ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL RI:PORT
No. 35

1984

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

1985

Anu]ual Report.:~~of \1:neNa\1:rronal Ins~itute of Genetics
NiDJ. 35, 1984

Published by

The National Institute of Genetics, Japan
1985

CONTENTS
General statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council and Advisory Committee
Association for propagation of the knowledge of genetics
Projects of research for 1984
Research activities in 1984

1. Molecular Genetics
Promoter selectivity of E. coli RNA polymerase. I. Promoter
strength recognized by regular holoenzyme. NOMURA, T.,
FUJITA, N. and ISHIHAMA, A.
Promoter selectivity of E. coli RNA polymerase. II. Structural andfunctional interconversion of RNA polymerase by nucleotide
factors. ISHIHAMA, A., NOMURA, T. and FUJITA, N.
Promoter selectivity of E. coli RNA polymerase. III. Structural andfunctional interconversion of RNA polymerase by protein
factors. NOMURA, T., ISHIHAMA, A. and FUJITA, N.
Promoter selectivity of E. coli RNA polymerase. IV. Transcriptionof a heat shock gene. FUJITA, N. and ISHIHAMA, A"
Cloning of the genes for the possible transcription factors of E.
coli. Analysis of the gene for stringent starvation protein (SSP).
FUKUDA, R., SERIZAWA, H. and YANO, R.
..
Structure and function of RNA-dependent RNA polymerase associated with influenza virus. I. Isolation and characterization.
KATO, A., UEDA, S. and ISHIHAMA, A.
Structure and function of RNA-dependent RNA polymerase associated with influenza virus. II. Possible proof-reading
function, ISHIHAMA, A., MIZUMOTO, K. and KATO, A. .....
Analysis of temperature-sensitive mutants of influenza virus: The
mutants of RNA segment 8. HASEGAWA, M., FUKUDA, R.
and SHIMIZU, K.
Structure and function of reverse transcriptase associated withavian myeloblastosis virus. KATO, A., NODA, A., UEDA, S. and

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

ISHIHAMA, A.
Analysis of mitochondrial DNA in cytoplasmic male sterile rice.
NAwA, S., SANO, Y. and FUJII, T.
Concerted evolution of the mouse immunoglobulin gamma chain
genes. HAYASHIDA, H., MIYATA, T., YAMAWAKI-KATAOKA,
Y., HONJo, T., WELS, J. and BLATTNER, F.

II.

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Immunogenetics
A lethal gene mapped within the mouse major histocompatibility
complex. SHIROISHI, T., SAGAI, T. and MORIWAKI, K. . . . . ..
Cytoplasmic gene flow in Japanese mice Mus musculus molossinus.
YONEKAWA, H., GOTOH, 0., TAGASHIRA, Y., MIGITA, S., Yu,
Z.-C., Lu, D.-Y., CHO, W.-S., MIYASHITA, N. and MORIWAKI,
K

IV.

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Microbial Genetics
Purification and sequencing of the active site tryptic peptide frompenicillin-binding protein 1b of Escherichia coli. NICHOLAS,
R. A., SUZUKI, H., HIROTA, Y. and STROMINGER, J. L.
Identification of the active site in penicillin-binding protein 3 ofEscherichia coli. NICHOLAS, R. A., STROMINGER, J. L., SUZUKI,
H. and HIROTA, Y.
A comparison of amino acid sequences of the active centers of
penicillin binding protein (PBP) -1 b, -3, carboxy peptidase(CPase), and p-lactamase.
SUZUKI, H., HIROTA, Y.,NICHOLAS, R. A. and STROMINGER, J. L. .. . . . . . . . . . . . . . . . ..
Sites of dnaA protein-binding in the replication origin of the
Escherichia coli K-12 chromosome. MATSUI, M., OKA, A.,
TAKANAMI, M., YASUDA, S. and HIROTA, Y.
A mutation uncouples DNA replication and cell division in Escherichia coli. NISHIMURA, A.
Effect of the sacUh 32 mutation on the growth and spore outgrowthof the div-341 strain of Bacillus subtilis. SADAIE, Y. and
KADA, T.

Ill.

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Developmental and Somatic Cell Genetics
In vitro formation of adult structures from lethal embryonic cells of
Drosophila melanogaster. KURODA, Y.
35

CONTENTS

Mutagenic activity of cytidine analogs in cultured Chinese hamster
cells. KURODA, Y., NEGISHI, K. and HAYATSU, H.
Studies on freezing of Drosophila embryos. KURODA, Y. and
TAKADA, Y.
Genetical studies on the hereditary mosaic (mo) strain of Bombyx
mori: mosaicism and fashion of meiotic divisions in oogenic
cells. MURAKAMI, A.
Genetical studies on the hereditary mosaic (mo) strain of Bombyx
mori: the appearance of polyploids. MURAKAMI, A.
A strain with highly .parthenogenic tendency in Bombyx mori.
MURAKAMI, A.
The second case of a chromosome specific instability in the silkworm, Bombyx mori. MURAKAMI, A.
Lethal phases of X-linked lethal mutations in Drosophila melanogaster. MINATO, K. and YAMADA, M. A.
Nematocyte differentiation in mutant strains of hydra with altereddevelopmental gradients. NISHIMIYA, C., FUJISAWA, T.,
WANEK, N. and SUGIYAMA, T.
Elimination of excess epithelial cells by phagocytosis in a mutant
strain of hydra (L4). KOBATAKE, E. and SUGIYAMA, T. .....
V.

Cytogenetics
Karyological survey of Indian ants. IMAI, H. T., BARONI URBANI,C., KUBOTA, M., SHARMA, G. P., NARASIMHANNA, M. N.,
DAS, B. c., SHARMA, A. K., SHARMA, A., DEODIKAR, G. B.,
VAIDYA, V. G. and RAJASEKARASETTY, M. R.
Chromosome observations on tropical ants from Indonesia. IMAI,
R. T., KUBOTA, M., BROWN Jr., W. L., IHARA, M., TOHARI,
M. and PRANATA, R. I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
The reproductive cycle of the queenless ant Pristomyrmex pungens.
ITow, T., KOBAYASHI, K., KUBOTA, M., OGATA, K., IMAI, R.
T., and CROZIER, R. Roo...
.. ..
Molecular analyses of transposable elements in Drosophila simulans.
INOUE, Y. R., WATANABE, T. K., MORIWAKI, K. and YAMAMOTO, M.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

VI.

Mutagenesis and Radiation Genetics
So called "Kada effect" of the tritium. KADA, To and SADAIE,
Y. . o.
o'
In vitro and in vivo analysis of antimutagenesis. KADA, T..
Gamma-induced chromosome aberrations in Caenorhabditis
elegans. SADAIE, Y. and SADAlE, To
Age-dependent and tissue-specific expression of a DNA repair
enzyme in the wasted mouse, a model animal of a human
genetic disease ataxia telangiectasia (AT). TEZUKA, H.,
INOUE, To, NOGUCHI, T., KADA, T. and SHULTZ, L. D ..
Effect of DNA damaging agents on isolated spleen cells and lungfibroblasts from the mouse mutant "wasted", a putative animalmodel for ataxia-telangiectasia. INOUE, T., TEZUKA, H.,
KADA, T., AIKAWA, K. and SHULTZ, L. Do ., o. o.
Mutagenic activity of 3'-deoxyadenosine in the soybean test system.
FUJII, T.
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Population Genetics
Evolution of an altruistic trait through group selection as studiedo.
by the diffusion equation method. KIMURA, Mo ...
Population genetics theory of concerted evolution and its application to the immunoglobulin V gene tree. OHTA, T. .
Population genetics of transposable elements. OHTA, T
Some models of gene conversion for treating the evolution ofmultigene families. OHTA, T. .
The cohesive population genetics of molecular drive. OHTA, T.
and DOVER, G. A.
F ST and G ST statistics in the finite island model. TAKAHATA, N.
and NEI, M
Mitochondrial gene flow. TAKAHATA, N. and SLATKIN, M. .
A model of extranuclear genomes and the substitution rate undero' .
within-generation selection. TAKAHATA, N. .
A quantitative genetic model of two-policy games between relatives.
AOKI, K ...
A population genetic model of the evolution of oblique culturaltransmission. AOKI, K. .
Evolution of alliance in primates: a population genetic model.
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CONTENTSv

AOKI, K.
Average coefficient of relationship within troops of the Japanesemonkey and other primate species with reference to the possi.
bility of group selection. AOKI, K. and NozAwA, K.
Group selection for a polygenic behavioral trait: estimating thedegree of population subdivision. CROW, J. F. and AOKI, K.

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Mathematical model of rapid invasion of the P-M type transposon
to a local population of Drosophila melanogaster. MUKAI, T.
and TAJIMA, F. .
.
Population bottlenecks and nonequilibrium models in population
genetics. I. Allele numbers when populations evolve from zerovariability. MARUYAMA, T
.

VIII.

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Evolutionary Genetics

A mathematical model of codon substitution and the constant of
evolutionary rate. GOJOBORI, T.
Rates of nucleotide substitution for cellular and viral oncogenes.
GOJOBORI, T. and YOKOYAMA, S.
Concerted evolution of the immunoglobulin V H gene family.
GOJOBORI, T. and NEI, M.
Classification and measurement of DNA polymorphism. NEI, M.,
TAJIMA, F. and GOJOBORI, T. .
.
Evolutionary phylogenies of the Drosophila montium subgroup.
KIM, B. K. and WATANABE, T. K. .
.
Genetic profile of wild mouse subspecies collected from South
Pacific Islands. MORIWAKI, K., MIYASHITA, N., YONEKAWA,
R., YOSIDA, T. R., SAGAI, T., SUZUKI, R., KURIHARA, Y. and
WATANABE, T.
Allelic constitution of the hemoglobin beta chain in wild population of the house mouse, Mus musculus. MIYASHITA, N.,
MIGITA, S. and MORIWAKI, K. .
.
Restriction fragment length polymorphism of rDNA in European
wild mice with Robertsonian translocations. KURIHARA, Y,SUZUKI, R., MORIWAKI, K., WINKING, R., KOMINAMI, R. and
MURAMATSU, M.
A 5'-end sequence of the fibroin gene from wild mulberry silkworm,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Bombyx mandarina. KUSUDA, J., NINAKI, 0., SUZUKI, Y. and
TAZIMA, Y. '" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 75

IX.

Human Genetics
Dse of retinoblastoma and Wilms' tumor as sentinel phenotypes for
population surveillance. MATSUNAGA, E. and MINODA, K. ..
Mitochondrial DNA polymorphism in Japanese. 1. Analysis withrestriction enzyme of six base pair recognition. HORAI, S.,
GOJOBORI, T. and MATSUNAGA, E.
High-resolution studies in patients with aniridia-Wilms tumor association. NAKAGOME, Y. and NAGAHARA, N.
High-resolution study in patients with Prader-Willi syndrome.
NAKAGOME, Y., TAKANO, T., NAGAHUCHI, S., HIBI, I., TANAKA,
F., NAKAMURA, Y. and TANAE, A. . . . . . . . . . . . . . . . . . . . . . . ..
Origin of restriction sites in a simple repeated DNA cloned fromhuman Y chromosome. NAKAHORI, Y and NAKAGOME, Y...

X.

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Behavioral Genetics
Effects of feeding experience before weaning on food preferance inmice. FunsHIMA, T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Effects of wave length of light on sexual maturity in female Japanesequails. FunsHIMA, T. and SAITO, M. ..... . . . . . . . . . . . . . . .. 84

XI.

Ecological Genetics
Monitoring of the wild-rice populations in Thailand. MORISHIMA,
H., SHIMAMOTO, Y., SANO, Y. and SATO, Y.-I.
85
Comparison between potential and realized genetic variability inthe wild-rice populations with different life-histories. BARBIER,
P. and MORISHIMA, H.
86

XII.

Applied Genetics
Genetic diversity in Indonesian native rice cultivars with reference
to the Indica-Japonica differentiation. GADRINAB, L. D.,
SATO, Y-I. and MORISHlMA, H,. 88
Frequency changes in isozyme genes observed in a hybrid population derived from a cross between an Indica and a Japonica
rice varieties. SANO, R. and MORISHlMA, H. . . . . . . . . . . . . . . . 89

CONTENTS

Cytoplasm substitution between an Indica strain of Oryza sativa
and O. glaberrima. SANO, Y.
Correlations between the amounts of amylose and Wx protein inrice endosperm. SANO, Y, KATSUMATA, M. and AMANO, E...
Varietal variations in basic vegetative phase and photosensitive
phase, and genetic control of basic vegetative phase in Japanesenative cultivars. SATO, Y-I.
Genetic control of apiculus hair length in rice. SATO, Y-I.
Two-dimensional gel electrophoresis of four kinds of fractionatedreserve proteins in rice endosperm. ENDO, T.. . . . . . . . . . . . ..
Sample size and number of loci to investigate for estimating effective
population number. IYAMA, S.
Surveys on the experimental biological stocks. lYAMA, S.
publications
Abstracts of diary for 1984
Foreign visitors in 1984
Author index

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108

GENERAL STATEMENT
In introducing the Annual Report of our institute that covers 1 year
from January to December 1984, I am pleased to begin by noting that, ourlong-cherished desire to reorganize the institute into a national center forjoint use by universities, has been realized by amendment of the related
law which took place on April 12 as a result of energetic efforts of the
Ministry of Education, Science and Culture.
Looking back upon the history of the institute, its foundation in 1949
was primarily based on the zealous wishes of the Genetics Society of Japanto create a central institute for studies in various branches of genetics,
putting emphasis on free scientific and personnel exchanges among universities and institutions without bias towards a particular university or
faculty. As such, the institute was placed under the direct control of the
Ministry of Education, Science and Culture. (At that time, a national
institute for joint use by universities did not exist within the scheme ofjurisdiction. The first example of this category was the Institute for HighEnergy Physics that was established in Tsukuba in 1971.) For 35 years
thereafter the institute has been making hard and steady efforts to produce
many significant achievements, through which it has become widely known
among the scientific community of the world. In the meantime the progressof genetics as the core of life science has been accelerated by various epochmaking developments in biotechnology such as recombinant DNA technique. In accordance with this tendency, it is becoming increasingly necessary to promote collaborative studies among scientists with different disciplines in different institutions. To meet this need, however, there were anumber of restrictions imposed by the administrative category to which ourinstitute belonged, so that we had been preparing in the past several years,
under the leadership of my predecessor Dr. Y. Tazima, for shifting it to thatfor joint use by universities. In the course of this preparation many people
including related academic societies rendered help and support to us, forwhich I wish to express my heartfelt thanks.
The main points of reorganization were as follows. Firstly, the preexisting 10 departments were rearranged into 5 according to the levels of research subjects, that is, Department of Molecular Genetics, Cell Genetics,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Ontogenetics, Population Genetics, and Department of Integrated Genetics.
Each department consists of three laboratories, two for regular members
and one for visiting professors. This year 3 new laboratories for visiting
professors were approved. Secondly, as facilities for joint use, DNA Research Center (DNA Structure Section and Recombinant DNA Section)
was newly established, and the Genetic Stock Research Center was expandedfrom 3 sections (plant, animal and bacteria) to 5, that is, Genetic Resources
Section was added and the animal section was divided into Mammalian andInvertebrate Sections. Thirdly, the occupational category of research staff
was changed, that is, after due review by an external committee, the department heads, laboratory heads and researchers were appointed Kyoju (professor), Jo-kyoju (associate professor) and Jo-shu (research member, aposition to which there is no equivalent in USA), respectively. Fourthly,technicians who had been belonging to each laboratory were put together
into Technical Section for the efficiency of labor.
The total expenditure in the 1984 FY was ¥917 million, about 55 % ofwhich were for personnel expenses; with respect to the amount allocated forresearch and related activities, there was a net increase by about ¥83 million
as compared with the 1983 FY, which accrued concomitantly with the reorganization. Besides, grants-in-aid in the amount of ¥106 million wererendered to selected staff members by the Ministry of Education, Science
and Culture. However, despite increased demand for new activity, there
was no increase at all in the full number of regular staff (currently 92), reflecting the present severe state of administrative and financial constraint.
The missions of the new institute, which should be open to scientists invarious universities and institutions, are to promote comprehensive andcollaborative studies in genetics, taking advantage of the facilities for joint
use including various genetic stocks, to cooperate in teaching students in
doctor course, and to promote international cooperation and exchange of
information. Through these activities we wish to contribute to the development of genetics and related sciences not only in Japan but also in the world.
In the past year two of our colleagues were loaded with honors. Professor Tomoko Ohta, Laboratory of Population Genetics, was elected to a
Foreign Honorary Member of the American Academy of Arts and Sciences
for her outstanding achievement in theoretical studies on population genetics
at the molecular level. Dr. Tadashi Inoue, Laboratory of Mutagenesis,
was given Encouragement Award of the Agricultural Chemical Society ofGENERAL STATEMENT

3

Japan for his biochemical studies on DNA repair and mutagenesis.
Speaking of personnel change, Dr. Tosihide H. Yosida, Head of the
Department of Cytogenetics, retired on April 1. Dr. Yosida has been
devoting himself enthusiastically to cytogenetic studies of Rodents for about
30 years, during which he produced energetically more than 500 papers.
In particular, his extensive studies on karyotype evolution of black rats,
developed on the basis of multiple field investigations in South Asia andOceania, are highly evaluated. Dr. Yosida also served as head of the
Genetic Stock Research Center and contributed to its development. The
institute conferred on him the title of Honorary Member. He was succeededby Professor Kazuo Moriwaki. Drs. Akira Ishihama and Ryuji Fukuda
were appointed professor and associate professor, respectively, of the Laboratory of Molecular Genetics; they both have been transferred from Institute for Virus Research, Kyoto University. Dr. Yasuo Nakagome waspromoted to professor of Laboratory of Human Genetics. Incidentally,
under the new system decisions about important items are made by theAdvisory Committee which consists of 10 members outside the institute and10 within the institute.
On April 21, the institute was opened to the public as usual. Some of theresearch activities of each laboratory were exhibited, and movie films wereshown. Double cherry blossoms in the campus were at their best and some
3,000 visitors enjoyed them. On October 27, public lectures were given
at the National Science Museum in Tokyo; the titles were "The ecology
and evolution of Drosophila" by Associate professor Takao K. Watanabe,and "Damage and repair of DNA" by Dr. Tadashi Inoue. In spite of
Saturday afternoon nearly 100 eager people listened to the lectures thatwere followed by lively discussions.
Two occasional meetings took place in the institute. One, entitled "DNAsequencing and its application" with lectures and technical training course,
was organized and held by Dr. E. Soeda of the DNA Research Center on
February 14 to 17, as part of activities of a specific research group (chairman: Prof. Y. Takagi of Kyushu Univ.) supported by a grant-in-aid from
the Ministry of Education, Science and Culture; there were 121 participantsfrom various parts of Japan, of whom 20 took the training course. The
other was an "Oji International Seminar" entitled "Population Genetics
and Molecular Evolution"; this meeting was organized and held by staffof the Laboratory of Population Genetics on November 13 to 16, and 33

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

participants including 9 distinguished foreign scientists discussed the subject
most actively. Then the 56th Annual Meeting of the Genetics Society ofJapan took place on November 23 to 25 in the campus of Nihon University,
Mishima, and most members of our institute served for its organization andoperation. It was well attended by about 440 participants from all overJapan. By the way, this was the 4th meeting that was held in Mishima
since 1953.
About 80 scientists visited our institute from abroad this year, with whom
information and views on recent studies were exchanged actively. Somedistinguished guests, including Dr. Linus Pauling, delivered stimulating
lectures at Biological Symposium. Those who stayed rather long for technical training and cooperative work were: Dr. P. Thipayathan, Associate
professor of Chulalongkorn University, Thailand (accepted by Laboratotyof Microbial Genetics); Dr. N. Houba-Herin, FRFC-Fellow researcher,Liege University, Belgium (Laboratory of Microbial Genetics); Miss P.
Barbier, graduate student of Universite des Sciences et Techniques due
Languedoc, Montpellier, France (Laboratory of Agricultural Genetics);
Mr. I. Loekman, Researcher and associate investigator, National Atomic
Energy Agency, Indonesia (Laboratory of Mutagenesis); Miss L. U.
Gadrinab, Scientist II of SEAMEO Regional Center for Tropical Biology,
Indonesia (Laboratory of Mutagenesis and Genetic Stock Research Center);Mr. Nai-Kai Zhu, Research associate, Institute of Environmental Chemistry,
Chinese Academy of Science, the People's Republic of China (Laboratoryof Mutagenesis); and Mr. Bong-Kee Kim, Teaching assistant, Dan Kook
University, Korea (Laboratory of Evolutionary Genetics).
The institute has just made the first step toward an interuniversity research institute. Visiting professors and associate professors for the 3
new laboratories have been appointed. We have accepted 17 collaborative programs, 4 workshops, 7 graduate students, 10 research fellows fromprivate corporations, and 3 scholarships from industries. Yet, there remain a number of problems to be solved in order to achieve the reality ofreform. Among others, the followings are of the highest priority: completion of DNA Research Center including the establishment of DNA
data bank, which should meet the high demand of scientists in our country,
and the construction of a second main building to accommodate the new
Research Center and various facilities for joint use, together with lodgings
for visiting researchers. We are all eager to do our best to accomplish theGENERAL STATEMENT

5

missions of the new institute. We wish to have continued encouragement
and support of all the persons concerned.

STAFF
Director

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

1. Department of Molecular Genetics
KADA, Tsuneo, 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.
Laboratory of Mutagenesis
KADA, Tsuneo, D. Sc., Professor
SADAIE, Yoshito, D. Sc., Associate professor
INOUE, Tadashi, D. Ag.
TEZUKA, Hideo
Laboratory of Nucleic Acid Chemistry (Guest Member)
MIURA, Kin-ichiro, D. Sc., Professor
YAMANE, Kunio, D. Sc., Associate professor

2. Depm·tment of Cell Genetics
HIROTA, Yukinori, D. Sc., Head of the Department
Laboratory of Cytogenetics
MORIWAKI, Kazuo, D. Sc., Professor
IMAI, Hirotami, D. Sc.
YAMAMOTO, Masatoshi, Ph. D.
Laboratory of Microbial Genetics
HIROTA, Yukinori, D. Sc., Professor
YASUDA, Seiichi, D. Sc., Associate professor
NISHIMURA, Yukinobu, D. Sc.
HARA, Hiroshi, M. Sc.

STAFF

Laboratory of Cytoplasmic Genetics (Guest member)SUZUKI, Hideho, D. Sc., Associate professor
YONEKAWA, 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.
Laboratory of Phenogenetics
KURODA, Yukiaki, D. Sc., Professor
MURAKAMI, Akio, D. Ag., D. Sc., Associate professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-Aki, M. Sc.

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.
AOKI, Kenichi, Ph. D.
Laboratory of Evolutionary Genetics
MARUYAMA, Takeo,. Ph. D., D. Sc., Professor
WATANABE, Takao K., D. Sc., Associate professor
TUTIKAW A, Kiyosi, Associate Professor
GOJOBORI, Takashi, D. Sc.
Laboratory of Theoretical Genetics (Guest member)
MUKAI, Terumi, Ph. D., D. Sc., Professor
MIYATA, Takashi, D. Sc., Associate professor

5.

Department of Integrated Genetics
MATSUNAGA, Ei., D. Med., D. Sc., Head of the Department
Laboratory of Human Genetics
NAKAGOME, Yasuo, D. Med., Professor
HORAI, Satoshi, D. Med.
NAKAHORI, Yutaka

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Laboratory of Agricultural Genetics
MORISHIMA-OKINO, Hiroko, D. Ag., Professor
ENDO, Toru, D. Ag., Associate professor
FUJISHIMA, Tohru, D. Ag.
SATO-HIRAOKA, Yoichiro, M. Ag.

6.

Research Facilities
Genetic Stock Research Center
SUGIYAMA, Tsutomu, Ph. D., Head of the Center
FUJII, Taro, D. Ag., Associate professor
IYAMA, Shin-ya, D. Ag., Associate professor
KUSUDA, Jun, D. Ag.
INOUE, Yutaka, D. Sc.
SANO, Yoshio, D. Ag.
NISHIMURA, Akiko
DNA Research Center
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Department
SOEDA, Eiichi, D. Ag.
Experimental Farm
FUJII, Taro, D. Ag., Head of the Farm
MIYAZAWA, Akira

7.

Technical Section
MARUYAMA, Takeo, Ph. D., D. Sc., Chief of the Section

8.

Department of Administration
AKATsuKA, Takao, Head of the Department
TAwARA, Koichi, Chief of the General Affairs Section
aIDE, Yukio, Chief of the Finance Section

COUNCIL
Chairman
YAMAMURA, Yuichi; President, Osaka University
Vice-chairman
MOROHOSHI, Seijiro; President, Tokyo University of Agriculture andTechnology

COUNCIL

9

Members (Alphabetical order)

EGAMI, Nobuo; Dean, Faculty of Science, University of Tokyo
IINO, Tetsuo; Professor, Faculty of Science, University of TokyoINOUYE, Eiji; Emeritus professor, University of TokyoNAGAKURA, Saburo; Director-General, Institute for Molecular Science
NAKAJIMA, Tetsuo; Professor, Faculty of Agriculture, University ofTokyo
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; Director, Institute of Economic Research, Kyoto University
OOSAWA, Fumio; Professor, Faculty of Engineering Science, Osaka
University
OZEKI, Hamo; Professor, Faculty of Science, Kyoto University
SAITO, Hiuga; Director, Institute of Applied Microbiology, Universityof Tokyo
SAKAI, Fuminori; Director, Japan Society for the Promotion of Science
SASA, Manabu; President, Toyama Medical and Pharmaceutical University
TAZIMA, Yataro; Director, Institute of Silkworm Genetics and Breeding
WATANABE, Itam; Professor, Kitazato University

ADVISORY COMMITTEE
Chairman

MATSUNAGA, Ei; Director
Vice-chairman

TANAKA, Ryuso; Professor, Faculty of Science, Hiroshima University
Members (Alphabetical order)

FUKUDA, Ichiro; Professor, Faculty of Liberal Arts and Sciences, Tokyo
Women's University
MIURA, Kin-ichiro; Professor, Faculty of Engineering, University ofTokyo
MUKAI, Temmi, Professor, Faculty of Science, Kyushu University

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

OHBA, Shigeru; Professor, Faculty of Science, Tokyo Metropolitan
University
OKADA, Masukichi; Professor, Institute of Biological Sciences, University of Tsukuba
OSAWA, Shozo; Professor, Faculty of Science, Nagoya 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
HIROTA, Yukinori; Professor, Department of Cell Genetics
ISHIHAMA, Akira; Professor, Department of Molecular Genetics
KADA, Tsuneo; Professor, Department of Molecular Genetics
KIMURA, Motoo; Professor, Department of Population Genetics
KURODA, Yukiaki; Professor, Department of Ontogenetics
MARUYAMA, Takeo; Professor, Department of Population Genetics
MORIWAKI, Kazuo; Professor, Department of Cell Genetics
SUGIYAMA, Tsutomu; Professor, Department of Ontogenetics
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro; President, Emeritus Professor of Tokyo Metropolitan
University
KADA, Tsuneo; Managing Director, Professor of National Institute of
Genetics
KURODA, Yukiaki; Managing Director, Professor of National Institute of
Genetics
SINOTO, Yosito; Manager
WADA, Bungo; Manager, Emeritus Professor of University of TokyoTAZIMA, Yataro, Manager
OSHIMA, Chozo; Manager
YOSIDA, Tosihide H.; Manager
MATSUNAGA, Ei; Manager, Director of National Institute of Genetics

PROJECTS OF RESEARCH FOR 1984
1.

