eggs, both maturation divisions take place (e.g., Astaurov, 1967). Either non-seminated orinseminated ovum in the ovaries of adult moths corresponding to late prophase I oocytes or those of the preceding stage prior to the resumption of
the 1st meiotic division. Anaphase I resumes while the pass through theovaries. Newly deposited eggs on the egg-card are at anaphase I and immediately undergo telophase I about 10 min thereafter. The completionof the 1st division occurs ca. 20-30 min after oviposition. Successively,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

the secondary oocyte goes on to interphase II and the 2nd meiotic divisionappears 90-100 min after oviposition. The completion of the 2nd meiotic
division occurs about 10-20 min thereafter.
To clarify which meiotic division type the oogenesis of the Bombyx
silkworm fits, 0-1 and 5 day-old ova (or secondary oocytes) laid fromvirgin F 1 female moths of a cross between JI06 and Cambodge males werecollected and immersed for 15 min into hot-water kept at 46°C. After
treatments the ova were then transfferred to water kept at a room temperature of 25°C for 15 min. The eggs were taken from the water and dried
naturally in a temperature controlled room at 25°C and 60-70 % relative
humidity. Those artificially-induced parthenogenetic eggs in a diapause
state were kept under natural conditions until the next generation. A verysmall number (less than 0.1 %) of the diapausing parthenogenetic eggs were
hatched regardless of the treated age of unfertilized eggs. Most hatched
insects were grown until the last instar. All larvae observed showed female
sex characteristics. Judging from morphological inspections of eggs and/orof larvae produced by the parthenogenetic females which had already beenmated with normal diploid males, most (95 % or more) of the parthenotes
were regarded as diploid, and the rest as triploid. If the secondary meiotic
division were blocked in the case of pre-reductional division types the
majority of parthenotes would be appeared in the case of the post-reductional type, Thus, the present findings suggest that the oogenesis of Bombyx
mori follows the post-reductional division process.
Embryonic Development of a Maternally Affected EmbryonicLethal Mutant in Drosophila melanogaster (II)Kiyoshi MINATO and Masa-Aki YAMADAIn the previous study on external morphology observed under an optical
microscope, eggs laid by female flies homozygous for a maternally affected
embryonic-lethal gene, fs(1)MAY-263 (Yamada, M. A., 1978) in Drosophilamelanogaster, proved to develop into embryos with some teratogenic anomalies possibly caused by abnormal morphogenesis at an early stage of development. To investigate in further detail, histological preparations oflethal embryos at various stages of development were made to compare withthose of normal embryos. Results showed from external morphology, thatembryos developed normally until the end of the cellular blastoderm stage,RESEARCH ACTIVITIES IN 1987

73

but thereafter, the formation of the inner layer of the germ band producedby the formation of the ventral furrow was not seen in mutant embryos.
Instead, many transverse invaginations of the external layer, which were
never seen in normal embryos, were formed here and there in abnormal
embryos. The dorsal extension of the germ band did not occur and theposterior midgut rudiment appeared to invaginate at the posterior pole ofembryos.
At a later stage, the abnormal embryos had the hypoderm constricted
here and there possibly owing to the remnants excessive furrows in theexternal layer formed at the early stage. In those embryos, the visceralnerve system was formed but somewhat disorganized by the constrictionsof the hypoderm. The muscle and fat body were scarcely seen in thoseembryos, and the midgut remained as a sac-like one without developing
into a coiled gut.
lt is interesting that the abnormal morphogenesis seen early in the development of the mutant embryos and the formation of excessive transverse invaginations of the external layer without the formation of the ventral furrow
resulting in the formation of an inner layer of germ band are not an isolatedcases but are seen in many lethal mutants, maternally or zygotically. The
above syndrome commonly seen in these mutants may give us some important information about morphogenesis at early stages of embryonicdevelopment in Drosophila.
Isolation and Characterization of a Maternally Affected
Embryonic-lethal Mutant Rescued by Transplantationof Normal Egg Cytoplasm inDrosophila melanogaster
Masa-Aki YAMADA and Saburo NAWA

In order to study the effects of the oocytoplasm on the gene expressionof zygotonucleus in the early development in D. melanogaster, maternallyaffected embryonic-lethal mutants (29 complementation groups) were isolated through the treatment of X chromosomes with EMS. From thesemutants, a mutant (fs(1)MY-18) rescued by the transplantation of normal
egg cytoplasm was obtained. This mutant gene was located at 1-12.5 andlocated in 4F7-8-5AI-2 bands on the salivary gland chromosome. Homozygous females had normal viability (over 90% when compared to hetero-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

zygous females) and the sex ratio of progeny produced by heterozygous
females was 0.52. Homozygous females were anatomically normal. Although they were completely sterile, they had normal ovaries and produced
morphologically normal eggs. However, these eggs were lethal when fertilized with sperm bearing a normal allele. But, the progeny (2 % of treatedeggs) were rescued with transplantation of the cytoplasm of normal eggs.
These results suggest that this mutant gene had no effect on late development and the viability of the fly but was effective on the generation ofoocytoplasm concerned in the early development of eggs during oogenesis.
Embryonic developmental features of this mutant were observed by timelapse photographs taken through a phase-contrast microscope. Eggs laidby homozygous females developed normally to the syncitial blastoderm
stage, but at the cellular blastoderm stage the formation of pole cells was
reduced compared to that in normal embryos. Thereafter the extention of
the germ band was weak and therefore the anterial elongation of the dorsal
plate stopped at about 50 % of egg length, although in normal embryos thetip of the dorsal plate reached about 75 % of egg length and hence, little
formation of the cepharic furrow was observed. The formation of thecrypeolabrum and the mid gut was later than in normal embryos. Although
the embryos moved actively within the vitellin membrane, they neverhatched. They also had a normal cuticular pattern. These observations
suggest that the maternal expression of this gene is required for the morphogenetic movements of embryonic cells in the early development.
A Novel Mobile DNA Element Consisting of Tandem Repeats
at the White Locus of Drosophila simulans
Yoshihiro H. INOUE and Masa-Toshi YAMAMOTO

According to the detailed characterization of individual mobile elements
isolated from D. melanogaster, they are classified into four families, copia,
P, FB and F. It has also been demonstrated that some mobile elements
such as the copia-group, are commonly found in various species belonging
to the D. melanogaster subgroup. However, those which are species specificor restricted to only a few species have also been reported.
white-milky (w mkY) was the first mutant of Drosophila simulans demonstrated
to be moderately unstable, and to carry a 16 kb DNA sequence inserted at
the white locus. The spontaneous occurrence of wmky and its revertability

RESEARCH ACTIVITIES IN 1987

75

suggest that the long insertional DNA belongs to a class of mobile elements.
We cloned the entire insertional sequence and analyzed the molecular structure. The insertional DNA consisted of three tandem arrays of approximately 6.2 kb in size, although the most proximal repeating unit to thecentromere had 2.5 kb deleted at the end. There were no detectable sequence homologies between both termini. It is not yet understood whether
the basic structure of the mobile element is 6.2 kb or 16 kb, since variously
fragmented internal sequences of the element exist in the genome. Since
no homology was detected with some mobile elements isolated from
Drosophila, the insertional DNA sequences were considered to be a novel
dispersed repetitive DNA element, and named "ninja". This element
exhibited intra-specific variation in genomic copy numbers.
Genetics of an Unstable white Mutant in Drosophila simulans:
Reversion, Suppression and Somatic Instability
Yoshihiro H. INOUE, Toshifumi TAIRA and Masa-Toshi YAMAMOTO

A spontaneous white mutation, white-milky (w mkY ) of D. simulans ismoderately unstable and is associated with a 16 kb long DNA insertion into
the white gene. wmky , which is an unstable mutation found in D. simulans,has been genetically analyzed. Among nine spontaneous, partial reversions toward wild type, five were white locus mutations. They arephenotypically different from each other and three show eye color sexual
dimorphism indicating a failure of the dosage compensation mechanism.
Two w locus mutations whose eye color appeared identical between males
and females were also isolated. Of the other back-mutants, three wereassociated with a recessive suppressor of lVmky and one was a semidominant
suppressor. These suppressor loci are located on the third chromosome at
map positions of about 90 and 120 respectively. The suppressor mutations demonstrate specific effects on w locus mutations derived from wmky
which lack gene dosage compensation. Somatic instability was detected
at a frequency of 5.6 x 10- 4 in wmky flies heterozygous for the recessive suppressor and the frequency was increased lO-fold when the suppressor mutation was placed in a different genetic background.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

The ninja Mobile Element is Abundant Specifically in a
Strain of Drosophila simulans
Masa-Toshi YAMAMOTO and Yoshihiro H. INOUE

Debates on the evolutionary history of mobile DNA elements, continue
and experimental evidence available to date upon which the major hypotheses stand do not seem supportive enough to reach a firm conclusion.
For elucidating the origin and subsequent dynamics of mobile elements,
a strain with specific dispersed repetitive DNA sequences is of crucial importance.
We previously identified a 16 kb mobile element, ninja, in a moderately
revertable mutant, white-milky (w mky ) of Drosophila simulans. The entire
insertional DNA, ninja, was isolated and the molecular analysis indicatesthat ninja is a mobile element whose structure is a novel type consisting of
tandemly triplicated DNA sequences with no terminal repeats larger than
100 bp. Ninja did not show sequence homology for most of the well characterized mobile elements isolated from both D. melanogaster and D. simulans.
We examined the DNA sequences homologous to ninja in nineteen D.
simulans strains (KI, w S , W2 • 4C , wR , WI, wmky , wmkY _2, w NIG , wL, Leiden, Riverside, y wf, Rakujuen, 0-9, SM1, K3, All Saint and XX, y w f), six strainsof D. melanogaster (en' bw" en 38j bW38j , Canton-S, M56i, St Hellens andUman) and six species of the melanogaster subgroup (D. mauritiana, D.
seehellia, D. teissieri, D. yakuba, D. ereeta and D. orena) by either Southernblot hybridization or in situ hybridization to the salivary gland chromosomes. The results consistently showed that ninja DNA is abundant specifically in the W mkll strain, and the copy number in other strains is miniscule.
Of significance is strain specificity in such a proportion that only one outof nineteen D. simulans strains posseses ninja in copy numbers. It is therefore very likely that the dispersed ninja elements in wmky have evolved very
recently in evolutionary history.
Grandsonless Strain of Drosophila simulans Carries MaternallyInheritable Sexratio Factor which Specifically
Dysfunctions Y-bearing Sperms
Masa-Toshi YAMAMOTO and Takao K. WATANABE

Prezygotic "sex-ratio" traits so far isolated from wild populations of

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various species of Drosophila in most cases deviated toward the direction
favoring females, and the genetic factors responsible for the deviation werelinked to either the X chromosome in the D. obscura species group or the
3rd chromosome in D. simulans. Postzygotic modifications of sex ratio
come a wide variety of develpmental problems associated with sex specificgene regulations, such as a series of genes involving sex determination anddosage compensation mechanisms. In addition to the above examples ofchromosomal sex-ratio genes, cytoplasmic factors, which distort the sex
ratio postzygotically, have also been identified as mycoplasmas and labeled
as sex-ratio organisms (SRO). However, cytoplasmic factors which strongly modify sex ratio prezygotically have not been previously reported.
In Drosophila simulans, we found a new type of sex-ratio distorter strain,
and studied its genetic and cytological characteristics. Crosses between
wild type males and females who are carriers of the distortion factor produced progeny at a sex ratio 1: 1. The daughters, when crossed with wild
type males, produced equal numbers of males and females, but the sonsproduced predominantly female offspring. Effects of the distortion factor
are phenotypically expressed in the F2 generation, and thus the number ofgrandsons is reduced significantly to about 10% of the total number ofprogeny. We call the strain Grandsonless (GS). The factor causing thephenotype, Grandsonless factor (Gsf), is maternally transmitted and nota chromosomal genic property. Being different from SRO, the factor
seems resistant to the antibiotic tetracycline.
Spermiogenesis was examined in males produced by a mother who wasa carrier of the factor Gsf In the strain exhibiting the extreme distortion,
the number of sperm heads were reduced to about half of the normal numberof sperm in a sperm bundle. Since the first and the second meiotic divisions are confirmed to be normal cytologically, the distortion is attributed
to prezygotic breakdowns of Y-bearing sperms at a stage of sperm elongation in spermiogenesis. Normal "egg to adult" viability supports thiscytological observation.
We are currently in the process of isolating the cytoplasmic factor Gsf
from the strain.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Studies on Gene Expression in Mouse Cerebellum
S. AOTA and T. IKEMURA

Gene expression in mouse cerebellum was studied using a mutant mouse(nervous) that is known to be deficient in normal cerebellum development.
We constructed a eDNA library from normal mouse cerebellum and isolated clones that are specifically expressed in cerebellum (e.g., not expressedin cerebral cortex and in liver) but not in the nervous cerebellum. Theseclones are useful developmental markers for the cerebellum. We determined
nucleotide sequences of several eDNA clones thus isolated and found that
one clone has a significant homology for an amino acid sequence withcarbonic anhydrase I and II (about 40 % homology with both of them at
the identical amino acid sequence level). We think this sequence corresponds to a new type of carbonic anhydrase that is specifically expressed in
cerebellum. Although physiological roles for each type of carbonic anhydrase are not well understood, interesting variations in tissue- and cell-type
distributions of the isozymes are known. At present it is not certain whether
the nervous mutation affects directly or indirectly the expression of thegene isolated.

Myofibril Assembly is linked with Vinculin, a-Actinin andCell-Substrate Contacts in Cardiac Myocytes in vitro
Yutaka SHIMADA

The relationship of nascent myofibrils with the accumulation of adhesion
plaque proteins and the formation of focal cell contacts was studied inembryonic chick cardiac myocytes in vitro. The cultures were doublestained with various combinations of the specific antiactin drug phalloidin
and antibodies against vinculin, a-actinin, connectin (titin), myosin heavy
chain, fibronectin and desmin, and examined under fluorescence and interference reflection microscopy.
Filament bundles with and without cross-striations in cardiac myocytes
were reactive to antibodies against muscle-specific proteins and, thus wereregarded as nascent myofibrils. The distribution pattern of these fibrillar
structures was generally in correlation with the shape of the cells. They
were aligned parallel to the long axis of the cells within their straight parts.
In polygonal areas, they were prominent as spanning cellular diagonals but

RESEARCH ACTIVITIES IN 1987

79

some were also observed along perimeters. Both ends of the nascent
myofibrils terminated at areas where vinculin and a-actinin, proteins thought
to be involved in the attachment of actin to the membrane, were concentrated. These areas overlapped with dark images seen with the interference
reflection. Fibronectin was located coincidentally in proximity to thesedark areas.
These results indicate that the formation of focal contacts and the accumulation of adhesion plaque proteins at these sites are prerequisite to or,at least, somehow related with the polymerization and regular alignment ofsarcomeric proteins. This suggests that the distribution of these protein
molecules must be polarized along lines that are generated between the sitesof focal cell contacts and, consequently, myofibrillogenesis occurs along
these inferred lines. Additionally, connectin and desmin do not appear toform the initial scaffold for sarcomeres.
Myogenesis in vitro as Seen with the ScanningElectron Microscope
Yutaka SHIMADA

We describe in this paper our recent observations by scanning electron
microscopy (SEM) on the differentiation of cell surface and cytoplasmic
organelles in embryonic chick skeletal muscle cells in vitro. The changes
in surface structures of myoblasts during mitosis were essentially similar to
those of other cell types, but the characteristic spindle shape of myoblasts
did not change throughout most of this period. Cytoskeletal structures
under the sarcolemma were examined by Triton extraction and metal coating. Cells in S, G 2 and M possessed a dense filament network, and thosein G l a loose one under the membrane. Myotubes possessed a dense
network under the sarcolemma. In the fusion area between a myoblast anda myotube, the cytoskeletal domain of the former could be distinguishedfrom the latter because of the mosaic appearance of the subsarcolemmal
cytoskeletal network. This network was composed predominantly of 1013 nm filaments; they were identified as actin filaments because of theirdecoration with myosin subfragment-l. Triton treatment and thiocarbohydrazide-osmium staining allowed us to visualize myofibrils. They ran in the
direction of inferred stress lines brought about by elongation and adhesionof the cells to the substrate. Intracellular membranous organelles could

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

be seen by the freeze-polishing and osmium-maceration procedure. Mitochondria exhibited complex irregular branchings with T system tubules
running a tortuous course. Sarcoplasmic reticula with occasional dilatations were found to be connected to each other. These results are of sufficient promise to encourage more extensive analysis of myogenesis by SEM.

RESEARCH ACTIVITIES IN 1987

v.

81

CYTOGENETICS

Expression of Ribosomal RNA Gene Clusters in Laboratory
Strains of Mice Demonstrated by Cytogenetic Techniques
Yasuyuki KURIHARA, Dong Sang SUB, Hitoshi SUZUKI'"
and Kazuo MORIWAKI

Several cytogenetic techniques can be applied to detect ribosomal RNA
gene (rDNA) clusters on chromosome preparations. The most commonlyused method is Ag-NOR staining which is specific for transcriptionallyactive rDNA clusters. Chromosome location of rDNA clusters can bealso demonstrated directly by in situ hybridization, irrespective of gene expression. By combining these two techniques, therefore, it is possible todistinguish transcriptionally active rDNA from inactive ones. In the present
study, we examined chromosome locations and the expression of rDNA
clusters in twelve laboratory strains of mice.
For chromosome preparations, short term cultures of spleen cells wereused. After G-banding for chromosome identification, each preparation
Table 1. Chromosome locations of ribosome RNA gene clusters demonstrated
by Ag-NOR staining and in situ hybridization
Ag-NOR staining

in situ hybridization

StrainAKR/J
BALB/cAnN
BALB/cHeA
BALB/cJBALB/cUCSD
C57BL/lOSnJ
C57BL/6J
CBA/CaHN
CBA/StMs
SM/J
SWR/J
129/J

12

15

+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+
+

'" Jikei Medical School.

