NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 191968

Published bll

THE NATIONAL INSTITUTE OF GENETICS
Misima. Sizuoka·ken, Japan1969

Annual Reportof theNational Institute of Genetics
No. 19, 1968

Published by

The National Institute of Genetics, Japan
1969

CONTENTS
General statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Research member..................................................
Council............................................................
Projects of research for 1968.......................................
Researches carried out in 1968 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

Cytogenetics
Scientific expedition for the study of rodents to South East Asiaand Oceania. I. Members, aims and schedule of the scientific expedition. YOSIDA, T. H.
Scientific expedition for the study of rodents to South East Asiaand Oceania.
II. Species and chromosome numbers ofrodents collected from South East Asia and Oceania. YOSIDA,
T. H., TSUCHlYA, K. and IMAI, H. T.........................
Scientific expedition for the study of rodents to South East Asiaand Oceania. III. Chromosomal polymorphism and new
karyotypes of black rat, Rattus rattus, collected in South East
Asia and Oceania. YOSIDA, T. H. and TSUCHIYA, K.........
Scientific expedition for the study of rodents to South East Asiaand Oceania. IV. Comparative idiogram analysis in several
species belonging to genus Rattus collected from South East
Asia and Oceania. YOSIDA, T. H. and TSUCHlYA, K.........
Scientific expedition for the study of rodents to South East Asiaand Oceania. V. Breeding of wild rodents collected fromSouth East Asia and Oceania. TSUCHIYA, K., YOSIDA, T. H...
Rat metaphase chromosomes incorporated into mouse metaphasecells. YOSIDA, T. H. and SEKIGUCHl, T......................
Autoradiographic analysis of the rate of protein synthesis in
diploid and tetraploid MSPC-l myeloma cells. MORIWAKI,
K. and IMAI, H. T..........................................
Chromosome alterations in vitro and in vivo of cultured hamster
cells transformed by chemical carcinogen. SEKIYA, K......
Aggregate-forming activity of rat hepatoma cells induced by
DAB. KURODA, Y..........................................
Pachytene pairing in a translocation heterozygote of maize having a huge knob close to the breakpoint. OHTA, Y.........
Preferential pairing of chromosomes in a tetraploid hybrid between Oryza glaberrima and O. sativa. OKA, H. I............

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

II.

Physiological and developmental genetics
Differentiation of ommatidium-forming cells of Drosophila melanogaster in culture. KURODA, Y. . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of ecdysone analogues on differentiation of eye-antennal
discs of Drosophila melanogaster in culture. KURODA, Y....
The relationship between cell growth cycle and molting cycle
in insect epidermis. MINATO, K. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cytoplasmic male sterility and virus inoculation in Capsicum.
OHTA, Y....................................................
Organ differentiation of isolated Crepis leaves in vitro. YONEDA Y.

III. Biochemical genetics
Genetic effects of DNA in Ephestia. NAWA, S. and YAMADA, M. A.
Further evidences of uptake of DNA by Ephestia eggs. YAMADA,
M. A. and NAWA, S..................... . . . . . . . . . . . . . . . . . . . .
Genetical and biochemical studies of lethal lemon silkworm
larvae. TSUJITA, M. and SAKURAI, S........................
Genetical and biochemical studies of lethal albino silkworm
larvae. TSUJITA, M. and SAKURAI, S........................
Preliminary examination of the cofactor role of sepiapterin forphenylalanine hydroxylase. TSUJITA, M. and SAKURAI, S....
Genitical and biochemical studies of pteridine granules in hypodermal cells of silkworm larvae: Significance of polypeptides inside the granules. SAKURAI, S. and TSUJITA, M. .
Genic analysis for peroxidase isozymes in Oryza sativa and O.
perennis. SHAHI, B. B., ENDo, T. and OKA, H. I.
Isozyme variations due to organ specificity in Oryza perennis.
SHAHI, B. B., ENDo, T. and OKA, H. I.
Partial purification and characterization of genetically differentacid phosphatase of human red cells. SHINODA, T. . . . . . . . . .
Amino acid sequences around cystine residues and COOHterminus of rM-globulin. SHINODA, T.
Peroxidase isozymes in four strains of morning glory. YONEDA Y.
IV. Evolutionary genetics
Intraspecific differentiation of cytoplasm in Aegilops caudata.
KIHARA, H. and OHTA, Y.
Interspecific hybrid between the two species of the genus Taeniatherum of the tribe Triticeae. SAKAMOTO, S. . . . . . . . . . . . . . . .
Scientific expedition for the study of rodents to South East Asiaand Oceania. VI. Serum transferrin polymorphism in Rattus

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CONTENTS

rattus collected in South East Asia and Oceania. MORIWAKI,
K., TSUCHIYA, K. and SAKATA, H.
Differentiation of pathogenic races of Piricularia oryzae into twogroups, "Indica" and" Japonica." MORISHIMA, H...........
Genetic basis of the weakness of Pi plants in Oryza breviligulata
and O. glaberrima. CHU, Y. E. and OKA, H. 1..............
A crossing barrier isolating Oryza perennis subsp. barthii fromits related taxa. CHU, Y. E. and OKA, H. 1.................
Variations in peroxidase, acid phosphatase and esterase isozymesof wild and cultivated Oryza species. SHAHI, B. B., MORISHIMA,
H. and OKA, H. 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V.

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Mathematical and statistical studies on population genetics
The evolutionary rate at the molecular level. KIMURA, M......
The average number of generations until fixation of a mutant
gene in a finite population. KIMURA, M. and OHTA, T. .....
Linkage disequilibrium due to random genetic drift. OHTA, T.
and KIMURA, M.............................................
Rate of decrease of genetic variability in a linearly subdivided
population. MARUYAMA, T..................................
One-dimensional stepping stone model of finite length. MARUYAMA,T.
Distribution of parent-offspring distance in Mishima district.
YASUDA, N.

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VI. Experimental studies on population genetics
Significance of loci of the persisting lethal genes. OSHIMA, C.
and WATANABE, T. K.
Allelic relations between lethal genes distributed in natural
populations. OSHIMA, C. and WATANABE, T. K.
Relationship between the dispersal of flies and the frequency ofallelism of lethals in a natural population. OSHIMA, C. and
WATANABE, T. K............................................
Frequency of sterile chromosomes concealed in a natural population. OSHIMA, C. and WATANABE, T. K.. . . . . . . . . . . . . . . . . . . . .
Persistence of a visible mutant in natural populations of Drosophila melanogaster. WATANABE, T. K.

VII.

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Radiation genetics and chemical mutagenesis in animals
Repair in the mutation process studied in low and high radiosensitivity strains of the silkworm. T AZIMA, Y.............

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

Comparison of mutagenic effects of 14 MeV neutrons, r-rays andsome chemical mutagens upon silkworm spermatogenic cells.
T AZIMA, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Frequency pattern of mosaic and whole-body mutants inducedby ionizing radiations in post-meiotic cells of the malesilkworm. T AZIMA, Y. and ONIMARU, K.....................
Mutagenic action of mitomycin-C and EMS on pre-meiotic germ
cells of the male silkworm. T AZIMA, Y. and ONIMARU, K...
Delayed mutagenic effect of mitomycin-C and EMS observed insilkworm. ONIMARU, K. and T AZIMA, Y.....................
The effect of post-treatment with low temperature on the frequencies of radiation-induced mutation on the silkworm
oocytes. MURAKAMI, A.
Studies on strain differences in radiosensitivity in the silkworm.
VI. Further report on the screening of sensitive and resistant strains to embryonic radiation killing. MURAKAMI, A.
Effects of X-irradiation on aggregate-forming activity andsorting-out property of HeLa cells in rotation culture.
KURODA, Y.
Postspermatogonial rate for 14.1 MeV neutron-induced dominant
mutations affecting the skeleton of mice. TUTIKAWA, K. . . .
Lack of effect of urethane on the induction of dominant lethal
mutations in male mice. TUTIKAWA, K.....................

VIII. Radiation genetics in microorganisms and plants
A mutant of Bacillus subtilis possessing modified specificities toirradiated transforming DNA. SADAIE, Y. and KADA, T....
32P-decay and mutations in Escherichia coli B/r WP2 try-.
HAYASHI, M. and KADA, T.
Mechanisms of radio-sensitization with iodine compounds. KADA,
T. and NAMIKI, M.
High bacteriocidal action of iodine compounds irradiated inacidic solution. KADA, T...................................
Action spectrum of photoreactivating light in UV irradiatedmaize pollen. AMANO, E.
Wave length effect on the photoreactivation of UV-induced
mutations in maize. FUJII, T.
Relative biological effectiveness of 14 MeV neutrons on mutation
frequency in maize. FUJII, T.
Fractionation experiments with gamma-rays and neutrons inmaize. FUJII, Too..........................................

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Determination of the length of flagella in Salmonella. IlNo. T.
Polarity of flagellar growth in Salmonella. IlNo, T.............
A method for concentrating paralyzed mutants from nonflagellated cells of Salmonella. ENOMOTO, M. . . . . . . . . . . . . . . .
Further studies on different frequencies of cotransduction in
Salmonella. ENOMOTO, M. and YAMAGUCHI, S................
Stability and phenotypic expression of mutation to arginine
sensitivity in Salmonella. ISHIDSU, Joo......................
High frequency mutations, drug sensitivity and it-lysogenizationin Escherichia coli K12. KADA, T.

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IX. Microbial genetics

X.

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Human genetics
Delayed fertilization and Down's syndrome. MATSUNAGA, E. and
MARUYAMA, Too.............................................
Maternal age of chromosome translocations with Down's syndrome. KIKUCHI, Y., OISHI, H., TONOMURA, A. and MATSUNAGA,

E.
Clinical conditions of patients with apparently normal chromosomes. V. OISHI, H., KIKUCHI, Y., KUMAGAI, M. and
SHIBATA, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cytogenetical studies of human ova. I. A preliminary note on
the oocytes liberated from the ovary. OISHI, H. and KIKUCHI,Y.
Studies on several genetic hematological traits of the Japanese.
SHINODA, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polymorphism of prealbumin fraction in human serum. OGAWA,
Y.
Allo-albumin F Mishima, a variant of human serum albumin.
OGAWA, Y.

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Applied genetics
Cluster distribution of related individuals in a natural forest of
Thujopsis dolabrata. SAKAI, K. I. and MIYAZAKI, Y. . . . . . . . .
A study on oligogenic and polygenic mutations in the flower
organs of Citrus. SAKAI, K, I., NISHIDA, T. and OHBA, K .. ,Use of zymography for clone identification in forest trees.
MIYAZAKI, Y. and SAKAI, K. I.
When do trees start competing in a forest? HAYASHI, S. and
SAKAI, K. I.
X-ray induced variation in the quantitative characters of rice.
IYAMA, S.

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A comparative analysis of productive traits in wild and domesticated Japanese quails. KAWAHARA, T. and MITA, A.
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Directional and fluctuating asymmetry of ribs of cervical vertebrae in fowls. KAWAHARA, T. and SAKAI, K.1.
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Developmental stability of American and Japanese rice varieties
tested in Kathmandu. SHAHI, B. B. and OKA, H. 1.......... 100
Books and papers published in 1968 by research members. . . . . .. ..
Abstracts of diary for 1968
Foreign visitors in 1968............................................
Acknowledgement.................................................
Author index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

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GENERAL STATEMENT
It is customary that this statement is written by the director. Therefore this will be my last statement for the annual report of the National
Institute of Genetics, as my wish for resignation from the director's
post was accepted by the Council and I shall retire by the end of the
fiscal year (March 1969).
It was in the fall of 1955 when I came to Misima from Kyoto, where
I served 35 years as assistant, assistant professor as well as professor at
Kyoto University. Since then almost 14 years passed away.
Thanks to the collaboration of our staff members and through thegood offices of the government officials, we could complete the newconcrete main building and we succeeded in establishing 10 departments
including molecular genetics, whose budget will be allocated from thenext fiscal year. Thus our goal set at the start had been attained.
Though the performance of our plans often lagged behind the set
standards, we should be at least content with our present scale of workand our endevours to complete the departments. Our thanks are due toforeign organizations, especially the Rockefeller Foundation who provided
us with many grants for genetic studies and funds for building a ricelaboratory and seed storage room. We are also grateful to the National
Institutes of Health for various grants for researches in special fields.
The year 1968 confronted the geneticists of Japan with a busy anddifficult task, namely the organization of the XII International Congressof Genetics. We could welcome some 1,500 colleagues from abroad andthe meetings were held in an atmosphere of true friendship and understanding thanks to our foreign colleagues, who cooperated with us tomake the Congress a success.
In our institute, Dr. M. Kimura, head of the Department of Population
Genetics was given an award from Japan Academy in May 1968. In
November Dr. F. A. Lilienfeld was decorated with the Fourth Order of
Sacred Treasure by His Majesty the Emperor of Japan for her services
of many years.
Dr. Lilienfeld's first stay in Japan (Kyoto) was from 1929-1936 andthe second one from 1950 up to the present. She worked with me firston wheat and is now interested in the study of chimeras induced byalien cytoplasms. She has read and corrected almost all of English
manuscripts written by us, for which I want to thank her heartily onbehalf of the institute.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

RESEARCH MEMBER
Director

KIHARA, Hitoshi, D. Sc., Member of Japan Academy, Emeritus Professorof Kyoto 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.
ONIMARU, Kimiharu
The 2nd Laboratory
KURODA, Yukiaki, D. Sc., Head of the Laboratory
MINATO, Kiyoshi

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
IMAI, Hirotami T., D. Sc.; SEKIYA,* Kunio
The 2nd Laboratory
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
YONEDA, Yoshiaki, D. Sc.; SAKATA,* Harumi

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.
The 2nd Laboratory
KIHARA, Hitoshi, D. Sc., Head of the Laboratory
SAKAMOTO, Sadao, D. Ag.; OHTA,* Yasuo, D. Ag.

4. Department of Biochemical Genetics
TSUJITA, Mitsuo, D. Ag., Head of the Department* Research members under grant from other organizations or visiting researchers.

RESEARCH MEMBER

The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory
YAMADA, Masa-Aki

The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
ENDO, Toru, D. Ag.

The 3rd Laboratory
TSUJITA, Mitsuo, D. Ag., Head of the Laboratory
SAKURAI, Susumu; KOJIMA, * Kunihiro

5. Department of Applied Genetics
SAKAI, Kan-Ichi, D. Ag., Head of the Department

The 1st Laboratory
SAKAI, Kan-Ichi, D. Ag., Head of the Laboratory
KAWAHARA, Takatada, D. Ag.; FUJISHIMA, Tohru, D. Ag.;INOUE,* Teruo

The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
HAYASHI,* Shigesuke; MIYAZAKI,* Yasusada;
MATSUURA,* Takashi; HIGUCHI, * Seiichiro

The 3rd Laboratory
aKA, Hiko-Ichi, D. Ag., Head of the Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag.; CHU,* Yaw-En; SHAHI,* B. B.

6. Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department

The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory

The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Department
FUJII, Taro, D, Ag.; KATSUYA, * Keizo, Ph. D.

The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
AMANO, Etsuo; SADAIE, Yoshito; HAYASHI, Masaru

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; MATSUDA, Ei

The 2nd Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

KIKUCHI, Yasumoto, D. Sc.; OISHI, Hidetsune, D. Sc.;SHIBATA,* Kunihiko; KUMAGAI,* Masaru

8. Department of Microbial Genetics
IINo, Tetsuo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
lINO, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
ENOMOTO, Masatoshi, D. Sc.; YAMAGUCHI,* Shigeru, D. Sc.
The 2nd Laboratory
IINo, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
SUZUKI, Hideho, D. Sc.; ISHIDsu, Jun-ichi
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
MARUYAMA, Takeo, Ph. D.; OHTA,* Tomoko
The 2nd Laboratory
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
YASUDA, Norikazu, Ph. D.
10. Experimental Farm
SAKAI, Kan-Ichi, D. Ag., Head of the Farm
MIYAZAWA, Akira
Honorary Members

KOMAI, Taku, D. Sc., Member of Japan Academy, Emeritus Professor of
Kyoto University
KUWADA, Yoshinari, D. Sc., Member of Japan Academy, Emeritus Professor of Kyoto University
LILIENFELD, Flora A., Ph. D.
OGUMA, Kan, D. Ag., Emeritus Professor of Hokkaido University
TANAKA, Yoshimaro, D. Ag., D. Sc., Member of Japan Academy, Emeritus
Professor of Kyushu University
Department of Administration

OYAUCHI, Toru, Head of the Department
ANDO, Yoshiichi, Chief of the General Affairs Section
KATO, Shigeo, Chief of the Finance Section

COUNCIL

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Association for Propagation of the Knowledge of Genetics

KIHARA, Hitoshi, President, Director of the Institute
TAZIMA, Yataro, Managing Director, Head of the Morphological Genetics
Department
OSHIMA, Chozo, Manager, Head of the Physiological Genetics Department
MATSUNAGA, Ei, Manager, Head of the Human Genetics Department
SINOTO, Yosito, Manager, Professor of International Christian University
WADA, Bungo, Manager, Emeritus Professor of University of Tokyo

COUNCILOKADA, YO, Chairman, Emeritus Professor of University of Tokyo
MORIWAKI, Daigoro, Vice Chairman, Professor of Tokyo Metropolitan
University
BABA, Isamu, Director of National Institute of Agricultural Sciences
FURUHATA, Tanemoto, Director of Scientific Research Institute of Police
KAYA, Seiji, Emeritus Professor of University of Tokyo
KIKKAWA, Hideo, Professor of Osaka University
MAKINO, Sajiro, Professor of Hokkaido University
MATSUO, Takane, Professor of University of TokyoMISONOO, Keisuke, Director of National Institute of Radiological Sciences
OCHI, Yuichi, President of Azabu University of Veterinary Science
OGUMA, Kan, Emeritus Professor of Hokkaido University
SAKATA, Takeo, President of T. Sakata Company
T ACHI, Minoru, Director of Institute of Population Problems
UEMURA, Teijiro, Director of Institute of Applied Microbiology, Universityof Tokyo
WADA, Bungo, Emeritus Professor of University of TokyoWATANABE, Yasusi, President of Shizuoka University

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

PROJECTS OF RESEARCH FOR 1968
Department of Morphological Genetics

Genetics of the silkworm (TAZIMA and ONIMARU)
Repair processes in radiation mutagenesis (TAZIMA and ONIMARU)
Genetic studies of radiosensitivity in the silkworm (MURAKAMI)
Chemical mutagenesis in the silkworm (TAZIMA and ONIMARU)
Genetic studies on insect cells in tissue culture (KURODA and MINATO)
Developmental genetic studies on carcinogenesis in tissue culture (KURODA)
Effects of radiation on cells in tissue culture (KURODA)
Department of Cytogenetics

Cytogenetical and biochemical studies on tumor cells (MoRIWAKI, IMAI,SEKIYA and YOSIDA)
Cytogenetical and biochemical studies of rodents in Southeast Asia andOceania (YOSIDA, MORIWAKI, IMAI and TSUCHIYA)
Studies on chromosomal and biochemical polymorphism of black rats
(Rattus rattus) (YOSIDA, MORIWAKI and TSUCHIYA)
Study on incorporation of isolated chromosomes into cultured mammaliancells (YOSIDA)
Experimental breeding and genetics of mice and rats (YOSIDA, MORIWAKI,
TSUCHIYA, SAKAKIBARA and SONODA)
Morphological and genetical studies on some plant tumors (YONEDA and
CHU)
Cytogenetical and biochemical studies on morning glory (YONEDA)
Department of Physiological Genetics

Population genetics of deleterious genes in natural populations of Drosophila melanogaster (OSHIMA and WATANABE)
Mechanisms of persistence of some lethal genes (OSHIMA and WATANABE)
Physiological and population genetics of sterile genes in natural populations of Drosophila melanogaster (OSHIMA and WATANABE)
Analysis of fitness in a fluctuating environment (OSHIMA and WATANABE)
Analysis of inversion chromosome in natural populations of Drosophliamelanogaster (OSHIMA and WATANABE)
Nucleus substitution in wheat and related species (KIHARA, SAKAMOTO andOHTA)RESEARCHES CARRIED OUT IN 1968

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Basic studies on hybrid wheat breeding (KIHARA)
Cytogenetic studies in the tribe Triticeae (SAKAMOTO)
Collection and preservation of Oryza species (KIHARA)
Studies on cytoplasmic inheritance in higher plants (OHTA)
Department of Biochemical Genetics

Studies on transformation in higher organisms (NAWA, YAMADA and
TSUJITA)
Genetical and biochemical studies of pteridine metabolisms in insects(NAWA and TSUJITA)
Studies on a gene for retarded moulting (rm) in the silkworm (TSUJITA)
Studies on the pteridine granule formation in larval hypodermal cells of
the silkworm (TSUJITA and SAKURAI)
Analysis of gene action on cell differentiation in higher organisms(TSUJITA and NAWA)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Genetical and biochemical studies of human serum proteins (OGAWA)
Genetical and biochemical studies on Japanese middle size dog (OGAWA)
Genetical and biochemical studies of the membrane protein of pteridine
granules in the silkworm (SAKURAI and TSUJITA)
Genetics on isozymes in plants (ENDO)
Enzyme regulation in cultured organ of morning glory (ENDO)
Department of Applied Genetics

Quantitative genetic studies in poultry (SAKAI, KAWAHARA and FUJISHIMA)
Genetic studies in wild populations of Japanese quails (KAWAHARA)
Theoretical studies on breeding techniques (SAKAI and IYAMA)
Studies on competition in plants (SAKAI, hAMA and MIYAZAKI)
Developmental genetics of quantitative characters in plants (SAKAI and
HIGUCHI)
Studies on the effects of X-ray irradiation on quantitative characters ofrice (hAMA)
Genetic studies in natural stands of forest tree species (SAKAI and MIYAZAKI)
Zymographic studies in forest trees (SAKAI and MIYAZAKI)
Simulation studies on artificial selection (hAMA)
Genetic studies of isolating barriers in Oryza (aKA aud CHU)
Numerical taxonomic studies in Oryza perennis (MORISHIMA and aKA)
Experiments on natural selection in wild and cultivated rice forms

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19(MORISHIMA and aKA)
Analysis of sterility genes in Oryza (aKA and MORISHIMA)
Analysis of genetic plant types in rice (MORISHIMA and aKA)
Department of Induced Mutation

Radiation genetics in mice (TUTIKAWA)
RBE and dose rate effects in higher plants (FuJII and AMANO)
Radiation genetics in Arabidopsis (FuJII)
Fine structure analysis in maize (AMANO)
Biological effects of ultraviolet radiation (KADA, FUJII, AMANO and HAYASHI)
Radiation-induced and chemical mutagenesis in microorganisms (KADAand SADAIE)
Department of Human Genetics

Genetic consequences of population trends (MATSUNAGA)
Dermatoglyphics (MATSUNAGA and MATSUDA)
Down's syndrome in Japan (MATSUNAGA, OISHI and KIKUCHI)
Cytogenetics in man (OISHI, KIKUCHI, SHIBATA and KUMAGAI)DNA replication in human chromosomes (KIKUCHI and OISHI)
Biochemical studies on plasma proteins and enzymes (SHINODA)
Chemical modification of ribonucleic acids and their constituents (SHINODA)
Department of Microbial Genetics

Genetic fine structure analysis on microorganisms (lINO, lSHIDSU and
YAMAGUCHI)
Genetics of cellular regulatory mechanisms (SUZUKI and lSHIDSU)
Genetics of bacterial flagella (lINO, ENOMOTO and SUZUKI)
Genetics of motility in bacteria (ENOMOTO)
Genetics of host range in bacteriophages (lINo, ENOMOTO and YAMAGUCHI)
Department of Population Genetics

Theoretical studies of population genetics (KIMURA)
Uses of computers in the theoretical studies of population genetics(KIMURA and MARUYAMA)
Studies on the genetic structure of human populations (YASUDA)RESEARCHES CARRIED OUT IN 1968I.

CYTOGENETICS

Scientific Expedition for the Study of Rodents to South East Asiaand Oceania. I. Members, Aims and Scheduleof the Scientific Expedition
Tosihide H. YOSIDA

Supported by a grant-in-aid for scientific research from the Ministryof Education, we had organized a scientific expedition for the study ofrodents to South East Asia and Oceania from September 20 to November
8, 1968. The members of the expedition were as follows:
Tosihide H. Yosida (Head)
Kazuo Moriwaki
Department of Cytogenetics,
Hirotami, T. ImaiNational Institute of Genetics
Kimiyuki Tsuchiya
Tatsuo Udagawa--Forest Experimental Station,
Ministry of Agriculture, Tokyo
Many species belonging to Muridae inhabit South East Asia andOceania, but informations on these animals are not sufficient. Therefore,
their study was undertaken with the following aims.
a) Research of polymorphism of the black rat, Rattus rattus: Presenceof chromosomal polymorphism and transferrin of blood protein in theblack rat was first detected in the Japanese population by Yosida et at.
(this report, No. 15-18, 1965-1968) and Moriwaki et at. (this report, No.
18, 1968). The main purpose of our expedition was to examine whether
such polymorphism should be found in the rats collected from several
localities of South East Asia and Oceania.
b) Karyotaxonomical studies of rodent species: Karyological studiesof species inhabiting South East Asia are very few. The second aim of
the expedition was to survey the karyotypes of these animals and to
determine the taxonomical relationships in this group.
c) Detection of suitable laboratory animals among the wild ones
collected in South East Asia and Oceania: As to the rodents living inSouth East Asia, they were not yet tested for their usefulness as laboratory animals. In order to find this out, we had sent to Misima living
animals of several species of rodents and we are breeding them in ourlaboratory.

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19d) Research on the damage to cultivated plants and forests by rodents:
The rodents cause tremendous damage to cultivated plants and forests
in several countries of South East Asia. The research was carried out
mainly by Dr. Udagawa, one of our team, to devise appropriate methodsof protection from these pests.
Schedule of the expedition was as follows:
Tokyo (left Sept. 20)~Tainan (Formosa)~Luzon (Philippines)~Bangkok(Thailand)~Kuala Lumpur (Malaya)~Singapore~Kotakinabalu(Borneo)~Bogor (Java)~Makassal (Celebes)~Brisbane and Cairns~(Australia)~
Wellington (New Zealand)~Pt. Moresby (New Guinea)~Mindanao (Philippines)~Hong Kong~Okinawa~Tokyo (arrived Nov. 8).

