NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 251974

Published by
THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan
1975

Annual Report-of theNational Institute of Genetics
No. 25, 1974

Published by

The National Institute of Genetics, Japan
1975

CONTENTS
General statement
Staff
Council......................................................
Association for propagation of the knowledge of genetics. . . . . . . . . . . .
Projects of research for 1974. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Researches carried out in 1974. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Molecular Genetics
A novel modified structure at the 5'-terminus of messenger RNAof cytoplasmic polyhedrosis virus. FURUICHI, Y. and MIURAK.
A modified 5'-terminal structure of messenger RNA of vaccinia
virus. URUSHIBARA, T., FURUICHI, Y., SHIMOTOHNO, K. and
MIURA, K.
Nucleotide sequence at the 5'-terminus ofreovirus messenger RNA.
SHIMOTOHNO, K. and MIURA, K.
Phosphohydrolase activity in the virion of cytoplasmic polyhedrosis virus. SHIMOTOHNO, K. and MIURA, K.
Modified structure in the 5'-terminus of genome double-stranded
RNA of reovirus. MIURA, K. and WATANABE, K.
Transcription of double-stranded RNA by the DNA-dependent
RNA polymerase from E. coli. SUGIURA, M. and MIURA,
K.

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II. Microbial Genetics
A mutation which causes a change in the structure of a lipoprotein
bound to peptidoglycan. SUZUKI, H., NISHIMURA, Y.,
IKETANI, H. and HIROTA, Y.............................

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III. Biochemical Genetics
Detection of hybrid enzymes specified by ACPl alleles in wild andcultivated rice strains. PAl, C., ENDo, T. and OKA, H. I... . .

IV. Developmental and Somatic Cell Genetics
Polymorphic characters of the external orifice of uterus in inbred

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

strains ofrats and their heredity. YOSIDA, T. H. and SANO, K.
Isolation of nematocyte deficient mutants of hydra. FUJISAWA,
T. and SUGIYAMA, T. .
.
Regeneration mutants of hydra. SUGIYAMA, T. and FUJISAWA, T.

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Analysis of time specificity of dor + gene action in embryonic deve.
lopment of Drosophila melanogaster. KURODA, Y
Effective substances having a repair action of dor embryonic de.
fects in Drosophila melanogaster. KURODA, Y.
Induction of 8-azaguanine resistant mutations by phloxine in.
embryonic human diploid cells. KURODA, Y
Protection by vitamin E against bisulfite-caused defect in growth ofcultured human cells. KURODA, Y.
Isolation and characterization of variant clones of Chinese hamster
cells after treatment with irradiated 5-iodouridine. KURODA,
Y., YOKOIYAMA, A. and KADA, T.
Effect of serum on proliferation of mammalian cells in culture.
MINATO, K.

v.

Cytogenetics
Frequency of A/A, A/S and SIS chromosomes in pairs no. 1,9 and
13 in 133 black rats collected in various localities. YOSIDA,
T.H.
Segregation of chromosomes in the second spermatocytes in F I
hybrids between Asian and Oceanian type black rats. YOSIDA.
T.R.
Relation between the decrease of C-band heterochromatin and dif.
ferentiation of Rattus species. YOSIDA. T. H
Effects of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) on bonemarrow chromosomes of the rat at different ages. YOSIDA,
T. H. and TSUJI, H.
.
Evidence against that the sister chromatid exchange is single-strand.
exchange. KATO, H
Chromosome analysis of sublines of a mouse hepatoma (MH134)
with a ribosomal RNA variation. MORIWAKI, K., IMAI, H.T.,
KOMINAMI, R. and MURAMATSU, M.
Genetical analysis of a newly gamma-ray induced translocation

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CONTENTS

between Wand V chromosomes in pupal oocytes of thesilkworm. MURAKAMI, A and OHNUMA, A. . . . . . . . . . . . . .
VI.

VII.

Mutation and Mutagenesis in Animals
Attempts to induce non-disjunction by means of irradiation andchemical treatment in the silkworm. TAZIMA, Y. and OHNUMA, A.
Mutagenic effectiveness of an internal f3 emitter, tritium. (1) A
preliminary experiment on somatic mutation in the silkworm.
TAZIMA, A, ONIMARU, K. and FUKASE, Y.. . . . . . . . . . . . . . . .
Effects of low-temperature post-treatment on the repair of premutational damage in spermatogonia of the silkworm.
TAZIMA, Y. and FUKASE, Y.. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Silkworm oocyte system for testing mutagenicity. TAZIMA, Y. and
ONIMARU, K.
Embryonic lethality in progeny of X-irradiated silkworm moths:
inherited lethality and sex-ratio distortion. MURAKAMI, A.. .
Mutagenicity of diethylsulphate in spermatozoa of the silkworm.
I. Delayed mutagenic effect. MURAKAMI, A. . .. . . . . .. . . ..
Mutagenicity of diethylsulphate in spermatozoa of the silkworm.
II. Dose-mutagenicity relationship. MURAKAMI, A. . . . . . . .
Lack of recombinogenicity of ethyl methanesulphonate in thesilkworm oocytes. MURAKAMI, A. and ARIGA, I.. . . . .. . . ..
Radiation Genetics and Chemical Mutagenesis in Micro-organisms
and Plants
Radiation and drug sensitivities of Bacillus subtilis rec strains.
SADAIE, Y, INUKAI, H. and KADA, T.. . . . . . . . . . . . . . . . . . . . .
Prophage induction in rec strains of Bacillus subtilis. SADAIE, Y. . .
Improved procedures of the rec-assay for rapid detection of chemical mutagens. KADA, T. and SADAIE, Y. . . . . . . . . . . . . . . . . .
AF2 mutagenicity and reductase activity of E. coli B/r WP2 resistant to Cr+6. KADA, T. . . . . . . . . . . . . . . . . . . .. .. . . . . .. ..
Ultraviolet light-induced mutagenesis in Bacillus subtilis spores.
SADAIE, Y, SAITO, Y. and KADA, T.. . . . . . . . . . . . . . . . . . . . . .
Involvement of a common enzyme in the repair of deprinated andr-ray-irradiated DNA of Bacillus subtilis. NOGUTI, T. and
KADA, T.............................................

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

Induction of wx mutant in an inbred line of maize. AMANO, E.. . .
Induction of waxy starch mutant in rice. AMANO, E.. . . . . . . . . .
Chromosome of cultured Haplopappus cell. AMANO, E.. . . . . . . .
Effects of fast neutrons on dry and wet seeds in maize. FUJII, T.. .
Sex expression in the offspring of gamma-ray induced monoeciouslike plants in cucumber. FUJII, T.. . . . . . . . . . . . . . . . . . . . . . .

VIII. Population Genetics (Theoretical)
Some models of allelic mutation in molecular population genetics.
KIMURA, M...........................................
Simulation studies on electrophoretically detectable genetic variability in a finite population. OHTA, T. and KIMURA, M. . . . .
Probability of gene fixation in an expanding finite population.
KIMURA, M. and OHTA. T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Detrimental genes with partial selfing and effects on a neutral
locus. OHTA, T. and COCKERHAM, C. C...................
Speed of gene frequency changes in reverse directions in a finite
population. MARUYAMA, T. and KIMURA, M.. . . . . . . . . . . . .
A Markov process of gene frequency change in a geographicallystructured population. MARUYAMA, T...................
A proof that certain quantities are independent of the geographicalstructure of population. MARUYAMA, T.. . . . . . . . . . . . . . . . .
IX.

X.

Population Genetics (Experimental)
Similarity of gene frequencies in different Drosophila species:
evidence for the neutrality hypothesis of protein polymorphisms. YAMAZAKI, T. and MARUYAMA, T................
Negative correlation between lethal genes and polymorphic inversions. WATANABE, T. K. and YAMAZAKI, T.. . . . . . . . . . . . . .
Electrophoretic protein polymorphism ina view of the stepwise
mutation model of neutrality theory. MARUYAMA, T. and
YAMAZAKI, T....
Evolutionary Genetics
On some principles governing molecular evolution. KIMURA, M.
and OHTA, T... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Distribution of antigenic specificities of mouse H-2 among wildrodents and its evolutionary implication. MORIWAKI, K. . . . .

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CONTENTS

XI.

Human Genetics
Indirect inguinal hernia: a multifactorial threshold trait. MATSUNAGA, E., SAWAGUCHI, S. and HONNA, T...................
Possible genetic consequences of relaxed selection against common
disorders with complex inheritance. MATSUNAGA, E.. . . . . .
Isozyme variations in man. SHINODA, T., MATSUNAGA, E. and
KOSHINAGA, J.........................................
Studies of Q-variants among patients with Down's syndrome andtheir families. IINUMA, K. and MATSUNAGA, E. . . . . . . . . . . . .
Non-random distribution of exchange points in patients withstructural rearrangements. NAKAGOME, Y. and CHIYO, H.. .
A 7p monosomy resulting from a stable dicentric chromosomewith reference to the mechanism of inactivation of a centromere- NAKAGOME, Y., TERAMURA, F., KATAOKA, K. and
HONoso, F............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structural studies on human immunoglobulins. SHINODA, T.. . . .

XII.

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Behavioral Genetics
Circadian locomotor rhythm of an adult fly of Drosophila melanogaster. OSHIMA, C.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of light, temperature and noise environments on longevity
and productivity of Drosophila melanogaster. OSHIMA, C.
and WATANABE, T. K...................................
Effects of light and noise environments on adult emergence rhythmof Drosophila melanogaster. OSHIMA, C. and WATANABE,
T.K.
Strain difference in larval phototactic behavior of the silkworm.
MURAKAMI, A. and KOTOKU, K.. . . . . . . .. . . . . . . . . . . . . . . . .
Strain differences in discriminated avoidance learning curve in
mice. FUJISHIMA, T...................................
Diallel cross experiment on the discriminated avoidance learning
abilities of mice. FUJISHIMA, T.. . . . . . . . . . . . . . . . . . . . . . . . .
Complete blindness of an inbred strain of mice, DI03/Ms. FUJISHIMA, T.

XIII. Applied Genetics
Breeding of isogenic lines of rice carrying various gene markers
and reciprocal translocations. OKA, H. 1. and MORISHIMA,

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H.
Genic analysis for the sterility of hybrids between Oryza sativa
and O. glaberrima. aKA, H. 1. and MORISHIMA, H.. . . . . . . .
A competition experiment between rice cultivars of different ages.
aKA, H. 1. and MORISHIMA, H.. . . . . . . . . . . . . . . . . . . . . . . . . .
Mortality and adaptive mechanisms of Oryza perennis strains.
aKA, H. 1. and MORISHIMA, H.. . . . . . . . . . . . . . . . . . . . . . . . . .
Competitive abilities of barnyard grass and dallis-grass against
finger grass. MORISHIMA, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Copper tolerance of barnyard grass strains. MORISHIMA, H. andaKA, H. 1.
Variations in heavy metal tolerance of weedy plants in the paddy
field. MORISHIMA, H. and aKA, H. I.. . . . . . . . . . . . . . . . . . . .
The mechanisms of weediness found in dallisgrass populations.
MORISHIMA, H',' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Developmental instability and phenotypic plasticity in Arabidopsis
thaliana. BAGCHI, S. and IYAMA, S.. . . . . . . . . . . . . . . . . . . . . .
Varietal difference in cadmium uptake ability in fodder turnip,
Brassica rapa. IYAMA, S,. . . . . . . . . . . . . . . . . . .
The effect of intensive inbreeding on the weights of various organs
in Japanese quails. KAWAHARA, T.. . .. . . . . . . . . . . . . .. . . ..
Properties concerning egg production of Japanese quails. KAWAHARA, T. and IYAMA, S.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bilateral asymmetry in the transverse processes of cervical vertebrae in chicken. KAWAHARA, Too..
..

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XIV. Miscellaneous
A modified analysis of two-treatment switchback design with different numbers of individuals in replications. FUJISHIMA, T.
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Pollen characters of Chrysanthemum species.

NAGAMI, S

Books and pap,ers published in 1974 by research members
Abstracts of diary for 1974
Foreign visitors in 1974
Author index

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GENERAL STATEMENTThe National Institute of Genetics attained the twenty-fifth anniversaryof its birth. The main commemorative event was the issue of the Comlllemoration Number of the Twenty-Fifth Anniversary, and we had a smallllleeting on November 9th, inviting Dr. H. Kihara, the former director, togive us a commemorative lecture. Besides, the annual public lectures weredt<livered in Tokyo by Dr. Y. Tazima, Head of the Department of Morphological Genetics, and Dr. M. Kimura, Head of the Department of Population Genetics. In the year 1949, when the institute was founded, modern
genetics just started to breathe energetically, succeeding "heroic age" ofclassical genetics. In the past twenty-five years of the institute we can trace
a history of the accumulation of endeavor, on the basis of classical genetics,
to develop the whole field of genetic studies in the current of modern genetics.
On August 8th we had the honor to have a visit of Prince and Princess
Hitachi. Prince Hitachi is majoring in biochemistry and he showed keeninterest in the study of cancer cells.
It is worth mentioning that Plant Section of Genetic Stock Center has
started. So far important strains of experimental materials such as wheat,rice, morning glory, mouse, silkworm, Drosophila, bacteria, have beenlllaintained in each department, respectively. However, considering increasing burden to the departments to take charge of the increasing strains ofgenetic importance, we have decided to establish Genetic Stock Center,aiming at "maintenance and storage of the materials necessary for genetic
studies and basic investigations in regard to their genetic characters." Asa first step the establishment of the plant section of the center has been approved. To this, we intend to add sections for animals and bacteria, so
that, while we may be sure to maintain important inherited characters, wewill be able to promote studies of higher level by making good use of them.
International exchange of scientists was also active this year. Among
many visitors from abroad the following three stayed rather long for cooperative studies: Dr. Montgomery Slatkin of the University of Chicago (June 1August 31), Professor James F. Crow of the University of Wisconsin (July16-December 23), and Professor A. Lima-de-Faria of the University of Lund
(October 31-December 28). From our side 14 members visited U. S. A.,
Canada, U. S. S. R., France, Austria, etc., some attending international

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

conferences, and others for cooperative works.
At the meeting of our Board of Trustees held lately it was approved that
I retire in March, 1975, from the director's post. I should like to express
hearty thanks to all persons who extended cooperation and encouragement tome during my six year service. I also wish for the great contribution,
expansion of research activities, and for further development of the institute.

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STAFF
Director

MORIWAKI, Daigoro, D. Sc., Emeritus Professor of Tokyo Metropolitan
University
Members

1. Department of Morphological Genetics·
TAZIMA, Yataro, D. Ag., Head of the Department
The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
MURAKAMI, Akio, D. Ag., D. Sc.
ONIMARU, Kimiharu
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. Sc., Head of the Laboratory
WATANABE, Takao K., D. Sc.
The 2nd Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
NAGAMI,* Shuzo, D. Ag.

* Research members under grant from other organization or visiting researchers.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

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, Toru, 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
BAGCHI, * Sujit
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
NOGUTI, Takehiko, D. Sc.
The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
AMANO, Etsuo, D. Ag.
SADAIE, Yoshito, M. Sc.

STAFF

7. Department of Human Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department
The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
SHINODA, Tomotaka, D. Sc.
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
SUZUKI, Hideho, D. Sc.
The 2nd Laboratory
ENOMOTO, Masatoshi, 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
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
OHTA, Tomoko, Ph. D., D. Sc.
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
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
FURUICHI, Yasuhiro, D. Med.
SHIMOTOHNO, Kunitada, D. Med.
11.

Genetic Stock Center
FUJII, Taro, D. Ag.

12. Experimental Farm
OKA, Hiko-Ichi, D. Ag., Head of the Farm
MIYAZAWA, Akira

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

31. Department of AdministrationTEZUKA, Asaichi, Head of the Department
YUHARA, Tokusaburo, Chief of the General Affairs Section
TAMATE, Shigeo, 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., Professor of Kagoshima University
TSUJITA, Mitsuo, D. Ag.

COUNCILKIKKAWA, Hideo, Chairman, Professor of Hyogo College of Medicine
FUJII, Takashi, Vice Chairman, Emeritus Professor of University of Tokyo
EGAWA, Tomoji, Director of National Institute of Agricultural Sciences
IZEKI, Shoei, Director of National Research Institute of Police Science
KAYA, Seiji, Emeritus Professor of University of Tokyo
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
KURODA, Toshio, Director of Institute of Population Problems
MAKINO, Sajiro, Emeritus Professor of Hokkaido University
MISONOO, Keisuke, Director of National Institute of Radiological Sciences
OeHI, Yuichi, President of Azabu University of Veterinary Science
SAKATA, Takeo, President of T. Sakata Company
TAKAHASHI, Ryuhei, Professor of Okayama University
TANAKA, Nobunori, Professor of Teikyo University
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Director of the National Institute of Genetics
TAZlMA, Yataro, Managing Director, Head of the Morphological Genetics
Department

COUNCIL

7

OSHIMA, Chozo, Managing Director, Head of the Physiological Genetics

Department
J{IHARA, Hitoshi, Manager, Emeritus Professor of Kyoto University
SINOTO, Yosito, Manager, Professor of International Christian University

WADA, Bungo, Manager, Emeritus Professor of University of Tokyo

PROJECTS OF RESEARCH FOR 1974
Department of Morphological Genetics

Genetic studies of the silkworm (TAZIMA and ONIMARU)
Repair processes in radiation mutagenesis (TAZIMA and ONIMARU)
Studies on radiosensitivity in the silkworm (TAZIMA and MURAKAMI)
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 andMINATO)
Department of Cytogenetics

Studies on chromosomal evolution in rodents (YOSIDA and KATO)
Chromosome study on experimental tumors (YOSIDA)
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,
and TSUCHIYA)
Immunogenetical study on the histocompatibility antigens of wild rodents(MORIWAKI)
Experimental breeding and genetics of mice, rats and other wild rodents(YOSIDA and MORIWAKI)
Department of Physiological Genetics

Studies on effects of noise environment to Drosophila (OSHIMA and WATANABE)
Behavior genetics of Drosophila (OSHIMA and WATANABE)
Analysis of deleterious and inversion chromosomes in natural populationsof Drosophila melanogaster (WATANABE)
Studies on fitness of Drosophila under controlled environment (OSHIMA andWATANABE)
Ecological genetic studies on the differentiation of Chrysanthemum species(NAGAMI)PROJECTS OF RESEARCH FOR 1974

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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 andODAKI)
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)
Genetic 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 (IYAMA, SAKAI, andKUDO)
Simulation studies on artificial selection (IYAMA)
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 (PAl and ENDO)
Genetic effects of environmental pollution on plants (IYAMA, MORISHIMA
and aKA)
Department of Induced Mutation

Molecular mechanisms of spontaneous, chemical- and radiation-induced
mutations (KADA, SADAIE and NOGUTI)
Environmental mutagens and carcinogens (KADA, SADAIE and TUTIKAWA)
Radiation genetics in mice (TUTIKAWA)
Biochemical factors involved in cellular repair of genetic damage (NoGUTI
and KAnA)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

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

Genetic studies on common disorders with complex inheritance (MATSUNAGA)
Genetic effects of family planning and population planning (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)
Structural studies on human immunoglobulins (SHINODA)
Isozyme variation in man (SHINODA and MATSUNAGA)
Department of Microbial Genetics

Genetic regulatory mechanism of DNA replication in E. coli (HIROTA andNISHIMURA)
Genetic regulatory mechanism of cellular division in E. coli (HIROTA)
Genetics of bacterial flagella (ENOMOTO and SUZUKI)
Transduction mechanism of phages (ENOMOTO)
Flagellar synthesis and its regulation in a cell-free system (SUZUKI)
Molecular genetics of flagellar synthesis and its regulatory mechanism(SUZUKI, ENOMOTO and HIROTA)

Department of Poputation Genetics

Theoretical studies of population genetics (KIMURA, MARUYAMA and OHTA)
Studies on molecular evolution from the standpoint of population genetics(KIMURA and OHTA)
Mathematical studies on the genetics of structured populations (MARUYAMA)
Linkage disequilibrium in finite populations (OHTA and KIMURA)
Experimental studies on protein polymorphism in Drosophila (yAMAZAKI)PROJECTS OF RESEARCH FOR 1974

11

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

Studies on the chemical structure of genome of viruses containing doublestranded RNA (MIURA, SUGIURA, FURUICHI, SHIMOTOHNO, WATANABE,
URUSHIBARA, YAZAKI, MUROFUSHI and KIMURA)
Studies on the interaction between RNA polymerase and template nucleic
acid (MIURA, SUGIURA, SHIMOTOHNO, FURUICHI and RORI)
Genetics Stock Center

Studies and conservation of germplasm resources in rice and wheat species(FuJII and OKA)
On the sex expression in monoecious plants (FuJII)
Specificity of mutagen tolerance in higher plants (FuJII)

RESEARCHES CARRIED OUT IN 1974I.

MOLECULAR GENETICS

A Novel Modified Structure at the 5'-terminus of Messenger RNA
of Cytoplasmic Polyhedrosis Virus
Yasuhiro FURillcm and Kin-ichiro MIURA

The messenger RNA of cytoplasmic polyhedrosis virus (CPV) containing
double-stranded RNA can be synthesized in vitro with the use of the substrates
XTP and the virion itself. The addition of a methyl donor S-adenosylmethionine (SAM) to the reaction mixture enhances this RNA synthesis andcauses methylation of the product mRNA. The structure of the methylatedmRNA was analysed.
The RNA was synthesized in the presence of [3H methyl] SAM and (3[32P] ATP. The product RNA was digested with ribonucleases T I and T 2,
pancreatic ribonuclease lA, alkali, and Penicillium nuclease Pl. The digests
were analysed on DEAE-urea column using saIt concentration gradient in
7M urea solution. Both the radioactivities of [3H] and [32P] were detected in
the same peak of an oligonucleotide in any case of the digestion samples.
The smallest labeled oligonucleotide component was obtained by Penicillium nuclease Pl' Since the [(3_3 2p phosphate] attached to adenylic acidshould locate at the 5'-terminus of the synthesized RNA, the methylation by
pH methyl] must occur only at the 5'-terminal part of the RNA. The [3 2P]
phosphate in the oligonucleotide component was not released by phosphomonoesterase treatment. Therefore, the (3-phosphate of the 5'-terminal
adenylic acid should be blocked by some material as X32ppA. Then thesmallest oligonucleotide component obtained by the Penicillium nuclease PI
digestion was further treated with venom phosphodiesterase. Three radioactive components were detected by paper electrophoresis at pH 3.5: [32P]_
inorganic phosphate, [3H]-2'-O-methyl-adenylic acid (pAm), and a [3H]_
containing neutral component (X) which did not move from the starting
point. Upon digestion of the last component with phosphomonoesterase,
pH methyl] was found in a component carrying a plus charge. This was

RESEARCHES CARRIED OUT IN 1974

HN~~;

H NANJ-N

2

OH

OH

13

N~N~

OH

b:,NJ-N

I/O---J.5CH2-0-P-0-P-O-P-O-H2V0",-1
~

~

888

OH OH

/0

OCH3

T:'N~>
O-H2~
o OH

1~ ,"~
0-H2~/OOH
O=P-OHI°
I

5'_ Terminal part of CPVmRNA

:
I
I

I

I

I

identified as 7-methylguanosine by paper-chromatographic analysis using
several kinds of solvent. The previous neutral component (X) was thenidentified as 7-methylguanosine-5'-phosphate (pm 7 G) by comparison withauthentic material on chromatography and by proof of presence of a cis-diol(free OR groups in the adjacent positions in 2' and 3'). As the phosphates
in the original material (XppA) were not released by phosphomonoesterase,
the original terminal component should be in the structure ofm7 G 5'ppp5'Am.
7-methylguanosine-5'-phosphate is linked to the .a-phosphate of ppAm
through a pyrophosphate linkage. The terminal nucleotide sequence of
CPV mRNA is concluded as m 7 G 5'ppp5'Am-G_U_ (see illustration),judging
from the above mentioned ribonuclease digestion experiment and the former
experiments with non-methylated CPV mRNA.
The blocked and methylated structure at the 5'-terminus of CPV mRNA

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

is the first indication as a modified structure in the messenger RNA molecule.
This structure could play an important role in the performance of mRNA
synthesis and/or the function of mRNA. This modified structure would
be resistant to exonucleolytic attack, so that it may be able to control a life
ofmRNA.
This work was published in Nature 253: 374-375 (1975).
A Modified 5'-terminal Structure of Messenger RNA
of Vaccinia Virus
Toshiyuki URUSIDBARA, Yasuhiro FURUICHI, Kunitada SHIMOTOHNO,
and Kin-ichiro MIURA

