ISSN 0077-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 31

1980

Published by
THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan1981

Annual Reportof theNational Institute of Genetics
No. 31,1980

Published by

The National Institute of Genetics, Japan198 ]Photo by Y. Nakagome

CONTENTS•

General statement
Staff
Council
Association for propagation of the knowledge of genetics
Projects of research for 1980
Researches carried out in 1980

I.

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Molecular Genetics
Relationship between structure of the 5'-noncoding region ofviral mRNA and efficiency in the initiation step of protein
synthesis in a eukaryotic system. YAMAGUCHI, K., HIDAKA,
S. and MIURA, K.-I.
Terminal part structure of genome RNA of cucumber mosaic
virus. HIDAKA, S. and MIURA, K.-I.
Sequence of a putative promoter region for the rRNA genes oftobacco chloroplast DNA. TOHDOH, N., SHINOZAKI, K.
and SUGIURA, M.
The complete nucleotide sequence of a 16S ribosomal RNA genefrom tobacco chloroplast. TOHDOH, N. and SUGIURA, M.. .
The nucleotide sequence of 4.5S ribosomal RNA from tobacco
chloroplasts. TAKAIWA, F. and SUGIURA, M.
The nucleotide sequences of two tRNA Asn genes from tobacco
chloroplasts. KATO, A., SHIMADA, H. and SUGIURA, M.. . . .
Purification of the T4 DNA ligase by blue sepharose chromatography. SUGIURA, M.
.. .. . .. .. . . .. .. .. .. .. .. .. ..
Cloning of mitochondrial DNA from silkworm eggs. KUSUDA,
J. and ONIMARU, K.

II.

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Microbial Genetics
On the biological role of penicillin-binding proteins 4 and 5. De
PEDRO, M. A., SCHWARZ, D., NISHIMURA, Y. and HIROTA,Y.
On the process of cellular division in Escherichia coli: Isolationand characterization of penicillin-binding proteins la, Ib,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

and 3. TAMURA, T., SUZUKI, H., NISHIMURA, Y., MIZOGUCHI,
J. and HIROTA, Y
A mutant of Escherichia coli defective in penicillin-binding protein
5 and lacking D-alanine carboxypeptidase IA. NISHIMURA,
Y., SUZUKI, H., HIROTA, Y. and PARK, J. T. o.
Synthetic ColEl plasmids carrying genes for penicillin binding
proteins in Escherichia coli. TAKEDA, Y., NISHIMURA, A.,NISHIMURA, Y., YAMADA, M., YASUDA, So, SUZUKI, H. and
HIROTA, Y
Cloning of pan Band fts I gene of Escherichia coli and purificationof penicillin-binding proteins 18 and 3. KRAUT, Ho, KECK,
W. and HIROTA, Y. . ..
Isolation of an Eo coli DNA topoisomerase I mutant. STERNGLANZ, R., DINARDO, S., WANG, J. C., NISHIMURA, Y. and
HIROTA, Y
Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition. STERNGLANZ, R., DINARDO, S., VOELKEL, K. A., NISHIMURA, Y.,
HIROTA, Y., BECHERER, K., ZUMSTEIN, L. and WANG, J. c.. ..
Characterization of the dnaA gene carried by lambda transducing
phage. MURAKAMI, Ao, INOKUCHI, H., HIROTA, Y., OZEKI,
H. and YAMAGISHI, Ho" ..
Globomycin sensitivity of Escherichia coli and Salmonella typhimurium: Effects of mutations affecting structures of mureinlipoprotein. LAI, J.-S., PHILBRICK, W. M., HAYASHI, S.,
INUKAI, M., ARAI, M., HIROTA, Y. and Wu, H. C. 0.......
Cloning and nucleotide sequence of asparagine synthetase gene of
Escherichia coli. NAKAMURA, M., YAMADA, M., HIROTA, Y.,
SUGIMOTO, K., OKA, A. and TAKANAMI, M
Conjugative transfer of pBR322 with a chromosomal DNA of
Escherichia coli by Hfr. YAMADA, M. and HIROTA, Y.....
Isogenic and allogenic suppression of fts mutation of Escherichia
coli. YAMADA, M. and HIROTA, Y. . . . . . . . . . . . . . . . . . . . .
Electron micrographic observation of E. coli flagellar precursors.
KOMEDA, Y.
Characterization of ribosomal RNA genes from blue-green algae.
TOMIOKA, N. and SUGIURA, M. .
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CONTENTS

III.

Biochemical Genetics and Immunogenetics
Effect of major histocompatibility gene complex (MHC) on thesusceptibility to urethane induced lung tumors in mice.
MORIWAKI, K. and MIYASHlTA, N.
High frequency of intra-H-2 recombination in BIO.AjBIO.MOLSGR heterozygote. SHlROISHl, T., SAGAI, T. and MORIWAKI,

K.

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Allozyme studies on the Japanese wild populations of medaka,
Oryzias latipes. SAKAIZUMI, M., EGAMI, N. and MORIWAKI,

K.
Conventional and developmental null forms coded by the Acp-llocus in rice. ENDO, T.
An extraction system for leaf zymographic analysis in woody
plants. ENDO, T.
..........
Modification gene for peroxidase isozymes coded by Px-l locus
in rice. PAl, C. and ENDO, T.

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Developmental Genetics and Somatic Cell Genetics
The growth and differentiation of germ cells of Drosophila melanogaster in culture. KURODA, Y.. . . . . . . . . . . . . . . . . . . . . . .
Effect of lectins on the cartilage nodule formation of embryonic
quail limb bud cells in culture. KURODA, Y. and MATSUTANI,
E.
Mutagenic activity of Trp-P-2 and Glu-P-l on embryonic human
diploid cells in culture. KURODA, Y.
Metabolic activation of tryptophan pyrolysis products by S-9
mix in induction of mutations in cultured Chinese hamster
cells. KURODA, Y. and ASAKURA, M.. . ..... . .. . . .. .. .. . .
Developmental effects of radiation on embryos in the silkworm(Bombyx mori L.) MURAKAMI, A.. . . . . . . . . . . . . . . . . . . . . . .
Roles of the head-activation potential and head-inhibition potential gradients in hydra pattern formation. SUGIYAMA, Y.,
ACHERMANN, J. and TAKANO, J.
Morphogenetic potentials of chimeric hydra strain. WANEK, N.
and SUGIYAMA, T. . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .
An SR-spiroplasma unable to kill Drosophila males. YAMADA,
M. A., NAWA, S. and WATANABE, T. K.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Hatchability and some developmental characters of flies in natural
population of Drosophila melanogaster. MINATO, K.. . . . . . .
Sex-specific lethals of Drosophila melanogaster. OISHI, K. and
WATANABE, T. K.
Transplantability of teratocarcinomas derived from 6 day embryos of BlO H-2 congenic mice in aIIogeneic hosts. NoGUCHI, T. and TAYA, C. .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .
Aggregation chimeras between embryonal carcinoma ceIIs of atesticular teratocarcinoma (STT-2) and 8-ceII mouse embryos. HANAOKA, K. and NOGUCHI, T.

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Cytogenetics
Segregation of F 2 hybrids between Mauritius and Oceanian typeblack rats. YOSIDA, T. H.
Spontaneous occurrence of a mosaic rat carrying translocation/
normal and translocation/inversion pair no. 1 and its geneticalsurvey. YOSIDA, T H.
Segregation and fecundity of offspring from the 1/12 translocation
heterozygotes of the Norway rat. YOSIDA, T. H.
Segregation and fertility of the Norway rats with metacentric pairno. 1 derived by pericentric inversion. YOSlDA, T H.. . . . . .
Frequency of sister chromatid exchanges in lung primary culturesin the Indian spiny mouse, Mus platythrix. YOSIDA, T. H.
and INOUE, T.
Precocious X-Y chromosome dissociation in Mus musculus subspecies, inbred laboratory mice and their F 1 hybrids. MATSUDA, Y., IMAI,H. T, MORIWAKI,K., KONDO,K. and BONHOMME,

F.
Multiple sex chromosome mechanism (XXY) in the filefish,
Stephanolepis cirrhifer. MUROFUSHI, M., OIKAWA, S., NISHIKAWA, S. and YOSlDA, T. H.
Cytological studies on male crossing over in Drosophila ananassae.
MATSUDA, M., IMAI, H. T. and TOBARI, Y. N.

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Mutation and Mutagenesis in Animals
Rat-liver S-9 mediated mutagenesis in germ-ceIIs of the silkworm
by benso(a)pyrene. MURAKAMI, A.... , . ... . . . . . . . . . . . . ..

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CONTENTS

Specific locus mutations induced in the silkworm by diethynitrosamine. MURAKAMI, A.
Lack of effects on the mutagenicity with procarbazine in silkworms. MURAKAMI, A.
Dominant lethal mutations induced by some indirect-acting
mutagens in the silkworm. Bombyx mori. MURAKAMI, A. ..

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Radiation Genetics and Chemical Mutagenesis in Microorganisms andPlantsGenetic study on the initiation of sporulation in Bacillus subtilis.
SADAIE, Y.
On the process of competent cell formation in Bacillus subtilis.
SADAIE, Y. and KADA, T.
Antimutagenic effect of cobaltous chloride on Bacillus subtilis
and Escherichia coli mutator strains possessing error-prone
DNA replicating enzymes. INOUE, T., SHIZAKI, M., KATO,
M. and KADA, T.
Antimutagenic activities of human placental extract on radiationinduced mutations in bacteria. KADA, T. and MOCHIZUKI,
H. ..
Antimutagenic factor in leaves of camelia families. KADA, T.
and KANEKO, K.
Effects of p-irradiation from 3H-water on transforming DNA of
Bacillus subtilis. KADA, T., MOCHIZUKI, H., INOUE, T. andSADAIE, Y.
Human enzymes functioning in repair of DNA damage induced
by gamma-irradiation. INOUE, T. and KADA, T.
On the expression of waxy starch mutants induced by thermal
neutrons in rice. AMANO, E.
Induction of waxy starch mutants in rice. AMANO, E.
Induction of waxy mutants in maize by gamma-ray. AMANO, E.. .
Automated analysis of maize pollen. AMANO, E.
Trial to induce waxy starch mutant in diploid wheat. AMANO,
E.
Effect of tumor promoter in soybean. FUJII, T.

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VIII.

Population Genetics (Theoretical)

Average time until fixation of a mutant allele in finite population
under continued mutation pressure: Studies by analytical,
numerical, and pseudosampling methods. KIMURA, M ....
A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. KIMURA, M.
Genetic variability and effective population size when local extinction and recolonization of subpopulations are frequent.
MARUYAMA, T. and KIMURA, M.
Amino acid diversity of immunoglobulins as a product of molecular evolution. OHTA, T. 0..........................
Linkage disequilibrium between amino acid sites in immunoglobulin genes and other multigene families. OHTA, T.. . . .
Two-locus problems in transmission genetics of mitochondria
and chloroplasts. OHTA, T. .. . . . . . . . . . . . . . . . . . . . . . . . . . .
Composite stepwise mutation model under the neutral mutation
hypothesis. TAKAHATA, N.
Polymorphism and loss of duplicate gene expression: A theoretical
study with application to tetraploid fish. TAKAHATA, N.
and MARUYAMA, T.
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Population Genetics and Evolutionary Genetics (Experimental)
Molecular evolution in papova viruses and their host species, andin bacteriophages. MARUYAMA, T. and SOEDA, E.
Stasipatric speciation in Drosophila. WATANABE, T. K. and
KAWANISHI, M
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Directional change in the frequencies of cosmopolitan inversionsof D. melanogaster. INOUE, Y. and WATANABE, T. K.......
Molecular phylogenies of Drosophila: Protein differences detectedby two-dimensional electrophoresis. OHNISHI, So, KAWANISHI, M. and WATANABE, T. K.
Geographical and ecological variations of Oryza perennis at six
isozyme loci. SECOND, G. and MORISHIMA, H.
Isozyme polymorphism in natural populations of Oryza perennis.
PAl, C. and MORISHIMA, H
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CONTENTS

X.

Human Genetics and Human Cytogenetics
Hereditary retinoblastoma: Host resistance and second primarytumors. MATSUNAGA, E.
Retinoblastoma: Host resistance and 13q-chromosomal deletion.
MATSUNAGA, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ectopic pregnancy and myoma uteri: Teratogenic effects andmaternal characteristics. MATSUNAGA, E. and SHIOTA, K. . .
Search for maternal factors associated with malformed humanembryos: A prospective study. MATSUNAGA, E. and SHIOTA,
K.
Inactivation centers in the human X chromosome. NAKAGOME,Y.
The "loss" of kinetochore from X chromosomes of aged females.
NAKAGOME, Y. and IINUMA, K.
High-resolution banding. A very simple procedure. NAKAGOME, Y. and MATSUBARA, T.
Preservation of whole blood for chromosome analysis. NAKAGOME, Y., YOKOCHI, T., MATSUBARA, T. and FUKUDA, F. ...
Two cases of Down syndrome with unusual karyotypes-A psu
dic (21) (q22) and a rob (14q2lq) in cousins, and maternallytransmitted extra ring (21). MATSUBARA, T., NAKAGOME, Y.,
IINUMA, K., OKA, S., OGASAWARA, N. and YOKOCHI, T. . ....

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Behal'ioral Genetics and Ecological Genetics
A parasitic wasp of Drosophila.

KAWANISHI, M. and WATANABE,

T. K.
Development of resistance to organophosphorous insecticides in
Drosophila. INOUE, Y.
Selection for the compound learning ability of mice. FUJISHIMA,
T.
The behavioral changes in mice continuously reared under noise
conditions. FUJISHIMA, T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Niche differentiation between perennial and annual types within
populations of Oryza perennis. MORISHIMA, H. and SANO,
Y.
Effect of experimental soil pollution on weed communities in rice
fields. MORISHIMA, H.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Studies on pioneer trees and the pioneer index: Comparison between the tropics and the temperate zones. SAKAI, K. I. and
IYAMA, S.

XII.

Applied Genetics
Nitrogen fixing activity of bacterial strains In association withrice plants. FUJII, T.
Further evidence for "one locus sporo-gametophytic interaction"model for F 1 sterility genes between Oryza sativa and O.
glaberrima. SANO, Y.
The direction of pollen flow between two cultivated rice species,
Oryza sativa and O. glaberrima. SANO, Y.
Mendelian segregation analysis for three isozyme loci in ricecultivars. SECOND, G. and MORISHIMA, H.
An introgressed electromorph found in an isogenic line of Oryza
sativa with a sterility gene from O. glaberrima. SECOND, G.
and SANO, T.
Simulation experiment on the mass selection for a quantitative
character when the effect of genes is not equal. IYAMA, S.. .

Books and papers published in 1980 by staff members
Abstracts of diary for 1980
Foreign visitors in 1980
Author index

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GENERAL STATEMENT•

Science of genetics has made remarkable progress in recent years. Since
the foundation in 1949, the Institute has devoted itself to the promotion of
fundamental researches on genetics, taking up research subjects of wide
range. So far, it has been operated under a policy to keep a balance among
each of ten research departments. Such policy permitted us to cope withmost research subjects in relevant fields. However, amazingly rapid
progress in this science has obliged us to reconsider the policy hitherto
adopted. Furthermore, we have been pressed by another unavoidable
circumstances. Staff members of the Institute has been reduced in number
year by year due to budgetary limitation of the government. Thus, theInstitute has confronted serious problems.
In order to find a new turn of the situation we have been discussing forseveral years about switching over from the present system to other one as
research institute for joint use by the universities. There had been several
arguments for and against the reorganization. After full discussion, theFuture Planning Committee of the Institute concluded at the meeting on
June 3rd that adoption of the new system should not be retarded in orderto keep up with the rapid progress of the genetic studies. The matter was
taken up into deliberation at the annual meeting of the Council held on
June 20th. The Council decided that it would be advisable for the Institute to take the new system. Then, having obtained the agreement of all
strata of the Institute, I offered our proposal to the Ministry of Education,
Science and Culture, and at the same time I requested our Future Planning
Committee to embody the plan in detail.
The number of staff members who went abroad decreased to 18 from 32of the preceding year, however, the visitors to our institute from othercountries came up to 62, twice as many as last year. Among them visitorsfrom People's Republic of China were the most in number. Those whohave been working as long term visiting researchers under the grant in aid
from the Japan Society for the Promotion of Science or with the aid fromscientific exchange fund of their own countries are: Dr. Gerard Second
from France in Department of Applied Genetics, Dr. C. Pai from Taiwan
in Departments of Applied Genetics and Biochemistry, Dr. Josef Acher-2

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

mann from Switzerland and Dr. Nancy L. Wanek from U.S.A. in Department of Biochemistry, Dr. Harold W. Keck and Dr. Hildegard Kraut from
West Germany in Department of Microbiology. The Institute is varied in
the nationality of the visiting researchers.
Dr. Hiko-Ichi Oka, the head of the Department of Applied Genetics,
retired on April 1st by reason of the age limit. He has long been working
in this institute as a competent leader of rice research group. I feel regret
at his retirement. Dr. Takeo Maruyama, the chief of the first laboratoryof the Department of Population Genetics was appointed to the post of thehead of the Department of Physiological Genetics which had been left
vacant for a year. It is regretful that Dr. Mitsuo Tsujita, honorary member,the former head of the Department of Biochemistry, succumbed to an
untimely death on April 7th. May his soul rest in peace!

..

STAFF
Director.

TAZIMA, Yataro, D. Ag.
Members

1. Department of Morphological Genetics
KURODA, Yukiaki, D. Sc., Head of the Department
The 1st Laboratory
MURAKAMI, Akio, D. Ag., D. Sc., Head of the Laboratory
The 2nd Laboratory
KURODA, Yukiaki, D. Sc., Head of the Laboratory
MINATO, Kiyoshi, M. Sc.
2.

Department of Cytogenetics
YOSIDA, Tosihide H., D. Sc., Head of the Department
The 1st Laboratory
YOSIDA, Toshihide, H., D. Sc., Head of the Laboratory
YAMAMOTO, Masatoshi, Ph. D.
The 2nd Laboratory
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
IMAI, HIROTAMI, D. Sc.

t

3.

Department of Physiological Genetics
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
WATANABE, Takao, K., D. Sc., Head of the Laboratory
The 2nd Laboratory
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Laboratory

4.

Department of Biochemical Genetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

YAMADA, Masa-Aki, M. Sc.
The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
ENDO, Toru, D. Ag.
The 3rd Laboratory
SUGIYAMA, Tsutomu, Ph. D., Head of the Laboratory
FUJISAWA, Toshitaka, Ph. D.

•
5.

Department of Applied Genetics
TAZIMA, Yataro, D. Ag., Head of the Department
The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
KAWAHARA, Takatada, D. Ag.
FUJISHIMA, Tohru, D. Ag.
The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
The 3rd Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag., Head of the Laboratory

6.

Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department
The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
INOUE, Tadashi, D. Ag.
The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
AMANO, Etsuo, D. Ag.
SADAIE, Yoshito, D. Sc.

7.

Department of Human Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department
The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
The 2nd Laboratory
NAKAGOME, Yasuo, D. Med., Head of the Laboratory

STAFF

8.

Department of Microbial Genetics
HIROTA, Yukinori, D. Sc., Head of the Department
The 1st Laboratory
HIROTA, Yukinori, D. Sc., Head of the Laboratory
NISHIMURA, Yukinobu, D. Sc.
The 2nd Laboratory
YASUDA, Seiichi, D. Sc., Head of the Laboratory
YAMADA, Masao, D. Sc.

9.

Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
OHTA, Tomoko, Ph. D., D. Sc., Head of the Laboratory
The 2nd Laboratory
KIMURA, Motoo, Ph, D., D. Sc., Head of the Laboratory
TAKAHATA, Naoyuki, D. Sc.
AOKI, Ken-ichi, Ph. D.

10.

Department of Molecular Genetics
MIURA, Kin-ichiro, D. Sc., Head of the Department
The 1st Laboratory
MIURA, Kin-ichiro, D. Sc., Head of the Laboratory
SOEDA, Eiichi, D. Ag.
SHIMOTOHNO, Kunitada, D. Pha.
The 2nd Laboratory
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
SHINOZAKI, Kazuo, D. Sc.

11.

Genetic Stocks Center
YOSIDA, Tosihide H., D. Sc., Head of the Center
Animal Section
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
NOGUCHI, Takehiko, D. Sc.
INOUE, Yutaka, D. Sc.
KUSUDA, Jun, D. Ag.
Plant Section
FUJII, Taro, D. Ag., Head of the Laboratory

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SANO, Yoshio, D. Ag.
Microbial Section
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
KOMEDA, Yoshibumi, D. Sc.
NISHIMURA, Akiko
12.

Experimental Farm
FUJII, Taro, D. Ag., Head of the Farm
MIYAZAWA, Akira

13.

•

Department of Administration
KITAHARA, Kunio, Head of the Department
lORI, Toshiteru, Chief of the General Affairs Section
KIMURA, Susumu, Chief of the Finance Section
Honorary Members

KIHARA, Hitoshi, D. Sc., Director of the Kihara Institute for BiologicalResearch, Member of Japan Academy, Emeritus Professor of Kyoto
University
SAKAI, Kan-ichi, D. Ag.
MORIWAKI, Daigoro, D. Sc., Emeritus Professor of Tokyo Metropolitan
University
OSHIMA, Chozo, D. Sc.
OKA, Hiko-Ichi, D. Ag.

COUNCIL
FUJII, Takashi, Chairman, Emeritus Professor of University of TokyoINOUYE, Eiji, Professor of University of Tokyo
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
KONDO, Norio, Professor of Tokyo University of Agriculture
MISONOO, Keisuke, Member of Atomic Energy Commission
MORIWAKI, Daigoro, Emeritus Professor of Tokyo Metropolitan University
MOROHOSHI, Seijiro, President of Tokyo University of Agriculture andTechnology
MUKAIBO, Takashi, President of University of Tokyo•

COUNCIL

7NAGAKURA, Saburo, Professor of University of TokyoOOSAWA, Fumio, Professor of Osaka University
SASA, Manabu, Emeritus Professor of University of TokyoSHINOZAKI, Nobuo, Director of Institute of Population Problems
TAKAHASHI, Man-emon, Professor of Hokkaido University
UMEZAWA, Hamao, Emeritus Professor of University of Tokyo•

ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Emeritus Professor of Tokyo Metropolitan
University
MATSUNAGA, Ei, Managing Director, Head of the Human Genetics Department
YOSIDA, Tosihide H., Managing Director, Head of the Cytogenetics Department
SINOTO, Yosito, Manager, Professor of International Christian University
WADA, Bungo, Manager, Emeritus Professor of University of TokyoTAZIMA, Yataro, Manager, Director of the National Institute of Genetics
OSHIMA, Chozo, Manager,

PROJECTS OF RESEARCH FOR 1980
Department of Morphological Genetics

Genetic studies on insect cells in tissue culture (KURODA and MINATO)
Developmental genetic studies on carcinogenesis in tissue culture (KURODA)
Genetics of somatic mammalian cells in culture (KURODA and MINATO)
Cytogenetics in the silkworm (MURAKAMI)
Studies on recombination in the silkworm (MURAKAMI)
Genetics of the silkworm (MURAKAMI, FUKASE and OHNUMA)
Radiation and chemical mutagenesis in the silkworm (TAZIMA and MURAKAMI)
Department of Cytogenetics

Studies on chromosome evolution and species differentiation in rodents andother small mammals (YosIDA)
Chromosome study on experimental tumors (YoSIDA)
Experimental breeding and genetics of mice, rats and other wild rodents(YOSIDA and MORIWAKI)
Genetic study on the subspecies differentiation of mouse (MORIWAKI)
Immunogenetical study on the histocompatibility antigens of wild rodents(MORIWAKI)
Cytogenetical study of ants (IMAI)
Cytogenetical studies on Drosophila (YAMAMOTO)
Cytogenetical study of fishes (YOSIDA)
Department of Physiological Genetics

Behavior genetics of Drosophila (WATANABE and OSHIMA)
Evolutionary and ecological genetics of Drosophila (WATANABE and KAWANISHI)
Population genetics of Drosophila (WATANABE, INOUE, and TsuNo)
Theory of population genetics and speciation (MARUYAMA)PROJECTS OF RESEARCH FOR 1980

9

Department of Biochemical Genetics

Studies on transformation in higher organisms (NAWA and YAMADA)
Genetical and biochemical studies of pteridine metabolisms in insects(NAWA)
Analysis of gene action on cell differentiation in higher organisms (NAWA
and YAMADA)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Genetical and biochemical studies of human serum protcins (OGAWA)
Genetical and biochemical studies on Japanese middle size dog (OGAWA)
Genetics of isozymes in plants (END0)
Genetic analysis of developmental mechanisms in hydra (SUGIYAMA andFUJISAWA)
Department of Applied Genetics

Quantitative genetic studies in poultry (KAWAHARA)
Genetic studies in wild populations of Japanese quails (KAWAHARA)
Behavioral genetic studies in mice (FUJISHIMA)
Theoretical studies on breeding techniques (IYAMA)
Genetic studies of trees in natural forest (IYAMA)
Evolutionary studies in wild and cultivated rice species (MORISHIMA andSANO)
Ecological genetic studies in weed species (MORISHIMA)
Genetic effects of environmental pollution on plant population (NAMA andMORISHIMA)
Department of Induced Mutation

Molecular mechanisms of spontaneous, chemical- and radiation-induced
mutations (KADA, SADAIE and INOUE)
Environmental mutagens and carcinogens (KADA, SADAIE, TUTIKAWA and
HARA)
Radiation genetics in mice (TUTIKAWA)
Biochemical factors involved in cellular repair of genetic damage (INOUE
and KADA)
Genetics of Bacillus subtilis (SADAIE and KADA)

10

ANNUAL REPORT OF NATIONAL lNSTITUTE OF GENETICS NO. 31

Molecular mechanisms of unicellular differentiation in Bacillus subtilis(SADAIE)
Radiation and chemical interaction in the cells (KADA)
Genetic fine structure analysis in maize (AMANa)
Department of Human Genetics

Genetic and epidemiologic studies on certain malformations In humanembryos (MATSUNAGA)
Genetic studies on retinoblastoma (MATSUNAGA)
Clinical cytogenetic studies of congenital abnormalities in man (NAKAGOME)
Molecular cytogenetic studies of human chromosomes (NAKAGOME)
Studies on human chromosome variants (NAKAGOME and MATSUNAGA)
Department of Microbial Genetics

Genetic regulatory mechanisms of DNA replication in E. coli (YASUDA)
Genetic regulatory mechanism of cellular division in E. coli (HIROTA,NISHIMURA and YAMADA)
Molecular genetics on DNA replication (YASUDA and HIROTA)
Genetic studies on bacterial cell envelope (HIROTA and NISHIMURA)
Synthetic bacterial plasmid (YASUDA and NISHIMURA)
DNA replication origin of E. coli (YASUDA, YAMADA and HIROTA)
Department 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 (ORTA)
Theoretical studies on altruism (AOKI)
Department of Molecular Genetics

Studies on the structure of genome RNA of viruses (MIURA)
Studies on the primary structure of DNA (SOEDA and MIURA)
Studies on structure and function of messenger RNA (MIURA).,PROJECTS OF RESEARCH FOR 1980

11

Genetical and enzymological studies on E. coli polymerase (SUGIURA)
Studies on T 4 RNA ligase (SUGIURA)
Cloning of eukaryotic genes and their structural analysis (SUGIURA andSHINOZAKI)
Genetic Stocks Center

Studies and conservation of germplasm resources in rice and wheat species(FuJII and SANa)
Specificity of mutagen tolerance in higher plants (FuJII)
Exploitation of genetic ability of nitrogen fixation in Graminae (FuJII, SANa,
IYAMA and HIROTA)
Studies on genetic differentiation in rice (SANa)
Cytogenetic studies of Norway rats and establishment of the chromosomal
mutant stocks (YOSIDA)
Experimental breeding of wild rodents for estabilshment of the new experimental animals (YOSIDA)
Developmental genetic studies on mouse teratomas (NOGUCHI)
Studies on chromosomal polymorphism in Drosophila (INOUE)
Analysis of fibroin genes of silkworm and its relatives (KUSUDA)
Genetical study of flagellar formation in Escherichia coli K-12 (KOMEDA)
Basic studies on the gene purification and the construction of DNA banks(SUGIURA)RESEARCHES CARRIED OUT IN 1980

I.

MOLECULAR GENETICS

Relationship between Structure of the 5'-noncoding Region ofViral mRNAs and Efficiency in the Initiation Step ofProtein Synthesis in a Eukaryotic System
Kazuko YAMAGUCHI, Soh HIDAKA and Kin-ichiro MIURA

To determine whether the rate of protein synthesis is controlled by the
structure of mRNA near its 5'-terminus, protein synthesizing ability, especially in its initial stage, was compared among RNAs of plant viruses.
Those viruses used here contain several definite pieces of single-stranded
RNA. Each of these RNAs acts as a messenger. Cucumber mosaic virus(CMV) RNA 5 synthesizes a small amount of a protein, molecular weight
7000, in an ill vitro protein synthesizing system from wheat germ or reticulocyte. Brome mosaic virus (BMV) RNA 4 synthesizes a large amountof a coat protein under the same conditions. Both RNAs carry the same5'-cap structure and a short noncoding region (ten nuc1eotides in CMV
RNA 5 and nine in BMV RNA 4) from the 5'-terminus to the initiation
codon AUG. A sequence complementary to the 3'-terminal of ISS ribosomal RNA is contained in BMV RNA 4, but not apparent in CMV RNA 5.
Formation of the initiation complex for protein synthesis by the 5'-terminallabeled mRNA of cytoplasmic polyhedrosis virus was inhibited by theaddition of cold BMV RNA 4, whereas it was only slightly inhibited bycold CMV RNA 5. BMV RNA 4 which has a sequence complementary
to rRNA can form the initiation complex more easily than CMV RNA 5.
It is concluded that an apparent complementary sequence to the 3'-terminalof ISS rRNA in the 5' noncoding region of eukaryotic mRNA as well as
the 5'-cap structure enhances the rate of initiation complex formation inprotein synthesis.
If the 5'-noncoding region is large, tertiary structure of this region seems
to arrange mutual relationships among these functional sites. Large hairpin structures can bring the initiation codon near the 5'-cap and/or the

RESEARCHES CARRIED OUT IN 1980

13

complementary sequence to the 3'-terminal of 18S ribosomal RNA. An
example of the possibility is seen in CMV RNA 4 that codes a coat protein
and carries 76 bases in the 5'-noncoding region (succeeding report, S. Hidaka
and K. Miura). Another example is seen in mRNA of a precursor of
corticotropin/,3-lipotropin, in which the 5'-noncoding region consists of
128 bases (Eur. J. Biochem. 113, 531-539 (1981), A. Inoue, M. Nakamura,
S. Nakanishi, S. Hidaka, K. Miura and S. Numa).
Terminal Part Structure of Genome RNA of Cucumber
Mosaic Virus
Soh HID AKA and Kin-ichiro MIURA

A genome of cucumber mosaic virus (CMV) is consisted of four pieces(strain 0) or five pieces (strain Y) of single-stranded RNA. Molecularweights of these RNA segments are RNA 1: 1.23 X 106 , RNA 2: 1.13 X 10 6 ,RNA 3: 0.83x10 6 , RNA 4: 0.33x10 6 and RNA 5: 0.1 X 10 6 , respectively.
Nucleotide sequence near the 5'-terminus and the 3'-terminus of these RNA
segments was analyzed. Every RNA segment of CMV carries the same5'-cap structure m 7 G 5 'pppG_.
After the cap part is deleted chemically, the 5'-terminus of RNA is labeledwith [32 P] phosphate by nucleotide kinase. The RNAs were digested
partially by weak alkali and ribonucleases (TI, D 2). The resulting oligoand poly-nuc1eotides were separated by gel electrophoresis and autoradiographed on a X-ray film. By this analysis the sequence of guanine(G), adenine (A) and pyrimidine (Y) was determined. The partial alkaline
digest was electrophoresed in pH 3.5 and pH 7 two-dimensionally in a gel,and autographed. By this analysis cytosine (C) and uracil (U) are distinguished. The 3'-terminus of RNA was bound with [32P]pCp by RNA
ligase. The 3'-labeled RNA was digested partially by chemical methods
and analyzed by gel electrophoresis as carried out in the nucleotide sequencing near the 5'-terminus.
The 5'-terminal nucleotide sequence including the 5'-cap, m 7G 5 'pppG_
D-D- is common for all the RNA segments of CMV, but the first initiation
codon for protein synthesis, A-D-G, appears at 56th in RNA 1 and 2, at
77th in RNA 4, and at 11th in RNA 5. It is known that RNA 4 carries acoat protein information. The amino acid sequence of the N-terminal of
the coat protein was analyzed to determine the location of initiation codon

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

of coat protein cistron. Since CMV coat protein is acetylated at the Nterminal amino acid as other plant viruses, N-acetylated oligapeptides
obtained by pronase digestion were analyzed. The N-terminal aminoacid sequence was identified as Ac-Met-(Asx), here Asx means asparagine(Asn) or apartic acid (Asp). In CMV RNA 4, the first initiation codon
A-U-G appears at 77th from the 5' -terminus, and the nucleotide sequence
following to A-U-G just corresponds to the N-terminal of coat protein:
AUG (Met)-GAU (Asp)-AAU (Asn). Therefore, the initiation codon at
the 77th nucleotide is confirmed as the true initiation codon for protein
synthesis. Upstream (5'-side) of the initiation codon contains nucleotide
sequences complementary to the 3'-terminal part of ISS rRNA in CMV
RNAs 1,2 and 4, but not in RNA 5. The 5'-noncoding region of RNA 1,
2 and 4 is the almost same between 0 strain and Y strain of CMV, though
there are several base changes.
The 3'-terminal nucleotide sequence was very similar among the RNA
segments of CMV, 10 nucleotides sequence from the 3'-terminus is perfectly
common for RNAs 1 to 4. It would mean that the structure near the
3'-terminus functions for template recognition of the RNA replicase.
The N-terminal analysis of protein was coworked with the late Prof.
Kozo Narita and Dr. Susumu Tsunazawa of Osaka University. CMV wasprepared by Drs. Susumu Kubo and Yohichi Takanami of Central ResearchInstitute of the Japan Tobacco and Salt Public Corporation.

