NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 271976

Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka.ken, Japan
1977

Annual Repor~of ~heNational Institute of Genetics
No. 27, 1976

Published by

The National Institute of Genetics, Japan
1977

CONTENTS
General statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council
Association for propagation of the knowledge of genetics
Projects of research for 1976
Researches carried out in 1976

I.

Molecular Genetics
Cloning of dnaA gene in E. coli TAKEDA, Y. and HIROTA, Y.
Construction and characterization of an F-ColEl hybrid plasmid.
TAKEDA, Y., YASUDA, S. and HIROTA, Y.
Cloning and mapping of the replication origin of Escherichia coli.
YASUDA, S. and HIROTA, Y.
The 5'-terminal structure of bacterial messenger RNA. MIURA,
K. and lMAMOTO, F.
Mechanism of formation of the modified structure at the 5'terminus of mRNA of reovirus. SHIMOTOHNO, K. and
MIURA, K.............................................
Modified structure at the 5'-terminus of low molecular weight
RNA in nucleus of animal cell. SHIMOTOHNO, K., URUSHIBARA, T. and MIURA, K.
Research on the enzyme cleaving the 5'-terminal methylated
blocked structure of messenger RNA. MIURA, K. and
SHIMOTOHNO, K.
Enzymatic removal of the 5'-terminal methylated blocked structureof tobacco mosaic virus RNA and its effect on infectivity
and reconstitution with coat protein. MIURA, K. and SHIMOTOHNO, K.
Role of the 5'-terminal modified structure of mRNA in protein
synthesis. SHIMOTOHNO, K. and MIURA, K.
A procedure for the isolation of temperature sensitive RNA
polymerase mutants of E. coli. SUGIURA, M. and
YOSHINAGA, K.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

II.

Microbial Genetics
Mutants of Escherichia coli altered in penicillin binding proteins.
HIROTA, Y., NISHIMURA, Y. and SUZUKI, H.
Synthetic ColEI plasmids carrying genes of cell division in
Escherichia coli. NISHIMURA, Y, TAKEDA, Y, NISHIMURA, A.
SUZUKI, H. and HIROTA, Y.. ,..........................
Penicillin binding proteins involved in cell elongation. SUZUKI,
H., NISHIMURA, Y. and HIROTA, Y.
Differential solubilization of membrane proteins of Escherichia
coli with detergents. YAMADA, M., SUZUKI, H. and HIROTA,Y.
Isolation of specialized transducing bacteriophage A carrying
leuA-ftsI-murEFC-ddl-ftsA region of Escherichia coli chromosome. YAMADA, M., YASUDA, S. and HIROTA, Y.

Ill.

IV.

Biochemical Genetics and Immunogenetics
A comparison of heat sensitivity of serum esterase (Es-I)
in different inbred mice, Mus musculus. AOTSUKA, T. and
MORIWAKI, K.
......................
Distribution of Hbb (Hemoglobin beta-chain) alleles in Japanesewild mice, Mus musculus molossinus. MINEZAWA, M., MoRIWAKI, K. and KONDO, K.
Suppression of erythrocytic H-2 expression in F 1 hybrid between
two mouse subspecies, Mus musculus domesticus and molossinus. MORIWAKI, K. and SHIROISHI, T.
The effect of K-region in H-2 complex on expression of H-2.5
antigen on erythrocytes. SHIROISHI, T. and MORIWAKI, K.. .
Developmental and Somatic Cell Genetics
In vitro analysis of tissue-specific defects in rudimentary embryoniccells of Drosophila melanogaster. KURODA, Y.
Muscle and cartilage differentiation in cultured mesenchymal cells
from embryonic quail limb-buds. KURODA, Y. and MATSUTANI, E.
Induction of 8-azaguanine-resistant mutations by sulfite in cultured embryonic human diploid cells. KURODA, Y.
.
Dose-rate effects of ethyl methanesulfonate on survival and mutation induction in cultured Chinese hamster cells. KURODA,

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V.

CONTENTS

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Y. and SUGIURA, K.
Selection and characterization of a 5-iodouridine-resistant variant
in cultured Syrian hamster cells. KURODA, Y., YOKOIYAMA,
A. and KADA, T.
Effects of SR and da on the viability of the hybrid males from
Drosophila melanogasterxD. simulans cross. WATANABE,
T. K. and YAMADA, M. A.
Characterization of a regeneration deficient strain of hydra.
SUGIYAMA, T. and FUJISAWA, T.
Isolation and characterization of an interstitial cell deficient strainof hydra. SUGIYAMA, T. and FUJISAWA, T.
Characterization of a nematocyte deficient strain of hydra.
FUJISAWA, T. and SUGIYAMA, T.
The relationship of mitotic activity in primordial germ cells to
testicular teratocarcinogenesis in strain 129 mice. NOGUCHI,
T. and STEVENS, L. C. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Cytogenetics
Karyotypes of four Myotis bats in Japan. HARADA, M. and
YOSlDA, T. H.
Geographical distribution of No. 18 chromosome polymorphismin the Japanese feral mouse, Mus musculus molossinus.
MORlWAKl, K. and MlNEZAWA, M.
Hybrids among three geographical variants of the black rats, I.
Hybrids between Asian and Ceylonese type black rats, andtheir karyotypes. YOSIDA, T. H.
. . . . . . . . . . . . ..
Hybrids among three geographical variants of the black rats, II.
F 1 and F 2 hybrids between Oceanian and Ceylonese typeblack rats and their karyotypes. YOSIDA, T. H.
Hybrids among three geographical variants of the black rats, III.
An XO female occuring in the F 2 hybrid between Oceanianand Ceylonese types. YOSIDA, T.H.
Candidates for a new experimental animal, I. The soft-furredrat, Millardia meltada. YOSIDA, T. H.
Subtelocentric No.3 chromosome in the Japanese black rat-a
new inversion. YOSIDA, T. H. and OCHIAl, Y.
An experiment on artificial insemination between the Norway rat

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VI.

Mutation and Mutagenesis in Animals

and the black rat.

YOSIDA, T. H. and TAYA, C.

Genetic analysis of spontaneously derived mosaics from abnormal
fertilization in the silkworm. MURAKAMI, A. and FUJIOKA,
H.
Chemically-induced transmitted translocations in the silkworm.
MURAKAMI, A. and MUROTA, T.
Drug metabolic activation in the silkworm. SAWAMOTO, K.,
MUROTA, T. and MURAKAMI, A.
Comparison of some nitrosonitroguanidines in respects to theirefficiency in inducing recessive visible mutations in silkworm
germcells. MURAKAMI, A. and NAKADATE, M.
Enhancing effect of caffeine on X-ray-induced dominant lethal
mutations in silkworm sperm. MURAKAMI, A. and NISHIJIMA, H.
Appraisal of silkworm oocyte system for mutagenicity testing ofenvironmental chemicals. TAZIMA, Y. and MURAKAMI, A...
Mutagenic effectivenss of an internal f3 emitter, tritium. (3)Effectiveness of f3-irradiation at low dose-rate range. TAZIMA, Y., ONIMARU, K. and FUKASE, Y.
Nature of chemically induced mutations obtained by oocyte
system in the silkworm (continued). TAZIMA, Y. ONIMARU,
K. and OHNUMA, A.
High incidence of translocation between Wand the fifth chromosomes among mutants detected by specific loci method in thesilkworm. TAZIMA, Y, ONIMARU, K. and OHNUMA, A. ....

VII.

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Radiation Genetics and Chemical Mutagenesis in Microorganisms
and Plants
Effect of gamma-irradiation on growth of cultured Haplopappus
cells. AMANO, E.
Measurement of waxy starch phenotype in cereal crops. AMANO, E.
Isolation of a new tester strain .of Bacillus subtilis for the screening of environmental mutagens and carcinogens by bacterialcell fusion. HIRANO, K., ASANO, T., SADAIE, Y. and KADA,

T.

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Highly sensItIve rec-assay using spores of Bacillus subtilis.
HIRANO, K., MATSUMOTO, H. and KADA, T.
DNA repair enzymes in ataxia telangiectasia and Bloom's syndrome fibroblasts. INOUE, T., HIRANO, K., YOKOIYAMA, A.,
KATO, H. and KADA, T.
Purification and properties of Bacillus subtilis endonuclease
specific for apurinic sites in DNA. INOUE, T. and KADA, T.. .
Utilization of a mutator strain for the isolation of mutants in
Bacillus subtilis. SADAIE, Y., OHTA, Y. and KADA, T. ....
The induction of monoecious mutants in cucumber. FUJII, T.. .

VIII. Population Genetics (Theoretical)
Population genetics and molecular evolution. KIMURA, M. ....
Role of very slightly deleterious mutations in molecular evolution and polymorphism. OHTA, T. . . . . . . . . . . . . . . . . . . . . . .
IX.

X.

Population genetics (Experimental)
Elimination of the polymorphic inversions from the laboratory
populations of Drosophila melanogaster. INOUE, Y. and
WATANABE, T. K.
Equilibrium frequency of lethal genes in cage populations ofDrosophila melanogaster. LEE, W. H. and WATANABE, T. K.
Enzyme and chromosome polymorphisms in natural populationsof Drosophila melanogaster. WATANABE, T. K. and WATANABE, T.
Evolutionary Genetics
Geographical distribution of Drosophila simulans in Japan. WATANABE, T. K. and KAWANISID, M.
How genes evolve: a population geneticist's view. KIMURA, M...
Archeology, population genetics and studies of human racial
ancestry. MARUYAMA, T.
Simple model for treating evolution of multigene families.
OHTA, T.
Simulation studies on the evolution of amino add sequences.
OHTA, T.
Reproductive barriers distributed in Melilotus species and their
genetic bases. SANO, Y. and KITA, F.

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Analysis of genes responsible for the F 1 sterility between Oryza
sativa ang. O. glaberrima. SANO, Y. and OKA, H. I.

XI.

XII.

Human Genetics
Hereditary retinoblastoma: penetrance, expressivity and age of
onset. MATSUNAGA, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A survey on maternal age and karyotype in Down's syndrome inJapan, 1947-75. MATSUNAGA, E. and FUJITA, H. .. . . . . . . . .
LBA technique in the detection of chromosome variants in man.
NAKAGOME, Y, OKA, S., MATSUNAGA, E. and ARIMA, M. ..
Holoprosencephaly in human embryos: an epidemiologic study.
SHIOTA, K. and MATSUNAGA, E.
Behavioral Genetics
Genetic variation of walking ability in Drosophila melanogaster. AKAI, S. M. and WATANABE, T. K.
Adult emergence rhythm of homozygous strains of Drosophilamelanogaster. LEE, W. H. and OSIDMA, C. . . . . . . . . . . . . . . . .
Circadian locomotor activity of Drosophila melanogaster undernoise environment and walking behavior of a group undershake environment. OSIDMA, C. and LEE, W. H.
Selection for the discriminated avoidance learning ability of mice.
FunsHIMA, T.
...................................

XIII. Ecological Genetics
Submergence tolerance of wild and cultivated rice strains. MoRISIDMA, H.
Variations in copper tolerance of Alopecurus aequalis strains.
MORISHIMA, H. .
,...........
The impact of copper pollution on barnyard grass populations.
MORISIDMA, H. and OKA, H. I.
The pollination .systems of Melilotus species. SANO, Y.
Resource allocation patterns in Oryza perennis strains. SANO, Y.
and MORISIDMA, H" ....................
Variations in competitive ability among Oryza perennis strains.
SANO, Y. and YAMAGISID, H. ......................•...
An experiment on the interaction between cultivated rice· andbarnyard grass at different planting densities. YAMAGISHI,

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CONTENTS

H., MORISHIMA, H. and OKA, H. 1.

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XIV. Applied Genetics
Population size maintained for the bulk method of breeding in
autogamous plants. IYAMA, S.
95
Difference in noise sensitivity affecting egg laying performance
between wild and domestic Japanese quails. KAWAHARA
T. .
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A breeding experiment in Oryza glaberrima. OKA, H. 1.
Plasma albumin polymorphism in Japanese quails. SHIBATA, T.,
WATANABE, S. and KAWAHARA, T.
97
The growth pattern and competitive ability of six rice varieties.
YAMAGISHI, H., MORISHIMA, H. and OKA, H. 1. .... . . . . . . . .
98
Nitrogen-fixing ability in rice strains. Fum, T., SANO, Y. and
IYAMA, S.
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Selection of induced mutants in Oryza glaberrima. SANO, Y. .. 101
Breeding of isogenic lines of rice carrying marker genes in interchanged segments. SANO, Y. and OKA, H.1.
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Books and papers published in 1976 by research members .. . . . . . . ..
Abstracts of diary for 1976
Foreign visitors in 1976
Author index ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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GENERAL STATEMENT
During the past 27 years since the foundation of the Institute, a large
number of scientific papers have been published by our staffs and various
prizes have been awarded to them from several learned societies. This
year an Order of Cultural Merits, one of the most glorious awards inJapan, was conferred upon Dr. Motoo Kimura, the Head of the Departmentof Population Genetics. As a pioneer of the population genetics in thiscountry, Dr. Kimura has contributed a number of valuable theoretical
papers. His article on neutral theory in evolution which was proposed on
his penetrated insight into the random modification of genetic code has
been highly evaluated in and outside Japan and won the great honor for the
first time in this new field of genetics.
I realize that it is not easy to accomplish distinguished research works,
but taking this opportunity, I would like to request earnestly to our staff
members to follow him in contributing excellent papers of full originality.
Hopefully, they should keep in mind that the government has been investinga huge amount of money on their studies.
Genetics, being a nucleus of biological sciences, has developed at surprisingly quick tempo. Among others it is of great importance to recognizethat recombination of DNA molecules has become technically feasible
between different species. The desire to create new superior organisms bycombining genes from different species had long been a subject of biology,
and gave birth to the science of genetics. I cannot help exciting when
I think over that man has just developed a technique for realizing his longcherished desire. Needless to say this technique offers a very promisingprospect for practical application. I should like, however, to emphasize
the importance of this technique in the field of pure genetic researches. For
instance, mechanisms of gene action can be analyzed at biochemical level if
genes of higher organisms could be cloned by connecting them with bacterial
plasmids. This will undoubtedly bring about in near future a revolution in
the methodology of genetic studies. Though there are many problems tobe solved by that time, we realize that genetic studies have entered in a newera.
The Institute held two international meetings in the fiscal year of 1976:
one was a symposium on "Molecular Evolution and Polymorphism" as

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

the Second Taniguchi International Symposium on Biophysics organized by
Dr. M. Kimura, and the other was East Asian Training Course on Mutagenicity Testing of Chemicals organized by Dr. Tsuneo Kada. The former
symposium, being participated by reputed scientists from several countries,
discussed strategies for further advancement of genetic studies. The latter
aimed to provide senior scientists of East Asian countries with a balanced
comprehensive overview of chemical mutagenesis and mutagenicity testing of'chemicals. There were 14 participants and 22 lecturers from six countries.
I am convinced that both meetings will prove to be significant for the
future development of our science.
As regular annual events, the Institute was opened to the public on April
17th, having as many visitors as we had last year. Public lectures weredelivered on November 13th at the lecture hall of the National Science
Museum in Tokyo. Speakers and topics were: Dr. Hideho Suzuki,
Department of Microbial Genetics, "Regulation and flagella formation inbacteria," and Dr. Chozo Oshima, the Head of the Department of Physiological Genetics, "Insect bioclocks." Both gave deep impression to theaudience.
The purchasing program of the neighboring area has been concluded, andthere will be accommodated buildings for Genetic Stocks Center. The oldannex building has completely been remodelled for low level radioisotope
laboratories.

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STAFF
Director

TAZIMA, Yataro, D. Ag.
Members

1. Department of Morphological Genetics
KURODA, Yukiaki, D. Sci., Head of the Department

The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
MURAKAMI, Akio, D. Ag., D. Sc.

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, Tosihide H., D. Sc., Head of the Laboratory
KATO, Hatao, D. Sc.

The 2nd Laboratory
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
IMAI, Hirotami, D. Sc.

3. Department of Physiological Genetics
OSHIMA, Chozo, D. Sc., Head of the Department

The 1st Laboratory
OSHIMA, Chozo, D. S., Head of the Laboratory
WATANABE, Takao K., D. Sc.

The 2nd Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
OISHI,* Kugao, Ph. D.

* Visiting researcher

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

4.

Department of Biochemical Genetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory
YAMADA, Masa-Aki, M. Sc.
The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
ENDO, TOfU, D. Ag.
The 3rd Laboratory
SUGIYAMA, Tsutomu, Ph. D., Head of the Laboratory
FUJISAWA, Toshitaka, Ph. D.

5.

Department of Applied Genetics
OKA, Hiko-Ichi, D. Ag., Head of the Department
The 1st Laboratory
OKA, Hiko-Ichi, 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
OKA, Hiko-Ichi, D. Ag., Head of the Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag.

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. Sc.
The 2nd Laboratory
KAnA, TSUNEO, D. Sc., Head of the Laboratory
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
AMANO, Etsuo, D. Ag.
SADAIE, Yoshito, M. Sc.

7.

Department of Human Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department

STAFFThe 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
SHIOTA, Kohei, D. Med.
The 2nd Laboratory
NAKAGOME, Yasuo, D. Med., Head of the Laboratory
IINUMA, Kazuso, B. Med.

8. Department of Microbial Genetics
HIROTA, Yukinori, D. Sc., Head of the Department
The 1st Laboratory
HIROTA, Yukinori, D. Sc., Head of the Laboratory
YASUDA, Seiichi, D. Sc.
The 2nd Laboratory
SUZUKI, Hideho, D. Sc., Head of the Laboratory
NISHIMURA, Yukinobu, 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
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Laboratory
YAMAZAKI, Tsuneyuki, Ph. D.
10. Department of Molecular Genetics
MIURA, Kin-ichiro, D. Sc., Head of the Department
The 1st Laboratory
MIURA, Kin-ichiro, D. S., Head of the Laboratory
FURUICHI, Yasuhiro, D. Pha.
SOEDA, Eiichi, D. Ag.
The 2nd Laboratory
SUGIURA, Masahiro, D. Sc. Head of the Laboratory
SHIMOTOHNO, Kunitada, D. Pha.
11.

Genetic Stocks Center
Plant Section
FUJII, Taro, D. Ag., Head of the Laboratory
SANO, Yoshio, D. Ag.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

Animal Section
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
NOGucm, Takehiko, D. Sc.

12. Experimental Farm
OKA, Hiko-Ichi, D. Ag., Head of the Farm
MIYAZAWA, Akira
13. Department of Administration
OIsm, Tatsunori, Head of the Department
OHTSUKA, Haruichi, 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
LILIENFELD, Flora A., Ph. D.
SAKAI, Kan-ichi, D. Ag.
TsunTA, Mitsuo, D. Ag.
MORIWAKI, Daigoro, D. Sc., Managing Director of the Institute of Physicaland Chemical Research

COUNCILKIKKAWA, Hideo, Chairman, Professor of Hyogo College of Medicine
FUJII, Takashi, Vice Chairman, Emeritus Professor of University of TokyoIZEKI, Shoei, Director of National Research Institute of Police Science
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
KONDO, Norio, Professor of Tokyo University of Agriculture
MISONOO, Keisuke, Director of National Institute of Radiological Sciences
MORIWAKI, Daigoro, Managing Director of the Institute of Physical andChemical Research
MORoHosm, Seijiro, Professor of Tokyo University of Agriculture andTechnology

COUNCIL

7NAGAKURA, Saburo, Professor of University of Tokyo
SASA, Manabu, Vice-Director, National Institute for Environmental Studies
SHINOZAKI, Nobuo, Director of Institute of Population Problems
TAKAHASHI, Ryuhei, Professor of Okayama University
TANAKA, Nobunori, Professor of Teikyo University
UMEZAWA, Hamao, Head of the Department of Antibiotics, National
Institute of Health
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Managing Director of the Institute of Physical and Chemical Research
MATSUNAGA, Ei, Managing Director, Head of the Human Genetics Department
YOSIDA, Tosihide H., Managing Director, Head of the Cytogenetics Department
KIHARA, Hitoshi, Manager, Emeritus Professor of Kyoto University
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, Head of the Physiological Genetics DepartmetentPROJECTS OF RESEARCH FOR 1976
Department of Morphological Genetics

Genetic studies of the silkworm (TAZIMA and ONIMARU)
Radiation and chemical mutagenesis in the silkworm (TAZIMA and ONIMARU)
Cytogenetics in the silkworm (MURAKAMI and IMAI)
Studies on recombination in the silkworm (MURAKAMI)
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)
Department of Cytogenetics

Studies on chromosomal evolution in rodents (YosmA)
CHROMOSOME study on experimental tumors (YosmA)
Experimental breeding and genetics of mice, rats and other wild rodents(YosmA and MORIWAKI)
Cytogenetical study on sister chromatid exchange in mammalian cells (KATO)
Cytogenetical and immunological studies on mouse plasma cell tumor(MORIWAKI)
Biochemical studies on serum transferrin variations in rodents (MORIWAKI)
Immunogenetical study on the histocompatibility antigens of wild rodents(MORIWAKI)
Cytogenetical study of ants (IMAI)
Department of Physiological Genetics

Behavior genetics of Drosophila (OSHIMA and WATANABE)
Analysis of de1ecterious and inversion chromosomes in natural populationsof Drosophila melanogaster (WATANABE)
Studies on fitness of Drosophila under controlled environment (OSHIMA andWATANABE)
Genetic studies on the effects of adverse environments on Drosophila flies(OSHIMA and WATANABE)

PROJECTS OF RESEARCH FOR 1976

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 proteins (OGAWA)
Genetical and biochemical studies on Japanese middle size dog (OGAWA)
Genetics of isozymes in plants (ENDO)
Effects of exogenous DNA on plant seed formation (ENDO)
Genetic analysis of developmental mechanisms in hydra (SUGIYAMA andFUJISAWA)
Department of Applied Genetics

Quantitative genetic studies in poultry (KAWAHARA and FUJISHIMA)
Genetics studies in wild populations of Japanese quails (KAWAHARA)
Theoretical studies on breeding techniques (IYAMA)
Behavioral genetic studies in mice (FUJISHIMA)
Genetic studies in natural stands of forest tree species (lYAMA)
Simulation studies on artificial selection (lYAMA)
Evolutionary studies in wild and cultivated rice species (aKA and MORISHIMA)
Ecological genetic studies in some grass species (MORISHIMA)
Genic analysis for isozyme variations in rice (ENDO and aKA)
Genetic effects of environmental pollution on plants (lYAMA, MORISHIMA
and aKA)
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
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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

Mechanisms of recombination repair (SADAIE and KADA)
Mutation and differentiation studies of plant tissue culture (AMANO and
KADA)
Radiation and chemical interaction in the cells (KADA)
Genetic fine structure analysis in maize (AMANO)
Department of Human Genetics

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

Genetic regulatory mechanism of DNA replication in E. coli (HIROTA andNISHIMURA, and YASUDA)
Genetic regulatory mechanism of cellular division in E. coli (HIROTA)
Molecular genetics on DNA replication (YASUDA and HIROTA)
Molecular mechanisms of cell division in E. coli (SUZUKI, NISHIMURA andHIROTA)
Genetics of bacterial cell envelope (HIROTA, SUZUKI, and NISHIMURA)
Molecular genetics on the regulatory mechanism of flagella formation(HIROTA and SUZUKI)
Synthetic bacterial plasmid (YASUDA)
Department of Population Genetics

Theoretical studies of population genetics (KIMURA, MARUYAMA and OHTA)
Studies on molecular evolution from the standpoint of population genetics(KIMURA and OHTA)
Theoretical studies on the evolution of multigene family (OHTA)
Mathematical studies on the genetics of structured populations (MARUYAMA)
Experimental studies on protein polymorphism in Drosophila (YAMAZAKI)PROJECTS OF RESEARCH FOR 1976

11

Statistical studies on protein polymorphisms in natural populations (MARUYAMAand YAMAZAKI)
Department of Molecular Genetics

Studies on the chemical structure of genome of viruses containing doublestanded RNA (MIURA, SUGIURA, SHIMOTOHNO)
Studies on the interaction between RNA polymerase and template nucleic
acid (MIURA, SUGIURA, SHIMOTOHNO, and SOEDA)
Studies on structure and function of messenger RNA (MIURA, SHIMOTOHNO)
Genetical and enzymological studies on E. coli RNA polymerase (SUGIURA)
Genetic Stocks Center

Studies and conservation of germplasm resources in rice and wheat species(FuJII, SANO and OKA)
On the sex expression in monoecious plants (FuJII)
Specificity of mutagen tolerance in higher plants (FuJII)
Exploitation of genetic ability of nitrogen fixation in Graminae (FuJII,
SANO, IYAMA and HIROTA)
Studies on teratocarcinogenesis in mice (NoGucHI)RESEARCHES CARRIED OUT IN 1976I.