It is known for vaccinia virus carrying double-stranded DNA genome
to contain various kinds of enzymes in a coat. When the virion is treatedwith trypsin, it is able to synthesize mRNA in vitro. If SAM was added
to this system, mRNA was methylated. The methylation was restricted to
the 5'-terminalpart of the mRNA. Two 5'-terminal nucleotide components
were isolated by chromatography or paper electrophoresis after digestion
with Penicillium nuclease Pl' The [3H] methyl-labeled mRNA were digested
with venom phosphodiesterase into pm 7 G, pGm and pAm in the ratio of
5: 3: 2. When [,B_ 32 P] ATP or [,B_3 2P] GTP was used for mRNA synthesis,[32P] inorganic phosphate was obtained by the same digestion methods.
Finally two kinds of the 5'-terminal components were identified as
m7G5/ppp5'Gm and m 7 G5'ppp5'Am (in the ratio of 3: 2). Vaccinia virus
genome contains several or more cistrons, so that some mRNAs carrym 7 GpppGm while other mRNAs carry m 7 GpppAm at the 5'-termini. These
unusual modified structures at the 5'-terminal of vaccinia mRNAs are thesame to the the mRNA of CPV. The commonness of these abnormal
structures suggests that the modifications at the 5'-terminus are necessary
for the function and/or the control of metabolism of mRNA.
The preliminary report was published in FEBS Letters 49: 385-389 (1975).
Nucleotide Sequence at the 5'-terminus of Reovirus Messenger RNA
Kunitada SHIMOTOHNO and Kin-ichiro MIURA

The 5'-terminal nucleotide sequence of mRNA synthesized in vitro byreovirus was determined. The fact that the 5'-terminal nucleotide is ppG

RESEARCHES CARRIED OUT IN 197415

was known by the incorporation of [.B_ 32 P] GTP into the RNA. From thedigestion of the labeled material with pancreatic ribonuclease, the second
nucleotide was analyzed as a pyrimidine nucleotide. [a_ 32 p] CTP and [a32P] UTP were prepared as the substrates of this part.
The labeled material
was digested with pancreatic ribonuclease and then digested with spleen
phosphodiesterase. These treatments yield mononucleotides from innerpart of RNA and a terminal oligonucleotide, because only the nucleotide
carrying the 5'-phosphate resists to the attack of spleen phosphodiesterase.
Only when [a_ 32 p] CTP was used, the terminal oligonucleotide was labeled.
[a_ 32 P] of UTP was not incorporated in the terminal oligonucleotide. The
labeled terminal oligonucleotide was isolated by chromatography and analyzed. Its structure was determined as ppG 32 pC 32 p. As only [a_ 32p] CTP
was used as a source of 32p in this experiment, the neighbor of the 3' -side
should be C. Therefore, the 5'-terminal three nucleotide sequence is
ppGpCpC------. Thus the 5'-terminal sequence of reovirus mRNA is thesame to the 5'-terminal modified strand in the genome double-stranded RNA
as the case of another double-stranded RNA containing virus: CPV.
Phospbobydrolase Activity in tbe Virionof Cytoplasmic Polybedrosis Virus
Kunitada SmMoToHNO and Kin-ichiro MIURA

Messenger RNA synthesized in vitro by CPV particle carries the following
5'-terminal structure: ppA-G-Y-------. There is no r-phosphate at the
5'-terminus. An enzyme activity which deletes r-phosphate was found in
the CPV particle.
The optimum conditions for this enzyme activity were pH 9.5, 45°C, and4~8 mM Mg++. The activity was inhibited to 50% by 2.5 mM inorganic
phosphate, 5 mM pyrophosphate, 0.1 M KCl, or 0.15 M NaCl. Only rphosphate of nucleoside triphosphate (NTP) was released by the enzyme,
and nucleoside diphosphate (NDP) was left. Among the four kinds of
NTP, ATP was readily hydrolysed in a few times higher rate than otherNTPs. The RNA sample, which contains pppA- as a 5'-terminus and has
4 S size, was hydrolysed only 1/30 in rate to the case of ATP. The rate ofhydrolysis was further lower for the 30 S RNA sample (below 10% of NTP).
Therefore, this enzyme acts on the r-phosphate of small nucleotide, which
can enter into the virus particle, but not on the terminal r-phosphate in along RNA chain. When the relationship between deletion of protein

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

subunits from a virion by pronase treatment and loss of enzyme activity wasstudied, it was suggested that the phosphohydrolase does not face to theoutside of a virion.
Modified Structure in the 5'-terminus of Genome
Double-stranded RNA of Reovirus
Kin-ichiro MIURA and Kumiko WATANABE

When the 5'-terminal nucleotide sequence of human reovirus doublestranded RNA was determined, removement of a few nucleotides fromthe 3 /-terminal was required prior to [32P] labeling of the 5'-termini bypolynucleotide kinase. If the 5'-terminal phosphates of the double-stranded
RNA is blocked by 7-methyl-guanylic acid as shown in mRNAs of CPV andreovirus, the 2'- and 3 /- positions in the ribose moiety of this 7-methylguanosine should form cis-diol and should be oxidized by periodate and labeledwith [3H] by the borohydride reduction. This method is the same to the 3'terminal labeling. When the 3'-terminal nucleosides of CPV and reovirus
were analyzed, an unknown [3H]-labeled material was detected in about half
amount of the sum of the [3H] trialcohols from the 3'-termini. Now we havedetermined this material as the trialcohol of 7-methylguanosine.
Reovirus RNA was labeled by [3H] using borohydride as mentioned above,and digested with Penicillium nuclease PI and phosphomonoesterase. Besides [3H] nucleoside trialcohols from the 3'-termini, a [3H]-labeled material
similar to the modified structure obtained from the 5'-end of mRNA wasdetected. On hydrolysis of this material with phosphodiesterase, [3H]
trialcohol of 7-methylguanylic acid was obtained. These results led us tothe conclusion that the 5'-terminus of one RNA chain in a double-stranded
RNA involves a modified structure same to the 5'-terminus of mRNA as
follows: m7 G 5'ppp5' Gm-C-------.
Thus we can write a structural relationship between genome double-stranded RNA of reovirus and its mRNA as follows:~--- A-V-C3 'm7 G 5'ppp5'Gm-C
3'C - G~
V-A-G 5 ,1transcription
1modification
m7 G5'ppp5'Gm-C
3'
Reovirus synthesizes single-stranded mRNA transcribing one strand of

RESEARCHES CARRIED OUT IN 1974

17

genome and releases only the completed mRNA outside of the VIrlon.
After the mRNA has finished a role for protein synthesis, it functions as atemplate for synthesis of the counterpart strand. Here, the latter (minus
strand) would combine with mRNA to make a double-stranded state andenter into a progeny virion. This idea for the multiplication process ofreovirus is suggested from the fact that both the plus strand in genome and themRNA contains the same modified structure at the 5'-terminus. This supports the asynchronous replication mechanism proposed by Acs et al. (Proc.
Nat. Acad. Sci. U. S. A.: 68, 505 (1971)), which is different from the replication mechanism of DNA.
Transcription of Double-stranded RNA by the DNA-dependent RNAPolymerase from E. coli
Masahiro SUGIURA and Kin-ichiro MIURA

In order to test the possibility to analyze the signals for transcription-operator, promotor, terminator and so on-the in vitro transcription of
double- stranded RNA by E. coli RNA polymerase was studied, as the nupleotide sequence is readily analyzed for RNA. Formerly RNA synthesis
was not detected in this system (Virus, 18: 555 (1968)). For the present
experiments, highly purified DNA-dependent RNA polymerase from E.
coli was prepared according to the Burgess' method followed by two-cycle
chromatography on Agarose and DNA-cellulose chromatography. Whenthe in vitro RNA polymerizing system was added with Mn++ ion instead of
Mg+ +, a little but definite amount of RNA was synthesized, depending on
the addition of double-stranded RNA of CPV as a template. This RNA
synthesis required addition of four kinds of NTP. It was inhibited byrifampicin, streptoligidin, and ethidium bromide, but not by deoxyribonuclease and actinomycin D, which does not bind with double-stranded RNA.
The synthesized RNA was 13 S in size and was able to hybridize with doublestranded RNA of CPV specifically. Since this RNA synthesis depended
completely on the sigma factor, the E. coli RNA polymerase would recognize
a specific part, which would correspond to the promotor site in DNA, tostart RNA synthesis.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

II.

MICROBIAL GENETICS

A Mutation which Causes a Change in the Structureof a Lipoprotein Bound to Peptidoglycan
Hideho SUZUKI, Yukinobu NISHIMURA, Hiroko IKETANI and Yukinori HIROTA

The lipoprotein bound covalently to peptidoglycan of Escherichia coli
was found by Braun and his co-workers (1970, Eur. J. Biochem. 13: 336).
A mutant of E. coli Kl2 which produced an altered lipoprotein was found.
The mutation appeared to result in the failure of a transfer reaction of fatty
acid diglyceride to the SH group of N-terminal cystein in the lipoprotein.
From the analyses by crosses and transduction, it was concluded that thelocation of the gene responsible for this reaction was around 31.5 min, between gurA and aroD, on the chromosome map of E. coli (A. Taylor 1972,
Bacteriol. Rev. 36: 504).
When the peptidoglycan-lipoprotein complex of the mutant was digested
with egg white lysozyme and subjected to the SDS-gel electrophoresis
followed by staining with Coomassie Blue, there appeared no band which
represented a lipoprotein with a minimal unit of peptidoglycan resulted fromlysozyme action, but instead, two new bands were observed at positions ofhigher molecular weights. These bands, which were absent in the electrophoretic pattern of the preparation from the wild type cells, disappeared
upon reduction of the lysozyme digest with mercaptoethanol to give rise to
two bands corresponding to a wild type lipoprotein with a peptidoglycan
unit and its free form. The lipoprotein of the mutant was reactive withmonoiodoacetate when exposed for an hour at pH 8.5, but the authentic
lipoprotein was not under the same condition. In the former nearly 1 moleof carboxymethyl group was incorporated into 1 mole of lipoprotein. The
presence of one cysteinyl residue per lipoprotein molecule of the mutant was
detectable by a common procedure for an amino acid analysis. In theauthentic lipoprotein, the sulfhydryl group of the N-terminal cystein is
known to be occupied by diglyceride of fatty acids through thioether bondresistant to acid hydrolysis. In order to examine if the mutant-lipoprotein
lacked this diglyceride leaving an SH group free, the cellular lipid waslabelled by addition of 2-(BH)-glycerol to the growth medium. The incor-

RESEARCHES CARRIED OUT IN 1974

19

poration of the glycerol into the lipoprotein of the mutant was absent whencompared with that into the lipoprotein of the wild type cells.
Possible phenotypic changes resulted from this mutation were examinedwith respect to the following points: sensitivities to bacteriophages, Tl,
T5, T4, T6, "t, PI, P2; to growth inhibitors, penicillin, methylene blue; todetergents, deoxycholate, SDS; to a chelating agent, EDTA; and to compounds of mercury, mercuric chloride, p-chloromercuribenzoate, phenylmercuric acetate, including seven additional compounds, and permeation forsugar such as lactose. The physiological characters related to the outermembrane as represented by the passive transport, receptors for bacteriophages, etc. were not changed by this mutation, but the mutant exhibited the
pronounced sensitivity for the compounds of mercury.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

III.

BIOCHEMICAL GENETICS

Detection of Hybrid Enzymes Specified by Acpt Alleles in Wild andCultivated Rice Strains
Chiang PAl, Tom ENDo and Hiko-Ichi OKA

It was concluded from genetic experiments that leaf acid phosphatase
zymograms observed among the rice strains belonging to Oryza perennis
Moench (wild) and O. sativa L. (cultivated) were specified by alleles at threeloci, ACPI, ACP2 and Acpa. At ACPI were detected seven codominant alleles,
Acpl 17 , Acp1 9 , ACPl\ Acpi, Acpi, Acpi 2 and Acpi\ each producing a group of
three major (A, M and C) and three minor (a, m and c) bands on the starchgel. The alleles determined mobility of the band group, in which the relative position of the six bands was almost constant. In addition, a null form
lacking all these bands was found in two cultivated strains which had a recessive allele, Acpr u1 •We once considered that hybrids between strains with different mobilities
produced three major hybrid bands, so that they had nine major bands intotal though some of them overlapped one another (Pai and Endo 1972;
Ann. Rep. 22). This year we examined four hypothesis, i. e., 1) no hybrid
bands occur in heterozygotes, 2) three major and three minor hybrid bands
are produced each by a set of corresponding parental bands, 3) corresponding
parental major bands produce three major hybrid bands (our previous assumption), and 4) corresponding minor band produce three minor hybrid
bands. Statistical analysis of the data taken from densitometric tracings ofzymograms indicated that the second hypothesis was most plausible i. e.,
the PI hybrids produce three major (AH, M H and C H) and three minor (a H,
mH and cH) hybrid bands.

RESEARCHES CARRIED OUT IN 1974

IV.

21

DEVELOPMENTAL AND SOMATIC CELL GENETICS
Polymorphic Characters of the External Orifice of Uterus
in Inbred Strains of Rats
Tosihide H. YOSIDA and Kunitoshi SANO

It has been known as an established fact that the uterus of the rat has two
external orifices; namely the cervice of the uterus opens directly to thevagina by two orifices. Morphology of the external orifice of the rats,
however, differs makedly from strain to strain. In some strains of rat the two
external orifices are observed as shown in the textbook, but in some other only
one orifice is observable. In the latter case, two cervices of the uterus join
together to constitute one external orifice that opens to the vagina. To know
the strain differences of the morphology of the external orifice, nine inbred
strains of rats, ACI, Albany Buffalo, Fisher, Long Evans, NIG, Wistar-M(WM), Wistar-King-A (WKA) and Wistar-King-S (WKS) were examined.
Among them only two strains (WKA and WM) showed two external orifices,
but the other 6 strains (ACI, Albany, Buffalo, Long Evans, Fisher and NIG)
had one external orifice. Only one strain (WKS) showed an intermediatetype of the above two forms. Morphology of the external orifice does notchange through the sexual cycle or the other physiological condition of thehost. It seems to be a rigid genetical characteristic of each strain of the rat.
Cross experiments between one- orificed and two-orificed rats and between
two orificed and intermediate type rats were carried out to know their genetical characters. All F I hybrids between them showed two orifices. Thus,
two-orificed type of the rat seems to be inherited as a dominant character tothe one-orificed and intermediate types.

Isolation of Nematocyte Deficient Mutants of Hydra
Toshitaka FUJISAWA and Tsutomu SUGIYAMA

Nematocytes are hydra's stinging cells and they are located mainly on thetentacles where they are used primarily to capture and paralyse prey. Hydra
magnipapillata, a common species in Japan, has four types of nematocytes(type A, B, C and D) which together account for one-third to one-fourth of

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

the total body cells.
The nematocyte deficient mutants of H. magnipapillata were isolated bycrossing male and female strains collected from the same ponds and examin.
ing their FI and F2 progenies for the food capturing ability and the nemato.
cyte compositions.
Nem-4, one of the mutants found by this procedure, was able to capture thelaboratory food, Artemia salinas nauplii, but was unable to paralyse them.
Miscroscopic examination revealed that the type A nematocyte (stenotele)
which contained neurotoxin to paralyse prey was absent from the tentaclesof this strain, but that the same nematocyte was present in its body region.
Since it is known that the differentiation of the nematocytes from the interstitial cells takes place in the body region and that only matured nematocytes
migrate to the tentacles, the defect of nem-4 seemed to be in the mechanism
for the migration of the newly-differentiated type A nematocyte from thebody region to the tentacles.
Other nematocyte mutants isolated included a strain which lacked type B
nematocyte and a strain which lacked all four types of nematocytes in thetentacles, and characterization of these mutants are currently in progress.
Regeneration Mutants of Hydra
Tsutomu SUGIYAMA and Toshitaka FUJISAWA

The regeneration deficient strains of Hydra magnipapillata were isolatedby the inbreeding of field strains as described in the preceeding paper.
After amputation of its head and foot, a wild type hydra regenerated a
new head and a new foot in their original places within a few days. In
contrast, the mutant strains exhibited greatly different patterns of regeneration under the same coneditions. For example, one of the mutant, reg-I,
regenerated the foot normally but it frequently failed to regenerate thehead. Another mutant, reg-8, regenerated the head normally but it frequently produced an extra head in place of the foot, thereby producing adouble-headed, or bipolar, animal. The third type of mutant was unable toregenerate any visible structures at all.
It is generally believed that hydra has polarity gradient (s) along its bodycolumn that specifies the distal to proximal nature of its body. The mutants
are being examined with the view that they may have abnormal polarity
gradient (s).

RESEARCHES CARRIED OUT IN 1974

23

Analysis of Time Specificity of dor + Gene Action in
Embryonic Development of Drosophila melanogasterYukiaki KURODA

In a previous work, it has been found that embryonic cells homozygous or
hemizygous for an X-linked (1-0.3) recessive lethal gene, deep orange (dar)were maintained in a chemically defined medium with some macromolecularsupplements for a relatively extended period over the prospective lethal phaseof the embryos, although some defects in properties such as syncytium formation of muscle cells, formation of cellular spheres, proliferation of small
cells, and droplet formation on nerve fibers were found. It has been also
found that the addition of unfertilized wild-type egg extract to the culture
medium of dar embryonic cells resulted in repair of their defective properties(for details, see Nature 252: 40-41, 1974).
In the present experiment dar embryonic cells were cultured to determine
the specific time of dar+ gene action introduced by the wild-type sperm inembryonic development. Embryos obtained from matings of dar/dar
females and dar + /Y males were allowed to develop to the stage of blastoderm
formation (3 hours after fertilization), gastrulation (5 hours after fertilization),head and trunk segmentation (8 hours after fertilization), and muscular movement (14 hours after fertilization), and used as donor embryos from which
extracts were prepared.
The all defective behavior and functions of dar embryonic cells were not
repaired by the extract from embryos at the stage of blastoderm formation.
The extracts from embryos at the stages after gastrulation were effective inrepairing the defective properties of dar embryonic cells. This suggests thatsome effective substances having a repair effect may be produced by paternal
dar + gene between 3 hours and 5 hours after fertilization.
Effective Substances Having a Repair Action of dor
Embryonic Defects in Drosophila melanogasterYukiaki KURODA

It has been previously found that dar embryonic cells had some defects inproperties such as syncytium formation of muscle cells, formation of cellularspheres and droplet formation on nerve fibers when they were cultured invitra and that these defects were repaired by the addition of unfertilized wild-

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

type egg extract to the culture medium of dor embryonic cells.
The effective substances having this repair effect were contained only in thewild-type egg extract, and not in unfertilized dor egg extract. The behavior
and functions of embryoic wild-type cells were not inhibited by the additionof dor embryonic egg extract. These results suggest that the repair action of
the wild-type egg extract may be due to the action of some effective substances
contained in the extract and not due to the removal of inhibitory substancesin embryonic dor cells.
When the wild-type egg extract was heated at 80°C for 10 minutes, its activity was lost. This indicates that the effective substances in the wild-type
egg extract may be some enzymes or heat-labile substances.
Isoxanthopterine and xanthopterine added to culture medium had noeffects on repair of the defective properties of embryonic dor cells, although
2-amino-4-hydroxypteridine had a slight effect on stimulation of droplet formation on nerve fibers of dor cells. Further investigation is required to
identify the chemical nature of the effective substances.
Induction of 8-Azaguanine Resistant Mutations by
Phloxine in Embryonic Human Diploid Cells
Yukiaki KURODA

The red dye, phloxine, is widely used as an additive in cakes and fish pastes
in Japan. It has been reported that in the "rec-assay" phloxine showed
a greater lethal effect on Rec- than on Rec+ strain of Bacillus subtilis andit had a mutagenic action on Escherichia coli B/r WP 2 try+.
In the present experiment the mutagenic effect of phloxine on 8-azaguanine(8AG) resistant mutations in cultured embryonic human diploid cells was
examined. Phloxine had a severe cytotoxic effect on the cells at concentrations of 1 to 10 ,ug/mI. At concentrations of more than 30 ,ug/ml of phloxine
no further decrease in cell survival was found. This cytotoxic effect ofphloxine was not dependent on the duration of treatment. After treatment
with phloxine for 2 hours division of cells in normal medium was inhibitedfor 120 hours.
When cells were treated with phloxine at various concentrations for 2
hours, cultured in normal medium for 48 hours, and then selected with 30,ug/ml of 8AG, an increase in the induced mutation frequency was found.
This increase in mutation frequency was dependent on the concentration of

RESEARCHES CARRIED OUT IN 1974

25

phloxine used as a mutagen and treatment with 100 .ug/ml of phloxine increased the frequency to six times that in untreated cultures. For details,
see Mutation Research 30: 239-248.
Protection by Vitamin E Against Bisulfite-Caused Defectin Growth of Cultured Human Cells
Yukiaki KURODA

It is known that bisulfite induces gene mutations in bacteria, yeast andbacteriophages. The induction of these gene mutations is produced by atransition process from cytosine to uracil in DNA molecules. At low concentrations bisulfite was found to inactivate the transforming activity ofbacterial DNA by free radical-mediated reactions in the co-existence withoxygen. On the other hand, it is generally accepted that vitamin E has anantioxidant activity in various metabolic processes in higher animals.
The present experiment reveals that vitamin E protects against a bisulfitecaused defect in growth of cultured human cells. The cells used were the
8-3 subline of HeLa cells, human cervical carcinoma cells. Triplicate
inocula of 103 cells were incubated for 2 hours at 37°C in 60-mm petri dishes,
and then bisulfite and/or vitamin E at various concentrations were added
to the medium. After incubation for 10 days at 37°C, the colony-forming
activity of cells was determined.
Bisulfite had a severe cytotoxic effect on HeLa 8-3 cells at a concentrationof 10- 4 M, causing complete inhibition of colony formation. A concentration of 10- 5 M bisulfite had a moderate cytotoxic effect on the cells, reducing
their colony-forming activity to one third of that of untreated control cultures. Addition of vitamin E at a concentration of 10- 7 M restored thecolony-forming activity of 10- 5 M bisulfite-treated cells to 71 % of that ofcontrol cultures. Addition of 10- 6 M vitamin E resulted in almost complete
recovery of colony-forming activity of bisulfite-treated cells to that of control
cultures. The counteraction of vitamin E to the cytotoxic effect of bisulfite
on human cells was suggested. For details, see Experimental Cell Research
94: 442-445.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

Isolation and Characterization of Variant Clones ofChinese Hamster Cells after Treatment with
Irradiated 5-Iodouridine
Yukiaki KURODA, Akiko YOKOIYAMA and Tsuneo KADA

Previously we reported that the colony-forming ability of Chinese hamster
cells decreased on treatment of the cells with r-irradiated 5-iodouridine atacidic pH's. We suggested that this might be due to radiation-induced
cytotoxic iodine radicals.
In the present work some variant clones were isolated from Chinese hamster
cells treated with irradiated 5-iodouridine. The following characters of a
primary variant clone, C-Il and a secondary variant clone, C-24 werecompared with those of the original clone C-l: colony-forming activity,
growth rate in the presence of irradiated and unirradiated 5-iodouridine,
distribution of chromosome numbers and cell cohesion.
The variant clone C-ll and C-24 were partially resistant to unirradiated
5-iodouridine at lower concentration and C-24 cells were slightly resistant toshort-term treatment with irradiated 5-iodouridine. Unlike clones C-l andC-ll the variant clone C-24 showed no lag phase on growth in 5-iodouridine
medium. The modal numbers of the chromosomes of all three clones were
22, like that of normal Chinese hamster diploid cells.
Of the three clones, the variant C-24 cells showed the least mutual cohesion
and the original C-l cells showed the most. The possibility that an alteration in cellular membrane might be related to an increase in the resistance toradiosensitizing agents was suggested. For details, see Mutation Research
33: 285-298.
Effect of Serum on Proliferation of Mammalian Cells
in Culture
Kiyoshi MINATO

It has been previously found that the cumulative number of cells whichwere maintained without medium change was dependent on the concentration of serum supplemented initially. In further studies on this phenomenon,a stable proliferation of cells was not obtained very frequently by unknown
reason.
In the present experiment, the reason why the stable proliferation of cells

RESEARCHES CARRIED OUT IN 1974

27

had not been obtained was investigated. This unstable cell growth was
related to a low quality of serum which was dependent on its lots. Considerable differences were found in population-dependent requirement (PDR)
for cell growth, doubling time, saturation density of cells, when cells were
cultured in different lots of serum; serum with low quality had bad effects
on these properties of cells.
It was also found that serum with low quality brought about an incomplete
repair of mechanical damages produced in the process of dissociation of cells,
and that this resulted in unstable proliferation of cells in further cultivation.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25v.

CYTOGENETICS

Frequency of A/A, A/S and SIS Chromosomes in Pairs No. 1,9
and 13 in 133 Black Rats Collected in Various Localities
Tosihide H. YOSIDA

Chromosome polymorphism of the largest pair no. 1 with respect toacrocentrics (telocentrics) and subtelocentrics has been previously reported ina small population of Japanese black rats (Yosida et at. 1965, Chromosoma
16: 70-78). Thereafter, the frequency of A/A, A/S and SIS pairs no. 1
in 453 rats corrected in 19 localities in Japan has been published (Yosida et at.
1971, Chromosoma 33: 30-40). Appearance of chromosome polymorphismin pairs no. 9 and 13 in regard to the acrocentrics and subtelocentrics has also
been reported by Yosida et at. (1969, this report 19: 12; 1970 this report 20:
10; 1971, this report 21: 55-56) in Japanese population, and Gropp et at.
(1970, Z.f. Siiugetier. 35: 363-371) in Thailand rats. Statistical survey of thefrequency of chromosome polymorphism of the pairs no. 9 and 13 has not been
carried out yet, except a study of 16 rats collected in Misima, Japan (Yosida
et at. 1971, this report 21: 55-56). To know the frequency of the polymorphism of pairs no. 1, 9 and 13, 133 rats were examined. Among them 95
rats were R. rattus tanezumi collected in Japan, 6 rats were R. rattus diardii
from Malaysia, 19 rats were R. rattus mindanensis from the Philippines, and theremaining 13 rats were R. rattus rattus from Oceania. In Japan rats werecollected in four localities; Hokkaido (22), Niigata (15), Misima (35) andNagasaki (23). In Hokkaido population nos. 1 and 9 were all acrocentrics,
but no. 13 were polymorphic. In Niigata, no. I were all acrocentrics but
nos. 9 and 13 were polymorphic. In Misima and Nagasaki populations
pairs no. 1, 9 and 13 were all polymorphic. In R. rattus diardii, pairs no. 1
and 13 were polymorphic, but no. 9 was all SIS type. In R. rattus mindanensis pair no. 1 was SIS type, except one rat with A/S, while pairs no. 9 and 13
were polymorphic. Pairs no. 1 and 9 of R. rattus rattus were SIS, except one
with A/A pair no. 9. Most of pair no. 13 were acrocentrics, but 2 were
heteromorphic A/S type. Besed on the above investigation, it can be said
that the frequency of polymorphism in the smallest pair no. 13 is higher thanthose of the pairs no. 1 and 9, and inversion of the no. 13 chromosome could
occur considerably earlier than the others.