DEPARTMENT OF MOLECULAR GENETICS
laboratory of Molecular genetics

Studies on regulatory mechanisms of gene expression in E. coli (ISHIHAMA,FUKUDA and FUJITA)
Studies on molecular mechanisms of transcription and replication of animalviruses (ISHIHAMA and FUKUDA)
laboratory of Mutagenesis

Molecular mechanisms of radiation- and chemical-induced mutations (KADA,SADAIE, INOUE and TEZUKA)
Environmental mutagens, desmutagens and antimutagens (KADA)
Biochemical factors involved in cellular repair of genetic damage and induced mutagenesis (INOUE and KADA)
Genetics of Bacillus sUbtilis (SADAIE and KADA)
Molecular mechanisms of unicellular differentiation in Bacillus subtilis(SADAIE)
laboratory of Nucleic Acid Chemisry

Studies on the relationship between nucleic acid conformations and biological activities (MIURA)
Expression of B. subtilis genes (YAMANE)
Expression of the foreign genes in B. subtilis cell (YAMANE)
2.

DEPARTMENT OF CELL GENETICS
laboratory of Cytogenetics

Studies on species differentiation of mouse from cyto- and molecular genetic
view points (MORIWAKI)
Cyto- and immunogenetical mechanisms for regulating tumor development

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

in the laboratory and wild mice (MORIWAKI)
Immunogenetical studies on the mouse MHC (SHIROISHI* and MORIWAKI)
Development of new mouse strains from wild populations (MORIWAKI andSHIROISHI*)
Theoretical bases for chromosomal evolution in mammals and ants (IMAI)
Cytogenetical analysis of meiotic mechanisms in mice (IMAI and MORIWAKI)
Cytogenetical studies on Drosophila (YAMAMOTO)
Laboratory of Microbial Genetics

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

Peptidoglycan biosynthesis in E. coli (SUZUKI and HIROTA)
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 andFUJISAWA)
Studies on transformation and cell differentiation in higher organisms (NAWA
and YAMADA)
Mitochondrial DNA organization in male-sterile cytoplasms of rice (NAWA,
SANO and Fum)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)
Genetics and cytogenetics of the silkworm (MURAKAMI)* Genetics Stock Center

PROJECTS OF RESEARCH FOR 1984

13

Mutagenesis in germ cells of the silkworm (MURAKAMI)
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)
Theoretical studies on variation and evolution of extranuclear DNA (TAKAHATA)
Theoretical studies on the evolution of altruism (KIMURA and AmG)
Population genetical studies on gene-culture coevolution (AOKI)Laboratory of Evolutionary Genetics

Theory of population genetics and evolution (MARUYAMA)
Studies on molecular evolution (GOJOBORI)
Evolutionary genetics of Drosophila (WATANABE)
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 (MIYATA)
5.

DEPARTMENT OF INTEGRATED GENETICS
Laboratory of Human Genetics

Genetic and cytogenetic studies on retinoblastoma and Wilms' tumor(MATSUNAGA, NAKAGOME and HORAl)
Clinical cytogenetic studies of congenital abnormalities in man (NAKAGOME)
Molecular cytogenetic studies of human chromosomes (NAKAGOME)
Studies on DNA polymorphisms in human populations (HORAl and
MATSUNAGA)

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

laboratory of Agricultural Genetics

Evolutionary studies in wild and cultivated rice species (MORISHIMA and
SATO)
Ecological genetic studies in weed species (MORISHIMA)
Genetic studies on rice reserve proteins (ENDO)
Behavioral genetic studies in animals (FUJISHIMA)
6.

RESEARCH FACILITIESGenetic Stock Research Center

Studies and conservation of germplasm resources in rice and wheat species(Fum and SANO)
Specificity of mutagen tolerance in higher plants (Fum)Exploitation of genetic ability of nitrogen fixation in Gramineae (Fum,SANO and IYAMA)
Studies on genetic differentiation in rice (SANO)
Theoretical studies on breeding techniques (IYAMA)
Genetic studies of trees in natural forest (IYAMA)
Genetic effects of environmental pollution on plant population (IYAMA andMORISHIMA)
Studies on gene transfer in Gramineae (SANO and Fum)
Documentation of genetic stocks in Japan (hAMA)Studies on the management system of genetic stocks information (IYAMA)
Studies on chromosomal polymorphism in Drosophila (INOUE)
Analysis of fibroin genes of silkworm and its relatives (KUSUDA)
Molecular studies on the origin of silkworm (KUSUDA)
Coordination of DNA synthesis and cell division in E. coli (NISHIMURA andHIROTA)
Synthetic ColEl plasmids carrying genes for cell division in E. coli (NISHIMURA)
DNA Research Center

Studies on primary structure of DNA (SOEDA)
Rugulatory mechanisms of gene transcription (ISHIHAMA)DNA data analysis (MARUYAMA)RESEARCH ACTIVITIES IN 1984I.

MOLECULAR GENETICS

Promoter Selectivity of E. coli RNA Polymerase, I.
Promoter Strength Recognized by Regnlar Holoenzyme
Teruaki NOMURA, Nobuyuki FUJITA and Akira ISHIHAMA

Promoter strength is determined by two factors: i) the binding affinity to
RNA polymerase and ii) the rate of isomerization from "closed promoterRNA polymerase complex" to transcriptionally active "open complex".
Previously we developed an in vitro mixed transcription system for quantitative determination of the promoter strength leading to productive transcription (for details, see Nucleic Acids Res. 11, 671-686; ibid. 11, 38733889). This year we determined the transcriptional organization and thepromoter strength of dnaQ and rnh genes, which codes for the DNA polymerase III e subunit and the ribonuclease H, respectively, both involved in
DNA replication. Transcription starts from two promoters for dnaQ anda single promoter for rnh, and proceeds on the same DNA region but into
opposite directions (for details, see J. BioI. Chern., 260, 7122-7125). The
three promoters carry different levels of the promoter strength (Fig. 1).
The regular holoenzyme appears to recognize the three promoters differentially because the relative level of transcription initiation among the threepromoters varied depending on the enzyme concentration. After separation of the three promoters into individual DNA fragments, the activity ofrnh promoter increased markedly (Nucleic Acids Res., in press). This implies
a promoter interference between the convergentIy transcriped genes.
The promoter strength is being determined for increasing numbers of E.
coli promoters including those involved in the three mixed operons (nusA,
tulE and divE, each composed of tRNA and protein genes), a heat-shock
gene (groE), two aminoacyl-tRNA synthetase genes (alaS and gInS), a
suppressor tRNA gene (leuX) and two catabolite-sensitive genes (malK andmalE).

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

4.0

~

laeP

3.0

~ recAp

= 2.0

2
E
'"
o
~

o

0...

laePUV5

1.0

lufBp
@

o

o

1.0
Parameter I

Fig. 1.

(k)

2.0

Map of promoter strength.

Promoter Selectivity of E. coli RNA Polymerase, It
Structural and Functional Intercmrversion of
RNA Polymerase by Nucleotide Factors
Akira ISHIHAMA, Teruaki NOMURA and Nobuyuki FUJITA

Increased numbers of evidence indicate that the control of gene transcnption in E. coli occurs through regulation of the level and specificity of functional RNA polymerase. Some mutations of the genes coding for the four
enzyme subunits, p', p, IX and (J, affect the promoter selectivity of the RNA

RESEARCH ACTIVITIES IN 198417

polymerase (for details, see Mol. Gen. Genet. 193, 8-16). RNA polymerase
seems to be interconvertible between various forms or states through physicalinteraction with a number of accessory proteins, termed transcription factors,
and nucleotides such as ppGpp and specific tRNA. Using the in vitro
mixed transcription system, we demonstrated that RNA polymerase lostthe recognition property to some specific promoters in the presence ofppGpp, a chemical mediator of stringent control. For example, transcription initiation from the strong promoter PI of an rRNA gene (rrnE) is inhibited by ppGpp but that from P2 is insensitive to it (for details, see J.
BioI. Chem. 259, 1951-1957). Likewise, the upstream promoter PI, oneof the two major promoters of the ribosomal protein Sl gene (rpsA) , issensitive to ppGpp but the downstream P3 is insensitive to it. These observations indicate that not all the promoters belonging to stringently controlled genes are sensitive to ppGpp. A discrimination mechanism seems
to operate such that the basal level of gene expression under a poor supplyof nutrients is maintained through operation of only the ppGpp-insensitive
weak promoters within multiple promoters of stringently controlled genes.
Studies on possible influence of specific tRNA on the promoter selectivityof RNA polymerase is also in progress, in particular with respect to therecognition of promoters within tRNA genes.
PI
v

P2

rrnE

'II'

[ppGpp)

rpsA[ppGpp)Fig. 1. Differential stringent control.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Promoter Selectivity of E. coli RNA Polymerase, III.
Structural and Functional futerconversioJll of
RNA Polymerase by Protein Factors
Teruaki NOMURA, Akira ISHlHAMA and Nobuyuki FUJITA

Our systematic search for protein factors was based on the idea that suchproteins with regulatory functions might form complexes with RNA polymerase prepared under mild conditions. Attempts were made to isolate
and characterize proteins copurified along with RNA polymerase under mild
conditions. More than 10 protein species have been identified as possible
accessory proteins of RNA polymerase, including NusA and p factors,
both involved in transcription termination, GroE ATPase and some otherheat-shock proteins, and SSP (stringent starvation protein). Among thehigh molecular-weight protein fraction, we identified a translational initiation factor IF2. Alteration of the promoter selectivity of RNA polymerase,when interacted with these proteins, is being examined using the in vitro
mixed transcription system.
Phosphorylation of RNA polymerase might also be involved in thecontrol of RNA polymerase activity (for details, see J. BioI. Chern. 259,
526-533). To test this possibility, attempts are being made to develop an
in vitro system for quantitative phosphorylation of RNA polymerase andto isolate E. coli mutants defective in protein phosphorylation.
Promoter Selectivity of E. coli RNA Polymerase, IV.
Transcription of a Heat Shock Gene
Nobuyuki FUJITA and Akira ISHIHAMA

When E. coli cells are exposed to high temperature, the synthesis of a groupof proteins, called heat-shock proteins, increases transiently. To approach
the molecular mechanism by which the heat-shock response is achieved,transcription of the groE gene, whose products were the most prominent
among heat-shock proteins in E. coli, was analyzed in vivo and in vitro.
Transcriptional start site in vivo was determined for the groE gene by Sl
nuclease mapping and reverse transcriptase mapping. In addition, theseexperiments clearly showed that the level of groE mRNA probe was markedly increased after heat shock and well paralleled the increased rate of protein synthesis, indicating that the regulation of the heat-shock response is

RESEARCH ACTIVITIES IN 198419

mainly achieved at the level of transcription. An in vitro system, in whichtranscription is initiated from the groE promoter, was constructed using
core RNA polymerase and purified htpR (rpoH) gene product, the positive
regulator of the heat-shock response in E. coli. Using this in vitro system,
further studies on the mechanism of the action of htpR protein, along withthe functional relationship between htpR protein and sigma factor, are in
progress.
Cloning of the Genes for the Possible Transcription
Factors of E. coli--Analysis of the Gene for
Stringent Starvation Protein (SSP)
Ryuji FUKUDA, Hiroaki SERIZAWA and Ryoji YANO*

Until now, up to ten polypeptides have been reported as the possible transcription factors of E. coli. In addition, we have found more than ten polypeptides in the enzyme-bound forms in RNA polymerase preparations atthe various purification steps. In order to address the physiological functions of these proteins, we have started to clone the genes for these proteins.
Cloning allows mapping of the gene on the E. coli chromosome, and permits
the introduction of mutations useful for understanding the physiologicalrole of these proteins.
This year we tried to isolate the genes for two polypeptides, SSP (stringent
starvation protein, 22.5 K) and (J) (10 K), which were stably associated with
RNA polymerase. Until now, we succeeded to clone the gene for SSP.
Usually E. coli cells contain about several thousand molecules of SSP.
However, when cells are exposed to extreme nutrient starvation, the protein
is synthesized predominantly, sometimes occupying more than 50 % of total
protein synthesis. Reconstitution experiments revealed that SSP binds to
RNA polymerase holoenzyme, forming an equimo1ar complex at the saturation. In contrast, no complex was formed between SSP and core enzyme.
Purified SSP considerably influences (mostly reduces) the activity of holoenzyme but not of core enzyme in an in vitro transcription system using
phage DNAs or synthetic polymers as template.
For isolation of clones containing the SSP gene, we employed a method
developed by Suggs et al. (1981). We first determined the sequence of the
N-terminus of SSP as well as the sequence of two cyanogen bromide-ge-* Institute for Virus Research, Kyoto University.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

nerated peptide fragments. We then chemically synthesized four sets of
mixture of oligodeoxyribonucleotides, representing all possible codon combinations for parts of these amino acid sequences. Restriction endonucleasefragments of E. coli DNA that hybridized with these probes were cloned
into pBR322. After analyses of partial base sequences for the cloned DNA
fragments and of proteins encoded by the DNA fragments, we identified aplasmid carrying a complete structural gene of SSP. The complete sequence
was determined using various restriction fragments subcloned into the pUC-9
vector.
For mapping of the SSP gene locus on the E. coli chromosome, we haveintegrated the recombinant plasmid containing Amp gene into the chromosome of E. coli Hfr stain carrying polA mutation. The plasmid was integrated into the chromosomal SSP gene by homologous recombination.
Location of the integrated Amp gene and the SSP gene is being determinedby Hfr mating and PI transduction technique (in colaboration with Dr. A.
Nishimura, Genetic Stocks Center of this institute).
To elucidate the function of SSP, we are now studying the regulation of
SSP synthesis in vivo and in vitro, and trying to determine whether the protein
is essential for cell growth or not, employing the SSP plasmid integration
technique. Studies are also in progress to isolate mutants of the SSP gene,using the SSP plasmid.
Structure and Function of RNA-dependent RNA Polymerase
Associated with Influenza Virus, I.
Isolation amI. Characterization
Atsushi KATO, Susumu UEDA* and Akira ISHlHAMA

The RNA-dependent RNA polymerase associated with influenza virus
plays an essential role in transcription and replication of viral genome ininfected cells. To determine the structure and function of the virion-associated RNA polymerase, we made a systematic attempt to isolate the enzyme
in an active form from influenza virus A/PRS. The procedure includes: i)disruption of virus particles with a non-ionic detergent; ii) removal of envelope proteins, HA and NA, by cesium trifluoroacetate centrifugation; andiii) removal of M and NP proteins by repeated chromatography on phosphocellulose columns. The resulting RNA polymerase complex is able
* Nippon Institute for Biological Science.

RESEARCH ACTIVITIES IN 198421

to catalyze not only dinucleotide-primed RNA synthesis but also endonucleolytic cleavage of capped RNA and initiation and elongation of cappedoligonucleotide-primed RNA synthesis. This enzyme complex contained
only three P proteins, PB 1, PBl and PA, but lacked NP protein. Taken
together we concluded that the RNA polymerase is composed of the three P
proteins and that NP is no required at least for primary transcription. Purification of the RNA polymerase devoid of viral RNA is in progress. For
details, see Virus Research 3, 115-127.
Structure amI Function of RNA-dependent RNA Pol.ymerase
Associated with Influenza Virus, n.
Possible Proof-Reading Function
Akira ISHIHAMA, Kiyohisa MIZUMOTO* and Atsushi KATO

The RNA-dependent RNA polymerase of influenza virus cleaves capped
RNA of host cells at the 5' side of either A or U residues located about 11
to 13 bases from cap termini, and utilizes the resulting capped RNA fragments as primers for the synthesis of viral mRNA (for details, see Nucleic
Acids Res. n, 3637-3649). Transcription commences with the addition of
GMP residue, which is complementary to the second nucleotide at the 3'termini of all eight viral RNA segments, to the capped primers. When highconcentrations of GTP are added as a sole substrate, however, multiple GMP
residues are polymerized to the primers. As illustrated in Fig. 1, the erroneously polymerized GMP residues, other than the first one, were foundto be removed prior to incorporation of eMP, the nucleotide complementary
0

Cap·1

e
0

@

PolyU

~
~ GTP

!

crp

t

pGpG
A

pGpC
6

---------

Fig. 1.

Proof-reading reaction.

* Institute of Medical Sceince, University of Tokyo.

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

to the third nucleotide at the 3'-termini of viral RNA. The results suggestthat the RNA-dependent RNA polymerase carries a proof-reading function
similar to that of DNA polymerases. In contrast to the proof-reading
reaction by DNA polymerases, the reaction by the influenza viral RNA
polymerase takes place only in the presence of substrates, implying a coupled
reaction between the removal of misincorporated nucleotides and the polymerization of correct nucleotides.
Analysis of Temperature-sensitive Mutants of Influenza Virus:
The Mutants of :RNA Segment 8
Masakazu HASEGAWA, Ryuji FUKUDA and Kazufumi SHIMIZU*

Temperature-sensitive (ts) mutants of influenza A virus are useful inunderstanding the genetic organization of the virus genome and the mechanism of virus replication. Previously Shimizu et al. (1982) isolated 83
mutants of influenza AjUdornj72 virus that were ts on primary rhesus
monkey kidney (RMK) monolayer cultures. These mutants had beenclassified into 13 complementation groups and 8 recombination groups
corresponding to each of the eight genomic RNA segments. As the firststep of this research, we have analyzed these ts mutants defective in thesegment 8.
Among these groups, the recombination group H had ts lesion(s) on the
RNA segment 8, as indicated by segregation analysis. The segment 8
encodes two nonstructural polypeptides, NSI and NS2, the functions ofwhich are entirely unknown. The mRNA encoding NS2 is thought to be
produced by splicing of NSI mRNA. However, the mutants of group H
were classified into 4 complementation groups on RMK cells, and 2 or 3
complementation groups on Madin-Darby canine kidney (MDCK) cells.
To address the uncertainties in the complementation analysis and assign
the mutants to each of the two polypeptides encoded by the segment 8, we
performed nucleotide sequence analysis of three mutants. Double-stranded
cDNAs prepared from genomic RNA of these mutants were cloned into
pBR322. Sequencing of the cloned DNA fragments revealed base substitutions in the segment 8 RNA. This was confirmed by directly sequencing that region of viral RNA, employing the chain terminator method, inwhich the RNA was transcribed with AMV reverse transcriptase and 5'* Nihon University, School of Medicine.

RESEARCH ACTIVITIES IN 1984

23

3zp_labeled restriction fragments as the primer. Two of these mutants, ICR
1629 and SPC 45, have defects in the NS1 coding region which is spliced out
for NS2 mRNA. The third mutant, ICR 516 has a defect in the NS2 coding
region but downstream of the NSI coding region. The synthesis of Ml
protein, which normally accumulates late in infection, was greatly reduced
in MDCK cells infected with those NSI ts mutants at 30°C compared to at
34°C. No significant difference was seen with NS2 ts mutant ICR 516 at
40°C on the synthesis of virus-specific protein. The results may indicatethat NSI participates in the switch from early to late protein synthesis.
Using an improved hybridization method, we are measuring the synthesis
ofmRNA, cRNA and vRNA of the eight RNA segments, separately, during
the infection of these mutants.
Structure and Function of Reverse Transcriptase Associated with
Avian Myeloblastosis Virus
Atsushi KATO, Akihiro NODA*, Susumu UEDA**
and Akira ISHIHAMAThe RNA genome of retroviruses is reverse-transcribed into doublestranded DNA, which is integrated into the host chromosome as the provirus, and both viral mRNA and progeny RNA are produced through transcription of the provirus DNA by DNA-dependent RNA polymerase II ofhost cells. The reverse transcriptase associated with retroviruses are believed
to catalyze not only the multiple step reactions leading to the synthesis of
double-stranded DNA from viral RNA but also the integration of viral
DNA into host chromosome. Using a new strategy for enzyme purification,
we established that most, if not all, avian retroviruses contain three forms,
a, a/3 and [32' of reverse transcriptase. To identify the role of each enzyme
form, we improved the purification procedure, which allowed us to purify
large amounts of the three enzyme forms of AMV reverse transcriptase to
the same extent of homogeneity (for details, see J. Virol Meth. 8, 325-339).
Comparison of the catalytic properties of the three enzyme forms indicatedthat the /32-form enzyme catalyzed the RNA-directed synthesis of DNA
efficiently whereas the a-form enzyme was the most active in the ssDNAdirected synthesis of dsDNA. Using a purified preparation of pl5 endo-* Central Research Institute, Takara Shuzo Co.
**

Nippon Institute for Biological Science.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

peptidase, we succeeded in processing in vitro of ,B2-form enzyme to a,Band a-form enzymes. Preliminary analysis of alteration of enzyme function indicated that both DNA-dependent DNA polymerase and DNA
endonuclease activities increase concomitantly with the processing of ,B
subunit.
Analysis of Mitochondrial DNA in Cytoplasmic
Male Sterile Rice
Saburo NAWA, Yoshio SANO and Taro FUJI!

Recently, Yamaguchi and Kakiuchi (1983, Japan. J. Genet. 58: 607)
discovered mtDNAs, B-l and B-2, in a cytoplasmic male sterile (cms) strainof rice. To obtain further information on the behavior of mtDNA, induction of a fertile revertant from a cms-strain of rice was investigated using
mutagen treatments. Seeds of a cms-strain, (cms-boro)rf1rft, were treatedby gamma-rays, ethyl methanesulphonate (EMS) and acridine orange withvarious intensities or concentrations. Two fertile plants were obtainedwith EMS treatments among 6500 treated seeds in total. These reverted
plants, designated 80-1 and 81-3, indicated reversion of the cytoplasmic
factor from cms to normal state, because the revertants showed complete
sterility when crossed as the pollen parent to the original cms-strain.
The following 5 rice strains, viz., (cms-boro)rftrft, (cms-boro)Rf1Rft,(n-boro)rf1rfb 80-1 and 81-3, were used for examination of mtDNA. Because few seeds are available in the ems-strain, a tissue culture technique wasutilized with B-5 medium, and callus cells propagated from seeds were analyzed. Calluses were homogenized in mannitol solution and mitochondria
were fractionated by repeated centrifugations. DNAs were extracted fromthe mitochondria in sodium sarkosyl-pronase, followed by purification withphenol, chloroform and RNAse treatments. Mitochondrial DNAs wereanalyzed in agarose gels. The gel electrophoresis revealed a main highmolecular DNA band in preparations from all of 5 strains. Preparations
from male sterile cytoplasms, (cms-boro)rf1rfl and (cms-boro)Rf1Rfl' were
found to contain two additional fast-migrating bands (B-l and B-2) whereas
no such DNAs were detected in preparations from normal cytoplasm[(n-boro)rf1rflJ and 2 revertant cytoplasms, indicating that reversion of cms
to fertile cytoplasm was associated with the disappearance of mitrochondrial
plasmid DNAs. This suggests a specific involvement of these plasmids in

RESEARCH ACTIVITIES IN 198425

the male sterility of rice.
The plasmids from cms cytoplasm were further purified by equilibrium
centrifugation in CsCl-ethidium bromide gradients. Both of B-1 and B-2
were recovered from the lower bands while the main mitochondrial DNAs
were recovered from the upper band. B-1 DNA treated with a restriction
enzyme moved slower than untreated DNA in agarose gel electrophoresis.
The same was in the case of B-2. From these results, B-1 and B-2 were
estimated to be supercoiled circular DNAs (cccDNA) having molecularweight of 2.3 and 1.6 kb, respectively.
Concerted Evolution of the Mouse Immunoglobulin
Gamma Chain Genes
H. HAYASHIDA, T. MIYATA, Y. YAMAWAKI-KATAOKA,
T. BONJO, J. WELS and F. BLATTNER

The nucleotide sequences of the immunoglobulin heavy-chain constantregion genes, Cr3, Crl, Cr2b and Cr2a, of the mouse, together with that ofa human equivalent Cr4 were compared. It was shown that all the six
pairs of genes within the mouse Cr gene family contain DNA segments thatexhibit marked homology, whereas no such segmental homology was foundin the case of inter-species comparison. This result indicates that the four
Cr gene of the mouse evolved concertedly by exchanging parts of their genetic
information with each other through mechanism of either gene conversion
or double unequal crossing-over. Another example for such concerted
evolution was found in gene regions encoding membrane domains of themouse Cr chains. We further searched such segmental homologies in othermammalian Cr gene families and found at least two more examples in man
and guinea-pig. In the mouse Cr gene family, the silent positions of an
exon encoding the third domain of Cr chains show divergence in sequencemuch more strongly than other regions, indicating that the genetic information encoded by this gene region was least scrambled throughout recent
evolution. A phylogenetic tree constructed from the nucleotide differencesof this exon demonstrates that at least two Cr genes had already existed
before mammalian radiation. Based on these results, evolution of mammalian
Cr gene families was discussed. For details, see EMBO J. 3, 2047-2053.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

II.

MICROBIAL GENETICS

Purification and Sequencing of the Active Site Tryptic
Peptide frl()ID Penicillin-binding-Protein Ib
of Escherichia coli
Robert A. NICHOLAS, Hideho SUZUKI, Yukinori HIROTA
and Jack L. STROMINGER

This paper reports the sequence of the active site peptide of PBP 1b.
Purified PBP 1b was labeled with [14C] penicillin G, digested with trypsin,and partially purified by gel filtration. Upon further purification by HPLC,
two radioactive peaks were observed, and the major peak, representing over
75 % of the applied radioactivity, was submitted to amino acid analysis andsequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The
active site nucleophile was identified by digesting the purified peptide withaminopeptidase M and separating the radioactive products on HPLC.
Amino acid analysis confirmed that the serine residue in the middle of thesequence was covalently bonded to the (14C]penicilloyl moiety. A comparison of this sequence to active site sequences of other PBPs and p-lactamases is presented. (For detail, see Biochemistry 1985, 24, 3448-3453)
Identification of the Active Site in Penicmin-binding
Protein 3 of Escherichia coli
Robert A. NICHOLAS, Jack L. STROMINGER, Hideho SUZUKI
and Yukinori HIROTA

We report the sequence of the active site tryptic peptide of penicillinbinding protein 3 from Escherichia coli. Purified penicillin-binding protein
3 was laveled with [14C]penicillin G, digested with trypsin, and isolated by
a combination of gel filtration and high-pressure liquid chromatography.
The major radioactive peak from high-pressure liquid chromatography wassequenced, and the sequence Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-SerThr-Val-Lys, which comprises residues 298-310 in the gene sequence, wasobtained. This sequence is compared to the active site sequences fromother penicillin-binding proteins and p-lactamases. (For detail, see J.