•"
Sequence of a Putative Promoter Region for the rRNA
Genes of Tobacco Chloroplast DNA
Naaki TOHDoH, Kazua SHlNOZAKI and Masahiro SUGIURA

The nucleotide sequence of the segment of tobacco chloroplast DNA
adjacent to and including the start of the 16S rRNA gene has been determined (766 base-pairs). The region just preceding this gene was foundto contain a gene (see Fig. 1, pp 15) and promoter-type sequences similar
to those which occur in E. coli were found before this tRNA gene. E. coli
RNA polymerase can recognize these sequences and in vitro co-transcribes
the tRNA and rRNA genes.

15

RESEARCHES CARRIED OUT IN 1980
BeU:nHil'\fI,I

CTI'ACC'AT TATCCT'l"f'fT CTACTCACG.\

AT~TT(::;C'~

TGTTCCT.o\AG AAAACCAlITT 'I'GTCCATITT TCSCGCTCTC AMCG-:;CCCT GGMhCCCAT AAG/lhCTCTT CIlATCCMhA CAGA'l'GTMC,,

JllnfI

-500

HindI

,

J!lnfI

1'CCACTTCCr TCGCAATCCG 'rAGTCAATAA TA'ITTCCGAT ACGGCCAr::rr CACA.j'\r~AA TCCGATTTTC ACCATTAl'TT T'::hTATCCCT AAThCT'JCGA

HnIl!

1

- ~ 00lt paII
AAACAhGCCC CCGCTCCAAC TTCTTCMCA ATAC'C-::;CCC'f 'l'CACTTTCTC CACCCTTfCA CTTAGChTTA CTCACTTCTJ\ T:T(;'fr.:c,\l':'; CC:GI:S::SGi:.~~lH

1RNAValGGGATATAAC TC"GCCGTAG hc'reTCACe! TCACGTGGTC ~_~.CTCATCA GTTCCACCCT CATTAT~:~""CCCC!IATG'f':;N:;TTT~T(; C'!,\';'j"j'r;r;!,'[-HO

lIr,II

TfGCTCCCCC ecce'fCcTTe AA'I'GAGAATG CATAACACGC TCCTGGCAIT GhCGTGAGGG GGCAGCSATG CCTlITATTTC rC:C:C:"'';CGAJ\

C'l"':~':GGGC{;A.

HhaI
llinn
I\hI
lJlnrItttl
ATATCMCCC CATGGATACA AGTTATGCCT TGCAATGAAA CACUTTCCG AA'fCC~C1TT CTGTflCCAAG AACGAlIC';TA TAASTM'fC:C AJ\STATGA!IT

-laD

Sa.'A

'.6StRNA..
.~l~er":TCA1'..~CAGA GTTCGATCCT GGC'l'CAGCAT CMCCCTGGC GCr:A'l'CCTTA ACACA";"GCAA GTCCGACGGG AAC1Y.;(..:r~:TT 'f~~C!,I":'lfCCG Cf,C(;C:S~A'~..fl

PvuII(AluI)'rA,\~~CGT/...l\. CAACCl'CCCC 'nCGCAGGGC AACAACM~tFig. 1. Nucleotide sequence in the region at the start of tobacco chloroplast
16S rRNA gene.

The Complete Nucleotide Sequence of a 16S Ribosomal RNA
Gene from Tobacco Chloroplast
Naoki TOHDOH and MASAHIRO SUGIURA.

The complete nucleotide sequence of a 16S ribosomal RNA gene fromtobacco chloroplast has been determined (Fig. 1, p. 16). The coding region of
the 16S rRNA gene is 1485 base-pairs long. Its nucleotide sequence has 96 %homology with that of maize chloroplast 16S rRNA gene and 74 %homology
with that of E. coli one. The 3' terminal region of this gene contains thesequence ACCTCC which is complementary to sequences found at the 5'
termini of prokaryotic mRNAs. The large stem structure can be constructed from the sequences surrounding the 5' and 3' ends of the 16S gene.
The Nucleotide Sequence of 4.5S Ribosomal RNA
from Tobacco Chloroplasts
Fumio TAKAIWA and Masahiro SUGlURAThe 4.5S RNA has recently been found in the large subunit of the chloroplast ribosomes from a number of higher plants. In tobacco chloroplasts, the 4.5S RNA is present in approximately equimolar amount as

16

ANNUAL REPORT OF NATIONAL INSTITURE OF GENETICS NO. 31

GCAAGCGTT~tGG''''AnrA rGGGCGTAAAGC
I~+-=="",~~~",-,-,""",,,,,,,,,,,',"-,G,,,C,,,AA,,,G,,,C",G.!.TT"-,,,CCGGMTI":;r\I.GGGCGTA.l\.AGC

A~TAAGT
rc

I

G

TMCT

AAGC1' '!l.G' CT~GffT~~GCGGTGAAATGCGT

!\AGe

c~r

!l.G

Cc}~.$A@!l.G~I~hkb~GT

GCGGTGAAATGCGTTGC/V\CTCcrPj1nTGM9f -'GGMTCGCT,\GTfV\TC If FCt~~\ flcfflGGTGJ\l'J tcc:'TCCCGGGCCTTGTACACACCG'r~ TGC;V'l.CTCd:~ATG!v\gcGGAATCGCTAGTAATCGCC~tI.f!lg::cMre:;GGTGAA

GTTCCCGGGCCTTGT1\CACACCG

Fig. 1. Nucleotide sequence of tobacco chloroplast 16S rRNA gene. For
comparison, sequence of E. coli 16S rDNA (upper row) is presented and homologous regions are boxed.

the chloroplast 5S RNA and is coded for by the chloroplast DNA between
the 23S and 5S rRNA genes. The nucleotide sequence of tobacco chloroplast 4.5S ribosomal RNA has been determined. The 4.5S RNA is 103
nucleotides long and its 5 ' -terminus is not phosphorylated. A possible
secondary structure for the 4.5S RNA can be constructed as shown in Fig. 1.
(p. 17). This work was published in the Nucleic Acids Research 8, 41254129 (1980).

RESEARCHES CARRIED OUT IN 1980

17......cUG
70A
A§,CG GGC
U A 80

AU
SOG

U
G

GUAUC C

UA
A
C

A

A

G

G

C

C

A

U
GA

C90
GG

U
AG
AU
U A 50 GUAG
C
GC
CG
AU
UA
AU

30A
AC
G

G A20
AU
C G100,GCCGUUUAGGUGUAAC OH (3)C GG CA
40
AG
10 C
U

G
G
A

A
OHG

( 5')
Fig. 1.

A possible secondary structure of tobacco chloroplast 4.5S RNA.

The Nucleotide Sequences of two tRNAAsn Genes from
Tobacco Chloroplasts•

Akira KATO, Hiroaki SHIMADA and Masahiro SUGIURA

Recombinant plasmids which contain EcoRI fragments of tobacco chloroplast DNA carrying tRNA genes were constructed. Plasmids pTC2l1
and pTC293 contain the base sequences for tRNAAsn in their 1.4 and 1.1
Md EcoRI fragments, respectively. These two tRNA sequences are identical. Fig. 1 (p. 18) shows the cloverleaf structure that can be deduced forthe tRNAAsn based on the DNA sequence. Each tRNAAsn gene is locatedat about 0.9 kb apart from the distal end of each 5S rRNA gene and iscoded for by the DNA strand opposite from that of the rRNA genes.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

A

C
C
A

pA-U

G-C
G-C
G-C

A-U
U-A

CGACUCAG

A-U UAGUCCG A
I I I Ii
G
AU
AUCAGT1UCCu

I I , IGUAGAGU G

U-G
C-G

A

A

f

T

G

A-U

C-G

C-G
U
U
G
U

GA C
Fig. 1. Tobacco chloroplast tRNAA,n (right) and tRNA Val (left).

Purification of the T4 DNA Ligase by Blue Sepharose
Chromatography
Masahiro SUGIURA

T4 DNA ligase catalyzes the formation of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxylends in nicked duplex DNA.
In addition, it catalyzes the joining of duplex DNA molecules at completely base-paired ends. These activities of T4 DNA ligase have beenused to synthesize DNA with defined sequences and to construct recombinant DNA molecules in vitro.
As T4 DNA ligase was found to bind to Blue Sepharose CL-6B, Blue
Sepharose CL-6B chromatography was introduced at the last step of purification procedure. The purified T4 DNA ligase preparation gave one
band on SDS-polyacrylamide gel electrophoresis and the molecular weight
was estimated to be 60,000. Thus Blue Sepharose chromatography removed
at least nuclease(s) active on (dT)lo, which could not completely be removed
by DEAE- and P-cellulose chromatography and gel filtration. This work
was carried out with assistance of Mrs. Mie Kusuda and was published in
the Analytical Biochemistry 108, 227-229 (1980).

•

RESEARCHES CARRIED OUT IN 1980

19

ThE' physical map of silkworm mt DNA.

•

Cloning of Mitochondrial DNA from Silkworm Eggs
Iun KUSUDA and Kimiji ONIMARU•

Several hundred of silkworm mutants were isolated and these relative
genetic loci were determined on chromosomes as a linkage maps. In
contrast to extensive analysis of chromosomes of silkworm, genetic andchemical characters of the extrachromosomes is little known. Silkworm
mitochondria show heterologous forms in the midgut or Malpighian tissues.
Elucidation of the gene organization of mitochondria has been difficult
because of its incapability of mating. Recent advance of gene cloning andnucleotide sequencing techniques allow us to analyse the fine structure ofthis organella DNA and estimate the genes coded on it. We have started
to study the structure of silkworm mitochondrial DNA by the applicationof these techniques. Mitochondrial DNA isolated from silkworm (strain
P-22) eggs were cleaved by Pst I into three fragments of 7.1 kiro base pairs(kbp), 6.2 kbp and 1.2 kbp long. Genome size of this DNA, estimatedfrom the summation of the size of Pst I fragments, was 14.5 kbp. Threefragments were cloned into E. coli plasmid pBR322 in order to obtain large
quantities of DNA homologous to mitochondrial DNA for further analysis.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Pst I fragments of the mitochondrial DNA were joined to pBR322 cleavedwith Pst I using T, ligase and transformed E. coli HBlOl. Transformants
containing recombinant plasmids were selected by the colony hybridization
using 32P-labeled mitochondrial DNA from the Tetracycline resistant and
Ampicirine sensitive colonies. Seven colonies hybridized to 32p labeledprobe. Gel analysis of Pst I digests of recombinant plasmids revealed thatthe three types of clones were obtained. pBmt24-2, pBmt13 and pBmt30
contain the Pst I fragments A, Band C, respectively. pBmt24-1 contains
both Pst I-B and Pst I-C fragments. pBmt24-1 and pBmt24-2 DNA weredigested with Sal I, Bam HI, Bgi IT, Sst I, Pst I, Kpn I, EcoRI and Hind III.
The location of cleavage sites were deduced from the electrophoretic patterns of double and triple digests with these enzymes. A physical map ofwhole mitochondrial DNA was constructed by the combination of pBmt24-1
and pBmt24-2 maps. As illustrated, mitochondrial DNA from silkworm
eggs contains 23 cleavage sites of 8 restriction enzymes tested. Sixteen ofthem were located on the region of 7 kbp long corresponding to half genome.
Since 6 among 8 enzymes used are specific for hexanucleotides including
4 GC base pairs, we assumed that the nucleotide sequence of this region
was rich in GC base pairs and the replication origin could be located in theanother half region containing few restriction sites, because this region isassumed to be rich in AT base pairs.

•

RESEARCHES CARRIED OUT IN 1980

II.

21

MICROBIAL GENETICSOn the Biological Role of Penicillin-binding Proteins 4 and 5
Miguel Angel de PEDR0 1), Uli SCHWARZ,l) Yukinobu NISHIMURA,
and Yukinori HIROTA•

Recent studies in vivo of murein biosynthesis in E. coli show that newly
synthesized murein differs in its chemical composition from preexisting
murein. Murein newly inserted into growing cells is less cross-linked thanthe preexisting murein and it carries pentapeptide subunits. The pentapeptide subunits act as donors in a secondary maturation process which
involves further transpeptidation by which the final stage of cross-linkage
in murein is achieved. This reaction seems to involve penicillin-binding
protein 4. In mutants with a defective penicillin-binding protein 4, thesecondary transpeptidation is abolished and the murein, in this case, is notprocessed further and remains poorly cross-linked. This indicates thatpenicillin-binding protein 4, which in vitro has carboxypeptidase IB and
DD-endopeptidase activity, may act as a transpeptidase in the intact cell.
First hints on the possible role of penicillin-binding proteins 5 and/or 6,
which show carboxypeptidase IA activity in vitro come from an accumulation of pentapeptide side chains in intact sacculi. These pentapeptide
subunits were identified by their strong and stable interaction with vancomycin. Exponentially growing cultures of appropriate mutants were
either pulse-(5 min) or uniformly labelled with tritiated diaminopimelicacid and the amount of pentapeptide-containing subunits and the extent ofcross-linkage in the murein were determined.
As listed in Table 1, mutants defective in penicillin-binding protein 5
show an increase of pentapeptide subunits, both after pulse- and uniformlabelling of the murein. All mutants tested with defective carboxypeptidase IA show a dramatic accumulation of pentapeptide in the murein.
Thus, penicillin-binding protein 5 seems to function as a carboxypeptidase
not only in vitro but also in vivo. It converts pentapeptide subunits, theactivated substrate for transpeptidation, into tetrapeptides.
The double mutants with defects in penicillin-binding proteins 4 and 5--_.~_ . . .

1)

Max-Planck-Institut fUr Virusforschung.

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31
Table 1. Cross-linkage and accumulation of pentapeptide subunits
in the murein of wild-type and mutant strains of E. coli
Cross-linkage"

Pentapeptide b

StrainPulse-labelled
5min

Uniformlylabelled

Pulse-labelled
5min

Uniformlylabelled

0.22±0.012
0.20±0.01
0.20±0.009
0.22±0.014
0.27±0.002

0.28±0.013
0.27±0.011
0.21±0.011
0.29±0.003
0.29±0.006

3 ±0.452.6±0.6
10 ±1.4
29 ±6.042 :l:0.88

0.3±0.18
0.4±0.15
0.9±0.1
5.6±1.49
15 ±0.96

0.26±0.01

0.27±0.009

36

15

~_

W-7 (Wild type)5509 (Wild type)DL64 (dacB) 4
1191 (dacA) 5
5683 (ponA, dacA,
dacB)C5684 (dacA, dacB)-----------

•
b
C

±1.05

..

_.-~-

•

±0.4
----~-----

Cross-linkage is expressed as molar fraction of cross-linked dimers in total murein.
Expressed as percentage of total monomeric lactyl-peptides.
Leaky for carboxypeptidase 1b activity; no penicillin-binding protein 4 detectable.

as well (strain JE5683 and JE5684) have a highly cross-linked murein both
after pulse- and uniform-labelling of the murein. This is unexpected
because a defect in penicillin-binding protein 4 alone results in decreased
cross-linkage (Table 1); maturation of murein via transpeptidation is abolished. However, the enormous accumulation of pentapeptide subunits inthe mutants may favour an abnormal formation of peptide cross-links in
the initial step of murein insertion which under normal conditions yields
poorly cross-linked murein. (For details, see FEMS Microbiology Letters9,219-221 1980).
On the Process of Cellular Division in Escherichia coli:
Isolation and Characterization of Penicillin-binding
Proteins la, 1b, and 3
Toshihide TAMURA, Hideho SUZUKI, Yukinobu NISHIMURA,
Junzo MIZOGUCHI, and Yukinori HIROTA

Multiple mutants of Escherichia coli defective in penicillin-binding proteins (PBPs) were constructed, and into these strains ColE! plasmids carrying the genes for PBP-la, -lb, or -3 were introduced. From these plasmidcarrying strains, PBP-l a and -1 b were purified by ampicillin-Sepharoseaffinity chromatography and PBP-3 by cephalexin-Sepharose affinity chromatography. Improved purification was achieved by differential elution"

RESEARCHES CARRIED OUT IN 1980•

23with NH 2 0H. Purified PBP-Ib synthesized murein when added to themembrane fraction of a PBP-Ib-defective mutant, which by itself failed tosupport murein synthesis in vitro. The PBP-l b preparation was able tosynthesize murein from the lipid intermediate extracted with chloroform/
methanol but was unable to utilize UDP-linked precursors for murein
synthesis. Murein synthesis was inhibited by vancomysin, ristocetin,
moenomycin, and endruacidin, but not by p-lactam antibiotics. The
synthesized murein was shown to contain crosslinked muropeptides. Theircrosslinking was abolished by action of p-lactam antibiotics. The PBP-laand -3 preparations showed substantially no activity for murein synthesisin the same reaction system. None of the three PBPs showed D-alanine
carboxypeptidase activity with UDP-N-acetylmuramyl-pentapeptide as
substrate or endopeptidase activity with bis(disaccharide-peptide) as substrate. (For detail, see Proc. Nat!. Acad. Sci. USA 77, 4499-4503, 1980)
A Mutant of Escherichia coli defective in Penicillin-binding. Protein 5 and Lacking D-alanine Carboxypeptidase IA
Yukinobu NISHIMURA, Hideho SUZUKI, Yukinori HIROTA,
and James T. PARK 1)•

A mutant of Escherichia coli defective in penicillin-binding protein 5
activity was isolated. The mutation (pfv) was shown to be located at
14.0 min on the E. coli chromosome map. Loss of penicillin-binding
protein 5 in the pfv mutant was associated with the loss of D-alanine carboxypeptidase IA activity and increased sensitivity to p-lactam antibiotics. We
conclude that penicillin-binding protein 5 catalyzes the major D-alanine
carboxypeptidase IA activity and that the enzyme activity, in vivo, protects
E. coli cells from killing by low inhibitory concentrations of p-lactam antibiotics. (For detail, see Journal of Bacteriology, July 1980, 531-534)
Synthetic ColEI Plasmids Carrying Genes for Penicillin-Binding
Proteins in Escherichitt coli
Yutaka TAKEDA, Akiko NISHIMURA, Yukinobu NISHIMURA, Masao YAMADA,
Seiichi YASUDA, Hideho SUZUKI and Yukinori HIROTA

Clarke and Carbon's collection of 2,000 Escherichia coli strains which
1)

Tufts Univ. Sch. of Med., Boston.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

harbor ColEl plasmids carrying small random segments of the E. coli
chromosome was screened for the correction of mutational defects in penicillin-binding proteins (PBPs): ponA (PBP-Ia), ponE (PBP-Ib), dacE (PBP-4)
and pfv (PBP-5). We found the plasmids carrying the chromosomal segments containing ponA + -aroB+ (pLC29-47), ponB+ -tonA + (pLC4-43, pLC-4
44 and pLCI9-19) and argG+ -dacB+ (pLClO-46 and pLCI8-38). Charactersof these plasmids were analyzed. Two other plasmids (pLC26-6 andpLC4-14) previously found to correct an ftsl mutation (Nishimura et al.,1977) were also investigated further. Restriction maps of chromosomal
DNAs carried by pLC29-47, pLC4-44, pLCI9-19, pLCI8-38, pLC26-6 andpLC4-14 were constructed. The regions of ponB-tonA on pLC4-44 andpLCI9-19, and of leuA-ftsI-murE & F on pLC26-6 were located on the
restriction maps. Although both pLC26-6 and pLC4-14 corrected a thermosensitive mutation,ftsl, which causes a defect in cell division due to abnormal
PBP-3, only pLC26-6 led an ftsl mutant to restore production of PBP-3,
while pLC4-14 did not. Restriction and hetero-duplex analyses of pLC26-6
and pLC4-14 have shown the absence of nucleotide sequence homology
between them. The plasmids, pLC29-47 carrying ponA+ and pLC4-43,
pLC4-44 and pLC19-19 carrying ponB+ led the host cell to overproduce
the respective PBP. (For details, see Plasmid, 1981)
Cloning of pon Band fts I Gene of Escherichia coli and Purificationof Penicillin-Binding Proteins 1B and 3
Hildegard KRAUT, Wolfgang KECK and Yukinori HIROTA

In the inner membrane of Escherichia coli at least 7 penicillin-binding
proteins were detected, to which penicillin binds covalently (Spratt, E.G.
1975, Proc. Natl. Acad. Sci. 72: 2999-3003). Since ,3-lactam antibiotics are
known to inhibit cell division and cell elongation, the role of these penicillinbinding proteins with respect to the murein metabolism has been investigated.
In the meantime, with the exception of penicillin-binding protein 6, mutants
for all these PBPs had been isolated (Suzuki et al. 1978, Proc. Natl. Acad.
Sci. 75: 664-668, Spratt, E.G. 1977, J. Bacteriol. 131: 293-305, Matsuhashi
et al. 1978, Proc. Natl. Acad. Sci. 75: 2631-2635). By studying thesemutants it was suggested that PBP-IB is directly involved in the final polymerization steps in murein biosynthesis (Suzuki et al. 1977, Ann. Rep.
Natl. Inst. Genet. 28: 23-24). An enriched fraction of PBP-lB synthesi:z;ed•

RESEARCHES CARRIED OUT IN 1980

"

•

25

crosslinked murein from lipid linked precursors (Tamura et al. 1980, Proc.
Natl. Acad. Sci. 77: 4499-4503).
The activity of PBP-3 is essential for cell division, which can be demonstrated by the follwing evidence. Specific p-lactam antibiotics, like benzylpenicillin or cephalexin which cause filament formation, bind at the effective
concentration preferentially to PBP-3 in the inner membrane (Spratt, B.G.
1977, Eur. J. Biochem. 72: 341-352). Mutants with a genetic defect in
PBP-3 possess a thermolabile PBP-3 and a temperature sensitive defect inseptum formation in parallel. Our main effort focused on these two PBPs
because of their essential functions in cell elongation and cell division.
Clarke and Carbon's synthetic Col El plasmid collection was screened forthe correction of mutational defects in penicillin-binding proteins and therestriction maps of the plasmids were established (Takeda et al. 1981,
Plasmid, in press). Escherichia coli strains carrying pLC 19-19 (pon B)
or pLC 26-6 (Its I) show about lO-fold overproduction of PBP-IB or -3
respectively (Tamura et al. 1980, Proc. Natl. Acad. Sci. 77: 4499-4503).
Starting from these plasmids we cloned the two structural genes pon B
and jts I independently into a plasmid with temperature dependent copy
number. This high copy number plasmid pSY 343 is derived from theminiplasmid pBED 17 which was constructed from a runaway replication
mutant pKN 402 of the antibiotic resistance factor Rl (Dhlin et al. 1979,
Gene 6: 91-106). Seiichi Yasuda exchanged the ampicillin resistance in
pBED 17 with kanamycin resistance to get the plasmid pSY 343. This
plasmid replicates at temperatures above 35°C without copy number control
during 2-3 hours. This gives a 500-1000 fold increase in gene dosage ofplasmid-coded genes compared to an average chromosomal gene. During
gene amplification, cell growth and protein synthesis continue at the normal
rate and lead to an overproduction of plasmid gene products.
The jts I gene was cloned from pLC 26-6 into the high copy number
plasmid pSY 343 by using the Bam HI and Eco RI site (Fig. 1). A mutant
strain (JE7627) with defective PBP-IE and -4 was transformed with thisnew plasmid pWK 7. For overproduction of PBP-3 the cells were grownin the presence of kanamycin at 30°C, shifted to 37°C in the early logarithmic phase and cultivated for 4 more hours. During this time the cells
reach 150-fold overproduction of PBP-3 compared to wild type cells, all ofwhich is inserted into the inner membrane.
. The cloning of pon B gene was a little more complicated, because there

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Pst I
Eco RI

Hind III

.
Smal

BamHI

Hind III

Pst I

Sma I

Fig. 1.

Cloning of jis I gene into the high copy number plasmid pSY 343.

are no suitable restriction sites in the plasmid pLC 19-19, which could be
used for direct cloning into the high copy number plasmid pSY 343. We
first used the Pst I sites to clone the pon B gene from pLC 19-19 into thePst I site of the vector pBR 322 (Fig. 2). Plasmids with both possible
orientations of the integrated fragment could be isolated (pHK 1811, pHK1824). Both types of plasmids lead to an unexpected high overproductionof PBP-1B, about 120-fold compared to the wild level. For the vectorpBR 322 only 30 copies per cell are normally described. Recently it was
found that the deletion of a given non-essential region from Col El-likeRESEARCHES CARRIED OUT IN 1980

27

Hind III•

6g1 II
Pst I
Sal I

pLC19-19

Pst I
Sma I
Pst I
Sa I I Pst I
Pst I

~

\..5l

Hind IIISal I

p6R 322

Hind III
6g1 II
Pst I
Sal I

Sal I

~
Fig. 2.

Cloning of pan B gene into the plasmid pBR 322.

plasmids results in an increased copy number. Moreover, other Col £1
sequences, which might be lost in the plasmids pHK 1881 and pHK 1824,
are involved in determining plasmid copy number (Twigg, A. J. and D.
Sherratt, 1980, Nature 283: 216-218). For further cloning of pan B genefrom pHK 1811 into the high copy number plasmid pSY 343 the Hind III
sites were used (Fig. 3). The plasmid pHK 231, which contains the complete DNA ofpHK 1811, pBR 322 and pSY 343, was obtained. A multiple

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Pst I

Sa I I~•

Hind III

Hind IIIBgi II

Pst I

Ps t ISal I

Eco R I
Sal I

Sma I
Smal

Bglli

Pst I

Smal

Bam HI

Kpn I
Eco RI
Sal I

Fig. 3.

Recloning of pOll B gene into the high copy number plasmid pSY 343.

mutant strain with temperature sensitive PBP-IA, which was also defectivefor PBP-IB and -4, was transformed with this new plasmid pHK 231.
Cells are grown as already described in the case of PBP-3. In these cells
PBP-IB was overproduced ISO-fold or more, compared to wild type level.
All this protein was membrane bound.

..

RESEARCHES CARRIED OUT IN 1980

29

By cloning and amplification of these two structural genes and the overproduction of the gene products we are able to isolate both penicillinbinding proteins and study them on the protein level, and we can isolate
the DNA-fragments, that code for the structural genes of these two proteinsand study them on the DNA level. For the purification of PBP-IB and -3
we used overproducing cells with defects in other penicillin-binding proteins.
After opening the cells the PBPs were extracted from the membrane fraction
and the crude extract was applied to covalent-affinity-chromatography.
For the isolation of PBP-IB ampicillin was coupled to activated CRSepharose 4B (Pharmacia) and for PBP-3 cephalexin was used. Afterloading the columns with crude extracts the columns were washed extensively with buffer containing 2M NaCI. The covalently bound penicillinbinding proteins are then eluted with buffer containing 1M hydroxylamine.
Both proteins could be obtained by this single step purification in pureform. Starting with 25 g wet weight of cells we could obtain, in a typicalpurification procedure, about 5-10 mg of pure PBP-IB and about 2 mg ofpure PBP-3.
With the purified proteins we are now able to study the structural properties, the binding-sites and hydrophobic sequences of these two proteins.
We will also continue to unravel their enzymatic activities, their bindingand complex-stability with different p-Iactam antibiotics. All this information will provide insight into the very complex process of cell growth andcell division in Escherichia coli.

.

Isolation of an E. coli DNA Topoisomerase I Mutant
Rolf STERNGLANZ,l) Stephen DINARDO,l) James C. WANG,2)
Yukinobu NISHIMURA and Yukinori HIROTA

A collection of temperature-sensitive E. coli mutants has been screenedin order to find a DNA topoisomerase I (w) mutant. Two such mutants
have been found, both with a significant reduction in w activity. One
mutant has about 10 % residual topoisomerase activity and the other has
1-5 % the activity of wild-type. The remaining activity is resistant to oxolinic acid and therefore is presumably due to w. The temperature sensitivity of the mutants can be removed by PI transduction or conjugation,
1)

2)

State Univ. of New York, Stony Brook.
Harvard Univ., Cambridge.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

and the resultant strains still lack (j) activity. Therefore, either (j) is notessential for E. coli or the residual activity present in the mutants is sufficient for viability. The mutation causing the lack of (j) activity maps near
trp at about 27 min on the E. coli genetic map. (For details, see Mechanistic
Studies of DNA Replication and Genetic Recombination, 1980)•

Mutations in the Gene Coding for Escherichia coli DNA
Topoisomerase I affect Transcription and Transposition
Rolf STERNGLANZ,!) Stephen DINARDO,!) Karen A. VOELKEL,!)
Yukinobu NISHIMURA, Yukinori HIROTA, Kathleen BETHLERER,2)Louis ZUMSTEIN,') and James C. WANG 2 )Mutations in top, the structural gene for Escherichia coli DNA topoisomerase I, have been identified and mapped at 28 min on the chromosome,near cysB. Strains carrying deletions of the top gene are viable. The top
mutations, however, do exert pleiotropic effects on transcription and transposition. Mutants lacking DNA topoisomerasc I have a more rapid rateof induction and a higher level of catabolite-sensitive enzymes including
tryptophanase and p-galactosidase. This general activation of transcription by top mutations can be attributed to an increase in the negative superhelicity of the DNA in vivo when the topoisomerase activity is abolished.
The frequency of transposition of Tn5, a transposon carrying kanamycin
resistance, is decreased by a factor of 40 or more in top mutants. A direct
or indirect role of the topoisomerase in transposition is discussed. The
transposition frequency of Tn3, however, is not dependent on top. Basedon the studies of the E. coli top mutants, it appears that the supX gene,which was originally studied in Salmonella typhimurium [Dubnau, E. &Margolin, P. (1972) Mol. Gen. Genet. 117,91-112] is likely to be the structural gene for DNA topoisomerase 1. (For detail, see Proc. Natl. Acad.
Sci. USA 78,2747-2751, ]981)

1)

2)State Univ. of New York, Stony Brook.
Harvard Univ., Cambridge.

..

RESEARCHES CARRIED OUT IN 1980

31

Characterization of the dnaA Gene Carried by Lambda
Transducing Phage
Akira MURAKAMI, Hachiro INoKucHI, Yukinori HIROTA,
Haruo OZEKI, and Hideo Y AMAGISHI•

Specialized transducing phages AdnaA were obtained by inducing lysogens
in which J..tna was integrated at the tnaA region of the Escherichia coli
chromosome; the tnaA region is located in the vicinity of the dnaA gene.
The dnaA- deletion derivatives of J..dnaA were isolated from the lysate ofJ..dnaA grown on bacteria carrying a transposon Tn3.
The structures of various transducing phages thus obtained were determined by heteroduplex DNA mapping. From these results, the transducing fragment of l3.8-kb-Iong was divided into nine domains. Upon
infection of UV-irradiated cells with the phage, production of polypeptidesof 49 kD and 42 kD was specifically associated with infections by the dnaAand recF transducing phages. Polypeptides of 49 kD and 42 kD appeared
to be coded for by dnaA and recF genes, respectively. The dnaA gene was
assigned to the region of 2.8-kb-Iong which extends by 2.4 kb in the counterclockwise direction on the E. coli genetic map and 0.4 kb in the opposite
direction, as measured from the nearest HindUI site close to the tnaA gene.
The recF gene was also discovered to lie very close to dnaA in the order of
tnaA-dna A-recF.