29

RESEARCHES CARRIED OUT IN 1974

Segregation of Chromosomes in the Second Spermatocytes in F I
Hybrids between Asian and Oceanian Type Black Rats
Tosihide H. YOSIDAF I hybrids between Asian (2n=42) and Oceanian type black rats (2n=38).are characterized by having 40 chromosomes in diploid number which consists of each haploid set from Asian and Oceanian rats. Therefore, the F I
rats have one large metacentric (MI ) which is derived from fusion of acrocentric pairs no. 4 and 7, and another large metacentric (M 2) arising fromfusion of acrocentric pairs no. 11 and 12. The rat also had unpaired acrocentrics no. 4, 7, 11 and 12 (Yosida et al. 1969, Jap. J. Genet. 44: 84-91;
Yosida et al. 1971, Chromosoma 34: 40-50). Idiogram of the F I hybrid inrespect of pairs no. 4, 7,11 and 12 is formulated as M I (A4 +Ar )jA 4 , A r , M 2(A ll +A12)/All , A I2 (M and A mean large metacentrics and acrocentrics,
respectively). In meiosis of the hybrid, synapsis between M 2 (A4 +Ar )/A 4
and A r, and between M 2(A ll +Al2)/All and AI2 can be performed, and in thefirst spermatocytes 17 bivalents and 2 trivalents are produced. At the firstmaturation division the following four second spermatocytes are expected to
be produced by segregation of the metacentrics M I and M 2 and acrocentrics
A 4 , A r , All and A 12 .
(A) MI(A4+Ar) and M2(All +A12).
(B) M 1 (A4 +Ar ), All and Al2 •(C) A 4, A r and M 2(A ll + A I2 ).
(D) A 4 , A r , All and A 12 .
Owing to the distribution of the large metacentrics (M 1 and M 2), thehaploid chromosome number in the second spermatocytes is expected toTable 1. Frequencies of the second spermatocytes with four different
chromosome types in the FI hybrids between Asian and Oceaniantype black rats
Chromosome types (pair no.)No.4
Ml (A4+Ar)
Ml (A4+Ar)
A4
A4

No.7

No. 11Ar
Ar

M2(An + A12)An
M2(An + A12)
An
Total

n

No. of cells
observed

19
20
20
21

13
17
14
21

No. 12
A1 2
A12

65

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

vary from 19 to 21. Microscopically, the second spermatocytes with 19
chromosomes having two metacentrics, with 20 chromosomes posessing one
metacentric and with 21 chromosomes having no metacentrics (Fig. 3) werereally observed. The second spermatocytes with the above four types are
expected to be equal in their frequencies. In 65 spermatocytes analysed thefour types were observed in almost equal number, although the frequency ofcells with 21 chromosomes was slightly higher than that of the others (Table
1).
Relation between the Decrease of C-band Heterochromatin
and Differentiation of Rattus species
Tosihide H. YOSIDAIn the previous report the author (Yosida and Sagai 1974, this report 24:
24-25; 1975, Chromosoma 50: 283-300) described that the black rat (Rattus
rattus) showed clear-cut and large-sized C-bands in the centromeric region of
all chromosome pairs, but polymorphism of the bands involving the presence
and absence or their size was observed in some subspcies of the animals.
From a view point of karyotype evolution of the animals the author suggested
that the C-bands show a tendency toward the decrease, so far as the closely
related subspecies of the black rat are concerned. In order to know whether
the tendency found in subspecies of the black rats occurs in the case of differentiation of species in genus Rattus, C-bands in 8 species, R. r. argenti-

venter, R. r. jalorensis, R. norvegicus, R. annandalei, R. exulans, R. muerelli,R.fuscipes and R. conatus; were investigated by way of comparison with thatof R. rattus.
Results of observations showed that all 8 species examined here presented
small sized or vaguely stained C-bands in contrast to those of R. rattus.
From view points of karyotype evolution in genus Rattus the author (Yosida
1973, Chromosoma 40: 285-297) suggested that the karyotype of R. rattus
with 2n=42 is an ancestral form, from which the karyotypes of the other 8
Rattus species investigated here have differentiated by pericentric inversion
and Robertsonian fusion. Based on these evidences a following suggestion
presents that the size of heterochromatic C-bands has also a tendency todecrease in the closely related species of the genus Rattus.

RESEARCHES CARRIED OUT IN 197431

Effects of 2-(2-furyl)-3-(5-nitro-2-furyl)-Acrylamide (AF-2) on
Bone Marrow Chromosomes of the Rat at Different Ages
Tosihide H. YOSIDA and Hideo TSUJI

Cytogenetical effect of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2,
a food preservative) on rat bone marrow cells has been reported by Sugiyama(1975, Mut. Res. 31: 241-246). The present authors reinvestigated theeffect of the chemical on bone marrow cells of the rat and found that theeffect was considerably different depending on the age (body weight) of the rat
used. Two rat strains (Wistar King-S and Long Evans) weighing 60 to 420 gr
were used in the present study.
The chemical (AF-2) was used at a concentration of 300 mgjk dissolvedin physiological saline. According to Sugiyama, the effect of the chemical
to bone marrow chromosomes is most prominant at 6 hours after injection.
As we also obtained the same result in a preliminary study, chromosome
observations were done exclusively on the 6 hour sample. The experiment
was carried out with 6 young rats weighing 60 to 190 gr and 3 old rats
weighing 380 to 390 gr.
Results of experiment are as follows: (1) Chromosomal abnormalities
caused by the chemical were all chromatid breaks and gaps, and no otherabnormalities were detected. The chemical might affect cells at the G 2stage of the cell cycle because the abnormalities was observed 6 hours after
treatment. (2) The effect was not different in two rat strains used. (3) Inyoung rats (60-190 gr) chromosomes of 600 cells were examined. Amongthem 42 cells (7 %) showed chromatid aberrations such as chromatid breaks(5 cells) and chromatid gaps (42 cells). In old rats (280-390 gr) 300 cells
were counted, and only 5 cells (1.6 %) had chromatid aberrations. The
difference between these aberration frequencies is statistically significant,but the latter value is not significantly different from that obtained in thecontrol. From the above experiments it can be said that effect of AF-2
differs markedly depending on the age of rats used.
Evidence against that the Sister Chromatid Exchange
is Single-Strand Exchange
Hatao RATO

Phenotypic similarities between the induction of sister chromatid exchanges

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25(SCEs) in mammalian chromosomes by ultraviolet (UV) irradiation andrecombinational repair of UV-induced DNA damage in E. coli led previous
workers (Bender, M. A. et al. 1973 Mutation Res. 20: 387) to postulate thatchromosomal aberrations are outcome of incomplete SCEs arising throughmolecular processes akin to those involved in recombinational repair.
However, the fact that the recombinational repair mechanism involves
exchange of single polynucleotide chains of DNA seems to offer various
difficulties in the explanation for the SCE induction based on the context
mentioned above (Kato, H. 1974 Exptl. Cell Res. 85: 239; 1974 Nature
249: 552). Firstly, in recombinational repair the size of a gap to be filled inby a segment of single strand transferred from the sister molecule is believed
to be about 1000 nucleotide long. This size seems to be too small to be
detected as SCEs in mammalian chromosomes. Secondly, single-strand
exchange requires denaturation of very long segments of DNA and theireventual annealing with the complementary strands in the sister chromatid.
These processes may be achieved without unwinding difficulty by crossed
strand migration (Lee, C. S. et al. 1970 J. Mol. BioI. 48: 1). However, thedistance for the crossed strand to migrate is considered to be unrealisticallylong in such a giant molecule as the mammalian chromosome: Suppose if
one single strand exchange is initiated at the midpoint of a Chinese hamster
No. 1 chromosome which is about 6 X 108 nucleotide pair long, the crossed
strand has to migrate through 3 X 108 nucleotide pairs to accomplish the SCE
process. Meselson (1972 J. Mol. BioI. 71: 795) calculated that at 37°C
about 20 seconds would be needed for such migration through 1000 nucleotide
pairs. This means that the completion of the given SCE needs 6 X 108
seconds, i. e. 167 hours.
Recently a new method was developed to distinguish sister chromatids
without the usage of tritium label. The method involves labeling of cellswith 5-bromo-2'-deoxyuridine (BUdR) and staining of fixed chromosomeswith acridine orange (Kato, H. 1974 Nature 251: 70), and can demarcate
unequivocally chromatids substituted unifilarly with BUdR from unsubstituted as well as bifilarly-substituted chromatids. By using this technique, thequestion as to whether the SCE is single-strand exchange or not was reexamined. If cells are labeled with BUdR for one round of cell cycle, a
BUdR-labeled metaphase chromosome would be composed of sister chromatids each containing unifilarly-substituted DNA. Exchange of any single
strands of the same polarity would result in the appearance of chromosomal

RESEARCHES CARRIED OUT IN 1974

33

segments which would be now composed of an unsubstituted DNA duplex
in one chromatid and a bifilarly-substituted one in its sister chromatid atcorresponding regions. These segments would be distinguished clearly
from each other as well as from the rest of chromosome regions by acridine
orange staining. On the other hand, if exchange occurs at a double strand
level, recombined chromatids would be still composed of unifilarly-substituted DNA along their entire length and the chromosome would be stained
uniformly.
Chinese hamster cells, D-6, were exposed to either 5 jjm 2 UV or 1O- 6M
mitomycin C (MMC) and then grown in culture medium containing 0.5,ugjml
BUdR for 8 h before harvest. It was confirmed previously that UV and
MMC at these doses induced SCEs at frequencies of 2.5 and 3.1 per No.1
chromosome, respectively, when they were applied during the 2nd postlabeling
S. Over a hundred metaphases were examined carefully for the presenceor absence of differentially stained segments but all the chromosomes were
found to be stained uniformly as expected if the SCE had occurred at doublestrand level. It is thus very likely that even if the initial step of the SCE
formation can be exchange of single strands arising from each sister chromatid,
breakage of the remaining strands would soon follow enzymatically at ornear the exchange site, resulting in double strand exchange as postulated byprevious workers (Whitehouse, H. L. K. 1963 Nature 199: 1034; Holliday,
R. 1964 Genet. Res. 5: 282). Obviously these processes are different from themechanism proposed for recombinational repair of DNA damage in UVirradiated E. coli (Rupp, W. D. et al. 1971 J. Mol. BioI. 61: 25).
Chromosome Analysis of Sublines of a Mouse Hepatoma (MH134)
with a Ribosomal RNA Variation
Kazuo MORIWAKI, Hirotami IMAI, Ryo KOMINAMI*
and Masami MURAMATSU*

It has already been reported that two sublines (C and Os) of mouse hepatoma MH134 are lacking in a spot-20 (CUAACA¢CUG) on the fingerprint
pattern of 28 S RNA, though either the other subline (Y) of this tumor andnormal liver have it (Kominami, Hashimoto and Muramatsu, Proc. Jap.
Cancer Assoc., 33: 37, 1974). In order to trace the possible position during
serial propagation where the ribosomal RNA variation may have occurred,* Department of Biochemistry, Tokushima University Medical School.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

the pedigree of eight sublines of this tumor was surveyed. Since 1955, thistumor line has been maintained by Prof. H. Sato in the Tohoku University
from where four sublines, Ms, Se, Y and Ib, originated after 1967. In 1959,
a part of this tumor was transferred to the Tokyo University Institute of Me.
dical Science (Prof. T. Yamamoto) from where the other four sublines, 165, Os, C and If, started after 1965. The cytogenetical survey by G-band
analysis on the karyotypes of those subline cells well approved this pedigree.
As summarized in Table 1, M4 and M5 marker chromosomes are common to
all the sublines, whereas Ml and M2 markers are observed only in the four
sublines directly originated from the Tohoku University-line. The OCCUr.
renee of the ribosomal RNA variation seems to be limited to the sublines
started from the Tokyo University-line. Furthermore, in sublines Os andC the extreme eleongation of the secondary constriction in a No. 15 chromo.
some was observed. Considering the repetitive nature of ribosomal genes in
the higher organisms, one may not explain simply the cause of the variationin ribosomal RNA.
Table l.

Occurrence of Specific Marker Chromosomes in MH134 Sublines
Frequencies of the marker chromosomes(% of 50 cells)

Sublines
Ms

Y

Se

Ib

Os

If

C

1-65

Modal chromosome No.

39

38

44

43

43 41/4242

42Marker chromosomesM1 (6-2 TRL.)
M2 (7-8 TRL.)
M3 (15 ISO.)
M4(A)
M5(B)
M6 (4 ISO.)
M7(C)
M8(D)
M9 (15 2nd Const. Elg.)

100
100
0
94
96
0
18
58
82

100
100
0
100
100
0
0
40
82

100
100
94
98
0
0
0
0
0

98
98
92
100
0
96
0
0
0

0
0
0
100
20
0
0
0
100*

0
0
0
94
94
0
48
58
100*

0
0
0
100
65
0
0
0
90

2

3

+

+

No. 16 Chromosome

1

Ribosome 28S-RNA-Spot
20**

+

+

0
0
0
100
72

0
0
0
90

1

* Extreme elongation ** Kominami, Hashimoto and Muramatsu, 1974

1

RESEARCHES CARRIED OUT IN 1974

35

Genetical Analysis of a Newly Gamma-ray Induced Translocation
between Wand V Chromosomes in Pupal Oocytesof the Silkworm
Aldo MURAKAMI and Aldo OHNUMAIn the course of progeny tests for 137Cs gamma-ray induced egg-color
specific locus mutations in pupal oocytes of the silkworm, a red egg (re,V-3I.7) locus mutation was detected. Genetical analysis suggests that thismutant resulted from a break in the vicinity of re locus on chromosome-5
and that one part of the chromosome including pe (pink egg, V-O.O) wastranslocated on to the W (or Y) chr.omosome. In the silkworm, the male ishomogametic ZZ or XX and the female is heterogametic ZW or XY depending on the nomenclature used for the sex chromosome.. This stock also
has a section of chromosome-5 bearing the marker oc (chinese oily, V-40.8)
gene locus. This translocation line therefore has the chromosome constitution Zjwvpe;voc+jV (Figure 1).

w
z

oc

pe

v

Fig. 1. Chromosome constitution of a newly gamma-ray induced translocation
between Wand V chromosomes.

This translocation line also produces a number of non-disjunctional individuals. In the female progeny of a cross between a female of tIns line and amarker strain male, individuals of three different chromosome types occurred(ZjViVpe;voc+jV, ZjViVpe+jV and Zjwvpe-;VjV) while in the male progeny,
there were also three types of individuals (ZjZ VjV, 'ljZ voc-;YjY and ZjZ yoc+jV). Some embryos of male progeny with ZjZ yoc+jY in a cross between thetranslocation line female and the marker male died, probably due to the lossof a large part (ca. 60 %) of chromosome-5. Similarly, in the progeny of across of the ZjViVpe-;YjV~ X ZjZ VjW!J, neither disjunctional males fromnormally colored eggs (+) nor females from yellowish-white egg (pe) wereobserved, while in a cross between ZjViVpe;voc+jV and ZjZ YjY a small

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

number of disjunctional products segregated. These observations suggest
to be nondisjunctional events in meiotic divisions because of the complex.
chromosome constitution of this translocation line.

RESEARCHES CARRIED OUT IN 1974

VI.

37

MUTATION AND MUTAGENESIS IN ANIMALS

Attempts to Induce Non-disjunction by Means of Irradiation
and Chemical Treatment in the Silkworm*
Yataro TAZIMA and Akio OHNUMA

Since the induction of aneuploidy by radiation attracted our great concern
from the view point of genetic hazard to man, experiments have been carriedout in our laboratory with silkworm. The results obtained hitherto indicatethat non-disjunction can hardly be induced by direct means, although innumerable trisomic strains have been known in this organism. The mutagenic agents used so far are ionizing radiations, high temperature, CO 2 gas,organic mercury compounds and MMC. Both sex-chromosome and autosome systems have been employed. The latter system, in which egg color
genes were used as markers, permitted to deal with a large number of experimental individuals with relative ease.
Females heterozygous for pe+/+re were subjected to mutagenic treatment and crossed to non-treated pere males. Individuals due to nondisjunction, if occurred, could be recovered as a wild type among F I eggs
which comprise I pe: I reo The incidence of wild type was fairly high bothin the control and treated groups: for instance, 13/59120 in the control,
25/99765 in X-irradiated groups and 41/123413 in methyl mercuric chloridegroups. Wild type individuals analysed so far were 70 females and I male.
70 females comprised I 4n, 63 3n and 6 2n and 2n females were confirmed as
the products of recombination. One male was 2n but its genotype was notdetermined. Thus, even a case of non-disjunction of marked chromosomes
has not been recovered yet, contrasting sharply to the findings in Drosophila.
Almost all trisomies thus far obtained in the silkworm occurred as a consequence of chromosomal unbalance due to deficiencies, inversions and/ortranslocations. Why non-disjunction can hardly be induced by directmeans may be ascribed to the holokinetic nature of the chromosome. Further
analyses are in progress.

* Presented at the fifth International Congress of Radiation Research held at Seattle,
July 19, 1974.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

Mutagenic Effectiveness of an Internal fl Emitter, Tritium.
(1) A Preliminary Experiment on Somatic Mutationin the Silkworm*
Yataro TAZlMA, Kimiharu ONIMARU, and Yosoji FUKASE

With the purpose of developing a new experimental system suitable for theassessment of mutagenicity of internal emitters, studies have been carried out
with silkworm, in which duration of egg stage, hence the duration of expOsureof embryo, can be controlled by experimental conditions. In this prelimin_ary experiment 3H_TdR was injected into the body cavity of wild type femalesof a strain C108 at a mid-pupal stage, so as to allow the incorporation of the
3H_TdR efficiently into the egg. Injected dose was 50 ,uCi per individual
pupa in 0.05 ml of physiological saline. The average radioactivity transmitted to the deposited egg was 0.002 ,uCijegg.
Injected females were crossed, after emergence, to pe ok males for the detection of induced somatic mutations. Mutations were expected to appear as
tiny dots either of pe-cells among wild type serosa cells or of translucent cells
among opaque larval integument cells. There were 103 days from deposition
to hatching, during which the embryos were exposed to p-rays. The resultsof the experiment are shown in Table 1.
Table 1. Somatic mutations induced by 3H-TdR injection++~ xpe, ok (5 (740)Treatment
ControlFrequency
3H-TdR
Frequency

Total
No.
obsvd.
7161
6381

No. of ok mutants
dotmosaic55
00.77 (0. 59-0. 99)*407
16.38 (5.80-7.05)*

whole

Total
No. ofdots

Dots permut. bearing larva

0

83

1.51

515

1.27

C108 ~, average body wt.; 1.33 g
Date of oviposition; Dec. 11, 1974: Date of hatching; March 16, 1975
* 95 % confidence interval

No significant increase was observed in the incidence of eggs with pe dotsin the injected group but the injection of 3H_TdR was distinctly effective inthe production of ok dots in larval integuments.
* Presented at the 17th meeting of the Japan Radiation Research Society, held at
Tokushima, Oct. 7, 1974.

RESEARCHES CARRIED OUT IN 1974

39

These results clearly show that the method of administration of radioisotope could be used effectively for the assessment of the genetic effects of internal emitters.

Effects of Low-temperature Post-treatment on the Repairof Premutational Damage in Spermatogoniaof the Silkworm
Yataro T AZIMA and Yosoji FUKASEAs a part of serial work to elucidate the nature of radiation-induced
damages on DNA, an experiment has been carried out by applying lowtemperature treatment after X-irradiation of the silkworm spermatogonia.
It has been known that enzymes that concerned with repair of DNA
damages are suppressed of their activity at cold temperature as O°e. It isinteresting to know whether the suppression of repair may result in the
failure of rejoining of broken ends of DNA strand or lead to successful
repair via slow acting but exact error correction process. If the former
holds true, it can be expressed as an increase in mutation frequency. Whilein the latter case we may observe the decrease in mutation frequency.
Wild type larvae of strain CIOS were used as materials. The newly hatchedlarvae were divided into three groups: non-irradiated control, irradiated andpost-treated at 27°C, and irradiated and post-treated at 5°e. For irradiation
larvae were contained in petri dishes, one with ice and the other with solid
paraffine, both rotating simultaneously under X-ray (300 Rjmin) beam.
The radiation dose was 1000 R. Immediately after irradiation irradiatedgroup on ice were kept at 5°C in ice jar for five hours and those irradiated onparaffine were kept in 27°C room for the same duration. After treatment
Table 1. Results of low-temperature post-treatmentof silkworm spermatogonia (742)Mutation frequency(x 10- 5)

No. of mutants
Treatment

Control
27°C
SoC

Total No.
observed

pe

pe
mos

re

re
mos

pe

pe
mos

re

re
mos

74,98883,51686,292

7
16
35

3
1
7

0
1
24

2
2
4

9.3
19.1
40.6

4.0
0
1.2 1.2
8.1 27.8

2.7
2.4
4.6

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

irradiated and control groups were raised in rearing room at 25°C throughout
their life cycle. For the measurement of mutation frequency specific locimethod using pe and re was employed. The results are given in Table 1.
The results clearly show that low-temperature post-treatment increased
the radiation induced mutation frequency at both loci. An average effectiveness was -1 X 1O-5/locus per 1°C. Analysis of induced mutants isin progress.
Silkworm Oocyte System for Testing Mutagenicity*
Yataro TAZIMA and Kimiham ONIMARU

As reported previously, a highly sensitive system has been developed for thedetection of mutation in the silkworm in which oocytes are treated withchemicals. The system proved itself to be very effective for the demonstration of mutagenicity of several nitrofuran derivatives including furylfuramide. In these experiments injection of the test chemical was performed
five days before emergence, because at this stage maximum mutation yield
had been revealed by our previous experiments with mitomycin C.
It was also known that silkworm oocytes are extremely sensitive to ionizing
radiation as to mutation induction around meiotic metaphase, which takes
place immediately after egg deposition. If the same is true for chemicalmutagens, the chemical must remain active in silkworm hemolymph formore than five days until deposition. This is quite dubious for furylfuramide,
because the compound is known to be easily metabolized in bacterial cells or
mammalian metabolic systems. The evidence that furylfuramide exhibited
clearly mutagenicity even when injected five days before emergence could be
ascribed to two alternative mechanisms: either the injected chemical reacts immediately upon genetic materials or the chemical remains unchanged until
it reacts upon genetic materials at their surmised most sensitive stage, perhaps
around meiotic metaphase. To determine which of the mechanisms holds
true, an experiment was carried out using EMS, of which metabolic halflife has been reported to be fairly short. EMS was injected in saline into thefemale pupae of a wild-type strain C108 at different developmental stages,
every 24 or 48 h. The administered dose was 20,ug/pupa. Mutation
frequency was estimated by the specific loci method using pe and re as* Presented at the Third Annual Meeting of The Japanese Environmental Mutagen
Society, held September 28, 1974.