RESEARCH ACTIVITIES IN 1984

27

Bacterial. 1985)
A CmnparisOlI:l. of Amino Add Sequences of the Active
Centers of Pemdmn Binding Protein (PEP -lb, -3,
Carboxy Peptidase (CPase) and fi-Iadamase
Hideho SUZUKI, Yukinori HIROTA, Robert A. NICHOLAS
and Jack L. STROMINGER

Enzyme Bacteria Amino acid sequence of the active center
CPase

B. subtilis

CPase
PBP-lbB. stcarotlzermophilus
E. coli

PBP-3

E. colij;'laetamaseS. (lureus

Phe(ClassA)

B. sereus

Phe

B. licheniJor11lis

Phe

E. coliPhe

Leu-AlaAla

Pro-Mel-MetSer

Thr Val

Lys

Ser

Thr

Ser

Lys

Ser

Tht"

Tyr

Lys

Set"

Tht"

Ile - Lys

Set"

Thr

Phe

Lys

* Ser and Lys residues of the penicillin reacting peptides are conunon in all the proteinsused. Boxed amino acids, show amino acid residues in common at the relative positions. (1) Waxman, Strominger: J. BioI. Chern., 1980. (2) Yocum, Rasmussen, Strominger: J. BioI. Chern., 1980. (3) Nicolas, Suzuki, Hirota, Strominger: Biochemistry, 1985.
(4) Keck, Glauner, Schwarz, Broome-Smith, Spratt: Proc. N. A. S. 1985. (5) Nicolas,
Strominger, Suzuki, Hirota: J. Bacteriol, 1985. (6) Ambler: Phil. Trans. R. Soc. Lond.,
1980.
Sites of dnaA ProteiJffi-binding in the Replication
Origin of the Escherichia coli K-12 Chromosome
Minami MATSUI, Atsuhiro OKA, Mitsuru TAKANAMI,
Seiichi YASUDA and Yukinori HIROTA

On the basis of the observation that dnaA protein binds preferentially to
DNA fragments carrying the Escherichia coli chromosomal replication origin(oriC), the binding sites were investigated by DNase I footprinting. As aresult, three strong binding sites were identified in the minimal oriC sequence.
The respective binding sites were 16 to 17 base-pairs long and contained acommon sequence (5') T-G-T-G-(GjT)-A-T-A-A-C (3') in the middle, although their polarities were not the same. Since mutants defective in func-

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

tion for autonomous replication have been isolated in the corresponding
positions of the common sequence at each binding site, dnaA protein-bindingat these sites seems to be significant for replication initiation. (For detail, see
J. Mol. BioI. 184: 529-533, 1985)
A Mutation Uncouples DNA Replication AmI Cell
Division in Escherichia coli
Akiko NISHIMURA

I have isolated a mutant of E. coli whose DNA replication and cell division are uncoupled.
Bacterial cell division is tightly coordinated with DNA replication.
PAT42, a thermosensitive mutant of DNA replication (dnaB42) forms filamentous cells when DNA synthesis is blocked at 41 ° (Hirota et al. 1968,
Cold Spring Harbor Symp. Quant. BioI. 33: 677).
A culture ofPAT42 was treated with 3 cycles of heat pluse (41 ° for 2 hours)
and survival colonies were isolated at 30°. One of these survivors, strain
J£6009, had the following properties; (1) JE6009 cells continued to divide
at 41° for several hours. Whereas the parental strain PAT42 grew as long
filamentous forms (longer than 10 ,um) when incubated at 41 ° for 2 hours,
the JE6009 culture contained 30 % normal-sized cells (1.5-2 ,urn) and 70 %short filamentous cells (shorter than 6,um). (2) During this time, the number of survivals were counted by colony forming ability at 30°. Survivals
ofPAT42 decreased logarithmically after 30 minutes lag, but those of J£6009
were constant over 2 hours at 41°. (3) DNA synthesis of both strains stopped
immediately after being shifted to 41°. (4) dnaBts mutation was co-transduced with malB+ by PI-phage from JE6009 to W3876 (malB-) strain. Thesetransductants formed long filamentous cells at 41
These results indicate that JE6009 carries a mutation, which uncouples
DNA replication and cell division. Furthermore, cell division of JE6009
also occurred when hydroxyurea (1.5 mg/ml) or nalidixic acid (10 ,ug/ml)
was used to stop DNA synthesis, whereas division of PAT42 was inhibitedcompletely under these conditions. Colony size of PAT42 at 30° was uniform but size-distribution, containing mini-colonies smaller than that of
PAT42, was found in JE6009. The number of colonies at 30° per a given
cell, from 30° cultures of 1£6009 was less than 1/12 that of PAT42. The
mutation in JE6009 might result in the initiation of cell division before
0.

RESEARCH ACTIVITIES IN 1984

29

completion of DNA replication and in reduction of survival cells even under
permissive conditions for DNA replication.
This mutant gene in JE6009 was not involved in SOS-pathway because
the phenotypes mentioned above did not change when recA, lexA, sfiA, orsjiB genes of strain JE6009 were substituted for the genes known to be wild
by PI-transduction. The ftsA protein has been suggested as being involvedin a mechanism that coordinates DNA replication and cell division througha pathway independent of the SOS-induced response (Tormo et al. 1985 J.
Gen. Microbial. 131: 239). The mutation in JE6009 was mapped close to
the thyA gene, and far from the ftsA gene by mating experiments with
Hfr-T42 (dnaBtS).
The relationship between the expression of a mutatnt gene in JE6009 andftsA gene, and/or the SOS-mediated inhibition of division remains to beelucidated.
Effect of the sacUh 32 Mutation 011 the Growth and Spore
Outgrowth of the div-341 Strain of Bacillus subtilis
Yoshito SADAIE and Tsuneo KADA

To elucidate the genetic control of asymmetric forespore septum formation in B. subtilis, we examined the effect of temperature sensitive septum
initiation mutations on sporulation and sporulation associated events in
B. subtilis. One of such mutations, div-341, showed early spoO phenotypes
at an intermediate permissive temperature (37°C) and its genes product was
assumed to be involved in a step required for the excretion of some exoenzymes and cell surface proteins. The sacU h 32 mutation on the other
hand showed a hyperproduction of exoenzymes and derepressed sporulation in the presence of excess nutrients. Both mutations are closely linked
by transformation. A double mutant with div-341 and sacU h 32 was constructed to examine the interaction of both genes. Although the filamentous
growth of the div-341 strain at 42°C was not recovered in the mutant, theslower growth and defective spore outgrowth (twisted outgrowing spore) of
the div-341 strain at 37°C were recovered in the double mutant. Thesesuggest that the sacU h 32 gene interacts with the div-341 gene directly or
indirectly in some step of cell growth or spore outgrowth (J. Bacteriol. 163(2), 1985, 648).

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

III.

IMMUNOGENETICS

A Lethal Gene Mapped within the Mouse Major
Histocompatibility Complex.
Toshihiko SHIROISHI, Tomoko SAGAI and Kazlio MORIWAKI

We have produced a series of new congenic strains to introduce the H-2
complex of Japanese wild mouse on the genetic background of C57BL/IOJ
strain. Some of them show enhanced intra-H-2 recombination (Shiroishi
et at. Nature 300: 370-372, 1982). More than twenty recombinant H-2
haplotypes were generated from one of such congenic strains, BIO. MOLSGR. A recombinant haplotype awl8 is derived from BIO. MOL-SGR/
BIO. A heterozygote. For the purpose of obtaining awl8 homozygote andfixing this haplotype, we made intercross of awl8/k and awl8/b heterozygotes. Two hundred fourty progeny were subjected to H-2 typing by cytotoxicity test, but none of them was homozygous for the awl8 haplotype.
The segregation ratio of each genotypes was; awI8/awI8: awl8/k or awI8/b:
k/k or b/b=O: 168: 72. These results strongly suggest that the awl8 haplotype carries recessive lethal gene. Since the lethality is tightly associated withthe homozygous state for the awl8 haplotype, it is likely that the lethal geneis linked to the H-2 complex. However, the possibility that this gene is located outside of the H-2 complex can not be ruled out. To make it clear if
the gene is mapped within the H-2 complex and to determine the fine location of the gene, we produced further recombinant haplotypes from the heterozygotes of awl8/k and awI8/b. Finally, eight independent recombinants
were generated. Their recombination break points were determined basedon the identification of the origin of genes within the new recombinant H-2
haplotypes. Subsequently, the lethality of the mouse homozygous for therecombinant haplotype was examined. The lethality was judged fromabsence of the homozygote in the progeny generated from the intercross ofthe heterozygotes of the recombinant and k or b haplotype at the age offour weeks after birth. The results are summerized in Table 1. Since a
recombinant haplotype aw26 whose proximal region to E" locus is derived
from k haplotype retains lethality, the lethal gene is mapped to distal partto E". A haplotype aw20, in which recombination occurred between the

RESEARCH ACTIVITIES IN 1984
Table 1.

-31

The lethality of the homozygote for the aw18-derived
recombinant haplotype
Genotype

Haplotype
wm?!)
aw18
aw20
aw21
aw22
aw23
aw24
aw25
aw26
aw2?

!)
2)

3)

Lethality2)

K

A~

Aa

E~

Ea

Sip

Ss

D

w
w
w
w
k
w
w
w
b
w

w
w
w
k
w
k
b
b
b
w

w
w
w
k
w
k
b
b
b
w

w
w
w
k
w
k
b
b
b
w

w
w
w
k
w
k
b
b
b[
w

w
d
d
k
d

w
d
d 13)
k
d
k
b
b
d
? I

w
d
k
k
d
k
b
b
d
k

k
b

b
d
dl

ViableLethalLethalViableLethalViableViableViableLethalLethalOriginal H-2 haplotype from which aw18 was derived.
Lethality was judged from the absence of recombinant homozygote in the offsprings
from the cross of Rib x Rib or R/k x R/k at 4 weeks after birth.
Vertical bar indicates the position of recombination.

locus of sIp and D, and distal region to the D locus came from k haplotype
still keeps lethality. Therefore, the lethal gene is located in the proximal
region to the D locus. Taken otgether, this gene appears to be mapped
to the E,,-D interval of the H-2 complex. All data obtained from the otherrecombinant haplotypes were completely consistent with the above conclusion. At present, there is no evidence that initial genetic recombinationin awl8 directly caused the lethal mutation, but it seems highly likely thatthe recombination event is related to the emergence of the lethality, because
the recombination break point is also located in the E,,-D interval.
In order to determine the stage that the lethality appears, we made theintercross of awl8/b heterozygote. The progeny was scored for the H-2
genotype at different three postnatal stages. As shown in Table 2, thirty
six percent of progeny were scored as homozygote at one day after birth.
After this stage, the frequency of the homozygote decreased, and none of
the homozygotes failed to survive beyond 15 days after birth. The result
led to the conclusion that the lethal gene begins to act just after birth. Although we have made preliminary characterization of this lethal mutation,
pathological investigation has not been done yet to elucidate the mechanism
to cause the lethality. Identification of the product of this lethal gene may

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35
Table 2. The segregation of the H-2 genotypes in the viable progeny
generated from the intercross of aw18/b at postnatal stages
Postnatal
stage

Litter No.

Genotype/Segregation

1 day

4 (x=8.3±1.5)5 days4 (x=7.7±1.7)15 days

6 (x=7.0±2.0)

aw18/aw18: aw18/b or bib
1221
aw18/aw18: aw18/b or bib
3
23
aw18/aw18: aw18/b: bib
30
0
13

%

Homozygote
36.4
11.5
0.0

x: Mean number of the viable progeny per litter

shed light on understanding the biological function of this gene and thecause of the lethality.
Cytoplasmic Gene Flow in Japanese Mice
Mus musculus molossinus
Hiromichi YONEKAWA, Osamu GOTOH, Yusaku TAGASHIRA,
Shunsuke MIGITA, Ze-Chang Yu, De-Yyan Lu, Wang Su CHO,
Nobumoto MIYASHITA and Kazuo MORIWAKI

Taxonomists propose that there are two subspecies of house mouse Mus
musculus in Eastern Asia; i.e. M. m. castaneus and M. m. molossinus. The
former occupies Southeastern Asia and South China, whereas the latter isfound in the Far East, especially Japan. The mice belonging to these two
subspecies can be easily distinguished from each other by restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA)(Yonekawa et al. Jpn. J. Genetics 55,289-296: 1980; Genetics 98: 801-816,
1981) as well as by biochemical markers encoded by the nuclear genome(Bonhomme et al. Biochem. Genet. 22: 275-303, 1984).
As for Japanese mice, we previously showed that there is no heterogeneity
in RFLP of mtDNA of mice collected at 15 localities distant from each other(Yonekawa et al. Differentiation 22: 222-226, 1982), although these mice
have extensive polymorphism of nuclear genes. On the other hand, considerable polymorphism in both cytoplasmic and nuclear genes are foundin other Asiatic subspecies such as M. m. castaneus and M. m. bactrianus.
These results suggest that some special events took place in Japanese mice.
In order to clarify this matter, we collected mice in 17 additional localities

RESEARCH ACTIVITIES IN 1984
Table 1.

subspecies
Collection
locality33

The subspecies of Mus musculus used, their sources andthe set of restriction enzyme cleavage patterns of
mtDNAs obtained from each sample
Restriction patterns*

Nos.

_

used Bm Ec H2 H3 Hp 1 He 2 Ps Bg Hp 2 He 3 Tq Hf Mb

M. m. bactrianus
Afganistan
Kabul
PakistanLahol
M. m. castaneus
Indonesea
Bogor
Malaysea
Kota Kinabal
Philippines***
Quezon city
TaiwanTaichunM. m. molossinus
Japan***
castaneus-type
mtDNA
molossinus-type
mtDNA
Korea
SuweonKojori
China
Changchun
PekingNankingShanghai
CengtuLanzhou
Jiayu-guang
TurfanUrumuchi

-**

C

C

B

B

B

B

B

B

B

B

C

C

B

B

B

B

B

B

C

C

C

B

C

B

C

C

B

A

K

L

C B

C B

C

C

B

A

L

M

C

B

C

B

C

C

BAD

L

J

C

B

C

B

C

C

B

L

I

C B C B

C

C

BAD

J

I

DBA

B

A

C

B

A

DAB
DBA

A
B

C
A

B
E

A
B

A
A

C
B

B
B

DBA B
DBA B
D DAB
DBA B
DBA B
DBA B
DBA B
DBA B
DBA B

A
A

C
C

B

D

B

A
A
A
A
A
A

C
C
C
C
C
C

B A
A
B A
C
B A
A
BAG
B A
A
BAH
B A
I
B A
J

C

G C

C

E

C

C E

C

A

E

F

G

C

H

C

E

B

E

F

I

F

* Abbreviation used are Bm: BamHI, Ec: BcoRI, H 2: HindU, H 3: HindIII, He 2:
HaeII, Hp 1: HpaI, Ps: Pst!, Bg: Bg 1 I, Hp 2: HpaII, He 3: Haem, Tq: TaqI, Hf: HinfIand Mb: MboI.
** "-" represent "not done".
*** The molossinus mice with castaneous-type mtDNA were collected at 9 different
localities, Le. Nemuro, Nakashibetsu, Sapporo, Teine, Oma, Kurihara, Koriyama andKagoshima. The mice with molossinus-type mtDNA were collected at 28 localities,
Le. Ohzuchi, Niigata, Shinanomachi, Wajima, Kuki, Inamachi, Omiya, Ichikawa, Yotsukaido, Koajiro, Mishima, Numazu, Nishio, Anjo, Mizuho, Gobo, Momoyama, Osaka,
Kochi, Mine, Hakozaki, Sasaguri, Izumi, Kagoshima, Miyazaki, Tsushima, Ogasawara
and YonagunL34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

in Japan (32 in total), 9 localities in China and 2 in Korea. Then we examined the RFLP of their mtDNA. We found two types of mtDNA in
Japanese mice, one of which is closely related to that Chinese mice (M. m.
molossinus mtDNA), while the other is related to that of the Southeastern
Asiatic subspecies M. m. castaneus. This shows that some Japanesemolossinus mice have been invaded by cytoplasmic genes from the subspecies
M. m. castaneus.
These two types of mtDNA are geographically well separated in Japan:
the mice with molossinus type mtDNA occupy Central Japan, whereas themice with castaneus type mtDNA are distributed in the two distal ends ofJapan. Furthermore, neither of the two mouse populations has any heterogeneity in RFLP of their mtDNA, suggesting that these two populations
might have gone through a severe founder effect.
From these results, we propose that Japan suffered two invasions of micefrom neighbouring contries, the first from Southeast Asia or South-East
China and the second from East-Central China. Since human population
movements in Japan are thought to have followed the same pattern, these
two mouse populations were probably brought from their native habitat byhumans.

RESEARCH ACTIVITIES IN 1984

IV.

35

DEVELOPMENTAL AND SOMATIC CELL GENETiCSIn Vitro Formation of Adult Structures from Lethal
Embryonic Cells of Drosophila melanogasterYukiaki KURODA

Embryos, homozygous for the sex-linked recessive lethal gene, dor (deeporange; 1-0.3) die during embryogenesis. When undifferentiated cells
from embryos at the stage of the post-gastrulation of this mutant were cultured in ecdysterone-free medium, some defects were observed in cell membrane-associated functions such as the syncytium formation of muscle cells,
the formation of cellular spheres and the neural secretion of nerve cells.
On the other hand, other cells from these lethal embryos developed normallyand showed no detectable defects in their morphology and functions.
The author has previously established a procedure for obtaining the invitro formation of adult structures from undifferentiated embryonic cells of
the wild type by their cultivation in the presence of ecdysterone. In thepresent experiment, the ability of dor embryonic cells cultured in ecdysteronecontaining medium to differentiate into adult structures was examined.
When cells from post-gastrula embryos of the dor strain were cultured in
the medium K-17 supplemented with 15% fetal bovine serum, 100,ugfml
fetuin and 10 ,ugfml ecdysterone, muscle cells differentiated, connected witheach other, and formed net-work structures. Some epithelial cells also
formed a structure of the adult leg bud.
When embryonic cells of dor embryos were cultured in medium containing
the wild-type egg extract and ecdysterone, the defects in the adult structures
formed were partially repaired. This indicates that undifferentiated cells
from dor embryos may have an ability to differentiate into adult structuresin the presence of the wild-type egg extract and ecdysterone.
Mutagenic Activity of Cytidine Analogs inCultured Chinese Hamster Cells
Yukiaki KURODA, Kazuo NEGISHI ' ) and Hikoya HAYATSU ' )Among various analogs of nucleic acid bases and nucleosides, 5-bromo1)

Faculty of Pharmaceutical Sciences, Okayama University, Okayama.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

2'-deoxyuridine, 2-aminopurine, 5-azacytidine and 6-mercaptopurine weremutagenic in cultured mammalian cells. These chemicals induced chromosome aberrations, sister-chromatid exchanges and gene mutations in mammalian cells. Recently, it has been found by the authors that a reaction
product of cytidine with hydrazine, N4-aminocytidine was strongly mutagenic to bacteria and phages. In the present experiment, the mutagenic
activity of N4-aminocytidine and N 4-amino-2'-deoxycytidine in cultured
Chinese hamster V79 cells was examined.
The cytotoxic effects of both nucleoside analogs on V79 cells were determined by scoring the colony-forming activity of cells treated with the analogs
for 3 hours. The LD 50 value of N4-aminocytidine was 22 ,ug/ml and that of
N4-amino-2'-deoxycytidine was 50 ,ug/ml.
Cells were treated with each of the analogs for 3 hours, cultured in normal
medium for 6 days, replated in fresh dishes, and cultured in 5 ,ug/ml 6thioguanine(6TG)-containing medium for 10 days. The number of 6TGresistant cell colonies formed was scored. N4-Aminocytidine had a moderatemutagenic activity, giving the induced mutation frequency of O.l2x 10- 6/,ug-hr/ml. N4-Amino-2'-deoxycytidine was more strongly mutagenic andshowed the induced mutation frequency of 1.1 X 10- 6 / ,ug-hr/ml. In microorganisms, N4-aminocytidine was twenty times more strongly mutagenic
than N4-amino-2'-deoxycytidine. It was suggested that the difference inthe mutagenic response of microorganisms and mammalian cells to theseanalogs is due to the difference in metabolic activity in these organisms.
Studies on Freezing of Drosophila Embryos
Yukiaki KURODA and Yuko TAKADA

Drosophila is one of the materials which have been the most extensively
used in various fields of genetics. All stocks of wild-type and mutant strainsof Drosophila are maintained at 18°C to 25°C in most laboratories. The
maintenance of many strains in a laboratory consumes a lot of money,
time and labor. In the present experiment, an attempt was made to freeze
Drosophila eggs at -80°C in an electric freezer.
Male and female flies of the Oregon-R strain of D. melanogaster were
kept in glass tubes at 25°C for 4 hours to collect eggs. Eggs were dechorionated by treatment with 3 %sodium hypochloride solution for 30 minutes.
Eggs at the appropriate stage of development were selected under a binocular

RESEARCH ACTIVITIES IN 198437

microscope. They were transferred to salt solution containing 10 %glyceroland frozen in small glass vials at - 80°e. After several days, eggs wererapidly defrosted, washed by salt solution, and incubated in salt solution at
25°C for 24 hours. Eggs hatched at a very low rate of 0.6 %. Hatchedlarvae developed normally to fertile adult flies.
The low survival of frozen eggs might be due to a low permeability of thevitelline membrane to glycerol and a limited developmental stage appropriate for freezing. Dechorionated eggs were divided into several groups
at different developmental stages and frozen with the addition of glycerol.
Eggs frozen within 1 hour after fertilization, showed the highest survival
after defrosting from -80°C. Studies to enhance the permeability of thevitelline membrane by treatment with chemicals are now being carried out.
Genetical Studies on the Heredihuy Mosaic (mo) Strainof Bombyx mori: Mosaicism and Fashion of Meiotic Divisions
in Oogenic Cells
Akio MURAKAMIIn the domesticated silkworm (Bombyx mori), Goldschmidt and Katsuki(1927) found a heritable mosaic strain in which a number of mosaics forboth somatic and sexual traits appeared as they were mixed with normal
individuals in one batch. The mosaicism is caused by a single recessive
gene (mo) possessed by the mother silkworm (Goldschmidt and Katsuki,1928). In cytological observations it was further found that two egg nuclei(or polar-bodies) appeared and each of them was fertilized with a sperm
nucleus (Goldschmidt and Katsuki, 1928, 1931). Each fertilized nucleus
might develop into each side of the insect body. The presence of two
genetically different, fertilizable maternal nuclei within one egg seems to be asophisticated tool to analyze a fashion of maturation divisions. In Bombyx
mori, the type of maturation divisions in oogenic cells has not yet been established: some geneticists support a pre-reductional division type andcytogeneticists a post-reductional one.
If the female is heterozygous for the gene involved in the appearance ofthe serosa-membrane cells, pe (yellowish-white) and pe+ (black), the two
egg nuclei and/or polar-bodies may be either alike, pe+ and pe+, or pe andpe, or different, pe+ and pe. Because femininity in Bombyx is based on aZW (or XY) sex chromosomal constitution, two nuclei or polar-bodies may

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

also either be alike, Z and Z, or Wand W, or unlike, Z and W. Consequently,
nine different combinations of the autosomal and sexual genotypes of thetwo nuclei are possible in the case of a post-reductional division type, while
there are seven combinations in the case of a pre-reductional type. Thereis a striking difference in the sex of egg-colour mosaics between the two cases:
three different mosaics, are possible, females, males, and gynanders for the
post-reductional division type, while there is only one possible in gynanders
for the pre-reductional type. From the cross between the hereditary mosaic(mo/mo) strain females heterozygous for pe+ andpe genes and males homozygous for the egg-colour gene pe, a hundred and twelve mosaic eggs wereobtained. According to their anatomical observations, the three different
types of mosaics in females, males, and gynanders were detected with moremale types than the other two types. The number of gynandromorphic
eggs was the same as that of female eggs. The present preliminary dataclearly indicate that in Bombyx the fashion of meiotic divisions of theoogenic cells is the post-reductional division type (or pre-equational separation in the first meiotic division and post-reductional separation in the second
division) rather than the pre-reductional one. However, Kobayashi andEbinuma (1984) have analyzed this subject by using a hereditary strain, andthey tentatively concluded that the type of meiotic divisions in the silkworm
females is ofthe pre-reductionaL The discrepancy may be due to a difference
in strains used for the experiments.
Genetical Studies on the Hereditary Mosaic (mo) Strainof Bombyx mori: The Appearance of Polyploids
Akio MURAKAMI

There are some reports in which the hereditary mosaic gene (mo) may
give rise to the polyploid, besides the mosaics either for sexual or somatic
traits. Tanaka (1928) first observed exceptional females which were regarded as due to polyploidy in a sex-mosaic (probably corresponding to
the hereditary mosaic) strain. Goldschmidt and Katsuki (1928, 1931) observed cytologically the case in which a female triploid nucleus was present
in an egg cytoplasm of a hereditary mosaic strain. Hashimoto (1934) actually detected several tetraploid females and analyzed their origin. However, detailed understanding of the function of the gene (rna) responsible forthose two striking biological phenomena is still lacking.

RESEARCH ACTIVITIES IN 1984

39

In the F , offspring of the hereditary mosaic strain females heterozygous
for the egg-colour gene (pe+ /pe) crossed with the males homozygous forpe, some triploid and tetraploid insects were detected besides a number ofsomatic or sexual mosaic and normal diploid insects. These tetraploid
females appeared as an exceptional egg either black or yellowish-white(hereafter yellow). The progeny from crossing the black tetraploid females
to the diploid marker males homozygous for the pe was black and yellow
eggs at a ratio close to the expected 1: 1. This suggests that the tetraploidis produced by the fusion of two diploid egg nuclei (+ /pe and pe/pe) fertilizedwith the sperm rather than the fertilization of a triploid egg nucleus with asperm. Thus, it can be said that the tetraploid insect has a +/pc/pe/peconstitution for the chromosome-5 and that two of the three chromosome-5
marked with the pe gene might be derived from the sperm. A segregation
with a 5: I ratio of black and yellow eggs was also rarely detected, suggestingthat the origin of this type of tetraploid might be also produced by the fusionof double nucleus eggs (+/+ /pe/pe) already fertilized with sperm. Tetraploid females with all yellow genes (pe/pe/pe/pe) were also rarely detected.
Even if triploids were detected, the analysis of their segregation ratio isdifficult because of their high sterility. Practically, there were two types of
triploids with black or yellow eggs. On crossing the black triploid female
to the marker pe male a segregation at a ratio of ca. I: I for black and yellow
eggs, occurred, while a cross of the yellow triploid female to the pe maleproduced only offspring of yellow eggs. From these observations, at least,
one chromosome-5 with either + or pe of the triploid females may be contributed from the female parent and also one chromosome-5 with the pc
gene from the male. The presence of +/+/pc triploids suggests that theremaining one would be derived from the female parent rather than the male.
It should be noted, however, that the tetraploids might primarily occur in
the F, offspring of the cross between the hereditary mosaic strain female
and marker male, but the triploid has a tendency to secondarily occur as the
product of a cross between tetraploid females and normal diploid males.
Considering the appearance of both mosaics and polyploids in the hereditary mosaic strain females, the gene responsible for those two biologicalphenomena may be ascribed to the high affinity among gametic nuclei, ova,
polar-bodies, sperm and/or synkaryons, so that the egg nucleus and its sister
nucleus can be fused to develop into diploidy in an egg. In Bombyx,
polyspermy is the rule and the two sperm are readily available for the two

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

egg nuclei. Consequently, it is also likely to expect that the diploid nucleus
can be fertilized with a haploid sperm and leads to a triploidal nucleus.
Briefly, in the hereditary mosaic strain, several combinations among the egg
nucleus, cellular polar-bodies and sperm in an egg can occur and in consequence form polyploids as well as mosaics.