Scientific Expedition for the Study of Rodents to South East Asiaand Oceania. II. Species and Chromosome Numbers of Rodents
Collected from South East Asia and Oceania
Tosihide H. YOSIDA, Kimiyuki TSUCHIYA,
Hirotami T. IMAI and Tatsuo UDAGAWA11

Species of rodents, their chromosome numbers and localities of collection in South East Asia and Oceania are listed in the following Table(Table 1).
Table 1. Names of species, collection localities and diploid chromosome
numbers of rodents collected from South East Asia and Oceania
Species
RattU8 annandalei
Rattu8 conatu8
Rattu8 canU8
Rattu8 exulan8/I/I

Rattu8 !u8cipe8
Rattu8 hung
Rattu8 muelleri
Rattu8 norvegicu8
Rattu8 rajah
Rattu8 rattu8/I/I

Localities of collection
Kuala Lumpur, Malaysia
Cairns, Australia
Kuala Lumpur
Luzon, Philippines
Makassar, Celebes
Bangkok, Thailand
Brisbane, Australia
Hong Kong
Kuala Lumpur, Malaysia
Taiwan, Philippines, Thailand,
Australia, New Zealand
Bangkok, Thailand
Okinawa, Japan
Tainan, Taiwan (Formosa)11 Forest Experimental Station, Tokyo.

Chromosome number (2n)

42
32
46
42 (44)4242
38 (39)42424252
42* (44)
42*

RESEARCHES CARRIED OUT IN 1968

11

Table 1 (continued)
Species

Rattus rattus
1/

/I

II

II

II

R. rattus rattus
/I

II

"

"

R. rattus argentiventer
R. rattus diardi
R. rattus jlavipectus
R. rattus Jalorensis
Rattus sp.
Rattus sp.
Rattus surifer
Rattus sabanus
Bandicota nemorivaga
Melomys cervinipes
Melomys littoralis
Mus thai
Mus caroli
Uromys caudimaculatus

Localities of collection

Chromosome number (2n)Khao Yai, Thailand
Luzon, Philippines
Mindanao, Philippines
Bogor, lava
Makassar, Celebes
Brisbane and Cairns, Australia
Wellington, New ZealandPort Moresby, New Guinea
Kuala Lumpur, Malaysia
Kuala Lumpur, Malaysia
Hong Kong
Kuala Lumpur, Malaysia
Hong Kong
Port Moresby, New Guinea
Kuala Lumpur, Malaysia
Kuala Lumpur, Malaysia
Tainan, Formosa
Brisbane, Australia
Cairns, Australia
Bangkok, Thailand
Bangkok, Thailand
Port Moresby, New Guinea

42*
42**
42**
42**
42**
38
38
38
42**
42* (43, 44)
42*
42**
42
34
52
42
44
54
52
40
40
54

* Polymorphism was found in No. 1 chromosomes (telocentrics and subtelocentrics).
** No polymorphism was found in No.1 chromosomes (subtelocentrics). Seldom
occorring chromosome number is given in parenthesis.

Scientific Expedition for the Study of Rodents to South East Asiaand Oceania. III. Chromosomal Polymorphism and New
Karyotypes of Black Rat, Rattus rattus, Collectedin South East Asia and Oceania
Tosihide H. YOSIDA and Kimiyuki TSUCHIYA

It was already reported that black rats, Rattus rattus, collected inJapan and Korea were characterized by a polymorphism of the largest
No. 1 chromosome pair, present either as a telocentric homomorphic

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

pair (TIT), or a telocentric and subtelocentric heteromorphic pair (TIS)or a subtelocentric homomorphic pair (SIS). (Yosida et al., 1965, Chromosoma 16: 70; 1968, this Ann. Rept. 18: 51). Recently we found thatNo. 9 and No. 13 chromosomes showed also similar polymorphism by
the presence of telocentric homomorphic (TIT), subtelocentric homomorphic (SIS) or subtelocentric and telocentric heteromorphic pairs (TIS).
A similar chromosomal polymorphism as in Japan was found in blackrats collected in Okinawa, Hong Kong, Bangkok and Kuala Lumpur.
On the other hand, all of Rattus rattus collected in paddy fields ofPhilippines, Java, and Celebes, showed that chromosomes No.1, No.9
and No. 13 were represented by SIS, SIS and TIT pairs. No chromosomal polymorphism was found in the rats collected in those countries.
Black rats collected in all localities of Far and South East Asia had
the diploid chromosome number of 42, while all animals collected inAustralia, New Zealand and New Guinea showed 38 diploid chromosomes.
Two pairs of large metacentric chromosomes, one larger than the other,characterized the karyotypes of these animals. No.1 and No.9 chromosome pairs were usually of SIS type. The other chromosomes weresimilar to those of the Asian black rats. From the results of comparisonof idiograms between Japanese and Australian black rats it is suggested
that two arms of the large metacentric element in the latter might behomologous with No.4 and No.7 telocentric elements of the former,
and two arms of the other smaller metacentrics might correspond to
No. 11 and No. 12 telocentrics.
F 1 hybrids between Australian (2n=38) and Japanese rats (2n=42)
showed 40 diploid chromosomes. It appears that among them 19 chromosomes came from the Australian rat and 21 chromosomes from theJapanese rat. Test crosses of F 1 animals and segregation of chromosomes
are under investigation.

Scientific Expedition for the Study of Rodents to South East Asiaand Oceania. IV. Comparative Idiogram Amalysis in Several
Species Belonging to Genus Rattus Collectedfrom South East Asia and Oceania
Tosihide H. YOSIDA and Kimiyuki TSUCHIYA

16 species belonging to genus Rattus were collected from South East
Asia and Oceania. They showed various diploid chromosome numbers,
such as 32, 34, 38, 42, 46 and 52. Among them, species with 42 chromosomes were found most frequently. From the above results it isRESEARCHES CARRIED OUT IN 196813strongly suggested that the basic chromosome number in genus Rattus
is 2n=42, and the basic karyotype consists of 13 pairs of telocentrics, 7
pairs of metacentrics and telocentric X and Y elements, which were observed most frequently in Rattus rattus with chromosomal polymorphism.
The relationship between the karyotypes varying from the basic patternand differentiation of species in genus Rattus was investigated.
(1) The following species had a basic chromosome number, but theirkaryotypes deviated slightly from the basic one. Rattus no rvegicus ,Rattus jalorensis, Rattus argentiventer, Rattus anandalei, Rattus exulans,
Rattus mulleri and Rattus sp. Hong Kong.
(2) The following species had a smaller chromosome number thanthe basic one due to a change of Robertsonian type. Rattus rattus rattus(2n=38), Rattus fuscipes (2n=38), Rattus sp. New Guinea (2n=34), andRattus conatus (2n=32).
(3) The following species had larger chromosome numbers than thebasic one by non-disjunction. Rattus rajah (2n=52) and Rattus surifer(2n=52).
(4) Lastly, species with a markedly different karyotype from the basic
one were Rattus sabanus (2n=42); 19 pairs of telocentrics and 2 pairs ofmetacentrics. Rattus canus (2n=46); 20 pairs of telocentrics and 3 pairsof metacentrics. Rattus hung (2n=46); 20 pairs of telocentrics and 3
pairs of metacentrics.

Scientific Expedition for the Study of Rodents to South East Asiaand Oceania. V. Breeding of Wild Rodents Collectedfrom South East Asia and Oceania
Kimiyuki TSUCHlYA and Tosihide H. YOSIDA

In order to find useful laboratory animals and to examine their behaviour, we had sent live specimens of several species of rodents collectedfrom South East Asia and Oceania to our institute and we are breedingthem in the laboratory. Names of species, number of propagated animals
and breeding generations are given in the following table (Table 1).
All animals of Table 1 have been bred in the laboratory. Amongthem up to date in four species we were successful in breeding theanimals to the first and second generations. We could also obtain hybrids
between two subspeices of Rattus rattus. We found that Rattus exulans
is a good experimental animal due to its small body and easy breeding.
Rattus conatus is also good for cytogenetical study, because its chrosome
number is smaller (2n=32) than that of other animals in the Rattus group.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19
Table l.

Species and number of animals breeding in laboratory

Collected locality
andspecies name

No. of animals at

No. ofcollected
animals

1 st gener.

2nd gener.

?

0

?

?

1
1

?

0

1
1

1
1

1
1

2

1

2

1

2

3

2

3

1

2

1

2

2
1
2
1
1
1

2
0
1
1
1
1

15
1
2
1
1
1

19
0
1
1
1
1

3
2
1

3
1
1

3
13
1

3
14
1

2
2
3
2
2

2
2
1
2
1

2
7
3
13
12

2
10
1
12
12(3)

1
2

1
1

33
2

24
1

7

5

5

2

0

0

Total no. ofanimals
under breed.

Okinawa (Naha city)

Rattus rattus
Mus servicolor
Formosa (Tainan)

Bandicota indica
nemorivage
Rattus rattus
ThailandRattus rattus
Malaysia (Kuala Lumpur)

Rattus rattus diardii
R. r. Jalorensis
R. sabanus
R. rajah
R. mulleri
R. bowersi

13

15

11

13

5

8

11
10

10
11

32

32

0

2

Indonesia (Celebes)

Rattus rattus
R. exulans (Java)
Rattus rattus
Australia (Brisbane)

Rattus rattus rattus
R. juscipes
Melomys cervinipes (Cairns)
R. r. rattus
R. conatus

3
Newly
born

New Guinea (Port Moresby)

R. r. rattus
Mus musclus
Fl hybrid between
R. r. tanezumi (Japan)
x R. r. rattus (Aust.)
R. r. rattus (Aust.)
x r. r. tanezumi (Japan)

3 litters
7
5
1 litter
2
5

RESEARCHES CARRIED OUT IN 1968

15

Rat Metaphase Chromosomes Incorporated into
Mouse Metaphase Cells
Tosihide H. YOSIDA and Toyozo SEKIGUCHlll

The present paper deals with the observation of rat metaphase chromosomes incorporoted into mouse metaphase cells. As recipient cells a
primary culture of mouse embryonic cells obtained from C57BL strain
was used. Most of the cells within one week after cultivation showednormal diploid chromosomes (2n=40), characterized by telocentrics or
acrocentrics. Donor chromosomes were isolated from the cells of rat
ascites hepatoma AH-130. The rat cells had hypotetraploid chromosome
number (s=75), including chromosomes of diverse shapes, such as meta-,
submeta-, subtelo- and acrocentrics.
One, two and four days after addition of rat chromosomes to theculture, they were observed to be incorporated into the metaphase of
the mouse cells at the rate of 0.3, 1.2 and 0.2 per cent, respectively.
The incorporated rat chromosome could be distinguished from the mouse
chromosomes by their characteristic shape, or by the occurrence of one
or two extraordinary elements. The incorporated chromosomes sometimes showed signs of degeneration or were unusually slender, but inother cells they appeared quite normal.
The percentage of mitotic cells in proportion to interphase cells, fourhours after treatment with colchicine, was one, two and four days after
the addition of chromosomes, 7.2, 0.7 and 5.0, respectively. From theabove results, it seems that some of mouse cells that had received rat
chromosomes may have degenerated and therefore the frequency of chromosome integration into mouse cells was just opposite to the mitotic
rates of the recipient cells.

Autoradiographic Analysis of the Rate of Protein Synthesis in
21
Diploid and Tetraploid MSPC-l Myeloma Cells
Kazuo MORIWAKI and Hirotami T. IMAIAs previously reported (K. Moriwaki et al. 1968, this Annual Report
18: 18-20), MSPC-l mouse myeloma cells exhibited an obvious tendency
to increase the rate of total protein synthesis per cell in parallel with
the increase of tetraploid cells until 40 per cent in metaphase count,
National Cancer Center Research Institute, Tokyo.
This study was supported by Public Health Service research grant CA 07798-05
from the National Cancer Institute, U. S. A. (T. H. Yosida).
11
21

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

beyond which it reached a plateau. In that case, however, the rate ofprotein synthesis was determined from a mass of tumor cells of theorder of 106 • In the present study a further attempt was made to observe
the variation in the rate of protein synthesis in individual tumor cells
following tetraploidization.
The tumor cells used in this experiment were suspended in MEM
medium with 5 percent calf serum and consisted of a mixture of diploidand tetraploid cells at the density of about 106 per m!. Tritiated leucine
was added to the suspension at the concentration of 50 pC per m!. After
two hour incubation at 25°C, the tumor cells were spread on microscopic
slides by air drying method. After dipping in NR-M2 autoradiographic
emulsion, the microscopic specimens were stored in a refrigerator for athree week exposure. The average number of deposited grains in atetraploid cell was approximately two times larger than in a diploid cell.
This finding suggests a gene-dose dependent increase of the rate ofprotein synthesis in tumor cells. The discrepancy between our previous
and present results might be explained by the following assumptions:
During the transient stage from diploidy to tetraploidy the actual percentage of tetraploid cells is somewhat larger than that estimated from
metaphase count, because in this stage a conciderable amount of binucleate cells appears in the tumor cell population and most of them aresecored as diploid from metaphase figures. This could bring about anunderestimation of tetraploid cell percentage. Furthermore, the partcomposed of binucleate cells can stay at interphase for a length of timeseveral generation cycles of the mononucleate cells. Such a retardationof mitosis in the binucleate cells might also contribute to an increase of
the actual percentage of tetraploid cells.
Consideraing the above assumptions, the percentage of tetraploid cells
seems to show an almost linear relationship between gene dosage andthe rate of total protein synthesis in MSPC-l tumor cells as a criterionof gene action.

Chromosome Alterations in vitro and in vivo of Cultured Hamster
Cells Ttansformed by Chemical Carcinogen"Kunio SEKIYA

Neoplastic transformation of golden hamster embryonic cells was induced in vitro by treatment of the cultures for 10 days with 4HAQO
11 This study was supported by Public Health Service research grant CA 07798-05
from the National Cancer Institute, U. S. A. (T. H. Yosida).

RESEARCHES CARRIED OUT IN 196817

(4-hydroxy-aminoquinoline-1-oxide) at a concentration of 10- 5 M. The
present report describes the neoplastic development of 4HAQO-treatedcells and chromosomal alterations which had taken place due to thetreatment.
Morphological features of transformation first appeared in culturesapproximately 20 days after carcinogen administration, as indicated by acriss-cross arrangement of cells and loss of contact inhibition resulting
in a "piling up" of cells. These cells, however, failed to give rise totumor when transplanted either to the subcutis or the cheek pouch ofadult hamsters. To acquire transplantability, the carcinogen-treated cells
seemed to require further cultivation in vitro. About 150 days after
treatment, the cultures started to show 100% transplantability to recipientanimals; an inoculum of 10 6 cells was capable to produce tumors in all
animals examined.
Chromosomes of these cells were studied at various stages of cultivation after treatment with the chemical. The modal chromosome numberof the transformed cells was found to shift gradually from the normal
diploid number, 44, to a hypodiploid 43. It seemed to be of fundamental
importance to find out if this karyotypical change had anything to dowith the cause of the neoplastic transformation of the culture or was
merely brought about by environmental factors operating during thecultivation. To obtain critical information regarding this alternative,
cells were transferred to various environmental conditions, and theirkaryotypes were carefully examined at certain periods after the transfer.
The environments employed were as follows: 1) cultivation in vitro, 2)cultivation in the subcutis of the flank of adult hamsters and 3) cultivation in adult hamsters that had received whole-body irradiation withr-rays. Cells grown under tissue culture conditions showed the mode
at 43 chromosomes as expected, while those transplanted to hamsters,
both irradiated and unirradiated, were found to possess a modal numberof 45. The percentage of cells showing 43 chromosomes was higher incells populations proliferating in irradiated animals than that in unirradiated ones. This fact may indicate that the irradiated hamsters offer
conditions resembling more those in vitro than do the unirradiated animals,
at least in terms of the maintenance of cells with a given karyotype.
The alterations in karyotype was mostly characterized by a missing oran additional chromosome in comparison with the normal karyotype.
Lack or addition of one chromosome seemed to occur randomly for any
merI' ber of the chromosome complement. Therefore, these changes may
be explained as resulting of non-disjunction of chromosomes in the
mitotic processes.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

It was found that cells transplanted to unirradiated hamsters showeda longer time lag before the production of a tumor as compared withthose transferred to irradiated animals. It was also found that viabilityof transplanted cells was higher in the irradiated animals than in theunirradiated ones, and this tendency was emphasized when a smaller
inoculum was used.
These results seem to be in favor of the idea that environmental
factors play a key role in the establishment of a new karyotype. Some
immunological mechanism might be involved in the in vivo alteration of
the cromosome complement of the transformed cells.

Aggregate-Forming Activity of Rat Hepatoma Cells
Induced by DAB
Yukiaki KURODA

In the process of malignant transformation of normal cells, changes
in aggregate-forming activity which is a useful indicator of cell function
determined quantitatively under strictly standarized conditions were examined. With the cooperation of Dr. Sato, Okayama University Medical
School, liver cells from Donryu Rats, fed with 4-dimethylaminoazobenzene(DAB) for various days, were tested for their malignancy (ability to
produce tumors in newborn rats), chromosome number and their aggregate-forming activity (aggregation pattern of the cells in rotation culture).
When strain dRLa-74 cells, which were derived from a male rat fed
with DAB for 191 days were retransplanted into newborn rats intracerebrally, intraperitoneally or subcutaneously with an inoculum containing 10 5 to 5 X 10 5 cells per rat, the rats died with tumors at the rates
of 4/9, 1/3 or 0/3, respectively. The number of chromosomes in straindRLa-74 cells had a main diploid mode and another small triploid mode.
The aggregate-forming activity of dRLa-74 cells was compared under
the standard conditions of rotation culture with that of strain C251 cells
which were derived from normal newborn rat livers. dRLa-74 cells
formed after 24 hours of rotation culture aggregates which were significantly larger in diameter than those obtained from C251 cells.
C83T cells, which were derived from the liver of a female rat fed
with DAB for 264 days, showed a moderate malignancy when they wereretransplanted into newborn rats. C84AT cells, which were derived
from the liver of a female rat fed with DAB for 312 days, showed ahigh malignancy. C84AT cells formed after 24 hours of rotation culture
significantly larger aggregates than those of C83T cells.

RESEARCHES CARRIED OUT IN 1968

19

In some other rat liver cells tested a close correlation was foundbetween the ability of cells to produce tumors and their aggregateforming activity. These facts coincide with the results which had beenpreviously obtained with RSV-infected chick cells, mouse mammary glandtumor cells (Kuroda, Y. 1968. GANN 59: 281), and mouse plasma tumor
cells (Kuroda, Y. 1968. Ann. Rep. Natl. Inst. Genet. Japan 18: 10). The
increase in aggregate-forming activity of cells in carcinogenesis may
provide an experimental clue to an explanation of the loss of contact
inhibition (growth inhibition due to cell contact) and loss of histoformative structure of tumor cells.
Pachytene Pairing in a Translocation Heterozygote of
Maize Having a Huge Knob Close to the Breakpoint
Yasuo OHTA

Chromosome knobs of maize in pachynema are a hereditary trait.
Their position, size and shape are very constant features of the karyotype
and can serve as cytological markers. Chromosome knobs consist mostlyof heterochromatin, and are considered to be genetically inert (except
for the abnormal chromosome 10).
An exceptionally huge knob (3L l l l ) was found on the long arm ofchromosome 3 (position .58) in an inbred line, N. C. No. 12, derived fromthe variety Jarvis Golden Prolific (Ohta 1965). To examine the effectof this huge mass of heterochromatin on the synapsis during meiotic
prophase, this inbred line was crossed to a translocation homozygote,
T3-8h (furnished by Dr. E. B. Patterson), having the breakpoint at .53
on the long arm of chromosome 3.
Pachytene pairing in a translocation heterozygote, 3e ll jT3-8h, wasanalysed. Three major types were found: 1) complete homologous pairing
throughout the cross-shaped configuration, 2) complete asynapsis or asynapsis in each arm of the cross-shaped configuration in the segments
adjacent to the breakpoint, and 3) partly homologous pairing and partly
asynapsis. The observed frequency of each of the three types was
about 1 : 1 : 1.
Preferential Pairing of Chromosomes in a Tetraploid Hybrid
between Oryza glaberrima and O. sativa
Hiko-Ichi OKA

A tetraploid hybrid was obtained from a cross between induced tetra-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

ploid strains of Oryza sativa L. and O. glaberrima Steud., which differedin glutinous (waxy) vs. non-glutinous starch (gl: +) and other genes. In
the hybrid, as compared with the parental strains, there was a reduced
number of quadrivalents and a relatively high degree of fertility. Segregation ratios for the glutinous gene observed in the back-cross and F 2
generations appeared to be modified not only by preferential homogenetic
pairing but also by gametic selection. Taking into account the effect ofgametic selection estimated from simplex heterozygotes, it was suggested
that preferential pairing could take place to a certain extent. It seems
to be difficult to measure quantitatively the degree of preferential pairingin tetraploid species hybrids, unless the effect of various segregationdistorting factors can be accounted for. (Published in Canadian ]. Genet.
& Cytol. 10: 527-535)

RESEARCHES CARRIED OUT IN 1968

II.

21

PHYSIOLOGICAL AND DEVELOPMENTAL GENETICS

Differentiation of Ommatidium-Forming Cells of Drosophilamelanogaster in Culture
Yukiaki KURODA

In holometabolic insects such as Hymenoptera, Lepidoptera and Dipteraa drastic change, called "metamorphosis," takes place from the larval
into the adult stage through the pupal stage during which certain larval
tissues and organs are destructed (histolysis) and adult structures arereorganized from primitive cell complexes, the imaginal discs. The eyeantennal discs are those from which the adult ommatidia, antenna, head
hypodermis and perhaps the outer eye ganglion are differentiated during
the pupal stage.
Eye-antennal discs which were dissected from mature third-instar
larvae of Drosophila melanogaster grown under sterile conditions were
cultured in chemically defined medium (Kuroda, Y. and Tamura, S. 1956.
Med. J. Osaka Univ. 7: 137) and examined for the mechanism of differentiation of ommatidium-forming cells. It has previously been foundthat a marked differentiation of ommatidium-forming cells was observed
when ring glands with brains and ventral ganglions or ecdysone analogues
were added to the medium in which the eye-antennal discs were cultured(Kuroda, Y. and Minato, K. 1968. Ann. Rep. Natl. Inst. Genet. Japan
17: 28). In the presence of ecdysone and its analogues, ommatidiumforming cells in eye discs formed many clusters of several cells each
after 24 hours of cultivation. The number of these cell clusters seemed
to correspond to that of adult ommatidia.
When 5-bromodeoxyuridine, actinomycin D or puromycin was added
to the medium at the concentrations of 10- 5 M, 1 pg/ml or 10 pg/ml, respectively, the hormone-dependent differentiation of ommatidium-forming
cells in eye-antennal discs was not affected after 24 hours of cultivation.
This indicates that ecdysone and its analogues may induce the migrationand orientation of ommatidium-forming cells by which morphologicallydistinct cell clusters are organized and that no new synthesis eitherof RNA or protein may be needed for the induction of cell orientationby the hormones.
Eye-antennal discs were irradiated with OR, 500R, 1,000R, 1,500R, and2,OOOR of X-rays (175 KVp, 25 mA, distance 40 cm, filter 1.0 mm AI, dose
rate 300 R/min), and examined for the effects of X-rays on the differentiation of ommatidium-forming cells in the presence of ecdysone analogue.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

In eye-antennal discs irradiated with less than 1,000 R of X-rays nomarked change was observed in the differentiation of ommatidium-forming
cells after 24 hours of cultivation. With 2,OOOR of X-ray their differentiation was almost completely inhibited when eye-antennal discs were
irradiated at the start of the culture. 2,000 R of X-ray, however, had
no effect on eye-antennal discs when they were irradiated after 4 hoursof cultivation.
These results suggest that some substance(s) necessary for the differentiation of ommatidium-forming cells had preexisted in the eye discs
in an unstable state at the start of the culture when the substance(s)was sensitive to 2,OOOR of X-ray and became stabilized after 4 hours ofcultivation although the ommatidium-forming cells were morphologicallynot yet differentiated.

Effects of Ecdysone Analogues on Differentiation of Eye-Antennal
Discs of Drosophila melanogaster in Culture
Yukiaki KURODA

It has previously been found that various steroids having ecdysone
activity which have been isolated from plants were highly active inpromoting the differentiation of eye-antenna I discs cultured in chemically defined medium (Kuroda, Y. and Minato, K. 1968. Ann. Rep. Natl.
Inst. Genet. Japan 18: 28). In a medium containing these steroids eyeantenna I discs dissociated from mature third-instar larvae of Drosophilamelanogaster showed a morphologically pronounced organization of ommatidium-forming cells which formed many cell clusters corresponding
in number to adult ommatidia.
Table 1 shows the effects of ecdysone, inokosterone, ponasterone C
and rubrosterone at various concentrations on the differentiation of ommatidium-forming cells in eye-antennal discs cultured in vitro.
Among ecdysone analogues tested, ecdysterone, inokosterone, andponasterone C showed rather lower activity than rubrosterone, which had
the highest activity on promoting the differentiation of ommatidiumforming cells in eye-antennal discs. This fact suggests a possibility thatin the intact insect ecdysone may metabolite through several ecdysone
analogues into rubrosterone or a related substance which may act on thetarget organs.

23

RESEARCHES CARRIED OUT IN 1968
Table 1.