Merogenotes heterozygous for the dnaA gene were constructed by introducing F'lOO-12 carrying AdnaA into the recipients with different mutations at or near dnaA. For combinations, F'(AdnaA+)jdnaA46 andF'(J..dna+)jdna-83, dnaA+ was trans-dominant, whereas the dnaA+ wasrecessive for F'(AdnaA+)jdna-5. For F'(AdnaA+)jdna-167, the result of thetransdominance test was affected by the growth media employed; dnaA +was dominant on a J..-broth plate, and dna-167 was dominant on an M9minimal plate. Thus, transdominance of dnaA+ in heterozygotes is affectedby difference in mutations and growth media. (For details, see Mol.
Gen. Genet. 180, 235-247, 1980)

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Globomycin Sensitivity of Escherichia coli and Salmonella
typhimurium: Effects of Mutations Affecting Structuresof Murein Lipoprotein
Jiunu-Shyong LAI,l) William M. PHILBRICK,l) Shigeru HAYASHI,!>Masatoshi INUKAI,2) Mamoru ARAI,2) Yukinori HIROTA,
and Henry C. Wu 1)

The sensitivity of strains of Escherichia coli and Salmonella typhimurium
to globomycin is increased in mutants defective in the lipopolysaccharide
structure. E. coli mutants altered in the structures or biosynthesis ofmurein lipoprotein are more resistant to globomycin than the parental
strains. (For details, see Journal of Bacteriology, Jan. 1981, 657-660)

Cloning and Nucleotide Sequence of Asparagine Synthetase
Gene of Escherichia coli
Masataka NAKAMURA, Masao YAMADA, Yukinori HIROTA,
Kazunori SUGIMOTO, Atsuhiro OKA and Mituru T AKANAMI

We have subcloned the asnA gene of E. coli K-12, a gene coding for
asparagine synthetase, from a previously cloned 6 magadalton segment of
E. coli chromosome containing the DNA replication origin, ori, and asnA
(Yasuda, S. and Y. Hirota. 1977 Proc. Natl. Acad. Sci. USA 74: 5458).
The complete nucleotide sequence of the asnA gene was determined: theregion of the structural gene extends 990 base-pairs which codes for apolypeptide of 330 amino acid residues with a molecular weight of 36,688
daltons. The nucleotide sequences of the promoter and the ribosomebinding site of the gene are also assigned. (For details, see Nucl. Acid
Res. 1981)
Conjugative Transfer of pBR322 with a Chromosomal DNA
of Escherichia coli by Hfr
Masao YAMADA and Yukinori HIROTA

It is well known that the non-autotransmissible plasmid, ColE!, can betransferred efficiently in the presence of F plasmid, while the common
1)

2)

Uniformed Services Univ. of Health Sci., Bethesda.
Res. Laboratories, Sankyo Co. Ltd., Tokyo.

•

RESEARCHES CARRIED OUT IN 1980•33

cloning vectors derived from ColEl-like plasmid, such as pBR322, pBR3l3
and pMB9, could be transferred barely in the presence of F, since they lack
the region corresponding to mob (mobility) gene of ColEl (Clarke, L. and
J. Carbon, 1976, Cell 9: 91, Young, I. G. and M. 1. Poulis, 1978, Gene 4:175). However, we found that such cloning vectors could be transferredfrom Hfr to recipient cells by a bacterial mating if they had a chromosomal
DNA of E. coli. When the chromosomal DNA in the plasmid corresponded
to the region transferred early by the Hfr, the frequency of the transfer was
significantly high. The transfer depended on the recA function of the donor
and recipient cells.
We speculate the transfer mechanism mediated by Hfr as follows. All
the transfer machinary is provided by the Hfr. Hybrid plasmid may associate with the specific region of the chromosome with the homology between them. When the chromosome was transferred by Hfr, the associatedplasmid simultaneously transferred to recipient cells. Any non-autotransmissible plasmids may be transferred to recipient cells by mating, aslong as they have the homologous sequence with the DNA to be transferredby another plasmid or Hfr.
This method will be a powerful tool for determination of chromosomal
site of the unidentified fragment cloned on plasmids, since we can test
whether or not the plasmid in question is transferred by a brief mating witha particular Hfr. Many Hfr strains with different transfer origin anddirection are available for the mating. This method will be also useful toenrichment of desired plasmids. Passage of cloned plasmids from appropriate Hfr strain to any recipient, enriches the plasmids with downstream
region of transfer origin of the Hfr.
Isogenic and Allogenic Suppression of Its Mutation ofEscherichia coli
Masao YAMADA and Yukinori HIROTA

In order to elucidate the molecular mechanism of cell division, we are
studying on the temperature-sensitive (ts) mutation of E. coli defective incell division and the hybrid plasmids suppressing the ts mutation.
Nishimura et al. (1977, Plasmid 1: 67) found 5, 4 and 2 plasmids correctingftsB, E and I mutaions, respectively, among the gene bank of E. coli
prepared by Clarke and Carbon (1976, Cell 9: 91). We analyzed the plasmid

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31
Table 1.

pLC plasmids that suppressed ts phenotype of fts mutation.
-~----------~---------

fts mutant

pLC plasmid

isogene- --------------

----

----

pLC 3-46
pLC 19-24
pLC 42-17
pLC 1-41
pLC 6-16

ftsB
ftsB
ftsB

pLC 31-16
pLC 31-32
pLC 19-48

ftsE
ftsE

all ogeneother genecarried by
the plasmid- - - - - - - - - _ .....

chromosomal
location_ - _ ... _ - - - - -(IlrdB) glpT
IlrdAB glpT
(IlrdB) glpT
sufB
sufB

"------------

48 min53- - - - - - - - - - - - - - - - - - - - " - - - _ _------ - - - - ...

74-79
slifE

aceE

3
--~------~

JE 10730 (ltsI-)

pLC 26- 6
pLC 4-14

ftsl

leliA murEF
sufI

2
64

DNAs with restrIctIOn enzymes, constructed their restriction map andsubcloned appropriate fragment of the plasmids. By these experiments,
we found that the plasmids correcting jts mutation could be classified into
2 groups. One had the gene itself that was defective in particular jts mutant(isogenic suppression). The other had an entirely different segment fromthe isogene with respect to the restriction map and the homology revealed
by hybridization analysis. It was confirmed that the segment was derived
from chromosome of E. coli by Southern blotting technique. The chromosomal location site of the segment was determined. The summary of theresults is shown in Table 1.
We named this phenomenon as allogenic suppression and the gene responsible for the suppression as allogene. It is probable that the allogene could
suppress ts mutation because of the elevated gene dosage of the cloned
material or that it was the normally silent gene which turned on as a consequence of being cloned.
Electron Micrographic Observation of E. coli
Flagellar Precursors
Yoshibumi KOMEDA

Escherichia coli mutants with defects in 29 flagellar genes identified so
far were examined by electron microscopy for possession of incomplete
flagellar structures in membrane-associated fractions.

•

RESEARCHES CARRIED OUT IN 1980•

35

In vivo gene fusion technique has revealed the transcriptional interactionof these flagellar genes in E. coli K-12 (Komeda, US-Japan Cooperative
Microbiology Congress, 1979). The flagellar genes were divided into six
groups for their transcriptional control. The hag gene is expressed after
the expression of other flagellar genes. The mutants with defects in thehag genes have the most complex structures with hook-basal body structures. Accordingly, it was plausible to expect that the early genes in thetranscription sequence were responsible for the simpler structure of aflagellum; the late genes were responsible for more complex structure.
Identification of the precursor structure of each mutant will elucidate therole of transcriptional control for flagellar assembly. Therefore, precursor
structures of each mutant were examined on the basis of their possible roleas assembly intermediates of a flagellum. Suzuki et al. (1978) succeeded
to identify these precursor structures of flagella by the aid of electron
microscope in S. typhimurium. So far, the electron microscopy has given
us the most reliable data for analysis of flagellar precursors. We applied
this method to identify precursor structures of E. coli flagellar mutants.
Hook-basal body structures were detected in flaD, flaS, flaT, flbC, andhag mutants. The flaE mutant had a polyhook-basal body structure. An
intact basal body appeared in flaK mutants. Putative precursors of thebasal body were detected in mutants with defects in flaM, flaU, flaV, andflaY. No structures homologous to the flagellar basal body or its parts
were detected in mutants with defects in flaA, flaB, flaC, flaG, flaH, flaI,
flaL, flaN, flaO, flaP, flaQ, flaR, flaW, flaX, flbB, and flbD. One flaZ
mutant had an incomplete flagellar basal body structure and another formed
no significant structure, suggesting that flaZ is responsible for both basal
body assembly and the transcription of the hag gene.
The result was compared with that offla-Iac study (last issue of this report).
Except the flaU mutants, it was noted that defects of the genes in the lategroup (hag, flaS, flaT, flbC) had the most complex precursor: hook-basal
body structure. I believe the scheme (last issue of this report) reflects atleast some aspects of the interaction of flagellar gene products for theirsynthesis and assembly.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Characterization of Ribosomal RNA Genes from
Blue-Green Algae
Noboru TOMIOKA and Masahiro SUGIURA

Blue-green algae are micro-organisms with chlorophyll-a as a photosynthetic pigment. Many species possess the ability of N 2-fixation, though
they evolve O 2 by their photosynthesis. About two thousand species arewidely distributed over the world and the algae have a long history during
the biological evolution on the earth. An endosymbiotic theory explainsthat chloroplasts were derived from an ancestral photosynthetic prokaryotes
related to the blue-green algae. In spite of their interesting characters, thepresent knowledge of genetics in the organisms is limited. Recent progressin molecular genetics, however, enable us to analyze fine gene structures.
Thus, we began to study rRNA genes of the organisms using gene cloning
and DNA sequencing techniques. First, we isolated total DNAs from
Anacystis nidulans, Anabaena variabilis and A. cylindrica, then purified by
CsCl equilibrium density centrifugation. Purified DNAs are digested withseveral restriction enzymes and fractionated by 0.7 % agarose gel electrophoresis. DNA fragments containing rRNA genes were detected bySouthern hybridization method. (5'3 2 P) rRNA was used as a probe. The
results showed that 5.5 and 4.2 Md Pst I fragments of Anacystis nidulans,
2.6 and 2.4 Md EcoRI fragments of Anabaena variabilis and 1.9 and 1.7 Md
EcoRI fragments of A. cylindrica contained rRNA gene(s). For further
analysis of the DNA fragments, it is necessary to clone these genes.

••RESEARCHES CARRIED OUT IN 1980

III.

37

BIOCHEMICAL GENETICS AND IMMUNOGENETICS

Effect of Major Histocompatibility Gene Complex (MHC) on the
Susceptibility to Urethane Induced Lung Tumors in Mice
Kazuo MORIWAKI and Nobumoto MIYASHITAIn addition to a pulmonary tumor resistance (ptr) gene probably locatedon chromosome 11, MHC seems to function in the processes of tumorigenesis. This study aims to reveal the effect of MHC on urethane-induced
lung tumor incidence by using A H-2 congenic strains. In susceptible AIJ
mouse, average number of lung tumor foci is 30.6. If H-2 a complex of
AIJ strain is replaced by H-2 b , H-2 f and H-2 s , those number reduced toabout 15 (Table 1). These results clearly show the profound regulatory
function of MHC for the lung tumor development by urethane in mousestrains with susceptible ptr gene. Suppressive effect of wild-derived chromosomes is also investigated in F i hybrids between ICR mouse and MOL.
TEN inbred strain derived from Japanese wild mouse.
Table 1.

Strains

Effect of H-2 complex on the lung tumor incidence
induced by urethane

H-2 haplotypes

Number of pulmonary tumor fociper mouse
FemaleMale~---_._-"---

A
ASW
A BY
A.CA
BI0. A
BIO
BI0. TENI
mo. TEN2abfab
W mo1 - 1
W mol - 2

26.7±4.6(12)9. 8±3 .4(17)14.9±5.3(19)17.5±4.5(l4)1.9±1.5( 8)1.0±1.0(l2)0.9±1.2( 7)0.1±0.2( 8)34.5±6.1(l2)13.6±5.3(12)13 .0±5. 2(25)15.3±6.5(14)1.7±1.5( 7)0.5±0.9(10)1.1±0.9( 9)
( 7)
0

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

High Frequency of Intra-H-2 Recombination in
BIO.AjBIO.MOL-SGR Heterozygote
Toshihiko SHIROISHI, Tomoko SAGAI and Kazuo MORIWAKI

Intra-H-2 recombinant has been one of the most powerful tools for thecurrent genetic analyses of the H-2 complex and traits it controls.
We have produced several BlO.H-2 congenic strains which carry the H-2
complex derived from Japanese wild mouse. Although these strains arecharacteristic and useful materials themselves, further precise studies require
intra-H-2 recombinants in which one end of H-2 complex is derived fromwell known H-2 haplotype of inbred strains and the other end from thoseof wild mouse. For this reason, we attempted to produce the intra-H-2
recombinants, using BIO.A and BlO.MOL-H-2 congenic strains.
First, the males of three BlO.MOL-H-2 congenic strains, BlO.MOLTENI, BIO.MOL-SGR and BlO.MOL-YNG2 were crossed with BlO.A
females. Then, females of the F 1 hydrids were backcrossed to the respective parental BlO.MOL-H-2 congenic mice. The offsprings obtained inbackcross were tested for H-2 genotype by hemagglutination. H-2 antisera
used in H-2 typing were D-23 (anti-H-2K.23) and D-4 (anti-H-2DA), which
direct private antigens encoded in H-2K and H-2D end of BIO.A strain.
Since each BI0.MOL-H-2 strain does not have both of H-2K.23 and H2DA antigens, backcross offsprings carrying either one of them are recombinants.
We have screened 722 mice and already found 13 recombinants. Recombination frequency in three combinations of H-2 heterozygotes aredifferent remarkably each other (Table 1).
Table 1.

Frequency of intra-H-2 recombination. ._ - _ . _ - . _ . _ .

H-2
heterozygous
parents

No. of micescreened(%).

BlO. l/BlO. MOL-TEN 1
BI0. AlB 10. MOL-SGR
BI0. A{BlO. MOL-YNG2

209
396
117

1
12

o

---

Recombination
frequency

No. of micerecombinants_._----------

0.48
3.0
0.0

Among them, BlO.AjBlO.MOL-SGR heterozygote shows extraordinaryhigh recombination frequency. On the other hand, the other two combinations show ordinary value.

..

RESEARCHES CARRIED OUT IN 1980
Table 2.

39

H-2 genotype of new intra-H-2 recombinants

H-2 heterozygous
parents

H-2K

H-2D

BlO. AjBlO. MOL-TEN1
BIO. AjBlO. MOL-SGR
BIO. A/BlO. MOL-SGR

w*
w*
k

d
d

1
7

w*

5

w*:

Genotype

No. ofrecombinants

H-2 genotype derived from Japanese wild mouse.

The H-2 genotypes of new recombinants obtained in the present experiment are shown in Table 2.
In BlO.AjBlO.MOL-SGR heterozygote, the reciprocal coss-over types,
KW_D<l and Kk_Dw occurred with approximately equal frequency.
Klein (1975) reported that overall frequency of intra-H-2 recombination
is 0.41 percent based on 12,629 mice. Accordingly, the value of recombination frequency observed in BlO.AjBlO.MOL-SGR heterozygote isalmost one order higher than that in previous reports. Since all mice usedin the present experiment were BlO.H-2 congenic strains, the difference ofrecombination frequency observed here can be attributed exactly to the H-2
region on 17th chromosome.
Allozyme Studies on the Japanese Wild Populationsof Medaka, Oryzias latipes
Mitsuru SAKAIZUMI, Nobuo EGAMI,l) and Kazuo MORIWAKI

Allozymic variation at 21 loci in Japanese wild populations of the freshwater fish, Oryzias latipes, collected at 47 localities, was studied. By meansof unique alleles at the Adh, Pgm, Sdh, and Sod loci, the Japanese wild
populations could be divided into two major groups, the 'Northern Population' (northern coast of the Sea of Japan) and the 'Southern Population'(the rest part of Japan). No clinal distribution was observed at these loci,but the boundary of two regions was very distinct. The Southern Population was further divided into five subpopulations (the Eastern Subpopulation, the Inland Sea Subpopulation, the San-in Subpopulation, the Kyushu
Subpopulation, and the Ryukyu Subpopulation) by means of the uniquealleles at Acp, Amy, Got, Mod, and Pgd and the electrophoretic pattern ofmuscle lactate dehydrogenase. In this case also, the boundary was clear.
1)

Zoological Institute, Faculty of Science, University of Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

The heterozygosity value of the Northern Population was very small(H=0.034). On the other hand, the Southern Population was variable asa whole, though the values were different among subpopulations (0.0460.095). The genetic identity values between the Northern Population andeach subpopulation in the Southern Population were 0.71-0.75. These
values between four subpopulations in the Southern population were 0.870.93.
These results show that Japanese wild populations of medaka are remarkably differentiated regionally. As little clinal distribution is observed,
it is supposed that the differentiation is primarily due to geographical isolation. Based on the results, the origin of the orange-red variety of madeka
is thought to be the Eastern Subpopulation.
Conventional and Developmental Null Forms Coded by the
Acp-1 Locus in RiceTom ENDo

A new type of null form isozymes was discovered in rice and was termed"developmental null form," though several other kinds of null forms, either
associated with active forms or not have been previously known. Each of
the active alleles at the Acp-1 locus produces a different set of three major
and three minor isozyme bands of acid phosphatase in the leaf and someother organs (Endo, Biochem. Genet. 19: 373, 1981). Two null strains,
1707 (Indica type: spontaneous origin) and N8-409 (Japonica type: induced
origin), did not show any band coded by the Acp-1 locus in old mature
leaves. However, in young leaves of the former strain three appreciably
intense but blurred bands were present near the position of the major bandA specified by the active allele of ACp-4. The intensity of the three bands,
however, gradually decreased during the maturation of the leaves.
This difference between the strains described above may be explained asfollows: in N8-409 a termination mutant site was formed at the proximal
region in the base sequence of the structural gene locus. On the other
hand, in 1707 the mutant site was formed at the distal region in the basesequence of the locus, and so an incomplete polypeptide subunit containing
only a part of the active site for acid phosphatase was produced. This
incomplete phosphatase might have formed dimers via incomplete modification and expressed atypical bands in young leaves. However, this in-•

RESEARCHES CARRIED OUT IN 1980

41

complete enzyme molecules could not have lasted until the leaf maturation, removing the bands from old leaves. The phosphatase might nothave formed in N8-409 since the termination occurred before the synthesis.
Under this assumption, the alleles for null forms of N8-409 and 1707 were
designated as the conventional null form (Acpnull) and the developmental
null form (Acpdev-nUll), respectively.

•

An Extraction System for Leaf Zymographic Analysis
in Woody Plants
Tom ENDO

There are many reports on gel electrophoresis for plant isozyme analysis
describing buffer systems for the gel and electrode as well as a number ofreacting systems for the band development of each enzyme species. Extracting systems for enzymatic ingredients from plant materials, however,
have not yet been developed sufficiently to cover a range at different materials. In general, distilled water or simple buffer solutions around neutralin acidity have been successfully used as extractants for enzymes fromannual crop plants.
On the other hand, water and the common buffers were not efficient as
extractants for several woody plants such as Camellia, Prunus and Shorea.
Therefore, I have examined about one hundred extraction systems for leafof Shorea. At present, the extractant called EE 7.5, a modified Berthou's
buffer for coffee leaves (Communication at 9th ASIC Colloquium, London1980), appears to be most useful. Its composition was 10 % Triton X-lOO,
0.5 M sodium chloride, 0.5 M Tris, 0.2 M ascorbic acid, 10 mM EDTA and
1 mM potassium ferricyanide adjusted at pH 7.5 with glacial acetic acid of
1.6 % at final concentration. Leaf cuts of 0.1 g were mixed with 0.5 ml of
the EE 7.5, 0.1 g quartz sand and 0.05 g polyvinylpolypyrrolidone (PolyclarAT), and were crushed thoroughly in a mortar. The resultant exudate waseither diretly adsorbed with filter paper strips or adsorbed after centrifugation with hematocrit tubes. In some cases, Tergitol NP-40 may be substituted for Triton X-lOO, though the zymographic patterns were slightly
different in Shorea.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Modification Gene for Peroxidase Isozymes Coded by Px-1
Locus in Rice
Chiang PAl and Toru ENDO

There are several reports showing the appearance of only the S band in
F I hybrids when two allelic isozyme bands, F (faster moving to the anode)and S (slower moving), are present in different homozygous strains: thatis, the case of alkaline phosphatase and aminopeptidase in fowl. Thisphenomenon was found to be due to the presence of modifier enzymes suchas neuraminidase, by which the F band is changed to a S band, and thegene specifying the modifier enzyme is assumed to be dominant in inheritance (Law, Science 156: 1106,1976).
Another such case was detected in the Px-l locus specifying peroxidasein rice. This was that the bandmorphs under the presence of a modifier
enzyme were unique in the heterozygotes at the Px-llocus, of which isozymes
were of dimeric form. Four alleles, Px 2 A, Px 4 A, Pxo c and Px null , havebeen detected so far at this locus. Under the dominant modifier gene,
Mo-Px, these alleles produce 2A, 4A and OC bands, as well as null form,
respectively. While under the recessive modifier gene, mo-Px, the nullallele does not code for any band and the rest produce 2mA, 4mA and OmC,
the migration rates of which are down about 4-5 mm from each of therespective normal bands.
In the cross between 2mA, and 2A strains, the F I showed only 2A andthe segregation of 3(2A): 1(2mA) was seen in the F 2 generation. From thecross between 2mA and null form strains, the F 1 showed only 2A, and thesegregation ratio in the F 2 was 9(2A): 3(2mA): 4(null). This segregation
ratio agrees with that expected when recessive epistasis is present. In the
cross between 2mA and 4A, the F I produced 2A-3A-4A bandmorph, thesame as that from the Px 2A jPx 4A heterozygote. This ratio is due to the recombination of codominant and conventional heterozygotes, and may be
a new type of Mendelian segregation regarding the interaction of two
genes of different loci.

RESEARCHES CARRIED OUT IN 1980

IV.

43

DEVELOPMENTAL GENETICS AND SOMATIC
CELL GENETICS

The Growth and Differentiation of Germ Cells of Drosophilamelanogaster in Culture•

Yukiaki KURODA

Analysis of the process of germ cell maturation in Drosophila is fundamentally essential to understand the nature of the maternal effect in thecytoplasm for some sex-linked recessive lethal genes and to analyze thelocalized specificity in the egg cytoplasm in embryogenesis. In the present
study, the testes and ovaries obtained from pupae were cultured in vitro
and the process of germ cell maturation was examined.
Testes and ovaries were obtained from 48-hr pupae which were grownunder sterile conditions. The testes had attached to the vas deferens
derived from the genital discs and were in the spiral shape. When thesetestes isolated were cultured at 28°e in T-5 flasks in medium K-17 supplemented with 0.1 fig/ml fetuin and 15 % fetal calf serum, they grew andincreased in length and thickness.
Spermatogonia in the anterior part of testes differentiated to form thecyst of spermatocytes. A single cyst of the primary spermatocytes was
isolated from testes by using the fine needles and cultured by the sameprocedure. The first meiotic division was observed to occur synchronously
in all spermatocytes in the cultured cyst. The cysts of spermatids in culture
elongated extensively from the initial round shape to a rod-like shape.
Each process of spermiogenesis which involves an extensive elongation of
sperm bundles was clearly observed under a phase contrast inverted microscope.
On the contrary, in ovaries obtained from 48-hr pupae no noticeable
growth and differentiation were observed in the inside germ cells under thesame culture conditions, Muscle cells and fibroblastic cells migrated fromthe peritoneal sheath of testes and ovaries, and grew rapidly around theoriginal explants. Some mutant strains relevant to the germ cell maturation are used to analyze the gene actions in the development of germ cells.

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Effect of Lectins on the Cartilage Nodule Formation of Embryonic
Quail Limb Bud Cells in Culture
Yukiaki KURODA and Etsuya MATSUTANIIn the process of limb morphogenesis of avian embryos, the condensationof cells occurs in the central region of limb buds, and then cells in high celldensity region differentiate into cartilage cells. This suggests that the cellcondensation may be prerequisite to cell differentiation in the process oflimb morphogenesis. In our previous works, we have analyzed the earlystep in cartilage differentiation, and found the role of cell aggregation inthis step. In the present work, we examined the effect of several lectins,
which had been known to promote cell aggregation in various systems,on the cartilage nodule formation of mesenchyme cells from limb buds.
Mesenchyme ce1ls were obtained from hind limb buds of quail embryos(Columix cotumix japonica) at stages 20-21. An inoculum of 5 X 10" cells
in 10 ,ul of medi um 199 with IS % serum was placed on a plastic perti dish.
After allowing 2 hr for cell attachment, the dish was flooded with 2 ml ofculture medium. Lectins used were ConA, succinyl ConA (s-ConA), DBA
and WGA. Cells were fixed on the fourth day and stained with 0.05 %toluidine blue for 4-5 hours. The cartilage nodules were stained purplishred with toluidine blue, then the number of cartilage nodules stained wasscored to estimate the degree of cartilage differentiation.
First, the treatment period of cells with lectins was determined using
s-ConA. It was found that the 24-hr treatment after the initial 24 hours
of incubation was most effective in promoting the cartilage differentiationof mesenchyme cells.
Next, four lectins were tested at various concentrations (5, 10, 20, 50 and
100 pg/ml) for their effect on cartilage nodule formation. The results
showed that ConA and DBA had almost no effect at low concentrations,while s-ConA and WGA increased the number of cartilage nodules at middle
range of concentrations. However, in the case of WGA, the arrangementof cells in cartilage nodules was abnormal. More detailed effects of lectins
were examined with s-ConA.
The effect of s-ConA on the aggregation of mesenchyme cells was examined by measuring the diameters of cell aggregates formed in gyratory
shaking cultures. The results indicated that s-ConA promoted cell aggregation of mesenchyme cells. More detailed examination on the time-lapse

RESEARCHES CARRIED OUT IN 1980

45

changes in the number of cartilage nodules indicated that the lectins had an
activity to reduce the time required for the appearance of cartilage nodules.
This supports the idea that the lectin may promote cell condensation during
the early step in the cartilage differentiation.
The effect of the lectin on cell proliferation was also examined. Cells
were cultured in 50 ,ugjml of s-ConA medium for 24 hours at different
times after inoculation. The results showed that the treatment at an initial
24 hours caused a decrease in the number of cells. The treatment at a
middle 24-48 hours and late 48-72 hours period had no effect on cell proliferation. As a result, it is suggested that lectins may have an activity toenhance the cell adhesion of mesenchyme cells, then their tight contacts
may be established in cell aggregates, and resulted in the increase of thenumber of cartilage nodules.
Mutagenic Activity of Trp-P-2 and Glu-P-l on Embryonic
Human Diploid Cells in Gulture
Yukiaki KURODA

Trp-P-2 and Glu-P-l are pyrolysis products of trypotphan and glutamic
acid. Both have strong flame-shift mutagenic activities and weak basechange mutagenic activities in the Salmonella system. In the present experiments, the effects of Trp-P-2 and Glu-P-l on survival and mutationinduction in embryonic human diploid cells were examined.
Cells were treated with Trp-P-2 at various concentrations for 1, 2, 4, 8,
16 and 24 hours and the colony-forming activities of cells after 14 days innormal medium were determined. Surviving fractions of cells decreased
depending to concentrations of Trp-P-2. The cytotoxic effect of Trp-P-2
giving the same product of concentrations and exposure times was stronger
in cells treated with higher concentrations for shorter exposure times thanin cells treated with lower concentrations for longer exposure times, as
shown previously in the effect of Trp-P-l. Since Trp-P-2 is relatively
stable for incubation at 37°C, this dose-rate effect suggests the presence ofrepair activity of cells from sublethal damages produced by chemicals.
Cells were treated with Trp-P-2 at various concentrations for 4 hours
and cultured in normal medium for the mutation expression time of 7 days.
The cells were replated and selected with 30 ,ugjml of 8-azaguanine (8AG).
After cultivation for 14 days, the frequency of 8AG resistant mutations

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

was calculated. In cells treated with Trp-P-2 at concentrations of 0.1 and
0.3 ,ug/ml, no 8AG-resistant mutations were induced. At cencentrationsof 1.0 and 3.0 ,ug/ml, the induced mutation frequencies of 8AG-resistantcells were 2.8 and 1.4 per 10 5 survivors, respectively.
Glu-P-l had a weak cytotoxic effect on cultured human diploid cells.
Cells treated with Glu-P-l at a concentration of 100,ug/ml for 4 hours,
still gave the surviving fraction of cells of 0.92. When cells were treatedwith Glu-P-l at various concentrations ranging from 0.3 to 30,ug/ml for
4 hours, the induced mutation frequencies of 8AG-resistant cells were 0.9
to 2.7 per 10 5 survivors. These results suggest that both Trp-P-2 and GluP-I may require the metabolic activation by microsomal enzymes for revealing their active mutagenicity.
Metabolic Activation of Tryptophan Pyrolysis Products by
8-9 Mix in Induction of Mutations in Cultured
Chinese Hamster Cells
Yukiaki KURODA and Masurni ASAKURAFor quantitative studies on dose-response relationship of chemicalmutagens, cultured Chinese hamster cells have some advantages: 1) They
maintain normal diploid or ncar diploid karyotype for relatively long
period. 2) They show the high growth activity and colony-forming activity. In the present experiment, the effect of 8-9 Mix on the frequencyof mutations induced by Trp-P-l and Trp-P-2 was examined in Chinese
hamster V79 cells.
The concentrations of Trp-P-l and Trp-P-2 lowering the colony-forming
activity of cells to 50 % of control cultures were 2.6 and 12.3 ,ug/ml, respectively. When the rat liver microsomal fraction and coenzymes (8-9Mix) were added to medium, no noticeable enhancement of cytotoxic
effects of Trp-P-I and Trp-P-2 was detected.
Cells were treated with Trp-P-I and Trp-P-2 for 4 hours and the frequencyof 8-azaguanine (8AG) resistant mutations induced by both chemicals was
examined. At a concentration of 0.02 ,ug/ml, both chemicals induced
mutations slightly, but at higher concentrations than 0.06 ,ug/ml no dosedependent increase in induced mutation frequency was observed. The
addition of 8-9 Mix to medium enhanced the frequency of mutants inducedby Trp-P-l three times that of cultures without 8-9 Mix.

•RESEARCHES CARRIED OUT IN 1980•

47

When ouabain (OU) resistance was used as the genetic marker for detecting mutations induced by Trp-P-l and Trp-P-2, the frequency of induced
mutations was approximately one tenth the frequency of 8AG resistantmutations induced by these two chemicals at the same concentrations.
No marked dose-response was observed between concentrations of Trp-P-l
and Trp-P-2 and the frequency of OU resistant mutations. When 8-9 Mix
was added to medium in which cells were treated with chemicals, the frequency of OU resistant mutations increased approximately five times thatin cultures without S-9 Mix. These results indicated that mutagenic activity of Trp-P-l and Trp-P-2 was enhanced by metabolic activation with:
S-9 Mix.