MOLECULAR GENETICS

Cloning of dnaA Gene in Escherichia coli
Yutaka TAKEDA and Yukinori HIROTA

It has been shown that the gene product of dnaA is required in theinitiation step of E. coli chromosome replication. We report here successful cloning of the dnaA gene.
E. coli DNA was prepared from W3110 and fragmented by digestion
with EcoRI. The DNA fragments were linked with a ColEl vector, pMB9
or pCRI, by T4 DNA ligase. The CaCl 2-treated cells of CRT46recA(temperature sensitive dnaA mutant) were transformed with the ligation
mixtures and the transformants which became temperature and drug resistant were selected. From these transformants, two plasmids were isolated:
one synthesized by ligation with pMB9 and the other with pCRI, and,referred to as pYT47 and pYT48, respectively.
The DNA of pYT47 was cleaved into two fragments by EcoRI digestion,

srnOl
BOrnl

pYT47

RESEARCHES CARRIED OUT IN 1976

13

producing'the linear molecule of pMB9 and the fragment originated from
E. coli DNA. The DNA of pYT48 was cleaved similarly, but producedpCRl instead of pMB9. The E. coli DNA fragments of these plasmids
were identical with each other, as shown by the restriction mapping (Fig. 1)
and heteroduplex analyses. Molecular weight of the E. coli DNA fragment
obtained from either plasmid was ca. 5.5 X 10 6 daltons. These plasmids
were confirmed to contain dnaA by transformation, but contained no known
genes around dnaA, such as tna, asn, bglA, rbs, and uncA. About 10
copies of the plasmid per chromosome were found in the wild type cells.
They were able to replicate in the presence of chloramphenicol, as known
in the ColEl replicon.
Construction and Characterization of an F-CoIEl Hybrid Plasmid
Yutaka TAKEDA, Seiichi YASUDA and Yukinori HIROTA

We have constructed an F-ColEl hybrid plasmid by means of in vitro
recombination between pVH51, a mini-ColE! plasmid, and pSC138, a miniF-ampicllin plasmid.
This plasmid, named pSYlll, can replicate under conditions where thereplication function of F or ColEl is inhibited. For example, pSYlll
was maintained stably in male strains or in the presence of acridine orange,
although replication of F factor is known to be inhibited under these
conditions. pSYlll could also replicate in a polAl strain or a polAl2
strain at high temperature, although ColEl could not. When both F and
ColEl replicon were inhibited in an Hfr polAl2 strain at high temperature,
this plasmid could not replicate autonomously.
We have examined the compatibility properties of pSYlll. This plasmid
was fully compatible with Flac and ColE1 as examined by transformation
frequency and segregation kinetics. These results are different from thatreported by Cabello et al. (Cabello, F., K. Timmis & S. N. Cohen. 1975
Nature 259: 285), who observed that pSC134, a pSClOl-ColEl hybrid
plasmid, was incompatible with both pSClOl and ColE!. The discrepancy
could be due to the difference in the parental plasmids used to construct
the hybrid plasmids. Cabello et al. have suggested that their results support
the model of negative control of the initiation of DNA replication. However, the nature of DNA replication of pSYlll could not be explainedby the negative control model. We conclude, therefore, that the model is

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

not applicable to all replicon and the detailed experiments are needed toobtain the generalized model of the plasmid replication.
Cloning and Mapping of the Replication Originof Escherichia coli
Seiichi YASUDA and Yukinori HIROTA

The replication origin of Escherichia coli has been cloned on a nonreplicating DNA fragment coding for ampicillin resistance. This recombinant DNA, named pSY211, replicates depending on the presence of thereplication origin and can be recovered as a closed circular plasmid DNA of
10.7 Mdal. A restriction map has been constructed. EcoRI cleaves pSY211
into two fragments: One is the ampicillin fragment of 4.5 Mdal and theother is a chromosomal fragment of 6 Mdal, which contains the origin.
The 6 Mdal EcoRI fragment has four BamHI sites, three HindUI sites and
one XhoI site. A mutant of pSY211 has been isolated which is deleted of
the Bam HI fragments of the chromosomal fragment. In recA hosts pSY211
is lost at a high frequency. In recA + hosts pSY211 is integrated into thechromosome due to nucleotide-sequence homology between pSY211 and thereplication origin of the E. coli chromosome. The integration site has
been mapped. We conclude that the replication origin is located at a site
between uncA and rbsK, at about 83 min on the genetic map of E. coli.
f

The 5 -Terminal Structure of Bacterial Messenger RNA
Kin-ichiro MIURA and Fumio IMAMOTOThe Sf-terminal methylated, blocked structure has now been detectedin eukaryotic mRNAs commonly since our discovery of this structure inmRNA of silkworm cytoplasmic polyhedrosis virus (Annual Rep. No. 25(1974) p. 12). In order to clarify whether this modified structure is present
in prokaryotic mRNA or not, the Sf-terminal structure was studied on
mRNAs transcribed from tryptophan operon in Escherichia coli by labelling with [32P] phosphate and [methyl 3H] methionine. Total RNA was extracted from the cell and fractionated in glycerol density gradient. The
fractions containing mRNAs were digested with ribonuclease T2 and thenwith Penicillium nuclease Pl' The blocked structure NpppN was seeked
in the enzymatic digests by chromatographic procedures. Any blocked

RESEARCHES CARRIED OUT IN 1976

15

structure was not found. Instead, nucleoside triphosphate pppN anddiphosphate ppN were detected as the 5'-terminal nucleotides of E. colimRNA.
When the [3H] methylated RNAs were fractionated by electrophoresis inpolyacrylamide gel, any methylation was not detected in mRNA fractions.
Ribosomal RNAs, including their precursor RNAs, and transfer RNAs
were methylated.
Mechanism of Formation of the Modified Structure
at the 5'-Terminus of mRNA of Reovirus
Kunitada SHIMOTOHNO and Kin-ichiro MIURAAt the 5'-terminus ofmRNA of reovirus, the modified structure including
the 7-methylguanosine blocking through 5'-5'-pyrophosphate linkages andribose-methylation at 2'-position of the first nucleotide was found (Annual
Rep. No. 26 (1975) p. 12). It was expressed as m7 G5'pppGm-C-C- ---.
Reovirus, which contains double-stranded RNA as a genome and enzymes
including RNA polymerase, phosphohydrolase and methylases, is able tosynthesize mRNA carrying the 5'-terminal modification in vitro in the presence of a methyl-donor S-adenosylmethionine (SAM). The process of theformation of the modified structure at the 5'-terminus of this mRNA wasstudied and compared with the case of silkworm cytoplasmic polyhedrosis
virus (CP virus) (Annual Rep. No. 26 (1975) p. 14).
The limited RNA synthesis in vitro was carried out with the pretreatedreovirus particle in the presence of pH methyl]SAM by controlling theamount of substrates NTP and the reaction temperature. Oligonucleotides
thus produced were separated by DEAE cellulose-urea column chromatography in order of size. The rates of methylation at the 5'-terminal partin these oligonucleotides were analysed. The following conclusion wasderived from these experiments: at first the confronting nucleotide structure
G5'ppp5'G is constructed, then one of guanine residues is methylated at the
7 position, forming m7 GpppG, and methylation at the 2' position in riboseof the other G occurs at the same time to the starting of chain elongationof RNA. The latter 2'-methylation has been performed until elongationof one or two nucleotides in the present case of reovirus, whereas threeor four nucleotides elongation is required for the completion of methylationof CP virus.

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

In order to confirm the steps of methylation the confronting nucleotide
structure without methyl groups, G 51 ppp51 G and G5lppp5lA, were preparedby chemical synthesis and added to the reaction mixture of RNA synthesis.
GpppG can be a substrate for the methylase associated to reovirus, but
GpppA can not. This is not conflict with the 5' -terminal structure ofreovirus mRNA, m7GpppGm. The situation in CP virus is opposite: CPvirus methylates GpppA, but not GpppG. The methylation enzyme inreovirus as that in CP virus seems to recognize the substrate structure
strictly and to methylate after the blocked structure is constructed. In fact,
GTP in the reaction mixture was not methylated. The methyl group incorporated into GpppG by reovirus was found only in 7-position of one ofguanine bases, but not in 2 /-position of ribose. Here, again it is clear thatmethylation at the 7-position of the blocking quanosine residue is carriedout at first, and the ribose moiety in the G-residue is not methylated before
one or two nucleotides link to it.
Modified Structure at the 5 '-Terminus of Low Molecular Weight RNA
in Nucleus of Animal Cell
Kunitada SHIMOTOHNO, Toshiyuki URUSHIBARA and Kin-ichiro MIURA

In the research on the gene structure and its expression of the doublestranded RNA-containing virus, especially silkworm cytoplasmic polyhedrosis virus (CPV), a novel structure was discovered at the 5' -terminusof the messenger RNA strand. The internucleotide linkages (linkages
between pentose and phosphate in a backbone chain) in a nucleic acidmolecule are repetition of the 5' -3 ' linkage, but 7-methylguanylic acid combines to the 5' -terminal nucleotide of the mRNA strand in a confronting
state between 5' and 5' of the two nucleotides, inserting two pyrophosphate
linkages as follows: m7G5Ippp5/Am-G-U-. The similar structure has beenfound thereafter for many kinds of messenger RNA (mRNA) of cells aswell as viruses of eukaryote organisms. Excepting some instances, themodified structure of the 5' -terminus of mRNA seems to be common foreukaryotic organisms, and it is expressed generally as follows: m7G5/pppN(m)N(m)-N-. The presence of such a modified structure was surveyed forother cellular RNAs.
In order to detect small amount of a modified structure in a long RNA
molecule, an efficient labelling with radioisotopes is required. The culture

RESEARCHES CARRIED OUT IN 1976

17of the baby hamster kidney (BHK) cell in the logarithmic phase was addedwith [3H] methionine as a methyl donor and [32P] orthophosphate. It was
incubated further at 37°C for 12 hours. After cells were disrupted by homogenizing in a hypotonic buffer, nucleus and cytoplasm were separated.
RNA extracted by phenol treatment was purified by DNase and fractionatedin a glycerol density gradient.
The 5/-5/ confroning nucleotide structure was explored as following: At
first an RNA preparation was digested with ribonuclease TI, which hydrolyzes
RNA into 3/-nucleotides except the special places containing 2'-0-methyl
nucleoside and pyrophosphate linkage as the 5/-5' confronting structure.
The digest was analyzed by DEAE cellulose column chromatography using
7 M urea solvent. Two kinds of oligonucleotide containing modified struc~ture were detected for nuclear small RNA. These components weredesalted and then digested with Penicillium nuclease PI, which cleaves
internucleotide bonds to yield 5'-monophosphate regardless of 2'-0-methyl
ribose residue. The nuclease PI digest was further incubated with phosphomonoesterase to leave only the 5'-5/ confronting nucleotide structure as
it is. The confronting nucleotide component was then isolated by column
chromatography using anion exchange resin Bio Rad AG-l.
The confronting nucleotide structure was identified by cleaving it withtobacco phosphodiesterase, which splits pyrophosphate linkages but not thenormal 5/-3' internucleotide linkages. The resulting products were analyzed.
Based on these results, the 5'-terminal modified structure of the nuclear
low molecular weight RNA was concluded as m;,2,7G5'pppAm.
In the RNase T z digest of the RNA, there were two oligonucleotides
which were resistant to this enzyme. From both the oligonucleotides thesame confronting nucleotide structure was detected. As the second oligonucleotide is one nucleotide longer than the first one according to the
DEAE-urea chromatography, these are judged as m;,2,7G5/pppAm_Np andm~,2,7G5'pppAm-Nm-Np,respectively. Such a confronting nucleotide structure was detected only in the nuclear low molecular weight RNA fraction(4S-lOS) either in the normal BHK cell or the adeno-transformed BAT cell.
These RNA fraction was further separated by polyacrylamide-gel electrophoresis. The 5'-5' confronting nucleotide structure was found only in the
5.7S RNA as m~,2,7G5/ pppAm-N- ... and in the 5.9S RNA as m~,2,7G5/_
pppAm-Nm-N-' . '.
A part of this work was published in Proc. Japan Acad. 52, 563-566

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27(1976).
Research on the Enzyme Cleaving the 5'-Terminal
Methylated Blocked Structure of Messenger RNA
Kin-ichiro MIURA and Kunitada SHIMOTOHNO

A new phosphodiesterase was isolated from tobacco cell culture in
Sugimura's laboratory (National Cancer Center, Tokyo), where the formation and degradation of poly (ADP-Ribose) are studied (H. Shinshi, M.
Miwa, K. Kato, M. Noguchi, T. Matsushima & T. Sugimura (1976) Bio,chemistry 15, 2185-2190). The enzyme hydrolyses phosphodiester linkages
in NDP, NTP, inorganic pyrophosphate, cyclic nucleotides, NAD+ andpoly(ADP-Ribose), but it does not cleave backbone of DNA and RNA.
The tobacco phosphodiesterase seems to be specific for pyrophosphate
linkage except for cyclic phosphate bonding.
Our recent studies show that the tobacco phosphodiesterase can delete
5'-terminal blocked structure in a eukaryotic mRNA and low molecularweight RNA without splitting a main chain in an RNA molecule. For
instance, when [3H methyl] labeled mRNA of CPV (cytoplasmic polyhedrosis
virus) was incubated with tobacco phosphodiesterase, only [3H methyl]
pm7 G was released into an acid-soluble fraction as a labeled material. The
rest of the radioactivity in CPV mRNA was remained in an RNA chain in
an acid-insoluble fraction. Since the CPV mRNA carries the structure
m 7G5'ppp5'Am_G_···· at the 5'-terminus, tobacco phosphodiesterase splits,only pyrophosphate linkages in the terminal blocked structure. During
incubation with tobacco phosphodiesterase the chain length of RNA did
not change. Thus, tobacco phosphodiesterase can be used for detection or!identification of the blocked terminal structure containing pyrophosphate
linkages in a nucleic acid molecule.
This work was collaborated with Drs. Masanao Miwa, Hideaki Shinshi
and Takashi Sugimura, and a part of it was published in FEBS Lett. 65,
254-257 (1976).

RESEARCHES CARRIED OUT IN 1976

19

Enzymatic Removal of the 5'-Terminal Methylated
Blocked Structure of Tobacco Mosaic Virus RNA
and its Effects on Infectivity andReconstitution with Coat Protein
Kin-ichiro MIURA and Kunitada SHIMOTOHNO

The 5'-terminus of tobacco mosaic virus (TMV) RNA, long thought
to be an unphosphorylated A residue (Sugiyama, Y. and Fraenkel-Conrat,
H. (1963) Biochemistry 2, 332-334; Fraenkel-Conrat, H. and Fowlks, E.
(1972) Biochemistry 11, 1733-1736), was recently identified as m 7 G 5'ppp5'Gp(Zimmern, D. (1975) Nucleic Acids Res. 2,1189-1201; Keith, J. and FraenkelConrat, H. (1975) FEBS Lett. 57, 31-33). Elimination of the 3'-terminal
nucleoside of TMV RNA by a chemical procedure (periodate oxidation andsubsequent cleavage by aniline) was reported to cause marked loss of
infectivity (Steinschneider, A. and Fraenkel-Conrat, H. (1966) Biochemistry
5, 2735-2743). However, this chemical procedure probably eliminated boththe 5'- and 3'-ends of TMV RNA, so it is uncertain which elimination
reaction was responsible for loss of infectivity. On the other hand, theassembly reaction of TMV RNA with TMVprotein has been thought tostart at the 5' end and proceed to the 3' end along the RNA chain (Ohno,
T., Nozu, Y. and Okada, Y. (1971) Virology 44, 510-516; Butler, P. J. G.
and Klug, A. (1971) Nature New Biology 229, 47-50; Thouvenel, J. C.,
Guilley, H., Stussi, C. and Hirth, L. (1971) FEBS Lett. 16,204-206). Now
it is a problem whether the odd structure at the 5'-terminus is related to theassembly reaction.
These considerations prompted us to investigate the removal of the 5'terminal blocked structure of TMV RNA and its effects on infectivity andassembly reaction. Recently a novel phosphodiesterase was purified from
cultured tobacco cells (Shinshi, H., Miwa, M., Kato, K., Noguchi, M.,
Matsushima, T. and Sugimura, T. (1976) Biochemistry 15,2185-2190). As
shown in the preceding report, this enzyme releases pm7 G from the 5'terminal blocked structure of cytoplasmic polyhedrosis virus mRNA, butdoes not attack the polynucleotide chain of the mRNA. We used thisenzyme to delete the 5'-terminal modified structure from a TMV RNA
molecule.
The infectivity of TMV RNA which had been treated with tobacco phosphodiesterase was assayed on tobacco plants as free RNA or after recon-

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

stitution with TMV protein. TMV RNA lost its infectivity almost cOmpletely on removal of the 5'-terminal blocked structure. This is the first
demonstration that the blocking structure of virus RNA is essential for
infectivity.
In order to see the ability of reconstitution of TMV particle after
treatment of TMV RNA with tobacco phosphodiesterase, the enzymetreated RNA was incubated with TMV protein under the conditions for thereconstitution reaction, and analyzed by sucrose density gradirnt centrifugation. The results show that TMV RNA lacking the 5'-terminal blocked
structure assembled with coat protein as well as native TMV RNA. Considering with other observations on the reconstitution process, the present
result supports the idea that assembly does not start from the 5' end of
TMV RNA.
This work was collaborated with Drs. Takeshi Ohno, Yoshimi Okada,
University of Tokyo, and Hideaki Shinshi, Masanao Miwa and Takashi
Sugimura, National Cancer Institute. The contents were published in
FEBS Lett. 67, 209-213 (1976).
Role of the 5'-Terminal Modified Structure ofmRNA in Protein Syntbesis
Kunitada SHIMOTOHNO and Kin-ichiro MIURA

Since our discovery of the methylated blocked structure-the confronting
nucleotide structure-at the 5'-terminus of the mRNA strand of CPV,
m7 G 5l pppAm_ (Furuichi, Y. and Miura, K, Nature, 253: 375 (1975); Miura,
K, Furuichi, Y, Shimotohno, K, Urushibara, T. and Sugiura, M., Proc.
10th FEBS Meeting, 95-108 (1975)), the presence of similar modified structures generally in eukaryotic mRNA's has been suggested (Review by
Griffin, B., Nature, 255: 9 (1975)). Although there are some exceptional
cases, the confronting structure has been considered to function in protein
synthesis. Some experiments have already supported this possibility (Both,G. W., Banerjee, A. K and Shatkin, A. J., Proc. Nat. Acad. Sci. U. S. A.,
72: 1189 (1975)), though there are some conflicting data (Review by Griffin,B., Nature, 263: 188 (1976)).
Recently it was shown that a pyrophosphatase purified from tobacco
cell culture specifically splits 7-methylguanylic acid and phosphate at the
pyrophosphate linkages from the 5'-terminus of eukaryotic mRNA without

RESEARCHES CARRIED OUT IN 197621

any scission at other parts of the RNA molecule (Shinshi, H., Miwa, M.,
Sugimura, T., Shimotohno, K and Miura, K, FEBS Letters, 65: 254(1976); Ohno, T., Okada, Y, Shimotohno, K., Miura, K, Shinshi, H.,
Miwa, M. and Sugimura, T., FEBS Letters, 67: 209 (1976)). Using thisenzyme, we have explored the function of the confronting nucleotide structure at the 5'-terminus of mRNA.
The mRNA's of CPV, TMV (viral RNA) and globin were treated withtobacco phosphodiesterase, and their template activities for peptide synthesis were compared with that of untreated mRNA in the cell-freeprotein synthesizing system of wheat germ. In every case, enzyme-treatedmRNA in which the confronting nucleotide structure at the 5'-terminus had
been completely deleted lost the ability to incorporate [3H]leucine into the
acid-insoluble peptide fraction.
The requirement for 7-methylguanylic acid in the confronting nucleotide
structure at the 5'-terminus of mRNA was confirmed by the following experiments. When 7-methylguanylic acid (5 '-phosphate) was added to thereaction mixture, the protein synthesis capability of native mRNA wasinhibited markedly. The inhibitory effect of pm7 G was highly specificamong analogous derivatives of guanylic acid.
In every case, analysis of the product proteins by gel electrophoresis
showed that the synthesis of proteins was inhibited equally for every kindof protein.
The initiation complex for protein synthesis of CPV mRNA and TMV
RNA was not obtained on using tobacco phosphodiesterase-treated mRNA.
Formation of the initiation complex of intact mRNA was also inhibitedstrongly by addition of pm7 G.
These experiments show unambiguously that the 7-methylguanylic acidblocking structure at the 5'-terminus of eukaryotic mRNA is necessary forprotein synthesis, especially for the step of formation of the initiation complex. This is common for three different kinds of mRNA. When m7 G at
the 5'-terminus is removed by ,B-elimination after oxidation of the freeribose moiety, the 3 /-terminal nucleotide is also deleted. As the tobacco
phosphodiesterase can remove only 5'-terminal modification, the effect ofdeletion of this structure can be clearly tested by the use of this enzyme.
There is a possibility that the confronting nucleotide structure is necessary
to ensure the stability of mRNA, protecting it from exonucleolytic degradation. This was tested by incubating the CPV mRNA, with or without

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

pretreatment with tobacco phosphodiesterase, in wheat germ extract. Boththe S-30 fraction (soluble fraction after centrifugation at 30,000 g) andS-100 fraction (soluble fraction at 100,000 g) were used for this experiment.
The intact CPV mRNA was stable on incubation for 3 min, whereas thetobacco phosphodiesterase-treated mRNA was broken down within 1 min.
Similar results were obtained for S-30 and S-100 fractions, and on adding
pm7 G. These results clearly show that the confronting nucleotide structure
functions to stabilize the mRNA, preventing its degradation.
A Procedure for the Isolation of Temperature
Sensitive RNA Polymerase Mutants of E. coli
Masahiro SUGIURA and Koichi YOSHINAGA

DNA-dependent RNA polymerase of E. coli consists of four subunits:
lX, /3, /3' and a.
As an approach toward understanding the precise role ofeach subunit in RNA polymerase during transcription, we undertook isolation of temperature sensitive (ts) mutants with altered RNA polymerases
which expressed their temperature sensitivity in the usual in vitro reaction
systems.
Ts lethal mutants derived from E. coli K12 strain PA3092 after mutagenesis with nitrosoguanidine were obtained from Dr. Hirota. All cells
were grown in L-broth supplemented with 50 ,ugjm1 thymine.
The rates of uridine incorporation at 30°C and 43°C were measured for
all the ts mutants. Cells were grown at 30°C in 2 ml medium for 2-5 hr
until an exponential phase was reached. 0.5 ml of the culture was removed
and incubated for 2 min at 30°C with [3H]uridine (0.2,uCi, 41.5 Cijmmol).
Rest of the culture was immediately transferred to 43°C. After incubation
at 43°C for 16 min, 0.5 m1 of the culture was removed and incubated for
2 min at 43°C with [3H]uridine as above. Pulse-labeled cells were precipitated with 2 m1 cold 10% TCA containing 1 mM uridine for over 30 min
and the precipitates were collected on Whatman GFjF filters and theradioactivities were counted. The ratio of the rate of uridine incorporation at 43°C to that at 30° was obtained for each ts mutant. The parent
strain had the ratio of 1-2. 280 st~ains which were low in these ratios (lessthan 0.5) were selected from 1200 ts mutants.
Toluene-treated cells of these strains were prepared according to Peterson
et al. (1971, J. Bact. 107: 585) with modifications. Cells were grown inRESEARCHES CARRIED OUT IN 1976

23

5 ml medium to a density of 5 x lOB cells/ml. The cells were chilled quickly.
havested by centrifugation and suspended in 0.5 ml of ice-cold 0.05 M Tris
(pH 7.4) -0.1 M KCl. The cold suspension was made 1% (v/v) in toluene
and shaken 5 times by a vortex for 5 sec every 3 min. The suspension was
kept at 0° during 3 min-intervals. After centrifugation, the cell pellet andthe wall of tube were rinsed with the cold above buffer and resuspended in
0.2 ml of the same buffer. For RNA synthesis assay, reaction mixtures.
contained (0.2 ml) 0.04 MTris (pH 7.9 at 25°C), 8 mM MgC1 2, 0.1 mM DTT.
0.1 mM EDTA, 0.1 M KCl, 2 mM spermidine, 0.8 mM K 2HP0 4 , 0.4 mM
each of ATP, CTP, GTP and [3H]UTP (10,uCi/,umol) and 20,ul toluenetreated cells. Incubations were for 10 min at 30°C and 43°C and acidinsoluble radioactivities were counted. Spermidine stimulated the incorporation of UTP. The ratio of UTP incorporation at 43°C to that at 30°
was obtained for each selected mutant. The parent strain had the radio of
about 0.6. 50 strains low in these ratios (less than 0.4) were selected.
Crude RNA polymerase preparations from these 50 mutants were obtainedby the simplified procedure as follows. Cells were grown in 300 ml medium
at 30°C till late log-phase and harvested by centrifugation. 0.5 g of frozen
cells were disrupted by sonication in 2 ml of 0.05 M Tris-HCl (pH 7.9).
0.01 M MgC1 2, 0.1 M KCl, 0.1 mM DTT, 0.1 mM EDTA, 10% glycerol and20,ug/ml DNase I, and centrifuged at 100,000 g for 1 hI'. To the supernatant added an equal volume of saturated (NH4)2S04 solution. The resultant precipitate was collected by centrifugation and washed with 8 mlof 45% saturated (NH4)2S04 solution made in 0.01 M Tris-HCl (pH 7.9).
0.1 mMDTT, 0.1 mM EDTA and 10% glycerol (buffer D). The washedprecipitate was dissolved in buffer D so that its conductivity was less thanthat of buffer D+O.l M KCl (9 mQ at 0°) and applied onto a 1 ml DEAEcellulose column (Whatman DE-52) equilibrated with buffer D+O.l MKCl.
The column was washed with 8 ml buffer D+O.l MKCl followed by 1 ml ofbuffer D+O.3 M KCl. The RNA polymerase was eluted with the next I ml
of buffer D+0.3 M KCl. Reaction mixtures for RNA polymerase assay
contained (0.2 ml) 0.04 M Tris-HCl (pH 7.9 at 25°C), 8 mM MgCI 2, 0.1 mM
DTT, 0.1 mM EDTA, 0.2 mM each of ATP, CTP, GTP and [3H]UTP(10 ,uCi/,umol), 0.2 M KCl, 0.4 mM K 2HP0 4, 100 ,ug BSA, 5 ,ug TrDNA and
30,u1 enzyme preparation (approximately I OD 2Bo /ml). Incubations werefor 10 min at 30°C and 43°C, and acid-insoluble radioactivities werecounted. The ratio of RNA polymerase activity at 43°C to that at 30°

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

was obtained for each preparation and was about 0.5 for the parent enzyme.
We obtained 4 RNA polymerase mutants from 1200 original ts strains. Oneof them, called JElO092, was identified as a ts (3' subunit mutant (Sugiura
et al. 1977 BBRC 76: 739, YOSHINAGA & SUGIURA 1977 BBA 479: 172).

RESEARCHES CARRIED OUT IN 1976

II.