A Strain with Highly Parthenogenic Tendency in Bombyx mori
Akio MURAKAMI

Natural parthenogenesis is one of potential tools for better understandingof the mechanism of sexual reproduction and ontogenesis in bisexual organisms. Spontaneous parthenogenic development of unfertilized eggs
takes place rarely in the silkworm (B. mori) as reported by several investigators. Accordingly, it is necessary to establish a highly spontaneous
parthenogenic line in the insect. In B. mori, the developing embryos or
eggs are easily discernible from the non-developing egg at the stage of serosa
membrane formation. The appearance of coloration or the formation ofpigment granules in the serosa membrane cells is generally connected withthe development of embryos. An unfertilized egg is light yellowish-white.
A fertilized egg on the 3rd day after oviposition starts growing darker andbecomes a dark grey peculiar to the silkworm egg around the 5th day. We
have made a selection with the intention of obtaining the highly parthenogenic line since 1980, but had not obtained such a line by 1983. In thepast year, eight different silkworm strains, ClO8, Aojuku, Jl06, Daizo, Uda
pe, od, pe: re and an F 1 hybrid between ClO8 and Aojuku were examined.
Consequently, the percentage of pigmented eggs or parthenotes per female
varied from individual to individual with a few fractions to a few percentof the number of unfertilized eggs per female. It also greatly varied in thestrain or stock line. This suggests that the spontaneous parthenogenic phenomena are under the control of genetic factor(s). Among them, a Chinesebivoltine race Daizo showed the highest potency of spontaneous parthenogenesis and the F 1 hybrid had a tendency to some extent. These findings
are in good accord with the view that naturally occurring parthenogenesis
is generally higher for bivoltine races and hybrid stocks than for univoltine
races. It is of interest to note that the egg-laying rate of Daizo was thehighest among the strains tested: Daizo completed the egg-laying within
one to two days when being place at a room temperature (25°C) after em-

RESEARCH ACTIVITIES IN 1984

41

ergence, while the other strains needed around 5-7 days (5 days on the
average). This means that unfertilized eggs may have a more vital potency
for the natural parthenogenesis in Daizo than those in the other strains usedin the experiment. These observations and other indicate that unfertilized
eggs may have fair chance for the completion of maturation divisions toremain for several days after oviposition without the penetration of spermatozoa.
In the experiment with the F 1 hybrid between C108 and Aojuku, only one
hatched embryo (ca. 0.002 %) from 150 naturally developed eggs was detected and it grew into a 3rd instar larva. The larva was male, indicatingthat the parthenote might have resulted from an egg-pronucleus passing
through both the reductional and equational maturation divisions. The
pronucleus might be divided into two haploid nuclei whose fusion into one
cleavage nucleus leads to diploidy in the egg.
The Second Case of a Chromosome Specific Instability
in the silkworm, Bombyx mori
Akio MURAKAMI

An egg-colour mosaic (pe+ /pe) female mutant was detected in chemicallyinduced mutagenesis experiments by the pe: re specific-locus method in which
wild-type F 1 hybrid (C108 X Aojuku) mid-stage female pupae were treatedwith ethyl methanesulfonate (EMS) and mated to the pe: re marker strain.
The moth that developed from the mosaic mutant was back-crossed to thepe: re marker male in order to analyze its genetic nature. When the blackegg (+ + /pe re) females appearing in the above cross were mated to males
homozygous for both pe and re loci, they laid a very small number of black(ca. 4.0%) eggs and an overwhelming number of yellowish-white eggs (ca.
90 % or more), differing from the expectation of a one to one ratio for blackand yellowish-white eggs. In addition, they also produced the same numberof red eggs (ca. 4.0 %) as black eggs. Furthermore, when the black female
eggs appearing in the preceeding generation were crossed with the pe: remarker males, they again laid a very small number of black eggs and theunexpected red eggs. Repetition of this mating procedure revealed a similartendency in the offspring of following generations. These findings suggestthat the unstable and/or mutable line, tentatively MVINSTA_2, may be classified
as a line having a chromosome specific genetic instability. It can be also

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

said that the instability of this line behaves as a semidominant one. Although the genetic instability in the present experiment on the silkworm
seems to be derived from EMS-treated oocytes, a possibility that the
MVINSTA_2 line might be of a spontaneous origin cannot be excluded. The
evidence that the instability affected both sexes is apparently based on thefact that both matings, black females and marker males and marker females
and black males, were carried out. No genetic effect of the MVINSTA_2
line on chromosome other than the chromosome-5 was detected, indicatingthat this line showed a chromosome-5 specific type instability similar to
MVINSTA (Murakami, 1975). However, the line did not induce a mosaic
type mutation as to egg-colour (serosa membrane) cells dissimilar to the
MVINSTA line. The egg-colour mutants detected in the line were of wholebody types for both pe and re loci. Theoretically, a germ cell mutation occurring or fixed at the stage of germ cells before (and during) DNA replication
should be expressed by a whole area of the egg (or a large mutant area),
while that occurring at a stage of germ cells after DNA replication should be
expressed by a small mutant area. This indicated that the genetic instabilityof the MVINSTA_2 line chromosome may be, at least, expressed at the germ
cell stage in prior to the completion of DNA replication or synthesis.
Lethal Phases of X-linked Lethal Mutations in
Drosophila melanogaster
Kiyoshi MINATO and Masa-Aki YAMADA

To investigate the mechanism of the expression of embryonic lethal genes,
the lethal phase of development was examined on 28 strains of X-linked
recessive lethals induced by ethyl methanesulfonate (2.5 X 10- 3 M) in Drosophila melanogaster.

Twenty-two strains among 28 strains died during larval stage, 6 strainsdied during pupal stage. No strains, however, died during embrionic stageof development. As compared to the previous reports (Hadorn and Chen,
1952, Oster, 1952, and D. T. Suzuki, 1970), the ratio of embryonic lethals
was extremely low. This may be partly due to a mild procedure for inducing
mutation used in our experiment, since it has been found that the milder
the procedure for inducing mutation was, the less the embryonic lethals
were obtained (Seta, 1954). Because the defects in gene activity produced
bv a mild procedure may reflect the defects in the stage-specific gene ac-

RESEARCH ACTIVITIES IN 1984

43

tivity, the above low ratio of embryonic lethals is of interest, suggesting therelative independency of embryonic development from the control by thezygotical genes.
Nematocyte Differentiation in Mutant Strains of
Hydra with Altered Developmental Gradients
Chiemi NISHIMIYA, Toshitaka FUJISAWA, Nancy WANEK
and Tsutomu SUGIYAMA

Nematocyte differentiation from the interstitial stem cells in hydra occurs
non-uniformly along the body column. The relative ratios of the 4 nematocyte types produced vary gradually from head to foot along the body axis
(Bode and Smith, 1977). To find out whether this regional variation innematocyte differentiation is related to the gradients of the head-activationand head-inhibition potentials, nematocyte differentiation patterns were
examined in strains which have significantly different developmental gradients along their body columns.
It was found that the regional variations in the nematocyte differentiation
were similar in all the strains examined, and that no significant differencesof the variation existed that could be attributed to the differences of themorphogenetic potentials in these strains. This suggests that nematocyte
differentiation is strongly affected by the axial position along the bodycolumn, but that the gradients of the morphogenetic potentials involved inhead formation are not involved in this effect. Instead, some other parameter(s) of axial position not directly associated with these gradients must
be responsible for the positional effect on nematocyte differentiation.
Elimination of Excess Epithelial Cells by Phagocytosis ina Mutant Strain of Hydra (L4)
Eucaly KOBATAKE and Tsutomu SUGIYAMA

The mechanism of removal of "excess" epithelial cells was examined ina mutant hydra strain which has a very reduced budding capacity (L4).
In a well-fed and exponentially growing population of normal hydra, theepithelial cells produced by cell division are largely utilized to increase thetissue mass (David and Campbell, 1972; Otto and Campbell, 1977). In
contrast, these cells produced in the mutant strain L4 are not effectively

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

utilized to increase the tissue mass (Takano et al., 1980). Instead, a large
number of them are apparently eliminated from tissue by some mechanisl1l(Takano, 1984).
In order to elucidate the mechanism of elimination of excess epithelial
cells in strain L4, its tissue was macerated and the resultant dissociated cells
were examined after Feulgen staining. It was found that a large numberof L4's epithelial cells had in their cytoplasm large phagocytic vacuoles that
contained partially-degraded cells, whose nuclei had highly condensed andintensely Feulgen-positive chromatin granules. This and other features ofthe entire phagocytic structure were very similar to those of the phagocytic
structure recently found in the starved animals of a normal strain by BOschand David (1984).
The incidence of epithelial cells having phagocytic vacuoles was relatively
high when L4 animals were cultured under the conditions which allowed alarge number of excess epithelial cell production, but relatively low when
cultured under other conditions allowing little or no excess epithelial cell
production. These results suggest that epithelial cells produced but not
utilized in strain L4 were removed, at least in part, by phagocytosis by neighbouring cells.

RESEARCH ACTIVITIES IN 1984v.

45

CYTOGENETICS

Karyological Survey of Indian Ants
H. T. IMAI, C. Baroni URBANI ' J, M. KUBOTA2), G. P. SHARMA3 J,
M. N. NARASIMHANNA4 ), B. C. DAS 4l, A. K. SHARMA5 ),A. SHARMA 5 ), G. B. DEODIKAR 6 J, V. G. VAIDYA 6),
and M. R. RAJASEKARASETTy 7 )The karyotypes of 94 species of Indian ants were examined. Their chromosome numbers range almost continuously between n=5 and 38, though
the frequency distribution is bimodal with a remarkable antimode at n=l1
and two modal points at n= 10 and 15. Based on this bimodal distribution,
Indian ants were classified into two groups; Lower-numbered species (n::;; 11)
and higher-numbered species (n> 11), the former being characterized bymetacentric-rich karyotypes, and acrocentrics predominating in the latter.
The three major subfamilies (Ponerinae, Myrmicinae, and Formicinae)
showed a highly divergent distribution in chromosome number, ranging
between n=7-38, 6-35, and 8-27, respectively, suggesting a convergence inkaryotype evolution of each subfamily. Another three subfamilies, of which
only a few species were examined, had moderate or lower numbers, i.e.,
n=5-14 in Dolichoderinae, n=14 in Cerapachynae, and n=12 in Dorylinae.
We found four Robertsonian polymorphisms, two pericentric inversion
polymorphisms, and four reciprocal translocations, three of which were
fixed. Robertsonian polymorphisms were found only in higher-numberedspecies, while translocation were restricted to lower-numbered species. A
possible biological significance for this nonrandom distribution of rear1)
Zoological Institute of the University, Rheinspring 9, CH-4051 Basel, Switzerland,and Naturhistorisches Museum Basel, CH-4001 Basel, Switzerland.
2)
Nakasone 13, Odawara, Kanagawa-ken 250, Japan.
3)
Department of Zoology, Panjab University, Chandigar-160014, India.
4)
Central Tasar Research Station, Ranchi-834005, India.
5)
Department of Botany, University of Calcutta, 35 Ballygunge Circular Road,
Calcutta-700019, India.
8)
Department of Zoology, Maharashtra Association for the Cultivation of Science,
Law College Road, Poona-411004, India.
7)
Department of Zoology, Manasa Gangotori, Mysore-560006, India.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

rangements is discussed with reference to karyotype evolution in ants.
details see Jpn. J. Genet. 59: 1-32, 1984.

For

Chromosome Observations on Tropical Ants from Indonesia
Hirotami Y. IMAI, Masao KUBOTA1), William L. BROWN Jr. 2 ),Masaaki IHARA3 ), Machmud TOHARI 4 ), and Rafael I. PRANATA4 )The senior author has been promoting a karyological survey of world
ants. As a part of this project, IMAI and KUBOTA had a chance to survey
chromosomes of Indonesian ants in cooperation with IHARA, TOHARI
and PRANATA. Collection of ants and chromosome preparations weremade from March 3 to March 15 in 1984. A total of 88 colonies including
4 subfamilies, 32 genera, and 64 species were examined. The colonies collected were labelled as HI84-(l '" 88) and their localities are as follows:
Bogor (HI84-1 '" 15, 44", 52, 54"'62, 71", 88), Chibodas (HI84-16 "'43, 53),
and Darmaga (HI84-63 '" 70). The identification of species was mademainly by BROWN and partly by BOLTON (British Museum) for Tetramorium. One set of alcohol specimens was deposited with BIOTROP,
which is maintained by PRANATA. The results are summmarized inTable 1.
Table 1.

Chromosome numbers of Indonesian ants

Taxa (Colony number)
PONERINAE
Discothyrea sp. near bryanti (HI84-56)
Leptogenys kraepelini (HI84-l7, 24, 30, 31)
L. diminuta (HI84-7)
L. iridescens (HI84-57)
L. myops (HI84-13, 62)
L. peuqueti (HI84-6, 75)
Diacamma sp. (HI84-4)
Odontoponera transversa (HI84-86)
Pachycondyla astuta (HI84-25)
P. astuta (HI84-54)
1)

2)
3)
4)

Chromo number(n) 2n

30
26
32
46
48
54
66
42
18
22

Nakasone 13, Odawara, Kanagawa-ken, 250, Japan.
Department of Entomology, Cornell University, Ithaca, New York, 14853, U.S.A.
JICA Scientific Expert, Tropical Forest Program BIOTROP, Bogor, Indonesia.
Tropical Agricultural Pest Biology, BIOTROP, Bogor, Indonesia.

47

RESEARCH ACTIVITIES IN 1984
Table 1.

(Continued).

Taxa (Colony number)

P. rubra (HI84-16, 34)
P. sp. near obscurans (HI84-64)
Ponera sp. (HI84-28)
Hypoponera pruinosa (HI84-58)
H. confinis (HI84-61)
Odontomachus latidens (HI84-23, 33)
O. simillimlls (HI84-12, 44)
Anochetlls modiclls (HI84-22)
A. graeffei (HI84-5, 76)
MYRMICINAE
Pheidole capellinii (HI84-2)
P. hortensis (HI84--37, 53)
P. binghami (HI84--40)
P. plagiaria (HI84-60)
P. sp. 5 (HI84-66)
P. sp. 6 (HI84-84)
P. sp. 7 (HI84-38, 43)
P. sp. 8 (HI84--39)
Crematogaster sp. 1 (HI84-74, 77)
C. sp. 2 (HI84--88) B-chromosome polymorphism
Meranopilis bicolor (HI84-11, 15)
Monomorillm sp. 1 (HI84-47)
M. sp. 2 (HI84-51)
Oligomyrmex sp. 1 (HI84-69, 80)
O. sp. 2 (HI84--49)
Vollenhovia sp. (HI84--29)
Myrmicaria sp. (HI84--18, 21)
Myrmecina sp. 1 (HI84-32)
M. sp. 2 (HI84--19)
Tetramorillm kheperra (HI84-81)*
T. brevidentatllm (HI84-35, 36)*
T. adelphon (HI84-10)*
T. insolens (HI84--9, 14)*
T. pacificllm (HI84--87)*
T. smithi (HI84--8)*
Smithistrlllna sp. (HI84--27, 71)
Kydris mlltica (HI84-73)
Strumigenys doriae (HI84-20)
S. godeffroyi (HI84--72)

Chromo number(n) 2n(10) 20
44
12
24
38
32
(22) 44
30
38

20
20
20
20
18
18
18
32
24
56/58
16
22
22
34
42
36
44
66
68
14
(10) 20
22
22
22
26
38
36
22
44

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35
Table 1. (Continued).
Taxa (Colony number)
DOLICHODERINAE
Dolichoderus bituberculatus (HI84--50)
lridomyrmex anceps (HI84-48)
Tapinoma melanocephalum (HI84-3, 67)
Technomyrmex sp. I (HI84-83)
T. sp. 2 (HI84-78)
FORMICINAE
Anoplolepis longipes (HI84-82)
Acropyga sp. (HI84-63)
Plagiolepis sp. (HI84-70)
Pseudolasius sp. (HI84-46)
Paratrechina longicornis (HI84-l)
P. sp. 2 (HI84-41)
P. sp. 3 (HI84--42, 68, 79, 85)
Camponotus sp. 1 (HI84-45, 52)
Robertsonian polymorphism
C. sp. 2 (HI84-65)
Polyrhachis gribodoi (HI84-26)
P. illaudata (HI84-55, 59)

Chromo number(n) 2n

30
48
10
28
30
34
32
18
30
(16)
30
30
38/39
38
48
(14) 28

The Tetramorium species with asterisks indicates samples for which the determination
has been checked and confirmed by Bolton.

The Reproductive Cycle of the Queenless
Ant Pl'istomyl'mex pungens
Tomio ITOWl), Kazuo KOBAYASI ' \ Masao KUBOTA2 ), Kazuo OGATA,3)
Hirotami T. IMAI, and Ross H. CROZIER 4)

The life cycle of the myricine ant Pristomyrmex pungens was investigated.
Colonies of this species are usually composed of several thousand small
workers, although a few males (2-3 %) occasionally appear during June andJuly in mature colonies, and large workers with ocelli and abortive sperl)
Department of Biology, Faculty of Education, Shizuoka University, Shizuoka 422,
Japan.
2)
Nakasone 13, Odawara, Kanagawa-ken, 250, Japan.
3)
Faculty of Agriculture, Kyushu University, Fukuoka, 812, Japan.
4)
School of Zoology, University of New South Wales, P. O. Box 1, Kensington,
N.S.W. 2033, Australia.

RESEARCH ACTIVITIES IN 198449

mathecae (here termed ergatoid queens) were observed extremely rarely.
We found that the virgin small workers can lay eggs and that these develop
into further small workers. Cerebral ganglion cells and oogonial cells had
the diploid chromosome number (2n=24), but the haploid number (n=12)
was observed in oocytes at pachytene, and also in spermatocytes from therare males. Males have functionally normal copulatory organs and theirspermatogenesis is normal, but they probably do not mate, because small
workers have no spermatheca and their copulatory organs are degenerative.
These observations indicate that reproduction in P. pungens is carried out
predominantly or, probably, wholly by the small workers and hence is thelytokous. We discuss the evolution of the P. pungens life cycle, pointing out
the difficulty of applying the "queen" concept to this species or even in deciding whether or not it is "eusocial". In view of the apparent genetic
isolation between colonies, the mode of selection maintaining sociality inthis case is probably interdemic group rather than kin selection. For details
see Insectes Sociaux 31: 87-102, 1984).
Molecular Analyses of Transposable Elements in
Drosophila simulans
Y oshihiro H. INOUE, Takao K. WATANABE, Kazuo MORIWAKI,

and Masatoshi YAMAMOTO

Transposable elements have been studied in most detail in Drosophila
And it is considered that the biological role of transposable
elements is to increase genetical variability by spontaneous mutations andchromosomal rearrangements. In order to examine whether this applies
to other organism, its sibling spieces D. simulans was studied. Although
D. simulans is morphologicaly similar to D. melanogaster, it has some different genetical characters from D. melanogaster; The frequency of chromosomal rearrangements found from wild population is lower than that ofD. melanogaster. And further, the dispersed middle repetitive DNA, towhich transposable elements belong, is possesed about one seventh fewer
than D. melanogaster. Here we report an unstable white allele, wmky in D.
simulans, which was spontaneously isolated from an isofemale line of wild
population in Japan.
The wmky of D. simulans is an unstable w allele and a few new eye colour
mutants were derived from the inbred line stock. The mutation frequency
melanogaster.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

of wmky was measured at 4 X 10- 5• From the wmky subline 1, wcho and wapI
were isolated. On the other hand, wpsm1 , wpsm3 , wpsm4 were obtained fromthe wmky subline 2. The genetical analyses showed that the derivatives fromthe subline 1 were the mutation at the w locus and those derived from thesubline 2 were atributed to su(w mkY ) on the third chromosome. The su(w mky)were closely linked to cu(3-90.0). This is the first evidence of an unstablegene locus in D. simulans. A molecular analysis of wmky indicated that an
about 15 Kb long DNA fragment of unknown origin was inserted into the
w locus. These genetical and molecular data suggest that the insertion is atransposable element which causes the instability of the w allele.
Thus, a transposable element which was able to induce an unstable mutation was found in D. simulans. This shows that the active transposable
element which contributes to induction ofthe spontaneous mutation is carriedin not only D. melanogaster but D. simulans.

RESEARCH ACTIVITIES IN 1984

VI.

51

MUTAGENESIS AND RADIAnON GENETICS
So called "Kada Effect" of the Tritium
Tsuneo KADA and Yoshito SADAlE

We previously reported about the in vitro inactivation efficiencies oftritiated water on DNA (Proc. 7th ICRR, BI-18, 1983). Transforming
DNA was extracted from B. subtilis cells (Arg+), purified, dissolved in SSC(5,ug/ml) containing tritiated water of different concentrations (100 ,uCi/ml,
10 ,uCi/ml, 1 ,uCi/ml ... etc.) and kept at 4°C. When the transforming
activities (Arg- -+ Arg+) were determined after different periods of thetritium treatment, we found that the inactivation efficiencies increased
markedly by decreasing the concentrations of tritiated water. Such a concentration effect of tritium as to the in vitro inactivation of DNA was called
the "Kada effect".
Characteristics of the above observation may be the following.
(1) When damages are produced in DNA by p-irradiation from tritum,
they should remain unchanged in a buffer solution at low temperatures,
there is no chance of being repaired.
(2) To obtain similar levels of the radiation exposure of different doses thatare proportional to the values ,uCi/ml X (days of treatment), it takes,
for example, 10 times more days in a treatment sample having 10 times
more diluted tritium concentration. Therefore the increase of inactivation efficiencies should be proportional to the period of treatment.
We previously supposed that certain stable trit um-generated radicals
might be generated and playa role in the "Kada effect". If this is the case,the inactivation event may be proportional to the period of treatment andnot to the concentration of tritium. Recently Dr. Watanabe and his collaborators (Tokyo Isotope Inst.) and Dr. Yamamoto (Hiroshima Univ.)
found generation of hydrogenperoxide (H 20 2) of considerable levels in
the course of stocks of tritiated water. We carried out experiments to check
if H 20 Z might inactivate DNA in a similar fashion as tritiated water, andfound very similar effects both for H 20 2 and tritiated water.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

In Vitro and In Vivo Analysis of Antimufagenesis
Tsuneo KADA

We carried out screenings of bio-antimutagens from tissues of different
plants using radiation and chemically treated bacterial strains such as E.
coli Blr WP2, Salmonella TAlOO, etc. Results of our studies on isolation of
bio-antimutagens in plants and animals and their actions indicated thefollowing mechanisms.
1) Activation of error-free DNA repair:In the case of cobaltous chloride, the spectrum of bio-antimutagenic
activity is very very wide. It reduced the induced mutations of UV, ,-ray,
MNNG, Trp-P-l, etc. Such a wide spectrum can not be explained by theaction of the agent on the path-way specific to each mutagen. We found thatthe survivals of 4NQO-treated bacteria were higher in the presence of bioantimutagens than in their absence. Similar observations were made withcinnamaldehyde. The increase in survival was dependent on the recA+
function. In vitro formation of D-loop from homologous double strand
DNA and single strand DNA by means of the recA protein was promotedby the presence of cobaltous chloride.
2) Promotion of DNA repair-pathways specific to UV and UV-mimetic
chemicals: Collaborative studies with K. Shimoi and 1. Tomita (Shizuoka
College of Pharmaceutical Sciences) showed that tannic acid reduced theUV-indiced mutation when present for as long as 10 minutes after irradiation. Liquid-holding experiments as well as other experiments indicatedthat this chemical might promote excision repair of DNA possessing UVinduced lesions.
We are also studying mechanism of other bio-antimutgens such as humanplacenta factor and green tea factor. It is interest to us whether these antimutagens might also work in higher plants and animals (Mutation Res.
150: 127, 1985; Proc. 4th Inter. Com. Env. Mutagens).
Gamma-Induced Chromosome Aberrations in
Caenorhabditis elegans
Yoshita SADAlE and Tamiko SADAlE

A simple method for observing chromosome aberrations in Caenorhabditis
elegans was established. Gravid worms were irradiated with gamma-rays

RESEARCH ACTIVITIES IN 198453

and metaphase chromosomes of early embryonic origin were examined.
The frequency of chromosome aberrations increased with increasing dosesof gamma-rays in C. elegans var, Bristol strain N2, and decreased upon incubation of the irradiated gravid worms. About 5 times as many spontaneous chromosome aberrations were observed in C. elegans var. Bergerac
strain BO which carries ten times more transposons than the Bristol strain.
Age-dependent and Tissue-specific Expression of a DNA
Repair Enzyme in the Wasted Mouse, a Model Animalof a Human Genetic Disease Ataxia Telangiectasia (AT)
Hideo TEZUKA, Tadashi INOUE, Takehiko NOGUCHI 1) ,Tsuneo KADA and Leonard D. SHULTZ 2 )Biological, cytogenetic and biochemical methods were used to study
various tissues from a stock of mouse mutant, wasted, after weaning. In
affected homozygotes, a marked age-dependent decrease in the ratio of spleenor thymus to body weight was observed. This tendency was, however,observed neither in the liver nor in the kidney. An age-dependent increase
was observed in the frequency of both spontaneous and gamma-ray-induced
chromosomal aberrations in bone marrow cells of wasted mice. In littermate
control mice, neither of these alterations was observed in an age-dependent
manner. Activities of primer activating enzyme and apurinic endonuclease
were measured in extracts from spleens or livers of affected homozygotes
and control mice. The only alteration was an age-dependent decrease inprimer activating enzyme activity in the spleens but not in the livers of homozygotes. No change in primer activating enzyme activity was found in thecontrol group while neither group showed alterations in apurinic endonuclease activity.
The present results suggest that this mouse mutant may serve as a useful
animal model for studying the relationship between DNA metabolism
and lymphoid tissue differentiation.

1)

2)

Genetic Stocks Research Center; deceased on November 24, 1983.
The Jackson Laboratory, Bar Harbour, Maine 04609, USA.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Effect of DNA Damaging Agents on Isolated Spleen CeUs
and Lung Fibroblasts from the Mouse Mutant "Wasted", aPutative Animal Model for Ataxia-Telangiectasia
Tadashi INOUE, Hideo TEZUKA, Tsuneo KADA, Katsuhiro AIKAWAl)and Leonard D. SHULTZ')

Ataxia-telangiectasia (A-T) is a human autosomal recessive disease characterized by a progressive neurological disorder, a marked IgA deficiency,
an increased predisposition to cancer, and a spontaneous chromosomal
instability. The hypersensitivity of A-T to ionizing radiation, expressed as
reduced viability and increased cytogenetic damage in cultured fibroblasts,
has suggested defective DNA repair in this disease. We have previously
demonstrated the reduced level of the primer activating enzyme in cellular
extract from A-T cells which enhances the priming activity of gamma-irradiated DNA for purified DNA polymerase, thus indicating that one of
the causal factors of A-T is a DNA repair defect.
Recently, Shultz et al. have described a mouse mutant, wasted (wst), whichshows pathological changes in both central nervous and lymphoid systems,
and exhibits increases in the frequency of spontaneous, as well as gammaray-induced, chromosomal aberrations. These findings prompted us toevaluate the mouse mutant as an animal model for A-T.
Isolated cells of wasted mouse were examined as to their responses to DNA
damaging agents including bleomycin and gamma-rays. DNA synthesis
was found to be more resistant to bleomycin, gamma-rays and 4-nitroquinoline I-oxide in wasted mouse spleen cells than in control mouse spleen cells
whereas both types of cells exhibited similar responses to UV light. The
amount of bleomycin-stimulated repair synthesis in wasted mouse spleencells was quite similar to that in control cells. Primary lung fibroblasts
derived from wasted mouse were not hypersensitive to the killing effect of
gamma-rays. These data indicate that the defect in DNA metabolism ofspleen cells of wasted mouse, if any, is specific for damages induced by
gamma-rays or by an agent which mimics gamma-rays and that the expression of defects is somewhat different between wasted mouse cells and ataxiatelangiectasia fibroblasts.

1)

2)

Institute for Animal Industry, Kukizaki, Ibaraki-ken 305 (Japan).
The Jackson Laboratory, Bar Harbor, Maine 04609 (U.S.A).