Developmental Effects of Radiation on Embryos of the Silkworm(Bombyx Mori L.)
Akio MURAKAMI

As a laboratory insect, the domestic silkworm posseses various attractive
features for biological studies of embryos because of their structural simplicity, independence, and minor sensitivity to the influence of environment.
In addition, it can be easily collected a large number of synchronized cell
populations during the early embryonic development stage providing a
useful in situ system for the quantitative analysis of the cellular phenomenain the insect. These silkworm advantages which make it possible to analyze
the change in stage-radiosensitivity, either embryonic mortality or mutagenicity, within the nuclear division cycle that occurs during the early
embryonic development, as well as the change in age-radiosensitivity
throughout the whole course of embryogenesis and/or morphogenesis takeplace from simple to complex form. In the silkworm egg, the serosa membrane of some mutant stocks in the all embryonic development is colorlessand transparent as well as that of wild-type stocks in the early embryonicdevelopment permitting effective exposure to the UV light. This makes
possible to compare the biological effects of UV light and ionizing radiation
on embryonic nuclei and other cellular constituents. Moreover, comparison of the radiosensitivity among different X-ray-sensitive strains is
possible and thus may shed light on mechanisms for the changes in radiosensitivity within the time course of cleavage division cycles.
In this paper, I shall attempt to assemble and present the available in-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

formation in the silkworm obtained by myself and by others with regard to
the relationship between radiosensitivity and nuclear division cycles, thenuclear-cytoplasmic interaction, and the relationship between radiosensitivity and embryonic morphogenesis et cetera. I shall further refer to the
aspects of radiation effects on determination and differentiation in theembryonic process of silkworms, if possible. (Abstarcts of a paper presented at the Kyoto University Radiation Biology Center Workshop "Radiation Effects on Development and Differentiation". August 18-20, 1980:
Misaki Marine Biological Station, Miura-shi)
Roles of the Head-activation Potential and Head-inhibition
Potential Gradients in Hydra Pattern Formation
Tsutomu SUGIYAMA, Josef ACHERMANN and Jun TAKANO*

Hydra has a strong regenerative capacity. After amputation of thehead and foot, the end of the body column which originally had the headalways regenerates a new head, while the other end which originally had
the foot always regenerates a new foot. It has been generally thought that
this rigid polarity of regeneration is governed by a "polarity gradient"which exists along the hydra body axis (Child, 1941).
Wolpert et al. (1974) proposed that the polarity gradient in hydra consistsof at least 2 factors. One is the potential to activate the formation of ahead (head-activation potential) and the other is the potential to inhibit
head formation (head-inhibition potential). Both potentials are high inthe head region and become gradually lower toward the foot. In normal
hydra tissue, the activation potential is not expressed due to the counteracting power of the inhibition potential. After amputation of the head,
however, the head-inhibition potential falls rapidly at the cut surface due
to its instability (diffusibility), allowing the head-activation potential toexpress itself to regenerate a new head. This model can successfully explain the results of various types of regeneration and grafting experiments
carried out with wild type hydra strains. A similar model was also proposedby Gierer and Meinhardt (1974) on a more theoretical basis. They expressed the interaction of the head-activation and head-inhibition potentials
in mathematical terms, and showed by computer simulation how the gradients of the two potentials are produced and maintained in normal, budding* Dept of Biology, Nagoya University

RESEARCHES CARRIED OUT IN 1980

49

and regenerating hydra.
In an attempt to test these models experimentally, we have devised a
procedure to semiquantitatively compare the relative strengths of the headactivation and head-inhibition potentials of different hydra strains. The
procedure used is a modification of the lateral tissue grafting method usedby Webster (1966).
Four different strains have been examined. They are 105, a wild type
standard strain, mh-l which shows an unusually strong budding capacityand produces many abnormal buds along its body column in positions
outside of the normal budding zone, reg-16 which is defective in headregeneration after head amputation, and L4 which has a very low buddingrate.
H was found that mh-l has a singificantly higher head-activation and asignificantly lower head-inhibition potential than 105. This suggests thatthe abnormal budding in mh-l is caused by the imbalance between the headactivation and the head-inhibition potentials of this strain. In contrast,
strains reg-16 and L4 were found to have significantly lower head-activation
potentials and significantly higher head-inhibition potentials than 105.
These strains, therefore, also show an imbalance of the two potentials, but
in the opposite direction from mh-l, and this imbalance appears to be responsible for their morphogenetic deficiencies.
These observations suggest that (1) the head-activation and head-inhibition potentials play crucial roles in hydra head morphogenesis as proposedby Wolpert et al. (1974) and by Gierer and Meinhardt (1974), and that (2)an imbalance between the two potentials leads to morphogenetic abnormalities. At present, however, it is not clear why a similar imbalanceleads to different abnormalities in reg-16 (defective head regeneration) andin L4 (defective budding rate).
Morphogenetic Potentials of Chimeric Hydra Strain
Nancy WANEK and Tsutomu SUGIYAMAIn order to determine which cell types in hydra are responsible for morphogenetic potentials, two types of chimeric hydra have been constructedfrom the standard wild type (105) and the regeneration-deficient strain(reg-l 6). Reg-16 has a greatly reduced head regenerative ability, and has
a very low head-activation potential and a high head-inhibition potential

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

when compared to 105 (see the preceeding paper).
Hydra tissue consists of three self-renewing cell lineages: the ectodermal
epithelial cell lineage, the endodermal epithelial cell lineage and the interstitial cell lineage (which includes interstitial stem cells and their differentiation products, nerve cells and nematocytes).
Chimeric strains consisting of epithelial cell lineages (both ectodermaland endodermal) from 105 and the interstitial cell lineage from reg-16, andvice versa, were prepared using standard methods for eliminating and reintroducing interstitial cells (Marcum and Campbell, 1978).
Chimeric strains consisting of ectodermal epithelial cells from 105 andendodermal epithelial cells from reg-16, and vice versa, were prepared by a
newly-developed procedure (Wanek and Campbell, 1981). In this method,a ring of vitally stained tissue (ectoderm and endoderm) is grafted into an
unmarked polyp. Differential movement of the ectodermal and endodermal
layers at the graft junction results in chimeric regions which are visible asmarked ectoderm overlying unmarked endoderm (or unmarked ectoderm
overlying marked endoderm). These chimeric regions are then excisedand allowed to regenerate into whole chimeric animals. (These chimericstrains probably contain a mixed population of interstitial cell lineages
from both parental strains).
The head regenerative capacity of the epithelial celli interstitial cell andectoderml endoderm chimeras was examined in detail and compared tothat of 105 and reg-16. The [105 epithelial celli reg-16 interstitial cell]chimera shows normal regenerative capacity when compared to 105. In
contrast, the [reg-16 epithelial celli 105 interstitial cell] chimera shows
greatly reduced regenerative capacity comparable to reg-16.
The [reg-16 ectl 105 end] chimera shows almost normal regenerative
ability, whereas the [105 ectl reg-16 end] chimera has regenerative ability
which is significantly lower than 105, but slightly higher than reg-16.
These results indicate that the regenerative defect of reg-16 resides in its
epithelial cells but not in the interstitial cell lineage, and that the endodermal
epithelial cells have more influence than the ectodermal cells on head regeneration. Analysis of the head-activation and head-inhibition potentialsof the chimeric strains is now in progress.

RESEARCHES CARRIED OUT IN 1980

51

An SR-spiroplasma unable to Kill Drosophila Males
M. A. YAMADA, S. NAWA and T. K. WATANABE

NSR-spiroplasmas, originally isolated from the hemolymph of Drosophila
nebulosa and transplanted to D. melanogaster females, usually kill the maleprogeny in the embryo stages. Among the NSR carrying D. melanogasterstocks, one (strain-A) changed to produce males in the progeny. Bothmales and females of this strain-A had the spiroplasmas in the hemolymph,
which were transmitted transovarially into the next generation. This
spiroplasma has neither male-killing ability nor any apparent deleterious
effects on the fecundity and longevity of host flies. When the standard
NSR females (strain-B) which had the NSR-spiroplasmas and no maleprogeny were back-crossed to the males of the strain-A for 4 generations,no males were produced in their progeny. When the spiroplasmas ofstrain-A were injected into the spiroplasma-free females of both strain-B
and Oregon-R flies, they were transmitted transovarially without the malekilling effect. But when the spiroplasmas of strain-B were injected into the
spiroplasma-free females of strain-A, they were transmitted and completely
killed the male embryos.
The spiroplasmas of strain-A were not discriminated morphologicallyfrom the NSR-spiroplasmas of strain-B under the dark field microscope.
Since the SR-spiroplasmas from various origins can be distinguished byforming clumps if the hemolymphs of flies carrying different SR-spiroplasmas are mixed on a slide glass, the spiroplasmas of strain-A were mixed
with those of WSR (D. willistoni origin), HSR (D. hydri origin; non-malekilling spiroplasma) and the original NSR, respectively. Both spiroplasmasof WSR and HSR reacted with the spiroplasmas of strain-A but the NSR
spiroplasmas did not. These results suggest that the fly itself of strain-A
has not changed in the resistance to the SR-spiroplasma action but the
NSR-spiroplasmas of strain-A has lost the male-killing ability.

Hatchability and Some Developmental Characters of Flies in
Natural Population of Drosophila melanogaster
Kiyoshi MINATO

There were large differences in the oviposition rate, the hatchability ofeggs, and the average pupation period and so on among various laboratory

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

stocks of the Oregon-R strain of Drosophila melanogaster. Therefore, toknow which stock of the Oregon-R strain shows the similar characters to
those of natural population, the wild flies were collected to investigate somecharacters to be compared with Oregon-R flies.
Sixteen fertlized female flies of D. melanogaster were collected at Misima
and the same numbers of iso-female lines were established through brothersister mating of each offspring. The flies were analyzed after three generations of culture in the laboratory with food of agar-cornmeal-yeast-sucrose.
The oviposition rates of the lines of wild flies were relatively low (theaverage of 5.5 eggs/female' day) compared to those of three lines of OregonR (each 7.3, 9.6 and 10.6 eggs/female· day), under present conditions. In
the egg mortality, some lines of wild flies showed the rates of more than
10 % with the average of 6.5 %, which was higher compared with those of
three lines of Oregon-R (each 1.8, 2.4 and 5.3 %). In the larval mortality,
the lines of wild flies also showed fairly higher rate (some showed more than
20 % with the average of 12.2 %) than those of Oregon-R (average, 2.4 %).
Thus, so far as iso-female lines were used, the wild flies appeared to be accompanied with rather high degree of lethality in both egg and larval periods.
In the pupal mortality, there was little difference between the wild andOregon-R flies (both 1-3 %).
The average pupation periods of the lines of wild flies were widely ranged
from 5.5 days to 7.3 days, while those of Oregon-R were equally near 6.1
days. Furthermore, it was remarkable that each different value of theformer lines was maintained almost unchanged over at least ten generations.
Sex-specific Lethals of Drosophila melanogaster
Kugao OISHI and Takao K. WATANABE

All the sex-specific lethals so far reported are either on the X or on thesecond chromosome. A third chromosome (named #132) extracted froma natural population of Katsunuma in 1979 was found to be male-specificlethal when homozygous if mothers are heterozygous, and lethal for both
females and males when mothers are homozygous. Two recessive genes
were identified on the #132 chromosome, maleless (3)132 [mle(3) 132, 325.8] which is responsible for the male-specific lethality irrespective ofwhether mothers are heterozygous or homozygous, and lethal (3) maternal
132 [1(3)mat132] which causes maternal effect lethality on the homozygous

RESEARCHES CARRIED OUT IN 1980

53

progeny (thus it is a rescurable maternal effect lethal mutant). No further
pursuit on the latter, which is located between red (3-53.6) and e (3-70.7),
was attempted. Homozygous mle(3)132 males die at various larval stages
but never pupariate. An extreme delay in development was observed: at
100 hr after egg laying when other genotypes were 3rd instar larvae, surviving homozygous males were mostly still in the 2nd instar and some evenin the 1st instar stage. The lethality is exerted only on the single-X individuals regardless of the phenotypic sex, changes of which can be caused
by various sex-transformation mutants such as dsx, tra, tra-2 and tra_2 0TF •No interactions with a second chromosome male-specific lethal, mle(2)27,
was observed. Gynandromorph analysis showed that only about 5 % of
the pupative sex mosaics homozygous for mle(3)132 ec1osed. Thosemosaics which did appear were with very small XO patches in which structures themselves were reduced in size. These and other observations seemto indicate that the primary effect of mle(3) 132 is the retardation in the rateof cell division. If the gene is acting, as in other male-specific lethals, tocancel the dosage compensation, Minute (M) effect (retardation in cell
division rate as well as some morphological effects) may result which would
also explain the lethality.
TransplantabiIity of Teratocarcinomas Derived from 6 day
Embryos of B10 H-2 Congenic Mice in Allogeneic Hosts
Takehiko NOGUCHI and Choji TAYA

We produced teratocarcinomas, OTTlOA-5 and OTTlOSn-3 from 6 day
embryos of strains, BlO.AjSgSn(H-2") and BIOjSn(H-2 b ), respectively.
These teratocaricnomas obtained from BlO H-2 congenic mice allowed us
to examine the effect of difference in H-2 haplotype on the transplantabilityof embryonal carcinoma cells.
Solid tumors of the teratoma lines were transplanted subcutaneously intosyngeneic and allogeneic H-2 congenic mice. The result (summarized inTable 1) indicates that these teratocarcinomas could not grow beyond theH-2 barrier. When OTTlOSn (H-2 b ) tumors were transplanted into
BlO.129(6M)jSn(H-2 bC ), some of them were accepted probably because of
the similarity in H-2 haplotype between the grafts and the hosts.
Teratocarcinoma lines, 402A and OTT6050, or the derivative cell lines
have been reported to grow beyond H-2 barrier. It is also well established

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31
Table 1.
Teratocarcinoma(H-2 haplotype)
OTTlOA(H-2 a )Rejection of OTTlOA and OTTlOSn tumors by allogeneic
BlO H-2 congenic mice
Host strain(H-2 haplotype)Transplantation
site

Sensitivity
to tumor

BI0.A (H-2")
BlO.BR (H-2 k )BI0/Sn (H-2 b )BI0.l29 (6M) (H_2 bc )subcutaneous

15/15
0/8
0/5
0/3

intraperitoneal

5/5
0/4
0/5

- - - - - - - - - - - --------

OTTlOA(H-2 a )BI0.A (H-2 a )BlO.BR (H-2 k )BI0/Sn (H-2 b )-------~-----

OTTlOSn(H-2 b )-----------------------

BI0/Sn (H-2 b )BI0.l29 (6M) (H_2bc )BI0.A (H-2 a )BlO.BR (H-2 k )subcutaneous

21/22
8/15
0/5
0/4

that murine embryonal carcinoma cells are B-2 antigen negative. Transplantation behavior of BlO-derived teratocarcinomas was unexpectedly
different from that of l29-derived teratocarcinomas. The B-2 specificitydependent transplantation behavior of the BlO teratocarcinomas suggeststhat the charactor of the stem cells of these teratocarcinomas may be somewhat different from those of l29-derived teratocarcinomas, especially inrespect to B-2 antigen expression.
Aggregation Chimeras between Embryonal Carcinoma Cells of a
Testicular Teratocarcinoma (STT-2) and 8-cell Mouse Embryos
Kazunori HANAOKA and Takehiko NOGUCHI

Some embryonal carcinoma cells (EC cells) can be incorporated into
blastocysts by aggregating with blastomeres of 8-cell embryos (Fujii andMartin, Develop. BioI. 74: 239, 1980), or with 8-cell embryos themselves(Stewart, J. Embryoi. Exp. Morph. 58: 289-303, 1980).
We reinvestigated these results using teratocarcinoma cells with different
origins. The teratoma lines used were STT-2, OTTlOA-5 and OTTlOSn-3
which were derived from mice strains, l29/Sv-ter, BlO.A/SgSn and BlO/Sn
respectively. Clonal cell lines were also used. A211, A323, B~72l andB~826 were all derived from OTTlOA-5 (Nishimune et al., Annual Rep. 30:
50, 1979). lOSn-l and lOSn-2 were from OTTlOSn, and 311 from STT-2.

RESEARCHES CARRIED OUT IN 1980

55

Stem cells of these teratoma lines were enriched by passaging every 2 to 3
days on feeder layer (3T3-A31, mitomycin C treated). Clumps of EC cells(10 to 20 cells) were aggregated with 8 to 12-cell stage embryos by essentiallythe same method as Stewart, except that aggregation was carried out in
modified PBS containing phytohemagglutinin P. Presence of cells derived
from EC cells was checked using glucose phosphate isomerase as a genetic-marker. F 1 hybrid embryos between C3H and C57BL (Gpi-P) were thepartners for EC cells of STT-2 and 311(Gpi-1 U), while F 1 embryos between
Balblc and DBA(Gpi-P) were the partners for EC cells of OTTlOA,
OTTlOSn and their derivative cell lines (Gpi-P).
EC cells of these teratocarcinoma lines could form aggregates with normal
embryos, and about 80 % of the aggregates developed into blastocysts within
48 hours. Electrophoretic analysis of Gpi-1 revealed that blastocysts
developed from aggregates between EC cells of STT-2 or 311 and the partner
embryos were chimeric, but we failed to get any evidence for the presenceof EC-derived cells in blastocysts developed from cleaving embryos aggregated with EC cells of OTTlOA, OTTlOSn and their derivative celllines. The charactor of the black mice-derived EC cells might differ fromthose of STT-2. They probably failed to participate in the normal development and eventually died.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31v.

CYTOGENETICS

Segregation of F 2 Hybrids between Mauritius and OceanianType Black Rats
Tosihide H. YOSIDAThe Mauritius type black rats occurring in Mauritius island were characterized by having 42 chromosomes, among which extra 8 small acrocentries having been included in the karyotype of the Oceanian type black rat.
Comparative karyotype analyses between the Mauritius and Oceanian typeblack rats showed that the small extra acrocentrics found in the former
black rat resulted from the Robertsonian fission of the metacentric pairs
no. 14 and no. 18 occurring in the latter black rat with 38 chromosomes(Yosida et al. 1979, Chromosoma 75: 51-62). From the laboratory matings
between the Mauritius type and the Oceanian type black rats F 1 hybrids
were obtained. They were characterized by 40 chromosomes, among thempairs no. 14 and no. 18 were heteromorphic each consisting of metacentricand acrocentrics (Yosida 1980, Proc. Jap. Acad. 55(B): 557-561). In mating
between the F 1 hybrids, F 2 offspring were also obtained successfully.
The average litter size of the F 2 hybrids was 4.3±1.7, slightly smaller
than that of F 1 hybrids and their parental Mauritius and Oceanian typerats, but higher than those of F 2 hybrids between Oceania X Ceylon andOceania X Asia. Among the 17 F 2 offspring of Mauritius X Oceania,
karyotypes of 16 rats were availably analysed. If the no. 14 and no. 18
chromosomes in both F 1 parents are consisted of one metacentric and two
acrocentrics, and if they segregate regularly in the meiotic division, F 2 rats
with 9 different karyotypes are expected to be produed theoretically. The
segregation ratio of the karyotypes in these 16 F 2 rats was not significantly
different from the expected value.
Based on the present hybridization experiments the following conclusion
was drown that; (1) the extra small acrocentrics included in the Mauritius
black rats, which were derived from the Robertsonian fission of small
metacentrics, inherited normally to offspring through all dividing processesof the mitosis and the meiosis. (2) the Mauritius type black rat is moreclosely related to the Oceanian type rat than the Ceylonese and Asian typeones.

RESEARCHES CARRIED OUT IN 1980

57

Spontaneous Occurrence of a Mosaic Rat Carrying
Translocation/Normal and Translocation/Inversion
Pair no. 1 and Its Genetical Survey
Tosihide H. YOSIDA

Spontaneous occurrence of the 1/12 chromosome translocation in afemale Lewis strain rat was previously reported (Yosida 1980, Proc. lap.
Acad. 56B: 268-272). The no. 1 chromosome of the rat was heteromorphic
for the translocation and its normal partner, and the no. 12 was also heteromorphic for a normal chromosome and a smaller partner by deletion.
Among 17 offspring obtained by mating between the female with the translocation and a normal male, 9 rats were normal homomorphic, but theremaining 8 were heteromorphic. One female among the latter rats wascharacterized by mosaic in somatic cells consisting of two kinds of the
karyotype; one of them was heteromorphic for pair no. 1 consisting of the
1/12 translocation and normal partner (t/+), while the other showed theheteromorphic pair no. 1 resulted from 1/12 translocation and the inversion
pair no. 1 (t/i). The inversion chromosome was characterized by thesubmedian centromere, and its length was similar to the normal no. 1.
This means that the submetacentric member might have been produced by
pericentric inversion in the normal chromosome partner to the translocation
pair no. 1. By G-banding staining it was confirmed that the submetacentrichas been derived by the pericentric inversion.
The chromosome mosaic of the present rat was initially found in tailcultured cells. In tail and ear cultures, the frequency of t/ + and t/i cells
was nearly identical, but in bone marrow that of the t/i cells was considerably
higher than in that of the t/ + cells.
In the mating of the mosaic female rat with the normal male, 6 offspring(4¥ : 26) were obtained. If the female germ cells would be mosaic asfound in the somatic cells, the offspring with three cells types, (+ / +),(+ It), and (+ Ii), are expected at ratio of 1: 2: 1. The 6 offspring showed
the segregation ratio to be expected. Based on these results it can be said
that the chromosome mosaicism of the female rat occurred in both somaticand germ cells, the two typed cells (+ /t and i/t) were included almost equallyin the female gonad, and they transmitted to offspring through regularmeiosis. As the cause of occurrence of the mosaic rat it is considered thatthe 1/12 translocation should have first occurred in a Lewis rat, and the58

ANNUAL REPORT OF NATIONAL INSTITURE OF GENETICS NO. 31

pericentric inversion could have been taken in the normal chromosome
partner in the heteromorphic pair no. 1. At the early stage of embryogenesis the inversion should have occurred in one zygote resulting in theestablishment of mosaic rat with two cell types.
Segregation and Fecundity of Offspring from the 1/12
Translocation Heterozygotes of the Norway Rat
Tosihide H. YOSIDA

Translocation between pair no. 1 and 12 chromosomes in a female Lewis
strain rat has been described in the previous paper. This rat had heteromorphic pair nos. 1 and 12 by translocation and deletion. By mating
between the female translocation hcterozygotes (t/ +) and the normal male(+ / +), 89 rats (~ 46 and d 43) were obtained. They segregated into
53 rats wi th the normal karyotype (+ / +) and 36 with the translocation
heterozygotes (t/+). Fifty nine offspring (~ 33+ c) 26) obtained by thereciprocal mating segregated into the normal (+ If-) and the heterozygotes(t/ +) at almost the same frequency. The segregation ratio in total 148
rats was not significantly different from the expected value.
From the mating between female and male heterozygotes (t/+), 68 rats
(Q 33: d 35) were obtained. They segregated into the three types, normal
karyotype (+ /+), heterozygotes (t/ +) and the translocation homozygotes(t/t). The number of rats with translocation heterozygotes was significantly
higher, but those with the translocation homozygotes was lower than thatof the expected one. All 148 rats from 16 litters obtained by mating between translocation homozygotes (t/t) had always the 1/12 translocation
homologous pair (t/t). Average litter size of the rats with transolcation
was very high (9.2±3.2), and the sex ratio was the female 4.9 to male 4.3.
In the control rats having the normal karyotype which were establishedfrom the translocation heterozygotes, litter size was 7.4±3.3 in average of
11 litters, with the same number of females and males. From these studies
it can be said that the rats with 1/12 translocation have not any deriterious
effect on ability of the reproduction. The rats with 1/12 translocation thus
Dbtained was named the LET-stock.

RESEARCHES CARRIED OUT IN 198059

Segregation and Fertility of the Norway Rats with Metacentric
Pair no. 1 Derived by Pericentric Inversion
Tosihide H. YOSIDA

A female LEW (Lewis) strain rat with the chromosome mosaic consistingof two cell types was originally obtained in this laboratory (Yosida 1980,
Proc. Jap. Acad. 56: 322-327); from this rat offspring with the normal andthe inversion pair no. 1 (+ Ii) were obtained. By use of these rats, thesegregation and fertility of rats with the inversion pair no. 1 was examinedby the following combinations of matings; females with inversion heteromorphic pair no. 1 and normal males (+fiCjl X +1+8"), its reciprocal(+I+CJl X +/i8"), and females and males with inversion hetermoorphic
pair no. 1 (+/iCjl X +/i6). Eighteen offspring (9 females: 9 males) obtained by mating between the (il -+) females and the normal (+1+) males
segregated into 7 normal (+ 1+) and 11 with the heteromorphic pair no. 1(il +). Thirty-two offspring (9 females: 23 males) obtained by the reciprocal
matings (+1+ CJl xi/+ 8") segregated into 13 normal (+/+) and 19 heteromorphic pair no. 1 (il -+). Fifty rats (CJl 23: 8" 27) obtained by mating between the females and males with inversion heteromorphic pair no. 1 segregated into 19 normal (-+/-+), 22 inversion heteromorphic (il +) and 9 inversion homomorphic (iii). These segregation ratios were not significantly
different from the expected ones.
Fertility of the rats with inversion pair no. 1 was almost normal in all
matings, although it was slightly smaller in the case of matings between
inversion heteromorphic and normal rats (6.3±3.3 in average litter size),
and also betwccn inversion heteromorphic females and males (5.7 ±2.3).
Average litter size of the 16 litters obtained by matings between the inversion
homomorphic females and males was 7.4±2.8, which was not significantly
different from the control. Based on the above study it can be said thatin the Norway rat the individuals with inversion pair no. 1 seemed to exert
no effect on the fertility so far as the breeding has been taken in the laboratory. In the case of the transolcation pair no. 1, the fertility was
rather better than the control rats with the normal karyotype as describedin thc above paper of this report. If such karyotype rearrangements occurred in natural population, the rats with the altered karyotype might
have taken over those with the normal karyotype. The Norway rats withthe metacentric pair no. 1 resulted from the pericentric inversion were

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

named the LEM-stock.
Frequency of Sister Chromatid Exchanges in Lung Primary
Cultures in the Indian Spiny Mouse, Mus platytltrix
Tosihide H. YOSIDA and Tort! INOUE l )It has been shown that the karyotype of the Indian spiny mouse (Musplatythrix) is characterized by 12 acrocentric autosome pairs and XY sex
chromosomes, and further that several pairs of the chromosome complement of this animal would be produced as a result of tandem fusion ofcertain acrocentric chromosomes in the house mouse (M. musculus) (Yosida
1979, Proc. Jap. Acad. 55(B): 270-274). The number of sister chromatid
exchanges (SCEs) of the house mouse in the presence of 0.5 ,ltg/ml BrdU isreported to be 8.5±OA/cell in the second cell cycle (Kato 1977, Intern.
Rev. Cytol. 49: 55-97). Since there is no information on the SCE value
for the Indian spiny mouse, studies on the SCEs of this animal were carriedout under the treatment with several concentrations of BrdU.
Exponentially growing lung cultured cells were treated with BrdU atconcentrations from 0.1 to 25.0 flg/ml in the dark for 24 to 36 hrs. The
highest frequency of labeled cells with BrdU at the stage of the second cell
cycle was found to occur at 30 hrs inoculation (72.2 %). The number of
SCEs increased gradually from 7.6:::1=0.75 to 18.1 ::1::0.93 at the above concentrations, but the curve became flat in the samples under the dose of
0.25 to 1.0 ,ug/ml. As the occurrence of SCEs in the dose of 0.5 ,ug BrdU
was considered as of spontaneous frequency in the house mouse (Kato
1977), that of the SCEs in the Indian spiny mouse seemed to be l1.1/cell.
Clear heterochromatic C-band has been shown in the centromeric regions
in almost all chromosomes of the house mouse, whereas it was very small
and not clear in the Indian spiny mouse. The small amount of the C-band
heterochromatin in the Indian spiny mouse was considered as a cause ofhigher frequency of SCEs in this animal than in the house mouse.

1) National Institute of Animal Health, Tsukuba.

RESEARCHES CARRIED OUT IN 198061

Precocious X-V Chromosome Dissociation in Mus musculus
Subspecies, Inbred Laboratory Mice and Their F, Hybrids
Yoichi MATSUDA, Hirotami T. IMAI, Kazuo MORIWAKI,
Kyoji KONDO ' ) and Francois BONHoMME 2 )Frequencies of the X and Y chromosome dissociation in the primary
spermatocytes were examined with six Mus musculus subspecies, fifteen
laboratory inbred mouse strains and their F, hybrids. Three different
types (type I, II and III) in the mode of X-Y dissociation were identified.
The type I was found in inter-subspecies hybrids (BALAjc X molossinus,
castaneus, urbanus or bactrianus), where the X-Y dissociation was lessthan 20% in each subspecies but 60-80% in the F , hybrids. The type II
was observed in DBAj2J and DDK strains, in which the dissociation frequency was about 30 % in the homozygous condition and increased up to
50 % in inter-strain hybrids (C57BLj6J X DBAj2J or DDK). The type III
found in WBjRe strain also showed high dissociation frequency (51.7 %)in the homozygous condition but the value reduced remarkably (51.7-+
10.9 %) in (C57BLj6J X WBjRe)F , . We suggested that some Asian wild
mice would have the type II or III as well as the type 1. Mus musculus
species can be classified into two groups in terms of the X-Y dissociation
frequency of the type I; (1) brevirostris, domesticus and inbred laboratory
mouse strain, and (2) molossinus, castaneus, urbanus and bactrianus.

Multiple Sex Chromosome Mechanism (XXY) in the Filefish,
Stephanolepis cirrhifer
Makoto MUROFUSHI,S) Sin OIKAWA,") Shohei NISHIKAWA c)and Toshide H. YOSlDA

It is already reported that the chromosome number of the filefish,
Stephanolepis cirrhifer, was 212=34, and all of the chromosomes wereacrocentric (Murofushi and Yosida 1979, Jap. Jour. Genet. 54: 191-195).
Department of Animal Breeding, Faculty of Agriculture, Nagoya University.
Laboratoire d'Evolution des Vertebres, Universite dcs Sciences et Techniques duLanguedoc, Montpellier, France.
S)
Mishima Junior College, Nihon University.
4)
Department of Agriculture, Kyushu University.
5)
Shimonoseki University of Fisheries.
1)

2)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

The sex chromosome, however, could not be identified because the specimens used were only females. The male and female specimens were newly
obtained and their karyotypes were reexamined. The diploid chromosome
number in all females was 34 as already described, while that in all males
was 33, among thcm one large metacentric chromosome was always foundin their karyotypes. Such large metacentric clement was never found in
the female specimens. If this species was characterized by the usual XY
sex determing mechanism, the chromosome numbers in female and malewould be the same. However, one chromosome was always less in themale than in the female specimens. These chromosome complementssuggest that the male in this species has the X j X 2Y sex determination mechanism, and the large metacentric chromosome should be the Y element.
To confirm the above finding, the spermatocytes were observed. In thesecondary spermatocytes two typed cells, n=16 and n=17, were found.
In cells with 16 chromosomes one metacentric was always included, whilein those with 17 chromosomes such element was never observed. Basedon these findings a conclusion was drown that this species has the X X 2 Y
sex chromosome complex in the male and the X X X 2 X 2 in the female.
Unfortunately we could not distinguish the X chromosomes from theautosomes in mitosis and meiosis, because they arc not morphologicallydifferentiated. Finding of the multiple sex chromosome seems to be important for the study on differentiation and evolution of the sex chromosomes in the fish, because it is regarded as that the morphological differentiation does not occur in this animal group.
jjj

Cytological Studies on Male Crossing Over in Drosophila ananassae
Muneo MATSUDA, Hirotami T. IMAI and Yoshiko N. TOBARI!)

Recently we have succeeded to obtain high quality of meiotic chromosome figures in D. ananassae by using the air drying methods improved by
Imai et al. (Chromosoma 59: 341-393, 1977). To determine the mechanismof male crossing over in D. ananassae, cytological studies were carried out
using two different heterozygous males between a marker strain (b se; briru) and two wild strains, TNG and L8. TNG F (b se; bri ruj TNG) males
are characterized by a high frequency (about half value of females) andL8 F 1 (b se; bri ruj L8) males are characterized by an extremely low frejj)Department of Biology, Tokyo Metropolitan University.

RESEARCHES CARRIED OUT IN 1980

63

quency (0-0.1 %) of male recombination. We demonstrated the presenceof chiasmata in TNG F 1 males and a positive correlation between chiasma
frequency and genetic recombination value (average chiasma frequency:
0.63/bivalent in TNG FI, O.lO/bivalent in L8 F 1 ). These results strongly
suggest that male recombination in D. ananassac is meiotic in origin. In
addition to chiasma, a new type of chromosome aberration characterizedby two chromosome breakages occurring simultaneously at comparablesites along a homologous pair and disordered reunion of the breakage
points was observed, particularly in the spermatocytes of TNG F 1 (9.2 %).
Because there was a parallelism between chiasma and chromosome aberration in distribution pattern, we proposed that the chromosomal aberration
might be closely related to formation of the chiasma, and under the control
of a common causative factor.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

VI.

