ISSN OOn-4995

NATIONAL INSTITUT~ OF G~N~TICS
JAPAN

ANNUAL REPORT1985

No. 36

Publiahed bll
THE NATIONAL INSTITUTE OF GENETICS
Mi.ima, Sizuoka-ken, Japan1986

Annual Reportof theNational Institute of Genetics
No. 36, 1985

Published by

The National Institute of Genetics, Japan
1986

CONTENTS
General statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council and Advisory Committee
Association for propagation of the knowledge of genetics
Projects of research for 1985
Research activities in 1985 ....................................•.

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I. Molecular Genetics
Promoter selectivity of Escherichia coli RNA polymerase: localization of a region of the f3 subunit involved in promoter selection. GLASS, R. E., JONES, S. T., NENE, V., NOMURA, T.,
FUJITA, N. and ISHIHAMA, A.
Promoter selectivity of Escherichia coli RNA polymerase: recognition of a heat shock promoter. FUJITA, N. and ISHIHAMA, A...
Promoter selectivity of Escherichia coli RNA polymerase: regulatory role of fMet-tRNAr et • NOMURA, T., FUJITA, N. and
ISHIHAMA, A.
Promoter selectivity of Escherichia coli RNA polymerase: effect ofpromoter location. NOMURA, T., FUJITA, N. and ISHIHAMA,A
Promoter selectivity of Escherichia coli RNA polymerase: effect of
DNA supercoiling. FUJITA, N., OSATO, K., KANO, Y., IMAMOTO, F. and ISHIHAMA, A.
Promoter selectivity of Escherichia coli RNA polymerase: correlation between promoter strength and DNA structure. TACHIBANA, H. and ISHIHAMA, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Analysis of the gene for the stringent starvation protein of Escherichia coli. A novel transcriptional control during the stringent
response. SERIZAWA, H. and FUKUDA, R.
Genetic mapping of the Escherichia coli stringent starvation
protein gene. FUKUDA, R., NISHIMURA, A. and SERIZAWA,
H
Mechanisms of transcription initiation by influenza virus-associated

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

RNA polymerase. HONDA, A., MIZUMOTO, K. and IsmHAMA, A
In vitro transcription and replication of influenza virus RNA.
TAKEUCHI, K., NAGATA, K. and ISHIHAMA, A.
In vitro splicing of influenza viral mRNA. NAGATA, K. and
ISHIHAMA, A.
Small RNA and CCA-enzyme like activity associated with influenza
virus. KATO, A., VEDA, S. and IsmHAMA, A.
Analysis of temperature-sensitive mutants of influenza virus.
Effect of ts lesions on the segment 8. HATADA, E., FUKUDA,
R., SmMIZU, K. and HASEGAWA, M.
Function of nuclear factor I. NAGATA, K. and ISHIMI, Y.
Cloning of the 3IB-D region of Drosophila melanogaster chromosome. INOUE, Y.
Codon usage and tRNA content in unicellular and multicellularorganisms. IKEMURA, T.
Codon usage and chromosomal banding in human genome. IKEMURA, T. and AOTA, S.-1.
Codon usage, tRNA content, and rate of synonymous substitution.
IKEMURA, T.
Organization of mitochondrial DNA from the male sterile cytoplasm of rice. NAWA, S., SANO, Y. and FUJII, T.

II.

III.

Microbial Genetics
Mutants of Escherichia coli defective in penicillin-insensitive murein
DD-endopeptidase. IIDA, K., HIROTA, Y. and SCHWARZ,
V
Binding of penicillin to thiol-penicillin-binding proteins 3 ofEscherichia coli: identification of its active site. HOUBAHERIN, N., HARA, H., INOUYE, M. and HIROTA, Y.
Dispensability of either penicillin-binding protein-Ia or -lb involved in the essential process for cell elongation in Escherichia
coli. KATO, J.-I., SUZUKI, H. and HIROTA, Y.
Expression activities of Bacillus subtilis a-amylase secretion
vectors. YAMANE, K.
Immunogenetics
A lethal gene-deletion in the mouse H-2 class III region associated

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CONTENTS

with meiotic recombination. SHIROISHI, T.. SAGAI; T.,
NATSUUME-SAKAI, S. and MORIWAKI, K.
Effect of H-2 complex and non-H-2 background on urethan-induced chromosomal aberrations in mice. MIYASHITA, N.
and MORIWAKI, K.
Effect of H-2 complex on the development of carcinogen-induced
pulmonary adenoma in mice. MIYASHITA, N. and MORIWAKI,
K
Serological survey of alphaprotein-l in wild mice. HARADA, Y.,
TOMITA, T. and MORIWAKI, K.
Characterization of Ly-2.1 w antigenicity newly found in Asianmice. KURIHARA, Y. and MORIWAKI, K.

IV.

Developmental and Somatic Cell Genetics
Effects of hormones on growth pattern of human diploid cells in
culture. KURODA, Y.
Antimutagenic activity of vitamin C on mutations induced by
EMS in cultured Chinese hamster cells. KURODA, Y.
Combined effects of EMS and MMS on mutation in Chinese
hamster cells in culture. KURODA, Y., KOJIMA, H. and KoNISHI, H.
................
Enhancing effect of Ara-C on mutations in cultured Chinese hamster cells treated with EMS. KURODA, Y., SHINKAWA-TAcm,
K., MORIMOTO, K. and KOIZUMI, A.
Freezing of Drosophila embryos after treatment with some chemicals. KURODA, Y. and TAKADA, Y.
Differentiation of embryonic cells of Drosophila melanogasterstudied with the electron microscope. SmMADA, Y. and
KURODA, Y.
Brown-embryo lethals in Drosophila melanogaster. MINATO, K. ..
The role of the cephalic furrow during the embryonic developmentof Drosophila melanogaster. MINATO, K.
Genetical studies on the parthenogenesis in Bombyx mori: a selection of highly parthenogenetic lines. MURAKAMI, A., OHTSUKI, Y. and KITAZAWA, T.
Genetical studies on the parthenogenesis in Bombyx mori: thermal
activation and its significance. MURAKAMI, A.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Sex and strain differences in lifespan of the adult silkmoth, Bombyx
mori. MURAKAMI, A., KURODA, Y. and FUKASE, Y.
49
Genetical studies on the hereditary mosaic (mo) strain of Bombyx
mori: function of polar-body. MURAKAMI, A. and KOBAYASHI, M.
50
Sex reversal by heterosexual parabiosis in hydra. SUGIYAMA, T.
and SUGIMOTO, N.
52
Zygotic effect of the daughterless gene in Drosophila melanogaster.
INOUE, Y.
52
Ovary freezing for the maintenance of silkworm genes. KUSUDA,
J. and ONIMARU, K.
54

V.

Cytogenetics
Karyological survey of wild mice in Ogasawara Chichi Island.
KURIHARA, Y., SAKAIZUMI, M., TSUCHIYA, K., HARADA, Y.
and MORIWAKI, K.
55
A genetic model for X-Y dissociation and testis weight in intersubspecies hybrids between M. m. domesticus (BALB/c)and M. m. molossinus. IMAI, H. T., LEE, W. H., TIAN, K. N.
and MORIWAKI, K.
56
Chromosome observations of Sarawak ants. TIAN, K. N., IMAI,
H. T., KUBOTA, M., BROWN, W. L. Jr., GOTWALD, W. H. Jr.,
YONG, H.-S. and LEH, C.
57
Modes of species differentiation and karyotype alteration in ants
and mammals. IMAI, H. T.
58
The exceptionally low chromosome number n=2 in an Australianbulldog ant, Myrmecia piliventris Smith (Hymenoptera:
Formicidae). IMAI, H. T. and TAYLOR, R. W.
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VI.

Mutagenesis and Radiation Genetics
Evidence suggesting high sensitivity of bone marrow cells to radiation-induced cell killing in the "wasted" mouse. TEZUKA,
H., INOUE, T. and KADA, T.
The effect of age on the frequency of spontaneous and mutageninduced micronuclei in mouse bone marrow. TEZUKA, H.,
TAMAI, K., MURAKAMI, K. and KADA, T.
Cancer susceptibility of the "wasted" mouse. INOUE, T., TAKAHASHI, J., OOTSUYAMA, A., TEZUKA, H. and KADA, T.

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Effects of low-level tritiated water ingestion and age on the numberof sister chromatid exchanges in mouse bone marrow cells.
TEZUKA, H., BENZ, R. D., CARSTEN, A. L. and IKUSHIMA,
T
64
Radiation induced chromosome aberrations in the radiation sensitive mutants of Caenorhabditis elegans. SADAIE, Y. andSADAIE, T.
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Mechanisms of bio-antimutagenesis. KADA, T. and INOUE, T.
66
Dietary desmutagens. KADA, T.
Mutagenic activities of EMS on somatic and recessive mutationsin the soybean test system. FUJII, T. and TANO, S.
66
Origin and genetic background of three substrains in inbred PW
mouse strain. TUTIKAWA, K., HARADA, Y. and MORIWAKI,
K
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VII.

Population Genetics
The role of compensatory neutral mutations in molecular evolution.
KIMURA, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Variances and covariances of identity coefficients of a multigene
family. OHTA, T.
A model of duplicative transposition and gene conversion for re. . . . . . ..
petitive DNA families. OHTA, T.
Reciprocal altruism and reciprocal alliance between relatives.
AOKI, K.
Gene diversity in finite populations. TAKAHATA, N.
Gene genealogy and variance of interpopulational nucleotide differences. TAKAHATA, N. and NEI, M.
The average frequency of private alleles in a partially isolated
population. SLATKIN, M. and TAKAHATA, N.
Extranuclear differentiation and gene flow in the finite islandmodel. TAKAHATA, N. and PALUMBI, S. R.
Introgression of extranuclear genomes in finite populations:
nucleocytoplasmic incompatibility. TAKAHATA, N. . ... . . . ..
Genic homozygosity in populations which experience periodic bottlenecks. MARUYAMA, T. and FUERST, P. A.
Number of alleles in a small population that was formed by arecent bottleneck. MARUYAMA, T. and FUERST, P. A.

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Dynamics of homozygous genetic loads in connection with the.
invasion of transposons. MUKAI, T

VIII. Evolutionary Genetics
Homology between P-transposable element of Drosophila melanogaster and bacterial transposase gene of Tn3. SATTA. Y.,
GOJOBORI, T., MARUYAMA, T., SAlGO, K. and CHIGUSA, S.1. ..
Tn3 resolvase-like sequence in P transposable element of Drosophila melanogaster. SATTA, Y., GOJOBORI, T., MARUYAMA, T.
and CHIGUSA, S. I.
.
Evolutionary features of oncogenes. GOJOBORI, T.
Rates of evolution of the retroviral oncogene of Moloney murine
sarcoma virus and its cellular homologues. GOJOBORI, T.
and YOKOYAMA, S.
Complete nucleotide sequence of an infectious clone of human Tcell leukemia virus type II: an open reading frame for theprotease gene. SHIMOTOHNO, K., TAKAHASHI, Y., SHIMIZU,
N., GOJOBORI, T., GOLDE, D. W., CHEN, I. S. Y., MIWA, M.
and SUGIMURA, T.
DNA polymorphism of AIDS virus. GOJOBORI, T., YOKOYAMA,
S. and MARUYAMA, T.
Homology of kringle structures in urokinase and tissue-type plasminogen activator: the phylogeny with the related serine
proteases. TAKAHASHI, K., GOJOBORI, T. and NAORA, H ....
Chromosome regions affecting hybrid sterilities of Drosophila
simulansjD. mauritiana and D. simulansjD. sechellia males.
KIMURA, K., LEE, W. H., WATANABE, T. K. and HATSUMI, M.

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Notes on two interspecific hybrids of the Drosophila melanogaster
subgroup: simulansjteissieri and teissierijmauritiana. LEE,
W. H. and WATANABE, T. K.
Cytogenetical and biochemical characters of wild mice collectedfrom Kunming area. MORIWAKI, K., IMAI, H., KURIHARA,
Y., YONEKAWA, H., QIN, C.-G. and SHI, L.-M.
Chromosome C-banding patterns of Korean wild mice. MORIWAKI, K., SUZUKI, K., LEE, W. H. and CHO, W. S.
The relationship between an isozyme locus Pox-l and life-histroy

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traits in wild rice. MORISHIMA, H. and SANO, R.
89
Computer studies on the molecular evolution. MIYATA, T.
90
Application of mitochondrial DNA restriction fragment length
polymorphism to genetic monitoring of laboratory mice.
YONEKAWA, H., KATOH, H., SAGAI, T. and MORIWAKI, K.
92

IX.

X.

Human Genetics
Mitochondrial DNA polymorphism in Japanese. HORAI, S. and
MATSUNAGA, E'.' . . . . . . . .
Analysis of mitochondrial DNA polymorphisms in Japanese monkeys. HORAI, S., MATSUNAGA, E., HAYASAKA, K., SHOTAKE,
T. and NozAwA, K.
Characterization of 2,7-anhydro-N-acetylneuraminic acid in human
wet cerumen. SUZUKI, M., SUZUKI, A., YAMAKAWA, T. and
MATSUNAGA, E.

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99

Applied Genetics
The Indica-Japonica differentiation of rice cultivars in Thailand
and its neighbouring countries. SATO, Y.-I., CHITRAKON, S.
and MORISHIMA, H
100
Variation in grain shape of the Indica and Japonica rice cultivars.
SATO, Y.-I.
100
Distribution of genes for F 1 weakness in Asian native rice cultivars.
SATO, Y.-I. and MORISHIMA, H.
101
Effect of a semi-dwarfness gene on morphological characters in ricewith different genetic backgrounds and environmental condi102
tions. SATO, Y.-I. and MORISHIMA, H. .
Genic analysis of isozyme genes in rice. SANO, R., ISHIKAWA, R.,
BARBIER, P. and MORISHIMA, H
102
Electrophoretic characterization of rice endosperms of Indica andJaponica with four kinds of protein fractions. ENDO, T.
103
Conservation methods of crop populations with mixed selfing
and outcrossing. YONEZAWA, K. and OKA, H.-I.
104
Evolutionary significance of regulatory changes in the Wx gene106
expression of rice. SANO, Y.
Genetic control of alcohol dehydrogenase and estimation of some
population parameters in Hopea odorata Roxb. (Dipterocarp-

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aceae). !HARA, M., GADRINAB, L. D., SIREGAR, D. J. and!YAMA, S.
107
Publications from Genetic Resources Section, Genetic Stocks Research Center. !YAMA, S.
108
Data base design of DNA base sequences. MIYAZAWA, S
108
Publication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 110
Abstract of diary for 1985
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 119
Foreign visitors in 1985
122
Author index
125

GENERAL STATEMENT
This report gives an outline of research activities carried out in our institute during one year from January to December 1985. The total expenditure in the 1985 fiscal year amounted to 946 million yen, about 52 %of which was personnel expenses. In addition, grants-in-aid in the amountof 140 million yen were rendered to selected staff members by the Ministryof Education, Science and Culture. New positions for two associate professors for the DNA Research Center, and new laboratories for visiting
professors for the Departments of Ontogenetics and Integrated Genetics
were approved in April. However, according to the 6th curtailment planof the Government two members were cut, so the total number of regularstaff remained the same as in the preceding year, that is, 92 including 53
research staff.
I am glad to note that three of our colleagues and associates were honoredin the past year. First, Prof. Tomoko Ohta was awarded the Japan Academy prize for her "Theoretical studies on population genetics at the molecular level." Following Prof. Motoo Kimura who received the same
prize in 1968, she is the second staff member of our institute to receive thisaward. Since she joined us in 1969, she has been devoting herself to challenging a number of extremely hard problems in population genetics andhas succeeded in solving them quantitatively. In particular, Ohta's theories
on linkage disequilibrium, on the role of slightly deleterious mutations inmolecular evolution and on the concerted evolution of multigene families,
are well known in the world. The award of this time in summer attracted
additional public attention as she was the first woman to receive the prize
singly. In autumn she won the Prime Minister's official commendation incommemoration of the United Nation's Women's Decade because of her
outstanding contribution to raising the status of women. Secondly, ourold friend, Prof. James F. Crow of the University of Wisconsin (U.S.A.)
was elected Foreign Honorary Member of the Japan Academy because of
his distinguished services in the promotion of the development of genetics
in Japan. Dr. Crow has visited our institute almost every year since 1957,
sometimes staying for a month, and he still continues collaborative studies
with the staff of our institute. Among them M. Kimura, T. Maruyama,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

T. Mukai and K. Aoki, have studied under Dr. Crow in his laboratory inMadison. It is remarkable that the collaborative studies have continued
for more than 30 years, resulting in many significant papers in the field of
population genetics. Third, Dr. Yataro Tazima, the former director andhonorary member of the institute, was decorated with the Second Order of
the Sacred Treasure for his contribution to academic as well as industrial
societies. Before joining the institute, Dr. Tazima succeeded in the breeding of a new strain of silkworm with a sex-limited marker produced byirradiation-a pioneer work with chromosome engineering-and, taking
advantage of this strain he elucidated the mechanism of sex determinationin the silkworm; for this work he was awarded, together with Dr. Haruo
Hashimoto, the Japan Academy prize in 1954. In the contemporary silkworm industry there are 68 commercially designated strains, of which 33
originated from a sex-limited Sable marking strain developed by Dr. Tazima.
There have been several active personnel changes in the last year. Prof.
Yasuo Nakagome of the Laboratory of Human Genetics left in April,
together with Dr. Masao Yamada and Yutaka Nakahori, fOJ; the newly
established Children's Medical Research Center of the National Children's
Hospital in Tokyo. While working with human cytogenetics for about
15 years in Mishima, Nakagome produced a number of solid achievements;for example, he identified several new syndromes of structural aberrations
by using the banding technique, elucidated the distribution of break points
with respect to the nature of chromosome bands, and reported the interesting phenomenon of the loss of centromeres with aging. Dr. Eiichi
Soeda of the DNA Research Center was transferred in June to the Instituteof Physical and Chemical Research. While working in the institute for10 years, Soeda became the first scientist in the world to determine thesequence of all the bases of polyoma virus and clarified the structure andfunction of its genes. He also improved the method of shotgun DNA
sequencing by simplifying and speeding up its procedures.
Dr. Hiroko Morishima was promoted to professor of the Laboratory ofAgricultural Genetics (March), and Dr. Hirotami Imai to associate professorof the Laboratory of Cytogenetics (July). Dr. Toshimichi Ikemura (theFaculty of Science, Kyoto University) was appointed as associate professor
for the newly established Recombinant DNA Section of the DNA ResearchCenter (April), and Dr. Sanzo Miyazawa (the National Institutes of Healthin U.S.A.) to associate professor for the DNA Data Analysis Section of

GENERAL STATEMENT

3

the Center (December). Dr. Kyosuke Nagata (Research fellow of theMemorial Sloan-Kettering Cancer Center, U.S.A.) joined us as a researchstaff member of the Laboratory of Molecular Genetics (February), and Dr.
Hiroshi Shimizu (graduate student of the Faculty of Engineering, Tohoku
University) as a research staff member of the Laboratory of Developmental
Genetics (June).
As a result of personnel changes during the last two years, the staff of theLaboratory of Molecular Genetics has wholly shifted, and they have actively started studies on the regulatory mechanisms of gene expression under
the leadership of Prof. Akira Ishihama. Prof. Takeo Maruyama and Dr.
Takashi Gojobori are constructing a DNA data bank, periodically sending"Newsletter" to the interested workers in Japan to inform them of the
progress of the works and of the availability of the data. In the next fiscal
year the scheduled upgrading of the electronic computer in the institute,and the addition of Miyazawa will help speed up the preparatory work inthis area.
The Genetic Stock Research Center supplies, as far as possible, uponrequest of competent researchers in and outside of Japan, specific strains ofmouse, Drosophila, silkworm, E. coli, B. subtilis, etc. that are kept in theCenter. The total number of cases supplied in 1984 amounted to 282 (63sent abroad) and the total number of strains supplied was 1733 (371 sent
abroad). Data-base information about a variety of experimental organisms
preserved in universities and research laboratories in Japan is being systematized by Dr. Shinya Iyama; he compiled the "Drosophila Genetic
Stock List in Japan (1985)", which is available for distribution to related
researchers in this country.
On April 20, the institute held its yearly open house for the public. Someof the research activities of each laboratory were exhibited, and movie
films were shown. Double cherry blossoms on the campus were at thierbest and some 1500 visitors enjoyed them. On October 26, public lectures
were given at the National Science Museum in Tokyo; the titles were
"Regulatory mechanisms of gene expression" by Prof. A. Ishihama, and"Evolution of mitochondrial DNA" by Dr. Naoyuki Takahata. In spiteof it being a Saturday afternoon about 120 eager people listened to thelectures, which were followed by rather specialized questions and lively
discussions.
Two occasional meetings took place in the institute. One was a technical

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

training course for "Shotgun DNA sequencing with personal computer
analysis" organized by Dr. E. Soeda on January 9-11, as one of the 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 andCulture. Among the many applicants, 8 who urgently needed to masterthe techniques were chosen and trained intensively. The other meeting
was the 25th meeting of the directors of national institutes for joint use heldon May 2. Twenty-three institutes participated and discussed various problems in advancing research activities under the present severe state ofadministrative and financial constraints.
International personnel exchanges were very active in the past year.
Members of our staff went abroad on 39 occasions for the purpose of presenting research results at various scientific meetings, exchanging information, and carrying out collaborative studies or field investigations; two ofthem stayed longer than 3 months. On the other hand, 52 scientists visited
our institute from abroad, with whom information and views on recent
studies were actively exchanged. Some distinguished guests, including
Professor Dr. F. Vogel of Heidelberg, delivered stimulating lectures at the
Biological Symposium. Those who stayed longer than 1 month for cooperative studies were: Dr. Paul A. Fuerst, Ohio State University, U.S.A.;
Dr. Robert E. Glass, Nottingham University, U.K.; Dr. Pierre Boursot,
Universite Montpellier II, France; Miss Pascale Barbier, Universite desSciences et Techniques du Languedoc, Montpellier, France; Dr. Nicole
Houba-Herin, Liege University, Belgium; Dr. Lee Won Ho, Pusan National
University, and Mr. Kim Bong-Kee, Dan Kook University, Korea: Mr.
Irwansyah Loekman, National Atomic Energy Agency, and Miss Lilian
Ungson Gadrinab, SEAMEO Regional Center for Tropical Biology, Indonesia; Dr. Ajay Kumar Jain, King George Medical College, India; Dr.
Yi-de Huang, Shanghai Institute of Plant Physiology, the Chinese Academyof Sciences, and Dr. Yuan shen Qiu, Guangdong Microbiology ResearchInstitute, People's Republic of China.
Two years have passed since the administrative category of our institutewas switched over to one for joint use by universities. Under the new
system, visiting professors for 5 laboratories have been appointed. We
have accepted 30 collaborative programs, 8 workshops, 12 graduate students,
10 research fellows from private corporations, and 3 scholarships fromindustries. Yet, there remain a number of problems to be solved in orderGENERAL STATEMENT

5

to achieve the reality of reform. Among others, the followings are of thehighest priority: completion of the DNA Research Center including theestablishment of the DNA data bank, which should meet the high demandof scientists in our country, and the construction of a second main building
to accommodate the new Research Center and various facilities for jointuse, together with lodgings for visiting researchers. We are all eager to do
our best to accomplish the missions of the new institute. We wish to havecontinued encouragement and support of all the persons concerned.

STAFF
Director

MATSUNAGA, Ei, D. Med., D. Sc.
Members1.

Department of Mole~ular Genetics
KADA, Tsuneo, D. Sc., Head o(the Department
Laboratory of Molecular Genetics
ISHIHAMA, Akira, D. Sc., Professor
FUKUDA, Ryuji, D. Med., Associate professor
FUJITA, Nobuyuki, D. Sc.
NAGATA, Kyosuke, D. Pharm.
Laboratory of Mutagenesis
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.

Department of Cell Genetics
HIROTA, Yukinori, D. Sc., Head of the Department
Laboratory of Cytogenetics
MORIWAKI, Kazuo, D. Sc., Professor
IMAI, Hirotami, D. Sc., Associate professor
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, D. 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.
SHIMIZU, Hiroshi, D. Eng.
Laboratory of Phenogenetics
KURODA, Yukiaki, D. Sc., Professor
MURAKAMI, Akio, D. Ag., D. Sc., Associate professor
MINATO, Kiyoshi, M. Sc.
YAMADA, Masa-Aki, M. Sc.
Laboratory of Physiological Genetics (Guest member)
SHIMADA, Yutaka, D. Med., Professor
KIJIMA, Hiromasa, D. Sc., Associate professor

4.

Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department
Laboratory of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Professor
OHTA, Tomoko, Ph. D., D. Sc., Professor
TAKAHATA, Naoyuki, D. Sc.
AOKI, Kenichi, Ph. D.
Laboratory of Evolutionary Genetics
MARUYAMA, Takeo, Ph. D., D. Sc., Professor
WATANABE, Takao K., D. Sc., Associate professor
TUTIKAWA, 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

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Laboratory of Human Genetics
HORAI, Satoshi, D. Med.
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.
Laboratory of Applied Genetics (Guest member)
YONEZAWA, Katsuei, D. Ag., Associate professor

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
SHIROISHI, Toshihiko, D. Sc.
DNA Research Center
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Center
IKEMURA, Toshimichi, D. Sc., Associate professor
MIYAZAWA, Sanzo, D. Sc., Associate professor
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
MATSUMURA, Tateo, Chief of the Finance Section

COUNCIL

9COUNCIL
Chairman
YAMAMURA, Yuichi; Emeritus professor, Osaka University
Vice-chairman
MOROHOSHI, Seijiro; Emeritus professor, Tokyo University of Agriculture and Technology
Members (Alphabetical order)
EGAMI, Nobuo; Director, National Institute for Environmental Studies
IINO, Tetsuo; Professor, Faculty of Science, University of TokyoINOUYE, Eiji; Emeritus professor, University of TokyoNAGAKURA, Saburo; President, Okazaki National Research Institutes
NAKAJIMA, Tetsuo; Professor, Tamagawa University
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, Haruo; 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, Itaru; Professor, Kitazato University

ADVISORY COMMITTEE
Chairman
MATSUNAGA, Ei; Director
Vice-chairman
TANAKA, Ryuso; Dean, Faculty of Science, Hiroshima University
Members (Alphabetical order)

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

FUKUDA, Ichiro; Professor, Faculty of Liberal Arts and Sciences, Tokyo
Women's University
MIURA, Kin-ichiro; Professor, Faculty of Engineering, University ofTokyo
MUKAI, Terumi, Professor, Faculty of Science, Kyushu University
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, Laboratory of Microbial Genetics
ISHIHAMA, Akira; Professor, Laboratory of Molecular Genetics
KADA, Tf>uneo; Professor, Laboratory of Mutagenesis
KIMURA, Motoo; Professor, Laboratory of Population Genetics
KURODA, Yukiaki; Professor, Laboratory of Phenogenetics
MARUYAMA, Takeo; Professor, Laboratory of Evolutionary Genetics
MORISHIMA-OKINO, Hiroko; Professor, Laboratory of Agricultural
Genetics
MORIWAKI, Kazuo; Professor, Laboratory of Cytogenetics
SUGIYAMA, Tsutomu; Professor, Laboratory of Developmental Genetics
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF 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

ADVISORY COMMITTEE

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

11

PROJECTS OF RESEARCH FOR 1985
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, FUKUDA and NAGATA)
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 developmentin the laboratory and wild mice (MORIWAKI)

PROJECTS OF RESEARCH FOR 1985

13

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 FUJII)
Laboratory of Phenogenetics

Genetic studies on insect cells in tissue culture (KURODA and MINATO)
Developmental genetic studies on animal cells in tissue culture (KURODA)
Genetics of somatic mammalian cells in culture (KURODA)
Genetic studies on abnormal fertilization in the silkworm (MURAKAMI)
Mutagenesis in germ cells of the silkworm (MURAKAMI)* Genetic Stock Research Center

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Studies on the male-specific killing activity of SRO in Drosophila (YAMADA)
Laboratory of Physiological Genetics

Electron microscopic studies on differentiation of animal cells (SHIMADA
and KURODA)
Nerve network formation in Hydra (KIJIMA)
Genetic analysis of nervous system development in hydra (KIJIMA andSUGIYAMA)

4.

DEPARTMENT OF POPULATION GENETICS
Laboratory of Population Genetics

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

Theory of population genetics and evolution (MARUYAMA)
Studies on molecular evolution (GOJOBORI)
Evolutionary genetics of Drosophila (WATANABE)
Radiation genetics in mice (TuTlKAwA)
Laboratory of Theoretical Genetics

Theoretical and experimental studies of transposons in population of Drosophila (MUKAI)
Computer studies on the molecular evolution (MIYATA)PROJECTS OF RESEARCH FOR 1985

5.

15

DEPARTMENT OF INTEGRATED GENETICS
Laboratory of Human Genetics

Studies on DNA polymorphisms in human populations (HORAI and MATSUNAGA)
Genetic studies on retinoblastoma (MATSUNAGA and HORAI)
Mitochondrial DNA restriction analysis of non-human primates (HORAI,
MATSUNAGA, HAYASAKA, SHOTAKE and NozAwA)Laboratory of Agricultural Genetics

Evolutionary studies in wild and cultivated rice species (MORISHIMA and
SATO)
Ecological genetic studies in weed species (MORISHIMA)
Genetic studies on rice reserve proteins (ENDO)
Behavioral genetic studies in animals (FUJISHIMA)
Laboratory of Applied Genetics

Methodology for the conservation of genetic resource populations (YONEZAWA)
6.

RESEARCH FACILITIESGenetic Stock Research Center

Studies and conservation of germplasm resources in rice and wheat species(FuJII and SANO)
Specificity of mutagen tolerance in higher plants (FuJII)
Exploitation of genetic ability of nitrogen fixation in Gramineae (FuJII,SANO and IYAMA)
Studies on genetic differentiation in rice (SANO)
Theoretical studies on breeding techniques (IYAMA)
Genetic studies of trees in natural forest (IYAMA)
Studies on gene transfer in Gramineae (SANO and FUJII)
Documentation of genetic stocks in Japan (IYAMA)
Studies on the management system of genetic stocks information (IYAMA)

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

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)
Synthetic ColEl plasmids carrying genes for cell division in E. coli (NISHIMURA)
Development of mouse embryo freezing system (SHIROISHI and MORIWAKI)
Molecular mechanism of high frequency recombination in mouse MHC
region (SHIROISHI and MORIWAKI)
DNA Research Center

Studies on primary structure of DNA (SOEDA)
Regulatory mechanisms of gene transcription (ISHIHAMA)DNA data analysis (MARUYAMA)
Studies on codon usage (IKEMURA)
Studies on RNA molecules of mouse brain (IKEMURA)
Database design for DNA base sequences (MIYAZAWA)RESEARCH ACTIVITIES IN 1985
I.