RESEARCHES CARRIED OUT IN 1974

41

markers. Induced frequencies varied with two peaks, major and minor.
Mutation response was lowest three or four days before emergence andthen it rose slightly showing a minor peak one-three days before emergence.
This finding seemed to support the first hypothesis, i. e., reaction takes placearound meiotic metaphase. A major peak of one or two orders of magnitude
higher was found at the earlier mid-pupal stage, which seemed to support thesecond hypothesis, i. e., immediate reaction. Perhaps the latter may represent the main response.
Thus the system appears to be applicable also to the detection of mutagensof short,life activity.
Embryonic Lethality in Progeny of X-Irradiated Silkworm Moths:
Inherited Lethality and Sex-Ratio Distortion
AIda MURAKAMI

Germ-cells of the silkworm moths were irradiated with X-rays at different
doses ranging from 0.5 to 21 kR and then were crossed with unirradiatedmoths in the opposite sex. The deposited fertilized eggs were examined for
either hatched or non-hatched eggs. The latter incidence was regarded asembryonic lethal mutations. The larvae were further examined for theirmortality and sex-ratio. The surviving PI progeny was again crossed withnormal moths of the opposite sex. The embryonic lethality and other biologival incidences of F I progeny were also investigated in the same manner as
did in the PI.progeny.
The results obtained so far clearly indicated that dose-response curves in
the PI progeny as well as in the F I progeny were linear at lower doses andnon-linear at higher doses. The embryonic lethality of the F I progeny fromthe treated male (XX) moths (or mature sperm) was markedly increased ascompared with that of the PI progeny at corresponding dose, regardless ofmating types. This inherited lethality was clearly dose-dependent. Thisphenomenon was interpreted as a delayed lethal effect. It should be noted,
however, that the lethality of the BFI progeny showed a decrease of about
50 percent as compared with the preceeding progeny. In contrast to thesefindings, when the female (XY) moths or prophase I oocytes have been irradiated with X-rays no remarkable such the inherited lethality was observedin F I and BFI progeny. The cause of these embryonic lethalities observedin these F I and BFI progeny from the treated male moths would be due to42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

translocations which may be able to pass through meiotic divisions.
Treated male moths produced much more male than female progeny (orlost much more female than male progeny in the course of embryogenesis).
This sex-ratio distortion was depended on the dose. However, irradiatedfemale moths produced the same number of male and female progeny. The
possible cause of this phenomenon would be explained as being due to sexlinked (recessive) lethal mutations. In addition to this class mutation,
mutational events as an autosomal factor (s) interfering with the X-chromo_
some inactivation mechanisms in the normal oogenesis should be also considered to be one of possible interpretations for the sex-distortion. Thesefindings suggested that embryonic lethal mutations in the PI progeny from thetreated males would be included in dominant lethal mutations as chromosomal abnormality, sex-linked recessive lethal mutations and others.
Mutagenicity of Diethylsulphate in Spermatozoa of the Silkworm
I. Delayed Mutagenic EffectAIda MURAKAMI

A high incidence of mosaic mutations is a remarkable characteristic featurein chemically-induced mutations in the silkworm. It is generally thoughtthat the mosaic mutation would be expressed as a delayed mutagenic (fixation) effect on the induced pre-mutational lesion. However, a question
arised whether the delayed mutagenic effect could be separated from a delayed
drug-action effect depending on the half-life of chemicals. The latter
effect will be more pronounced for chemicals with a long half-life than thosewith a short one.
The mutagenicity of diethylsulphate (DES) in pupal spermatozoa of thesilkworm was investigated to achieve a better understanding of the natureof chemically-induced delayed mutagenic effects. DES was selected by areason of its short half-life (1.8 hr at 25°C) and strong mutagenicity. Itshould be of interest to note that DES is extensively used in a variety of
ethylation processes in organic synthesis and in various commercial areas.
Detection of mutational incidences was made by the egg-color specific locus
methods. 0.025 ml per pupa of a freshly prepared DES emersion at different
concentrations from 0.1 to 1.0 % in 0.85 % NaCI solution was injected into
the dorsal abdomen of ll-day old male pupa (or 2 days before emergence),
which was 0.75 g in body weight.

RESEARCHES CARRIED OUT IN 1974

43

The result of experiments indicated that DES induced a markedly highincidence of simple mosaics regardless of locus, whereas the incidence ofcomplete mutations was rare. Other type mutants such as complex double
and triple mosaics were also detected slightly. Although the testis in pupae
at this stage contains various stages of germ-cells ranging from primordial
spermatogonia to spermatozoa, the most advanced germ-cell or mature spermatozoon would be only concerned in fertilization. Therefore it would
take at least 50 hours between the DES treatment and the syngamy under theexperimental condition at 25°C. Considering the short half-life of DES inaqueous media and the pupal age (or germ-cell stage) tested it is very unlikely
to suspect that the chemical was mutagenic in either sperm pronuclei orcleavage nuclei. Consequently, the high incidence of mosaic mutations
would be interpreted on the assumption that DES which induce premutational damage in spermatozoa is fixed and/or expressed as mutations bypassing through one or more cleavage divisions.
Among various types of mosaics, half-and-half type mosaics were detected.
If this type mutant were what is called the real one-half type, this mutant
would arise from a permanent change in one strand of an already exisiting
double strand DNA molecules in mature sperm. The complete mutation
would arise from simultaneous two-hit events in the paired nucleotide chainsof DNA in the locus. Double mosaics would arise from an independent
two-hit event at the pe locus in one strand of chromosomes and that at there locus in another strand in the mature sperm. The triple mosaic mutation
would arise as the mutational events both in mature sperm and in earlycleavage cells.
In any case, the result of the present experiment clearly suggested thatabout 90 % of DES-induced egg-color mutations in mature sperm would
have been resulted from the delayed mutagenic effect, but remaining about
10% (half-and-half type mosaics, complex mosaics and completes) would
have been resulted from the permanant genetic change in an already exisiting
gene DNA of the sperm:
Mutagenicity of Diethylsulphate in Spermatozoa of the Silkworm
II. Dose-Mutagenicity Relationship
Akio MURAKAMI

Analysis of dose-mutagenicity relationship would provide some useful

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

informations for better understanding the nature of chemically-induced
mutations as did for that of radiation-induced mutations. It would also
provide an important information to asses the dose-level at which thechemical is likely to be used in view of the beneficial judgement on environ_mental chemicals. Although a number of reports on the chemically-induced
mutations little informations on the dose-mutagenicity relationship havebeen communicated. In the relations between diethylsulphate (DES) dose.
egg-color mutagenicity in silkworm spermatozoa, the frequency was in.
creased slowly at first and then rapidly to the dose level at 0.6 %, regardlessof the locus. Over this dose level the frequency of mutations was slowly
decreased at the pe locus or kept at 0.6 % level at the re locus. The frequency of complete mutations was too rare that the dose-mutagenicity
relation curve could be regarded as that of the mosaic mutations for themost part. In other words, the DES dose-response curve of mosaic mutamutations is sigmoidal or logarithmic in shape, regardless of the locus.
This observation is well agreed with that in the DES-induced sex-linked
recessive lethal mutations of Drosophila spermatozoa (Pelecanos and Alderson1964). The DES dose at which the maximum mutation frequency was
shown in silkworm spermatozoa was about 0.6 %, indicating that it is about
1.5 times as high as that in Drosophila spermatozoa (0.4 %) (Pelecanos andAlderson 1964). In E. coli an exponential increase was observed in streptomycin-resistant mutations after treatment with mono-functional alkylating
agents, MMS and DDVP (Wild 1973).
Reasons why the DES dose-mutagenicity curve was sigmoidal could beinterpreted by at least two possibilities as follows: one is characteristics ofchemical reaction of DES in ethylation of sperm DNA. Indeed, Sega et al.
(1973) indicated that in the dosimetric study on mouse sperm DNA the ethylation per nuclectide increased at a rate greater than the first power of the dose.
Another is the nature of biological interaction of DES in vivo: cellular repairability of ethylated sperm DNA at lower doses and cell killing (or toxic)
effect at higher doses. Furthermore, a compromized mechanisms of thosepossibilities could not be completely ruled out.

RESEARCHES CARRIED OUT IN 197445

Lack of Recombinogenicity of Ethyl Methanesnlphonate
in the Silkworm Oocytes
Akio MURAKAMI and Isao ARIGA1 )In the previous paper, it was reported that a bifunctional as well as monofunctional alkylating agent, Mitomycin C (MC), slightly but significantly
induces much more interchange type recombinants than exchange type ones inearly growth stage oocytes of the 5th instar silkworm larvae (Ariga and Murakami, J. Sericult. Sci. Japan 44: 154-160, 1975). To account for the finding
a tentative interpretation was proposed that MC produces both DNA strand
breakages (or cut) in the larval oocytes due to the formation of cross-linking
between the paired DNA strands and which would reunion in turn withrecombinational repair mechanism. The purpose of the present experiment
was intended to test whether a monofuctional alkylating agent ethyl methanesulphonate (EMS) can induce recombinants in the larval silkworm oocytes.
The incidence of recombinations was scored by the egg-color method.
0.025 ml per larva of EMS solution at three different concentration (0.25,
0.50 and 1.00 %) dissolved in 0.85 % NaCI solution was injected into 5 and 7
day female larvae at the 5th instar heterozygous in the trans type of two
egg-color genes, pe (V-O.O) and re (V-31.7).
The frequency of recombinants recovered was very low and not significantly different from that of the control, regardless of EMS doses and stages
at which larvae were treated. It should be noted, however, that at 7 day inthe 5th instar (or mature) larvae EMS at a dose of 0.1 % slightly but clearly
increased the frequency of mosaic or fractional-body mutations when compared with the control in the egg-color specific locus method, but not increased the incidence of complete or whole-body mutations probably corresponding to double strand DNA changes. Such being the case, the doseof EMS used in the experiment might be not insufficient for the induction ofrecombinants. Current thought indicates that EMS induces a predominant
number of single strand breaks (or nick). EMS-induced single strand DNAchanges (including DNA strand breaks) would not be converted into double
strand changes since the germ-cell at this larval stage has already been finishedDNA replication. It is very likely to note that this type single strand damage
would be repaired by certain mechanisms, resulting in the low incidence ofmosaic mutations.
1)

Kanagawa-ken Sericutural Research Center, Ebina, Kanagawa-ken 243.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

From these results in the present and previous experiments, recombino.
genicity of the alkylating agents may depend on the number of functionalsites and it may require at least two functional sites. In other words, bothstrand breakages in the complementary DNA strands may be essentiallynecessary for the induction of recombinations in prophase I oocytes of thesilkworm.

47

RESEARCHES CARRIED OUT IN 1974

VII.

RADIAnON GENETICS AND CHEMICAL MUTAGENESISIN MICRO-ORGANISMS AND PLANTS
Radiation and Drug Sensitivities of Bacillus subtilis rec Strains
Yoshito SADAIE, Hatsuo INUKAI and Tsuneo KAnA

Lack of genetic recombination capacity usually makes strains manifest
different phenotypes. Recombination-deficient strains are usually sensitive
to UV, gamma-rays and certain chemicals. Recombination repair processes are postulated to be efficient mechanisms assuring cellular resistance to anumber of DNA-damaging agents. We examined radiation and drug
sensitivities of ree mutants of Bacillus subtilis carrying one of sevendistinctive ree mutations. The results indicated that the radiation (UV or
gamma-rays) sensitivities are roughly correlated to the sensitivities to typicalchemical mutagens. The reeE locus seems to be essential and the reeA andreeG loci may be also required generally for DNA repair. Some chemicals
such as 4NQO or potassium bichromate are more specific to reeB or reeD
Table 1.
Strains
rec+
recAl
recB2
rece?
recD3
recE4
recF16
recG13rec+rec-43
rec-45

Drug sensitivities of Bacillus subtilis rec strains
MC(0.3 pg)
1
7
9
3
8
9
7

7
1
12
14

Length of inhibition (mm)MMS
4NQO
KZCrZ07(119 pg)(4.8 pg)(30 pg)
022
13
6
8
14
11
16
426
13
7
16
19
5
10
14
5
10
6
9
16
021
16
12
12
16
8
13

DAPA
(194 pg)
2
8
7
1
6
11
8
9

o
11
14

Bacteria were streaked radially from the center of broth agar.
Paper disc was placed with indicated amounts of drug on the center of the plate.
After overnight incubation, the length ofgrowth inhibition was measured (Mutrition Res.
16 165, (1972».
MC: mitomycin C, MMS: methyl methanesulfonate, 4NQO: 4-nitroquoline-N-oxide,
DAPA: sodium-p-dimethylaminobenzene-diazosulfonate

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

mutants. SP02 lysogens of recE, reeF and recG strains are not inducible.
These loci may be required for their inducible repair functions. Many ofthese rec strains were kindly supplied by Dr. Dubnau (J, Bacteriology II 7,
488).

Some of the results are described in Table 1.
Prophage Induction in rec Strains of Bacillus subtilis
Yoshito SADAlE

Isolation and characterization of bacterial recombination-deficient (rec)mutants revealed involvement of certain rec gene products in prophage
induction. The prophage SP02 inducibility was examined in rec mutantsof Bacillus subtilis Marburg NIG43 (rec-43) and NIG45 (rec-45) isolated
in our laboratory. As indicated in Fig. 1, the rec-43 mutation locatesright to dna-8132 (Hara et al. Nature New BioI. 244, 200 (1973») with a cotransfer index of about 75 % by transformation and the rec-45 tightly linked
to recAl. SP02 lysogens of these mutant strains did not produce phage
replication
originFig. 1. Genetic map of Bacillus subtilis showing rec loci and somereference markers.

RESEARCHES CARRIED OUT IN 1974

49

particles after UV or mitomycin C treatment, though SP02 transfectionof both mutants was normal, indicating possible involvement of ree-43+
and ree-45+ gene products in destruction of the repressor system of SP02
(J. Bacteriology, in press).
Recombination-deficient strains so far isolated in other laboratry wereobtained, lysogenized and treated with UV or mitomycin C. Among sevendistinctive ree mutations, only three (reeE4, recF16 and reeG13) conferred
the lysogens uninducible. Genetic analysis revealed that the mutations
ree-43 and ree-45 located very probably in the reeG and reeE loci respectively.
The ree-45 and reeE4 lysogens produced very reduced numbers of phage
particles both spontaneously and upon artificial induction. The above
bacterial strains were kindly supplied by Drs. C. Anagnostopoulos and D.
Dubnau. Figure 1 was drawn based on the original maps of LepesantKejzlarova et al. (J. Bacteriol. 121, 823) and Dubnau et al. (J. Bacteriol. 117,
488).
Improved Procedures of the ree-assay for Rapid Detectionof Chemical Mutagens
Tsuneo KADA and Yoshito SADAIE

Since most chemical mutagens produce damages in DNA that are subjected
to cellular recombination repair, those substances showing increased lethal
activity on Rec+ over Rec cells may have damaged cellular DNA and such
DNA-damaging agents are very often mutagens. The ree-assay utilizing
B. subtilis strains Hl7 Rec+ and M45 Rec (Mutation Res. 16, 165, 1972)
was useful for detection of upto date more than 30 new chemical mutagens in
the environment including phloxin, AF2, Dexon, NBT, etc. In the currently adopted mothod, the Rec+ and Rec- bacteria are grown overnight inliquid broth, each streaked on the "dry" surface of broth agar, the starting
points are covered by a round paper disk containing each drug, the plates
are incubated overnight at 37°C, and the inhibition zones are measured.
We have recently performed the following examinations.
A: Strains: We collected more than 50 repair-deficient strains of B.
subtilis, E. coli and Salmonella possessing impairements in ree, her or/
and pol functions and studied about their sensitivities to 30 typicalchemical mutagens. A set of HI7 Rec+ and M 45 Rec- strains of B.
subtilis showed so far the highest and widest sensitivity spectra.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

B:Preparation of bacteria: Practically no difference in the sensitivity
was found in exponential and stationary bacterial cultures. On theother hand, the stocked cultures (at -80°C, with 12.5 % glycerol)
showed considerably higher sensitivity than freshly grown cultures.
C: Cold incubation: Usually bacteria and drug were placed on theplate at the same time and incubated overnight at 37°C. We recently
found that if one lets diffuse the drug first on the plate at cold and thengrow the bacteria, the drug inhibition zones were much larger thanthose in the usual case.
Considering the above observations, the improved new procedures are thefollowings; by these procedures, the sensitivity increased about 30 times.
Strains H 17 Rec+ and M 45 Rec- are grown overnight in broth (meatextract 10 g, polypeptone 10 g, NaCI 5 g, water 1000 ml, pH adjusted to7.0). One ml of 50% glycerol (W/V) was added into 3 ml of full-grownbacterial broth culture and stocked at -80°C. On the day of experiments,
each culture was streaked on the "dry" surface of broth agar (15 gil of agar
added in the above liquid broth) and the drug paper disk is placed. All theplates are kept at 4-5°C for 24 hours then incubated at 37°C for about 20
hours. The length of inhibition zone is then measured.
Table I
Chemical agent(I' g per disk)
4NQO (002)(0.002)AF2 (0.02)(0.002)

Inhibition zones in mm
Old procedures
New procedures
HI7 Rec+
M45 Rec
HI7 RecM45 Rec-

o

1

o

o

o

6
1

o

o
o

15

o

16
9

o

1

AF2 Mutagenicity and Reductase Activity of E. coli Blr WP2
resistant to Cr+6
Tsuneo KADA

It has been previously reported that derivatives strains resistant to AF2[2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, or furylfuramide] were isolated
from E. coli Blr WP2 try and that supplementation of rat liver homogenate(S9) was recessary for mutations (Try- -)Try+) to occur in them (cited in

RESEARCHES CARRIED OUT IN 1974

51

Mutation Res. 32 1975, p. 74). As supposed by the above observation andalso shown experimentally by Dr. K. Tsuji (Institute of Physical and Chemical Research), cellular reductase activity was found to be deficient in the AF2resistant bacteria than in the original sensitive bacteria, since DNA-reactive
metabolites may be produced by reduction of AF2. The reduction capacityof S9 is thus involved in production of an active form of AF2. We haverecently observed that though many derivative strains of E. coli WP2 resistant to potassium chromate (K 2 Cr 2 0 7) were also resistant to AF2, about
10% of Cr+6 resistant isolants became sensitive to AF2. Since Cr+6 gave
positive results in the B. subtilis rec-assay and showed mutagenec effects on
E. coli WP2 strains but Cr+ 3 did not (R. Nishioka, Mutation Res. 31, 185,1975), we suppose that there may exist at least two or three types of reductases
whose increased activities are correlated with the cellular Cr+ 6 resistance.
The one of them may be involved in reduction of AF2 as well as in reduction Cr+ 6--+Cr+ 3.
Ultraviolet Light-induced Mutagenesis
in Bacillus subtilis Spores
Yoshito SADAIE, Yukio SAITO and Tsuneo KADA

Studies on ultraviolet light (UV) mutagenesis in germinating spores ofBacillus subtilis have some unique features. Semi-conservative DNA
synthesis begins synchronously about 2 hours after the commencement ofgermination. It may be interesting to know if photoproducts in spores are
mutagenic or not and how pre-mutational damages are fixed. Strains
Table 1. Frequency of His+ mutations induced
by UV irradiation of spores

Strainpo[+

polA59

UV dose(ergs/mm2)

No. ofsurviving
spores/ml(x 107)

No. of His+
mutants
found/ml(x 102 )

0
300
900
1800
0
300
600
900

9.5
7.7
3.0
0.2
9.2
3.1
0.5
<0.01

1.8
11.9
60.1
40.8
2.7
70.1
21.5
2.7

Frequency of
induced His +mutations/spore(x 10- 5)

1.36
19.8
204.0
22.3
24.7
>270.052

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

HA101 (leu met his) and HA101 (59) F (leu met his po1A59) are isogenic andcarry Pol+ and Pol- characters and a suppressible histidine auxotroph.
Their spores were irradiated with UV and plated on selective agar containing
small amounts of broth for detection of mutations to histidine-independence.
We observed that UV induced mutations in spores and their degree Was
higher in Pol- than Pol+ spores (Table 1). It has been also confirmed invegetative cells that Pol- cells produce more reversions than Pol+ cells.
The frequency of mutations declined considerably when UV-exposed spores
were incubated in liquid both before plating on selective agar. Therefore,
it seems that photoproducts in spores serve as premutational damages andtheir fixation process is hampered by polymerase I function.
Involvement of a Common Enzyme in the Repairof Deprinated and r-ray-irradiated DNA
of Bacillus subtilis
Takehiko NOGUTI and Tsueno KADA

We have isolated from Bacillus subtilis cells an enzyme which makes rray-irradiated DNA a better primer for DNA-polymerase I and named "primer activating (PA) enzyme" (Biochim Biophys. Ada 395,284 & 294, 1975).
The enzyme has been partially purified by DEAE and phosphocellulose
column chromatography. It was shown that this enzyme introduced new
nicks in DNA of phages T7 in addition to those introduced by r-ray irradiation. Verly W. G. et al. (Nature New BioI. 244. 1973, 67-69) isolated an
endonuclease specific to apurinic sites of DNA and demonstrated its generaloccurrence in bacteria, plants and animal cells. Because both the apurinic
sites and certain portion of r-ray-provoked lesions are known to be alkalilabile, we examined if the PA enzyme might be also specific to the deprinatedsites. Depurination was carried out by heating T7 DNA at 70°C by themethod of Lindahl, T. and Nyberg, B. (Biochemistry 11, 1972, 3610-3617).
DNA was then treated with the PA enzyme in a buffered solution (pH 8.0).
The priming activity was determined as to the above DNA samples after
purification with phenol treatment. In order to observe the effect of our
enzyme on the alkali-labile sites, denaturation of DNA was carried out in
two ways, treatment with formamide or exposure of a portion of the treated
DNA to alkali. The treated DNA was thereafter analyzed with sucrose
gradients containing formalin. The profiles obtained with formamide treat-

RESEARCHES CARRIED OUT IN 1974

53

ment indicated that the enzyme introduced cuts into the heated DNA possessing apurinic sites. The subsequent alkali treatment reduced the molecularweight of the heated DNA close to a level found with the enzyme treatedDNA. The results show that the primer activating (PA) enzyme introduces
nicks at the alkali-labile region of heated DNA, probably at its apurinic
sties. It is thus indicated that certain portion of r-ray provoked lesionsof DNA may be repaired by the apurinic site specific enzymes.
Induction of wx Mutant in an Inbred Line of Maize
Etsuo AMANO

The waxy starch (wx) locus of maize is one of a few loci suitable for genetic
fine structure analysis. Its phenotype can be examined in endosperm and inpollen grains which are in haploid generation. Thus the fine structure analysis of the locus can be done by pollen analysis in which scoring of 106 pollen
grains is not so difficult.
, To obtain useful wx mutants, several mutagens have been tried and EMS
was found to be the most effective. However, since maize has unisexual
flowers, segregation of the mutants by self-pollination can not be expected
like barley or rice plant when seeds are treated with the mutagen. To detect
wx mutants, the test cross with a standard wx tester stock is a very convenientand common method. However when hybridized by the test cross, the genetic background may be disturbed; moreover it becomes very difficult to identify a newly induced wx mutant gene from the tester wx in the segregatedprogenies. To establish inbred line mutants in maize, useful method has
been proposed previously (Ann. Rep. N. 1. G. No. 23 pp. 84-85). It utilizes
mixed pollen of the tester wx and the non-treated parental Wx line for pollination of mutagen treated Wx plants. If a cob was found to have some wx
mutant kernels, theoretically a third of the W x kernels or a half of the kernels
with the parental Wx phenotype on the cob would be heterozygous for the
newly induced wx mutant gene. By self pollination of such inbred line
heterozygotes, three wx mutant lines have been established in 1974. Also
another additional ten mutant carrying cobs have been detected. These
three inbred line mutants induced by EMS were normal in fertility and inother viability characters. These inbred line mutants will be used in studies
on fine structure analysis of the wx locus and on characterization of theinduced mutants.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

Induction of Waxy Starch Mutant in Rice
Estuo AMANO

Waxy (wx) mutants were obtained at a high frequency in maize by treating
seeds with a chemical mutagen, ethyl methanesulfonate (EMS). Ionizing
radiations have failed to induce wx mutants having good viability or fertility.
To study fundamental factors in mutation breeding, seeds of Oryza sativa
var. Norin No.8 were treated with EMS to induce waxy starch mutants.
Norin No.8 is one of the typical paddy field rice in Japan and its starchcharacter is non-waxy (Wx). Seeds were soaked in 0.05 M or 0.1 M aquous
solution of EMS for five hours at 27°e in a shaking incubator. After thetreatment, seeds were rinsed three times with water. In these treatments,
distilled and deionized water was used. The rinsed seeds were sown in soil ofwooden flats in green-house. Some of the seedlings were transplanted tothe soil bed in a greenhouse, but most of them were left in flats densely
planted. Mature panicles were harvested and dried. Each panicle wasthreshed separately and more than ten hulled kernels were examined visually.
Possible wx mutants were further examined by means of iodine staining.
Among 1535 panicles thus examined, seven panicles were found to segregate
waxy starch kernels. The overall frequency of wx mutants was 0.45 %,being about a half of that of the maize experiment. Although the value wassmaller in rice than in maize, it is still very high for a single locus. According
to the results in maize experiments, most of the mutations induced by EMS
might be point mutations, but still another deleterious mutations mightoccur simultaneously in these mutants, because the high efficiency of themutagen might be also true in other loci. Studies on these simultaneous
mutations and on characteristics of the induced mutants will be continued
further.
Chromosome of Cultured Haplopappus Cell
Etsuo AMANa

The cultured callus cell of Haplopappus gracilis (2n=4) is soft and fast
growing. Five cell lines have been maintained on agar slant medium for
more than three years. The medium (EFR) used is a modified Erikson's
which is based on Murashige and Skoog's medium and contains 2.4 D (2ppm)
and yeast extract (0.3 %) besides inorganic salts, vitamines and sucrose (2 %).