MUTAnON AND MUTAGENESIS IN ANIMALS

Rat-Liver S-9 Mediated Mutagenesis in Germ-Cells of the
Silkworm by Benzo(a)pyrene
Akio MURAKAMI

Benzo(a)pyrene [BP] is an aromatic hydrocarbone that has been a wellknown environmental agent. BP has also been a potent carcinogen inexperimental animals and a mutagen which requires to be metabolically
activated to the reactive ultimate formes) in order to exert its mutagenic or
carcinogenic activities. This aromatic compound was selected as a test
chemical for the short-term carcinogenic compounds screening programmein the Ministry of Health and Welfare of Japan. Although BP was proved
to be mutagenic in most test organisms, this carcinogenic polycyclic hydrocarbon has been tested for mutation induction in the silkworm withnegative results for the egg-colour specific locus test. We tested BP for
mutagenic effect by suspending it in 0.1 % carboxylmethyl cellulose (CMC)
solutions. BP was injected into pupal females. There was no significant
increase for the specific locus mutation over controls. As we have alreadybeen reported in this Report that mutagenicity of a promutagenic polycyclic hydrocarbon Dimethylbenzanthracene (DMBA) was only detectedin pupal oocytes of the silkworm when the parent compound was in vitro
incubated with S-9 fractions from rat-liver homogenates before injection.
It is very likely to forecast that BP could not be detected the mutation insilkworm test systems unless the metabolic activation system is supplemented.
This report concerns the positive mutagenic activity of BP in pupal oocytesof the silkworm by the egg-colour specific locus test coupled with the invitro mammalian metabolic activation system.
Several concentrations of BP dissolved in dimethylsulfoxide (DMSO)
solution were incubated with the incubation mixture at 30 C for 20 min.
The incubation mixture in 0.025 ml per capita was made up of a BP in
0.017 ml DMSO solution, 0.0166 ml S-9 fractions from livers of rats whichwere pretreated with polychlorinated Biphenyl (PCB) and 0.0067 ml cofactor
systems containing NADPH, p-NADH, G-6-P, ATP, and others as describedby Sugimura et al. (1976). After injection with BP without the S-9 treatD

RESEARCHES CARRIED OUT IN 1980

65

ment, no detectable mutation frequency was still found in pupal oocytes
after all. While, the result of the experiments with the S-9 treatment indicated that BP was clearly mutagenic to pupal oocytes. The increase of
mutation frequencies was significant when compared to the control (e.g.
2.9 X 10- 5 at re locus) without an apparent relationship to the exposure
dose level used.
The frequencies of re locus mutations detected were
46.8 X 10- 5 in 75,ug/pupa, 61.2 X 10- 5 in lOOlig/pupa and 46.0 in 125 ,ug/
pupa. This inconsistency is to be expected with a limited yeild-rate for the
mutagenic intermediates produced under the described experimental condition.
The present observation confirmed that the silkworm egg-colour specificlocus test method coupled with the in vitro rat-liver metabolic activation
system-the silkworm/rat-liver S-9 system-is useful to test the mutagenicity
for promutagenic polycyclic hydrocarbones.
Specific Locus Mutations Induced in the Silkworm by
Diethylnitrosamine
Akio MURAKAMI

Several dialkylnitrosamines are well-known mutagenic agents in various
test organisms and potent carcinogenic agents in experimental animals.
The dialkylnitrosamines are one of typical indirect mutagens which require
metabolic activation to mutagenic derivatives. In the silkworm, Dimethylnitrosamine (DMNA), one of dialkylnitrosamines, has been tested formutation production in germ-cells with negative results for egg-colour
specific locus mutations and for dominant lethals. So far as we know,
ethyl compounds for methansulphonates, nitrosoguanidines and others are
more effective for the production of mutations in germ-cells of the silkworm
than these methyl compounds. In the present short communication, we
present data on the mutagenic effects of Diethylnitrosamine (DEN) in thesilkworm, the results for which may be compared with that of DMNA.
Several different concentrations of DEN were made up in solution with
0.85 % NaCl. They were tested by injection into the abdomen of pupal
females and males. The injected pupae were then mated individually to
pe: re marker stocks each for the specific locus tests. DEN treatments athigh doses ranged from 1.5 to 2.0 ,ul per female pupa (or oocyte) produced
a significant increase in egg-colour specific locus mutations compared to66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

controls, but no increase of the mutations was detected at low dose treatments. There was no detectable increase over controls for specific locus
mutations in male pupae (or spermatozoa) even after high dose treatmentswith DEN as well as DMNA. In the dominant lethal experiments in parallel with the speciilc locus tests, DEN affected only pupal oocytes at thehigh dose experimcnt groups, but not affectcd spermatozoa. At up tonear mortal concentrations for pupae, DMNA did not produce the dominantlethal mutations in any of the pupal germ-cells. This finding clearly indicates that premeiotic oocytes in the pupae presumably have the capacity
to metabolize the dialkylnitrosamines to mutagenic intermediates, butmature sperm have not the capacity as well as other pupal tissues or organs
regardless of the sexes. Namely the active metabolites of dialkylnitrosamines may be short-lived so that damage in the pupae would be only
produced in target cells or oocytes that come into direct vicinity to the siteof metabolic activation. From the present finding and others, it can bealso said that the large amounts of mutagenic metabolites would be required
to induce the specific locus mutations as well as dominant lethal mutations.
The failure to detect the mutational events with DMNA in pupal oocytes
might be resulted from some un-explained reasons other than the metabolic
activation. In the silkworm, differential mutagenic sensitivity to the
dialkylnitrosamines, DMNA and DEN, is very similar feature to that of
the monoalkyl methanesulphonates, MMS and EMS. In this connection,
it is worthwhile to note here that methyl compounds, MMS, MNNG and
DMNA are generally more toxic to silkworms than ethyl compounds,
EMS, ENNG and DEN. Accordingly, it can be explained that the absenceof mutagenicity to DMNA may be due to a lack of sufficient amounts ofeffective mutagenic intermediates for the induction of mutations because
the dosage is restricted by the high mortality of the treated insects at thehigher concentrations. It can not completely exclude a possibility that thelack of mutagenic activity have probably resulted from a repair of altered
DNA before mutation detection.
Lack of Effects on the Mutagenicity with Procarbazine
in Silkworms
Akio MURAKAMI

Procarbazine has been used as a carcinostatic agent.

This chemical

RESEARCHES CARRIED OUT IN 1980

67

agent is known to be as a promutagen and that is metabolically activatedby oxidation to azoprocarbazine. Mutagenic tests with Salmonella andSaccharomyces for reversion or foward mutations did not show a positive
response to Procarbazine regardless of whether they were performed with
the S-9 fractions from rat livers, while this drug gave a positive response in
mammalian cells in culture, in mammals and Drosophila for various genetic
endpoints. In Drosophila, both sex-linked recessive lethal mutations anddominant lethals were induced by this carcinostatic agent in male postmeiotic germ-cells or spermatids. Thus, it can be expected that in thesilkworm, procarbazine would be effective on the induction of recessive
visible mutations and dominant lethal mutations. This communication
reports the result of mutagenicity tests of Procarbazine' HCI in the silkworm.
Sample Procarbazine' HCI was the gift of Nippon Loche, Japan. The
compound was dissolved in a 0.85 % NaCl or physiological saline for thesilkworm was tested at various concentrations up to 750,ugjcapita. Each
concentration of the compound was administered by the injection method
into the abdomen of wild-type male larvae and both sex pupae. The
recessive visible mutation was detected by the egg-colour specific locus
method. The treated individuals were mated to non-treated opposite sexesof the marker stock homozygous for pe and re genes.
We found that Procarbazine was not effective for the induction of specificlocus mutations in larval spermatocytesjspermatids, pupal spermatozoa
and oocytes. There is no evidence that dominant lethal mutations at the
reduction of embryonic hatchability were induced by this drug in any ofthese germ-cells.
The failure to detect the mutational events with Procarbazine in thesilkworm is to be expected with a lack of effective metabolic activation
systems. Accordingly, the mutagenicity test of the drug in the S-9 mix isin progress. Hydrazone of monomethyl hydrazine and formyl isopropylbenzamid are biological active intermediates of procarbazine. It may besuggestive to note that mutagenicity of hydrazines is not yet detected in thesilkworm test system. The induction of dominant lethals in Drosophila
was observed only at the high dose of the drug (Biljleven, W.G.H. and E.
Vogel, 1977). This indicates that large amounts of the chemical or its
metabolites would be required to induce the specific locus mutations as wellas dominant lethal mutations in the silkworm test system as well as Drosophila.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Dominant Lethal Mutations Induced by Some Indirect-Acting Mutagens
in the Silkworm, Bombyx Mori
Akio MURAKAMI

The mutagelllcity of chemical compounds with a special emphasis on
the indirectly acting mutagens was studied by dominant lethal tests as the
reduction of egg-hatchability in pupal pre-meiotic oocytes and sperm of thesilkworm. Wild-type (C10S x Aojuku) F 1 hybrids at the mid-stage pupa
were given a single dose of at least five different doses for each chemical andmated with the opposite sex moths of a different stock.
Among chemicals tested two mycotoxins, Aflatoxin B1 and Sterigmatocystine, gave a positive response in the dominant lethal test for both oocytes
and sperm as has been reported to be positive by the specific locus test.
A minor tranquillizer Diazepam also gave a positive response for bothgerm-cells by the dominant lethal test, although it has been shown to be anegative response by the specific locus test. Chlorodiazepoxide, a diazepin
derivative and a minor tranquillizer, was also mutagenic in oocytes of thesilkworm. 2-Acetylaminofluorene, Caffeine, Theophylline, and p-Ethoxyacetanilide (or Phenacetine) were all mutagenic for oocytes by the dominantlethal test, but not for sperm regardless of suspiciously positive or negativein the specific locus test for both germ-cells. The nitrofurane compounds,
Furylframide or AF-2 and Nitrofurazone, were not mutagenic in the induction of dominant lethals in pupal oocytes at up to near killing doses, while
Panfurane, 3-Amino-6-[2-(5-nitro-2-furyl)vinyl]-I, 2, 4-triazine, was only
mutagenic in the oocytes at the high doses ranged from 250 to 500 ,ug/pupa.
It has been reported that Panfurane is mutagenic in the oocytes of thesilkworm by the specific locus test at the low dose (ca. 5 ,ug/pupa) (Tazima,
1974: Ind. J. Genet. & Plant Breeding, 34A, 302-310). The shape of thedose-response curves on the induction of dominant lethals in pupae treatedwith these indirect mutagens as well as the positive control, Ethylmethanesulphonate (EMS) and tris[1-(2-Methylaziridinyl) phosphine oxide](meTEPA) was almost the same and sigmoidal fashion.
From the present study and others, it may safely be said that chemicals
having the ability to induce dominant lethal mutations tend to induce
recessive visible mutations detected by the egg-colour specific locus method.
In addition to accuracy of the dominant lethal test method, simplicity
makes it possible to test the mutagenic activity in a large number of environ-RESEARCHES CARRIED OUT IN 198069

mental chemicals. Thus, it can be stated the dominant lethal test in thesilkworm may be a useful short-term assay system for emvironmental
mutagens. (This work was supported by in part a Grant-in-Aid for Cancer
Research from the Ministry of Health and Welfare of Japan.)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

VII.

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICROORGANISMS AND PLANTS
Genetic Study on the Initiation of Sporulation in
Bacillus suhtilis
Yoshito SADAIE

Ten isogenic strains of different spoD loci have been constructed to study
the mechanism of initiation of sporulation in Bacillus subtilis, which is thebest studied microorganism for differentiation. Because sporulation isunder the control of catabolite repression, we examined the ability of these
spoD strains to use different carbon or nitrogen sources. They grew wellon the minimal agar supplemented with different carbon sources including
glucose, mannose, maltose, sorbitol, arabinose, fructose, glycerol, sucrose,or trehalose. SpoD mutants examined were not mutants concerning catabolite repression of sugar utilization. Catabolite repression working on
sporulation appears to be very similar to that on histidine utilizing operon.
SpoD mutants grew well in the minimal medium with histidine as sole carbon
and nitrogen sources. The first enzyme of Hut operon, histidase, was examined in toluene treated cells and ten spoD mutants all showed higher
activities suggesting derepression of Hut operon in spoD mutants.
The effects of spoD mutations on vegetative growth were examined withthese mutants. They grew well at 37°C and 48°C in a complex sporulation
medium or in a minimal medium. They were resistant to various kinds ofantibiotics including MC, Rif, Nal, Km, CM, Amp, PenG, and EtBr, andmutable toward DNA damaging agents. SpoD mutations so far examined
did not affect vegetative growth. Strains used were spoDA34, spoDB136,
spoDC9, spoDD8, spoDE81, spoDF221, spoDGI4, spoDHI7, spoDJ87, and
spoDKI41 mutants.
On the Process of Competent Cell Formation in Bacillus suhtilis
Yoshito SADAIE and Tsuneo KADA

The process of competent cell formation for transformation has beenstudied with early stationary phase cells of Bacillus subtilis which had been

RESEARCHES CARRIED OUT IN 1980

71

grown in an enriched Spizizen minimal medium and transferred to a second
synthetic medium. The formation of competent cells took place around50 min after transfer of the early stationary phase cells into the second
medium in accordance with the onset of cell division of total population.
Rifampicin, chloramphenicol, penicillin G and tunicamycin were strong
inhibitors of competent cell formation as well as vegetative cell division.
Competent cells formed were no longer sensitive to the above agents. An
inhibitor of chromosomal replication, hydroxyphenylazouracil did notinhibit development of competency. A D-alanine requiring mutant strain
developed competency even in the absence of D-alanine in the second
medium. These results suggested that competent cells were formed withless cross-linked cell walls and by a factor(s) whose gene(s) was activatedduring cell division. Competent cell formation was completely blocked
by 0.7M ethanol, which is a specific inhibitor of early events during sporulation. Competent cells were formed even in media which supported sporulation. Development of competency was also studied with the spoO mutantsof ten different loci. Most spoO mutations reduced development of competency except for spooe, spoOG and spoOJ.
These results suggest that there are common steps involved both inforespore septation and in competent cell formation.

Antimutagenic Effect of Cobaltous Chloride on Bacillus subtilis
and Escherichia coli Mutator Strains Possessing
Error-Prone DNA Replicating EnzymestTadashi INOUE, Masumi SHIZAKI, Masayuki KATO
and Tsuneo KADA

We previously reported that cobaltous chloride markedly diminished thespontaneous mutation frequency to histidine prototrophy in a Bacillus
subtilis mutator strain NIG1125 (his mut-l) whose mutator activity has been
ascribed to its temperature-sensitive DNA replicating enzyme which iserror-prone at the permissive temperature (Inoue et al. 1981 Mutation Res.
91: 41). In order to confirm that the cobaltous chloride interferes withmutagenesis itself, we attempted to determine the effect of the metal compound on mutation rate, not on mutation frequency, by adopting the method
originally developed by Matney (1955. J. Bacteriol. 69: 191) which is basedon the fact that bacterial populations growing on the surface of membrane

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

filters can be easily transfered from one solid medium to another without
disturbing the spacial relationship among the members of the population.
The experiments yielded the results that the treatment of the cells with
40 ,ug/ml of cobaltous chloride diminished the mutation rate from 2.33 X
10- 5 mutation/cell/generation to 0.30 X 10- 5 mutation/cell/generation. These
results strongly indicated that the metal compound really interfered withmutagenesis itself acting on the error-prone DNA replicating enzyme.
To investigate further the antimutagenicity of the metal compound, we
examined the effect on another mutator strain, Escherichia coli SG902(leu dnaEtS), which has similar mechanisms to B. subtilis NIG 1125 for its
mutator activity. The spontaneous mutation to leucine prototrophy was
also remarkably supressed in this strain by the metal compound. In addition, we found that the strain SG902 was far more sensitive to cobaltons
chloride than its parental strain DG17 (dnaE+). This fact suggests that thetarget of the metal compound is dnaE gene product (DNA polymerase III).
This was also confirmed by the following two lines of evidence. First,
high proportion (4/42) of spontaneously arisen cobaltous chloride-resistantclones from SG902 was non-mutator. Second, high proportion (3/24) ofspontaneously arisen temperature-resistant clones was cobaltous chlorideresistant. These results indicate that temperature-sensitivity, cobaltous
chloride-sensitivity and mutability are closely linked to each other in thestrain SG902 and that antimutagenicity of the metal compound is expressedby some mechanisms which involve DNA polymerase III.

Antimutagenic Activities of Human Placental Extract on
Radiation-Induced Mutations in Bacteria
Tsuneo KADA and Hajime MOCHIZUKI

It is interesting to know whether human cells possess factors antagonizing with radiation-induced cellular mutagenesis. We report here that thehuman placental extract is working as a strong antimutagen in UV- andgamma-ray-induced mutagenesis in bacteria.
Cells of Escherichia coli B/r try were grown overnight in broth, washedby phsophate buffer and exposed to 3.45 J/m 2 of UV or 10 kR of gammaray (137Cs); 0.1 ml or 0.2 ml portion of the irradiated suspension together
with 0.1 ml of the placental extract were then incorporated into 3 ml of

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RESEARCHES CARRIED OUT IN 1980

molten solf agar mixed well, and poured onto the semi-enriched minimum
agar. At the same time, the irradiated suspension was diluted 10- 5 or
10- 6 and 0.1 ml portion of each diluted solution was over-layered on the
MB agar by means of the 3 ml soft agar. Plates were incubated at 37°C
for 3 days and the number of induced mutant colonies and that of survivingcolonies were scored.
Both for irradiation with UV- and gamma-rays, the number of induced
mutant colonies decreased remarkably, whereas thc effects of the extract
on the survival were not so significant. It was shown that the presence ofthe placental extract did not modify significantly the number of viablebacteria which were not treated with radiation but the number of induced
Trp+ colonies per plate decreased considerably. The placental extract
itself was not mutagenic. We confirmed that the established Try+ cells
were not particularly sensitive to the placental extract either in the case of
UV or gamma-ray induction. These results indicate that the human placental extract contain factor(s) antagonizing with UV- or gamma-rayinduced mutations in this bacterial system.
The antimutagenic actions of the placental extract was also confirmed inother bacterial systems where the frequencies of mutations were reduced
remarkably by it. We are examining if the present placenta factor might
be involved in proofing spontaneous or induced mutagenic errors that are
produced in the course of embryogenesis. (in J. Rad. Res. 22, 297, 1981)

Antimutagenic Factor in Leaves of Camelia Families
Tsuneo KADA and Kenji KANEKO

It was previously suggested that anitmutagenicity of cobaltous chloride
may be explained by its proofing activity in error-prone replication of DNA
by a modified DNA polymerase (Kada, T. and Kanematsu, N. 1978, Proc.
Jap. Acad. 54: 234; Inoue, T. et al. 1981, Mut. Res. 41: 91). We recently
found that leaves of the Camelia plants contain a similar factor.
Since our daily consumed green tea is produced from plants of the family
Camelia japonica, its antimutagenicity was studied.
Fresh plant leaves were homogenized with an equal weight of cold waterand the homogenate was centrifuged at about 1 X 10 4 xg. The sterilesupernatant was collected and stocked at -40 ~ - 80 C. The antimutagenic potency was calculated in a similar way in which that of cobaltous
G

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

chloride was determined by Inoue et al. (1981). When B. subtilis cells of
NIGl125 (his met mut-l) grew in semi-enriched agar containing cobaltous
chloride at a concentration of 1 ,ug/ml, 821 His+ mutants disappeared in
the course of 2.8 X 10' cellular divisions taking place. It comes out thatabout 30 mutations are lost per 1 X 106 divisions due to presence of theantimutagen. Doing similar experiments, we could calculate the antimutagenicity level of samples. The specific activity of antimutagenicity represents a concentration Cug/ml) of cobaltous chloride whose antimutagenic
activity is equivalent to that of the test sample. That of Camelia japonica
ranged between 10 and 20.
Effects of .a-irradiation from 3H-Water on Transforming
DNA of Bacillus subtilis
Tsuneo KADA, Hajime MOCHIZUKI, Tadashi INOUE
and Yoshito SAD AlE

In order to elucidate mechanisms and efficiencies of DNA inactivationof tritium, the bacterial transforming system was studied. DNA wasextracted from cells of B. subtilis Marburg (arg+), purified by the phenol
method, dissolved in SSC buffer solutions containing different concentrations of 3H-water ranging 0.001 /tCi/ml to 10,uCi/ml. The solutions werethen kept at 4°C for p-irradiation. After different periods, portions of thesolutions were sampled, dialyzed, against cold SSC to eliminate radioactivity and exposed to competent cells of B. subtilis H17 arg- to measureinactivation effects of the arginine marker by ,e-rays. We found that, when
% inactivations were plotted against 3H-dose (/tCi/ml x days), effects of
p-irradiation increased drastically by decreasing 3H concentrations. For
example the D 37 dose for 10 ,uCi/ml teratment was 18 ,uCi/ml X day, wherasthat of 0.1 ,uCi/ml treatment was about 0.4 ,uCi/ml X day.
These results showed that the inactivation efficiency of p-irradiation oftritium became progressively higher by lowering concentrations of tritium
water. It is indicated that the RBE of tritium irradiation may be very
high (more than 100) at least in the case of in vitro DNA inactivation under
the above experimental conditions (Related results in press in J. Rad. Res.)RESEARCHES CARRIED OUT IN 1980

75

Human Enzymes Functioning in Repair of DNA Damage
Induced by Gamma-Irradiation
Tadashi INOUE and Tsuneo KADA

Ionizing radiation produces many kinds of lesion in DNA including
strand breaks and modification of base as well as of sugar moieties. Livingorganisms can, however, repair most of these damages efficiently. Our
systematic studies on the repair mechanisms operating on gamma-rayinduced DNA damages using permeabilized cells of Bacillus subtilis revealed
that some repair processes are necessary prior to those involving DNA
polymerase and DNA ligase. Further studies showed that there exist
enzymes (primer activating enzymes) which convert some of the gammaray-induced lesions to effective priming sites (i.e., 3'-0 H terminals) for
repair replication by DNA polymerase.
We recently demonstrated that the level of the primer-activating enzyme
activity was lower in the extracts from ataxia-telangiectasia fibroblasts thanthose from normal fibroblasts (Inoue et al. 1981, Biochim. Biophys. Acta
655: 49). Ataxia-telangiectasia is an autosomal recessive disease of man
characterized by telangiectasia, neurological degeneration, immunologicaldeficiency, predisposition to malignancy, and radiation hyper-sensitivity.
It is therefore of interest to examine the relationship between these symptoms and defects in the primer-activating enzymes. Tissue culture cells,
however, provide too little materials for extensive enzymatic characterization, so we have thus turn to human placenta to study further the physicaland catalytic properties of human repair enzymes.
The primer activating enzyme activity in the placental extracts was separated into two fractions by means of DEAE-cellulose chromatography.
Both fractions were further purified by affinity chromatography on gammairradiated DNA-cellulose. This procedure was found to be extremely
effective for purification, giving an overall purification of 800-fold for both
fractions. Preliminary characterization revealed that one of the primer
activating enzyme fraction had a catalytic activity to enhance the priming
activity of gamma-irradiated DNA without any effect on unirradiatedDNA, and neither phosphatase nor exnonuc1easc activity was detected in
the preparation.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

On the Expression of Waxy Starch Mutants Induced by
Thermal Neutrons in Rice
Etsuo AMANO

Many waxy (lI'x) starch mutants have been induced and examined in rice,
Oryza sativa var. Norin 8. When a chemical mutagen, ethyl methanesulfonate was used, half of the lI'X mutants induced were more or less intermediate between H'X and normal non-waxy (Wx). To compare between
the mutagenic treatments, H'X mutants were induced by thermal neutronsof D 2 0 (deuterium water) facility of KUR (Kyoto University Reactor).
In 1978, six lI'X like mutants were obtained from thermal neutron irradiation, which suppressed seedling height to 60 % of the non-irradiation control. All mutants produced enough M s seeds for measurements of wxindices. Visual examination of M 3 seeds revealed that only two of themutants were full mutants and the rest four seemed to be very weakly waxy.
Two wave lengths photometry was used to measure the waxyness expression quantitatively. Hulled brown rice grain was crushed with pliers andput into a test tube, one grain for each tube and more than 100 grains for aplant. 3 ml of water was added to each tube and then autoclaved for
15 min at 1 atm pressure. Starch solutions were stirred while warming.
After overnight cooling down to room temperature, supernatant fluids were
measured photometrically after iodine staining. Concentration of the
starch solution, or amylopectine, stained with iodine, was monitored by
430 nm blue light during the stained sample solution was diluted by water inthe measuring cell. When transmittance of the 430 nm blue light increased
to 50 % by dilution, the transmittance of 660 nm red light was measured andregistered by a digital printer as index value to express waxyness of the
starch. The visually confirmed two full H'X mutants were proved to bealso full wx mutants photometrically. Other four mutants were very
weakly waxy.
Example of intermediate wx mutants in rice induced by ionizing radiation
has been reported, but such a high proportion as four intermediate mutantsin six induced mutant lines could not be expected. These results and thepresence of gene dosage effect of Wx-wx character in rice endosperm would
suggest some kinds of specificity in the expression pathway for waxy starch
In nee.

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77

Induction of Waxy Starch Mutants in Rice
Etsuo AMANO

To investigate the relationship between mutagen treatment and characteristics of structural alterations in induced mutants, many waxy (wx)starch mutants have been induced in Oryza sativa var. Norin 8. Mutagenic
treatments by ethyl methanesulfonate exhibited same high efficiency inmutation induction as the cases in maize. Presence of many intermediate
phenotypes in the wx starch character might suggest that considerable
proportion of these wx mutants might be induced as point or missence
mutations. As another type of mutagenic treatment, irradiation of seeds
by reactor thermal neutrons in the DzO facility of KUR (Kyoto University
Reactor) was successful to induce some waxy starch mutants. Some characters of these wx mutants were described elsewhere in this Annual Report.
In the present experiments, gamma-ray and a chemical agent, acridine
orange, were tested to see activity to induce waxy starch mutations. M 1
plants were grown in plastic flats filled with ordinary soil, so densely thateach M 1 plant could yield only a few panicles. From the seeds which were
irradiated by 30 kR of 137Cs gamma-ray, 106 panicles with one to nine
fertile seeds and 252 panicles with ten or more seeds could be harvested.
From 40 kR treatment, 959 and 1545 panicles respectively could be harvested. With 40 kR treatment, 1012 sterile panicles were also scored.
As a chemical agent, acridine orange was selected expecting to induce someframe shift mutations by hypothetical mechanisms of insertion of the molecules into DNA double helix. Rice seeds were soaked into 2 % water solution of the dye without any buffering salts, for 48 h at n°e. Coleoptiles
and some other embryonic tissues were stained in dark red color, but aftersome growth, young leaves seemed to be normal in apperance. The sameplanting method of M 1 was followed and yielded 213 panicles with one tonine seeds and 317 panicles with ten or more seeds.
Harvested materials were examined according to standard detection
procedure for waxy starch mutants. After drying well the harvested materials, segregation of waxy starch mutant was surveyed on panicle bases.
Seeds from a single panicle were hulled and endosperms were examinedvisually first. Opaque endosperms of any degree or other questionablegrains were tested further by scraping off a small area of endosperm surface
and staining by iodine reagent. Out of the two mutagen treatments, gamma-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

ray and acridine orange dye, in the present experiments, 3392 fertille panicles
were examined but no waxy starch mutant including detectable intermediateor leaky waxy mutant could be obtained. Reaction of rice to gammairradiation seemed to be very alike to maize in which viable mutant wasdifficult to induce by gamma-ray. Mutagenicity of acridine orange should
be carefully examined further before omitting from mutagen list, because
under the treatment condition used here, visible light activation of the dyeDNA complex might not be expected since embryo might be kept lightshielded by hulls.
Induction of Waxy Mutants in Maize by Gamma-Ray
Etsuo AMANO

To investigate the relationship between mutagenic treatments and induced genetic alterations, waxy (wx) mutants have induced and maintainedfor detailed analyses. Treatments by a chemical mutagen, ethyl methanesulfonate (EMS) have yielded many viable wx mutants. Germicidal
ultraviolet light applied to pollen grain have also yielded three viable wx
mutants. Among ionizing radiations so far tested, fast and thermal neutrons
induced some wx mutants but viabilities of these mutants were very low.
The most popular ionizing radiations, X- and gamma-rays have also been
used, but the mutagenic activities for wx locus were very low. Only a few
wx mutants were induced in some ten years of trials. All of them were
not viable. To confirm the previous results and to obtain, if possible,
viable wx mutants for further analyses, seed irradiation experiment was
carried out.
Seeds of multiple dominant inbred line (Cl Sh Bz Wx, Y)were irradiatedby 20 kR of l37es gamma-ray and planted in field. At flowering time, all
the irradiated plants were detasseled. In order to obtain induced mutants
with their inbred line genetic background, mixed pollen method was used
to pollinate the treated ears. Male tassels were collected on the previous
day from untreated control and a multiple recessive tester (C sh bz wx, Y)
separately. Tassels of each groupe were placed in a bucket of water on alarge sheet of paper. On the day of pollination, pollen were shaked offfrom tassels onto the sheet of paper and sifted with a fine mesh sieve. Pollenof the two strains were then weighed and pollen of equal weight were mixed
together in a large glass beaker. They were brought to the field in PetriRESEARCHES CARRIED OUT IN 1980

79

dishes. Pollen were placed on silks of the treated plants using a small
brush. By this method, mutant F I kernels would be segregated in 1: 3
ratio within the mutant sector. Among the non-wx kernels in the sector,
half of the inbred line type kernels, or 1/4 of the total kernels, within the
sector would be expected to be heterozygous for the mutant gene with inbredline background. From these heterozygotes, new wx mutant with theoriginal inbred line background would be isolated in the next generation.
F I hybrids with the tester line or with any contaminating pollen would be
distinguished by out-growth due to heterosis. After harvesting and drying,kernels were examined visually on the female ear bases. Opaque endosperms were tested by iodine staining for waxyness. 530 ears carrying nineor less kernels and 2,568 ears with ten or more kernels were examined andthree ears segregated wx kernels. The frequency on ear bases amounted
to 0.1 % which was comparable to previous results of X- and gamma-rays.
In the present experiments, several wx kernels were segregated suggesting
better survival than the previous cases; Three sh i and six non-CI mutants
were also detected. As a trend, radiations induced more mutations in
distal loci than proximal ones and chemical mutagen, EMS, in reverse.
The difference in this trend might be statistically insignificant, but reproducibility of the results seemed to be high after several years of experience.
Automated Analysis of Maize Pollen
Etsuo AMANOPollen grains are haploid gamete cells of uniform shape and size, and canbe obtained and handled in large quantity. If appropriate genetic traits(ex. Wx-\Vx or Ae-ae) are used, they can be an excellent material for investigation of rare but important biological events like intracistronie recombinations or mutations induced by very low level of mutagens. Thisadvantage will be further improved, if the laborious counting and examination can be made automatically. Objectivity of the observed results will bealso increased.
For automation of pollen analysis, techniques of flow analysis and image
analysis would be applicable. In image analysis, samples are fixed on aslide glass, and then each picture frame taken by a TV camera is scanned
for analysis. In flow system, sample grains are suspended in solution andpass through a narrow detection area and signals generated by the detector

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

MCA
r

I

CRT monitor

I

Microscope r
I

M

S.mp"

: ,

",.,

Fig. 1.