25

MICROBIAL GENETICS

Mutants of Escherichia coli Altered in
Penicillin Binding Proteins
Yukinori HIROTA, Yukinobu NISHIMURA and Hideho SUZUKI

Five thousand mutants of Escherichia coli thermosensitive in growth wereisolated from independent origin and stocked as the source of mutations
with which the mechanisms of cell division were investigated. Five hundredof these mutants were examined with respect to the defect in penicillin
binding proteins (PBP), which were numbered as PBP-l, PBP-2, etc.,
according to Spratt and Pardee (1975 Nature 254: 516). In the course ofthis screening for PBP mutations, we found a number of mutants defective
in PBP-l and these mutants fell into two categories. This led to the conclusion that the PBP-l contained two different gene products designated as
PBP-la and PBP-lb. Mutations leading to the defects in these PBPs werereferred to as ponA and ponB (penicillin binding protein one A or B), respectively. Consequently, we found eleven mutants defective in PBP-la,four in PBP-lb, three in PBP-2, one in PBP-3 and three in PBP-4. In
addition, we found a mutant defective in activity of D-alanine carboxypeptidase Ia (Matsuhashi et al. in preparation). This enzyme is an identicalentity with PBP-5/6 (Spratt and Strominger, 1976 J. Bacteriol. 127: 660).
By using these mutants, the map position of the mutations for each PBP
was determined: ponA (PBP-la) 73.5 min, ponB (PBP-lb) 3.3 min, rodA
(PBP-2) 14.4 min, fts! (PBP-3) 1.8 min, dacB (PBP-4) 68 min and dacA
(PBP-5/6) 13.7 min.
One of the mutants for PBP-la carried thermosensitive defect in penicillin binding ability of la (la-ts); i.e., the PBP-la did not bind penicillin at
42°, but did at 30°. All of the ponA mutations examined, including thisthermosensitive defect, were separated from the defect in thermosensitive
growth of the mutants and cotransduced with aroB at a frequency of 8090%.
Among four mutants of PBP-l b, three showed no penicillin binding bandfor PBP~lb and one showed faster migration of the electrophoretic bands
of PBP-lb. The PBP-lb gave three electrophoretic bands in the wild typeand in this mutant. All of the mutation in PBP-lb (ponB) were separated

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

from thermosensitive mutation in growth and cotransduced with tonA at afrequency of about 82 %. The mutants which lost the PBP-l b were hypersensitive to j3-lactams. The hypersensitivity was strictly linked with theloss of the band of PBP-l b.
The trials to construct the double mutant defective in both PBP-la and
PBP-l b were unsuccessful. This result suggested the leathality in the ponA
pon B double mutant. The mutation ponAts (la-ts) was introduced into theponB mutant by a cross. Although both of the parental strains were
thermoresistant, 19 % of the recombinants became thermosensitive and lysedat the restrictive temperature. The thermosensitive loss of the binding
ability of PBP-la parallelled with the thermosensitive growth in the double
mutant. Therefore, it is concluded that the PBP-la and PBP-lb may share
a common part of the reaction required for cell elongation so that the lossof the function of PBP-lb may be bypassed by PBP-la.
The mutations in PBP-2 and PBP-3 were linked with thermosensitivity
in colony formation. The mutants defective in PBP-2 formed ovoid cells
and those in PBP-3 showed filamentation at the restrictive temperature.
These results agree with the previous works of Spratt (1975 Proc. N. A. S.
U.S. 72: 2999).
The PBP-4 and PBP-5/6 were identified as D-alanine carboxypeptidase
Ib and la, respectively (Matsuhashi et al. 1977 Proc. N.A.S. U.S. 74: 2976,
Matsuhashi et al. in preparation). The mutations, dacB (PBP-4) anddacA (PBP-5/6) were cotransducible with argG and leuS, respectively, at afrequency of about 87 %. The double mutant lacking both enzyme activities grew normally at 40° as well as at 30°, indicating that these enzymes
were dispensable for cell growth and division.
The extensive studies on the isolation and characterization of the PBP
mutants allowed us to determine the chromosomal location of all the genes
for the PBPs. This opens the way to construct multiple mutants defective invarious PBPs and to dissect the roles of the PBPs in cell growth and division.
Synthetic ColEl Plasmids Carrying Genes of
Cell Division in Escherichia coli
Yukinobu NISHIMURA, Yutaka TAKEDA, Akiko NISHIMURA,
Hideho SUZUKI and Yukinori HIROTA

To clarify the functions of the genes involved in the processes of cell
division, the elucidation of the gene products and their properties must be

RESEARCHES CARRIED OUT IN 197627

of crucial importance. The amplification of the gene involved in cell division may facilitate the identification and biochemical characterization of thegene product. Clarke and Carbon prepared a collection (1976, Cell 9: 91)
of Escherichia coli strains which harbored ColE1 plasmids carrying small
random segments of the E. coli chromosome. This collection was utilized
to search the plasmid carrying the genes for the process of cell division andfor the murein-lipoprotein.
The 2000 strains in the collection were screened for the correction of thefollowing genetic defects: par (partition of daughter nuclei),fts (cell division),rod (determination of a cell shape) and lpo (synthesis of the mureinlipoprotein). We found plasmids carrying E. coli chromosomal segments
containingftsB+ , ftsE+ ,ftsI+ , ftsM+ and parA +. However, none was foundto transfer ftsA+ ftsC+, ftsF+, ftsG+, ftsJ+, ftsK+, ftsL+, parB+, rod+and lpo+. Among the fts mutations, ftsI was identified as a mutation
affecting penicillin-binding protein (PBP) 3 (according to the nomenclatureby Spratt 1975, Proc. N. A. S. U.S. 72: 2999). The PBP was examined in
the strains carrying the plasmid which corrected the ftsI defect. One of
the strains carrying pLC26-6 was found to overproduce the PBP-3 by about
10-fold.
Meanwhile, it was found that the gene for PBP-la (ponA) was cotransducible with aroB and the defect in PBP-lb (ponB) was strictly correlated
to the bacterial phenotype of hypersensitivity to cephaloridine (Suzuki et al.,in this Ann. Rep. p 27). These findings led us to search the synthetic plasmids
carrying the genes for PBP-la and for PBP-lb by examining the correctionof aroB and of hypersensitivity to cephaloridine. One strain carrying theplasmid pLC29-47 which corrected the aroB mutation was found to overproduce the PBP-la and four strains carrying the plasmid; pLC4-43,
pLC4-44, pLC19-19 and pLC26-43, which corrected hypersensitivity tocephaloridine, overproduced the PBP-l b.
Penicillin Binding Proteins Inovlved in Cell Elongation
Hideho SUZUKI, Yukinobu NISHIMURA and Yukinori HIROTA

At least six penicillin binding proteins (PBP) exist in Escherichia coli andthey are numbered as PBP-l, PBP-2, etc., in the order of their molecularweights (Spratt and Pardee, 1975 Nature 254: 516). Among them, PBP-l,-2 and -3 were inferred to be involved in cell elongation, cell shape determina-

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

tion and cell division, respectively (Spratt, 1975 Proc. N.A.S. U.S. 72: 2999).
We found that the PBP-l contained two components designated as laand 1b and isolated mutants defective in each component. The defective
mutations of la and Ib were mapped at separate loci on the chromosome.
The defect either in la or in 1b, per se, did not render the cell thermosensitivein growth. All the mutants defective in PBP-l b became hypersensitive to
j3-lactam antibiotics. The minimal inhibitory concentration of cephaloridine
and cephalothin was in the range of 0.1-0.2 ,ugjml for the PBP-l b mutant,while it was 2-4 ,ugjml for the wild type (Table 1). The affinities of PBP-laand -1 b for these antibiotics were determined by competition experiments.
Table 1. The binding of cephalosporins to the penicillin binding proteins
Concn. required to reduce[14C]penicillin-bindingby 50% (flgjml)*

Cephaloridine
Cephalothin

PBC-la

-lb

-2

-3

0.11
0.04

2.7
20

45
30

3.4
1.0

Minimal inhibitory conCR.
(flgjml)

Mutant Isogenic Mutant Isogenic
laIbwild typewild type
2

2
4

5

..

0.1
0.2

2
3

-"--"--* Determined by competition experiments.
As shown in Table 1, PBP-la showed a conspicuously high affinity forthese antibiotics. Cephalothin induced filamentation of wild type cells at
5-10 ,ugjml. At this concentration range, cephalothin binds to PBP-l a
and -3 but does not to PBP-lb (Table 1). On the other hand, cephalothin
was effective at a concentration far below that required to bind with PBP-I b,
in the mutant defective in PBP-l b. From these observations, it was inferredthat the biochemical reactions for cell elongation can proceed normally in thepresence of either PBP-la or -lb and cell elongation in the PBP-lb mutant
may be sustained by PBP-la which is very sensitive to the j3-lactams, andtherefore, cell elongation becomes hypersensitive to these antibiotics. These
interpretations were compatible with the genetic analyses of the lajlb
mutant.
The double mutant carrying the thermosensitive defect in PBP-la (la-ts)
and no binding ability of PBP-l b showed thermosensitive growth, lysing at
the restrictive temperature. The parental strain carrying either the la-ts or
the 1b mutation was thermoresistant.
Murein-synthetic activity of the mutant defective in PBP-la or -lb was

RESEARCHES CARRIED OUT IN 1976

29

measured in vitro by incorporation of radioactive N-acetylmuramyl pentapeptide. The activity for murein synthesis in vitro was decreased to about
1/10 in the mutant defective in PBP-lb, although the lipid-linked precursor
was formed in a considerable amount. Thus, PBP-lb is involved in thereaction at the final stage of murein synthesis for cell elongation. The cellsof the mutant defective in PBP-l b were able to propagate normally. Presumably PBP-la took over the reaction required for cell elongation, as a
substitute for PBP-l b.
Differential Solubilization of Membrane Proteinsof Escherichia coli with Detergents
Masao YAMADA, Hideho SUZUKI and Yukinori HIROTA

Recently O'Farrell devised a high resolving technique for separation ofproteins by combination of isoelectric focusing and SDS-gel electrophoresis(two dimensional electrophoresis) (O'Farrell, P. H. 1975, J. BioI. Chem.
250: 4007). He applied it to resolving of the whole cell extract of E. coli,
and about 1,100 protein spots were displayed on a 12 X 16 cm plate. This
technique is a powerful tool for studying the correlation of genes to geneproducts and the regulation of protein synthesis at a cellular level. We
adopted the procedure to study membrane proteins.
Envelope fraction of E. coli was obtained by differential centrifugation
after disruption of the cells by a sonic disintegrator, solubilized with 0.5-2 %various detergents at 30-37°C, and then analyzed by the two dimensional
electrophoresis. The number of spots of proteins resolved on the electrophoretogram depended on the detergent used, and increased in the following
order: sodium deoxycholate, Triton X-IOO, Brij 35, Brij 58, sodium dodecyl
sulfate (SDS), sodium N-lauroyl sarcosinate (Sarkosyl). Major proteinsin the outer membrane were not extracted with any detergent without heat
treatment. Sarkosyl and SDS almost completely solubilized only the inner
membrane while Triton X-IOO solubilized it partially. These results are notcompatible with previous observation by Filip et at. (1973, J. Bacteriol. 115:717) in which Sarkosyl solubilized the inner membrane selectively but Triton
X-IOO and SDS solubilized both the inner and outer membranes.
Some proteins altered their isoelectric point by heating in the presenceof detergent resulting in appearance of multiple spots along the isoelectric
focusing dimension. So, a method to obtain the highest resolution for the30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

separation of membrane proteins is as follows: First, envelope was solubilized with 2 % Sarkosyl and then Sarkosyl-insoluble fraction was solubilizedwith 2 % SDS at 100°C for 2 min. Each fraction was analyzed separately
by essentially the same procedure reported by O'Farrell. Using this technique, at least 250 and 50 proteins were found to exist in the inner and outermembrane of E. coli, respectively.
Isolation of Specialized Transducing Bacteriophage A Carrying leuAftsI-murEFC-ddl-ftsA Region of Escherichia coli Chromosome
Masao YAMADA, Seiichi YASUDA and Yukinori HIROTA

Thermosensitive mutants of cell division of E. coli K-12 were isolated in
our laboratory whose penicillin binding component 3 could not bind [14C]benzylpenicillin. The mutation, designated as fts!, was mapped at about
1.8 min of E. coli chromosome (Nishimura, Y. et al. 1977, Plasmid 1: 67).
Around this locus, many other genes concerning cell wall synthesis andseptum formation have been mapped. They include ftsA, temperaturesensitive filament formation (Richard, M., and Y. Hirota 1973, J. Bacteriol.
116: 314), murCEF and ddlwhich are involved in the formation of nucleotide
sugar intermediate of peptidoglycan synthesis (Wijsman, H. J. W. 1972,
Genet. Res. 20: 65), and mra, a gene cluster coding the enzymes of peptidoglycan synthesis (Miyakawa, T. et al. 1972, J. Bacterial. 112: 950).
In order to determine the precise map in this region and to study on theregulatory mechanism of these genes, we have isolated phage A carrying thisregion.
Strain JE5631 (Its!) was transformed to temperature-resistant with UVlysate of strain JE6600 (leuA, ..d (pro-lac), ..d (gal-uvrB), thi) with ApleuA i 21
nin 5 integrated at a secondary attachment site in leu operon. Among
about 200 temperature-resistant colonies isolated, 22 colonies gave highfrequency transducing phages to fts! mutant upon UV-induction. Lambda
phages from the primary heterogenotes were classified into 5 groups according to the transducing ability to other loci. One phage transduced leuA andfts! (class I), 3 phages transduced leuA, fts! and murE (class II), 16 phages
transduced leuA, fts!, murE and murF (class III), 1 phage transduced leuA,
fts!, murE, murF and murC (class IV), and 1 phage transduced leuA, fts!,
murE, murF, murC, ddl andftsA (class V). No phage that transduced othercombination of these genes was obtained. One of the transducing phages

RESEARCHES CARRIED OUT IN 197631

belonging to class III was a plaque-former and others were defectives. From
the results, it is concluded that the order of genes on the E. coli chromosome
is leu-ftsI-murE-murF-murC-(ddl-ftsA).

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

III.

BIOCHEMICAL GENETICS AND IMMUNOGENETICS
A Comparison of Heat Sensitivity of Serum Esterase(Es-1)
in Different Inbred Mice, Mus musculus
Tadashi AOTSUKA1 and Kazuo MORIWAKI

Recent studies of Drosophila by Bernstein et al. (Proc. Nat. Acad. Sci. 70:
3928, 1973) and Singh et al. (Proc. Nat. Acad. Sci. 71: 1080, 1974. Genetics
80: 673, 1975) have given the experimental verifications of hidden genetic
variability in electrophoretic isozyme classes by a heat inactivation technique.
Such new physicochemical variabilities of isozymes would supply more informations about the genetic divergence occured in the process of species
formation. The present paper presents the results of heat inactivation experiments of serum esterase (Es-l) in laboratory inbred strains of house
mouse (Mus musculus). Two alleles (Es-la, Es-lb) which produce isozymes
having different electrophoretic mobilities were detected at the Es-l locus
from the inbred strains (Popp and Popp, J. Hered. 53: Ill, 1962. Petras andBiddle, Can. J. Genet. Cytol. 9: 704, 1967).
Total of 21 inbred strains were analysed in the present investigation(Table 1). Blood was obtained by inserting a heparinized capillary tube
into the suborbital canthal sinus. After centrifugation, the serum was pretreated at various temperatures (0-70°C) for 7 minutes before subjected toelectrophoresis or the colorimetric assay of esterase activity.
Table 1 summarizes the results. The faster migrating isozyme (Es-la)Table 1.

Types of heat sensitivity of Es-1 in 21 inbred mice, Mus musculus.
Alleles fixed in each strain are also represented
Inbred strains

C57BL/10Sn, C57BL/6J, B10. BR, B10. D2
NZB/Es-1a, HTI
C3H/HeJ, BALB/c, DDY, DDN, 129/J
AKR/J, SM/J, CBA/CaHN.T-6, DBA/2J
A/J, SWR/J, SJL/], HTG, PL, NZB/Es-1b

AlleleHeat sensitivity*aStableb

Sensitive* The determination of the types of heat sensitivity was based on residual activity at
58°C.
1)

Tokyo Metropolitan University,

RESEARCHES CARRIED OUT IN 1976

33

was more heat-stable than the slower migrating isozyme (Es-lb). At 58°C,
the Es-1a retained 60% of its total activity, whereas the Es-1b lost morethan 80% of the total activity. T1 / 2 (Temperature at which one-half of thetotal activity is lost) of the sensitive Es-l b was lower than that of the stable
Es-1a by about 2.5°C. Further investigations are now in progress to answer
the question whether the difference in the heat sensitivities between two
types of Es-1 isozymes is due to the genetic alteration only on the Es-1
locUS.
Distribution of Hhh (Hemoglobin beta-chain) Alleles inJapanese Wild Mice, Mus musculus molossinus
Mitsuru MINEZAWA1 ), Kazuo MORIWAKI and Kyoji KONDal)

In wild mouse populations of Western hemisphare and also in laboratory
strains, Hbb d and Hbb s alleles are common and Hbb P allele is rare. Onlyan AUjSs strain carries the allele Hbb P (Petras 1969, Can. J. Genet. CytoI.
11: 497~513; Staats 1976, Cancer Res. 36: 4333-4377). In contrast, Hbb p
and Hbb d alleles were observed commonly in Asian wild mice and several
inbred strains originated in Japanese mice (Mus musculus molossinus, petmice) possess Hbb p allele (Gilman 1974, Ann. N. Y. Acad. Sci. 241: 416433; Watanabe et al. 1976, Japan J. Genet. 51: 377-383).
But, the three hemoglobin beta-chain alleles, Hbb P , Hbb d and Hbb s , wereobserved in several populations of Japanese wild mice, Mus musculus molossinus (Minezawa et al. 1976, this report 26: 23-25).
Therefore, 259 mice were collected from 13 geographical regions in Japan
with the object of doing a survey on the alleles. Hemolytic samples,
untreated and treated with iodoacetic acid, were separated by starch gelelectrophoresis. The numbers of examined mice and allelic frequencies atthe Hbb locus in each region are demonstrated in Table 1. In Japan, on
the whole, the Hbb P and Hbb d alleles were common and the Hbb s allele wasrare. The frequencies of Hbb P and Hbb d in toto were 62.3 and 36.9%,
respectively, and interregional differences were also observed. The Hbb pallele predominated or fixed in Shikoku and Mainland facing the Pacific
Ocean, namly, San-yo, Kinki, Tokai and Kanto. The Hbb d allele predominated in Hokkaido, Okinawa and Oosumi IsIs. The frequencies of the1)

Department of Animal Breeding, Faculty of Agriculture, Nagoya University,
Chikusaku, Nagoya.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27
Table 1.

Localities
collected

Allelic frequency at hemolobin beta-chain (Hbb)
locus in Japanese wild mice population
Allelic frequencies of Hbb(%)

No. ofanimals
examinedp

Okinawa
Amami Isis.
Oosumi Isis.
KyushuShikoku
San-inSan-yoKinki
Tokai
Hokuriku
Kanto
TohokuHokkaido

4
9
7
42
12
23
15
15
44
33
13
23
19

61.1
21.4
38.1
100.0
56.5
93.3
86.7
97.7
43.9
100.0
63.0
7.9

Total of all
localities

259

62.3

d

s

100.0
38.9
78.6
53.3

3.6

41. 3
6.7
13.3
2.3
56.1

2.2

37.0
92.1
36.9

0.8

two alleles in the other regions, Kyushu, San-in, Hokuriku and Tohoku,
were intermediate between the two region groupes mentioned above. The
Hbb s allele was found in 4 mice in a heterozygous state. The frequency
was only 0.8 % in toto.
From the results, it could be concluded that the Japanese wild mice,
Mus musculus molossinus, was constructed originally of Hbb p and Hbb dalleles and that Hbb s was not an original allele, but intruded into the ancestral
Japanese population of Hbb P and Hbb d• In both Western and Asian wild
mice, Hbb d is a basic allele, whereas Hbb P allele is a characteristic marker
for Asian wild mice. The origin of Hbb s allele remains unexplained but
it is worth noting that the allele is common in Japanese fancy mice andlaboratory strains (Mizuno et al. 1977, Exp. Anim. 26: 43-49).

Suppression of Erythrocytic H-2 Expression in F1 Hybrid between
Two Mouse Subspecies, Mus musculus domesticus and molossinus
Kazuo MORIWAKI and Toshihiko SHIROISHI

In general mouse histocompatibility genes are considered to be expressed

35

RESEARCHES CARRIED OUT IN 1976

in the codominant fashion on most adult tissues. However,in the F l hybrid
between two kinds of mouse subspecies, the European feral mouse Mus
musculus domesticus from which most of the present laboratory mouseprobably originated and the Japanese feral mouse Mus musculus molossinus,
expression of H-2 antigen on erythrocytes is shown to be markedly suppressed in the present study.
This phenomenon was first observed in a hemagglutination test for H-2.5
antigenic specificity of the F l hybrid between the BlO.A laboratory strain and
the Mol Sg line derived from M.m. molossinus. Maximal antiserum
dilution to give a definitely positive hemagglutination was 256 in BlO.A,<8 in BlO.D2, <8 in Mol Sg, 256 in (BlO.AxBlO.D2)Fl and <8 in (BlO.A
X Mol Sg)Fl . Thus, expression of H-2.5 specificity on erythrocytes isobviously suppressed in (BlO.AxMol Sg)Fl, whereas never in (BlO.Ax
BlO.D2)Fl .
These results have been further confirmed by the quantitative absorptionof anti H-2.5 and anti H-2.23 sera with erythrocytes to be examined. Afterabsorption, the residual antibody activity in the serum which is inversely
proportional to the degree of H-2 expression, was assayed by the cytotoxicity
method using 5ler-Iabelled lymphocytes. Table I demonstrates the apparently reduced expression of H-2.5 (public) and H-2.23 (private) specificities on the erythrocytes of (BlO.A X Mol Sg)Fl hybrid quantitatively.
No suppressed expression of H-2 antigens was observed in spleen cells of
this intersubspecies hybrid. Mechanism of this type of suppression remains
open.
Table 1. Suppression of H-2 antigen expression in F l hybrid
between M. m. domesticus and M.m. molossinus
Relative absorbing capacity (%)
Cells
examined

H-2 antigenic
specificities
testedMouse strains and their hybrids employed
BIO.A
BIO.A
Mol-SgIx
x
BlO.A
BIO.D2
BIO.D2
Mol-SgI

RBC

K5
K23

0
8.6

64.4
62.1

0
14.6

20.6
33.5

54.3
56.1

Spleen
cells

K5
K23

85.7
43.0

94.9
95.6

2.3
30.0

77.3
74.9

80.6
78.7

Mol SgI: Mus musculus molossinus line
BI0.A, BI0.D2: BIO congenic strains probably developed from Mus musculus domesticus.

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

The Effect of K-region in H-2 Complex on Expression of H-2.5 Antigen
on Erythrocytes
Toshihiko SHIROISHI and Kazuo MORIWAKI

Some H-2 antigens are detectable by cytotoxicity much more readily
than by hemagglutination (Demant et al. 1971). It is also known that the
C57BLjlOJ erythrocytes show a poor expression of H-2.5 antigen, althoughspleen cells and other cell types express it strongly. However it remains
unanswered whether the presence or absence of the H-2 antigens on erythrocytes is controled by the H-2 complex itself, or by the other genotype.
We made an attempt to answer this question and to identify the fine region
controlling the expression of H-2 antigens on erythrocytes by using various
B10 congenic strains.
Following strains were used: C57BLjlOJ (H-2 b ) (abbreviated symbol
BIO), BlO.A (H-2 a ), BIO.A(5R) (H_2 i5), BlO.A(2R) (H_2h2 ), BlO.BR (H-21'),and BlO.D2 (H-2d ).
Alloantiserum, D-5bAF, supplied from NIH, which mainly directed H2.5, was employed. Erythrocytes and spleen cells were examined for H-2.5
by the quantitative absorption and subsequent cytotoxicity test.
The results are summarized in Table 1.
Table 1. Quantitative absorption test with erythrocytes and spleencell of various BI0 congenic strains
BIO
congenicstrainsH-2
haplotype

BI0.BR
BI0.A
BI0.A(2R)
BI0
BI0.(5R)
BI0.D2

k
a
h2
b
i5
d

H-2 complex
Absorption capacity (%)
Parentalfor H-2.5
H-2
haplotype K IAIB IC S GD Erythrocyte Spleen cell
kid
alb
b/a

k
k
k
b
b
d

k
k
k
b
b
d

k
k
k
b
b
d

k
d
d
b
b
d

k
d
d
b
d
d

k
d
?
b
d
d

k
d
d
b
d
d

88.6
90.3
62.8
20.9
9.0
0.0

64.3
76.0
81.1
79.5
82.1
6.5

These data indicates the followings:

1) The degree of an expression of the H-2.5 antigen on erythrocytes isdifferent among these various strains, but that on spleen cells is not.
2) The strains having a k-haplotype in H-2K, lA, and IB regions,

RESEARCHES CARRIED OUT IN 1976

37

showed a strong expression of the H-2.5 antigen on erythrocytes, while thosewith a b-haplotype in these regions expressed it poorly.
All these data suggest that the degree of erythrocytic expression of theH-2.5 antigen is controled by an allele within the H-2 complex, probably in
K and neiboring regions.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS
In Vitro Analysis of Tissue-Specific Defects in
rudimentary Embryonic Cells of
Drosophila melanogasterYukiaki KURODA

In the series of experiments on tissue-specific defects of cells from developmentally lethal embryos in Drosophila melanogaster, some defects in differentiation of muscle cells, imaginal disc cells and nerve cells have beenfound in cultured deep orange embryonic cells (Kuroda, Nature 252, 40,
1974; Develop. Growth Different. 19, 57, 1977). A similar defect in nerve
cells has been found in cultured fused embryonic cells.
In the present work, tissue-specific defects of rudimentary39lc (r 39lc) embryoniccells were investigated in culture. The r 39lc gene is located at 54.5 on the X
chromosome. When the lethal effective phase was examined on 926 embryos
collected from matings of r39lc/r39lc females and r 39lc /Ymales, 4.0 %died before
gastrulation, 9.3 %died by abnormal gastrulation, 13.2 %died after head andtrunk segmentation, 14.8 % died after sac-like midgut formation, 40.7 %died after entrance of air into trachea, 10.3 % died after the stage of active
movement and 5.7 % died immediately after hatching.
When cells from r 39lc embryos which developed beyond gastrulation were
cultured, the pulsation and syncytium formation of muscle cells, the proliferation of fibroblastic cells, the sphere formation of prospective imaginal
disc cells, the extension and branching of nerve fibers of nerve cells and thedroplet secretion on nerve fibers were normally observed as the samemanner as those in wild-type embryonic cells.
On the other hand, epithelial cells grew and increased in size, but thebrown chitinous pigmentation which was usually observed in wild-type
embryonic epithelial cells was not observed in r 39lc epithelial cells.
Muscle and Cartilage Differentiation in Cultured
Mesenchymal Cells from Embryonic
Quail Limb-Buds
Yukiaki KURODA and Etsuya MATSUTANI

Mesenchymal cells in avian embryonic limb-buds differentiate into muscle

RESEARCHES CARRIED OUT IN 1976

39

or cartilage in normal development. They are used for analyzing themechanisms by which a common precursor cell is determined to differentiate
into two phenotypically different types of cells. In the present study various
culture conditions under which mesenchymal cells differentiated into muscle
or cartilage were examined.
Limb-bud mesenchymal cells from 4 day (Stage 20-22) quail embryos
were dissociated. into single· cells by treatment with EDTA and trypsin at
37°C after removal of the epidermis by cold incubation in trypsin solution.
The cells were cultured in 35-mm petri dishes in various media supplemented
with 15% fetal calf serum.
The growth of cells was largely dependent on the number of cells
inoculated initially. When 4 X 105 cells in petri dishes were cultured for
6 days in Medium No. 199 with serum, the number of cells increased as
many as 4.4 times. When the number of inoculated cells decreased by half,
no cell growth was found in the same incubation period. When the numberof inoculated cells decreased to one-fourth, the number of cells decreased to
one-tenth during incubation period of 4 days. The growth rates of cells
were best in Medium No. 199 with serum, and then in Eagle's MEM withserum. No cell growth was found in Ham's Medium F12 with serum.
The differentiation of muscle cells and the formation of cartilage nodules
were markedly accelerated by covering the cell layer in petri dishes with acellophane membrane. With a cellophane membrane the differentiation ofmuscle cells was well progressed in Medium No. 199 with serum or Eagle's
MEM with serum. The formation of cartilage nodules was accelerated in
Ham's Medium F12 and Medium No. 199, each supplemented with serum.
Induction of 8-Azaguanine-Resistant Mutations by
Sulfite in Cultured Embryonic Human
Diploid Cells
Yukiaki KURODA

Bisulfite is ,used' as a preservative in wines, juices, foods and pharmaceutical products. It intereacts with nucleic acid components and produces
gene mutations in bacteriophage lambda, T4, Escherichia coli and yeast.
In the present experiment the mutagenic effect of bisulfite on 8-azaguanine(8AG)-resistant mutations in cultured embryonic human diploid cells was
examined.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

Bisulfite reduced the colony-forming activity of cells to 65 % and 0 %of that of untreated cells at concentrations of 10- 2 M and 10- 1 M, respectively. The Do value calculated from a concentration-survival curve forbisulfite was 6.5 X 10- 2 M.
Triplicate inocula of 105 cells were treated with bisulfite at various concentrations for 2 hours, cultured in normal medium for a mutation expression
time of 48 hours, and then selected with 30 pg/ml of 8AG. The frequency
of 8AG-resistant mutations increased as an increase in the concentrations ofbisulfite employed for treatment. At a concentration of 10- 2 M of bisulfite
the induced mutation frequency increased three times that in untreated control cultures.
The effect of mutation expression times on the frequency of induced
mutations was examined. Among mutation expression times of 0, 24, 48,
72, 96 and 120 hours, a mutation expression time of 72 hours was mosteffective in inducing mutations by bisulfite. It was found that the number. of cells in population increased 1.4 times during this mutation expression
time.
Dose-Rate Effects of Ethyl Methanesulfonate on
Survival and Mutation Induction in Cultured
Chinese Hamster Cells
Yukiaki KURODA and Katsura SUGIURA