55

RESEARCHES CARRIED OUT IN 1974

Chromosomes of these five cell lines and a cell line (HB) of one year old
were examined by aceto-orcein staining. Squash preparations were madefrom freshly growing callus. To examine the ploidy of the cell, anaphase
chromosome numbers were converted to metaphase numbers. The results
are shown in Table 1. In five out of the six cell lines, most of the cells were
diploid. However, in a cell line HL, more than half of the cells examined
were tetraploid. When the chromosome number was examined all of thefive cell lines were diploid as reported last year. Further examinations weremade about two HL derivatives used in an experiment which might not
disturb the ploidy. Results included in Table 1 indicate that one of themhad the same proportion of ploidy as the parental HL line, but the other wasvery alike to the other diploid cell lines. Such a fluctuation of the ploidy
might be caused by chance during transferring the callus to a new medium,
although further investigations are necessary.
In the course of these chromosome studies, certain difference in the karyotype was noticed. The cell lines, HR (originated from root), HS (stem)
HNC (spontaneously occured on stem) and HB (flower bud), were all inhomomorphic with two submedian and two subterminal chromosomeswith satellite. In two cell lines, HL (leaf) and Hse (seed) which were derived
from different individual plants, had an identical submedian chromosome pair,but one of the satellite chromosome was different in shape. It seemed to haveelongated short arm and lack the satellite, giving a significantly heteromorphic configuration. Such heteromorphism in H. gracilis was reported by
Kamra (Om P. Kamra 1963 Chromosoma 13: 540-544). Effect of theheteromorphism or of this chromosome on callus culture was not significant
Table 1.

Cell line
715 HR
715 HS
7110HNC
716 HL705 Hse736 HB716 HL716 HL

Number of chromosomes in cultured Hap[opappus cells
on EFR agar medium.

Transfer
generation
74
73

Cells with chromosome No.
4

8

58
78
116

68

17

2
1
13
22

(SGC24)(SGC48)

71
8
17
114

21
6

12

16

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

as far as morphology and growth of the callus were concerned.
an useful marker in cytological studies of these cultured cells.

It may be

Effects of Fast Neutrons on Dry and Wet Seeds in Maize
Taro FUJII

To investigate the effect of water content upon gamma-ray or neutron_
induced mutation, seeds heterozygous for the Yg 2-gene were used. Moisturestabilized (13 %) or 24 hrs water-steeped seeds were exposed to 137Cs gammarays or 14 MeV neutrons. Mutation from Yg 2 to yg2 was detected in theseedlings as color change, green to yellow-green. Although the mutation
frequency enhanced by increasing the dose almost in a linear relationship, theaverage number of yg-stripes per leaf differed considerably between dry andwet seed lots of gamma-rays. The frequency in each 3 different doses ofwet seeds was about 1.5 times higher than that of corresponding lot of dry
seeds. Experiments were repeated twice using similar doses of neutrons inorder to check carefully the mutation frequency at respective doses as well as
to determine the difference in frequencies between dry and wet seeds. However, unlike the case with gamma-rays, no clear difference in the frequency
between wet and dry seeds was observed. Namely, a frequency of 2 % was
observed with around 20 rad exposures; frequencies of 6-7% at the highest
dose lots were observed throughout two experiments using dry and wet seeds.
It may be noted that the no change or no recovery in neutron induced mutations was seen in the dry or wet seeds treatments. Though the mechanism ofneutron mutagenesis lacking repairability is not clear, the present observation had led to a general conclusion that the effect of neutron is less modifiable
by exposure conditions or by environmental changes.
Sex Expression in the Offspring of Gamma-ray Induced
Monoecious-like Plants in Cucumber
Taro FUJII

Effect of gamma-irradiation on the sex expression in the F 1 plants obtained
from the crosses between original gynoecious strain (MSU) with male flowersof monoecious-like plant (MSU-713-5M) (cf. Ann. Rep. 24, 45) has beenstudied. F 1 plants came from 6 F1-fruits were examined so far. Observation was carried out up to 50th nodes in each individual since some plants

57

RESEARCHES CARRIED OUT IN 1974

did not survive beyond ca. 55th nodes. As shown in Table 1, most of thestrains segregated monoecious-like plants. The number of male flowers insegregants from the F]-fruits No.1 and 3 was merely 2 or 3 on the basal
nodes in each plant. On the other hand, most segregants from Fr-fruit No.
S had developed many male flowers. For instance, 116 male flowers in total
had developed over 19 nodes in some plants; even in the least case of thesame strain, 26 male flowers developed over 4 nodes. Differences in thenumber of male flowers in segregants among the Fl-strains might be correlated to certain genic alterations, since sex expression in cucumber is controlled
mainly with 2 genes, Acr and G. Examination of F 2 and Fa generations has
been done with several F 2 seeds obtained from selfed Fl-segregants up to date,and segregation of monoecious-like plants was observed successively. Thus
the monoecious-like behaviour of the radiation treated MSU strain might bedue to certain heritable change.
Table 1.
F-fruits number
1
2
3
4
5
1

Segregation of monoecious-like plants in Fl
No. of Fl plants
23
5

14
no germination
41
1

No. of monoecious-likeplants
6

o
2

18

o

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

VIII.

POPULATION GENETICS (THEORETICAL)
Some Models of Allelic Mutation in
Molecular Population Genetics
Motoo KIMURA

In order to analyse theoretically how much genetic variability is maintained
within a finite population at the molecular level (i. e., at the level of theinternal structure of the gene), two models of allelic mutation are considered.
In the first model, it is assumed that the number of possible allelic states at agene locus is so large that whenever mutation occurs it leads to a new, notpreexisting allele; both selectively neutral and overdominant cases are considered. In the second model, allelic states are expressed by integers andstepwise production of alleles by mutation is assumed.
Analytical treatments based on the diffusion equation method are supplemented by numerical analyses and Monte Carlo experiments. Formulae
are derived which give the effective number of alleles maintained in a population at equilibrium. For details, see "Lectures on Mathematics in the Life
Sciences" (1974, American Math. Soc.): 1-23.
Simulation Studies on Electrophoretically Detectable
Genetic Variability in a Finite Population
Tomoko OHTA and Motoo KIMURA

Using a new model of isoalleles, extensive Monte Carlo experiments were
performed to examine the pattern of allelic distribution in a finite population. In this model it was assumed that the set of allelic states is represented
by discrete points on a one-dimensional lattice and that change of state by
mutation occurs in such a way that an allele moves either one step in the positive direction or one step in the negative direction on the lattice. Such amodel was considered to be appropriate for estimating theoretically the
number of electrophoretically detectable alleles within a population. The
evenness of allelic distribution was measured by the ratio of the effective tothe actual number of alleles (ne/n a ). The results of the Monte Carlo experiments have shown that this ratio is generally larger under the new model of
isoalleles than under the conventional Kimura-Crow model of neutral isoal-

RESEARCHES CARRIED OUT IN 197459

le1es. In other words, the distribution of allelic frequencies within a population is expected to be more uniform in the new model. By comparing theMonte Carlo results with actual observations, it was concluded that theobserved deviation from what is predicted under the new model with selective neutrality is not in the direction of conforming to the overdominancehypothesis but is, in fact, in the opposite direction. For details see Genetics
76: 615-624.
Probability of Gene Fixation in an Expanding Finite Population
Motoo KIMURA and Tomoko OHTA

A mathematical theory was developed, based on diffusion models, thatenables us to compute the probability of a rare mutant allele eventually
spreading through a population when the population size changes with time.
In particular, we elaborated the case in which the mutant allele has a definite
selective advantage and the population expands following the logistic law.
In this case, the probability of ultimate fixation of a single mutant is given by
u=2s(ZjN), where s is the selective advantage and ZjN is a factor by which
the probability of fixation is modified through population expansion.
Analytical expression was obtained for ZjN, and the validity of the formula
for u was checked by Monte Carlo experiments. For details, see Proc.
Nat. Acad. Sci. 71: 3377-3379.
Detrimental Genes with Partial Selting andEffects on a Neutral Locus
Tomoko OHTA and C. Clark COCKERHAM

Gene and genotypic frequencies for a deleterious mutant in mutation selection balance are derived for an infinite population undergoing partial selffertilization. These provide formulations of mean survival and the mutational load. Obtained also are the average number of mutant genes andaffected individuals stemming from a single mutant.
As a concomitant effect on frequencies at a neutral locus the mutationalload is distributed disproportionately among the neutral genotypes. For
partially recessive mutant genes on the 1, l-sh, l-s scale, the effect is to increase the frequency of the heterozygote and to decrease the frequencies ofhomozygotes at the neutral locus relative to the frequencies expected with

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

complete neutrality. This apparent overdominance at the neutral locus has
been shown to be connected with identity disequilibrium rather than linkage
disequilibrium. It increases generally as sand h decrease, and as the pro.
portion of self-fertilization and the degree of linkage increase. The apparent
overdominance with complete linkage is generally less than double that for
free recombination. For partially dominant mutant genes, h ~ 1/2, theeffects on the frequencies of heterozygote and homozygotes at the neutral
locus are reversed. For details, see Genet. Res. 23: 191-200.
Speed of Gene Frequency Changes in Reverse Directions
in a Finite Population
Takeo MARUYAMA and Motoo KIMURA

It is shown that in finite populations the average length of time which it
takes for an allele to increase its frequency from p to q (>p) on the way tofixation is equal to the average length of time which the same allele takes
when its frequency decreases from q to p on the way to extinction, although
the probabilities of occurrence of these two events can be very different. In
addition to the means, the sojourn times in each specified gene frequency
interval are shown to be the same for the two reverse processes. For details
see Evolution 28: 161-163 (1974).
A Markov Process of Gene Frequency Change in a
Geographically Structured Population
Takeo MARUYAMAA Markov process (chain) of gene frequency change is derived for a geographically-structured model of a population. The population consists ofcolonies which are connected by migration. Selection operates in eachcolony independently. It is shown that there exists a stochastic clock thattransforms the originally complicated process of gene frequency change to arandom walk which is independent of the geographical structure of the
population. The time parameter is a local random time that is dependent
on the sample path. In fact, if the alleles are selectively neutral, the timeparameter is exactly equal to the sum of the average local genetic variation
appearing in the population, and otherwise they are approximately equal.
The Kolmogorov forward and backward equations of the process are obtain-

RESEARCHES CARRIED OUT IN 1974

61

ed. As a limit of large population size, a diffusion process is derived. The
transition probabilities of the Markov chain and of the diffusion process are
obtained explicitly. Certain quantities of biological interest are shown to be
independent of the population structure. The quantities are the fixation
probability ofa mutant, the sum of the average local genetic variation and thevariation summed over the generations in which the gene frequency in thewhole population assumes a specified value. For details see Genetics 76:
367-377 (1974).
A Proof that Certain Quantities are Independentof the Geographical Structure of Population
Takeo MARUYAMA

A proof is given to that certain quantities are independent of the geographical structure of a population. The quantities are: (1) the fixation
probability of a mutant; (2) the sum of the quantity x(l-x), where x is themutant frequency, while the mutant is segregating; and (3) the quantity x(lx) summed over the generations during which the gene frequency in thewhole population assumes a specified value. The independence of geographical structure for the latter two quantities is not exact if there is selection,
but is a close approximation. The model is a geographically structured
version of Moran's haploid overlapping generation model. The population consists of colonies connected genetically by migration. Each individual
has the same negative exponential lifetime distribution. When an individual
dies, it is immediately replaced by an individual born in the same colony
with a probability proportional to the frequency and fitness of the type giving
birth. In a diploid population the guantity x(l-x) is proportional to theheterozygosity. For details see Theo. Pop. BioI. 5: 148-154 (1974).

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

IX.

POPULATION GENETICS (EXPERIMENTAL)

Similarity of Gene Frequencies in Different Drosophila Species:
Evidence for the Neutrality Hypothesis ofProtein Polymorphisms
Tsuneyuki YAMAZAKI and Takeo MARUYAMA

Ayala and Gilpin (proc. Nat. Acad. Sci. 71: 4847, 1974) applied a genetic
distance method to test the validity of the neutrality hypothesis of protein
polymorphisms advanced by Kimura and Ohta. Based on the data of several
Drosophila species they have concluded that the theoretical prediction of themeasure is incompatible with the data and therefore the neutrality hypothesis
is proved to be incorrect. Here, however, we show that Ayala and Gilpin's
conclusion is unwarrented and more importantly that their measure is indeed
consistent with the neutrality hypothesis and the test supports the hypothesis.
Consider two isolated populations (species) which were identical to eachother at time t=O (say), and assume that the two populations proceedindependent evolution of gene frequency change under the influence of genetic
random drift, natural selection, mutation etc. The frequencies of various
alleles at a given locus become different in the two populations as time goeson. To this situation, Ayala and Gilpin used a genetic distance between
populations, say 1 and 2, defined by
d= {El(X1i-X2i)2}!/ V2.

where Xii is the frequency of allele j in population i, and k is the number ofalleles at a locus under consideration. The distance d takes values between 0
and 1. If two populations have identical gene frequencies d=O, and if two
have no allele in common d= 1.
Assuming no selection (neutrality), no mutation or migration from outside
and finite population size (N), Ayala and Gilpin have calculated the statistical
distribution of the d-values for a case of three alleles with equal initial frequency (1/3 each). The distributions are graphically presented for t=O.IN,
0.2N, 0.5N and N, but no further time. The distributions of d given in
their figure are all bell shaped, though for larger tthe variance is larger. It
is important to note that the distribution is not given for t greater than N,

RESEARCHES CARRIED OUT IN 197463

the population size. There seems to be no reason to assume that history oftWO separated species is always less than N generations.
In order to test the prediction derived from the neutrality hypothesis,
they used data from Drosophila species. The d-values were measured among
different species at three different degrees of evolutionary level; among subspecies, among sibling species and among fullspecies. They used threesibling species D. willistoni, D. equinoxialis and D. tropicalis; one nonsibling
species D. nebulosa; two pairs of subspecies D. w. willistoni, D. w. quechua
and D. e. equinexialis, D. e. caribbensis. They assayed electrophoretic alleles at 36 gene loci. At each of the three evolutionary levels, the distribution is strongly concaved downward and slightly distorted V-shaped withpeaks at the d=O and d= I classes. As the evolutionary degree proceedsfrom subspecies level to full species level, the d=O class becomes less frequentand the d= I class increases, and the intermediate classes also decreases,(see Fig. I-A which is reproduced from Fig. 2A in their paper). The
graphs of the histogram contrast strongly with the presented predictions
derived from the neutrality hypothesis. Ayala and Gilpin claim that contrast
between the data and the prediction proves the failure of the neutral hypothesis. They further argue that more consistent explanation is some form of
normalizing selection which maintains quasi-stable polymorphic equilibria.
Having strong reservation, we have made calculations for two different
cases. The first is exactly the same as Ayala and Gilpin; no mutation,
selective neutrality and three alleles of initially equal frequency (1/3 each).
The distance d was calculated among ten separated populations. This enables us to make 190 pairwise comparison. The results of the distribution of
the d-values are presented in Fig. I-B for t=N, 4N, 5N. We can clearly
see a remarkable agreement between the data presented in Fig. I-A(the same as Ayala and Gilpin's Fig. 2A) and the prediction derived fromthe neutrality hypothesis. This comparison of Fig. I-A and I-B alone issufficient to unwarrent Ayala and Gilpin's conclusion. The disagreement
pointed out by them is simply due to insufficient separation time used for theprediction. When it is taken long enough as we did, the theoretical distribution agrees very well with the data. Our present finding can be regarded
as supporting evidence for the neutrality hypothesis.
However we realize that this can not be taken too strongly for two reasons.
The evolutionary time for the Drosophila species is not known and thus weare not certain as to the appropriateness of the generations used in our

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

calculations. In the absence of mutation, the genetic variability in each population decreases at a rate of Ij2N in each generation and eventually it becomes monomorphic. But the Drosophila populations studied are verypolymorphic and it is not unreasonable to assume they are in some equilibrium states. To take this into account, we introduced mutation and kept
the populations always in equilibrium states. A single equilibrium population was copied into 5 identical populations and thereafter each population

0.3

A Subspecies
"'55

c

B
No mutation

HON
With mutation

02
0.1

0.3
Sibling species

0.2

n-219

0.1

0.5 .
Full species

0,4

t'30N

n-149

0.3
0.2
0.1

Fig. 1. (A) Distribution of loci relative to genetic distance between subspecies(upper), sibling species (middle), and non-sibling species (lower) of the Drosophila
wiIIistoni group. n is the number of pairwise comparison. Redrawn from Fig. 2 (A)of Ayala and Gilpin (1974).
(B) and (C) Distribution of genetic distance simulated by Computer TOSBAC 3400.
The population size N = 100. (B) is the result of simulation done under the same
assumption as that of Ayala and Gilpin (1974): neutrality, no mutation and initiallythree alleles with equal frequency (1/3). In (C) mutation is incorporated, (4Nu=0.15).

65

RESEARCHES CARRIED OUT IN 1974

proceeded independent evolutionary process. At three different generation
times, the d-values were calculated among populations. We made 20
reprications and altogether 10 X 20=200 pairwise comparisons, (4Nu=0.15). The distribution of the d-values are presented in Fig. I-C. It is Ushaped and in early generations the d=O class is more numerous than thed= 1 class, but the situation is reversed in later generations. There is again aremarkable agreement between the data of Fig. I-A and the prediction presented in Fig. I-C. This case of prediction seems more realistic for the presence of mutation and thus each population itself is in a steady state. Therefore we present this analysis as supporting evidence for the neutrality hypothesis.
A similar point has been made by Nei and Tateno (Proc. Nat. Acad. Sci.
72; 2758-2760 (1975).
Negative Correlation between Lethal Genes
and Polymorphic Inversions
Takao K. WATANABE and Tsuneyuki YAMAZAKI

It has recently been shown that lethal frequency on chromosome 2 innatural populations of Drosophila melanogaster in Japan has increased rather
drastically within last ten years: from 15 to 30 % in Katsunuma population,and from 10 to 20% in Hiroshima population. Moreover it was noted in
Katsunuma population that this increase of lethal was accompanied by adecrease of polymorphic inversions on the same chromosome.
Through examination of all available data on lethal and inversion frequencies on the second chromosome in natural populations of D. melanogaster, we have discovered that there is a clear negative correlation between
the two quantities. Lethal genes are located more densely on the regions ofTable 1. Viability of inversion heterozygotes with or without induced mutations.
Genotype

No. of cross

+ jIn
+'jIn
+ jIn'

64
58
59, indicates EMS treated chromosome.

* significant at 5 % level.
** significant at 1 % level.

Viability ± s. e.
1.000±O.025**.980±O.035*.887 ±O.026*.**

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

standard gene arrangement than the inverted regions, and this accounts forthe negative correlation.
To reveal the underlying mechanism of the phenomena, we have carriedout an experiment and found that effect of EMS induced mutations on the
inversion-carrying chromosome is more severe than that on the standard
chromosome (Table 1). We interpret these results as evidence for coadaptation or position-effect within the inversion chromosomes. New mutations
within the coadapted gene complex are quickly eliminated from the population and polymorphic inversions are kept free of mutants through selective
elimination.
Electrophoretic Protein Polymorphism in a View of the Stepwise
Mutation Model of Neutrality Theory
Takeo MARUYAMA and Tsuneyuki YAMAZAKI

Protein variations are detected by differences in electrophoretic mobility.
The mobility is believed to be determined by differences in the numbers of
positively and negatively charged amino acids in the protein under examination. Some amino acid alterations, but not all, change the electric charge byunit amount in a positive or negative direction. Taking these properties ofelectrophoretically demonstrable mutants into account, Ohta and Kimura(Genet. Res. 22; 20, 1973) put forward the "stepwise mutation" model andadvanced its theory. They have derived a formula for the probability(H o) that two homologous genes are of the same electric charge, and therefore
operationally identical and the probability (C k ) that two homologous genes
taken from a population are alleles which differ by a given amount of electric charge;
Ho=l/V 1+8Nu ,Ck=HoA 1 k

(1)

(2)

where N=population size, u=mutation rate and1+4Nu-.v 1+8Nu
4Nu

On the other hand, the data in various speices are now available to test
this stepwise mutation model. We collected data, from published literature,
on about 200 loci. (The data source is given at the end of this paper.)RESEARCHES CARRIED OUT IN 1974

67

The homozygosity and the probabilities C k for k=l, 2, ... , 5 were calculated for each of the loci. For example, if PI, P 2, ... , Pn are the frequencies of alleles arranged in a sequential order in accordance with electrophoretic mobility, then HO=P12+P22+ ... +P2n, Ck=PIPk+l +P2P k+2+... +Pn-kPn' Since the C k in formula (2) depends on the Nu, it is not
appropriate to combine data of loci with different Nu values. Thus weclassified the loci into four groups of similar heterozygosity; heterozygosity
range 0"'0.2, 0.2",0.4, 0.4"'0.6 and 0.6"'0.8.
The purpose of this note is to compare these C k for the appropriate theoretical expectation derived from the neutrality model. Since the theoretical
Ck is a function of Nu, we need to know it for each class. At present the true
values are not known, but an estimation can be obtained from observed H ovalue and formula (1). Namely, since
Expectation (Ho)=l/V 1+8Nu ,
2

Nu=Ho

8

1

(3)

Using the observed mean H o and this estimate of Nu, we calculated Cl, C2 ,... , C5 from formula (2). The comparisons are made in Table 1. Since
Ho's are not independent in the present analysis, they are not a subject of
the comparison.
Data source: Prakash, S., Lewontin, R. c., and Hubby, J. L. (1969)
Genetics 61, 841. Kojima, K, Gillespie, J. and Tobari, N. (1970) Biochemical Genetics 4,627. Prakash, S. (1969) Proc. N. A. S. 62, 778. Lakovaara,
S. and Saura, A. (1971) Genetics 69, 377. Lakovaara, S. and Saura, A.
(1971) Hereditas 69, 77. Kojima, K, Smouse, P., Yang, S., Nair, S. andBrnic, D. (1972) Genetics 72, 721. Yang, S. Y, Wheeler, L. L. and Bock,
1. R. (1972) Studies in Genetics Univ. Texas Publ. 7213. Rockwood-Sluss,
E. S., Johnston, J. S. and Heed, W. B. (1973) Genetics 73, 135. Saura, A.
(1974) Hereditas 76, 161. Ayala, F. J., Tracey, M. L., Barr, L. G. andPerez-Salas, S. (1974) Genetics 77, 343. Gillespie, J. H. and Kojima, K(1968) Proc. N. A. S. 61, 582.

0\
00

~

-z

~
~

Table 1. Theoretical and observed values of Ck, the probability that two homologous genes areof aneles k-step apart. The numbers in the parentheses are the theoretical values.
The mean 8Nu was estimated from the mean Ho and formula (3). Comparison for
Ho was not made for this reason. A value in data represents the averageof the corresponding probabilities for the number of loci givenin the rightmost column.
Class
Heterozygosity
I-Ho, between

o

El
o

'TI

~

~

Mean
8Nu

Ho

Cl

Cz

C3

C4

C5

Number of locibelonging to
the class

"'0.2

0.085

0.960

0.018
(0.019)0.002(0.000)0.000(0.000)0.000(0.000)0.000(0.000)400

r.n.

0.2"'0.4

1.094

0.691

0.118
(0.126)0.030(0.023)0.006(0.004)0.001(0.001)0.000(0.000)58

0.4"'0.6

3.114

0.493

0.178
(0.167)0.060(0.057)0.010(0.019)0.003(0.007)0.001(0.002)

68~

0.6"'0.8

6.506

0.365

0.205
(0.170)0.084(0.079)0.019(0.037»0.008(0.017)0.001(0.008)

12

0

Z
~

o'TI

~
j

(j

r.n.

~

IV

Vl

RESEARCHES CARRIED OUT IN 1974

X.

69

EVOLUTIONARY GENETICSOn Some Principles Governing Molecular Evolution
Motoo KIMURA and Tomoko OHTA

The following five principles were deduced from the accumulated evidence
on molecular evolution and theoretical considerations of the population
dynamics of mutant substitutions: (i) for each protein, the rate of evolution
in terms of amino acid substitutions is approximately constant per site per
year for various lines, as long as the function and tertiary structure of themolecule remain essentially unaltered. (ii) Functionally less important
molecules or parts of a molecule evolve (in terms of mutant substitutions)faster than more important ones. (iii) Those mutant substitutions that
disrupt less the existing structure and function of a molecule (conservative
substitutions) occur more frequently in evolution than more disruptive ones.
(iv) Gene duplication must always precede the emergence of a gene having
a new function. (v) Selective elimination of definitely deleterious mutants
and random fixation of selectively neutral or very slightly deleterious mutants
occur far more frequently in evolution than positive Darwinian selection ofdefinitely advantageous mutants. For details see Proc. Nat. Acad. Sci.
71: 2848-2852.
Distribution of Antigenic Specificities of Mouse H-2 among Wild
Rodents and Its Evolutionary Implication
Kazuo MORIWAKI

Serological survey of erythrocyte antigens crossreacting with mouse H-2
alloantisera has been carried out by using various wild rodents collected fromJapan, Southeast Asia and India. An improved PVP-hemagglutination
method (Kaliss, N. Transpl. 15: 251,1973) which modified PVP specificationfrom K60 to K90, was employed in this work. Alloantisera for various
H-2 specificities given by Transplantation Immunology Branch, NIH(D. S. A.), have been diluted serially with 0.6 %PVP solution containing 5 %BSA and phosphate buffered isotonic saline. The mixture of antiserum andred blood cells kept in the microtiter plate was incubated at 37°C for one
hour and 5°C for 15 hours. Degree of hemagglutination was estimated

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

under the microscope. The results of the present survey are summarized inTable 1.
The remarkable findings in the present study are as follows: (1) Mus
musculus molossinus almost failed to show crossreaction with H-2 speci.
ficities (D 3, 4,13, 19,41,42,43,44 and K 5,23) which appeared in the labora.
tory mice, Mus musculus musculus. In the feral population of M. m. muscu.
Ius subspecies collected from Europe, several H-2 specificities have been
demonstrated as well (Mickova, M. & P. Ivanyi, In "Immunogenetics of theTable 1.