[Il

x- Y plotter~-- Printer

U~~L E,h.", ",.,

~AVR

Schematic diagram of instrumentation of the flow system.

are examined. Here, transportation system of the sample grains is important but fixation of samples on a slide glass is not necessary. Unfixedsample pollen grains can be analyzed repeatedly or analyzed by othermethod like ordinary visual analysis or by image analysis. Presently aflow analysis system with an optical detector was tested using maize pollen.
The flow system used is shown schematically in Figure 1. Sample pollen
grains were suspended in iodine solution and agitated by a small stirring
blade. They were transported through a glass capillary (0.8 mm diameter)
by gentle suction of air using a mierotube pump. The capillary was fixed
between a slide glass and a cover slip. Space around the capillary was filledwith balsam. The capillary was positioned precisely in the light path of a
microscope. Although the capillary used for maize pollen grain was quite
large compared to diameter of a pollen grain, particles flowed at the centerof the capillary when the suspension was transported at the rate of 2 ml/min
or 4 ml/min. This centering of pollen grains in flow was an unexpectedadvantage of the present system and made focusing of pollen images on the

RESEARCHES CARRIED OUT IN 1980

81

photocell easy. Interuptions of the light path by pollen grains were detectedby a silicon photocell after optical magnification of about ten times. Electricpulses generated by the photocell were amplified and sent to a multichannel
pulse height analyzer (MCA, Series 30 by CANBERRA). MCA used wascapable of storage of data up to 10 6-1 counts in each of its 1024 channels.
Each input pulse was assorted to corresponding channel according to its
pulse height which would reflect the genetic character. The frequency
distribution of pulse height can be displayed on a TV screen of the MCA.
Graphic display and digital data including integrated data of region of interest (ROI) can be printed out by a X-Y plotter and a line printer. Extra
care was taken to stabilize the light source to illuminate the capillary.
Mechanical fixation, soldered leader wires, and an electronic voltage regulator were used.
At the flow rate of 2 ml/min, count rate of 500 pulses/sec (3 X 10 4 pulses/min) seemed to be optimal for pulse separation and scoring speed. In thisconcentration and speed of flow, 106 pollen grains may be analyzed in some
half an hour. Staining and ratio of transmitting and reflecting light arevery important to differentiate the pulse height generated by the sample
pollen grain with different genetic traits. For good discrimination of thegenetic traits, like Wx-wx, illumination of the sample grains must be improved.
Trial to Induce Waxy Starch Mutant in Diploid Wheat
Etsuo AMANO

Waxy (wx) starch mutants have been found in many crops like rice,maize, barley, sorghum and millets. These mutants have been picked up
and maintained in the long agricultural history in eastern Asia. Recognition and preference for waxy starch might closely relate to the cooking
habit of the people in this area. In rye which is diploid but did not becomea main food crops in this area, and wheat or oats both of which are polyploid, no waxy starch mutant has been reported. However, starches ofthese rye, wheat and oats also consist of both amylose, straight chain molecules, and amylopectine, branched molecules, in the same normal proportion as rice, maize or barley. If genetic alterations which would disturb
amylose synthesis pathway could be induced, it would become a low amylose
waxy starch mutant. Possibilities of inducing waxy starch mutants in rye,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

wheat and oats would be suggested by the fact that waxy starch mutants
were found in barley which is closely related to these three crops. Anothersupport for the possibilities would be the case of Oryza glaberrima, an
African species of cultivated rice in which no waxy starch mutant had beenreported. The first waxy starch mutant in this species was induced by achemical mutagen, ethyl methanesulfonate (EMS). Breaking through thegenetic barrier of polyploidy would not be impossible, if the techniques ofsynthesis of hexploid bread wheat are followed, waxy starch mutations
being induced step by step in the constituting genomes. Encouraged bythese examples, experiments to induce waxy starch mutation in wheat werecarried out.
A diploid spcies of wheat, Triticum monococcum, was selected to use in
the experiments. Preliminary experiments suggested that soaking treatment of seeds of barley or wheat should be made at lower temperature thanrice or maize, the summer crops. After presoaking in distilled water,
seeds were soaked in 0.1 M solution of EMS for 20 hours at 15°C. During
the soaking treatments, seeds containing Ehrenmyer flasks were gently
shaked in the water bath for uniform contact of seeds with the treatment
solution. After rinsing and post-soaking in distilled water for 24 hours,
seeds were sown in wooden flats. Seedlings were transplanted to field in
November. On M 1 plants, many chlorophyll mutation stripes could beobserved indicating that the treatments might be adequate. Two spikes
were harvested from each of M 1 plant. Well dried endosperms were examined for segregation of possible waxy starch phenotype, on spike bases.
The material used in the present experiments, T. monococcum fiavescens,had relatively transluscent appearance in its endosperm. However, thetransluscence was not enough, compared to rather transparent rice or maize,
to distinguish opaque apperance of waxy starch phenotype by visual examination as the first screening procedure. Each opaque or questionable
seeds were further tested by iodine staining for confirmation. Higher
protein content in wheat should be also considered for the phenotypic
expression. In the present experiment, 400 spikes harvested from thetreatment by 0.1 M solution of EMS were examined but no waxy starchmutant was detected. In case of maize and rice, the mutant segregating
spikes would be found, after optimal treatments, in about a half to one
percent of the spikes examined. It would be suggested that grain to grain
tests for iodine staining to more than some hundreds of spikes might be

RESEARCHES CARRIED OUT IN 198083

required for induction and detection of the waxy starch mutant III thediploid wheat.
Effect of Tumor Promoter in Soybean
Taro FUJII

l2-0-tetradecanoyl-phorbol-13-acetase (TPA) isolated from oil of theseeds of Croton tiglium is a very strong tumor promoter. Another indol
alkaloid compound, teleocidine isolated from Streptomyces mediocidicus isalso suspected to be tumor promoter. To examine the effects of thesechemicals in higher plants, seeds of soybean T219 were treated and frequency of spot per leaf was investigated. (Chemicals were kindly supplied
by Dr. H. Fujiki of the National Cancer Center Research Institute).
Chemicals were disolved in DMSO and diluted to given concentrations.
The amount of DMSO added for disolve (5 ,ugjml) has no effect on mutation induction. Different concentration of chemicals, viz., 1, 5 and 10 ,ugjml
of TPA, and 1, 10 and 50,ugjml of teleocidine were applied to dry seeds,
Mutagenicity tests of these chemcials were repeated twice with similar concentrations, however, spotting frequency did not increase at least concentions tested. It is known that about 80 % of the carcinogens induce mutagenic effects. Although both chemicals has a potency to promote oftumors in rats under the cooperation with initiating substance(s), no mutagenic activity has been reported in single treatment on Ames' spot test
method. Experimental results with soybean seeds indicated that bothchemicals has no mutagenic effect in plants.
Combination treatment of caffeine with TPA and teleocidine was carriedout to see the effect of these chemicals on the caffeine mutagenesis. Single
treatment of 0.02 % caffeine induced about 10 spots per leaf (control value
is about 1.5 per leaf). Unexpectedly, such large number of mutant spots
induced by caffeine treatment decreased to about a half by mixture of5,ugjml of TPA or 50,ugjml of te1eocidine, respectively. Combination
treatment of gamma-rays and TPA was also conducted to see the effect ofchemical on the radiation induced mutational damage. However, nodifference in spotting frequency was observed between single radiation
treatment of 200 or 500 R and combination of chemical with radiations.
Mechanism is not well explained at present, but some sort of repair process
might be involved in the combination of two chemicals, and some hidden

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

action of promoters may act to caffeine mutagenesis but not to radiation
mutagenesis.

RESEARCHES CARRIED OUT IN 1980

VIII.

85

POPULATION GENETICS (THEORETICAL)

Average Time until Fixation of a Mutant Allele in a Finite
Population under Continued Mutation Pressure: Studies
by Analytical, Numerical, andPseudo-sampling Methods
Matoo KIMURA

We consider a single locus, and denote by A the wild-type allele and by
A' the mutant allele that is produced irreversibly in each generation from
A at the rate v. Let 1+s, 1+h, and 1 be respectively, the relative fitnessesof mutant homozygote A'A', mutant heterozygote A'A, and wild-typehomozygote AA. Then, it is shown, on the basis of the diffusion equation
method, that the average time until fixation of the mutant allele (A ') in arandomly mating population of effective size N" given that the initial frequency is p, is_.
(I(YeBIX)xV-1
T(P)=4Nc )p e-BlY)y-Vdy )o-i~x-dx,
in which B(x)=(S/2)x 2 +Hx(l-x), S=4Nc s, H=4Nc h, and V=4Nc v. Of
particular interest are the cases in which the mutant allele is deleterious(s=-s', s'>O). Three cases are considered; the mutant is: (i) completely
dominant s=h=-s', (ii) completely recessive s=-s', h=O, and (iii)
semidominant s= -s', h= -s' /2, in which s' is the selection coefficierit
against the mutant homozygote. It is shown that the average time until
fixation is shorter when the deleterious mutant allele is dominant thanwhen it is recessive if 4Nc v is larger than 1. On the other hand, the situationis reversed if 4Ncv is smaller than 1. It is also shown that for a mutant
allele for which N c s'>10, it takes such a long time until fixation that wecan practically ignore the occurrence of random fixation of a deleterious
allele under continued mutation pressure. To supplement the analyticaltreatment, extensive simulation experiments were performed by using adevice called the pseudosampling variable, which can enormously accelerate
the process of simulation by a computer. This method simulates the diffusion process itself rather than the binomial sampling process (in population genetics the diffusion model is usually regarded as an approximation

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

of the discrete binomial sampling process).
Acad. Sci. USA 77, 522-526.

For details, see Proc. Natl.

A Simple Method for Estimating Evolutionary Rates of Base
Substitutions Through Comparative Studies
of Nucleotide Sequences
Motoo KIMURA

Some simple formulae were obtained which enable us to estimate evolutionary distances in terms of the number of nucleotide substitutions (and,also, the evolutionary rates when the divergence times are known). In
comparing a pair of nucleotide sequences, we distinguish two types of differences; if homologous sites are occupied by different nucleotide bases but
both are purines or both pyrimidines, the difference is called type I (or"transition" type), while, if one of the two is a purine and the other is apyrimidine, the difference is called type II (or "transversion" type). Letting
P and Q be respectively the fractions of nucleotide sites showing type I andtype II differences between two sequences compared, then the evolutionary
distance per site is K=-(lj2) In {(l-2P-Q),J1-2Q}. The evolutionaryrate per year is then given by k=Kj(2T), where T is the time since the divergence of the two sequences. If only the third codon positions are compared, the synonymous component of the evolutionary base substitutions
per site is estimated by K s '=-(lj2) In (l-2P-Q). Also, formulae forstandard errors were obtained. Some examples were worked out using
reported globin sequences to show that synonymous substitutions occur atmuch higher rates than amino acid-altering substitutions in evolution.
For details, see J. Mol. Evol. 16, 111-120.
Genetic Variability and Effective Population Size When Local
Extinction and Recolonization of Subpopulations are Frequent
Takeo MARUYAMA and Motoo KIMURA

If a population (species) consists of n haploid lines (subpopulations)which reproduce asexually and each of which is subject to random extinction and subsequent replacement, it is shown that, at equilibrium in which
mutational production of new alleles and their random extinction balanceeach other, the genetic diversity (1 minus the sum of squares of allelic fre-

RESEARCHES CARRIED OUT IN 198087

quencies) is given by 2Nev/(1+2Nev), whereN e =N+n/(2J.) +nNv/J.,

in which N is the harmonic mean of the population size per line, n is thenumber of lines (assumed to be large), A is the rate of line extinction, andv is the mutation rate (assuming the infinite neutral allele model). In a
diploid population (species) consisting of n colonies, if migration takes
place between colonies at the rate m (the island model) in addition to extinction and recolonization of colonies, it is shown that effective population
size isN e =N+n/[4(v+A+m)]+nN(v+m)/(v+A+m).
If the rate of colony extinction (A) is much larger than the migration rateof individuals, the effective population size is greatly reduced comparedwith the case in which no colony extinctions occur (in which case Ne=nN).
The stepping-stone type of recolonization scheme is also considered. Bearing of these results on the interpretation of the level of genetic variability
at the enzyme level observed in natural populations is discussed from thestandpoint of the neutral mutation-random drift hypothesis. (Proc. Natl.
Acad. Sci. USA 77: 6710-6714.)

Amino Acid Diversity of Immunoglobulins as a Productof Molecular Evolution
Tomoko OHTA

Based on population genetics theory of the evolution of multigene families,
the sequence variability of the variable regions of immunoglobulins compiled by Kabat et al. (1976) has been analysed. An amino acid identity
coefficient either within or between species is calculated separately for boththe hypervariable and the framework regions. Under the somatic mutation
hypothesis, the somatic component of amino acid diversity exists in addition
to the germ line component and should contribute an amount of change
between the hypervariable and framework regions that is independent of
the time since the divergence of any two immunoglobulin gene families.
The relationship between the identity coefficient of the hypervariable region
and that of the framework region is shown to be not in accord with suchprediction. The result indicates that the rate of evolutionary accumula-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

tion of amino acid replacements in the hypervariable region is roughly threetimes more rapid than in the framework region and the hypervariability
within a species is a necessary consequence of the high evolutionary rate.
For details, see J. Mol. Evol. 15, 29-35.
Linkage Disequilibrium between Amino Acid Sites in
Immunoglobulin Genes and Other Multigene Families
Tomoko OHTA

Linkage disequilibrium between segregating amino acid sites in repeatedgenes which form a multigene family was investigated by using the population genetics theory. The degree of non-random association of aminoacids is measured by the disequilibrium coefficient which is determined by
the balance among various forces; unequal and equal crossing-over, mutation, random genetic drift and recombination which takes place between
the two sites in question. Another measure of disequilibrium, 'standardized identity excess', represents excess probability of simultaneous
identity at the two sites over that expected from random association ofamino acids. Sequence data of variable region of immunoglobulins provide
most interesting example of multigene family to apply the theory. Statisticalanalyses on identity excess for various groups and subgroups of variable
region sequences of immunoglobulins suggest that a multigene family suchas human f( or mouse K gene family consists of several subfamilies between
which recombination is limited. The analyses also indicate that the recombination may take place between any gene members in one subfamily.
For details, see Genet. Res. Camb. 36, 181-197.
Two-Locus Problems in Transmission Geneticsof Mitochondria and Chloroplasts
Tomoko OHTA

A problem in transmission genetics was studied theoretically by regarding
a cell as a population of organelle chromosomes. A hybrid cell of thetwo strains is assumed to consist initially of equal numbers of the two
types of nucleoids, each containing an equal number of chromosomes.
The two parent strains have different alleles at the two loci, and the twolocus dynamics of the hybrid cell line was analyzed. Within each nucleoid,

RESEARCHES CARRIED OUT IN 198089

stochastic matching and gene conversion are assumed to occur between
the chromosomes, and at a certain rate fusion and division are assumed totake place between the nucleoids within the cell. Under this model, thechanges of average genotype frequencies at the two loci within a nucleoid
and within a cell were formulated, and the final frequencies of recombinant
chromosomes were obtained. Random segregation of nuc1eoids at cell
division was incorporated into the analyses; in addition, the increase of
homozygosity in the cell was formulated. The results may be useful forinterpreting the observed data on organelle genetics of some species ofalgae and yeast. For details, see Genetics 96, 543-555.
Composite Stepwise Mutation Model under the Neutral
Mutation Hypothesis
Naoyuki TAKAHATA

As an extension of the conventional (Ohta-Kimura) stepwise mutation
model, a new model is proposed. In this model, it is assumed that eachcharge state (electromorph) is represented by K alleles and that a mutation
changes an allele either by one step in the charge space or to one of theother members of the identical electromorph. It is shown that the net
genetic variability within a population is similar to that predicted by theinfinite allele model rather than to that predicted by the stepwise mutation
model, and the K-dependence of genetic variability is rather weak whenK~2 and the effective population size is not much greater than the reciprocal of mutation rate. The results are compared with the recent observations at the xanthine dehydrogenase locus in Drosophila pseudoobscura.
For details, see J. Mol. Evol. 15, 13-20.
Polymorphism and Loss of Duplicate Gene Expression: A
Theoretical Study with Application to
Tetraploid Fish
Naoyuki TAKAHATA and Takeo MURUYAMA

We studied the fixation of null alleles at independent duplicate loci, assuming that wild-type active alleles mutate irreversibly to nonfunctionalnull alleles and that the population is finite and panmictic. Solving thetwo-dimensional Kolmogorov backward equation numerically, we obtained

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

the rate at which one of the active genes is lost and the amount of heterozygosity at specified times. Previously harmful genes, including recessive
lethals, can be fixed at one of the duplicate loci, which would not happen
with a single locus. Examination of data from several fish families showedthat the rate of fixation of null alleles is too slow and the amount of heterozygosity too small to be compatible with complete recessivity at all loci.
Our conclusion differs in this regard from that of Bailey et al. (Proc. Natl.
Acad. Sci. USA 75, 5575-5579). They also reported that the time taken
for 50% of the loci to be fixed for null alleles is approximately l5N+v- 3 / 4 ,in which N and v are the effective population size and mutation rate, when
the alleles are completely recessive and the double homozygote is lethal.
We found that the fixation rate depends not only on N, but also on Nv.
For details, see Proc. Natl. Acad. Sci. USA 76, 4521-4525.

RESEARCHES CARRIED OUT IN 1980

IX.

91

POPULATION GENETICS AND EVOLUTIONARYGENETICS (EXPERIMENTAL)

Molecular Evolution in Papova Viruses and Their Host
Species, and in Bacteriophages
Takeo MARUYAMA and Eiichi SOWA

Comparing homologous genes of three papova viral genomes, we showed
the very close relative phylogeny among the viral species and their hostspecies, and therefore the viral species seem to have evolved with their hostorganisms (Soeda et aI. 1980). Additionally, the DNA-sequence data ofbacteriophages tP X174 and G4 and their overlapping genes were examinedfor evolutionary patterns. It was made evident that overlapping genes havea quite different substitutional pattern with respect to the position of nucleotides in the codons than do non-overlapping sequences. Namely, inoverlapping regions the third positions are usually substituted fastest, followed by the first positions, while the second positions are slowest in eachgene, though different genes may have different rates of nucleotide substitution. With overlapping genes, this pattern does not apply, but rather isaltered because of an interaction between the substitution rates in the twogenes involved in an overlap. (Publication, Stadler Symp 12: 83-96.)
Stasipatric Speciation in Drosophila
Takao K. WATANABE and MASAOKI KAWANISHIWhite (1968, Science 159: 1065) proposed a model of speciation named"stasipatric speciation" from the karyological studies of a grasshopper
species group. That is, a new species arose within the old species range
and took over whatever area it could by eliminating the old species. Thismodel can be applicable to some Drosophila species evolution if we takeour hypothesis of interspecific mating preference (1979, Science 205: 906)
into account for the geographical distribution of some sibling species ofDrosophila. Our hypothesis insists that a new species female rejects an old
species male but the old species female accepts the new species male. Evolutionary sequence of the D. takahashii group can be speculated from our92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

hypothesis as follows; D. pseudotakahashii (New Guinea-Australia)----o>D.
trilutea (Taiwan)----o>D. lutescens (Japan)----o>D. takahashii (India-Southeast
Asia-Japan). We can suppose that the older species which had ever evolvedin the center of the Southeast Asia having spread over to the present marginalareas such as Australia, Taiwan and Japan, then the newest species (D.
takahashii) finally occurred in the center and is now spreading its distribution by eliminating the older species around the center. In anotherspecies group we can show the direction of species evolution as follows:
D. pseudoananassae (from Thailand, Borneo to New Guinea, Australia)----o>
D. malerkotliana (from India to Borneo)----o>D. bipectinata (from India, Japan
to Fiji, Australia)----o>D. parabipectinata (around Borneo). All the four
species now coexist in Borneo which is the probable center of this group.
The newest species (D. parabipectinata) had evolved recently in Borneo
where already the other three older species existed, although it did not rid
the older species yet. The reason why our hypothesis is favorable to infer
the evolutionary change of the species distribution is as follows: (1) A new
species can establish itself in a nonperipheral local colony previously occupied by the older species if she has an ability to discriminate and reject
old species males. (2) Sinee the new species males are accepted by the old
species females, old species population retreats its previous territory showing
a hybrid zone when the new species adapts to the environment. Thus,
asymmetrical mating preference between sibling species supports the stasipatric speciation model.
Directional Change in the Frequencies of Cosmopolitan
Inversions of D. melanogasterYutaka INOUE and Takao K. WATAKABEPolymorphic inversions in several world populations of D. melanogasterwere compared. Frequencies of two common cosmopolitan inversions on
the second chromosome (In(2L)t, In(2R)NS) are listed in Table 1. The
Old World and Australian populations generally showed the relation ofIn(2L)t>In(2R)NS, but the New World populations rather showed aIn(2R)NS> In(2L)t relation.
A Japanese(Katsunuma) population has been surveyed for 16 years. It
was clearly 2Lt>2RNS in the sixties but now is in 2Lt;:::;;2RNS due to a
rapid decrease of In(2L)t. The relation of In(2L)t>2R(NS) was still

RESEARCHES CARRIED OUT IN 1980
Table 1.

Population

Frequencies (%) of the second chromosomal inversions per
arm of the several world populations
No. oftestedchromosomes

2Lt

Reference

2RNS..

- - ~ ~ - _Japan (Katsunuma)
Japan (Katsunuma)
Korea
Papua New Guinea
Crete
GreeceGreeceIsraelEgypt
South-Africa
Australia
Columbia
U.S.A.
U.S.A.

93

1200
2081
1720
52
64
32
426
32
1340
90
68
10
1504
4323

31.7
13.8
9.8
53.8
6.3
4.7
2.3
21.9
7.4
18.8
26.5
10.0
17.2
13.0

22.8
11.9
7.5
42.3
1.6
0.9
0.9
3.1
4.1
0
16.2
30.0
8.6
16.0

_-,~-

The present study 1963-1969
The present study 1970-1980
Paik 1979
Inoue and Watanabe 1980
Ashbumer and Lemeunier 1976
Alahiotis 1977
Zachalopourou 1974
Ashburner and Lemeunier 1976
Mourad and Mallah 1960
Inoue and Watanabe 1980
Ashburner and Lemeunier 1976
Ashburner and Lemeunier 1976
Warters 1944
Yang and Kojima 1972
Ashburner and Lemeunier 1976
Stalker 1976
Mettler et al. 1977

observed in other Japanese populations than Katsunuma. On the other
hand, the past U.S. populations, about thirty years ago, had shown therelation of In(2L)t>ln(2R)NS like the Old World populations, although
the recent U.S. populations are in In(2R)NS>ln(2L)t. Thus, it can be
speculated that the original type of In(2L)t>ln(2R)NS relation which wasobserved over the world has been changing to the new type of In(2R)NS>In(2L)t relation and that the U.S. population is an outrider.
Molecular Phylogenies of Drosophila: Protein Differences
Detected by Two-dimensional Electrophoresis
Seido OHNISHI, Masaoki KAWANISHI and Takao K. WATANABE

Many abundant proteins (ca. 100) of Drosophila adults were investigatedby an O'Farrell's technique (two-dimensional electrophoresis; 2DE).
Eighteen species belonging to three taxonomically different groups (sixspecies of the D. melanogaster subgroup, four of the D. auraria complex
and eight of the D. virilis group) were used. The number of charge change

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

aGenetic Distance
1.0
1.5

0.5

D. quadraria ]D. lri(lllr~lria .Jl------,=------.Jf----------------~: :;:::~;::~:-,

D. leissieri
D. yakubalJ. creeta------,[ D, (wrona[lJ--------,

D. m((uriti(ln(l-'-~
1.),'

2.5

2.0

I

nl(_,htnOg(l.sler--.----J~I~- - - - - - - - - - - -..
!

~j: ~~;~~~l~:.an(( e t =D. viTilis
D. nOl'amexieana-----,'-~ID. [ummel~,
D. e;:oana~

z: U~~~~

....1

~

1"

t

i

1

Local Sub- or Sibling
Population Semi- Species
SpeciesMorphologicallyDistinguishable Species

Fig. 1. The phylogenie tree of three Drosophila groups constructed from dataon protein differences detected by two-dimensional electrophoresis. Arrows show
the level of speciation according to allozymes' data of Ayala et al. (1974). Brackets
at left margin indicate taxon in a conventional taxonomy.
Table 1. Correlation coefficients between genetic distances obtainedby 2DE, SDE and the percent of successful mathings (PSM) in
three taxonomically different groupsGroup----_._--.

D. melanogaster subgroup
D. auraria complex
D. virilis group

*
**

d.f.

20E vs.
SOE
0.819**
0.504*
0.782**- ~ - - - - - _ .

13
14
8

SOE vs.
20E vs.
PSM- - - - -PSM----0.755**-0.889**-0.734**-0.720**-0.715*-0.900**- - _....

__..

~-

Significant at 5 % level.
Significant at 1 % level.

differences in the proteins was compared in all combinations of species pairs
within the group (or complex). A dendrogram was constructed fromgenetic distances (Nei, M., Amer. Nat. 106: 283, 1972) which were calculated from the above differences (Fig. 1). This phylogenie relationship wasconsistent with the results obtained from the percent of successful matingsor conventional (one-dimensional) electrophoresis (SDE) as shown in
Table 1, and also with the morphological and cytological phylogenies.

RESEARCHES CARRIED OUT IN 1980

95

Therefore, if we consider that genetic variation within species estimatedby 2DE is lower than that by SDE (Ohnishi et al., Genetics, in press) andthat many protein loci can be analyzed by a single gel in 2DE, this technique
is a more useful tool for the taxonomical studies of genetically differentiatedspecies.
Geographical and Ecological Variations of Oryza perennis
at Six Isozyme Loci
Gerard SECOND and Hiroko MORISHIMAIn Asia, Oryza perennis is found mainly in the vicinity of rice fields, from
West India to southern China and Indonesia, showing a range from perennial to annual ecotypes. In the line of previous investigations of O. sativa,
the genetic diversity of O. perennis was surveyed at three isozyme loci,
Cat-A (catalase), Pgi-A and Pgi-B (phosphoglucose isomerase), with regardto i) electrophoretic mobility and ii) heat stability (for Pgi only). About
300 strains were studied. In addition, the original data recorded by Drs.
B. B. Shahi and C. Pai on three more loci, Acp-l (acid phosphatase), Px-l
and Px-2 (peroxidase), were reexamined with the same aim. The results
are summarized as follows:
1) Pgi electromorphs were more numerous in O. perennis than in O.
sativa. In all geographical areas and ecotypes of O. perennis, the mostfrequent electromorphs were those characterizing the Japonica type of O.
sativa, although Indica cultivars are predominant in the distribution areasof O. perennis.
2) Similar bands of Pgi in different individuals rarely showed differential
heat stability. The most frequent band was most thermo-resistant.
3) Two electromorphs only were found at the Cat-A locus. They werecharacteristic of the Indica and Japonica types of O. sativa, respectively,
and showed a geographic cline clearly: The "Japonica" electromorph was
frequent in southern China including Taiwan, and the "Indica" electromorph was frequent in other regions. A similar cline seemed to exist
among the acid phosphatase electromorphs specified by the Acp-Ilocus.
4) All annual types of O. perennis so far studied lacked the "Indica"
electromorph specified by the Pgi-A locus.
In interpreting these data, it must be taken into consideration i) that thecurrent populations of O. perennis are relicts of larger populations that

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

should have existed before domestication by man, ii) that they have hybridized with O. sativa cultivars which might have originated in different
geographical areas, and iii) that some of the present races of O. perennis
may represent a return to wild type following hybridization between independently domesticated cultivars, as was observed in Africa. The data
indicated a geographical pattern of variations in isozyme markers which
are presumably neutral in seletion, among the populations of O. perennis.
This pattern is related to the Indica-Japonica differentiation, but not to thepresent distribution of Indica and Japonica cultivars. The barriers to land
migration of O. perennis like the Himalayan range and the ocean seem toaccount for the geographical pattern of variation. Thus far, no particular
ecotype of O. perennis nor the populations in any particular geographicalregion, may be assigned to be the direct progenitor of O. sativa. Yet, thedata are in agreement with the hypothesis of independent primary domestication of the Indica and Japonica types of O. sativa. An additional survey
for a total of 25 isozyme loci including these six is under way.
Isozyme Polymorphism in Natural Populations of Oryza perennis
Chiang PAl and Hiroko MORISHIMA

Plants raised from the seeds of natural populations of wild rice, Oryza
perennis Moench, collected in India and Thailand in 1979 (Morishima et al.
1980), were investigated with regard to peroxidase and acid phosphataseisozymes. The data were analyzed together with those for other O. perennis
populations from different parts of the world so far observed. A total of
78 populations (60 Asian, 13 African, 4 Australian, and 1 American) werestudied, and heterozygosity and gene diversity (as shown by I-Ix;", Nei
1975) were computed from the frequencies of alleles at the PX 1 and ACPlloci. It was found that:
1) Three codominant alleles, OC, 2A, and 4A, are known at the PX 1
locus. Both 2A and 4A were frequent in Asian perennial populations
while many of Asian annual populations were monomorphic with 2A.
Allele 4A was predominant in African (perennial) and American (perennial)
populations; allele OC was fixed in the Australian (annual) populations.
2) At the ACPl locus, seven codominant alleles controlling the electrophoretic mobility of the AMC band-group are known, which were designated as -17, -9, -4, 4, 9, 12, and 24 according to the running distanceRESEARCHES CARRIED OUT IN 198097

of band C (Pai et al. 1975). Indian perennial populations were polymorphic
and generally showed high frequencies of alleles -4 and 9; Indian annual
populations showed fixation of either -4 or 9. In Thailand, allele 9 wasrare. Thailand perennial populations were polymorphic and showed ahigh frequency of allele -4; this allele was fixed in Thailand annual populations. African populations had fixed allele 4, while American as well asAustralian populations had fixed allele 12.
3) The level of isozymic variability was generally higher in the perennial
than in the annual type. Among Asian populations, the mean frequencyof heterozygotes and mean gene diversity for the PX 1 locus were 0.201 and
0.273 for the perennial type, and 0.002 and 0.049 for the annual type, respectively. African populations, rhizomatous and strongly perennial,
were rather monomorphic although they are almost completely outcrossed.
Their heterozygosity was 0.036 and their gene diversity was 0.034 when afew populations which had coexisted with other Oryza species were excluded.
This suggests that the African populations consisted of genetically identiacl
clones propagating by rhizomes. The gene diversity at PXl and that at
ACPl were intercorrelated among the populations examined, although theformer locus was less polymorphic than the latter.
4) Almost all cultivars of O. sativa uniformly had allele 2A at the PX 1
locus and either allele -4 (Indica) or 9 (Japonica) at the ACPl locus. Wild
populations, particularly those of perennial types, are more polymorphic.
Geographic differences in the frequency of ACPl alleles were also detected.

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31x.

HUMAN GENETICS

Hereditary Retinoblastoma: Host Resistanceand Second Primary Tumors
Ei MATSUNAGA

Data for 26 cases of nonradiogenic osteosarcomas that developed inpatients with retinoblastoma and in their close relatives were collected fromthe literature and analyzed. The distribution of unaffected and unilaterally
and bilaterally affected cases of retinoblastoma agreed with the expectationthat risk to osteosarcoma did not differ by the phenotypes of the gene carriers. This finding indicated not only that increased risk to second primarytumors was a pleiotropic effect of the retinoblastoma gene but also thathost resistance to the gene effects was tissue-specific. The risk of genecarriers to nonradiogenic osteosarcoma was estimated at 1.2 %, and therelative risk was estimated at 230. Liability of the gene carriers to osteosarcoma could be regarded as a multifactorial threshold character, andheritability was estimated at 71 %. Results of analyses of ages at diagnosis
suggested that nonradiogenic osteosarcoma developed slightly earlier incarriers of the retinoblastoma gene than in children not carrying the gene.
These findings are discussed in the light of the host resistance model in whichgenes involved in tissue resistance must be suppressors to an error in differentiation (presumably a crucial event in malignant transformation) andradiogenic osteosarcoma in gene carriers is ascribed to the accumulation of
induced mutations in that suppressor system. For details, see J. Natl.
Cancer lnst., 65: 47-51, 1980.
Retinoblastoma: Host Resistance and 13q- Chromosomal Deletion
Ei MATSUNAGA

Of genetic importance is the question as to whether the retinoblastoma
gene is located on that segment of 13q, which when deleted, would develop
the tumor at high frequency. While the question may eventually be answered by linkage studies of familial retinoblastoma using biochemical or
chromosomal markers, we may take a different approach by comparing

RESEARCHES CARRIED OUT IN 198099

the variation in expressivity or laterality among patients with hereditary
retinoblastoma with that among patients with 13q-. This approach isvalid provided that the host resistance that is largely controlled by genetic
factors other than the major gene or deletion of 13q can be adjusted between
the two groups of patients.
Data for 27 cases of retinoblastoma that developed in patients with 13qwere collected from the literature and analyzed. The distribution of unilateral and bilateral cases of retinoblastoma differed significantly from theexpectation that the degree of expressivity does not differ between theretinoblastoma gene and deletion of l3q. The excess of unilateral cases
among the patients with 13q-, which could not be accounted for by ascertainment bias, was attributed to somewhat lowered carcinogenic potentialof deletion of 13q14 as compared with the retinoblastoma gene. It wasargued that the retinoblastoma gene is probably not located on l3q, andperhaps 20 % or more of the individuals with a deletion of l3q14 would not
develop retinoblastoma. The normal allele at the retinoblastoma locus,
the haplicon in the segment of l3q14, and the suppressor genes as definedby the host resistance model, may be all concerned, in their function additively and without dominance, with normal differentiation of the embryonicretinal cells. For details, see Hum. Genet. 56: 53-58, 1980.
Ectopic Pregnancy and Myoma Uteri: Teratogenic Effects andMaternal Characteristics
Ei MATSUNAGA and Kohei SHiOTA

Environmental factors responsible for congenital malformations include
a variety of physical, mechanical, biochemical, chemical, and biologicalagents which can affect the organogenesis of an embryo. Among these,
little attention has been paid by teratologists to the potential teratogenic
effect of spatial restrictions imposed by an altered micro-environment surrounding the developing embryo. The most clear-cut example of this istubal pregnancy. Even if the tube does not rupture but distends, accommodating the growing conceptus with placenta formation, conditions in
the wall of the tube, which are totally different from those in the uterus,
must be adverse for the development of the embryo. The same could
apply, to a lesser degree, to cases of pregnancy complicated by large myomas;
small myomas are generally considered to be clinically insignificant.

100 ANNUAL REPORT OF NATIONAL INSTITURE OF GENETICS NO. 31

Morphological data from 3,614 well-preserved human embryos derived from
artificial termination of pregnancy, collected by Nishimura and his associates
in Kyoto University, were used to determine whether ectopic implantation or
enlarged myomas could enhance the prevalence of localized malformationsof the embryo. The frequency of malformed embryos was 11.6 % among
43 recovered from ectopic pregnancies, 6.2 % among 97 from myomatous
pregnancies, and 3.3 % among 3,474 from normally implanted pregnancies
not complicated by myomas. Unilateral amelia in the ectopic cases andcaudal dysplasia in the myoma cases were significantly increased. Bothmalformations were quite unusual in the control group. It is argued thatspatial restrictions could be a teratogenic agent in human embryos. Mothersof both ectopic and myoma cases were on average much older than mothersof the control specimens, and those with myomas had a higher frequency
of previous pregnancy wastages. Ectopic pregnancy was found to be
associated with lowered parity, previous ectopic pregnancy, and maternalsmoking and drinking. These associations are discussed and interpreted
in relation to etiology of each of the two conditions. For details, see
Teratology 21: 61-69, 1980.

Search for Maternal Factors Associated with Malformed
Human Embryos: A Prospective Study
Ei MATSUNAGA and Kohei SHIOTA

Using data from 3,474 human embryos derived from induced abortionin the Nishimura collection, various maternal factors ascertained prior tomorphologic examination of the specimens were analyzed in association
with the frequency of malformed embryos. The overall frequency of malformed embryos was 3.3 %; polydactyly, holoprosencephaly, exencephaly
with or without myeloschisis, and cleft lip accounted for 88 % of the total
malformations. Mothers of low parity showed an increased frequency ofmalformed embryos. There were nine embryos from mothers with thyroid
disorders, of which one was affected with exencephaly and myeloschisis andanother with holoprosencephaly and polydactyly; this finding may support
the relation of maternal hyperthyroidism with anencephaly suggested byprevious case reports. The frequency of malformed embryos from mothers
who drank alcohol was significantly lower than from those who did not.