+

16

+
+
+
+
+

18

+
+
+
+
+
+
+
+
+
+
+

19

12

15

16

18

19

+
+

+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+
+
+
+
+

+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+

+
+
+

+
+
+

+

+
+
+
+
+

+
+ +
+ +
+ +

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

was subjected to either Ag-NOR staining or in situ hybridization. Results
are shown in Table 1. Chromosome locations of rDNA clusters are closer
to the centromere on chromosomes 12, 15, 16, 18, and 19. Moreover
seven out of twelve strains have one or two inactive rDNA clusters, inaddition to an active one. The same activity patterns observed in different
individuals of the same strains, suggest this is a fixed genetic character.
Tissue specificity of expression was also examined in bone marrow cells
and meiotic cells from CBA/J male mice, The rDNA clusters on chromosome 16 were not stained by Ag-NOR staining in all preparations. The
mechanism of inactivation for the rDNA clusters as a genetic trait remain
under study.

Chromosomal Polymorphism in the Ant Myrmecia (pilosula) n=l
Hirotami T. IMAI, Robert W. TAYLoR*, Ross H. CROZIER**,
Michael W. L. CROSLAND** and Graeme P. BROWNINO***

Ants of the species group, Myrmecia pilosula (Fr. Smith), are commonlyreferred to in Australia as 'Jack-jumpers', and are among the most primitiveof all living formicids. Their chromosome numbers were first reported
to include 2n=9, 10, 31-32, by Imai et al. (1977, Chromosoma, 59: 341393). At that time only one species was recognized in taxonomic literature.
More recently a 'pilosula' colony collected at Tidbinbilla near Canberra,
and reported by Crossland and Crozier (1986, Science 231: 1278), has
yielded the lowest chromosome number known among higher organisms(or indeed possible), namely n= I and 2n=2.
Encouraged by these findings we have established an international cooperative programme aimed at the karyological and taxonomic survey ofthe genus Myrmecia, especially, at this stage, of the M. pilosula group, thespecies now referred to collectively as Myrmecia (pilosuIa). Large-scale
field studies were carried out in 1985 and 1987, supported by the Overseas
Visitors Programme of the CSIRO Division of Entomology (Australia),
and by a Grant-in-Aid for Overseas Scientific Research from the Japanese
Ministry of Education. During these surveys more than 150 M. (pilosuIa)* Division of Entomology, C.S.I.R.O., Canberra.
** School of Biological Science, The University of New South Wales, Sydney.
*** Department of Zoology, University of Adelaide.

RESEARCH ACTIVITIES IN 1987

83

c

a
M M

M

ST A

ST SM

A A

ST

A

M

Fig. 1. Polymorphic karyotypes of Myrmecia (pilosula) n=1. a: 2n=2. b: 2n=3.
c: 2n=4. d: A chromosomal rearrangement (tandem fusion) involved in thispolymorphism. Darkly stained regions indicate C-banding. Arrow heads
indicate the presumed breakage points.

colonies were collected in New South Wales, the Australian Capital Territory, Victoria, South Australia and Tasmania. Species of the groupwere found to be karyologically highly differentiated. The range of chromosome numbers observed was 2n=2, 3, 4, 9, 10, 15, 17-32. Significant
but subtle morphological divergence has also been observed, often involving
characteristics previously accorded little taxonomic importance. This has
improved taxonomic acuity for revisionary studies on Myrmecia at large,
which probably includes over 200 Australian species (only about half ofwhich have scientific names), and one from New Caledonia.
It is now clear that the M. (pilosula) complex includes at least 5 separate,
similar biological species. Details will be published elsewhere. We provide here a brief summary of chromosomal observations on the species
currently denoted M. (pilosula) n=l, which was mentioned above.
M. (pilosula) n= 1 is distinguished from other members of its group by
its stocky form and details of cuticular sculpture and pilosity. It builds
relatively small nest mounds compared to most other species. A total of
11 colonies (with the code numbers indicated) have been collected from
Tidbinbilla, A.C.T. (HI87-151, 157), Canberra City, A.C.T. (HI87-165, 235),
the Mongarlowe/Charleyong district, N.S.W. (HI87-136, 148, 150, 151,
153, 154, 157) and South Warrandyte, Vic. (HI87-213).
We have found that this species is in fact chromosomally polymorphic,with 2n=2, 3 or 4. Five karyologically different colony series have been
discriminated, as follows: 2n=2 (HI87-151, 157); 2n=2/3 (HI87-148, 150,

84

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

153, 213); 2n=3/4 (HI87-136, 154); 2n=4 (HI87-l65); and 2n=2/3/4(HI87-235). In the last colony the three karyotypes formulated are 2K=
2M (Fig. la), 2K=lM+lST+lA (Fig. lb), and 2K=lSM+lST+2A(Fig. Ie) respectively. The C-banding patterns suggest a strong homology
between the long arm of the large M of 2n=2 cells and ST of 2n=4 cells,
and likewise between the short arm of M and A of the latter. This indicatesthat the large M chromosome probably originated secondarily following atandem fusion which occurred between the short arm terminal of ST and aproximal region of the long arm of chromosome A (Fig. 1d).
A Note on Trophic Egg-feeding by the Queen of the Primitive
Ant Myrmecia (pilosula) n=l
Keiichi MASUKO

The early history of social evolution in ants is obscure. This is due tothe lack of an adequate fossil record of possible wasp-like ancestors for the
fully-eusocial formicids, and because no living non-eusocial sister groupis known. Studies of the social behaviour of primitive species in genera
such as Amblyopone and Myrmecia can, however, assist in the reconstruction of possible evolutionary events. The feeding behaviour of the queen
is a key attribute for such an approach. It is well known that Myrmecia
workers lay non-reproductive eggs which are passed as food to the colony
queen, and to larvae and other workers. These are called 'trophic eggs'.
By observing a mature laboratory colony of Myrmecia (pilosula) n=l
I have sought to understand how the queen obtains trophic eggs fromdonor workers, and how the eggs contribute to her nutrition. Despite thecontinuous accessibility of captured insect remains in the nest, the queen
was not observed to feed upon prey. She fed only on worker-laid trophic
eggs, which were consumed at the rate of 1.0±O.9 (SD) per hour. Whensoliciting a trophic egg the queen always approached a potential donor
worker and stroked its body strongly with her antennae, after which theworker sometimes laid a trophic egg. This response by workers was notalways positive, because most moved away from the queen when solicited,or remained still and unresponsive until the queen ceased stroking andmoved away herself. Thus, the queen obtained eggs for only 7.5 % of allsolicitations. The queen was never observed to obtain eggs without soliciting. Oral regurgitative feeding between workers and the queen (which has

RESEARCH ACTIVITIES IN 1987

85

been noted in other Myrmecia species (R. W. Taylor pers. com.)) was notseen. The queens in mature colonies of M. (pilosula) n=l are thus apparently completely dependent upon worker-laid trophic eggs for theirnutrition.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

VI.

MUTAGENESIS AND RADIATION GENETICS

Inactivation of Transforming DNA with Gamma-rays at
Low Dose Rates
Y oshito SADAIE and Tsuneo KADAAs tritiated water inactivates bacterial transforming DNA more efficiently
at lower concentrations of the tritium atom, a similar effect was hypothesizedfor gamma-rays at lower dose rates. Transforming DNA from the Bacillus
subtilis wild type strain was dissolved in xO.l SSC (standard saline citrate)
at a concentration of 5 flgjml. The sample was irradiated with gamma-rays
at room temperature from 137CS (33kRjh-O.2KRjh) and 6 0 CO (40RjhO.2Rjh) and the remaining Arg+ transforming activity was examined withthe arginine requiring B. subtilis mutant strain. The LD 37 obtained varied
from lllR (33KRjh) to 300R (O.2Rjh), suggesting no dose rate effect for
DNA inactivation with gamma-rays.
Tissue-specific Radiation Sensitivity and Its Relation to
Differentiation in a Mouse Mutant, Wasted
Hideo TEZUKA and Tadashi INOUE

Tissue specificity on radiation sensitivity was investigated in the mousemutant, wasted. The two endpoints used were the induced frequency ofchromosomal aberrations examined 18 or 24 h after gamma-ray-irradiationand changes in survival of cultured cells.
Studies for the former consisted of in vivo- and in vitro-irradiation treatments: in vivo, bone marrow and spermatogonia; in vitro, erythropoietinstimulated bone marrow cells and fibroblasts in culture. Radiation sensitivity was observed in bone marrow cells in both in vivo and in vitro cases.
No differences in sensitivity were shown in the other cases between wasted
and littermate control mice. Studies for the latter used bone marrow cells
in culture, and revealed that differentiated erythropoietic stem cell, CFU-E,
was sensitive, while the granulopoietic stem cell, CFU-C, was not sensitive.
These results suggest that the effect of the wst gene is tissue- and evencell-specific in the wasted mouse and that radiation sensitivity in the bonemarrow may be based on changes in the erythropoietic cell lineage itself.

RESEARCH ACTIVITIES IN 1987

87

Mutagen-Modifying Effects of Vitamin A and Vitamin E
in Cultured Chinese Hamster Cells
Yukiaki KURODA

Most mutagens present in our environment are modified in their mutagenic
activity by other substances in their presence. In the previous report, it
was found that vitamin C and its derivatives reduced the cytotoxicity andmutagenicity of ethyl methanesulfonate (EMS) in Chinese hamster cells.
In the present study, vitamin E which is an antioxidant and has a stabilizing
effect on cell membrane, and vitamin A, which shows various physiologicalactivities in living organisms, were examined for their activity modifying
the mutagenicity of EMS.
Cells used were a clonal line of Chinese hamster V79 cells. Mutations
induced by EMS were detected by using the 6-thioguanine (6TG)-resistantmarker. Cells were treated with EMS alone or together with vitamins for
3 hours. The cytotoxicities and induced mutation frequencies were determined by the colony-forming activity of treated cells in normal medium
or in 6TG-containing medium. EMS alone had a cytotoxic effect on thecells, showing an LD 50 of 540 flg/ml. Vitamin E had a moderate cytotoxicity, with the LD 50 of 710 flg/ml. In the presence of vitamin E, thecytotoxicity of EMS increased, and showed a greater value than the sum ofcytotoxicities of both substances. Vitamin A had a strong cytotoxic effect,
showing an LD 50 of 302 flg/ml. In the presence of vitamin A at a concentration of 100 flg/ml, which had no cytotoxicity, the cytotoxicity of EMS
was slightly reduced.
EMS alone showed a strong activity in inducing 6TG-resistant mutations
in V79 cells. At a concentration of 1,000 flg/ml, EMS induced 6TGresistant mutations at a frequency of 88 X 10- 5 • Vitamin E alone had no
mutagenic effect on the cells. However, in the presence of vitamin E, the
EMS-induced mutations increased markedly, depending on concentrationsof vitamin E. Vitamin E at a concentration of 100 flg/ml enhanced thefrequency of EMS-induced mutations three times.
This enhancing effect of vitamin E was detected only when vitamin E
coexisted with EMS: a pretreatment with vitamin E had no effect on EMSinduced mutations. Since vitamin E wIth an antioxidant activity was not
antimutagenic, it suggests that the antimutagenicity of vitamin C may be
due to some mechanism other than antioxidant activity.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Vitamin A alone also had no mutagenic effect on cells at concentrations
up to 100 pg/ml. In the presence of vitamin A, the EMS-induced mutations reduced to 1/2-2/3, depending on the dosage of the vitamin A (200600 pg/ml).
Mutagenicity of N4-Aminocytidine and Its Derivatives
in Chinese Hamster V79 Cells
Yukiaki KURODA, Akinori NOMURA*, Kazuo NEGISHl*
and Hikoya HAYATSU*

Most mutagens are DNA-attacking agents causing damage in the structure of the nucleic acid, leading to the production of mutated progenies
through the process of DNA replication. On the other hand, pyrimidine
and purine bases structurally different from DNA bases can be mutagenic
by incorporating into DNA and causing mutations due to their erroneousbase-pairing properties. In the present study, DNA-precursor type mutagens, N4-aminocytidine and its derivatives were tested for their mutagenicity
in cultured Chinese hamster V79 cells.
N4-aminocytidine and N'-methy-N4-aminocytidine showed a strong mutagenicity in inducing 6-thioguanine (6TG)-resistant mutations. N' -(2-hydroxyethyl)-N4-aminocytidine, which had an alkyl group on the N'-nitrogenof the hydrazino group at position 4 of N4-aminocytidine also had a strong
mutagenic activity. On the other hand, N4-aminocytidine derivatives withan alkyl group on the N4-nitrogen and N4-aminodeoxycitidine were notmutagenic for V79 cells.
To investigate the mechanism of the strong mutagenic activity of N4_
aminocytidine, the incorporation of H3-labeled N4-aminocytidine into DNA
bases was examined. As a result of this experiment, the H3-labled compound was incorporated into a N4-aminodeoxycitidine fraction. This suggests that N4-aminocytidine may be de-hydrozinated to form uridine, followed by metabolic conversion of the uridine into deoxycitidine triphosphate.
We previously found that N4-aminocytidine incorporated into DNA
produced base-pair transitions, i.e., TA to CG and CG to TA in phage andbacteria. It is likely that this type of mutation is also taking place in
V79 cells. For details, see Mutation Research 177: 283-287 (1987).
* Faculty of Pharmacentical Sciences, Okayama University, Okayama.

89

RESEARCH ACTIVITIES IN 1987

Major Genetic Loci Regulating Susceptibility to Pulmonary
Adenomas in CXB Recombinant Inbred Strains of Mice
Nobumoto MIYASHITA and Kazuo MORIWAKITo determine the major genetic loci regulating susceptibility to pulmonary
adenomas, CXB recombinant inbred (RI) strains of mice, A/Wyand their
Fl progenies received a single s.c. injection of 1.5 mg urethane/g bodyweight. These mice were sacrificed in the 5th month after treatment.
After the lungs were fixed in ethanol/formaldehyde (9: 1), the number of
adenoma foci was assessed. The progenitor strains, BALB/c and C57BL/6,
and 7 recombinants could be divided into two groups with respect to theTable 1. Urethane-induced pulmonary adenomas in CXB recombinant inbred,
A/Wyand their F1 hybrids
CXB strain

No. of tumors(mean±SEM)

Incidence:
No. of mice withtumors/total no.
of surviving mice

BALB/c
B6
CXBG
CXBH
CXBE
CXBJ
CXBD
CXBK
CXBI

6.76±0.81
0.53±0.17
18.17±2.07
to.29±1.60
9.33±1.02
7.80±0.88
1.30±0.35
0.86±0.34
0.50±0.29

21/21
7/17 (41 %)
6/ 6
7/ 7
15/15
15/15
7/10 (70%)
4/ 7 (57%)
2/ 4 (50%)

F1 hybrids
between A/Wy
and CXB RI
strains

No. of tumors(mean±SEM)

Incidence:
No. of mice withtumors/total no.
of surviving mice

A/WySnJ(A xBALB/c)F1(A xB6)F1(A x CXBG)F1(AxCXBH)F1(AxCXBE)F1(A x CXBJ)F1(AxCXBD)F1(AxCXBK)F1(AxCXBI)F1

27.15±1.0218.42±1.8518.71±0.81
24.60±1.24
17 .80±1.07
15. 30± 1. 75
16.64±1.73
24.86±1.08
18.40±1.4017.29±1.22

46/46
7/ 7
31/31
to/10
30/30
10/tO

11/11
14/14
5/ 5
17/17

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

average number of adenoma foci per mouse: one labeled susceptible, towhich strains BALB/c, CXBE, CXBG, CXBH and CXBJ belong, and theother resistant, which included strains CXBD, CXBK, CXBI and C57BL/6(Table 1). The average number of adenoma foci of the former group andthe latter one ranged from 6.76 (BALB/c) to 18.17 (CXBG) and from 0.50
(CXBI) to 1.30 (CXBD), respectively. The strain distribution pattern of
adenoma susceptibility were exactly the same as that of the locus at majorurinary protein-l (Mup-l) loci on chromosome 4 in CXB RI strains. Minor
differences in susceptibility were found in each group. The average number of adenoma foci in CXBG and CXBD is twice that of the other 4 susceptible and 3 resistant strains, respectively. In (A x B6)Fl and (A x
BALB/c)Fl, intermediate responses were observed. The number of adenoma foci in Fl hybrids between strain A and the CXB strains were alsointermediate except for (AxCXBG)Fl and (AxCXBD)Fl, which showedsusceptible phenotypes like A/Wy strains. From these results, it can beargued that one major gene responsible for adenoma susceptibility is locatednear Mup-l locus on chromosome 4. In addition, other minor genes werealso found to exist, but not located near Mup-l.
Genetic Analysis of the Reciprocal Translocation between
a Sex Chromosome (Y) and an Autosome (5)in the Silkworm
Akio MURAKAMI

Many translocation chromosomes have been detected, and research has
been carried out chromosome markers containing the visible character geneon sex chromosome (Y). Therefore, few approaches have been made to
study the mechanism of the production of translocation chromosomes, andthere has only been an analysis of a strain with reciprocal translocations of
the zebra gene on the Y chromosome and an autosome (3) (Hashimoto,1957). He reported that most of the visible character genes were attachedto the translocated chromosome.
We treated female pupae (oocytes) labeled by the Y translocation chromosome with ethyl nitrosoguanidine, and mated them with males with anautosome (5) labeled by pe and re egg color genes immediately after emergence. We detected mosaics with black egg color and yellowish whitecolor (pe), which can be used to estimate the mutation in the pe locus of the

RESEARCH ACTIVITIES IN 1987

91

egg. The mutant egg was subjected to the progeny test.
Males and females were found to derive from black and white eggs, respectively. The viability (hatchability) of the black eggs tended to besignificantly higher than that of the yellowish white eggs. The percentage
of zebra larvae was the same as that of plain larvae for both females andmales. A female (or male) zebra mutant with normal epidermis was matedwith a male (or female) labeled with the recessive pe, ok or re gene, and thecharacteristics of the next generation were analyzed. Females were derived
from black eggs and males from yellow eggs, respectively. The ok character
was observed in all male larvae, but not in any female larvae. Since no
red eggs appeared in the males or females, judging from the present mating
scheme, the fragment of the 5th autosome translocated on the Y chromosome
was found to contain the ok+ (4.7) locus as well as the pe+ (0.0) locus.
Male larvae in the yellowish white eggs of the present translocated strain
were crossed with females of the pe: re marker stock. About 25 % of theeggs (embryos) obtained by mating among the zebra larvae were dead.
Therefore, this strain was confirmed to be deficient in the autosome (5).
At the same time, the zebra part fragmented from the yze translocation
chromosome was found to be translocated to the deficient part. That is,in the present translocation chromosome induced by a chemical mutagen,we concluded that fragmentation occurred simultaneously in the Y chromosome and the autosome (5), and the fragments showed reciprocal translocation with mutual translocation of the fragmented part.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

VII.