MOLECULAR GENETICS

Promoter Selectivity of Escherichia coli RNA Polymerase:
Localization of a Region of the fJ Subunit
Involved in Promoter Selection
Robert E. GLASS·, Steven T. JONES·, Vishvanath NENE**,
Teruaki NOMURA, Nobuyuki FUJITA and Akira ISHIHAMARNA polymerase of E. coli is complex in structure, consisting of at least
four different subunits, £1',13,13' and one of sigma subunits. The catalytic
site for RNA synthesis is located on the 13 subunit. This subunit is also
involved in the recognition of promoters (Nomura et aI., Mol. Gen. Genet.
193,8-16). Glass and his colleagues have set up a genetic system to analyze
structure-function relationships of the 13 subunit (Nene and Glass, Mol.
Gen. Genet. 194, 166-172). This system is based on the isolation of a strain
collection synthesizing several hundred variants of RNA polymerase, eachvariant carrying a known amino acid substitution at a known site in the 13
subunit.
Analysis of the altered functions of these mutant RNA polymerase has
allowed us to locate regions required for specific functions. For example,
we identified relaxed RNA polymerases, which were insensitive to ppGpp
both in vivo and in vitro (Glass et aI., FEES Lett. 153, 307-310; Mol. Gen.
Genet., 203, 265-268). This indicates that a small region of RNA polymerase 13 subunit is a target for ppGpp.
Among pseudo-revertants of the rpoE (13 subunit gene) amber mutants
carrying internal deletions, we found a mutant RNA polymerase withaltered promoter selectivity (Glass et aI., Mol. Gen. Genet., 203, 487-491).
The mutant enzyme lacks 55 amino acid residues near to the C-terminus of 13
subunit and strains carrying this mutation exhibit slower growth in minimal••*

Department of Biochemistry, Queen's Medical Centre, University of Nottingham,
Nottingham, England.
Department of Pathology, University of Cambridge, Cambridge, England.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

medium. The results allow localization of a region in the f3 subunit involvedin promoter selection.
Promoter Selectivity of Escherichia coli RNA Polymerase:
Recognition of a Heat Shock Promoter
Nobuyuki FUJITA and Akira ISHIHAMA

The htpR gene, the positive regulatory gene of heat shock response in
E. coli, codes for a sigma-like protein (a 32 ) and is supposed to participate in
the specific recognition of heat shock promoters by RNA polymerase. We
have purified RNA polymerase associated with a 32 (Ea 32 ) to apparent homogeneity from heat-shocked E. coli cells, and analyzed its molecular andfunctional properties.
The novel holoenzyme Ea 32 correctly recognized a heat shock promoter
involved in the groE gene and efficiently initiated transcription at the samesite as that found in vivo. However, Ea 32 could never recognize the promoters which are usually transcribed by regular holoenzyme (Ea 7D ), including
lacUV5, nusA, trp, recA, rplJ and rpsA promoters. On the other hand,
Ea 7D could not recognize the groE promoter, showing that strict difference
exists in the promoter selectivity between two molecular species of the RNA
polymerase holoenzyme. Several lines of in vitro evidence indicated that thetwo sigma subunits were easily exchanged between the two holoenzyme
species. This finding supports the view that the spectrum of gene expressionin E. coli is under dynamic control by intracellular levels of individual sigma
subunits.
Promoter Selectivity of Escherichia coli RNA Polymerase:
Regulatory Role of fMet-tRNA}let
Teruaki NOMURA, Nobuyuki FUJITA and Akira ISHIHAMAIn Escherichia coli, the rate of RNA synthesis is related to the level of
intracellular amino acid pool. This observation raised a hypothesis thattRNAs are involved in the control of RNA synthesis, their activities being
regulated by charging amino acids. On the basis of the observation that the
inhibition of the formylation of Met-tRNA}let reduced not only the rate ofprotein synthesis but also that of RNA synthesis, the hypothesis was modified

RESEARCH ACTIVITIES IN 1985

19

such that tRNAfet is a specific regulatory molecule which mediates thecoupling of transcription to translation. Using the in vitro mixed transcription system (Kajitani and Ishihama, Nucleic Acids Res. 11, 671-686; ibid.
11, 3873~3889), we found that ppGpp, the nucleotide factor involved instringent control, interacts with purified RNA polymerase holoenzyme andalters its promoter selectivity (Kajitani and Ishihama, J. BioI. Chern. 259,
1951-1957). On this line, we examined the effect of charged and uncharged forms of individual tRNAs on the transcription of specific genes.
The results showed that both charged and uncharged forms of specifictRNAs except tRNAfet inhibited RNA polymerase by competing with
DNA templates although the inhibitory activity was different among tRNA
species examined and promoters used.
Both tRNA~et and Met-tRNA~et were inhibitors of RNA polymerase.
However, formylation of Met-tRNAfet not only relieved this inhibition
but also stimulated the transcription from some promoters (Nomura et aI.,
Nucleic Acids Res. 14, 6857-6870). This result supports the notion that
RNA~et plays a regulatory role in transcription as well as in translation.

Promoter Selectivity of Escherichia coli RNA Polymerase:
Effect of Promoter Location
Teruaki NOMURA, Nobuyuki FUJITA and Akira ISHIHAMA

A single promoter of the rnh gene coding for RNase H and two promotersof the dnaQ gene coding for DNA polymerase III e subunit are located ina small vicinity of the E. coli chromosome and direct transcription in opposite
direction (Nomura et aI., J. BioI. Chern. 260, 7122-7125). This year, we
focused our studies on possible interference among these closely locatedpromoters. For this purpose, we employed the in vitro mixed transcription
system and used either a truncated DNA template carrying the three promoterson a single and the same DNA fragment or a mixture of truncated templates,
each carrying a single promoter. The results indicated that the rates ofopen complex formation (parameter II of "promoter strength") for the threepromoters were unaffected depending on whether they were located on thesame DNA strand or separated into individual fragments but the level ofopen complex (parameter I of "promoter strength") increased for the rnh
promoter when it was separated from the strong dnaQ promoters (Nomura

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

et al., Nucleic Acids Res. 13, 7647-7661). This suggests a promoter interference between the convergently transcribed genes.
Promoter Selectivity of Escherichia coli RNA Polymerase:
Effect of DNA Supercoiling
Nobuyuki FUJITA, Katsuaki OSATO"', Yasumasa KANO"',
Fumio IMAMOTO'" and Akira ISHIHAMA

In this laboratory, promoter strength has been determined for a numberof E. coli promoters in an in vitro mixed transcription system, in which
mixtures of linear DNA fragments, each carrying a specific promoter, wereused as the templates. Several lines of evidence, however, suggest that thestrength of some promoters is affected by formation of DNA supercoilingboth in vivo and in vitro. To quantitatively determine the influence ofDNA supercoiling, two forms of circular DNA templates, relaxed andnegatively supercoiled, were prepared for tryptophan (trp) promoter, tryptophan-lactose hybrid (tac) promoter, and tac promoter with UV5 mutation (tacUV5). The levels of transcription in vitro from these circulartemplates were determined by hybridization with single-stranded DNA
probes followed by 81 nuclease digestion and gel electrophoresis. To
improve the assay, we measured transcript of the p-Iactamase (amp) geneon plasmid vector as an internal standard during RNA synthesis and hybridization.
DNA supercoiling differently affected the strength of the three test promoters, especially the rate of open complex formation (parameter II).
Transcription from trp and tac promoters was stimulated whereas that from
tacUV5 and amp promoters was not significantly affected. These findings
suggest that the activity of the "-10" signal is affected by DNA supercoiling.
Promoter Selectivity of Escherichia coli RNA Polymerase:
Correlation between Promoter Strength and DNA Structure
Hideki TACHIBANA'" and Akira ISHIHAMA

Using the "in vitro mixed transcription" system, we have determined two'"

Laboratory of Molecular Genetics, Institute of Physical and Chemical Research,
Wako, Saitama.
'" Graduate School of Science and Technology, Kobe University, Kobe.

RESEARCH ACTIVITIES IN 1985

21

parameters of the promoter strength, i.e., the level (parameter I) and therate (parameter II) of open complex formation, for more than 20 promotersfrom our collection of E. coli promoters (Kajitani and Ishihama, Nucleic
Acids Res. 11, 671-686; ibid. 11, 3873-3888; Nomura et al., Nucleic Acids
Res. 13, 7647-7661). As an attempt to reveal correlation between the
promoter strength and the DNA structure, we estimated the "opening
potential" of DNA segments around promoters using the known thermodynamic parameters, which are based on the stabilities of nearest neighborbase pair doublets against thermal melting. A positive correlation was
found between the calculated opening potentials in the -9 to +3 region(+ 1 represents the position at which transcription is initiated) and the rateof open complex formation (parameter II) (Tachibana and Ishihama,
Nucleic Acids Res. 13,9031-9042).
Analysis of the Gene for the Stringent Starvation Proteinof Escherichia coli.-A Novel Transcriptional
Control dOling the Stringent Response
Hiroaki SERIZAWA and Ryuji FUKUDA

Up to 10 polypeptides associated with RNA polymerase and which may
have some functions modulating RNA polymerase during transcription
have been identified. Stringent starvation protein (SSP) is one such polypeptide. It forms an equimolar complex with the RNA polymerase holoenzyme, and is synthesized occupying more than 50 % of the total protein
synthesis when cells are exposed to amino acid starvation. To study thephysiological function of SSP, we cloned the gene for the protein (Fukuda,R., et al. Mol. Gen. Genet., 201, 151-157, 1985).
This year, we determined the sequence of 1616 bp, which contained thecomplete coding region, as well as the flanking regions, and obtained thefollowing results. I. SSP has 212 amino acid residues and a molecularweight of 24,304.56. 2. At 350 bp upstream from the initiation codon of
SSP, we found a stem and loop structure of a typical rho-independent transcription termination signal. The signal is located about ISO bp downstream
from the three termination codons, which are arranged in phase following along coding sequence. It was thus considered that the transcription termination signal was that of the preceding gene. 3. A long coding sequence
starts from 45 bp downstream from the termination codon of the SSP gene

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

with a SD sequence and AUG initiation codon. 4. No obvious transcription termination signal was identified in the 3' flanking region of the SSP
gene.
These results suggest that the SSP gene is a member of a multicistronic
operon, and the regulation of its synthesis is not simple.
As the next step, we carried out nuclease S1 mapping of the in vivo transcripts to identify the transcription start site(s). During the ordinary growthof cells, the transcript of the SSP gene starts from 158 bp upstream fromthe initiation codon. We could identify a Pribnow sequence but not a-35 sequence corresponding to the transcription initiation. In contrast,
when the cells were exposed to amino acid starvation, the transcription was
initiated from a starting point further upstream, about 185 bp from theinitiation codon, and the amount of SSP transcripts greatly increased. We
could not identify the ordinary promoter sequences around the transcription
starting site. These results indicate the existence of two different promotersfor the SSP operon. The upstream promoter controls the SSP transcription during the stringent response of the cells, while the downstream promoter
is used during the ordinary growth of cells.
Genetic Mapping of the Escherichia coli Stringent
Starvation Protein Gene
Ryuji FUKUDA, Akiko NISHIMURA· and Hiroaki SERIZAWA

To determine the map position of the SSP gene, the following approach
was undertaken. 1. The SSP gene was cloned to the plasmid pBR322.
The resulting recombinant plasmid carried the intact AmpR gene. 2. The
recombinant plasmid was transfected and multiplied at 30 e in a temperature-sensitive polA Hfr strain, where it could replicate autonomously at
30 e, but could not at the nonpermissive temperature of 42 e. 3. The
transformants were grown at 42°e in the presence of ampicillin. Stable
ampicillin-resistant cells were those cells in which the plasmid had integrated
into the chromosome at the SSP gene by homologous recombination. 4.
The chromosomal site of drug resistance (and thus the integration site of theplasmid carrying the SSP gene) was mapped by Hfr matings and PI transduction. 5. Plasmid integration at the chromosomal SSP gene was confirmed by the change in the restriction pattern of the SSP gene as indicated
D

D

•

D

Genetic stock center of this institute.

RESEARCH ACTIVITIES IN 1985

23

by the Southern hybridization method.
The gene for SSP was mapped to the region between gltB and glnF at
min 69.5 on the E. coli chromosome. It was thus established that the SSP
gene is a hitherto unknown gene.
We are now trying to determine whether SSP is essential for cell growthor not, employing a similar plasmid integration technique.
Mechanism of Transcription Initiation
by Influenza Virus-Associated RNA Polymerase
Ayae HONDA, Kiyohisa MIZUMOTO* and Akira ISHIHAMA

RNA-dependent RNA polymerase associated with influenza virus plays
an essential role in transcription and replication of the viral genome. For
initiation of transcription, the RNA polymerase cleaves host cell mRNAat specific sites (Kawakami and Ishihama, Nucleic Acids Res. 11, 36373649) and uses the resulting capped RNA fragments as primers. The
capped RNA-dependent transcription initiation can be by-passed if highconcentrations of oligonucleotides were added as primers. A systematic
analysis of the effect of the sequence and length of oligonucleotides on
their priming activities indicated that the transcription initiation took place
within a small region near the 3' termini of viral RNA segments only whenprimers were hybridizable at their 3' termini with templates (Honda et aI.,J. Bioi. Chern., 261,5987-5991). This suggests that the RNA polymerase isassociated near the 3' termini of viral RNAs. Analysis of the RNA polymerase-binding site(s) on viral RNA is in progress, using RNA polymeraseviral RNA complexes isolated by the CsTFA centrifugation method (Katoet aI., Virus Research 3, 115-127).
In vitro Transcription and Replication of Influenza Virus RNA
Kaoru TAKEUCHI, Kyosuke NAGATA and Akira ISHIHAMA

We have developed an in vitro transcription and replication system using
isolated nuclei prepared from influenza virus-infected cells. In this system,
two species of positive-sense RNA transcripts, i.e. mRNA and cRNA (fullsized complementary RNA without poly(A) tail), were found to be synthesized when analyzed by RNA-RNA hybridization using negative-strand* Institute of Medical Science, University of Tokyo, Tokyo.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

RNA probes from viral RNA segment 8 and gel electrophoresis. Inhibitorsof DNA-dependent RNA polymerases such as a-amanitin and actinomycin
D did not inhibit the reaction. Dinucleotide ApG, which is the best primer
in RNA synthesis by the virion-associated RNA polymerase (Honda et aI.,J. BioI. Chern., 261, 5987-5991), enhanced the extent of RNA transcription.
By fractionating and reconstituting the system, we plan to identify factors
responsible for transcription and replication of influenza RNA.
In vitro Splicing of Influenza Viral mRNA
Kyosuke NAGATA and Akira ISHIHAMA

Influenza virus RNA segment 7 codes for at least two polypeptides, MI
and M2, translated from separate mRNA species. Segment 8 also codes for
two nonstructural polypeptides, NSI and NS2, translated from separate
mRNA species. The Ml and NSl mRNAs are unspliced primary transcripts
of viral RNA segments 7 and 8, respectively, whereas the M2 and NS2
mRNAs are supposed to be formed from their unspliced precursors after
splicing.
To elucidate the molecular mechanism of splicing, attempts were made toestablish an in vitro system using nuclear extracts prepared from influenza
virus-infected human cells. A precursor RNA substrate was prepared in
an in vitro transcription reaction using influenza virus segment 8-specific
cDNA template and SP6 RNA polymerase.
In influenza-infected cells, two species of positive-strand RNA, viralmRNAs and template RNAs for RNA replication, are formed. In spiteof the fact that signals for splicing are present in both RNA species, only
mRNAs are spliced. Analysis on the molecular basis of this discrimination
is in progress.
Small RNA and CCA-Enzyme Like Activity
Associated with Influenza Virus
Atsushi KATO, Susumu UEDA* and Akira ISHIHAMA

The gemone of influenza virus consists of 8 segments of single-stranded
RNA ranging from 890-2340 nucleotides in length. Defective interfering* Nippon Institute for Biological Sciences, Tokyo.

RESEARCH ACTIVITIES IN 1985

25(DI) particles of influenza virus contain DI RNAs of 440-690 nucleotidesin length, which are derived from the RNA polymerase genes (segments
1-3). Besides we found novel species of small-sized RNAs, referred to as
smRNAs, of 80-100 nucleotides in length. Several species of smRNA
were purified from viral particles. Sequence analysis of isolated smRNAs
and of cDNAs reverse-transcribed from smRNAs revealed that smRNAs
are similar to tRNA in their structure. These smRNAs were good substrates for the cellular CCA enzyme, which is involved the modification of
the 3' termini of tRNAs.
In addition, we found a virion-associated enzyme activity, by which CMP
was added to smRNAs at their 3' termini. The enzyme partially purified
from virions catalyzed the cytidylation of not only smRNAs but also cellular
tRNAs. Possible roles of the smRNAs and the smRNA-cytidylating
enzyme in transcription or replication of influenza RNA are being studied.
Analysis of Temperature-sensitive Mutants of Influenza Virus-Effect of ts Lesions on the Segment 8
Erika HATADA, Ryuji FUKUDA, Kazufumi SHIMIZU
and Masakazu HASEGAWA

The RNA segment 8 of influenza virus encodes two proteins that are only
found in infected cells but not in virions, and have been designated as NSI
and NS2 proteins. The NS2 mRNA is produced by the splicing of NSI
mRNA, but the function of these proteins is still unknown. In order tostudy the role of NSI and NS2 proteins in virus multiplication, we analysed
temperature-sensitive (ts) mutants derived from the A/Udorn/72 (H3N2)
strain, which have defects in segment 8.
As previously reported, we determined the base sequence change in segment 8 for three of these mutants. Two of them, ICR1629 and SPC45,
have a single point mutation only in the NSI protein, while the third mutant,
ICR516, has an amino acid change only in the NS2 protein. The synthesisof the Ml protein, which normally accumulates late in the course of infection, was greatly reduced in MDCK cells infected with both of these
NSI mutants at the nonpermissive temperature of 40°C as compared to
34°C. Further studies revealed the reduced synthesis of the NSI protein
at 40°C for both NSI mutants. In addition, NS2 synthesis was greatly
decreased in SPC45 but normal in ICR1629. NSI protein synthesized at

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

40°C was unstable in SPC45, while it was stable in ICR1629. The reduced
synthesis of the NS 1 protein in SPC45 could not be explained solely by the
instability of the de novo synthesized protein. No significant differenceswere seen in the synthesis of virus proteins for the NS2 mutant, ICR516.
Which steps in the virus multiplication is impaired in these mutant virusinfected cells? For analysis, we have established a system for measuring
the quantities of mRNAs, cRNAs (the template for vRNA replication), andvRNAs of eight genome segments separately. To get RNA probes forquantitative hybridization in the system, 170-500 bp fragments of individual
viral ds-cDNAs were recloned in the SP-6 vectors, and the DNA fragments
were transcribed by SP-6 RNA polymerase. To detect mRNA and cRNA,
the RNA probe was obtained by transcribing the SP-6 recombinant DNA,
which gave the intact 5'-end sequence of vRNA. As mRNA deletes 16
nucleotides of the 3'-end sequence, the RNA hybrid formed with mRNA isshorter than the cRNA hybrid. We can thus discriminate cRNA frommRNA on polyacrylamide gel electrophoresis. Transcription of the opposite strand of the SP-6 DNAs gave probes to analyze vRNAs.
Function of Nuclear Factor I
Kyosuke NAGATA and Yukio ISHIMI*

The in vitro synthesis of full-length adenovirus DNA requires three viral
coded proteins and two host-derived proteins. One of the latter proteins,designated nuclear factor I (NFl), has been purified as an essential factor
involved in initiation of adenovirus DNA replication and shown to be asite-specific DNA binding protein (Nagata et aI., Proc. Natl. A cad. Sci.
USA 80, 6177-6181). Isolation and analysis of viral as well as cellular DNA
sequences bound by NFl indicates that the sequence motif, TGG(N)6_7
GCCAA, is a minimum requirement for the binding. An interesting featureis that DNase-hypersensitive sites are detected around putative NFl-bindingsites in isolated nuclei. Such DNase-hypersensitive sites are often observed in the transcriptionally active chromatin and the origin of some
replicons.
Using an in vitro reconstituted chromatin from purified histones, assembly
protein, NFl and DNA containing the NFl binding sequence, we proved* Faculty of Pharmacential Sciences, University of Tokyo, Tokyo.

RESEARCH ACTIVITIES IN 1985

27

that DNase-hypersensitive sites were generated in the neighborhood of aThis suggests that NFl is one of the transacting factors that facilitate DNase-hypersensitive sites in chromatin.
Cloning of DNA sequences flanking to chromosomal NFl-binding sites andstudies on possible functions of NFl in transcription are in progress.
bona fide NFl-binding site.

Cloning of the 3lB-D Region of Drosophila melanogaster
Chromosome
Yutaka INOUE

Some spontaneous mutations are caused by insertion of the transposable
elements in Drosophila. The chromosome of one spontaneous mutant,daughterless (da), was examined by hybridizing with several transposon
DNA. The da locus is supposed to be 31B-D of the second chromosome
by the deficiency-mapping. The six kinds of plasmid DNA containing
Drosophila transposons (P, Copia, Gypsy, Hobo, Roo, 412) were labeled by
3H-nick translation and hybridized to the control and mutant polytene
chromosomes. The P element and Hobo didn't hybridize with the dachromosome. The Copia, Roo and 412 hybridized in 10-30 sites in the
genome including the chromocenter, but none of them were specific to the
3IB-D region of the da chromosome. The Gypsy-insertion was found tobe specific to the 31B-D region of the da mutant chromosome. There werealso 4-6 sites of Gypsy-insertion including the centromere in the dachromosome. Then a genomic library from one da mutant strain wasmade by inserting DNA fragments generated by partial Mbol endonuclease
digestion into BamHI site of Lambda cloning vector EMBL4. From ascreening of approximately 20,000 plaques, 40 clones were purified whichhybridize to a plasmid containing Gypsy-DNA. To identify plaques
carrying genomic sequences from the polytene chromosome region of 31B-D,
the DNA was isolated from each clone for in situ hybridization to the
Canton-S polytene chromosomes which have no Gypsy-insertion at 3IB-D,
and one clone hybridizing to 31B-D was identified. This cloned Drosophila
DNA insert was about 11 kb in length. From Southern gel analysis withnick-translated Gypsy-DNA as the hybridization probe, an approximately
4 kb fragment was found to be the Gypsy-free genomic part.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Codon Usage and tRNA Content in Unicellular
and Multicellular Organisms
Toshimichi IKEMURA

Choices of synonymous codons in unicellular organisms were extensively
analyzed using a DNA Sequence Data Bank, and differences in synonymouscodon usages between Escherichia coli and the yeast Saccharomyces cerevisiae
were attributed to differences in the actual population of iso-accepting
tRNAs. There is a strong positive correlation between codon usage andtRNA content in either organism, and the extent of this correlation relates
to the protein production levels of individual genes. Codon-choice patternsof multicellular organisms were also studied, and a diversity in G+C %at the 3rd position of codons in vertebrate genes was revealed. The highest
G+ C % was 98 % (for chicken histone H2A), and the lowest was 35 % (forhuman blood Christmas factor IX); genes for the abundant cellular "housekeeping" proteins (e.g. actin, histone, tubulin) usually have a high G+C %at this position. It was also found that genes with a high G+C % at the
3rd position (e.g. more than 80 % G+ C) are usually flanked by a wide genome
portion (far larger than the coding region, e.g., >10 kb) of high G+C%
(60-70%), and genes of low G+C% at the 3rd position (less than 50%)
are flanked by a wide portion of low G+C % (usually less than 45 %). It isinteresting that large genome portions surrounding protein genes which are
far larger than the coding region are kept either G+C-rich or A + T-rich,in accord with the G+C content of the 3rd positions. This may be related
to some gross genetic information on gene expression lying in the large DNA
segment, and/or the regional chromosome structure. The variation of
G+C% distribution throughout the vertebrate genome should be a causative factor in generating peculiar codon-choice patterns, either extremely
G+ C-rich or A + T-rich, at the codon 3rd position and, therefore, resultsin the observed diversity of G+C % at this position of vertebrate genes.
For details, see Mol. BioI. Evol. 2 (1985): 13-34.
Codon Usage and Chromosomal Banding in Human Genome
Toshimichi IKEMURA and Shin-ichi AOTA

During the course of our studies to elucidate the biological significanceRESEARCH ACTIVITIES IN 1985

29of the segmental distribution of G+C content throughout the vertebrate
genomes, computer analyses using a DNA Data Bank showed that the
A+T-rich segments (e.g. 40-50 G+C%) of human genomes are mainly
associated with chromosomal G (Giemsa) banding and the G+C-rich segments (e.g. 50-65 G+C %) with R (Reverse) banding. On the basis ofcytological studies, G bands have been believed to correspond to A + Trich, late replicating DNA segments, and R bands to G+C-rich earlyreplicating ones. Our findings based on the computer analyses are consistent with this previous knowledge. Thus we think that chromosomal
banding is a factor in generating peculiar codon-choice patterns, either
G+C-rich or A+ T-rich at the codon 3rd position, and therefore the diversity of G+C % at this position of human genes, as well as presumably ofother vertebrates. For details, see, Nuc!. Acids Res. 14 (1986): 6345-6355.
Codon Usage, tRNA Content, and Rate of Synonymous
Substitution
Toshimichi IKEMURA

The non-random choices of synonymous codons in Escherichia coli andyeast genomes have been previously attributed to the availability of tRNA
molecules within a cell. The tRNA quantification data of Salmonella
typhimurium in the present work revealed that the same is also true for thisenterobacteria. There is a strong positive correlation between codon usage
and isoaccepting tRNA content in these organism, and the extent of thiscorrelation relates to the protein production levels of individual genes. In
other words, codon choices of these organisms are constrained by tRNAavailability, and this constraint is more evident for highly expressed genes
than for moderately or poorly expressed genes. Extensive examination ofsynonymous substitutions occurring among Enterobacteriaceae showedthat the levels of synonymous substitution in the former group of genes areclearly lower than those of the latter group. We therefore concluded thatthe evolutionary constraint imposed by tRNA content on codon choicedecelerated, rather than accelerated, the synonymous substitution rate ofEnterobacteriaceae genes. For details, see T. Ikemura in "Population
Genetics and Molecular Evolution" (Eds. T. Ohta and K. Aoki). pp 385406, Japan Scientific Societies Press, Tokyo/Springer-Verlag, Berlin.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Organization of Mitochondrial DNA from the Male
Sterile Cytoplasm of Rice
Saburo NAWA, Yoshio SANO and Taro FUJll

Rice mitochondria of the male sterile cytoplasm, (ems-boro)rf,rfr, contained two supercoiled circular DNAs, designated B-1 and B-2, whereas nosuch DNAs were detected in the normal fertile cytoplasm, (n-boro)rf1rfr,
showing that the male sterility is associated with the presence of the plasmidlike DNAs.
Whole sequences of B-1 and B-2 were cloned in the plasmid PUC 12.
The cloned B-1 contained one restriction site for Aval, AvaIl, EeoRI, HindIand Stul, and more than two sites for Hhal and Rsal, but none for BamHI,
BglI, BglII, Dral, Hindlll, Nael, Pst!, Pvull, Sad, Sall and XbaI. The
cloned B-2 contained one restriction site for BgllI and EstEll, and morethan two sites for Fokl, Hinfl, Hpal, Mspl, Rsal, Sau3A, Seal, Taql andXbal, but none for Ball, BamHI, BglI, EeoRI, Haelll, Hindlll, Pstl, Pvull,
Sad, Smal and StuI.
The distribution of sequences homologous to B-1 or B-2 in nuclear or
mitocondrial DNAs from the male sterile strain, the normal fertile strain
and the two fertile revertants derived from the male sterile cytoplasm was
examined by using these cloned B-1 and B-2 as probes. Preparations of
total DNAs were digested with restriction enzymes, fractionated by agarose
gel electrophoresis and transferred to nitrocellulose filters. Hybridization
was carried out by using biotin-labelled B-1 or B-2. The labelled B-1 hybridized to at least four of the Hindlll restriction fragments of male sterile
DNA, showing that the chromosome, either nuclear or mitochondrial, contained sequences homologous to the free plasmid-like DNA in various regions.
DNA from the normal fertile strain contained three Hindlll fragments
homologous to B-1 which were identical with those found in the sterile DNA.
However, one homologous fragment, which was most strongly labelledin the steile DNA, was missing in the normal fertile DNA. The similar
results were obtained when EeoRI, HindI and BamHI restriction fragments
were examined. DNAs prepared from the two fertile revertants, which havebeen induced from the male sterile strain with EMS treatment, produced thesame restriction patterns as the normal DNA. Thus, the reversion to
fertility in the male sterile cytoplasm resulted in the loss of the chromosomal
sequences homologous to B-1 and B-2, as well as the loss of the free plasmid-

RESEARCH ACTIVITIES IN 198531

like DNAs. However, the strain, (cms-boro) Rf1Rf;., restored to fertility
by a nuclear restoring gene Rfl contained the free plasmid-like DNAs andthe same chromosomal sequences homologous to B-1 or B-2 as the malesterile strain, (cms-boro)rf;.rf;.. Therefore, the presence of the homologous
sequences alone, either in the integrated forms or free forms, is not sufficient
to cause male sterility.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

II.

MICROBIAL GENETICS

Mutants of Escherichia coli Defective
in Penicillin-Insensitive Murein DD-Endopeptidase
Kyoko IIDA, Yukinori HIROTA and Uli SCHWARZ

The mutants of Escherichia coli that are deficient in the penicillin-insensitive DD-endopeptidase have been isolated. The strain JEI0874 (mepA)
has about 10 %-20 % of the residual activity and another strain, JEI0368(mepB) has 40 %-50 % of the activity found in the wild-type, parental strain,
PA3092. The penicillin-insensitive endopeptidase is a periplasmic enzyme.
Genetic mapping studies show that the mutation mepA is located close toaroC (50 min) and the other mutation, mepB, is very close to malE (91 min)on the chromosome. These mutants grow normally under a wide range of
growth conditions; other phenotypic properties of the mutants are very
similar to those of the parent strain. A double mutant (mepA mepB), anda triple mutant (mepA mepB dacB), deficient in both penicillin-insensitiveand penilillin-sensitive endopeptidases, were constructed. Again, thesemutants grew normally. We conclude that either the very low level ofresidual enzyme activity in the mutants is enough for their survival or thatthe penicillin-insensitive endopeptidase is not essential for survival underlaboratory conditions. (For detail, see Mol. Gen. Genet. 1983, 189, 215221.)
Binding of Penicillin to Thiol-Penicillin-Binding Protein 3of Escherichia coli: Identification of its Active Site
Nicole HOUBA-HERIN, Hiroshi HARA, Masayori INOUYE
and Yukinori HIROTA

In order to determine the active site of penicillin-binding protein 3 ofEscherichia coli (PBP3), the serine residue at position 307 was replacedwith alanine, threonine or cysteine by oligonucleotide-directed site-specificmutageneses. Since a unique BanII site exists at the position corresponding
to serine-307, BanII digestion of the plasmid DNA after mutagenesis resulted
in significant enrichment of the mutant plasmids. For mutagenesis, theRESEARCH ACTIVITIES IN 198533

gene coding for PBP3 (ftsI) was inserted into the expression cloning vector
pIN-lIB. The hybrid protein produced was able to bind penicillin whilemutant PBP3, in which serin-307 was replaced with either alanine or
threonine, did not lead to any detectable binding. However, contrary to
the report of Broome-Smith et al. (EMBO J. 1985,4,231-235) thiol-penicillinbinding protein 3, in which serin-30? was replaced with cysteine, was still
able to bind penicillin. Replacement of serine-445 with an alanine residue
had no effect on penicillin binding to PBP3. (For detail, see Mol. Gen. Genet.
1985, 201, 499-504.)
Dispensability of either Penicillin-Binding Protein-1a or -1b
Involved in the Essential Process for Cell Elongation in
Escherichia coli
Jun-ichi KATO, Hideho SUZUKI and Yukinori HIROTA

A strain of Escherichia coli lacking the entire ponB gene (penicillin-binding
protein one B gene) and a strain lacking the proximal part of the ponA gene(penicillin binding protein one A gene) were constructed by substitution witha drug resistance gene. These strains lost either penicillin-binding protein(PBP) -lb or -la totally and their growth was apparently normal at 30°C
and 42°C except that growth of the ponB deletion strain was poor on anutrient agar plate containing no NaCl at 30°C and 42°C. Transductional
experiments to introduce the ponB deletion into the ponA deletion strain,
and vice versa, showed that the ponA ponB double deletion was lethal unlessthe deletion was functionally compensated, e.g., by the presence of a plasmidcarrying either gene. Thus, either PBP-lb (ponB) or PBP-la (ponA), but
not both, is dispensable for cell viability, at least under ordinary culture
conditions. Transduction experiments also suggested that the r componentof PBP-l b or the PBP-l b lacking the C-terminal portion encoded in the
distal region to the SphI site on the ponB was sufficient for supporting growthof the E. coli cell. (For detail, see Mol. Gen. Genet. 1985, 200, 272-277.)
Expression Activities of Bacillus subtilis
a-Amylase Secretion Vectors
Kunio YAMANE

a-Amylase is one of the major extracellular enzymes of Bacillus subtilis.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

We cloned the a-amylase structural gene from an a-amylase hyperproducing
strain B. subtilis NA64. Then we constructed a B. subtilis secretion vector
using an AluI-DNA-fragment specifying the B. subtilis a-amylase promoter
and signal-peptide-coding regions. When the E. coli j9-lactamase structuralgene was ligated downstream of the DNA fragment and the resultant chimeric
plasmids were transferred into B. subtilis, the E. coli j9-lactamase structuralgene was expressed and the active j9-lactamase synthesized was secreted
into the culture medium from the B. subtilis cells.
In order to study the transcription initiation site of the secretion vector,
we extracted mRNA from B. subtilis cells containing the chimeric plasmid
at 6 and 12 h cultivation after inoculation. Then the site was determinedby the method of the primer extension. The site was residue A at the -121
position from the translation initiator ATG and it was 3 bp downstream
from the promoter region. Several artificial promoters were chemicallysynthesized and were inserted into the B. subtilis secretion vector to obtainthe j9-lactamase hyper-producing strains.