RESEARCH ACTIVITIES IN 1984

55

Mutagenic Activity of 3'-deoxyadenosine in the
Soybean Test System
Taro FUJIIThe study of somatic mosaicism in soybean plants offers a sensitive andreliable method for screening and monitoring environmental mutagens.
Mutagenic effects of some chemicals in higher plants were examined withthis test system and the mutagenicities of known carcinogens, such as AF-2
and L-ethionine etc., were clearly demonstrated. Mutagenic activity of
3'-deoxyadenosine (3'-dA) and 3'-deoxyguanosine (3'-dG), a known repair
inhibitor for radiation induced damage, were investigated with soybean
test system, and 2'-dA and 2'-dG were used as control drugs.
2'- and 3'-dA: Concentrations of 0 to 2 mM of 3'-dA were applied to seeds
in the pilot experiment. Seedling growth was inhibited with increasing
drug concentrations, gradually, and all seedlings at 1.0 and 2.0 mM treatments showed shrunken or deformed leaf development. Based on the above
information, much lower concentrations of drug, 0.05 and 0.1 mM, wereused in the next experiment. 2'-dA was used at the same concentrations.
Spotting frequencies of 1.1 and 1.0 per leaf obtained from 0.05 and 0.1 mM
treatments of 2'-dA, respectively, were similar to that of 0 mM treatment.
On the other hand, 3'-dA induced numerous spots with 4.7 and 8.5 per leaf
with 0.05 and 0.1 mM treatments, respectively, being observed. Moreover,some leaves at 0.1 mM treatment showed 30 or more spots per leaf. Further
experiments with more diluted concentrations, viz., 0.025, 0.05 and 0.075
mM, were conducted to check the dose versus frequency relationships.
The results clearly demonstrated mutation induction by 3'-dA, and when
the data are plotted on the figure, spotting frequency increased more effectively with increasing concentrations.
From the experiments, it is evident that 2'-dA is not mutagenic but 3'-dA
has strong mutagenic activity in the present test system. A severe toxic
effect of 3'-dA on plant growth was also noticed with concentrations higher
than 0.1 mM.
2'- and 3'-dG: To obtaine further information concerning the mutagenic
activity of 3'-dA, experiments were extended with another repair inhibitor,
3'-dG. Seeds were treated with 0.1, 0.25 and 0.5 mM solutions of 2'- and3'-dG. 2'-dG showed no mutagenic activity; spotting frequencies of the
0.1-0.5 mM treatments were not much different from that of the 0 mM

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

treatment. On the contrary, 3'-dG indicated mutagenic activity with thenumber of spots per leaf increasing with increasing of drug concentrations.
However, the activity of 3'-dG is much less than that of 3'-dA for inductionof somatic mutations.

RESEARCH ACTIVITIES IN 1984

VII.

57

POPULATION GENETICS

Evol.ution of an Altruistic Trait through Group Selection
as Studied by the Diffusion Equation Method
Motoo KIMURA

A diffusion model is formulated which incorporates the process of group
selection (i.e. interdeme competition) in addition to mutation, migration,individual selection, and random genetic drift. Let us assume a diploid
species consisting of an infinite number of competing groups ("demes")each having N e breeding members and in which mating is at random. Consider a locus at which a pair of alleles A and A' are segregating, where A' isthe "altruistic allele," which has selective disadvantage s' relative to A withrespect to individual selection within demes, but which is beneficial for ademe in competition with other demes; namely, we assume that a deme
having A' with relative frequency x has the advantage c(x-x) relative to theaverage deme, where c is a positive constant and x is the average of x overthe entire species. Let ¢=¢(x; t) be the distribution function of x at time
t such that ¢(x; t)L1x represents the fraction of demes whose frequency of
A' lies in the range (x, x+L1x). Then, we obtain the following diffusion
equation.

where
and

Mox=v'(l-x)-vx+m(x-x)-s'x(l-x) ,in which v' and v are mutation rates to and from the altruistic allele A', andm is the migration rate per deme per generation (assuming Wright's islandmodel).
By investigating the above diffusion equation, I obtained a condition forgroup selection to prevail over individual selection leading to the evolutionof an altruistic trait: Let

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

DK =c/(v+v'+m)-4Ne s' ,then, group selection overrides individual selection if DK>O, and the altruistic allele predominates in the species. It can also be shown that, if
DK<O, individual selection prevails so that the altruistic allele becomesrare and cannot be established in the species. For details, see Kimura, M.
(1984), IMA Jour. of Math. Appl. in Med. & BioI. 1: 1-15.
Popnlation Genetic:> Theory of Conceded Evolution and
Its Application to the ImmulJloglobulin V Gene Tree
Tomoko OHTA

The previous simple model for treating concerted evolution of multigene
families has been revised to be compatible with various new observations on
the immunoglobulin variable region family and other families. In the previous model, gene conversion and unequal crossing-over were considered,and it was assumed that genes are randomly arranged on the chromosome;neither subdivision nor correlation of gene identity and chromosomal distance were considered. Although this model satisfactorily explains theobserved amino acid diversity within and between species, it fails to predictthe very ancient branching of the mouse immunoglobulin heavy chain Vgene family. By incorporating subdivided structure and genetic correlation with chromosomal distance into the simple model, the date of divergence may be satisfactorily explained, as well as the rate of nucleotide substitution and the amino acid diversity. The rate at which a V-gene is duplicated or deleted by conversion or by unequal crossing-over is estimated by
the new model to be on the order of 10- 6 per year. The model may be
applicable to other multigene families, such as those coding for silkmoth
chorion or mammalian kallikrein. For details, see J. MoL EvoL 20: 274280.
Population Genetics of Transposable Elements
Tomoko OHTA

Transposable elements are DNA sequences that can move or duplicativelytranspose on the chromosome. A simple model of transposable elements
was studied, and the frequency spectrum, allelism, and identity coefficientsat equilibrium were obtained. In the analyses, the two cases of asexual

RESEARCH ACTIVITIES IN 198459

haploid with no recombination and diploid with free recombination weretreated separately. It was shown that the multiple allele theory of population genetics is applicable to the problems of frequency spectrum and allelism.
On the other hand, identity coefficients may be formulated analogously tothe treatment of concerted evolution of multigene families. Results indicate that, except for allelic identity, the model of transposable elements
and that of multigene families predict similar values of identity coefficients.
For details, see IMA Jour. Math. AppI. Med. & BioI. 1: 17-29.
Some Models of Gene Conversion for Treating
the EvoIllltlion of Multigene Families
Tomoko Of-ITA

A simple model of gene conversion presented earlier has been comparedwith a more general model under the assumption of no bias. The former isbased on a simple concept that, by conversion, a gene (or a segment of agene) is transferred from one locus to another in a multigene family. Intrachromatid symmetric conversion may result in reciprocal exchange of genes
within a multigene family, and the previous theory of identity coefficientsneeds modification when it occurs. The relationship of various parametersof the two models was made clear in formulating the transition equations ofidentity coefficients. As an example of extending the analyses, a model ofpreferential conversion that is relevant to the observed uneven polymorphisms of class I genes in the major histocompatibility complex has been
investigated. It differs from the biased conversion, in that directional
process is through location of genes on the chromosome. It is shown thatthe model satisfactorily explains the observed data, even though the effectof preferential conversion is difficult to distinguish from that of natural
selection. For details, see Genetics 106: 517-518.
The Cohesive Population Genetics of Molecular Drive
Tomoko OHTA and Gabriel A. DOVER"

The long-term population genetics of multigene families is influenced byseveral biased and unbiased mechanisms of nonreciprocal exchanges (geneconversion, unequal exchanges, transposition) between member genes, often* Dept. of Genetics, Univ. of Cambridge.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

distributed on several chromosomes. These mechanisms cause fluctuationsin the copy number of variant genes in an individual and lead to a gradualreplacement of an original family of 12 genes (A) in N number of individuals
by a variant gene (a). The process for spreading a variant gene through afamily and through a population is called molecular drive. Considerationof the known slow rates of nonreciprocal exchanges predicts that the population variance in the copy number of gene a per individual is small at any
given generation during molecular drive. Genotypes at a given generation
are expected only to range over a small section of all possible genotypes fromone extreme (12 number of A) to the other (12 number of a). A theory is developed for estimating the size of the population variance by using theconcept of identity coefficients. In particular, the variance in the courseof spreading of a single mutant gene of a multigene family was investigatedin detail, and the theory of identity coefficients at the state of steady decayof genetic variability proved to be usefuL Monte Carlo simulatiolls andnumerical analysis based on realistic rates of exchange in families of known
size reveal the correctness of the theoretical prediction and also assess theeffect of bias in turnover. The population dynamics of molecular drive in
gradually increasing the mean copy number of a variant gene without thegeneration of a large variance (population cohesion) is of significance regarding potential interactions between natural selection and molecular drive.
For details, see Genetics 108: 501-521.
F ST and GST Statistics in the Finite Island Model
Naoyuki TAKAHATA and Masatoshi NEIIn Wright'S island model of population structure it is assumed that a
population consists of an infinite number of subpopulations each of thesame size N, and in each subpopulation the proportion m of the total genepool is derived from immigrants that may be considered a random sampleof the entire population. The extent of genetic differentiation of subpopulations is measured by the fixation index (F ST ). The equilibrium value of
F ST is given by+

F ST = 1/[1 2N{ (1~ m)2 - 1} ]which has been derived by considering a pair of alleles at a locus without
mutation.

RESEARCH ACTIVITIES IN 1984

61

With the development of the infinite allele model of neutral mutations,a number of authors have extended this model to the case of a finite numberof subpopulations. Nei defined G ST as the ratio of the intersubpopulational
gene diversity to the total gene diversity. G ST is an extension of F ST to thecase of multiple alleles, but Nei's formula for G ST is quite complicated, andits relationship with F ST is not clear. Recently Takahata derived a simple
equation for G ST using diffusion approximations: yet, its relationship with
Wright's or Nei's is not immediately clear. We therefore examined Nei's
exact formula to clarify its relationship with other related formulas, andobtained that the exact formula for G ST becomes
GsT=1 /[

1+2NC~ 1){ (l-m):(1-v)2 -I}]

~1/[1+4N(s~l )(m+v)]
where v is the mutation rate per generation and s is the number of subpopulations. We also studied the non-equilibrium behavior of G ST for its use indata analysis. For details, see Genetics 107: 501-504.
Mitochondrial Gene Flow
Naoyuki TAKAHATA and Montgomery SLATKIN

To account for the transmission of mitochondrial DNA between conspecific species Drosophila pseudoobscura and D. persimilis in sympatry reported by J. R. Powell [Powell, J. R. (1983) Proc. Natl. Acad. Sci. USA 80:
492-495], a simple model of gene flow and selection in infinite populations isanalyzed. The model assumes two alleles at each of two loci, one of whichis coded by an autosome and the other by mitochondrial DNA. Viability
selection is presumed to be underdominant-i.e., heterozygous inferiority
to the homozygotes-at an autosomal locus, and neutral or deleterious at
a mitochondrial locus, with the combined action being multiplicative. Extremely strong selection against heterozygotes may prevent the transmissionof mitochondrial DNA between two species, but otherwise the transmission
can easily occur over species boundaries. The rate of approach to equilibrium is determined by the level of gene flow and is not affected much by
selection against an autosomal locus. The divergence of the nuclear genomesof the two species is reexamined. Based on published data on enzyme loci,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

we conclude that there has been mitochondrial gene flow between these
species for a long enough time that several nuclear loci examined could diverge because of accumulation of neutral mutations. For details, see Proc.
Natl. Acad. Sci. USA 81: 1764-1767.
A Model of Extranuclear Genomes and the Substitution Rateunder Within-Generation Selection
Naoyuki TAKAHATA

A single locus model of extranuclear genomes is developed under theassumption of the complete action of within-generation drift which is causedby random transmission of multiple copy genomes during cell division in a
generation. Within-generation drift segregates different copy genomes in acell into different cells, resulting in homoplasmic cells. Under some conditions, the present model reduces to that for haploid nuclear genomes. A
point overlooked in previous models is that the multiplicity also admits ofthe possibility of selection occurring within a cell or between cells in an
individual (within-generation selection). If there is selection mediated by,for instance, differential proliferation of genomes, then a haploid model nolonger explains the dynamics of extranuclear genomes. Rather a model
analogous to biased gene conversion at a single locus (Nagylaki, 1983;
Walsh, 1983) is more appropriate. An application of this model to either
the fixation probability or substitution rate of new mutations shows thatstrictly maternal inheritance does not allow the fullest use of mutations, as
it obscures the effect of within-generation selection. But if there is appreciable paternal contribution, within-generation selection could be a strong
evolutionary force to which nuclear genomes are never exposed. For details,
see Genet. Res., Camb., 44: 109-116.
A Quantitative Genetic Model of Two-policy
Games between Relatives
Kenichi AOKIIn a behavioral interaction between two diploid individuals of the samegeneration in which two policies are possible, assume that changes in fitness
proportional to the following occur: a for policy 1 against policy 1, (3 fOl
policy 1 against policy 2, r for policy 2 against policy 1, (5 for policy 2 agains1

RESEARCH ACTIVITIES IN 1984

63

policy 2. Let the probability that an individual adopts policy 1 as opposed
to policy 2 in any interaction be a quantitative genetic trait, C, determinedby many additively acting genes of small effects and an independent environmental component. If interactions occur at random between all membersof the population, the change per generation in C, the population mean ofC, is given by
LlC= Vt[(p-o)+(a-p-r+o)C]/W .

Vt and W stand for the total genotypic variance and the mean fitness of the
population, respectively. On the other hand, if interactions occur between
relatives of the average degree of relatedness, r, where r is the average correlation between their genotypic values, the corresponding equation is
LlC= Vt{p-o+(a-p-r+o)[C+Cov(C" Vs)/Vt ]+r[r-o+(a-p-r+O)(C+Tb/Vb)]}/W.
It is assumed that each group of relatives and the number of such groups
is sufficiently large. Here, Vb is the between group genotypic variance,
Cov(t" Vs) is the covariance of group means and within group genotypicvariances, and T b is the third central moment of group means. For aquantitative genetic trait with the additional assumption of unlinked loci,it is reasonable to assume that Cov(C s, Vs)/Vt and Tb/Vb are both negligible,in which case this becomes

LlC= V t {p+rr-(l +r)o+[(1 +r)a-(p+ rr )-(r +rp)+(1 +r)o]C}/ W .
This equation can be heuristically derived from the first by substituting thecorresponding inclusive fitness effects. Note that when per locus selection
coefficients are small and linkage equilibrium holds, r can be equated approximately with Wright's coefficient of relationship. Thus, the quantitative genetic model provides a genetic basis for the inclusive fitness approach
toward games between relatives. By contrast, in a monogenic system withmajor gene effects Cov(Cs, Vs)/Vt and Tb/Vb cannot be neglected, and we
obtain substantially different results which contradict those obtained by the
inclusive fitness approach in game theory. Applications are made to the
Hawk-Dove game, and the simple and iterated forms of the Prisoner's
Dilemma. For details, see J. Theor. BioI. 109: 111-126, 1984.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

A Population Genetic Model of the Evolutionof Oblique Cultural Transmission
Kenichi AOKILet there be two haploid asexual genotypes A and a, each capable ofexpressing either of two phenotypes 1 and 2, acquired by cultural transmission from the parental generation. I assume viability selection with selection coefficient s(>O) against phenotype 2. Among adults after viability
selection, denote the frequency of individuals of genotype A by p and phenotype 1 by y. The phenotype of a newborn of the next generation is determined as follows: If of genotype A, it expresses phenotype 1 with probabilityf( y) and phenotype 2 with probability 1-f( y); if of genotype a, it expresses
phenotype 1 with probability g( y) and phenotype 2 with probability l-g( y).
Then the coevolutionary recursion equations in y and pare
Wy' =pf(y)+(l-p)g(y)Wp'=pf(y)+(l-s)p[l-f(y)]where a prime indicates the next generation, and W=l-s[l-pf(y)-(l-p)
xg( y)] is the mean fitness. For example, let genotype A be imitative andgenotype a be contrary. We make the reasonable assumption that acquisition of phenotype 1 which confers a selective advantage is error prone,with respective error rates u and v (O~u, v<l) for the two genotypes. Thenthe transmission functions aref(y)=(l-u)y, g(y)=(l-v)(l-y).
When (1-s)(l-uv)-(l-u)2>O, we obtain the possibly counterintuitive
result that contrariness prevails over imitativeness. For details, see Proc.
Jap. Acad. 60(B): 310-313, 1984.
Evolution of Alliance in Primates:
A Population Genetic Model
Kenichi AOKI

Alliance behaviors in anubis baboons, chimpanzees, Japanese monkeys,
and rhesus monkeys are briefly described. Alliance differs from simple
altruism or cooperation between 2 individuals in that a third conspecificindividual, the common enemy, is adversely affected. Two models ofalliance formation are considered, one in which support is given unilaterally

RESEARCH ACTIVITIES IN 198465

and the other in which both parties can profit. It is assumed that the allies
are equally related to each other and to the common enemy. Using a quantitative genetic model, conditions are derived for alliance behavior to be selectively advantageous. The models are applied to reciprocal altruism between adult male anubis baboons and manipulation by adult male chimpanzees. It is argued that reciprocally altruistic alliance in baboons as
described is difficult to reconcile with theory. For details, see J. Ethol. 2:
55-61, 1984.
Average Coefficient of Relationship Within Troops of theJapanese Monkey and Other Primate Species with
Reference to the Possibility of Group Selection
Kenichi AOKI and Ken NozAwA

The average coefficient of relationship within troops of the Japanesemonkey (Macacafuscata) is .1667 ±.0433; this corresponds to an F ST between
troops of .0935±.0277. The estimate is based on polymorphic variation
at 12 blood protein loci in a sample of about 1,500 individuals distributed
over 33 troops. The average coefficient of relationship among adult females(the sedentary sex) is .1886±.0670 and that among adult males (immigrants)
is .1067 ± .0382. The difference, though not statistically significant, isin the direction expected from the observed sex asymmetry in dispersal.
We give an approximate formula for the coefficient of relationship between
specified relatives. The coefficient of relationship in the Japanese monkey
is compared with corresponding estimates in five other terrestrial primate
species and in tribal man. It is suggested that the best informed guess of
the average coefficient of relationship within bands of Pleistocene man may
have been about 10 to 15 %. This estimate is relevant to the possibility ofthe evolution of altruism in our ancestors by group selection. For details,
see Primates 25: 171-184, 1984.
Group Selection for 11 Polygenic Behavioral Trait:
Estimating the Degree of Population Subdivision
James F. CROW and Kenichi AOKIFor assessing the degree of population subdivision, and therefore theextent to which group selection might favor an altruistic trait, an appro-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

priate measure is Nei's G ST ' defined by (Fo-P)j(l-P). F o is the probability that two alleles drawn from the same group are identical in state and
P is the probability for two alleles drawn at random from the entire population. These probabilities can be assessed from molecular polymorphisms.
G ST has a number of properties that make it useful for empirical studies.
When the mutation rate is small relative to the migration rate and the reciprocal of the group size, G ST depends mainly on the absolute number of
migrants per generation, moves rapidly to near equilibrium, and is independent of the number of alleles. The relative homogenizing effect of migration in the island and stepping-stone models is not as different as might be
expected; one immigrant chosen randomly for the rest of the population is
only one to two times as effective as one from a neighboring group, appreciably exceeding 2 only when there are 1000 or more groups. The use of molecular data to estimate the degree of population subdivision may permit
testable predictions of the extent of altruistic behavior. For details, see
Proc. Natl. Acad. Sci. USA 81: 6073-6077.
Mathem.atical Model of Rapid Invasion of the P-M
Type TrausposoHl to a Local Populationof Drosophila melanogaster
Terumi MUKAI and Fumio TAJlMA

The following assumptions were made for cytotype: In the crosses of
P(~)XP(d), P(~)xM(c3"), M(~)xM(c3"), the cytotypes of the progeny are

the same as that of the mother and the mutation rate is not increased. (2)In the cross ofM(~) XP(c3"), the cytotype of a fraction of the progeny changes
to P, while that of (1-0') remains M, but the mutation rate in all the progeny
increases at the increment of v. If the frequency of the P cytotype is P andthat of M is (1- P) and generation is expressed by t, the following approximate equations can be obtained:~
Poe"'tP= l+Po(e"'t-l)~ 1
pel-po)t = ~ In (1-P)P o

where Po is the frequency of individuals carrying the P cytotype at the starting generation.

RESEARCH ACTIVITIES IN 1984

67

If we assume that the frequency of the transposon-induced detrimental
allele is q and that q is much smaller than 1, furthermore, if hs (selection
against heterozygote) is much smaller than a, then,txt~ -kst {v(1-Po)Po(e -l)}q=ea[l+Po(etxt-l)] +qo
where s is the selection coefficient against the mutant homozygote and qo
is the frequency of transposon-induced detrimental allele at the starting
generation.
U sing these formulae, the case of the Katsunuma population was analyzed.
The detail will be published in Proe. Natl. Acad. Sci. U.S.
Population Bottlenecks and Nonequi!ibrium Modellsin Population Genetics. I. Allele Numbers wileXl
Populations Evolve from Zero Variability
Takeo MARUYAMA

A simple numerical method was developed for the mean number andaverage age of alleles in a population that was initiated with no genetic variation following a sudden population expansion. The methods are used to
examine the question of whether allele numbers are elevated compared withvalues seen in equilibrium populations having equivalent gene diversity.
Excess allele numbers in expanding populations were found to be the rule.
This was true whether the population began with zero variation or with lowlevels of variation in either of two initial distributions (initially an equilibrium allele frequency distribution or initially with loci occurring in only
two classes of variation). Although the increase of alleles may persist for
only a short time, when compared with the time which is required for approach to final equilibrium, the increase may be long when measured inabsolute generation numbers. The pattern of increase in very rare alleles(those present only once in a sample) and the persistence of the original
allele were also investigated. (Genetics 108: 745-763.)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Viii.

EVOLUTIONARY GENETiCS

A Mathematical Model of Codon Substitution
and the Constancy of Evolutionary Rate
Takashi GOJOBOR!

A mathematical model for codon substitution is presented. This model
is constructed by a 61 X 61 transition probability matrix for the 61 nonterminating codons, taking into account unequal mutation rates among
different nucleotides and selective constraint against amino acid changes.
A computer simulation under this model is conducted to study the numbersof silent (synonymous) and amino acid altering (nonsynonymous) nucleotide
substitutions. It is shown that, when the mutation rates are not equalamong different nucleotides, the numbers of synonymous substitutions estimated by the methods of Perler et al. (CELL, 1980) and Miyata and Yasunaga (1. Molec. EvoL, 1980) both show a general trend of nonlinear increase
with the time, though the true number of synonymous substitutions increases
linearly. It is, therefore, possible that the 'saturation' of synonymous substitutions observed by Perler et al. is due to the inefficiency of their method
in detecting all synonymous substitutions. However, it is also shown that,when the pattern of mutation is similar to that of nucleotide substitution inpseudogenes, the estimate of synonymous substitutions obtained by Miyata
and Yasunaga's method increases linearly. This seems to be expected, for
Miyata and Yasunaga incorporated the empirically obtained pattern ofamino acid changes into their method. For details, see Proceedings ofthe XHth International Biometric Conference (Invited papers), pp. 287-297.
Rates of Nucleotide Substitution for Cellular
and Viral Oncogenes
Takashi GOJOBORI and Shozo YOKOYAMAl)

With the aim of quantitative analyses of evolutionary dynamics of oncogenes, we devised a method of computing the rates of nucleotide substitution for coone, v-one, and the retrovirus genome simultaneously. Using
1)

Washington University Medical School.

RESEARCH ACTIVITIES IN 198469

the method, we compared DNA sequences of an oncogene (v-mos) of
Moloney murine sarcoma virus (M-MuSV) and its cellular homologue(c-mo s), and gag genes of M-MuSV and Moloney murine leukemia virus(M-MuLV). The estimated rates (per site per year) of nucleotide substitution for c-mos, gag gene, and v-mos are the order of 10- 9 , 10-\ and 10- 3 ,respectively. The present results imply that v-oneS fit in with a generalevolutionary feature that the evolutionary rate of the RNA genome can be
more than a million times greater than that of the DNA genome because ofa high mutation rate in the RNA genome. A quick report for these results
has been published in Genetics 107: s39.
Concerted Evoll.l.tioJm of the Immunoglobulin V H Gene Family
Takashi GOJOBORI and Masatoshi NEI')

With the aim of understanding the concerted evolution of the immunoglobulin VH multigene family, a phylogenetic tree for the DNA sequences of 16
mouse and five human germline genes was constructed. This tree indicatesthat all genes in this family have undergone substantial evolutionary divergence. The most closely related genes so far identified in the mouse genome
seem to have diverged about 6 million years (MY) ago, whereas the most
distantly related genes diverged about 300 MY ago. This suggests thatgene duplication caused by unequal crossing-over or gene conversion occurs
very slowly in this gene family. The rate of occurrence of gene duplicationin the VH gene family has been estimated to be 5 X 10- 7 per gene per year,
which seems to be at least about 100 times lower than that for the rRNA
gene family. This low rate of concerted evolution in the VH gene family
helps retain intergenic variability that in turn contributes to antibody diversity. Because of accumulation of destructive mutations, however, about
one-third of the mouse and human VH genes seem to have become nonfunctional. Many of these pseudogenes have apparently originated recently,
but some of them seem to have existed in the genome for more than 10 MY.
The rate of nucleotide substitution for the complementarity-determining
regions (CDRs) is as high as that of pseudogenes. This suggests that there
is virtually no purifying selection operating in the CDRs and that germline mutations are effectively used for generating antibody diversity. For
details, see Mol. BioI. Evol. 1: 195-212.
1)

The University of Texas at Houston.

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Classification and Measurement of DNA Polymorphism
Masatoshi NEIl), Fumio TAJlMA 2 ), and Takashi GOJOBORI

Statistical methods for studying DNA polymorphism by using restriction endonucleases are presented. It is shown that the most fundamental
measure of DNA polymorphism is nucleotide diversity (heterozygosity per
nucleotide site), and this quantity can be estimated by several different
methods. Data on restriction site polymorphisms as well as on nucleotide
sequences indicate that nucleotide diversity is generally 0.002 to 0.02 ineukaryotic genes. In the influenza A virus hemagglutinin gene, however,it amounts to 0.51. This high degree of DNA polymorphism is apparently
caused by a high rate of mutation in RNA viruses. To evaluate the effects
of insertion and deletion, a method of estimating the number of gap l1ucleotides per nucleotide site is introduced. In some genes such as the alcohol
dehydrogenase gene in Drosophila melanogaster the effect of insertion anddeletion on nucleotide sequence variation is larger than that of nucleotide
substitution. Statistical analyses of available data suggest that a large partof DNA polymorphisms in natural populations are caused by mutation andrandom genetic drift. For details, see Human Population Genetics: The
Pittsburgh Symposium (ed. A. Chakravarti), pp. 307-330, Van Nostrand
Reinhold Company, New York.
Evolutionary Phylogenies of the Drosophila montium Subgroup
Bong K. KIM and Talmo K. WATANABE

Ohnishi and Watanabe (1984) constructed a biochemical phylogeny of
the D. montium subgroup by an electrophoretic technique (2DE). Twentynine species were classified to three complexes and others. Seventeen species
belonging to D. kikkawai complex (6 spp), D. jambulina complex (4 spp)
and D. auraria complex (7 spp) were genetically tested by crossing interspecifically. Mating successes during two days mating were examined bydissecting females. Percent of unsuccessful matings between species significantly correlated with the genetic distances estimated by 2DE (r=0.58,
d.f=79).
From the difference of successful matings between pairs of reciprocal
1)
2)

The University of Texas at Houston.
Faculty of Science, Kyushu University.