POPULATION GENETICS

Molecular Evolutionary Clock and the Neutral Theory
Motoo KIMURA

From the standpoint of the neutral theory of molecular evolution, it isexpected that a universally valid and exact molecular evolutionary clock
would exist if, for a given molecule, the mutation rate for neutral alleles
per year were exactly equal among all organisms at all times. Any deviation from the equality of neutral mutation rate per year makes the molecular clock less exact. Such deviation may be due to two causes: one
is a change in mutation rate per year (such as due to change in generationspan), and the other is the alteration of the selective constraint for eachmolecule (due to change in the internal molecular environment). A statistical method was developed to investigate the equality of evolutionary
rates among lineages. This was used to analyze protein data to demonstrate that these two causes are actually at work in molecular evolution.
It was emphasized that departures from exact clockwise progression ofmolecular evolution by no means invalidates the neutral theory. It waspointed out that experimental studies should be undertaken to settle theissue of whether the mutation rate for nucleotide change is more constant per
year or per generation among organisms whose generation spans are very
different. For details, see J. Mol. Evol. 26: 24-33 (1987).
A Stochastic Model of Compensatory Neutral Evolution
Motoo KIMURA

Using the diffusion equation method, I investigated the average time
until fixation of a mutant gene or genes in a finite population under continued (irreversible) mutation pressure. I mainly considered the situationin which the initial population consists exclusively of the wild type allele(or alleles). I denote by v the mutation rate per locus per generation.
The treatment for the single locus case (with a pair of alleles A and A')
is straightforward. It shows that for a slightly deleterious mutant the
average time taken for fixation is too long to be of practical significance inRESEARCH ACTIVITIES IN 1987

93

evolution unless 4Nes' < 10, where N e is the effective population size ands' is the selection coefficient against the mutant allele (A').
The main aim of this paper is to investigate the two-locus cases. I assumed a pair of alleles A and A' at the first locus, and Band B' at the second
locus, and investigated the situation in which mutations are individually
deleterious but become harmless (i.e., selectively neutral) in combination.
Such mutations may be termed 'compensatory neutral mutations.' I
assigned relative fitnesses 1, l-s', l-s' and 1 respectively to AB, A'B, AB'
and A'B'. Two extreme cases, i.e., (i) free recombination, and, (ii) complete
linkage between the loci were considered. Assuming 2Ne v= 1, where v isthe mutation rate per locus, the following results were obtained. (i) In thecase of free recombination, the average time until fixation (f') was about
5Ne generations for netitral mutations (s' =0). For slightly deleterious
mutations in the range <4NeS' ~ 10, l' was slightly shorter but not verymuch (e.g. 1'=4.5Ne for 4Nes' = 10). If 4Nes' was much larger, the fixation
time quickly became very large, and for mutations with 4Ne s'>40, l' became so enormously large that such mutations are unlikely to play a partin evolution. (ii) In the case of complete linkage, single mutations withmuch larger deleterious effects are allowed to participate in "compensatory
neutral evolution," namely, joint fixation of the selectively neutral double
mutant (A'B') occurs without having to wait an unrealistically long time.
In fact, even for 4Ne s' =400, the average fixation time was only 10 times aslong as the completely neutral case. The bearing of this finding on molecular evolution was discussed with special reference to coupled substitutionsat interacting amino acid (or nucleotide) sites within a folded protein (orRNA) molecule. It was concluded that compensatory neutral mutations
may play an important role in molecular evolution. For details, see in
Lecture Notes in Biomathematics Vol. 70, pp. 2-18 (1987).

°

Very Slightly Deleterious Mutations and the Molecular Clock
Tomoko OHTA

A model for very slightly deleterious mutations was examined from thestandpoint of population genetics in relation to the molecular evolutionary
clock. The distribution of selection coefficients of mutants (in terms ofamino acid changes) with little effect is thought to be continuous aroundzero, with an average negative value. The variance in selection coefficients

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

depends upon environmental diversity and hence on total population sizeof a species. By considering various examples of amino acid substitutions,the average and standard error of selection coefficients and the reciprocalof population size are assumed to have similar values. The model predicts
a negative correlation between evolutionary rate and population size. Thiseffect is expected to be partially cancelled with the generation time effectin intrinsic mutation rate. Implications of this prediction on the molecular evolutionary clock were discussed. For details, see J. Mol. Eval.
26: 1-6 (1987).
A Model of Evolution for Accumulating Genetic Information
Tomoko OHTA

By taking into account recent knowledge of multigene families and otherrepetitive DNA sequences, a model for evolution by gene duplication foraccumulating genetic information was studied. Genetic information isdefined as the sum of distinct functions that the gene family can perform.
A coefficient, "genetic diversity" defined and used in this study, was highly
correlated with genetic information. Initially, a multigene family with a
few gene copies was assumed, and natural selection started to work on thisgene family to increase genetic diversity contained in the gene family. As
an important mechanism, unequal crossing-over was incorporated. Together with mutation, it was responsible for supplying genetic variability
among individuals for selection to work. A specific model, in which individuals with less genetic diversity are selectively disadvantageous, hasbeen studied in detail. Through approximate theoretical analysis andextensive Monte Carlo studies, it has been shown that the system is anextremely efficient way to accumulate genetic information. For attaining
one gene, the genetic load is much smaller under this model than under the
traditional model of natural selection. The model may be applied to theprocess of origin of multigene families with diverse copy members such as
those of immunoglobulin or cytochrome P450. In general, the process ofcreating new genes by duplication might be somewhere between the present
and the traditional models. For details, see J. Theor. Bioi. 124: 199-211(1987).

RESEARCH ACTIVITIES IN 1987

95

Simulating Evolution by Gene Duplication
Tomoko OHTA

By considering the recent finding that unequal crossing-over and othermolecular interactions are contributing to the evolution of multigene families, a model of the origin of repetitive genes was studied using Monte
Carlo simulations. Starting from a single gene copy, how genetic systems
evolve was examined under unequal crossing-over, random drift and natural
selection. Both beneficial and deteriorating mutations were incorporated,
and the latter were assumed to occur ten times more frequently than theformer. Positive natural selection favors those chromosomes with more
beneficial mutations in redundant copies than others in the population, but
accumulation of deteriorating mutations (pseudogenes) have no effect on
fitness so long as there remains a functional gene. These results imply thefollowing: (I) Positive natural selection was needed in order to acquire
gene families with new functions. Without it, too many pseudogenes accumulated before attaining a functional gene family. (2) There was alarge fluctuation in the outcome even if parameters were the same. (3)When unequal crossing-over occurred more frequently, the system evolved
more rapidly. It was also shown, under realistic values of parameters, thatthe genetic load for acquiring a new gene is not as large as J. B. S. Haldane
suggested, but not so small as in a model in which a system for selection
started from already redundant genes. For details, see Genetics 115:207-213.

On the Overdispersed Molecular ClockNaoyuki TAKAHATA

Rates of molecular evolution at some loci are more irregular than described by simple Poisson processes. Three situations under which molecular evolution would not follow simple Poisson processes were reevaluatedfrom the viewpoint of the neutrality hypothesis: (i) concomitant or multiple
substitutions in a gene, (ii) fluctuating substitution rates in time caused bycoupled effects of deleterious mutations and bottlenecks, and (iii) changes
in the degree of selective constraints against a gene (neutral space) causedby successive substitutions. The common underlying assumption thatthese causes are lineage nonspecific excludes the case where mutation rates

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

themselves change systematically among lineages or taxonomic groups, andseverely limits the extent of variation in the number of substitutions among
lineages. Even under this stringent condition, however, the third hypothesis, the fluctuating neutral space model, can generate fairly large variations. This was described by a time-dependent renewal process, which did
not exhibit any episodic nature of molecular evolution. It was argued thatthe observed elevated variances in the number of nucleotide or amino acidsubstitutions do not immediately call for positive Darwinian selection inmolecular evolution. For details, see Genetics 116: 169-179, 1987.
Adult Lactose Absorption and Milk Use from theStandpoint of Gene-culture Theory
Kenichi AOKITwo major hypotheses proposed to explain the observed association between prevalence of adult lactose absorption and milk use in human populations are the "culture historical hypothesis" and the "calcium absorption
hypothesis". A rigorous formulation of the evolutionary problem calls
for an approach based on gene-culture theory. This work selectively reviewed data bearing on the coevolutionary approach. First, a reinterpretation of the data on Finnish school children suggested that lactose malabsorption is fully expressed by age 12 rather than after age 15. This result wasconsistent with a one-locus two-allele determination of the trait, and alsosuggested that lactase is not an inducible enzyme. Second, a comparisonof milk preferences in lactose absorbers and malabsorbers suggested that adifference in preference may manifest itself when malabsorbers are in the
majority, as was presumably the case in ancestral human populations.
Third, calcium absorption in the presence of lactose is more efficient inlactose absorbers than malabsorbers. This result supported the contention that malabsorbers were more susceptible to bone diseases and hence
at a selective disadvantage in northern Europe. Fourth, a simple modelof gene-culture coevolution was presented to show how the reviewed data
pertain. The time required for genetic change was computed as a functionof the intensity of selection. Strong selection pressures must be invoked
if the genetic change is to have occurred by natural selection during the6000 years since adoption of milk use. Finally, the incompleteness of theassociation between prevalence of adult lactose absorption and milk use

RESEARCH ACTIVITIES IN 1987

among the populations surveyed was discussed.
Genet. 62: 445-459.

97

For details, see Jpn. J.

Toward a Theory for the Evolution of Cultural Communication:
Coevolution of Signal Transmission and Reception
Kenichi AOKI and Marcus W. FELDMAN

A haploid sexual two-locus model of gene-culture coevolution was examined, in which a dichotomous phenotype subject to natural selection istransmitted vertically with probabilities dependent on the chosen parent's
genotype and phenotype and the offspring's genotype. Stability conditions
for the genetically monomorphic corner equilibria were obtained. In a
specialization of this general model, one locus controls the transmission
and the other controls the reception of adaptive information. The cornerand edge equilibria of this doubly coevolutionary model were fully analyzed,
and conditions for transmission and reception to coevolve were derived interms of the efficiency of vertical transmission, the selective advantage
gained from possessing the information, the costs of transmission andreception, and the recombination fraction between the two loci. Possible
applications of the model are to the evolution of semantic alarm calls invervet monkeys and the phonetic aspects of human language. In a thirdmodel with diploid genetics, we considered the initial increase in culturaltransmission from a mutation-selection balance in which the adaptive
phenotype is the consequence of a dominant gene at one locus. A second
gene controls the transmission of the phenotype in such a way that a new
mutant at this second locus permits learning of the adaptive phenotype froma parent who has it. This new mutant cannot increase when rare. For
details, see Proc. Nat!. A cad. Sci. USA 84: 7164-7168.
Gene-culture Waves of Advance
Kenichi AOKI

Major genetic and cultural changes may have been coupled duringhominid evolution. Since hominids have had a wide geographical distribution for about one million years, any mutant gene or cultural innovation that became established had to spread from its origin. A pair ofnonlinear diffusion equations was derived which models the propagation

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

of a mutant gene and a cultural innovation. Both were assumed to originate
in the same locality along a linear habitat. The mutant gene and its allele
are semidominant, and the two cultural choices are transmitted according to what I call the logistic attraction-repulsion model. The genes
influence cultural choice, and the two interact to determine fitness. Of
particular interest is the case in which mutant gene and cultural innovation
are mutually dependent, neither being able to spread without the other.
Each equation of the pair is similar in form to Fisher's equation, with alinear function of the other dependent variable replacing the constant coefficient in the reaction term. The partial differential equations were solved
numerically to obtain the asymptotic speeds. Their form also suggested
an heuristic argument which proved useful, but I was unable to obtain any
analytic results. The waves of the system were shown to be of two types,
synchronous and asynchronous. When genes and culture are mutuallydependent, synchronous traveling waves can exist. However, their existence is dependent on initial conditions, and the speed of propagation isslow. For details, see J. Math. BioI. 25: 453-464.

RESEARCH ACTIVITIES IN 1987

VIII.

99

EVOLUTIONARY GENETICS

Molecular Evolutionary Rates of Oncogenes
Takashi GOJOBORI and Shozo YOKOYAMA

Using nine sets of viral and cellular oncogenes, the rates of nucleotide
substitutions were computed using Gojobori and Yokoyama's (1985)
method. The results obtained confirmed our previous conclusion thatthe rates of nucleotide substitution for viral oncogenes are about a million
times higher than those for their cellular counterparts. For cellular oncogenes and most viral oncogenes, however, the rate of synonymous substitution is higher than that of nonsynonymous substitution. Moreover,the pattern of nucleotide substitutions for viral oncogenes is more similar
to that for functional genes (such as cellular oncogenes) than for pseudogenes. This implies that nucleotide substitutions in viral oncogenes may
be functionally constrained. Thus, our observations of nucleotide substitutions for oncogenes in those DNA and RNA genomes are consistent
with Kimura's neutral theory of molecular evolution (Kimura 1968, 1983).
For details, see J. Mol. Evol. 26: 148-156, and Jpn. J. Genet. 62: 163-177.