RESEARCHES CARRIED OUT IN 1980

101

Several possibilities were suggested to interpret this unexpected finding.
No association was verified with maternal age, irregular menstrual cycles,
irradiation, contraceptive methods, maternal occupation, and a smoking
habit, or with parental consanguinity. For details, see Teratology 21:
323-331, 1980.
Inactivation Centers in the Human X Chromosome
Yasuo NAKAGOME

According to Therman et al. (1974, 1979) and Summitt et al. (1978),there is only one inactivation center in the human X chromosome andwithout it an X chromosome or a segment thereof cannot be inactivated.
This inactivation center has been proposed to be located in the proximal
portion of the Xq and the presence of two Barr bodies in cases with duplication of this segment has been offered in support of this hypothesis.
In the present study, I compiled cases with a structurally abnormal X
that had been reported in the literature and compared the involved segmentsof X and their replication patterns to test this hypothesis. These included
17 balanced and 26 unbalanced X-autosome translocations, each with inactivation of either a derivative X or a derivative of any of the autosomes[abb. der(X) and der(A)]. A further 52 cases with various structural rearrangements were studied.
When a particular segment of X remains active, this does not necessarily
exclude the presence of an inactivation center(s) within it. On the other
hand, the occurrence of inactivation, even in a single case or in a small
proportion of cells, suffice to prove that the segment contains an inactivation center(s). In the present study, any segmcnts of X which replicatedlate were taken to be inactivated.
In a case of X-autosome translocation, a pter-~q112 segment was latereplicating. According to Therman et al. (Hum. Genet. 50: 59, 1979), X
inactivation center lies near the border of the Q-dark and the adjoining
bright band which presumably correspond to that of q 13 and q21. The
pter--->q112 segment does not include this site although it is late replicating,i.e., the short arm contains an inactivation center(s). The presence of aprobable case of i(Xp) is also in support of this conclusion. In an adjacent2 segregant of a t(X; 6) (p2l; p24) translocation, the X segment of der(6)replicated late in most of examined cells. It is obvious that distal half

102

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31(pter----*p21) of the short arm of X has an inactivation center and it can
suppress an autosome segment about 4 times larger. These findings do
not necessarily preclude the existence of another inactivation center(s) inthe proximal Xp. In a case of t(X; 14) (qI3; q32) translocation, a der(14)replicated late as a whole in all 25 cells examined. Therefore, a ql3----*qter
segment of X contains an inactivation cetner(s) and it can supress theautosomal segment of the derivative chromosome. It includes the proposedsite of inactivation center by Therman et al. The shortest late-replicating
segment of Xq reported to date involved q25 or 26----*qter. Late replicating pattern, however, was detected in only 2 out of 37 and none in 273
informative metaphases analysed by the BUdR-plus-thymidine and simple
BUdR protocols, respectively. Presumably, the segment was inactivatedin a small number of cells, i.e., it contains an inactivation center(s).
It appears reasonable to assume that in addition to proximal Xq, bothdistal short arm (pter----*p21) and distal Xq (q25 or 26----*qter) also include
an inactivation center(s). (For further details. see Nakagome, Y.: Amer.
J. Hum. Genet., in press).
The "Loss" of Kinetochore from X Chromosomes of Aged Females
Yasuo NAKAGOME and KAZUSO IINUMA1)

In 1974, Eiberg described a technique called Cd band which stained two
small dark spots on both sides of a centromere (Kinetochore). Later, it
was shown by Nakagome et al. that the presence of Cd-positive spots corresponded to that of a functioning centromere and that inactivation of thelatter was associated with the loss of Cd spots (Clin. Genet. 9: 621, 1976;
Ann. Rep. Nat. Inst. Genet. 30: 97, 1980). The results were confirmed byboth Daniel (1979) and Maraschio et al. (1980). On the other hand, it has
been known that the proportion of hypodiploid cells significantly increases
in aged individuals. According to Fitzgerald and McEwen (1977), an X
chromosome(s) of some elderly humans tends to separate prematurely at
the centromere.
In the present study, it was attempted to know if X chromosomes of aged
individuals lost function of their centromeres. Metaphase spreads wereobtained by the standard leukocyte-culture technique, from two females
both aged 81 years. Cd-band technique was that described by Eiberg
1)

Ebina Kosei Hospital, Fbina, Kanagawa-ken.

RESEARCHES CARRIED OUT IN 1980

103

(1974). In some of preparations, chromosomes were identified by DAPI
staining and then stained with Giemsa.
In a few percent of metaphases, one of two X chromosomes assumed an
appearance of two parallel bars without showing any constriction at thesite of kinetochore. In a small number of cells, both Xs showed parallel
appearance. The standard C-band technique (CBG) revealed small dark
spot on each parallel bar at or near the site of inactivated kinetochore. On
the other hand, Cd band revealed positive spot in none of the parallelbars (X).
It appears that in old women, an X chromosome(s) tends to lose Cdband positive material which is associated with the loss of the function ofa kinetochore.

High-resolution Banding. A Very Simple Procedure
Yasuo NAKAGOME and Takako MATSUBARA

The advent of banding techniques has made it possible to identify individual chromosomes or a part of it involved in various structural rearrangements. However, a still higher degree of resolution is urgently needed
as each band contains hundreds of genes and it is reasonably expected thatmany of small structural rearrangements remain to be detected due to lackof resolving power of the present banding techniques.
Yunis (Science 191: 1268, 1976) and Yunis et at. (Hum. Genet. 49: 291,
1979) developed a technique of "high-resolution" banding which made it
possible to increase the number of bands per haploid set from about 300 to
500-1000. However, it involves amethopterin black and later release bythymidine-rich medium. The entire process, including centrifugation, has
to be done at 37°C with utmost cautiousness, and still results are not always
satisfactory.
In the present communication, a technique of high-resolution banding is
described. It is very easy to do, very effectual and reproducible.
Methods: Leukocytes were cultured for 72 hours by the standard technique. Either RPMI 1640 or Eagle's minimum-essential medium (15 %fetal calf serum, half of it can be replaced with newborn calf serum) wasused. One hour prior to the harvest, colcemid (0.025-0.03 microgram/ml)
was added to the cultures. Thirty minutes later, acridine orange (20microgram/ml) was further added (Chroma, dissolved in Hanks' solution).

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Techniques of harvest was as usually we do except that hypotonic treatment
was 15-20 min (0.075 M KCl) rather than 6-7 min in our standard technique. Air-dried slides were aged for about 10 days at room temperature(3 to 4 days at 60°C), treated with trypsin (0.05 % in Hanks' balanced salt
solution) for about 10 sec and then stained with Giemsa.
Mitotic index of the preparations thus obtained was somewhat lessthan that in control preparations without acridine orange. Chromosomes
showed various extent of elongation, the total number of bands ranging
from 300 per haploid set to about 700 or up. The majority of mitotic
figures showed 450-500 bands/haploid set and roughly corresponded towhat was obtained in our laboratory by the Yunis' method. Further details
will be published elsewhere (MATSUBARA and NAKAGOME, in preparation).
Preservation of Whole Blood for Chromosome Analysis
Yasuo NAKAGOME, Tsunehiro YOKOCHI,I) Takako MATSUBARA,
and Fumio FUKUDA1)

During the course of chromosome studies on parients with congenitalabnormalities, it is sometimes necessary to start additional blood cultures.
The initial culture is usually made by the standard technique, however,more and more banding techniques require the treatment of cells while
they are in culture. Examples include DNA-replication study, the detection of sisterchromatid exchanges or fragile sites, and high-resolution banding. Additional cultures would also be needed upon the development of
new banding techniques which may be useful in the definitive diagnosis of"inconclusive" cases and upon failure of the initial culture.
We now describe a technique which facilitates leukocyte cultures using
blood samples kept in a freezer for months.
Up to 10 ml of heparinized blood was obtained from 12 normal individuals
and 3 with various chromosome abnormalities. Blood (1 ml) was poured
into several centrifuge tubes, the remaining blood was used for control
cultures. To each of the tubes we then added 1 ml of culture medium(RPMI 1640) supplemented with 20% fetal calf serum (FCS), 50 mcg/ml
Kanaymcin and 20 % dimethyl sulfoxide (DMSO). DMSO had been
autoclaved and stored in refrigerator until its addition to the medium.
The tubes were kept in a freezer (-80°C) for up to 12 weeks.
1)

Division of Clinical Pathology, Shizuoka Children s Hospital, Shizuoka.

RESEARCHES CARRIED OUT IN 1980

105

To start the cultures, the tubes were thawed in a 3T)C water bath, 10 mlof culture medium containing 10-15 % calf serum were added and the tubes
were centrifuged (1,000 rpm,S min). The sediment was rinsed with thesame amount of culture medium, transferred to a culture flask and 10 mlof culture medium containing 20% FCS, 50 mcg/ml Kanamycin and 0.3 mlof phytochemagglutinin were added. Then the flasks were placed in an
incubator (37°C) for 72 hours. Chromosome preparations were made by
the standard technique.
We froze 64 tubes from the 12 normal individuals and three tubes fromthe 3 individuals with chromosome abnormalities. Of 64 cultures fromnormal individuals, 56 (88 %) were successful. In most of these, chromosome preparations contained numerous metaphases, many of which weresuited for karyotyping. The mitotic indices of the successful cultures
ranged from 1.7 % to 11.9 %, the average was 6.5 % (control, 5.2± 1.3 %).
The cultures were also successful in all 3 cases with chromosome abnormalities (For further details, see Nakagome et al. Cytogenet. Cell Genet. inpress).
Two Cases of Down Syndrome with Unusual KaryotypesA psu dic(21) (q22) and a rob (14q21q) in Cousins,and Maternally Transmitted Extra ring (21)
Takako MATSUBARA, Yasuo NAKAGOME, Kazuso IIT\UMA,l)Shigehiro aKA,') Nobuaki OGASAWARA;')and Tsunehiro YOKOCHI 4 )Familial occurrence of Down syndrome is rare except for those causedby either translocation or mosaicism of one of the parents. A girl withtypical Down syndrome had 46, XX, -21, +psu dic(2l) (pter~cen~q22: q22~pter) karyotype. A maternal cousin was also Down syndrome
with 46, XV, -14, +rob(l4q21q) karyotype. It is noteworthy that two
different types of structural rearrangement arose in a family both being denovo. According to Hagemeijer et al. (Hum. Genet. 38: 15, 1977), thetriplication of the 21q22 band is essential for the phenotype of Down syn1)
2)

3)Ebina Kosei Hospital, Ebina, Kanagawa-ken.
Hamamatsu Medical University, Hamamatsu, Shizuoka-ken.
Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi-

ken.
4)

Shizuoka Children's Hospital, Shizuoka, Shizuoka-ken.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

drome. The triplication of the most distal part of it, however, may not benecessary (Poissonnier et al. Ann. Genet. 19: 69, 1976; and Raoul et al.
Ann. Genet. 19: 187, 1976). The psu dic(2l) (q22) case was trisomic forno. 21 except for the most distal part of q22 band and showed typicalfeatures of Down syndrome in agreement with the literature.
The trisomy on account of the extra ring chromosome is also extremelyrare (Cantu et al. Ann. Genet. 18: 193, 1975). In most cases with a ring,
one of the normal chromosome pair is substituted by the ring. A boy
with typical Down syndrome had 47, XY, +r(2l)(pll ?q22) karyotype andelevated SOD-l (the erythrocyte superoxide dismutase-l) activity. To oursurprise, the mother showed 46, XX, r(2l)(pll ?q22) karyotype. She had
normal phenotype with no sign common to the r(2l) syndrome. SOD-l
activity was within normal limits. The analysis of chromosomal heteromorphism revealed a normal 21 chromosome in addition to the r(2l) wastransmitted from the mother to the son. The proposed SOD-l locus iseither in sub-band 2lq22.l (Sinet et al. Exp. Cell Res. 97: 47, 1976) or inband 21q21q (Leshot et al. Hum. Genet. 57: 220, 1981). SOD-1 activity
and phenotype-karyotype correlation of the proband and the mother werecompatible with the presence of break point close to the distal end of 2lq22,
i.e., the ring was, infact, r(2l)(pllq22). (For further details, sse Matsubara et al. Jap. J. Hum. Genet. 26: 55, 1981; and Matsubara et al. in preparation).

RESEARCHES CARR1ED OUT IN 1980

XI.

107

BEHAVIORAL GENETICS AND ECOLOGICAL GENETICS
A Parasitic Wasp of Drosophila
Masaoki KAWANISHI and Takao K. WATANABE

One female of a Trichopria species (Diapriidae) was collected from abanana trap put in Katusnuma in 1978. This species name is still unknown
but this wasp was found to oviposit in pupae of many Drosophila species.
Ten Drosophila females were introduced into a vial with males and let themlay eggs for a day. When the eggs became to the late larvae (5 to 9 days
after the egg laying), three wasp females aged 4 days with males were put
into the vial and left for one day. Ten different species of Drosophila
were examined for the host of the wasp. The numbers of Drosophila flies
and wasps emerged from each vial were counted every day. Uninfected
Drosophila flies developed in regular days and the wasp's development wasdelayed a few days (18.3 days for males and 20.5 days for females). The
sex ratio of the wasps was distorted to female side significantly. The percent
of wasp among the emerged host and parasites varied from 69 % of D.
ananassae vial to 18 % of D. auraria vial. 1t was 61 % for D. melanogasterand 37 % for D. simulans, though they are closely related sibling species.
This wasp did not lay eggs on larvae but on pupae exclusively and the sizeof pupae was not the determinative factor. The most probable factor
seems to be the site Drosophila pupates. Highly infected Drosophila species
tend to pupate remote from food such as the wall of vial. The wasp may
prefer Drosophila pupae rather dry. The preference was examined fromthe view point of the coexistance of a sibling pair, D. melanogaster and D.
simulans, in a cage population. Equal number of both species were introduced into four population cages. The parasitic wasps were contaminatedinto two cages and the other two cages were maintained without wasps(control). D. simulans flies were finally eliminated from the latter cages
at 145 days as reported by many workers (see Kawanishi and Lee, 1978
lap. J. Ecol. 28, 213). However, the former cages have maintained D.
simulans until the end of experiment (225 days). This suggested that thewasps preferably attacked D. melanogaster pupae pupating at remote sites
from the food which resulted in the decrease of D. melanogaster to a good

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

balance of both Drosophila species in the cages. During the experiment
numbers of Drosophila flies and 'wasps fluctuated alternately. The reason
is probably as follows: When the wasp increases in number its food (Drosophila pupae) is limited, then the wasp's number reduces inevitably whiehallows Drosophila flies a chance of population growth. These results suggest
the natural balance of animals not only between competing sibling species
but also between food-chain-species.
Development of Resistance to Organophosphorous
Insecticides in Drosophila
Yutaka I"OUE

Drosophila mclanogastcr from a wineyard population showed a large
variation in susceptibility to organophosphorous insecticides, Diazinon and
Sumithion, though the average wild flies were more resistant to the insecticides than the laboratory flies probably due to an extensive use of these
insecticides. The wild strains showed a significantly positive correlationin the susceptibilities to Diazinon and Sumithion (rO.294, d.f.=86, p<0.01). This may be ascribed either to the application of both Diazinon
and Sumithion in the field, or to the development of mutual cross-resistances.
Flies cultured by the food containing Diazinon (30 ppm) or Sumithion(45 ppm) for 24 generations became resistant to each insecticide about
threefold or twofold, respectively. The development of cross-resistance tothe other insecticide was confirmed though it delayed 12 generations asshown in Table 1. Under the normal food these insecticide-resistant flies
Table 1.

The development of Diazinon- and Sumithion-resistanee
after 12 and 24 generations of treatment
Diazinon-resistance

Population
12 gen.

24 gen.

1.000:lc
0.033
1. 276- L
0.024***
0.695±
0.034***

1.00010.112
2.825+
0.098***
1.088:1=
0.116- - - - - - ------

ControlDiazinontreated (30 ppm)
Sumithion
treated (45 ppm)****

Sumithion-resistance----------- - - - ---------------

Significant at 5 % level.
Significant at 0.1 % level.

12 gen.

24 gen.

I.OOO±
0.009
0.969±
0.013*
1.019±
0.007

1.000.-1-:
0.080
1. 523±0.075***
1.921 +
0.059***RESEARCHES CARRIED OUT IN 1980

109

showed significantly lower fecundity than the control (trade-off), although there were no differences in viability. An interesting phenomenon
must be noted that flies from the insecticide-treated populations showeda significantly higher speed in development even in the normal food. It
can be speculated that the use of insecticide selected Drosophila which candevelop faster to get rid of insecticides in food.
Selection for the Compound Learning Ability of Mice
Tohru FUJISH1MA

A selection has been made bidirectionally for the discriminated avoidance
performance (DAR, %) of mice (60-70 days of age) in the second of two
daily training sessions, each comprising fifty learning trials. The fundamental stock was constructed from crossing of inbred mouse strains (C3H/
HeMs, SWM/Ms, C57L/JMs and Dl03/Ms), each having different characteristics of DAR measured with an automated Y-type maze apparatus(cf. Ann. Rep. 25: 85). Careful attention has been paid to keep the same
inbreeding coefficient in both selected lines.
The data for the seventh selected generation were obtained in this year.
They indicated that the cumulative genetic gain for the average of males
and females was 16.3, and the difference in avoidance between the DARHigh line and the DAR Low line (High line minus Low line) was 25.8,
while the difference in discrimination was -0.5. These results suggested
that the selection for higher DAR resulted in the higher avoidance abilityof mice rather than the higher discriminatory ability.
The Behavioral Changes in Mice Continuously Reared under
Noise Conditions
Tohru FUJISHIMA

Each full sib family of mice was divided into two groups; one was continuously reared under experimental conditions with noise treatment,
while another was reared without noise treatment as a control. They were
kept at 25°C with 12 hour light and 12 hour darkness. To the noise treatment group, noise (pink, 100 phon) was applied six times for one hour
each at one hour interval during the dark period (6 p.m. to 6 a.m.). The
avoidance and discriminatory learning performances of mice were measured

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

with an automated Y-type maze apparatus at approximately 60 days old aswell as their activity and body weight.
The results of the first generation obtained this year showed that there
were significant differences in activities and body weights between thetreated and control groups, that is, the noise reduced body weight andincreased activities of mice. These results indicated that the effect ofnoise might arouse the emotionality of mice.
Niche Differentiation between Perennial and Annual Types
within Populations of Oryza perennis
Hiroko MORISHIMA and Yoshio SANO

Thirty six populations of wild rice, Oryza perennis Moench, collected inIndia and Thailand, were studied on the relationship between perenniality
and habitat condition. In general, the perennial type was found in deep
swamps which could remain moist in the dry season and relatively stablethroughout the year, while the annual type was found in shallow, temporary
swamps ,vhich were parched in the dry season and more frequently disturbed
by man. There was a tendency for the perennial type to be associated withperennial companion species, and for the annual type with annual companion species. Intermediate perennial-annual types were found in or
around rice fields and did not seem to be fully adaptive in truly natural
habitats (Morishima et al. 1980).
Two populations in which the perennial and annual types grew side byside were studied in some more details to look into microhabitat separation.
Progeny lines from the collected seeds were tested in an experimental plot
with automatic shortday control for morphological traits and zymogramsof peroxidase, acid phosphatase, and phosphoglucose isomerase. In one
population found in a swamp near Bhubaneswar, Orissa, India, the plants
at the shallow-water periphery were an annual type, and those in the deepwater center were a perennial type. In the other population found in aroadside ditch near Chiangrai, Thailand, the plants collected from a strongly
disturbed spot were annual while those distributed nearby were perennial
to intermediate perennial-annual types, although habitat separation between
them was not so clear as observed in the Bhubaneswar population.
In both populations, the annual plants so categorized by their low regenerating ability of excised stem segments flowered earlier and produced

RESEARCHES CARRIED OUT IN 1980

111

more seeds of larger size giving a higher reproductive effort than the perennial plants. They also had other characteristics of the annual type, andappeared to be an r-strategist as compared with the perennial plants which
tended to be a K-strategist, as discussed by Oka (I 976). The genetic distances between the coexisting annual and perennial plant-groups, estimatedfrom the data for 8 isozyme loci, were quite small and indicated their close
relationship. They seem to have become differentiated in their present
habitat in response to different modes of selection, as was pointed out inVeronica peregrina in vernal pools (Linhart 1976).
The perennial and annual types differ in niche preference. If disruptive
selection is strong enough, sympatric differentiation may take place even
within a single gene pool. Microhabitat separation would depend on how
the impact of selection is localized and predictable. Major agents ofr-selection for the annual type would be drought stress and disturbance inthe dry season.
Effect of Experimental Soil Pollution on Weed Communities
in Rice Fields
Hiroko MORISHIMA

Copper sulphate solution was applied to a concrete bed filled with fieldsoil and planted to rice in order to study the effect of soil pollution on weed
community. The application of copper was repeated several times since 1978,
and the soil copper content measured in 1980 was 110 ppm. Two strainsof barnyard grass, Echinochloa crus-galli, one being copper tolerant andthe other non-tolerant, were sown in a 1: 1 mixture in the 1978 season.
Since then, rice was planted every year, but no weeding was made. An
observation of the summer weeds in 1980 showed that barnyard grass dominated over other weed species in the copper plot while in the control
plot, several weed species coexisted more equitably and the copper-tolerantstrain of barnyard grass disappeared as replaced by the non-tolerant strain.
The non-tolerant strain had a higher competitive ability than the tolerantstrain (Morishima and Oka 1977). The trend of species diversity to decrease in response to pollution was also observed in the winter weed community. In winter, Poa annua was dominant in the copper plot. Total
weed biomass per unit area tended to be greater in the copper-treated thanin the control plot.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 3I

A herbicide, Propanil, has been applied successively in an experimental
field since 1974. Soil samples were taken from the PropaniI-treated andcontrol (non-weeding) plots, and the plants raised from the soil-buried seeds
were observed. In the treated plot, Lindernia procumbens obviously dominated over other species, but its domination was not distinct in the control plot. Total plant number as well as total biomass per unit area showedno significant differences between the treated and control plots.
The experiments thus indicated that soil pollution affects the structure of
weed community resulting in a decrease of species diversity and dominanceof certain species, although it does not affect the overall growth of weeds.
This is in good agreement with the author's observations in copper polluted
rice fields (Morishima and Oka 1977, 1980). The evolution of tolerance
in the weed species grown in the experimental plots will be investigatedlater.
Studies on Pioneer Trees and the Pioneer Index: Comparison
between the Tropics and the Temperate Zones
Kan-Ichi SAKAI and Shinya IYAMA

It is interesting in relation to ecological genetics of natural forest as wellas tree breeding to investigate the characteristics of pioneer trees in a naturalforest. In the present report, pioneering ability of tree species was numerically expressed by a pioneer index which was computed so as to makecorrelation between linear combination of component traits and pioneering
ability maximum. Two indices were computed using 27 tree species in
Hokkaido, Japan and 34 in Jave Island, Indonesia, each representing temperate and tropical forests, respectively. The following 14 traits were
included in the calculation, each being scored 0 to 3 or 0 to 5: (1) lightdemand for germination, (2) germination speed, (3) dormancy, (4) germination percentage, (5) seed production, (6) seed fillingness, (7) annual productivity of seed, (8) flight ability of seed, (9) seed size, (10) flight apparatus of
seed, (11) early seed production, (12) longevity, (13) initial growth and (14)wood decay. Four traits of largest contribution among the 14 traits were
initial growth, early seed production, light demand for germination andannual productivity of seed for temperate forest and light demand, flightability of seed, early seed production and flight apparatus for tropical ones.
Light demand and early seed production were common in both cases, but

RESEARCHES CARRIED OUT IN 1980

113

early growth and annual seed production were important for temperate
forests and flight ability of seed for tropical ones. Seed dormancy did notseem to have an important role to the pioneering ability.

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

XII.

APPLIED GENETICS

Nitrogen Fixing Activity of Bacterial Strains in Association
with Rice Plants
Taro FUJII

In order to investigate the nitrogen fixing activity of bacteria in combination with rice plants, pure inoculation of nitrogen fixing bacterial strain
into rice seedlings was conducted. Rice strain C5444 which showed highnitrogen fixing ability was used as a host plant. Six nitrogen fixing bacterial
strains so far isolated from the rhizosphere soil of C5444 was used for the
inoculation experiments. Pure culture of each bacterium was inoculated
into the rhizosphere soil of germ free rice seedlings which were grown ina test tube filled with sterilized soil and reached two months old. Acetylene
reducing activity was measured every two weeks after inoculation. No
activity was observed in a seedling without inoculation. Plants inoculatedwith bacterial strains COCoS and NG-13 showed appreciable acetylene
reducing activity three months after inoculation. Plants inoculated withother four bacterial strains, however, did not show the activity during repeated measurements up to four months after inoculation (Table 1). Fromthese results, the followings were concluded: Though various nitrogen
fixing bacteria were existing in the rhizosphere soil of rice, only particularstrains among them were able to manifest nitrogen fixing activity in association with rice roots, suggesting the importance of interaction between
genotypes of bacteria and rice in regulation of "symbiotic" nitrogen fixation.
Table 1. Acetylene reducing activity of various nitrogen fixing
bacteria inoculated into germ free rice rhizosphere*
Date ofmeasurement
May 6
May 14
May 23Control(no inoculation)

Clostridium

CO-C-8

NG-13

0-0-2

0-20

N-SG-47

0
0
0

0
0
0

1.5
3.6
8.4

0
2.3
2.8

0
0
0

0
0
0

0
0
0

* Acetylene reducing activity was expressed by the amount of C 2 H 2 produced (nmol/h/plant).

RESEARCHES CARRIED OUT IN 1980

115

Fruther Evidence for "One Locus Sporo-gametophytic Interaction"
Model for F 1 Sterility Genes between Oryza sativa
and O. glaherrima
Yoshio SANOAs reported previously (Ann. Rep. 30: 113), isogenic F ,-sterile lines
having the genetic background of O. sativa and O. glaberrima parent wereisolated from BsFe plants, respectively. Experiments with those lines
indicated that the F1 sterility between the two species could be explained bya "one locus sporo-gametophytic interaction" hypothesis, which assumes
that the parental species has Sa sa and SS, respectively, and when a S geneis present in the maternal tissue, gametes with sa deteriorate. To test thishypothesis, an experiment was carried out as follows:
An isogenic line having gene S3 (from O. glaberrima, W025) in the genetic
background of Taichung 65 (0. sativa) was crossed with another isogenic
line of Taichung 65 having the glutinous gene (wx). The glutinous vs.
non-glutinous character of pollen grains is testable by iodine reaction, andheterozygotes for the locus (-I- Iwx) are expected to produce the two kindsof pollen grains in a 1: 1 ratio. In the F1 plants between S3/8:3 a and -I- 1wx,
however, pollen grains with wx were found to be eliminated by gametic
selection. Out of 3,780 pollen grains observed, only 128 were glutinous.
This indicates that S3 and wx were closely linked and the wx gene was eliminated together with the Ssa gene. In the selfed progeny, heterozygotes
with Ss-ll'xISsa--I- were obtained. As expected, the heterozygotes showed
only 107 non-glutinous pollen grains out of 2,845 observed. The recombination value between S3 and wx was calculated from the data to be 0.035.
The experimental result strongly supports the hypothesis that the F 1sterility is controlled by sporo-gametophytic interaction of alleles at a
certain locus. The data so far obtained indicate that there are a numberof such F1-sterility gene loci between O. sativa and O. glaberrima. Possibly,
such allelic changes have been accumulated during the porcess of species
differentiation. Their fixation will be rapid because of strong gametic
selection, which would also affect the genic content of the chromosomes
which carry such sterility genes. This type of sterility genes may be regarded
as "selfish" genes.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

The Direction of Pollen Flow between Two Cultivated Rice
Species, Oryza sativa and O. glabcl'rima
Yoshio SANa

Oryza glaberrima is endemic in West Africa, while O. sati\'a is of tropicalAsian origin. Prior to the introduction of O. sativa into Africa, the cultivation of O. glaberrima was well estabilshed (Porteres 1950). O. sativa,
after being introduced, spread across the West African rice zone with selection for adaptability. At present, the two rice species are often grown in
mixture in farmers' fields. They can be distinguished only by a few distinct
characteristics like short and tough ligules and few secondary panicle
branches of O. glaberrima which make contrast with long ligules and many
secondary panicle branches of O. sativa. Although their F 1 plants arehighly pollen-sterile, backcrossing is possible with the pollen grains ofeither parent. During the 1977 study-tour in West Africa, I have observed
a number of F 1 plants in rice fields where the two species were planted in
mixture. A basic problem that needs to be solved is whether or not introgression between the two species has played a role in their adaptation to
the growing conditions in West Africa. T herewith report the result of anexperiment on the extent of natural hybridization and which parent contributes its pollen in natural hybridization"
The seeds of coexisting plants of the two species, sampled from an upland
and a lowland field in northern Nigeria, were used for the present experiment. The material consisted of two pairs of a sativa and a glabcrrima
strain. Tn J978, each pair of strains was planted in a J: I mixture, in a
concrete bed with automatic shortday control, with a spacing of 10 X JO cm.
They flowered from late July to early August and their flowering periods
overlapped one another. Their seeds were harvested randomly fromabout 20 plants of each strain. In 1980, about 800 plants per strain were
tested for the percentage of F 1 plants between the two species, which were
easily distinguished by the occurrence of long awns, pollen sterility, andan intermediate ligule shape. Hybrid plants were observed in both strainsof O. glabcrrima with a relatively high frequency (8.9 % in 0 1 and 1.6 %in 0,). Tn contrast, no hybrid was found in either strain of O. sativa.
The result of this experiment indicates that when introgression takes
place, pollen flow occurs from O. sativa to O. glaberrima. This might bedue to differences in flowering behavior and in the amount of pollen prod-

RESEARCHES CARRIED OUT IN 1980

117

uced. If the hybrids were backcrossed successively by O. sativa, the resulting plants would have genes of O. sativa in the cytoplasm of O. glaberrima. Crossing experiments to detect cytoplasmic differences are in progress.
Mendelian Segregation Analysis for Three Isozyme Lociin Rice Cultivars
Gerard SECO>iD and Hiroko MORISHIMA

Three F 2 populations involving five parental strains of Oryza sativa wereanalysed at the germination stage for the segregation of two contrasting
electromorphs which were presumed to be specified by the Pgi-A, Pgi-B,
and Cat-A loci, respectively. Two of the crosses also involved glutinousand non-glutinous parents. As shown in Table 1, the observed segregation
patterns were in good accordance with the presumed genic systems giving
I: 2: 1 ratios. In one of the crosses, a distorted ratio was observed for theF 2 segregation for Pgi and Cat electrornorphs

Table 1.

Pi type

Locus & Cross
-

Pgi-A

1
2
Total

Pgi-B

1

Cat-A

2
3
Total
1
2

3
Total

F, typc

P 2 type

x2

88
65
153
88
79
22
189
55
74
12
141

46
39
85
48
31
12
91
29
20
8
57

1.45
1. 18
2.27
.94
2.05
.93
.13
.97
9.07*
.93
6.34*

._---

54
39
93
52
31
8
91
34
44
9
87

---,..-'
~vx

2

Total

75
72
147

204
255
459

.52
1.55

.17

* ;(, for 1 : 2: 1 ratio, significant at 5 % level.
Cross 1:
2'
3:

Ace. 130 x Ace. 221-(Pgi: Ai B 2 : Cat: Ai: Non-waxy) x (Pgi: A 2 B, ; Cat:A 2 : Waxy),Ace. 143 x Ace. 221-Ibid.
T65xC5444-(Pgi: A 2B , ; Cat: A 2)x(Pgi: A 2 B 2 ; Cat: Ai)'

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

Cat-A locus, which is not unusual in crosses between rice cultivars. No
linkage relationship was found between any of the four loci investigated.
An Introgressed Electromorph Found in an Isogenic Line of
Oryza sativa with a Sterility Gene from O. glaherrima
Gerard SECO:"D and Yoshio SANO

Two isogenic lines of O. sativa with a sterility gene introduced from astrain of O. glaberrima were analysed for phosphoglucose isomerase (PGI)
loci at the germination stage. The isogenic lines had been isolated fromBsF 2 populations using Acc. 108, an Indica strain of O. sativa as the reccurent parent (Sana, Chu, and Oka, Jap. J. Genetics 54: 121-132). The
parents, Acc. 108 and W025, and the F , plants between the isogenic lines
and Acc. 108 were examined. According to Second and Trouslot(ORSTOM, TD 120, 1980), the genic interpretations for Pgi zymograms of
Acc. 108 and W025 were A'/A' B 2 /B 2 and AS/AS B'/B" respectively. Oneof the isogenic lines was like the O. sativa parent but the other had A3 andB 2 alleles in homozygous state, suggesting that A 3 had been introgressedfrom O. glaberrima. The zymogram observed in this isogenic line is uniquesince the combination of genes AS and B 2 has never been found in any ricestrain. The A' and A 3 electromorphs exhibit only a small difference in theirPGI mobilities. The F , hybrids between the isogenic line and Acc. 108
showed a band located in between A' and AS and slightly broader than theparental bands. It may be presumed that Pgi-A locus is located on thechromosome on which a sterility gene from O. glaberrima is also located.
Recombination between them was detected in the F 2 • Further investigation of the F 2 population is in progress.
Simulation Experiment on the Mass Selection for a Quantitative
Character When the Effect of Genes is not Equal
Shinya IYAMA

Computer simulation was performed in order to visualize the effect ofmass selection on the change of gene frequency when the effects of genes
affecting a quantitative character were not equal. The results showed thatthe magnitude of additive effect of the genes and the joint effect of dominanceand gene frequency had important roles in the response of the individual

RESEARCHES CARRIED OUT IN 1980

119

genes to the selection. When the genes had not equal effect, selection for
the genes having small effect was delayed compared with the genes of large
effect. Similarly. when selected from the low gene frequency together with
non-recessive genes, recessive genes stayed at the initial level of low frequency longer than other genes. Since those genes of slow response to
selection stayed at low gene frequency longer, they were likely to be underserious risk of being lost from a population due to random genetic drift
when the population was relatively small. It was suggested from the results that population size should be chosen carefully to avoid the loss ofgenes, especially ofr those genes as of small effect or recessive.