RESEARCH ACTIVITIES IN 198471

hybrid matings, we speculated the direction of species evolution in each complex according to Watanabe and Kawanishi (1979); that is females of a
derived species do not mate well with the ancestoral males but females of
the ancestral species readily mate with derived males. In the D. kikkawaicOl11plex, species evolved as kikkawai ----.. leontia ----.. pennae ----.. lini-like ----"lini~ bach In the D. auraria complex, it was punjabiensis ----.. punjabiensis-like~ jambulina ----.. barbarae. In the D. auraria complex, it was quadraria-)
asahinai ----.. rufa ----.. subauraria ----.. biauraria ----.. triauraria ----.. auraria.
Ancestral species such as kikkawai and punjabiensis distributed in the widest
area among the D. kikkawai complex and the jambulina complex. In caseof D. auraria complex, rufa is found in the wide area of Asia, althoughquadraria and asahinai are restricted in Taiwan and its near islands. These
results support the stasipatric speciation model by Watanabe and Kawanishi(1983) in which old species distribute in wide or restricted area and young
species in a small and central area of each sibling group.
Genetic PJ:'Ofile of Wild Mouse Subspecies CoHectedfrom South Pacific Jrsiamls
Kazuo MORIWAKI, Nobumoto MIYASHITA, Hiromichi YONEKAWA,
Tosihide YOSIDA, Tomoko SAGAI, Hitoshi SUZUKI,
Yasuyuki KURIHARA and Tomomasa WATANABE""The scientific survey on the wild rodents in the Pacific Islands" supported
by a Grant-in-Aid for the Overseas Scientific Survey was held from July toAugust in 1984, in which three members, Dr. Udagawa (Azabu Univ.), T.Y.
and K.M., joined. In this survey, we collected a number of mice, Mus
musculus, in New Caledonia, Fiji and Tonga and analyzed various genetic
characters of them. Those are chromosome C-band patterns, antigenic
specificities of lymphocytes, RFLPs of mitochondrial DNA and ribosomal
DNA, electrophoretic mobilities of hemoglobin-beta, Proteinases andAmylases as summarized in Table 1. In these mice, C-bands of each chromosomes are almost positive as similar as European and Central- and Southeast Asian subspecies, except negative C-bands of No. 16 and X chromosomes. Anti-H-2 monoclonal antibodies, Hd-l, Hd-37 and Hd-38, wellreacted with H-2 public specificities in the European subspecies in our previous survey. Hd-39 exhibited much crossreaction with the Asian subspecies.

* Aichi Coloney Research Institute.

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35
Table 1.

SampleNo.

Genetic characters of wild mice collectedfrom South Pacific Islands

Place ofcollection
Port Lagoerre(New Caledonia)

2
3

/I

Katiramona(New Caledonia)

4
5
6

Chromosome C-bands pattern

Sex
F

1-11+/+, 12+/±, 13-19+/+, X+/±F

1-15+/+,16+/-,17-19+/+, X±/±
1-19+/+, X-V

M

/I

M

Lakena (Fiji)Longo (Tonga)

FF

1-16+/+, 17±/±, 18-19+/+, X-V
1-19+/+, X+/1-/-,2+/-,3+/+,4+/-,5-15+/+,16-/-,17-19+/+, X+/-

mAb anti-H-2

Sample
No.

2
3
4
5
6

Hd-l

Hd-37

Hd-38

weak

3+

+5

+3

+4

+4
+4

+4
+4
+3
weak

+5
-1-5
+5
+5
+4

mtDNA
RFLP

Hd-39

rDNA
RFLP

Hbb

domesticus- domesticustype
brevirostristype
weak,
+3

1/

1/

1/

1/

1/

1/

1/

1/

brevirostristype

Lymphocyte Antigens

Sample
No.

Thy-1

Ly-1

Ly-2

Ly-3

1
2
3
4
5
6

2
2
2
2
2
2

2
2
2
2
2
2

1
1
1
1
1,2
1

2
2
2
2
2
2

S
S
DS
S
S
D

1/

Proteinases

AmylasePrt-1 & 3 Prt-2

Amy-2

BB
BB
BA
BB
BA
BB

A
A
A
A
A
A

B
B
B
AB
A
A

MtDNA and rDNA mostly demonstrated M. m. domesticus-nature ofthose mice. Hbb s allele is restricted to the European subspecies. All the
lymphocyte antigenic specificities, Proteinases (Prt-l & 3 and Prt-2) andAmylase (Amy-2) were European type. Thus, wild mouse subspecies inSouth Pacific Islands here examined is almost similar to the European sub-

RESEARCH ACTIVITIES IN 198473

species M. m. domesticus, though those in Tonga may closely related to M.
m. brevirostris. We could have hardly found the effect of Southeast Asiansubspecies, M. m. castaneus in these area.
Allelic Constitution of the Hemoglobin Beta Chain in Wild
Populations of the House Mouse, Mus musculus
Nobumoto MIYASHITA, Shunsuke MIGITA* and Kazuo MORIWAKI

We surveyed the allelic constitution of the hemoglobin beta chain locus
in the wild populations of house mouse in Asia and surrounding areas. A
total of 222 mice at 45 localities was examined to the electrophoretic analysis(Table 1).
One hundred seventeen mice were collected in 22 localities in Asia excluding Japan. In these populations, Hbb p and hbb d were common
although their frequency distribution was rather heterogeneous. In sevenlocalities in northern China and two localities in Korea, solely Hbb p homozygotes were observed. Both Hbb P and Hbb d were common in the othereight localities in Asia. In five localities of Southeast Asia and Sri Lanka,
only Hbb d was observed.
Sixty-three wild mice were caught in 16 localities in Japan. Threephenotypes, Hbb-P, -PD and -D, were observed in the wild populations ofTable 1.

The allelic frequency of the Hbb locus in wild
subspecies of Mus musculus
AIIelic frequency

No. of
mice

p

d

s

63
23
10
7

0.86
0.33
0.35
0.57

0.14
0.67
0.65
0.42

0.00
0.00
0.00
0.00

58
19
18
12
12

1.00
0.66
0.00
0.00
0.00

0.00
0.34
0.28
0.08
0.46

0.00
0.00
0.72
0.92
0.54

Subspecies
M. m. molossinus
M. m. castaneus
M. m. urbanus
M. m. bactrianus(unidentified)Central and northern China, and Korea
China, coastal areas (Beijing and Shanghai)
Chichi Island (Ogasawara Is.)
Australia (Sydney) and South Pacific Is.
Seychelles and Mauritius* Cancer Research Institute, Kanazawa University.

47

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Japanese house mouse, Mus musculus molossinus, as well as Asian house
mouse subspecies. The overall allelic frequencies of Hbb P and Hbb d were
86 and 14 %, respectively. Moreover, interregional differences in thesefrequencies have been observed. Geographically, the Hbb p allele was prevalent in the central part of Japan, while the Hbb d was common in the
peripheral regions.
Forty-two wild mice were collected from Chichi Island (the Ogasawara
Is., Japan), Sydney (Australia), Numea (New Caledonia), Suva (Fiji),Nuku'alofa (Tonga), Mahe Island (Seychelles) and Reduit (Mauritius), in
which only three phenotypes, Hbb-S, -SD and -D were identified. The
Hbb phenotypes of these wild mice were similar to those of European wild
mice, M. m. domesticus and M. m. brevirostris (Miyashita et al., Biochem.
Genet. 23: 975-986 (1985)).
Restriction Fragment Length Polymorphism of rDNA
in European Wild Mice with Robertsonian Translocations
Yasuyuki KURIHARA, Hitoshi SUZUKI, Kazuo MORIWAKI,
Heinz WINKING ' ), Ryo KOMINAMI2 ) and Masami MURAMATSU 2 )Genetic diversity of ribosomal DNA (rDNA) in wild mice (Musmusculus) with Robertsonian translocations (Rb) was estimated by southernblot analysis using l8S- and 28S rDNA probes, and the data were comparedwith our previous study (Ann. Rep. Natl. Inst. Genet. 33: 26, 1982) about
restriction fragment length polymorphism of rDNA in various mouse subspecies. The comparison revealed that restriction patterns could be interpreted as a mixture between two types; one of which is found in Asian subspecies and the other is European subspecies. For example, CB mice from
central Italy exhibited a 5.5 kb band, specific to M. m. brevirostris from
France, and a 9.0 kb band, characteristic of Asian mice, when hybridized
with the 28S rDNA probe after EcoR I digestion. Moreover, Zadar micefrom Yugoslavia exhibited a 2.3 kb band, characteristic of European mice,and a band over 20 kb, characteristic of Asian mice, when hybridized with
288 rDNA probe after BamH I digestion. Similar results were obtainedin the other Rb-mice. Although more detailed works are desired, we may
assume that a genetic introgression from Asian subspecies could have
1)

2)

Medizinische Hochschule Lubeck, Lubeck.
Tokyo University.

RESEARCH ACTIVITIES IN 198475

occurred in European subspecies.
On the other hand, rDNA clusters which are cytologically identified as
NORs are generally located at centromeric regions on several chromosomesin Mus musculus. But, exceptionally, in some Rb-mice named WMP and
Cremona (Rb-mice t ), one of the rDNA clusters is located in the telomericregion of chromosome No.4. We have also studied these Rb-mice t in thesame way: commonly in Rb-micet, our 18S rDNA probe hybridized withan 8.7 kb band after Pvu II digestion, and our 28S rDNA probe hybridized
with an 18- and a 14 kb bands after Hind III digestion. These bands were
not observed in the other Rb-mice. Conceivably, the structures of thetelomeric rDNA units are largely different from those of the centromericrDNA units.
A 5 ' ·end Sequence of the Fibroin Gene from Wild Mulberrysilkworm, Bomhyx mandallina
Jun KUSUDA, Osamu NINAKI, Yoshiaki SUZUKI
and Yataro TAZIMA

Morphological and cytological studies have suggested that Bombyx
mandarina is a possible ancestor of domesticated silkworm, B. mori. In
order to elucidate their evolutionary relationship, the structures of theirfibroin genes were compared. The fibroin genes of B. mori have alreadybeen cloned from some strains and the sequences around 5'-ends are known.
We constructed the DNA library of B. mandarina DNA and screened its
fibroin gene hybidizing with the cloned DNA containing the fibroin gene of
B. mori. One clone (J1Ma3) contained 14.5 Kb B. mandarina DNA andthe insert was cut out for three fragments with BamHI; 5.1 Kb, 4.9 Kb and3.5 Kb.
Since the probe of B. mori hybridized only with the 4.9 Kb fragment, theregion inserted to vector seems to be the end of fibroin gene or the junctionof exon-intron. To analyse this region precisely, 4.9 Kb BamHI fragment
was recloned into pUC12. One clone (pMaf7) was cleaved with several
restriction enzymes and the physical map was constructed. The restriction
sites resembled those around 5'-end of fibroin gene of B. mori. A 2.6 Kb
fragment corresponding 5'-end of B. mori was sequenced by M13 vectordideoxy method. The nucleotide sequences of both fibroin genes were
almost identical except for some base exchanges and deletions or insertions.

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Comparison of the base exchanges of B. mandarina with those of B. moristrains revealed that B. mandarina is closer to Chinese bivoltine strains than
European univoltine strains. These results support the idea that B.
mandarina is a closely related species to B. mori and a probable proto-typeof domesticated silkworm.

RESEARCH ACTIVITIES IN 1984

IX.

77

HUMAN GENETICS

Use of Retinoblastoma and Wilms' Tumor as Sentinel
Phenotypes for Population Surveillance
Ei MATSUNAGA and Kensei MINODA *A vast majority of retinoblastoma (RB) and Wilms' tumor (WT) occur
sporadically, and they are generally arising from a germinal or somatic mutation. Although the two tumors are not usually detectable at birth, incountries where systematic registration of childhood malignancies has been
established, data on the incidence of the two tumors may readily be used for
population surveillance of environmental mutagens.
In Japan, the ongoing nation-wide registration of all childhood malignancies is supported by a private foundation and maintained by voluntarycooperation of physicians. The current data, however, are not satisfactory
because of underreporting (Hanawa, Y. All Japan Children's Cancer Registration, 1969-73. Children's Cancer Assoc. Japan, Tokyo, 1975), withthe exception of those for RB reported in the initial stage of registration(Minoda, K. 1976. Acta Soc. Ophthalmol. Jpn. 80: 1648). On the other
hand, fairly reliable data at the prefectural level are available in Kanagawa,where the ascertainment is made by reviewing the application form submitted by the patients to the local government for defraying the cost of
medical treatment (Nishihira, K. 1983. Kanagawa Children's Med. Center
Jour. 12: 62); since 1971 childhood malignancies have been among a numberof "designated diseases" for which, upon application, such assistance isrendered by central and local governments. From these data the live-birthfrequencies of RB and WT are estimated to be about 1: 15,000 and 1: 20,000
in Japan, respectively.
With approximately 1.5 million births a year, the annual number of newcases of the two tumors combined would be about 175; if this is taken as astandard, then the border-line above which a rise in the incidence is statistically significant at the 1 % level would be 210. If a significant rise in theincidence of sporadic cases could be detected, a case-control study has to beundertaken. In bilateral cases either paternal or maternal exposure prior* Tokyo Metropolitan Yoiku-In Hospital.

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

to the conception of the index child should be suspected, whereas in unilateral cases the child's exposure before or after birth should be suspected.
The Ministry of Health and Welfare should encourage and promote theregional registration of childhood malignancies on the population basis.
Mitochondrial DNA Polymorphism in Japanese
I. Analysis with Restriction Enzymes of Six
Base Pair Recognition
Satoshi HORAI, Takashi GOJOBORI and Ei MATSUNAGA

Since Potter et at. (1975) showed that a restriction enzyme can detectvariability in mammalian mitochondrial DNA (mtDNA) sequences, restriction enzyme analysis has become a powerful tool for detecting genetic
variations in human (Brown and Goodman 1979; Brown, 1980). This
paper deals with the results of analysis of 120 mtDNAs from Japanese digested with 15 restriction enzymes that recognize six base pairs.
The digestion patterns with HincH and Hadl are highly polymorphic.
Seven distinct cleavage patterns were observed for the HincH digestion andfive different patterns were found for the HaeII digestion. In addition
EcoRV was found useful to detect polymorphism in our sample, because
5 % of individuals showed an atypical cleavage pattern. In digestions with
StuI, HindIII, Scal and XhoI, three different cleavage patterns were observed for each enzyme respectively. While StuI and ScaI were not examinedin previous studies, digestion patterns with HindIII, XhoI, Sac!, EcoRI
and Pvull were reported to be monomorphic (Brown, 1980). In the present
samples, an atypical cleavage pattern was observed for each of Sad, EcoRI
and PvuH. The analysis with four additional restriction enzymes, BamHI,
Xbal, Kpnl and Dral showed monomorphic patterns in all samples.
Based on the combination restriction enzyme morphs observed for eachof the 120 individuals, we found 22 distinct mtDNA restriction types.
Seventy-two percent of the individuals showed the cleavage pattern identicalto that of the published mtDNA sequence except the HincH and Stul digestions (type 1). Although other types occurred at low frequencies, it isinformative to measure the index of nucleotide diversity. The averageamount of nucleotide change in human mtDNA can be estimated by themethod of cleavage site comparisons (Nei and Li, 1979). In the present
study, an average of 62 sites (equivalent to 372 base pairs, or 2.2 % of the

RESEARCH ACTIVITIES IN 198479

whole mtDNA sequence) could be compared. Estimating the number (0)of nucleotide substitutions per nucleotide site between any two types, we
obtained the mean value of 0.0042. Because we used restriction enzymes
that recognize six base pairs, a nucleotide substitution by site gain can beeasily deduced from the published sequence. In the present study 11 site
gains were observed, of which 8 occurred in the protein coding regions,
resulting in amino acid substitution in at least 4 cases. Of the 11, 7 weretransitions and 4 were transversions. The apparent ratio of transitions totransversions if 1.75: 1, which is slightly less than the ratio (2.5: 1) obtained
by Cann et at. (1984). These are, however, significantly lower than theratio obtained by sequence comparisons among higher primates mtDNAs(11: 1, Brown et at. 1982) and among a 900 base pair segment of the noncoding region from several human mtDNAs (32: 1, Aquadro and Greenberg
1983). A phylogenetic tree was constructed by genetic distances among the
22 restriction types, suggesting that the Japanese population conceals aconsiderably high degree of mtDNA diversity. For details, see Human
Genetics 68: 324-332.
High-resolution Studies in Patients with Aniridia-Wilms
Tumor Association
Y. NAKAGOME 1 ,2) and N. NAGAHARA 3)

In a recent issue of Human Genetics, we described results of high-resolution studies in patients with aniridia-Wilms tumor association and a few
related conditions (Nakagome et at. Hum. Genet. 67: 245, 1984). It included
a boy with aniridia-Wilms tumor association (case 2545) with IIp deletion.
The mother (case 2580) also had an apparently identical IIp deletion andno insertion could be detected at that time. Both the father and maternal
grand mother had a normal karyotype.
Recently, we have had an opportunity to examine phenotypically normal
sister as well as to re-examine the mother by the acridine-orange high-resolution technique. Both were balanced carriers of an insertion between
a no. 11 and a no. 12, karyotype being 46, XX, ins (12; 11) (p. 11. 2; p. 12
p. 14.2) (Nakagome & Nagahara: Hum. Genet. in press).
1)

2)

3)

Dept. Hum. Genet., Nat. Inst. Genet., Mishima.
Dept. Congo Abn. Res., Nat. Child. Med. Res. Ctr., Tokyo.
Dept. Surgery, Osaka Child. Med. Ctr., Osaka.

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

High-resolution Study in Patients with Prader-Willi Syndrome
Y. NAKAGOME1 ,2), T. TAKANo3 !, S. NAGAHucHl4), L HIEl 4),
F. TANAKA 5 ), Y. NAKAMURA6 ) and A. TANAE4)

For many years, Prader-Willi syndrome has remained to be a typicalexample of malformation syndrome with unknown etiology. In increasing
number of cases chromosome abnormalities involving proximal l5q havebeen described. However, in still considerable number of cases chromosome
abnormality remains undetected. The lack of resolution in cytogenetic
analysis is one possibility. The other is variation of criteria in the clinicaldiagnosis of the syndrome.
In the present study, 27 clinically diagnosed cases ofPrader-Willi syndrome
were studied by the technique of acridine-orange high-resolution banding(Matsubara and Nakagome, Cytogenet. Cell Genet. 35: 148, 1983). In
one case, number of mitotic cells was not satisfactory. In 6 cases, karyotype analysis was possible at or lower than 400-band per haploid-set level.
In the remaining 20 cases, chromosome analysis was carried out at about
550-band per haploid set or higher. In 14 of them, small deletion involving
proximal 15q is detected. In the remaing 6, no delection was detected.
Clinical evaluation of patients in the two groups is in the progress to detect
difference, if any, between them.
Origin of Restriction Sites in a Simple Repeated DNA
Cloned from Human Y Chromosome
Yutaka NAKAHORI* and Yasuo NAKAGOME*A DNA fragment was cloned from EcoRI-digested and gel-electrophoresed
DNA from a normal man. It was 3.5 kb in length, highly repeated andmale specific (Nakahori and Nakagome, this report, No. 34). Restriction
analysis revealed that there were enzymes which cleaved it at many sites
Dept. Congo Abn. Res., Nat. Child. Med. Res. Ctr., Tokyo.
Dept. Hum. Genet. Nat. Inst. Genet., Mishima.
3)
Dept. Hum. Genet., Tsukuba Univ.
4)Nat. Child. Hosp., Tokyo.
5)
Dept. Pathol., Univ. Tokyo.
6)
Dept. Pediat., Metropolitan Huchu Hosp., Tokyo.
* Dept. Hum. Genet., Nat. Inst. Genet., Mishima and Dept. Congo Abn. Res., Nat.
Child. Med. Res. Center, Tokyo
1)

2)

RESEARCH ACTIVITIES IN 1984
Table 1.

----

Number of restriction sites in pHY6

4b p')-----:raqF) many (1)
4 (2)
Rsa I
2 (2)
Sau3AI
1 (2)RaeIII
o (2)
RpaU
AluI

o (3)

81

5bp

6bp

HinfI

many (1)

EcoRI
BanIU

2 (2)
1 (2)

DdeI

1 (3)

BamHI
SalI
AatI
BanU

0(3)
0(3)
0(4)
0(4)

Length of recognition sequence.
For each enzyme, observed number of sites in pHY6 and number of base substitutions needed to generate them (in parenthesis), are shown. There is no EcoR! site within
the fragment but both ends of it are marked with them.
1)

2)(Taq I and Hinf I), 4 sites (Rsa I), two sites (Sau 3AI and Eco RI; at bothends) and one site (Hae III, Dde I and Ban III). No site was detected as
to Hpa II, Alu I, Bam HI, Sal I, Aat I and Ban II (Table 1).
A small segment of the fragment was sequenced by the dideoxy method
using M13 phage. It was basically composed of repeat of a 5-base-pairunit TTCCA.
Assuming (TTCCA)n to be the original sequence of the entire fragment,
number of base substitution(s) necessary to generate each type of site in thetable 1 was counted and was also included in the table (in parenthesis). Only
two types of restriction sites, TCGA (Taq I) and GANTC (Hinf I), were possible to occur through a single base change. They can be generated in an
overlapped form within the (TTCCA)n tract by a single base substitution;from TCCATTC to TCGATTC. It is noteworthy that they were frequently
detected on the fragment. There are other 4- or 5-base pair sequences which
can be generated by a single base change, however, none of them represented
to recognition sites of known restriction enzymes. Five of six restriction
sites that can be generated by the substitution of two base pairs were actuallydetected in the fragment (Table 1). None of the available enzymes thatneeded three or more base substitution actually cut the fragment with the
exception of Dde 1.
Possible mechanisms for all observed restriction sites are summarized in
the Table 2. Among the two-base change sites, it is expected that either
Rsa I or Sau 3AI has better chance of cutting it than either of Rae III or
Hpa II as the former two can be generated through two different mechanisms.

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35
Table 2.

Possible restriction sites in a (TTCCA)n tract byone- or two-base substitutions
Base changes needed

Original
sequences

Generated
restriction
sites

Enzymes

TCCN)
CATTC
TTCC
ATTC
ATTC
CATT
TTCC
CCAT
CCATTCTTCCAT
CCATTC

TCgA2)
gATTC
gTaC
gTaC
gaTC
gATc
ggCC
CCgg
gaATTCaTCgAT
gCATgC

Taq 1
HinfI
Rsa P)
Rsa 14)
Sau3AI4)
Sau3AP)
Rae III
Hpa nEcoRI
Ban III
Sph I

transversion 1

transitiontransversion II

C-G3)
C-G
T-G,C-A
T-A
T-A
C-G

A-G
A-G
T-C
A-G

C-G
T-A, C-G
C-G

T-G,T-G
T-G
C-A
T-G

1) A segment of a tract (TTCCA)n.
2) Eleven cleavage sites for 9 different enzymes areshown. Substituted bases are represented in a small letters. 3) C-G denotes a C in
the original sequence was replaced with a G. 4) An identical site can be generated by 2
different mechanisms.

This was the case as shown in the Table 1. Out of three Rsa I sites sequenced, two were due to the one shown on the 3rd line and the other wason the 4th (Table 2).
As a whole, observed number of sites is in agreement with expectations if
we assume that the fragment started as (TTCCA)n and they have been
generated more or less randomly. Further sequencing of the fragment isin progress.

RESEARCH ACTIVITIES IN 1984

x.

83

BEHAVIORAL GENETICS

Effects of Feeding Experience before Weaning on
Food Preference in Mice
Tohru FUJISHIMA

Clarifying the mechanism controlling the food preference of animals
will be meaningful not only for animal husbandry but for human beings.
The study has been carried out to investigate the roles of genetical and environmental factors in determining the food preference of mice. The present study was conducted to determine which time period in development(if any) was most effective in controlling the food preference of adult mice.
Three groups of male inbred mice, each having been reared with one ofthree conventional foods (A, B and C), were divided into three sub-groups
at weaning time of 21 days of age, respectively. Animals in each subgroup were fed one of the three foods from weaning to the food preference
test. Individual food preferences were assessed with the cafeteria feeding
test of the three foods for over 60-day-old mice. In the preference test the
weekly amounts of each consumption of the three foods were measured during the 4-week test period.
Food consumption in the preference test was highest in B food and lowest
in C food in all t<"'1e sub-groups, no matter what food mice had been fed
until the test time. This may be suggestive of the fact that mice used in thepresent experiment genetically prefer B food best of the three foods. However, the ratio of each food consumption to the total amounts of food ingested during the test period was highest in the food of all the groups withwhich mice had been reared before weaning, suggesting that feeding experience
before weaning, or nursing period, plays the most important environmentalrole in determining the food preference of adult mice. The extents of genetical and environmental factors involved in the determination of thefood preference of mice were also estimated with the analysis of variancetechnique. They were 29.3 % for genetical and 70.7% for environmental
factors. In the environmental factors the factor due to before-weaning
feeding was 60.7%.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Effects of Wave Length of Light on Sexual Maturity
in Female Japanese Quails
Tohru FUJISHIMA and Masami SAITO

Environmental light influences a number of physiological mechanisms inbirds, including reproductive development, growth and endocrine function.
The mechanism by which light acts on the reproductive system has not been
resolved. It has been only demonstrated that the long wave length· of lightaccelerates sexual maturity in birds, while the short wave length has nosuch an effect. This study has been conducted to genetically approach
resolving the mechanism of light affecting sexual maturity through thenervous system in the Japanese quail.
Four replicate experiments with 250 Japanese quails in total were conducted to obtain the preliminary information on the selective experiment
for the sexual maturity of quails under different colored-light conditions.
Female birds were housed in battery brooders under a white light condition
for 5 weeks after hatching and then randomly allotted to three light-controlled chambers. The light environments were 16L: 8D white (Toshiba
white fluorescent lamp, FLlOD-EDL-56K), 16L: 8D red (National purered colored fluorescent lamp, FL40S R-F, 9 lux, 590-700 nm) and l6L: 8D
blue (National pure-blue colored fluorescent lamp FL40S S-F, 9 lux, 380540 nm). During the experimental period, the age in days at first egg wasmeasured for each bird in each group.
The age at first egg was 55.3 for the white, 55.7 for the red and 72.5 for theblue light groups. Female quails kept under blue light had significantly
lower sexual maturity than birds kept under red or white light (P<.OI).
There were no familial correlations of the age at first egg between different
colored-light groups, suggesting that the mechanism of light affecting sexual
maturity in quails differs with different wave lengths of light. Significant
differences in sexual maturity among families were found in the white and the
red light groups, but not in the blue light group.

RESEARCH ACTIVITIES IN 1984

XI.