Effects of ecdysone analogues on the differentiationof eye-antennal discs in culture

Substance

Concentration(pgl pi)

No. ofexplants
testedNo. of explants inwhich ommatidia
were differentiated

Per cent ofdifferentiation

Control
Ecdysterone

0
10.0
1.0
0.1
1.0
0.1
0.01
1.0
0.1
10.0
1.0
0.01
0.00010.000001
0.00000001

16
16
8
6
3
9
8
8
8
8
20

0
15
7
2
3
4
3
5
2
6
14
10
12
6
2

0
94
88
33
100
44
38
63
25
75
70
91
92
50
22

Inokosterone

Ponasterone C
Rubrosterone

11

13
12
9

The Relationship between Cell Growth Cycle and Molting
Cycle in Insect Epidermis
Kiyoshi MINATO

To elucidate further the mechanism by which the molting of the larval
epidermis takes place through the action of a molting hormone, ananalysis was carried out on the cell kinetics during the molting processof Philosamia cynthia ricini larvae.
It has previously been found that larval epidermal cells of this insect
grew very synchronously for a relatively short period before molting.
A detailed examination unexpectedly revealed that the cell division of
the epidermis takes place not after but before DNA synthesis during thefourth larval instar. In the present investigation it was found that theoccurrence of cell division prior to DNA synthesis also was taking placein the epidermis of the third instar larvae. These facts suggest thatepidermal cells may not divide after DNA synthesis but continue intetraploid state through the molting period and divide immediately before
DNA synthesis in the next larval instar.
To ascertain this possibility the epidermal cells were labeled with apulse of 3H-thymidine at various periods in the third instar. Incorpo-

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

ration of 3H-thymidine was found in mitotic figures of epidermal cells
in the fourth instar larvae. When epidermal cells were continuously
labeled with 3H-thymidine throughout the fourth instar larval period, no
grain was found in mitotic cells of the fourth instar larvae.
These results indicate that epidermal cells have a peculiar growth
cycle in which they do not divide after their DNA synthesis in thesame larval instar but divide after a long G 2 period (about 48 hours) inwhich they pass through molting to the next larval instar.

Cytoplasmic Male Sterility and Virus Inoculation in Capsicum
Yasuo OHTA

Phenotypical similarity between cytoplasmic and virus induced malesterility in certain plants is well known.
Cytoplasmic male sterility in Capsicum annuum L. is controlled by asterile cytoplasm (5) and a male sterile gene (rf, original symbol, ms).
(5) rfrf plants are pollen sterile, (5) RfRf or (5) Rfrf plants are pollen
fertile, as are plants having normal cytoplasm (N).
In the first experiment, (5) Rfrf plants obtained from a cross (5) rfrf
Fresno Chile x (N) RfRf Fushimiamanaga and (N) RfRf plants (Fushimiamanaga) were inoculated with an ordinary strain of cucumber mosaic
virus (CMVo), ordinary and yellow strains of tobacco mosaic virus (TMVoand TMVy), and broad bean wilt virus (BWV) (designated as tobaccoringspot virus (TRSV) in previous reports).
(5) Rfrf plants inoculated with CMVo or BWV showed reduced pollen
fertility which was 64.2 to 68.0 per cent on the average. On the other
hand, (N) RfRf plants showed little or practically no change in pollen
fertility. In the second experiment, both (5) Rfrf and (N) RfRf plants
were inoculated with three strains of cucumber mosaic virus (ordinary(CMVo), necrotic spot (CMVn), and leguminous (CMVl», BWV, alfalfa
mosaic virus (AMV), and a mottle strain of potato virus X (PVXm).
Again, CMVo- and BWV inoculation produced a reduced fertility in (5)Rfrf plants which was 72.5 to 75.3 per cent on the average. A similar
result was obtained also by CMVI inoculation in which pollen fertility
was reduced to 77.6 per cent in (5) Rfrf plants.
Selfing of those inoculated plants in the first experiment was successful, and the progenies were examined for pollen fertility. Since theinoculated plants were heterozygous for the fertility gene, a 3 fertile:
1 sterile ratio was expected. Two lines from a plant inoculated with
CMVo having lowest pollen fertility (42.8%) and a line from a TMVy·RESEARCHES CARRIED OUT IN 196825

infected plant having normal pollen fertility (95.1%) produced many
more sterile plants than expected. The observed ratio for the above
three lines is more likely to be the 1:1 ratio expected from a backcrossto (N) rfrf plants. But such a possibility was refuted by the segregation ratio for two characters of the fruit, erect vs. pendent, and oval vs.
round. In addition to steriles, some plants showed reduced pollen fertility (semi-fertiles and semi-steriles). Thus in the progeny of a (5)Rfrf plant inoculated with CMVo and a (5) Rfrf plant inoculated with
TMVy, pollen fertility tended to be lower.
It may be said, therefore, that a particular cytoplasm (5) or at leastcytoplasm (5) together with Rfrf genotype is responsible in some way
for the production of reduced pollen fertility by specific viruses.

Organ Differentiation of Isolated Crepis Leaves in vitro
Yoshiaki YONEDAIn many plant tissues cultured in vitro, externally supplied plant hormones have been found to induce callus or organs differentiation such as
rooting and bud formation. This phenomenon could present a suitablemeans for analysing the hormonal mechanism of differentiation. In thepresent study, it is described how isolated leaf blades of Crepis capillaris
were responding to different hormones or hormone combinations.
As a basal medium, Linsmaier & Skoog (1950)'s mineral salts containing organic constituents without hormones were used. Agar was used
at 0.7% concentration. Crepis plants were cultured on the basal medium containing 0.5 ppm kinetin (KT medium), and leaf blades (leaf terminal, 1 cm long) were severed for inocula which were transplanted tovarious hormones containing media. For differentiation experiments, indoleacetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT)and yeast extract (YE) were added singly or in combinations. The leaf
blades were incubated for one month at 27°C in the dark and examinedfor differentiation.
On the basal medium, leaf blades showed no morphological changes.
On the medium containing 0.1 to 10 ppm of IAA (IAA medium), the leaf
blades became transparent and soon many roots appeared. Over theveinlets of these leaf blades occurred many protrusions consisting of small
seemingly meristematic cells, some of which seemed to produce roots.
In contrast, KT (0.05-0.5 ppm) did not produce any morphological change
in the leaves. On IAA-KT combined media, rooting and bud initiation
was observed depending on the concentration of both hormones. For

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

bud initiation, the most effective concentration of KT was 0.5 ppm whencombined with IAA at 1.0 ppm. The general tendency observed was that
KT increasingly suppressed root initiation with increasing concentration,and that increasing concentration of IAA reduced the inhibitory effect ofkinetin.
When 2,4-D or NAA were added instead of IAA and combined withKT at 0.5 ppm, bud initiation was also observed at 0.1 ppm of 2,4-D and
0.1 to 1 ppm of NAA. Higher concentration of 2,4-D completely suppressed bud initiation. On media containing 2,4-D at 0.1 ppm and KT at
0.1 ppm, only undifferentiated growth took place, resulting in slowly
growing firm, solid callus. Addition of YE (0.5%) to this medium (2,4D-KT-YE medium) also produced callus, though rootings were observedin this case. The callus was soft and friable and grew very rapidly.
These results indicate that rooting, callus formation and bud initiation
seem to be controlled by the kind and concentration of the hormone.
Kinetin favors induction of bud initiation and IAA stimulates rooting,while undifferentiated callus growth is observed by 2,4-D.

RESEARCH CARRIED OUT IN 1968

III.

27

BIOCHEMICAL GENETICS

Genetic Effects of DNA in Ephestia
Saburo NAWA and Masa-Aki YAMADA

A heritable change induced by DNA in Ephestia had been already reported (this Annual Report, No. 18). The method used in these experiments consists of the treatment of larvae or eggs, having the recessive genotype a, with DNA from wild type or DNA from a-genotype.
Adults developed from treated larvae or eggs were mated to a/a animals
to observe possible changes in eye color in their progeny (BF 1)' The
appearance of wild type eyes in BF 1 was repeatedly observed, confirming
the previously reported results. In the overall experiments to date, 19
cases of black-eyed mutant out of 138,917 BF 1 were obtained in the caseof +DNA treatment, while no black-eyed mutant out of 46,229 was observed in the treatment with homologous DNA extracted from a-strain.
The difference between the effect of +DNA and a-DNA is significant
2(x =5.5, P:::::0.02). Black-eyed mutants obtained by +DNA treatment
were successively backcrossed to a/a until segregation of black and redeyes occurred. Then, in successive sister-brother matings, black-eyedanimals were bred to each other and have been kept as mutant lines.
Since cases of nonspecific mutations induced by DNA have been
known in Drosophila, a possibility might be considered that the changeof red to black in our experiment is due to an accidental mutational
effect of DNA such as replacement of a nucleotide or nucleotides in the
a-locus leading to something like an intra-cistronic suppressor mutation.
If so, amino acid compositions of tryptophan pyrrolase (TP) of mutant
lines would be altered, since the gene a + is considered to be the structural gene for TP. Therefore, properties of the enzyme from mutant
lines were compared with that from wild type. It was found that all
mutant lines had TP activities nearly the same as the wild type. On
starch gel electrophoresis, TP activity was detected as a single, slightly
diffuse band. It was observed that TP from all of the mutant lines had
the same mobility as TP of wild type, suggesting identical amino acidcomposition. Although it became possible to purify the enzyme about
80 fold from a homogenate of adults by ammonium sulfate fractionation,
treatment with calcium phosphate gel, followed by chromatography on
DEAE-Sephadex, an attempt to get an antibody for the enzyme was
unsuccessful.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

Further Evidences of Uptake of DNA by Ephestia Eggs
Masa-Aki YAMADA and Saburo NAWA

Evidence of DNA uptake by Ephestia eggs was previously reported(this Annual Report, No. 18). The 3H-labelled DNA (1.5xl0 6 -6.2x105
cpm/ flg) was prepared from a thymineless mutant of Salmonella cultured
on a medium containing 3H-thymidine. Eggs ranging from 0 to 18 hours
after they had been spontaneously laid in plastic boxes were dechorionated. After washing with saline, they were immersed in the 3H-labelled
DNA solution for a definite time and treated with DNase to remove the
DNA attaching to the egg surface. After washing with saline, DNA
was extracted from the eggs by SDS-phenol method. The radioactivity
was measured by a low back gas flow counter. When eggs were immersed in the DNA solution (300 flg/ml) for 2, 4 and 6 hours, the radioactivities per 500 flg DNA extracted from the eggs were 99, 296 and 339
cpm respectively. When eggs were treated with 100, 200 and 300 flg/ml
DNA solutions, the radioactivities per 500 flg DNA extracted from thetreated eggs were 672, 1529 and 2171 cpm respectively. The repeatedexperiments gave similar results, showing that the uptake of DNA by
eggs depends on the concentration of the DNA solution and the time of
their immersion in DNA. DNA extracted from the eggs treated with
3H-DNA for 2, 4 and 6 hours was submitted to fractionation by centrifuge chromatography on DEAE-celullose paper pulp. The elution patternof the extracted radioactivities is given in Table 1. In the eggs treatedfor 2 hours, radioactivities were found in the fractions of high molecularweight. Radioactivities extracted from the eggs treated for 4 or 6 hours
shifted to the fractions of lower molecular weight. These results indicate that the foreign DNA is incorporated into the eggs in high molecular state.
Table 1.

Fractionation of the DNA extracted from the eggs treated with
3H-DNA

2 hours

4 hours

6 hours

No.

(Average
Molecular
weight)Radioactivity(%)

O.D.

Radioactivity(%)

O.D.

Radioactivity (96)

O.D.

1
2
3
4
5
6

(5 x 102)(2 x 103)(5 x 104 )(5x 105 )(1 x 106)(5 x 106 )

2.4
5.4
19.3
46.8
22.8
3.6

0.65
0.92
2.01
3.15
4.13
1.80

10.4
1.2
21.9
67.1
6.5
3.7

0.61
1.03
0.96
1.29
5.60
1.75

0
0
73.6
17.9
8.5
0

0.81
0.80
0.98
1.20
3.81
1.31

Fraction

RESEARCH CARRIED OUT IN 1968

29

Genetical and Biochemical Studies of Lethal Lemon
Silkworm Larvae
M. TSUJITA and S. SAKURAI

It had been reported (Tsujita 1956) that normal (+ lem), lemon (tem)and lethal lemon (tem l ) are multiple alleles of the gene located at 22.3
units on chromosome 3.
In the lethal lemon larvae melanin pigment is scarcely produced in
the cuticular layer of the hypodermis and also their mandibular cuticle
does not sufficiently develop owing to incomplete hardening of cuticularscleroprotein, so that they cannot chew up mulberry leaves and starve
to death after the first moulting. They exhibit a distinctly yellow bodycolor and in their hypodermis a large amount of pteridine granules containing sepiapterin and uric acid is produced.
Using normal, lemon and lethal lemon larvae the activity of tyrosinase
in larvae immediately after the first moulting was examined by discelectrophoresis. The activity of protyrosinase in lethal lemon larvae
could not be discriminated from that of normal larvae. However, inlethal lemon larvae a weaker tyrosinase activity was detected than innormal larvae.
It has been known that the conversion step from sepiapterin to tetrahydropteridine is catalysed by pterine reductase. The activity ofpterine reductase of normal, lemon and lethal lemon larvae was reported
in our previous paper (Tsujita 1963). According to experimental results
normal larvae show a strong activity of pterine reductase and lemon
larvae also have some though weaker activity. However, lethal lemon
larvae lack the activity of this enzyme so far as the method which weused is concerned. In spite of this fact, some amount of isoxanthopterinin addition to a large amount of sepiapterin could be detected in lethallemon larvae.
It was proved that a much smaller amount of alkali isoluble matterand tyrosine metabolite incorporated into this insoluble matter was foundin lethal lemon larvae than in normal larvae. And it seems that the
principal components of this alkali insoluble substance is scleroprotein of
the cuticular layer of larval hypodermis.
It has been known (Tsujita 1956) that embryos homozygous for thel
fem gene, usually develop normally to black young larvae and die immediatly after the 1 st moulting. Namely, in the cross +Ilem l X +Ilem lor + Ilem l X lemllem l , the yellow lethal larvae can be recognized in the
2nd instar. On the contrary, in the cross lemllem1x +Ilem l or lemllemlx
Jemllem l , however, all homozygous lemlilem i individuals die within the

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

eggs which show through the semi-translucent shells the yellowish-brown
dying larvae.
In my previous paper (Tsujita 1963) the following relationship was
found between the presence or absence of pterine reductase in eggs immediately after they were laid by their mother moth and the stage at
which the homozygotes for lem 1 die. When these homozygotes develop
from the biginning in the presence of the enzyme, they become normal
black young larvae and they are able to hatch out by chewing the egg
chorion. However, when they develop in the absence of the enzyme,
they become yellowish-brown young larvae and cannot hatch out owing
to incomplete differentiation and insufficient hardening of their mandible
cuticle. It may be safely said that enzyme activity is directly controlled by the genes +lem, lem and lem 1•
According to Kaufman (1962) sepiapterin and its hydrated derivatives
act as cofactor in the hydroxylation of phenylalanine to tyrosine. Therefore a hypothesis based on Kaufman's finding was considered for the
explanation of the cause of lethality of the mutant larvae. Namely, innormal and lemon larvae tyrosine is sufficiently produced before and at
the time of new formation of the cuticular layer because phenylalaninehydroxylase stimulated by the cofactor activity of sepiapterin Gan normally catalyse the conversion of phenylalanine to tyrosine. On the contrary, in the lethal lemon larvae sepiapterin loses its cofactor activity
owing to the lack of pterine reductase, so that phenylalanine hydroxylase cannot sufficiently catalyse the conversion from phenylalanine totyrosine, resulting in the production of a small amount of melanin pigment and incomplete differentiation, i.e. insufficient hardening of the
cuticular layer of the larval hypodermis.

Genetical and Biochemical Studies of Lethal Albino
Silkworm Larvae
M. TSUJITA and S. SAKURAI

The experimental results of linkage test (Tsujita 1956) showed that thelethal albino gene (al) is not located on chromosome 3, and is inherited
independently of lem 1 gene. However, the phenotype of lethal albino
larvae resembles that of lethal lemon larvae. Namely, melanin pigmentof both is scarcely produced in the cuticular layer of the hypodermis
and their mandibular cuticle does not sufficiently develop due to incomplete hardening of cuticular scleroprotein, so that they cannot chew up
mulberry leaves and starve to death after the first moulting. The lethal

RESEARCH CARRIED OUT IN 1968

31

albino larvae have partly light brown and partly white body color andtheir hypodermal cells contain a large amount of pteridine granules filledwith pteridine metabolic products and uric acid. A weaker tyrosinase
activity is detected in them than in normal (+al) larvae, although theactivity of protyrosinase is normal. Furthermore, a very low activity ofphenylalanine hydroxylase was detected in the larvae of this mutant.
0.2 g of frozen albino larvae immersed in 1 ml of 30% alcohol washomogenized in a glass homogenizer and heated for 20 minutes in an
water bath at a temperature of 95-100°C in order to extract pteridine
compounds. After centrifugation at 3000 rpm for 10 minutes, 0.15 ml ofsupernatant was used for the separation of several pteridines by twodimensional paper chromatography. According to experimental results amuch smaller spot of isoxanthopterin was observed than in normal larvae(+ al). However, a very large spot showing light greenish-blue fluorescence which was not observed in normal larvae appeared in the placeof isoxanthopterin.
The activity of pterine reductase and pterine dehydrogenase was normal. Also, according to determination of the amount of uric acid byuricase method, it is normally produced in the mutant larvae.
Based on these experimental results, it is assumed that the formation
step of some precursor of sepiapterin is blocked in the mutant larvae
and that this abnormal pteridine metabolism have an intimate relation
to the weak activity of tyrosinase and phenylalanine hydroxylase.
Preliminary examination of the Cofactor Roleof Sepiapterin for Phenylalanine Hydroxylase
M. TSUJITA and S. SAKURAI

Materials and methods: normal larvae (+ ai, + I,m), lethal albino (at)larvae and lethal lemon larvae (fern l ) were used as materials.
A mixture of 1 ml of 0.01 mole phosphate buffer solution (pH 6.8) and
0.5 g of frozen larvae was homogenized under ice cooling and centrifuged at 10,000 rpm for 30 minutes. The supernatant was used as crude
enzyme solution.
The following modified reaction solution obtained after the method
used by Kaufman (1959) was prepared. Namely, a mixture of i) 0.5 mlof 0.5 m mole L-phenylalanine solution (L-phenylalanine was dissolved in
0.01 mole phosphate buffer at pH 6.8), ii) 0.2 ml of sepiapterin solution
containing 100 fig pigment per ml, iii) 0.2 ml of NADPH* solution con* Abbreviations used: NADP, nicotinamide-adenine dinucleotide phosphate; N ADPH, reduced form of NADP.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

taining 100 pg reduced NADP* per ml, and iv) 0.2 ml of crude enzyme
solution was incubated at 25°C for 30 minutes and then 2 ml of 12%
TCA was added to the solution to stop the reaction. After centrifugation of the solution at 3000 rpm for 10 minutes, the supernatant wasused for the determination of the amount of formed tyrosine.
0.05 ml of 0.1 mole iodine solution was added to a mixture of 1 ml ofsupernant and 0.4 ml of 2 mole tris buffer solution. After keeping the
mixture at room temperature for 2 minutes, 0.2 ml of 0.1 mole sodium
thiosulfate was added and then the solution was diluted to 3 ml of the
final volume by addition of distilled water. The amount of formed tyrosine was determined by measuring with Beckman's spectrophotometer at
310 mp wave length.
Results: tyrosine formation was recognized in the reaction solution
containing the crude enzyme solution of normal larvae (+ lem, + a\ Further, almost the same results were obtained in both cases of reaction
solutions with addition of sepiapterin or without it.
Next, in the reaction solution containing the crude enzyme solution
prepared from lethal lemon larvae the formation was found of a muchsmaller amount of tyrosine than in the reaction solution of normal larvae.
In the reaction solution of albino lethal larvae formation of a muchsmaller amount of tyrosine was recognized than that of the normal
strain (+al). Besides, it is a remarkable fact that the production oftyrosine increased in the reaction solution by addition of sepiapterin.
Thus, so far as the present in vitro obtained results are concerned,
lethal lemon larvae showed low activity of phenylalanine hydroxylase incomparison with the normal larvae (+ lem). However, it was difficult toconfirm the cofactor role of sepiapterin for phenylalanine hydroxylasefor these mutant larvae.
On the contrary, the lethal albino larvae exhibited not only weaktyrosinase activity but also low activity of phenylalanine hydroxylase.
Thus, the fact that the amount of tyrosine in the reaction solution of
the mutant larvae was increased by addition of sepiapterin seems toindicate the cofactor role of sepiapterin for phenylalanine hydroxylase.

RESEARCH CARRIED OUT IN 1968

33

Genetical and Biochemical Studies of Pteridine Granules in
Hypodermal Cells of Silkworm Larvae: Significance of
Polypeptides inside the Granules
S. SAKURAI and M. TSUJITA

Comparative studies of peptidase activity in the body fluid and hypodermal tissue of larvae during the period from 4th instar to mounting
stage of the 5th instar were carried out using as materials C-124, a hybrid(Daizo x W-C) with normal hypodermis and WO" oily mutant strain.
Body fluid was taken from the caudal horn by cutting off the apex of
the horn.
1 g of fresh larvae hypodermis together with 3 ml of 0.02 mole verona 1
buffer (at pH 7.6) was homogenized in a glass homogenizer and centrifuged at 10,000 rpm for 30 minutes. The supernatant was used as hypodermis extract.
Substrates used for the detection of peptidase were leucyl-glycyl-glycine
in case of aminopeptidase and glycyl-tyrosine, alanyl-histidine, glycylglycine, alanyl-alanine and alanyl-glycine in case of dipeptidase.
Mixture of 0.05 ml of body fluid or hypodermis extract and 0.3 ml of
0.04 mole substrate solution (substrate was dissolved in 0.02 mole veronal buffer at pH 7.6) was incubated at 37°C for 2 hours and the activityof enzymes was analysed by paper chromatography. Chromatograms
were run in n-butanol-acetic acid-water (100: 20 : 4 v/v) for 20 hours anddeveloped with 0.296 ninhydrin-acetone solution.
In both, body fluid and hypodermis extract, the activity of amino- anddipeptidase was detected. So far as the present experimental results areconcerned, the extract of hypodermal tissues showed a stronger activityof alanyl-glycine dipeptidase than the body fluid. Furthermore it was
found that the activity of amino- and dipeptidase of hypodermal tissue
and body fluid gradually increased with time during the period from the
5th day of the 5th instar to mounting stage.
It is well known that the silkprotein, fibroin, is a very specific protein
composed of 4 main amino acids, i.e. glycine, alanine, serine and tyrosine, and that it is synthesized in gland cells of the posterior division of
the silkgland, especially in a later larval stage, i.e. mounting stage.
Although amino acids required for fibroin formation are partly synthesized in silk gland cells, it is not sufficient to provide a large amount ofamino acids which is necessary for rapid synthesis of fibroin. Consequently, there must be some other source of those amino acids.
Each of two materials used, normal (Daizo x W-C) and WO" strain, has
the following characteristics; in hypodermal cells of the former a large

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

amount of pteridine granules is produced, and in those of the latter suchgranules are scarcely formed. Using two strains, dry weight of the
middle and posterior division of the silkgland was measured at a definite
time during the period from the 5th day of the 5th instar to mounting
stage in order to examine the activity of fibroin secretion in silkglandcells.
It was found from experimental results that secretion of fibroin insilkgland of normal larvae was in the mounting stage more rapidly increased than in the wax mutant larvae.
Our previous report showed that a large amount of granules containing pterine pigment-polypeptide complexes and a uric acid-polypeptide
complex lose their function in larval mounting stage, and that polypeptides combined with pigment or uric acid are released from granules andexcreted from hypodermal cells into the body fluid. It is consideredthat in hypodermal cells and body fluid these polypeptides are decomposed by peptidases and that free amino acids derived from polypeptides
are absorbed into the gland cells of the posterior division of the silkgland and are an important source of the materials used for fibroin
synthesis.

Genic Analysis for Peroxidase Isozymes in Oryza sativa
and O. perennis
B. B. SHAHI, Toru ENDO and Hiko-ichi OKA

Eight strains belonging to the Asian form of Oryza perennis and O.
sativa were inter-crossed, and peroxidase isozymes of the hybrid plants
were observed by the starch-gel electrophoresis method. Isozyme bands
C2 and C4 were found to be specified by dominant genes pe cz + and pe C4+,
respectively. Bands A2 and A4 were allelic and controlled by co-dominant allels, pe AZ and pe A4 , respectively. Their heterozygotes showed zymogram A2-A3-A4, in which A3 was a hybrid dimer enzyme. From diallel
crosses among ten plants of a population of O. perenis, a regulatory geneRC4 was found which suppressed band C4.

Isozyme Variations to Organ Specificity in Oryza perennis
B. B. SHAHI, T. ENDO and H. 1. OKA

Variations in peroxidase and acid phosphatase isozymes among thevegetative and reproductive organs of three Oryza perennis strains wereRESEARCH CARRIED OUT IN 1968

35

observed by the starch-gel electrophoresis method. Each organ showed
its characteristic zymogram which was more or less modified with aging, while the variation pattern differed among strains. In the stigmas
before pollination, both peroxidase and acid phosphatase isozymes weresilent, but some of them became active after pollination. The acidphosphatase isozymes of the anthers containing pollen grains showedtrailing nature. In plants which were heterozygous for a locus specifying a set of allelic isozymes of peroxidase (pe A2 ; pe A4 ), the isozyme bands
were fully expressed only in a certain organ but were suppressed inother organs in different manners, and the pattern of suppression differedaccording to strains. Further, the lemma and palea and some other flower
organs showed particular bands which were absent in the vegetative
organs of the plant but were found in those of another distantly related
strain. It was suggested that the synthesis of isozymes in an organ
might be controlled by some inducer-producing genes which take partin organ differentiation.

Partial Purification and Characterization of Genetically Different
Acid Phosphatase of Human Red Cells
Tomotaka SHINODA

Partial purification and comparison of general characteristics weremade using three genetically different types of acid phosphatase [EC 3.l.
3.2, orthophosphoric monoester phosphohydrolase] isolated from normal
human red cells. Heterogeneity was observed on chromatography as wellas on starch gel electrophoresis made with CM-or DEAE-cellulose and
NaCl gradient. The partially purified enzyme appeared to show essentially the same electrophoretic pattern on starch gel as the fresh hemolyzate, except that a faster cathodal component was absent in the
purified material. Heat stability of acid phosphatase carried out at 50°C
was found to be different according to the phenotypes of the enzyme,
the order of their stability being B>AB>A. Average times at whicha half amount t 1/ 2 , of the original enzyme activity of types A, AB andB remained, were 4.1, 4.5 and 6.8 minutes, respectively. A change inelectrophoretic pattern on starch gel was noticed either by heat treatment or by incubation of the sample with oxidized glutathione. A newzone with slow mobility and reduced activity appeared by these treatments. The effect of guanidine on the enzyme was tested as a functionof its concentration, but no significant difference was observed among
different types of enzyme. Upon incubation with guanidine a rapid fall

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

of activity took place at a relatively low concentration of guanidine and,with increasing of its concentration the activity gradually decreased to1096 of its original strength. The partially purified sample was slightly
more susceptible to the treatment as compared with the enzyme ofstroma free hemolyzate.
When stroma free hemolyzate was submitted to gel filtration on a
Sephadex G-75 column, the enzyme, regardless of its type, was eluted
as a single peak slightly behind hemoglobin. This suggests that multiple isozyme components of each type are similar in molecular size. By
gel filtration, the specific activity was found to be increased several
times with respect to enzyme units per gram of hemoblobin. By combination with several procedures, types AB and B were purified approximately 1,500 fold.
Further characterization of purified enzyme is now going on.