In the field of radiation biology, the dose-rate effects on cell survivals andthe induction of mutations have been extensively studied. On the other
hand, a few reports have been made on the dose-rate effects of chemicals
on cell survivals and the induction of mutations. In the present experiment
the dose-rate effects of ethyl methanesulfonate (EMS) on the survival andthe induction of mutations in Chinese hamster cells were examined.
The dose-rate effects of EMS on cell survivals were determined by thecolony-forming activity of cells. Cells were treated with EMS at various
concentrations for I, 2, 3, 4, 4.5, 8, 16 and 20 hours, and then incubated innormal medium for 7 or 8 days. The most effective exposure time of EMS
in reducing the survival fraction of cells was 4 hours. At shorter or longer
exposure times than 4 hours EMS was less effective in reducing cell survivals. A threshold or minimum concentration of EMS giving a surviving
fraction of 0.5 was found to be 0.1 mg/m!. No further decrease against thisRESEARCHES CARRIED OUT IN 1976

41

concentration giving a surviving fraction of 0.5 may be shown even if cells
might be exposed for infinite time.
Cells were treated with EMS at various concentrations for 1, 2,4,8, 16 and20 hours, incubated in normal medium for 48 hours, and then selected in
medium containing 0.1 p,gjmI6-thioguanine (6TG) for 10 days. An exposure
time of 4 hours produced lower frequency of6TG-resistant mutations thanshorter or longer exposure times of EMS giving the same surviving fractionof cells. An exposure time of 20 hours produced the highest induced
mutation frequency, suggesting that it may be useful as more sensitive procedure for detecting the mutagenic action of the chemicals using cultured
Chinese hamster cells.
Selection and Characterization of a 5-IodouridineResistant Variant in Cultured Syrian
Hamster Cells
Yukaiaki KURODA, Akiko YOKOIYAMA and Tsuneo KADA

A variant cell line was obtained from Syrian hamster BHK 21 (C-13)
cells after twice treatments with ethyl methanesulfonate and cyclic andcontinuous selections with 10- 2 M 5-iodouridine for 5 months. The colonyforming activity, growth rates in the presence of 5-iodouridine, 5-iododeoxyuridine (IUdR) and 5-bromodeoxyuridine (BUdR), distribution of chromosome numbers, uptake of 3H-thymidine eH-TdR) and cell surface properties of the variant cell line were compared with those of the original
BHK cell line.
The variant cells were highly resistant to 5-iodouridine and showed crossresistances to IUdR and BUdR. They grew more slowly and showed ashort lag phase in normal medium. They showed a higher growth rate anda shorter doubling time in the presence of 5-iodouridine than in its absence.
The variant cells were more severely affected by short-term treatment
with irradiated 5-iodouridine than the original BHK cells. The variant
cells also showed less uptake of 3H-TdR into the cold TCA-insoluble fractions than the original cells and differed from the latter in morphology andmutual cohesion. This may be the first observation on the marked crossresistances of the variant cell line to 5-iodouridine, IUdR and BUdR incultured mammalian cells. For details, see Japan. J. Genetics 52: 133-147,
1977.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

Effects of SR and da on the Viability of Hybrid Males from
Drosophila melanogaster X D. simulans Cross
Takao K. WATANABE and Masa-Aki YAMADAThe NSR-spirochetes injected to D. simulans killed sons in the embryonicstages and the SR condition was established in this species .(simulans-NSR)
by transmission of the spirochetes from mother to daughter through generations;Hybrid progenies from normal simulans mothers and melanogaster fathers(control cross) were mostly (99.7 %) males as expected, while those fromsimulans-NSR mothers, though the number was very small (2.0 per cross,when control was 37.0), were 35 % males and 65 % females. All these males
and females carried SR-spirochetes when their hemolymph was examined.
Since NSR-spirochetes usually killed males in the embryonic stages inmelanogaster as well as in simulans, the appearance of adult males suggeststhat the hybrid males are somewhat resistant to the male-killing effect ofSR agent.
A slight resistance to NSR killing effect on the hybrid males was recognized in, the cross of melanogaster attached-X (yf:=) females X simulans
males. Normal (yf:=) females produced 99.9 % hybrid sons, but SR (yf:=- NSll) females did not have any adult offspring. Many larvae and pupae
died on the waH of vials in the latter cross, and some (eye pigmentation
stage) were proved to be males from their sex combs and genitalia.
The homozygous da (daughterless, 2-39 of melanogaster) females produce
only sons regardless of the husband genotype, since the daughters die in theembryonic stages. When the dajda females were crossed with simulans
males, all hybrid progenies died as larvae. Salivary gland chromosomes
proved them to be males. Thus, sons were killed by the hybrid effect anddaughters were killed by the da gene. For details, see Japan, J. Genetics
52: 9-14 (1977).

Characterization of a Regeneration Deficient Strain of Hydra
Tsutomu SUGIYAMA and Toshitaka FUJISAWA

A mutant strain of Hydra magnipapillata (reg-l6) with regeneration deficiency was characterized in detail.
The number of the tentacles regenerated per animal was used as a

RESEARCHES CARRIED OUT IN 1976

43

measure of the regeneration capacity. Wild type strains generally had
about 6 tentacles per animal and after amputation of the head and the foot,they regenerated practically the same number of the tentacles within 5 to 6
days. In contrast, reg-16 regenerated only about 10 % of the originaltentacles during the same period of time. The tentacle regeneration, however, was substaintially enhanced when the regenerating fragment was subdivided longitudinally.
Lateral tissue implants that induced head formation in wild type hydra
either regressed or induced foot formation in reg-16 polyps. This suggeststhat the regeneration deficiency in reg-16 is due to a defective polarity
gradient.
A chimera strain was produced by introducing reg-16 interstitial cells
into nf-l strain (see next report). The chimera regenerated normally,suggesting that the defect of reg-16 is not located in the interstitial cells
or nerve cells but probably in the epithelial cells.

Isolation and Characterization of an Interstitial
Cell Deficient Strain of Hydra
Tsutomu SUGIYAMA and Toshitaka FUJISAWA

A mutant strain of Hydra magnipapillata (nf-l) was isolated that contained no interstitial cells and their derivatives, nerve cells and nematocytes.
This strain appears spontaneously in a mass culture of a strain (sf-I) whichappears normal in every respect except in one: it occasionally gives riseto nf-1. Although nf-l is unable to capture food by itself, it can grow andmultiply when force-fed. Thus nf-l is a suitable strain to study the roles ofinterstitial cells and their derivatives, especially the roles of nerve cells inmorphogenesis.
Nf-l regenerated normally retaining its original polarity when the heador the foot or both were amputated. The polarity gradient levels in nf-l
and its parental strain, sf-I, when examined by lateral grafting experiment,
showed no significant difference.
A chimera strain was produced by repopulating interstitial cells from awild type strain into nf-l. The comparison of growth rates between
the wild type, sf-I, nf-l and the chimera strain showed that the chimera
grew as fast as the wild type and faster than sf-l and nf-1.
These results suggest that interstitial cells and their derivatives play

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

very limited role in morphogenesis, but that nerve cells may play some"fine tuning" roles in hydra development which are basically establishedby epithelial cells.

Characterization of a Nematocyte Deficient Strain of Hydra
Toshitaka FUJISAWA and Tsutomu SUGIYAMA

Nematocytes are hydra's stinging cells and they are located mainly in thetentacles where they are used primarily to capture and paralyse prey.
Hydra magnipapillata has four types of menatocytes (stenotele, holotrichousisorhiza, atrichous isorhiza and desmoneme) which together account for one
third to one fourth of the total body cells.
The nematocyte deficient strains can be isolated by sexual crosses. One
such strain, nem-4, has virtually no stenoteles in its tentacles, but contains
them at a normal level in the body column. And the stenotele nematocytesof nem-4 turns over normally.
Grafting experiments between the nem-4 head region and the wild typebody column (and vice versa) showed that the wild type stenotele nematocytes
can migrate into the nem-4 tentacles but the nem-4 stenotele nematocytes
cannot move into the wild type tentacles. These observations suggest thatthe stenotele nemaotyctes of nem-4 are produced normally by differentiationfrom the interstitial cells in the body column, but that they are somehow
prevented from migrating into the tentacles in this strain.

The Relationship of Mitotic Activity of Primordial Germ Cells
to Testicular Teratocarcinogenesis in Strain 129 Mice
Takehiko NOGUCHI and Leroy C. STEVENS*

Teratomas are tumors which have several unusual and interesting characters. They are usually composed of many different kinds of tissues.
Teratomas are most commonly found in gonads. Spontaneous testicular
teratomas are seldom found in laboratory mice except in the inbred strain
129. At present several substrains of 129 showing different teratoma
incidences have been developed by introducing some mutant genes onto thegenetic background of original 129. Testicular teratomas derive fromprimordial germ cells (PGC) in embryonic testes. Very small fractions of* The Jackson Laboratory, Bar Harbor, Maine, U.S.A.

RESEARCHES CARRIED OUT IN 197645

these PGC begin to develop, presumably through a process akin to parthenogenesis. Many non-genetic as well as genetic fractors (or conditions)influencing the teratocarcinogenesis have been known. But little is known
how these factors (or conditions) stimulate or inhibit the parthenogenesislike activation of PGc.
In order to find a clue to this problem, developmental biological approach
was made. Several experimental evidences indicate that the activation of
PGC may occur during the developmental stages ranging from 12 days to
16 days of gestation. During this period the sex cord of male genital ridges
develops into seminiferous tubules. PGC become mitotically very queit
a couple of days after they are enclosed into seminiferous tubules. As
PGC under mitosis can easily be distinguished from the other cells, themitotic rate of PGC was chosen as a marker of PGC differentiation during
the period in question. The mitotic rate of PGC in fetal testes was determined with hematoxylin-eosin stained praffin sections for several substrainsof 129 mice that differ in incidence of testicula teratomas. If the mitotic
rate is expressed by the number of mitoting PGC per testis, it peaked at
13 days of gestation and then decreased precipitously. At 15 to 16 days
when early teratomas start to appear, the mitotic rate of PGC became very
low. Testes of 129/Sv-ter which has the maximum incidence (33 %) of
teratomas, however, quite often contained larger number of PGC undermitosis at this late stage than usual. Probability for testes to have teratomas
was several times higher in testes showing unusually high level of mitosis
of PGC than in those showing normal level of mitosis. The incidence oftestes containing unusually larger number of PGC under mitosis at thelate stage was far less in the other substrains of 129 which have much lower
incidences of teratomas.
When 12-1/2 day male genital ridges of 129 x A/He are grafted toscrotal testes, they develop into testes and most of them have teratomas.
When they are grafted to cryptorchid testes or to kidneys, the incidence
becomes lower, although they develop into testes. In kidneys few of themhas teratomas. In grafts in scrotal testes, the time when PGC became
mitotically quiescent was considerably delayed. When 12-1/2 day genitalridges were grafted for 4 days in these adult sites, the mitotic rate of PGC
was the highest in scrotal testes and was the lowest in kidneys. The
delay in entering in a mitotically quiescent stage of PGC is well correlatedwith the high incidence of testicular teratomas.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

V.

CYTOGENETICS

Karyotypes of Four Myotis Bats in Japan
Masashi HARADA and Tosihide H. YOSIDA

Cytotaxonomical investigations of four Myotis bats collected in Japan
were carried out by conventional, G- and C-band staining techniques. The
four species all had 2n=44 and their karyotypes were, excepting one
chromosome pair, identical to each other. The only difference in theirkaryotypes was found in the morphology of the chromosome no. 5. A
minute acrocentric (A) was observed in M. nattereri and a polymorphic (A)and (Mh ) which is a minute metacentric with a totally heterochromatic shortarm was found in M. hosonoi. In M. frater kaguyae the pair no. 5 was asmall submetacentric with a totally heterochromatic long arm (SMh).
Polymorphic (SM h) and (M), which is a small metacentric induced from(SMh ) by pericentric inversion, were seen in M. macrodactylus. Such amorphological differentiation of the no. 5 was interpreted by assuming theinversion and also duplicating growth of the constitutive heterochromatin.
Their evolutional pathway in genus Myotis, thus, is assumed to be as
follows: (A)~(Mh)~(SMh)~(M). This assumption was supported by thegeographical evidence that the species with the (A) type of the pair no. 5
widely distributed throughout the world but the others are restricted to Asia
(M h type) and Japan in particular (SM h and M types). Based on the differentiation of the chromosome no. 5, it is suggested that M. nattereri and
M. hosonoi are phylogenetically closely related, while M. f kaguya andM. macrodactylus are of another kin relationship.
Geographical Distribution of No. 18 Chromosome Polymorphismin the Japanese Feral Mouse, Mus musculus molossinus
Kazuo MORIWAKI and Mitsuru MINEZAWA *Chromosome C-band patterns of the Japanese feral mice, Mus musculus
molossinus, have been already observed by Dev et al. (Chromosoma 53: 335,
1975) who revealed that their bands in Nos. 1, 2 and 4 chromosome werelarger than those of M.m. domesticus and those in Nos. 13 to 17 were smaller.

* Department of Agriculture, Nagoya University.

47

RESEARCHES CARRIED OUT IN 1976

We collected molossinus mice from various localities in Japan and examinedthe chromosome G- and C-band patterns. Characteristic features in theirC-band patterns were almost similar as those reported by Dev et al.
Moreover, an interesting C-band polymorphism in No. 18 chromosome
has been found out. There were three types of the C-band pattern in theNo. 18 chromosome; positive band (+), large positive band (+1) and positive band in the middle of the arm (+m). Results obtained from ten
individuals are summarized in Table 1.
Table 1.
Sample No.
1
2
3
4

5
6
7
8
9
10

C-band pattern polymorphism in No. 18 chromosomes ofMus musculus molossinus
Place of collection
Teine, Hokkaido
Johoji, Northeastern Mainland
Gotenba, Middle Mainland
Anjyo, Middle Mainland
Kanazawa, Middle Mainland
Mine, Southwestern Mainland
Hakozaki, Kyushu Island
Sasaguri, Kyushu Island
Yonakuni Island
Okinawa Island+: Positive C-band in No. 18.
middle of No. 18.

C-band of No. 18 chromosome+1/+ 1

+1/+1
+1/+1
+m/+m
+m/+m
+m/+m
+/+
+/+
+m/+m
+J/+ 1+1: Large positive C-band. +m; Positive band in the

The geographical distribution of those polymorphic chromosomes likely
suggests possible immigration processes of molossinus mouse into Japan.
Probably the first immigrants with a large positive C-band in No. 18 chromosome came to this country through the Korea Peninsula and spread all overJapan. The next was those with positive C-band in the middle of No. 18
and the latest those with positive C-band of No. 18 as similar as that ofM.m. domesticus. The last ones are distributed still in the restricted areaof Northern Kyushu. Geographical distribution of No. 18 polymorphism
summarized in Table I may reflect the possible processes of repeated immigration of M.m. molossinus into the southwestern Japan as proposed
above.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

Hybrids among Three Geographical Variants of the Black Rats, 1.
Hybrids between Asian and Ceylonese Type Black Rats,
and Their Karyotypes
Tosihide H. YOSIDA

Delivery of hybrids in the laboratory matings between the Asian (Rattus
rattus tanezumi) (2n=42) and the Oceanian type black rats (R. rattus rattus)(2n=38) and their karyotypes have already been reported (Yosida et ai.,
1971, Chromosoma 34: 40; Yosida, 1976, Proc. Jap. Acad. 52: 304). However, any hybrids between the Asian and Ceylonese types (2n=40) and also
between Oceanian and Ceylonese types have not yet been reported. The
present report deals with the F 1 hybrids between Asian (2n=42) and Ceylonesetype black rats (2n=40) and their karyotypes. The Asian type (R. rattus
jlavipectus) and Ceylonese type (R. rattus kandianus) were collected in Hong
Kong and Sri Lanka, respectively, and then bred in this laboratory forseveral years.
Ten F 1 hybrids were obtained by matings between Ceylonese type (~)
and Asian type (6) black rats. They invariably had 41 chromosomes,
among which pair no. 11 was composed of one large metacentrics (M2)and one acrocentrics. The acrocentric no. 12 was not paired. The partnerof the no. 12 is homologous to the short arm of the M 2 chromosome. Pairs
no. 1 and 9 were acrocentric and subtelocentric heteromorphism. It is
obvious that the subtelocentric pairs no. 1,9 and the large metacentrics (M2)originated from the Ceylonese type, while the acrocentrics in the pairs no.
1, 9, 11 and 12 were derived from the Asian type rat. All chromosome
pairs were detected by the conventional and G-band sequential stainings.
The karyotype of the F 1 hybrid was then formulated as follows: 2n=41(4S+20A+14SM+IM+XY) (S=subtelocentrics, A=acrocentrics and SM=small metacentrics). Although the effort to get the F 2 hybrid from themhad been taken place, it has not yet been obtained until this time. The
F 1 hybrids seem to be semisterile as seen in the hybrids between Asian andOceanian type black rats.

RESEARCHES CARRIED OUT IN 1976

49

Hybrids among Three Geographical Variants of the Black Rats, ll.
F 1 and F 2 Hybrids between Oceanian and Ceylonese TypeBlack Rats and Their Karyotypes
Tosihide H. YOSIDA

By laboratory matings between Oceanian type (Rattus rattus rattus, 2n=38)
originally from the United States and Ceylonese type black rats (R. rattus
kandianus, 2n=40) originally from Sri Lanka, 15 hybrids (~ 8: 6 7) wereobtained. They invariably showed 39 chromosomes. The karyotype wasremarkable in having 3 large metacentrics. One of them was the M 1chromosome resulting from Robertsonian fusion of the pairs no. 4 and 7
and the other two are the M 2 pairs resulting from the fusion of the pairs no.
11 and 12. Pair no. 4 is hetermorphic by the M1 (4/7) and one acrocentric no. 4. Pair no. 7 was acrocentric and not paired. The partner ofthe pair no. 7 is translocated to the M 1 chromosome. Pairs no. 1, 9 and 13
were subtelocentrics which were derived from both parents. Seven small
metacentric (SM) pairs (no. 14--20) were not different from those of theother black rats. Thus, the karyotype of the F 1 hybrids was formulated
as follows; 2n=39 (6S+ l4A+IM1 +2M 2 + 14SM+XY).
A total of the 12 (6~: 6 6) F 2 offspring were obtained in three littersfrom the hybrids matings. The average litter size of the F 2 was 4.0 which
was an almost normal condition. Karyotypes of 9 (6 ~: 3 6) rats among
them were analysed. Three karyotypes, namely Oceanian type (2n=38),
F1 type (2n=39) and Ceylonese type (2n=40), were expected theoreticallyat the ratio of 2.25: 4.5: 2.25. The observation, therefore, resulted in 6 F 1
types and 3 Oceanian types. No Ceylonese type was observed at this time.
F 2 offspring were not obtained from the hybrids between Asian and
Ceylonese types, but the hybrids between Ceylonese and Oceanian types wereapparently fertile. Based on these findings, it is suggested that the Oceaniantype is more closely related to the Ceylonese type than the Asian type black
rat. This result seems to coincide with the karyotype evolution in thesethree geographical variants already postulated by the present author (Yosida
et al., 1974, Chromosoma 45: 99; Yosida 1977a, Chromosoma 60: 391,
Yosida 1977b, Cytogenet. Cell Genet., 18: 149).

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

Hybrids among Three Geographical Variants of the Black Rats, ID.
An XO Female Occuring in the F 2 Hybrid between Oceanianand Ceylonese Types
Tosihide H. YOSIDA

Among 6 females in F 2 offspring from the hybrids between Oceanian and
Ceylonese type black rats, one female showed 37 chromosomes. The
fundamental karyotype of the female was the Oceanian type. All 20 cells
from bone marrow and cultured tail tip tissue of the rat had invariably 37
chromosomes. By analysis of G-band staining it was clearly shown thatthe sex chromosome mechanism of this rat was of the XO type. The rat
was apparently a normal female, but its fertility was not confirmed because
it was killed for the chromosome observation. The other 5 female and 2
males born of the same parents which produced the XO female showednormal XX (~) and XY (8') types. Both parents had also a typical karyotype comprising the regular sex chromosome mechanism with the XX (~)
and XY types (8').
Presence of an XO female has already been reported in one Oceanian typeblack rat obtained in Davis, California (Yosida et al., 1974, Jap. J. Genet.
49: 49). The monosomic X has been confirmed by G-band staining. The
fertility of the animal was also unknown, because the rat died about 6
months after capture without reproducing. Therefore, whether the XO
female of the black rat is fertile or not is uncertain at present. Anotherexample of the XO female was found among the black rats collected in
New Guinea in 1968. This rat was also basically an Oceanian type
(Yosida, unpublished). Based on these findings, it can be said that thepresent XO female found in the F 2 offspring is not a specific case in the
hybridization, but rather a natural and sporadic case as seen in the otherblack rats.

Candidates for a New Experimental Animal, I.
The Soft-Furred Rat, Millardia meltada
Tosihide H. YOSIDA

The soft-furred rat, Millardia meltada, collected in India has been bred
successfully in the laboratory by brother and sister matings for about 10
generations by use of our breeding system. The relative size of thisRESEARCHES CARRIED OUT IN 1976

51

animal is about midway between the mouse and the rat. The mean bodyweight of 30 males and 30 females in 12 weeks old is 82.7±16.0 g and59.9±8.I g, respectively. The number of mammae in the animal is eight(2: 0: 2). They are easily handled in the laboratory due to its gentle
behaviour. Laboratory breeding of the animal is easy by use of the usual
rat cage. Average litter size is 4.09 (based on 21 litters observed). The
gestation period of this animal is 20 days on the average, and about 12 days
after the birth the eyes open. Diploid chromosome number of the animal
is 50 among which two autosome pairs (pairs no. 1 and 2) are large
metacentrics, 21 autosome pairs (nos. 3 to 23) are acrocentrics, and the one
autosome pair (no. 24) is a small metacentric. The X is a large metacentricand Y a median sized acrocentric.
Some interesting characteristics of this animal were as follows: 1) The
new born baby within one day has thin hair on its body. 2) Welldeveloped inciser teeth have been found in the new born. 3) The ageof the animals seems to be shorter (about one year) than the other rodent
species. 4) Individuals with low blood presure are often found among theanimals. Based on the above characteristics, this animal is one of the more
promising candidates for a new experimental animal.
Subtelocentric No.3 Chromosome in the JapaneseBlack Rat-a New Inversion
Tosihide H. YOSIDA and Yuriko OeRIAl

The Japanese black rat, Rattus rattus tanezumi, was generally characterized as having 42 chromosomes (21 pairs). Among them pairs no. 1, 9
and 13 were characterized by acrocentric and subterocentric polymorphism.
Pair no. 3 in about one thousand black rats collected in several countries in
the world, however, was always characterized by an acrocentric centromere.
One male black rat collected in the field of Gotenba, Shizuoka-ken, Japan,
had 42 chromosomes as the other Asian type rats. Pairs no. 1 and 9 ofthis rat were acrocentrics, but pair no. 13 was an acrocentric and subtelocentric heteromorphism. Out of the heterologous pair no. 13, the pairno. 3 was remarkable because it had an unexpected acrocentric and subtelocentric heterologous pair. As the length of the acrocentrics and subteleocentrics was almost similar, the subtelocentrics would seemed to have
:leveloped by pericentric inversion of the originally acrocentric chromosome.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

From the G-banding analysis, it was proved that pericentric inversion could
have occurred in about one-third of the acrocentric no. 3, and then thesubtelocentrics developed.
On the origin of polymorphism in pairs no. 1, 9 and 13 in the black rat,
the present author suggested that the original type of these chromosomes
were acrocentrics. The subteolocentrics of these members have developedby pericentric inversion. New observation of the subtelocentric no. 3 seems
to prove the above suggestion. The rat demonstrating thus is being
maintaining to observe fertility and no. 3 chromosome behavior.
An Experiment on Artificial Insemination between the
Norway Rat and the Black Rat
Tosihide H. YOSIDA and Choji TAYATo ascertain the relation between the black rat and the Norway rat,
artificial insemination between these two species was taken. As the female
recipient, inbred strains of the Norway rats (Rattus norvegicus) were used.
Semen was obtained from the dissection of the cauda epididymis of theblack rat (Rattus rattus jlavipectus). When the female rat was at the proestrus stage of the estrus cycle, the cervical uterus was stimulated by faradic
current. Then the semen was injected into the uterine horns through each
cervical canal by the hypodermic needle. For the control experiment,
the semen from the Norway rat was inseminated artificially.
Among 20 Norway rats which were inseminated artificially with thesemen of the black· rat, 9 rats were successfully fertilized. In 5 of the rats
the impregnation was confirmed by dissection. Between one and 4 embryos(2.2 on average) were confirmed in the impregnated rats. Offspring, however, were never born in the case of interspectific hybridization, and theembryo always died at a maximum age of about two weeks after the insemination. In the control, 25 rats were treated and 23 among them were impregnated successfully. Seven impregnated females were dissected and thenumber of impregnated embryos were counted. In this case there were
between 3 and 11 embryos (6.3 on average). From the other females
impregnated young were born normally. Thus, the number of embryos in
the case of interspecific hybridization was significantly less than the control.
For the observation of chromosomes, the blastocysts were obtained fromthe uterus 5 days after insemination, and then chromosomes were observed

RESEARCHES CARRIED OUT IN 1976

53

by drying technique. The Norway rat and the black rat together have 42chromosomes and their karyotypes are similar (Yosida 1973, Chromosoma
40: 285). Pairs no. 1,9 and 13, in the black rats (R. rattusflavipectus) wereacrocentrics, but those in the Norway rat were subtelocentrics. Fivehybrid biastocysts examined showed invariably 42 chromosomes, among
which the nos. 1,9 and 13 chromosomes were remarkable as having heteromorphic pairs. The blastocysts from the control had the regular karyotypeof the Norway rat which consisted of the subtelocentric pairs no. 1,9 and 13.
Based on the above investigations, it can be said that although the Asiantype black rat has the same chromosome number and a similar karyotype to
the Norway rat, evolutional difference between these two species is muchlarger than those among the geographical variants in the black rats, eventhough each of them is characterized by having a different chromosome
number and karyotype.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

VI.