85

ECOLOGICAL GENETICS

Monitoring of the Wild-Rice Populations in Thailand
Hiroko MORISHlMA, Yoshiya SHiMAMOTO*, Yoshio SANO
and Yo-Ichiro SATO

Effect of environmental stress on population dynamics has been a central
theme of population biology. Little is known about wild-rice populations.
Environmental constraints for survival of the Asian common wild rice
Oryza perennis (= O. rujipogon) might be water stress, habitat disturbanceand gene flow from cultivated rice growing nearby. To make a long-term
observation for a demographic-genetic study, seven study-sites of wild rice
populations were designated in the suburb of Bangkok during our studytour in 1983, some of which were repeatedly observed since 1973. Those
sites were revisited in June and August in 1984, and January in 1985.
Various items regarding life-history characters and habitat conditions wererecorded. Preliminary results on plant demography and population flux
are summarized below. Genetic change in population structure associatedwith demographic process is reported elsewhere in this issue (Barbier andMorishima).
a) By checking juvenile plants, it was confirmed that four populations
propagate mainly by seeds (annual type, sexual reproduction), one population by ratoons (perennial type, asexual reproduction), and two populations
both by seeds and ratoons (intermediate or mixed type). b) Annual types
are essentially inhabitants of shallow swamps which are parched in thedry season, while perennial types are found in the sites which retain soil
moisture during the dry season. The sites inhabited by the intermediatetypes are characterized by strong habitat disturbance and introgression from
cultivated rice. c) June and August census indicated that high mortality
occurred at the juvenile stage in seedlings as well as in ratooned tillers. d)Annual populations were fugitive. During our observation period, one
populationbecame almost extinct showing decrease in genic diversity possibly
due to change in water regime in the area. Another population rapidly
expanded on a huge area after destruction of its vegetation. e) In the inter-* Hokkaido University.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

mediate populations, a trend of habitat segregation between seedlings andratoons was often recognized on a fine scale. This could be explained byenvironmental heterogeneity within a site.
Together with the results of our general survey on wild and cultivatedrices, details of the above study-sites are given in our Trip Report "Observation on Wild and Cultivated Rices in Thailand for Ecological-Genetic Study"
pp. 82, 1984).
Comparison between Potential and Realized Genetic
Variability in the Wild-Rice Populations withDifferent Life-Histories
Pascale BARBIER * and Hiroko MORISHIMA

Populations of Asian wild rice Oryza perennis (=0. rujipogon) show acontinuous variation in the degree of perenniality and associated characters. Seven populations (4 annual, 1 perennial and 2 intermediate types)
found in the suburb of Bangkok are continually studied to monitor theirdemography, genetic structure and population flux. In the present study,
plants collected from these populations were examined to compare genetic
structure of population at different stages of life cycle. In each population,
the plants examined consisted of two groups sampled in the different seasons:
The first group was raised from seeds collected at maturity stage (December
1983, early dry season), and the second group from seedlings and/or ratoons
collected at juvenile stage (June 1984, early rainy season). The former isconsidered to represent potentially available variability and the latter as
actually realized variability.
Genotypes at 6 isozyme loci (Pox-I, Sdh-I, Est-2, Pgi-I, Pgi-2, Pgd-I)
and several life-history characters (reproductive allocation, regenerating
ability, anther length, awn length, flowering time) were recorded on an
individual basis. Average genic diversity was calculated for each sample
using allozyme data. In the populations raised from seeds, the annualtypes showed a low genic diversity mainly due to fixation of alleles at manyloci, while the perennial and intermediate type showed higher genic diversity
with high heterozygosity. When compared with this potential variability,
significant drops in the level of genic diversity were found in realized variability observed in juvenile-derived populations for the perennial and some

* Universite des Sciences et Techniques du Langudoc, Montpellier.

RESEARCH ACTIVITIES IN 198487

annual types. This might be because the perennial types propagate mainly
by vegetative means though they are highly allogamous, while the annualtYpes are predominantly selfing and propagate by seeds. The intermediatetypes showed high genic diversity in realized as well as in potential variability. This can be explained by their partial allogamy combined with
mixed mode of sexual and vegetative reproduction. Such intermediatetype could have a high evolutionary potential.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

XII.

APPLIED GENETICS

Genetic Diversity in Indonesian Native Rice Cultivars with
Reference to the Indica-Japonica Differentiation
L. U. GADRINAB*, Y. 1. SATO and H. MORISHIMA

Asian rice cultivars are known to be differentiated into the Indica andJaponica types. However, there is very meager information on the Indonesian rices in this regard. Two-hundred thirty seven accessions collected in
Java, Kalimantan and Sumatra were investigated to examine whether or notthey were differentiated into these two types. Three diagnostic characters,
KC10 s resistance, phenol reaction and apiculus hair length, were examinedfor each accession. A bimodal distribution of the discriminant scores
combining the above three characters showed a variation ranging from the
Indica to Japonica types, though accessions with Japonica-specific characteristics were more frequent. Based on this score, 160 accessions wereclassified as the Japonica type and 77 as the Indica type. Relative frequenciesof the two types did not differ much according to the water regime in thefields; 68 %of upland and 59 %of lowland varieties were Japonica types. A
trend of differentiation into two types was observed similarly in the materials collected in different islands, although number of accessions collected
in Sumatra and Kalimantan was not many.
An electrophoretic survey of 6 isozyme loci of 5 enzyme species was
carried out. All loci assayed were polymorphic and their allelic frequencies
significantly differed between the Indica and Japonica types except for one
locus (Est-2). Among 96 possible genotypes expected from the alleliccombinations at 6 loci, 33 genotypes were found. A genotype with alleles
2 (Cat-i), +9 (Acp-l), 2 (Pgi-l), 0 (Pox-2) and 1 (Pgi-2) was most predominant in the present materials (68 %). Most accessions classified as the
Japonica type were found to have this or its similar genotype. Among thoseclassified as the Indica type, in addition to this same genotype, contrasting
genotypes with different alleles at most loci were frequently found. It was
found, further, that in allozyme variation lowland rices showed higher varietal
diversity than upland rices, and that in lowland as well as in upland rices* BIOTROP, Indonesia.

RESEARCH ACTIVITIES IN 198489

the Indicas were more diverse than the Japonicas.
Frequency Changes in Isozyme Genes Observed in a Hybrid
Population Derived from a Cross between an Indica
and a Japonica Rice Varieties
Reiko SANO and Hiroko MORISHIMA

Studies of multilocus polymorphism for isozymes in Asian rice cultivars
revealed that two major varietal groups, the Indica and Japonica types,
tend to have different allelic combinations for several loci. It is not known,
however, how such non-random association in isozymes was established in
Asian rices. To look into this problem, genotypes of 6 isozyme loci whosegene frequencies significantly differ between the Indica and Japonica types
were examined in a hybrid population derived from a cross between an
Indica (130, Taiwan) and a Japonica (221, the Philippines) varieties. Plants
examined consisted of two F r populations raised from F s populations whichwere established in Fa and thereafter separately maintained in bulk without
conscious selection in Japan as well as in the Philippines. The loci assayedwere Cat-l, Acp-l, Pgi-l, Pgi-2, Pox-2 and Est-2, and the parents carrydifferent alleles at all these loci. Among these 6 loci, Est-2 and Pgi-2 arelinked with 14 % recombination and Acp-l and Pox-2 with 31 %, and Cat-land Pgi-l seem to be independent of each other as well as of the above 4
genes.
The results obtained are summarized as follows: 1) When compared withFa, frequencies of alleles derived from an Indica parent increased consistently at all loci examined. 2) Nonrandom associations between independent
loci (parental as well as recombined types) were detected in some cases. 3)
Increasing trend of parental genotypes at the cost of recombined genotypes
was not observed. 4) Two populations maintained in Japan and the Philippines showed similar trends. Thus, an increasing trend of allelic frequenciesof genes derived from the Indica parent, which is often observed in the IndicaJaponica crosses, was clearly demonstrated. But, mechanisms whichbrought about differentiation into the Indica- and Japonica-specific isozyme
genotypes could not be detected in the scope of this experiment.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Cytoplasm Substitution between an Indica Stminof Oryza sativa and O. glaberrima
Yoshio SANO

To look into differential nucleus-cytoplasm interactions between the two
cultivated rice species, Oryza sativa and O. glaberrima, cytoplasmic substitution lines have been made by using a glaberrima strain (G) and an
Indica strain of sativa (B). The G cytoplasm had no adverse effect on pollen
development when combined with the nucleus of S. On the other hand,when the S cytoplasm was combined with the G nucleus, the substitution
line showed no seed set due to male sterility although the pollen grains werenormally stained with 12 - KI solution. A dominant gene derived from S
strain seemed to cause anther indehiscence in the substitution line. Further,a restorer gene (Rfj) from Akebono of Japonica type was effective on pollen
restoration in the male sterile line, suggesting that the S cytoplasm is thesame as those of Japonica type in terms of a fertility-restoring system.
(Euphytica in press)
Correlations between the Amounts of Amylose
and W x Protein in Rice Endosperm
Yoshio SANO, Mitsuko KATSUMATA and Etsuo AMANO

Amylose content and Wx protein level were analyzed in 25 nonwaxy ricecultivars and in F 2 endosperms of a cross of 74-5 (dull) X T65wx (glutinous)
in order to look into the relationship between the two traits. Regression
analysis indicated that 69 % (among 25 cultivars) and 77 % (in F 2) of thevariation in amylose content were associated with the variation in the Wx
protein leveL This implied that in rice endosperm amylose content increases
as the Wx protein increases. Further, the difference in amylose content
between Indica and Japonica types appeared to be well explained by theallelic difference at the waxy locus. (SABRAO J. in press)
Varietal Variations in Basic Vegetative Phase andPhotosensitive Phase, and Genetic Control of Basic
Vegetative Phase in Japanese Native Cultivars
Yo-Ichiro SATO

Heading time of rice plant is an important character from evolutionary

RESEARCH ACTIVITIES IN 198491

as well as from agronomic standpoints. The number of days from seeding
to heading is considered to be determined mainly by the lengths of the photosensitive phase (PSP) and basic vegetative phase (BVP). In order to look
into the origin and dissemination of early-maturing cultivars in Japan, thevariations in the above two phases were examined among 93 native cultivars
collected from various localities in Japan, and allelism test of the genes controlling the length of basic vegetative phase was carried out. They weregrown under a shortday (30°C phytotoron in winter) and a longday (a paddy
field, in summer) conditions. The lengths of BVP and PSP were estimatedfor each cultivar by the number of days to heading in shortday condition andby the difference in the days to heading between longday and shortday conditions, respectively. The cultivars tested were divisible into 2 groups
with a long (75-95 days) and a short (38-75 days) BVP. Among the cultivars with short BVPs, the distribution of PSP, varying from 0 to 72 days,
was trimodal. Among those collected from areas north of 32°N in latitude,
PSP decreased with increasing latitude of collection site. But no correlation between PSP and latitude was found among cultivars from the southern
islands. The early-heading cultivars (with short BVP and PSP) were foundthroughout Japan although they were infrequent. The pattern of distribution of native rice cultivars with varying BVP and PSP periods seems toreflect the variations in natural environments and cropping system underwhich they have been cultivated so far.
The early-heading cultivars were divisible into 2 groups, early-A (extremely early) and early-B (about 10 days later than early-A). To test allelic relationship of genes carried by Japanese early cultivars with a known earlinessgene which shortens BVP (Ef-lb), twelve early cultivars were each crossedwith a test-strain carrying Ef-lb, and a modifier (m) emphasizing its effect.
Segregation patterns of heading date in F 2 showed that the early-A types
carried Ef-lb, and m and/or another modifier with stronger effect than m.
Many of the early-B types possessed the same genes as of the test-strain (Eflb m), but a few others seemed to carry a different earliness gene. The
Ef-lb gene was found in many of early-maturing cultivars distributed in different localities of Japan. This suggests that those cultivars were differentiated in an early period of diversification of rice in Japan. (Japan. J.
Breed. 35: 72-75, and 35: 160-166).

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

Genetic Control of Apiculus Hair Length in RiceYo-Ichiro SATO

The length of apiculus hair is known to serve as an Indica-Japonica
discriminating character as it is longer in Japonica than in Indica. No
major genes have been yet reported for this character except for gl-l andgl-2 for the absence of hair or glabrousness. The mode of inheritance of
this character was studied in randomly selected Fa plants and their derivativelines from an Indica X Japonica cross. The parents used are f. 145 (Indica,
black hull, apiculus hair 0.32 mm) and T65 (Japonica, non-black hUll,
apiculus hair 0.90 mm).
The F 4 plants derived from 15 F g with apiculus hairs shorter than 0.7
mm showed an apiculus hair length ranging from 0.1 to 0.7 mm. Almost
all F 4 plants derived from 14 Fa plants with apiculus hair longer than 0.7
mm had the same range of hair length as of the F g plants. The F 4 plants
derived from other 7 Fa plants with similarly long apiculus hair segregatedinto 2 classes of apiculus hair length, shorter and longer than 0.7 mm. The
numbers of plants with short and long apiculus hair were 37: 96, which
fitted the 1: 3 ratio expected when monogenic segregation was assumed(t=0.56, non-significant). The numbers of F 4 lines with all long, all shortand segregating apiculus hair were 14, 15 and 7, respectively, which appeared
to fit a 3: 3: 2 ratio (x2=0.63, non-significant). These data suggested thatthe apiculus hair length is controlled by a major gene, although there areother modifier genes. Tentatively this gene is symbolized as Aph/aph (t).
Of 21 Fa plants with long (longer than 0.7 mm) apiculus hair, only one
had the black hull, whereas 8 out of 15 Fa plants with short (shorter than
0.7 mm) apiculus hair showed black hull. Since the black hull is controlledby 3 genes, Ph, Bh-a and Bh-b gene, aph would be linked with one of thethree genes. Among 26 Fa plants with yellow hull, 18 had Ph and 9 ofthem had apiculus hairs longer than 0.7 mm and the other 9 had shorter
apiculus hairs. Therefore, the aph gene does not seem to be linked with
Ph. It would linked with either Bh-a or Bh-b.

RESEARCH ACTIVITIES IN 198493

Two-dimensional Gel Electrophoresis of Four Kinds ofFractionated Reserve Proteins in Rice Endosperm
Tom ENDO

Four kinds of protein molecular species, i.e., albumin, globulin, prolaminand glutelin, were sequentially extracted from rice endosperm, where theiryields for 10 g of the powdered endosperm (150 mesh) were, in one case, 14ll1g, 202 mg, 19 mg and 393 mg, respectively. Extraction of glutelin was
carried out as follows: endosperm was macerated with ten volume of 0.1 M
NaOH for 15 min. and centrifuged. The supernatant was neutralized with
1 M ascorbic acid and 0.1 M 2-mercaptoethanol to about pH 6.5. Mostof glutelin fraction was obtained as precipitate. The dry precipitate wasfound to be soluble in 8 M urea solution. Other kinds of neutralization
procedures produced an insoluble form in urea solution.
One or two hundred micrograms of each protein fraction were applied
to O'Farrell's system (1975 and 1977), where albumin and glutelin wereanalysed with equilibrated pH gradient IEF method, because most polypeptide components of both the fractions were in acidic isoelectric points.
Also, globulin and prolamin were applied to non-equilibrated method,
because of their alkalinity in their major components. All polypeptide
ll1aps were obtained by silver staining method due to Oakley et al. (1980).
Although the number and the configuration of polypeptide spots in thell1aps are not completely reproducible at present, there are detected morethan 40 spots in albumin fraction, 30 in globulin, 20 in prolamin and 15
in glutelin. It was noted that the albumin fraction showed the maximum
number of spots though the total amount extracted was the least and, in
contrast, the glutelin fraction produced the minimum number despite being
the most abundant in rice endosperm.
Sample Size and Number of Loci to Investigate for
Estimating Effective Population Number
Shin-ya IYAMA

Based on the well-known theoretical relationship between genetic drift
and population size, effective population number of fish populations inhatchery was estimated from the amount of genetic drift based on the frequencies of various isozyme loci (p. 88, Ann. Rep. 1982).

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

For the estimation, frequencies of various isozyme markers were estimatedon the basis of N s individuals sampled from the population and the varianceof gene frequency (Vd ) was calculated on the basis of those of n1 loci (Xi)after standardized with the initial gene frequencies (Pi):di=(xi-pi)/p;(l-pi) .
The range of confidence at 95 %probability for estimated N e was calculatedusing the confidence intervals of estimated variance V/

Sd//x2.025<Vd<Sd//x2.975,
Numerical calculation of the intervals with various N s and n1 revealed
that width of the range is dependent on the sample size N s and number ofloci examined (n 1 ). Results of computation showed that more than tenloci at least may be desired for estimating the variance of genetic drift andthat estimated N e becomes erroneous if N s is as small as N e •Surveys on the Experimental Biological Stocks
Shin-ya IYAMASurvey on the various experimental biological stocks including animals,
plants, microorganisms and cultured cells maintained in the universities
and other related institutes was done in cooperation with other staffs in
Genetic Stocks Research Center. The purpose of this survey is to collectthe informations about experimental biological stocks maintained in Japanand construct a useful database for biological research. So far the replies
to the inquiry have been obtained from 187 universities and institutes andthey are under data-processing. Preliminary results showed that the numberof stocks maintained at various locations were 6,800 animal stocks at 316
locations, 80,000 plant stocks at 73 locations, 205,000 microbial stocks at
310 locations and 3,200 cultured cell stocks at 240 locations.

PUBLICAnONS

95

PUBLICATIONSAOKI, K: A quantitative genetic model of two-policy games between relatives. J. TheoL
BioI. 109: 111-126, 1984.
AoKI, K: Evolution of alliance in primates: a population genetic model. J. Ethol.
2: 55-61, 1984.
AoKI, K: A population genetic model of the evolution of oblique cultural transmission.
ProC. Jap. Acad. 60(B): 310-313, 1984.
AOKI, K and NOZAWA, K: Average coefficient of relationship within troops of theJapanese monkey and other primate species with reference to the possibility of group
selection. Primates 25: 171-184, 1984.
BONHOMME, F., CATALAN, J., BRITTON-DAVIDIAN, J., CHAPMAN, V. M., MORIWAKI, K.,
NEvo, E. and THALERE, L.: Biochemical diversity and evolution in the genus Mus.
Biochem. Genet. 22: 275-303, 1984.
BOURSOT, P., BONHOMME, F., BRITTON-DAVIDIAN, J., CATALAN, J., YONEKAWA, H., ORSINI,
P., GUERASIMOV, S. et THALER, L.: Introgression differentielle des genomes nucleaires et mitochondriaux chez deux semi-especes Europeennes des souris. C. R.
Acad. Sci. Paris 299: 365-370, 1984.
CROW, J. F. and AOKI, K: Group selection for a polygenic behavioral trait: estimating
the degree of population subdivision. Proc. Natl. Acad. Sci. USA 81: 6073-6077,
1984.
ENAMI, M. and ISHIHAMA, A.: Protein phosphorylation in Escherichia coli and purification of a protein kinase. J. Bio!. Chern. 259: 526-533, 1984.
FUJISAWA, T. and DAVID, C. N.: Loss of differentiating nematocytes induced by regeneration and wound healing in Hydra. J. Cell Sci. 68: 243-255,1984.
FUKUI, K., HAMAGUCHI, Y., SHIMIZU, A., NAKAI, S., MORIWAKI, K. and HONJO, T.: Duplicated immunoglobulin gamma-2a genes in wild mice. J. Mol. Cell Immuno!.
1: 321-330, 1984.
GOJOBORI, T.: A mathematical model of codon substitution and the constancy of evolutionary rate. Proceedings of the XUth International Biometric Conference (Invited
papers), 287-297, 1984.
GOJOBORI, T. and NEI, M.: Concerted evolution of the immunoglobulin V H gene family.
Mol. Bio!. Evo!. 1: 195-212, 1984.
GUSHIMA, H., YASUDA, S., SOEDA, E., YOKOTA, M., KONDO, M. and KIMURA, A.: Complete nucleotide sequence of E. coli glutathione synthetase gsh-II. Nucleic Acids
Res. 12(24): 9299-9308, 1984.
HARA, H. and SUZUKI, H.: A novel glycan polymerase that synthesizes uncross-linked
peptidoglycan in Escherichia coli. FEBS Lett. 168: 155-160, 1984.
HARADA, K., YUKUHIRO, K. and MUKAI, T.: Modifying mutations of the specific activity of alcohol dehydrogenase caused by a putative transposon in Drosophila metanogaster. Jpn. J. Genet. 59: 411-415, 1984.

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HASEGAWA, M., YANO, T. and MIYATA, T.: Evolutionary implications of error ampli_
fication in the self-replicating and protein-synthesizing machinary. J. Mol. Evo!.
20: 77-85, 1984.
HASHIMOTO, Y., SUZUKI, A., YAMAKAWA, T., WANG, C. H., BONHOMME, F., MIYASHITA,
N. and MORIWAKI, K.: Expression of GMI and GDla in liver of wild mice. J.
Biochem. 95: 7-12, 1984.
HAYASHIDA, H., MIYATA, T., YAMAWAKI-KATAOKA, Y., HONJO, T., WELS, J. and BLATTNER,
F.: Concerted evolution of the mouse immunoglobulin gamma chain genes. EMBo
J. 3: 2047-2053, 1984.
HORAl, S. and MATSUNAGA, E.: Differential enzyme activities in human esterase D
phenotypes. Human Genet. 66: 168-170, 1984.
HORAl, S., GOJOBORI, T. and MATSUNAGA, E.: Mitochondrial DNA polymorphism inJapanese. I. Analysis with restriction enzymes of six base pair recognition. Human
Genet. 68: 324-332, 1984.
HIDAKA, S., ISHIKAWA, K., TAKANAMI, Y., KUBO, S. and MIURA, K.: Complete nucleotide sequence of RNA5 from cucumber mosaic virus (strain Y). FEBS Lett.
174: 38-42, 1984.
IKEGAMI, M., MORINAGA, T. and MIURA, K.: Infectivity of virus-specific double-stranded
DNA from tissue infected by mung bean yellow mosaic virus. Virus Res. 1: 507512, 1984.
IMAI, H. T., URBANI, C. BARONI, KUBOTA, M., SHARMA, G. P. ,NARASIMHANNA, M. N.,
DAs, B. C., SHARMA, A. K., SHARMA, A., DEODIKAR, G. B., VAlDYA, V. G. and
RAJASEKARASETTY, M. R.: Karyological survey of Indian ants. Jpn. J. Genet.
59: 1-32, 1984.
INOUE, Y., TOBARI, Y. N., TSUNO, K. and WATANABE, T. K.: Association of chromosome
and enzyme polymorphism in natural and cage populations of Drosophila melanogaster.
Genetics 106: 267-277, 1984.
INOUE, Y., WATANABE, T. and WATANABE, T. K.: Evolutionary change of the chromosomal polymorphism in Drosophila melanogaster populations. Evolution 38: 753765, 1984.
ISHII, R., YOSHIKAWA, K., MINAKATA, H., KOMURA, K. and KADA, T.: Specificities ofbio-antimutagens in plant kingdom. Agric. BioI. Chern. 48: 2587-2591, 1984.
ITO, T., KADA, T., OKADA, S., HIEDA, K., KOBAYASHI, K., MAEZAWA, H. and ITO, A.:
Synchrotron system for monochromatic UV irradiation (140 nm) of biological material. Radiation Research 98: 67-73, 1984.
ITOW, T., KOBAYASHI, K., KUBOTA, M., OGATA, K., IMAI, H. T. and CROZIER, R. H.:
The reproductive cycle of the queenless ant Pristomyrmex pungens. Insectes Sociaux.
31: 87-102, 1984.
IZUI, K., MIWA, T., KAJITANI, M., FUJITA, N., SABE, H., ISHIHAMA, A. and KATSUKI, H.:
Promoter analysis of the phosphoenolpyruvate carboxylase gene of Escherichia coli.
Nucleic Acids Res. 13: 59-72, 1984.
JUKES, T. H. and KIMURA, M.: Evolutionary constraints and the neutral theory. J.
Mol. Evol. 21: 90-92, 1984.
KADA, T., MOCHIZUKI, H. and MIYAO, K.: Antimutagenic effects of germanium oxide

PUBLICAnONS

97

on Trp-P-2-induced frameshift mutations in Salmonella typhimurium TA98 and TA
1538. Mutation Research 125: 145-151,1984.
KAVA, T., KATO, M., AIKAWA, K. and KIRIYAMA, S.: Adsorption of pyrolysate mutagens
by vegetable fibers. Mutation Research 141: 149-152, 1984.
KAVA, T., INOUE, T., YOKOIYAMA, M. MOCHIZUKI, H., NAKATSUGAWA, S. and SUGAWARA,
T.: DNA-repair and cancer radiotherapy. Strahlentherapie 160: 695-699, 1984.
KAVA, T., SADAIE, Y. and SAKAMOTO, Y.: Bacillus subtilis repair test. "Handbook of
Mutagenicity Test Procedures" (Eds. B. J. Killbey, and others), 13-31, Elsevier Science
Publ.,1984.
KADA, T., SADAIE, Y., SAKAMOTO, Y. and HIRANO, K.: Use of the Bacillus subtilis recassay in environmental mutagen studies. "Mutation, Cancer, and Malformation"(Eds. E. H. Y. Chu and W. M. Generoso), 197-216, Plenum Publ. Corp., 1984.
KAVA, T., INOUE, T., YOKOIYAMA, M., MOCHIZUKI, H., NAKATSUGAWA, S. and SUGAHARA,
T.: Repair of DNA damage induced by ionizing radiation in ataxia telangiectasia
cells and potentially lethal damage repair inhibitors. "Modification of Radiosensitivity in Cancer Treatment", 251-263, Academic Press Japan, 1984.
KAJITANI, M. and ISHIHAMA, A.: Promoter selectivity of Escherichia coli RNA polymerase: Differential stringent control of the multiple promoters from ribosomal
RNA and protein operons. J. BioI. Chern. 259: 1951-1957, 1984.
J(AKINUMA, K., OKADA, Y., IKEKAWA, N., KADA, T. and NOMOTO, M.: Antimutagenic
diterpenoids from a crude drug Isodonis herba (enmei-so). Agric. BioI. Chern. 48:
1647-1648, 1984.
KAKINUMA, K., KOIKE, J., KOTANI, K., IKEKAWA, N., KADA, T. and NOMOTO, M.: Cinnamaldehyde: Identification of an antimutagen from a crude drug, Cinnamoni cortex.
Agric. BioI. Chern. 48: 1905-1906, 1984.
KATO, A., ISHIHAMA, A., NODA, A. and UEDA, S.: Improved purification and enzymic
properties of three forms of reverse transcriptase from avian myeloblastosis virus.
J. Virol. Meth. 9: 325-339, 1984.
KATO, J., SUZUKI, H. and HIROTA, Y.: Overlapping of the coding regions for IX and rcomponents of penicillin-binding protein 1b in Escherichia coli. Mol. Gen. Genet.
196: 449-457, 1984.
KATO, Y., HASEGAWA, T., SUZUKI, T. and FUJII, T.: Changes in the contents of free andprotein amino acids in Hiproly barley callus during differentiation. Agric. BioI.
Chern. 48: 1405-1410, 1984.
KIHARA, H., TOI, K., SAIGO, S., KAWAKAMI, K., HONDA, Y., ISHIHAMA, A., NAGAMURA,
T., FURUNO, T., WAKABAYASHI, K. and AMEMIYA, Y.: Association and dissociationof ribosomal subunits monitored by stopped-flow x-ray scattering. PF-Activity
Report 1984.
KIKUNO, R., TOH, R., HAYASHIDA, R. and MIYATA, T.: Sequence similarity between
putative gene products of geminiviral DNAs. Nature 308: 562, 1984.
KIMURA, M.: Neutral evolution as an innevitable process of change at the molecularlevel. "Darwin a Barcelona" (Ed. Josep Sancho), pp. 231-252, Promocions Publicadons Universitaries, Barcelona, 1984.
KIMURA, M. (ed.): Introduction to Molecular Evolution (in Japanese), Baifukan, Tokyo,