Cross reaction with various rodent erythrocytes of anti-H2 alloantisera
Maximum dilution (Log2) of antisera for positive
hemagglutination

Alloantisera

C3b

D4

D5AF

DB

D23

Antigen specificities

D3,19

D4

K5,52

D4, 13,41
42,43,44

K23

Control
BlOD2
BlOBRSpecies tested
Family Muridae
Subfamily Murinae
Mus musculus molossinus
M. caroli
M. dunni
M. platythrix
Rattus rattus
R. sabanus
R. f1avipectus
R. norvegicus (LE)(WKS)
Millardia meltada
Vandeleuria oleracea
Micromys minutus
Family Cricetidae
Subfamily Cricetinae
Peromyscus leucopus
Cricetus griseus
Subfamily Microtinae
Microtus montebelli
Clethrionomys rufocanus
bedfordiae

9.0

13.0
13.0

0.0
0.0

0.0
0.0

0.0
0.0

0.0

3.0
0.0

0.0

0.0

0.0 (3)0.0

0.0 (8)0.0
5.5
2.8 (4)
0.0

0.0
0.0

0.0
0.0

0.0

0.0
0.0

0.0
0.0
0.0
0.0
0.0

0.0

0.0

0.0

4.0
0.0
0.0
4.0

7.0

0.0 (lO)
0.0
5.7 (3)5.1 (8)4.3 (4)3.5
5.7(3)
0.0
0.0

1.4 (8)0.0
5.5
2.5 (4)
0.0

2.0

13.0
0.0

0.0 (8)0.0
3.0
2.5 (4)
0.0

0.0
4.0

Log2 value represents mean of two individuals, unless number is indicated in the parenthesis.

RESEARCHES CARRIED OUT IN 197471

B-2 system" 1971). (2) Unexpectedly, some species belonging to genus
Rattus and Mus other than Mus musculus have exhibited the considerable
degree of crossreaction with the mouse H-2 antigens. (3) Most rodent
species other than genus Rattus and Mus could not show any detectable
positive hemagglutination.
One of the possible explanation for those findings might be given from theevolutionary view point of H-2 genes. The broad distribution of H-2
antigens in genus Mus and Rattus allows us to assume that those antigens
appeared in a common ancester of the both genera. Though these antigens
have been retained until today in such species as Mus musculus musculus,
M. dunni, M. platythrix, Rattus rattus, R. jlavipectus and R. sabanus,
they seem to have been altered in M. m. molossinus. Considering that theEuropian sbuspecies is inhabiting in rather peripheral region than the Asian
one in the geographical distribution of this species, the former, M. m. musculus, could be somewhat older than the latter, M. m. molossinus. Probably,
the alteration of the H-2 genes might have occurred during the differentiation process of those subspecies from musculus to molossinus.

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

XI.

HUMAN GENETICS

Indirect Inguinal Hernia: A Multifactorial Threshold Trait
Ei MATSUNAGA, Shigenori SAWAGUcm* and Toshiro HONNA*

Among 1,723 probands who had been operated because of indirect inguinal
hernia, the ratio of males to females was 2.8: 1, being the same as that in thegeneral population, and in both sexes the ratio of bilateral to unilateral
hernia was 1: 4. Analysis of family history showed that the incidence ofhernia among parents and elder siblings relative to that in the general population varied to some extent according to the sex of the probands and thelaterality of hernia. The relative incidence tended to be higher, thoughslightly, if the proband was a female than ifit was a male, and if the condition
was bilateral than if it was unilateral, suggesting that predisposition to hernia
is determined by polygenic inheritance with a threshold beyond which
individuals are at risk. However, no indication was found for an increase inparental consanguinity. The heritability of liability to indirect inguinal
hernia was estimated as 64±3 %from the regression coefficients of parents on
probands. There were more twins among the probands than would be
expected, suggesting that twins are especially prone to hernia. Details of
the study will be published in the Jap. Journ. of Human Genet.
Possible Genetic Consequences of Relaxed Selection against
Common Disorders with Complex InheritanceEi MATSUNAGAWith increasing success in medical treatments for many common disorders
with complex inheritance, it is expected that the incidence of patients requiring treatments will gradually rise from generation to generation. While
this is theoretically true for a long range of future, the pattern of possible
increase in the frequency of a disorder resulting from complete cure has been
examined with special reference to the next few generations. It was shownthat the maximum possible increment will take place in the first generation,with additional increment rapidly diminishing with subsequent generations.
* National Children's Hospital, Tokyo.

RESEARCHES CARRIED OUT IN 197473

For congenital heart disease, for example, the restoration of f11ll fertility to allof the patients would incur an increase in the frequency at birth of the disease
by about 10 % after five to ten generations. Details of the study will be
published elsewhere.
Isozyme Variations in Man
Tomotaka SmNoDA, Ei MATSUNAGA and Jushiro KOSI:llNAGA*

In order to assess the degree of genetic variation of individuals some 30
enzyme loci were analyzed using tissue extracts by means of gel electrophoresis and specific histochemical staining. These loci were randome1y selected.
Enzymes tested in this study included several groups such as kinases, dehydrogenases, oxidases, reductases, transaminases and deaminases.
Of 33 different loci examined, 8 were found to be polymorphic with practically the same frequencies as in the previous study. Besides these, somevariations in zymogram were seen for several enzymes, but it was not certain
if these variations were genetic in origin. The sample size was relatively
small and some enzymes easily produced artifacts which had the similar
substrate specificity but had different mobility in gels. A part of the data waspublished in Japan. Journ. Human Genet., 19: 243-250.
Studies of Q-variants among Patients with Down's Syndrome
and Their Families
Kazuso IINUMA and Ei MATSUNAGA

Q-variants of chromosome no. 21 detected by fluorescent staining wereclassified as either 'F' or 'f', according to the intensity of fluorescence in theregions of the short arms or satellites. A total of nine cases with standard
trisomy 21 and their parents were analysed under the classification and it
was possible to identify one case suggesting nondisjunction at the second
meiotic division in the mother. There was another case where maternal
nondisjunction was most likely. Meiotic errors at maternal second divisionwere excluded in 3 cases and paternal first division in 2 cases, respectively.
The results were non-informative in two cases. It was found that one of thelast cases had a de novo translocation t(6q+; l5q-). Q-variants of chromosome no. 15 of the patient and his parents were analysed. The derivative* Medical Examinar's Office, Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

no. 15 had a medially fluorescing satellite and this characteristic satellite Was
also observed in one of the mother's no. 15. Thus, the formation of thetranslocation was considered to have occurred in the maternal germ cells(Jap. J. Hum. Genet., 20: 147, 1975).
It was examined whether 'F' of the no. 21, defined as an intense fluorescenceof the short arms or satellites, has any correlation with the predisposition tomeiotic nondisjunction of chromosome no. 21. Thirty-seven males and 27
females, whose karyotypes were proved to be normal, were selected as acontrol group. Twenty-seven parents of standard 21-trisomic infants werealso confirmed to have a normal karyotype. The third group were 5 boys
and 14 girls with standard trisomy 21. The frequencies of 'F' in the control
group, the parents, and 21-trisomic infants, were 0.18, 0.20 and 0.19, respectively. No significant differences in the frequencies of 'F' were observed
among these groups.
A part of this study was reported in the 19th annual meeting of the Japan
Society of Human Genetics (1974).
Non-random Distribution of Exchange Points in Patientswith Structural Rearrangements
Yasuo NAKAGOME and Hideaki CHIYO

A total of 248 breakage points from 130 unrelated cases with structural
rearrangements were studied to determine ~hether they were preferentiallylocated within any particular type of band. Thirty of them (15 cases) wereidentified by the authors and 218 (115 cases) were collected from 51 different
publications. Only cases with identification of each breakage within asingle band were included in the study. Positions of breakages were classified into light, variable and dark bands, based on their G-band patterns.
Alternatively, sites of breakages were classified according to their positions
within a chromosome arm, i. e., terminal, centromeric and intermediateparts. Expected numbers of breakages were based on an assumption thateach unit length of band had an equal chance of being broken.
Of248 breakages, 178 were from reciprocal translocations, 32 from inversions
and 38 from various other types of rearrangements. In reciprocal translocations, breakages were more frequently observed in the light and variable bands
than expected numbers, while those in dark bands were rare (p~O.OOI).
In inversions, an excess of light bands and a lack of dark bands were ob-

RESEARCHES CARRIED OUT IN 197475

served (p<O.005). There was no excess of variable bands. In other types ofrearrangements, the observed numbers of breakages in each type of bands
were very close to the expected.
In both reciprocal translocations and rings, the middle part of a chromosome arm seemed to have less chance of being the site of an exchange incontrast to terminal and centromeric parts (for details: Nakagome & Chiyo,Amer. J. Hum. Genet. in press).

A 7p Monosomy Resulting from a Stable Dicentric Chromosome-with Reference to the Mechanism of Inactivationof a CentromereYasuo NAKAGOME, Fumio TERAMURA, Kenkichi KATAOKA
and Fumitoshi HOSONO

Partial monosomies of any of C group chromosomes have rarely been
described. They included rings of nos. 6, 7 and 9 as well as partial monosomies of 9p, lOp, 11q and 12p due either to a translocation or a deletion.
Propositus was a 3-month-old girl. Main clinical features were: failure
to thrive, markedly retarded development, prominent forehead, hypertelorism
a downward slant of eyes, low set and malformed ears, high arched palate,micrognathia, funnel chest and pes varus.
Cytogenetic examinations were carried out by the standard leukocyteculture technique. Her two brothers and parents showed a normal karyotype. The propositus had only 45 chromosomes. One chromosome was
missing from each of the C and the D group and there was an extra "no. 2"
chromosome. Metaphases were examined further by various banding techniques. The detailed studies of the G-band pattern revealed that the exira"no. 2" consisted of a no. 7 and a no. 15. The break points were in a 7p21
and a 15pll bands respectively, i. e., the derivative chromosome had atdic (7; 15) (p21 ; P11) constitution. The patient was monosomic for both the
7p22 and a part of the 7p21 bands.
Although the derivative chromosome had two centromeres, none of themshowed a typical appearance of a dicentric chromosome. By the C-band
technique, intensely stained spots were observed at two different sites on
the derivative chromosome, both of them being corresponded to the presumed
sites of centromeres. The Cd-band method stained only one of them, i. e.,
the centromere derived from the no. 7. It was assumed that the centromere

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

derived from the no. 15 lost its component parts and was inactivated.
details: Nakagome et at., CHn. Genet. in press).

(for

Structural Studies on Human Immunoglobulins
Tomotaka SHINODA

Sequence analyses were carried out on a variety of human immunoglobu.
lins of different types and classes, which were isolated from plasmas Or
urines of patients with multiple myeloma or heavy chain disease. These
results ununbiguously indicated that sequences of the first 110 residues Wererandomly varied from one to another regardless of the types of the L chainsor the classes of the H chains. However variable in this region, these wereclassified into several subgroups based on certain degree of sequence simi.
larlity among them. Subgroups for the L chains were type-dependent
whereas those for the H chains were independent of its classes. In connection with subgroup specificity, we found a L chain whose variable region
sequence shared a part of two different subgroups. Thus, it was reasonably
assumed that such a sequence had arisen as a consequence of crossing-overbetween two parent subgroups genes.
Assuming that each subgroup was encoded by a few different germ line
genes, then at least 12 major genes should be considered for the variable
regions of the Land H chains. Similarly, at least 14 different genes can beassumed for the constant regions of immunoglobulins. Still it seems difficult
to interpret the mechanism of structural variabilities of immunoglobulins,
unless some new concepts,for example, two gene-one immunoglobulin are
introduced. Our data suggested that much variabilities in sequences had
resulted from somatic events rather than from those in germ line genes.
Parts of data were published in J. Biochem., 75: 23-44, 77: 1277-1296.

77

RESEARCHES CARRIED OUT IN 1974XII.

BEHAVIORAL GENETICS

Circadian Locomotor Rhythm of an Adult Fly of
Drosophila melanogaster
Chozo OSHIMA

Many kinds of actograph have been designed in order to record the circadian locomotor rhythm of insects. In this experiment, an electric actograph
was originally designed; especially a Light Emitting Diode (LED) and a
Sharp Photo Diode (SPD) were used as a senser part. A small glass box(5 X 10 X 50 mm) was put between these Diodes for a beam of red light topenetrate it. When a fly moved and crossed the beam, the voltage on SPD
was changed. The small change was enhanced by an amplifier and recorded
as a single vertical line on film in Micro Pen-oscillograph. The film band ismoving continuously with a definite speed. The senser part was put in an
incubator, controlling temperature and light conditions. The environmentof this experiment was set up as follows; temperature was constant at 24 C
and circadian light (1500 lux) and dark condition LD 13: 11 (L consisted ofdawn-l hr, dusk-2.5 hr and light-9.5 hr periods) was repeated every day by
the program photocontroller. A small glass box has a small amount of poor
medium, containing only red sugar and agar, and the unstop was covered
with gauze. A fly could survive for 2-3 days and if the culture medium was
exchanged with new one every several days, the circadian locomotor rhythmof a fly during a considerable time could be recorded.
Among individual circadian locomotor rhythms of many wild flies in anatural population, a fairly amount of variations would be found on period
between acrophases and locomotor activity (amplitude). However, thecircadian locomotor rhythm of a wild male fly, extracted from Ishigakijima population, was recognized to be bimodal from results, obtained by theactograph mentioned above, and the high locomotor activity was displayed
most frequently at dusk period and another locomotor activity was displayed
before or at dawn period. Such a circadian locomotor rhythm would be
regulated by an endogenous function of the bioclock in brain and it is possible to assume that its function would be daily readjusted at dusk period.
Q

This work was supported by a grant (No. 930403) from the Ministry of Education.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

Effects of Light, Temperature and Noise Environments on Longevity
and Productivity of Drosophila melanogaster
Chozo OSHIMA and Takao K. WATANABE

Many flies of one day age of Oregon R strain were collected, and many
sets, consist of one female and two males, were respectively introduced
into small vials, which have standard corn meal, sugar and yeast medium.
These 200 vials were divided into four groups and each group was put separa_tely under different light and temperature environments; 1) constant light(LL) 2000 lux and constant 25°C (C), 2) constant light (LL) and circadianfluctuated between 20 an<;l 30°C (F), 3) circadian light and dark (LD 12: 12)and constant 25°C (C), 4) circadian light and dark (LD 12: 12) and fluctuatedbetween 20 and 30°C (F). Flies of each vial were transferred into a newvial two times a week and dead females were scored. When a male fly was
found to be dead, it was supplied with a new one of the same age. Such procedures were carried for 30 days. On the other hand, all vials were put
under constant light (LL) and temperature (25°C) and all emerged flies werecounted to estimate productivity under different environments. The experiments were replicated for several times for estimating the longevity under
different environments and the results were summed up in Table I.
Table 1. Longevity (LD 50) of female flies under four kinds of different
environments.
Environment
Light

Temperature

Number of
replications

LD 50 (day)LL
LL
LD 12: 12
LD 12: 12

C 25°CF 20~30°C
C 25°CF 20~30°C

42
33

16.7±1.1
17.2±0.2
21.2±2.3
25.3±1.6

Ratio
1.00
1.03
1.27
1.52**The next experiment was carried in order to estimate the effect of noise(2000 helz, 100 phone) on longevity of flies. The material and method were
the same to the previous experiment, but two kinds of noise environment;
the circadian noise and quiet condition (NQ 12: 12) and the constant quiet
condition (QQ) were added to the constant light condition (LL). The
experiment was replicated two times and the results were presented in Table 2
The productivity was similarly estimated to the previous experiment.
This work was supported by a grant from the Ministry of Environmental Agency.

RESEARCHES CARRIED OUT IN 1974
Table 2.

--Longevity (LD 50) of female flies under two kinds ofdifferent environments.

Environment
Light
Noise

LL
LL

79

QQ
NQ 12:12

Number of
replications

LD 50 (day)

Ratio

2
2

23.1O±4.122.40±1.9

1.00
0.97

Two characters, longevity and productivity (number of eggs), are assumed
to be highly correlated, and the results showed the assumption to be correct.
However, the experimental results, obtained at different time, could not becompared with each other. In the previous experiment, the circadian lightand dark and fluctuated temperature environments were most favourable forthe longevity of fly, and the mean number of offspring per female for life
time was about 1.3 times more than that under the constant light and temperature condition. On the other hand, the circadian noise and quiet environment was not so unfavourable for the longevity of fly, and the mean
number of offspring per female for life time was unexpectedly more than that
under the constant quiet condition.
Effects of Light and Noise Environments on Adult Emergence Rhythmof Drosophila melanogaster
Chozo OSIllMA and Takao K. WATANABE

One thousand and five hundred eggs, laid for 12 hours (first period) andanother 1500 eggs laid for following 12 hours (next period) by many fiies ofOregon R strain, were divided into three groups respectively and each group(50 by 50 eggs put on culture medium of 10 vials) was allowed to develop in
incubators, having three kinds of light (about 2000 lux) environments;
constant light condition (LL), circadian light and dark condition (LD 12: 12)
and mixed circadian light and constant dark conditions (LD 12; 12 forearly 7 days and DD). The temperature was kept constant at 25°C in allcases. After 7.5 days, adult flies of the first group began to emerge and they
were scored two times a day.
Frequency curves of adult emergence, connecting several scoreing points of
two groups, were parallel under the constant light condition and the distance
between peaks of these emergence rhythms was half a day. However, theseThis work was supported by a grant from the Ministry of Environmental Agency.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

frequency curves was transformed under circadian and mixed light conditions.
About 50 percent flies of the first group emerged for light period of 8.5 day
and about 50 percent flies of the next group emerged for light period of 9.5
day. The emergence rhythms of two groups under the constant light condition were very similar, but the phases of such rhythms were shifted faster orslower under circadian and mixed light conditions and the distance between
peaks was spread to be a day. Such a phase shift was also observed under themixed light condition and then, it is assumed that the developmental rhythm
during larval and early pupal periods was individually readjusted every day
to the circadian light and dark condition. Consequently, the synchronization of adult emergence of many flies was enhanced. The adult eye was notjet developed during the period, then, the readjustment of rhythm would becontrolled by a bioclock located in a main lobe of the brain.
Materials and procedure of the noise experiment were similar to those oflight experiment mentioned above. One thousand and five hundred eggs
were allowed to develop in incubators, having constant light (LL) and temperature (25°C) and three kinds of noise environments; circadian noise andquiet condition (NQ 12: 12), mixed circadian noise and constant quiet (NQ
12: 12 for early 7 days and QQ) and its reversed conditions (QQ for early 7days and NQ 12: 12). The noise was pure sound having 2000 helz and
100 phone. The unstop of vial was covered with gauze for penetrating thesound. About 70 percent flies of the first group emerged for quiet period of
8.5 day, but about 60 percent flies of the next group emerged for noise periodof 9.5 day. The phase of emergence rhythm was not so remarkably shifted
under the circadian noise and quiet condition as shown in the light experiment. However, the readjustment of a bioclock in this case was presumed to
be clearly weakened by the constant light condition. The grade of synchronization of adult emergence in two conditions (NQ 12: 12, NQ 12: 12 forearly 7 days and QQ) was much higher than that of another condition (QQ
for early 7 days and NQ 12: 12). As a matter of fact, both light and noise
environments accelerate the development and they would have a bad effectfor the life of fly, especially when they disturve the circadian rhythm of bioclock.

RESEARCHES CARRIED OUT IN 1974

81

Strain Difference in Larval Phototactic Behavior of the Silkworm
Akio MURAKAMI and Kosuke KOTOKU 11

A comparison of phototactic behaviors between strains (or stocks) showing
a different phototaxis may help to better understand the genetical control
mechanism of such a biological phenomenon. From this view point, thesenior author has been carrying out extractions of the different phototactic
strains among 200 silkworm stocks being maintained in his institute. Consequently, four silkworm stocks manifesting differential phototactic responses
at the mounting stage under a light condition in an ordinary silkworm rearing
room were extracted: two stocks, pe; ok and nb, are gathered in crowds tothe light part (photopositive) and other two stocks, Ze ;pe; re and sf, aregathered to the dark part (photonegative). The other stocks showed noclear cut phototactic behaviors.
Using those photosensitive stocks, further experiments have been carriedout to throw light upon the phototactic behavior in relation to the growthof larvae throughout the whole larval stage from newly hatched larvae tomature larvae. The measurement of the larval behavior was made by amean walking distance (in centimeter) from the center (or neutral part) inthe silkworm phototatic measurement box to either light or dark direction.
The box was composed of two parts: one part is illuminated with an ordinary
fluorescence light (20 W) and another is covered with dark paper to shade thelight.
The result of experiments indicated that larval stages other than the stageof mature larva are insensitive to phototactic response. At the stage ofmature larvae, however, the previous observation was confirmed that pe; ok
and nb strains showed the positive phototaxis and the st and Ze ;pe; restrains showed the negative one. Among them the pe; ok and sf strains showed so clear cut phototaxis that factor (s) manifesting their phototactic response
seemed to be controlled by a few major genes. Not yet completely analyzed,
however, the result of analysis of phototactic behaviors in hybrids between
photopositive and negative strains indicated that phototactic response of the
PI hybrid was somewhat photonegative rather than neutral, regardless ofparent phototaxis and the F 2 (or F 1) extended to both directions. Thissuggests that photonegative nature is more dominant than photopositive
11

Ehime-ken Sericultural Experiment Station, Ohzu, Ehime-ken 795.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

one and that factor (s) manifesting phototactic response is controlled bypolygenes.
Strain Differences in Discriminated Avoidance Learning
Curve in Mice
Tohru FUJISffiMA

Fifteen inbred strains of mice were tested for discriminated avoidance
responses (in percentage) with the aid of an automated Y-maze apparatus,
and the performance of each strain was shown by a learning curve. The
operating procedure was to use a lamp and a buzzer for 5 seconds as theconditioned stimulus, which was immediately followed by an electric shockfor 5 seconds as the unconditioned stimulus, with intertrial intervals of 30
seconds. Two daily sessions, each consisting of 50 trials, were carried out.
The results indicated that C3H/He acquired the highest learning effect(52.3 %), while D103 did not show any acquisition of learning effects, andC57L, C57BR and DBA/2 were superior in reminiscence. It was also foundthat, although one might expect discrimination to be more difficult to learn
than avoidance, some strains showed the reversed trend; SWM and RF
prefered avoidance to discrimination, while DIm prefered discrimination
to avoidance. On the other hand, C3H/He and C57BL/6 learned approximately equally both of the learning tasks.
Dianel Cross Experiment on the Discriminated Avoidance
Learning Abilities of Mice
Tohru FUJISffiMA

A diallel cross experiment was conducted with four inbred strains selected
for the memory patterns of discriminated avoidance learning abilities measured with an automated Y-maze apparatus. The strains used were C3H(a high and high line), SWM (a high and low line), C57L (a low and highline) and D103 (a low and low line) for the short-term and long-term memory
effects obtained from discriminated avoidance learning tests, respectively.
The data generally indicated that the memory pattern found in the progeny was consistent with that of the parents. This suggests that the memory
pattern as measured by the present method is genetically controlled. The
general and specific combining abilities had significant variances, but the

RESEARCHES CARRIED OUT IN 197483

maternal effects were insignificant. For the general combining abilities,
SWM was the highest (6.42 %) and D103 the lowest (-6.13 %) in short-term
memory effect; C57L was the highest (4.58 %) and D103 the lowest (-5.80 %)in long-term memory effect.
Complete Blindness of an Inbred Strain of Mice, DI03/Ms
Tohru FUJISIDMA

A mouse inbred strain, D103/Ms, derived from strain DM, has a visualsensory defect though its eyes appear to be normal. The sensory defect wasinvestigated by inserting a bi-polar electrode into the non-specific sensory
system of the cerebral cortex and observing the evoked electroencephalogram
responses to light. It was compared with C3Hs (adult), which had beenreported to have visual sensory defects, and also with DM, the parental strain
ofD103.
The result showed that D103 had a complete blindness, while C3H/He
was not so completely blind as D103, and DM had no visual defects. It
was also found from a preliminary crossing experiment that the blindness
gene of D 103 was recessive.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

XIII.