Amino Acid Sequences Around Cystine Residues and COOHterminus of rM·g1obulin
Tomotaka SHINODA

Chemically pure K-type human rM-globulin was submitted to structure
analysis with special interest in the position of cystine residues in themolecule. Pepsin digest of intact rM-globulin was first treated with
Sephadex G-lO in N acetic acid, and the fractions containing disulfide
bridges were pooled and lyophilyzed.
Purification of the peptides was performed by column chromatography
and high voltage electrophoresis in a volatile buffer system. Amino acidsequences of respective peptides were carried out by Edman's degradation method. At present four disulfide bridges were identified, and theirsequences were as follows: 1) Val-Cys-Leu: Ala-Cys-Glu, 2) Ile-Thr-CysLeu: Tyr-Thr-Cys-Val-Ala-Val, 3) Tyr-Cys-Ala-Val-Val-Asn: Thr-Cys-ThrPhe-Ser-Gly, 4) Thr-Cys-Leu: Thr-Cys-Thr (Asx, Thr 2, Ser, Pro2' Val,
Leu, His, Lys) Leu-GIn. The COOH-terminus of ,u-chain was sequenced
as Met-Ser-Asx-Thr-Ala (Gly, Thr)-Cys-Tyr-COOH. The cystine residue
in this sequence was recovered in the S-carboxy-methylated form, indicating that it was involved in an interchain disulfide bond that was
selectively cleaved and alkylated during chain separation. The fragment
does not have the reported sequence adjacent to the disulfide bonding of
the heavy and light chains of a rM-globulin; and it is not analogous to
the proline rich sequences adjacent to the H-H interchain bonds of human and rabbit r-chains. Hence, the unique cystine residue of ,u-chins

RESEARCH CARRIED OUT IN 196837

is probably involved in the intermolecular disulfide bridge linking five 7
S rM monomer units to form polymeric 19 S macroglobulins. Indeed,
when CNBr fragments were prepared from the whole rM-globulin, the
COOH-terminal fragment was not eluted at the position of the S-carboxymethylated fragment, presumably because it was in the dimeric form.
Fragment 10-3 isolated from peak 4 (sequence: Gln-Arg-Gly-Glx-Pro-LeuSer-Pro-Gln-Lys-Tyr-Val-Thr-Ser-Ala-Pro-Met) has a strong homology insequence to residues 108 to 125 of the Fc region of rabbit r-chain. Although human p-chains appear to have an identical sequence for the
COOH-terminal pentapeptide, there is no similarity to the corresponding
portion of either human or rabbit r-chains.
The entire work was carried out at Indiana University, U.S.A.

Peroxidase Isozymes in Four Strains of Morning glory
Yoshiaki YONEDA

Among the stocks of morning glory, Pharbitis nil, we have in the
National Institute of Genetics, four geographical strains: TKS strain, anormal standard strain selected by Dr. Y. Takenaka from a Japanesecultivariety; Peking Tendan strain collected by Dr. H. Kihara in Peking
in 1938; Nepalese strain collected by Dr. S. Nakao in Nepal in 1952;
African strain collected by Dr. K. Furusato in Africa in 1959. This laststrain could be easily crossed with the other three strains, but the morphology of its leaf showed a reduced type and its flowers opened in late
September. In this report, the peroxidase isozyme patterns of leaves ofthese four strains were compared by the method of starch gel electrophoresis (Endo, T. 1968, Plant & Cell Physiol. 9:333). Peroxidase isozymes were detected by H 20 2 -benzidine staining.
As previously reported (Yoneda and Endo, 1967, this report 18:43), theTKS strain had 7 anodal peroxidase bands, designated as 1 A to 7 A fromthe original line, and one sharp cathodal band, designated as 1 C. The
1 C band appeared in leaves, stems and roots irrespective of age, withthe exception of seeds and young cotyledons. One line was found which
had no 1 C band (Endo, T. 1966, this report 17: 50).
The isozpme pattern of Peking Tendan strain was very similar to thatof TKS strain. It had a sharp cathodal 1 C band. The Nepalese andthe African strains had also a similar isozyme pattern to that of TKS,
except on the following points. The Nepalse strain had the same sharp
cathodal isozyme, 1 C, but one more band appeared between the fastest
moving anodal band (7A) and the second one (6A). In the African strain,

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

a few more bands were also recognized between 6A and 7A bands.
Another difference was found in that a sharp cathodal moved at 22 mmfrom the start line, in contrast to the 1 C band at 18 mm in the otherthree strains in four hour electrophoresis under 10 V/cm voltage gradient.
This band was designated as 2 C.
The African strain was crossed to TKS and Peking Tendan strains andthe F 1 hydrids were analysed for the cathodal band. All hybrids had
both 1 C and 2 C isozyme bands and no hybrid enzymes were observed.
This suggests that the cathodal isozyme bands, 1 C and 2 C, are controlled by different genes.
A new finding was that the TKS, Peking and Nepalese strains had incommon 1 C isozyme, while the African strain had 2 C isozyme.

RESEARCHES CARRIED OUT IN 1968

IV.

39

EVOLUTIONARY GENETICS

Intraspecific Differentiation of Cytoplasm in Aegilops caudata
Hitoshi KIHARA and Yasuo OHTA

Aegilops caudata L. includes two varieties, vars. typica and polyathera
Boiss. The former is awnless and is found mainly in Greece and Crete,
while the latter is awned and is distributed solely in Turkey.
The reciprocal hybrids between two varieties were easily obtained.
The chromosome behavior was normal throughout meiosis in F b but the
hybrids were almost completely pollen sterile.
As expected, with the advance of restoration backcrosses (RB) in the
F I between typica (9) and polyathera (0), the pollen fertility was strikingly increased until it became complete in RB 3 • On the contrary, thesubstitution backcross lines (SB c SB 3 ) were totally pollen sterile.
The reciprocal hybrids having the cytoplasm of polyathera gave quite
unexpected results. Both restoration and substitution backcrosses gave
rise to fertile offspring. A difference is only in the speed of recovering
full fertility. Contrary to our expectation, the RBI and RB 2 were completely sterile and from RB 3 fertility was getting higher until in RB 4 oneplant had completely dehiscent anthers with 94.4% fertile pollen and theremaining 14 had an average fertility of 55.796.
From these investigations, it may be concluded that two varieties ofAe. caudata are slightly different in their genome and also in the cytoplasm.

Interspecific Hybrid Between the Two Species of the Genus
Taeniatherum of the Tribe Triticeae
Sadao SAKAMOTO

The genus Taeniatherum comprises two diploid annual species (2n=14),
Tn. asperum (Simonk.) Nevski and Tn. crinitum (Schreb.) Nevski, distributing widely from Central Europe to Central Asia. A strain of Tn.
asperum (strain no. 7065) collected at Pul-i-Khumri, Afghanistan, wascrossed reciprocally with a strain of Tn. crinitum (no. 7064) found at
Karaji, Iran. Only three F I plants, one from Tn. asperum x Tn. crinitum
and two from the reciprocal combination, were obtained.
F 1 plants showed vigorous growth and marked heterosis was observedin the number of tillers, plant height, and length of top internode, flag

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

leaf and spike. However, the number of spikelets per spike was intermediate between the parents.
Average chromosome pairing per cell of PMC's of the F 1 was 0.2IV+
O.lm+5.81I+1.9I. Pollen fertility of non-dehiscent anthers of the F l wasvery low (0.2-0.5%) and no seeds were obtained at maturity.
From these observations it is concluded that the two diploid species of
Taeniatherm contain very similar but structurally differentiated genomes.

Scientific Expedition for the Study of Rodents to South East Asiaand Oceania VI. Serum Transferrin Polymorphism in Rattus
rattus Collected in Southeast Asia and Oceania
Kazuo MORIWAKI, Kimiyuki TSUCHIYA and Harumi SAKATA

Recently a genetic polymorphism of serum transferrin in Rattus mttus
has been found in the natural population of Japan (Moriwaki, K. ct at.
1969. Genetics, in press). Three types of transferrin pattern, TfR, TfRN
and TfN, could be identified by starch gel electrophoresis. TfR was
characterized by two fast moving bands, TfN by two slowly moving
bands and in TfRN all four bands were evident. These bands migrated
into the region between slow Q'l-globulin and fast Q'l-globulin. The overall frequencies of TfR, TfRN and TfN in the natural population of Japan
were 0.74, 0.21 and 0.05, respectively.
A similar kind of electrophoretic survey has been extended to serum
specimens of Rattus rattus collected in South East Asia and Oceania. The
same type of Rattus rattus as that in Japan was obtained in the former,
not in the latter. The localities and frequencies of TfR, TfRN and TfN
were as follows: Okinawa (32 samples) 0.75, 0.22, 0.03; Luzon (44 samples) 0.62, 0.33, 0.05; Thailand (39 samples) 0.15, 0.26, 0.59. Apparently
a higher occurrence of Tf N gene in the Thailand population seems to be
an important finding with regard to the origin of this gene in the species

Rattus rattus.
In Java and Makassar Battus rattus argentiventer is widely distributed.
All specimens collected in Java (16 samples) and Makassar (57 samples)
showed an similar pattern of transferrin as TfR of Rattus rattus in Japan.
A serum transferrin type, tentatively designated as TfRo, which exhibited a smaller mobility on starch gel than TfN of Japanese Rattus
rattus was observed in Rattns rattus of Oceania. All individuals collectedfrom New Zealand (22 samples), Australia (28 samples) and New Guinea(13 samples) were of this TfRo type. In F 1 hybrids obtained in our laboratory between Rattus rattus of Japan and that of Australia, a mixture

RESEARCHES CARRIED OUT IN 1968

41

of TfR and TfRo emerged, though they were somewhat faint.

Differentiation of Pathogenic Races of Piricularia oryzae
into two Groups, "Indica" and "Japonica"
Hiroko MORISHIMA

To obtain a summarized picture of host-parasite relationships, the datareported by Kozaka and Matsumoto (1967) for 484 varieties of Oryza sativa
from various countries of the world, each inoculated with 21 pathogenic
races of Piricularia oryzae collected from eight countries, were analysedfrom the viewpoint of numerical taxonomy. The two methods used,pattern analysis and principal component analysis, gave consistent results. The pathogenic races examined were clearly divided into twogroups, one mainly pathogenic to rice varieties of the Indica type andthe other to the Japonica type. They were called the "Indica" and"Japonica" race groups. The rice varieties could also be divided into
two major groups of differential reactions to the races, which were represented by the Indica and Japonica types. In addition, variations ingeneral resistance of rice varieties and in general pathogenecity of blast
races were observed. An about 40% fraction of the multi-dimensionalvariation was estimated to be due to general resistance, but the fraction
due to general pathogenecity seemed to be of lower magnitude. The
variation pattern in pathogenecity of the fungus seems to reflect that inresistance of the host, as if a shadow follows the shape of a body.
(Published in SABRAO Newsletter 1: 81-94.

Genetic Basis of the Weakness of F I plants in Oryza
breviligulata and O. glaberrima
Yaw-En CHU and Hiko-Ichi OKA

The weakness of F I plants was frequently found between strains ofO. glaberrima Steud. (cultivated) and O. breviligulata Chev. et Roehr.
(wild), which are endemic to West Africa. From the segregation patternof normal and weak plants in three-way crosses, a set of complementary
dominant lethals, WI and W 2 , was found to control the F I weakness.
About one half of breviligulata strains tested had either WI or W 2 , whilemost of glaberrima and semi-wild strains had WI only or neither. They
showed no particular trend of localization in geographical distribution.
The F I weakness was found to be due to a disturbance in tissue differ-

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

entiation of the adventitious roots, which was directly controlled by thecomplementary lethals. The undifferentiated tissues of induced callus,
derived from weak plants, grew as well as those from normal plants.

A Crossing Barrier Isolating Oryza perennis subsp. barthii
from its Related Taxa
Yaw-En CHU and Hiko·Ichi OKA

The African subspecies of Oryza perennis Moench, called barthii does
not easily produce viable F 1 seeds when crossed with its wild and cultivated relatives (other forms of perennis, O. sativa L., O. breviligulata
Chev. et Roehr., and O. glaberrima Steud.). When barthii is used as thepollen parent, the F 1 embryos and endosperms begin to deteriorate about
six days after fertilization, resulting in shrunken and inviable F 1 seeds.
When barthii is used as the maternal parent, the F 1 zygotes deteriorate
about three days after fertilization. In both directions of the cross, tissue differentiation in the zygotes was disturbed. The chance to obtain
viable F 1 seeds was in both less than 596. The results of crossing experiments using exceptionally obtained F 1 plants indicated that the crossing barrier was controlled by a set of complementary dominant lethals.
The difference in the stage of zygote deterioration between reciprocal
crosses was explained by the assumption that the genes would primarily
interrupt tissue differentiation in the endosperm. As barthii is a crosspollinated plant sympatrically growing with its wild and cultivated relatives, this crossing barrier may protect it from frequent hybridization.

Variations in Peroxidase, Acid Phosphatase and Esterase
Isozymes of Wild and Cultivated Oryza Species
B. B. SHARI, Hiroko MORISHIMA and Hiko-Ichi OKA

A total of 541 strains belonging to 21 Oryza species were observed forleaf-blade isozymes of peroxidase, acid phosphatase and esterase. The
species markedly differed in zymogram variability. Widely distributed
wild species were generally highly variable, while localized wild species
as well as cultivated ones showed a limited variation. Though certain
species had isozyme bands peculiar to them, identification by zymogram
seemed to be difficult on account of wide variations within species. Inview of the complicacy of the data, the technique of pattern analysis wasused for obtaining an integrated picture. In the scatter diagram thus

RESEARCHES CARRIED OUT IN 1968

43

obtained, closely related species were placed near each other, thoughstrains of a species were distributed in a wide range. In the scatter
diagram for acid phosphatase zymograms, species of the Sativa and Officinalis Groups were separated and strains of the latter were divided
into two groups having the bands of odd and even numbers. In both
peroxidase and acid phosphatase, the cultivated species, sativa and glaberrima, had no zymogram peculiar to them as all their zymograms were
present among those of their respective wild progenitors, perennis andbreviligulata. The zymograms of sativa varieties were only two which
represented the Indica and Japonica types, respectively, and it appeared
as if a part of perennis strains having certain isozymes could have been
domesticated. However, sativa varieties showed 19 different zymogramsof esterase, and the Indica and Japonica types differed in the frequenciesof those peculiar to sativa.

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

V.

MATHEMATICAL AND STATISTICAL STUDIESON POPULATION GENETICSThe Evolutionary Rate at the Molecular Level
Motoo KIMURA

From the data of amino acid differences between homologous proteins
and the total number of nucleotide pairs making up the genome, it wasestimated that in the evolutionary history of mammals, nucleotide substitution has proceeded roughly at the rate of one substitution every two
years.
On the other hand, based on the concept of the cost of natural selection (substitutional load), Haldane (1957) estimated that, in the standard
rate evolution, a new allele may be substituted in the population roughly
every 300 generations.
The large discrepancy between these two estimates may most easily
be interpreted by assuming that a majority of the observed differencesin amino acids between homologous proteins are the result of random
fixation of selectively neutral mutations.
If this interpretation is correct, we may conclude that random genetic
drift is playing a very important role in forming the genetic structureof biological populations.
For details, see Kimura (1968).

The Average Number of Generations until Fixation ofa Mutant Gene in a Finite Population
Motoo KIMURA and Tomoko OHTA

Since gene substitution plays a key role in the evolution of the species,
it is desirable to know the average number of generations until fixationof a mutant gene in a finite population (excluding the cases of eventualloss).
By using the diffusion models in population genetics, we have foundthe solution to this problem.
Let
Tj(p)= ~~ taU(ft, t) dt ,(1)

where u(P, t) is the probability that the mutant allele becomes fixed by

45

RESEARCHES CARRIED OUT IN 1968

the t-th generation, given that its frequency is p at t=O. Then, it canbe shown that T\(p) satisfies the following differential equation,(2)

where Mop and V op are the mean and variance of the rate of change inthe frequency of the mutant gene per generation and u(p)=lim u(P, t)
is the probability of ultimate fixation. The mean time until fixation,
t\(P), which is equal to T 1(p)/u(P), can be obtained as the solution ofequation (2), namely,
t~=

- L,I
t\(P)=¢(;)u(;){l-u(;)}d;+l-u(P)u(P),P

Jo ¢(;)u (;)d;.
2(3 )In the above formula,u(P)=~~ G(X)dX!~: G(x)dx and ¢(X)=2~:G(X)dX!WoxG(X)} ,in which
G(X)= exp{-~: 2~~;< d;} .

If the mutant gene is selectively neutral, formula (3) reduces to-1t\(P)= --p{4Ne (1-P) log (l-p)} .

(4)

For the cases of additive gene effect and overdominance, formula (3) was
evaluated by numerical integration. Generally, the time until fixation
becomes smaller with additive advantageous effect and larger with overdominance. The analytical results were verified by Monte Carlo experiments.
The details will be published in GENETICS.

Linkage Disequilibrium due to Random Genetic Drift
Tomoko OHTA and Motoo KIMURA

Linkage disequilibrium between 2 linked loci in finite populations may
be caused by epistatic interaction in fitness and/or by random genetic
drift. The latter effect on linkage disequilibrium was investigated byusing the diffusion models. In the present investigation the variance ofthe linkage disequilibrium coefficient was worked out, assuming no selec-

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

tion.
Let N e be the effective population number and c the recombination
fraction between the 2 loci, with alleles Al and A 2 in the first locus, andB I and B 2 in the second. We will denote by gt, g2' g3 and g4 the initialfrequencies of 4 gamete types, AIBt, A IB 2, A 2B I and A 2B 2 respectively.
Let P=gl+g2' q=gl+g3 and D=glg4-g2g3' Then the expected value ofthe square of linkage disequilibrium at time t can be expressed as follows,
E{D/}=t Ci[ pq(1-P)(1-q)+-..L(3+4R+22 i )
2(1+2

i=1

4

i)

X D(1-2P)(1-2q)+D2]eiifloVe(1)

In the above formula, 2;'s are the 3 eigenvalues of the Kolmogorov
forward equation involved. The coefficients C;'s are the functions of 2;'s
as follows,

1+2 3

•

C 3=- (21- 23)(22-2 ) (1. 1 +2 2+2Nec+2.5)
3Some numerical values of 2;'s are listed in the following table.
Table l.

Values of 2;'s for various values of Nec,
ranging from Nec=O to N ec=5.

Nec

-h

-22

-23

0.0
0.2
0.4
0.6
0.8
1.0
2.0
3.0
4.0
5.0

0.50
0.67
0.78
0.84
0.88
0.91
0.97
0.98
0.99
0.99

1.50
1.66
1.88
2.13
2.39
2.66
3.96
5.13
6.22
7.28

3.00
3.27
3.55
3.83
4.12
4.42
6.07
7.89
9.79
11.73

RESEARCHES CARRIED OUT IN 1968

47

From direct evaluation of formula (1), it was found that the variance ofthe linkage disequilibrium coefficient is fairly large with small Nec. Details will be published in GENETICAL RESEARCH.

Rate of Decrease of Genetic Variability in a Linearly
Subdivided Population
Takeo MARUYAMA

The asymptotic rate of decline of heterozygosity in a linearly subdivided population, in which migration occurs only between adjacentcolonies, was investigated. If we denote the number of colonies in the
population by n, the number of diploid individuals in a single colony by
N and the migration rate between adjacent colonies by m/2, the rate isapproximately equal to
mrr

2n

2

(1)

2

when
mrr

2

1
2Nn'

- -2 < - 2n

(2 )Thus for these cases the rate is proportional to migration rate but independent of colony size. When the inequality (2) is reversed, the rate
is approximately equal to
1

(3 )

2Nn'

Thus it is equal to the rate in a panmictic population of nN diploid individuals, and therefore the effect of subdivision of the population disappears as far as the rate of decrease in heterozygosity is concerned.
Approximation formulas for the eigenvectors associated with the dominant eigenvalues were also obtained. Let fii be the probability that
two homologous genes taken one from the i-th colony and the other fromthe j-th colony are identical by descent, and let h ii =1-fij. Then theasymptotic form of the matrix [h ii ] is given by

I· n+

i - j . i +j
ij oc
[h]
[ sm---sm--1

n+ 1I]"." ..

t, J= 1, 2 , ... n,(4)

when the dominant eigenvalue is most closely approximated by (1), andthe associated eigenvector of the eigenvalue approximated by (3) is

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

1 1
1 1[h'il= ; ;

(5 )

r

These results were checked by numerical calculations.
are presented in Tables 1 and 2.

A few examples

Table 1. Comparison of eigenvalues obtained by approximation formula(1), or (3), with exact values obtained numerically
Number ofcolonies
(n)
30
20
20
10
10
10
10
10
10
10
10

Colony
size
(N)
2
2
5
5
5
5
5
5
50
50
50

Migration
rate
(m)
0.4
0.4
0.1
0.05
0.05
0.03
0.01
0.005
0.005
0.003
0.001

10
10
10
10
10
10
10
10
10

10
50
50
150
500
1500
5000
1000
5000

0.5
0.2
0.15
0.05
0.05
0.05
0.05
0.005
0.005

Dominant Eigenvalue
Approximation Exact val ue by theby (1)
power method
0.002160.002130.00474
0.004360.001230.001100.002450.002140.002450.002410.001310.001480.000471
0.000494
0.000240
0.000247
0.000213
0.000247
0.000131
0.000147
0.0000472
0.0000490
by (3)
0.004640.000924
0.000901
0.000300
0.0000967
0.0000329
0.00000989
0.0000428
0.00000974

0.00500
0.001000
0.001000
0.000333
0.0001000
0.0000333
0.00001000
0.0000500
0.00001000

49

RESEARCHES CARRIED OUT IN 1968
Table 2. Comparison of eigenvector (matrix) given by (4) with exact
eigenvector obtained numerically by the power method. Becauseof identity hij=hji=hn-i+b n-j+l=hn-j+l, n-i+l of the elementsin the vector, only one quarter of them are presented.
n=10, m=O.OI, N=5.

~~

1

2

3

4

0.00050.01430.01080.03670.0296
0.06490.05490.09610.08430.12730.11490.15540.14380.17780.16820.19210.18590.19710.195100.00140.02230.02010.05050.04540.08170.07490.11290.10550.14110.13440.16340.15890.17780.176600.00220.02820.02740.05940.05680.09010.08760.11880.11660.14110.141100.00270.03120.03200.06240.06280.0906
0.0919

:J_'=

5_ _

i=

4

By formula (4)By power method
By formula (4)By power method
By formula (4)By power method
By formula (4)By power method5

By formula (4)By power method

1
2

3

6
7

8
9

10

By formula (4)By power method
By formula (4)By power method
By formula (4)By power method
By formula (4)By power method
By formula (4)By power methodo00.00300.03120.0336

One-dimensional Stepping Stone model of Finite Length
Takeo MARUYAMA

The so-called "stepping stone model" of population structure proposedby Kimura [1] was analysed in a one-dimensional case of finite length,with special reference to variance and correlation of gene frequencies
between colonies.
The analysed model is as follows. A population consists of n colonies
of finite size, each located at grid point of one-dimensional lattice of finite
length. A non-terminal colony receives immigration from each of its
adjacent colonies at the rate m/2 per generation, and a terminal colony
receives immigration only from its corresponding subterminal colony at
the rate m. We consider a locus segregating two alleles At and A 2 , andassume that each colony receives immigration, at the rate m= per gene-

c.n

0

>
Table l. Comparison of values by theoretical formulas (1) and (2), and by Monte Carlo method.
The diagonal entries of the table are variances and the off-diagonal entries are genetic correlations between colonies specified. n=10, m==O.OI, ml=O.I, 2N=50 and q=0.5
Colony

1

2

3

4

5

6

7

8

9

10

1

.0559
.0585

.7466
.7964
.0545
.0554

.5252
.5676
.6904
.6957
.0499
.0463

.3517
.3645
.4627
.4732
.6556
.6908
.0478
.0461

.2303
.2514
.3030
.3048
.4297
.4437
.6399
.6699
.0469
.0438

.1499
.1507
.1927
.1938
.2797
.2923
.4169
.4303
.6355
.6398
.0469
.0450

.0982
.1158
.1292
.1372
.1833
.1904
.2732
.2720
.4169
.4107
.6399
.6450
.0478
.0415

.0659
.0932
.0867
.1175
.1230
.1212
.1833
.1547
.2797
.2219
.4297
.3902
.6556
.6306
.0499
.0421

.0465
.0295
.0611
.0982
.0867
.0782
.1292
.1131
.1972
.1909
.3030
.3367
.4627
.4008
.6904
.6392
.0545
.0473

.0354
.0491
.0465
.0800
.0659
.0932
.0982
.1287
.1499
.1131
.2303
.2385
.3517
.3367
.5252
.5090
.7466
.7650
.0559
.0512

2
3
4
5
6
7
8
9
10

Z
Z

e:::

>
t""
::0
tIl

"0

0

(Theoretical)(Monte Carlo)(Th.)(M.C.)(Th.)(M.C.)(Th.)(M.e.)(Th.)(M.C.)(Th.)(M.C.)(Th.)(M.C.)(Th.)(M.C.)(Th.)(M.C.)(Th.)(M.C.)