MUTATION AND MUTAGENESIS IN ANIMALS

Genetic Analysis of Spontaneously Derived Mosaics from
Abnormal Fertilization in the Silkworm
Akio MURAKAMI and Hisashi FUJIOKA

In the course of experiments on the spontaneously-induced egg-colour
specific locus (pe: re) mutations of the silkworm females, the mosaic mutant
having yellow (pe) and black (pe+) serosa cells at the ratio of
1: 1 has repeatedly been detected at the high frequencies (103-645 X 10- 5)
in wild type (Cl08 or Aojuku strain) female pupae treated with (or without)0.85 % NaCI solution. These treated females were mated to marker strain(pe: re) males. However, the frequency of the other type mosaics having
red (re) and black (re+) serosa cells (0.0-2.1 X 10- 5) was not significantly
differed from usual ones. In addition, such the phenomenon was notobserved when the marker females were mated to the wild type males.
Hatchability of these mosaic mutant eggs derived from the former mating
cross was approximately 5 %and this was corresponded to about one sixteenthof non-mutated F 1 eggs. Of nineteen mosaic eggs hatched so far, six were
gynoandromorphs and two females and eleven males. Both females andmales were again mated to a marker strain homozygous for the pe gene andthey were produced without exception yellow and black eggs with a 1: 1
ratio, indicating that no germ-cell of the yellow part (pe: re/pe: re or pe/pe)might be produced or survived probably due to lethal events caused by androgenesis. It should be emphasized that in this mutation test system the eggcolour of the non-mutated F 1 (+: + /pe: pe) individual is black and the pegene is epistatic to the re gene, so that the eggs homozygous for both genes(pe: re/pe: re) are yellow. Thus, it is likely that the black part of themosaics (+: + /pe: re) may be formed by normal fusion of egg (+: +) andsperm (pe: re) nuclei and would be expressed the maleness and femalenesswith an equal probability, while the yellow part may be formed via either
fusion of two sperm nuclei (pe: re/pe:re) or development of one sperm(pe: re) nucleus showing only the maleness. When cleavage begins, thederivatives of the two resulting cleavage nuclei with different gene constitutions (either pe+ /pe or pe/pe) populate the opposite (right and left, ingeneral) sides of the ovum (or egg). If the derivatives of the two cleavage

RESEARCHES CARRIED OUT IN 1976

55

nuclei having different sex-chromosome constitutions (XX for male, XY
for female) were populate the opposite sides resulting in a sexual mosaic or
gynandromorph. Lack of BPi progeny produced only yellow eggs may besupported the view that germ-cells of the yellow part (probably maleness)
is formed as the result of androgenesis and that would be showed an extremely high lethality by receissive lethal mutations on the X chromosome.
As to the origin of the black part, it can be rule out a possibility of autogramic fusion of two haploid derivatives in products of female meiotic divisions, since both yellow and black eggs were observed in BPi progeny,
regardless of sex of the mosaics analyzed.
To sum up, the origin of the half-and-half type mosaics can be interpreted
on the assumption that one part of an ovum is formed via normal fertilization between oocytes (pe+: re+) and sperm (pe: re) and the other part isformed by either homozygous diploid or monospermic androgenesis, suggesting that the origin of these mosaics is essentially differed from that of ahereditary mosaics (rna), due to fertilization of an egg-nucleus and one ofthree polar-bodies (Goldschmidt and Katsuki, 1931). Unfortunately, themechanism(s) of spontaneous appearance of the mosaics are not known.
It is more likely, however, that the phenomenon may depend on a functional
disorder of cytoplasm in oocytesjeggs being due to genetic or infectious
factors.
Chemically-Induced Transmitted Translocations in the Silkworm
Akio MURAKAMI and Tetsuro MUROTA1)Transmitted translocation, that is translocations induced in the postmeiotic germ-cells and subsequently transmitted to the progeny leading toa reduction in fertility owing to abnormal meiotic divisions, can be able todetect by either genetic or cytogenetic method. In lepidopterous insects,
this type genetic incidence is simply detected by an increase of embryoniclethality (or sterility) of the Pi progeny so that the test method described inthis communication would be recommended for the detection of chemicals
inducing such the translocations. A practical procedure for the detectionof this type genetic damage is to measure the reduction of hatchability infertilized eggs of the first generation offspring of treated parents or the Pi
progeny. This report describes the result of transmitted translocation tests
1)

Hatano Institute of the Food and Drug Safety Center, Hatano, Kanagawa-ken.

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

in the sperm after treatment of the mid-stage male pupae with mitomycin C(MC).
In the present test, wild type (e108 strain) male pupae were injected witha 10 ,ug/pupa of MC, mated to a different strain (pe: re) females to minimize a noise being resulted from recessive lethal mutations and survived PI
individuals were further mated to the marker (pe: re) strain to determine theembryonic lethality as the reduction of egg-hatchability of the F I progeny
as being due to the incidence of heritable tranlocations.
The results of the experiments summarized in Figure 1 showing a typicalcase of the transmitted lethality in which hatchabilities of the F I progeny
derived from the treated male parents were lower than those of the PI
progeny. However, the hatchability of the BFI progeny was higher thanthose of both PI and F I progenies. This recovered hatchability in the BPI
progeny may be interpreted by the selection of the cells having translocations
lead to lethality during the course of gametogenesis in the back cross PI
generation (or F2).
BF1

MC
~

pe: re~ x ++c3'(85.7%)

F 1 ~ xnontreatedpe: re c3'(73 ..3%)
nontreatedpe: re ~ xF1 c3'(64.9%)BFz
BFl ~ xnontreatedpe: re c3'(92.2%){
nontreatedpe:re ~XBFlc3'(87.0%)
BF1 ~ xnontreatedpe : re c3'(106.8%){
nontreatedpe: re ~ xBF1 c3'(88.2%)

Fig. 1. Changes in hatchabilities of eggs for three generations after treatmentof the mid-stage male pupae (or sperm) with 10,ug/pupa of mitomycin C. The
numbers in parentheses indicate the hatchability in percent of controls.

As communicated in the previous report (this Report No. 26: 42-44(1976)) that the sperm in the mid-stage pupae was the least sensitive to MC
for the induction of dominant lethal mutations during the post-meiotic stageof this insect male. The present and other findings tend to suggest that thelow sensitivity of the sperm at this pupal stage may be at least in part due
to effective repairability for broken DNA molecules or chromosomes. From
this context, it can be possible to said that the inherited lethality caused bytranslocations may be formed via incorrect repair processes for broken
DNA.

RESEARCHES CARRIED OUT IN 1976

57

Drug Metabolic Activation in the Silkworm
Kyoko SAKAMOT01), Tetsuro MUROTA1) and Akio MURAKAMI

For the past few years, we have been studied on the production of the
egg-colour specific locus mutation in pupal oocytes and sperm with anumber of known indirect mutagens (such as benzo(a)pyrene, BP; dimethylnitrosamine, DMNA; 4-nitroquinoline-N-oxide, 4NQO; 2-acetylaminofluorene, AAF and so on) which require metabolic activation to exert theirmutagenic activity. The first three mutagens so far tested have not yet
been shown to be a positive result in these germ-cells, while AAF has beenshown to be a very weak mutagenicity in pupal oocytes. This may beinterpreted as the deficiency of these drug metabolic activation systems in
pupal silkworm.
This communication reports the activity of AAF metabolic activation in
pupae as well as other developmental stages of the insect. The metabolic
activity for AAF was measured by a slight modification of the Salmonella/
microsome test (cf., Ames et al., 1973, Proc. Natl. Acad. Sci. U.S. 70: 2281).
Each biological sample was homogenized in three volumes of ice-cooled
1.15 % KCl solution with a Teflon homogenizer. The homogenate wascentrifuged at SoC and 9000 g for 30 min. The resulting supernatant withthe cofactor system was used for the measurement as an S-9 reaction mixture(S-9 mix). The S-9 mix, Salmonella tester strains (TA 98 and TA 100) andAAF is incubated at 37°C for 20 min with shaking before pouring on aminimal glucose agar plate with aid of molten top agar. The cofactor
system is contained NADPH, NADP, G-6-P, MgCl2 and is further added
G-6-P- dehydrogenase. The plates are incubated at 37°C for 48 hrs and therevertant colonies to His+ prototrophy are counted.
The result of experiments clearly indicated that the activity of the AAF
metabolic activation by homogenates derived from various developmental
stage silkworms was markedly changed with the stage. The activity wasincreased with the larval growth or the amount of mulberry leaves ingested.
The marked activity was observed in the homogenate from the last (5th)instar larvae, not in embryos and pupae. It is of worthwhile to note thatthe activity of AAF metabolic activation by the last instar larvae was almost
the same or somewhat higher than that by the rat liver in which rats had
been pretreated with polychlorinated biphenyl (KC-500). Homogenates of1)

Hatano Institute of the Food and Drug Safety Center, Hatano, Kanagawa-ken.

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

mid-gut and blood in the last instar larvae were shown to be the same activity
as the whole insect.
No significant mutagenicity of the induction of the egg-colour specificlocus mutations in spermatocytes/spermatids after treatment of even the last
instar larvae with AAF was observed, therefore, suggesting that the lack ofmutagenicity of AAF in such the test system may be related to the nature ofmutants detected by the specific locus method. Namely, much higher dosesof active metabolites which interact with DNA would be required to induce
the specific locus mutations including deletion and chromosome breakage incontrast to the molecular incidence detected by the Salmonella/microsome
test system. By the way, one of possible mechanisms responsible for the
lower mutagenicity of AAF to pupal oocytes is that this parent compound
itself may interact with DNA presumably through interacalation leading toframeshift type mutations.
In any case, the assay method for AAF metabolic activations may be
useful to other indirect mutagens in this insect.
Comparison of Some Nitrosonitroguanidines in Respects to
Their Efficiency in Inducing Recessive Visible Mutationsin Silkworm Germ-Cells
Akio MURAKAMI and Masanori NAKADATE1)

It is a well known fact that N-methyl-N'-nitro-N-nitrosoguanidine or
Methylnitrosonitroguanidine (MNNG or MeNNG) is a highly mutagenic
and carcinogenic chemical in various biological organisms including Salmonella, cultured cells, Drosophila and so on. In Drosophila, it has beenreported that MeNNG induces a broad mutational spectrum in all germcell stages with a higher frequency of gene or point mutations than that ofother class ones (Browning, 1969). According to Nakao and his associates(1966), however, they have reported that in silkworm MeNNG is a low
mutagenic chemical for inducing egg-colour specific locus mutations inmale silkworms. The present experiment was undertaken to obtain more
information for the mutagenicity of MeNNG as well as four other nitrosonitroguanidines (Ethylnitrosonitroguanidine, ENNG; Propylnitrosonitroguanidine, PNNG; Butylnitrosonitroguanidine, BNNG; isobutylnitrosonitroguanidine, iBNNG) to germ-cells of male and female silkworms. The
1)

National Institute of Hygienic Sciences, Tokyo.

RESEARCHES CARRIED OUT IN 1976

59

mutagenicity was measured by the egg-colour specific locus method. The
late stage pupae (or the last few days before emergence) were injected with
the nitrosonitroguanidines which had been dissolved in 0.85 % NaCl solution.
The incidence of fractional-body or mosaic mutations in pupae treatedwith nitrosonitroguanidines was more frequently induced than that of who1ebody ones regardless of the compounds tested and germ-cell types. The
incidence of the latter type mutations in both oocytes and sperm after
treatment of pupae with ENNG was slightly higher than that of controls,
but not significantly increased with MeNNG, BNNG and iBNNG. On an
equimo1ar basis at the time of injection, a comparison of the frequency forthe fractional-body mutations induced with these five nitrosonitroguanidinesin pupal oocytes and sperm was made: the mutagenicity was follows, ENNG,
PNNG, MeNNG, BNNG and iBNNG in this order of effectiveness. Thus,
it can be said from this experiment that MeNNG is not always highly
mutagenic for production of the egg-colour specific locus mutations in bothoocytes and sperm of the silkworm. This experimental result is in goodaccord with that after Nakao et al. (1966). In any case, it should be said
that mutagenicity of ENNG is equivalent to the known potent mutagens suchas ethyl methane sulphonate (EMS) and mitomycin C (MC) in this insect.
It is of interest to note that MeNNG and methyl methane sulphonate (MMS)
are more highly toxic, but lower mutagenic to silkworm pupae than ENNG
and EMS. This difference may be caused by lack of an excision repair
system in germ-cells of the silkworm as in an excision deficient stock of
Salmonella in which ENNG is more mutagenic than MeNNG (Matsushima
et al. 1977).
Enhancing Effect of Caffeine on X-Ray-Induced DominantLethal Mutations in Silkworm Sperm
Akio MURAKAMI and Hiyoriko NISHIJIMA

Silkworm females at the stage of mid-pupae or prophase I oocytes weretreated with a purine analogue, caffeine (1,3,7-trimethy1xanthine) then matedto male moths or mature sperm that had been irradiated with X-rays.
The mutation frequency of X-ray-induced dominant 1etha1s in the sperm wasmeasured. The frequency of this class lethal mutation was determined by
dividing the number of dead embryos or non-hatched eggs by the total

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

number of fertilized eggs. For caffeine treatments, one of various dosesof caffeine which had been dissolved in 0.85 %NaCl solution or physiologicalsaline was injected into the abdominal cavity of the pupae. Controls were
injected with 0.85 % NaCl solution without caffeine. In the irrdiation ,male moths were exposed to various doses of 180 kVp X-rays (25 rnA, 1.0 mm
Al filter and a dose-rate at 300 R/min) ranging from 2,500 to 15,000 R.
When female pupae at the mid-stage were treated with caffeine at thedose over 200 ,ug/pupa, she showed extremely high sterility such as inability
to mate and infecundity. However, no dominant lethality was induced insperm after treatment of the mid-stage male pupae with caffeine of doses
varying from 0.1 to 300 ,ug/pupa. When various doses of X-rays were
exposed to male moths and then mated to female moths which had beentreated with caffeine (25 to 250,ug/pupa), frequencies of X-ray-induced
dominant lethal mutations in the sperm were clearly increased with thedose of caffeine. The enhancing effect of caffeine on the radiation-induced
dominant lethals in sperm was an approximately 1.3-1.5 times at a caffeine
dose of 200 ,ug/pupa as compared with the control regardless of X-ray doses(10,000 and 15,000 R). This observation may be interpreted as due to theinhibition of a maternal cytoplasmic and/or probably dark repair systemthat is responsible for repair of X-ray-induced chromosome (DNA) breaks
in sperm. If so, it can be said that most of breaks induced in sperm would
stay open prior to syngamy or DNA synthesis at the early cleavage division
stages. It is of interest to note here that no significant enhancing effect wasobserved when a radiosensitive stock, Kojiki, female pupae were treatedwith caffeine. This finding suggests that this stock may be lacking in thecytoplasmic repair system in oocytes.
Appraisal of Silkworm Oocyte System for Mutagenicity
Testing of Environmental Chemicals
Yataro TAZIMA and Akio MURAKAMI

For the purpose of mutagenicity testing of chemical compounds, wedeveloped in silkworm a sensitive test system named "oocyte system," whichis essentially a specific loci method using egg color genes as markers (Ann.
Rept. Nat. Inst. Genet. (25): 40-41). Using this system, we have performed,
as a part of cooperative research project under a grant from Ministry of
Welfare, mutagenicity testing of various compounds including known muta-

RESEARCHES CARRIED OUT IN 1976

61

gens, carcinogens and their allied compounds. The results so far obtained
indicated that the system could be adequately applicable to the testing of
direct mutagens and/or carcinogens, but not necessarily to those compounds----

Table 1.

Mutagenicity testing of chemical compounds with silkworm~~~~~~~~~~~~~~~~-~~~~~~~~~---

Compounds

Mutageneicity

DAtiridine and derivatives
Ethylene imine
TEM
Mitomycin C*
2) Mustards
Nitrogen mustard
Nitromine
ICR-170
3) Nitrosamines and Nitrosamides
73-20 Dimethylnitrosamine
74-10 Di-n-butyl-nitrosamine
73-22 Butylbutanolnitrosamine
74-14 N-methyl-N-nitrosourea
73-29 N-n-butyl-N-nitrosourea
74-12 N-n-butyl-N-nitrosourethane
Mitomycin C'"
MNNG
ENNG
75-06 Urethane
75-16 Methylazoxymethanol(MAM)4) Amines and Amides
74-09 Di-n-butylamine
73-21 Butylbutanolamine
74-13 Methylurea
73-23 Butylurea
74-11 N-n-butylurethane
76-08 Thiourea
76-07 Thioacetamide
Hydroxylamine
5) Exoxide, Aldehyde, Lactones
Formaldehyde
75-13 p-Propiolactone'" Appearing in two places.

+
+
+

+
+
+
±

+
+

+
+
+
+
±

Compounds

Mutagenicity

75-12 e-Caprolactone
76-21 Epichlorohydrin+
6) Alkylsulfates, Alkane sulfonic esters+
Diethyl sulfates (DES)
Methylmethanesulfonate
+
(MMS)
EMS
+
75-28 Propane sultone+
7) Nitrofuranes
3-Amino-6-2-(5-nitro+
2-furyl) vinyl-1,2,4triazine, (NFT)
AF2+
Nitrofurazone+
Furazolidone
8) Heterocyclic compounds
75-25 Pyridine
75-26 Quinoline
74-274 NQO
74-28 4 NH2 QO
9) Acridines and Acridineum salts+
Acridine Orange
Acridine Yellow+
Profla~ne±±+10) Polycyclic hydrocarbons
73-11 AAF
74-02 Benz(a)pyrene
73-05 Dimethylbenzanthracene
Phenanthrene11) Aflatoxines
12) Base analogues
BUdR
13) Others
73-18 Nitrous acid

+
+

+
+'"

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

acting indirectly.
In the Table listed are the results of mutagenicity testing of chemical compounds, the majority of which were obtained by applying oocyte system.
Those are prefixed with experimental number to their chemical name. In
order to make possible the detection of indirect mutagens several measures
are now being investigated in the silkworm.
Mutagenic Effectiveness of an Internal fJ Emitter, Tritium.
(3) Effectiveness of fJ-irradiation at Low Dose-Rate Range
Yataro TAZIMA, Kimiharu ONIMARU and Yosoji FUKASE

Mutagenic effectiveness of very low dose-rate exposure to mutagens has
been investigated using tritiated thymidine. The assay system applied wasa silkworm oocyte system which was developed especially for chemicalmutagenesis study in this insect. The injected compound was taken into
egg plasm and then into embryo and excreted mostly soon after feeding of
hatched larva. The duration of egg stage was controlled artificially.
3H-TdR in aqueous saline was injected into the body cavity of female
pupae of a wild type strain (20 and 100 ,uCi per head). Emerged females
were mated to males of another wild type strain to allow deposition.
Average radioactivity transmitted to the deposited egg were 2.28 and 12.73,uCi/g, which corresponded to ca. 0.66 and 3.7 rad/day, respectively. Halfof those egg layings were subjected to acid treatment for artificial hatching,whereas the rest half was allowed to hibernate. The durations of egg stage
were 29 and 167 days respectively.
Combination of two dose-rates and two durations gave four dose groups;
for 29 days, a) 19.3 rad and b) 107.3 rad, and for 167 days, c) 109.0 rad andd) 612.6 rad. Both sexes of those treated and non-treated groups werecrossed to pe re partners and mutation frequencies were assayed. The
induced mutation frequencies were: almost control level in a) and markedly
enhanced in d), while those in c) were very low being about half of thoseobtained in b), although the total dose was almost same. The results indicated the existence of dose-rate effect even at a very low dose-rate range as
0.66 rad/day (J. Rad. Res. 18: 32).

RESEARCHES CARRIED OUT IN 1976

63

Nature of Chemically Induced Mutations Obtained by Oocyte
System in the Silkworm (continued)
Yataro TAZIMA, Kimiharu ONIMARU and Akio OHNUMAIn continuation of the work reported previously (this Annual Rep. (26),40-41) analyses of the nature of chemically induced mutations have been

carried out in the silkworm.
1) Lethality test in homozygote
Test for lethality of chemically induced mutations in the homozygote has
been continued using the same procedure as reported previously. The whole:lata including the previous one is given in the Table 1, in which the number
Jf tested mutants is doubled. It is noted that proportion of viable mutants
Ire more than half of total tested.
Table 1.

Results of lethality test for chemically induced
mutations in homozygotes
Homozygote

Mutagen
EMS,DES

ViableLethal35

25

Semi-L

Trans!.

Total

2

63

2) Complementation test
Complementation test was performed among similar type of induced
nutants in two groups; i.e. lethal red and viable red. Samples were exracted one per each mutant strain. In the lethal red group 11 mutant lines
I'ere tested, among which 10 were complete lethal and one was semi-lethal
ly hatching time. Those strains were crossed each other in all combinaions. Mutant red phenotypes were segregated in all crosses, which diedluring embryonic stage excepting crosses with a semi-lethal strain. The
lnding indicated that no complementing relationship exists among those 10~thal red strains. This may be accounted for by assuming that all thosemtant strains have a deficiency in common in addition to red mutation.
Similar test performed on viable red mutants brought about different~sults. Nine mutant lines were crossed each other. In all those crosses~d eggs were segregated as expected. They were viable. But in someross combinations frequency of the incidence of batches in which pink
ggs are segregated were anusually high, suggesting that those were trisomies
1 regard to their chromosome constitution. Among tested 5 mutant lines

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

were assumed to be trisomic and the rest 5 mutant lines were disomic.
Taking into consideration the genotypic classification of the recovered
mutants and the results of complementation tests, the proportion of trisomics
among viable red mutants was roughly estimated to be 13/35, which was
unexpectedly high.
In summary, most lethal mutations and some viable mutations were assumed to be disomic but there were a fairly large proportion of trisomics
among visible mutations. In this regard, a mere comparison of the viablewith the lethal is mere superficial.
High Incidence of Translocation between Wand the Fifth Chromosomes
Among Mutants Detected by Specific Loci Method in the Silkworm
Yataro TAZIMA, Kimiharu ONIMARU and Akio OHNUMAExperimental induction of a translocation involving W chromosome andan autosome has been reported by several workers in the silkworm.
Although such tanslocations had been successfully induced, the frequencyof the incidence was fairly low in those experiments excepting a case reported by Hasimoto (1948). We have observed in this connection cases ofsurprisingly high incidence of such translocation among mutants recovered
as a visible recessive mutation at the marker gene locus.
Wild type females of the silkworm were treated by either ,-rays or achemical mutagen, EMS, and mated to males of a marker strain with pe reo
In their F r , mutants were obtained either as a whole (pe or re) or as amosaic (pe, + or +, re) with regard to the expression of marker genes.
These were subjected to lethality test in homozygous condition. For thispurpose a mutation bearing chromosome was led to homozygous condition
via appropriate genetic procedures. For instance, in a case of pe mutation,
the mutant females were mated to +re males and in F r only individuals ofwild phenotype were crossed inter. se to produce F 2, and homozygotes formutation bearing chromosome were obtained as one of the segregants in
F 2 : 1 pe+: 2+ + : 1+re. In some cases of F r batches of whole mutants
or BFr batches of mosaic mutants, all wild type individuals gave rise tofemales. Further analysis revealed that those females were carriers oftranslocation chromosome between Wand the fifth chromosomes.
The incidence of such translocation was observed in five different experimental series and the cu1culated frequency of such incidence was

65

RESEARCHES CARRIED OUT IN 1976
Table 1. Translocations involving Wand the fifth chromosome discovered
among mutations at loci marked with +pe and/or +re genes
Name oftrans!.

OriginalstrainMutagenic
agent

Phenotype ofmutants captured

Frequency
of incidence

Year

aT
M28
ON
EM21
EM30

C108
C108
C108
AojukuAojukuSpont.
Spont.
7-ray
EMS
EMS

pe
pe
re
pemos
remos

1/12
1/5
1/24
1/17
1/8

1961
1975
1972
1975
1975

found surprisingly high (Table I).
The over-all frequency of the translocation was 5/66, or 1/13 per detectedmutants at specific locus. This is surprisingly high. The expected frequency
is calculated to be 1/27, even when all recovered mutants were assumed tobe due to translocation. The discrepancy is obvious and seems to suggestthat there might exist some kind of special prejudice working between the W
chromosome and the fifth chromosome in the silkworm.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

VII.

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICRO-ORGANISMS AND PLANTS
Effect of Gamma-Irradiation on Growth of Cultured
Haplopappus Cells
Estuo AMANOSoft and friable callus of Haplopappus gracilis (2n=4) can grow in liquidmedium. Suspension culture of cell line HSe was used to obtain gammaray dose response curve for cell growth. Liquid culture medium used was
1/2 strength of modified Murashige and Skoog's medium without agar.
This medium contains I ppm of 2.4 D, 0.15 % of yeast extract, besides
vitamines and inorganic salts. Under rotation culture (4 rpm) at 25°C
and initial cell concentration of more than 2 mg/ml by fresh weight, cells
grow at the rate of about 1.4 fold increase a day in fresh weight, up to 9th
day and reach to plateau on about 20th day. 1 ml of cell suspension
cultured for 14 days was inoculated into 20 ml of liquid medium in 100 ml
Ehrenmyer flask and irradiated by mcs gamma-rays up to 25 kR. Freshweight was measured on the 14th day of rotation culture at 25°C. Freshweight in mg/ml decreased as the gamma-ray dose increased. 50 %reduction
dose for fresh weight was estimated to be about 6 kR. 10 kR irradiation
reduced the fresh weight to about 15 % of a kR control. Cultured cells
seemed to be resistant compared to normal fresh tissues. This might bedue to the number of active cells in the population or tissues responsible forthe growth.
Measurement of Waxy Starch Phenotype in Cereal Crops
Etsuo AMANO

In the studies of waxy starch mutation in maize and rice, it became
necessary to compare the phenotypic expression of waxy starch mutation
quantitatively, among mutants as well as kernels. For this purpose, blue
value method was modified to fit for analysis of a single kernel and forlarge number of samples. Normal endosperm starch contains about 25 %amylose which stains blue black by iodine, and 75 % amylopectine whichstains reddish purple to brown. As the color of iodinated amylose is very

RESEARCHES CARRIED OUT IN 1976

67

dark, normal starch shows blue black color by iodine stain. Waxy starchmutants delete, or contain reduced amount of amylose, express waxy or
opaque appearance in endosperm, and stain purple to brown color byiodine.
In the colorimetric analysis used here, two wave lengths were adopted.
Blue light (430 nm) which is absorbed by major component of starch,
amylopectine, after iodine stain, is used to adjust concentration of starchsolution. Transmission (T value) of red light (660 nm) of the iodine stained
starch solution adjusted to the concentration of 50 %transmission at 430 nm,
was used as index of amylose content, or waxy phenotype. The apparatus
developed for this measurement include microtube pumps and a small mixing motor mounted to suit for dilution in the measuring cell. Changes of
the T values for the two wave lengths by dilution of the sample solution
were recorded on a X-Y recorder. The index of waxy phenotype of eachsample was determined from this dilution curve.
Starch solution was prepared in a small test tube from a single kernel.
Each kernel was crushed and 5 ml of water per tube was added. Test
tubes were then heated in an autoclave for 15 min. at 1 atm. After coolingto room temperature, 1 ml of supernatant was taken into measuring celland mixed with 0.1 ml of 0.2% I~KI solution. Non~waxy starch showedvery dark blue color and waxy mutant starch showed reddish purple. For
the preparation of the starch solution, alkali dispersion was tested but it
required neutralization before iodine staining, and moreover, difference inthe waxy indices was reduced.
T value of normal non-waxy kernels distributed from 10 % to 20 % andwaxy starch around 60 %for large kernels of maize as well as small grains ofItalian millet (Setaria italica). Among six EMS induced waxy mutants inrice so far examined, two showed reduced waxy indices around 40 %,indicating they were intermediate or leaky waxy mutants. These were alsointermediate in their appearance of homozygote endosperm. Besides thesehomozygotic intermediate phenotype, often the waxy indices of normal
non-waxy kernels in segregating F 2 population distributed from 10 % to40 %. This might be due to gene dosage effect in triploid endosperm. In
fact, in some mutants, kernels of intermediate or semi-waxy appearance
segregated waxy kernels in the progeny indicating that they were heterozygotes. Measurement of mutant and heterozygous kernels will be continued
to characterize the mutant phenotype and to confirm the gene dosage effect.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

Isolation of a New Tester Strain of Bacillus subtilis for the
Screening of Environmental Mutagens and Carcinogens
by Bacterial Cell Fusion
Koichi HIRANO, Taiji ASANO, Yoshito SADAIE and Tsuneo KADA

Salmonella typhimurium strains of Ames system are widely used as tester
strains for detecting environmental mutagens and carcinogens (B. N. Ames
et al. 1973 Proc. Natl. Acad. Sci., 70: 782, 1975 Proc. Natl. Acad. Sci.,
72: 979), and Bacillus subtilis strains have also been utilized in many labora_
tories to· examine the hazardous nature of chemicals by means of recassay, prophage induction assay, forward mutation assay using spores, andso on.
Recently, a new tester strain of B. subtilis was isolated by Sadaie et al.
(1976 J. Bacteriol., 126: 1037). It was constructed by introduction of
dna-8132 (Hara, H., Nature New BioI., 244: 200) into the polA59 strain(Gass, K. B., 1971 J. Bacteriol., 108: 364). The constructed strain, NIG
1124 (his met dna-8132 poIA59), showed an enhanced mutability for basechange type mutagen such as AF2.
In this case, nonsense mutation of his gene might be reverted by theerror-prone repair function of the dna gene product(s) in the absence of the
polA+ gene function(s).
To obtain a more sensitive strain, we constructed a new strain by meansof the bacterial cell fusion technique developed by Schaeffer et al. (1976
Proc. Natl. Acad. Sci., 73: 2151). The fusion was performed between B.
subtilis strains NIG 1122 (his met dna-8132) and UVSS-19-8 (met thy trp
uvr asp) isolated by Munakata et al. (1969 Mutation Res., 7: 133).
The newly isolated strain BKH 5002 (trp dna-8132 uvr) showed a markedly increased response to ICR-170, a typical frameshift type mutagen, in
producing an increased number of Trp+ revertant colonies. This strain
was also reverted by a base change type mutagen such as AF2.
Furthermore, when the strain was lysogenized with temperate phage SP02,
it was found that dimethyl nitrosamine, which is one of the presumed humancarcinogens showing a weakly positive effect in the Salmonella system, could
be detected at a markedly lower doses by pre-incubation with the 89 mixture.