Organization and Nucleotide Sequence of a Densovirus
Genome Imply a Host-Dependent
Evolution of the Parvoviruses
Hisanori BANDO, Jun KUSUDA, Takashi GOJOBORI
Takeo MARUYAMA and Shigemi KAWASE

The genome structure of a densovirus in silkworm was determined bysequencing more than 85 % of the complete genome DNA. The genome
organization of an insect parvovirus was deduced from the DNA sequence.
In the viral genome, two large open reading frames designated 1 and 2 andone smaller open reading frame designated 3 were identified. The first two
open reading frames shared the same strand, while the third was found in
the complementary sequence. Computer analysis suggested that open
reading frame 2 may encode all four structural proteins. The genome
organization and a part of the nucleotide sequence were conserved among

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

the insect densovirus, rodent parvoviruses, and a human dependovirus.
These viruses may have diverged from a common ancestor. For details,
see J. Virol. 61: 553-560.
Evolutionary Origin of Pathogenic Determinants inEnterotoxigenic Escherichia coli
and Vibrio cholerae 01
Tatsuo YAMAMOTO, Takashi GOJOBORI and Takeshi YOKOTA

Three families of the pathogenic determinants in enterotoxigenic
Escherichia coli and Vibrio cholerae 0 I are evolutionarily related to eachother. These three families are (1) a family of cholera enterotoxin (CT)and heat-labile enterotoxin (LT) including CT, LTh, and LTp, (2) a familyof heat-stable enterotoxin I (STI) including STIa and STIb, and (3) a familyof K88 enteroadhesion fimbriae including K88ab, K88ac, and K88ad.
They were analyzed for synonymous (silent) nucleotide substitutions, using
gene nucleotide sequences of earlier reports and a newly determined LTp
gene nucleotide sequence. The results obtained suggest that the divergences
between LT and CT and between STIa and STIb occurred in the remote
past, whereas those between LTh and LTp and between members of theK88 family occurred very recently. We therefore concluded that the LT
gene is a foreign gene that has been acquired by E. coli to form an enteropathogen. This provides evolutionary evidence of species-to-species
transfer of pathogenic determinants in prokaryotes. For details, see J.
Bact. 169: 1352-1357.
Molecular Evolution and Phylogeny of the Human AIDS
Viruses LAV, HTLV-ID, and ARV
Shozo YOKOYAMA and Takashi GOJOBORI

A phylogenetic tree for human lymphadenopathy-associated virus (LAV),
human T-cell lymphotrophic virus type III (HTLV-III), and acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV) has been
constructed from comparisons of the amino acid sequences of their gag
proteins. A method was proposed for estimating the divergence times
among these AIDS viruses and the rates of nucleotide substitution for their
RNA genomes. Analyses indicate that the LAV and HTLV-III strains

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101

diverged from one another after 1977 and that their common ancestor
diverged from the ARV virus no more than 10 years earlier. Hence, theevolutionary diversity among strains of AIDS viruses apparently has been
generated within the last 20 years. It is estimated that the genome of the
AIDS virus has a nucleotide substitution rate on the order of 10-3 per site
per year, with the rate in the second half of the genome being double thatin the first half. For details, see J. Mol. Evol. 24: 330--336.
Molecular Phylogeny of the Human Immunodeficiency
and Related Retroviruses
Shozo YOKOYAMA, Etsuko N. MORIYAMA and Takashi GOJOBORI

Human immunodeficiency virus (HIV) is the aetiological agent of acquired
immune deficiency syndrome (AIDS). It is known that HIVs are moreclosely related to lentiviruses than to oncoviruses (Sonigo et al. 1985; Chiu
et al. 1985; Gonda et al. 1985). In these phylogenetic analyses, heterogeneity of HIV strains has been neglected. We constructed a phylogenetic
tree of different HIV strains, lentiviruses, and oncoviruses, by comparing
amino acid sequences for the reverse transcriptase (RTase) and endonuclease/integrase (ENase) domains. This phylogenetic tree shows that among the
HIV strains, BH5, BRIO, pv. 22 isolated in New York and LAV BRu in
France, diverged most recently. Their common ancestor diverged from
ARV2 in San Francisco somewhat earlier, and LAVMAL and LAVELl in
Africa are the most distantly related. It also confirmed that HIVs are
related to the lentiviruses more closely than to oncoviruses. Using therate of amino acid substitution for the gag protein, we also estimated thedivergence times among the HIVs. For details, see Jpn. A cad. Sci. 63(B):147-150.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Six Chloroplast Genes (ndhA-F) Homologous to Human
Mitochondrial Genes Encoding Components of the
Respiratory Chain NADH Dehydrogenase are
Actively Expressed: Determination of the
Splice Sites in ndhA and ndhB pre-mRNAs
Tohru MATSUBAYASHI, Tatsuya WAKASUGI, Kazuo SHINOZAKI
Kazuko YAMAGUCHI-SHINOZAKI, Norihiro ZAITA
Tadashi HIDAKA, Bing Yuan MENG, Chikara OHTO
Minoru TANAKA, Akira KATO, Takeo MARUYAMA
and Masahiro SUGIURA

Sequences (ndhA-F) homologous to human mitochondrial genes forcomponents of the respiratory chain NADH dehydrogenase have beenfound in the chloroplast DNA of tobacco. The ndhA, D, E and F sequences corresponding to mitochondrial URFl, 4, 4L and 5 are located ina small single copy region, the ndhB sequence corresponding to URF2 in
the inverted repeat and the ndhC sequence corresponding to URF3 in thelarge single copy region of the chloroplast DNA. Northern blot hybridization revealed that all six ndh sequences are actively expressed in the chloroplasts. The ndhA and ndhB sequences contain single introns and thesplice sites of their pre-mRNA were determined by reverse transcription
analysis. These findings suggest that potential components of an NADH
dehydrogenase are synthesized in the chloroplasts. For details, see Mol.
Gen. Genet. 210: 385-393.
Male-driven Molecular Evolution Demonstrated by Different
Rates of Silent Substitutions between Autosomeand Sex Chromosome-linked Genes
Takashi MIYATA*, Hidenori HAYASHIDA, Kei-ichi KUMA*
and Teruo YASUNAGA**

It is generally believed that the number of cell divisions differs between
sperms and eggs (Winter, R. M. et al. (1983) Hum. Genet., 64, 156-159).
This factor is important in relation to the rate of molecular evolution,
because errors in DNA replication and repair are probably the major
source of mutations contributing to molecular evolution (Wilson, A. C.
***

Department of Biology, Faculty of science, Kyushu University.
The Institute of Physical and Chemical Research.

RESEARCH ACTIVITIES IN 1987

103

et al. (1977) Ann. Rev. Biochem., 46, 573-639, Britten, R. J. (1986) Science,
231, 1393-1398), as suggested by rapid evolutionary rates of viral-coded
genes and mammalian mitochondrial genes (Hayashida, H. et al. (1985)
Mol. Bioi. Evol., 16, 23-36). We hypothesized the different numbers ofgerm-cell divisions between males and females result in different mutation
frequencies between autosome and sex choromosomes, if replication errors
are the major source of mutations.
Nucleotide sequence analyses for the highly divergent portions of autosome-, X- and Y-linked genes revealed a strong correlation between theobserved rates of evolution and mutation frequencies expected from themodel, when the male to female ratio of the number of germ-cell divisions
is very large. These results suggest that males serve as a mutation generator
in molecular evolution. For details, see Proc. Japan Acad., 63, 327-331.

Nucleotide Sequence of Immunoglobulin Epsilon Genesof Chimpanzee and Orangutan: DNA Molecular
Clock and Hominoid Evolution
Yasuhiko SAKOYAMA*, Kyong-Ja HONG*, Si Myung BYUN*
Hiroshi HISAJIMA*, Shintaroh UEDA**, Yoshio YAOITA***
Hidenori HAYASHIDA, Takashi MIYATA****and Tasuku HONJO***To determine the phylogenetic relationships among hominoids and thedates of their divergence, the complete nucleotide sequences of the constantregion of the immunoglobulin epsilon-chain (Cel ) genes from chimpanzee
and orangutan were determined. These sequences were compared withthe human epsilon-chain constant region sequence. A molecular clock(silent molecular clock), measured by the degree of sequence divergence atthe synonymous (silent) positions of protein-encoding regions, was introduced for the present study. From the comparison of nucleotide sequences of alphal-antitrypsin and beta- and delta-globin genes between
humans and Old WorId monkeys, the silent molecular clock was calibrated:the mean evolutionary rate of silent substitution was determined to be
*
**
**.
••••

Department of Genetics, Osaka University School of Medicine.
Department of Anthoropology, Faculty of Science, The University of Tokyo.
Department of Medical Chemistry, Kyoto University Faculty of Medicine.
Department of Biology, Faculty of Science, Kyushu University.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

1.56 X 10- 9 substitutions per site per year. Using the silent molecular
clock, the mean divergence dates of chimpanzee and orangutan from thehuman lineage were estimated as 6.4±2.6 million years and 17.3±2.6
million years, respectively. It was also shown that the evolutionary rateof primate genes is considerably slower than those of other mammaliangenes. For details, see Proc. Natl. Acad. Sci. USA 84, 1080-1084.
Global Variation in G+C Content along Genome DNA
Sequences with Reference to Chromosome Banding
Structures of Vertebrate Genomes
Toshimichi IKEMURA and Shin-ichi AOTA

The global, rather than local, variation in the G+C content along thenuclear DNA sequences of various organisms was studied using GenBank
Sequence Data. When genome portions of E. coli or of the yeast S.
cerevisiae which had been sequenced for a long distance were examined,
the levels of their G+C percentages were found to be within a narrow range
around the overall genome G+C %. The G+C % levels of vertebrates,
however, were found to lie over a wide range, showing that their genomes
are mosaics of sequences with different G+C % levels, in which each of thesequences is fairly homogeneous in its G+C % for a very long distance.
Through surveying the Human Genetic Map (HGM8, 1985; McKusic, 1986)
and GenBank DNA sequences, we were able to connect the global variation in G+ C % along the human genome at the sequence levels to thechromosomal G (Giemsa) and R (Reverse) band structures. For details,
see J. Mol. Bioi (in press).
Codon Usage Tabulated from the GenBank Genetic
Sequence Data
Shin-ichi AOTA, Takashi GOJOBORI, Fumie ISHIBASHI
Takeo MARUYAMA and Toshimichi IKEMURA

Codon usages in 3681 genes were analyzed using all nucleotide sequence
data obtained from the GenBank Genetic Sequence Data Bank (Release
50.0, 1987). This extensive survey of codon usage has recently been registered as a member of Molecular Biology Databases (LiMB Database by
Los Alamos National Laboratory of USA) being designated CUTG (CodonRESEARCH ACTIVITIES IN 1987

105

Usage Tabulation from the GenBank, by T. Ikemura).
To reveal the characteristics of the codon usage pattern for individualorganisms, the frequency of codons in each organism was calculated byadding codon uses in all genes of the organism. The resultant codonchoice patterns are very similar among mammals, although the codonchoices (e.g. G+C % at the codon third letter) in individual genes of one
mammal are often very different from each other. The codon-choicepatterns, that are roughly common among mammals, do not depend on
the choice of genes added; i.e., when the codon frequencies for ten or moregenes with varying functions are summed up for each mammal, they usuallyconverge into a very similar pattern regardless of the genes added. This
converged pattern seems to relate both with the dinucleotide frequency of
mammalian genomes and with their cellular tRNA content, although
the pattern is clearly modulated by the segmental G+C % distribution along
the genomes (J. Mol. BioI., in press, 1988). See details, Nucl. Acids Res.
16, Supplement, r315-r402.
Serological Survey of Mouse Complement Factor H in
Common Laboratory and Wild-derived Strains;
A New Allotype H.3
Yoshi-nobu HARADA, Kazuo MORIWAKI and Takeshi TOMITA*

Antigenic polymorphism of a serum protein migrating electrophoretically in the p region of mice has been reported as SAS-2 (serum
antigenic substance-2) (Harada et al. J. Immunogenet. 14: 33-41, 1987).
By using serological analyses, SAS-2 was identified with complement factor
H, which is a plasma glycoprotein of p-mobility functioning as a cofactor
for the conversion of C3b to iC3b by serine protease 1. Two allotypes ofthe factor H, H.l and H.2, have been previously reported (NatsuumeSakai et al. J. Immunol. 134: 2600--2606, 1985). In the present study, a new
allotype of complement factor H, H.3, was identified in BFM/2Ms and
BFM strains derived from European wild mice. Using three different alloantisera for each mouse factor H allotype, a serological survey was carriedout on common laboratory strains and wild-derived strains from Mus
musculus and its relatives, Mus spretus, M. spretoides, and M. spicilegus.
All the common laboratory strains examined in this survey had H.l except*

Faculty of Agriculture, Nagoya University.

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38
Table 1. Strain distribution patterns of factor H allotypes among common
laboratory strains
Allotypes offactor H

Strains

H.I

A/J, AKR/J, BALB/c, BALB/cAnN, BALB/c-Aph-2 b CBA/J,
CE/J, CFW, C3H/HeJ, C57BL/6J, C57BL/IOJ, C57BR, C57L,
C58/J, DBA/I, DBA/2, DM, HRS/J, HTH, HTI, HTG, lITES,
ITES, IXBL, KK, KR, KSB, LP/J, MA/MyJ, NC, NZB, P/J,
PL/J, SM/J, SJL/J, SM/J, SWR/J, TF/GnLe, WB/ReJ, WHT,
WN,129/J
STR/N, BALB/c-H.2
not observed

H.2
H.3

for STR/N which had H.2 (Table 1). Geographical distributions of factor H
allotypes in M. musculus were specific to the subspecies. Mice from M. m.
domesticus and M. m. castaneus had the H.l allotype. Mice from M. m.
musculus, M. m. bactrianus, and M. m. molossinus had H.2. Only the
BFM/2Ms and BFM strains derived from M. m. domesticus had H.3. Seraof strains derived from M. spretoides and M. spicilegus cross-reacted with
anti-H.2 serum and those from M. spretus cross-reacted with anti-H.3
serum. A possibility was suggested that the gene for H.3 allotype in BFM/
2Ms and BFM strains was derived from M. spretus by gene introgression.
Genetic Characterization of the Mouse Strain with
Morphological Phenotypes Suggesting a Descendantfrom Ancient Oriental Stocks of Laboratory
Mice (Japanese Waltzing Mice)
Hiromichi YONEKAWA, Jorn T. NIELSEN, Yoshibumi MATSUSHIMA
Shigeharu WAKANA, Hideki KATOH, Nobumoto MIYASHIMA
and Kazuo MORIWAKI

By the use of diagnostic genetical markers to distinguish subspecies ofhouse mouse, Mus musculus, the origins of laboratory mice have beenshown to be intersubspecific hybrids between the European subspecies,
such as M. m. domesticus and an Asiatic subspecies such as "M. m.
molossinus" or the Asiatic race of M. m. musculus". This conclusion isbased on the following experimental evidence. Restriction analysis of
mitochondrial DNA (mtDNA) showed that most of the laboratory strains

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107

of mice have only one haplotype of mtDNA, suggesting that these strains
including the strains called "old inbreds", descended from (a) small ancestralcolony (ies) as (a) maternal founder(s). Restriction fragment length polymorphisms (RFLP) of nuclear genome using a DNA probe specific to the
Y-chromosome, showed that the RFLP of most strains examined was identical to that of M. m. musculus but not of M. m. domesticus, suggesting that
extensive introduction of paternal genes from the Asiatic subspecies hadtaken place during the establishment of recent laboratory mouse inbred
strains. One remaining question about the origin of the Asiatic mice.
whether the mice have a Japanese origin or a Chinese one.
Today there are many strains which show genetic characteristics very
close to "M. m. domesticus", whereas no strain has been thought to exist
which shows the characteristics being obviously descended from Asiaticmice. In the beginning of the 19th century, several investigators reportedon laboratory stocks showing a distinctly Asiatic origin and they werecommonly used for transplantation experiments for tumor cells. They
were however thought to have become extinct during the development ofresearch on laboratory mice, because there have been no reports on Asiaticmice since about 1940.
Two years ago, one of the authors, K.M., found a unique strain of laboratory mice, which had been bred by one of the authors, J.T.N., in the animalroom of the University of Aahrus. The coat color and other morphologicalcharacteristics are very similar to those of ancient oriental stocks of laboratory mice described by Little and Tyzzer (1916) and Gate (1926) and they
were called Japanese waltzing mice. If so, this strain would be importantfor following the history of laboratory mice. Thus we started to examine
their genetic characteristics.
The restriction phenotypes of mtDNA show that the haplotype is identicalto that of the Japanese wild mice "M. m. molossinus" but not those of any
Chinese mice. A biochemical survey with mouse major tear protein complex (Mtp) and major salivary protein complex (Msp) also showed thatboth protein complexes of this strain were specific to Asiatic mice and not
to any European mice, although this assay system could not distinguishbetween Japanese mice and other Asiatic mice. With a genetic survey ofother biochemical markers specific to each chromosome, we found that
this strain has Hbbp and Pgm-2b, which have hardly been found in laboratory mice. Other alleles examined were common among many other108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

strains of laboratorymice. These results strongly suggest that this mouseoriginated from Japanese mice but not from Chinese mice and that the strain
is descended from the ancient stocks Asiatic mice such as the Japanesewaltzing mice although this strain showed no waltzer phenotype.
Ribosomal DNA Spacer-length Polymorphisms in Rice
Yoshio SANO

Recent molecular studies have revealed that repetitive DNA families arewidespread in genomes of higher organisms. One gene family that has been
extensively studied in plants is the ribosomal gene family. The intergenic
spacer regions of ribosomal RNA show extensive sequence divergence
relative to the conserved coding regions. Both restriction-site variation
and, more commonly, length variation occur, suggesting that the intergenic
spacer regions in rDNA may evolve rapidly. Therefore, these intergenic
spacer regions might be useful for tracing population level phenomena.
We decided to study the evolution of this gene family in order to complement biosystematic studies of wild and cultivated rice. In rice, little is
known about the extent of genetic variability and genetic behavior of rDNA
repeats.
Spacer-length variation in rDNA was surveyed by Southern blotting
analysis. Among 140 strains of wild and cultivated rice, 27 spacer-length
phenotypes containing 13 different spacer-length variants have been detected
so far. Length heterogeneity was observed in the spacer within and among
species as well as within individuals. Asian cultivars and their wildprogenitor were highly polymorphic for rDNA repeats whereas African
cultivars and their wild progenitor were monomorphic. Ribosomal DNA
repeats were differentiated among species as well as among varietal groups
within Asian cultivars. However, all variants observed in cultivated species
were found in wild progenitors, indicating that no variant is specific tocultivars. This implies that rDNA spacer-length variants have remained
stable during the domestication process.
Rice Carries rDNA Repeats at Two Unlinked Loci
Yoshio SANO

Eukaryotic ribosomal DNA (rDNA) is organized into families of tam-

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109

demly repeated genes, some or all of which may comprise the nucleolarorganizer regions of chromosomes. Spacer-length heterogeneity within
an individual was often observed in rice, especially in Asian wild rice.
When heterogeneity was observed within the individual, these were twodifferent size classes of rDNA repeats in most cases. In addition, the intensity of hybridization suggests that the classes are of roughly equal abundance. The question arose whether the two repeat lengths represent heterogeneity within the chromosome or are located in different chromosomes,
each being homogenous for one class. In order to study the inheritance ofrDNA spacer-length variants, F 2 segregations were observed in two selectedcrosses. Results indicated that at least 4 variants behave as Mendelianfactors and rDNA repeats are located at two unlinked loci. Each variant
specifes an array of tandemly repeated units as codominant allele. If rice
carries rDNA repeats at two unlinked loci as shown here, it would appear
that Asian cultivars tend to carry the same class of rDNA repeats at the
two loci.
A Chromosomal Interchange Induced in a Near-isogenic
Line Carrying an Alien Sterility Gene
Yoshio SANa

To understand the genetic mechanisms controlling sterility barriers between rice species, alien genetic factors which disturb the normal development of gametes were introduced into the genetic background of a standard
strain by successive backcrossings, as previously reported (Ann. Rep. 33).
A near-isogenic line of Taichung 65 was established from BC sF 2 • No
other gene responsible for infertility was detected in BC7 F 2 and BCsFz.
The extracted sterility gene (designated S,) acted as a gamete eliminator in
the heterozygous condition (S,/S,a) and was tightly linked with wx. To
purify the S, gene, backcrosses were continued. A different type of sterility
factor was detected in BCgF,. The semi-sterile plants showed a normal
segregation for waxy and nonwaxy pollen grains, suggesting that any gamete
eliminator linked with wx no longer existed in the semi-sterile plants. The
semi-sterile plants always segregated into a ratio of I fertile to 1 semisterile. A series of test crosses showed that the semi-sterility might be
explained by the one-locus sporophytic interaction model. The model
assumes that sporophytic semi-sterility occurs only in the heterozygote

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38(Sjsa) and no gametic selection occurs. Of paticular interest is the originof the semi-sterility. It is apparent that a mutational event occured during
the BCg to BCij transition.
The segregation patterns observed in the case of the sporo-gametophytic
interaction model are also expected to occur in chromosomal interchanges
although the two original cultivated species show no such chromosomal
difference. If the semi-sterile plant showed no chromosomal abberation,
an interchange of small segments might explain the infertility and, if so,
the mutational event might involve a transposition of chromosomal segments. However, the semi-sterile plants showed a quardrivalent in thecourse of meiosis. This indicates that an interchange between non-homologous chromosomes occurred during the BCg to BCij transition.