..,

::0

0

"I1

Z

..,>
.0

Z

>

t""

.-

..,Z...,..,e:::
Ul

tIl

0

"I1

0
tIl
Z

..,

tIl

n
Ul
Z
0

.....
<D

RESEARCHES CARRIED OUT IN 1968

51

ration, from an external colony in which the frequency of allele Al isconstant with time and equal to ij.
An approximation of the covariance of the frequencies of allele Al between colonies at equilibrium is given by
2n-2

Cij=C

L:

k~l

cos

(i-1)(k-1);r(j-1)(k-1);r
n-1
cos
n-1

1- [ (l-m=) { 1-m ( 1- cos (k-1);rn-1)}]2 i, j=l, 2, ... nin which C is a constant and the subscripts indicate colonies.
the genetic correlation between colonies is

Therefore

Cij

(1)

1/ CiiCjj

And the variance III the frequencies of allele Al III the i-th colony isgiven byij(l-ij)

1+2N{1-2f32- a2-4apY1 ,2lij(l-ij)

Vi= 1+2N{1-2p2- a2-2ap(Yi, i-1+ Yi+l, i)-2f32Yi~l, i-Ii
fori=2,3,oo.n-1

(2)

and
where a=(l-m=)(l-m) and p=(1-m=)m/2.
The validity of the analysis was checked by Monte Carlo experiments.
One example is presented in Table 1.
[1]

Kimura, M., 1953. "Stepping stone" model of population.
Inst. Genetics, Japan 3: 62-63.

Ann. Rept. Nat.

Distribution of Parent-offspring Distance in Mishima District
Norikazu YASUDA

The previous report (Ann. Rep. No. 18, pp. 68-70, 1967) was about astudy on the distribution of matrimonial distance in Mishima district.
The frequency of couples whose marital distance is Y was derived froma square-root-exponential function; namely, C·y· exp(-kv'r), whereC=
k 4 /12 and k is a constant. The present report is concerned with the dis-

52 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

tribution of parent-offspring distance; namely, distance between birthplaces of parent and their offspring. This type of distribution in man
is important in studies on gene dispersion or migration.
Two histograms, each consisting of father-offspring and mother-offspring
distances, were preliminarily obtained from the Nakazato area of Mishima
district. Some characteristic features of the two distributions, in comparison with those of matrimonial distance, are summarized in Table 1.
Table 1. Some features of distributions of parent-offspring andmarital distance in Mishima district
Type of distanceFather-offspring
Mother-offspring
Matrimonial

Frequency ofzero distanceMode ofdistribution(km.)

442/988o74/988

2

11944

0.5

Type of migratory
function*
normalnormalsquare-root-exponential* Tentative.

Although conclusions are still tentative, the results suggest that theform of distributions between marital and parent-offspring distances seems
to be essentially different, particularly in the neighborhood of zero distance. Columns 2 and 3 of the table also suggest that migratory activity
is much higher in females than in males even in a sedentary population
like Nakazato.
The project will be continued to clarify the above points.

RESEARCHES CARRIED OUT IN 1968

VI.

53

EXPERIMENTAL STUDIES ON POPULATION GENETICS
Significance of Loci of the Persisting Lethal Genes
Chozo OSHIMA and Takao K. WATANABE

From the results of our experiments during the past five years, six
kinds of clustering lethal genes were confirmed to have been maintained
for more than three years in natural populations in Kofu and Katsunuma.
The loci of these lethal genes on the second chromosome were determinedby using dominant marker genes Sp (Sternopleural), BI (Bristle) and L(Lobe). They were divided into three groups; the first located at theterminal region of the left arm, the second located at the mid-left arm
and the third located near the centromere region. The results werecompared with that obtained by Spiess et al. (1963 Genetics 48: 13771388) and the similarity was found between the loci of persisting lethals
in natural populations and those of lethals clustering in a cage population. Since 1965, a visible recessive mutant gene, named reduced bristle(rb/), has been extracted every year at a frequency as high as 3-4 percent. The viability of homozygous flies for the gene was reduced tosemilethality. The locus was determined to be 82.3 by using two dominant marker genes, Land bw D (brown dominant).
About ten kinds of autosomal inversions were observed in natural
populations in Kofu and Katsunuma. Six of them have been maintained
at a relatively high frequency. The equilibrium frequencies of In(2L)B,
identical to In(2L)t and In(2R)C, identical to In(2R)NS were about 35 percent and 25 per cent respectively. In(2L)B suppresses completely the crossing
over between loci 0 and 48.5 and In(2R)C acts in similar way between loci70 and 100. The relationship between the mitotic chromosome and thegenetic map has been established (A. Hannah 1951 Advances in Genetics
4: 87-125, T. Hinton 1942 Genetics 27: 119-127). The terminal regionsof both arms and that near the centromere of the mitotic chromosome
were assumed to be the regions of infrequent crossing over. On thecontrary, two regions between 10 and 45 and between 60 and 100 lociof the genetic map were thought to be regions of frequent crossing over,because genes in these regions were concentrated in small parts of bothdistal portions of the mitotic chromosome. Loci 32 and 82 of the genetic
map were about at the midpoints of the frequent crossing over regions,but these two points might be included in the relatively infrequent partdue to interference with crossing over.
An adaptive gene complex might result from natural selection in such

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

crossing over infrequent regions. When a deleterious gene arises as
mutation in such regions, its deleterious effect in the heterozygote might
be covered by the epistatic actions of an adaptive gene complex of the
background.
The hypothesis that the paracentric inversions In(2L)B and In(2R)C
were adaptive gene complexes, may be supported by their persistence
and wide distribution in natural populations. If a lethal gene arose by
mutation in such inversions, it would be maintained naturally, and if it
occurred between the centromere and the end of inversions, it would bealso maintained. From the fact that I 201 gene (47.9) has been maintained for at least three years together with In(2R)C, an adaptive genecomplex may have been formed in the region induding the inversion
and both sides of the centromere.
Allelic Relations between Lethal Genes Distributedin Natural Populations
Chozo OSHIMA and Takao K. WATANABE

In late October of 1967, a particular collection of flies was carried out
Table 1.

Frequencies of intrapopulational aIIeIisms
and interpopulational aIIelisms.
Frequency
of intrapopulational
allelism

Population

Distance
betweenpopulations

Frequency
of interpopulational
allelism

Locality

Population

No. oflethalchromosome

Katsunuma

A
B
C
D

34
27
16
25

5.3596
3.70
2.50
13.33

A-B
B-C
C-D
B-D
A-C
A-D

430m
450
450
630
880
950

5.3 4 96
4.40
5.00
6.81
5.70
8.82

Mean

6.22%

Mean

632m

6.01%

10

6.67

E-A
E-B
E-C
E-D

14000 m
14000
14000
14000

6.47
7.04
3.75
9.60

Mean

14000 m

6.72%

E
Kofu

Overall allelic rate: 6.4496

RESEARCHES CARRIED OUT IN 1968

55

simultaneously at four sites in Katsunuma and one site in Kofu. The
distances between four sites; A, B, C and D were relatively short between 430 and 950 meters, and the distance between them and site E in
Kofu was about 14 kilometers. The allelism test was performed byintercrossing 112 Cy/lethal balanced strains extracted from 5 populations
located at A, B, C, D and E sites.
From the results of a total of 6216 crosses, allelic rates within the five
populations and between them were obtained as shown in Table 1.
The overall allelic rate (6.44%) was the highest among those obtained
for the past five years, but the relationship between allelic rate anddistance reported by Wallace (1966) could not be recognized. Almost
equal allelic rates between and within populations should be attributed
to many clustering and wide spread lethals. Most of them may havebeen persisting lethals for a long time in the natural populations. Either
such wide spread lethals were transplanted from a population to anotherone by migratory flies or have persisted already in those populations
could not be ascertained. However, most of them could be rather persisting ones.

Relationship between the Dispersal of Flies and the Frequencyof Allelism of Lethals in a Natural Population
Chozo OSHIMA and Takao K. WATANABE

One day of the beginning of October 1968, Drosophila flies were collected by 10 traps with the mixture of banana and dry yeast. These
traps were put almost linearly at intervals of 30 meters from the firstone which was put in a large natural population of D. melanogaster.
This site was located in the locality of Katsunuma.
All flies were caught by net four times between 10 AM and 3 PM fromthese traps except the first trap and adequate number of flies were caught
from the first trap. Flies totaled to 2699. Several species were foundamong them and their frequencies were as follows; D. melanogaster75.5.%. D. immigrans 17.9%, D. hydei 3.0%, D. lutea 2.796 and D. auraria
0.7%.
The dispersal of male flies of D. melanogaster was assumed to be more
active than female flies by the mean sex-ratio (2.16) of flies collectedfrom the second to tenth traps, but the sex-ratio of flies collected in thelarge population was 1.08. Results of the collection were shown in
Table 2.
As B. Wallace (1966 Amer. Nat. 100: 551-563) discussed on the dis-

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19
Table 2.

Number of D. melanogaster flies collected from traps and theresults of viability test of second chromosomes and ofallelism between lethal genes.

D. melanogastercollected
Trap

No. ofsecondchromosomes
testedNo. oflethalchromosomes

88
103
101
95
73

5
20
16
21
13

0.95 5.24 5.36 5.24
0.00 3.46 2.88 4.00 0.00

44

90.00 2.22 2.08 1.06 0.83 0.00

~ } 0.003.330.007.942.563.70 0.00
87 (15 .296)

1
2
3
4
5
6
7
8
9
10

230
139
100
118
42
17
8
26
29
0

249
342
245
237
91
33
17
42
2

37
30
0

Total

709

1330

571

72

Distance between traps
Allelic rate (916)

0-30m
4.63

Allelic rate (96) within (italic)
and between traps
1

2

3

4

5

6-7 8-90.00
1.006.32

2.507.195.00

60-90m3.33

120-150m
1.64

persal of Drosophila, the dispersal of a natural population decreased
linearly with the square root of distance. In our experiments, fairly
number of flies unexpectedly came into the eighth and nineth traps, but
its cause could not be found out.
Six hundred and eleven male flies among collected flies were matedwith virgin Cy/Pm female flies and viabilities of homozygotes for each571 second chromosome could be estimated in the F 3 generation. From
the results, 87 chromosomes (15.2%) carried a lethal gene and four chromosomes among them were presumed to have two different lethal genes
respectively by the allelism test.
Among a total of 91 lethal genes, 58 (63%) lethaIs were clustering ones
and classified into 14 allelic groups and other 33 lethal (3796) were single
ones. In the half diallel cross between these lethal strains, 143 crosses
among 3741 crosses were allelic and its frequency was 3.82 per cent.
The allelic rates between lethals isolated from flies collected from thesame and different traps were obtained as represented in Table 2. The
relationship between the dispersal of flies and the frequency of allelism

57

RESEARCHES CARRIED OUT IN 1968of lethals in a natural population of D. melanogaster in the locality of
Katsunuma was found to be similar to that observed by B. Wallace (1966
Amer. Nat. 100: 565-578) in a tropical population of the same species.

Frequency of Sterile Chromosomes Concealed
in a Natural Population
Chozo OSHIMA and Takao K. WATANABE

Total 473 non-lethal second chromosomes were isolated from about 600
male flies each, collected by 10 traps described in the previous report.
They were classified into three grades of viability in homozygous state
as follows; 31 semilethal, 36 subvital and 406 normal chromosomes. Then.
homozygous male and female flies for these individual chromosomes werecrossed with heterozygous female and male flies for Cy and the same
chromosomes respectively. Such experiments containing 473 x 2 crosses
were repeated for two generations. When no offspring could be obtained.
the chromosome was assumed to have a recessive sterile gene. The
results were given in Table 3.
Table 3.

Frequency of sterile chromosomes among non-lethal chromosomes.
Classes of viability in homozygote

No. of chromosome
No. of sterile chromosome(96)

No. of chromosome manifesting male sterility
No. of chromosome manifesting female sterility
No. of chromosome manifesting sterility of both sexes

semilethals

subvitals

normals

Total

31
13
(41.1)

369(25.0)

406
36
(8.9)

573
58
(12.3)

7

4

24

3

4

10

3

1

2

35
(60.4)
17(29.3)
6(10.3)

From the results, the interrelation between such physiological charactersas viability and fertility was thought to be intimate. About 41 per centsemilethal chromosomes were assumed to have also sterile gene and thefrequency of sterile chromosomes among subvital and normal chromosomes decreased. It should be analyzed whether the semilethality andsterility are manifested by different genes or by pleiotropic action of thecommon genes. The number of sterile chromosomes manifesting only

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

male sterility was about twice that of ones manifesting only female
sterility and about six times that of ones manifesting sterility of both
sexes.
Egg formation in the ovary of sterile female flies was remarkably
reduced, but the testis of sterile male flies could not be distinguishedmorphologically from the normal one.

Persistence of a Visible Mutant in Natural Populationsof Drosophila melanogastert
Takao K. WATANABE

A recessive visible mutant named reduced bristle (rbl) has been frequently extracted from the natural populations of Kofu-Katsunuma since1965. Its frequency in the population was 3.8, 3.1 and 3.5 per cent in
the successive annual samplings of 1965, 1966 and 1967 respectively. The
locus of rbl was determined to be 82.3 on the right arm of second chromosome and the mutant gene associated often with an heterotic inversion,
In(2L)B, on the left arm. The homozygous flies (rbl/rbl) had lower viability, developmental rate, female productivity and fecundity than normal
heterozygous flies (+ / +). However, heterozygous flies (rbl/ +) showedremarkable heterosis in productivity and fecundity though they were
neutral in viability and developmental rate (Table 4). The role of inversion, In(2L)B, was assumed to enhance the heterosis of rbl gene at leastin the cis-phase (In(2L)B rbl/ + +). In such a relation we may see themechanism of persistence of rbl gene in the natural populations.
Table 4.

Genotype

Viability

Developmental
rate (day)Productivity

Fecundity

+1+
rbll+

1.00
1.01
0.36**

10.7
10.6
12.2**

1.00
1.31**
0.66**

1.00
1.19**
0.64**

rbllrbl**t

Viability, developmental rate, productivity
and fecundity of rbl bearing flies.

Highly significant.

Watanabe, T. K., Japan. J. Genetics 44: 15-22 (1969).

RESEARCHES CARRIED OUT IN 1968

VII.

59

RADIATION GENETICS AND CHEMICALMUTAGENESIS IN ANIMALS

Repair in the Mutation Process Studied in Low and HighRadiosensitivity Strains of the Silkworm *Yataro TAZIMA

Based on our experimental results obtained recently with the use of
low and high radiosensitivity silkworm strains, the repair of radiationinduced premutational damages was briefly discussed.
A direct evidence of repair of premutational damages was obtained by
split-dose irradiation to spermatogenic cells, mostly spermatids, of fullgrown larvae. Applying similar treatments to several strains differingin radiosensitivity, we discovered that only low sensitivity strains werecapable to repair radiation induced pre-mutational damages, while sensitive
strains lacked this ability.
In order to gain better insight into the nature of repair, the effects ofpre- or post-irradiation treatment were compared between nitrogen andoxygen by subjecting mature larvae to those gases for 25 minutes before(including period of irradiation) or after irradiation. Post-irradiation
treatment with nitrogen or oxygen affected only slightly the mutation
frequency. In contrast, nitrogen pretreatment and irradiation in nitrogen
atomosphere (anoxic irradiation) decreased the frequency to an appreciable extent. The effect of dose-reduction of anoxic irradiation varied
depending on the type of mutation, being more pronounced for mosaics
than for whole-body mutants.
These findings seem to suggest that most premutational damages produced by anoxic irradiation on one of the double helices of DNA are
more likely to be repaired than those produced on both helices.

* Summary of a paper presented at the Small Symposia of the Twelfth International Congress of Genetics, 1968 held in Tokyo.

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

Comparison of Mutagenic Effects of 14 MeV Neutrons,
r-Rays and Some Chemical Mutagens Upon
Silkworm Spermatogenic Cells*
Yataro TAZIMA

Effects of different mutagens can be compared in several ways. Since
the double helix model of DNA suggests that there could be two typesof mutations concerning the site of occurrence, i.e. those occurring in
one of the double helices and those occurring in both helices, comparative
studies have been carried out with different mutagens with special regard
to the ratio of mosaics to whole mutants.
As reported previously, the mutation response of silkworm germ cells
to r·rays varied markedly with the advancement of the stage of gametogenesis not only with respect to induced frequencies but also with respect
to the proportion of mosaic mutants. The incidence of mosaics, when
expressed in terms of the ratio of mosaics to whole mutants, was very
low before meiosis, reached to the unity around V·4.5 stage and thereafter increased rapidly with the progress of spermiogenesis. Experimental
data used for the construction of the curve were obtained, for the mostpart, from 1,000 R irradiation but some were obtained from experiments,
in which higher doses were given: i.e. 3,000 R at mid·pupal stage and
5,000 R at the adult stage. It is known that the incidence of wholemutants increases linearly with increasing dose, while that of mosaics
increases more rapidly than linearly. However, since the mutation re·sponse is relatively low at such advanced stages as pupa and moth, thecurve can be utilized as a convenient standard for the comparison ofmutagenic effects among different mutagens.
Mutation spectra have been obtained at several developmental stages
by treating the insects with neutrons and chemical mutagens. 14 MeV
neutron beams were administered to the insects at V·1 and mid-pupal
stages. Radiation doses were 500 rad and 1,000 rad. For both doses
calculated ratios of mosaics to whole mutants did not appreciably differ.
They were clearly lower than those obtained for r·rays. Contrary to theeffect of neutron, chemical mutagens gave very high figures in the mosaic
ratio. The chemicals used were Mitomycin-C and EMS. Those chemicals
were administered to the insects by injecting their aqueous solution into
the body cavity of the insect at several developmental stages: i.e. III-I,
IV-3, V-I, mature larvae and early pupae. The injection was kept

* Summary of a paper presented at the International Symposium on Genetic
Effects of Radiation and Radiomimetic Chemicals held in Kyoto during the periodof August 30-31, 1968.

RESEARCHES CARRIED OUT IN 1968

61

constant for its concentration but varied in quantity according to thestage of the insect. Throughout all stages examined the incidence ofmosaics was extraordinarily high. For instance, injection of MitomycinC into IV-3 larvae (0.04%, 0.01 ml per head) gave the mosaic ratio as highas 11.8 and the ratio increased at more advanced stages. Injection at III-lsatage gave a somewhat lower but still significantly higher than thoseobtained for i-rays.
Above results are consistent with the expectation that mosaics inducedat postmeiotic stages of spermatogenesis are due to a mutational event
occurred in 0ne of the double helices of DNA.

Frequency Pattern of Mosaic and Whole-Body Mutants
Induced by Ionizing Radiations in Post-Meiotic
Germ Cells of the Male Silkworm *Yataro TAZIMA and Kimiharu ONIMARU

This work was undertaken in order to investigate the mutation spectra
induced by i- or X-rays in post-meiotic germ cells of male silkworm
with special regard to the change in the frequency pattern of mosaic(fractional) and whole-body (complete) mutants.
When silkworm spermatozoa were irradiated with i-rays, the frequencyof whole-body mutants increased linearly with increasing dose, whereasthat of mosaics increased exponentially. The latter relation is in striking
contrast to that observed in Drosophila, where the incidence of mosaics
has been known to reach a plateau at a relatively low dose level. Several
possibilities were considered for the interpretation of the difference.
Mosaics produced after irradiation of silkworm spermatozoa appeared toform a mixed class of at least two types, single-helix type and chromosome-aberration type. The proportion of both was assumed to varydepending on the irradiated dose.
The incidence of mosaics in comparison to that of whole-body mutantsvaried considerably with the progress of spermatogenesis. On a per Rbasis, spermatogonia yielded the lowest frequency of mosaics; thefrequencies of both complete and mosajc mutations increased sharply
through meiotic prophase up to V-4.5 (fifth instar larvae, day 4.5). Until
this stage relatively more completes than fractionals were produced.
Around V -4.5 the ratio of mosaics to completes reached unity. From
this stage onward, during spermiogenesis, relatively more mosaics thancompletes were produced, although the absolute frequencies of their*

Summary of a paper published in Mutation Research 8 (1969): 177-190.

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

induction was far lower than in V-4.5.
This observation may be interpreted as resulting from a greater capacity
for repair of premutational damage leading to mosaic mutations in theearly than in the late stages of spermatogenesis.

Mutagenic Action of Mitomycin-C and EMS on Pre-Meiotic
Germ Cells of the Male Silkworm
Yataro TAZIMA and Kimiharu ONIMARU

The double helix model of DNA can satisfactorily explain the highincidence of mosaic mutants in the F 1 obtained from the individuals
whose germ cells had been treated with Mitomycin-C or EMS at theirpost-meiotic stage. Results of such experiments had been briefly reported (this Annual Report 17: 94-95). The present report is concerned witha similar treatment of young germ cells of the male silkworm at premeiotic stages, L e. spermatogonia and spermatocytes. The chemicaltreatment was performed at the stages of the first, second or third instar.
Mitomycin-C and/or EMS were administered to larvae of the wild typestrain C108 either by adding the chemical to food (for first and second
instar treatment) or by injection (for third instar treatment). Afteremergence the treated males were mated to pe re females which permitted
to assess in the F 1 the induced frequencies both for whole and mosaic
mutations. The amount of in-take of those chemicals was compared on
per gram body weight basis.
The incidence of mosaics was negligibly small in both groups treatedat the first or the second instar but it was significantly high in the third
instar group. In contrast, the appearance of whole type mutants washighest in the first instar group and decreased as the treatment stage
advanced further. The latter observation was closely related to the sizeof the mutant cluster. The distribution of cluster size, Le. number ofsame kind mutants per batch, followed Poisson's equation in MitomycinC group but significantly deviated from it showing a long tail in EMS
group.
Those results are consistent with the expectations that the chemicals
produce mutations by affecting one of the double helices of DNA in
dividing cells as well as in non-dividing cells and that the affected cells
give rise to whole type mutants instead of mosaics, since in those cells
DNA multiplication occurs several times after mutation had taken place.
The evidence that the frequency of mosaics increased in the third instar

RESEARCHES CARRIED OUT IN 1968

63

treatment group seems to suggest that by this stage some fractions ofmale germ cells have already completed DNA synthesis.

Delayed Mutagenic Effect of Mitomycin-C and EMS
Observed in Silkworm
Kimiharu ONIMARU and Yataro TAZIMA

It has been reported by Alderson (1965) that EMS manifests its mutagenic action on Drosophila germ cells even at later cell generations
following the treatment. In our experiments with the silkworm we haveobserved a similar effect not only for EMS but also for Mitomycin-C.
Male larvae of wild type strain ClO8 were injected with 0.05~0.025ml
of physiological saline solution of Mitomycin-C (0.04%) or EMS (0.055'6)
at V-I (day-1 of the 5th instar) or V-7. Those males were crossed tope re females after emergence. The F 1 from those crosses comprised,in addition to the expected wild type, a fairly large number of wholetype mutants for pe and re and mosaic type mutants for pe+ and re+.
Among those F lJ only wild type insects were bred to the next generation.
Females of those wild type individuals, were back-crossed again to pe remales. Since those females are considered to be + +/pe re, the segregation of 1 pe : 1 + could be expected in the next generation, because pe isepistatic to reo Experimental results, however, showed that re eggs, inaddition to the expected pe and +, actually segregated in some batches.
The frequencies of appearance of such batches were 14 among 224 total
batches observed for MMC-V-1, 12/223 for EMS-V-1, 15/194 for MMC-V7 and 1/234 for EMS-V-7. The number of re eggs segregated varied
from 1 to 87 per batch. Further, the number of wild type eggs wasobserved to decrease below unity, in comparison to pe eggs in thosebatches where a large number of re appeared.
Since crossing over does not take place in the female of the silkworm,
it seems reasonable to attribute the appearance of re eggs among theoffspring of F 1 females to the delayed action of the mutagenic agents.
The observation that there was a considerable variation in the numberof segregated re eggs seems to suggest a difference in the time of actionof those agents during the successive cell generations of gametogenesis.

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

The Effect of Post-Treatment with Low Temperature on
the Frequencies of Radiation-Induced Mutationon the Silkworm Oocytes
Akio MURAKAMI

Repair of pre-mutational state is terminated by mutation fixation. This
process is invariably associated with DNA replication. There are many
reports with different organisms that in some experiments post-treatment
with metabolic inhibitors lowers the mutation-frequency. These findings
were interpreted as an effect of delation of mutation-fixation: the inhibitors, apart from actively affecting repair of premutational damages, slow
down the rate of mutation fixation, and thus give more opportunity forrepair to occur. There are many metabolic inhibitors known among
which low temperature is considered as a typical one. The present
experiment investigated the effect of low temperature treatment on the
X-ray induced mutation frequencies of the silkworm oocytes.
As the experimantal materials prophase I oocytes in moth are usedbecause the oocytes of this stage were well known to keep for long times
under cold temperature conditions. Furthemore, the oocytes in silkworm
have characteristics that make it possible to expect that the first DNA
replication after irradiation takes place at least 100 to 150 minutes after
oviposition (or irradiation): which correspond to stage from the latesecond meiotic division of oocytes to the first mitotic division stage ofzygotes.
The oocytes in wild-type C10B strain moths which were mated with
pe; re marker strain males for 5 hours after emergence were irradiatedby X-rays (180 kVp, 25 rnA, 10 mm Al filter and dose-rate at 250 R/min)with three different doses of 500, 1,000 and 1,500 R. Just after irradiation,these moths (or oocytes) were divided into two groups, one of them was
subjected to post-treatment with the temperature of 5°C for 5 hours inan ordinary ventilated refrigerator and the other under room temperatureof 25°C. All experimental procedures were done at 25°C except for thetime of irradiation. The mutational responses were estimated by the
egg-colour specific locus method. These data show a linearity on semilogarithmic plot in the relation between dose and frequency for wholebody mutation. This is in agree with the observation by Tazima (1964).
The experimantal data also indicated that radiation-induced mutation
frequencies were less in the group treated with low temperature of 5°Cthen in the non-treated group. By the way, significant differences havenot been shown in fractional-body mutation between X-ray alone andpost-treatment with low temperature groups because of the frequencies

RESEARCHES CARRIED OUT IN 196865

for this type of mutation found rarely to be observed for both experimental groups. In any case, these findings seem to agree with our
expectation suggesting that post-treatment with low temperature caneffect both repair and mutation fixation process. Further examinations,for example the changing the duration of cold-treatments and changing
the phase of irradiations, are in progress to gain an insight into themechanisms involved.

Studies on Strain Differences in Radiosensitivity in the Silkworm
VI. Further Report on the Screening of Sensitive andResistant Strains to Embryonic Radiation Killing
Akio MURALAMI

In a previous report (Annual Report, No. 17; 98-100, 1967), we showedthat there were striking strain differences in radiation induced embryonickilling. These findings indicated that among the 30 strains observed that
Table 1.