RESEARCHES CARRIED OUT IN 1976

69

Highly Sensitive Rec-assay Using Spores of Bacillus subtilis
Koichi HIRANO, Hisako MATSUMOTO and Tsuneo KADA

The rec-assay procedure is widely used for mutagen screening. Thismethod uses a pair of Bacillus subtilis strains M45 (arg trp rec-45) and H17(arg trp). When a drug produces damage in DNA, it may show increased
lethality upon Rec- strain over Rec+ strain (T. Kada et al. 1970 Ann.
Rept. Natl. Inst. Genet. 21: 72). Indeed, a wide variety of environmental mutagens were detected by this method, including AF2, Dexon,
phloxin and 2,4-dinitrophenyl thiocyanate, etc. Moreover, a wide drug
spectrum containing frameshift type or base-change type mutagens could
be detected by this method. Recently, using this method, many pesticides
were screened by Shirasu et al. (1976 Mutation Res., 40: 19). Theirresults showed the existence of close correlation between the rec-assay
positiveness and mutation inducibility.
B. subtilis is a unique bacterium, compared with enteric bacteria such as
Salmonella typhimurium or E. coli on account of permeability and spore
formability. Since spores are very stable at 4°C in water suspesion, it may
be possible to do quantitative analysis.
Our improvements on this assay were as follows;
1) Sensitivity
Using spore plates which contained about 1 X 106 spores in 10 ml of brothagar in a 90 mm plastic dish for M45 and 1 X 105 for H17, 10 or more foldhigh sensitivity was obtained compared with the current method using vegetative cells.
2) Reproducibility and quantitativity
Because of constant viability of spores, it is possible to obtain reproducible results about the differences between diameters of the inhibition zone
for the M45 plate and those of the H17 plate. Moreover it is also possible
to obtain quantitative values for the minimal inhibition concentration(MIC), determined by means of extrapolation of the dose response curveto the drug concentration producing no inhibition zone.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

DNA Repair Enzymes in Ataxia Telangiectasia and Bloom's
Syndrome Fibroblasts
Tadashi INOUE, Koichi HIRANO, Akiko YOKOIYAMA, Hatao KATO
and Tsuneo KADA

Ataxia telangiectasia (AT) and Bloom's syndrome (BS) are autosomal
recessive diseases of man characterized by telangiectasia, immunodeficiency,
and an increased frequency of malignancy. The ionizing radiation sensitivity of AT patients has been indicated by in vivo and in vitro studies ofskin fibroblasts. On the other hand, fibrolasts from BS patients have beenreported to be ultraviolet light sensitive. An enhanced level of spontaneous chromosome instability was also observed in both AT and BS cells.
These features suggest that some kinds of impairment in DNA repair are
the molecular bases of the diseases. From this point of view, we have compared the activities of "primer activating" enzyme as well as the apurinicsite specific endonuclease in extracts from AT strain and BS cells with thosefrom normal cells. The primer activating enzyme was assayed by a new
system in which the capacity of cellular extract to enhance the priming activity of gamma-irradiated colicin El DNA for purified type I DNA polymerase
was measured (Inoue, T. and T. Kada 1977 Biochim. Biophys. Acta 478:234-243). The experiments yielded the results that the primer activating
enzyme activity of AT cells was substantially lower than normal cells, andthat BS cells had a slightly lower level of the activity. These results indicatethat some kinds of enzymes which function in the excision-type repair
prior to repair replication by DNA polymerase are defective in the AT and
BS cells. However, cellular extracts from these three strains had similar,
if not identical, activities of the apurinic site specific endonuclease, suggesting that the decreased activity of the primer activating enzyme in AT and BS
cells was not due to the defect in the endonuclease (1977 Biochim. Biophys.
Acta 479: 497-500).
Purification and Properties of Bacillus suhtilis Endonuclease
Specific for Apurinic Sites in DNA
Tadashi INOUE and Tsuneo KADA

Considering the multiplicity of lesions created in gamma-irradiated DNA,
it is reasonable to expect that a wide variety of enzymes have evolved, eachcapable of recognizing a different class of lesions and catalizing the important

RESEARCHES CARRIED OUT IN 1976

71

step in an excision-type repair pathway. Indeed, we have identified several
kinds of enzymes which may function in the repair of gamma-irradiatedDNA in extracts of Bacillus subtilis cells using a new assay system in whicha capacity of cellular extracts to enhance the priming activity of gammairradiated DNA for purified type I DNA polymerase was measured. Preliminary experiments revealed that one of them was an endonuclease specificfor apurinic sites (Noguti, T. and T. Kada 1975 Biochim. Biophys. Acta
395,294-305) and the other was 'cleaning' exonuclease (Inoue, T. and T. Kada
1977 Biochim. Biophys. Acta 478: 234-243). The former enzyme waspurified to near homogeneity and had properties described below.
The enzyme had a molecular weight of about 53,000 as determined by gel
filtration and SDS-polyacrylamide gel electrophoresis. It had a pH optimumof 8.0 and has no MgH requirement, and was fully active in the presence of
EDTA. The depurinated DNA was an especially good substrate, however,alkylated DNA was also attacked to some extent. Associated activities of
exonuclease and ribonuclease were not detected. The enzyme appeared to
produce 3'-OH near apurinic sites in DNA, since the priming activity of
DNA for DNA polymerase was enhanced by the treatment of the enzyme.
Utilization of a Mutator Strain for the Isolation of
Mutants in Bacillus suhtilis
Yoshito SADAIE, Yoshiko OHTA and Tsuneo KADA

Utilization of strong mutator strains may be useful to isolate mutants in
Mutations produced can be easily transferred, purified
and stabilized by transformation of a second strain by DNA from original
mutants because transforming DNA integrated into host genome usuallycomprises a small segment of whole chromosome coding for about one
cistron. Revertant analysis on the constructed mutants is necessary to see
whether or not the mutants carry a single mutation. A strong mutator strain
NIG1125 (met his mut-l) of B. subtilis was constructed by transformation.
Two mutations, met and his, appeared to be suppressible by a nonsense
suppressor mutation (Okubo and Yanagida, J. Bacteriol. 95: 1187-1188;
Shub, J. Bacteriol. 122: 788-790) and the mutation mut-l reduces DNA
polymerase III activity and greatly enhances spontaneous mutation frequency(Bazill and Gross, Nature New BioI. 243: 241-243). We used this mutator
strain for the isolation of mutants in B. subtilis and the isolated mutations
Bacillus subtilis.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

were transferred by transformation into the second strain.
(1) Twenty four RifrGspts mutations were isolated at a frequency
of about 1.8 X 10- 7 per cell. Nine mutants among them grew well at the
restricted temperature. All the mutants carried two mutations rif and gsp,
most of which was linked to cysAl4.
(2) Temperature sensitive nonsense suppressor mutations were isolated
from colonies grown on minimal agar at 42°C (or 30°C) at a frequency of
about 1.6x 10- 5 (or 3.2x to- 6) per cell. DNA was isolated from themutants and used to transform NIG1121 (met his) to prototroph at 42°C
(or 30°C) in order to fix the suppressor mutation.
The Induction of Monoecious Mutants in Cncumber
Taro Fum

A female strain (MSU) of cucumber was exposed to 50 kR of gammarays, and among 28 M 1 plants observed, three were found to develop male
flowers, which were designated as MSU-50A, SOB and SOC. The M 2 plants
raised from their selfed seeds segregated into monoecious and gynoecious
types giving 19: 15, 32: 27 and 23: 11 ratios, respectively. The monoecious
segregants showed a wide variation in the number of nodes with male
flowers; a plant had only one such node at the basal part, while anotherhad male flowers on 36 of 50 nodes observed. The monoecism: gynoecism
ratios in the M 3 generation derived from MSU-50A were 8: 15 in a line and
16: 1 in another. The M 4 plants from the latter M 3 line were exclusively
monoecious (35: 0).
The data suggest that monoecism was promoted by selling, though thenumber of plants observed was too small to consider segregation ratios.
The tendency of seIfing to promote monoecism was also observed in a
backcrossing experiment carried out previously.
A backcrossing experiment was made again in which 50A, 50B and 50C
were crossed with the control plants of MSU. The B1 generation showed
8: 9, 17: 17 and 11: 16 ratios of monoecious and gynoecious plants, respectively. The control MSU plants showed no monoecism in the same culture(0: 34). Although the incidence of monoecious plants in the M 1 generation, 3/28, was too high to consider it to be a mutation, the inheritance ofmonoecism in the progeny suggests that some heritable change in themechanism of sex dtermination has resulted from irradiation.

RESEARCHES CARRIED OUT IN 1976

VIII.

73

POPULATION GENETICS (THEORETICAL)
Population Genetics and Molecular Evolution
Motoo KIMURA

Recent studies of evolution and polymorphism at the molecular level(i.e., at the level of internal structure of the gene) have brought many puzzling as well as enlightening results. It appears that, in addition to natural
selection, chance (in the form of random gene frequency drift) is playing
a much larger part than previously considered. Furthermore, natural
selection is likely to be based more directly on the secondary and the higher
order structure of informational macromolecules rather than ecological conditions, usually in the form of negative selection. In this respect, there is
growing evidence suggesting that very slightly deleterious mutations whose
selective disadvantages are not excessively large as compared with themutation rates are playing a significant role in variation and evolution at themolecular level. For details, see Johns Hopkins Medical Journal 138 (6),253-261.
Role of Very Slightly Deleterious Mutations in
Molecular Evolution and Polymorphism
Tomoko OHTA

Several models of multiple slightly deleterious alleles are reviewed andtheoretical consequences of slightly negative selection are discussed in conjunction with evolution and variation at the molecular level. Facts areorganized which may be satisfactorily explained by the hypothesis of veryslightly deleterious mutations. They are: (1) There appears to be an
upper limit in heterozygosity for protein loci as measured by electrophoresis.
(2) The excess of rare alleles is more pronounced in Drosophila than in man.
(3) Correlation of heterozygosities at a locus among sibling species of the
Drosophila willistoni group is too high compared to what is expected by thestrict neutral theory, while it is not so among human races and between man
and chimpanzee. (4) The rate of protein divergence is exceptionally high in
Hawaiian Drosophila. For details, see Theoretical Population Biology 10,
254-275.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

IX.

POPULATION GENETICS (EXPERIMENTAL)

Elimination of the Polymorphic Inversions from the Laboratory
Populations of Drosophila melanogasterYutaka INOUE and Takao K. WATANABE

When natural populations of Drosophila have been transferred to alaboratory environment, the relative frequencies of the founder chromosomal
variants may change. Three natural populations (Akayu, Katsunuma,
Ishigaki) of D. melanogaster were transferred to 14 laboratory cages. The
initial frequency of the polymorphic inversions per autosome arm was 12.0 %for Akayu, 16.6% for Katsunuma and 53.0% for Ishigaki populations. For
the Akayu and Katsunuma populations, only 40 generations of cage life
were quite enough for the reduction of the frequency to less than 1%. For
the Ishigaki population, the average frequency of the polymorphic inversions
reduced to be 0.4 % at the 70th generation.
Thus, we can conclude that the polymorphic inversions found highly
polymorphic in natural populations in Japan turn deleterious when they aretransferred to laboratory cages. The magnitude of the selection coefficientis about 5 percent.
On the other hand, the frequency of the rare inversions was found to be
0.26 % for the major autosome arm both in natural and cage populations.
This suggests that newly arisen chromosomal mutations are very deleterious
in natural and cage environments enough to be selected against soon after
the mutation occurred.

Equilibrium Frequency of Lethal Genes in Cage Populationsof Drosophila melanogaster
Won Ho LEE and Takao K. WATANABE

Lethal and sterility mutations were accumulated in a cage population
which was initiated with lethal- and sterility-free second chromosomes ofD. melanogaster. It took about 2,000 days for the frequencies of thesegenes to rach equilibrium levels, i.e. the frequencies of 18 % lethal and 9 %male-sterile chromosomes. Two other cage populations which were initiatedwith random chromosomes sampled from natural populations and kept for

RESEARCHES CARRIED OUT IN 1976

75

more than 11 years in the laboratory showed 19-20 % lethal content.
In the course of accumulation of mutations, the frequency of lethals wasalways higher than that of male-steriles, and the frequency of male-steriles
was always higher than that of female-steriles.
The elimination rates of lethal chromosomes by homozygosis (0.08-0.19 %)in these populations were smaller than the mutation rate (0.73 %). Therefore, a large part of selection against lethal genes must occur in heterozygous
condition. By using Nei's formulae, the deleterious effect of a lethal genein the heterozygote (h) was estimated to be 0.035.
The effective population number in the cage population was estimated tobe 1,000-2,900, while the actual population number was 3,500-7,800.
Thus, about 30 % of the censused adult population contributes to the next
generation.
Enzyme and Chromosome Polymorphisms in Natural Populationsof Drosophila melanogaster
Takao K. WATANABE and Taishu WATANABE

Collections of D. melanogaster from three natural populations (Katsunuma, Akayu, Ishigaki) and three cage populations were analyzed for
enzyme and chromosomal polymorphisms. Allelic frequencies at the Adh(2-50.1) and a-Gpd (2-17.8) loci were compared with polymorphic inversion,
In(2L)B (22D-34A) and In(2R)C (52A-56F), frequencies. There was asignificant positive correlation between the frequencies of Adh s and In(2L)B,
and a-Gpd F and In(2L)B, caused by linkage.
Two fitness components of these enzyme and chromosomal variants were
measured in two different environments; neither of the two loci showed
heterozygote superiority in viability or productivity, while the inversion
heterozygotes showed a superior productivity compared to the correspond~
ing homozygotes. Therefore, allelic frequencies at the Adh and a-Gpd lociseem to be determined by the frequency of In(2L)B in natural populations
where the invesrion appears to be maintained by selection, but not by theselection on the enzyme itself.
On the other hand, inversion-free cage populations maintained in thelaboratory for a longe time showed considerably larger variation in thefrequencies of these enzyme alleles, which seem very likely to be a consequence of random drift.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

Although the history of the polymorphic inversion remains unknown,
we can speculate that this adaptive chromosomal rearrangement, In(2L)B,
has occurred in one standard sequence chromosome which had Adh s anda-Gpd F alleles and spread throughout the world. For details, see Genetics
85: 319-329 (1977).

RESEARCHES CARRIED OUT IN 1976

X.

77

EVOLUTIONARY GENETICS

Geographical Distribution of Drosophila simulans in Japan
Takao K. WATANABE and Masaoki KAWANISm

A census of domestic or semi-domestic species of Drosophila has beentaken throughout Japan in the fall of 1976, attempting to know the present
distribution of D. simulans which is a recently immigrant species. A totalof 85 collection sites throughout the country was chosen in parks, temple or
shrine backyards and similar places which were nearby human habitation.
D. simulans was already distributed in the two wide areas; Kanto-Tokai
district and Kyushu district. The intervening area, Western mainland, isalmost lacking simulans except for the west end (Shimonoseki). Shikoku
is an interesting area in the mosaic occurrence of this species. Other
places are at present free from simulans with a few temporary introduced
individuals.
The two years survey (1975-1976) of the domestic species at Yamanashi
and eastern Shizuoka areas suggested that the invasion of simulans from the
Pacific coast to the inland area was not so fast.
The colonization of simulans remarkably affected on other domestic species,
especially on melanogaster. In the simulans lacking sites, melanogasterwas the most abundant species (33.6 %), followed by immigrans (28.6 %)and lutescens (17.0 %). While in the simulans abundant sites, simulans was
the most abundant species (36.7 %), followed by immigrans (27.0%), lutescens(12.5 %) and melanogaster (11.8 %).
How Genes Evolve; A Population Geneticist's View
Motoo KIMURA

This paper is based on the author's lecture delivered at the College deFrance on May 14th 1976. After a brief discussion of historical development of evolutionary theories, a history of population genetics itself isreviewed. It is then pointed out that, during the last decade, with the adventof molecular genetics, studies of evolution has been revolutionized. This
led to the theoretical framework called "Molecular population genetics" inwhich the mechanisms of evolution and variation at the molecular level are

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

treated using the diffusion equation methods in population genetics ("diffusion models"). The neutral mutation-random drift hypothesis ("the neutral
theory") and controversies following its proporsal in 1968 are reviewed.
Also, evidence supporting the neutral theory is detailed particularly in
relation to structure and functional constraints of the evolving molecules.
The present paper consists of the following sections;(1) From Lamarck to the three savants, (2) Development of theoretical
population genetics, (3) Dynamics of mutant substitution and the rate ofevolution, (4) Progress and degeneration in evolution, (5) Our past andfuture. For details, see the original paper [Kimura, M. (1976) Ann.
Genet. 19(3): 153-168.]
Archeology, Population Genetics and Studies of
Human Racial Ancestry
Takeo MARUYAMA

Using traditional classification, many people have tried to determine theplace and time of origin of the major human races. Two basically conflicting views have prevailed, one asserting that races developed as local variations in a species evolving phyletically over much of the Old World for up
to one million years, and the other view holding that present races are
local variations on populations recently expanding into most areas andreplacing the previous hominid inhabitants, this occurring since the timeof the Neanderthals. Both views are based on the same archeological data.
Gene frequency data and time-calibrated genetic distance measures haverecently been applied to this problem. Here we show that the probabledemographic nature of Pleistocene populations has obscured genetic distances to such an extent that they cannot be used to discriminate between
the two viewpoints of racial origins. The racial classifications themselves
are probably not useful in this context and obscure the question, so that
we presently do not have a scientifically valid understanding of racial
origins. Published in American Journal of Physical Anthropology 44: 3150.

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79

Simple Model for Treating Evolution ofMultigene Families
Tomoko OHTA

The evolution of repeated genes forming a multigene family was studied
by assuming unequal crossing-over. As a first step, the single chromosomalline is considered. It was shown that the contraction-expansion of mutant
genes due to unequal crossing-over within a chromosome may be treated by
the diffusion model developed by Kimura for analysing the behavior ofmutant genes in finite populations, if the number of gene units in the multigene family stays constant (n). Two consecutive crossovers (involving
duplication and deletion) is called one cycle of the process. Then, one
cycle is shown to be equivalent to 4Ne /n 2 generations of random genefrequency drift, in which N e is the effective population size. For details,
see Nature 263, 74-76.
Simulation Studies on the Evolution of
Amino Acid Sequences
Tomoko OHTA

A model of molecular evolution in which the parameter (intrinsic rateof amino acid substitution) fluctuates from time to time was investigated bysimulating the process. It was found that the usual method of estimationsuch as Poisson fitting underestimates this variation of the parameter whenremote comparisons are made. At the same time, four distance measures(minimum base difference, Poisson fitting, random nucleotide substitutions
and negative binomial fitting) were tested for their accuracy. When thesubstitution rate is not uniform among the amino acid sites, the negative
binomial fitting gives most satisfactory results, however, one needs to know
the parameter beforehand in order to use this method. It was pointed outthat the fluctuation of the evolutionary rate is expected if the nearly neutral
but very slightly deleterious mutations play an important role on molecular
evolution. For details, see J. Mol. Evol. 8, 1-12.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

Reproductive Barriers Distributed in Melilotus Species andTheir Genetic Bases
Yoshio SANO and Fumiji KITA

In order to look into species relationships, crosses were made in all possible combinations of 9 species belonging to the subgenus Eumelilotus; 504
crosses were made among 53 strains in total. The reproductive barriers
found among the species were a crossing barrier (death of young F 1 zygotes),
F 1 inviability and weakness due to chlorosis, F 2 breakdown due to the occurrence of chlorotic segregants (classified into lethal and weak types),partial F I sterility, and a segmental interchange of chromosomes. All the
hybrids within species were normal. In crosses between M. alba and M.
suaveolens, the hybrids of annual strains showed more pronounced F I and F 2
chlorosis than those of biennial strains. The hybrids of self-fertile species
or forms tended to have a low reproductive potential. The F I chlorosis
between the above two species was considered to be controlled by a set ofcomplementary dominant lethals, ChI and Ch 2 • The F 2 chlorosis in thesame species hybrid was controlled by the sets of duplicate recessive genes,
chll and chl2 for lethal type and chwi and chw2 for the weak type. The
chl2 and ChW2 loci were linked with a recombination value of about 13
percent. (Submitted to Can. J. Genet. Cytol.; this experiment was carriedout at the Hokkaido University.)

Analysis of Genes Responsible for the F I Sterility between
Oryza sativa and O. glaberrima
Yoshio SANO and H. 1. OKA

This experiment has been continued since 1966 (B I generation) to observevarious crosses between isogenic Fl-sterile lines with the genetic backgroundof either sativa or glaberrima parent (Ann. Rep. 24: 67 & 25: 85). The
genic system responsible for the F I sterility, so far obtained was as follows:
The sativa parent has AI/AI and its isogenic Fl-sterile lines have Als/Als
introduced from the glaberrima parent. When an AISgene is present in the
sporophytic tissue (AI/AIS), the gametes (both pollen and embryosac) carrying
Al deteriorate during the course of development. The same hypothesis also
applied to the isogenic lines with the genetic background of glaberrima
parent, for which genes A 2 : A 2 s were assumed.

RESEARCHES CARRIED OUT IN 1976

81

A new evidence supporting this hypothesis was that in a AI/Als hybrid,a gene controlling apiculus coloration which· was introduced from theglaberrima parent and tightly linked with Als showed no P 2 segregation(all P 2 plants were colored) since all Al genes were eliminated by gametic
selection. However, the presence of other genic systems conditioningsterility was also suggested; for instance, a true-breeding semi-sterile line withsativa background was obtained, which seemed to be homozygous forcomplementary genes controlling sporophytic sterility. To look moreclosely into the genic systems, a backcrossing experiment was initiated using
a sativa isogenic line with AIs/AIs and a glaberrimaisogenic line with A 2s/
A2 s; their intercross produced highly sterile PI plants.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

XI.

HUMAN GENETICS

Hereditary Retinoblastoma: Penetrance, Expressivity
and Age of Onset
Ei MATSUNAGA

It is now well recognized that there are two etiologically distinct forms ofretinoblastoma, one due to an autosomal dominant gene with incomplete
penetrance, and the other being non-hereditary and perhaps due to somatic
mutation. As to the hereditary form, the degree of penetrance has been
generally supposed to be about 80 %. However, analysis of family data
published in Japan concerning 29 kindreds with two or more cases ofretinoblastoma revealed that, in the children who received the gene from acarrier parent, both penetrance and expressivity increase with increasing
degree of expressivity in the parent. This suggests that suppressor genes atother loci play a significant role in manifestation of the major dominantgene for retinoblastoma. The estimate for the average degree of penetrance
varies from 0.7 to 0.9, depending upon the method employed. It is certainthat the value will be increased with increasing number of survivors ofhereditary retinoblastoma. Furthermore, there was some evidence that in
the hereditary form of retinoblastoma onset tends to be later in unilateralthan in bilateral affection, though in the non-hereditary form on;>et will befurther delayed. For details, see Humangenetik 33: 1-15, 1976.