RESEARCH ACTIVITIES IN 1985

III.

35

IMMUNOGENETICS

A Lethal Gene-Deletion in the Mouse H-2 Class III Region
Associated with Meiotic Recombination
Toshihiko SHIROISHI, Tomoko SAGAI, Shunnosuke NATSUUME-SAKAI'"
and Kazuo MORIWAKI

A haplotype awl8 is one of the H-2 recombinants generated from theB1O.AjB1O.MOL-SGR heterozygote. Based on the genetic constitutionof the awl8 haplotype, the recombination point was mapped to the Ea-Slp
interval in the H-2 class III region. Mating experiment revealed that thisrecombinant haplotype carries a single recessive lethal gene located in the
Ea-Slp interval.
The H-2 class III region corresponding to the Ea-Slp interval includes
genes encoding four complement components, such as C2, Facter B (Bf),
SIp and C4. Recently, two steroid 21-hydroxylase genes, 21-0H.A and
21-0H.B, were also mapped in this region. To examine whether thesegenes are intact in the awl8 haplotype, southern blot analysis was carriedout using DNA probes for 2I-hydroxylase, C4 and Bf genes. When hybridized with a C4 probe, Hind III digestion of DNAs extracted from B1O.A
and BIO.MOL-SGR, which are parental strains of the awl8 recombinant,yielded two fragments of 4 kb and 23 kb corresponding to C4 and SIp genes
respectively. Likewise, the same DNAs digested with Bgi II yielded nonpolymorphic 8.8 kb fragment and polymorphic (9.8 kb in B1O.A and 10.5
kb in BIO.MOL-SGR) fragment when hybridized with 21-hydroxylaseprobe. In contrast, the awl8 homozygote exhibited only one fragmentwith either of these two probes, losing 4 kb C4 fragment and polymorphic
9.8 kb 21-hydroxylase gene fragment which is specific to the B1O.A strain.
The result suggested that gene deletion occurred in the C4 and one of the
21-hydroxylase genes. When hybridized with the Bf probe, digestion of the
awl8 homozygote DNA with BamHI, EcoRI, Bgi II and HindIII showed
non polymorphic fragments indistinguishable from those of the parental
strains, indicating that there is no detectable gene deletion and gene rearrangement in the Bf gene of the awl8 recombinant.
'"Department of Immunobiology, Cancer Research Institute, Kanazawa University.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

The protein production of complement components encoded in the class
III region in the awl8 haplotype was then tested by immunodiffusion for
the C4 and SIp, and by tryptic peptide pattern analysis for Factor B, using
plasma of the awl8jk heterozygote. The male produced SIp but neither
male nor female produced C4 protein. This result supports idea that the
awl8 haplotype has an intact SIp gene but not a C4 gene as expected fromthe southern blot analysis. The plasma of the same heterozygote showed
production of Factor B of d and k haplotypes, indicating that the awl8
haplotype has an intact Bf gene derived from the BlO.A strain.
All data obtained in this study clearly demonstrated that gene deletionoccurred in the class III region being coupled with meiotic recombination
between homologous chromosomes of BlO.A and BlO. MOL-SGR andcaused recessive lethality due to this gene deletion.
Effect of H-2 Complex and Non-H-2 Background on
Urethan-Induced Chromosomal Aberrations in Mice
Nobumoto MIYASHITA and Kazuo MORIWAKIThe incidence of in vivo urethan-induced chromosomal aberrations was
examined in H-2 congenic strains of mice with BlO and A backgrounds.
Chromosome analysis of bone marrow cells could divide seven lines of A.H-2
congenic strains into two groups; one with a higher frequency of chromosomal aberrations such as AjWy (H-2 8-), AjJ (H_2 8-), AAL (H-2 aI) and A.TL
(H_2 tl ), and the other with a relatively lower frequency such as A.TH (H_2 t2),
A.CA (H-2 f ), A.BY (H-2b ) and A.SW (H-28 ). The same tendency wasobserved in the spleen cells. Among BlO.H-2 congenic strains of mice,
BIO.A (H-2"), BlO.BR (H-2 k ), BIO.A(3R) (H_218), BlO.A(5R) (H-2 15) and
BIO.S(9R) (H_2t4 ) and BlO.S(7R) (H_2 t2 ). F 1 hybrids between BlO (resistant) and BlO.A (susceptible) were susceptible.
To determine the effect of non-H-2 genetic backgrounds on urethaninduced chromosomal aberrations, four pairs of strains which have thesame H-2 haplotypes such as BIO vs. A.BY (H-2b ), BlO.A vs. AjWy (H-2"),
BlO.S vs. ASW (H-2S), and BlO.S(7R) vs. A.TH (H_2 t2 ) were compared.
The strains with a B10 background exhibited the significantly higher frequencies of deletions and heavily damaged cells and the lower frequenciesof exchanges than the strains with an A background.
These data suggested that at least two genes are involved in the regulation

RESEARCH ACTIVITIES IN 1985

37

ofurethan-induced chromosomal aberrations in mice, one of which is mapped
between Sand D in the H-2 complex, and another not belonging to H-2(Jpn. J. Cancer Res., 76, 1141-1145 (1985), Mutation Res., in press).
Effect of H-2 Complex on the Development of Carcinogen-Induced
Pulmonary Adenomas in Mice
Nobumoto MIYASHITA and Kazuo MORIWAKIThe present study was performed to determine the locus in the H-2 complex which affects the development of pulmonary adenomas. For that
purpose, a single s.c. injection of either urethan or 4-nitroquinoline I-oxide(4-NQO) was achieved in 7 H-2 congenic strains with an AjWy backgroundand 12 strains with a BIO background.
In H-2 congenic strains with an AjWy background, the number of adenoma
foci per mouse was checked 5 months after the urethan treatment, and 7
months after the 4NQO treatment. The average number of adenoma fociper mouse was significantly higher in H-2 haplotypes a, tl and aI, than in b,s, f and t2. These data showed that the gene (or genes) in the H-2K-H-2D
interval determined the susceptibility to the development of the carcinogeninduced pulmonary adenomas. In the case of H-2 congenic strains with a
BIO background, the average number of adenoma foci per mouse induced
by urethan was also higher in H-2 haplotypes k, a and h2 than in b, d, f, s, r,
i3, M, i5 and t4. The number of foci in F Ihybrids, between the susceptible
and resistant strains, such as (A x A. BY)F1 and (BIO X BlO.A)F 1 hybrids,
was intermediate between those of the two parental strains. On the other
hand, in (BIO.A(4R) x BlO.A(5R»F 1 mice, the number of foci was higher
than those in the resistant parental strains both of which are recombinantsof H-2 a and H-2 b •These patterns in the response to carcinogens paralleled those obtainedfor immune response to lactate dehydrogenase-beta (LDH-B) and immunoglobulin gamma 2a (IgG2a) proteins. The difference in susceptibility to
the development of pulmonary adenomas among H-2 congenic mice is likely
to be due to the polymorphism of the I-E molecule, which is encoded by
the Ea and Ej3 loci in the H-2 complex. This possibility was also supportedby the fact that the injection of anti-I-Ek monoclonal antibody could inhibit
the growth of AjJ derived transplantable lung tumor in vivo. These results
suggest that the I-P molecule has a function of suppression of immune

38

ANNUAL REPORT OF NATIONAL INSTITUEE OF GENETICS NO. 36

surveillance system against the development of pulmonary adenomas.
Serological Survey of Alphaprotein-l in Wild Mice
Yoshi-nobu HARADA, Takeshi TOMITA· and Kazuo MORIWAKI

Alphaprotein-l (Aph-l) is an alloantigen which migrates immuno-electrophoretically to the a-region. Two allelic forms of this alloantigen,
Aph-IA and Aph-IB were shown by reciprocal alloimmunizations between
Mol-A strain (a strain derived from Japanese wild mice) and BALB/c strainof mice. The Aph-IA (BALB/c type) and Aph-lB (Mol-A type) are controlled by a codominant autosomal locus designated Aph-l (Harada et aI.,
Immunogenet. 24: 47-50, 1986). All of the common laboratory strains examined in the former study exhibited Aph-1A, whereas all derived fromJapanese wild mice Aph-IB. This unique distribution of the alloantigens
could probably be a reflection of evolutionary divergence occurring inmouse sub-species.
Table 1. Crossreaction of the wild mouse sera against
anti-Aph-lB monospeciuc alloantiserum
Strain of
PositiveNo.
examined

Strain of
NegativeNo.
examined

M. Mol-Msm
M. Mol-Kgs
M. Mus-Big 1
M. Mus-Big 2
M. BRV-MPL (BRVj2)
M. Sub-las 2
M. Sub-las 3
M. Sub-Kjr 2
M. Sub-Cht
M. Sub-Shh 1
M. Sub-Shh 2
M. Sub-Ac 1
M. Sub-Jyg
M. Sub-Bjn 2
M. Sub-Bjn 3
M. Sub-Tch

2
2
3
2
2*
2
3
2
3
2*
1
2
2
2
2
1

M.DOM-PGN2
M.Dom-Blg
M. Cas-Tch 1
M. Cas-Bgr 1
M. Mus-Njl

2
2
1
2

* Weak reaction.
•

Faculty of Agriculture, Nagoya University.

RESEARCH ACfIVITIES IN 1985

39

Sera from 21 wild derived strains were surveyed by gel precipitation with
anti-Aph-IB monospecific alloantiserum (Table I). Sera from 16 strainsof mice including all of strains derived from Eastern Asia (Japan, Korea andChina) crossreacted with the antiserum. Sera from two M. m. domesticus
strains (M.DOM-PGN 2 and M. Dom-Blg) did not crossreact with it asexpected.
Sera from M.BRV-MPL and M.Sub-Shh I crossreacted weaker thanthose of the other positive strains. The precipitin lines of these sera against
anti-Aph-IB did not fused completely with that of control serum inOuchterlonyanalysis. Mice of M.BRV-MPL and M.Sub-Shh I may havethe third allele at the Aph-llocus.
Characterization of Ly-2.1 Antigenicity
Newly Found in Asian MiceW

Yasuyuki KURIHARA and Kazuo MORIWAKI

Ly-2 antigen is expressed on thymocytes, suppresser T cells and cytotoxic
T-cells, discriminating them from the other kind of lymphocytes. So far,
two allelic forms of it, Ly-2.1 and Ly-2.2, have been known in laboratory
strains of mice. Our previous study (Immunogenet. 22: 211-218, 1985)
demonstrated that thymocytes of Mus musculus castaneus and the related
mice failed to react with Ly-2.2 and expressed very weak reaction to Ly-2.1specific monoclonal antibody. One hybridoma clone, designated YKI2-601,
secreting monoclonal antibody specific to Ly-2.1 w antigen was establishedfrom spleen lymphocytes of C57BLj6 mice immunized by castaneus thymocytes. This monoclonal antibody strongly reacted with the Ly-2.1 W antigen,but weakly with Ly-2.1 antigen. The weak reaction for Ly-2.1 observedin the castaneus mice might have resulted from a minor structural change ofthe genuine Ly-2.1 antigen, suggesting that Ly-2.1 w could be the third allele.
Another monoclonal antibody, designated YK12-457 was isolated fromthe same immunization as YK12-601. This monoclonal antibody positively
reacted with the thymocytes of twelve out of thirteen standard mouse strains
including the recipient strain C57BLj6. Only exception was DMjShi strain
which was obtained from Aburabi Laboratories of Shionogi PharmaceuticalCo. (Dr. S. Makino). Thus, YK12-457 monoclonal antibody seems to be akind of cytotoxic autoantibody for syngenetic thymocytes.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS
Effects of Hormones on Growth Pattern of
Human Diploid Cells in Culture
Yukiaki KURODA

Normal diploid fibroblasts obtained from human embryos die after about
fifty population doublings in culture. This phenomena are useful for investigating the mechanism of aging by cultured human diploid cell as a model
system. In the present study, the effect of hormones such as testosterone,
insulin, gonadotropin and thyroxine on growth pattern of human diploidcells was examined in clonal cultures.
Normal diploid fibroblasts were obtained from a human abortive embryoof 10-week gestation. When cells at the sixth population doubling level
were cultured as a clone from single cells, they showed a peculiar growth
pattern. Some cells in the cell population grew exponentially, and othercells stopped to divide after several times of cell divisions. Some part ofcells in population was maintained in an active state without any cell division
during culture period of one week.
When testosterone, insulin or thyroxine was added to culture medium atconcentrations of 0.05 to 0.5 p.gjml, it enhanced divisions for both dividing
cells and non-dividing cells. Gonadotropin had stimulating effects only
on dividing cells, resulting in shortening the cell cycle time, but they had
no effect on non-dividing cells.
In our previous study, it has been found that growth factors and hormones
could be divided into two groups: one stimulated all cells to divide in population, and other stimulated only dividing cells in population. The former
substances such as epidermal growth factor (EFG) and fibroblast growth
factor (FGF) had no effect on the increase in the number of population
doublings of human fibroblasts. On the other hand, the latter substancesuch as hydrocortisone was effective in expanding the population doubling
number. In this context, it was suggested that testosterone, insulin andthyroxine may have no effect on in vitro cell aging, while gonadotropin may
have some effect on expanding the cell aging of human cells in culture.

RESEARCH ACTIVITIES IN 1985

41

Antimutagenic Activity of Vitamin C on Mutations Induced
by EMS in Cultured Chinese Hamster Cells
Yukiaki KURODA

Vitamin C is abundantly contained in green tea, fruits, and vegetables.
It is effective in preventing scurvy, and its antioxidant activity is useful for
detoxification of some drugs and for protection against rancidity in foodstuffs. In the present experiment, it has been found that vitamin C had amarked effect in reducing 6-thioguanine (6TG) resistant mutations inducedby ethyl methonesulfonate (EMS) in cultured Chinese hamster V79 cells.
Vitamin C alone had no detectable effect on cell survival at concentrationsof less than 100 flgJml. At a concentration of 300 flgJml, the colonyforming activity of cells was completely inhibited. On the other hand,
EMS had cytotoxic effect on survival of V79 cells. The value of LD 50 of
EMS was 541 flgJml. In the presence of 100 flgJml vitamin C, the cytotoxic
effect of EMS decreased markedly, giving the LD 50 of more than 1,000 flgJml.
Vitamin C also had a marked effect in reducing 6TG resistant mutations
induced by EMS. Vitamin C alone had no detectable activity in inducing
6TG resistant mutations. EMS had a strong inducing activity of 6TG
resistant mutations in the absence of vitamin C. At a concentration of
1,000 flgJml, EMS induced 6TG resistant mutations at a frequence of
87.5 x 10- 5 • In the presence of 100 flgJml vitamin C, the mutagenic activity of EMS reduced to about one third or one fourth. We are taking
vitamin C from the diet at the rate of 20 to 500 flgJkg body weight. Without
extra vitamin C in the diet, the concentrations in human tissue are reported
to range from 2 to 50 flgJ100 g. Vitamin C at these concentrations may beplaya role in the prevention of mutagenic action of chemicals taken into thebody from our environment. The inhibitory effects of vitamin C on thecytotoxic action and mutations induced by EMS in the present experimentare an interesting problem in understanding the mechanism of vitamin C.
Combined Effects of EMS and MMS on Mutation in
Chinese Hamster Cells in Culture
Yukiaki KURODA, Hajime KOJIMA'" and Hiroaki KONISHI'"

Many chemicals present in our environment have been examined for their'" Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd., Ogaki, Gifu.

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

mutagenic activity by various test systems. Most of these tests were dealt
with the effects of the single chemicals on organisms. In our environment,
however, may chemicals were present together. It is necessary to examine
the combined effects of different chemicals. In the present experiment, thecombined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on 6-thioguanine (6TG) resistant mutations in Chinese hamster
V79 cells were examined.
When EMS at a concentration of LD 20 was combined with MMS atvarious concentrations and used for treatment of cells for 3 to 12 hours,some synergistic effects of both chemicals on the induction of 6TG resistantmutations were found, compared with the theoretical values calculated fromthe mutation frequencies obtained by single chemicals. However, when thecytotoxic effect of both chemicals was considered, the combined effects were
not synergistic, but additional. When MMS at a concentration of LD 20
was combined with EMS at various concentrations and used for treatment ofcells, no definite results on combined effects of both chemicals were obtainedin the induced mutation frequency, due to a strong lethal effect of MMS.
When cells were successively treated with EMS and MMS each for 3
hours, the induced mutation frequency varied, additional or synergistic,
depending on the sequence of treatments with both chemicals and the concentrations and times of chemicals. When an interval of cultivation for 3
hours in normal medium was inserted between two treatments with EMS
and MMS each for 3 hours, some repair phenomena in induced mutation
frequency were observed. When cycloheximide, an inhibitor of protein
synthesis, at a concentration of 10 flg/ml, was added in the medium during
the above interval, the induced mutation frequency was slightly reduced.
Hydroxyurea at a concentration of 50 flg/ml had no effect on the induced
mutation frequency. The study to examine the combined effects of bothchemicals on chromosome aberrations is also progressed.
Enhancing Effect of Ara-C on Mutations in Cultured
Chinese Hamster Cells Treated with EMS
Yukiaki KURODA, Kanako SHINKAWA-TACHI*
Kanehisa MORIMOTO* and Akira KOIZUMi*

In addition to the investigations on the combined effects of chemicals having'"

Department of Public Health, Faculty of Medicine, University of Tokyo.

RESEARCH ACTIVITIES IN 198543

the same target bases in DNA, it is important to know the combined effects
of more than two chemicals which are different in their action mechanism
to DNA in the cells. In the present experiment, the effect of l-fi-D-arabinofuranosyl-cytosine (Ara-C), an inhibitor of DNA polymerase, on 6-thioguanine (6TG) resistant mutations in Chinese hamster V79 cells treated with
ethyl methanesulfonate (EMS) was examined.
The treatment time of cells with both chemicals was 6 hours, in consideration of their cytotoxicity and the cell cycle time. The concentrations ofchemicals were fixed for one chemical at that giving the LD 20 (EMS: 4 X 10- 3
M, Ara-C: 2x 10- 6 M) and varied for the other.
EMS had a strong mutagenic effect on V79 cells. As for Ara-C, however,there has been a controversy in the previous reports as to whether it is mutagenic or not. In the present experiment, Ara-C had no detectable effectin inducing 6TG resistant mutations of V79 cells up to a concentration of
5x 10- 4 M.
The mutation frequencies of cells treated simultaneously or successively
with EMS and Ara-C were higher than those obtained by treatment with
EMS alone. These results indicate that Ara-C had an enhancing effect on
mutations induced by EMS in Chinese hamster cells, although Ara-C alone
was not mutagenic to the cells.
Freezing of Drosophila Embryos after Treatment
with Some Chemicals
Yukiaki KURODA and Yuko TAKADA

In a series of experiments to freeze embryos of Drosophila melanogasterat -SO°C, one of the most difficult problems was the fact that the vitelline
membrane of embryos was resistant for freezing-protective agents to penetrate
into embryos. It is necessary to permit the penetration of these agents suchas glycerol or dimethyl sulfoxide, to avoid the formation of ice in theembryos at -SO°C. The treatments of embryos with various chemicals
were attempted to enhance the permeability of the vitelline membrane.
Eggs collected from adult flies of the Oregon-R strain of D. melanogasterwere dechorionated by treatment with 3 % sodium hypochloride solution
for 10 minutes. Selected embryos at the appropriate stage of development
were treated with pentaethyleneglycol-monododecyl ether (PEGE) at various
concentrations for various times of incubation. When embryos were treated

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

with 20 % PEGE for 30 minutes, neutral red, which was used for detecting
the change in the permeability of the vitelline membrane, entered into embryos. These embryos were transferred to salt solution containing 10 %glycerol and frozen at -80°C in an electric refrigerator. When they weredefrosted and incubated at 25°C, they continued to develop and hatch tolarvae at a low frequency.
Since PEGE had a toxic effect on embryos, some other non-toxic enzymes,
which were effective in enhancing the permeability of the vitelline membrane,
were tested. Embryos were treated with lipase, pancreatin, pectinase andtrypsin at various concentrations. After treatments with these enzymes,
their toxic effects on survival of embryos and the hatchability of treatedembryos after freezing at -80°C were examined. When embryos weretreated with concentrations as low as 0.01 to 0.1 % of trypsin for 18 to 24
hours, their toxic effect was low with an increase in the hatchability of treatedembryos after freezing at -80°C. During treatment for these long periods,
however, embryos proceeded their development. More improved procedures for treatment of embryos with enzymes are now in progress.
Differentiation of Embryonic Cells of
Drosophila melanogaster Studied with
the Electron Microscope
Yutaka SHIMADA and Yukiaki KURODA

In the series of tissue culture studies of embryonic cells from Drosophilamelanogaster, we have examined the fine structure of these cells cultured
with and without ecdysterone.
When undifferentiated cells dissociated from post-gastrula embryos of
the wild-type strain (Oregon-R of D. melanogaster) were cultured in medium
K-17 (supplemented with 15 % fetal calf serum and 0.1 ftgJml fetuin) without
ecdysterone at 25°C, they differentiated into various embryonic or larval
cells such as muscle, epithelial and nerve cells. In muscle cells, the formation of typical myofibrillar structure was observed; A, I, Z and M bands
were clearly distinguished. In epithelial cells, interdigitations, gap junctions and desmosomes were formed between adjacent cells. Nerve cells
extended many cytoplasmic processes, in which neurofilaments and neurotubules were found.
When these cells were incubated in the same medium with addition of

RESEARCH ACTIVITIES IN 1985

45

10 ftgfml ecdysterone, two cell types could be distinguished at early stagesof culture, i.e., dark and light cells. The former cells possessed a dark
nucleus and a cytoplasm containing numerous endoplasmic reticula, mitochondria and ribosomes. The latter cells had a lightly stained cytoplasm
with fewer endoplasmic reticula, mitochondria and ribosomes. After one
week in culture, differentiation of muscle and nerve cells was evident. The
former cells with the typical myofibrillar structures had adherent junctions
between the same cell types. As in the nerve cells cultured without
ecdysterone, the latter cells had neurotubules and neurofilaments in theirprocesses. Tracheae with numerous bifurcations and infoldings were
found in many cell types. Virus-like particles were observed in nuclei ofmany cells. After 2 weeks of culture, cells with vacuoles and degenerated
endoplasmic reticula increased.
The results suggest that the fine structure of the cells cultured with andwithout ecdysterone may be useful for analyzing the more detailed characteristic action of the specific genes during normal development.

Brown-embryo Letbals in Drosophila melanogaster
Kiyoshi MINATO

Some embryos in Drosophila melanogaster have been known to die inbrown embryos (BR) when eggs are irradiated with X-rays. This BR isthought to reflect dead embryos with some developed organogenesis.
The similar, but inherited syndrom was found and has been maintained
stably for many generations in a sub-line of the Oregon-R strain in ourlaboratory. In this sub-line a high proportion (about 30 %) of embryos
died in BR. Furthermore, in some other strains maintained in our laboratory, the BR syndrome appeared possibly owing to sub-optimal culture
conditions including bacterial infection of foods. In almost BR except
one, however, dissapeared gradually from populations of flies according
to the improvement of culture conditions. In only one strain,fs(I)MA Y-263 ,BR has been maintained stably for many generations without dissapearing,in which about 30 % of embryos died in brown embryos.
The external and internal morphology in brown embryos was observed
microscopically in the above two lines showing the stable genetical syndromeof BR. As the results, the characteristics of BR in the two lines were very
similar to each other. That is, some segmentation of the integument was

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

found, whereas the trachea, the gut and the head were not formed. Since
most of yolk, however, had been consumed, the development of other organs
may proceed considerably in brown embryos. Furthermore, a characteristic
Malpighian tubule, which can not be seen externally in the normal embryos,
was observed as the dark one in brown embryos. This darkeness of Malpighian tubule may be somehow associated with the phenomenon of brownish
change of dead embryo.
The Role of the Cephalic Furrow during the Embryonic
Development of Drosophila melanogaster
Kiyoshi MINATO

The oblique cleft of the cell layer is temporarily found laterally at about
one-third distance from the anterior end of the embryo at the late-gastrula
stage (3.5 hours at 25°C) of embryonic development of Drosophila melanogaster. As to the roles of this oblique cleft or "cephalic furrow" (CF),there are two concepts: (1) CF absorbs temporarily a stress derived froman intense increase of the cell layer : (2) CF is present as a boundary between
the head and the body regions.
To know which of the two is the case, the mitotic frequency of cells invarious regions was examined around the period of CF formation in histologic;;!.l preparations in the Oregon-R strain of D. melanogaster. It was foundthat mitosis was frequently seen not in the exterior but in the interior regionof the CF. Although mitotic divisions in the insect embryogenesis is not
generally followed by an increase in cell mass, the above finding may suggest some opposite situations to the concept of "absorvance of stress".
Genetic Studies on Parthenogenesis in Bomhyx mori:A Selection of Highly Parthenogenetic Lines
Akio MURAKAMI, Yoshiki OHTSUKI* and Toshio KITAZAWA*

For several years, we have been attempting to isolate highly spontaneousor natural parthenogenetic lines in Bombyx. In the previous communication (this Report No. 35, 1985), we reported that a Chinese bivoltine strain,
Daizo, showed a high rate of natural parthenogenesis. To investigate•

Breeding division, the National Sericultural Experiment Station, Tsukuba.

RESEARCH ACTIVITIES IN 198547

these results further, the natural parthenogenicity of the strain Daizo andtwo other strains, J106 and Cambodge, which are often used for experiments
on artificially-induced parthenogenesis, were investigated colouring serosa
cells with purplish black as an indication of embryonic development. Unfertilized or non-developmental eggs remained light yellowish-white.
Results showed that the frequency of natural parthenogenesis in eggs laidfrom virgin moths of the strain Daizo (D) was 3.6 %, and those of J106 (J)and Cambodge (C) were 8.7 and 16.4 %, respectively (bluish eggs or embryos
seen through the egg-chorion in the latter strain were judged as the developed
embryos). This contrasts with our previous conclusion that the Daizo
strain has the highest frequency of natural parthenogenesis. Surprisingly,
a remarkably high frequency of natural parthenogenesis was detected ineggs laid from some F , hybrids, DxC, CxD, and CxJ, ranging from
16.0 to 24.9 %. In particular the F , hybrid of J106 female and Cambodge
male showed the highest frequency, 32.1 % of all strains in this experiment.
These findings show that the frequency of natural parthenogenesis varies
from as lower 3.6 % to as high as 32.1 % depending on the stock or strain.
The frequency of natural parthenogenesis appeared to be generally higher
for the multivoltine strains and for crossed hybrids than for the bivoltine
strains.
Generally, embryos developed from natural parthenogenesis died at theearly embryonic diapause stage so that the frequency of hatching wasalways very low. Consequently, phenotypic analyses of natural parthenotes
different from those of artificially-induced parthenotes appeared to be
impossible. Either coloured or non-coloured egg or embryo from the F ,virgin female moths in a cross, J106 female and Cambodge male, was deprived of egg-chorion and then transferred one by one in drops of Grace's
medium to make hanging-drop cultures, and cultivation was continued at
25°C without renewing the medium. Results showed that all coloured
embryos grew the stage of bristle formation or of the late stage of organogenesis, and that most of the embryos from the non-coloured eggs also developed to the stage of bristle formation. A similar tendency was observedin eggs from the virgin moths of F 1 hybrid of the different crosses, Cambodge
female and J106 male, and Daizo female and Cambodge male. It, thus,
can be tentatively concluded that eggs laid from virgin moths in Bombyx
mori regardless of strains or stocks may have a high potentiality for naturalparthenogenesis.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Genetic Studies on Parthenogenesis in Bomhyx mor;:
Thermal Activation and Its SignificanceAkio MURAKAMIIn Bombyx, it is very rare for eggs to develop without the participationof sperm and these spontaneous parthenogenetic eggs usually do not survive
to hatching. Theoretically, in parthenogenesis, a meiotic haploid oocyte
duplicates before and/or during the first mitotic division, and the zygote
has two sets of chromosomes derived from its mother. This may explain
the lack of vigor in natural as well as induced parthenotes.
In this insect, artificial parthenogenesis can be easily induced by an appropriate treatment of unfertilized eggs or oocytes by various means. Among
successful parthenogenetic methods, the thermal activation of ovarianeggs dissected from virgin moths (Astaurov, 1940) is the most common anda majority of the parthenotes survive to hatch. Some of them develop to
adults with a normal reproductive function. It is of particular interest
to note that the sex of these parthenotes is female. When the first meiotic
division was blocked by exposure of ovarian eggs to a high temperature, thechromosome number was not reduced, diploidy (or ameiotic parthenogenesis)
was maintained. Recently, Strunnikov and his colleagues (e.g., 1983)
devised another method combining activation of newly deposited unfertilized eggs with low and high temperatures. This method is characterized
by the use of deposited unfertilized eggs. It is, therefore, of interest tocompare hatchabilities (or viabilities) of artificially-induced and naturallyoccurring parthenotes to understand the mechanism(s) of artificial activation of unfertilized eggs.
Hybrids between CI08 females and Aojuku males were used in the present
experiment. For the combined activation, the newly deposited unfertilized
eggs aged one to five days after natural laying were kept in a refrigerator
at -11 °C for 120-150 minutes and then immersed in warm water at 46°C
for 12 minutes. After natural drying, eggs were treated similarly to normally-fertilized eggs. For thermal activation, ovarian eggs obtained fromvirgin moths were treated in the usual manner according to Astaurov (1940).
Hatchabilities of parthenotes obtained from combined activation varied
from a few per cent up to several per cent depending on the treatment, compared to 30-50 % for those obtained from the thermal activation. Hatchabilities of normally-fertilized eggs used as the control was over 95 %.