APPLIED GENETICS

Breeding of Isogenic Lines of Rice Carrying Various Gene Markers
and Reciprocal Translocations
Hiko-Ichi OKA and Hiroko MORISffiMA

As reported last year (Ann. Rep. 24, p. 66), isogenic lines having various
gene markers in the genetic background of Taichung 65 (T65) were isolated,each from recurrent backcrossing with T65 repeated 7 times or more. The
genes incorporated are: wx (or gl, glutinous), dz (Ebisu dwarf), 19 (liguleless),
Ph (phenol reaction), Rd (red pericarp), g (long empty glume), d 1 (Daikoku
dwarf), la (lazy habit), ne (neck leaf or bract development), be (brittleculm), gb (glabrous leaf), etc. The purpose of this work is to use the isogeniclines for genic analysis and to investigate the pleiotropic effects of respective
genes.
For the latter purpose, the effects of those genes on heading date and on
the size of various organs of seedlings and adult plants were observed. The
ne and dz genes delayed heading about seven and four days, respectively.
Genes g, gb and d1 also delayed heading slightly. The dwarfness genes (d1and dz) reduced the size of organs giving rise to particular allometric patterns.
In addition, it was found that Ph reduced the number of secondary rachilla
per rachis, g reduced primary rachilla per panicle, be increased secondaryrachilla per rachis but reduced primary rachilla per panicle, etc. However,these genic effects were generally small in magnitude and could be overlookedwithout a careful statistical test. Therefore, these marker genes excepting
those causing dwarfism seem to be useful in genic analysis for various quantitative characters.
Reciprocal translocation homozygotes of T65 under observation are 30
lines, each having been purified by three times backcrossing to eliminate
radiation-induced mutant genes. They were each crossed with isogeniclines carrying 19 and g, and ne and be, respectively, to identify the chromosomes involved in translocations. The seeds of these isogenic lines will bemailed to research workers interested in rice genetics upon request.

RESEARCHES CARRIED OUT IN 1974

85

Genic Analysis for the Sterility of Hybrids between Oryza sativa
and O. glaberrima
Hiko-Ichi OKA and Hiroko MORISIDMAThe F I hybrid between the two rice species is highly pollen-sterile, though
their chromosomes normally pair in meiosis. In order to look into the genetic basis of the sterility, isogenic Frsterile lines having the genetic backgroundof sativa and glaberrima parents, respectively, were isolated from Bs F 2
plants. Their crossing experiments are under way. The results so far obtained indicate that the FI-sterility genes from both parents cause deterioration of the gametes through an interaction of their effects on the sporophyte
and gametophyte; gametes of both sexes carrying a deteriorate in the plantwith A. Evidence supporting this hypothesis has been accumulated, while
the presence of other types of genes was also suggested.
A Competition Experiment between Rice Cultivars of Different Ages
Hiko-Ichi OKA and Hiroko MORISIDMA

It has been advocated by Sakai (1965, in The Genetics of Colonizing
Species, etc.) and coworkers that competition does not take place between
plants with the same genotype. However, this assertion should be interpreted as: plants with the same genotype and at the same age do not express theeffect of competition since they have the same competitive ability, even though
they compete each other. Two pure lines of rice (Oryza sativa), Taichung
65 and Peiku, were each seeded on May 8 (early) and May 28 (late), and theseedlings raised separately were mix-planted at a spacing of 20 X 20 cm indifferent combinations. The results showed that when early and late-seeded
plants of the same line were mix-planted, the early plants had an advantage incompetition over the late ones. Peiku has a stronger competitive ability thanTaichung 65. This relation was reversed when early Taichung 65 and late
Peiku were grown in mixture. The experimental results suggest that competition depends upon the growth rate at a certain stage when the leaves ofneighboring plants come into contact.

Mortality and Adaptive Mechanisms of Oryza perennis Strains
Hiko-Ichi OKA and Hiroko MORISIllMA

Six strains of Oryza perennis Moench and two strains of cultivated rice

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25(0. sativa L.) were tested in various "semi-natural" (partly weeded noncultivated field) conditions and in a paddy field (control) for survivorship,
phenotypic plasticity, and seed maintenance in the soil, at the Central Luzon
State University, Philippines. The perennis strains, ranging from perennial
to annual types, showed wide variations in the characters observed. The
perennial type showed lower mortality in the seedling stage but a higher
mortality in the adult stage than annual types. The perennial type also
showed greater phenotypic plasticity in seedling height and a lower degreeof it in panicle length than annual types. Examinations of soil samples
about one month after maturity proved that annual strains maintained moreseeds in the soil than perennial ones. This difference was also recognizedfrom the number of plants occurring in the next season. Further, experiments made at Misima showed that the seeds of O. perennis remained alive
for more than three years in water or in moist soils at 25°C. When newly
harvested seeds were dibbled or scattered on the soil surface in a greenhouse,a part of them germinated in ten months as their dormancy was overcome.
It was pointed out that various attributes were inter-correlated resulting indifferentiation of the plants in their adaptive strategies.

Competitive Abilities of Barnyard Grass and DalIis-Grass against
Finger Grass
Hiroko MORISHIMA

In an attempt to look into the adaptability of weedy plants, five strains ofbarnyard grass (Echinochloa crusgalli) and three of dallis-grass (Paspalum
di/atatum) were tested for their competitive ability against the finger grass(Digitaria sanguinalis). The field plot to be used was tilled and the soil was
pulverized on April 20 (1974), and young seedlings of barnyard grass anddallis-grass were transplanted on May 17 (early planting) and June 10 (lateplanting). Four treatments were made each with two replications, i. e.,
weeding by hand, no weeding, early seeding (broadcasted, April 24) of finger
grass, and late seeding (May 22) of finger grass. At maturity in September,the barnyard grass and dallis-grass strains were measured for plant heightand panicle number per plant. The fresh weight of other weeds per square
meter (mainly finger grass) was also recorded.
The barnyard grass strains significantly differed in the competitive ability
against finger grass and other weeds. Those from copper-polluted sites

RESEARCHES CARRIED OUT IN 197487

tended to have lower competitive ability than those from unpolluted sites.
The dallis-grass strains showed no significant differences among them, but
generally expressed a higher degree of phenotypic plasticity than barnyard
grasses.
Copper Tolerance of Barnyard Grass Strains
Hiroko MORISHIMA and Hiko-Ichi OKA

Seeds were collected from barnyard grass (Echinochloa crusgali) populations growing on a slag deposit at Hanaoka copper mine (Akita-ken), paddy
fields at Morita (Ohta, Gunma-ken, where the people have suffered fromcopper pollution of the Watarase River from Ashio mine since 1880's), and inother unpolluted places. Young seedlings from the seeds were cultured
in testtubes containing Hyponex solution and CUS04 at the rates of 0.5 and2 ppm copper. Root elongation was found to be a good indicator of thereaction to copper toxicity. The lines tested were 34 from polluted and 26
from unpolluted sites. Lines from the same population showed a wide rangeof reactions suggesting that barnyard grass populations would be highly
heterogeneous. Nevertheless, it was recognized that populations at polluted
sites had a higher mean tolerance and a higher frequency of tolerant plants
than those at unpolluted sites.
Perhaps, metabolic systems increasing copper tolerance have been selectedin those plants growing in the polluted area. This work was undertaken as
a part of a research project financially supported by the Bureau of Environment, "Genetic influences of environmental pollution on plants and animals",
and is still under way.
Variations in Heavy Metal Tolerance of Weedy Plants
in the Paddy Field
Hiroko MORISHIMA and Hiko-Ichi OKA

In order to observe variations in the tolerance of paddy weeds to copperpolluted soils, soil samples were taken from paddy fields at 8 polluted (containing more than 200 ppm copper) and 4 non-polluted sites nearby Takara
copper mine (Tsuru, Yamanashi-ken). The soil samples taken in late May(before transplanting rice) were put in trays and kept moist in a greenhouse to
obtain plants from the buried seed population. The seedlings of paddy

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

sedge (Cyperus difformis) at about one month after germination were tran.
splanted into pots containing field soil (control) and toxic soil (a half-to-half
mixture of field soil and the soil from a precipitation pond of the mine,
containing 1,160 ppm copper and 6,080 ppm zinc). The growth, root develop_
ment and seed production of the sedges were then observed. This experi_
ment was repeated twice in 1973 and 1974. The results in both years consistently showed that the plants from polluted sites apparently had higher
tolerance to the toxic soil than those from unpolluted sites. Some plantsfrom polluted sites tended to grow better on the toxic soil. This suggeststhat tolerant genotypes have been selected in response to the pollution ofsoils by the mine.
In parallel to this, the plant species obtained from the buried seed populations (15 sites in total) were recorded and association analysis was made.
The association of major paddy-weed species was found to be almost at
random. Yet their relative frequencies significantly differed between
polluted and unpolluted sites. Polluted soils carried a larger number ofFimbristylis miliacea, Vandellia angustifolia and Rotala indica plants but asmaller number of Mazus japonicus plants than unpolluted soils. Cyperus
difformis seemed to be more frequent in unpolluted than in polluted soils,
though it is highly tolerant to heavy metals as mentioned above.
The Mechanisms of Weediness Found in Dallisgrass Populations
Hiroko MORISillMA

The dallisgrass, Paspalum dilatatum Poir., is an apomictic perennial grass.
It has been a weed in some areas of Japan long before its introduction as aforage crop. Recently, its remarkable spread was noticed in southern
Kyushu. They grow in the habitats disturbed by man, such as roadside,
dikes of farm field, waste places in town, etc. In order to look into the adaptive mechanisms of this species, seeds were collected from natural populations
in four localities (two in Miyazaki-ken, one in Kumamoto-ken, and one inSizuoka-ken), and the plants from the seeds were observed at Misima. The
following points were concluded from the results.
(1) Genetic variability as shown by character variations: 180 lines belonging to 20 populations were tested in a plant-to-row experiment to takemeasurements of several morphological traits. The amount of genetic variations observed was obviously smaller than those in other sexually reproducing

RESEARCHES CARRIED OUT IN 1974

89

grassy plants. Yet, the differences between localities, between populations
within locality, and between lines within population (between individuals ofa natural population) were in most cases significant. Parent-offspring regressions also indicated the existence of intra-populational genetic variability.
It was pointed out, further, that intra-populational genetic variance wasnegatively correlated to phenotypic plasticity among populations.
(2) Breeding systems and colonizing ability: It was assumed that sexual
reproducing potential can be rougWy estimated from pollen development.
The sexual ability estimated in each population was negatively correlatedwith seed propagating ability, and was positively correlated with intrapopulational variability. This suggests that the genetic variations observedin populations were at least partly due to segregation after sexual reproduction. It was commonly found in three localities that periphery populations
contained a larger amount of genetic variations than those remaining at thecenter of distribution. Releasing genetic variability might have been necessary for the plants to colonize themselves in new habitats.
(3) Responses to environmental stresses: With a part of the collectedmaterials, responses to various environmental stresses were tested. The
plants under a stress condition (walked over in summer, non-weeded) better
survived winter than those under a protected condition. A mix-planting
experiment with "Suzumenohie" (Paspalum thunbergii Kunth; a closely related species native to Japan) showed that dallisgrass was a strong competitor
at a relatively low density of planting. In competition with the finger grass(Digitaria sanguinalis L.), dallisgrass showed a high mortality and phenotypic
plasticity as compared with barnyard grass (Echinochloa crusgalli Beauv.).
It was suggested from these observations that the apomictic dallisgrasshas a different adaptive strategy from that of annual weeds. Dallisgrassseems to have a "general-purpose genotype" consolidated by virtue ofheterozygosity. Its weediness would depend upon colonization by its apomictic
seeds, tolerance to various environmental stresses, and pronounced phenotypic plasticity. The adaptive genotype is multiplied by apomixis withoutchange.

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

Developmental Instability and Phenotypic Plasticity
in Arabidopsis thaliana
Sujit BAGCm and Shin-ya IYAMAIn order to inquire into the relationship between developmental instability
and phenotypic plasticity, high and low instability lines of Arabidopsis thali.
ana, derived from the progeny of gamma-ray irradiated seed were grownunder different environmental conditions. Twelve lines each of high andlow instability groups for days to first flower were tested under five different
conditions (combinations of nutritional levels and temperatures). Also,
10 high and 11 low instability lines for plant height were tested under sevenconditions (Table 1). Developmental instability was measured by withinline standard deviation. The sensitivity to environment or phenotypic
plasticity of a line was expressed by the coefficient of regression of its performance on environmental values which were given by the average of alllines grown under the environments.
The response of each line to environments varied among the lines, butshowed no significant difference between high and low instability groups(b= 1.02 and 0.98 for plant height and b=0.997 and 1.003 for days to flower).
This indicates that developmental instability is a different property fromphenotypic plasticity. The expression of developmental instability differedaccording to environments. As to plant height, developmental instability
was least under a varying temperature condition, and showed higher values
under constant temperature conditions (Table 1). The value obtained from
Table l. Average within-line standard deviations of high and low
instability line groups under different environments.
Environments
Nitrogen
El
E2
E3
E4
Es
E6
E7

Days to first flower

Plant height

Temp.

High

Low

Mean

High

Low

Mean

Oppm
25°C
40
25
30
25
20
30
30
30
30
20/30
30
(10/30)*

2.94
2.14
2.16
3.02
2.07

2.30
1.81
1.98
2.43
1.61

2.62
1.98
2.07
2.73
1.83

1.91
2.33
2.44
6.21
4.22
1. 56
3.45

1.60
1.90
1.74
4.67
3.49
1.43
3.53

1.75
2.11
2.09
5.44
3.86
1.49
3.49

2.47

2.02

2.25

3.16

2.62

2.89

Average* Approximate range in uncontrolled glasshouse.

RESEARCHES CARRIED OUT IN 1974

91

a 10-30°C treatment (in a glasshouse), however, was high perhaps because of
too wide temperature range. It seems that developmental instability is a
genetic character and expresses itself under constant temperature andunfavorable condition.
Varietal Difference in Cadmium Uptake Ability in Fodder
Turnip, Brassica rapa
Shin-ya IYAMA

To investigate genotypic differences in cadmium uptake ability, ten varieties
of fodder turnip were tested. Five plants per variety were grown individually in a pot containing 20 ppm Cd in 4.5 kg field soil together with untreatedcontrol. At maturity, they were harvested and their leaves and roots weredried separately. The samples were ashed by a low temperature dry asher
and then amount of extracted cadmium was measured by atomic absorption
spectrophotometry. The plants from Cd-containing soils apparently had ahigher cadmium content in both the leaves and roots than the control plants.
Average cadmium contents in the control were 0.62 ppm in leaf and 0.21 ppm
in root, whereas the treated plants had a mean of28.51 ppm with a range from
20.13 to 40.73 ppm in the leaves and a mean of 9.17 ppm with a range from
6.41 to 14.62 ppm in the roots. Analysis of variance of the data showedthat there were significant differences among varieties in cadmium contents
in both leaf and root, suggesting that the cadmium uptake ability is a genetic
trait. No correlation was found between cadmium content and total dry
weight.
The Effect of Intensive Inbreeding on the Weightsof Various Organs in Japanese Quails
Takatada KAWAHARA

When the Japanese quails are propagated by successive full-sib mating,complete loss of reproductive ability is observed in between the third andfifth generation (F=50-67 %). Body weight also decreases with inbreeding.
This report deals with the difference in the weight of various organs foundbetween inbred and non-inbred strains. Data were collected from domestic
Japanese quails at three levels of inbreeding, namely, non-inbred control(F=O %), and second (F=38 %) and third (F=50 %) generations of full-sib

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

mating. Female and male birds at the age of25 weeks were observed. Their
numbers were 35, 55 and 37 for the three groups, respectively. The weightsof 16 different organs were measured seperately after dissection. The effectof inbreeding was shown by linear regression of the weight of an organ on
inbreeding coefficient (F).
The mean reduction of organ weight due to 10 %increase ofF was obtained
as follows: -4.80 g in total body weight (mean weight for female and male:
123.65 g), -0.10 g in bone (4.00 g), -2.64 g in muscle (73.78 g), -0.02 g inheart (1.07 g), -0.04 g in lung (1.16 g), -0.13 g in liver (3.93 g), -0.07 g
in intestine (2.51 g), -2.56mg in spleen (42.96mg), -0.03 g in kidney (1.17 g)
and -0,01 g in brain (0.67 g). The weight of sexual organs also decreased
with inbreeding at the rate of -0.13 g in testis (mean wt.: 2.47 g), -0.95 g inovary (4.83 g), and -0.21 g in oviduct (5.65 g) per 10 % increase of F. All
these regressions were significant at 5 % or higher level. The regression
obtained in gizzard (-0,01 g), eye balls (-0.001 g) and pancreas (+0.001 g)
were insignificant.
The result of this experiment indicates that the effect of inbreeding differs
according to organs; it was strongest on spleen and testis.
Properties Concerning Egg Production of Japanese Quails
Takatada KAWAHARA and Shin-ya IYAMA

Variation pattern in the traits concerning egg production of Japanesequails was investigated. Phenotypic and genetic correlations among various
traits were estimated in a random bred population kept in this Institute. The
characters measured at the age of 23 weeks were: egg weight (Xl)' albumen
weight (X2), yolk weight (Xs), shell weight (X4), shell thickness (X5), egg
length (X 6), egg width (X 7) and body weight (Xs). Those at sexual maurity
were: body weight (X g), egg weight (X lO) and age in days (Xu). Hen-day
egg production rate (X12) was recorded up to 120 days after the first egg. The
birds were killed at the age of 25 weeks in order to measure the weights ofoviduct (X13) and ovaries (X14). Data were collected from 305 female birds
originated from 68 single pair matings between male and female randomly
chosen from the population.
Principal component analysis was applied to the correlations obtained.
On the whole, extracted components based on the phenotypic and genetic
correlations were in good accordance with each other. First four com-

93

RESEARCHES CARRIED OUT IN 1974

ponents explained 70 to 80 % of total variations found in the population asshown in Table 1. The first component was apparently a size factor. The
second one would reflect egg production, poor egg production (X12) being
associated with poor development of ovaries (X14) and late sexual maturity(X9 , X IO , Xu), and vice versa. In the third component, large number of
eggs produced was associated with large body (XS, X 9) and small eggs (Xl,
X2' X s, X o, X 7), suggesting that the component would be related to anotherpattern of egg productivity. The fourth one was assumed to reflect mainly
egg shell property.
Table 1. First four principal components extracted from the phenotypicand genetic correlations among various traits of Japanese quails.
Principal components based on
I

Xl
X2
Xs
X4
X5
X6
X7

Xs
Xs
XlO

Xu
X12
XIS
X14

Genetic correlations

Phenotypic correlations

CharacterII.411 -.039.380 -.024.334 -.030.300 -.094.044 -.100.325 -.034.359 -.029.269 -.054
.196
.351
.198
.513
-.005
.638
-.008 -.320.242 -.042.200 -.277

PVAC* 37.74

11.21

III

IV

III

IV

I

-.168
-.131
-.258
.096
.314
-.055
-.249
.377
.459
.073
.091
.550
.167
.140

.032
.013
-.059
.437
.735
.063
.042
-.238
-.264
.063
.108
-.010
-.127
-.316

.409
.360
.289
.290
.063
.312
.327
.226
.211
.239
-.066
-.008
.362
.199

.040 -.148.073 -.136-.023 -.065-.124 -.122
-.026
.076
-.036 -.011.169 -.279
.011
.551
.141
.576
.051
.531
.110
.585
-.172
.431
-.185 -.058
-.489
.138

.016
.111
-.340
.273
.632
.145
.065
-.114
-.109
.069
-.183
.425
-.116
-.341

9.88

9.22

42.70

14.23

11.89

II

12.78

* Percent of variance accounted for by the component.

Bilateral Asymmetry in the Transverse Processes ofCervical Vertebrae in Chicken
Takatada KAWAHARA

In order to elucidate the occurrence of developmental instability in chicken,
observations of transverse processes have been continued since 1963 (Ann.
Rept. 15 & 19). Based on the data so far obtained, a concluding review on

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

the problem is presented in this report. The asymmetry was classified into
two categories, directional and fluctuating. Directional asymmetry Was
measured in each strain by the mean left-right difference in transverse
process length. Fluctuating asymmetry was measured in each bird as theresidual value given by (left-right difference)-(mean left-right difference).
A total of 507 female birds of White Leghorn at the age of 16 months Wereobserved, which belonged to a closed flock strain (F=6 %), four inbredlines (F=57-64%), and five strains of F 1 hybrid between the inbreds.
In directional asymmetry, significant differences were found between thefour vertebrae, but were not found between strains. The left transverse
processes were usually longer than the right, the mean difference being 0.126
mm. In fluctuating asymmetry, the 10 strains significantly differed. The
F 1 hybrids were less asymmetric than their inbred parents. This will he
expected if the fluctuating asymmetry results from developmental instability
and is mitigated by heterozygosity.
The values showing directional and fluctuating asymmetry were positively
inter-correlated among strains (r=0.696), though their correlations with thelength of transverse processes itself were insignificant. The asymmetry
values showed no significant correlations with body weight, with which thelength of transverse processes was correlated (r=0.789). (Published in Jap.
J. Genet. 49: 1-9, 1974).

RESEARCHES CARRIED OUT IN 1974

XIV.

95

MISCELLANEOUS

A Modified Analysis of Two-Treatment Switchback
Design with Different Numbers of Individuals in
Replications
Tahru FUJISHIMA

An attempt was made to propose an analytical procedure for the twotreatment switch-back design with replications consisting of unequal numbersof individuals.
Let Yijkmn be the performance of the k th individual of the mth treatment in
the nth experimental period of the ph treatment sequence group in the jthreplication. Then the statistical model for Yijkmn may be expressed asfollows:
Yijlcmn=,u+Bi+Sj+(BS)ij+Cijk+Tm+Pn+(BT)im+(BP)in +(ST)jm+ (SP)jn +(TP)mn +eijkmm
where ,u is general mean, B i is jth replication effect, Sj isphtreatment sequencegroup effect, Cijk is ijkth individual effect, T m is mth treatment effect, P" isnth experimental period effect, and eijkmn is random error, and (BS)ij, (BT)im,(BP)in, (ST)jm, (SP)jn and (TP)mn are the corresponding interaction effects
to the respective elements. All elements are fixed constants, except Cijk andeijkmn which are random variables.
Now we compute for each individual a quantity(m=1= m'),and calculated the following sums of squares:

L.d~jk( L . d 'k)2

TSS=(~ ~ f d~jk)/6, TCT= ~ if L1~' SSt= ~ if kni;3~(L. 1;:i.jk )2/2 '6, CT=(l; L. 1;:i. k \)2/2 .6 .b,
j

SSb=

~

:J

1,3

'It

di jk
and tSS= r; .4 ( 1;n . )2/6
'It:1

1,3

J

1,)

/6·b,

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

where nij denotes the number of individuals of the ph treatment sequencegroup in the i th replication and b the number of replicaions.
Thus, we obtain the analysis of variance table given in Table 1. This
procedure was found to be useful after a trial with the data for milking at
unequal intervals in dairy cows.
Table 1. Analysis of variance for two-treatment switch-back design
with unequal cell sizes.
d. f.

Source of variation

Sum of squares
SSt-CT

Treatments

b-1
b-1

BlocksTreat. X blocks

SSb-CTtSS-SSt-SSb+CT
TSS-TCT

N .. -2b
N .. -2b

Error

Expected mean square
6;;2+b(TP)2/6+3b(SP)2/2+8bP/3
6;;2 + 3(BP)2

6;;2 + 8(BT)2/3
68 2
6;;2

6;;2=(1/2b) L.L.(1/mj)6e2ijT2= L. T m2, (SP)2= L. (SP)j2 2, (TP)2= L. (TP)m2 2,
m

m

3"

(BP)2= L. (BP)i2 2/(b-1), (BT)2= L. L. (BT)im 2/(b -1).
ii mPollen Characters of Chrysanthemum Species
Shuzo NAGAMI1 1

The pollen characters, especially the diameter and spine number, of 16
Chrysanthemum species were studied, by using a scanning electron microscope(Table 1). The conclusions are as follows: (1) All pollen grains of these
species were spherical (mean value of diameter, 27.7 fl), and had small spines
on the surface (mean value of spine number on the outline of microscopicalfigure, 16). (2) Gradual increase of the diameter was observed in proportion
to the increase of Chrysanthemum polyploidy, but no increase was found inspine number (NAGAMI, 1972). (3) In the pollen characters, relationshipsof L> D > N were found, but no remarkable relationships between the characters and distributional conditions were observed.
11
Professor, Department of Biology, Faculty of Education, Yakohama National
University.

RESEARCHES CARRIED OUT IN 1974

97

Table 1. Species name (H: Hagi strain, Y: Yuhara str., T: Tsubakidomaristr., A: Awasima str., M: Manazuru str., K: Kamakura str.), subsection name (SN. D: Dendranthema, L: Leucanthemum, N: Nipponicae),
distribution (d. CJ: Central Japan, NJ: North Japan, SJ: South Japan,
c: coast species, f: field sp.), chromosome number (cn. X=9), and pollen
characters (pd: pollen diameter, sn: spine number) of chrysanthemums.
* exceptional data.
species name

SN

d

cn

pd

sn

1. C. Makinoi
2. C. boreale
3. C. nipponicum

D
D
N

CJ-f
CJ-f
NJ--c

2X
2X
2X

22.4/1
21.5
22.8

20
16
14

4.

C. indicum

D

CJ-f

4X

28.0*

15

5.
6.

D
D

D

CJ--c
CJ--c
CJ--c

6X
6X
6X

24.6
28.3
29.4

13
13

7.

C. indicum var. hexaploid (H)C. indicum var. hexaploid (Y)C. japonense

8.
9.
10.
11.
12.

(T)
C. Shiwogiku(A)
C. Shiwogiku
C. Shiwogiku var. kinokuniense
C. Weyrichii
C. japonense var. octoploid

D
D
D
L
D

CJ--c
CJ--c
CJ--c
NJ--c
CJ--c

8X
8X
8X
8X
8X

26.2
26.4
26.6
28.5
29.0

21*

13.