A Survey on Maternal Age and Karyotype in Down's
Syndrome in Japan, 1947-75
Ei MATSUNAGA and Hiroko FUJITA1)

In the absence of any nation-wide monitoring system in Japan, we
analyzed data concerning karyotype and maternal age of 1,954 cases of
Down's syndrome to see if the rate of chromosome mutations leading tothis abnormality has been enhanced in recent years. Comparison of datafor patients born in 1947-60 with those born in 1961-75 revealed littlechange with time in the proportions of cases due to different karyotypes,
the overwhelming majority being of 21 trisomy type in both periods. Howl)

Department of Child Health, Osaka City University, OsakaRESEARCHES CARRIED OUT IN 1976

83

ever, there has been a remarkable decline in the mean maternal age from 33.1
years to 29.7 years as well as in the variance from 50.5 to 29.4. While therate of decline in the variance was almost the same as that for all births
occurring in the same periods, the decline in the mean maternal age wasmuch greater for the patients than for all births, suggesting that the rate ofnondisjunction might have increased in younger rather than in older
mothers. However, when the risk of bearing a child with Down's syndrome
for mothers aged 40-44 is taken as unity, no evidence was found for an
increase with time in the relative risk for younger mothers. Moreover,results of surveys made in 1960 and thereafter in different parts of Japan
indicate that the crude incidence rate of Down's syndrome at brith has beenaround 0.10 %, giving no indication for an upward trend. These findings
seem to be rather remarkable when we consider the serious environmental
pollution, including possible genetic hazards, with which Japan has been
faced in the 1960s. For details see Humangenetik 37: 221-230, 1977.
LEA Technique in the Detection of Chromosome Variants in Man
Yasuo NAKAGOME, Shigehiro OKA, Ei MATSUNAGA and Masataka ARIMA

Both Q- and C-band techniques have widely been used in the detection ofchromosome variants (polymorphism) in man. However, many problems
remain still open as only very limited number of variants are detectable bythem. In the present report, a technique is described which uses DNAreplication pattern for the purpose of identifying new variants which may
not be detected by the two conventional techniques.
BUdR (30 ,ug/ml) was added to leukocyte cultures (standard 3-day culture)
24h prior to the harvest. Fourteen hours later, cells were rinsed and fed
with preconditioned medium supplemented with thymidine (3 ,ug/ml). The
preparations were stained with quinacrine mustard, photographed under afluorescence microscope, destained with 1: 1 mixture of ethanol and acetic
acid, stained with acridine orange (100 ,ug/ml in Sorensen's buffer solution at
pH 6.5 for 5 min) and then photographed again. Both photographs fromeach metaphase were compared each other.
Segments of chromosomes which replicated at the last part of the S-phaseshowed intense fluorescence in acridine stained preparations. These segments will be referred as LBA bands and the procedure as LBA technique(late-replicating bands detected by the BUdR-acridine technique) based on

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

the suggested system of nomenclature in the Paris Conference (supp!. 1975).
Most of the LBA bands corresponded to Q-positive bands. Exceptions
included qh regions of nos. 1, 9 and 16 pairs, centromeres of most pairs ofchromosomes and several other polymorphic regions. All of them fluoresced
intensely.
A total of 41 variants were detected in 40 acrocentrics from 4 unrelated
individuals. Twenty-eight of them were detected only by the LBA-technique, 11 of them being in short arms and 17 of them in centromeres. Seven
variants, including 4 of those in satellites, were detected only by the Qtechnique. Six short arm variants were detected by both methods. As tocentromeric variants of nos. 3 and 4, both of them worked equally well.
In the evaluation of qh variants in pairs 1, 9 and 16, the present (LBA)
technique proved itself to be a very effective implement. Because of
intense fluorescence, it was very easy to recognize and evaluate them. The
technique was practically free of technical variables coherent with the C-band
technique and therefore was possible to use the size of a C-negative euchromatic segment of a chromosome as a reference standard. LBA variants
were observed in centromeres of about 50 % of the members of the remaining 12 pairs of autosomes, i.e., nos. 2, 5, 6, 7, 8, 10, 11, 12, 17, 18, 19 and 20.
It appears that the LBA technique is especially useful in the detectionof variants of short arms and centromeres of acrocentrics. It is also usefulin the evaluation of qh and centromeric variants of non acrocentrics. On
the other hand, the Q technique is more efficient in the detection of variantsof satellites. It has been known that C treatment sometimes fails to work
after the Q staining. On the other hand, the LBA bands are not inpaired at
all by the preceding Q treatment. Thus, it appears that a sequential QLBA technique is one of the most efficient procedure available for the
purpose of detecting chromosome variants in man (for further details, see
Nakagome et al. and aka et al.: Human Genetics 38; 307 and 39; 31, 1977).
Holoprosencephaly in Human Embryos:

An Epidemiologic Study

Kohei SHIOTA and Ei MATSUNAGA

Among the large collection of human embryos in the Human EmbryoCenter for Teratological Studies of Kyoto University, 150 cases of holoprosencephaly (cyclopia-arhinencephaly series) were found during the period
between January 1962 and December 1974. The incidence of the defect inRESEARCHES CARRIED OUT IN 197685

the embryos was 4.1 per 1000 against 0.06 per 1000 newborns, which implies
that almost all of the affected embryos are destined to be eliminated prenatally. The occurrence was largely at random through time, and no'epidemic' was noted in particular years or months, but there appeared morecases derived from conceptions in winter than in summer months. The meanmaternal age did not differ significantly from that of the general embryonic
population, indicating that, although none of our cases were karyotyped,
chromosome aberrations such as trisomies 13 and 18 that are closely
associated with maternal age may not constitute a major part as causes ofholoprosencephaly in human embryos. Maternal age did not differ by thepresence or absence of associated external anomalies. No association was
found with paternal age, parental consanguinity nor with maternal medical
history, including irregularity of menstrual cycles, and smoking and drinking
habits. There was an indication that the mothers were prone to have repeated miscarriages, supporting the view that some kind of maternal predisposition is responsible for the causation of holoprosencephaly. Apartfrom various chromosome aberrations well documented as causes of thismalformation, polygenic mechanism probably accounts for the majority of
the cases with normal karyotype. Full data are published in Teratology
16: 261-272, 1977.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

XII.

BEHAVIORAL GENETICS

Genetic Variation of Walking Ability in Drosophila melanogaster
Sumio M. AKA! and Takao K. WATANABE

Genetic variation of walking ability in D. melanogaster was measured
by a Benzer countercurrent apparatus. A total of 74 homozygous lines
for the second chromosomes were obtained from Ishigaki population. The
mean walking ability of each line was measured in the dark (DW), in thelight (LW) and to the light (toL) conditions. Variances due to lines, sex
and line X sex were highly significant for each environment. Males walked
much more than females but correlation coefficient between sexes was
about 0.8.
Flies walked active in the light (LW) and it was more accelerated toward
the light source (toL). However, the walking abilities in the three light
conditions positively correlated. The correlation coefficient was 0.58 for
DW-LW, 0.43 for DW-toL and 0.85 for LW-toL.
Heterozygous effect of the walking genes was examined by 80 offspringmidparent combinations. Correlation coefficient was 0.74 for DW, 0.81
for LW and 0.68 for toL which were all highly significant.
These results suggested that the walking ability of Droosophila was controlled by the additive polygenes located in the second chromosomes.
Adult Emergence Rhythm of Homozygous Strains ofDrosophila melanogaster
Won Ho LEE and Chozo OSHIMA

Fifty strains, homozygous for each different second chromosome, wereisolated by Cy/Pm method from a natural population of Katsunuma in
autumn of 1975. Five crosses were prepared from a strain and each cross
was composed with 7 females and 5 males. They were permitted to lay eggs
on cultural medium of 5 vials for 12 hours under constant light condition
and thereafter transferred into new vials and permitted to lay eggs on cultural
medium for successive 12 hours. The same procedure was repeated more
two times simultaneously with 5 replications prepared from each strain.
The first A group of 5 vials, including total about 250 eggs on cultural
medium, was introduced into the Insectron at the beginning of light period

RESEARCHES CARRIED OUT IN 197687

of LD 12: 12, 25°C constant condition. The next B group of 5 vials,
including similar number of eggs on cultural medium, was introduced into
the Insectron at the beginning of dark period of LD 12: 12 condition. The
following C and D groups of vials were introduced into the Insectorn at thesame light condition as A and B groups respectively. When about 7.5 days
passed, adult flies began to emerge. After that, two times per day, that is,flies emerged for dark 12 hours and for light 12 hours were counted
respectively. Such counts of flies were repeated for at least 4 days. Mean
rates of flies emerged for light and dark periods were calculated by pooled
results of four groups (A-D) and mean rates of 5 replications were also
obtained. It has been investigated as a normal function of bioclock in brainthat most flies emerged synchronously for several hours after dawn period.
Then, the difference between mean rates of adult emergence for light anddark periods would indicate the synchronization of bioclock of the homozygous strain. Values of most strains, showing adult emergence rhythms,
distributed normally among 0.956-0.500 and four strains showed low values
below 0.50. The mean value of 50 homozygous strains was 0.731. From
the result, the function of bioclock of Drosophila fly would be polygenic
character and a few strains would have any defective genes.
Circadian Locomotor Activity of Drosophila melanogaster
under Noise Environment and Walking Behavior of a Group
under Shake Environement
Chozo OSHIMA and Won Ho LEE

Many homozygous strains for the second chromosome were extracted by
CyjPm method from a natural population of Katsunuma in 1975, and someof them were used in this experiment. Locomotor activity of a male flyin a small glass cell (5 x 10 x 50 mm) during several days was recorded
continuously by the electronic actograph. The locomotor activity was
found to be circadian rhythmic and two high peaks of locomotion appearedat dusk and dawn periods of the program controlled LD 14: 10 and NQ
12: 12 environments. The locomotion of a male fly under the constantdark environment was inhibited at low level and continued for about
twelve hours. On the other hand, the locomotor activity of a male fly
under NQ 6: 6 (white noise 100 phon) and constant dark environments
This research was supported by a grant from Ministry of Environment Agency.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

increased and continued for about twelve hours at high level. The excitement of a fly, stimulated simultaneously by circadian rhythmic change oflight and noise environments, was pacified after several hours and suchstates were repeated for several days, but the excitement, stimulated byacircadian rhythmic noise, continued without pacification for a week.
Thirty flies (N) of either male or female of each strain were introduced
into a terminal tube (dia. 12 mm, leng. 150 mm) and after 15 minutes,
number of flies (P) distributed in five connected tubes (a: 1-5) was counted.
Walking index (I;ap/N) was estimated. Walking activities of 61 homozygrous strains were estimated under light (L) and dark (D) environments
and shake (S) (5 cm cross ways 70 times/minute) environment. Total mean
walking index of 61 strains was obtained under each SL, QL, QD and SD
environments as follows; 1.79, 1.69, 1.44 and 1.34. The correlations
between all walking indexes of these strains under two environments except
for SL and SD, were highly significant. The effect of shake accelerated
walking activity of a group of flies under light environment, but inhibited it
under dark one.
Locomotor activities for six days of a male fly of strain 108, showing fast
walking index 3.63 under SL environment, and of a male fly of strain 40,
were recorded by a new actograph. The activity of the former was lower
than the latter, and then these characters, locomotion and walking behaviors,
would be regulated by different nerve centers. The former's bioclock lostits function as a nerve center under the constant dark environment.
Selection for the Discriminated Avoidance Learning
Ability of Mice
Tohru FUJISHIMA

The avoidance, discriminatory and discriminated avoidance performancesof mice were measured in percentage with an automated Y-type mazeapparatus, using buzzer and lamp as conditioned stimuli and electric shock
as an unconditioned stimulus (cf. Ann. Rep. 25: 82).
Selection was made bi-directionally for discriminated avoidance performance on the within-litter basis. The present report deals with thefirst generation of selection. A significant selection effect was observed in
the first generation. The selection differential, i.e. the difference between
the high and low lines selected, was 29.9, and the selection response was

RESEARCHES CARRIED OUT IN 1976

89

6.1. Accordingly, the realized heritability with standard error was 0.204±
0.0493. The correlated response for avoidance ability was positive andstatistically significant, but that for discriminatory ability was significantly
negative.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

XIII.

ECOLOGICAL GENETICS

Submergence Tolerance of Wild and Cultivated Rice Strains
Hiroko MORISHIMA

Seventy eight rice strains collected from different tropical countries (wild44 strains of Oryza perennis and 7 of O. breviligulata; cultivated-16 of
O. sativa and 11 of O. glaberrima) were tested for submergence tolerance.
Their seedlings at an age of 30-35 days, raised in a seed-bed box, were cut
at a height of 15 cm, and were submerged for 14 days in a water tank(water temperature being 21 Qq. Seven days after they were taken out of thetank, their survival was recorded. This experiment was carried out in
May-June of 1975 and 1976 with two replications. The wild species showeda wide range among strains. Among Asian strains of O. perennis, theannual type tended to have higher submergence tolerance than the perennial
type. The American strains of O. perennis has a low tolerance even though
they grow in deep water in their natural habitats. Strains of O. sativa also
showed a wide range but no particular difference between the Indica andJaponica types. O. glaberrima seemed to have a low tolerance. It waspointed out that submergence tolerance and floating ability were independent characters.
Variations in Copper Tolerance of Alopecurus aequalis Strains
Hiroko MORISHIMA

The seeds were collected from copper-polluted and normal rice fields at
Ohta, Gunma-ken and Tsuru, Yamanashi-ken. A total of 28 strains wereused for this experiment, each representing more than 20 plants standing at
the same site. They were tested in pots (20 cm in diameter) at three levelsof copper concentration in the soil, i.e., 0, 200, and 400 ppm (in the formof CUS04), with two replications. About 500 seeds per pot were sown inearly November. In December-January, the seedlings were killed by frost
needle at different rates; copper treatment affected the restoring ability offrost-damaged roots and reduced survival. Percent survival was recorded in
March for each pot, and the survival rate of a strain in copper-treated potsin percent of that in control (no copper) pots was considered as showing

RESEARCHES CARRIED OUT IN 197691

its copper tolerance. The ratio of survival rates ranged from 26 to 77
percent among the 28 strains tested, and showed a significant correlation(r=0.50) to the copper content in surface soil (4-160 ppm) of the fields fromwhich the strains were collected.
The number of plants of this species arising from the buried seed populations in soil samples, in percent of the total plant number including otherspecies, was exclusively high (above 90 percent) in the soil samples from
polluted fields (above 50 ppm copper), though it ranged from 20 to 97
percent in the soil samples from normal fields. This indicates the dominanceof Alopecurus aequalis in copper-polluted fields. The partial correlation
coefficient between dominance and copper tolerance was significant (r=0.61)
when copper content in the· soil was used as a fixed variable. It may besuggested that the evolution of copper-tolerant ecotypes plays a role in thedominance of this species in copper-polluted fields.

The Impact of Copper Pollution on Barnyard Grass Populations
Hiroko MORISHIMA and H. I. OKA

To look into the microevolution of weed populations in copper-polluted
rice fields, we have been engaged in observations of weed communities.
This report deals with the results so far obtained in the barnyard grass(Echinocloa oryzicola, 4X, and E. crus-galli, 6X). A total of 210 barnyard
grass strains (progenies of single plants) collected from copper-polluted (25310 ppm) and control (1-18 ppm) fields were tested for copper tolerance
and other characters. Copper tolerance was evaluated by the ratio of
performance in copper-treated plot to that in control (no copper) plot,
mainly in gravel culture and partly in water culture. The 4X species tended
to be more tolerant than the 6X species. It was also found that 6X strains
resembling the 4X species morphologically had higher tolerance than typical
6X strains. In both species, the populations in polluted fields showed ahigher frequency of tolerant plants or a higher mean tolerance than thosein control fields. But the populations were polymorphic not only morphologically but also in copper tolerance. There were tolerant plants innormal fields and sensitive plants in polluted fields. The tolerant plants
were generally small in the size ofvarious organs and inferior in performance
when tested in normal conditions. They were also inferior in competitive
ability. The tolerant strains showed a lower copper content in the leaves

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

and stems than normal strains in the copper-treated plot of gravel culture.
The data were largely comparable with those reported by British workers inperennial grass species growing on strongly polluted sites, though the level ofcopper pollution in Japanese rice fields was much lower.
The Pollination Systems of Melilotus Species
Yoshio SANO

To look into the pollination systems of Melilotus species, variations in
self-fertility, floral characters and in the attractiveness of flowers for bees
were observed using 87 strains belonging to 19 species. Self-fertility ranged
from 99 to 2 percent among the strains. In four species in which bothannual and biennial forms were present, the annual forms had higher selffertility than the biennial forms. Other species which were annual showed
high self-fertilities. Nine species of subgenus Eumelilotus each represented
by a biennial strain significantly differed in the number of bees visiting aplant per minute. The number of bees and self-fertility were negatively
correlated. The attractiveness for bees seemed to depend on the number offlowers per raceme and petal size, which also were negatively correlated with
self-fertility. These characters might have been selected so as to maintaina balance among them in accordance with the pollination strategy adopted.
(Submitted to OEco!. Plantarum; this experiment was carried out at the
Hokkaido University.)
Resource Allocation Patterns in Oryza perennis Strains
Yoshio SANO and Hiroko MORISHIMA

The photosynthetic product in the leaf of a plant species is distributed
to various organs according to a certain scheme to achieve its inherent
pattern of growth and development. The allocation of resources for reproductive organs, called "reproductive effort", is considered to be an important factor in determining adaptive strategy. The present experiment
was carried out with the view to looking into the variations in allocation
rates among 38 strains of Oryza perennis, which show a continuum between
the annual and perennial types. The plants were grown in two concretebeds with automatic shortday controls, and the size, number and dry weightof various organs were recorded. The proportion of seed weight to total

RESEARCHES CARRIED OUT IN 197693

dry weight ranged from 1 to 50 percent, being high in Asian annual strains
as well as in cultivated (sativa) strains tested for comparison.
Correlations were computed among 45 attributes recorded, 35 from thepresent experiment and the other 10 obtained from other experiments on thebreeding systems, competition, etc. Those 45 attributes were divisible into
two groups showing positive correlations within each, between which thecorrelations were exclusively negative. One group included indices forreproductive effort, seed productivity, seed dormancy, seed dispersal,
drought tolerance, submergence tolerance, etc. The other group included
vegetative performance, culm length, regenerating ability, outcrossing rate,anther weight in percent of flower weight, competitive ability, etc. It wassuggested that the pattern of resource allocation was related to other ecological characters like tolerances and competitive ability, and could be anagent conditioning the differentiation of O. perennis strains in adaptive
strategy.
Variations in Competitive Ability among Oryza perennis Strains
Yoshio SAND and Hiroshi Y AMAGISHI

On the competitive abilities of wild rice strains, no published papers arefound except for a report that a strain of Oryza perennis was inferior to acultivated strain (0. sativa) when tested in Ceylon (Narise and Sakai 1960,
Ann. Rep. 10: 66). We have tested 18 Asian, 9 American and 2 African
strains of O. perennis, 2 sativa strains and a glaberrima strain in a mixplanting experiment with a test-strain belonging to O. sativa (Taichung 65).
The plants were harvested at the end of maximum tillering stage to measure
dry weight, and the competitive ability of a strain (1) against the test-strain(2) was evaluated by the differences in dry weight between mix-planting(12; 21) and pure stand (11; 22), as (X12-XU)-(X21-X22).
The variance of strains was highly significant indicating their differencesin competitive ability. Among Asian strains of O. perennis, the perennial
type tended to have higher competitive ability than the annual type. Theintermediate annual-perennial strains of Asia as well as American strains
showed a wide range of variation, their means being intermediate between
those for the annual and perennial types of Asia. One of the two sativa
strains tested showed a stronger competitive ability than all perennis strains.
Based on covariations between competitive ability and other measurements

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

of vegetative growth, path coefficients were computed to estimate the effectof growth rate on competition. The result showed that 63 percent of thevariance of competitive ability could be accounted for by those measurements of growth in pure stand, and that the numbers of tillers and leaves
at 60 to 70 days after germination and their increasing rate played a majorrole in determining competitive relations.
An Experiment on the Interaction between Cultivated Ri.ce and Barnyard
Grass at Different Planting Densities
Hiroshi YAMAGISHI, Hiroko MORISHIMA and H. 1. OKA

To examine the relation between density effect and species interaction, arice and a barnyard grass strain were tested in pure and mix (l : I) plantings
at 5 different densities. In pure stands, the data for dry weight per plantand other characters gave a good fitness to the logarithmic regression equa"tion on plant density (derived from WNa=K). As for mix-planting, aparameter showing neighbor effect was added to the equation, and its
values were estimated. Rice and barnyard grass had different signs for thisparameter. The trend of competition effect to increase with density was
not obvious. Both species tended to reduce reproudctive effort at highdensities. Rice showed logarithmically linear density responses in both size
and number of stems, while the barnyard grass showed it only in stem
number. (Submitted to Agro-ecosystems)

RESEARCHES CARRIED OUT IN 1976

XIV.

95

APPLIED GENETICS

Population Size Maintained for the Bulk Method of
Breeding in Autogamous Plants
Shinya IYAMA

Bulk method of breeding is widely used for selection of quantitative
traits in autogamous crop plants, i.e. after crossing between two parental
genotypes, progenies are propagated by selfing withowt selection for several
generations during which recombination between genes takes place and highhomozygosity is attained. For the success of selection, one has to havedesirable recombinant genotypes in a final population.
Probability that the population includes at least one desirable recombinant
was calculated for various propagation scheme mentioned as follows: thepopulation is to be propagated by taking n; progeny from each parental
plant at F;_l generation. Then the population size at F; generation isn2n3' .. n;. The results of calculation showed that (1) at a given criterion ofrisk, the size of final population maintained by single seed decent method(SSD for which n 3 = ... =n;=I) is the smallest, (2) expanding a population
at later generation is not effective to reduce the risk, and (3) under proper
scheme of propagation one can reduce the size of F 2 population to about
20 % of SSD population so as to keep the final size not considerably larger
than that of SSD.

Difference in Noise Sensitivity Affecting Egg Laying Performance
between Wild and Domestic Japanese Quails*
Takatada KAWAHARA

A wild strain of Japanese quail propagated for 10 generations in cages
after being captured in the field showed changes in various productive andbehavioral characters toward the domestic type (Ann. Rept. 24: 72; Kawahara, T. 1976, Experimental Animals 25: 351). The wild strain and a domestic strain were investigated with regard to their sensitivity to nose. The
birds to be tested, 92 in total, were reared under a noise treatment with* This research was supported by a grant from the Ministry of Environment Agency.

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENFTICS NO. 27

a buzzer of 95 phons working one hour 12 times a day for 20 days. The
photoperiod applied was 18-hour light and 6-hour darkness throughout theexperiment.
The egg laying performance decreased in both strains under the noise
treatment, but the wild strain showed more remarkable depression thanthe domestic strain. The egg production rate of the wild strain decreased
by 23.8 percent (82.9 % in the noiseless period before treatment), while thatof the domestic strain decreased by 7.1 percent (90.0% in the noiseless period
before treatment). But, such a depressing effect of noise did not lastlong. In the domestic strain, the egg production rate was recovered inthree days even though the noise treatment was continued for 20 days. In
the wild strain, the recovery took 9 days. The incidence of shell-less andsoft-shelled eggs slightly increased under the noise treatment in both wildand domestic strains. There was no perceptible effect of noise on the"time-of-lay" in both strains.
The wild strain has been domesticated by means of "unconscious selection" under domestic condition. Therefore, those birds may be regarded
as semi-domestic, but they have maintained some noise sensitivity of thewild birds.
Noise: buzzer treatment(95 phon every other hour)100--.----------------...-------- Domestic strain

/-_
//
'"'\
,
'.,,'
'/'
,~--~--...
\ .."

90

,

2

E

"
~

"0

-treatment{n:3S)

Wild strain-control {n:3031
Wild
strain80.~u-control (n:191l
Domestic
strain-treatment (n:S7)

70

o

a.

....~

60

1

:3

6

9

12

15

18

21

24

27

30

33

36 39

42 45

48

Days from the beginning of examination

Fig. 1. Egg production curves in noise treated and noiseless control groupsof wild and domestic strains.

RESEARCHES CARRIED OUT IN 1976

97

A Breeding Experiment in Oryza glaberrima
H. I. OKA

Oryza glaberrima, cultivated by the native people in West Africa fromancient times, has never been subjected to varietal improvement by themodern methods. Most of the varieties have red pericarp and are strongly
sensitive to photoperiod. Looking for plants with colorless pericarp or whitegrain, three were found out of about 400 strains examined, which were from
Gambia. Search for photoperiod non-sensitive plants gave 8 out of 84,
which were from Guinea. From hybridization of these two exceptional
types (17 crosses in total, in 1970), 37 F 2 plants were selected which wereinsensitive to photoperiod and had white grain. In a part of the crosses,
insensitivity was monogenic recessive, while it was digenic (15: 1) in theothers. Colorless pericarp was monogenic recessive in all crosses. However, the selected plants badly lodged at maturity when heavily fertilized.
With the hope of selecting non-lodging plants, the F s lines were inter~crossed in 35 random combinations, and their progeny lines were tested fortwo seasons. Thirty non-lodging F 3-F2 plants were selected from about 1,000,
but their progeny lines showed that the selection was unsuccessful (1974).
Their grain yield was 301 g/m 2 on the average (10-6-6 NPK, g/m2), while
an improved sativa variety (Taichung 65) in an adjacent plot gave 447 g/m2 ;Then, the bulked seeds of the selected plants were irradiated with 20 kR
gamma-rays, and about 10,000 M 2 plants (by one-panicle-one~kernel method,from about 1,000 M 2 plants) were observed, and 21 non-lodging plants wereselected. But their M s lines lodged again (1976); we had to give up ourtrial to select non-lodging glaberrima strains. Possibly, the lodging of theglaberrima rice is related to its annual habit; not only the leaves but also
the straw shows senescence with seed-ripenning. It seems difficult to obtaina non-lodging mutant unless the annual habit is modified.

Plasma Albumin Polymorphism in Japanese Quails
Takeshi SHIBATA,1) Seiki WATANABE1) and Takatada KAWAHARA

The blood plasma from six Japanese quail strains (4 domestic, 1 wild,and 1 hybrid; 936 birds in total) were examined for albumin polymorphism.
1)

Laboratory of Animal Breeding, Tokyo University of Agriculture, Setagayaku,
Tokyo.

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

.

."

oC

<

Origin

88

AC

A8

AA

Fig. 1. Diagrammatic patterns of plasma albumin in Japanese quail.

Four band forms, named AA, BB, AB and AC were detected (Fig. 1). The
result of mating experiments indicated that these bands were controlled
by three codominant autosomal alleles, AlbA for the fast band (A), Alb B
for the medium band (B), and Albo for the slow band (C). The albumin
polymorphism was first reported by Haley (1965, Genetics 51: 983) who
found two codominant autosomal alleles and their resultant three bandforms. The present experiment proved the presence of the third allele,Alb,o though its frequency was very low and its homozygote was not
obtained. The gene frequencies in the present material were 0.980 for
AlbA, 0.016 for Alb B , and 0.004 for Albo. All 256 wild birds were homozygous for AlbA; Alb B was found in domestic strains preserved in the Tokyo
University of Agriculture, and Albo was found in a domestic strain of thisInstitute.

The Growth Pattern and Competitive Ability of Six Rice Varieties
Hiroshi YAMAGISHI, Hiroko MORISHIMA and H. I. OKA

The "Rice Adaptation Experiment" was conducted in 1968 to 1972
under the International Biological Programme (JIBPjUM), testing a numberof rice varieties for their yielding potential and yield stability at different
stations distributed in the world. From a part of the data, we have pointed
out the variation between the "early vigor" and "late vigor" types andRESEARCHES CARRIED OUT IN 1976

99

discussed their bearings on yielding potential (Morishima and Oka 1974,
Jap. J. Breed. 24: 226). In the present experiment, this variation was reexamined with six selected varieties which had been used in the Rice
Adaptation Experiment and their competitive abilities against a test-strain(Taichung 65) were also observed. The experiment was laid out according
to the randomized blocks design with two replications.
The data for growth pattern were generally in good agreement with thosefrom the Rice Adaptation Experiment (r=0.89 in multiple correlation forvarious indices showing growth). Competitive ability was evaluated by theincrement or decrement in performance of two mix-planted strains (1, 2) ascompared with their values in pure stand (X12 - X u - X21 +X22 ). It markedly differed among the varieties tested, and showed strong correlations
with the growth rate at 70 to 90 days after germination in high-fertilizer
plot (r=0.96), or at 90 to 100 days in low-fertilizer plot (r=0.89). Competitive ability was also strongly correlated with percent cover of soil surface by the leaves at an age of 75 days (r=0.99) and with its increasing rate
per day (r=0.99). This indicates that in rice competition takes place forlight by means of growth rate, and that the "early vigor" type has higher
competitive ability than the "late vigor" type.
Nitrogen-Fixing Ability in Rice Strains
Taro FUJII, Yoshio SANO and Shin-ya IYAMAThe capacity to assimilate aerial nitrogen is limited to certain species ofbacteria and algae, and to legumes and some other plants with which
nitrogen-fixing bacteria are symbiotic. If the nitrogen-fixing ability was
introduced into a non-leguminous crop plant by genetic procedures, a great
advantage would be expected in crop production. With the hope of exploring such possibilities, we have initiated experiments on the nitrogen-fixing
ability in rice strains. The acetylene reduction assay by gaschromatography(Shimadzu GC-3BF) was adopted for evaluating the ability for nitrogen
fixation by the roots with which some bacteria might coexist. Ethylene
yield was assessed by conversion from gas-chromatographic profile to a
figure in n moljg fresh root/hour, using the value obtained with soybean
roots (T 202) as the standard. In soybean roots, acetylene reduction started
immediately after incubation in an aerobic gas phase (10% C2 H 2 +90% air),and an average of 1,400 n moljg/h of ethylene yield was obtained.