RESEARCH ACTIVITIES IN 1985

49

Combined activation, although essentially analogous to natural parthenogenesis with respect to using deposited unfertilized eggs, produced parthenotes which survived to larvae with a sex ratio of 1: 1. The differencein the sex-ratio between larvae obtained from these two activation methods
is clearly due to the stage at which the eggs are treated: the deposited eggs
had nearly completed the meiotic division forming four haploid chromosome
sets or nuclei. In this method, diploidy may result from a fusion between
two haploid nuclei (or meiotic parthenogenesis).
From these observations, it can be concluded that exposure to temperatures whether high or low is effective in inducing either meiotic or ameiotic
parthenogenesis leading to the restoration of the diploid number of chromosomes. It is also very likely that high temperature treatment acts as spermatozoa do in fertilization in inducing cell divisions or differentiations.
Sex and Strain Differences in the Lifespan of the
Adult Silkmoth, Bombyx mori
Akio MURAKAMI, Yukiaki KURODA and Yosoji FUKASE

In the silkworm, the entire lifespan takes about one year from egg toadult death after laying eggs. However, the greatest part of the lifespanis spent in the egg stage. From newly hatched larvae to the emergence ofan adult takes only about 40 days when reared at the standard temperatureof 25°C with a relative humidity of about 70-80 %. Information on theadult lifespan is meagre, but preliminary observations indicate that it takes
1-3 weeks depending on the strain and sex. In spite of its brevity, the adultstage has advantages for studies on the lifespan and aging because duringthat time they feed on nothing, and their body is composed entirely of postmitotic cells and so on (e.g., Osanai, 1985). This report describes one of aseries of investigations on the genetics of longevity or lifespan in Bombyx
mori.
Several silkworm strains, Cl08, Aojuku, Daizo, Cambodge, od, pe:re andsome their F 1 hybrids, reared on mulberry leaves, were used in the present
experiment. Newly emerged virgin moths were collected at about 10 a.m.
every day. Each moth was kept separately on an egg-card covered withmoth cover in a rearing room controlled at 25°C with a relative humidityof about 70-80% under dark conditions. Twice daily, observations werecarried out, at 10 a.m. and 4 p.m.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Survival curves for each strain proved to be of a typical sigmoidal fashion
regardless of the strain. The mean lifespan of each strain varied from
3-21 days. The lifespan of females was about 1.5 times longer than that ofmales for all strains tested except for the Daizo, a Chinese bivoltine stock.
In the Daizo, the lifespan was so short, 3-5 days, that no marked sex-differences were observed. Virgin female moths of the JI06 laid over 90 %of their eggs during the 4-5 days after the beginning of oviposition, while
those of the Daizo laid over 90% of their eggs during the first 1-3 days andthey bigin oviposition, about 8 hr after eclosion from the pupae. The
lifespan of the JI06 strain was found to be 15 days. The pupal stage took
10-14 days in the JI06 and Daizo strains. No significant differences in the
duration of embryonic (about 10-14 days) and larval (about 21-25 days)
stages were observed when using strandard rearing conditions. This indicates that the lifespan in the Daizo is shorter than the JI06 only in regards
to the adult stage. It appeares that the Daizo strain is regarded as a mutant
for a short adult lifespan in Bombyx.
The mean adult lifespan of the F, hybrid obtained from a cross between
Daizo and JI06 was somewhat longer than that of the JI06. In their backcross, [Daizo X JI06] X Daizo, two types for the adult lifespan corresponding
to the JI06 and Daizo types with the ratio of 1: 1 were observed. This observation indicates that the Daizo strain has a recessive genetic factor responsible for a short lifespan, whereas the JI06 has a dominant factor for along one. This finding may rise a question as to whether or not the shortlifespan factor in the Daizo strain is adult lethal. To get a final conclusion,
further studies are required.
Genetic Studies on the Hereditary Mosaic (mo) Strain of
Bomhyx mori: Function of the Polar-body
Akio MURAKAMI and Masahiko KOBAYASHI*

In Bombyx mori, it is known that a hereditary mosaic (mo) gene responsible
for the double-nucleus fertilization in an egg produces various types ofmosaicisms (e.g., Goldschmidt and Katsuki, 1928, 1931). In the courseof a neurogenetic investigation using a hereditary mosaic strain, we obtained 201 larvae, primarily egg-colour mosaics from a cross between the* Faculty of Agriculture, University of Tokyo, Tokyo.

RESEARCH ACTIVITIES IN 1985

51

hereditary mosaic strain females heterozygous for two genes in egg-colour(pe) and translucent larva (ok) and males homozygous for these two recessive genes, pe and ok. These larvae were further classified into either

nonmosaic or mosaic in regard to both sex and the autosomal traits, or nonmosaic for the sex and mosaics for the autosomal traits, and vice versa.
As a result of classifications, there were 37 females, 28 males and 136
gynanders with a ratio of I : I: 2. This observation suggests that the fashion
for meiotic divisions in Bornbyx females supports the post-reductional types
rather than the pre-reductional one.
If the pre-reductional division is the case, gynanders may raise throughthe two combinations of double-nucleus fertilization, either one of two
primary polar-bodies and an ovum, or a primary polar-body and a secondary
polar-body. While in the case of the post-reductional division type, both
females and males may appear in addition to the gynanders. Two different
combinations are also possible: an ovum and a secondary polar-body,and a pair of primary polar-bodies. It, thus, appears that the appearanceof each female, male and gynander is caused by one of twelve random com
binations between two nuclei among the four haploid nuclei formed by the
meiotic divisions in oogenic cells. In other words, all four haploid nuclei
from the primary oocyte have the same function in the rno strain and two ofthese four haploid nuclei have an equal function (or chance) to fuse witheach sperm. In the wild strain, however, one of the meiotic products would
differenciate into an oocyte pro-nucleus having a function to selectively
receive and/or recognize a sperm in the course of the meiotic division, while
the other three haploid nuclei would have no such function.
It is well known that in the insect egg, there is no morphological differencesamong the four haploid nuclei formed from a primary oocyte, besides thenucleus arranged in the innermost of ooplasm functions as the oocyte pronucleus. The remaining three nuclei in ooplasm are not usually concernedin fertilization. In consequence, polar-bodies in the strict sense are notformed in the insect. However, each of the three haploid nuclei in females
possessing a recessive gene rno may have an equal function to participate
in syngamy with sperm. Moreover, these meiotic products seem to have atendency to a high affinity for sperm. In any case, it is suggested that therno gene may be deficient in the function for differentiating the oocyte pronucleus from the three other polar-body nuclei.

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Sex Reversal by Heterosexual Parabiosis in Hydra
Tsutomu SUGIYAMA and Norio SUGIMOTO

Sex change from female to male occurs at a high rate in hydra when afemale polyp is exposed to a male polyp by parabiosis for a limited periodof time (1-3 days). In contrast, sex change in the opposite direction does
not occur when a male polyp is parabiotically exposed to a female polyp(Tardent, 1968).
The mechanism involved in this masculinization process by heterosexual
parabiosis was investigated. When a male strain which produces immotile
sperm was used to masculinize female strains, the resultant masculinizedanimals produced only immotile sperm. Evidence was also obtained whichindicated that the original female interstitial cells remained in large numbersin the masculinized animals but did not differentiate into gametic cells.
These observations suggest that masculinization is achieved not by the
hypothetical "masculinizing factor" (Wiese, 1953; Brien, 1963), but by the
migration of the male interstitial cells from the male to the female tissueduring parabiosis (Tardent, 1968). For details, see Developmental Biology,
110,413-421 (1985).
Zygotic Effect of the daughterless Gene in
Drosophila melanogasterYutaka INOUE

The sex specific lethal effect of the daughterless (da: 2-40.3) gene is recessive, maternal and temperature-sensitive. The da/da homozygous females
produce no daughters at 25°C and 29°C, but some daughters are rescued at
the lower temperature of 18°C. The heterozygous females produce daugh·ters at any temperature. To test the zygotic effect of da, the females heterozygous with the Curly-balancer chromosomes (Cy/da) were mated withda/da males for the four different da strains (#47, #46, JP83, #233), and thesex ratios (<;(./<3) were examined in each type of progenies (Cy/da and da/da).
The parental flies were placed at 18°C, 22°C and 29°C, respectively, for
4-5 days before letting them deposit eggs. The sex ratios of the Cy/daprogenies were normal; 0.92 for #47, 0.94 for #64, 1.00 for JP83 and 1.00
for #233 on the average for the three temperatures. At the 18°C and 22°C
conditions, although the #47 strain showed a slight significant differenceTable 1. The sex ratio (No. of femalesjNo. of males) of Cyjda and dajda progenies from the
Cyjda females mated with dajda males in the four strains at three temperature
Tempt.

#47
No.")

Cyjda

18°C

6417

1.07

22°C

5813

0.86

I

*

a)

4962

0.84

tTl
tTl

>

#233

:::c

No.

Cyjda

dajda

No.

Cyjda

dajda

No.

Cyjda

da/da

0.87

3980

0.94

0.83

2299

0.97

0.94

4190

0.95

1.00

0.95

4528

0.94

0.86

3436

1.09

0.93

5987

1.04

0.98

dajda

I

1- *-1
29°C

JP83

#64

:::c
CIl

0.73

2055

0.94

0.58

Number of flies counted.
* and ***: Significant at 0.05 and 0.001 level, respectively.

1-*"-1

1-***-1

1-***-1
2301

0.93

0.46

5833

1.00

0.58

g
>
Q
....
~
..-j
....
~
....

Z

\0

00
VI

VI

I,H

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

between Cyjda (1.07) and dajda (0.87), 7 cases out of 8 showed no significant
differences between the sex ratios of Cyjda and dajda progenies. However,at a higher temperature of 29°C, all strains showed a lack of females in the
dajda progenies; 0.73 for #47, 0.58 for #64, 0.46 for JP83 and 0.58 for #233.
Compared with their Cyjda sibs, all four cases showed statistically significant differences, and three of them were highly significant (p<O.OOl).
This data suggests a zygotic deleterious effect in the dajda daughters at
29°C.
Ovary Freezing for the Maintenance of Silkworm Genes
Jun KUSUDA and Kimiharu ONIMARU

We previously reported that the silkworm ovaries preserved in liquid
nitrogen produced mature eggs retaining the capacity for embryogenesis
and hatching.
To apply this method for stocking mutant genes of silkworm, we attempted
to freeze ovaries of Moricaud silkworm (PM) which has a dark epidermis.
The pM was crossed with Cambodge (Camb) to enhance the ability ofparthenogenesis. Ovaries taken from larvae of the Fl hybrid, pMjCamb,were cooled at a rate of lOCfmin to -35°C in the presence of 1.5 M glycerol
and preserved in liquid nitrogen. After 2 days, they were rapidly thawed(500°Cfmin) and the glycerol was removed. Forty three frozen thawed
ovaries were transplanted into wild type female larvae. Some ovaries
developed to generate mature eggs according to the state of th host development. When emerging as adults, the moths were dissected to obtain mature
eggs with a chorion. Collected eggs (536) were immersed in a warm water
bath (46°C) for 18 min to induce parthenogenetic development. This wasfollowed by the treatment with HCl solution (specific gravity 1.075 at 25°C)
to prevent diapause initiation. After about 10 days, 14 larvae hatched fromthe eggs, of which 10 developed to the 5th instar larvae. Among them,
five had clear dark epiderms as the donor worms did, and the remaining
five had normal epiderms like the hosts. Thus, ovary cells carrying pMmaker gene survived under sub-zero temperatures, and this technique could
be applied for the maintenance of other mutant genes of silkworms.

RESEARCH ACTIVITIES IN 1985

v.

55

CYTOGENETICS

Karyological Survey of Wild Micein Ogasawara Chichi Island
Yasuyuki KURIHARA, Mitsuru SAKAIZUMI 1),
Kimiyuki TSUCHIYA 2 ) Yoshinobu HARADA
and Kazuo MORIWAKIIn 1977, we found mice with a Robertsonian (Rb) translocation (9, 15) in
the Ogasawara Chichi island located about 1000 km south of Tokyo (Genet.
Res. Camb. 43: 277-287, 1984). To know the present status of Rb-translocation in this island, the mice were collected in December, 1985 and March,
1986. Total number of the mice collected are 89 from five localities in the
Chichi island as summarized below (distance and direction from "Kitafukurozawa" where the Rb-mice were previously collected, were shown in
the parentheses);1)

2)

Locality

No. micecollected

Date ofcol1ection

Mikazuki Yama (15 km, NW)
Suzaki (1 .5 km, NW)
Sub-Tropical
Agriculture Center (1 km, N)
Kitafukurozawa
Kominato (500 m, W)

12 (m: 6, f: 6)
10 (m: 5, f: 5)

Dec., '85
Dec., '85

20 (m: 10, f: 10)
32 (m: 14, f: 18)
15 (m: 8, f: 7)

Dec., '85
Mar., '86
Mar., '86

Tokyo Metropolitan Institute of Medical Science.
Miyazaki Medical Col1ege.

Chromosome preparations were made from testis in male and tail skinculture in female. No chromosomal variant was observed in the mice tested.
The mice with Rb (9.15) inhabited 9 years ago have probably been eliminatedfrom the natural populations in Ogasawara Chichi island, suggesting ageneral genetic instability of Rb-variants in wild populations.

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

A Genetic Model for X-V Dissociation and Testis Weigbt
in Intersubspecies Hybrids between
M. m. molossinus (BALB/c) and
M. m. molossinus
Hirotami T. IMAI, Won Ho LEE 1), Kiauw Nio TJAN 2)
and Kazuo MORIWAKI

A linkage test of genetic factors controlling the terminal association of
X-Y chromosomes and the testis weight was made by using Japanese wild
mice (Mus musculus molossinus), BALB/c, and their hybrids (Flo N 2 _ 5).
When male mice were classified by the frequency of precocious dissociationof X and Y at metaphase I (X//Y) and the relative testis weight (RTW;
Testis weight/body weight) as High X//Y having X//YZ.50%, Low X//Y
(X//Y <50 %), large sized testis (Lt; RTW>3.0), and small sized testis (St;
PTW <3.0), both molossinus and BALB/c were characterized by (Low X//Y,
Lt) whereas their F 1 hybrids were (High X//Y, St). Four types of progeny(High X//Y, St), (High X//Y, Lt), (Low X//Y, St), (Low X//Y, Lt) weresegregated at the 2nd backcross generation, though only (Low X//Y, Lt)males were obtained from (Low X//Y, Lt) sires. We assumed two genes Sxa(sex chromosome association) and Twr (testis weight regulator), adjoined in
the common part of the X and Y chromosomes and controlled the end-toend association of the sex chromosomes and the size of testis, respectively.
The Sxa and Twr alleles of BALB/c were denoted as (Sxa a, Twr a) and thoseof molossinus were (Sxa b, Twr b), where the terminal association of the X andY chromosomes dissociates precociously at metaphase I in heterozygotes
for Sxa (i.e., Sxaa/Sxa b), and Twr b is dominant to Twr a. The genotype
expected in (High X//Y, St) males was (Sxa a, Twra)/(Sxa b, Twr b), and thatof low X//Y males and their parental stocks was either (Sxa a, Twra)/(Sxa a,
Twr a) or (Sxa b, Twrb)/(Sxa b, Twr b). The segregation of (Low X//Y, Lt)males from (High X//Y, St) sires was interpreted simply as a single recombinant, and (High X//Y, Lt) and (Low X//Y, St) as double recombinant in
the mating of BALB/c females X F 1 males.

1)2)

Department of Biology, Pusan National University, Pusan 607, Korea.
Department of Biology, Bandung Inst. of Technology, JL. Gamesa 10, Bandung,Indonesia.

RESEARCH ACTIVITIES IN 1985

57

Chromosome Observations of Sarawak Ants
Kiauw Nio DAN 1), Hirotami T. IMAI, Masao KUBOTA2)
William L. BROWN Jr. 3), William H. GOTWALD Jr. 4 )Hoi-Sen YONG') and Charles LEB6)

This is a preliminary report on the chromosomes of Sarawak ants.
Collection of ants and chromosome preparations were made at Bako National Park (Kuching, Sarawak) from June 9 to June 13, 1985. A total of
37 colonies including 5 subfamilies, 21 genera, and 29 species were examined.
The colonies collected were labelled as HI85-(19-54, 67). The identification of species was made by Brown and Kubota, and the chromosome
observations were made by Tjan. One set of dry specimens was deposited
with the Sarawaku Museum.
Table 1. Chromosome numbers of Sarawak ants
Taxa (Colony number)
PONERINAE
Mystrium camillae (HI85-19)
Proceratium sp. (HI85-29)
Cryptopone testacea (HI85-41, 43)
Leptogenys myops (HI85-23, 25)
Leptogenys sp. (H185-53)
Pachycondyla rubra (HI85-44)
Diacamma sp. (HI85-51)
Odontomachus simillimus (HI85-27)
Anochetus sp. (HI85-50)

I)

2)
3)
4)

')
6)

Chromosome number(n) 2n32
48(9) 18(24) 485420
5844(17) 34

CERAPACHYINAE
Cerapachys sp. (H185-47)

50

MYRMICINAE
Crematogaster sp. (HI85-45)

26

Department of Biology, Bandung Inst. of Technology, JL. Gamesa 10, Bandung,Indonesia.
Nakasone 13, Odawara, Kanagawa-ken, 250 Japan.
Department of Entomology, Cornell University, Ithaca, New York, 14853, U.S.A.
Department of Biology, Utica College of Syracuse University, Burrstone Road,
Utica, New York 13502, U.S.A.
Department of Zoology, University of Malaya, Kuala Lumpur 59100, Malaysia.
Sarawak Museum, Kuching, Sarawak, East Malaysia.

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36
Table 1. (Continued).
Taxa (Colony number)
Oligomyrmex sp. 1 (HI85-22)
Oligomyrmex sp. 2 (HI85-38)
Pheidologeton sp. (HI85-35)
Lophomyrmex sp. (HI85-32)
Tetramorium smithi (HI85-31)
Tetramorium sp. 2 (HI85-26, 24)
Tetramorium sp. 3 (HI85-28)
Acanthomyrmex sp. (HI85--42, 67)
Pheidole sp. 1 (HI85-39, 40)
Pheidole sp. 2 (HI85-21)
Pheidole sp. 3 (HI85--49)
Pheidole sp. 4 (HI85--46)
DOLICHODERINAE
Iridomyrmex cordatus (HI85-36, 37)
FORMICINAE
Anoplolepis longipes (HI85-54)
Acropyga sp. (HI85-20, 52)
Pseudolasius sp. (HI85-34)
Camponotus sp. 1 (Hl85-30)
Camponotus sp. 2 (HI85--48)

Chromosome number(n) 2n

36
44
42
38
26
20
24
24
(10) 2018(9) 181816(17)(15) 3030
40
20

Modes of Species Differentiation and Karyotype Alteration in
Ants and Mammals
Hirotami T. IMAI

Two closely related topics for speciation and karyotype evolution ofants and mammals were investigated. First, I attempted to analyze therole of karyotype alteration in speciation by using a two dimensional graph(S-K G graph). When the number of species and dissimilar karyotypes involved in a genus are denoted as S and KG, the genus can be represented asa point (S, KG) on the S-KG graph. If karyotype alterations always initiate
speciation as suggested by the stasipatric speciation model, and if speciation
occurs without karyotype alteration, the genus will fall in the area KG/S;;::: 1
and KG/S~I, respectively. Among 420 mammalian genera examined,
the number of genera showing K G/S>I, =1, and <1 was 21 (8.9%), 57(24.3 %), and 157 (66.8 %), respectively. The same result was obtained

RESEARCH ACTIVITIES IN 1985

59

also in ants, i.e., Ka/S> 1, = 1, and < 1 was 2, 6, and 23. These observations strongly indicate that visible karyotype alterations (i.e., Robertsonian
rearrangement, pericentric inversion, and tandem growth of constitutive
heterochromatin) do not always contribute to speciation.
Next, I analyzed the karyotype evolution of ants and mammals quantitatively, and found a general tendency for karyotypes to evolve as a wholetowards increasing chromosome numbers and/or arm numbers mainly bycentric fission and pericentric inversion changing from acrocentric to metacentric (p.i. (AM». Finally, I proposed the minimum interaction hypothesis
for interpreting such uni-directional alteration in ants and mammals. The
minimum interaction hypothesis assumes that the karyotype evolution proceeds toward minimizing the genetic risks due to reciprocal translocations,and that an increase in chromosome number by centric fission may be one
such mechanism utilized. The non-random distribution patterns of translocation polymorphisms and Robertsonian polymorphisms found in ants
[the former was found only in ants with low chromosome numbers (n< 12)
but the latter was chracteristic to high-numbered species (n> 12)], was proposed as supporting evidence for the minimum interaction hypothesis.
The Exceptionally Low Chromosome Number n=2 in
an Australian Bulldog Ant, Myrmecia piliventris
Smith (Hymenoptera: Formicidae)
Hirotami T. IMAI and Robert W. TAYLOR·

The Australian bulldog ants of the species rich genus Myrmecia areamong the most primitive of all extant Formicidae. Myrmecia is characterized by the exceptionally wide range of chromosome numbers among its
species. Known number include n=5, 6, 11, 16, 19, 25, 30, 33 and 42(Imai, Crozier and Taylor 1977, Chromosoma 59: 341). Also, it is clearthat morphologically very similar species may differ greatly in chromosome
number. We report here two karyotypically very different but morphologically very similar species, either of which would previously have been
identified as Myrmecia piliventris Smith on the conventional basis of worker
morphological characteristics.
During recent studies in southeastern New South Wales and near Canberra•

CSIRO, Division of Entomology, GPO Box 1700, Canberra, A.C.T., 2601, Australia.

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

we observed the chromosomes of two M. piliventris colonies from Black
Mountain, Canberra (35° ITS, 149°13'E), one of which (HI85-l7l) had
n=34, while the other (HI85-ln) had the very low number n=2, 2n=4
(Fig. 1). These two colonies must surely represent separate species, yet themorphological features which distinguish their workers are of a nature
conventionally recognized merely as intraspecific variation (see, for example,
the discussion of M. piliventris by Brown (1953, Bull. Mus. Compo Zool.
111: 20). This, of course, has important implications for future taxonomic
work on the genus. The species with n=2 has been confidently identified
by R. W. Taylor as M. piliventris.
The 2n=4 karyotype comprises two pairs of submetacentrics (2K=4ST),of which both chromosomes have well developed C-bands at the proximal
regions of their long arms. Homologues of chromosome 1 are homomorphic, but those of chromosome 2 show remarkable heteromeorphismin C-bands, and in the size of their short arms.
Three other colonies collected from Jerrabomberra Hill, near Queanbeyan(35°2l'S, l49°l4'E), N.S.W. (HI85-l88) and at a much lower elevationnear Nelligen Creek Bridge. W. of Batemans Bay (35°43'S, 150 0 1O'E),
N.S.W. (HI85-211 and -241) also had the same n=2 karyotype (K=2ST
and 2K=4ST), suggesting that the species involved is both stable and fairly
widely distributed.
The even more remarkable chromosome number n=l, 2n=2 was reported
recently for a species of the Myrmecia pilosula (Smith) group by Crosland
and Crozier (1986, Science 231: 1278). This karyotype comprises one
pair of submetacentrics (2K=2SM) with large C-banded blocks at theirpericentromeric regions. Among the smaller species of Myrmecia, M.
piliventris and M. pilosula are morphologically rather distinct from eachother, but their karyotypes can be systematically related. We assume
that the n= 1 pilosula karyotype would have been derived from n=2 karyotype by reciprocal translocation (though not necessarily in a lineage including
morphologically piliventris-like species). More information about thedetails of C-bands and the karyology of other related species is desirable.
M. pilosula, M. piliventris, and their many chromosomally unstudied relatives, must stand as prime analysis of karyotype evolution and speciation inants.
H. T. IMAI was supported in this investigation by the Scientific Visitors
Program of the Division of Entomology, CSIRO. Appropriately labeled

RESEARCH ACTIVITIES IN 1985

61

voucher specimens are deposited in the Australian National Insect Collection, Canberra.

Fig. 1. Karyotypes of Myrmecia piliventris. A and B. 2n=4 (worker). C and D.
n=2 (male). A and C. Conventional staining by Guimsa. Band D. Cbanded karyotypes.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

VI.

MUTAGENESIS AND RADIATION GENETICS

Evidence Suggesting High Sensitivity of Bone Marrow Cells
to Radiation-Induced Cell Killing in the "Wasted" Mouse
Hideo TEZUKA, Tadashi INOUE and Tsuneo KADA

Bone marrow cells of 26 day-old wasted mice were found to be susceptible
to gamma ray-induced chromosomal aberrations (Tezuka et al., MutationRes., 161, 83). We examined the dose-response relationship between
exposure to gamma irradiation and the induction of chromosomal aberrations in bone marrow cells (mainly proerythroblasts and erythroblasts),
and the subsequent development of the aberrations to micronuclei in bonemarrow polychromatic erythrocytes. The doses used were 0, 0.25, 0.5, 1
and 2 Gy. Normal appearing littermates served as controls. The frequency of cells with aberrations showed 2- to 4-fold increases in wasted
mice over that in controls. The frequency of micronuclei, however, was
50-75% lower in wasted mice than in controls.
This indicates that most aberrant bone marrow cells die before nuclear
expulsion in wasted mice. There may be a certain stage which is sensitive
to cell killing during erythropoiesis. This is in contrast with results ofin vitro experiments using wasted mouse-derived lung fibroblast cultures
where the lack of radiosensitivity was shown (Inoue et aI., Cancer Res.,46,3979).
The Effect of Age on the Frequency of Spontaneous andMutagen-Induced Micronuclei in Mouse Bone Marrow
Hideo TEZUKA, Koichi TAMArI), Kazuo MURAKAMr 2)
and Tsuneo KADA

In vivo mutagenicity tests in mammals are usually conducted on sexuallymature young animals, 9 to 12 weeks old. Consequently, the effects ofchemicals on very young animals are rarely observed. One way of over1)

2)

Health Science Research Institute, Yokohama.
Sanwa Kagaku Kenkyusho, CO., Nagoya.

RESEARCH ACTIVITIES IN 1985

63

coming this is to score the frequency of micronuclei appearing in bonemarrow polychromatic erythrocytes (BMPCE) of animals at various ages
after birth. The appearance of such cells reflects chromosomal aberrationinduction in erythroblasts.
We examined BMPCE of male mice at 1, 3, 6,9, 13 and 18 weeks of age.
The mice were sacrificed 24 to 30 hours after treatment with mutagens.
Unexposed animals of the same age served as controls. The frequency of
micronuclei in controls remained constant at low frequencies. In mutagentreated animals, the frequency of micronuclei was significantly higher thanthat of controls across all age groups. However, the effect of age on thefrequency of such mutagen-induced micronuclei differed with the type of
agent. A marked induction of micronuclei was observed in 1 week old micetreated either with a DNA crosslinking agent mitomycin C or with alkylating
agents such as methylmethanesulfonate or cyclophosphamide. In older
mice, the frequency of such micronuclei decreased with age. In contrast,
the frequency of micronuclei in mice treated with vincristine sulfate, aspindle poison, was low in 1 to 6 week old mice, but increased markedly in
9 week old mice, and thereafter remained constant.
The results presented suggest the involvement of multiple cellular mechanisms, including DNA repair, in the induction of micronuclei. The enzyme
activity involved in such mechanisms may be altered with developmental
stages.
Cancer Susceptibility of the "Wasted" Mouse
Tadashi INOUE, Junko TAKAHASHI, Akira OOTSUYAMA ' )Hideo TEZUKA and Tsuneo KADA

The mouse mutant "wasted" (wstjwst) is a putative animal model for
Ataxia-Telangiectasia (A-T), a human genetic disease characterized bychromosomal instability, radiation-hypersensitivity and increased predisposition to cancer. Although the wasted mouse shares many characteristics
with A-T, cancer susceptibility has not been examined because of the shortlife span of the mutant. To overcome this, we performed skin grafts fromnormal and wasted mice onto recipient normal mice. We then sequentiallyapplied the cancer initiator dimethylbenzanthracene and the cancer promoter TPA to the grafted areas of the recipients. While several papillomas
1)

National Cancer Center Research Institute.

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

developed on grafts from healthy controls, no tumors appeared on skinfrom mutant mice. This indicates that some functions required for cancer
induction are deficient in the wasted mouse.
Effects of Low-Level Tritiated Water Ingestion
and Age on the Number of Sister Chromatid
Exchanges in Mouse Bone Marrow Cells
Hideo l'EzUKA, Robert D. BENZ,') Arland L. CARSTEN ' )and Takaji IKUSHIMA2 )We quantitated the number of sister chromatid exchanges (SCEs) occurring in male BNL-H/S mice maintained on 3.0, 7.5, 15 or 30/lCi/ml
tritiated water (HTO) for 1, 2, 4, 8, 12, 18 or 22 weeks. Identically housed
mice kept on normal water served as controls. Following the predetermined exposure time, animals were infused with bromo-deoxyuridine andthen sacrificed. Their bone marrow cells were removed, processed andthe number of SCEs counted. In the control groups, the number of SCEs/
cell decreased slightly, but significantly with age. The number of SCEs
in animals exposed to the various levels of HTO was essentially identicaland remained constant with age. The difference in the numbers of SCEs
between control and exposed mice slowly increased with the duration of
exposure. We speculate that this "anti-aging" effect of low level tritium
irradiation may be caused by constant accumulation of trace DNA damage
or by constant stimulation/induction of enzymes involved in the repair ofradiation-induced damages.
Radiation Induced Chromosome Aberrations in the
Radiation Sensitive Mutants of
Caenorhabditis elegans
Yoshito SADAIE and Tamiko SADAIE

The fraction of aberrant metaphase chromosomes in gamma irradiatedearly embryos of C. elegans decreased substantially upon incubation of thegravid worms following exposure. The decrease was blocked by the rad-2
mutation but not by the rad-l mutation of the same epistasis group which
1)

2)

Medical Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Research Reactor Institute, Kyoto University, Osaka.

RESEARCH ACTIVITIES IN 1985

65

makes worms sensitive to radiation and chemicals. Eggs laid immediately
after irradiation by the rad-2 mutant showed low hatchability. However,those laid some time after exposure hatched normally. This suggests thatchromosomal aberrations are not fatal in C. elegans which has holocentricchromosomes.
Mechanisms of Bio-Antimutagenesis
Tsuneo KADA and Tadashi INOUE

Agents suppressing cellular mutagenesis have been known for some time
and their modes of action have been analyzed in the field of bacterial genetics. More recently, a number of mutagens have been detected in ourenvironment and their genotoxicities recognized. Therefore, it is necessary
to have knowledge about antimutagens and their modes of action in order
to assess the genotoxic nature of our environment.
The word antimutagen has an old origin and has been adopted for factors
which reduce the rates of spontaneous and induced mutagenesis by different
modes of action. We proposed a distinction among categories of antimutagens. The "desmutagens" relate to chemical or biochemical modification of mutagens outside the cells. On the other hand, factors, interfering with cellular functions producing genetically stable informative genes
from primary damages in DNA, should be distinguished from desmutagens
and named "bio-antimutagens".
Our studies on mechanisms of bio-antimutagenesis were recently reviewed(T. Kada, T. Inoue, T. Ohta and Y. Shirasu: in Antimutagenesis and Anticarcinogenesis: Mechanisms, Ed. D. M. Shankel et aI., Plenum Press, New
York, pp. 181-198, 1986). The followings are the major mechanisms so far
studied in vitro in the bacterial systems.
a) Increase in the fidelity of DNA replication (epigallo-catechin-gallate,EGCg, a green tea factor).
b) Repair promotion of DNA damage (cinnamaldehyde, cobaltous
cWoride, tannic acid, etc.). Involvement of recombination repair involving
recA protein as well as UV excision repair are shown.
c) Inhibition of error-prone (SOS) repair (5-fluoro-uracil and cordycepin(3'-dA)).