C. japonense var. crassum

D
L
D
D

SJ--c
NJ-c
CJ--c
CJ--c

lOX
lOX
lOX
lOX

36.0
29.6
31.5
32.1

14

14. C. arcticum ssp. Maekawanum(M)
15. C. pacificum(K)
16. C. pacificum

16
17

14
17

19
17

15
13

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

BOOKS AND PAPERS PUBLISHED IN 1974
BY STAFF MEMBERS
(1)
(2)

(3)
(4)
(5)

( 6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)

(15)
(16)
(17)

ANDERSON, W. W. and T. K. WATANABE 1974: Selection by fertility in Drosophila
pseudoobscura. Genetics 77: 559-564.
BRYANT, R. E., T. FUJISAWA and P. S. SYPHERD 1974: Ribosomal proteins andribonucleic acids of ribosome maturation mutants of Escherichia coli. Biochemistry
13: 2110-2114.
CHOO, J. K. and C. OSHIMA 1974: Phototactic selection and its effects on somequantitative characters of Drosophila virilis. Environ. Control in BioI. 12: 41-51.
ENOMOTO, M. and B. A. D. STOCKER 1974: Transduction by phage pike in Salmonella typhimurium. Virology 60: 503-514.
FURUICHI, Y. 1974: "Methylation-coupled" transcription by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA.
Nucleic Acid Res. 1: 809-822.
*HIROTA, Y. 1973: Analysis of cell division and DNA replication in Escherichia
coli by a genetic method. Folia Microbiol. 18: 269.
HIROTA, Y. 1974: On the analysis of bacterial division I. (In Japanese) Kagaku
44: 536--43.
HIROTA, Y. 1974: On the analysis of bacterial division II. (In Japanese) Kagaku
44: 694-700.
IINUMA, K., A. TANAE and G. TANAKA 1974: An XYY baby with Prader syndrome.
Clinical Genetics, 6: 323-325.
IKENAGA, M., S. KONDO, and T. FUJII 1974: Action spectrum for enzymatic photoreactivation in maize. Photochem. and Photobiol. 19: 109-113.
IMAI, H. T. 1974: B-chromosomes in the myrmicine ant, Leptothorax spinosior.
Chromosoma (Berl.) 45: 431-444.
JAMES, J. S. and T. YAMAZAKI 1974: Genetic background and the fitness of allozyme. Genetics 78: 1185-1189.
KAnA, T. 1974: Mechanisms of radiosensitization with iodine compounds. Excerpta Medica, Intern. Congr. Ser. 338: 569-573.
KADA, T., M. MORIYA and Y. SmRAsu 1974: Screening of pesticides for DNA
interactions by "rec-assay" and mutagenesis testing and frameshiftmutagens detected.
Mutation Res. 26: 243-248.
KATO, H. 1974: Differential chromosomal sensitivities of aneusomic cell clones to
UV light. Exp. Cell Res. 83: 55-62.
KATO, H. 1974: Induction of sister chromatid exchanges by chemical mutagens andits possible relevance to DNA repair. Exp. Cell Res. 85: 239-247.
KATO, H. 1974: Photoreactivation of sister chromatid exchanges induced byultraviolet irradiation. Nature 249: 552-553.

* Contributions from other institutes

BOOK AND PAPERS PUBLISHED IN 1974

99(18) KATO, H. 1974: Spontaneous sister chromatid exchanges detected by a BUdRlabelling method. Nature 251: 70-72.
(19) KATo, H. 1974: Is isolabeling a false image? Exp. Cell Res. 89: 416-420.
(20) KATO, H. 1974: A possible role of DNA synthesis in formation of sister chromatid
exchanges. Nature 252: 739-741.
(21) KATO, H, K. TSUCIDYA and T. H. YOSIDA 1974: Constitutive heterochromatin ofIndian muntijac chromosomes revealed by DNase treatment and a C-banding
technique. Canadian J. Cytol. Genet. 16: 273-280.
(22) KAWAHARA, T. 1974: Interstrain variation in gamma-ray induction of hindlimb
deformities in chick embryos. Radiation Res. 57: 332-341.
(23) KAWAHARA, T. 1974: Bilateral asymmetry in the transverse processes of the cervicalvertebrae in the chicken. Jap. J. Genet. 49: 1-9.
(24) KIMURA, M. 1974: Gene pool of higher organisms as a product of evolution.
Cold Spring Harbor Symposia on Quantitative Biology 36: 515-524.
(25) KiMURA, M. 1974: Some models of allelic mutation in molecular population
genetics. Lectures on Mathematics in the Life Sciences (American Mathematics
Society) 7: 1-23.
(26) KIMURA, M. (ed) 1974: Genetics and Future of Man. (In Japanese) Baifukan,
Tokyo, 219+ix.
(27) KIMURA, M. and T. OHTA 1974: On some principles governing molecular evolution.
Proc. Nat. Acad. Sci. U. S. A. 71: 2848-2852.
(28) KiMuRA, M. and T. OHTA 1974: Probability of gene fixation in an expanding finite
population. Proc. Nat. Acad. Sci. U. S. A. 71: 3377-3379.
(29) *KOIKE, K., M. KOBAYASID and T. FUJISAWA 1974: Novel properties of two classesof nascent mitochondrial DNA formed in vitro. Biochim. Biophys. Acta 361: 144147.
(30) KURODA, Y. 1974: Spermatogenesis in pharate adult testes of Drosophila in tissue
cultures without ecdysones. J. Insect Physiol. 20: 637-640.
(31) KURODA, Y. 1974: Ovarian cells from pharate adults of Drosophila in tissue culture.
Zool. Mag. 83: 203-206.
(32) KURODA, Y. 1974: Studies on Drosophila embryonic cells in vitro.!. Characteristics
of cell types in culture. Develop. Growth Different. 16: 55-66.
(33) KURODA, Y. 1974: In vitro activity of cells from genetically lethal embryos of
Drosophila. Nature. 252: 40-41.
(34) KURODA, Y. 1974: Mutagenesis in cultured human diploid cells.!. Effects ofsome mutagens and a selective agent on cell survival. Jap. J. Genet. 49: 379-388.
(35) KURODA, Y. 1974: Mutagenesis in cultured human diploid cells. II. Chemical
induction of 8-azaguanine-resistant mutations. Jap. J. Genet. 49: 389-398.
(36) KURODA, Y. 1974: Studies on a procedure for detecting somatic cell mutations in
cultured human diploid cells. Mutation Res. 26: 435-436.
(37) KURODA, Y. 1974: Inhibition by cyclic AMP and dibutyryl cyclic AMP on aggregation of embryonic quail liver cells in culture. Exp. Cell Res. 84: 303-310.
(38) KURODA, Y. 1974: Inhibitory effects of dextran sulfates on aggregation of embryonic quail liver cells in culture. Exp. Cell Res. 84: 351-356.

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25(39) KURODA, Y. 1974: Effects of hexosamines and their acetyl derivatives on aggre_
gation of rat hepatoma cells in rotation culture. Cancer Res. 34: 403-409.
(40) KURODA, Y. 1974: Differential inhibition by fucoses of aggregation of rat hepatoma
cells in rotation-mediated cell cultures. J. Nat. Cancer Inst. 52: 161,-166.
(41) KURODA, Y. 1974: Methods of Animal Tissue Culture. (In Japanese). (Modern
Biology Series 23), 406 pp, Kyoritsu Shuppan Sha (Tokyo).
(42) KURODA, Y. 1974: Embryological aspects-differentiation and tissue reconstruction.
(In Japanese). "Cell Society" (ed. by S. Sugino); 36-50, Kodansha, Scientific(Tokyo).
(43) KURODA, Y. 1974: Mutual recognition among animal cells in culture. (In Japanese). Heredity (Tokyo) 28 (10): 16-22.
(44) KURODA, Y. 1974: Differentiation onnsect cells in tissue culture. (In Japanese).
Shokubutsu B6eki (Tokyo) 28: 313-317.
(45) MARUYAMA, T. 1974: The age of an allele in a finite population. Genet. Res.
Camb. 23: 137-143.
(46) MARUYAMA, T. 1974: A Markov process of gene frequency change in a geographically structured population. Genetics 76: 367-377.
(47) MARUYAMA, T. 1974: A simple proof that certain quantities are independent of thegeographical structure of population. Theor. Pop. BioI. 5: 148-154.
(48) MARUYAMA, T. 1974: The age of a rare mutant gene in a large population. Amer.
J. Human Genetics 26: 669-673.
(49) MARUYAMA, T. 1974: Identity of genes in geographically separated individuals andthe genetic variability maintained in a population. In "Genealogical Mathematics"(ed. by P. A. Ballonoff), 291-311, Maison des Sciences de l'Homme (Paris).
(50) MARUYAMA, T. and M. KIMURA 1974: Geographical uniformity of selectively
neutral polymorphisms. Nature 249: 30-32.
(51) MARUYAMA, T. and M. KIMURA 1974: A note on the speed of gene frequencychanges in reverse directions in a finite population. Evolution 28: 161-163.
(52) MARUYAMA, T. and T. YAMAZAKI 1974: Analysis of heterozygosity in regard to theneutral theory of protein polymorphism. J. Mol. Evol. 4: 195-199.
(53) MATSUNAGA, E. 1973: Investigation of disputed paternity: theoretical and practicalaspects. (In Japanese) Jap. J. Legal Med. 27: 419-431.
(54) MATSUNAGA, E. 1974: Concept and role of eugenics: implications for family planning in Asia. Social Action 24: 33-48.
(55) MATSUNAGA, E. 1974: Are congenital malformations rapidly increasing in Japan?
(In Japanese) Kosei-no-Shihyo 21 (11): 11-21.
(56) MATSUNAGA, E. 1974: Genetics of diabetes mellitus. (In Japanese) Jap. J. Public
Health 21 (10) Suppl.: 98-99.
(57) MATSUNAGA, E. 1974: Family planning and its effect on the gene pool. Proc. XIV.
Intern. Congress of Pediat. (Buenos Aires) 10: 193-204.
(58) MIURA, K, K WATANABE and M. SUGIURA 1974: 5'-terminal nucleotide sequences of the double-stranded RNA of silkworm cytoplasmic polyhedrosis virus.
J. Mol. BioI. 86: 31-48.

BOOK AND PAPERS PUBLISHED IN 1974

101

MIURA, K., K. WATANABE, M. SUGIURA and A. J. SHATKIN 1974: The 5'-terminal
nucleotide sequences of the double-stranded RNA of human reovirus. Proc. Nat.
Acad. Sci. U. S. A. 71: 3979-3983.
(60) MORISIDMA, H. 1974: Differentiation of populations and phenotypic plasticity.
(In Japanese) "Gendai no Idengaku (Modern Genetics)" (ed. by C. Oshima): 145-174
Asakura-Shoten (Tokyo).
(61) MORISIDMA, H. 1974: Adaptation and evolution. (In Japanese) "Handbook of
Breeding" (ed. by T. Matsuo): 113-122, Yokendo (Tokyo).
(62) MORISIDMA, H. and H. I. OKA 1974: Analysis of genetic variation in plant type of
rice, VI. Intervarietal variation in growth pattern obtained from the International
Rice Adaptation Experiment. Jap. J. Breed. 24: 226-236.
(63) MORrwAKI, D. and M. TSUJITA 1974: Synaptonemal complex and male crossingover in Drosophila ananassae. Cytologia 39: 829-838.
(64) MORrwAKI, K., T. SADAIE and S. HIRASAWA 1974: Improved method for separationand identification of serum transferrins: Thin layer acrylamide-gel electrophoresis
with acrinol pretreatment. Experientia 30: 119-120.
(65) MUKAI, T., T. K. WATANABE and O. YAMAGUCID 1974: The genetic structure ofnatural populations of Drosophila me!anogaster. XII. Linkage disequilibrium in alarge population. Genetics 77: 771-793.
(66) MURAKAMI, A. 1974: The mutagenic activity of quinacrine hydrochloride in thesilkworm. Mutation Res. 22: 295-297.
(67) MURAKAMI, A. and H. T. WAI 1974: Cytological evidence for holocentric chromosomes of the silkworm, Bombyx mori and B. mandarina, (Bombycidae, Lepidoptera).
Chromosoma 47: 167-178.
(68) NAKAGOME, Y. 1974: Participation of D-group chromosomes in satellite associations. Humangenet. 25: 235-236.
(69) NISIDMURA, A., Y. NrSIDMURA and C. LUCIEN 1973: Isolation of Hfr strains fromR + and Co1V2+ strains of Escherichia coli and derivation of an R'!ac factor bytransduction. J. Bacteriol. 116: 1107-1112.
(70) OHTA, T. 1974: Mutational pressure as the main cause of molecular evolution andpolymorphism. Nature 252: 351-354.
(71) OHTA, T. and M. KIMURA 1974: Simulation studies on electrophoretically detectable
genetic variability in a finite population. Genetics 76: 615-624.
(72) OHTA, T. and C. C. COCKERHAM 1974: Detrimental genes with partial selfing andeffects on a neutral locus. Genet. Res. 23: 191-200.
(73) OKA, H.1. 1974: Analysis of genes controlling Fl sterility in rice by the use of isogenic lines. Genetics 77: 521-534.
(74) OKA, H. 1. 1974: A short history of the first hybrid semidwarf rice variety, Taichungnative 1. SABRAO J. 6: 241-243.
(75) OKA, H. 1. 1974: Experimental studies on the origin of cultivated rice. Genetics
78: 475-486.
(76) OKA, H.1. and F. F. CAMPOS 1974: Breeding behavior and genetic variations ofsunflowers observed in Central LUZON. SABRAO J. 6: 61-67.
(59)

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25(77) OSHIMA, C. and J. K. CHOO 1974: Studies on effects of urbanization on the habitatof Drosophila populations and of noise, illumination environments on development
copulation and oviposition of flies. (In Japanese) Environmental Research in Japan:
1973, 5-1~5-8., Environment Agency, Japan.
(78) OSHIMA, C. 1974: Chapter 3-5. "Gendai no Idengaku (Modern Genetics)" (ed.
by C. Oshima) Vol. 5: 59-108, Asakura-Shoten (Tokyo). (In Japanese)(79) OSHIMA, C. 1974: Phototaxis and biorhythm (Drosophila), (In Japanese) "Be
haviour and adaptation in Insects" (ed. by C. Oshima): 45-68, Baihu-kan (Tokyo)(80) SAKAI, K., S. HAYASHI and S. IYAMA 1974: Genetic studies in natural populationsof Pinus. I. Genetic variability in local populations from several prefectures.
Mem. Fac. of Agr., Kagoshima Univ. 10 (19): 37-49.
(81) SHAMA RAo, H. K. and T. KAnA 1974: Differential sensitivity of induced dwarf
rice mutants to gibberellin, fast neutron and gamma radiations. Radiation Bot. 14:
153-157.
(82) SHAMA RAo, H. K., T. FUJII and T. KADA 1974: Copper requirement for callus
induction in rice. Plant Sci. Letters 2: 177-183.
(83) SHIMOTOHNO, K. and K. MIURA 1974: 5'-terminal structure of messenger RNA
transcribed by the RNA polymerase of silkworm cytoplasmic polyhedrosis virus
containing double-stranded RNA. J. Mol. BioI. 86: 21-30.
(84) SHINODA, T. and Y. TSUZUKIDA 1974: Identification of rapidly trinitrophenylating
amino groups of human Bence-Jones proteins. J. Biochem. 75: 23-44.
(85) SHINODA, T., E. MATSUNAGA and J. KOSHINAGA 1974: Polymorphism at a second
structural locus for tetrazolium oxidase. Jap. J. Human Genet. 19 (3): 243-250.
(86) SHINODA, T. 1974: TNBS technique in immunology. (In Japanese) "Methods in
Immunology", Vol. 4: 871-876 (ed. Jap. Soc ImmunoI., Kanazawa).
(87) SHINODA, T. 1974: Peptide fractionation. (In Japanese) "Methods in Immunology", Vol. 4: 877-890 (ed. Jap. Soc. Immunol., Kanazawa).
(88) SHINODA, T. 1974: N-terminal determination by dansyl technique. (In Japanese)"Methods in Immunology", Vol 4: 891-898 (ed. Jap. Soc. Immunol., Kanazawa).
(89) SUZUKI, H. 1974: Regulation of flageIIin biosynthesis. Proc. 1st. Intersec. Congr.
Internat. Assoc. Microbiol. Soc.
(90) TAZIMA, Y. 1974: Naturally occurring mutagens of biological origin-A review.
Mut. Res. 26: 225-235.
(91) TAZIMA, Y. 1974: Some aspects of overdominance viewed from experiments onradiosensitivity of the silkworm. "Breeding Researches in Asia and Oceania,"
Ind. J. Genet. 34 A: 211-219.
(92) TAZIMA, Y. 1974: Some problems in the methodology of chemical mutagenesis
in the silkworm. "Breeding Researches in Asia and Oceania," Ind. J. Genet. 34A:
302-310.
(93) TAZlMA, Y. 1974: Attempts to induce non-disjunction by means of irradiation andchemical treatment in the silkworm. Rad. Res. 59 (1): 267.
(94) TAZIMA, Y. and K. ONIMARU 1974: Results of mutagenicity testing for some nitrofuran derivatives in a sensitive test system with silkworm oocytes. Mutation Res.
26: 440.

BOOK AND PAPERS PUBLISHED IN 1972103(95) TSAI, K. H., Y. C. Lu and H. I. OKA 1974: Mutation breeding of soybean for the
resistance to rust disease. SABRAO J. 6: 181-191.
(96) TuTIKAWA, K. and T. KADA 1974: Quantitative evaluation of the genetic toxicity
on Bacillus subtilis cells injected into the peritoneum of mice orally administrated
with furylfuramide, a food additive. Proc. 1974 Technicon Intern. Symp., 280-283(97) *VIRASORO, S., J. MORDOH and Y. HIROTA 1974: Further studies on DNA thermosensitive mutants of Escherichia coli using the toluenized cell system. Biochimie 56:
363-371.
(98) YAMAZAKI, T. 1974: Organization of linked genes under frequency-dependent
selection of minority advantage. Jap. J. Genet. 49: 33-36.
(99) YAMAZAKI, T. and T. MARUYAMA 1974: Analysis of neutrality in protein polymorphism. Science 183: 448.
(100) YAMAZAKI, T. and T. MARUYAMA 1974: Evidence that enzyme polymorphisms are
selectively neutral, but blood group polymorphisms are not. Science 183: 1091-1092.
(101) YAMAZAKI, T. and T. MARUYAMA 1974: Lack of evidence for the neutral hypothesisof protein polymorphism: A reply. J. Hered. 65: 376.
(102) YOSIDA, T. H., H. KATO, K. TSUCHIYA, T. SAGAI and K. MORIWAKI 1974: Cytogenetical survey of black rats, Rattus rattus, in Southwest and Central Asia, withspecial regard to the evolutional relationship between three geographical types.
Chromosoma (Berl.) 45: 99-109.
(103) YOSIDA, T. H., K. MORIWAKI and T. SAGAI 1974: A female black rat (Rattus rattus)
with a single X-chromosome. Jap. J. Genet. 49: 49-52.
(104) YOSIDA, T. H., K. MORIWAKI and T. SAGAI 1974: Oceanian type black rats (Rattus
rattus) with a subtelocentric M 2 chromosome and C-type transferrin obtained from
North America. Experientia 30: 742-744.
(105) YOSIDA, T. H., K. MORIWAKI, H. KATO, K. TSUCHIYA, T. SAGAI and T. SADAIE 1974:
Studies on the karyotype and serum transferrin in the Ceylon black rat, Rattus rattus,
having 40 chromosomes. Cytologia 39: 753-758.
(106) YOSIDA, T. H., 1974: Chromosome alteration in the course of serial transplantationsof experimental tumors and aging of tumor stemline cells. Rec. Result Cancer
Res. 44: 86-93.
(107) YOSIDA, T. H. 1974: The history of the rat inscribed in the chromosomes. (InJapanese). La Kromosoma 96-97: 2965-2973.
(108) YOSIDA, T. H. 1974: Animal chromosome banding. (In Japanese). The Cell(saibO) 6: 354-362.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

ABSTRACTS OF DIARY FOR 1974
December ('73)
March
April
May
June
July
AugustSeptember
October
December

22 1l0th Biological Symposium
22 218th Meeting of Misima Geneticists' Club
23 III th Biological Symposium
10 219th Meeting of Misima Geneticists' Club25 112th Biological Symposium
6 220th Meeting of Misima Geneticists' Club29 22lst Meeting of Misima Geneticists' Club12 222nd Meeting of Misima Geneticists' Club27 113th Biological Symposium
30 223rd Meeting of Misima Geneticists' Club
31 224th Meeting of Misima Geneticists' Club3 114th Biological Symposium
25 115th Biological Symposium
22 116th Biological Symposium
22 117th Biological Symposium
2 225th Meeting of Misima Geneticists' Club13 118th Biological Symposium

FOREIGN VISTORS IN 1974

105

FOREIGN VISITORS IN 1974
February 19-20 NACE, G., University of Michigan, U.S.A.
22-23 GRACE, T.D.C., C.S.I.R.O., Australia
April
18 SUEOKA, N., University of Colorado, U.S.A.
May
23 GROSSMAN, M., University of Illinois, U.S.A.
August30 WOLLMAN, E. L., Institut Pasteur, France
September 24----26 ABRAHAMSON, S., University of Wisconsin, U.S.A.
October 21-23 PETIT, C., Universite de Paris, France
October 22-23 ROWND, R. H., University of Wisconsin, U.S.A.
CURTISS, R. III, University of Alabama, U.S.A.

106

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 25

AUTHOR INDEXAMANO, E. • ••••••••••..••••••••. 53, 54

I.

.45
BAGCffi, S ••••••••••••..•.•••••••.... 90
CHIYO, H
74
COCKERHAM, C. C. • •••.•••••••••.... 59
ENDO, T .••••••••.•••..•••••.•••..•. 20
FUJII, T ••••••••••••••.....•••.••.•.. 56
FUJISAWA, T •.•.•.•••••..••...... 21, 22
FUJISffiMA,T..•••..••.......... 82, 83, 95
FUKASE, Y ...•.•.•....•.....•••.. 38, 39
FURUICHI, Y •...••.........•..... 12,14
HIROTA, Y .••...•..•......•......... 18
HONNA, T .••••.•...•............••• 72
HOSONO, F. • ••••••••.••.....•....•.• 75
IINUMA, K. • •..•...•...••••..••..••• 73
IKETANI, H .••.....•....•.•...•...... 18
IMAI, H. T ••. , •.......••.........•.. 33
INUKAI, H. .
.47
IYAMA, S .•••••••••••...••.... 90, 91, 92
KAnA, T26, 47, 49, 50, 51, 52
KATAOKA, K. • ••.••••••••••••••••••• 75
KATO, H .•••••••••.•.•.•.•...•.•.••• 31
KAWAHARA, T
91, 92, 93
KIMURA, M ••.•.......•..•. 58, 59, 60, 69
KOMINAMI, R ••.•••••.••••.••••.•••.. 33
KOSffiNAGA, J. .
73
KOTOKU, K. • •••••••.••.•••••••••••• 81
KURODA, Y ••...•.•...••... 23, 24, 25, 26
MARUYAMA, T •....••...... 60, 61, 62,66
MATSUNAGA, E.
72, 73
MINATO, K. • •••.••••..••••••••••••. 26
MIURA, K.
12, 14, 15, 16, 17
AIuGA.

MORISHIMA, H. " ••.•••• 84,85,86,87,88
MORIWAKI, K .•.••••••••.•.••..•. 33, 69
MURAKAMI, A.

35, 41, 42, 43, 45, 81

MURAMATSU, M •.••••••••.•.••.••..•• 33
NAGAMI, Soo ••..••.••.••....•...•. ".96

NAKAGOME, Y. " .••.•.•.•..•.•.•• 74, 75
NISHIMURA, Y •..••••......••••.•••.•. 18
NOGUTI, Too •.••..••.•.......•••.•... 52
OHNUMA, A ....•....••••.•......• 35, 37
OHTA, T58, 59, 69
20, 84, 85, 87
ONIMARU, K .•.•.•••.........••... 38, 40
OSHIMA, C .•.......•.......••. 77,78,79
PAl, C20
SADAIE, Y •.•.....•.••.... .47, 48, 49, 51
SAITO, Y .......•............•.••.••. 51
SANO, K .......•...•........••...••. 21
SAWAGUCHI, S ....•.........••....•.. 72
SHIMOTOHNO, K.
14, 15SHINODA, T. . .......••.•••••••••. 73, 76
SUGIURA, M .•••....•..•.......•..... 17
SUGIYAMA, T •.•••••••.•....••••.. 21, 22
SUZUKI, Hoo •••••••.•••••••••••••••.• 18
TAZIMA, Y •••••••••..••.•.• 37, 38, 39,40
TERAMURA, F .•••.......•••.•••.••••. 75
TSUJI, H ........•..••.••.••••••••.•• 31
URUSHIBARA, T •••..•.••••••••••••••. 14
WATANABE, K
16
WATANABE, T. K.
65, 78, 79
YAMAZAKI, T •••••••••••••••••• 62, 65, 66
YOKOIYAMA, A. • ..••.••••••••••••••• 26
YOSIDA, T. H ••••••••••. 21, 28, 29, 30, 31
OKA, H. I.

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