Race

Japanese

Chiness

Differences in radiosensitivity of the different
silkworm strains

Strain

LD 50 (R)Seihaku
Shoka
Kojiki (Beggar)
Kojiki (Matsumoto)
Chiyozuru
AgurisanKinkoshuOnichijira
Tanegashima
Okusa (Fujisawa)
Kokin
Amoi-moricaud
Hakuyoen
HekirenSisen-kinsan
Yoko
Asia-Oken
Shoko-abura

560
610
870
1,000
1,000
1,020
1,050
1,060
1,270
1,330
1,420
690
710
840
850
900
925
1,000

Race

ChineseSouthernasin

European

Strain

LD 50(R)

KaizyoKanko-syaken
KohokuKinto
No 5-Shokei
Nanko
Shinshocho
Ginto
RenshinTenmonTokuishin
Canbodge
BagdadCevennes
St. Julian
Szezard
Ascoli-oken

1,000
1,000
1,080
1,130
1,140
1,160
1,200
1,240
1,300
1,670
1,710
1,020
620
900
1,000
1,070
1,160
1,170

Spain

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

the LD 50 for the most resistant strain was approximately 1,500 R for
Kansen; while two strains were extremely sensitive, i.e., LD 50 were 240
R for Kojiki and 140 R for Ascoli.
Since we first reported on this subject, we have attempted to obtain
more additional information on radiation killing in different strains of
the silkworm. Thirty-six new different strains were selected and obtained to investigate their radiosensitivity to embryonic mortality of overwintered eggs. The strains used in this work were generously presented
by the National Sericultural Experiment Station, Japan. The experimental methods and conditions were quite the same, except for theexperimental year, as those reported in the preceeding paper.
The results obtained are summarized in Table 1. It may be seen fromthe table that there were differences in radiosensitivity among the defferent strains. The average LD 50 for 36 strains investigated was 1,040 R.
It seemed to indicate a somewhat higher LD 50 than 910 R of the previous
data in 1966. From this table it can be selected the two marked radiation resistant strains, i. e., Tenmon (1,670 R) and Tokuishin (1,710 R)
which are more resistant than those strains found in the previous report,
Aojuku (1,460 R) and Kansen (1,580 R). However, It failed to find out
the radiosensitive strain as had been found in the previous report, Ascoli
and Kojiki.

Effects of X-Irradiation on Aggregate-Forming Activity
and Sorting-Out Property of BeLa Cells
in Rotation Culture
Yukiaki KURODA

It has previously been suggested that in liver and heart cells dissociatedfrom chick and quail embryos low doses of X-rays may stimulate theactivation of aggregate-forming substances already present on the cellsurface and higher doses of X-ray may inhibit new production of thesesubstances through suppression of RNA and protein synthesis (Kurorla,
1968. Ann. Rep. Nat!. Inst. Genet. Japan 18 : 96). In the present investigation effects of X-irradiation on the function of HeLa cells were examinedby testing them for their aggregate-forming activity and sorting-out property from embryonic quail liver cells after irradiation with various
doses of X-ray.
Cell suspensions of HeLa S3 strain were obtained from monolayer
cultures at logarithmic growth phase, introduced into Petri dishes andirradiated with 0 R, 400 R, 800 R, 1,200 R, 1,600 Rand 2,000 R of X-rays

RESEARCHES CARRIED OUT IN 196867

(175 KVp, 25 mA, distance 40 em, filter 1.0 mm AI, dose rate 300 Rjmin).
After irradiation each cell suspension containing 106 cells in 3 ml culture
medium was transferred into a 25 ml Erlenmeyer flask and rotated on agyratory shaker at 70 rpm at 38°C.
Non-irradiated control HeLa cells formed after 24 hours of rotation
culture aggregates with an average diameter of 150 fl. HeLa cells irradiated with X-ray formed aggregates, whose shaps and average diameter were not significantly different from those of non-irradiated control
cells. Many cells irradiated with X-ray remained free as single cells inthe suspension after 24 hours of rotation culture. The number of thesesingle cells was gradually increasing with the amount of X-rays.
Irradiated HeLa cells were intermixed with liver cells dissociated from
7-day quail embryos, then tested for their sorting-out activity in rotation
culture for 24 or 48 hours by the standard procedure. Non-irradiatedcontrol HeLa cells became sorted-out from embryonic quail liver cells.
Both types of cells formed aggregates separately from each other andno chimaeral or mosaic tissues were formed. HeLa cells which wereirrradiated with various doses of X-ray were gradually losing their sorting-out property with the increase of X-ray dose. HeLa cells irradiatedwith 400 R still maintained their ability to form aggregates which werecomposed of HeLa cells alone. HeLa cells irradiated with 800 R or 1,200 R
formed aggregates in which many quail liver cells were interspersed.
HeLa cells irradiated with 2,000 R lost completely their sorting-out property from quail liver cells and formed chimaeral, or mosaic aggregatescomposed of both types of cells.
These changes in sorting-out activity of HeLa cells after irradiation
with X-rays may be useful as strictly controllable and allowing quantitative cell function to be detected after relatively short term cultures
following X-irradiation.

Postspermatogonial Rate for 14.1 MeV Neutron-Induced
Dominant Mutations Affecting the Skeleton of Mice
Kiyosi TUTIKAWA

The present study was designed primarily to obtain an estimate of
frequency of 14.1 MeV neutron-induced dominant mutations affecting theskeleton of mice. A method devised by Ehling was used (Ehling, U. H.,
1966, Genetics 54: 1381). The present experiment is an extension of an
earlier study (Tutikawa, K., 1967, Ann. Rep. Nat. Inst. Genet., Japan,
No. 18: 98).

68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

The methods were given in the previous report. In the present paper,
the progeny were selected for sampling gametes irradiated in postspermatogonial stages. Since the examination of the skeletons is still inprogress, some of the prepared specimens have been examined in detail.
The assumed mutations are briefly described as follows; 1) Control: (C188)
Anomaly of the skull (hydrocephalus); 2) Neutrons 242.5 Rad: (F73)
Longitudinal shortening of the skull, shape anomalies of the interfrontalbone and foramen ovale, presphenoid-basisphenoid fusion; (F1l7) Abnormal
frontals, presphenoid-basisphenoid fusion, dyssymphysis of the posteriorof C7 and Th1; (F161) Curvature and shortening of the radius and ulna
in both forelimbs; (F162) Absence of the left upper molar, dyssymphysisof the posterior of C5 and C7; (F230) Foramina transversaria imperfecta
in the C3 (both sides), C4 (both sides), C5 (both sides) and C6 (left side);(F245) Malformation of the sternum associated with whole intrasternalfusions and irregularities of the costosternal articulation, shape anomalyof the foramen ovale, shortening of the right tubercula anterius in theC6; (F272) Imperfect ossification of the right frontal bone, interfrontalfrontal fusion, right squamosal-parietal fusion. 3) Neutrons 485 Rad: (G5)Shape anomalies of the foramen ovale on both sides, foramina transversaria imperfecta in the C5 (both sides) and C6 (left side), fusion of
cervical ribs to the transverse process and centrum in the C7 (both sides);(G16) Imperfect ossification of the basisphenoid, shape anomaly of theforamen ovale (right side); (G17) Shape anomalies of the skull, interfrontalbone and foramen ovale, abnormal elongation of incisor, basisphenoidoccipital fusion, presphenoid-basisphenoid fusion, dyssymphysis of the C4,
foramina transversaria imperfecta in the C3 (both sides), C4 (left side)
and C5 (both sides); (G23) Longitudinal shortening and imperfect ossification of the skull, absence of the left tubercula anterius in the C6; (G133)
Foramen ovale open posteriorly (left side) ThlO-11 fusion; (G144) Shortening metacarpal and metatarsal bones, fusion of II-III metacarpal (bothmanus) and II-III metatarsal (left pes), malformation of all phalanges.
So far, one of the deviations assumed to be mutations (Gl44), has been
confirmed as such by breeding test. The estimate for the induced
dominant mutation rates was 7.3x 10- 5 per gamete per Rad for neutronsand 4.6 x 10- 5 for X-rays.

RESEARCHES CARRIED OUT IN 1968

69

Lack of Effect of Urethane on the Induction of
Dominant Lethal Mutations in Male Mice
Kiyosi TUTIKA W A

In the earlier studies (Tutikawa, K. and A. Akahori, 1964, Ann. Rep.
Nat. Inst. Genet., Japan, No. 15: 62; Tutikawa, K. and A. Akahori, 1965,
Congo Anom. 5: 166), a marked strain difference was found in the frequency of a certain kind of induced malformation between inbred strains
CBA and C3H~B/Fe, when the female mice were injected once with 1.5
mg per g of 1096 urethane after 8.5 days of pregnancy. However, thechanging pattern of urethane levels in blood withdrawn at each time up
to 15 h after injection of urethane did not reveal any difference between
those strains.
From the viewpoint that the active principle appears to be a metaboliteof urethane, an attempt was made for N-hydroxyurethan analysis, butfailed to reveal any N-hydroxyurethan in blood withdrawn after the
injection of 1.5 mg urethane per g.
Table l.

Analysis of matings of urethane treated male mice bydissection 14 days after mating
Week

StrainCBA

Controls
Corpora lutea (CL)
Implantations (IMP)
Early deaths (ED)Late deaths (LD)Living embryos (LE)ED/IMP)(ED + LD)/IMP 96
LE/CL
Corpora lutea (CL)
Implantations (IMP)
Early deaths (ED)C3H e B/Fe

Late deaths (LD)Living embryos (LE)1

ED/IMP(ED+LD)/IMP 96
LE/CL

1

2

137
132

110
104

3

4

5

131
122
115
104 107
116
8
8
8
3
6
2
2
3
1
2
122
94
105
100
99
7.7
2.9
6.1
6.9
5.6
7.6 9.6
9.5
3.9
7.5
89.1 85.5 80.2 82.0 86.1
52
43
5
1
37
11.6
14.0
71.2

35
33
2
0
31
6.1
6.1
88.6

63
54
7
1
46
13.0
14.8
73.0

26
25
2
1
22

34
33
5
1
27
8.0 15.2
12.0 18.2
84.6 79.4

6
36
35
4
0
31
11.4
11.4
86.1

218
204
18
2
184
8.8
9.8
84.4
323
292
29
0
263
9'9
9.9
81.4

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

The purpose of the present experiment was to provide an information
on mutagenic action of urethane in mice, using the dominant lethal
technique. Male mice of two inbred strains, CBA and C3H eB/Fe, were
injected once intra-peritoneally with 1.5 mg per g of 1096 urethane aqueous solution. After injection, each male was mated to 2 BALB/c females
each week for the next 5 to 6 weeks. The females were dissected on
the 14th day of pregnancy.
The results obtained from these males are given in Table 1. The
weekly frequencies of dominant lethals did not increase significantly overthe control values. Thus, it seems that urethane may lack the ability
to induce dominant lethals. I took notice of Bateman's report (Bateman,
A. J. 1967, Mut. Res. 4: 710) after my experiment started. His findings
are in agreement with mine.

RESEARCHES CARRIED OUT IN 1968

VIII.

71

RADIATION GENETICS IN MICROORGANISMS
AND PLANTS

A Mutant of Bacillus subtilis Possessing Modified Specificities
to Irradiated Transforming DNA
Yoshito SADAIE and Tsuneo KADA

A mutant named Sb-36 was isolated by treatment with N-methyl-N'nitroso-N-nitrosoguanidine of a substrain named H-17 derived from a
Marbourg strain of Bacillus subtilis. The parent H-17 and the mutant
Sb-36, both requiring arginine for their growth, have similar cellularradio-sensitivities to gamma-rays or ultraviolet light. However, whentransforming DNA extracted from a wild strain was irradiated withincreasing doses of gamma-rays and donated to H-17 or Sb-36, inactivation curves as to the arginine marker were much steeper by titrating on
Sb-36 than H-17. The observation is explained by assuming that the strain
Sb-36 lacks the repairing capacity of irradiated DNA whereas the strain
H-17 has it. This capacity was concerned with the transformation process but not with radiation-induced cellular lethality. Ultraviolet irradiation acted similarly to gamma-irradiation.
Similar specific sensitivities were also found with transforming DNA
treated with certain enzymes relating to DNA metabolism. Ando, T. and his
collaborators (J. Biochemistry, Tokyo, 66 (1969) 1) have recently isolated
from T4 infected cells of E. coli B two enzymes A and B having nicking
actions on DNA strands. Treatment of transforming DNA with enzyme
B reduced much more the yield of transformants using Sb-36 as therecipient strain than using H-17. Such specificity was more moderated
with enzyme A. Thus these enzymes aoted in a similar way to irradiations.
Above results indicate that gamma-irradiation as well as ultraviolet
irradiation may involve DNA damages of the similar nature as those due
to nicking action. It is supposed that the strain Sb-36 might lack arepair capacity on this point.

32P_Decay and Mutations in Escherichia coli B/r WP2 tryMasaru HAYASHI and Tsuneo KADA

Tryptophane reversions were studied in Escherichia coli B/r WP2

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

heavily labelled with 32p. The bacteria were grown in a modified Hmedium (Stent, G. and C. R. Fuerst, ]. Gen. Physiol. 38 (1955) 441),
contaning a low level of phosphorus supplemented with hot H 3 PO" formore than several generations, washed to eliminate extra-cellular material, and stocked at -20°C in H-medium suspensions without glucose butwith a 12.596 glycerine supplement.
The following points suggest that induction of mutations occurred inconsequence of decay events in addition to those induced by ,,-irradiation.
a) To observe mutations, labelled bacteria were washed and stocked
for different periods. Mutation inductions in such a stock were comparedwith a non-labelled control stock in which the bacteria were added only
to obtain radioactivity for example, approximately 300,uCi per ml which
is similar to that of the previous suspension of labelled cells. Higher
rate of mutation induction was observed in the course of stocking thelabelled cells.
b) When bacteria in stock were thawed and plated on minimal agar
plates with and without 2.596 supplementation of Difco broth, proportionsof mutants observed on these two kinds of media were different for ,8-ray
exposed cells and for 32 P-incorporated cells. Labelled cultures gave usually
over five times more mutants on broth-supplemented plates than on
minimal agar. Cultures exposed to ,,-irradiation gave only twice as manymutants with supplementation as those without it.
Many revertants were isolated and purified to study their genetic
nature. Approximately 7096 of mutants derived from a 32p labelled population could support the growth of the T4 mutant possessing an ochre
nonsense codon. No mutant permitted the growth of amber mutantsalone. This indicates that ochre suppressor mutations are predominantas Bridges, B. A. (Harwell) showed for mutations induced by ultraviolet
light or X-ray.
When efficiencies of decay events were calculated as to their lethal aswell as mutagenic effects it was found that the killing efficiency decreased
with increasing rate of labelling. On the other hand, the efficiency ofmutation induction was constant throughout the experiments when cells
were labelled with different rates. The higher was the level of labelling,
the more frequently were the reversions observed.

RESEARCHES CARRIED OUT IN 1968

73

Mechanisms of Radio-Sensitization with Iodine Compounds
Tsuneo KADA and Mitsuo NAMIKI*

Bridges, B. A. (Nature 188 (1960) 415) was the first to show that lethal
effects of irradiations are enhanced in the presence of certain chemicals.
Since then compounds containing iodine atoms such as iodoacetic acid
(Dean, C., and Alexander, P.; Nature 196 (1962) 1324), potassium iodide(Namiki, M., Okazawa, Y., and Matsuyama, A.; Agr. BioI. Chern. 25(1961) 108) or potassium iodate (Kada, T.; Int. J. Rad. BioI. 15 (1969) 271)
have been ascertained to be especially effective. In order to find if thesensitization involves enhancement of radiation damages in deoxyribonucleic acid, our studies have been carried out in the transformation
system of Bacillus subtilis.
Cells of B. subtilis 168 T- can be inactivated to the 50:'6 survival levelby 15 kR of gamma-rays of 137Cs. With this dose of irradiation the survival fractions were reduced to 1% in the presence of iodoacetic acid(0.3 mM), potassium iodide (2.5 mM) or potassium iodate (1.0 mM) in cellular suspensions in phosphate buffer solution (pH 7.0) prepared for irradiation. Detailed studies were carried out with potassium iodide. Deoxyribonucleic acid was extracted by phenol method from irradiated cells
and the transforming capacity was measured as to the arginine marker.
No remarkable difference in genetic capacity of DNA was observed
whether the bacteria were irradiated under nonsensitized or sensitizedconditions. Observations with alkali sucrose gradient technics showedthat the number of single strand breaks of DNA of irradiated cells was
not enchanced by addition of the sensitizer during irradiation. Whentransforming DNA was irradiated in vitro, inactivation of the tryptophanemarker was protected very much by addition of potassium iodide. Protective effect of the iodine compound was also found in radiolysis of four
DNA bases dissolved in water. These observations suggest that radiationinduced damages in DNA molecules were not enhanced by the presenceof the sensitizer, whether DNA was irradiated in vitro or in vivo. Therefore it is supposed that the radio-sensitization may not be due to theenhancement of non-specific damages in cellular DNA. These studies,briefly reported here, had been initiated in the Institute of Physical andChemical Research, Tokyo (Radiation Biology Lab.) and then continued
in this institute.

* Dept. of Agriculture, University of Nagoya.

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS ON. 19

High Bacteriocidal Action of Iodine Compounds Irradiatedin Acidic Solution
Tsuneo KADA

It is known that the cellular lethality induced by ionizing irradiationis very much increased by the presence of certain iodine compounds at
non-toxic levels in the bacterial solution. Most previous studies have been
carried out with neutral buffer solutions to which bacterial cells andchemical reagents, such as KI, KI0 3 or I·CH 2 ·COOH, were added andaltogether irradiated. Under these conditions, the presence of chemicals
at the time of irradiation was required in order to observe the radiation
sensitizing effect and the reagent solutions previously irradiated had little
toxicity. These observations indicated that radio-chemical reaction between some cellular key substances and the reagent were terminted in avery short time under irradiation.
Chemical reagent (1 mM)
None

KI

pH of reagent solution

Number of viable cells per ml

5.6
7.1
5.6
7.1
5.6
7.1
5.6
7.1

2.6xl0 5
3.0xl0502.4xl0 5

01.2x10501.2xl05

We have observed that iodine compounds irradiated in acidic solutions
were highly toxic. As Table indicates, bacteria cells were completely
inactivated at aoc by 1 mM reagent solutions previously irradiated with
10 kR of gamma-rays at pH 5.6. On the other hand no lethal action was
found at pH 7.6. Additional experiments have shown that the radiationproduced toxicity disappeared by heating to 37°C.
It was necessary to elucidate if similar mechanisms are actually involvedin the radiation-sensitization phenomena where cells are usually irradiatedin the presence of chemicals in neutral solutions. Studies are being
carried out on genetic effects of radiation products of certain stability
as found above.

RESEARCHES CARRIED OUT IN 196875

Action Spectrum of Photoreactivating Light in UV
Irradiated Maize Pollen
Etsuo AMANO

To study the mechanism of photoreactivation in maize pollen, kind of
mutation, and wave length dependence of UV induced mutation had
been investigated (Ikenaga et al. 1966, Amano et al. 1966 and Amano 1967,
the two last in the Ann. Rep. N.I.G. of 1966 and 1967). Both fractionaland entire endosperm mutations and both single and multiple locimutations of linked marker genes were induced by 250 mil UV light andphotoreactivated by post-irradiation with visible light.
The present report deals with the wave length dependence of photoreactivation of UV irradiated maize pollen. The materials used were
the same genetic stocks as used in the study on wave length dependenceof UV damages. Pollen of the multiple dominant stock, C I Sh Bz Wx,
was first irradiated in a single layer with 6 x 10 3 erg/mm 2 of UV fromfour 4 W germicidal lamps at the intensity of 100erg/mm 2 ·sec. Thenfollowed irradiation by monochromatic output of a UV monochrometer(Bausch and Lomb). A 500 W super high pressure mercury lamp (Philips,
SP-500) was used as a light source. PR light intensity as measured bya thermopile (Eppley) varied from 330 erg/mm 2 ·sec at 334 mil to 753 ergl
mm 2 ·sec at 365 mil. As a standard PR treatment, some of the pollen
grains were exposed to the PR light as in the previous experiments, i.e.
30 minutes, 6,000 lux of visible light from a high pressure mercury
fluorescent lamp. Variously treated pollen was used to pollinate an appropriate multiple recessive stock. Except during the treatment, pollen
was handled in a dark room illuminated by a yellow sodium lamp.
Pollinated ears were wrapped in alminum foil to prevent unnecessary
photoreactivation.
After harvest and drying, kernels showing mutations of any of thelinked marker genes (e I Sh Bz Wx) on more than 1/8 of the endosperm
surface were classified according to the expression pattern, area affected
and number of genes involved. Of the four near UV wave lengths
tested, none showed and ability to induce mutation in my experiment of
1967. At the comparable PR light dose (3 x 10 5 erg/mm 2 ) photoreactivation
was not significant at 365 mil and 405 mil. More effective photoreactivations were observed at 334 mil and 436 mil, the latter being the mosteffective.
Present results show the same spectrum for total mutant frequency,
single and multiple loci mutations, and entire and fractional mutations.

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

Wave Length Effect on the Photoreactivation of UV-Induced
Mutations in Maize
Taro FUJII

Photoreactivation of mutation induced by UV in maize pollen has been
recently observed (Ikenaga & Mabuchi 1966, Rad. Bot. 6: 165, Matsumura& Mabuchi 1966, Seiken Ziho 18: 1, Fujii 1968, Ann. Rep. 18: 102, Amanop. 75 in this issue). In order to check whether the PR of UV induced
mutation in maize pollen is of the direct or indirect type, the kineticsof the PR involved was compared at 3341 and 4047A. The method usedin this experiment is basically the same as that reported in the previous
paper. Pollen grains of a dominant line were exposed to 8000 erg/mm 2
of UV-light from a germicidal lamp emitting primarily 2537 A. Immediately after the UV irradiation, pollen grains were divided into four parts;
the first and the second parts were exposed to 3341A or 4047 A lightobtained from a Bausch-&-Lomb Monochrometor. The third part was
exposed for 30 minutes to visible light, emitting light from 4000 to 7000A,
and the last part was held for 30 minutes in the dark. After these postTable 1.

Decreasing UV-induced whole and partial mutations after photoreactivation
with luminescent-lamp light, 3341 or 4047 A.

UV-treatment PR-treatment(ergjmm 2 )(ergjmm 2 )Number
of
seeds
7964

8000

/I

4126
Visible*6.5x10'3341A
3 x 10'/I/I

1 x 105/I

3 x 105
4047A
3x 10'/I1 x 105II

3x 105*

Luminescent lamp light.

2303
2324
2753
1680
4014
2707
3016

Mutation frequency (96 ±S.E.)
Whole
1(0.01±0.01)
34(0.82±0.14)
9(0.39±0.13)
15(0.65±0.17)
23(0.84±0.17)
7(0.42±0.16)
20(0.50±0.11)
13(O.48±0.13)
9(0.30±0.10)

PartialTotal

103(2.50±0.24)
27(1.17±0.22)
46(1.98±0.29)
68(2.47±0.30)
36(2.14±0.35)
103(2.57±0.25)
56(2.07±0.27)
48(1.59±0.23)

1(0.01±0.01)
137(3.32±0.28)
36(1.56±0.26)
61(2.60±0.33)
91(3.31±0.34)
43(2.56±0.39)
123(3.06±0.27)
69(2.55±0.30)
57(1.89±0.25)

77

RESEARCHES CARRIED OUT IN 1968

treatments, the pollen grains were dusted on the silks of the recessive
female stock in a dark room. The experimental results are summarizedin Table 1. Two types of mutations, whole and partial, occurred andthe frequency of the latter was higher than of the former. As may beseen from the table, frequencies of whole and partial mutations at 8000
erg/mm 2 UV-exposure with dark treatment amounted to 0.82 and 2.50%,
respectively, and both kinds decreased by about 50,96 by visible lighttreatments. In the region of monochromatic light doses studied, it seemedthat 4047 A treatment reactivated the mutagenic lesion more steadily withincreasing doses than 3341A treatment. Further studies are required toascertain this point.

Relative Biological Effectiveness of 14 MeV Neutrons on Mutation
Frequency in Maize
Taro FUJIIThe frequency of gamma-ray and 14 MeV neutron induced endosperm
mutations (Bz-gene) was already reported by the present author (d. Ann.
Table 1.
Treatment (rad)0Gamma-rays
290
580
870
1450
Neutrons
69
127
154
284
295
545
2034
3700
10735

Mutated seedlings after pollen irradiation.
Number of seedlings(germination rate)

Number of mutants(96)1472 (97.7)

4 ( 0.27)

2601 (97.0)
1660 (96.1)
1370 (86.5)
2749 (79.9)

8 ( 0.31)
13 ( 0.78)
19 ( 1.39)
76 ( 2.76)

364 (87.5)
2454 (89.8)
4277 (92.1)
5894 (83.6)
4397 (82.3)
6160 (82.7)
339 (10.9)
9 ( 0.8)o( 0.0)4 ( 1.10)
25 ( 1.02)
50 ( 1.17)
115 ( 1.95)
101 ( 2.30)
157 ( 2.55)
40 (11.80)
5 (55.56)

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

Rep. 18: 103-105). In addition to Bz-gene, the materials used in thatexperiment had the dominant and the recessive Yg2 and yg2 genes (yellowish green character) in male and female stocks, respectively. Mutationfrom Y g2 to Yg2 was examined in F 1 seedlings and the results are given
in Table 1. In 1450 rad of gamma-ray treatment, germinability was only
slightly decreased, however a drastic effect on germinability was observedin the neutron treatments beyond 545 rad.
It is obvious that the mutation frequency after neutron irradiation isvery much higher than that after gamma-ray irradiation: by gamma-ray
irradiation 1.496 mutation was induced at 870 rad, while it could be induced
at less than 284 rad by neutron irradiation. From these results considering the preceding results (endosperm mutation), the RBE value of 14 MeV
neutrons after pollen irradiation was roughly assessed to be about 5.
Thus, a relatively low RBE value was observed in this material, since
it was pollen, being as such haploid in nature in contrast to the othermaterials which were diploid in nature somatic cells. Although theenvironmental variation is very small in high LET radiation, the low
RBE value of neutrons was also observed in the killing effect on presoaked
Arabidopsis seeds (d. Ann. Rep. 18: 107-108). Ploidy or water content
thus seems to be influence of RBE value.