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1984.
KIMURA, M.: Evolution of an altruistic trait through group selection as studied by the
diffusion equation method. IMA Journ. of Math. Appi. in Med. & BioI. 1: 1-15,
1984.
KURODA, Y.: Mutagenic activity of Ames test-negative carcinogens in cultured
mammalian cells. Mutation Res. 130: 369-370, 1984.
KURODA, Y., YOKOIYAMA, A. and KADA, T.: Assays for the induction of mutations to
6-thioguanine resistance in Chinese hamster V79 cells in culture. Progress in Muta.
tion Res. 5: 537-542, 1984.
KURODA, Y.: Dose-rate effects of chemicals on mutation induction in mammalian cells
in culture. "Problems of Threshold in Chemical Mutagenesis" (Ed. by Y. Tazima, S.
Kondo and Y. Kuroda), 99-108, Environ. Mutagen Soc. of Japan, Mishima, 1984.
KUSAKABE, S. and MUKAI, T.: The genetic structure of natural populations of Drosophilamelanogaster. XVII. A population carrying genetic variability explicable by the classicalhypothesis. Genetics 108: 393-408, 1984.
KUSAKABE, S. and MUKAI, T.: Ditto XVIII. Clinal and uniform genetic variation over
populations. Genetics 108: 617-632, 1984.
MARUYAMA, T.: Stochastic theory of population genetics. Bull. Math. BioI. 45: 521554, 1983.
MARUYAMA, T. and FUERST, L. A.: Population bottlenecks and nonequilibrium models
in population genetics. I. Allele numbers when populations evolve from zerovariability. Genetics 108: 745-763, 1984.
MATSUI, M., OKA, A., TAKANAMI, M., YASUDA, S. and HIROTA, Y.: Site of dna A protein·binding in the replication origin of the Escherichia coli K-12 chromosome. J. Mol.
BioI. 184: 529-535, 1985.
Matsunaga, E.: Introduction to Symposium on Monitoring of Birth Defects: Present
status and problems. Teratology 30: 4A, 1984.
MATSUNAGA, E. and MINODA, K.: Seasonal variation in the births of patients withsporadic retionoblastoma. (in Japanese). Jpn. J.Hum. Genet. 29: 237, 1984.
MATSUNAGA, E.: Genetics and Man. (in Japanese). Baifukan, Tokyo, 185 p., 1984.
MATSUNAGA, E.: Frequencies of hereditary diseases in Japan. (in Japanese). Shonika
MOOK 32, "Genetic Counseling" (Eds. K. Baba, N. Kobayashi and M. Higurashi),
1-11, Kanehara, Tokyo, 1984.
MATSUNAGA, E.: Multistage and multifactorial carcinogenesis in hereditary tumours,with special reference to retinoblastoma. "Models, Mechanisms and Etiology of
Tumour Promotion" (Ed. by M. Borzsonyi, K. Lapis, N. E. Day and H. Yamasaki),p. 373-383, IARC (Lyon), 1984.
MIYOSHI, J., KAGIMOTO, M., SOEDA, E. and SAKAKI, Y.: The human c-Ha -ras 2 is aprocessed pseudogene inactivated by numerous base substitutions. Nucleic Acids
Res. 12(4): 1821-1828, 1984.
MORISHIMA, H. : Wild plants and domestication. "Biology of Rice" (Ed. by S. Tsunoda
and N. Takahashi), 3-30, Jap. Sci. Soc. Press, Tokyo/Elsevier, Amsterdam, 1984.
MORISHIMA, H., SANO, Y. and OKA, H. I.: Differentiation of perennial and annual types
due to habitat conditions in the wild rice Oryzaperennis. PI. Syst. Evoi. 144: 119-

PUBLICATIONS

99135, 1984.
MORITA, K., KADA, T. and NAMIKI, M.: A desmutagenic factor isolated from brudock(Arctium lappa Linne). Mutation Research 129: 25-31, 1984.
MORIWAKI, K., YONEKAWA, H., GOTOH, 0., MINEZAWA, M., WINKING, H. and GROPP,
A. : Implications of the genetic divergence between European wild mice with Robertsonian translocations from the viewpoints of mitochondrial DNA. Genetical Res.
43: 277-289, 1984.
MUKAI, T., HARADA, K. and YOSHIMARU, H.: Spontaneous mutations modifying theactivity of alcohol dehydrogenase (ADH) in Drosophila melanogaster. Genetics
106: 73-84, 1984.
NAKAGOME, Y., ABE, T., MISAWA, S., TAKESHITA, T. and IINUMA, K.: The "loss" ofcentromeres from chromosomes of aged women. Amer. J. Hum. Genet. 36: 398404,1984.
NAKAGOME, Y., ISE, T., SAKURAI, M., NAKAJO, T., OKAMOTO, E., TAKANO, T., NAKAHORI,
Y., TSUCHIDA, Y., NAGAHARA, N., TAKADA, Y., OHSAWA, Y., SAWAGUCHI, S., ToYoSAKA, A., KOBAYASHI, N., MATSUNAGA, E. and SAITO, S.: High-resolution studies
in patients with aniridia-Wilms tumor association, Wilms tumor or related congenitalabnormalities. Hum. Genet. 67: 245-248, 1984.
NAKAHORI, Y. and NAKAGOME, Y.: A malformed girl with duplication of chromosome
9q. J. Med. Genet. 21: 387-388, 1984.
NAKATSUGAWA, S., KADA, T., NIKAIDO, 0., TANAKA, Y. and SUGAHARA, T.: PLDR
inhibitors: Their biological and clinical implications. Br. J. Cancer 49: 43-47,1984.
NEI, M., TAJIMA, F. and GOJOBORI, T.: Classification and measurement of DNA polymorphism. "Human Population Genetics: The Pittsburgh Symposium" (Ed. by A.
Chakravarti), 307-330, Van Nostrand Reinhold Company, New York, 1984.
NICHOLAS, ROBERT A., SUZUKI, H., HIROTA, Y. and STROMINGER, JACK, L.: Purificationand sequencing of the active site tryptic peptide from penicillin-binding protein Ib
of Escherichia coli. Biochemistry 24: 3448-3453, 1985.
NICHOLAS, ROBERT A., STROMINGER, JACK L., SUZUKI, H. and HIROTA, Y.: Identification of the active site in penicillin-binding protein 3 of Escherichia coli. Jour. Bacteriol. (in press).
NODA, M., SHIMIZU, S., TANABE, T., TAKAI, T., KAYANO, T., IKEDA, T., TAKAHASHI, H.,
NAKAYAMA, H., KANAOKA, Y., MINAMINO, N., KANGAWA, K., MATSUO, H., RAFTERY,
M. A., HIROSE, T., INAYAMA, S., HAYASHIDA, H., MIYATA, T. and NUMA, S.: Primary structure of Electrophorus electricus sodium channel deduced from cDNA
sequence. Nature 312: 121-127, 1984.
NOMURA, T., ISHIHAMA, A., KAJITANI, M., TAKAHASHI, T., NAKADA, N. and YOSHINAGA,
K. : Promoter selectivity of Escherichia coli RNA polymerase, II. Altered promoter selectivity of holoenzymes from temperature-sensitive mutants. Mol. Gen.
Genet. 193: 8-16, 1984.
OHNISI-ll, S. and WATANABE, T. K.: Systematics of Drosophila mont/urn species subgroup:
Biochemical approach. Zool. Sci. 1: 801-807, 1984.
OHTA, K., SANO, Y., FUJII, T. and IYAMA, S.: Variation in nitrogen fixing activity amongwild and cultivated rice strains. Japn. J. Breed. 34: 29-35, 1984.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

OHTA, T.: Some models of gene conversion for treating the evolution of multigene
families. Genetics 106: 516-528, 1984.
OHTA, T.: Population genetics of transposable elements. IMA Journal of Mathematics
Applied in Medicine and Biology 1: 17-29, 1984.
OHTA, T.: Population genetics theory of concerted evolution and its application to theimmunoglobulin V gene tree. Jour. Mo!. Evo!. 20: 274-280, 1984.
OHTA, T. and DOVER, G. A.: The cohesive population genetics of molecular drive.
Genetics 108: 501-521, 1984.
OHTA, T., NAKAMURA, N., MORIYA, M., SHIRASU, Y. and KADA, T.: The SOS-functioninducing activity of chemical mutagens in Escherichia coli. Mutation Research 131:
101-109, 1984.
PEDERSEN, S., SKOUV, J., KAJITANI, M. and ISHIHAMA, A.: Transcriptional organizationof the rpsA operon of Escherichia coli. Mo!. Gen. Genet. 196: 135-140, 1984.
ROBINSON, PETER J., STEINMETZ, MICHAEL, MORIWAKI, K. and LINDAHL, KIRSTEN FISCHER:
Beta-2 microglobulin types in mice of wild origin. Immunogenetics 20: 655-665,
1984.
SADAIE, Y., KADA, T., OHTA, S., KOBAYASHI, K., HIEDA, W. and ITo, T.: Induction ofprophages in spores of Bacillus subtilis by ultraviolet irradiation from synchrotron
orbital radiation. J. Radiat. Res. 25: 170-173, 1984.
SANO, Y.: Differential regulation of waxy gene expression in rice endosperm. Theor.
App!. Genet. 68: 467-473, 1984.
SANO, Y., SANO, R. and MORISHIMA, H.: Neighbour effects between two co-occurring
rice species, Oryza sativa and O. glaberrima. J. App!. Eco!. 21: 245-254, 1984.
SHIMIZU, M., YONEDA, S., MIURA, K., MIYOSHI, H. and WATANABE, K.: Theoretical andexperimental approach to recognition of amino acid by tRNA and nucleotide n.
Nucleic Acids Res. Symp. Ser. No. 15: 193-196, 1984.
SUGAWARA, K.l, YONEKAWA, H., TAGASHIRA, Y. and SEKIGUCHI, K.: Population genetics of horse shoecrab based on restriction analysis of mitochrondria DNA. (inJapanese) "Biology of Horse Shoe Crab" (ed. K. Sekiguchi), 304-310, 1984.
TACHIBANA, H. and ISHIHAMA, A.: Correlation of the formation rate of open promoter
complexes with the melting property of DNA. "Physical Foundation of Protein and
Nucleic Acid Functions" (Ed. by A. Wada), The Taniguchi Foundation, 1984.
TAKAHASHI, S., FUKUOKA, Y., MORIWAKI, K., OKUDA, T., TACHIBANA, T., SAKAI, S. and
TAKAHASHI, M.: H-2 controlled structural polymorphism of the second componentof mouse complement: analysis by microscale peptide mapping. J. Immuno!. 19:
493-501, 1984.
TAKAHATA, N.: A model of extranuclear genomes and the substitution rate under withingeneration selection. Genet. Res. 44: 109-116, 1984.
TAKAHATA, N. and NEI, M.: F ST and G ST in the finite island mode!. Genetics 107:
501-504, 1984.
TAKAHATA, N. and SLATKIN, M.: Mitochondrial gene flow. Proc. Nat!. Acad. Sci.
USA 81: 1764-1767, 1984.
TAKANo, J. and SUGIYAMA, T.: Genetic analysis of developmental mechanisms in hydra.
IX. Effect of food on development of a slow-budding strain (L4). Developmental

PUBLICATIONS

101

Biology 103: 96-108, 1984.
TAKANO, J. and SUGIYAMA, T.: Genetic analysis of developmental mechanisms in hydra.
XII. Analysis of chimeric hydra produced from a normal and a slow-budding strain(L4). J. Embryo!. Exp. Morpho!. 80: 155-173, 1984.
TATEI, K., TAKEMURA, K., MAEDA, A., FUJIWARA, Y., TANAKA, H., ISHIHAMA, A. andOSHIMA, Y.: Ul RNA-protein preferentially binds to both 5' and 3' splice junction
sequences in RNA or single stranded DNA. Proc. Nat!. Acad. Sci. USA 81: 62816285, 1984.
TUTIKAWA, K.: Developmental disorders in the offspring of male mice treated with radiation and chemicals. Teratology 30: 2A, 1984.
WATANABE, S., SOEDA, E., UCHIDA, S. and YOSHIIKE, K.: DNA rearrangement affecting
expression of the BK virus transforming gene. J. Viro!. 51(1): 1-6, 1984.
WATANABE, T. K., INOUE, Y. and WATADA, M.: Adaptation of Drosophila simulans inJapan. Jpn. J. Genet. 59: 225-235, 1984.
YAMAGUCHI, K., NAKAGAWA, 1., SEKINE, M., HATA, T., SHIMOTOHNO, K., HIRUTA, M.
and MIURA, K.: Chemical synthesis of the 5'-terminal part bearing cap structure ofmessenger RNA of cytoplasmic polyhedrosis virus (CPV): m 7G"' pppAmpG and
m 7G"' pppAmpGpU. Nucleic Acids Res. 12: 2939-2954, 1984.
YAMAMOTO, A., HIHARA, F. and WATANABE, T. K.: Hybrid dysgenesis in Drosophilamelanogaster: Predominance of Q factor in Japanese populations and its change inthe laboratory. Genetica 63: 71-77, 1984.
YAMAMOTO, T., NAKAZAWA, T., MIYATA, T., KAJI, A. and YOKOTA, T.: Evolution andstructure of two ADP-ribosylation enterotoxins, Escherichia coli heat-labile toxin
and cholera toxin. FEBS letters 169: 241-246,1984.
YASUDA, S., FURUICHI, M. and SOEDA, E.: A altered DNA sequence encompassing theras gene of Harvey murine sarcoma virus. Nucleic Acids Res. 12(14): 5583-5588,
1984.
YONEKAWA, H. and MORIWAKl, K.: Variation of mitochondrial DNA: a merkmal ofsubspecies differentiation. (in Japanese) "Jikken Seibutsugaku Koza" (Ed. K.
Moriwaki), 319-350, 1984.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

ABSTRACTS Of DIARY FOR 1984

Biological Symposium
215th meeting Jan. 11

2l6th

II

Mar. 102l7th"

Mar. 26

2l8th"

Mar. 28

2l9th"

Mar. 29

220th

"

Apr.

22lst

"

Apr. 13

222nd"

May 10223rd"

May 21

224th"

May 28225th"

Jun. 25226th"

Aug. 10227th"

Aug. 18

4

Restriction endonuclease study of the mitochondrial DNA of closely related frogs andfishes (David L. Jameson)
Chromosome polymorphism of a few mammals in South India (N. V. Aswathanarayana)
Mouse genetics; the last ten years (A. G.
Searle)Developmental genetics: present and future
prospects (R. S. Edgar)
No title of his talk was previously announced,
but he talked on his past research activities
which were related to genetics (Linus Pauling)
RNA-dependent RNA polymerases in plant
cells (H. Fraenkel-Conrat)
Generation of two different H-2K histocompatibility antigens through different splicing ofa unique H-2Kd gene (G. Gachelin)Studies of the mutation process in Drosophila
by means of MR-mediated P-transposition andemploying repair-deficient mutants. (F. H.
Sobels)
DNA sequences and a primeval genetic code
(John C. W. Shepherd)
Molecular analysis of an ecdysone regulated
gene from D. melanogaster (Kenneth C. Burtis)
Epigenetics of early development: biochemical
regulation of sperm behavior before and after
fertilization (sea urchin) (Bennett M. Shapiro)
Evidence for heterogeneity in the nerve cell
populations and neuronal plasticity in hydra(Hans R. Bode)
Isolated muscle cells of hydromedusae can

ABSTRACTS OF DIARY FOR 1984

1/

Aug. 30

1/

1/

230th

1/

Sep.

231st

II

Sep. 21232nd

II

Nov. 27

233rd

II

Dec. 12

228th
229th

6

103

undergo pluripotent transdifferentiation andform a complex regenerate (Volker Schmid)
Export of protein in E. coli (Linda L. Randall)
Structure of chemotactic transducers, proteinsconserved throughout bacterial evolution(Gerald L. Hazelbauer)
DNA comparisons of immunoglobulin kappa
genes of human, mouse and rabbit (Samuel
Karlin)Evolution of antigen binding sites (Susumu
Ohno)
Pheromone polymorphism in Drosophila (J.
M. Jallon)
Genetic control of developmental pathway(Antonio Garcia-Bellido)

Mishima Geneticists' Club
289th meeting Feb. 2 Cloning ofY-specific repetitive DNA sequences(Yutaka Nakahori)
Feb. 23 Cellular skeleton of cultured muscle cells
290th(Yutaka Shimada)Mar. 12 Transposon Tn5: Variation and its regulation
291st(Chihiro Sasagawa)Mar. 23 Numerical analysis of the experimental results
292ndabout hydra morphogenesis (Hiroshi Shimizu)
293rdMar. 29 Forty years of chromosome study-Retrospect and prospect-(Tosihide H. Yosida)
294th
Sep. 11 Analysis of biological functions of ras oncogene(Toru Kamata)
II

II

II

II

II

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

fOREIGN VISITORS IN 1984
January 1-31
January IS-June 20
January 14-29
January 30

February 1
March 1February 29-March 13
March 5-6
March 9March 13-14
March 26-27
March 28
II

March 29
II

April 1April 4
April 9-Jameson, David L., University of Houston
U.S.A.
'
Thipayathasana, Pairor, Chulalongkorn Uni_
versity, Thailand
Loresto, G. c., International Rice ResearchInstitute, The Philippines
Zhang, Yihua; Jiang, Yuming; Si, Yiping;Qiu, Lingcang; China National Rice Research Institute, China
Yang, Han Chul, Korea
Sved, John, University of Sydney, Australia
Aswashanarayana, N. V., University ofMysore, India
Houba, Chris, University of Liege, Belgium
Kim, Bong Ki, Don Kook University,
Korea
Huh, Hong Wook, Pusan National University, Korea
Searle, Antony G., Medical ResearchCouncil's Radiobiology Unit, U. K.
Edgar, Robert S., University of California,
U.S.A.
Bailey, Donald W., The Jackson Laboratory,
U.S.A.
Soerianegara, Ishemat, SEAMEO Regional
Center for Tropical Biology, Indonesia
Pauling, Linus; Zuckerkandl, Emile, Linus
Pauling Institute of Science & Medicine,
U.S.A.
Houba, Nicole, University of Liege, Belgium
Frankel-Conrat, Heinz, University of California, U.S.A.
Pascale, Barbier, Universite des Sciences et

FOREIGN VISITORS IN 1984

A.pril l3
April 23-24
May 9

May 10
May 21
May 26

May 28nJune 17-18
June 22-26
July 12
August 3
August 8-20
August 17-20
August 24
August 24-25

August 29-September 1n105Techniques du Languedoc, France
Gachelin, Gabriel, Pasteur Institute, France
Park, James T., Tufts University, U.S.A.
Inouye, M., State University of New York,U.S.A.
Sobels, F. H., Leiden University, The
Netherlands
Shepherd, John C. W., Biocentre of the
University of Basel, Switzerland
Wu, Weiguang; Tang Wei Liu; Wu, Peng
Tuan; Huang, Guan Xian, Chen, ,'luo Fu;
Asia and Oceania Silkworm and Mulberry
Training Center, China
Maas, Werner K., New York University
Medical Center, U.S.A.
Burtis, Kenneth C., Stanford University
School of Medicine, U.S.A.
Shankel, D. M., University of Kansas, U.S.
A.
Shapiro, Bennett M., University of Washington, U.S.A.
Temtamy, Samia A., National ResearchCenter, Cairo, Egypt
Chung, Yong-Jai, Ewha Women's University, Korea
Bode, Hans R., University of California,
U.S.A.
Schmid, Volker, University of Basel, Switzerland
Macgregor, Herbert c., University ofLeicester, England
Wongkaew, Wongchan c., South East AsianRegional Center for Tropical Biology,
Indonesia
Hazelbauer, Gerald L., Washington State
University, U.S.A.
RaldaH, Linda L., Washington State Uni-

106

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

August 30-31
September 6-7
September 10
September 10-11
September 11
September 14-16
September 16-November 14
September 17-18/I

September 21
September 29/I

October 8
October 9
October 18
October 25
October 23
October 31
November 6
November 13-16

versity, U.S.A.
Weir, Bruce S., North Carolina State Uni.
versity, U.S.A.
Karlin, Samuel, Stanford University, U.S.A,
Wu, J. S.; Shi, S. K.; Beijing Agricultural
University, China
Martin, Malcohn A., National Institutes of
Health, U.S.A.
Khush, G. S., International Rice ResearchInstitute, The Philippines
Cockerham, Clark, North Carolina State
University, U.S.A.
Baradjanegara, Abdal Aziz, National
Atomic Energy Agency, Indonesia
Wurster-Hill, D. H., Dartmouth Medical
School, U.S.A.
Ward, O. G., University of Arizona, U.S.A.
Ohno, Susumu, City of Hope Beckman Research Institute, U.S.A.
Loekman, Irwansyah, National Atomic
Energy Agency, Indonesia
Gadrinab, Lilian Ungson, SEAMEO Re.
gional Center for Tropical Biology, Indonesia
Kuliev, A. M., World Health Organization,
Switzerland
Tjio, J. H., National Institutes of Health,
U.S.A.
Ruffi6, Jacques, College de France, France
Cui, Taishan, Academia Sinica, China
Eschbach, F., Stuckenschmidt, D., DAAD,
Germany
Lederberg, J., The Rockefeller University,
U.S.A.
Duda, George, U.S. Department of Energy,
U.S.A.
Hartle, Daniel L., Washington University,

FOREIGN VISITORS IN 1984

/I

1/
/I
/I

/I
/I

/I

/I

November 23
November 27
December 12
December 24-

107

U.S.A.
Crow, James E, University of Wisconsin,
U.S.A.
Nei, Masatoshi, University of Texas, U.S.A.
Milkman, R, University of Iowa, U.S.A.
Selander, R K., University of Rochester,
U.S.A.
Li, W.-H., University of Texas, U.S.A.
Watterson, G. W., Monash University,
Australia
Charlesworth, B., University of Sussex,
England
Calder, N., EnglandHu, Han, Institute of Genetics, Academia
Sinica, China
Jallon, Jean-Marc, Laboratoire de Biologie
Genetique Evolutives, CNRS, France
Garcia-Bellido, Antonio, Univerided
Autonoind de Madrid, Spain
Lee, Won Ho, Pusan National University,
Korea

108

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 35

AUTHOR INDEXAIKAWA, K.

KIM, B. K . . . . . . . . . . . . . . . . . . . . . . . . • • 70

AMANO, E

57
43
KOBAYASHI, K
48
KOMINAMI, R
74
KUBOTA, M. .
45, 46, 48
KURIHARA, Y. .
71, 74
KURODA, Y. .
35, 36
Lu, D.-Y
32
KusuDA, J
75
MARUYAMA, T
67
MATSUI, M. .
27
MATSUNAGA, E .............•••. " 77, 78
MIGITA, S.
32, 73
MINATO, K. .
.42
MINODA, K.
77
MIYASHITA, N
32, 71, 73
MIYATA, T
25
MIZUMOTO, K
21
MORISHIMA, H
85, 86, 88, 89
MORIWAKI, K.
30,32,49,71,73,74
MUKAI, T
66
MURAKAMI, A
37, 38, 40, 41
MURAMATSU, M. .
74
NAGAHARA, N. .
79
NAGAHUCHI, S
80
NAKAGOME, Y
79, 80NAKAHORI, Y. .
80
NAKAMURA, Y
80
NARASIMHANNA, M. N
.45
NAWA, S
24
NEGISHl, K . . . . . . . . . . . . . . . . . . . . . . . . • . 35
NEI, M. .
60, 69, 70
NICHOLAS, R. A. .
26, 27
NINAKI, O. .
75
NISHIMIYA, C
43NISHIMURA, A
28
NODA,A
23
NOGUCHI, T
53

54
90
AOKI, K.
62, 64, 65
BARBIER, P. .
86
BARONI URBANI, C.45
BLATTNER, F
25
BROWN Jr., W. L.
.46
CHO, W.-S
32
65
Crow, J. F
CROZIER, R. H. .
.48
DAS, B. C
45
DEODIKAR, G. B.
.45
DOVER, G. A.
59
ENDO, T
93
FUJII, T. . . . . • . . . . . . . . . . . . . . . . . . 24, 55
FUJlSAWA, T • . . . . . . . . . . . . . • . . . . . . . . .43
FUJISHlMA, T.. . . . . . . . . . . . . . . . . . .. 83, 84
FUJITA, N. .
15, 16, 18
FUKUDA, R. .
19, 22GADRINAB, L. U
88
GOJOBORI, T
68,69,70,78
GOTOH, 032
HASEGAWA, M • . . . . . . . . . . . . . . . . . . . . . . 22
HAYASHIDA, H
25
HAYATSU, H. .
35
HIBI, I,80
HIROTA, Y. .
26, 27
HONJO, Too
25
HORAI, S
78
IHARA, M.46
IMAI, H. T
45,46, 48
INOUE, T. .
53, 54INOUE, Y. H.
49, 53, 54
ISHIHAMA, A. .
15, 16, 18,20,21,23
ITow, T
.48
IYAMA, S
93,94](ADA, T.
29, 51, 52, 53, 54
RATO, A.
20, 21, 23
KATSUMATA, M • . . . . . . . . . . . . . . . . . . . . . 90

KIMURA, M.

KOBATAKE, E.

AUTHOR INDEX

15, 16, 18
65
OGATA, K
48
OHTA, T. . . . . • . . . . . . . . . . . . . . . . . . 58, 59
OKA, A
27
PRANATA, R. J.
46
RAJASEKARASETTY, M. Roo
45
SADAIE, T . • . . . . . . . . . . . . . . . . . . . . . . . . . 52
SADAIE, Y • . . . . . . . . . . . . . . . . . . . 29, 51, 52
SAGA], T.
30, 71SAJTO, M
84
SANO, R···.·.···· .. ···· .. · .89
SANO, Y. .
24, 85, 90
SATO, Y.-I. .
85, 88, 90, 92
SERIZAWA, H
19
SHARMA, A.
45
SHARMA, A. K
.45
SHARMA, G. P
.45
SHIMAMOTO, Y
85
SHIMIZU, K. .
22
SHIROISHI, T. . . . . . . . • . . . . . . . . . . . . . . . 30
SHULTZ, L. D. .
53, 54SLATKIN, M
61
STROMINGER, J. L.
26,27
SUGIYAMA, Too . . . . . . . . . . . . . . • • . . . . . . .43
SUZUKI, H
26, 27, 71, 74
SUZUKI, Y. .
75
NOMURA, T.

NOZAWA, K.

.

109

TAGASHIRA, Yoo . . . . . . . . . . . . . . . . . . . . . • 32
T AJIMA, F.

. . . . . . . . . . . . . . . . . . . . .. 66, 70.
36
TAKAHATA, N. .
60, 61, 62
TAKANAMI, M
27
TAKANO, T . . . . . . . . . . . . . . . . . . . . . . • . . . 80
TANAE, A80
TANAKA, F.
80
TAZIMA, Y • . . . . . . . . . . . . . . . . . . . . . . . . • . 75
TEZUKA, H
53, 54TOHARI, M.46
VEDA, S. .
20, 23VAIDYA, V. G
45
WANEK, N.43
WATANABE, T. . . . . . . . . . . . • . . . . . . . . . . 71
WATANABE, T. K
49,70
WELS, J
25
WINKING, H. .
74
YAMADA, M. A
.42
YAMAMOTO, M. .
49
YAMAWAKI-KATAOKA, Y
25
YANO, R
19
YASUDA, S
27
YOKOYAMA, S
68
YONEKAWA, H
32,71
YOSIDA, T. H
71
Yu, Z.-c.
32
TAKADA, Y.

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