RESEARCH ACTIVITIES IN 1987

IX.

111

HUMAN GENETICS

Onocogene Activation by Philadelphia Translocationsin Chronic Myeloid Leukemia and Acute
Lymphoblastic Leukemia
Takashi IMAMURA and Hitoshi NAKASHIMA

The Philadelphia (Ph) chromosome is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22 t(9; 22)(q34; ql I).
The breakpoints on chromosome 22 band q11 occur in a small region of
5.8 kb, the breakpoint cluster region or ber. Subsequent cloning experiments and DNA sequence analysis revealed that the ber is part of a large
gene, which has been called the ber gene. The ber contains four exons(bl-b4), and chromosomal breaks occur in two introns between exons b2,
b3, and b4. Breakpoints on chromosome 9 are scattered over a distanceof at least 100 kb, but are all located 5' of the tyrosine kinase domain of thee-abl proto-oncogene. As a result of the Ph translocation, the e-abl geneis transferred from its normal position on chromosome 9 band q34 to the
Ph chromosome. This event creates a head to tail ber-abl juxtaposition on
the Ph chromosome, with the bcr gene closer to the centromere.
The presence of the Ph chromosome was thought to be pathognomonic
for CML. However, patients with acute lymphoblastic leukemia (ALL)have a high percentage ofPh+ marrow cells. To date, it has been reported
that 25 %-30 % of adult and 2 %-10 % of childhood ALL are Ph positive.
It is surprising that Ph + ALL and CML share the same marker chromosome, while they are clinically different diseases. Either the two diseases
are different expressions of one underlying malignant process, or the marker
chromosome is not the same at the molecular level. A factor obscuring
the difference between Ph+ ALL and CML is the fact that following thechronic phase, 30 % of the CML patients eventually develop a lymphoid
blast crisis that closely resembles de novo Ph+ ALL.
We report here an studies on the underlying genetic mechanism in Ph +bcr- ALL. Chromosomal breaks still occur within the bcr gene, butthey take place in the putative first intron of the bcr gene, which is 5' of theber involved in CML. In this wayan alternative ber-e-abl fusion gene is112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

created, which is transcribed into a chimeric 7 kb ber-e-abl mRNA. Most
likely the RNA encodes a Ph+ ALL-specific pl90 ber-abl hybrid protein
since this product was shown to be present in another Ph+ber- ALLpatient with a chromosomal breakpoint in the first ber intron. Thesefindings establish the existence of two molecularly distinct Ph chromosomesin Ph+ ALL versus CML.
Malignancy is probably a multistage process involving a series of genetic
changes rather than a single mutational event. One such genetic change inthe pathogenesis of CML appears to be the acquisition of the Ph. The
enhanced expression of abl-related RNA in the K562 cell line is associatedwith amplification and rearrangement of the e-abl gene in these cells. To
determine if the similarly enhanced expression of e-abl in the blast crisis
cells was also associated with e-abl gene amplification or rearrangement,
the v-abl probe was hybridized to Southern blots of DNA from 2 blastcrisis cells, digested with restriction endonucleases. In contrast to K562,
intense hybridization of the v-abl probe to DNA from these two cells was
not noted, indicating that e-abl sequences were not amplified in these cells.
Thus, the increased c-abl-related RNA in these CML blast crisis cell lines
may arise through different pathways: in some cells, including K562, e-abl
gene amplification may be the major pathway, whereas in others the pathway
is unknown.
We and others have showed that in Ph+ber- ALL, breakpoints inchromosome 22 occur within the same ber gene, but at 5' more of the ber.
This resulted in deletion of most of the exons of the ber gene from thee-abl-related mRNA because of translocation of the ber exon regions tochromosome 9. Thus the deletion of the rearranged ber/e-abl sequencesat the 5' side on the CML blast crisis may result in the manifestation of amuch more aggressive leukemic state which differs from those usuallyobserved in chronic phase of CML.
In a series of DNA transfection analyses using the tumorigenicity assay,
we observed no tumor induction in nude mice in four of the CMLs investigated, which is in contrast to the previous report in that four of seven
AMLs and all three AML cell lines tested actually induced tumor formations. The analysis for the presence of mutated RAS genes by means ofhybridization of mutation-specific oligonucleotide probes to genomic DNA
has been considerably improved by including an in vitro amplification stepof RAS sequences. Investigation of 6 CML in chronic acute or phase andRESEARCH ACTIVITIES IN 1987

113

the K562 cells, all positive for Ph chromosome, gave no indication formutated RAS genes. These results are comparable with DNA transfection
studies that likewise detected activated RAS sequences in CML very infrequently. These results indicate that in Ph positive CML and ALL
there appers to be no case which contains activated RAS sequences.
Evolutionary Implications of Mitochondrial DNA
Polymorphism in Human Populations:Comparison of Two Different
Populations in Japan
Satoshi HORAI, Kenji HAYASAKA, Takashi GOJOBORI
and Ei MATSUNAGA

In a previous study (Horai and Matsunaga 1986), we analyzed 116 samples
of mtDNAs from Japanese living in Shizuoka Prefecture located in central
Japan. A total of 95 different morphs were detected with 9 enzymes(HaeIII, HinfI, Sau3AI, HhaI, RsaI, TaqI, HpaIl, AvaIl and AccIl), 60
of which had not been documented previously. Based on a comparisonof the cleavage maps of all individuals, 62 types of different combinationsof the morphs were observed. A phylogenetic analysis of the 62 types
indicated that they are separated into at least two distinct groups withdifferent frequencies: a group with the smaller frequency (group I; 18 %)which first diverged from the other group with the larger frequency (group
II; 82 %). The divergence time between the two groups was estimated to
be as early as 125,000 years ago, assuming a rate of mtDNA divergence as
2 X 10- 8 per site per year. However, this dating seems too early, if we
accept that racial divergence took place about 120,000 years ago, as estimated from gene frequency data for blood proteins (Nei and Roychoudhury1974). Therefore we intended to investigate whether or not similar clustering patterns could be observed in Okinawa, which is located about 1,300 km
from Shizuoka.
A total of 82 mtDNAs from the Okinawa people (Ryukyuan) wereanalyzed with the same nine enzymes as used previously. Sixty different
morphs were observed in total, 19 of which were unique to that population.
Comparisons of the frequencies of each morph for the Okinawa population with those for the mainland Japanese revealed significant differencesin the frequencies of 10 morphs between the two populations. In particular,

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

HinfI morph 31 was observed at a frequency of 35 % in Okinawa, while thefrequency of this morph was only 11 % on the mainland. Moreover, in the
RsaI digestions, 17 different morphs were observed on the mainland, whereas
only 6 morphs were detected in Okinawa. Furthermore, HaellI morph 2
was found at the frequency of 5 % in Okinawa, while this morph was observed at the frequency of 16 % on the mainland. These observations
therefore indicate that a considerable genetic difference exists between the
Okinawa and mainland populations. However, all individuals who possessed HaellI morph 2 in the Okinawa population shared five additionalvariant morphs with those for the mainland. These five morphs are HaellI
morph 35, HinfI morphs 27 and 28, RsaI morph 13 or 17, and TaqI morph
15. As will be discussed later, these individuals belong to group I in thephylogenetic tree constructed for mainland Japanese.
Based on a comparison of the cleavage maps among the Okinawa samples,
39 types of different combinations of the morphs were observed. It isremarkable that only 11 of them have been found among mainland Japanese
while the remaining 28 types were unique to the Okinawa population. The
combination of two variant morphs (HaeIII morph 3 and HinfI morph 31)
were scored in 17 individuals, though none of the mainland Japanese showed
this type. We then estimated the numbers of nucleotide substitutions (d)for each pair of these types using the method of Nei and Li (1979). The
mean value for d was 0.0016, taking into account the frequency of each type,which is lower than that (0.0026) for mainland Japanese.
A phylogenetic tree among the 39 types within the Ryukyuans was also
constructed by the unweighted pair group (UPG) method (Sokal and Sneath
1963; Nei 1975). The overall pattern of the tree was very similar to thatfor the mainland Japanese. The Okinawa population can also be separated
into two distinct groups which correspond exactly to groups I and II fromthe mainland. Moreover, the divergence time between the two groups in
the Okinawa tree was estimated to be about 125,000 years ago (Genetic
distance D=0.005), which was virtually the same as that obtained from themainland Japanese. However, group I included 4 types (4 individuals)
while the remaining 35 types (78 individuals) belonged to group II. The
divergence time of the earliest branch point for group I of the Okinawa
population was estimated to be about 95,000 years ago (D=0.004), whereas
all types in group II seem to have diverged only 75,000 years ago (D=0.003). The recent divergence of all types within group II apparently

RESEARCH ACTIVITIES IN 1987

115

contributes to the low mean value of d in the Okinawa population.
Phylogenetic Relationships among Japanese, Rhesus, Formosan,
and Crab-eating Monkeys Inferred from Restriction
Enzyme Analysis of Mitochondrial DNAs
Kenji HAYASAKA, Satoshi HORAI, Takashi GOJOBORI
Takayoshi SHOTAKE, Ken NOZAWA and Ei MATSUNAGA

About 20 species of macaques, genus Macaca, are widely distributed
throughout Asia as well as neighboring islands having adapted to diverse
environments. While they can be distinguished from each other by theirmorphological traits, some of them inhabit sympatrically and interbreed.
This implies that they have not speciated completely and are in the processof speciation. Therefore, genetic studies of macaques are of interest forunderstanding the processes of evolution.
We, thus, analyzed mitochondrial DNA (mtDNA) polymorphisms infour species of macaques, i.e., ten Japanese monkeys, Macaca fuscata, one
rhesus monkey, M. mulatta, one Formosan monkey, M. cyclopis, and one
crab-eating monkey, M. fascicularis to study phylogenetic relationships.
Using 17 restriction enzymes of six base pair recognition, 42 to 49 sites
were observed in the samples. The estimated number of nucleotide substitutions per site among Japanese, rhesus, and Formosan monkeys ranged
from 0.0318 to 0.0396 and that between crab-eating and the other monkeys
from 0.0577 to 0.0653. The phylogenetic trees constructed from thesevalues suggested the following relationships; The four types of Japanesemonkeys form a culster and the crab-eating monkey appears the most
distantly related to the others. The Formosan monkey diverged from thelineage leading to Japanese and rhesus monkeys before the latter two species
diverged. However, an exact branching order for these three species cannot
be conclusively determined by this analysis, because of the small differencesin estimated nucleotide diversity among these species and the considerable
sampling error due to the small number of recognition sites. If we assume
an average nucleotide substitution rate of (2-4) x 10- 8 per site per year
(Wilson et al. 1985), the Japanese, rhesus, and Formosan monkeys would
have diverged approximarely 0.9-1.8 million years (Myr) ago and thedivergence time between these three monkeys and the crab-eating monkey
would be 1.5-3.0 Myr ago.

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

The phylogeny suggested from our analysis is compatible with that suggested from fossil records in terms of topology but not for divergence times.
This discrepancy might result from the ambiguity of the estimation ofdivergence times from fossils. Alternatively, it is also possible that weoverestimated the divergence times since what we estimated was the divergence time for genes but not for species.
The analysis of blood protein polymorphism suggested that phylogenetic
relationships differ from those we found. This discrepancy might havebeen caused by a difference in mechanisms of inheritance between nuclear
DNA and mtDNA since mtDNA is inherited maternally unlike nuclear
DNA.
Female macaques stay in their native populations for their entire lives,
while males migrate between populations. In the past, there might havebeen a large extent of overlapping distribution. This is partly because the
Asian continent had been contiguous to its neighboring islands from time
to time. Moreover, hybrids between rhesus and crab-eating monkeys havebeen observed in nature. Interspecific hybrids of macaques have also been
observed in captivity and are known to be fertile. It is possible that bisexually inherited nuclear genomes might have been homogenized by the
migration of males while maternally inherited mtDNA might reflect thepast events of population splitting more accurately. For details, see Mol.
Bio/. Evol. 5(3), 270-281.

RESEARCH ACTIVITIES IN 1987

X.

117

APPLIED GENETICS

Alien Pollen Primacy (APP) in the Rice Plant
Yo-Ichiro SATO, Ling-Hua TANG* and Ryuji ISHIKAWA**

Mendel's law assumes that all pollen grains deposited on stigma have thesame ability in terms of fertilization. Segregation ratios at a marker
locus will be distorted from the expected ratio 3: I, when pollen grains
having a certain allele at the locus show either a higher or lower ability for
fertilization than ones having different alleles. Until recently there havebeen three recognized cases when Mendel's assumption would be at
fault; 1) differential germination rates or elongation speeds of pollen tubes
are brought about by particular certation genes or various environments,
Alien Pollen Primacy (APP) was recently discovered as the fourth case inwhich Mendel's law could not be adopted.
APP was found in a mixed pollination experiment in rice conducted in
the summer of 1987. Four kinds crosses using two varieties, A and B, i.e.,Ax(A+B), Ax(B+A), Bx(A+B) and Bx(B+A) were made and alloffspring were grown in the winter season of 1987-1988. A+B denotes A
pollen being deposited five minutes earlier than B pollen on the same stigma.
The amount of A pollen and that of B pollen were approximately the same.
In each cross, progeny fertilized by A pollen or B pollen were estimatedusing particular gene markers. In the first cross, the number of offspring
fertilized by A and B pollen was approximately equal, even though A
pollen was deposited prior to B. In the second cross, offspring fertilized
by B were three times more frequent than those of A. These two crosses
indicated B pollen showed an advantage over A pollen for fertilization.
The latter two crosses in which B was used as the female parent, however,showed a strong advantage for A pollen. The data obviously indicatesthat between A and B, plants tended to accept alien pollen more frequently
than their own pollen. This feature of APP seems to be common to selfincompatibility by means of an advantage for alien pollen. In caseof self-incompatibility, own pollen cannot be accepted, and selfpollination results in failure. In common rice, all varieties are self-com* Jiangsu Academy of Agric. Science, Nanjing, China.
** Hokkaido Univ., Present address: Hirosaki Univ.

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

patible, and therefore, APP should be distingushed from self-incompatibility.

The Ph-Aph Association in Rice, and its Significance in
Indica-Japonica Differentiation
Yo-Ichiro SATO and Hiroko MORISHIMA

A complicated association between genes and characteristics was observed among native cultivars as well as in hybrid populations raised by
the single-seed-descent method (SSD) from an Indica x Japonica cross incommon rice. SSD is a breeding method that minimizes zygotic selection.
Many associations found among native cultivars disappeared in the F 2
generation, indicating that they were shaped through selection explored bycultivation under various environments. Association observed in F 2 were
due to pleiotropy or linkage of the genes concerned. Several associations
which were observed in native cultivars but not in F 2 , e.g., that between
positive phenol reaction (Ph) and short apiculus hair length (aph) (Ph-aph
association), were detected in F 5 raised by SSD. Ph and Aph loci arelocated on different chromosomes. Thus the Ph-aph association can not
be caused by zygotic selection nor linkage.
It was suggested that a particular gametophyic selection or certation
brought about this association.