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Dietary Desmutagens
Tsuneo KADA

The word "desmutagen" was proposed to indicate those agents which
inactivate mutagens by reacting directly with them (Kada et al., 1981). In
addition, many investigators are aware that factors exist which reduce theyield of mutations by indirect means, such as inhibition in the formationof mutagens from precursors or by inhibition of metabolic activation ofmutagens. These agents should be also classified as "desmutagens".
In earlier studies, we found that mutagens are produced by a reaction
between sorbic acid and nitrite and that the mutagenic activities were irreversibly lost by treatment with extracts obtained from different kinds ofvegetables. Crude extracts prepared from a number of vegetables suppressed, in vitro, the mutagenicities of pyrolysate mutagens formed in proteinous food through heating. Analysis of cabbage desmutagens showedthat a hemo-protein possessing peroxidase activity would enzymaticallyinactivate Trp-P-l or Trp-P-2 (Inoue et al., 1981). Later studies showedthat heat-resistant desmutagenic factors exist in vegetables. Studies on
burdock revealed that high molecular ingredients could adsorb mutagens,resulting in an irreversible loss of mutagenic activity (Morita et al., 1984).
More recently, we found that isolated fibers of many vegetables adsorb
pyrolysate mutagens such as Trp-P-I, Trp-P-2, Glu-P-l, Glu-P-2, IQ, etc.
(Kada et al., 1984). It seems that the bindings are very firm and thatmutagens can be recovered partially only under special conditions such as
grinding with special organic solvents.
When mutagens are working as initiators of cancer, many desmutagens
can be considered "anticarcinogens" whose modes of actions must involve
the above suggested models of desmutagenesis outside and inside the animaland human body. Obviously the in vivo events may be much more complex.
Mutagenic Activities of EMS on Somatic and Recessive
Mutations in the Soybean Test System
Taro Fum and Shigemitsu TANo*

Somatic mutation is an event occurring in differentiated somatic cells as* Faculty of Agriculture, University of Tokyo.

RESEARCH ACTIVITIES IN 198567

a result of mutagenic treatments or spontaneous mutation. Recessive
mutation which can be recognized in the M 2 generation is true genetic damage
since this event is inherited in the following generations by transmission
through germ cells. However, somatic mutation events and the inductionof genetic damage have positive correlations since the events take placerandomly among the cell population in the tissue, and some of the cell(s)involving the gene mutation will succeed to the germ line through differentiation. To study this problem, somatic mutations induced by ethyl methanesulfonate (EMS) in M 1 plants and the segregation of chlorophyll deficientmutants in the M 2 population were investigated with soybean plants (strain
T-2l9). Localization and distribution of the drug in the seed tissue weretraced using 14C-labeled EMS.
Treatment with 0.05 % EMS induced about 16 spots per leaf together
with more than 30 spots in other leaves of the heterozygotes. Most of theleaves at the 0.1 and 0.2 % treatment plots showed abnormal growth, andhad 30 or more spots, which were not scored accurately. Yellow green
spots appearing on the leaves of the dominant homozygotes were also examined. More than 20 spots per leaf were observed at the 0.1 % treatment
plot, and a large number of spots was observed with the highest EMS concentration treatment.
Segregation of the dominant and recessive homozygotes and heterozygotes
from the heterozygotic M 1 plants showed a nearly 1: 2: 1 ratio in all treatment plots. The results indicated no selective killing effect of the mutagen
for the two types of germ cells having Yll or Yll genes, indicating the dominant and recessive genes may respond equally to EMS. Some of theM 2 families had segregated mutants, for instance, at the 0.2 % EMS treatment plot, 10 (6.3 %) of the 146 M 2 plants, derived from 51 dominant homozygotes and 95 heterozygotes, had segregated mutant seedlings. Dose vs.
mutation frequency relationships were not similar for somatic and recessive
mutations, there was an almost linear relationship for the former and aleveling off at a high EMS concentration for the latter, respectively. In the
M 2 generation, chlorophyll mutation frequencies per pg of EMS were
estimated to be 0.14,0.61 and 0.41% at 0.05, 0.1 and 0.2% treatments,
respectively. The present results indicate that the scoring of somatic mutations can aid in the assessment of the extent of recessive mutational events.
However, more detailed studies are required to obtain the conversion factor
from somatic mutation to recessive mutation because the events may vary

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

with the kinds of mutagens and test systems.
Origin and Genetic Background of Three Substrains in
Inbred PW Mouse Strain
Kiyosi TUTIICAWA, Yoshimasa HARADA and Kazuo MORIWAKIIn order to develop the experimental basis for a standard protocol of themouse spot test using available strains of mice in Japan, the work group inwhich members from 10 laboratories collaborate, have started to study
with ethylnitrosourea by using the cross between any non-agouti strain and
PWfa substrain. In this report the origin and genetic background of three
PW substrains will be described briefly.
One of the authors (K.T.) received one adult male and two pairs of young
mice in the same litter which derived from the cross involving strains NB,
Table I. Alleles presented in PWja, PWjb and PWjc substrains of mice
Alleles present
Locus
Idh-I
Pep-3
Akp-I
He
a
Car-2
b
Gpd-I
Pgm-I
Ldr-l
Gpi-l
p
c
Hbb
Es-l
Es-2
d
se
Mod-I
Trf
Es-3

Chromosome
PWja

PWjb

PWjc

1

a
b
b

a
b
b

2
2
3
4
4
5
6
7
7
7
7
8
8
9
9
9
9
11

a
b
b
b
b
b
a
p
c· h
s
b
b
d
se
b
b
a

a
b
b
1
a
b
b
b
b
b
a
p

1

1

1

c· h
s
b
b
d
se
b
b
a

1

a
b
b
a
b
b
a
p
c· h
s
b
b
d
se
b
b
a

RESEARCH ACTIVITIES IN 1985

69

LV and AC from Dr. M. C. Green at Jackson Laboratory, Bar Harbor, in
October 1956. The progeny were transferred to National Institute of
Genetics, Mishima, in 1957. After six alleles fixed to aa, bb, cChpjcChp,
d sejd se, sib-mating started with two pairs from different litter in 1961.
Thereafter two substrains 'a' and 'c' inbred under full-sib mating regime
independently each other for 88 and 77 generations. Substrain 'b' separated from 'a' at 66 generation and maintained further twenty generations.
Three substrains of the PW strain were thus formed, designated PWja,
PW/b and PWjc.
As shown in Table 1, three substrains share the smae alleles at twenty
loci tested. Only one allele of Gpd-l (on chromosome 4) differ between
PWjc and the other two substrains. In addition, all substrains show theidentical chromosome C-band patterns consisting of all positively stained
chromosomes. The same H-2 haplotype (H-2 b ) carrying H-2K.33 andH-2D.2 is present in three substrains.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

VII.

POPULATION GENETICS

The Role of Compensatory Neutral Mutations
in Molecular Evolution
Motoo KIMURA

A pair of mutations at different loci (or sites) which are singly deleterious
but restore normal fitness in combination may be called compensatory
neutral mutations. Population dynamics concerning evolutionary substitutions of such mutants were developed by making use of the diffusion
equation method. Based on this theory and, also, by the help of Monte
Carlo simulation experiments, a remarkable phenomenon was disclosedthat the double mutants can easily become fixed in the population by random
drift under continued mutation pressure if the loci are tightly linked, evenwhen the single mutants are definitely deleterious. More specifically, I
consider two loci with alleles A and A' in the first locus, and alleles Band B'
in the second locus, and assign relative fitnesses I, l-s', l-s' and I respectively to the four gene combinations AB, A'B, AB' and A'B', where s' is theselection coefficient against the single mutants (s'>O). Let v be the mutation rate per locus per generation and assume that mutation occurs irreversibly from A to A' at the first locus, and from B to B' at the second locus,
where A and B are wild type genes, and A' and B' are their mutant alleles. In a
diploid population of effective size N. (or a haploid population of 2N. breeding individuals), it was shown that the average time (1') until joint fixationof the double mutant (A'B') starting from the state in which the populationconsists exclusively of the wild type genes (AB) is not excessively long evenfor large 4N.s' values. In fact, assuming 2N.v=1 we have 1'=54N. for
4N.s' =400, and 1'= 128N. for 4N.s' = 1000. These values are not unrealistically long as compared with 1'~ 5N. obtained for 4N.s' =0. The approximate analytical treatment has also been extended to estimate the effect of
low rate crossing over in retarding fixation. The bearing of these findings
on molecular evolution is discussed with special reference to coupled substitutions at interacting amino acid (or nucleotide) sites within a folded protein (or RNA) molecule. It is concluded that compensatory neutral mutants
may play an important role in molecular evolution. For details, see J.

RESEARCH ACTIVITIES IN 1985

71

Genet. 64, 7-19.
Variances and Covariances of Identity Coefficientsof a Multigene Family
Tomoko OHTA

By using a model of gene conversion for treating concerted evolution ofmultigene families, a theory for calculating the variances and covariances ofidentity coefficients has been developed. Six coefficients on triple identityand 15 coefficients on quadruple identity are needed. The variances andcovariances are obtained from the quadruple identity coefficients and divided
into within-population and between-population components. The former
represent the variation of identity coefficients among individuals within apopulation, whereas the latter represent the magnitude of fluctuation of
population averages. Monte Carlo simulations confirmed the theoreticalpredictions at equilibrium. For details, see Proc. Natl. Acad. Sci. USA
82, 829-833.
A Model of Duplicative Transposition and Gene
Conversion for Repetitive DNA Families
Tomoko OHTA

A model of duplicative transposition and gene conversion for the evolution of repetitive DNA families was studied. In this model, transpositionand conversion (both unbiased) are assumed to occur both within and between the genomes in a diploid cell, and any degree of linkage intensity isincorporated. The transition equations for allelic and nonallelic identity
coefficients have been formulated by using the previous results. The
results are widely applicable to many repetitive sequences, from dispersed
families like transposons to tightly linked multigene families. It has beenshown through extensive numerical studies on equilibrium properties that
duplicative transposition and gene conversion have very similar effects on
nonallelic identity coefficients, but that allelism and allelic identity are greatly
influenced by the relative rates of occurrence of the two processes. For
details, see Genetics 110, 513-524.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Reciprocal Altruism and Reciprocal Alliance
between Relatives
Kenichi AOKIThe inclusive fitness approach to two-policy games between relatives isshown to be approximately valid, based on a population genetic model
which assumes quantitative inheritance of the behavioral trait. The theory
is applied to the iterated prisoner's dilemma game, and a condition is derived
for TIT FOR TAT reciprocal altruism to evolve in terms of the requisite
degree of relatedness between the interacting reciprocal altruists. A simulation of reciprocal altruism between full sibs supports the approximate analytical results. An extension of the model to treat reciprocal alliance is thenpresented, in which the allies and the common enemy are equally related.
It is argued that Packer's description of reciprocal alliance among maleanubis baboons is difficult to reconcile with theory. For details see Ohta,
T. and Aoki, K. (ed.) Population Genetics and Molecular Evolution, pp.
429-441, Jpn. Sci. Press, Tokyo/Springer-Verlag, Berlin.
Gene Diversity in Finite Populations
Naoyuki TAKAHATA

DNA sequence comparison among homologous genes sampled at random
from one or two populations allows one to estimate the ultimate amount ofgenetic variation maintained in a population and to construct the genegenealogy within and between populations. Moreover, if one uses theconstant-rate or molecular clock hypothesis, it is possible to infer the divergence pattern and time of populations under study. In this note, various
statistics concerning the above are studied when only two genes sampled at
random from two related populations are involved. In particular, formulasfor estimating the mean and standard error of time, t, of population splitting
are derived asi = - -.l.-log
8fta(i)=[(1-(I - -±-p)
- 2N
3

+)

(2N)2++ p (I-p) j{2.u(l- ~ p)rr2

where N is the effective size of the ancestral populations, n the number

RESEARCH ACTIVITIES IN 1985

73

of nucleotide sites compared, p, the mutation rate per site and p the proportion of nucleotide differences per site. It is seen that the estimateddivergence time of homologous sequences always exceeds that of populations by 2N and that there is an intrinsic error in i even when DNA sequencesof infinite length are compared. These results have an important bearing
particularly on inference of divergence times of closely related species fromDNA sequence comparisons. For details, see Genet. Res. Camb. 46, 107113.
Gene Genealogy and Variance of Interpopulational
Nucleotide Dilferences
Naoyuki TAKAHATA and Masatoshi NEI

A mathematical theory is developed for computing the probability that m
genes sampled from one population (species) and n genes sampled fromanother are derived from I genes that existed at the time of population
splitting. The expected time of divergence between the two most closely
related genes sampled from two different populations and the time ofdivergence (coalescence) of all genes sampled are studied by using this theory.
It is shown that the time of divergence between the two most closely related
genes can be used as an approximate estimate of the time of population
splitting (T) only when T==.tJ(2N) is small, where t and N are the numberof generations and the effective population size, respectively. The varianceof Nei and Li's estimate (d) of the number of net nucleotide differences
between two populations is also studied. It is shown that the standard error(Sd) of d is larger than the mean when T is small (T ~ 1). In this case, Sd is
reduced considerably by increasing sample size. When T is large (T> 1),
however, a large proportion of the variance of d is caused by stochastic
factors, and an increase in the sample size does not help to reduce Sd' To
reduce the stochastic variance of d, one must use data from many independent unlinked gene loci. For details, see Genetics 110, 325-344.
The Average Frequency of Private Alleles
in a Partially Isolated Population
Montgomery SLATKIN and Naoyuki TAKAHATA

An analytic model is developed to explore the relationship between gene

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

flow, selection, and genetic drift. We assume that a single copy of a mutant
allele appears in a finite, partially isolated population and allow for theeffects of immigration, genic selection, and mutation on the frequency ofthe mutant. Our concern is with the distribution of the mutant's frequency
before it either is lost from the population or emigrates. Before either ofthese events, the allele will be a "private allele" and would be found in only
one of several populations in a larger collection. Slatkin [(1985) Evolution
39, 53-65] found several simple properties of private alleles in his simulations. We use the method developed by Karlin and Tavare [(1980) Genet.
Res. 37, 33-46; (l981a), Theor. Pop. BioI. 19, 187-214; (l981b) Theor. Pop.
BioI. 19, 215-229] for a model similar to ours to obtain a diffusion equation
with a "killing term", and obtain the mean and variance of the mutant's
frequency and its expected frequency in samples of a specified size. Thereis only fair agreement between the analytic results from this model andthose from Slatkin's (loc. cit.) simulations. The difference indicates thatthe distribution of private alleles in the simulation results is not due toalleles in the demes in which they first appeared, but due to private alleles
after they have spead to other demes. Also, the rescaling method used to obtain the results indicates that if emigration is relatively frequent, the distribution of rare alleles is governed largely by the balance between genetic drift
and emigration, with selection, mutation, and immigration playing a lesser
role. For details, see Theor. Pop. BioI. 28, 314-331.
Extranuclear Differentiation and Gene Flow in
the Finite Island Model
Naoyuki TAKAHATA and Stephen R. PALUMBI

Use of sequence information from extranuclear genomes to examine
deme structure in natural populations has been hampered by a lack ofclear linkage between sequence relatedness and rates of mutation and migration among demes. Here, we approach this problem in two complementary
ways. First, we develop a model of extranuclear genomes in a population
divided into a finite number of demes. Sex-dependent migration, neutral
mutation, unequal genetic contribution of separate sexes and random
genetic drift in each deme are incorporated for generality. From this model,
we derive the relationships between gene identity probabilities (between andwithin demes) and migration rate, mutation rate and effective deme size.

RESEARCH ACTIVITIES IN 1985

75

Secondly, we show how within- and between-deme identity probabilities
may be calculated from restriction maps of mitochondrial (mt) DNA.
These results, when coupled with our results on gene flow and genetic differentiation, allow one to estimate the relative interdeme gene flow whendeme sizes are constant and genetic variants are selectively neutral. We
illustrate the use of our results by reanalyzing published data on mtDNAin mouse populations from around the world and show that their geographic differentiation is consistent with an island model of deme structure.
For details, see Genetics 109, 441-457.
Introgression of Extranuclear Genomes in Finite Populations:Nuc1eo-Cytoplasmic Incompatibility
Naoyuki TAKAHATA

A 'two locus two allele' model is developed with special reference to theintrogression of extranuclear genomes between two species of finite size.
The model assumes that one locus, coded by a nuclear genome, causes the
reproductive barrier while the other locus, coded by an extranuclear genome,
causes nucleo-cytoplasmic incompatibility in particular genotypes. To
fully study this model, simulations are conducted, and a diffusion equation
is derived when introgression or extranuclear gene flow occurs in one direction. It is shown that although selection against the nuclear genome canreduce the levels of extranuclear gene flow and retard the introgression
process, the dynamics are very similar to those without such selection. In
contrast, the nucleo-cytoplasmic incompatibility directly affects the dynamics
of introgressing extranuclear genomes: in large populations the ability ofincompatibility to overcome extranuclear gene flow is conspicuous, but insmall populations it is overshadowed by random sampling drift. Paternal
leakage of extranuclear genomes, if present, is of evolutionary importanceonly when the male's migration rate is much larger than the female's. Whenthe sizes of two populations are unequal, the introgression is most likely tooccur from the larger population to the smaller one in the absence of mating
preferences of backcross progeny. Recent observations on interspecificmitochondrial transfer in various species do not support the ubiquitousness of nucleo-cytoplasmic incompatibility as an efficient reproductive
barrier. For details, see Genet. Res. Camb. 45, 179-194.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Genetic Homozygosity in Population which
Experience Periodic Bottlenecks
Takeo MARUYAMA and Paul A. FUERST

The amount of variability in a population that experiences repeated restrictions in population size has been calculated. The restrictions in size
occur cyclically with a fixed cycle length. Analytical formulas for describing
the gene identity at any specific time in the expanded and restricted phasesof the cycle, and for the average and second moment of the gene identity,
have been derived. It is shown that the level of genetic diversity depends
critically on the two parameters that account for the population size, mutation rate and the time of duration for each of the two phases in the cycle.
If one or both of these composite parameters are small, the gene diversity
will be much reduced, and population gene diversity will then be predictablefrom knowledge of the harmonic mean population size over the entire cycle.
If these parameters take on intermediate values, diversity changes constantly
during the cycle, fluctuating steadily from a high to a low value and backagain. If these parameters are large, gene diversity will fluctuate rapidly
between extreme values and will stay at the extremes for long periods of time.
For details, see Genetics 111: 691-703 (1985).
Number of Alleles in a Small Population thatwas Formed by a Recent Bottleneck
Takeo MARUYAMA and Paul A. FUERST

A model is presented in which a large population in mutation/drift equilibrium undergoes a severe restriction in size and subsequently remains at
the small size. The rate of loss of genetic variability has been studied.
Allelic loss occurs more rapidly than loss of genic heterozygosity. Rarealleles are lost especially rapidly. The result is a transient deficiency in the
total number of alleles observed in samples taken from the reduced population when compared with the number expected in a sample from a steadystate population having the same observed heterozygosity. Alternatively,
the population can be considered to possess excess gene diversity if thenumber of alleles is used as the statistical estimator of mutation rate. The
deficit in allele number arises principally from a lack of those alleles that are

RESEARCH ACTIVITIES IN 1985

77

expected to appear only once or twice in the sample. The magnitude of
the allelic deficiency is less, however, than the excess that an earlier study
predicted to follow a rapid population expansion. This suggests that
populations that have undergone a single bottleneck event, followed by rapid
population growth should have an apparent excess number of alleles, given
the observed level of genetic heterozygosity and provided that the bottleneck
has not occurred very recently. Conversely, such populations will be deficient for observed heterozygosity if allele number is used as the sufficient
statistic for the estimation of 4Nv. Populations that have undergone very
recent restrictions in size should show the opposite tendencies. For details,
see Genetics 111: 675-689 (1985).
Dynamics of Homozygous Genetic Loads in Connection
with the Invasion of Transposons
Terumi MUKAIFor the 1970 and 1984 Raleigh, N. C. population of Drosophila melanogaster, homozygous lethal loads (L) and detrimental loads (D) in the second
chromosomes were estimated. The results were: D=0.334 and 0.237;
L=0.501 and 0.275. The former figures are the estimates of 1970 (fromMUKAI and YAMAGUCHI 1974, Genetics 76: 339-366) and the latter are of
1984 (from Baba, H. et aI., unpublished). It is obvious that the amounts ofhomozygous loads became about one half after 14 years. Without knowing
the true nature of the P elements, we have reported the frequency of the P
element-carrying (mutator-carrying) second chromosomes to be 0.66±0.059
for the 1970 population (cf. YAMAGUCHI, CARDELLINO and MUKAI 1976,
Genetics 83: 409-422). Thus, the above decrease in homozygous loads
can be explained as due to the increase in the frequency of the P elementcarrying individuals to the level of 100 %, where increased mutation rate
as one of the syndrome of the P-M hybrid dysgenesis returned to the levelof the original mutation-selection balance. Under the above condition,
the formulae reported last year [Ann. Report 35: 66-67] were employed forthe lethal load and the time when the P elements immigrated into the Raleighpopulation was estimated to be about 40 (assuming a=0.25) and about 50
generations (assuming a=0.20) before the summer of 1970, where a is thefraction of individuals of cytotype P in the offspring of M (female) X P(male) crosses. This may suggest that the rapid invasion of the P element-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

carrying individuals into the Raleigh, N. C. population had occurred. In
this calculation, the frequency of individuals carrying the P elements (Po)
was assumed to be 0.0001, since the effective size of the population was
estimated to be ca. 20,000. The results of the above calculation accord
well with the change in the frequency of lethal-carrying second chromosomes
after the invasion of the P elements.
The present experimental results together with those of MUKAI et al.
(1985, PNAS USA 82: 7671-7675) suggest the following process after theinvasion of the P-M type transposons into the population consisting of the
M type individuals: When the transposons enter the population, they induce
viability mutations. As the results, homozygous loads increase. When
all the individuals become the P type, the mutation rate returned to theoriginal rate. Accordingly, the homozygous loads gradually return to thestandard level.

RESEARCH ACTIVITIES IN 1985

VIII.

79

EVOLUTIONARY GENETICS

Homology between P-Transposable Element of Drosophilamelanogaster and Bacterial Transposase Gene of Tn3
Yoko SATTA, Takashi GOJOBORI, Takeo MARUYAMA
Kaoru SAIGO and Sadao I. CHIGUSAWith the aid of a computer, each of the four open reading frames (ORFs)
in the P-transposable element of Drosophila melanogaster was aligned withthe amino acid sequence of the transposase (TnpA) of bacterial transposon
Tn3. The four ORFs, named ORFO, -I, -2, and -3 lined in this orderfrom 5' to 3' direction in the P element, turned out to have on the average
18 percent homologies with the Tn3 transposase in the same order without
much overlap between adjacent ORFs. Based on the comparison withconsensus sequences at exon-intron junctions, three possible introns werepredicted in the P element. Each of these putative introns covers the region
between adjacent ORFs and these splicings do not alter the initially predicted reading frames. It appears, therefore, that a mature mRNA processed from the transcript of the whole P element codes a single polypetide
having homology to TnpA. This suggests that the P element and the TnpA
gene may have diverged from a common ancestor. Taking account of ourprevious finding that the resolvase of Tn3 has a sequence homology with
ORFI of the P element, evolutionary relationships among the P element,
the resolvase gene and the TnpA gene were discussed. For details, see
Jpn. J. Genet. 60: 499-503 (1985).
Tn3 Resolvase-Iike Sequence in P Transposable Elementof Drosophila melanogaster
Yoko SATTA, Takashi GOJOBORI, Takeo MARUYAMA
and Sadao I. CHIGUSA

Using each of the four open reading frames in the P transposable elementof Drosophila melanogaster, computer search of homology was carried out
against the protein sequence data base of NBRF version 4. A deduced
polypeptide of about 210 residues in ORFI of the P element appears to have

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

a significant homology with the resolvases of bacterial transposable elements
Tn3 and rD. The polypeptide seems to possess a region consisting of 22
amino acid residues having homology with a DNA-binding fold of theresolvases of the bacterial transposons Tn3 and ro, which are known to havea repressor function of the transposable elements. There are a nine basepair region in ORF3 and also in the 31 base pair inverted terminal repeatof the P element having homology with a site in the promoter region of the
Tn3 and also of ro at which the resolvases bind and suppress transcriptionof transposases. Based on these findings, it was suggested that the ORFI
might code a resolvase protein which mediates site-specific recombination
and also suppresses the transposition of the P by binding at a specific site
in the ORF3 or in the terminal repeat. It was also suggested that the whole
P might code a transposase. For details, see Jpn. J. Genet. 60: 261-266(1985).
Evolutionary Features of Oncogenes
Takashi GOJOBORI

Although the rate of nucleotide substitution for the cellular oncogene iscomparable to that for many functional genes in DNA genomes, the rate
for the viral oncogene in the RNA genome is roughly a million times greater.
Nevertheless, the viral oncogene still retains the general feature of functional
genes that the rate of synonymous substitution is much higher than that ofnonsynonymous substitution. This suggests that nucleotide substitution
in the RNA genome is consistent with Kimura's neutral theory of molecular
evolution. Moreover, the phylogenetic tree of cellular oncogenes showsthat the src family of oncogenes including the c-mos gene may share acommon ancestral sequence with the yeast cell division control gene, suggesting that the function of oncogenes in the src family is related to thecontrol of cell division. For details, see Population Genetics and Molecular
Evolution (Eds. Ohta, T. and Aoki, K.), pp. 353-368.
Rates of Evolution of the Retroviral Oncogene of Moloney
Murine Sarcoma Virus and its Cellular Homologues
Takashi GOJOBORI and Shozo YOKOYAMA

A method is proposed for computing the rates of nucleotide substitution

RESEARCH ACTIVITIES IN 198581

for an oncogene of a retrovirus (v-one), its cellular homologue (c-one), andthe retrovirus genome simultaneously. The method has been applied to
DNA sequences of the v-mos gene of Moloney murine sarcoma virus (MoMuSY) and the c-mos and gag genes of Mo-MuSY and Moloney murine
leukemia virus (Mo-MuLY). The rates of nucleotide substitution for cmos, the gag gene, and v-mos are estimated to be 1.71 X 10- 9 , 6.3 X 10-4,
and 1.31 X 10- 3 per site per year, respectively. The rate of evolution ofc-mos is comparable to that of many functional genes in DNA genomes,suggesting some important biological function played by cellular oncogenes.
The rates of nucleotide substitution in the v-mos and gag genes are very
high and similar to those of RNA viral genes such as the hemaggulutinin
and neuraminidase genes in the influenza A virus. Thus, oncogenes seemto exemplify a general feature of genome evolution: the rate of evolution of
RNA genomes can be more than a million times greater than that of DNA
genomes because of a high mutation rate in the RNA genome. For details,
see Proc. Natl. Acad Sci. USA 82: 4198-4201.
Complete Nucleotide Sequence of an Infectious Cloneof Human T-Cell Leukemia Virus Type II:
An Open Reading Frame for the Protease Gene
Kunitada SHIMOTOHNO, Yuri TAKAHASHI, Nobuaki SHIMIZU
Takashi GOJOBORI, David W. GOLDE, Irvin S. Y. CHEN
Masanao MIWA and Takashi SUGIMURA

The entire nucleotide sequence of an infectious clone of human T-cellleukemia virus type II provirus was determined. This provirus consistsof 8,952 nucleotides. In addition to long terminal repeats and gag, pol,env, and X, a protease gene that is responsible for processing the gag precursor protein was found. The protease gene is encoded in a different
frame from gag and pol and was located between the gag and pol open reading
frames. Coding regions of the provirus show about 60 % homology withthose of human T-cell leukemia virus type I at the nuleotide level. Thisfinding suggests that human T-cell leukemia virus types I and II have diverged from a common ancestral genome. For details, see Proc. Natl.
Acad. Sci. USA 82: 3101-3105.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

DNA Polymorphism of AIDS Virus
Takashi GOJOBORI, Shozo YOKOYAMA and Takeo MARUYAMA

To quantify the degree of DNA polymorphism of human retrovirus isolated from patients with the acquired immune deficiency syndrome (AIDS),
population genetics theory was applied to restriction endonuclease mapsof four closely related clones of HTLV-III. In about 9 kb of the region
where the four clones of AHXB2, AHXB3, ABH5/8, and ABHlO share (Shawet aI., Science 226: 1165-1171, 1984), we computed the mean number ofrestriction-site differences (lJ=2.66), the number of segregating restriction
sites (k=5), and the nucleotide diversity (7!'=0.012). In particular, ourestimate of 7!' was confirmed by the nucleotide sequence comparisons among
the AIDS virus genomes (Ratner et aI., Nature 313: 636-637, 1985). Thesefindings provide two different views concerning evolution of the AIDS
virus. First, if the virus population is at a steady state, the estimated threequantities (lJ, k, and 7!') consistently imply that 2Nu is about 0.01, where N
is the effective population size and u is the mutation rate per nucleotide.
Hence, if mutation rate is of the order of 10- 3 , as reported, the effective
population size must be extremely small, possibly of the order of 100 or less.
Secondly, if the population is in a rapidly expanding state, these values oflJ, k, and 7!' reflect the history of the virus, and imply a very recent origin.
The probable divergence time appears to be only several years. A quick
report on these results has been published in Genetics 100; s91.