•

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

BOOKS AND PAPERS PUBLISHED IN 1980
BY S1 AFF MEMBERSARRAND, J. R., E. SOEDA, J. E. WALSH, N. SMOLAR and B. E. GRifFIN 1980: Polyoma
virus DNA. Scqucnce from the late region that specifies the leader sequence forlate mRNA and codes for VP2, VP3 and the N-terminus of VPI. J. Virol. 33:
619-630.
ASSEMAT, L. and H. r. OKA 1980: Neighbor effccts between rice (Oriyza sativa L.) andbarnyard grass (Eehinoehloa erus-galli Beauv.) strains. I. Performance in mixture
and aggrcssiveness as influenced by planting density. OEco!. Plant. 1(15): 371-393.
AZUMI, J., Y. NAKAGOMf, S. OKA and E. MATSUNAGA 1980: A new approach in theevaluation of chromosome variants in man. II. Pairs without Q or C (qh) variants.
Hum. Genet. 55: 75-79.
FUJII, T. 1980: Somatic mutations induced by furylfuramide (AF-2) in maize and soybean. Jap. J. Genet. 55: 241-245.
FUJISAWA, T. and T. SUGIYAMA 1980: Nematocyte differcntiation from interstitial cells
newly-introduced into interstitial cell-deficient hydra. "Dcvclopmental and Cellular
Biology of Coelenterates", 319-324. Elsevier (Amsterdam)
GRIFFIN, B. E., E. SOEDA, B. G. BARRELL and R. STADAN 1980: Sequcnce and analysis ofpolyma virus DNA, DNA tumor viruses. "Molecular Biology of Tumor Viruses"(ed. by J. Tooze, 2nd edition) Part II: 831-896. Cold Spring Harbor Lab. (Ncw
York).
HIRAI, B., K. MORIWAKI and T. UCHIDA 1980: Comparative analyses of Japanesc woodmicc from the Oki islands and the mainland of Japan based on biochcmical genetics
and cytogenetics. J. Fac. Agr. Kyushu Univ. 25: 1-8.
HIROTA, Y., M. YAMADA, A. NISHIMURA, A. OKA, K. SUGIMOTO, K. ASADA and M. TAKANAMI 1981: The DNA rcplication origin (ori) of Escherichia coli: structure andfunction of the ori-containing DNA fragmcnt. Progrcss in Nucleic Acid Research& Molecular Biology 26: 33-48.
IMAI, H. T. and R. B. CROZIER 1980: Quantitative analysis of directionality in mammalian karyotype evolution. Am. Nat. 116: 537-569.
KADA, T., K. HIRANO and Y. SHIRASU 1980: Screening of environmental chemical mutagens by the ree-assay system with Bacillus subtilis. Chemical Mutagens 6: 149-173.
KANEKO, Y., S. SAKAI, K. MORIWAKI and S. MIGITA 1980: Frhybrid resistance to murine
plasmacytoma MOPC-70A: Hybrid resistance to MOPC-70A controlled by a locus
linked to H-2. J. Immunol. 125: 2031-2038.
KANEMATSU, N., M. HARA and T. KADA 1980: Ree assay and mutagenicity studies on
metal compounds. Mutation Res. 77: 108-116.
KATO, H., M. HARADA, K. TSUCHIYA and K. MORIWAKI 1980: Absence of correlation
between DNA repair in ultraviolet irradiated mammalian cells and life span of thedonor species. Jap. J. Genet. 55: 99-108.

BOOKS AND PAPERS PUBLISHED IN 1980 BY STAFF MEMBERS•

121

KAWANISHI, M. and T. WATANABE 1980: Genetic variations of courtship song of Drosophila melanogaster and D. siml/lans. Jap. J. Genet. 55: 235-240.
KAwACHI, T., T. KOMATSU, T. KADA, M. ISHIDATE, M. SASAKI, T. SUGIYAMA and Y.
TAZIMA 1980: Results of recent studies on the relevance of various short-term screening tests in Japan. "The Predictive Value of Short-term Screening Tests in Carcinogenicity Evaluation", 253-267.
KAWACHI, T., T. YAHAGI, T. KADA, Y. TAZIMA, M. ISHIDATE, M. SASAKI and T. SUGIMURA 1980: Cooperative programme on short-term assays for carcinogenicity inJapan. "Molecular and Cellular Aspects of Carcinogen Screening Tests", 323-330.
KIMURA, M. 1980: Contributions of population genetics to molecular evolutionary studies.
"Genetics and Evolution of RNA Polymerase, tRNA and Ribosomes", (ed., S. Osawa,
and others): 499-518. University of Tokyo Press.
KIMURA, M. 1980: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120.
KIMURA, M. 1980: Average time until fixation of a mutant allele in a finite population
under continued mutation pressure: Studies by analytical, numerical, and pesudosampling methods. Proc. Natl. Acad. Sci. USA 77: 522-526.
KOMEDA, Y., K. KUTSUKAKE and T. IINO 1980: Definition of additional flagellar genes in
Escherichia coli K12. Genetics 94: 277-290.
KUDO, I., H. HAYATSU, A. YOKOIYAMA and Y. KURODA 1980: Effect of bisulfite on cellto-substratum adhesion of Chinese hamster cells in culture. J. Pharm. Dyn. 3: 74-79.
KURODA, Y. 1980: Cell differentiation and organogenesis in insects (in Japanese).
Gendai-Seibutsugaku-Taikei (NUMANOI, H. et al., eds). Nakayama-Shoten (Tokyo),lla: 243-254.
KURODA, Y. 1980: Procedure for assortment of chromosomes. (in Japanese). ShinShoniigaku-Taikei (ARIMA, M. et al., eds.) Nakayama-Shoten (Tokyo), 7b: 48-59.
KURODA, Y. 1980: Chromosome map. (in Japanese). Shin-Shoniigaku-Taikei (ARIMA,
M. et al., eds.) Nakayama-Shoten (Tokyo), 7b: 69-81.
KURODA, Y. 1980: Experimental system for detecting gene mutations in cultured mammalian cells. (in Japanese). Experimental Procedures for Environmental Mutagens(TAZIMA, Y. et al., eds.) Kodansha Scientific (Tokyo): 152-167.
KURODA, Y. 1980: Correlation between mutation and transformation in cultured mammalian cells. (in Japanese). Mutagens and Toxicology 10: 24-38.
KUSUDA, J., K. SHINOZAKI, F. TAKAIWA and M. SUGIURA 1980: Characterization of thecloned ribosomal DNA of tobacco chloroplasts. Molec. gen. Genet. 178: 1-7.
KUTSUKAKE, K., T. IINO, Y. KOMEDA and S. YAMAGUCHI 1980: Functional homology
of fia genes between Salmonella typhilnllriliin and Escherichia coli. Molec. gen.
Genet. 178: 59-67.
MARCUM, B. A., T. FUJISAWA and T. SUGIYAMA 1980; A mutant hydra strain (sf-I)
containing temperature-sensitive interstitial cells. "Developmental and Cellular
Biology of Coelenterates", 429-434. Elsevier (Amsterdam).
MARUYAMA, T. 1980: On the overdominant model of population genetics. Adv. Appl.
Prob., 12: 274-275.
MARUYAMA, T. and M. KIMURA 1980: Genetic variability and effective population size

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

when local extinction and recolonization of subpopulations are frequent. Proc.
Nat!. Acad. Sci. USA 77: 6710-6714.
MARUYAMA, T. and E. SOEDA 1980: Molecular evolution in papova viruses and their
host species, and in bacteriophages. Publication, Stadler Symp. 12: 83~96.
MATSUNAGA, E. 1980: Inherited tissue resistance to the gene for retinoblastoma. Genetic
and Enviro;'''lental Factors in Experimental and Human Cancer. (Edited by H. V.
GELBOIN et al.), 161-173, Japan Sci. Soc. Press (Tokyo).
MATSUNAGA, E. 1980: Hereditary retinoblastoma: Host resistance and second primarytumors. J. Nat!. Cancer Inst. 65: 47-51.
MATSUNAGA, E. and K. SHlOTA 1980: Ectopic pregnancy and myoma uteri: Teratogenic
effects and matcrnal characteristics. Teratology 21: 61-69.
MATSUNAGA, E. and K. SHlOTA 1980: Search for maternal factors associated with malformed human embryos: A prospective study. Teratology 21: 323-331.
MATSUNAGA, E. 1980: Retinoblastoma: Host resistance and 13q-chromosomal deletion.
Hum. Genet. 56: 53-58.
MATSUNAGA, E. 1980: On estimating penetrance of the retinoblastoma gene. Hum.
Genet. 56: 127-128.
MATSUTANI, E. and Y. KURODA 1980: Effect of cell association on in vitro chondrogenesisof mesenchyme cells from quail limb buds. Cell Structure and Function 5: 239-246.
MINEZAWA, M., K. MORIWAKI and K. KONDO 1980: The third allele of supernatantisocitrate dehydrogenase of mouse, Id~lc, originates from Asian continent. Jap. J.
Genet. 55: 389-396.
MORISHIMA, H. and H. 1. OKA 1980: The impact of copper pollution on water foxtail(Alopecurus aequalis SOBOL.) populations and winter weed communities in rice
fields. Agro-Ecosystems 6: 33-49.
MORISHIMA, H., Y. SANO and H.I. aKA 1980: Observations on wild and cultivated rices
and companion weeds in the hilly areas of Nepal, India and Thailand. Special
Report from National Institute of Genetics, pp. 97.
MORIWAKI, K., T. SHIROISHI, N. MIYASHITA, N. KANDA and H. T. IMAI 1980: Intersubspecies hybrid of mouse as a tool for studying genetic system governing tumor
development. Gann Monograph 25: 165-176.
MURAKAMI, A., H. INOKUCHI, Y. HIROTA, H. OZEKI and H. YAMAGISHI 1980: Characterization of the dnaA gene carried by Lambda transducing phage. Molec. gen.
Genet. 180: 235-247.
MUROFUSHI, M., S. OIKAWA, S. NISHIKAWA and T. H. YOSIDA 1980: Cytogeneticalstudies on fishes, Ill. Multiple sex chromosome mechanism in the filefish Stephanolepis cirrhifer. Jap. J. Genet. 55: 127-132.
NAKAGOME, Y. 1980: On the new policy for reports on chromosomal anomalies. Hum.
Genet. 53: 427.
NAKAGOME, Y. and Y. SUZUKI 1980: Reply to the letter of Prieto et al. concerning ourpaper on a case of 13q; 18q translocation. Hum. Genet. 53: 283.
NAKAGOME, Y., T. TANAKA, T. HASHIMOTO, M. KUYAMA and M. MARUYAMA 1980: Interstitial dcletion 6q in a malformed boy. Ann. Genet. 23: 49-51.
NAMIKI, M., S. UDAKA, T. aSAWA, K. TSUJI and T. KADA 1980: Formation of mutagens

BOOKS AND PAPERS PUBLISHED IN 1980 BY STAFF MEMBERS

123

by sorbicacid-nitrite reaction: effects of reaction conditions on biological activities.
Mutation Res, 73: 21-28.
NISHIMURA, Y., S. TAKAHASHI, T. YAMAMOTO, M. TSUBOI, M. HATTORI, K. MIURA, K.
YAMAGUCHI, S. OHTANI and T. HATA 1980: On the base-stacking in the 5'-terminal
cap structure of mRNA: a fluorescence study. Nucleic Acids Res. 8: 1107-1119.
NISHIMURA, Y., H. SUZUKI, Y. HIROTA and J. T. PARK 1980: A mutant of Escherichia
coli defective in penicillin-binding protein 5 and lacking D-alanine carboxypeptidase
IA. J. Bacteriol., 143: 531-534.
NOGUCHI, T. 1980: Primordial germ cell proliferation and its relation to teratocarcinogenesIs In mice. GANN Monograph Cancer Res. 25: 79-87.
OHTA, T. 1980: Evolution and Variation of Multigene Families. 131 p. (Lecture Notes
in Biomathematics, Vol. 37) Springer (Berlin).
OHTA, T. 1980: Amino acid diversity of immunoglobulins as a product of molecular
evolution. J. Mol. Evo!. 15: 29-35.
OHTA, T. 1980: Linkage disequilibrium between amino acid sites in immunoglobulin
genes and other multigene families. Genet. Res. 36: 181-197.
OHTSUKA, E., T. MIYAKE, K. NAGAO, H. UEMURA, S. NISHIKAWA, M. SUGIURA and M.
IKEHARA 1980: Elongation of oligonucleotides in the 3'-direction with activatedmononucleotides and their analogs using RNA ligase. Nucleic Acids Research 8:
601-610.
OKA, A., K. SUGIMOTO, M. TAKANAMI and Y. HIROTA 1980: Replication origin of the
Escherichia coli K-12 chromosome: the size and structure of the minimum DNA
segment carrying the information for autonomous replication. Molec. gen. Genet.
178: 9-20.
OKA, S., Y. NAKAGOME, J. AZUMI, E. MATSUNAGA and Y. IGARASHI 1980: A new approachin the evaluation of chromosome variants in man. III. Pairs with established Q or
C variable sites. Hum. Genet. 55: 327-331.
OONO, K. and M. SUGIURA 1980: Heterogeneity of the ribosomal RNA gene clusters inrice. Chromosoma (Berl.) 76: 85-89.
OSAWA, T., H. ISHIBASHI, M. NAMIKI and T. KADA 1980: Desmutagenic actions ofascorbic acid and cysteine on a new pyrrole mutagen formed by the reaction between
food additives; sorbic acid and sodium nitrite. Biochem.Biolphys. Res. Comm. 95:
835-841.
SAKAIZUMI, M., N. EGAMI and K. MORIWAKI 1980: Allozymic variation in wild populations of the fish, Oryzias tatipes. Proc. Jap. Acad. 56: 448-451.
SANO, Y. 1980: Adaptive strategies compared between the diploid and tetraploid formsof Oryza punctata. Bot. Mag. 93: 171-180.
SANO, Y., H. MORISHIMA and H. I. OKA 1980: Intermediate perennial-annual populations of Oryza perennis found in Thailand and their evolutionary significance. Bot.
Mag. 93: 219-305.
SANO, Y., Y. E. CHU and H. I. OKA 1980: Genetic studies of speciation in cultivatedrice, 2, Character variations in backcross derivatives between Oryza sativa and O.
glaberrima: M-V linkage and key characters. Jap. J. Genet. 55: 19-40.
SHIROISHI, T. and K. MORIWAKI 1980: Genetic regulation of the expression of H-2K.

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31
5 antigen on erythroid cells by H-2K end. J. Immunogenet. 7: 325-331.
SOEDA, E., J. R. ARRAND and B. E. GRIFFIN 1980: Polyoma virus DNA. The complete
nucleotide sequence of the gene which codes for the polyoma virus capsid protein
VP1 and overlaps the VP2/VP3 genes. J. Viro!. 33: 606-618.
SOEDA, E., J. R. ARRAND, N. SMOLAR, J. E. WALSH, B. E. GRIFFIN 1980: Coding potential and regulatory signals of the polyoma virus genome. Nature 283: 445-453.
SOEDA, E., T. MARUYAMA, J. R. ARRAND and B. E. GRIFFIN 1980: Host dependent
evolution of papova viruses, BKV, SV 40 and polyoma virus. Nature 285: 165-167.
SUGIURA, M. 1980: Existence of the gene for 4.5S RNA in broad bean chloroplast
DNA. Current Genetics 2: 95-96.
SUGIURA, M. 1980: Purification of the T4 DNA ligase by blue sepharose chromatography. Ana!. Biochem. 108: 227-229.
SUGIURA, M. and K. OONO 1980: Detection of the proplastid ribosomal RNA genes inrice embryos. Plant Science Letters 18: 13-17.
SUGIURA, M. and Y. HOTTA 1980: EcoRI analysis of nuclear and plastid ribosomal
DNAs in lily. Plant & Cell Physio!. 21: 1129-1132.
SUGIURA, M., J. KUSUDA, K. SHINOZAKI and F. TAKAIWA 1980: Cloning and characterization of chloroplast ribosomal RNA genes. "Genetics and Evolution of RNAPolymerase, tRNA and Ribosomes", 437-449. Tokyo Univ. Press.
SUZUKI, H., Y. Van HEIJENOORT, T. TAMURA, J. MIZOGUCHI, Y. HIROTA and J. Van
HIJENOORT 1980: In vitro peptidoglycan polymerization catalysed by penicillin
binding protein 1b of Escherichia coli K-12. FEBS Let. 110: 245-249.
TAKAHATA, N. 1980: Composite stepwise mutation model under the neutral mutation
hypothesis. J. Mo!. Evo!. 15: 13-20.
TAKAIWA, F. and M. SUGIURA 1980: The nucleotide sequence of 4.5S ribosomal RNA
from tobacco chloroplasts. Nuclcic Acids Research 8: 4125-4129.
TAKAIWA, F. and M. SUGIURA 1980: Nucleotide sequences of the 4.5S and 5S ribosomal
RNA genes from tobacco chloroplasts. Molec. gen. Genet. 180: 1-4.
TAKAIWA, F. and M. SUGIURA 1980: Cloning and characterization of 4.5S and 5S RNA
genes in tobacco chloroplasts. Gene 10: 95-103.
TAKAIWA, F., N. TODO and M. SUGIURA 1980: Molecular cloning of an EcoRI fragment
which contains 4.5S and 5S RNA genes in tobacco chloroplasts. Jap. J. Genet. 55:
121-125.
TAKAMURA, T. and T. K. WATANABE 1980: Further studies on the lethal hybrid rescue(Lhr) gene of Drosophila simulans. Jap. J. Genet. 55: 405-408.
TAKANO, J., T. FUJISAWA, and T. SUGIYAMA 1980: Growth rate and cell cycle of hydra.
"Developmental and Cellular Biology of Coelenterates", 409-414. Elsevier (Amsterdam).
TAMURA, T., H. SUZUKI, Y. NISHIMURA, J. MIZOGUCHI and Y. HIROTA 1980: On theprocess of cellular division in Escherichia coli: Isolation and characterization of
penicillin-binding proteins la, 1b, and 3. Proc. Nat!. Acad. Sci. USA. 77: 44994503.
TAZIMA, Y. 1980: Chemical mutagenesis in the silkworm, in "Chemical Mutagens"(eds. F. J. de Serres and A. Hollaender.) Vo!. 6: 203-238. Plenum Pub!. Corp.

BOOKS AND PAPERS PUBLISHED IN 1980 BY STAFF MEMBERS

125

YANAGISAWA, K., H. URUSHIHARA, H. FUJIMOTO, T. SHIROISHI and K. MORIWAKI 1980:
Establishment and characterization of cell lines from homozygous brachyury (TIT)
embryos of the mouse. Differentiation 16: 185-188.
YAZAKI, K. and K. MIURA 1980: Relation of the structure of cytoplasmic polyhedrosis
virus and the synthesis of its messenger RNA. Virology 105: 467-479.
YONEKAWA, H., K. MORIWAKI, O. GOTHO, J. WATANABE, J. HAYASHI, N. MIYASHITA,
M. L. PETRAS and Y. TAGASHIRA 1980: Relationship between laboratory mice andthe subspecies Mus musculus domesticus based on restriction endonuclease cleavage
patterns of mitochondrial DNA. Jap. J. Genet. 55: 289-296.
YOSIDA, T. H. 1980: Cytogeneties of the Black Rat-Karyotype Evolution and Species
Differentiation. 256 p. Univ. Tokyo Press (Tokyo) and Univ. Park Press (Baltimore).
YOSIDA, T. H. 1980: Karyotype of the Indian spiny mouse resulted from the tandem
fusion of some of the house mouse chromosomes. Cytologia 45: 753-762.
YOSIDA, T. H. 1980: Studies on the karyotype differentiation of the Norway rat, I.
Translocation between the pair nos. 1 and 12 chromosomes in the Lewis strain rat.
Proc. Japan Acad. 56(B): 268-272.
YOSIDA, T. H. 1980: Studies on the karyotype differentiation of the Norway rat, II.
A mosaic rat carrying the translocation and inversion of pair no. 1 chromosomeswith a note on their transmission to offspring. Proc. Japan Acad. 56(B): 322-327.
YOSIDA, T. H. 1980: Studies on the karyotype differentiation of the Norway rat, III.
Segregation of offspring from the 1/12 translocation heterozygotes and fertility of thetranslocation homozygotes. Proc. Japan Acad. 56(B): 437-442
YOSIDA, T. H. 1980: Studies on the karyotype differentiation of the Noway rat, IV.
Segregation and fertility of the Norway rats with inversion pair no. 1. Proc. Jap.
Acad. 56(B): 397-403.
YOSIDA, T. H. 1980: Segregation of karyotypes in the F 2 generation of the hybrids
between Mauritius and Oceanian type black rats with a note on their litter size.
Proc. Japan Acad. 56(B): 557-561.
YOSIDA, T. H., B. B. PARIDA 1980: Karyotype evolution, species differentiation andenvironmental mutagen. Proc. Japan Acad. 56(B): 79-84.
YOSIDA, T. H. and C. TAYA 1980: Studies on interspecific hybridization in the rodents.
III. Artificial insemination between Rattus norvegicus, R. annandalei and R. losea.
Proc. Japan Acad. 56(B): 141-145.

126

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

ABSTRACTS OF DIARY FOR 1980

OctoberNovember

14
21
24
2
14
17
27
28
15
12
20
28
17
31
13
1
6
27
14
12

December

13
28
18

January

February
March

April
May
June
July
AugustSeptember157th Biological Symposium
264th Meeting of Misima Geneticists' Club
265th Meeting of Misima Geneticists' Club
266th Meeting of Misima Geneticists' Club
158th Biological Symposium
159th Biological Sumposium
160th Biological Symposium
267th Meeting of Misima Geneticists' Club
268th Meeting of Misima Geneticists' Club
269th Meeting of Misima Geneticists' Club
161st Biological Symposium
162nd Biological Symposium
163rd Biological Symposium
164th Biological Symposium
165th Biological Symposium
166th Biological Symposium
167th Biological Symposium
168th Biological Symposium
169th Biological Symposium
170th Biological Symposium
171st Biological Symposium
172nd Biological Symposium
173rd Biological Symposium
174th Biological Symposium

ABSTRACTS OF DIARY FOR 1980

127

FOREIGN VISITORS IN 1980

January

February
March

April

May

June

CHAPEVILLE, Fran<;ois, Universite Paris VII,
FranceTSUGITA, S., European Molecular Biology
14
Laboratory, West Germany
NEUNGTON, Neelobo1, Mahidol University,
31-Feb. 15
ThailandTORREY, J. G., Harvard University, U.S.A.
21
STICH, F. H., B.c. Cancer Research Center,
12-14
CanadaGEORGIEV, G. P., Institute of Molecular Bio17
logy, U.S.S.R.
CROW, James F., University of Wisconsin,
21-Apr.9
U.S.A.
LEGAY, J. M., Universite de Lyon, France
20
NIERHAUS, Knud, Max-Planck-Institut fUr
27
Molekulare Genetik, West Germany
INOUE, M., State University of New York,
1-3
U.S.A.
ANDERSON, E. S., Central Public Health La9boratory, England
14-15
NAORA, H., Australian National University,
Australia
I-Aug. 6, '81 SECOND, Gerard, Office de la Recherche Scientifique et Technique Outer-mer, France
I-Apr. 30, '82 ACHERMANN, Joseph, Universitat Zurich,
Switzerland
4-May 12, '82 KECK, Wolfgang, Max-Planck-Institut fur
Virusforschung, West Germany
12-Jul. 11
ZAW, Khin Maung, Bio-Medical ResearchCenter, Burma
16
ALTuKHov, Yu. P., Institute of General Genetics, U.S.S.R.
28
David, Jean R., Laboratoire de Biologie et
14

128

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

July

4-5
IS-Sept. 15
16
30

August

1-2
2

1

9

9-10
13-14
September

1-2

Genetique Evolution, France
OHTSUBO, E., State University of New York,U.S.A.
Wu, Hsin-Kan, Institute of Botany, Academia
Sinica, Taiwan
LEVAN, Goren, University of Gothenburg,
SwedenSHIBATANI, A., Commonwealth Scientific andIndustrial Research Organization, Australia
RASMUSSEN, S(.iren Wilken, Carlsberg Laboratory, Denmark
GARAL, Jean-Pierre, Universite Claude Bernard
Lyon-I, France
FESTING, Michael F. W., Medical ResearchCouncil Laboratory Animals Centre, England
HEDRICH, Hans. J., Zentralinstitut fur Versuchstiere, W cst Germany
KOFFMAN, Harold A., National Institutes of
Health, U.S.A.
MOBRAATEN, Larry, The Jackson Laboratory,
U.S.A.
MOUTIER, R., Centre de Selection et d'Elevage
des Animaux de Laboratoire C.N.R.S., France
RADZIKOWSKI, Cz., Polish Academy of Sciences, Poland
KRISHNAMURTHY, N. B., University of Mysore,
India
CHOWDAIAH, B. N., Bangalore University,
India
CHEN, P. S., Universitiit Zurich, Switzerland
DUBENDORFER,
A.,
Universitiit
Zurich,Switezrland
De SERRES, F. J., Michael D. Shelby, National
Institute of Environmental Health Sciences,
U.S.A.
MENDELSOHN, M. L., Laurence Livermore
Laboratory, U.S.A.

FOREIGN VISITORS IN 1980

SHREFFLER, Donald c., Washington University,
U.S.A.
13
JIANG, Tong-Qing, South-West Agr. ColI.,
China
18-Sept. 16, '81 WANEK, Nancy, University of California,
U.S.A.
29
PAUN, Yen-Xang, South China Inst. of
Tropical Crops. China
2-Mar. 31, '81 PAl, Chian, Nat. Chung-Hsing University,
Taiwan
14-15
MOSCONA, A. A., University of Chicago, U.S.A.
16
HERZENBERG, L. A., Stanford University,
U.S.A.
20
FRANKEL, O. H. and W. J. PEACOCK, Commonwealth Scientific and Industrial Research
Organization, Australia
27
HSIEH, Sung-Ching, Taiwan Agr. Res. Inst.,
TaiwanPERNES, J., C.N.R.S., France
28
Xu, Yuntian, Inst. Crop Germplasm Resources, China
POULSEN, E., National Food Institute, Den6mark12
De NAVA, C. C., Universidad Nacional Autonomade Mexico, Mexico
12-13
GILES, Norman H., University of Georgia,
U.S.A.
13-14
OHNO, S., City of Hope National Medical
Center, U.S.A.
18
RILEY, Ralph, Agricultural Research Council,
England27
SANDBERG, A. A., Roswell Park Institute,
U.S.A.
18-19
MARKERT, C. L., Yale University, U.S.A.
5-7

OctoberNovember

December

129

130

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 31

AUTHOR INDEX.48
76, 77, 78, 79, 81
ARAI, M
32
ASAKURA, M.46
BECHERER, K. .
30
BONHOMME, F. .
61
DE PEDRO, M. A.
21
DINARDO, S
29, 30
EGAMI, N • . . . . . . . . . . . . . . . . . . . . . . . . . . 39
ENDO, T40, 41, 42
FUJII, T. .
83, 114
FUJISHIMA, T • . . . . . . . . . . . . . . . . . . . . . . 109
FUKUDA, E. .
104
HANAOKA, K. .
54
HAYASHI, S
32
HIDAKA, S.
.
12, 13
HIROTA, Y
21,22,23,24,29,30,31,32,33
IINUMA, K.
102, 105
IMAI, H. T
61, 62INOKUCHI, H
31
INOUE, T
60, 71, 74, 75
INOUE, Y.
.
92,108INUKAI, M
32
IYAMA, S. .
112, 118
KADA, T. .
70, 71, 72, 73, 74, 75
KANEKO, K. .
73
KATO, A.
17
KATO, M
71
KAWANISHI, M. .
91,93, 107
KECK, W
24
KIMURA, M.
.
85, 86
KOMEDA, Y
34
KONDO, K
61
KRAUT, H
24
KURODA, Y. .
.43, 44, 45, 46
KUSUDA, J
19
LAI, J. S
32
MARUYAMA, T • . . . . . . . . . . . . . . . 86,89,91
MATSUBARA, T.
.
103,104,105
ACHERMANN, J.

AMANo, E.

.

MATSUDA, Y.

.

61

MATSUDA, M . . . • . . . . . . . . . . . . . . . . . . . . 62
MATSUNAGA, E . . . • . . . . . . . . . . . 98, 99, 100
MATSUTANI, E. . . . • . . . . . . • • . . . . . . . . . . 44

51
12, 13MIYASHITA, N
37
MIZOGUCHI, J. .
22
MOCHIZUKI, H.
• • . . . . . . . . . . . . . . . 72, 74
MORISHIMA, H.
95, 96,110,111,117
MORIWAKI, K • • . . . . . . . . . . . 37, 38, 39, 61
MURAKAMI, A • . . . . . . 31, 47, 64, 65, 66, 68
MUROFUSHI, M
61
NAKAGOME, Y
101, 102, 103, 104, 105
NAKAMURA, M. . • . . . . . . . . . . . . . . . . . . . 32
NAwA, S • . . . . . . . • . . . . . . . . . . . . . . . . . . 51
NISHIKAWA, S
61
NISHIMURA, A
23, 29NISHIMURA, Y
21, 22, 23, 29, 30
NOGUCHI, T. .
53, 54
OGASAWARA, N
105
OHNISHI, S. .
93
OHTA, T. .
87,88
OISHI, K
52
OIKAWA, S • . . . . . . . . . . . . . . . . . . . . . . . . . 61
OKA, A
32
OKA, S105
ONIMARU, K.
19
OZEKI, H
31
PAl, C. .
.42, 96
PARK, J. T
23
PHILBRICK, W. M •..•........•........ 32
SADAIE, Y. .
70, 74SAGAI, T . . . . . . . • . . . . . . . . . . . . . . . . . . . . 38
SAKAIZUMI, M. . . • . . . . . . . . . . . . . . . . . . . 39
SANO, Y110, 115, 116, 118
SAKAI, K. I.
112
SCHWARZ, U", .•....... 21
SECOND, G
95,117,118
MINATO, K

MIURA, K. I.

AUTHOR INDEXSHIMADA, H.

.

17
14
SHIOTA, K.
.
99, 100
SHIROISHI, T ....••••.••.•............ 38
SHIZAKI, M. .
71
SOEDA, E
91
STERNGLANZ, R
29, 30SUGIMOTO, K. .
32
SUGIURA, M
14,15,17,18,36
SUGIYAMA, T • . • • . • . . . . . . . . . . • . . . .48, 49
SUZUKI, H. .
22, 23
TAKAHATA, N
89
TAKAIWA, F
15
TAKANAMI, M
32
TAKANO, J.48
TAKEDA, Y • . . . . . . . . . . . . . . . . . . . . . . . . . 23
TAMURA, T
22

SHINOZAKI, K.

p'.

.

TAYA, C

131

53
62
TOHDOH, N.
.
14, 15TOMIOKA, N
36
VOELKEL, K. A
30
WANEK, N
49
WANG, J. C. .
29,30
WATANABE, T. K. .. 51,52,91,92,93,107
Wu, H. C
32
YAMADA, M
23,32,33
YAMADA, M. A
51
Y AMAGISHI, H
31
YAMAGUCHI, K
12
YASUDA, S • . . . . . . . . . . . . . . . . . . . . . . • . . . 23
YOKOCHI, T. •104, 105YOSIDA, T. H
56,57,58,59,60,61
ZUMSTEIN, L. .
30

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