Intra-Populational Genetic Diversity Found in Indigenous
Rice Cultivars of Yunnan
Hiroko MORISHIMA

Two population samples of primitive rice cultivars were obtained froma farmer's seed stock in Yunnan, China. One is an upland population and
one a lowland population. Variation and covariation within populations
were examined regarding several agronomic characteristics supposedly
related to adaptability to water conditions, as well as diagnostic characteristics and isozymes which are useful for the classification of IndicaJaponica types representing the two major varietal groups of Asian common
rice.
The lowland population was relatively uniform, consisting of Indica plants

RESEARCH ACTIVITIES IN 1987

119

with lowland adaptability. In contrast, the upland population showed alarge amount of variability ranging from Indica to Japonica types, andcontained, in addition to upland-type plants, some lowland-type plants.
The average gene diversities computed from 13 polymorphic loci were0.099 and 0.153 for lowland and upland populations, respectively.
In each populatIOn, grain yield was evaluated under two different diversity
levels; first, as an average of component lines which were each derived fromoriginal plants and tested in pure stands, and secondly, as a population
composed of a mixture of one seed from one component line. The uplandpopulation, though it was a lower yielder than the lowland one under our
experimental conditions, tended to show an "populational buffering" oran advantageous effect due to genotype mixture.
Marked difference in diversity level found between two natural populations cultivated by the same farmer suggests a differential effect imposedby lowland and upland conditions on preserving genetic diversity.
Characteristics of Weed Rice Strains Collected from
Various Asian Countries
Ling-Hua, TANG and Hiroko MORISHIMA

A weed is defined as an unwanted plant which has adapted to habitats
disturbed by man. Many crops are accompanied by their related "weed"
types, in addition to their "wild" progenitors. The weed type of a certain
crop might be 1) a transitional state from wild to cultivated type, or 2) anescape from cultivars, or 3) derivatives of natural hybridization between
wild and cultivated types. In rice, the existence of weed types in andaround arable lands (mainly rice fields) was reported in many localities ofrice growing areas. Because weed rice can be said, in a sense, to be anintermediate state between wild and cultivated types, studies on its origin
and genetic nature might provide important information for understanding
the evolution of rice and also for practical rice breeding.
Twenty-four weed rice strains obtained from many Asian countries were
examined regarding various characteristics and isozyme loci. Each strain
was quantitatively evaluated on the axes of wild vs. cultivated variation
and Indica vs. Japonica variation which represents a major varietal differentiation in Asian common rice.
1) Weed rice strains possessed intermediate characteristics between wild

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

and cultivated types showing a wide variation. Some were similar to wild
types in that they have high degree of seed shedding, seed dormancy, andblack and light seeds, though they looked like cultivars at the stage ofvegetative growth. Others were very similar to cultivated types in many
respects, being distinguished only by a red pericarp. Such "mimics" ofcultivars are probably harvested and sown together with cultivated riceunconsciously.
2) Judging from several diagnostic characteristics and allozymes which
are useful for Indica-Japonica classification, weed strains collected from
China (Jiangsu) and Korea where Japonica types are cultivated were of the
Japonica type, while those from India and Thailand where Indica types arecultivated were close to the Indica type, though variation was large. Interestingly, those collected from hilly areas of Nepal, where both Japonica
and Indica types were grown within the same field, were found to be IndicaJaponica intermediates.
Weed rices found in humid tropics inhabited by wild rices originatedmost probably from the natural hybridization between wild and cultivatedtypes followed by selection for weediness. However, the origin of weed
rices found in areas where wild rice is not distributed remains unknown.
Isozyme Genes Useful for Indica-Japonica Classificationof Common Rice
Ling-Hua TANG and Yo-Ichro SATO

Two subspecies of common rice, Indica and Japonica types, differ not
only in so-called discriminating characteristics but also in allelic frequencies
at various isozyme loci (Glaszmann 1987; de Kochiko 1987). It is expectedthat an adequate combination of alleles at a fewer number of enzymecoding loci can effectively discriminate between Indica and Japonica types.
To look for such combinations alleles at twelve isozyme loci, Amp-I, Amp2, Amp-3, Acp-I, Cat-I, Est-2, Est-9, Pgd-I, Pgi-I, Pgi-2, Pox-2 and Sdh-I,of 104 cultivars from various localities in Asia were examined. The cultivars were classified into Indica and Japonica types prior to isozyme analysis, using Sato's discriminant function.
Z=Ph+l.313K-0.82Hr
where Ph, K and Hr indicate the phenotypic value of phenol reaction,

RESEARCH ACTIVITIES IN 1987

121

susceptibility to KCI0 3 and apiculus hair length respectively, (Sato et al.
1986). The hundred and four cultivars tested were classified into 51 Indicas
and 53 Japonicas.
It was pointed out that between the Indica and Japonica types allelicfrequencies significantly differed at 10 loci other than Amp-l and Sdh-l.
Particularly at Amp-2, Acp-l and Cat-l loci allelic frequencies were markedly
different between the two types. Allele combinations at these three locimay be used for making a rough estimate whether a cultivar falls into the
Indica or the Japonica type. Of 51 classified as Indica type from their Z
value, 47 commonly had Amp-2 2 , Acp-I-4 and Cat-Jl (designated as (2,-4, 1)). Fourty-seven out of 52 classified as Japonicas had an association (1, 9, 2) which was the diametric combination of (2, -4, 1). To confirm this trend, 48 random samples from upland native cultivars in Thailand
were used and subjected to the same analysis. Forty-four had (2, -4, 1)or (1, 9, 2), and the former largely belonged to the Indica and the latter to
the Japonica type. Probability of misjudgement was less than 10 percent.
Location of Pgd-l Locus in Rice
Ryuji ISHIKAWA*, Toshiro KINOSHITA* and Hiroko MORISHIMAOne of the rice isozyme loci, Pgd-l, was found to be located on chromosome 9 (linkage group VIII) by trisomic analysis (Ishikawa 1986). To
determine the location of Pgd-l on this chromosome by linkage analysis,
an Indica strain carrying allele 2 at the Pgd-l locus was crossed with a
Japonica tester strain FI-280 which has an alternative allele 2 at this locus
and two mutant genes, la (lazy) and v-4 (virescent) belonging to linkage
group VIII. F 2 segregation indicated Pgd-l was linked with these twoloci. The recombination value was calculated using the formula of Allard(1956). Pgd-l showed a recombination of 5.4 % with v-4, and 14.2 % withlao In addition, a linkage between Pgd-l and another isozyme locus Adh-l
was found, though the order of their sites has not yet been determined.
Thus, twelve isozyme loci were so far assigned in rice to their relevant
chromosomes and four of them were mapped.

* Hokkaido University.

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Genetic Aspects of Differentiation into Annuals
and Perennials in Asian Wild Rice
Pascale BARBIER* and Riroko MORISHIMA

Surveys of inter-populational variation in Asian common wild rice
Oryza rufipogon (Asian form of O. perennis) showed that various characteristics pertaining to the life-history and mating system are intercorrelated
in the following way: Early flowering, high fecundity, low post-reproductive
survival and selfing behavior on one hand (characterizing the annual type),
and late flowering, low fecundity, high post-reproductive survival andoutbrceding behavior on the other hand (perennial type).
To investigate the genetic basis of these characteristic associations, fourcrosses were made between individuals sampled from one annual population (used as females) and those from one perennial population (males),and the pattern of covariation between characteristics in F 2 was examined.
Many of the correlations mentioned above were not found in the F 2 progenyof these crosses. This indicates that such correlations may have no genetic
basis such as by linkage or pleiotropy. The corresponding trait associations which were observed in the interpopulation surveys probably resulted
from independent selection of these traits. This is consistent with therather low intra-population variance observed both in annuals and perennials. However, a positive correlation between early flowering and panicle
number (the main component of fecundity) was consistently found in F 2 sof all crosses (r=0.377-0.523, n=50-60). Other significant correlations
were found in some crosses; for example the fecundity component (panicle
number) and the longevity component (number of vegetative tillers developing after reproduction) were negatively correlated (r=0.314), longevity
and outcrossing rate (estimated from anther length) were positively correlated (r=0.390).
An investigation of relationships between these life-history traits andallozyme variation revealed an association between the presence of alternative alleles at locus Pox-l and early vs. late flowering. Interestingly,
this association is also found among natural populations; the mechanism
whereby such association persists in nature has however not been elucidated.

* Nagoya University.

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123

Electrophoretic Analysis by Immobilized pH Gradient ofSeven Protein Fractions Sequentially
Extracted from Rice Endosperm
Toru FNDO

From 10 g of 100-mesh rice endosperm powder, seven fractions of four
protein classes were sequentially extracted. In order of extraction, they
are as follows: acid-soluble (alb-I) and alkali-soluble albumin (alb-2), acidsoluble (glb-l) and alkali-soluble globulin (glb-2), alcohol-soluble prolamin(prl), and acid-soluble (glt-l) and alkali-soluble glutelin (glt-2). Each extract was lyophilized immediately, or following desalting, or was dried
after centrifugation. The protein contents of the powdered extracts were
estimated by the eBB method. The extracts were then dissolved in 8M
urea and subjected to electrophoresis in an acrylamide gel (4 % T, 3 % C)with an immobilized pH 4-10 gradient. A sample of the results are shownin Fig. 1.
From the figure, the numbers of bands in each of the seven fractions were

Fig. 1. Anode at top. From left, samples applied are alb-I, alb-2, glb-l, glb-2,
prJ, glt-l and glt-2 of rice endosperm of CY. Inakura, tropical type of Japonica.

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

counted. For the alb-1 through glt-2 fractions the numbers were: a minimum of 42 (including 3 major bands), 59(11), 66(14), 52(8), 32(6), 60(12),and 33(7), respectively. It can be seen that there are appreciable differences between the acid-soluble and alkali-soluble fractions of the threeprotein classes, i.e. albumin, globulin, and glutelin. Also, there are similarities between the band patterns of the alb-2 and glb-2 fractions as wellas those of the glb-2 and glt-l fractions. These observations suggest thatthe extractants used may be inadequate relative to the complexity of theendosperm protein molecules.
Multiple Alleles at the wx Locus as a Major Factor
for Amylose Content in Rice Grain
Yoshio SANO

Rice cultivars greatly differ in amylose content, the major determinantof grain quality. As reported already (Ann. Rep. 34), there are two alleles,
Wx& and Wx b , in addition to wx which control the quantitative level ofamylose as well as the gene product called Wx protein. Therefore, thetwo alleles of Wx& and Wx b were considered to be a major factor controlling
amylose content among nonglutinous cultivars. However, considerable
variations in both amylose content and Wx protein levels are found amongcultivars, with some cultivars showing an intermediate amount between
those of Wx& and Wx b • One possible explanation for this is that there
may be other genes which regulate both amylose content and Wx protein
level such as low amylose mutant genes. Another possible explanation isthat there are additional Wx alleles which produce an intermediate amountof amylose and Wx protein between those of Wx& and Wx b • To investigate
this, Wx genes were introduced into a standard glutinous strain (Taichung
65 Wx) by successive backcrosses from three cultivars which produced anintermediate amount of amylose and Wx protein. In BCs F 2 and BC4 F2 ,homozygous plants for Wx always showed an intermediate amount ofamylose and Wx protein. Wx& and Wx b were not detected in the derivatives. This implies that an additional Wx allele also contributes to variations in amylose content among nonglutinous cultivars.

RESEARCH ACTIVITIES IN 1987

XI.

125

DATABASE

Constructing a DNA Database
Sanzo MIYAZAWA

A data entry system and data search/retrieval system for DNA sequence
data were developed on a unix operating system. We built a data entry
system by utilizing the SCCS (Source Code Control System) available in
the unix operating system. The SCCS is a source code management system with such features as version control and exclusion control. Version
control means that a record is kept with each set of changes; of what thechanges are, why they were made, and who made them and when. Ecclusion control means that only one person can modify data at a time.
Both are critical in data entry with more than one user. Quality controlof data is also important in any database. We installed programs madeby Dr. J. Fickett in the GenBank for error checking of data. These programs check whether the following features are illegal or not; record format,
journal name, start and stop codons, and codon frame. A codon table
which depends on an organism is maintained as a database. Taxonomy
records in the data are automatically generated by using a taxonomy
database which is also maintained by GenBank. More extensive automatic
error-checking should be developed on the basis of studies of the telationshipbetween DNA sequence pattern and function. A simple search and retrieval system which uses flat files and therefore called "flat" has also beendeveloped. Some basic tools are available as filters in the unix system to
output specified types of records, to search strings expressed in regularexpression over database and output names of entries which include suchstrings, to take "and", "or" and "xor" over sets of entry names, and to
cut out specified entries from the database. By joining such filters together
with "pipe", one can search and retrieve entries from the database by
keywords such as author name, journal name, title, organism name, gene
name, and any combination of such items. This "flat" system is designed
to be portable and easy to maintain at the cost of speed; it is portable amongunix systems which are available for a wide range of computers from superto personal· computer.

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

Publication from Genetic Resources Section, Genetic
Stocks Research Center
Shin-ya IYAMA

The following publications were released from the Genetic Resources
Section this year.
1. "Experimental Mouse Strains Maintained in Universities in Japan.
1987." (Compiled by S. Iyama and K. Moriwaki) 177 pages (in Japanese).
This was completed in cooperation with the Mammalian Section of thisCenter. The catalogue listed 1032 entries of 621 genetic strains of mouse
maintained in various universities and research institutes in Japan under
the administration of the Ministry of Education and Culture. Each entry
was described with its strain name, location maintained, historical recordof acquisition, genetic characteristics, and information on maintenanceconditions and distribution.
2. "Rice Genetics Newsletter Volume 4". 131 pages (in English). Thisis published in cooperation with the Rice Genetics Cooperative, an international organization of rice geneticsts. This volume contained thefollowing:(1) Eleven newly registered gene symbols approved by the Committee
on Gene Symbolization, Nomenclature and Linkage Groups.
(2) Lists of newly adopted gene symbols (20) and marker genes (25)whose linkage groups have been confirmed. These are in supplement tolists appearing in previous issues.
(3) Current linkage maps of rice.
(4) A List of 60 recently published papers of rice genetics.
(5) 32 research notes of various fields of rice genetics.

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127

PUBLICATlONSAOKI, K.: Gene-culture waves of advance. J. Math. BioI. 25: 453-464,1987.
AOKI, K.: Adult lactose absorption and milk use from the standpoint of gene-culture
theory. Jpn. J. Genet. 62: 445-459, 1987.
AOKI, K. and FELDMAN, M. W.: Toward a theory for the evolution of cultural communication: coevolution of signal transmission and reception. Proc. NatI. Acad.
Sci. USA 84: 7164-7168, 1987.
AOTA, S., GOJOBORI, T., SHIGESADA, K., OZEKI, H. and IKEMURA, T.: Nucleotide sequence and molecular evolution of mouse retrovirus-like lAP elements. Gene 56:
1-12, 1987.
BONHOMME, F., GUENET, J.-L., DOD, B., MORIWAKI, K. and BULFIELD, G.: The polyphyletic origin of laboratory inbred mice and their rate of evolution. BioI. J. of theLinnean Soc. 30: 51-58, 1987.
BANDO, H., KUSUDA, J., GOJOBORI, T., MARUYAMA, T. and KAWASE, S.: Organizationand nucleotide sequence of a densovirus imply a host-dependent evolution of parvoviruses. J. Virology 61: 553-560, 1987.
BOURSOT, P., YONEKAWA, H. and BONHOMME, F.: Heteroplasmy in mice with deletionof a large coding region of mitochondrial DNA. Mol. BioI. EvoI. 4: 46--55, 1987.
ENDO, T.: Differential characteristics of endosperm protein fractions between Indica
and Japonica rice varieties. In "Crop Exploration and Utilization of Genetic Resources" (S.C. Hsieh, ed.), pp. 31-39, Taiwan Provo Taichung Dist. Agric. Imp.
Stat., 1987.
FISCHER-LINDAHL, K., LOVELAND, B., RICHARDS, S. and YONEKAWA, H.: In search of
the molecular basis for a mitochondrial transplantation antigen. Abstract of papers
presented at the 1987 meeting on molecular biology of mitochondria and chloroplasts.
p.205. Cold Spring Harbor Laboratory, 1987.
FUJII, T., HUANG, Y.-D., HIGASHITANI, A., NISHIMURA, Y., IYAMA, S., HIROTA, Y., YONEYAMA, T. and DIXON, R. A.: Effect of inoculation with Klebsiella oxytoca on dinitrogen fixation by rice-bacteria association. Plant and Soil 103: 221-226, 1987.
FUJII, T. and INOUE, T.: Modulating effect of dimethylbenzanthracene on gamma-ray
mutagenesis in the soybean test system. Jpn. J. Genet. 62: 425-430, 1987.
FUJISAWA, T.: Inhibition of stenotele differentiation by head tissue in hydra. J. Cell
Sci. 87: 315-322, 1987.
FUJISAWA, T.: An endogenous inhibitor involved in position-dependent stenotele differentiation in hydra. Dev. BioI. 122: 210-216, 1987.
FUJITA, N., NOMURA, T. and ISHIHAMA, A.: Promoter selectivity of Escherichia coli
RNA polymerase: Purification and properties of holoenzyme containing the heatshock sigma subunit. J. BioI. Chern. 262: 1855-1859,1987.
FUJITA, N., ISHIHAMA, A., NAGASAWA, Y. and UEDA, S.: RNA polymerase sigma-