Fractionation Experiments with Gamma-Rays and Neutrons in Maize
Taro FUJII

To investigate the fractionation effect of radiation treatments, 1400 radof gamma-rays and 600 rad of 14MeV neutrons, both adequate doses formutation experiments with maize pollen, were delivered as a single
fraction or in two fractions with 30, 60 and 120 minute intervals. Irradiated by gamma-rays and neutrons maize pollen with Bz-gene wascrossed to the recessive stock. Mutation from Bz to bz could be observedin the F 1 seeds with bronzy aleurone color and the results are given in
Table 1. In the gamma-ray lot, mutation frequency at single dose of
1400 rad was 2.25%, and frequency at two fractions each of 700 rad with30 minute interval was almost the same as that of a single dose. However, decrease of mutation frequency was observed with the increase ofinterval to 60 and 120 minutes as the table shows. In the neutron lot,on the other hand, mutation frequencies did not change within 30 to 120
minute interval with 300 rad as first and second dose; frequencies inthese lots are almost the same as that after a single dose of 600 rad.
The results suggest that some of premutational damage induced by gamma-

79

RESEARCHES CARRIED OUT IN 1968
Table l.

Relation between mutation frequency and fractionationof gamma-rays and neutrons.
Treatment

First dose (rad)Gamma-rays
1400
700
700
700
Neutrons
600
300
300
300

Interval (min) Second dose (rad)Numberofseeds

Mutationfrequency(;?6±S.E.)

10682

0

30
60
120

700
700
700

8574
7557
7341
15858

2.25±0.16
2.30±0.17
1.99±0.16
1.62±0.10

30
60
120

300
300
300

11086
9285
11056
15340

3.34±0.17
3.01±0.18
3.13±0.17
3.04±0.14

rays could be repaired during the interval, and recovery was enhanced
when the interval was extended. But such recovery did not occur whenneutron exposure was fractionated with up to 120 minute interval.

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

IX.

MICROBIAL GENETICS

Determination of the Length of Flagella in Salmonella
Tetsuo IINO

On Salmonella cells grown for 2 to 3 hr in a medium containing pfluorophenylalanine, the percentage of flagella with different numbers of
newly-formed curly waves was calculated for each group which had different numbers of pre-existing normal waves. All distributions fitted a
Poisson distribution. The average numbers of curly waves per flagellum
decreased with the increase in normal wave number and reached zerofor 5 to 6 normal waves. The lines plotted at different incubation times
were not parallel but convergent. The lower was the number of normal
waves in a flagellum the smaller was its whole length. The decrease in
curly wave number in the flagella with greater normal wave number is
attributed to the decline of growth rate with the increase in length.
When flagella were shortened by mechanical shaking of bacteria before
p-fluorophenylalanine treatment, the growth rate of flagella was not decreased as compared with that of intact ones of similar length. Thus
the decline of the growth rate is explained by a decrease in transportation efficiency of flagellin molecules with increasing length rather thanby ageing of the flagellum forming apparatus.
The length of flagella grown on bacterial bodies has an upper limit.
In Salmonella, the greatest number of waves seen on the wild type strain
was between 5 and 6 normal waves in ordinary culturing conditions.
This maximum corresponds to that at which the growth rate of flagellareached zero in the present experiment. It is likely that the mechanism
controlling the decline in growth rate of flagella also plays an importantrole in determining flagellar length in growing bacteria. (The detailsubmitted to J. gen. Microbiol.)

Polarity of Flagellar Growth in Salmonella
Tetsuo IINO

The phenomenon whereby Salmonella cells produce curly flagella inmedia containing p-fluorophenylalanine was used in an investigation of
the polarity of flagellar growth. S. tYPhimurium LT2 in logarithmic
growth in broth was transferred to minimal medium or saline. Then,after 2 to 3 hr at 37 C in a medium containing p-fluorophenylalanine the

RESEARCHES CARRIED OUT IN 196881

distributions of number, length and shape of their flagella were observed.
Under the condition where newly formed flagella were all curly, curly
waves appeared at the distal portions of flagella, indicating that a flagellum
grows at the end distal from the bacterial body. This agrees with thepolarity of flagellar growth in vitro.
A normal flagellar strain and a curly mutant strain of S. abortus-equi
were grown together in broth. Numbers of both types of cells andflagella increased to 1.5 fold in 3 hr. At this stage, neither heteromorphous
cells nor single flagella having both normal and curly waves were detected. Hence, flagellin molecules reach the top of a growing flagellum
without being excreted into the culture medium; the remaining pathway
by which flagellin molecules can reach the tip may be the tubular holeof the flagellum. (The detail submitted to ]. gen. Microbiol.)

A Method for Concentrating Paralyzed Mutantsfrom Non-Flagellated Cells of Salmonella
Masatoshi ENOMOTO

A method was invented for concentrating flagella-paralyzed (mar) cells
occurring in a large number of non-flagellated cells of Salmonella. The
mar cells expressing the phase-l flagellar antigen i were agglutinatedby anti-i serum with carriers, the dead cells of the wild-type TM2 expressing the phase-l antigen i; only the agglutinated cells were harvested
by low speed centrifugation and incubated in broth. About 10 fold concentration was achieved per one tretment which was repeated for further
concentration. By this method, the motA- motC- double mutant which
was produced by transduction at a frequency of about 3x 10- 6 per a nonflagellated recipient cell (motA - ahF) was isolated from the broth culture
concentrated five times, in which about 30% of the cells were the double
mutant. (The detail was submitted to Genetical Research, Vol. 14, 1969).

Further Studies on Different Frequencies of
Cotransduction in Salmonella
Masatoshi ENOMOTO and Shigeru YAMAGUCHIThe frequency of cotransduction of mote (motility) and H 1 (phase-I
flagellin) gene in Salmonella was investigated, using four Salmonella
species (S. dublin, S. abortus-equi, S. abony, and S. typhimurium)and many serotypic recombinants as donors and S. typhimurium mote82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

mutant as recipient. The frequency varied with the four species from
7 to 52%. By comparing these frequencies with those obtained fromserotypic recombinants, it was suggested that the difference in frequency
arose not only from differences in genetic homology between the chromosome of the recipient and the fragment from the donor, but also fromdifferences in genetic composition of the chromosome fragments carriedby the phage. The frequency of serotypic recombinants selected for
mot+ and Hi gene of the donor was generally higher than with recombinants selected for flaK+ and Hi. The difference in genetic homology
between S. typhimurium and other species was more marked in the region
between Hi and flaK than between mote and Hi. (The detail was submitted to Genetical Research vol. 14, 1969).

Stability and Phenotypic Expression of Mutation to
Arginine Sensitivity in Salmonella
Jun-ichi ISHIDSU

To elucidate the mechanisms of phenotypic expression of Salmonellamutants which show sensitivity both to arginine and to uracil, the patternof back mutation and the characters of the back mutants were examined
under various culture conditions.
Arg-s-3 was first grown in liquid minimal medium supplemented witharginine and uracil. After washing, cells were transferred into the following media: 1) minimal, 2) minimal plus arginine, 3) minimal plus
uracil, or 4) minimal plus arginine and uracil. Incubation was continued
for one to three hours, then after washing, samples were plated onminimal agar plates containing arginine or uracil. Number of colonies
which appeared after incubation was largest in the case of 1), where botharginine and uracil biosyntheses had been derepressed beforehand, and wassmallest in the case of 4), where the two pathways had been simultaneously repressed. When one of the two pathways had been repressed (inthe case of 2 or 3), the number was intermediate.
Such "revertant" colonies appearing on minimal medium supplemented
with either arginine or uracil were picked up and their characters were
further examined. In either case, only less than 10 per cent were true
revertants, losing sensitivity to both chemicals simultaneously. Manyof such "revertants" had lost the sensitivity to only one of the two
chemicals or had gained auxotrophic requirement for uracil. Unexpectedly,
most of the colonies that appeared on arginine-supplemented minimal
agar plates showed the original, unchanged property on further tests,

RESEARCHES CARRIED OUT IN 1968

83

namely they still carried the sensitivity to both chemicals.
These phenomena altogether suggest that the mechanisms of phenotypic expression of this mutation is very complicated and greatly affectedeven by slight changes in culture conditions. More detailed tests arebeing carried out and will be reported elsewhere.

High Frequency Mutations, Drug Sensitivity and ALysogenization in Escherichia coli K12
Tsuneo KADA

Previous studies of high frequency reversions in a threonineless strainof Escherichia coli K 12 Hfr H have shown that revertants due to aplural suppression had increased sensitivities to irradiations and the
majority of cells formed filaments during post-irradiation culture. Observations of the growth of T 4 nonsense mutants on typical bacterial
strains indicated that two suppressor genes involved, one (mod) locatingin the leu region and the other (fgr) near gal, were neither of amber
nor of ochre type. It was especially noted that mutations fgr- ~fgrt­
were very frequent (more than 10- 3 ) in mod+ strains.
In certain streptomycin-resistant and poly-auxotrophic F- strains having
mod- andfgr- genes, approximately 30% of lysogenized strains with phage
A became sensitive to the antibiotic. In these strains, reversion rates
increased considerably simultaneously at many loci. Many kanamycinresistant and neomycin-resistant strains were isolated from a typical above
mentioned F- strain and lysogenized with phage A. Approximately 10%of lysogenic strains became sensitive to kanamycin. Reversion-rates also
increased drastically at many loci in those strains whose sensitivities toantibiotics were modified. Phenotype of revertants was that of fgr+.
Above observations were interpreted as follow: Phage A might havetransduced specifically certain suppressor gene into the lysogenic strains,
and the rate of mutation fgr- ~fgr+ increased very much in its presence
as was the case of mod+ strains of Hfr H. Functions of these suppressor
genes may concern the ribsomes.

84

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

X.

HUMAN GENETICS

Delayed Fertilization and Down's Syndrome
Ei MATSUNAGA and Takeo MARUYAMA

German proposed that decreasing frequency of coitus with advancing
age which predisposes to delayed fertilization could account for the correlation of increased incidence of Down's syndrome with advancing maternal age. This hypothesis is especially interesting because it points
out a possible biological consequence of human sexual behaviour.
In order to test the hypothesis, we first analyzed the original data by
Kinsey et al. on the frequencies of marital coitus for females of different
age groups and found that their variation among individual couples fora given age group can be approximated by a Poisson distribution. Although this finding does not necessarily imply that coitions themselves
follow a Poisson process within individual couples, we could hypothesize
an "average" couple whose coitions follow a Poisson process with themean frequency of that group, the resulting variability within this couple
in the interval between coitions representing the corresponding variability
from couple to couple belonging to the group. It is further assumed
that ovulation occurs independently of the occurrence of coitus, so thatthe interval between an ovulation and the following or the preceding
coitus, t, also follows a Poisson process, its probability density being expressed byp(t)=J.e-J.t

where i. is the inverse of the mean interval between coitions for thegroup concerned.
The above assumptions led us to construct the following formula which
expresses the expected frequency of non-disjunction for a given age group:F().)~: J.e-J.tft(t)dt+~:J.e-l~2(t)dt-~:J.e-ltfl(t)dt~: J.e- uf2(t)dt

where a and (3 are the maximum life spans of the sperm and the secondary oocytes in the female reproductive tube respectively, fl(t) and f2(t)are respectively the probabilities that the male and female gametes remain fertilizable, and get) the frequency of non-disjunction. The valuesof a and (3 may be taken, according to German, as 48 hand 24 h respec-

85

RESEARCHES CARRIED OUT IN 1968

tively. Assuming that both fl(t) and f2(t) decrease linearly while g(t)increases either linearly or exponentially with t, various values of A calculated from the data of Kinsey et at. were applied to the above formula.
It was shown that the expected frequency of non-disjunction with advancing age was rather slow; the frequency in the oldest group was lessthan twice as high as that in the youngest group, indicating that German's
hypothesis cannot account for the well known steep rise in the incidenceof Down's syndrome in older mothers. The details of this work were
published in Nature 221: 642-644, 1969.
It may be worth while noting that although we have made several
different models for fl(t) and f2(t) other than those given above, the final
results were essentially the same. For example, if we assume that both
fl(t) and fit) decrease exponentially while get) increases in similar fashion,

Le.,
fl(t)

e t -1and g(t)=-~-1-ke"-e te -e"-1Table 1. Frequency of non-disjunction for different age groups, as expectedfrom delayed fertilization; the maximum life spans of sperm and oocytes
are assumed to be 48 hand 24 h, respectively, their capacity for fertilization decreasing exponentially while the frequency of
non-disjunction increases exponentially
Kinseys' data on
marital coitus
of females
Age group

16-20
21-25
26-30
31-35
36~40

41-45
46-50
51-55
56-60

Mean
frequency
per week

3.7
3.0
2.6
2.3
2.0
1.7

1.4
1.2

0.8

Expected
frequencies
of
non-disjunction

Meaninterval(in hours)

45.4
56.0
64.6
73.0
84.0
98.8
120.0
140.0
210.0

0.022
0.018
0.016
0.014
0.012
0.010
0.008
0.007
0.005
0.000

k X 10- 2
0.304
0.360
0.391
0.424
0.459
0.496
0.535
0.556
0.598
0.714

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

where k is a constant, we obtain,lk
F(J.)= (2-,l){e.l'a'fJl(1-,l)-l)

and its limiting value as ,l approaches to zero will bekF(,l~O)= 2(a+p-2)
Table 1 summarizes the results of computation of F(,l) according to a=
48 h, 13=24 h and different values of ,l based on the data by Kinsey et at.

Maternal Age of Chromosome Translocations with
Down's Syndrome
Yasumoto KIKUCHI, Hidetsune OISHI, Akira TONOMURA!l
and Ei MATSUNAGA

It is well established that the condition of the majority of patients with
Down's syndrome is due to trisomy for chromosome no. 21, and that thematernal age at the birth of the patients is, on the average, higher thanin the control population. On the other hand, cases with (DqGq) or (GqGq)translocations are less frequent, and information of maternal age for thetranslocations has so far been very scarce.
Thirty one cases of Down's syndrome with such translocations have beencollected from our consecutive survey. As shown in Table 1, mean maTable 1.

Mean maternal ages for the translocation types

Translocation type

Number of cases
examined

Mean maternal age

Sporadic
Inherited from motherInherited from father

227227.8±3.83
29.6±3.25
25.5±2.50

ternal ages for 22 sporadic, 7 maternally inherited and 2 paternally inherited translocations were 27.8 years, 29.6 years and 25.5 years, respectively.
The maternal age distribution for 22 sporadic cases is given in Table
2. The expected number for each maternal age group, classified by five1)

Department of Cytogenetics, Tokyo Medical & Dental University.

RESEARCHES dARRIED OUT IN 1968
Table 2.

Maternal age(years)87

Maternal age distribution of cases withsporadic translocation
Observed

Expected
0.26
6.06
10.05
4.30

40~

0
4
9
7
2
0

Total

22

22.00

Mean age
S.D.

27.82
3.83

27.14
4.61

15-19
20-24
25-29
30-34
35-39

1.11
0.22

year's interval, was constructed from vital statistics data and was corrected for the year of birth of the patients. According to this procedure,
however, the 6 cases born in the years 1966-1968 were included in thegroup born in 1965. Using the median value of each quinquennium, themean maternal age was estimated to be 27.1 years for the general population, which is almost the same as the mean age (27.8 years) for themothers of the patients. There was also no difference in the distributionof the maternal age between the observed and the general population.
The above results may suggest that the occurrence of sporadic translocation Down's syndrome is independent of maternal age at the patient's
birth. It is, however, necessary to separate cases of (DqGq) translocationfrom those of (GqGq), so that more data are needed in order to arrive ata definite conclusion.

Clinical Conditions of Patients with Apparently
Normal Chromosomes, V.
Hidetsune OISHI, Yasumoto KIKUCHI, Masaru KUMAGAI and
Kunihiko SHIBATA

Based on leucocyte cultures of peripheral blood, patients with various
pathological conditions were found to have 46 chromosomes of apparently
normal karyotype. Cases of some interest are listed below.

88

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

Name

Age Legal sex

Clinical conditionsa) Multiple deformations with mental retardation
84

J.M.

M

85 M.Y.

1 month

F

86

M.S.

2 months

F

87 T.O.

2 months

F

88

6 months

F

H.T.

89 Y.O.
90

Y.Y.

9 months F
1 year
M

91

K.T.

1 year

M

92

S.H.

9 years

M

93

M.W. 4 months F

94

K.K.

7 yearsF

95 S.S.

4 months M

96 H.I.

4 yearsF

Osteogenesis imperfecta; high-arched palate; malformed ears; transverse palmar
creasesHypertelorism; antimongoloid slant eyes;
malformed and low-set ears
Brachycephaly; hypertelorism; low-set ears;
short neck; congenital heart disease
Exophthalmos; epicanthus; low-set ears; higharched palate; transverse palmar creases
Cleft palate; congenital heart disease; mild
coarctation of aorta; overlapped fingers
Microcephaly; hemangioma; galactosemia
Antimongoloid slant eyes; low-set ears;
micrognathia; transverse palmar creases;
retentio testes
Cleft palate; malformed ears; congenital
heart disease; umbilical hernia
Hypertelorism; antimongoloid slant eyes;
large deformed ear lobes; low-set ears
Microcephaly; lacunal skull; exophthalmos;
overlapped toes; lipochondrodystrophy
Antimongoloid slant eyes; coloboma of theiris; nystagmus; epicanthus
Microphthalmia; incurved fifth finger withclinodactyly; transverse palmar creases
Prominent occiput; strabismus; nystagmus;
hypomyotonia; logopathyb) Sex anomalies

97 Y.K. 7 months F
98 K.T. 25 years
F
99 T.K. 30 years
M
100 M.S.

4 daysF

101 S.A.

3 yearsF

Female pseudohermaphroditism
Male pseudohermaphroditism
Slender body; short neck; shield chest; gynecomastia; small penis; atrophic testes(Klinefelter's syndrome ?)Webbed neck; excessive hair-growth(Turner's syndrome?)
Female pseudohermaphroditism; high-arched palate; excessive hair-growth

RESEARCHES CARRIED OUT IN 1968

102 I.M.

5 months F

89

Female pseudohermaphroditismc) Hereditary disease
103 -. S. 19 days
104 K.K. 1 year

F
F

Dysostosis cleidocranialis
Dysostosis cleidocranialis;
webbed neck; saddle nose

hypertelorism;

Cytogenetical Studies of Human Ova, I. A Preliminary Noteon the Oocytes Liberated from the Ovary
Hidetsune OISHI and Yasumoto KIKUCHI

In order to investigate the meiotic chromosomes of human ova, ovarian
oocytes were collected in collaboration with some university hospitals.
Six oocytes were obtained from 5 ovaries by puncturing large follicles
during gynecological operations of the patients for various clinical conditions. In other instances when whole ovaries were removed in theoperation, comparatively large follicles were dissected under a dissecting
microscope, and 53 oocytes were collected from 7 ovaries. These oocytes
were surrounded by several cell layers of corona radiata, and were similar
to those obtained by puncture. However, 4 of these collected from ovariotomy showed signs of degeneration; they were partially devoid of corona
cells, and the vitellus was deformed though the shape of zona pellucida
was spherical. An ovum with blastomeres was also found in 4-cell stage;
it was possibly formed by parthenogenetic cleavage.
Thirty oocytes were used for the examination of meiosis. In order toremove the corona cells, the oocytes were treated with hyaluronidase in
hypotonic solution. Air-dried preparations were made with a modificationof Tarkowski's method (Tarkowski, A. K. 1966. Cytogenetics 5:394) andstained with carbol fuchsin. All had a germinal vesicle in dictyotene
stage.
In vitro cultivation of the ova is under examination.

Studies on Several Genetic Hematological Traits of the JapaneseTomotaka SHINODA

Polymorphic genetic systems recognized in man have revealed striking
differences from one population to another, not only in specific gene frequencies but also in the presence or absence of particular genes.
Since 1963, I have analyved several polymorphic hematological traits

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19of the Japanese. The results were partly already reported.
Acid phosphatase in red cells determined by three autosomal allelicgenes, namely pa, pb and pc, occurs in six types. However, occurenceof phenotypes associated with the pc gene, such as AC, BC and C, seems
to be very rare in the Japanese population. With regard to this finding
analyses of a total of 612 samples collected from unrelated persons havebeen carried out.
Of 612 samples examined 26 (4.2,Q6) of type A, 192 (31.496) of type AB
and 394 (64.4;6) of type B were identified. Accordingly, the frequenciesof the genes pa and pb may be estimated as 0.199±0.011 and 0.801±
0.011, respectively. No single case of types AC or BC was found. A
complete lack (or an extreme rarity) of the gene pc seems to be a characteristic feature of the Japanese population. The weighted mean enzyme
activity attributable to each of the two alleles was estimated approximately as for pa 56.2 units and for pb 91 units, the ratio being close to 2: 3.
Phosphoglucomutase (PGM) occurs in multiple forms and a number ofinherited variants have been described. They appear to be controlled byalleles at three distinct and not closely linked autosomal loci, each ofwhich determines a separate group of PGM isozymes.
A total of 932 blood samples collected from unrelated adults have so
far been analyzed and the result is summarized in Table 1. From theTable 1.

Observed and Expected Numbers of PGM1 Types
among the Japanese
Expected*

Observed
No.

0/
/0

No.

0/

1
2-1
2

567
307
58

130.84
32.94
6.22

556.9
327.1
48.0

59.75
35.09
5.16

Total

932

100.00932.0

100.00* On the basis of Hardy-Weinberg law with allele frequencies:=0.773 and PGM12=0.227.

/0

PGMII

data of Table 1, the frequencies of the genes PGM/ and PGM1 2 may be
estimated as 0.773 and 0.227, respectively. The above value of PGM 1
appeared to be somewhat higher than that of British whites, but the difference was not significant. No variation was found in the PGM2 isozyme
region. Although variation was observed in the PGMg isozyme region,
the data were not sufficient for the estimation of gene frequency.

91

RESEARCHES CARRIED OUT IN 1968

The haptoglobin, a serum protein which binds with hemoglobin in vivo
and in vitro occurs in three common types, namely 1-1, 2-1 and 2-2, which
are determined by a pair of allelic genes Hpl and Hp". A total of 629
samples collected from unrelated adults were tested by starch gel electrophoresis. From the data summarized in Table 2, the frequencies of theTable 2. Observed and Expected Numbers of Haptoglobin Types
Assuming a Hardy-Weinberg Equilibrium with Gene Frequencies
H p l=0.242 and H p 2=0.758 among the Japanese
Hp typeObserved

Expected

No.

/Q

0/

No.

96

1-1
2-1
2-2

42
220
367

6.68
35.14
58.18

36.8
230.8
361.4

5.86
36.67
57.47

Total

629

100.00629.0

100.00genes Hpl and Hp 2 were estimated to be 0.242 and 0.758, respectively.
The frequency of the Hpl gene observed for the Japanese is somewhat
higher than that of the Indian population, but is significantly lower thanthose of American whites and Negroes. No incidence of a modified type,such as Hp 2-1 (Johnson) often reported for the Negroes was found in
this survey.

Polymorphism of Prealbumin Fraction in Human Serum
Yoshito OGAWA and Norio ODAKI

When human serum is subjected to zone electrophoresis in starch gelwith a sodium acetate-ethylenediaminetetraacetic acid buffer of pH 4.95,
several bands migrate in front of the albumin fraction. These rapidly
migrating fractions were named collectively "prealbumin" and five different phenotypes were reported in Norway. According to the genetic
theory of Fagerhol et al. (1964), three codominant alleles, designated bysymbols PrF Pr M and Prs, are responsible for the observed phenotypes.
The present writer found antitrypsin activity in some fractions of these
rapidly migrating region in starch gel and confirmed the identity of these
active fractions with lXI'antitrypsin in the serum by immunochemical examination. Furtheremore, six different phenotypes were recognized inJapan. These results agree with the finding of lXI-antitrypsin variation
found in Sweden by Eriksson et al. in 1963.

92

ANFUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

This finding shows that aI-antitrypsin fractions of human serum often
migrate into the so-called prealbumin fraction when starch gel electrophoresis is applied.

Allo-albumin F Mishima, a Variant of Human Serum Albumin
Yoshito OGAWA and Norio ODAKI

A second case of heritable allo-albumin F (a fast type of bis-albumin),an electrophoretic rapidly migrating variant of human serum albumin,
was found among thousand and fifty nine sera chosen at random from
specimens available in the departments of clinical pathology of the Mishima
hospitals.
Cellulose acetate (Separax) electrophoretic technique was performed at
0.8 rnA/em for 33 minutes at 28°C in 0.06 M barbiton buffer of pH 8.6.
The abnormal (fast) band accounted for approximately half of the total
albumin fraction when densitometric scanning of cellulose acetate strip
stained by Ponceau 3R was applied.
The propositus was a 48 yr old man treated in the hospital after accidental injury. He was otherwise healthy and so were the other members of his family. From the results of the family study, this albumin
variant seems to be inherited as a codominant autosomal allele.
The allo-albumin F Mishima, might be identical with one of the rapidly
migrating albumins demonstrated by Weitkamp et al., 1967, who usedbis-albuminaemic sera from nineteen unrelated families.

RESEARCHES CARRIED OUT IN 1968

XI.

93

APPLIED GENETICS

Cluster Distribution of Related Individuals in a Natural
Forest of Thujopsis Dolabrata
Kan-Ichi SAKAI and Yasusada MIYAZAKI

Variability in peroxidase isozyme pattern was investigated in a naturalforest of Thujopsis dolabrata. The forest consisted of forty five trees,
sporadically mixed with broad-leaved trees. The trees were of different
ages, the range being several hundred years from the youngest to theoldest. Leaves were sampled from each of the trees whose location had
been mapped on a graph paper. The collected samples were examinedelectrophoretically according to the method developed by Smithies in1965 and by Endo in 1968.
In the present species, more than fifty bands of peroxidase isozymes
were superficially identified. On the basis of those isozyme bands, eachtree was investigated for its resemblance to other trees in the forest. It
was found from this comparison that trees growing in proximity were
often very similar with each other in the isozyme pattern, whereas trees
distant from each other were always very dissimilar. If we assume thatenvironmental variation in the forest does not affect the isozyme patternof the tree and that variation in the isozyme pattern is genotypic, we
may interpret our finding as indicating that trees genetically related arelikely to grow rather in clusters than to be randomly distributed in theforest. Without doubt, the trees which are vegetatively propagatedfrom one tree should show approximately or exactly the same pattern of
isozymes. A few of supposedly such cases have been noticed. Most of
the trees we are interested in are, however, those which are more dissimilar in isozyme pattern than their vegetative sibs, but highly similar
in comparison with other trees in the forest. The occurrence of trees
more or less similar in isozymes growing in proximity may indicate thatthey are produced from seeds of single trees.