Homology of Kringle Structures in Urokinase andTissue-Type Plasminogen Activator:
The Phylogeny with the Related Serine Proteases
Kei TAKAHASHI, Takashi GOJOBORI and Hiroyuki NAORA

Twelve amino acid sequences of kringle-forming polypeptides were compiled from the known sequences of urokinase A-chain (human), a tissuetype plasminogen activator (human), prothrombin (human and bovine),and plasminogen (human). Their sequence homologies were examinedusing a computer program. Analyses of the homology alignment and dot
matrix showed that they had a great degree of homology. In particular,
all the cystein residues responsible for the kringle structures of urokinase

RESEARCH ACTIVITIES IN 198583

and the tissue-type plasminogen activator were completely preserved. A
phylogenetic tree constructed suggests that the kringe-related serine proteases have diverged from a common ancestral element around at 500
million years ago. Thus, the kringle-families seem to have a long evolutionary history. For details, see Cell Struc. Func. 10: 209-218.
Chromosome Regions Affecting Hybrid Sterilities ofDrosophila simulansjD. mauritiana andD. simulansjD. sechellia Males
Kiyoshi KIMURA, Won Ho LEE, Takao K. WATANABE
and Machiko HATSUMI

Takamura and Watanabe (1979, Ann. Rep. 30, 91) reported preliminary
results on the hybrid male sterility of D. simulansjD. mauritiana in which
it was noted that the sterility is markedly affected by genes on the X andthird chromosomes. Recently, Coyne (1984, PNAS 81,4444) pointed outthat such sterility may be attributable to interactions between X and Y
chromosomes. Both experiments, however, failed to deduce more information about the number and chromosomal location of loci which are responsible for hybrid male sterility, because only one marker gene was used
for the X chromosome.
In this study we focused only on the X chromosome which is thought tohave a chief effect on hybrid male sterility, and used two marker strains ofD. simulans, yv2f and m 65j'66 to study this phenomenon in more detail. The
precise map positions of these loci in D. simulans are not yet known so that
we assumed in the following data analysis that they were the same as those of
D. melanogaster, namely, yeO), v2(33), m 66 (36.1),f and f66( 56.7) and centromere(70). The above two strains were crossed with D. mauritiana males. The
Fl females were backcrossed to D. simulans males of the same strain. Eight(four) types of recombinant males for the yv2f(m65j'66) strain were examined
to determine whether or not seminal vesicles contained motile sperms. If a
recombinant male contained motile sperms, it was judged as fertile.
The frequency distribution of the fertile recombinants thus obtained was
examined by a chi-square test. The null hypothesis in this test was constructed on the assumptions that (1) there is a single locus on the X chromosome, (2) crossing over occurs twice at most, and (3) the centromere mayor
may not affect a hybrid fertility change of the map position of such a locus

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

and inclusion or exclusion of the centromere alters the chi-square value.
Through the simulation study which can identify the minimum chi-squarevalue, we found that the most probable location of such a locus is around40 of the X chromosome and that the proximity of the centromere also
plays an important role in hybrid fertility. Thus, it appears that hybrid
males become fertile as long as the two regions on the X chromosome, 40
and 70, are the same as those of the paternal species.
The same experiment was carried out using D. simulans and D. sechellia.
The strain we used first was yf; bw; st of D. simulans. The effect of the X
chromosome was larger than that in D. simulansjD. mauritiana, and theautosomal effect was almost negligible. Next we used yv2f and m 65j66 of D.
simulans for the precise regions of hybrid sterility on the X chromosome.
Again, the regions, 40 and 70, were detected as those involved in hybrid
male sterility of D. simulansjD. sechellia.
These two experiments indicate that hybrid males carrying the simulans
Y become fertile if they have the authentic simulans 40 and 70 regions evenwhen the rest of the X chromosome is descended from either D. mauritiana
or D. sechellia. However, when we studied the hybrid sterility of D.
simuransjD. sechel/ia males carrying the Y chromosome of D. sechel/ia, we
identified different regions. These regions are located around 50 and 70.
In other words, a different inner region of the X chromosome of D. sechellia
can recover fertility of hybrid males in this cross. Thus, interactions between X and Y chromosomes, or, more presicely the regions of the X
chromosome, which result in hybrid male sterility may be specifically determined by the Y chromosome.
Notes on Two Interspecific Hybrids of the Drosophilamelanogaster Subgroup: simulansjteissieriand teissierijmauritiana
Won Ho LEE and Takao K. WATANABE

A total of eight species are included in the D. melanogaster subgroupwhich can be divided into two complexes. With D. teissieri (yakuba
complex) it is usually difficult to obtain hybrids with D. simulans or D.
mauritiana (both melanogaster complex). Matings between D. simulans
females and D. teissieri males were successful only by 7 % of the cross while
reciprocal matings were unsuccessful. On the other hand, matings between

RESEARCH ACTIVITIES IN 198585

D. teissieri females and D. mauritiana males were 22.1 % successful but nosuccessful mating occurred in the reciprocal cross. Among the successful
crosses, we observed some sex-specific hybrid lethalities. Hybrids, simulansf
teissieri, were all females with reduced ovaries though the hybrid females
mated easily with D. teissieri males. Hybrids, teissierifmauritiana, showed
continuous variation in sex-ratio, from equal numbers of males and females
to all females lacking males. The variations came from using D. mauritiana
as the paternal strain. The average proportion of Fl males of 14 strains
was 12.2 %. Two typical D. mauritiana strains, one producing 50 % males
and the other producing no males, were selected and crossed reciprocallyto obtain heterozygotes. Then the heterozygous males were crossed with
D. teissieri females. The percentages of hybrid males were 25 % and 22 %which was not significantly different and they were close to the mid-parents
value (25 %). This result suggests that the viability of the hybrid males
between D. teissieri and D. mauritiana is controlled by some autosomal
genes of D. mauritiana.
Cytogenetic and Biochemical Characteristics of Wild Mice
Collected from the Kunming Area, China
Kazuo MORIWAKI, Hirotami IMAI, Yasuyuki KURIHARA
Hiromichi YONEKAWA, Chang-geng QIN*
and Li-ming SHI*In August 1985, we (K. M. and H. 1.) visited Kunming, China and collected a number of mice at two localities in that area with the help of theKunming Institute of Zoology. Taxonomic identification of these mice
was kindly achieved by Dr. Wang Ying-Xiang of the Institute, where theair-dried chromosome preparations and proteinase-K digested particulate
fractions of liver cells were made from the collected specimens. Later, thechromosome preparations and proteinase digested fractions were brought
to our laboratory for analysis of chromosome C-band patterns and RFLP
both in the mitochondrial DNA and the ribosomal DNA. Table 1 summarizes the results of these analyses.
Chromosome C band patterns of all mice indicated that they belong toneither Mus musculus musculus nor M. m. molossinus. This was also
demonstrated by the RFLP both in 28s rDNA-NTR and mtDNA. The* Kunming Institute of Zoology, Academia Sinica, China.

00

0'1

Table 1. Taxonomic, cytogenetic and biochemical characteristics of wild mice cellected from the Kunming area
Sample
No.
85016
85017
85018
85019
85020

Place ofcollection

Name ofspecies·Sex

Body
weightF

Wang Jiaqiao

Mus domesticus
homourus

MM

14.5 g
12.0
5.0

70mm
67
50

(n.t.)
70mm
75
1-19+/+, X+, y58
1-2+/+, 3±/+, 4-19+/+,X+,Y-Mus castaneus
tytleri

F
F

11.8
7.6

70
60

72

Xiao Ximen

Body
lengthTaillengthChromosome C-band

1-19+1+, X+/+
1±/±, 2-16+1+, 17±/±,
18-19+1+, X+I+

70

mtDNA restriction patterns

28S rDNA NTR RFLP

Sample
No.

Bm

Ec

H2

H3

Hpl

He2

Ps

Bg

EcoRI

BamHI

85016
85017
85018

C
C
C

B
B
B

C
C
C

B
B
B

C
C
C

C
C
C

B
B
B

A
A
A

7.0,9.0kb
7.0,9.0
7.0,9.0

6.4kb
6.4
6.4

85019
85020

C
C

C
C

B
B

B
B

B
B

B
B

B
B

B
B

7.0,9.0
7.0,9.0

6.4
6.4

•

Identified by Dr. Wang.

~

c:::

~
~

~

~
o>;j

~

~
~

~
~

Cl

~
n

tn

~

w

0'1

RESEARCH ACTIVITIES IN 1985

87

former could not discriminate between castaneus and bactrianus, but thelatter demonstrated that those from Wang Jiaqiao village were of the
castaneus type and those from Xiao Ximen village the bactrianus type,though both villages are located in the Kunming area. As yet, we do nothave any diagnostic markers which can clearly discriminate the nuclear
genomes of castaneus and bactrianus. It is, however, of great interest thattwo groups of wild mice having mtDNAs being genetically remote fromeach other inhabit a limited area such as Kunming city.
Chromosome C-Banding Patterns of Korean Wild Mice
Kazuo MORIWAKI, Kyoko SUZUKI, Won Ho LEE·
and Wang Su CHO·*

In the fall of 1984, a number of wild mice were trapped in the Suweon
area of S. Korea. The place of collection and body size of specimen collected are shown in Table 1. Chromosome C-bands of bone marrowcells were analysed by quinacrine Hoechst staining. C-banding patterns of
all individuals are summarized in Table 1. All mice examined exhibitedseveral chromosome pairs with quite faint C-bands as well as some pairs
with densely stained C-bands. This feature indicates that those mice belong to Mus musculus molossinus or Mus musculus musculus. Biochemical
analysis of RFLP in the mitochondrial DNA demonstrated the same line ofevidence as well (Yonekawa et ai., Ann. Rep. Nat. Inst. Genet. No. 35,
1985). Even within the musculus-molossinus group, minor RFLP of mtDNA
has been observed, among which the restriction patterns of Japanese andKorean wild mice are the most similar. The C-banding patterns of thetwo types of mice are similar as well. For instance, a dense C-band on thesubcentromeric region of No. 18 chromosome can be frequently observedin both Japanese and Korean populations. It is probable that musculuslike wild mice imigrated Japan through the Korean peninsula, and not
directly from northern China.

* Pusan National University, Pusan, Korea.
*. Institute of Agricultural Science, Suweon, Korea.

00
00

Table l.
SampleNo.

Place ofcollectionSex

1

KojuriAC-I
AC-II
lAS-III

F
F
F

2
3
4

Sample
No.
1

2
3
4

I

Taxonomical and cytogenetical characters of Korean wild mice trapped in Suweon area
Body
weightBody
lengthTaillength

11.4g
14.3
15.6
12.1

79.0mm
86.0
82.0
84.0

68.0mm
66.0
69.0
56.0

M

~

"t:I

o

C-band pattern

2

1

3

4

5

6

7

8

+/+ +/+ -/- -/- -/- +/+ -/- -/+/+ +/+ -/- -/- -/- +/+ -/- +/++/+ +/+ -/- -/- -/- +/+ -/- +/+/+ -/- -/- -/- -/- +/+ -/- +/+

10

11

±/±
+/±
±/±
+/+

+/+
+/+
+/+
+/+

-/-/+
-/-/-

AC: Agricultural college, Suweon.

12

13

14

15

16

17

-/-/-/-/--/-/-/-/--/-/-/-/-±/± +/+±/++/+±/± ++/+±/± +/+

-/-

-/-

-/-

-/-

-/-/-

-/-/-

~

o

~

Z
~
Vl

C-band pattern
9~~lAS: Institute of Agricultural Science.

18

19

X

+/+/+
±/±
+/-

-/-/-/-

Y

~

o"11
Cl

~

Q

z

9
w

0\

RESEARCH ACTIVITIES IN 1985

89

The Relationship between an Isozyme Locus Pox-land Life-History Traits in Wild Rice
Hiroko MORISHIMA and Reiko SANO

The evolution of life-history in plants has provoked the renewed interestof population biologists during the last decade. However, the genetic
control of life-history traits and the coherence mechanism of the relevent
genes has remained almost unexplored. The present study was designed
to investigate this problem.
In the Asian common wild rice, a perennial-annual continuum is foundwithin the species. The perennial type is characterized by low seed productivity, a high outcrossing rate, late flowering, tall stature, and othercharacteristics as compared with the annual type which propagates mainly
by seeds. In the past studies, we found a strong association between these
correlated traits and allelic variation at a polymorphic isozyme locus Pox-I,
in which three alleles OC, 2A and 4A have been identified. Results ofpast studies have shown that; 1) Populations of perennials are polymorphic,
while those of annuals are exclusively fixed for 2A (rarely oq. 2) In theexperimental populations segregating at Pox-l locus, the 2A allele tended toincrease when propagated by seeds, while the 4A increased when propagatedvegetatively. 3) The frequency of 2A increased in a subpopulation selected
for early flowering.
In the present study, we studied hybrid progeny obtained from a crossbetween an annual and a perennial strain of Asian wild rice. In F s lines(F 2 derived), the relationships among various life-history traits and allelicvariation at the 5 isozyme loci were examined. Most of the character correlations which are consistently found among natural populations bringing
about perennial-annual differentiation disappeared among F s lines, indicatingthat they are the product of natural selection. But, high efficiency in seedproduction, short anther (favoring selfing) and early flowering tended to
be associated among F s lines. This suggests that linkage or developmental
correlation could have partly played a role in perennial-annual differentiation. Further, 2A/2A plants showed a higher efficiency in seed production,shorter anther and earlier flowering than 4A/4A plants. This suggests theexistence of major gene or genes for those traits.
In order to search for the neighboring region of Pox-l locus, 7 pairs of
isogenic lines each of which carried 2A and 4A on the same genetic back-

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

ground were developed from the same cross by repeating selfing of 2AJ4A
heterozygotes. Comparison between isogenic lines in Fe indicated thatgenes underlying flowering time, anther length, panicle number and plantheight seemed to be located near this locus, although these linkages werebroken in more than half of the Fe lines. This chromosomal segment
marked by Pox-l may be said to be "an adaptive gene block." Why thisassociation persisted so widely and strongly in nature remains unsolved.
Computer Studies on the Molecular Evolution
Takashi MIYATA

The transfer of genetic information from RNA into DNA was thought
to be a unique feature of retroviruses, but there is growing evidence that it
may be a more general strategy. Both DNA viruses, hepatitis B virus(HBV) and cauliflower mosaic virus (CaMV) as well as eukaryotic transposons, Drosophila copia and yeast Tyl elements were found to have RNAintermediates that are reverse transcribed into DNA (Summers, J. and Mason,
W. S. 1982, Cell, 29: 403; Pfeiffer, P. and Hohn, T. 1983, Cell, 33: 781;
Shiba, T. and Saigo, K. 1983, Nature, 302: 119; Boeke, J. D. et al., 1985,
Cell, 40: 491). Furthermore, these DNA viruses and a Drosophila copialike element 17.6 have polymerases which share striking sequence homologies
with retroviral pol gene products for a region (RT domain) that is thought
to carry reverse transcriptase activity (Toh, H., Hayashida, H. and Miyata,T. 1983, Nature, 305: 827; Saigo, K. 1984, Nature, 312: 659).
Another line of evidence for the possibility of nonretroviral reverse
transcriptase came from a recent report by Michel and Lang (Michel, F.
and Lang, F. Nature, 316: 641). Following the suggestion for the possibility of a Neurospora mitochondrial plasmid being related to transposon,
they compared amino acid sequences predicted from the ORFs (open reading
frames) of fungal mitochondrial class II introns together with a mitochondrial plasmid with those of polymerases of retroviruses, CaMV, HBV
and a Drosophila transposon 17.6 and found remarkable homologies within
the RT domain. This finding added further evidence for the view that the
RT-like sequence can now be recognized as a ubiquitous one distributed
over many genetic elements of such a wide evolutionary distance. In addition, such a widespread occurrence of similar sequences suggests that these
homologies were derived by divergence, but not by convergence.

RESEARCH ACTIVITIES IN 1985

91

Apart from the RT-like sequence, the ORF product of copia-like element
17.6 contains another sequence related to the DNA endonuclease (EN)of retroviral pol gene products (Toh, H. et al., 1985, EMBO J., 4: 1267),
thought to be important in integration of viral DNA into host DNA. Thisstrongly suggests that the transposition mechanism of the copia-like element
is retrovirus-like. Interestingly CaMV polymerase exhibits a striking
homology with ORF2 product of the 17.6 over its entire region, but is completely lacking the EN-like sequence (Toh, H. et al., 1985, EMBO J., 4:1267). Also no EN-like sequence presents in HBV (Toh, H. et al., 1985,a)mt intron a1
rot intron a2
RSV

N[l]KIiJE G 5 T E
KLRNTGLSEHTLV- INPTSSGLVE
RPQSSGfOlVE

M-MuLV
Copia-like 17. 6
Copia

N~Q~QAMVET K T G V A~I E
T~QLMGVSEb)
I - i 100 residues"'RTDomain+ -EN Domain-

HBV
CaMV
Capia - like 17.6
M-MuL VHTLV- I
RSV
Capia
mt intra n a1
mt intra n a2
mt plas mid

w

"""j

J
"

- GXXERXN-~~- YXDD(hydraphabic)3-

Fig. 1. Alignment of amino acid sequences of viral and transposon polymerases.
a), Alignment for a highly conserved segment of the DNA endonuclease (EN)domain. Mt introns al and a2, amino acid sequences predicted from ORFs
in introns al and a2 of yeast mtDNA-coded cytochrome oxidase subunit I
gene. Mt plasmid, the sequence predicted from ORF in Mauriceville mitochondrial plasmid. Most common amino acids (identical or chemicallysimilar amino acids) were boxed. b), Homology map. Positions of highly
conserved stretches of amino acids in the RT (reverse transcriptase) and EN
domains were indicated by .., and \1, respectively. Their consensus sequenceswere also shown (Data from Miyata, T., Toh, H. and Saigo, K. 1985, Proc.
Japan Acad., 61B: 464).

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

EMBO J., 4: 1267). The Drosophila copia is distantly related to retroviruses
and its putative polymerase shares less extensive homology with retroviral
pol gene products (Emori, Y. et a!., 1985, Nature, 315: 773), but has both
highly conserved stretches of amino acids -YXDD- (X, anyone of aminoacids) flanked by three hydrophobic residues and -GXXERXN- which are
diagnostic of the RT and EN domains, respectively (Miyata, T., Toh, H.
and Saigo, K. 1985, Proc. Japan Acad., 61B: 464). Similar searches revealed
the presence of both sequences in ORFs of mitochondrial introns but the
mitochondrial plasmid lacks the latter sequence (Miyata, T., Toh, H. and
Saigo, K. 1985, Proc. Japan Acad., 61B: 464). Interestingly the RT and
EN domains present in reverse order in the copia and mitochondrial introns(Fig. 1).
In conclusion, of all the reverse transcriptase-containing viruses andtransposons examined so far, DNA viruses and plasmids lack an endonuclease-related domain, which is in sharp contrast to retroviruses andtransposons involving the endonuclease and intrachromosomal phase,thought to be important in their life cycle. Although HBV is known tohave an intrachromosomal phase, the mechanism of integration into hostDNA possibly differs from that of retroviruses. Mitochondrial plasmids
could be transposons that lack an intrachromosomal phase. It may be ofinterest to know whether or not the relationship between the presence ofDNA endonuclease and the chromosomal form (RNA or DNA) could be
extended to most reverse transcriptase-containing viruses. Further data
will clarify this point.
Application of Mitochondrial DNA Restriction Length
Polymorphism to Genetic Monitoring of Laboratory Mice
Hiromichi YONEKAWAl), Hideki KATOR2 ), Tomoko SAGAI3 )and Kazuo MORIWAKI3)

Our previous study showed that most strains of laboratory mice share asingle type of mtDNA, which was designated as mtDNA restriction phenotype d. However, there were some exceptions: most of the NZB strain
showed the another phenotype b and strain RR showed m (Yonekawa et al.,
1)

2)
3)

Laboratory of Cytoplasmic Genetics.
The Central Institute for Experimental Animals.
Laboratory of Cytogenetics.

RESEARCH ACTIVITIES IN 1985

93

1982, Differentiation 22, 222).
When we examined mtDNA phenotypes among several substrains of
NZB, two clearly different phenotypes were found among the substrains.
Out of 6 substrains examined, three had b-type mtDNA while the rest had
d-type (Table 1). This suggests that some NZB substrains had undergone
genetic contamination by other strains. This suggestion is supported by
the fact that only one type of mtDNA should have been detected in the
NZB substrains, since all of the substrains used have been maintained byfull sister-brother mating. Since the mice of this strain are used widely asmodels in studies on murine lupus, which is thought to be controlled by a
few genes, it then important to disclose the genetic characteristics of these
NZB substrains.
To do this, the authentic NZB strain maintained in the Walter and Elisa
Research Institute was used for the restriction analysis. Since the authentic
one shows b-type mtDNA, the NZB of d-type must be a genetic contaminant.
We compared nuclear biochemical markers of the authentic NZB and the
3 contaminated substrains had different chromosome distribution patternsof biochemical markers (Table 2). However, as all loci were fixed as homogeneous, the contamination probably occurred a long time ago. Furthermore,
Akp-la and Es-lb were the alleles which have never been found in any of the
NZB substrains with b-type.
Next the question arises whether the contamination in the NZB substrains was caused by an intercross between NZB and other NZ-strains,since the intercross between NZB and NZW were often carried out to enhance in vivo immunoreactions for murine lupus (Shirai, T. 1982, Immunology Today 3, 182). This possibility, however, was eliminated by the analysis of biochemical markers among four strains of NZ-stocks and NZW.
Namely, Akp-ll and Es-lt could not be found in any of the NZ-strains and
NZW.
We then compared biochemical markers between the contaminated NZB
substrains and four strains of black-coated mice, since genetic contamination often occurs between strains with the same coat-color and NZB has a
black coat-color. The experiments showed that the biochemical markers
of C57BL/Ks were very similar to those of contaminated NZB. Fromthese results, we finally concluded that the contamination was probably
caused by an intercross between NZB and some black-coated strains.
Here we show that the analysis of mtDNA RFLP is a useful tool for

\0

.j>.

Table I. Genetic profiles of NZB sublines, other NZ strains and the NZW strain>~

Strains*
Locus(Chr. No.)

NZB/
KIJms

NZB/
Nga

NZB/
Nrs

NZB/
BI

NZB/
MRC

(Contaminated)
Idh-I
Pep-3
ASp-I

Hc
Mup-I
Gpd-I
Pgm-I
Hbb

(I)
(I)

(I)
(2)
(4)
(4)
(5)
(7)

a
c
b
I
b
b
b
s

a
b***

a***
c

q

q

0

0
b

b
0

b
b
d

(8)

q

-a

q

(9)
Mod-I
(14)
Es-IO
H-2K (17)
H-2D- (17)
mtDNA (-)

b
b
d
d
d

b
b
d
d
d

b
b
d
d
d

Es-I

NZO/
BI

NZq
Bl

NZX/
BI

NZW/
Wehi

a
b
b
d
b
b
b
d
d

b

~
~

'"C

(Standard strains)
a
c

a
b
b
d

NZB/
San

0

a
c
b

a
c
b

b
c
b

a
c
b

0

0

1

0

a
b
b
d
b
b
b
d
d
b

a
b
b
d
b
b
b
d
d
b

a
a
a
s
b
a
a
u
ND**
b

b
a
a
d
b
b
b
d
d
d

a
c
b
I
b
b
b
s
b
b
a
d
d
d

b
b
b
I
a
b
b
d
b

a
a
z
z
d

* The details of the strains are given in Materials and Methods.
** ND: not determined.
*** Gothic and italic letters represent the alleles confirmed in ref. 12 and observed to be aberrant in contaminated NZB,
respectively.
Other markers examined are as follows: Car-2", Ldr-2", Lyt-2b, Gpi-I", Es-2 b , Thy-I b, Trf b , Es-3 c and Lyt-I b.

~

0

"r:1

~

>-i
......

0

Z

>
t'"

......

Zt;n>-i......
>-i

C

>-i

m

0

"r:1

0
m
Z

m

>-i
......

()
t;n

Z

9
w

C\

95

RESEARCH ACTIVITIES IN 1985
Table 2.

Comparison of allele distributions in NZB and otherblack-coated strains
Strain

Locus

Pep-3
Akp-l
Hc
Mup-l
Hbb
Es-l
H-2
mtDNA
reference

AU/SsJ

C58/J

C57BL/6J
andC57BL/I0Sn

C57BL/Ks

NZB(Contaminated)

NZB(Standard)b (+)*
a (+)
0(-)
?
p (-)
b (-)

a (-)
a (+)
1 (+)
b (+)
s (+)
b (-)
k (-)
d (+)
12,13

a (-)
a (+)
1 (+)
b (+)
s (+)
a (+)
b (-)
d (+)
12,13

a (-)
a (+)
1 (+)
b (+)
s (+)
a (+)
d (+)
d (+)
12, 13

b/c**
alb
1/0
b/a
sid
a
d
d
8

c
b
0
a
d
b
d
b
8

q (-)

d (+)
12,13

* (+) and (-) indicate respectively that this allele is "the same as" and "different
from" the aberrant allele of contaminated NZB.
** Aberrant allele (Gothic letter)/standard allele.

genetic monitoring of laboratory animals. However, this technique has aweak point in that the animals must be killed for analysis. If live animals
are necessary for use, we have to carry out a partial hepatectomic operation,
but this is quite time-consuming and not safe for the animals. To overcome
this problem, we have developed a very sensitive method of analysis, which
requires only 50,ul of blood to examine one restriction pattern. Fifty,ulof blood can be obtained from the retro-obital venous plexus and the animals,
even mice, will be still alive after this treatment. Thus, this technique enables us to use easily any animal samples for genetic monitoring.
We describe the details of this method below: 50 to 200 ,ul of blood wasremoved from retro-orbital venous plexus with a heparinized glass capillarytubing. Then the blood was spun down and the fraction of cellular components were collected. Five hundred ,ul of 0.5 M EDTA-l % sodium
lauroyl sarcosinate was added to the pellets. Then the sample was treatedwith 100 ,ug/ml (final concentration) proteinase K at 37°C overnight. Thenthe samples were treated with 3 % NaCI-saturated phenol twice (In thesesolution conditions, phenol phase was up on the aqueous phase.), 3 %NaCI-saturated phenol-chloroform mixture once and then chloroform once.
Whole nucleic acids were precipitated with 2 volumes of ethanol and thencollected with centrifugation at 10,000 X g for 20 min. The same procedure

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

was repeated three times in the presence of 2 M ammonium acetate. The
pellet of nucleic acids was washed with 90 % ethanol, dried under vacuum
and finally dissolved into 0.1 mM EDTA (pH 8.0) of an equal volume tooriginal blood. Forty-five,al of the nucleic acids solution was used for
restriction analysis. Digestion of samples with restriction enzymes andagarose gel electrophoresis were carried out as usual (Yonekawa et al., 1982,
Differentiation 22, 222). Southern blotting was performed on Pall Biodyne
membrane (BNNG). The conditions for blotting, prehybridization,
hybridization and membrane washing were those recommended by the supplier. MtDNA used for probe was purified by two or three cycles of CsClethidium bromide density gradient centrifugation. Mter linearization witha suitable enzyme, the mtDNA was labeled with 3ZP-a-dCTP by oligonucleotide random primer extension method (Feinberg and Vogelstein, 1984
Anal. Biochem. 137, 266).

RESEARCH ACTIVITIES IN 1985

IX.

97

HUMAN GENETICS

Mitochondrial DNA Polymorphism in Japanese
Satoshi HORAI and Ei MATSUNAGA

The mitochondrial DNA (mtDNA) of humans is a closed circular molecule
which has been completely sequenced for one individual (Anderson et al.,1981). The mtDNA of this individual comprises 16569 base pairs. In aprevious study on mtDNA restriction analysis with fifteen enzymes of six
base pair recognition, we suggested the existence of a considerably highdegree of mtDNA diversity within the Japanese population. In this study
an attempt was made to obtain sufficient purified mtDNA from humanplacenta in order to examine mtDNA polymorphism with a large numberof restriction enzymes without using Southern blotting analysis or the endlabeling method. In the present study, we have extended this study toinclude nine enzymes of four or five base pair recognition. The size of the
mtDNA fragments produced by digestion by each enzyme were comparedafter gel electrophoresis. A total of 95 different morphs were observedwith nine enzymes, 60 of which have not been so far reported in the previous
study by Cann (1982), which is the only comparable study to ours with respect to the population size examined and the number of enzymes used.
The high frequency of new polymorphisms is, however, due to the fact that
our survey is the first extensive analysis of mtDNA polymorphism in theJapanese population. Although Cann (1982) also analyzed mtDNA frommany Orientals, only two Japanese were included. Therefore, this finding
may reflect a unique profile of the Japanese population inferred from mtDNA
polymorphisms. This is supported by our previous analysis with enzymesof hexanucleotide recognition, where several new polymorphisms withvarious enzymes were detected. Based on the combination of restriction
enzyme morphs observed for each of 116 individuals, we found 62 distinct
mtDNA restriction types. Thirteen individuals showed the restriction
type derived from the dominant pattern for each digestion of the 9 enzymes(type 21). The next most frequent type was found in 12 individuals (type1). Although the remaining 60 types occurred at low frequencies, it isinformative to measure the index of nucleotide diversity. The average

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

amount of nucleotide change in human mtDNA can be estimated by themethod of cleavage site comparison (Nei and Li 1979). In the present study,
an average of 233 sites (equivalent to 932 base pairs, or 5.6 % of the wholegenome) could be compared. Using pairwise comparison of each restriction type, the average number of nucleotide substitutions per nucleotidesite (a) was estimated to be 0.0026. Phylogenetic analysis of the present
data indicates that at least two distinct lineages exist in the Japanese population. The first cluster consisting of 13 types (21 individuals) first diverged
from the second cluster. The branching point is estimated as early as about
125,000 years ago. For details, see Human Genetics 72: 105-117.
Analysis of Mitochondrial DNA Polymorphisms inJapanese Monkeys
Satoshi HORAI, Ei MATSUNAGA, Kenji HAYASAKA ' )Takayoshi SHOTAKEl) and Ken NozAwAl)

To investigate the genetic relationship between the species of genus
Macaca, mtDNA was purified from the liver of a Japanese monkey. It
was digested with 12 restriction enzymes (Eco RI, Bam HI, Pst I, Hind III,Hinc II, Ava I, Bst ElI, Bgl II, Acc II, Sac I, Xba I, Kpn I) and the restriction patterns were analysed by agarose gel electrophoresis. By double
digestion analysis, a restriction map with 32 sites was obtained. M. Goerge
Jr. (1982) reported mtDNA restriction maps in three species of Macaca
other than Japanese monkeys and four species of other genera of the Old
World monkeys. On the basis of these seven maps and that of the Japanesemonkey, we calculated, according to the method by Nei & Li (1979), theindex of nucleotide diversity by cleavage site comparison and the numberof nucleotide substitution per site (a) between each pair of the 8 species.
A phylogenetic tree was constructed from the values of a's by the unweighted
pair group method. As expected, the tree showed that the four species of
Macaca belonged to the same cluster but it also showed some differences
as compared with that constructed from the electrophoretic analysis of blood
proteins. Analysis with the restriction enzymes which recognize four basepair sequences or sequence analysis is needed in order to clarify their phylogenetic relationships more precisely.
1)

Dept. Variation Res., Primate Res. Inst., Kyoto Univ.

RESEARCH ACTIVITIES IN 1985

99

Characterization of 2,7-anhydro-N-acetylneuraminic
Acid in Human Wet Cerumen
Minoru SUZUKI*, Akemi SUZUKI*, Tamio YAMAKAWA*
and Ei MATSUNAGA

The dimorphism of human normal cerumen, wet (sticky) and dry (flaky),
is determined by a pair of autosomal alleles, the wet type being inherited as
simple dominant over the dry type. Although there are several reports on
the chemical nature of human cerumen, the biochemical basis to distinguishthe two types of cerumen is entirely obscure. We have isolated a molecularspecies of sialic acid in a free form from cerumen of the wet type, but notof the dry type, by an ion-exchange column chromatography and preparative
high-performance liquid chromatography. Structural analysis of this sialic
acid was performed by gas-liquid chromatography/mass spectrometry withchemical ionization (CI) and electron ionization (EI). In the CI mass
spectra, protonated molecular ion of the trimethylsilyl derivative was observed at m/z 580 and that of the methyl ester-trimethylsilyl derivative was
at m/z 522. In the EI mass spectrum, the methyl ester-trimethylsilyl derivative gave the characteristic ions at m/z 506, 462, 418, 416, 328, 316, 238,
228, 205, 186 and 173. This mass spectrum was identical to that of 2,7anhydro-N-acetylneuraminic acid, which was already reported by Lifely
and Cottee (Carbohydr. Res., 107, 187-197, 1982) as the mass spectrum of
a by-product prepared from N-acetylneuraminic acid by methanolysis.
These results indicate that the compound in the wet cerumen is 2,7-anhydroN-acetylneuraminic acid. Since this sialic acid species could not be detected in cerumens of the dry type, its formation in the wet cerumen may becontrolled by an autosomal dominant gene. For detail, see J. Biochem.
97: 509-515, 1985.

* Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113.

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36x.

APPLIED GENETICSThe Indica-Japonica Differentiation of Rice Cultivars
in Thailand and its Neighbouring Countries
Yo-Ichiro SATO, Songkran CHITRAKONl) and Hiroko MORISHIMA

Two hundred fifty eight native cultivars of the common rice, 97 fromThailand and the rest from Nepal, Bhutan, Northeast India, Bangladesh,
Burma and Laos were examined to learn the distribution pattern of the Indica
and Japonica types under the different habitat conditions, especially in relation to their photoperiodic sensitivity. The materials tested were divisible
into the Indica and Japonica types by a discriminant function which combined measurements of phenol reaction, KClO a resistance and apiculus
hair length. Japonica types were distributed in upland as well as lowland
fields with high elevations in a wide area. They seemed to be adapted inthese habitat, where early heading is required. They had weak photoperiodic
sensitivities. The glabrous or nuda cultivars were frequently found in uplandfields. They are also reported in Yunnan China, and Philippines. For
detail, refer Proc. Vth Congr. SABRAO (1986).
Variation in Grain Shape of the Indica andJaponica Rice Cultivars
Yo-Ichiro SATO

Grain shape has been used traditionally as a key character for distinguishing between the Indica and Japonica types of rice cultivars. The grain
shapes of the Temperate Japonica, Tropical Japonica and Indica types
have been recognized to be "round", "large" and "slender", as Matsuo(1952) classified them into a, band c types, respectively. To evaluate theusefulness of grain shape in the study of varietal differentiation, native
cultivars from various Asian countries, 277 in total, were classified into theabove-mentioned three types by Oka's (1958) criteria (discriminant scores),and also by grain shape in accordance with Matsuo's scheme, and the con1)

Pathum Thani Rice Research Center, Thailand.

RESEARCH ACTIVITIES IN 1985

101

cordance of the results obtained by two criteria were examined. Of 111
cultivars classified as Indicas, 83 were of slender or c type, and of 63 classified
as Temperate Japonicas, 49 were of round or a type, suggesting that thesetwo varietal groups could be recognized by their grain shape more or lesscorrectly. However, among 103 cultivars of the Tropical Japonica types,
50 were slender, 41 were large, and 12 were round types. On the whole,
the coefficients of concordance was 0.625. The concordance differed according to regions; it was very high (0.705-0.813) in Japan, Korea, China,
India (other than Assam) and Sri Lanka, but much lower (0.444-0.507) in
Assam, Himalayan hill regions, Philippines, Indonesia and Taiwan. For
rice cultivars from these regions in which varietal diversity is known to behigh, grain shape may not be a reliable criterion for Indica-Japonica classification.
Distribution of Genes for F 1 Weakness in AsianNative Rice Cultivars
Yo-Ichiro SATO and Hiroko MORISHIMAThe F l weakness occurring in crosses of a Peruvian cultivar "Jamaica"
with some Japanese rice cultivars, first reported by Amemiya and Akeminein 1963, is controlled by a set of complementary genes, Hwc-l and Hwc-2(formerly designated as L-2-a and L-2-b). To learn about the distributionof the genes, 348 Asian native cultivars were test-crossed with Jamaica,
Hwc-l carrier. Among the cultivars tested, 154 carried Hwc-2, while theremaining 194 had its recessive allele. One out of 115 Indica types (0.8 %)and 153 out of 233 Japonica types (65.8 %) were Hwc-2 carriers, indicatingthat this allele was Japonica-specific. The Hwc-2 frequency in the Japonica
types differed according to their origins, being high in Japan (94 %) andChina (92 %), and lower in Philippines (17 %) and Indonesia (13 %). Noneof 30 strains of the Asian common wild rice tested which were collected fromvarious localities had Hwc-2 gene. Further, 116 cultivars were also te8tcrossed with T65 or other Hwc-2 carriers. None of them had Hwc-l,
except for Jamaica. Probably, the Hwc-2 gene was born in the Japonica
types after they had evolved from their wild progenitor. This gene could
be closely linked with certain adaptive genes differentiating between the
Indica and Japonica types, or with some of the genes for intervarietal hybrid
sterility.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Effect of a Semi-Dwarfness Gene on Morphological
Characters in Rice with Different Genetic Backgrounds
and Environmental Conditions
Yo-Ichiro SATO and Hiroko MORISHIMA

Semi-dwarfness genes have much contributed to the improvement of thehigh yielding rice varieties in the world. Surprisingly, those genes proved
to be located on the same locus though their origins were different. In thepresent study, expression of such semi-dwarfness genes on morphologicalcharacters, such as culm length, internode length and leaf length were investigated under the different genetic backgrounds and environmental conditions. Materials used were 4 semi-isogenic pairs with and without thesemi-dwarfness gene under the genetic background of Taichung 65(T65),
Shiokari (SK), Fujiminori (FJ), and an American variety, Calrose (CR),respectively. In the former two pairs, a spontanous mutant gene carried
by Taichung native I (d-47) was introduced into T65 and SK. The latter two
were induced mutants (the allele are d-49 of FJ and sd-l of CR, respectively),
and their respective original varieties. The effects of these genes on theorgan length depended on genetic backgrounds as well as on environmental
conditions. The d-47 in T65 and SK seemed to give different expression
from d-49 in FJ or sd-l in CR in various aspects. The effect of d-47 was
expressed during whole growth duration, whereas d-49 and sd-l were notexpressed earlier than flowerbud initiation. This could imply the effect ofdifferent alleles at the same locus or the effect of different genetic backgrounds.
Genic Analysis of Isozyme Genes in Rice
Reiko SANO, Ryuji ISHIKAWA, Pascale BARBIER
and Hiroko MORISHIMA

More than 40 isozyme loci are presumed in rice, but formal genic analyses
have been conducted only on a limited number of loci. In the present
study, Mendelian segregations were confirmed for Amp-2, Amp-3, Est-ca,
Pgd-l and Sdh-l. Linkage relationships were examined for 10 isozyme
loci and some marker genes. We found a tight linkage between Amp-3
and Est-2 (0.7%). They are located on chromosome 6 in the following
Amp-3-Est-2
Pgi-2.
sequence, wx(31 %)(13%)

RESEARCH ACTIVITIES IN 1985

103

To determine chromosomal location of other loci, a series of trisomies
of a Japanese variety were crossed as females with a Vietnamese cultivar
and also with a strain of Asian common wild rice. So far, segregations of
9 isozyme genes were studied in F 2 s with 8 trisomies. The results obtained
indicated that Amp-2 and Pgd-l are located on chromosome 12 and 9, respectively. In other combinations of loci and trisomies, normal segregation or a deficiency of the alleles derived from the trisomic parent (Japoniea)
whieh is commonly observed in Indica-Japoniea crosses were found.
Electrophoretic Characterization of Rice Endosperms of
Indica and Japonica with Four Kinds of Protein Fractions
Toru ENDO

Rice cultivars (Oryza sativa) comprise two types, Indica and Japonica,
where the latter is believed to be derived from the former. The differences
between the two types were investigated with respect to several aspects,
including analyses of endosperm protein components, i.e., albumin, globulin, prolamin and glutelin. It is not so difficult to separate the first threeprotein fractions from rice endosperm as samples applicable to IEF-PAGE(isoelectrofocusing-polyacrylamide gel electrophoresis), but extraction ofglutelin as a fraction which can be dissolved in 8M urea solution has so far
been difficult.
Separation of the glutelin fraction was attempted as follows: after sequential extraction of the first three protein components, glutelin was extracted with 0.1 M NaOH and centrifuged, and the supernatant was neu-

Glutelin

T65108~;:f);lJ!~lii~~~lll~ ~l~ f if ~~

_

5.13

Fig. 1. Comparison of glutelin patterns from rice endosperms of T65 and 108
using the equilibrium system of IEF-PAGE. Arrows at top show majordifferences between both cultivars.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

tralized with 1 M ascorbic acid and centrifuged to get the precipitate of theglutelin fraction. The fraction was found to be soluble in 8 M urea solution,
but was insoluble when acids other than ascorbic acid were used.
The four kinds of protein fractions were examined in each of the varieties
of Indica (l08) and Japonica (T65) types. From the analysis of the experiments a total of at least 126 and 112 bands of the four fractions werecounted in T65 and 108, respectively. Characteristic differences wereobserved between the two types in the protein fractions. Major and mediumband differences are the fewest in albumin, are appreciable in globulin andglutelin (Fig. 1), and are considerable in prolamin fractions between bothtypes.
Conservation Methods of Crop Populations with
Mixed SeIfing and Outcrossing
Katsuei YONEZAWA and Hiko-Ichi OKA

Five different methods of seed propagation as defined in Table 1, i.e.,
SSD (single seed descent) method, two different types of pedigree and bulk
methods each, for rejuvenation of plant populations with mixed selfing andoutcrossing were compared with respect to the relative magnitude of random
genetic drift represented by the sampling variance of the frequency of genes
and genotypes. The result shows that, in predominantly selfing populations, the SSD method causes a much smaller sampling variance than thepedigree and bulk methods. The SSD method requires only about 1/4
as many plants as the bulk method, and about 1/2 as many plants as thepedigree method, to give the same level of random drift in relation to genefrequency. Partial sampling of the plants as harvesting only a small fraction of the plants grown may cause an irrecoverable genetic loss in predominantly selfing populations. The difference due to the propagation
methods is largely reduced in predominantly outcrossing populations, while
the order of the methods in relative efficiency remains the same as in selfing
populations. Controlled selfing by bagging would in many cases be theonly practicable way to avoid the contamination by alien pollens. In thissituation, the SSD method is recommended to be applied combined withbagging. Population size for a practical conservation project was briefly
discussed. (Proceedings of the 5th SABRAO Congress, 1986).

Table 1.
MethodPlantsharvested

Methods of seed propagation investigatedHarvest manipulation

Seeds/plant

Handling

Seeds forrejuvenation

SowingI. SSD method

All plants

One

BulkedAll seeds

several to many

A seed lot fromeach plant

One seed/plantseed lotor,Pedigree families
identified or not
identified

~

tT1
V1
tT1

>
~
(')

:I:
Pedigree method
Pol method
All plantsII.

P-2 method

One plant/
pedigoMany

Many

A bulked seed lot
for each pedigo
A seed lot fromeach plant (pedig.)A certain numberof seeds/pedigo
seed lot
A certain numberof seeds/plantseed lot

Pedigree families
identified
Pedigree families
identified

>
Q
....
-<
....
>-l

....tT1

V1

....
Z

\0
00

v.

III. Bulk method

B-1 method

All plants

Many

BulkedRandomly sampled

B-2 method

Some randomly
chosen plants

Many

BulkedRandomly sampled

Pedigree families
not identified
Pedigree families
not identified

SSD method: Single seed descent method.

oVI106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Evolutionary Significance of Regulatary Changes
in the Wx Gene Expression of Rice
Yoshio SANO

Recent interest in the theory of evolution has been focused on the importance of gene regulation. However, there is little evidence relating
regulatory changes at the protein level to gross phenotypic changes to be
selected for. To examine the importance of cis-acting regulatory elements
on racial differentiation in rice, the distribution of Wx& and Wx b was surveyed
in terms of the intensity of Wx protein. From the intensity of Wx protein
in mature endosperms, 193 from 207 strains examined were readily classified
as Wx& or Wx b and only 14 showed an intermediate intensity, with theirallelic states remaining uncertain. Wx& showed a Wx protein level ten foldhigher than that of Wxb as well as a higher level of amylose.
The four species, O. rufipogon (=0. perennis, a wild progenitor of O.
sativa), O. glaberrima, O. barthii (=0. breviligulata, a wild progenitor ofO. glaberrima) and O. longistaminata, all exhibited only the Wx& allele. On
the other hand, 103 strains of O. sativa had Wx& and 30 had Wx b • The
frequency distribution of the two alleles was markedly different among threeecogeographical races within O. sativa, Wx b was predominant in the Japonica
type while most strains of the Indica type had Wx&.
Racial differentiation within O. sativa has been extensively studied by anumber of researchers concerning geographical distribution and morphological, physiological and biochemical characteristics. In addition, theJapnica type is characterized by a lower content of amylose than the Indica
type (Lu and Chang 1980). The starch of nonglutinous (nonwaxy) endosperms contains both amylose and amylopectin and amylose content affects
the degree of viscosity of cooked rice, so glutinous (waxy) cultivars lacking
amylose are often called 'sticky rice'. Since amylose content is the major
determinant of the eating qualities in rice, factors controlling the amount ofamylose must have been selected for or against by farmers. In several
cereals including rice, maize, barley and so on, the glutinous (waxy) cultivars
lacking amylose are found mostly in Southeast Asia, especially in themountaineous regions. The geographical distribution of the wx allele inrice appears to be restricted to the regions where the Japonica type with low
amylose content prevalis. Recently, rice cultivars with an amylose content
lower than the Japonica type were widely found in the mountaineous regions

RESEARCH ACTIVITIES IN 1985

107

of Southeast Asia (Nakagahra et al., 1985). This leads us to consider thatthe current cultivation of glutinous or low amylose rice cultivars is primarily
due to acquired preference or traditional use in the local diet. Hence, an
artificial selection seems to strongly operate on differences in amylose
content.
Since the wild progenitor of O. sativa has Wx" only, a cis-acting regulatory
mutation must have produced Wx" from Wx b • The frequency of Wx"
which drastically reduces the Wx protein level as well as amylose content inrice endosperm probably increased during domestication leading to theestablishment of the Japonica type. The results obtained in this study
confirm that the regulatory change controlling the Wx gene expression isclosely related to an important agronomical trait which could be selected
for (Euphytica, 34: 587).
Genetic Control of Alcohol Dehydrogenase andEstimation of Some Population Parameters in
Hopea odorata Roxb. (Dipterocarpaceae)
Masaaki IHARA*, Lilian U. GADRINAB**, Ulfah J. SIREGAR**
and Shin-ya IYAMAThe ADH isoenzymes were investigated in seeds, seedlings and leaves ofmature trees in a tropical tree species, Hopea odorata. The materials werecollected from several places in Java, Indonesia as well as Thailand. The
isozymes were found to be dimeric and controlled by two loci; Adh-l consisted of 3 alleles designated as Adh-IS, Adh-IN and Adh-IF, and Adh-2
was isoallelic. Because of the differential gene expression of these loci in
cotyledons of mature seeds and in leaf blades of seedlings and trees, it was
concluded that Adh-l was expressed thoughout the whole life span of atree while Adh-2 only during a limited period in mature seeds.
Using Adh-l as a genetic marker, genotype frequencies in a plantationnear Bogor, Indonesia were investigated. The plantation consisted of two
separate stands, Site I where S/S, S/F and F/F genotypes were found andSite II where S/S, S/F and F/F were found and they were 700 m apart fromeach other. Genotypes of fallen seeds in Site I included N/F and S/F* Graduate Division of Biochemical Regulation, Faculty of Agriculture, Nagoya
University, Nagoya, 464 Japan.
** Tropical Forest Biology Program, BIOTROP, P.O. Box 17, Bogor, Indonesia.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

genotypes besides SIS, SIN and NIN, indicating the gne flow from Site II.
Based on the genotype frequencies of breeding trees in Sites I and II, andthose of seeds in Site I, interpopulational pollen flow rate (u=0.315) was
estimated together with outbreeding rate (t=0.634) and selection coefficientfor NIN genotype (s=0.486).
Publications from Genetic Resources Section,
Genetic Stocks Research Center
Shin-ya IYAMA

The following publications were released from Genetic Resources Section
this year.
1. "Stocks of experimental organisms maintained in the universities inJapan". 42 pages. (in Japanese).
Names of experimental organisms, location (laboratory) where the stocks
are maintained, person in charge, and number of stocks were listed according
to the universities. In total, 1141 locations in 172 universities and research
institutes were included: 336 locations for animal stocks, 125 for plantstocks, 318 for microorganism stocks and 362 for cultured cell stocks.
2. "Drosophila Stock List in Japan 1985." 61 pages.
This list contains 1360 drosophila stocks kept in various places in Japan.
Each stock is indicated with the place of maintainance. Wild and mutant
stocks of D. melanogaster, D. simulans, D. ananassae, D. hydei, D. virilis
and other species are included.
3. "Rice Genetics Newsletter Vol. 2". 122 pages. (in English).
This volume contains lists of recommended gene symbols and gene marker
stocks of rice, a list of about 240 recent publications on rice genetics and
46 research notes on rice genetics. Rice Genetics Newsletter will be published annually.
Data Base Design of DNA Base Sequences
Sanzo MIYAZAWA

Experimental analyses of DNA base sequences have been developed andknown DNA base sequences are rapidly growing. To make DNA sequencedata, published in scientific journals, readable by computer and accessible
to public domain, DNA sequence data banks have been established in Europe

RESEARCH ACTIVITIES IN 1985

109

and USA. This recent trend prompts biologists to use computers toanalyze DNA base sequences. Typical examples of common analyses arethose such as homology search, restriction enzyme site map, and open reading frame search. Search and retrieval of specific base sequences from DNA
sequence data are prerequisite to such analyses. Thus, a DNA data baseis demanded to efficiently manipulate sequence data. UNIX operating
system has been chosen as a target system to develope a data base, becauseof a portable operating system. By taking advantage of superior tools forinterprocess communication in UNIX such as shared memory and pipeline,
basic tools for data manupulation will be implemented as commands atthe shell level. In addition, a wide variety of application programs forsequence analyses will be incorporated into the data base system.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

PUBLICATlONS
ACKERMANN, J. and SUGIYAMA, T.: Genetic analysis of developmental mechanisms in
hydra. X. Morphogenetic potentials of a regeneration-deficient strain (reg-I6).
Develop. BioI. 107: 13-27, 1985.
AOKI, K.: Reciprocal altruism and reciprocal alliance between relatives. In "Population Genetics and Molecular Evolution" (ed. by T. Ohta and K. Aoki), 429-441,
Japan Sci. Soc. Press, Tokyo/Springer, Berlin, 1985.
ENAMI, M., FUKUDA, R. and ISHIHAMA, A.: Transcription and replication of eight
RNA segments of influenza virus. Virology 142: 68-77, 1985.
ENDO, T. and IHARA, M.: Notes on extractant-dependent leaf peroxidase and malate
dehydrogenase zymograms from twenty plant species. Jpn. J. Breed. 35: 340-345,
1985.
ENOMOTO, T., SUZUKI, M., TAKAHASHI, M., KAWASAKI, K., WATANABE, Y., NAGATA, K.,
HANAOKA, F. and YAMADA, M.: Purification and characterization of two forms of
DNA polymerase alfa from mouse FM3A cells. Cell Struc. Func. 10: 161-171, 1985.
FUJII, T. and INOUE, T.: Absence of mutagenic activity of benzo(a)pyrene in the soybean
test system. Env. Exp. Bot. 25: 139-143, 1985.
FUJITA, N. and ISHIHAMA, A.: Regulation of transcription initiation in prokaryotes.
(in Japanese). Jpn. J. Genet. 60: 505-525, 1985.
FUKUDA, M., WAKASUGI, S., TSUZUKI, T., NOMIYAMA, H., SHIMADA, K. and MIYATA, T.:
Mitochondrial DNA-like sequences in the human nuclear genome: Characterizations
and implications in the evolution of mitochondrial DNA. J. Mol. BioI. 186: 257266, 1985.
FUKUDA, R" YANO, R., FUKUI, T., HASE, T., ISHIHAMA, A. and MATSUBARA, H.: Cloningof the E. coli gene for the stringent starvation protein. Mol. Gen. Genet. 201: 151157, 1985.
FURUSATO, T., TAKANO, J., JIGAMI, Y., TANAKA, H. and YAMANE, K.: Two tandemly
located promotors, artificially constructed, are active in a Bacillus subtilis a-amylase
secretion vector. J. Biochem. (in press).
GOJOBORI, T.: Evolutionary features of oncogenes. In "Population Genetics and Molecular Evolution" (ed. by T. Ohta and K. Aoki), 353-368, Japan Sci. Soc. Press, Tokyo/
Springer, Berlin, 1985.
GOJOBORI, T. and YOKOYAMA, S.: Evolutionary rates of the retroviral oncogene of
Moloney murine sarcoma virus and the cellular homologues. Proc. Natl. Acad.
Sci. 82: 4198-4201, 1985.
GRONOSTAJSKI, R. M., AoHYA, S., NAGATA, K., GUGGENHEIMER, R. A. and HURWITZ, J.:
Site-specific DNA binding of nuclear factor I: analysis of cellular binding sites. Mol.
Cell. BioI. 5: 964-971, 1985.
HAYASHIDA, H. and MIYATA, T.: On the direction of gene conversion. Proc. Japan
Acad. 61 (B): 204-207, 1985.

PUBLICATIONS111

HAYASHIDA, H., TOH, H., KIKUNO, R. and MIYATA, T.: Evolution of influenza virus
genes. Mol. BioI. Evol. 2: 289-303, 1985.
HAYASHIDA, H., KIKUNO, R., YASUNAGA, T. and MIYATA, T.: A classification of geneconversion and its evolutionary implications. Proc. Japan Acad. 61 (B): 149-152,
1985.
HAYASHIDA, H. and MITAYA, T.: Sequence similarity between epidermal growth factor
precursor and atrial natriuretic factor precursor. FEBS Letts. 185: 125-128, 1985.
HIDAKA, S" TSUNASAWA, S., YOON, J., NARITA, K., TAKANAMI, Y., KUBO, S. and MIURA,
K.: Messenger RNA structure participating in the initiation of synthesis of cucumber mosaic virus coat protein. J. Biochem. 97: 161-171,1985.
HOUBA-HERIN, N., HARA, H., INOUYE, M. and HIROTA, Y.: Binding of penicillin tothiolpenicillin-binding protein 3 of Escherichia coli: identification of its active site.
Mol. Gen. Genet. 201: 499-504, 1985.
HURTWITZ, J., ADHYA, S., FIELD, J., GRONOSTAJSKI, R" GUGGENHEIMER, R. A., KENNY,
M., LINDENBAUM, J. and NAGATA, K.: Synthesis of adenoviral DNA with purified
proteins. In "Genetics, Cell differentiation and Cancer", 15-24, Academic Press,
1985.
IIDA, K., HIROTA, Y. and SCHWARZ, U.: Mutants of Escherichia coli defective in penicillin-insensitive murein DD-endopeptidase. Mol. Gen. Genet. 189: 215-221, 1983.
IKEMURA, T.: Codon usage, tRNA content, and rate of synonymous substitution. In"Population Genetics and Molecular Evolution" (ed. by T. Ohta and K. Aoki), 385406, Japan Sci. Soc. Press, Tokyo/Springer, Berlin, 1985.
IKEMURA, T.: Codon usage and tRNA content in unicellular and multicellular organisms.
Mol. BioI. Evol. 2: 13-34, 1985.
INOUYE, S., NOGUCHI, M., SAKAKI, Y" TAKAGI, Y., MIYATA, T., IWANAGA, S., MIYATA,
T. and TSUJI, F. I.: Cloning and sequence analysis of cDNA for the luminescentprotein aequorin. Proc. Natl. Acad. Sci. USA 82: 3154-3158, 1985.
ISHII, R., YOSHIKAWA, K., MINAKATA, H., KOMURA, H. and KADA, T.: Specificities ofbio-antimutagens in plant kingdom. Agric. BioI. Chern. 48: 2587-2591,1985.
ISHIKAWA, Y., KANO, M., TAMIYA, N. and SHIMADA, Y.: Acetylcholine receptors ofhuman skeletal muscle: a species difference detected by snake neurotoxins. Brain
Res. 346: 82-88, 1985.
IZUI, K., MIWA, T., KAJITANI, M., FUJITA, N., SABE, H" ISHIHAMA, A. and KATSUKI, H.:
Promotor analysis of the phosphoenolpyruvate carboxylase gene of Escherichia coli.
Nucleic Acids Res. 13: 59-72, 1985.
JASPERS, N. G. J., PAINTER, R. B., PATERSON, M. c., KmSON, C. and INOUE, T.: Complementation analysis of ataxia-telangiectasia. In "Ataxia-Telangiectasia: Genetics,
Neuropathology, and Immunology of a Degenerative Disease of Childhood" (ed. by
R. A. Gatti and M. Swift), Alan R. Liss Inc., New York, 147-162, 1985.
KADA, T., KANEKO, K., MATSUZAKI, S., MATSUZAKI, T. and HARA, Y.: Detection andchemical identification of natural bio-antimutagens. A case of the green tea factor.
Mutation Res. 150: 127-132, 1985.
KADA, T., SADAIE, Y. and INOUE, T.: Tritium effects on DNA. Tritium concentration
dependency of RBE in aqueous solution. NIRS-M-52 (proc. 2nd Workshop on

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36
Tritium Radiobiology and Health Physics): 64-74, 1985.
KAMIMURA, T., TSUCHIYA, M., URAKAMI, K., KOURA, K., SEKINE, M., SHINOZAKI, K.,
MWRA, K. and RATA, T.: Synthesis of a dodecaribonucleotide, GUAUCAAUAAUG, by use of "fully" protected ribonucleotide building blocks. J. Am. Chern.
Soc. 106: 4552-4557, 1984.
KATAGIRI, F., KODAKI, T., FUJITA, N., IZUI, K. and KATSUKI, H.: Nucleotide sequenceof the phosphoenolpyruvate carboxylase gene of the cyanobacterium Anacystis
nidulans. Gene 38: 265-269,1985.
KATO, A., MIZUMOTO, K. and ISHIHAMA, A.: Purification and enzymatic properties of
an RNA polymerase-RNA complex from influenza virus. Virus Res. 3: 115-127,
1985.
KATO, J-I., SUZUKI, H. and HIROTA, Y.: Overlapping of the coding regions for a and rcomponents of penicillin-binding protein 1b in Escherichia coli. Mol. Gen. Genet.
196: 449-457, 1984.
KATO, J-I., SUZUKI, H. and HIROTA, Y.: Dispensability of either penicillin-binding
protein-l a or -lb involved in the essential process for cell elongation in Escherichia
coli. Mol. Gen. Genet. 200: 272-277,1985.
KATOH, H., ESAKI, K., SHOJI, Y., NOMURA, T., MORIWAKI, K., YONEKAWA, H.: Demonstration of genetic profiles in various lines of AKR and NZB by a genetic monitoring system. In "The Contribution of Laboratory Animal Science to the Welfareof Man and Animals: 8th ICLAS/CALS Symposium, Vancouver, 1983" (ed. by J.
Archibald & others), 495-502, Gustav Fisher, Stuttgart, 1985.
KATOH, Y., HASEGAWA, T., SUZUKI, T. and FUJII, T.: Changes in the amounts of putrescine, spermidine and spermine in Hiproly barley callus after auxin withdrawal.
Agric. BioI. Chern. 49: 1027-1032, 1985.
KAWAKAMI, K., NOGUCHI, S., NODA, M., TAKAHASHI, H., OHTA, T., KAWAMURA, M.,
NOJIMA, H., NAGANO, K., HIROSE, T., INAYAMA, S., HAYASHIDA, H., MIYATA, T. and
NUMA, S.: Primary structure of the a-subunit of Torpedo californica (Na + + K +)ATPase deduced from cDNA sequence. Nature 316: 733-736, 1985.
KAWAKAMI, K., MIZUMOTO, K., IsHIHAMA, A., YAMAGUCHI-SHINOZAKI, K. and MIURA,
K.: Activation of influenza virus-associated RNA polymerase by cap-l structure.
J. Biochem. 97: 655-661, 1985.
KIKUNO, R. and MIYATA, T.: Sequence homologies among mitochondrial DNA-coded
URF2, URF4 and URF5. FEBS Letters, 189: 85-88, 1985.
KIKUNO, R., HAYASHIDA, H. and MIYATA, T.: Rapid rate of rodent evolution. Proc.
Japan Acad. 61 (B): 153-156, 1985.
KIMuRA, M.: Diffusion models in population genetics with special reference to fixation
time of molecular mutants under mutational pressure. In "Population Genetics andMolecular Evolution" (ed. by T. Ohta and K. Aoki), 19-39, Japan Sci. Soc. Press,
Tokyo/Springer, Berlin, 1985.
KIMuRA, M.: Genes, populations and molecules: a memoir. In "Population Genetics
and Molecular Evolution" (ed. by T. Ohta and K. Aoki), 459-481, Japan Sci. Soc.
Press, Tokyo/Springer, Berlin, 1985.
KIMURA, M.: Natural selection and neutral evolution. In "What Darwin Began" (ed.