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38
related proteins in Escherichia coli: Detection by antibodies against a synthetic peptide.
Mol. Gen. Genet. 210: 5-9, 1987.
FUJITA, N. and IsHIHAMA, A.: Heat-shock induction of RNA polymerase sigma-32
synthesis in Escherichia coli: Transcriptional control and a multiple promoter system.
Mol. Gen. Genet. 210: 10-15, 1987.
GLASS, R. E., JONES, S. T. and ISHIHAMA, A.: RNA polymerase is a target for ppGpp.
In "RNA Polymerase and the Regulation of Transcription", (W. S. Reznikoff, et al.,eds.), pp. 393-396, Elsevier Science Publ., New York, 1987.
GLASS, R. E., JONES, S. T., NOMURA, T. and ISHIHAMA, A.: Hierarchy of the strength ofEscherichia coli stringent control signals. Mol. Gen. Genet. 210: 1-4, 1987.
GOJOBORI, T., MORIYAMA, E. N. and YOKOYAMA, S.: Molecular evolution of oncogenes. Jpn. J. Genet. 62: 163-177, 1987.
GOJOBORI, T. and YOKOYAMA, S.: Molecular evolutionary rates of oncogenes. J. Mol.
Evol. 26: 148-156, 1987.
HARADA, Y.-N., HAYAKAWA, J.-I., NODA, E. and TOMITA, T.: A gene locus controllinga serum protein migrating electrophoretically in the f3 region of mice and detected byusing a strain derived from the Japanese wild mouse (Mus musculus molossinus). J.
Immunogenet. 14: 33-41, 1987.
HAYASHI, J.-I., YONEKAWA, H., MURAKAMI, J., TAGASHIRA, Y., PEREIRA-SMITH, O. M. M.
and SHAY, J.: Mitochondrial genomes in intraspecies mammalian cell hybrids display
codominant or dominant/recessive behavior. Exp. Cell Res. 172: 218-227, 1987.
HIAl, H., BUMA, Y. 0., IKEDA, H., MORIWAKI, K. and HISHIZUKA, Y.: Epigenetic control
of endogeneous ecotropic virus expression in SL/Ni mice. JNCI 79: 781-787, 1987.
HIROSE, S. and SUZUKI, Y.: In vitro transcription of eukaryotic genes are affected differently by the effect of DNA supercoiling. Proc. Natl. Acad. Sci. USA 85: 718722, 1988.
HIROSE, S., TABUCHI, H. and YOSHINAGA, K.: GTP induces knotting, catenation andrelaxation of DNA by stoichiometric amounts of DNA topoisomerase II from
Bombyx mori and HeLa cells. J. BioI. Chern. 263: 3805-3810, 1988.
HONDA, A., UEDA, K., NAGATA, K. and ISHIHAMA, A.: Identification of the RNA polymerase-binding site on genome RNA of influenza virus. J. Biochem. 102: 12411249, 1987.
HORAI, S., GOJOBORI, T. and MATSUNAGA, E.: Evolutionary implications of mitochondrial DNA polymorphism in human populations. In "Human Genetics" (F.
Vogel and K. Sperling, eds.), pp. 177-181, Springer-Verlag, Berlin, 1987.
HORAI, S., INOUE, T. and MATSUNAGA, E.: An apparent discrepancy between chain
length and electrophoretic mobility of restriction fragments: A case of human mitochondrial DNA. Human Genet. 75: 73-74, 1987.
HORI, H. and SATOW, Y.: IRIS: Integrated RNA information for systematics. In"Prospectives in Nucleic Acid and Protein Sequencing Data; Collection, Evaluation,
Distribution" (R. R. Colwell, ed.), Oxford Univ. Press, in press.
HORI, H. and OSAWA, S.: Origin and evolution of organisms as deduced from 5S ribosomal RNA sequences. Mol. BioI. Evol. 4: 445-472, 1987.
HORI, H., MUTO, A., OSAWA, S., TAKAI, M. and KAWAMATSU, M.: Molecular evolution

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129

of Turbellaria as deduced from 5S ribosomal RNA sequences. Prog. Zoo!. 36: inpress.
HORIKAWA, K., OTAFUJI, T., NAKAGAWA, R., SERA, N., KURODA, Y., OTSUKA, K. and
TOKIWA, H.: Mutagenicity of dinitrofluoranthenes (DNFs) and their carcinogenicity in F344 rats. Mutation Res., 182: 360-361, 1987.
IMAMURA, T.: Hemoglobinopathies. (in Japanese) In "Practice of Internal Medicine,
5th ed." (M. Abe et al., eds), 557-569, Igaku-Shoin, Tokyo, 1987.
IMAMURA, T.: Thalassemia genes. (in Japanese) In "Annual Review of Haematology
1987" (F. Takaku et al., eds), 48-57, Chugai-Igaku-Shoin, Tokyo, 1987.
IMAMURA, T.: Disorders of complement receptors in autoimmune diseases. (in Japanese)
Immune Haematology 8: 413-426, 1987.
IMAMURA, T.: Mendelism and clinical medicine. (in Japanese) Clinical Pediatrics 50:
573-575, 1987.
ISHIHAMA, A., FUJITA, N. and NOMURA, T.: Promoter selectivity of Escherichia coli
RNA polymerase. In "RNA Polymerase and the Regulation of Transcription" (W. S.
Reznikoff, et al., eds.), pp. 397-402, Elsevier Science Publ., New York, 1987.
ISHIHAMA, A. and NAGATA, K.: Viral RNA polymerases. In "CRC Reviews in Biochemistry", CRC Press, Florida, 1987.
ISHIHAMA, A., FUJITA, N. and GLASS, R. E.: Subunit assembly and metabolic stabilityof E. coli RNA polymerase. Proteins: Structure, Function and Genetics, 2: 42-53,
1987.
ISHIHAMA, A., HONDA, A., NAGASAWA-FuJIMORI, H., GLASS, R. E., MAEKAWA, T. and
IMAMOTO, F.: Multivalent regulation of the nusA operon of Escherichia coli. Mol.
Gen. Genet. 206: 185-191, 1987.
ISHIHAMA, A.: Transciption and RNA processing. (in Japanese) In "Medical Molecular
Biology" (M. Muramatsu, ed.), 36-41, Nanko-do, Tokyo, 1987.
ISHIHAMA, A.: Mechanism of gene transcription. (in Japanese) In "New Approaches inLife Sciences" (The Org. Committee of Symp. on Univ. and Sci., ed.), 235-255,
Shuppan-Kagaku, Tokyo, 1987.
ISHIKAWA, R., MORI, K., KINOSHITA, T. and MORISHIMA, H.: Expression pattern of
isozyme genes in rice caUi. (in Japanese) Memoirs of Fac. Agr., Hokkaido Univ.
15: 371-379, 1987.
ITOH, N., YAMADA, H., KAZIRO, Y. and MIZUMOTO, K.: Messenger RNA guanylyltransferase from Saccharomyces cerevisiae: Large scale purification, subunit functions, and subcellular localization. J. Bio!. Chern. 262: 1989-1995, 1987.
JAIN, A. K. and TEZUKA, H.: Effect of age on micronucleus frequency induced by mitomycin C in ddY male mice. Curro Sci. 56: 894-895, 1987.
JAIN, A. K., TEZUKA, H., KADA, T. and TOMITA, I.: Evaluation of genotoxic effects ofturmeric in mice. Curro Sci. 56: 1005-1006,1987.
KADA, T. and SHIMOI, K.: Desmutagens and bioantimutagens. Their modes of action. BioEssays 7: 113-116, 1987.
KIMURA, M., KALLIANPUR, G. and HIDA, T. (ed.): Stochastic Methods in Biology (Lecture Notes in Biomathematics 70). Springer-Verlag, Berlin, 1987.
KIMURA, M.: A stochastic model of compensatory neutral evolution. In "Stochastic

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38
Methods in Biology" (M. Kimura, et al., eds.), pp. 2-18, Springer-Verlag, Berlin,
1987.
KIMURA, M.: Die Neutralitatstheorie der molekularen Evolution. Verlag Paul Parey,
Berlin und Hamburg, 1987.
KIMURA, M.: Molecular evolutionary clock and the neutral theory. J. Mol. Evol. 26:
24-33, 1987.
KOJIMA, H., KONISHI, H. and KURODA, Y.: Combined effects of chemicals on mammalian cells in culture. Effects of order and intervals of administration of methyl
methanesulfonate and ethyl methanesulfonate. Mutation Res., 182: 364, 1987.
KOMIYAMA, K., ITO, K. and SHIMADA, Y.: Origin and development of the epicardium inthe mouse embryos. Anatomy and Embryology 176: 183-189, 1987.
KONDO, K., AOSHIMA, Y., HAGIWARA, T., VEDA, H. and MIZUNO, S.: Tissue-specificand periodic changes in the nuclease sensitivity of the fibroin gene chromatin in thesilkworm Bombyx mori. J. BioI. Chern. 262: 5271-5279, 1987.
KURODA, Y.: Antimutagenic mechanism of vitamin C and its derivatives in mammaliancells in culture. Mutation Res. 182: 365, 1987.
LEE, W. H. and WATANABE, T. K.: Evolutionary genetics of the Drosophila melanogaster
subgroup. I. Phylogenetic relationships based on matings, hybrids and proteins.
Jpn. J. Genet. 62: 225-239, 1987.
MATSUBAYASHI, T., WAKASUGI, T., SHiNOZAKI, K., YAMAGUCHI-SHINOZAKI, K., ZAITA,
N., HIDAKA, T., MENG, B. Y., OHTO, C., TANAKA, M., KATO, A., MARUYAMA, T.
and SUGIURA, M.: Six chloroplast genes (ndhA-F) homologous to human mitochondrial genes encoding components of the respiratory chian NADH dehydrogenase
are actively expressed: Determination of the splice sites in ndhA and ndhB pre-mRNAs.
Mol. Gen. Genet. 210: 385-393, 1987.
MATSUNAGA, E.: Hereditary diseases in Asia: Chairman's introduction. In "Human
Genetics" (F. Vogel and K. Sperling, eds.), p. 539, Springer-Verlag, Berlin, 1987.
MATSUNAGA, E.: Incidence of genetic disease in Japan. In "Human Genetics" (F.
Vogel and K. Sperling, eds.), pp. 540-546, Springer-Verlag, Berlin, 1987.
MATSUNAGA, E.: Possible effect of biotechnology development upon human gene pool.
(in Japanese) In "Biotechnology & Medical Care" (F. Takaku, ed.), 57-71, Tokyo
Univ. Press, 1987.
MATSUNAGA, E.: Statistics in human genetics, with special reference to the study ofretinoblastoma. (in Japanese) In "Statistics in Science" (H. Akaike, ed.), 181-216,
Kodansha, Tokyo, 1987.
MATSUNAGA, E.: Genetics and epidemiology of retinoblastoma. (in Japanese) Gan no
Rinsho 33(5): 507-513, 1987.
MINODA, K., HORAI, S., MATSUNAGA, E. and MOTEGI, F.: Esterase D and chromosome
13 in patients with retinoblastoma. (in Japanese) Rinsho Ganka 41: 943-947, 1987.
MIYASHITA, N., MIGITA, S. and MORIWAKI, K.: Effects of H-2 complex and non-H-2
background on urethane-induced chromosomal aberrations in mice. Mutation Res.
176: 59-67, 1987.
MIYASHlTA, N. and MORIWAKI, K.: H-2 controlled genetic susceptibility to pulmonary
adenomas induced by urethane and 4-nitroquinoline I-oxide in A/Wy congenic strains.

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131

Jpn. J. Cancer Res. (Gann) 78: 494-498, 1987.
MIZUMOTO, K. and KAZIRO, Y.: Messenger RNA capping enzyme from eukaryotic
cells. Prog. Nucl. Acid. Res. Mol. BioI. 34, 1987. (in press)
MORISHIMA, H. and GADRINAB, L. U.: Are the Asian common wild-rices differentiatedinto the Indica and Japonica types? In "Crop Exploration and Utilization of Genetic
Resources" (S. C. Hsieh, ed.), 11-20, Taiwan Provo Taichung Dist. Agric. Imp.
Stat., 1987.
MORIWAKI, K.: Genetic significance of laboratory mice in biomedical research. In"Animal Models: Assessing the Scope of Their Use in Biomedical Research" (1. Kawamata & E. C. Melby, eds.), pp. 53-72, Alan R. Liss, New York, 1987.
MORIYA, S., OGASAWARA, N., FUKUOKA, T. and YOSHIKAWA, H.: DnaA-boxes in theoriC region of the Bacillus subtilis chromosome are involved in regulation of chromosomal replication. Genetic and Biotechnology of Bacilli. (in press).
MORIYA, S., OGASAWARA, N., FUKUOKA, T. and YOSHIKAWA, H.: Regulation of initiationof the chromosomal replication by DnaA-boxes in the origin region of the Bacillus
suhtilis chromosome. EMBO J. (in press)
NAKASHIMA, H.: Genetic counselings. (in Japanese) JAMA 6: 120--122,1987.
NAwA, S., SANO, Y., YAMADA, M. and FUJII, T.: Cloning of the plasmids in cytoplasmic
male sterile rice and changes of organization of mitochondrial and nuclear DNA in
cytoplasmic reversion. Jpn. J. Genet. 62: 301-314, 1987.
NITo, S., OKANIWA, A. and MORIWAKI, K.: Effects of H-2 histocompatibility genecomplex on urethan and ethylnitrosourea (ENU)-induced malformations in mice.
Congo Anom. 27: 61-68, 1987.
NOMURA, A., NEGISHI, K., HAYATSU, H. and KURODA, Y.: Mutagenicity of N4- aminocytidine and its derivatives in Chinese hamster lung cells. Incorporation of N 4 _aminocytidine into cellular DNA. Mutation Res. 177: 283-287,1987.
NOMURA, T" FUJITA, N. and ISHIHAMA, A.: Expression of leuX gene in Escherichia coli.
Regulation at transcription and tRNA processing steps. J. Mol. BioI. 197: 659670, 1987.
OHTA, T.: A model of evolution for accumulating genetic information. J. Theor. BioI.
124: 199-211,1987.
OHTA, T.: Simulating evolution by gene duplication. Genetics 115: 207-213,1987.
OHTA, T.: Very slightly deleterious mutations and the molecular clock. J. Mol. Evol.
26: 1-6, 1987.
OHTA, T., WATANABE, M., SHIRASU, Y. and INOUE, T.: Post-replication repair andrecombination in uvrA umuC strains of Escherichia coli are enhanced by vanillin, an
antimutagenic compound. Mutation Res. (in press).
OHTSUBO, M., KAI, R., FURUNO, N., SEKIGUCHI, T., SEKIGUCHI, M., HAYASHIDA, H.,
KUMA, K., MIYATA, T., FUKUSHIGE, S., MUROTsu, T., MATSUBARA, K. and NISHIMOTO,
T.: Isolation and characterization of the active eDNA of the human cell cycle gene
(RCC1) involved in the regulation of onset of chromosome condensation. Genesand Development 1: 585-593, 1987.
PARK, Y. H., HORI, H., SUZUKI, K. I., OSAWA, S. and KOMAGATA, K.: Nucleotide
sequence of 5S ribosomal RNA from Rhodococcus erythropolis. Nucleic Acids Res.,

132 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38
15,365, 1987.
PARK, Y. H., HORI, H., SUZUKI, K. I., OSAWA, S. and KOMAGATA, K.: Phylogenetic
analysis of the coryneform bacteria by 5S rRNA sequences. J. Bacteriol. 169,
1801-1806, 1987.
SAKOYAMA, Y., HONG, K., BYUN, S. M., HISAJIMA, H., UEDA, S., YAOITA, T., HAYASHIDA,
H., MIYATA, T. and HONJO, T.: Nucleotide sequences of immunoglobulin epsilon
genes of chimpanzee and orangutan: DNA molecular clock and hominoid evolution. Proc. Natl. Acad. Sci. USA 84: 1080-1084, 1987.
SANO, Y.: Gene regulation at the waxy locus in rice. Gamma-Field Symp. 24: 63-79,
1987.
SATO, Y. I.: Character association patterns among Japonica rice varieties of Asianorigin. In "Crop Exploration and Utilization of Genetic Resources" (S. C. Hsieh,ed.), 21-29, Taiwan Provo Taichung Dist. Agric. Imp. Stat., 1987.
SATO, Y. I. and MORISHIMA, H.: Studies on the distribution of complementary genes
causing F 1 weakness in common rice and its wild relatives. 2. Euphytica 36: 425-531,
1987.
SERIZAWA, H. and FUKUDA, R.: Structure of the gene for the stringent starvation proteinof Escherichia coli. Nucleic Acids Res. 15: 1153-1163, 1987.
SHIMADA, Y., KOMIYAMA, M., SHIOZAKI, M., ISOBE, Y. and MASUKO, S.: Myogenesis
in vitro as seen with the scanning electron microscope. Scanning Microscopy 1:
1377-1386, 1987.
SHIROISHI, T., SAGAI, T. and MORIWAKI, K.: Sexual preference of meiotic recombination within the H-2 complex. Immunogenetics 25: 258-262,1987.
SHIROISHI, T., SAGAI, T., NATSUUME-SAKAI, S. and MORIWAKI, K.: Lethal deletion of thecomplement component C4 and steroid 21-hydroxylase genes in the mouse H-2 class
III region, caused by meiotic recombination. Proc. Natl. Acad. Sci. USA 84: 28192823, 1987.
SUH, D.-H. and MUKAI, T.: Mosaic lethal mutations as one of the syndrome of hybrid
dysgenesis due to the P element in Drosophila melanogaster. Jpn. J. Genet. 62: 5158, 1987.
SUH, D.-H. and MUKAI, T.: Genetic variability due to mutation-selection balance in anorthern population of Drosophila melanogaster in Japan. Jpn. J. Genet. 62: 95107, 1987.
SUZUKI, H., MORIWAKI, K. and NEVO, E.: Ribosomal DNA (rDNA) spacer polymorphism in mole rats. Mol. BioI. Evol. 4: 602-610, 1987.
SUZUKI, H., KATO, J., SAKAGAMI, Y., MORI, M., SUZUKI, A. and HIROTA, Y.: Conversion of the ex component of penicillin-binding protein 1b to the f3 component inEscherichia coli. J. Bacteriol. 169: 891-893, 1987.
TACHI-SHINKAWA, K., KURODA, Y., MORIMOTO, K. and KOIZUMI, A.: Enhancing effectsof cytosine arabinoside on ethyl methanesulfonate-induced 6-thioguanine resistancemutations in Chinese hamster V79 cells. Mutation Res. 191: 37-40, 1987.
TAKAHASHI, T., MATSUDAIRA, Y., OBATA, Y. and MORIWAKI, K.: An autoreactive H-2specific monoclonal antibody with allospecificity. Immunogenetics 26: 105-106,
1987.