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

This year, 50 wild (Oryzaperennis) and 150 cultivated (0. sativa) strains ofrice were selected at random from our genetic stocks collected from different
tropical countries, and used for the experiment. The seeds were sown in
seed-bed flats, and the seedlings raised in a green-house were transplanted
into an irrigated paddy field in May. Fertilizers applied were limited to
the fused phosphate fertilizer (25 gjm 2 ) and potassium sulfate (14.5 gjm 2),
as basic dressing. When the plants started heading, the roots were sampled
for assay. The whole plants were dug up, and the roots were excised and
immediately washed with tap water. The roots were cut into pieces 4-5 cm
long and 2 g in fresh weight were put into a 30 ml bottle with a rubber-needle_
puncture stopper. The rice roots showed no acetylene reduction underaerobic conditions.
Then the air in the bottle was evacuated and filled with a mixture of 90 %helium and 10 % acetylene. The bottles were then incubated at 30°C for
assay. One ml of incubated gas was taken out from the bottle by using a
glass syringe and was injected directly into the gas-chromatograph column.
As a control experiment, when acetylene was not added, no ethylene wasproduced. The assay of field soil gave no ethylene either. These demonstrate the role of roots in acetylene reduction.
The production of ethylene was observed after a lag time of 4 to 6 hours,
and increased with incubation time. Most of the strains tested showed about
5 to 15 n moljgjh. Three strains showed significantly high ethylene yieldsof 45 to 65 n moljgjh (Table 1). They were C5444 and C8005 from India
and C7588 from Kenya. The variation observed suggests that the nitrogenfixing ability of the root in the soil is genetically controlled. The assayof the whole root system with the basal part of stems and soils is under way.
Table 1. Variation of acetylene reduction ability in rice strains

Cultivated strains
Wild strains

Total No.
of strainstestedAcetylene reduction activity (n mol/g(h)
5

15

25

35

45

81
46

45
12

17
15

13
16

4
2

1

55

65
1

RESEARCHES CARRIED OUT IN 1976

101

Selection of Induced Mutants in Oryza glaberrima
Yoshio SANO

An examination of more than 400 strains of Oryza glaberrima collectedfrom West African countries gave quite a limited number of striking morphological differences useful for genic analysis. In order to obtain genemarkers and to compare their functions with those of similar genes of
O. sativa, the seed of a photoperiod insensitive strain with colorless pericarp(GMS, selected by Oka, presented in this Annual Report) was treated with
EMS (0.5 % solution for 5 hours at 27°C after 2-hour presoaking). The M 2
Table 1. Mutants selected from an M 2 population (7079 plants, EMS treated)of a glaberrima strain insensitive to photoperiod and with colorless pericarp
Type compared withsativa mutants
Dwarf, "Dwaikoku", tillering,others
Chlorosis
Narrow leaf
Broad leaf
Brown discoloration
Rolled leaf
Branching
Brittle culm
LigulelessLax panicle
Long empty glume
Double glume
Hairy glume
Awned
Apiculus coloration
Minute grain
Large grain
Round kernelLong kernel
Glutinous endosperm
Early heading
Early loding
Semi-sterile

No. ofplantsFrequency
X 10-4

10
8
18
13
4
1
9
1
1
2
12
1
1
1
1
1
1
2
2
1
1
1
3
1
2

14.1
11. 3
21.4
18 .4
5.6
1. 4
12.7
1. 4
1 .4
2. 8
17.0
1. 4
1.4
1.4
1. 4
1.4
1.4
2. 8
2.8
1 .4
1.4
2.6*
4.2
1 .4
2.8

* Frequency of heterozygotes; homozygote frequency is its 1/4.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

seeds were collected by "one-panicle-one-kernel" method from about 1,000
M I plants, and about 7,000 M2 plants were raised in an upland field.
The M 2 plants displayed a spectrum of morpholgoical changes. A totalof 96 plants were selected; their mutant characters were classified into 24
types, among which were found different types of dwarfisms, plants withnarrow or broad leaves, long empty glumes, and necrotic brown spots on theleaves, ligulelessness, early heading, etc. (Table 1).
Further, a glutinous (waxy) mutant was found from iodine test of the M 2
seeds on M I panicles (1/3760=0.026%), and glutinous M 2 plants wereobtained from the seeds. In heterozygous panicles, the non-glutinous:
glutinous ratio of M 3 seeds was 83: 25 indicating that the glutinous character
was a monogenic recessive. When crossed with a glutinous strain of
O. sativa, the F I seeds had glutinous endosperm suggesting that the induced
glutinous gene had a similar function as that of O. sativa.
As O. glaberrima is annual, all the plants die after seed maturity. To
search for mutations affecting the annual habit, the M 2 plants at maturity
were cut and ratooning stubbles were looked for. Though it was in late
October, 20 ratooning plants were found; they were transplanted in pots andwere cultured in a greenhouse. However, all of them died in one month,indicating that the search for perennial mutations was unsuccessful.
Breeding of Isogenic Lines of Rice Carrying Marker Genes inInterchanged Segments
Yoshio SANO and H. 1. OKA

The breeding of isogenic marker lines and isogenic translocation lines ofTaichung 65 was reported previously (Ann. Rep. 24: 66 & 25: 84). Fromintercrossing of the marker lines, isogenic lines carrying two independent
markers were obtained, that were T65-ne-bc (neck leaf and brittle culm),
T65-g-lg (long empty glumes and liguleless), and T65-la-gl (lazy habit andglabrousness). They were each crossed with respective translocation lines,
30 in total number, to identify the chromosomes involved in the reciprocaltranslocations. Linkage relations between a marker and the point of interchange were detected in 15 out of 55 F 2 populations so far observed. Fromeach F 2 population showing such a linkage, one or two plants homozygous
for a marker and heterozygous for an interchanged segment were selected,
and from their progenies plants homozygous also for translocation wereRESEARCHES CARRIED OUT IN 1976103

selected by test-crossing the Fa plants with the original strain of Taichung65. Thus, three translocation lines carrying different markers in the interchanged segment were obtained. They will be used for genic analysis. This
work is still under way to increase the number of translocation lines carrying
gene markers.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

BOOKS AND PAPERS PUBLISHED IN 1976
BY STAFF MEMBERS(1) AIuGA, I. and A. MURAKAMI 1976: Recombinogenic effects of ethyl methanesul.
fonate on the silkworm oocytes. (In Japanese) J. Sericult. Sci. Japan. 45: 219-224.
(2) BARADJANEGARA, A. A., T. FUJII and E. AMANO 1976: Effects of gamma-raySand neutrons on the seedling and callus growth in rice seeds. Radioisotopes 25:
209-214.
(3) CmYo, H., Y. KUROKI, 1. MATSUI, N. NIITSU and Y. NAKAGOME 1976: A case ofpartial trisomy 3q. J. Med. Genet. 13: 525-528.
(4) FUJITA, H. and E. MATSUNAGA 1976: Down's syndrome and materanal age: A
survey over 30 years in Japan. Excerpta Medica Intern. Congress Series No.
397: 72.
(5) HATA, T., I. NAKAGAWA, K. SHIMOTOHNO and K. MIURA 1976: The synthesis ofa,r -dinucleoside triphosphates. The confronted nucleotide structure found at the
5'-terminus of eukaryote messenger ribonucleic acid. Chern. Lett. 980-990.
( 6) IMAI, H. 1976: Further evidence and biological significance for non-random
localization of the centromere on mammalian chromosomes. J. theor.Biol. 61:
195-203.
(7) ISONO, K., J. KRAuss and Y. HIROTA 1976: Isolation and characterization oftemperature-sentitive mutants of Escherichia coli with altered ribosomal proteins.
Mol. gen. Genet. 149: 297-302.
'( 8) KAnA, T. 1976: Rec-assay with cold incubation with and without metabolic
reactivation in vitro. Mutation Res. 38: 340.
'( 9) KATO, H. and H. F. STICH 1976: Sister chromatid exchanges in ageing and repairdeficient human fibroblasts. Nature 260: 447-448.
(10) KAWAHARA, T. 1976: History and useful characters of experimental strains ofJapanese quail (Coturnix coturnix japonica). (In Japanese) Experimental Animals
25: 351-354.
(11) KAWAHARA, T. and K. SAITO 1976: Genetic parameters of organ and body weights
in the Japanese quail. Poultry Sci. 55: 1247-1252.
(12) K1MURA, M. 1976: Random genetic drift prevails. Trends in Biochemical Sciences
1 (No.7): NI52-N154.
(13) KIMURA, M. 1976: Population genetics and molecular evolution. Johns Hopkins
Medical Journal 138 (No.6): 253-261.
(14) KIMURA, M. 1976: How genes evolve; a population geneticist's view. Annales deGenetique 19: 153-168.
(15) KIMuRA, M. 1976: The neutral theory as a supplement to Darwinism. Trends in
Biochemical Sciences 1 (No. 11): N248-249.
(16) KIMURA, M. 1976: The origin of life and molecular evolution. (In Japanese)
Iwanami Lecture Series in Modern Biology 7 (ed. by M. Kimura and S. Kondo),

BOOKS AND PAPERS PUBLISHED IN 1976

105

Iwanami Shoten (Tokyo).
(17) KURODA, Y. 1976: Sensitization procedure for detecting drug-resistant mutations
in cultured human diploid cells. Mutation Res. 38: 341-342.
(18) KURODA, Y. 1976: Mechanisms of induction of somatic cell mutations in cultured
human diploid cells. (In Japanese) Tissue Culture (Tokyo) 2(1): 20-23.
(19) KURODA, Y. 1976: Rotation culture. (In Japanese) "Tissue Culture" 158-167,
Asakura-Shoten (Tokyo).
(20) KURODA, Y. 1976: Application of tissue culture in embryology. (In Japanese)"Tissue Culture" 418-451, Asakura-Shoten (Tokyo).
(21) KURODA, Y. 1976: Progress and application of procedures for detecting environmental mutagens by using cultured mammalian cells. (In Japanese) J. Environ.
Pollut. Contro112(4): 384-392.
(22) MATSUNAGA, E. 1976: Possible genetic consequences of relaxed selection against
common disorders with complex inheritance. Hum. Genet. 31: 53-57.
(23) MATSUNAGA, E. and H. OGYU 1976: Retinoblastoma in Japan: Follow-up surveyof sporadic cases. Jap. J. Ophthalmo!. 20: 266-282.
(24) MATSUNAGA, E. 1976: Hereditary retinoblastoma: Penetrance, expressivity andage of onset. Hum. Genet. 33: 1-15.
(25) MATSUNAGA, E. 1976: Introduction to a symposium on monitoring of birth defects.
Teratology 14: 231.
(26) MATSUNAGA, E. 1976: Are oral contraceptives mutagenic or clastogenic? (InJapanese) San-Fujinka no Chiryo 32(1): 88-93.
(27) MIURA, K. 1976: Viruses-chemistry of their structural unit. Methodicum
Chimicum. 11 Pt. 1: 84-102. Academic Press (New York)-Georg Thieme(Stuttgart)-Maruzen (Tokyo).
(28) MORISHlMA, H. 1976: Some problems of evolution and adaptation in wild andcultivated rice species. (In Japanese) In "Advances in Breeding Sciences" (Ed. by
Jap. Soc. Breed.): 13-18, Keigaku Pub!. Co. (Tokyo)(29) MURAKAMI, A. 1976: X-ray-induced recombination during oogenesis in the silkworm (Bombyx mori L). Rad. and Environm. Biophys. 13: 187-195.
(30) MURAKAMI, A., T. MUROTA, Y. SHIMADA and Y. TAZlMA 1976: Further studies on
the genetic effects of furylfuramide on mature spermatozoa of the silkworm.
Mutation Res. 38: 342-344.
(31) MURAKAMI, A. 1976: Recent advances in the technique for detecting environmental
mutagens in the insect test systems. (In Japanese) J. Environ. Pollut. Control(Tokyo) 12: 393-396.
(32) MURAKAMI, A. 1976: Mutagenicity of PCB to silkworm germ-cells. (In Japanese)
Chemistry and Biology (Tokyo) 14: 782-785.
(33) MUROTA, T. and A. MURAKAMI 1976: Induction of dominant lethal mutations by
Alkylating agents in germ-cells of the silkworm, Bombyx mori. Mutation Res.
38: 343-344.
(34) NAKAGOME, Y. 1976: Nonrandom distribution of exchange points in patients withstructural rearrangements. Amer. J. Hum. Genet. 28: 31-41.
(35) NAKAGOME, Y. 1976: Early replicating DNA and chromosome bands. Excerpta

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

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(38)

(39)

(40)

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(42)

(43)
(44)
(45)
(46)

(47)
(48)
(49)

(50)

(51)

Medl., Intern. Congr. Ser. No. 397, 5th Intern. Congr. Hum. Genet. 143.
NAKAGOME, Y., F. TERAMURA, K. KATAOKA and F. HosoNo 1976: Mental retardation, malformation syndrome and partial 7p monosomy 45, XX, tdic, (7; IS) (p21;pll). Clin. Genet. 9: 621-624.
NAKAnMA, S., M. YANAGISAWA, S. KAMOSIDTA, Y. NAKAGOME 1976: Mentalretardation and congenital malformations associated with a ring chromosome 9. Hum.
Genet. 32: 289-293.
NISHIDA, Y., S. YASUDA and M. SEKIGUCHI 1976: Repair of DNA damaged bymethylmethanesulfonate in bacteriophage T4. Biochim. Biophys. Acta 442: 208215.
OHNISHI, S. 1976: Effects of population density and temperature condition on
fitness in Drosophila melanogaster. I. Developmental time and pre-adult viability.
Japan. J. Genet. 51: 19-25.
OH:NISHI, S. 1976: Effects of population density and temperature condition on
fitness in Drosophila melanogaster. II. Fecundity and mortality. Japan J. Genet.
51: 305-314.
OHNO, T., Y. OKADA, K. SHIMOTOHNO, K. MIURA, H. SHINSHI, M. MIWA and
T. SUGIMURA 1976: Enzymatic removal of the 5'-terminal methylated blocked
structure of tobacco mosaic virus RNA and its effects on infectivity and reconstitution with coat protein. FEBS Lett. 67: 209-213.
OHNUMA, A. and A. MURAKAMI 1976: Genetical analysis of a newly gamma-ray
induced translocation between Wand 5th chromosomes in pupal oocytes of thesilkworm. (In Japanese) J. Sericult. Sci. Japan 45: 172-178.
OHTA, T. 1976: Simulation studies on the evolution of amino acid sequences.
J. Mol. Evol. 8: 1-12.
OHTA, T. 1976: Simple model for treating evolution of multigene families. Nature
263: 74-76.
OHTA, T. 1976: Role of very slightly deleterious mutations in molecular evolution and polymorphism. Theor. Pop. BioI. 10: 254-275.
OHTSUKA, E., S. NISHIKAWA, M. SUGIURA and M. IKEHARA 1976: Joining ofribooligonucleotides with T4 RNA ligase and identification of the oligonucleotideadenylate intermediate. Nucleic Acids Research 3: 1613-1623.
OKA, H. I. 1976: Mortality and adaptive mechanisms of Oryza perennis strains.
Evolution 30: 380-392.
SADAIE, Y. and T. KAnA 1976: Recombination-deficient mutants of Bacillus subtilis.
J. Bacteriol. 125: 489-500.
SADAIE, Y. and K. NARDI 1976: Repair deficiency, mutator activity, and thermal
prophage inducibility in dna-8132 strains of Bacillus subtilis. J. Bacteriol. 126:1037-1041.
SADAIE, Y., K. NARDI and T. KADA 1976: Enhanced ultraviolet mutagenesis.
repair deficiency, mutator activity, and thermal prophage inducibility in dna-8132
strains of Bacillus subtilis. Proc. of VII. Inter. Congr. on Photobiology (Roma)p. 151.
SAKUMA, K., R. KOMINAMI, M. MURAMATSU and M. SUGIURA 1976: Conservation

BOOKS AND PAPERS PUBLISHED IN 1976

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(55)

(56)
(57)
(58)

(59)
(60)
(61)
(62)
(63)

(64)
(65)

(66)

(67)

(68)

107

of the 5'-terminal nucleotide sequences of ribosomal 18-S RNA in eukaryotes,
Differential evolution of large and small ribosomal RNA. Eur. J. Biochem. 63:
339-350.
SmMADA, Y., A. MURAKAMI and Y. TAZIMA 1976: Dosimetric studies on the incorporation of furylframide into oocytes of the silkworm. Mutation Res. 38: 346347.
SHIMOTOHNO, K. and K. MIURA 1976: The process of formation of the 5'-terminal
modified structure in messenger RNA of cytoplasmic polyhedrosis virus. FEBS
Lett. 64: 204--208.
SHIMOTOHNO, K., T. URUSHIBARA and K. MIURA 1976: The N2,2,7-trimethylguanylic acid-blocking structure at the 5'-terminus of some low molecular weightRNAs in nucleus of an animal cell. Proc. Japan Acad. 52: 563-566.
SHINSHI, H., M. MIWA, T. SUGIMURA, K. SHIMOTOHNO and K. MIURA 1976: Enzyme
cleaving the 5'-terminal methylated blocked structure of messenger RNA. FEBS
Lett. 65: 254--257.
SHIOTA, K. 1976: Embryotoxic effects of polychlorinated biphenyls (Kanechlors
300 and 500) in rats. Okajimas Fol. Anat. Jap. 53: 93-104.
SmoTA, K. 1976: Postnatal behavioral effects of prenatal treatment with PCBs
(polychlorinated biphenyls) in rats. Okajimas Fol. Anat. Jap. 53: 105-114.
SHIOTA, K. 1976: Fetal accumulation of polychlorinated biphenyls (PCBs). A
consideration on the problem of human prenatal exposure to environmental
pollutants. Congenital Anomalies 16: 9-16.
SHIOTA, K., M. J. CHOU and H. NIsmMURA 1976: Factors associated with theoccurrence of early resorption of human embryos. Mutation Res. 38: 347-438.
SHIOTA, K. 1976: Human materials and study of pollutants. Lancet ii: 48.
SHIOTA, K. and E. MATSUNAGA 1976: Epidemiological study of holoprosencephaly
in human embryos. Teratology 14: 253.
SHIRASU, Y., M. MORIYA, K. KATO and T. KADA 1976: Mutagenicity screening ofpesticides in the microbial system. Mutation Res. 40: 19-30.
SUZUKI, H., Y. NISHIMURA, H. IKETANI, J. CAMPISI, A. HIRASHIMA, M. INOUYE and
Y. HIROTA 1976: A novel mutation which causes a structural change in a lipoproteinin the outer membrane of Escherichia coli. J. Bacteriol. 127: 1494--1501.
TAZIMA, Y. 1976: Methodology in mutagenesis. studies. (In Japanese) In "Prenatal
Medicine" (Ed. by U. Murakami et al.); 490-508, Igaku Shoin Ltd. (Tokyo).
TAZIMA, Y. 1976: Prevention of congenital anomaly due to environmental factorsmutagens. (In Japanese) In "Prenatal Medicine" (Ed. by U. Murakami et at): 887899, Igaku Shoin Ltd. (Tokyo).
WATANABE, T. K. and W. W. ANDERSON 1976: Selection for geotaxis in Drosophilamelanogaster: Heritability, degree of dominance, and correlated responces toselection. Behav. Genet. 6: 71-86.
WATANABE,T. K., O. YAMAGUCHI and T. MUKAI 1976: The genetic variability ofthird chromosomes in local population of Drospohila melanogaster. Genetics 82:
63-82.
WATANABE, T. K. and T. YAMAZAKI 1976: Evidence for coadaptation: Negative

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27
correlation between lethal genes and polymorphic inversions in Drosophila melano_
Genetics 82: 697-702.
WATANABE, T. K and M. KAWANISHI 1976: Colonization of Drosophila simulans
in Japan. Proc. Japan Acad. 56: 191-194.
WATANABE, T. K, T. WATANABE and C. OSHIMA 1976: Genetic changes in natural
populations of Drosophila melanogaster. Evolution 30: 109-118.
WEISS, K M. and T. MARUYAMA 1976: Archeology, population genetics andstudies of human racial ancestry. Amer. J. Physical Anthropo!. 44(1): 31-50.
YAGI, Y., K TUTIKAWA and N. SHIMOI 1976: Teratogenicity and mutagenicity ofa phthalate ester. Teratology 14: 259-260.
YASUDA, S. and M. SEKIGUCHI 1976: Further purification and characterization of
T4 endonuclease V. Biochim. Biophys. Acta 442: 197-207.
YOSIDA, T. H. 1976: Segregation on the No.1 chromosome pair in the black rat(Rattus rattus) maintained in a population room. Proc. Japan Acad. 52: 130-133.
YOSIDA, T. H. 1976: Karyotypes and meiotic segregation of hybrids between Asianand Oceanian type black rats. Proc. Japan Acad. 52: 304-307.
YOSIDA, T. H. 1976: Frequencies of chromosome polymorphism (pairs, No.1, 9
and 13) in the black rat of Japan. Proc. Japan Acad. 52: 405-408.
YOSIDA, T. H. 1976: Cytogenetics and evolution of rats. "Asian Rats and Their
Control," 3-10, ASPAC (Taipei)
YOSIDA, T. H. 1976: The chromosomes and evolution of rat. (In Japanese) Ann.
Rep. Res. Inst. Eva!. Bio!. 2: 3-20.
gastor.

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ABSTRACT OF DIARY FOR 1976

ABSTRACTS OF DIARY FOR 1976
January 16 23lst Meeting of Misima Geneticists' Club
February 9 232nd Meeting of Misima Geneticists' Club
4 233rd Meeting of Misima Geneticists' ClubMarch
5 234th Meeting of Misima Geneticists' Club8 123rd Biological Symposium
17 235th Meeting of Misima Geneticists' Club10 124th Biological Symposium
April
15 236th Meeting of Misima Geneticists' ClubMay
28 237th Meeting of Misima Geneticists' ClubJuly
28 238th Meeting of Misima Geneticists' Club
September 16 239th Meeting of Misima Geneticists' Club
October 15 240th Meeting of Misima Geneticists' Club109

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

FOREIGN VISITORS IN 1976
February

24
25

March

31

April

9-11
13

14
14
July
July
August

9

30-31
17
27
27

September

67
7
October

1-5
1-5
1-5

JACQUARD, P., Centre d'Etudes Phytosociologiques etEcologiques, Montpellier, France.
PUTMAN, U., Department of Botany, The Hebrew University, Israel.
PANAYOTOR, I., Institute for Wheat and Sunflower, Bulgaria.
COMSTOCK, R. E., Dept. of Genetics and Cell Biology,
University of Minnesota, U.S.A.
RAYCHAUDHURI, S. P., Haryana Agricultural University, India.
NYGREN, A, Department of Genetics and Plant Breeding, Agricultural College of Sweden, Uppsala, Sweden.
SAMMARRAI, S. M., College of Agriculture, Universityof Rizadh, Saudi Arabia.
CHUNG, Y. J., Ewha Women's University, Korea.
MACKAY, A, Dept. of Crystallography, Birkbeck College, University of London, England.
Ro, B. J., Ewha Women's University, Korea.
ORAM, M. N., C.S.I.R.O. Division of Plant Industry,
Canberra City, Australia.
LAWN, R. J., C.S.I.R.O. Division of Tropical Crops andPastures, St. Lucia, Queensland, Australia.
BLUNDELL, T. L., Dept. of Crystallography, Birkbeck
College, University of London, England.
RUFFlE, J., Centre d'Hemotypologie, CNRS, France.
KOWNACKI, M., Polish Academy of Sciences, Instituteof Genetics and Animal Breeding, Poland.
KING, J. L., Dept. of Biological Sciences, University ofCalifornia, U.S.A.
AYALA, F., Dept. of Genetics, University of California,
U.S.A.
HOOD, L., Division of Biology, California Institute ofTechnology, U.S.AFOREIGN VISITORS IN 1976

1-5
1-5

9

November 10-1118111

DICKERSON, R. E., Division of Chemistry and Chemical
Engineering, California Institute of Technology, U.S.A.
McLACHLAN, A. D., MRC Laboratory of Molecular
Biology, University Postgraduate Medical School,
Cambridge, England.
GANI, J., Division of Mathematics and Statistics, Commonwealth Scientific & Research Organization, Australia.
VERGNES, H., Centre d'Hemotypologie de Toulouse,
France.
CHU, E. H., Department of Human Genetics, Universityof Michigan Medical School, Ann Arbor, Michigan,
U.S.A.

112

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 27

AUTHOR INDEXAKAI, S. M.

86.

AMANO, E • • . . . . . . . . . . . . . . . . . . . . . . . . 66

32
83
ASANO, T
68
FUJII, T
72, 99
FUJIOKA, H. .
54
FUJISAWA, T • . . . . . . . . . . . . . . . . .42,43,44
FUJISHIMA, T. .
88
FUJITA, H
82
FUKASE, Y. .
62
HARADA, M. .
.46
HIRANO, K.
68, 69, 70
HIROTA, Y • . . . 12,13,14,25,26,27,29,30
IMAMOTO, F
14
INOUE, T
70
INOUE, Y
74
IYAMA, S
95, 99
KADA, T
.41, 68, 69, 70, 71
KATO, H70
KAWAHARA, T. "95, 97KAWANISHI, M. .
77
KIMURA, M. .
73,77
KITA, F
80
KONDO, K.
33
KURODA, Y
38, 39,40,41
LEE, W. H. .
74, 86, 87
MARUYAMA, T • . . . . . . . . . . . . . . . . . . . . . . 78
MATSUMOTO, H. .
69
MATSUNAGA, E. .
82, 83, 84
MATSUTANI, E
38
MINEZAWA, M • . . . . . . . . . . . . . . . . . . 33,46
MIURA, K.
14, 15, 16, 18, 19,20
MORISHIMA, H
90,91,92,94,98
MORIWAKI, K.
32, 33, 34, 36, 46
MURAKAMI, A. .
54, 55, 57, 58, 59, 60
MUROTA, T. .
55, 57NAKADATE, M. .
58
NAKAGOME, Y. .
83
AOTSUKA ,T.

ARIMA, M.

.

.

59
26
NISHIMURA, Y. .
25, 26, 27
NOGUCHI, T,
44
OCHIAI, Y
51
OHNUMA, A. .
63, 64
OHTA, T. .
73, 79
OHTA, Y
71
OKA, H. I.
80,91, 94, 97, 98, 102
OKA, S83
ONIMARU, K
62, 63, 64
OSHIMA, C
86, 87SADAIE, Y
68, 71SAKAMOTO, K.
57
SANO, Y. .
80, 92, 93, 99,101,102
SHIBATA, T. .
97
SHIMOTOHNO, K.
15, 16, 18, 19,20
SHIOTA, K. .
84
SHIROISHI, T. '"34, 36STEVENS, L. C
44
SUGIURA, K.
.40
SUGIURA, M
22
SUGIYAMA, T.
.
.42,43,44
SUZUKI, H
25, 26, 27, 29
TAKEDA, Y . . . . . . . . • . . . . . . . . . . . 12, 13,26
TAYA, C ..................••••••••.• 52
TAZIMA, Y
60, 62, 63, 64
URUSHIBARA, T. .
16
WATANABE, S. .
97
WATANABE, T
75
WATANABE, T. K.
.42, 74, 75, 77, 86
YAMADA, M
29, 30
YAMADA, M. A.
.42
Y AMAGISHI, H. .
93, 94, 98
YASUDA, S. .
13, 14, 30
YOKOIYAMA,A
41,70
YOSHINAGA, K . . . . . . . . . . . . . . . . . • . . . . . 22
YOSIDA, T. H
46,48,49,50,51,52
NISHIJIMA, H. .

NISHIMURA, A. .

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