PUBLICATIONS

It3

by L. R. Godfrey), 73-93, Allyn and Bacon, Boston, 1985.
KIMURA, M.: The neutral theory of molecular evolution. New Scientist, No. 1464
(It July): 41-45,1985.
KIMURA, M.: Neutralisme dans I'evolution. Sciences & Avenir, Numero Special: 2934, 1985.
KIMURA, M.: The role of compensatory neutral mutations in molecular evolution. J.
Genetics 64 (1): 7-19, 1985.
KITAMURA, N., KITAGAWA, H., FUKUSHIMA, D., TAKAGAKI, Y., MIYATA, T. and NAKANISHI, S.: Structure organization of the human kininogen gene and a model for
its evolution. J. BioI. Chern. 260: 8610-8617, 1985.
KOMINAMI, R., MURAMATSU, M., SUDO, K., YOSHIKURA, H., SUZUKI, H., MORIWAKI, K.
and HILGERS, J.: The origin and meiotic instability of a polymorphic repetitive
sequence RPI family. In "The BALB/c Mouse: Genetics and Immunology" (ed. by
M. Potter), 58-65, Springer-Verlag, Berlin, 1985.
KOMINAMl, R., SUDO, K., YOSHIKURA, H., SUZUKI, H., MORIWAKI, K., HILGERS, J. and
MURAMATSU, M.: A polymorphic repetitive-sequence PRI family evidence formeiotic instability. J. Mol. BioI. 183: 301-309, 1985.
KUBO, T., NODA, M., TAKAI, T., TANABE, T., KAYANO, T., SHIMIZU, S., TANAKA, K.,
TAKAHASHI, H., HIROSE, T., INAYAMA, S., KIKUNO, R., MIYATA, T. and NUMA, S.:
Primary structure of 0 subunit precursor of calf muscle acetylcholine receptor deduced
from cDNA sequence Eur. J. Biochem. 149: 5-13, 1985.
KURIHARA, Y., MIYASHITA, N., MORIWAKI, K., PETRAS, M. L., BONHOMME, F., CHO,
W. S. and KOHNO, S.: Serological survey of T-lymphocyte differentiation antigens
in wild mice. Immunogenet. 22: 211-218, 1985.
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 Mutation Research (ed. by J. Ashby & others), Vol. 5: 537-542, 1985.
KURODA, Y., HAYATSU, H. and NEGISHI, K.: Mutagenic activity of cytidine analogs incultured mammalian cells. Mutation Res. 147: 262-263, 1985.
KURODA, Y.: Aging of cells. (in Japanese) In "Development and Aging" (ed. by G.
Eguchi and N. Osawa), II, 89-100, Nakayama-shoten, Tokyo, 1985.
KURODA, Y.: Clonal culture of cells and growth factors. (in Japanese) In "Studies onControl of Aging" (ed. by Institute of Physical and Chemical Research), 218-223,
Sozo Life Science Kenkyukai, Tokyo, 1985.
KUSUDA, J., ONlMARU, K., NOGUCHI, T. and YAMASHITA, 0.: Maturation and hatchingof eggs from silkworm ovaries preserved in liquid nitrogen. J. Insect Physiol. 31:
963-967, 1985.
MARUYAMA, T. and FUERST, P. A.: Non-equilibrium models in population genetics:
The first arrival times and the number of alleles in evolving populations. In "Population Genetics and Molecular Evolution" (ed. by T. Ohta and K. Aoki), Japan Sci. Soc.
Press, Tokyo/Springer, Berlin, 1985.
MARUYAMA, T. and FUERST, P. A.: Population bottleneck and non-equilibrium models
in population genetics. II. Number of alleles when a small population is derived
from a large steady state population by means of a bottleneck. Genetics 111: 675-

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36
689,1985.
MARUYAMA, T. and FUERST, P. A.: Population bottlenecks and non-equilibrium models
in population genetics. III. Genetic homozygosity in populations which experience
periodic bottlenecks. Genetics 111: 691-703, 1985.
MATSUI, M., OKA, A., TAKANAMI, M., YASUDA, S. and HIROTA, Y.: Sites of dna A
protein binding in the replication origin of the Escherichia coli K-12 chromosome.
J. Mol. BioI. 184: 529-533, 1985.
MATSUNAGA, E.: Monitoring of birth defects: present status and problems. (in Japanese).
J. Jpn. Med. Assoc. 93: 1322-1324, 1985.
MINAKATA, H., KOMURA, H., TAMURA, S. Y., OHFUNE, Y., NAKANISHI, K. and KADA,
T.: Antimutagenic unusual amino acids from plants. Experientia 41: 1621-1623,
1985.
MIYASHITA, N., MORIWAKI, K., MINEZAWA, M., YONEKAWA, H., BONHOMME, F., MIGlTA,S., Yu, Z. C., Lu, D. Y., CHO, W. S. and THOHARI, M.: Allelic constitution of thehemoglobin beta chain in wild population of the house mouse, Mus musculus. Biochern. Genet. 23: 975-986, 1985.
MIYASHITA, N., SUZUKI, K. and MORIWAKI, K.: The H-2 complex affects the frequency
of urethan-induced chromosomal aberrations. Jpn. J. Cancer Res. 76: 1141-1145,
1985.
MIYATA, T., TOH, H., HAYASHIDA, H., KIKUNO, R., INOKUCHI, Y. and SAlGO, K.: Sequence homology among reverse transcriptase-containing viruses and transposable
genetic element: functional and evolutionary implications. In "Population Genetics
and Molecular Evolution" (ed. by T. Ohta and K. Aoki), 313-331, Japan Sci. Soc.
Press, Tokyo/Springer, Berlin, 1985.
MIYATA, T., HAYASHIDA, H., KIKUNO, R., TOH, H. and KAWADA, Y.: Evolution of interferon genes. In "Interferon 6" (ed. by Grosser), 1-30, Academic Press, London,
1985.
MIYATA, T., TOH, H. and SAIGO, K.: Reverse transcriptase-containing DNA virus andplasmids lack integrase. Proc. Japan Acad. 61B: 464-466, 1985.
MORISHIMA, H.: Habitat, genetic structure and dynamics of perennial and annual populations of the Asian wild rice Oryza perennis. In "Genetic Differentiation and
Dispersal in Plants" (ed. by P. Jacquard, & others), 179-190, Springer-Verlag, Berlin,
1985.
MORITA, K., NISHIMURA, Y. and KADA, T.: Chemical nature of a desmutagenic factor
from burdock (Arctium lappa Linne). Agric. BioI. Chern. 49: 925-932,1985.
MORIWAKI, K., MIYASHITA, N. and YONEKAWA, H.: Genetic survey of the origin oflaboratory mice and its implication in genetic monitoring. In "Contribution ofLaboratory Animal Science to the Welfare of Man and Animals: 8th ICLAS/CALS
Symposium, Vancouver, 1983" (ed. by J. Archibald & others), 237-247, Gustav
Fischer, Stuttgart, 1985.
MUKAI, T.: Experimental verification of the neutral theory. In "Population Genetics
and Molecular Evolution" (ed. by T. Ohta and K. Aoki), 125-145, Japan Sci. Soc.
Press, Tokyo/Springer, Berlin, 1985.
MUKAI, T., BABA, M., AKIYAMA, M., UOWAKI, N., KUSAKABE, S. and TAlIMA, F.: Rapid

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115

change in mutation rate in a local population of Drosophila melanogaster. Proc.
Natl. Acad. Sci. USA 82: 7671-7675, 1985.
MURAKAMI, A.: Dose-response relationships for mutations induced by chemicals in thesilkworm. In "Problemes of Thresholds in Chemical Mutagenesis" (ed. by Y. Tazima& others), Environm. Mutagen Soc. Japan, 5-13, 1984.
MURAKAMI, A.: A new mutant at the pe+ locus, black eyed light yellowish-brown egg
in the silkworm, Bombyx mori. Sericologia 25: 327-342, 1985.
NICHOLAS, R. A., STROMINGER, J. L., SUZUKI, H. and HIROTA, Y.: Identification of theactive site in penicillin-binding protein 3 of Escherichia coli. J. Bacteriol. 164: 456460,1985.
NICHOLAS, R. A., SUZUKI, H., HIROTA, Y. and STROMINGER, J. L.: Purification andsequencing of the active site tryptic peptide from penicillin-binding protein 1b ofEscherichia coli. Biochemistry 24: 3448-3453, 1985.
NOMURA, T., AlBA, H. and ISHIHAMA, A.: Transcriptional organization of the convergent
overlapping dnaQ-rnh genes of Escherichia coli. J. BioI. Chern. 260: 7122-7125, 1985.
NOMURA, T., FUJITA, N. and ISHIHAMA, A.: Promotor selectivity of E. coli RNA polymerase: analysis of the promotor system of convergently-transcribed dnaQ-rnh
genes. Nucleic Acids Res. 13: 7647-7661,1985.
OHNISHI, S. and WATANABE, T. K.: Genetic analysis of color dimorphism in Drosophila
montium subgroup. Jpn. J. Genet. 60: 355-358, 1985.
OHTA, T.: Variances and covariances of identity coefficients of a multigene family. Proc.
Natl. Acad. Sci. USA 82: 829-833, 1985.
OHTA, T.: A model of duplicative transposition and gene conversion for repetitive
DNA families, Genetics 110: 513-524, 1985.
OHTA, T. and AOKI, K. (eds.): "Population Genetics and Molecular Evolution", Japan
Sci. Soc. Press, Tokyo/Springer, Berlin, 1985.
OHTA, T.: Genetic variation of multigene families. In "Population Genetics and Molecular Evolution" (ed. by T. Ohta and K. Aoki), 233-242, Japan Sci. Soc. Press, Tokyo/
Springer, Berlin, 1985.
ONO, M., TOH, H., MIYATA, T. and AWAYA, T.: Nucleotide sequence of the Syrian
hamstar intracisternal A particle gene: Close evolutionary relationship of type A
particle gene with type Band D oncovirus genes. J. Virol. 55: 387-394, 1985.
SADAIE, Y. and KADA, T.: Bacillus subtilis gene involved in cell division, sporulation,and exoenzyme secretion. J. Bacteriology 163: 648-653.
SANO, Y., MAEKAWA, M., and KIKUCHI, H.: Temperature effects on the Wx protein
level and amylose content in the endosperm of rice. J. Heredity 76: 221-222, 1985.
SANO, Y., KATSUMATA, M. and AMANO, E.: Correlations between the amounts of amylose
and Wx protein in rice endosperm. SABRAO J. 1985 (in press).
SANO, Y.: Interspecific cytoplasm substitutions of an Indica strain of Oryza sativa L.
and O. glaberrima Steud. Euphytica 34: 587-592, 1985.
SATO, Y.-I. and HAYASHI, K.: Varietal variations in basic vegetative phase and photosensitive phase among Japanese native cultivars of rice. (in Japanese). Jpn. J. Breed.
65: 72-75, 1985.
SATO, Y.-I. and HAYASHI, K.: The genetic control of basic vegetative phase in early-116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36
maturing native rice varieties of Japan. (in Japanese). Jpn. J. Breed. 35: 16a-166,
1985.
SAlTA, Y., GOJOBORI, T., MARUYAMA, T. and CHIGUSA, S. I.: Tn3 resolvase-Iike sequencein P transposable element of Drosophila melanogaster. Jpn. J. Genet. 60: 261-266,
1985.
SAlTA, Y., GOJOBORI, T., MARUYAMA, T., SAIGO, K. and CHIGUSA, S. I.: Homology
between P-transposable element of Drosophila melanogaster and bacterial transposaseof Tn3. Jpn. J. Genet. 60: 499-503, 1985.
SmMADA, Y. and ISOBE, Y.: Cytoskeletal organization in embryonic chick skeletal muscle
cells in vitro revealed by the detergent-extraction, freeze-dry method. In "Molecular
Biology of Muscle Development" (ed. by C. Emerson, & others), (in press) Alan R.
Liss, New York, 1985.
SHIMIZU, N. and SHIMADA, Y.: Immunochemical analysis of tropoin T isoforms inadult, embryonic, regenerating and denervated chicken fast muscles. Develop. BioI.
111: 324-334, 1985.
SmMOI, K., NAKAMURA, Y., NORO, T., TOMITA, I., FUKUSHIMA, S., INOUE, T. and KADA,
T.: Methyl cinnamate derivatives enhance UV-induced mutagenesis due to theinhibition of DNA excision repairs in Escherichia coli B/r. Mutation Res. 146:
15-22, 1985.
SHIMOI, K., NAKAMURA, Y., TOMITA, I. and KADA, T.: Bio-antimutagenic effects oftannic acid on UV and chemically induced mutagenesis in Escherichia coli B/r. Mutation Res. 148: 17-23, 1985.
SHIMOTOHNO, K., TAKAHASHI, Y., SHIMIZU, N., GOJOBORI, T., CHEN, I. S. Y., MIWA, M.
and SUGIMURA, T.: Complete nucleotide sequence of an infectious clone of humanT-cell leukemia virus type II: A new open reading frame for the protease gene. Proc.
NatI. Acad. Sci. 82: 3101-3105, 1985.
SHIROISHI, T., EVANS, G. A., ApPELLA, E. and OZATO, K.: In vitro mutagenesis of amouse MHC class I gene for the examination of structure-function relationships.
J. ImmunoI. 134: 623-629, 1985.
SLATKIN, M. and TAKAHATA, N.: The average frequency of private alleles in a partially
isolated population. Theor. Pop. BioI. 28: 314-331,1985.
SRIMAL, S., MIYATA, T., KAWABATA, S., MIYATA, T. and IWANAGA, S.: The completeamino acid sequence of coagulogen isolated from southeast Asian horseshoe crab,
Carcinoscorpius rotundicauda. J. Biochem. 98: 305-318, 1985.
SUGIYAMA, T. and SUGIMOTO, N.: Genetic analysis of developmental mechanisms in
hydra. XI, Mechanisms of sex change by parabiosis. Develop. BioI. 110: 413-421,
1985.
SUZUKI, M., SUZUKI, A., YAMAKAWA, T. and MATSUNAGA, E.: Characterization of
2,7-anhydro-N-acetylneuraminic acid in human wet cerumen. J. Biochem. 97:
509-515, 1985.
TACHIBANA, H. and ISHIHAMA, A.: Correlation of the formation rate of open promoter
complexes with the melting property of DNA. In "Physical Foundation of Protein
and Nucleic Acid Functions" (ed. by A. Wada), Jpn. Sci. Soc. Press/Elsevier, 1985.
TACHIDA, H. and MUKAI, T.: The genetic structure of natural populations of Drosophila

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117

melanogaster. XIX. Genotype-environment interaction in viability. Genetics 111:
43-55, 1985.
TAKAHASHI, K., GOJOBORI, T., and NAORA. H.: Homology of kringle structures inurokinase and tissue-type plasminogen activator: The phylogeny to the related
serine proteases. Cell Struc. Func. 10: 209-218, 1985.
TAKAHATA, N.: Introgression of extranulcear genomes in finite populations: nucleocytoplasmic incompatibility. Genet. Res. Camb. 45: 179-194, 1985.
TAKAHATA, N.: Gene diversity in finite populations. Genet. Res. Camb. 46: 107-113,
1985.
TAKAHATA, N. and PALUMBI, S. R.: Extranuclear differentiation and gene flow in thefinite island model. Genetics 109: 441-457, 1985.
TAKAHATA, N. and NEI, M.: Gene genealogy and variance of interpopulational nucleotide
differences. Genetics 110: 325-344, 1985.
TAKAHATA. N.: Population genetics of extranuclear genomes: A model and review.
In "Population Genetics and Molecular Evolution" (ed. by T. Ohta and K. Aoki),
195-212, Japan Sci. Soc. Press, Tokyo/Springer, Berlin, 1985.
TEZUKA, H., SASAKI, Y. F., INOUE, M., UCHIDA, A., MORIYA, M. and SHIRASU, Y.:
Heritable translocation study in male mice with trimethyl phosphate. Mutation
Res. 157: 205-213, 1985.
TEZUKA, H., INOUE, T., NOGUTI, T., KADA, T. and SHULTZ, L. D.: Evaluation of themouse mutant "wasted" as an animal model for ataxia telangiectasia. I. Agedependent and tissue-specific effects. Mutation Res. (in press).
TOH, H., HAYASHIDA, H., KIKUNO, R., YASUNAGA, T. and MIYATA, T.: Sequence similarity between EGF receptor and a,-acid glycoprotein; Nature 314: 199, 1985.
TOH, H., ONO, M., SAIGO, K. and MIYATA, T.: Retroviral protease-like sequence inthe yeast transposon Tyl. Nature 315: 691, 1985.
TOH, H. and MIYATA, T.: Is the AIDS virus recombinant? Nature 316: 21-22,1985.
TOH, H., ONO, M. and MIYATA, T.: Retroviral gag and DNA endonuclease coding
sequences in IgE-binding factor gene. Nature 318: 388-389, 1985.
TOH, H., KrKUNO, R., HAYASHIDA, H., MIYATA, T., KUGIMIYA, W., INOUE, S., YUKI, S.
and SAIGO, K.: Close structural resemblance between putative polymerase of a
Drosophila transposable genetic element 17.6 and pol gene product of Moloneymurine leukaemia virus. EMBO. J. 4' 1267-1272, 1985.
TSUNEWAKI, K., SPETSOV, P. and YONEZAWA, K.: Increasing genetic variability incommon wheat by utilizing alien cytoplasms--Cytoplasmic effects on the performance and interplant variability of the F, and F 2 generations of the cross, Triticum
aestivum cv. Norin 26 xcv. Norin 61. Japan. J. Breed. 35' 398-412,1985.
WATANABE, K. and MIURA, K.: Specific interaction between tRNA and its cognateamino acid as detected by circular dichroism and fluorescence spectroscopy. Biochern. Biophys. Res. Comm. 129: 679-685, 1985.
WEIR, B. S., OHTA, T. and TACHIDA, H.: Gene conversion models. J. Theor, BioI.
116: 1-8, 1985.
YAMADA, M. A., WATANABE, T. K. and KOANA, T.: Absence of resistance genes against
male-killing action of the SRO in Drosophila melanogaster. Jpn. J. Genet. 60: 93-

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36
102, 1985.
YASUDA, S., NAKAYAMA, N., JIKUYA, H. and SOEDA, E.: An improved method for shotgun
DNA sequencing. Agr. BioI. Chem. 49: 1525-1526, 1985.
YONEKAWA, H., KATOH, H., EsAKI, K., SAGAI, T., MORIWAKI, K. and TAGASHIRA, Y.:
Maternal contamination of some NZB sublines and genetic profiles of NZ-strains.
Lab. Anim. Sci. (in press, 1985).
YONEZAWA, K.: A definition of the optimal allocation of effort in conservation of plant
genetic resources--with application to sample size determination for field collection. Euphytica 34: 345-354, 1985.
YOSHIMARU H. and MUKAI, T.: Relationships between the polygenes affecting the rateof development and viability in Drosophila melanogaster. Jpn. J. Genet. 60: 307334, 1985.
YOSIDA, T. H., UDAGAWA, T., ISHIBASHI, M., MORIWAKI, K., YABE, T. and HAMADA, T.:
Studies on the karyotypes of the black rats distributed in the Pacific and South PacificIslands, with special regard to the border line of the Asian and Oceanian type blackrats on the Pacific Ocean. Proc. Jpn. Acad. 61B: 71-74, 1985.
YUKUHIRO, K., HARADA, K. and MUKAI, T.: Viability mutations induced by the P
elements in Drosophila melanogaster. Jpn. J. Genet. 60: 261-266, 1985.

ABSTRACTS OF DIARY FOR 1985

119

ABSTRACTS OF DIARY FOR 1985
Biological Symposium
234th meeting Feb.

235th

236th
237th

238th

239th
240th

241st
242nd
243rd
244th
245th
246th

7 Molecular population genetics of Drosophila:transcriptional units and transposable elements (C. H. Langley)
Feb. 26 Molecular analysis of a "sex chromosome associated" repeated DNA sequences in Drosophila melanogaster (P. R. Simpson)Mar. 18 Genetic dissection of RNA polymerase (R. E.
Glass)
Mar. 18 Hereditary variation of the normal electroencephalogram (EEG): a model system inhuman behavior genetics (F. Vogel)
Apr. 17 Use of DNA probes to study the population
genetics of intracellular bacteria (genus Rickettsia) (P. A. Fuerst)
May 30 The role of calcium influxes during hydrozoan
development (G. Freeman)
June 7 Geographic origins, genetic diversity and themolecular clock hypothesis in the Oryzeae(G. Second)
Sept. 19 Human cancer susceptibility genes and oncogenes (W. F. Benedict)
Sept. 20 Analysis of the transcriptional signals ofbacterial rRNA genes (P. Venetianer)Oct. 21 Molecular genetics of the mouse major histocompatibility complex (J. A. Frelinger)
Nov. 20 Models of biological pattern formation (H.
Meinhardt)
Nov. 19 Genes phylogeny and species phylogeny in thegenus Mus (F. Bonhomme)
Dec. 5 Influence of DNA inhibitors on the biologicaleffects of physical and chemical mutagens(A. T. Natarajan)

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

Mishima Geneticists' Club
295th meeting Feb. 14 The structure of the gene for the human major
histocompatibility antigen, HAL Class II(Kiyotaka Okada)
296thFeb. 19 Game between males for possession of females--emergence curve of butterflies and sex ratioof frogs (Yoh Iwasa)
Mar. 11 Quantitative analysis of biological morpho297th
genesis (Hiroshi Shimizu)
Mar. 13 Reverse transcriptase involved in the DNA
298th
replication of cauliflower mosaic viruscloning of the gene for the reverse transcriptase
and its expression in yeast cells (Johe Ikeda)
Apr. 11 Immunological self-recognition in killer-T
299thcell as a model (Nobutaka Shinohara)
Apr. 25 An adenovirus vector for expression of cloned
300th
genes in mammalian cells (Masao Yamada)May 31 Bacillus subtilis spoO genes and the major
301stsigma factor (Fujio Kawamura)
June 25 Recent findings on MR system in Drosophila
302ndmelanogaster (Yuichiro Hiraizumi)
July 22 Analysis of molecular genetic variation at the
303rdloci encoding amylase and ADH (Hidenori
Tachida)Sep. 18 Theoretical analysis of promoter strength in
304thtranscription (Hideki Tachibana)Sep. 19 Search for new oncogenes; use of newly
305thdeveloped cDNA expression cloning vector
system (Hiroto Okayama)
Oct. 21 Altered expression of the human non-a-globin
306thgene complex by the mutation (Takashi Imamura)Nov. 28 Sexual differentiation and hormones of snales
307th(Naokuni Takeda)
Nov. 26 Mouse major histocompatibility class I gene
308th
expression in early-stage embryos (Keiko
Ozato)

ABSTRACTS OF DIARY FOR 1985

309th

121

Dec. 24 Expression of recombinant genetic element in
the teratocarcinoma stem cell (Makoto Taketo)122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

FOREIGN VISITORS IN 1985
March 9, 1984February 28, 1985
March 16, 1984April 30, 1985
December 22, 1984February 9, 1985
September 29, 1984December 19, 1985
September 29, 1984December 19, 1985
January 19
January 25-February 15
February 6-12
February 6-7
February 8
February 26March 1
March 18-19
March 18-19

March 21-28
March 26-May 4
April 1-4
April I-November 9Kim, Bong Kee, Dankook University, Korea
Houba-Herin, Nicole, Liege University, Belgium
Lee, Won Ho, Pusan National University, Korea
Loekman, Irwansyah, National Atomic Energy
Agency, Indonesia
Gadrinab, Lilian U., SEAMEO Regional Center
for Tropical Biology, Indonesia
Tetsuji, Matayoshi, Centro de Genetica Medicale,Argentina
Choo, Jong Kil, Chung Ang University, Korea
Simpson, Paul, Australian National University,
Australia
Paik, Yong Kyun, Hanyang University School of
Medicine, Korea
Langley, C. W., National Institute of Environmental Health Sciences, NIH, U.S.A.
Boursot, Pierre, Universite Montpellier, France
Huang, Yi-de, Shanghai Institute of Plant
Physiology, Academia Sinica, China
Glass, Robert E., University Medical School,
Nottingham, England
Vogel, F., Institut flir Anthropologie u. Humangenetik der UniversiHit Heidelberg, West
Germany
Boussy, Ian, Australian National University,
Australia
Fuerst, Paul A., Ohio State University, U.S.A.
Guhardja, Edi, Institut Pertanian Bogor, Indonesia
Jain, Ajay Kumar, King George Medical Col-FOREIGN VISITORS IN 1985

April 5
April 6
April 22

May 22
May 22-24
May 23
May 30June 3June 12-30
June 12-13
June 19
July 5
July 16
August 1
September 2
September 7-20
September 9123lege, India
Lu, Hong Sheng, Institute of Sericulture, Chinese
Academy of Agricultural Sciences, China
Majewski, Tomasz, Institute of Botany, Polish
Academy of Sciences, Poland
Zhang, Xu Jing, Qinghai Academy of Veterinary
Medicine, China
Fang, Xi Ye, Experimental Animal Center,
Chinese Academy of Medical Sciences, China
Zhang, Cheng Gui, Yunnan Primate Center ofExperimental Animals, China
Ruffie, Jacques, Laboratoire d'Anthropologie
Physique, College de France, France
Glass, Robert E., University Medical School,
Nottingham, England
Richards, S., British Council, Tokyo
Rabson, Robert, Office of Basic Energy Sciences,
U.S.A.
Second, Gerard, Centre d'Etudes Phytosociologiques et Ecologiques, France
Crow, James F., University of Wisconsin, U.S.A
de Azevedo, Joao Lucio, University of Sao
Paulo, Brazil
Haselkorn, Robert, University of Chicago,
U.S.A.
Warid, Warid A., University of Cairo, Egypt
Sudarwarti, Sri, Bandung Institute of Technology, Indonesia
Yung, Sun Kang, Seoul, Korea
Mukherjee, T. K., University of Malaya, Malaysia
Jacquard, Pierre, Centre d'Etudes Phytosociologiques et Ecologiques, France
Jia, Shi Rong; Wang, Ji Fang; Chuan, Huan
Ying; Vegetable Research Institute, Chinese
Academy of Agricultural Sciences, China
Ping, Ji Ming; Zhang, Ru Yu, Ministry of124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 36

September 11September 17
September 19
September 20
September 20-21
October 11October 15October 21
November 13
November 19
November 19-21 'November 26
December 4-6

Agriculture, Animal Husbandry and Fishery,
China
Lee, Won Ho, Pusan National University, Korea
Park, Hyeng Shik; Korea
Benedict, William F., Children's Hospital of Los
Angeles, U.S.A.
Fallon, Ann M., UMDNJ-School of Osteopathic
Medicine, U.S.A.
Ventetianer, Pal, Hungarian Academy of Sciences, Hungary
Barbier, Pascale, Universite des Sciences etTechniques du Languedoc, Montpellier, France
Qiu, Yuan Sheng, Guangdong Microbiology
Research Institute, China
Frelinger, Jeffrey A., University of North Carolina, U.S.A.
Riley, Ralph, Agricultural Research Council,
England
Bonhomme, Francois, Institut des Sciences del'Evolution, Universite Montpellier, France
Meinhardt, Hans, Max-Planck-Institut fUr Virusforschung, West Germany
Czarnomska, Alina, Institute of Oncology,
PolandNatarajan, A. T., University of Leiden, The
Netherlands

AUTHOR INDEX125

AUTHOR INDEXAOKI, K •••••••••••••••••••••••••••• 72

INOUYE, M •••••••••••••••••••••••••• 32

AOTA, S.-I•••••••••••••••••••••••••• 28

17,18,19,20,23,24
102
ISHIMI, Y. .
26
IYAMA, S •••••••••••••••••••••• 107,108
JONES, S. T
17
KADA, T•.••.............. 62, 63, 65, 66
KANO, Y •.....•.................... 20
KATo,A....•......................• 24
KATO, J.-I. • •••••••••••••••••••.•••• 33
KATOH, H •••••••••••••••••••••••••. 92
KIMURA, K. • ••••••••••••••••••••••• 83
KIMURA, M. .
70
KITAzAwA, T•..........'
46
KOBAYASHI, M •••••••••••.•••••••••• 50
KOIZUMI, A. • ••••••••••••••••••••••• 42
KOJIMA, H. • •••••••••••••••••••.•••. 41
KONISHI, H. • ••••••••••••••••••••••. 41
KUBOTA, M. • ••••••••.•••••••••••••• 57
KURIHARA, Y. .
,':39, 55, 85
KURODA, Y. .
40, 41, 42, 43, 44, 49
KUSUDA, J •••••••••••••••• ,' ••.•••••• 54
LEE, W. H ..•............. 56,83,84,87
LEH, C
57
MARUYAMA, T
76,79,82
MATSUNAGA, E•.............. 97, 98, 99
MINATO, K. • ••••••••••••••••••• 45, 46
MrwA, M:
81
MIYASHITA, N ••• ::.:•••••••••••• 36, 37
MIYATA, T
90
MIYAZAWA, S ••••••••••••••••••••.• 108
MIZUMOTO, K ••••••••••••••••••••••• 23
MORIMOTO,K•••••••••••••••••••.••• 42
MORISHIMA, H •••••••••• 89, 100, 101, 102
MORIWAKI, K ••••••••• 35,36,37,38,39,
55, 56, 68, 85, 87, 92
MUKAI, T•.......................... 77
MURAKAMI, A. • •••....•... 46, 48, 49, 50
MURAKAMI, K ••••••••••••••••••• 62,68

BARBIER, P.... :: ...•............... 102
BENZ, R. D. '................•..•.... 64
BROWN, W. L. Jr.
CARSTEN,A. L• •••••••••••••••••• 57

64

CHEN, I. S. Y•....................... 81
CHIGUSA, S. I •••••••••••••••••••••••. 79
CHIToRAKON, S •••••••••••••••••••.• 100
CHO, W.-S;...•..................... 87

ENDo, T •••.•.....•...... : .•......• 103
FUERST, P. A •••••••••••••••••••••••• 76

Fum, T.

30, 66

FUJITA,N••••••••••••••••• 17,18,19,20
FUKASE, Y

49

FUKUDA, R•................. 21,22,25
GADRINAB, L. U •................... 107
GLASS, R. E•••..•.•......••..•••.... 17
GOJOBORI, T. .
79, 80, 81, 82
GOLDE, D. W. .
81
GOTWALD, W. H. Jr•••••••••••••••••• 57
HARA, H ••••••••••••••••••••••••••. 32
HARADA, Y. . ....•........... 38, 55, 68
HASEGAWA, M'
25
HATADA, E. ','25
HATSUMI, M. .
83
HAYASAKA, K. • ••••••••••••••••••••. 98
HIROTA, Y.. :.'.. :32, 33HONDA, A. ..,'•••••••••••••••••••••• 23
HORAl, S
97,98
HOUBA-HERIN, N •••••••••••••.•••••• 32
lHARA, M
107
IDA, K•............................ 32
IKEMURA, T. .
28, 29lKuSHIMA, T
64
IMAI, H. T. •.......... 56, 57, 58, 59, 85
IMAMOTO, F. •20
INOUE, T•...............•.... 62, 63, 65
INOUE, Y,
27,52

IsHIHAMA, A •••••••ISHIKAWA, R.

.

126

ANNUAL REPORT OF NATIONAL INSTIT{)TE OF GENETICS NO. 36

NAGATA, K. . ..•............. 23, 24, 26
NAORA, H ....•..•...••....•........ 82
NATSUUME-SAKAI, S•..........•...... 35
NAwA, S
30
NEI, M....•..............•......•.. 73
NENE, V
17
NISHIMuRA, A. . ......•...... '22
NOMURA, T
17, 18, 19
NOZAWA, K •........................ 98
OHTA, T. . ....•.••..•.•...... :71
OHTSUKI, Y•........................ 46
OKA, H. I ............•............. 104
ONiMARU, K. .
::54
OOTSUYAMA, A•...•.•............... 63
OSATO, K
20
Qm, C.-G••........................ 85
PALUMBI, S. R
74
SADAIE, T .............•.....•...... 64
SADAIE, Y ..••.•..........• ;64
SAGAI, T............••.......... 35, 92
SAIGO, K
79
SAKAIZUMI, M. .
55
SANO, R. .
89, 102
SANO, Y. . .....•.....••....•••. 30, 106
SATO, Y.-I. .
100, 101, 102
SATTA, Y••...............•.•....... 79
SCHWARZ, U .............•.......... 32
SERIZAWA, H •....••....••....... 21,22
SHI, L.-M........•.•..•...••........ 85
SHIMADA, Y ..........•.••.••........ 44
SHIMIZU, K. .
25
SHIMIZU, N .........•............... 81
SHIMOTOHNO, K:
81
SHINKAWA~TACHI; K•••............. .42

SHIROISHI, T•.............•.......... 35
SHOTAKE, T•........................ 98
SIREG~' U. J. .
107
SLATKIN, M. . ..........•............ 73
SUGDdOTO, N.................•...... 52
SUGIMURA, T ...................•.... 81
SUGIYAMA, T
52
SuzuKI, A•.. .' . .': •• .':;99
SUZUKI, H. .
33
SUZUKI; K. . ...........••.. .' .•...... 87
SUZUKI, M
99
TACHIBANA, H •.•.... ' '" ..••.•...... 20
TAKADA, Y. ' ........•..... '..•...•.. .43
TAKAHASHI, J'..•....•. '.63
TAKAHASHI, K
82
TAKAHASHI, Y
81
TAKAHATA, N. . ......•.... 72, 13, 74, 75
TAKEUCHI, K
23
TAMAI, K
62
T;'NO, S.•......•............••..... 66
TAYLOR, R. W
: ....•.. 59
TEZUKA, H •.............••... 62, 63, 64
TJAN, K. N. . ...............•.•• 56, 57
TOMITA, T
38
TsuCHIYA, K. .
55
TUTIKAWA, K. .
68
UEDA, S•.•...••.•••••.............. 24
WATANABE, T. K
83,84
YAMAKAWA, T
99
YAMANE, K. .
33
YOKOYAMA, S
80,82
YONEKAWA, H
85,92
YONEZAWA, K
85, 104)TONG, H.-S•........•...•.•.•....... 57

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