PUBLICATIONS133

TAKAHATA, N.: On the overdispersed molecular clock. Genetics 116: 169-179, 1987.
TAKANO, T., KUSAKABE, S. and MUKAI, T.: The genetic structure of natural populations of Drosophila melanogaster. XX. Comparison of genotype-environment interaction in viability between a northern and a southern population. Genetics 117:245-254, 1987.
TAKEUCHI, K., NAGATA, K. and ISHIHAMA, A.: In vitro synthesis of influenza viral RNA:
Characterization of an isolated nuclear system that supports transcription of influenza viral RNA. J. Biochem. 101: 837-845, 1987.
TAKEUCHI, M., HARA, M., INOUE, T. and KADA, T.: Adsorption of mutagens by refinedcorn bran. Mutation Res. (in press)
TOMITA, T., TANAKA, T., KOGA, A., NAKAMA, T., KONDO, K., YAKENO, Y. and MUKAI,
T.: Strain difference in homozygous load due to induced mildly deleterious mutations in Drosophila melanogaster. Proceedings of the Japan Academy. 63: 151-154,
1987.
WATANABE, T., MIYASHITA, N., MORIWAKI, K. and HILGERS, J.: Evolutionary relationships between laboratory mice and subspecies of Mus musculus based on the genetic
study of pancreatic proteinase loci, Prt-l, Prt-2, Prt-3, and Prt-6. Biochem. Genet.
25:239-251,1987
Wu, c., WILSON, S., WALKER, B., DAwID, 1., PAISLEY, T., ZIMARINO, V. and UEDA, H.:
Purification and properties of Drosophila heat shock activator protein. Science 238:
1247-1253, 1987.
YAMAMOTO, T., GOJOBORI, T. and YOKOTA, T.: Evolutionary origin of pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerase 01. J. Bact. 169:
1352-1357, 1987.
YOKOYAMA, S. and GOJOBORI, T.: Phylogeny and molecular evolution of the human
AIDS viruses LAV, HTLV-III, and ARV. J. Mol. Evol. 24: 330-336, 1987.
YOKOYAMA, S., MORIYAMA, E. N. and GOJOBORI, T.: Molecular phylogeny of the humanimmunodeficiency and related retroviruses. Proc. Jap. Acad. 63(B): 147-150, 1987.
YONEKAWA, H., MORIWAKI, K., GOTOH, 0., MIYASHITA, N., MATSUSHIMA, Y., SHI, L.,
CHo, W. S., ZHEN, x.-L. and TAGASHIRA, Y.: Hybrid origin of Japanese mice"Mus musculus molossiums"; Evidence from restriction analysis of mitochondrialDNA. Mol. BioI. Evol. (in press)
YONEKAWA, H. and FISCHER LINDAHL, K.: Searching for the molecular basis of a mitochondrial transplantation antigen. Genetics 116: s41, 1987.
YONEKAWA, H. and FISCHER LINDAHL, K.: Molecular evolution of mtDNA amongclosely related Mus species. Abstracts of papers presented at the 1987 meeting on
molecular biology of mitochondria and chloroplasts. p. 205. Cold Spring Harbor
Laboratory, 1987.

134 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

ABSTRACTS OF DIARY FOR 1987
Biological Symposium
257th meeting Dec. 12 Is the classic case of natural selection a1986 classic case of another kind? (D. Lambert)
258th
Dec. 19 Adaptive evolution at the molecular level (M.
Nei)
259th
Mar. 18 Reduction of acrosomal protenase level in1987 mouse spermatozoa caused by partial deletion of Y-chromosome (J. Styrna)
260thApr. 9 MHC polymorphism (J. Klein)
261st
May 25 Molecular biology of T cell Iymphokines: A
model system for proliferation and differentiation of mammalian cells (K. Arai)
May 25 A novel method for transfection of DNA262nd
protein complex to eukaryotic cells (D.
Wienhues)
263rd
May 29 Consequences on brain function of in-utero
exposure to atomic bombing in Hiroshima
and Nagasaki (W. J. Schull)
264th
Jun. 20 Genetics and development of mice (Hee-Sup
Shin)
265th
Oct. 27 Phylogenetic origin of t-haplotypes specificmarkers (G. Gachelin)Nov. 17 High natural radiation area in Brazil (A.
266thFreire-Maia)
Nov. 30 Protein processing in picona virus-infected
267thcells (B. D. Korant)
268th
Dec. 4 Identifying domains in protein sequences(W. C. Barker)
Mishima Geneticists' Club
319th meeting Feb. 5 Modulation of the MHC class I gene expression and tumorgenesis (K. Tachida)
Apr. 17 Mammalian DNA replication (F. Hanaoka)
320th
321st
May 20 Regulation of the insect homeotic genes (H.

ABSTRACTS OF DIARY FOR 1987

322nd
323rd
324th

325th
326th

327th

328th
329th

330th
331st

332nd

135

Ueda)
9 Relational gene library of E. coli genome (Y.
Ohara)
Jun. 25 Chromosome translocations in human cancer
cells (Y. Tsujimoto)
JuI.
3 Theoretical study of protein conformationalchanges and dynamics by computer simulation (N. Go)
JuI. IO Mechanisms of DNA replication in the yeast
Saccharomyces cerevisiae (A. Sugino)
JuI. 24 Organization and expression of Marchantia
polymorpha chloroplast genome (K. Umezono)
Aug. 26 Induction of targeted point mutation by asite-specific acetylaminofluorene adduct to
DNA in bacteria and mammalian cells (M.
Moriya)Oct. 2 Replication initiation of plasmid pSCIOl (K.
Yamaguchi)
Oct. 2 Roles of transcription in initiation of DNA
replication: a case of plasmid ColEI (H.
Masukata)
Nov. 27 Probable heterosis and functional differencesof isozymes in aquatic animals (K. Fujino)Dec. IO A cell surface marker protein of murine
embryogenesis, fotomodulin, and its application to molecular embryogenetic study (M.
Imada)
Dec. 18 Permanency of response to selection in finite
populations and effect of identity disequilibrium on it (H. Tateda)Jun.

136 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

FOREIGN VISITORS IN 1987
April 9, 1984January 30, 1986January 29, 1987
April 22, 1986April 19, 1987
January 16
January 27
February 7-8
February 7-8
March 14
March 17-19
March 18
April 1April 9
May 6
May 6
May 25
May 25
May 27
May 29June 20
June 25

Barbier, Pascale, Universite des Sciences et Techniques
du Languedoc Montpellier, France
Styrna, Jozefa, Jagiellonian University, Poland
Hong, Nguen Xuan, Hanoi Universtiy, Vietnam
Chen, Zheng Hua, Genetics Institute, Chinese Academy of Sciences, China
Wu, Xiang Lin, Lanzhou Institute of Biological
Products, Ministry of Public Health, China
Bodmer, Walter F., Imperial Cancer Research FundLaboratories, U.K.
Yoo, Ick-dong, Genetic Engineering Center, Korea
Advanced Institute of Technology, Korea
Brenton, Nick, BBC Open University Production
Centre, U.K.
Styrna, Jozefa, Jagielonian University, Poland
Suh, Dong Sang, Genetic Engineering Center, Korea
Advanced Institute of Technology, Korea
Klein, Jan, Max-Planck-Institut flir Biologie, West
Germany
Sun, Chong Rong, Fudan University, China
Lu, Qun, Institute of Genetics, Fudan University,
China
Arai, Ken-ichi, DNAX Research Institute of Molecular
and Cellular Biology, U.S.A.
Wienhues, Ursula, University of Cologne, West
Germany
Pardo, Daniel, French Embassy, Tokyo
Schull, William Jack, Radiation Effects Research Institute, Hiroshima (University of Texas, U.S.A.)
Shin, Hee-Sup, Whitehead Institute, MIT, U.S.A.
Tsujimoto, Yoshihide, The Wistar Institute, U.S.A.

FOREIGN VISITORS IN 1987

July 10
July 22
August 4September 1October 9
October 27
November 10
November 14-22
November 16
November 16-17
November 17-22
November 30
December 4
December 22-

137

Sugino, Akio, Laboratory of Genetics, NIEHSjNIH,
U.S.A.
Grote, John, The British Council, Tokyo
Tang, Ling Hua, Institute of Food Crops, Jiangsu
Academy of Agricultural Sciences, China
Kim, Sam-Eun, Sericultural Experiment Station, Rural
Development Administration, Korea
Fouden, Leslie, Rothampsted Experimental Station,
Agricultural and Food Research Council, U.K.
Gachelin, Gabriel, Institut Pasteur, France
Pan, I-Hung, National Taiwan University, Taiwan
Yi, Hao Xiong, Jiang Xi University, China
Shao, Qi Quan, Institute of Genetics, Academia
Sinica, China
Freire-Maia, Ademar & Dertia Y., Sao Paulo State
University, Brazil
Nasution, Rusdy E., Bogor Botanical Garden, National Institute of Biology, Indonesia
Korant, Bruce D., E. I. du Pont de Nemours & Co.,
U.S.A.
Barker, W. C., Georgetown University Medical Center, U.S.A.
Wu, Xiao Mei & Zhao, He, Lanzhou Institute of
Biological Products, Ministry of Public Health, China

138 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 38

AUTHOR INDEXAlBA, H • . . . . • • . . . . . . . . . . . . . . . . . . . . . 17
AOKI, K. • ..••........•......•... 96, 97
AOTA, S.

.

78, 104

BANDo, H • . . • . • . . . . . . • . . . . . . . . . . . . . . 99
BARBIER, P

122
82
BYUN, S. M. .
103
CROSLAND, M. VV.J •••••.....•.•...•. 82
CROZIER, R. H. • •........•.....•.•.. 82
ENDO, S
17
ENDo, T123
FELDMAN, M. VV•.....•...•......•.•. 97
FunsAwA, T
67
FUJITA, N
16,17,19,21
FUKUDA, R
20, 28, 29, 30
MASUKO, K
84
GLASS, R. E • . . • . . . . . . • . . . . . . . . • . . . . . 16
GOJOBORI, T.... 99,100,101,104,113,115
GOTOH, H . . . . . . . • . . . . . . . . . . • . . . . 61,62
HANAOKA, F. .
31
HANKINS, R. VV• . . . . . . . • . . . . . . . . . . . . . 24
HANZAWA, N
60
HARA, H. •.48, 49, 52
HARADA, Y
105
HASEGAWA, M • • . . . . . . . . . . . . • . • . . 28, 29
HATADA, E • . . . • . . . . . . . . . . . . . . 28, 29, 30
HATANAKA, M . . . . . . . . . . • . . . . . . . . . . . . 29
HAYASAKA, K ••....••..•....... 113, 115
HAYASHIDA, H. .
102, 103
HAYATSU, H . • . . . . . . . . . . • . . . . . . . . . . . . 88
HIDAKA, T
102
HIROSE, S. .
32
HIROTA, Y
39, 40, 41, 43, 45, 48, 49, 52
HISAJIMA, H •..•...•.....•.......... 103
HONDA, A • . . • • . . . . . . . • . . . . . • . • . . . . . 23
HONG, K.-J• . . . . . . . . . . . • . . . . . . . . . . . 103
HONJO, T
103
HORAI, S
113,115
HORIKOSHI, K. .
39
BROWNING, G. P

HOSAKA, Y

27
78, 104
IMAI, H. T. .
82
IMAMURA, T. .
III
INOUE, T
86
INOUE, Y. H ••.....••...•..... 74, 75, 76
IROBE, R
57
ISHIBASHI, F •.••.•••.•••••••.••••••• 104
ISHIHAMA, A •.•••...•••• 16, 17, 19, 21-27
ISHIKAWA, R
117, 121IYAMA, S
126
KAnA, T86
KASUYA, T
64
KATO, A ••......•....•..•......•... 102
KATO, J
.41, 43, 45, 49
KATOH, H ••.............•.....•.... 106
KAWAMOTO, Y
61
KAWASE, S
99
KIMURA, M • . . . . . . . . . . • . . . • . . . . . . . . . 92
KINOSHITA, T
121
KUMA, K • . . . . . . . . . . . . . . . . • . . . . . • . . . 102
KURIHARA, Y
81
KURODA, Y
64,65,87,88
KUSUDA, J . . . . . . . . . . . . . . . • . • . . • • . . • . 99
MARUYAMA, I. N
39,40
MARUYAMA, T
99, 102, 104
MATSUBAYASHI, T
102
MATSUMOTO, K •...........•...•••••. 31
MATSUNAGA, E • • . . . . • . . . . . . . . . . 113, 115
MATSUSHIMA, Y
106
MENG, B. Y
102
MINATO, K ••.........•...•.••••••••• 72
MIYASHITA, N
89,106MIYATA, T
102, 103
MIYAZAWA, S
125
MIZUMOTO, K •...•.•......•...•.• 35,36
MORI, T
70
MORISHIMA, H
118, 119, 121, 122
MORIWAKI, K •.. 60, 61, 62,81,89, 105, 106
IKEMURA, T

139

AVTHORINDEXMORIYAMA, E. N
0.• 0
101
MUKAIGAWA, J
28,30
MURAKAMI, A
67,68,70,71,90
NAGASE, Y
39
NAGATA, K
23, 24, 25, 26, 27, 31
NAKASHIMA, H •.................. ., .111
NAKAYAMA, M. 017,21NAWA, S. 0" . 0.. 0..........•..... 33, 73
NEGISHI, K
88
NIELSEN, J. T•..• 0......••.•........ 106
NISHIMURA, A. 0.•... 0.• 0..•...•.... 057
NISHIMURA, Y.41, 43, 45, 48
NOBUHARA, M •...................... 39
NOMURA, A. . .•........••••......•.. 88
NOMURA, T.•.••..•.......
22
NOZAWA, K
000 •.•.. 0 115
OHISHI, K. .
65
OHTA, T••.• 0..•....•..•••.... 93, 94, 95
OHTO, C0o' .102
OSAWA, S•.....
21
SADAIE, Y. .
86
SAGAI, T,
60,62
SAKAGUCHI, B. .
65
SAKAKIBARA, Y. . ....•............
55
SAKOYAMA, Y •.•.... ,0 103
SANO, Y, ., .33, 108, 109, 124
SATO, Y.-I.
117, 118, 120
SERIZAWA, H •...............
20
SHIMADA, J
67,70
SHIMADA, Y •..................... 78,79
SHIMIZU, H,,
66
SHIMIZU, K.
28, 29, 30
SHINOZAKI, K
00.•....... 0102
SHIROISHI, T•........ 060, 61, 62
SHOTAKE, T. .
.,.,.,., ....• 115
SOMA, M39000•••••••••••••••••••••••••••••••••••••••

0••••••••••••••••••••

00•••••••••••••••••••••••••••••••••••

0

00

0

••

•••••••••••••••••••••••

0•••••••SUGIURA, M. . .....•..........•. 0... 102
SUGIYAMA, T..............•......... 66
SUH, D. S
81
SUZUKI, H.41, 43, 45, 48, 49, 52, 81
SUZUKI, Y.
32
TABUCHI, Ho . o.....
.32
TAIRA, T•..............
75
TAKADA, Y ...................•....•. 64
TAKAHATA, N.................•.••.•. 95
TANAKA, M •................. 0.0 ... 102
TANG, L.-H•• ., ... .,., . ., .,117, 119, 120
TAYLOR, R. W•.•.
82
TEZUKA, H .•..........
86
TOMITA, T •.•••.. 00
00•. 105
VEDA, K
23
VEDA, S.•..... 0.. 0
103
UESHIMA, R . .,.,.,.,., .,16,17,19
WAKANA, S...............•......... 106
WAKASUGI, T ........•........... 0•. 102
WATANABE, T. K
.76
YAMADA, M ...•..• 0.....•..... 0. .43, 45
YAMADA, M.-A. ....•.......... 33,72,73
YAMAGUCHI-SHINOZAKI, K. . ..... 0... 102
YAMAMOTO,Ao...•..... 40
YAMAMOTO, Mo-T.•............ 74, 75, 76
YAMAMOTO, T. . ..............•....• 100
YAMANAKA, K.26, 27
YAOITA, Y. . 0
103
YASUDA, S
39, 48, 53, 55
YASUNAGA, T•.. 0....••.•........... 102
YOKOTA, T•.............. 0......•.. 100
YOKOYAMA, S..... 0..... 0... 99, 100, 101
YONEKAWA, H •.........•••.•..•.... 106
YOSHIKAWA, H .................•.... 37
YOSHINAGA, K
32
ZAITA, T •.............. 0.. 00•... 1020•••••••••••••••••••••••••

0••••••••••••

0

0

'"

••••••••••••••••••••••••••••••

0•••••••••••••

00lEft~~~i'f~ffl~~

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