A Study on Oligogenic and Polygenic Mutations in the Flower
Organs of Citrus
Kan-Ichi SAKAI, Teruo NISHIDA and Kihachiro OHBA

The present report deals with the results of an investigation of ashort-styled mutation in a clone of Iyokan-orange, Citrus iyokan. Pro-

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

pagules from the clone were divided into two groups. One of them wassteadily irradiated by gamma-rays for 184 days in 1965, while anotherwas left non-irradiated as the control group. The dose rate applied was50, 100 and 200 R per day. The treated and control plants were grownin pots under glass for two and a half years before they were investigated in the spring of 1968. The investigation consisted in measuringpetal length, style length, stamen length and length of the peduncle withthe aim to detect the effect of irradiation. Examined were five to ten'flowers collected from each twig on each branch of irradiated and control
trees.
It was found that in the irradiated plots, the flower organs were highlyvariable in length in comparison with the control. Of special interest
was a distinctly bimodal distribution of style length in the irradiatedtrees. The average length of the mutated style was 2 to 3 millimeters,
whereas that of the normal style was 9-10mm. The frequency ofmutant flowers was about 8 percent in the 100 Rand 200 R treatments
though the rate of spontaneous mutation in the control plot was only
about 196. This mutation is understood to involve a major gene responsible for style length. This gene, however, is found to influence
pleiotropicaUy also other flower organs, i.e. petals, stamens and peduncles. The pleiotropic effect was found to be shortening of petal, stamen and peduncle by 2396, 1996 and 1496 respectively in comparison with
their normal size.
In addition to this major gene, mutated polygenes were found to participate in shortening the size of flower organs. Their effect was to reduce primarily style length and the length of other organs.

Use of Zymography for Clone Identificationin Forest Trees
Yasusada MIYAZAKI and Kan-Ichi SAKAI

Some species of forest trees are propagated vegetatively, yielding
superior cultivars for artificial plantation. The sugi-tree or Cryptomeria
japonica is one of those forest trees and we have at present a numberof cultivars, either of single clones or clone complexes consisting of several clones each. Since coniferous trees have apparently less variablegenotypes concerning the vegetative characters than broad-leaved trees,
it is often necessary to develop some techniques allowing us to discriminate easily and reliably one cultivar from others. This study deals
with an electrophoretic variation in the peroxidase among the genotypes

RESEARCHES CARRIED OUT IN 196895

of sugi-trees with the objective of establishing a zymographic techniquefor clone identification.
We have a famous clone-variety of Cryptomeria japonica named Kumotooshi which was originated from a single mother tree detected in the early
beginning of this century by a late forester in Kyushu. This clone showsa rapid growth in the early stage after planting, due to which it meetswith a favourable reception of forest people. The materials for the present study were "propagules" from this clone which are grown in various districts of Japan.
At first, needle-leaves were collected from different parts of a single
tree or from three trees which were genuine propagules of a Kumotooshi
tree in order to see to what extent isozyme patterns of a homologous
organ, for instance needle-leaves, vary among different parts of a single
individual or within a single clone. After confirming that the isozyme
patterns is accurately the same within a tree or a clone, needle-leaves
were collected from 51 trees which were more or less firmly believed to
be derivatives from the Kumotooshi cultivar. They were grown ineleven localities widely distributed in Kyushu island and Japan proper.
Sampling was made from May through October, 1968. Samples collected were stored in a freezer kept at -20°C until they were examinedelectrophoretically. The electrophoretic technique adopted was that developed by Smithies in 1965 and by Endo in 1968.
The electrophoretic test of the 51 trees showed that 45 of them were
genuine but nine were false. Thus, it is concluded that the electrophoretic variation of peroxidase is useful for determining if a given tree
is a member of a given clone or not.

When Do Trees Start Competing in a Forest?
Shigesuke HAYASHI and Kan-Ichi SAKAI

Intraspecific competition in a seed propagated forest of Cryptomeria
japonica is investigated on the basis of correlation of stem diameter at
breast height between the adjoining trees: Positive and high correlation
indicates none or a slight, if any, competition, whereas low positive,
zero or negative correlation is indicative of the occurrence of competition. Based on this observation, it may be possible to answer thequestion "When do the trees start competition?", if the correlation ofannual stem growth between adjoining trees is investigated in successive
years. The method of investigation consists in mapping the location ofeach tree on a graph paper, on the one hand, and measuring the annual

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

growth of the stem diameter of individual trees, on the other. The
measurement of annual growth was performed by measuring disks foryearly ring width in eight directions, from which the mean annual
growth was estimated. The total number of investigated trees was alittle less than 200, all of which being 50 years of age.
The correlation was calculated for total growth and annual growth of
the radius and total and annual growth of the basal area. It was foundfrom the result of computation of correlation coefficients that among thefirst six or seven year old seedlings no competition appears to occur,
the correlation coefficient being some +0.60. Later, however, the trees
start to compete against each other, with increasing intensity year after
year until they are about 30 years old, after which the competition remains the same.

X-ray Induced Variation in the Quantitative Characters of Rice
Shin-ya IYAMA

Seeds of rice variety Norin No.8 were irradiated by 2.5kr, 5kr, lOkr
and 20 kr of X-ray in 1959. Each treated population together with untreated control was maintained until the X s generation by one-parent-oneoffspring method in order to avoid as much as possible a bias due to natural
selection and sampling. At the X s generation, when most of the mutations induced were supposedly fixed, variation in the experimental populations was investigated on individual plant basis. Genetic variances ofthese populations in respect to the examined characters were estimatedby subtracting environmental variance from their variances (Table 1).
Table 1. Observed variances and estimated genetic variances in somequantitative characters of rice in control and X-ray treated populations
Plant weight
Treatment

Variance
961
614
677
1071
792

75.1484
81.193679.0052
77.371587.6930

Environmental 2576
variance

64.3263

Control
2.5 kr
5 kr
10 kr
20 kr

Plant height

Panicle wt./plant

d.f.
ag

2

10.822116.8673
14.6789
13.0452
23.3667

Variance

Varianceag

15.3563 5.5924
17.0010 7.2371
16.8181 7.0542
18.5513 8.7874
24.7647 15.0008

19.8094
20.6469
21.3889
21.517724.06402.91493.75244.49444.6232
7.16959.763916.8945

ag

2

2

RESEARCHES CARRIED OUT IN 1968

97

Environmental variance was estimated from the variance within lines
which were derived from 150 X 7 plants. The characters investigatedwere plant weight (g), plant height (em), panicle number and panicle
weight per plant (g). The results showed that 1) genetic variance wasrevealed even in the control population, 2) genetic variance was increased by X-ray irradiated except for panicle number and 3) induced genetic
variance increased with increasing dose of irradiation. The regressionsof induced genetic variance (Y) in the characters investigated on thedoses of irradiation (X kr) werePlant weight:
Y =0.4898X + 12.0823,
Plant height:
Y=0.4549X+ 5.3229,
andPanicle weight per plant:
Y=0.1973X+ 3.1112.
Though the difference between population means were not significant(Table 2), a comparison of the frequency table of control population withthose of the treated ones suggested that the irradiation treatments werelikely to induce mutations in the minus rather than in the plus direction.
Table 2.

Means of some quantitative characters of rice in control and
X-ray treated populations

Treatment

No. ofplant

Plant
weightPlantheightPaniclenumberPanicle weight
per plant

Control
2.5 kr
5 kr
10 kr
20 kr

968
620
683
1077
798

47.35 g
46.98
45.88
46.69
45.47

112.90 cm
113.00112.50112.60110.60

12.35
12.00
11.64
11.84
11.51

23.40 g
22.92
22.77
22.66
22.84

A Comparative Analysis of Productive Traits in Wild andDomesticated Japanese Quails
Takatada KAWAHARA and Akihiko MITA

Two hundred and sixty-eight wild quails captured at the foot of Mt.
Fuji were successfully propagated in cages. F 1 hybrids between wildand domesticated strains and backcrosses to the parental strains havebeen bred for our experiment. The birds were kept in a heated battery
brooder for the first 3 weeks after hatching and then were removed toan unheated one. After sexing at 5 weeks of age, the birds were transferred to individual or colony cages. They were given ad libitum acommercial feed and were kept under 24 hour light without dark period

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

conditions throughout the experimental period. Traits observed werebody weight and external shank length at various developmental stages,
age at sexual maturity, fertility, hatchability, viability, egg production
rate and egg weight.
Results of the experiment are summarized as follows: The wild strain
was consistently poorer in all traits than the domesticated line. Growth
curves of body weight showed that the wild line was always lighter
than the domestic one, the differences being 14.5% for females and 13.1%
for males on the average of various stages. The growth pattern of theF 1 hybrids and backcrosses almost agreed with the logical curves on theassumption that gene effects on the growth curve are additive. The external shank length showed the same genetic nature, the genetic controlof this character being clearer than for the body weight. There was afairly large difference between the body size of wild birds when captured and the plateaued size of the wild line bred in the laboratory, theformer being 97.6 g. for females and 92.6 g. for males and the latter
93.4 g. for females and 83.0 g. for males. The external shank length ofcaptured birds was longer than that of the laboratory wild strain. Such
differences may be due to the differences in ecological conditions. A
remarkable difference was found in the age at sexual maturity. Average number of days to attain sexual maturity was 48.3 days for domesticated and 117.0 days for wild lines. In addition, the appearance of nonlayers over 20 weeks after hatching amounted to 0% for the domesticatedstrain, whereas it was 47% for the wild line. Other differences between
wild and domesticated strains were: 51.7% in viability (up to 20 weeks
after hatching), 20.9% in hatchability, 22.5% in fertility, 44.196 in egg
production rate (up to one month after sexual maturity) and 0.6 g. in egg
weight (at sexual maturity), the wild strain always showing poorer performance than the domestic one. Backcross tests between wild and domesticated strains showed a tendency to approach the performance of therecurrent parent in all traits investigated except fertility. These results
suggested that there are distinct differences in genetic constitutions between wild and domesticated quails and such differences may be producedin the course of domestication, though the mechanism of domestication
is not yet known.

RESEARCH CARRIED OUT IN 196899

Directional and Fluctuating Asymmetry of Ribs of Cervical
Vertebrae in Fowls
Takatada KAWAHARA and Kan-Ichi SAKAI

It was demonstrated in the previous report (Sakai, K.1. and T. Kawahara, 1964, Ann. Rept. Nat. Inst. Genet. 15: 52) that the bilateral asymmetry of ribs of the cervical vertebrae of the fowl, measured by theabsolute difference between sinistral and dextral rib length, was significantly different among strains. This paper describes the results of afurther study on this subject. The asymmetry was classified into two
categories: directional and fluctuating. The directional asymmetry wasmeasured by the difference in the length of ribs, that is, sinistral minus
dextral. The fluctuating asymmetry, however, was defined as asymmetry due to chance fluctuation in the development of sinistral and dextral
ribs and was measured by the bilateral asymmetry remaining after subtraction of the directional asymmetry. Let the degree of fluctuating
asymmetry be a, then, a= I (sinistral-dextral)-average (sinistral-dextral)1 on a single bird basis. Since a was found not to distribute normally, we have employed the transformation, A= loglo[(a+0.l)2]. The birds
investigated were White Leghorn females of six inbred, one closed and5 F 1 hybrid strains, each at 16 months of age. Measured were lengthof sinistral and dextral ribs of four sixth to ninth cervical vertebrae.
Results of this study are summarized as follows:
1) The directional asymmetry was found to be statistically significant
for the vertebrae order, but not among strains. The sinistral rib wasusually longer than the dextral one, the mean difference being 0.13mm.
2) With regard to the fluctuating asymmetry, variation among strains
was highly significant. The F 1 hybrids were less asymmetric regarding fluctuating asymmetry than their parental inbreds. If we assume
that the fluctuating asymmetry in rib length of cervical vertebrae is
a result of developmental instability in the bird, it may be reasonable
to expect that heterozygosity might reduce the instability of thefluctuating asymmetry as was found in this study.
3) Directional and fluctuating asymmetries were positively correlated witheach other (r=0.472). In other words, those vertebrae which showed
higher directional asymmetry had simultaneously high fluctuating
asymmetry. Both asymmetries, however, were not significantly correlated with rib length. Correlation between body weight and rib
length was estimated to be 0.789, whereas body weight was not correlated with either directional or fluctuating asymmetry.

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

Developmental Stability of American and Japanese Rice
Varieties Tested in Kathmandu
B. B. SHARI and H. 1. OKA

From the data for one native, six American and six Japanese rice
varieties tested at Kathmandu, Nepal, standard deviations of individual
plants were studied with regard to panicle number per plant, single
panicle weight, total panicle length per plant, panicle weight per unit
length, and grain yield per plant. Among the American and native
varieties, some showed stability of panicle number and plasticity of single
panicle weight, while others showed the relation in opposite. A similarvariation pattern was also found in the relative stability of total
panicle length per plant and panicle weight per unit length. This indicates that the stability-plasticity pattern of yield characters differs according to varietal genotypes. Some Japanese varieties had a large
variability for all these characters, and among Japanese varieties stability was correlated to high character value. Varieties showing large
variability in many characters were considered inadaptive. It was suggested that the stability-plasticity pattern of character development could
be a factor conditioning yield stability. (Published in SABRAO Newsletter 1: 103-111)

RESEARCHES CARRIED OUT IN 1968

101

BOOKS AND PAPERS PUBLISHED IN 1968
BY STAFF MEMBERS( 1)

AMANO, E., 1968. Comparison of ethyl methanesulfonate- and radiation-induced
waxy mutants in maize. Mutation Res. 5: 41-46.
(2) ASAKURA, S., G. EGUCHI and T. IINO, 1968. Unidirectional growth of Salmonella flagella in vitro. j. Mol. BioI. 35: 227-236.
(3) ENDO, T., 1968. Indoleacetate oxidase activity of horseradish and other plant
peroxidase isozymes. Plant & Cell Physiol. 9: 333-341.
(4) FUJII, T., 1968. Killing efficiency of neutrons in Arabidopsis seeds. Arabidopsis Information Service 5: 47-48.
(5) FUJII, T., 1968. Whole and partial mutations induced in the developing embryo of maize by X-rays. Seiken Zih6 20: 55-61.
(6) GLASSMAN, E., T. SHINODA, E. DUKE and j. F. COLLINS, 1968. Multiple molecular forms of xanthine dehydrogenase and related enzymes. Ann. New York
Acad. Sci. 151: 263-272.
(7) HARTLE, D. L. and T. MARUYAMA, 1968. Phenogram enumeration: The numberof genotype-phenotype correspondences in genetic systems. j. Theoret. BioI.
20: 129-163.
(8) IINO, T., 1968. Bacterial classification from the view point of genetics (injapanese). jap. j. Bacteriol. 23: 109-114.
(9) IINO, T., 1968. Formation and differentiation of bacterial flagella (in japanese).
jap. j. Devel. BioI. 22: 7-11.
(10) KADA, T., 1968. Problems of gene mutations in carcinogenesis (in japanese).
The Saishin-Igaku 23: 425-431.
(11) KIHARA, H., 1968. Cytoplasmic relations in the Triticinae. A review. Proc.
III Internatl. Wheat Genet. Symp.: 125-134.
(12) KIHARA, H., 1968. History of biology and other sciences in japan in retrospect. Proc. XII Internatl. Congo Genet. III (in press).
(13) KIHARA, H., 1968. Use of interspecies hydridization in three cultivated plants.
Seiken Zih6 20: 1-14.
(14) KIKUCHI, Y., 1968. Autoradiography. (in japanese) Biological Techniques II
Yoshioka-Shoten, Kyoto, pp 495-506.
(15) KIMURA, M., 1968. Haldane's contributions to the mathematical theories of
evolution and population genetics, in "Haldane and Modern Biology" (ed. K.
R. Dronamraju), pp. 133-140. johns Hopkins Press, Baltimore.
(16) KIMURA, M., 1968. Evolutionary rate at the molecular level. Nature 217:624-626.
(17) KIMURA, M., 1968. Genetic variability maintained in a finite population due
to mutational production of neutral and nearly neutral isoalleles. Genetical
Research 11: 247-269.
(18) KOJIMA, K., M. TSUJITA and S. SAKURAI, 1968. Ribonucleic acid content in102 ANNUAL REPORT OF NATIONAL lNSTlNUTE OF GENETICS NO. 19

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the larval hypodermis of the silkworm. J. Serical. Sci. Japan 37(4): 263-273.
KURODA, Y., 1968. Technique of animal cell culture. I. (in Japanese). Heredity (Tokyo) 22(7): 75-79.
KURODA, Y., 1968. Technique of animal cell culture. II. (in Japanese). Heredity (Tokyo) 22(8): 74-79.
KURODA, Y., 1968. Technique of animal cell culture. III. (in Japanese). Heredity (Tokyo) 22(9): 74-79.
KURODA, Y., 1968. Technique of animal cell culture. IV. (in Japanese). Heridity (Tokyo) 22(10): 74-79.
KURODA, Y., 1968. Technique of animal cell culture. V. (in Japanese). Heredity (Tokyo) 22(11): 74-79.
KURODA, Y., 1968. Technique of animal cell culture. VI. (in Japanese). Heridity (Tokyo) 22(12): 108-114.
KURODA, Y., 1968. Selective sorting-out property of tumor cells for somespecific normal cells in rotation culture. (in Japanese). Japan. J. Develop.
BioI. 22: 98-99.
KURODA, Y., 1968. Preparation of an aggregation-promoting supernatant fromembryonic chick liver cells. Exptl. Cell Res. 49: 626-637.
KURODA, Y., 1968. Characteristic and selective aggregate-forming activity ofneoplastic cells in culture. GANN 59: 281-288.
KURODA, Y., 1968. Growth and differentiation of embryonic cells of Drosophila melanogaster in vitro. Proc. XII lnternatl. Congo Genet. II: 100-101.
LILIENFELD, F. A., 1968. Cytoplasm conditioned chimeras in a cross between
two races of Medicago truncatula Gaertn. Seiken ZihQ 20: 15-34.
MARUYAMA, T. and M. KIMURA, 1968. Development of temporary overdominance associated with neutral alleles. Proc. XII lnternatl. Congo Genet. II:
229.
MATSUMOTO, H., K. TAKATSUKI and E. MATSUNAGA, 1968. Gm factors inJapan: Population and family studies with statistical appendix. Jap. J. Human
Genet. 13: 10-19.
MATSUNAGA, E., 1968. Hereditary diseases and chromosome aberrations (inJapanese). Prenatal Medicine, cd. by MURAKAMI, U., M. SUZUKI and K. BABA:
381-400, Igaku-Shoin, Tokyo.
MATSUNAGA, E., 1968. Genetic load in human population (in Japanese). Clinical Genetics, ed. by E. INOUYE and T. YANASE, Asakura-Shoten, Tokyo.
MATSUNAGA, E., 1968. Genetic aspects of recent population trends (in Japanese)
Heredity (Tokyo) 22(4): 24-31.
MATSUNAGA, E., 1968. Better environments and better genes (in Japanese).
Jap. J. Public Health 15: 441-448.
MATSUNAGA, E., 1968. The concept of variation (in Japanese). Heredity(Tokyo) 22(10): 54-56.
MATSUNAGA, E., 1968. Birth control policy in Japan: A review from eugenic
standpoint. Jap. J. Human Genet. 13: 189-200.
MITANI, M. and T. IINO, 1968. Electron microscopy of Salmonella flagella in

RESEARCHES CARRIED OUT IN 1968

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103

methylcellulose solution. J. gen. Microbiol. 50: 459-464.
MORISHlMA, H. and H. I. OKA, 1968. Analysis of genetic variations in plant
type of rice. III. Variations in general size and allometric pattern among
mutant lines. Japan. J. Genet. 43: 181-189.
MORIWAKI, K., H. T. IMAI and T. H. YOSIDA, 1968. Gene dosage effect on
the rate of protein synthesis in the mouse plasma cell tumor, MSPC-1 (inJapanese). SABCO Journal (Osaka) 4: 59-75.
MORIWAKI, K., 1968. Plasma cell tumor (in Japanese). In "Developmental
biochemistry". Ed. by J. Ishida. Shokabo, Tokyo. p. 133-154.
MORTON, N.E., N. YASUDA, C. MIKI and S. YEE, 1968. Population structureof the ABO blood groups in Switzerland. Amer. J. Human Genet. 20: 420-429.
NAWA, S. and M. A. YAMADA, 1968. Hereditary change in Ephestia after
treatment with DNA. Cenetics 58: 573-584.
OGAWA, Y., 1968. On the analytical technique of serum lipoproteins on cellulose acetate strip (in Japanese). Physico-Chern. BioI. 93(4): 258-259.
OGAWA, Y., 1968. Cellulose acetate electrophoretic technique (in Japanese).
Jap. J. Clin. Path. Special Ed. 11: 46-67.
OGAWA, Y., 1968. Technical errors in the standard procedure established bysociety of electrophoresis in Japan for the analysis of serum protein fractions
by cellulose acetate electrophoresis (in Japanese). Physico-Chemical BioI. 13(2): 89-90.
OGAWA, Y., 1968. The cellulose acetate electrophoretic technique 12. Analytical results of BPB pre-stained human ·serum on cellulose acetate strips.
(in Japanese). Med. and BioI. 76(2): 59-61.
OGAWA, Y., 1968. Growth, differentiation and regeneration 44. Effect of
double X-irradiation on the actin formation in early Triturus embryo (inJapanese). Med. and BioI. 76(2): 81-84.
OGAWA, Y., 1968. a2-lipoprotein in human serum detectable with cellulose
acetate electrophoresis (in Japanese). Med. and BioI. 76(3): 118-120.
OGAWA, Y., 1968. A staining procedure of serum lipoproteins on cellulose
acetate strip (in Japanese). Med. and BioI. 76(5): 237··241.
OGAWA, Y. and A. SAKAMOTO, 1968. Normal values of serum lipoprotein
fractions on Separax in Japanese adults (in Japanese). Med. and BioI. 76(6):283-285.
OHTA, T., 1968. Effect of initial linkage disequilibrium and epistasis on fixation probability in a small population, with two segregating loci. Theoreticaland Applied Genetics 38: 243-248.
OHTA, T. and K. KOJIMA, 1968. Survival probabilities of new inversions inlarge populations. Biometrics 24: 501-516.
OHTA, Y., 1968. Effect of virus inoculation and thermoshock treatment oncytoplasmic male sterility in Capsicum. Seiken Zih6 20: 63-67.
OKA, H. I. and H. MORISHIMA, 1968. Analysis of genetic variations in plant
type of rice. IV. General growth rate, oscillating growth and allometricpattern. Japan. J. Genet. 43: 191-201.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19
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OKA, H. I. and K. H. TSAI, 1968. Genetic studies of yielding capacity andadaptability in crop plants. 3. Further observations on the effects of an earliness gene, E, in the genetic background of a rice variety, Taichung 65. Bot.
Bull. Acad. Sinica 9: 75-88.
OKA, H. I., 1968. Preferential paring of chromosomes in a tetraploid hybrid
between Oryza glaberrima and O. sativa. Can. J. Genet. Cytol. 10: 527-535.
OSHIMA, C., 1968. Persistence of some recessive lethal genes in natural populations of Drosophila melanogaster. Proc. XII Internatl. Congo Genet. II:
170-171.
OSHIMA, C., 1968. Recent activities of Japanese Geneticists 1967 (in Japanese)
KAGAKU 38: 447-451.
SAKAI, K. I., H. MUKAIDE and K. TOMITA, 1968. Intraspecific competition inforest trees. Silvae Genetica 17: 1-5.
SAKAMOTO, S., 1968. Cytogenetic studies in the tribe Triticeae. VI. Intergeneric hybrid between Eremopyrum orientale and Aegilops squarrosa. Japan.
J. Genet. 43: 167-171.
SAKURAI, S., 1968. Genetical and biochemical studies of chromogranules inlarval skin cells of the silkworm. Yokohama Med. Bul. 19(2): 29-43.
SANDBERG, A. A., K. YAMADA, Y. KIKUCHI and N. TAKAGI, 1967. Chromosomes and causation of human cancer and leukemia. III. Karyotypes of cancerous effusions. Cancer 20: 1099-1116.
SHINODA, T., 1968. Studies on genetically different acid phosphatase of human
red cells. J. Biochem. 64: 733-742.
SHINODA, T. and E. GLASSMAN, 1968. Multiple molecular forms of xanthine
dehydrogenase and related enzymes. II. Conversion of one form of XDH toanother by extracts of D. melanogaster. Biochem. Biophys. Acta 160: 178187.
STONIER, T., F. RODRIGUEZ-ToRMES and Y. YONEDA,1968. Studies on auxin
protectors. IV. The effect of manganese on auxin protector-I of the Japanesemorning glory. Plant Physiol. 43: 69-72.
STONIER, T., Y. YONEDA and F. RODRIGUEZ-ToRMES, 1968. Studies on auxin
protectors. V. On the mechanism of IAA protection by protector-I of theJapanese morning glory. Plant Physiol. 43: 1141-1145.
T AZIMA, Y., 1968. Repair in the process of mutation production by radiation(in Japanese). Japan. J. Genet. 43: 456.
TAZIMA, Y., 1968. Biological effectiveness of radiations of different quality(in Japanese). Radioisotopes 17: 125-138.
TSUJITA, M. and S. SAKURAI, 1968. Reconsititution of vesicular membranefrom degraded subunits of pteridine granule membrane in the silkworm.
Proc. Japan Acad. 44(10): 1048-1053.
WATANABE, T. K., 1968. Persistence of a lethal gene associated with SD genein natural populations of Drosophila melanogaster. Proc. XII Internatl. Congo
Genet. I: 232.
YAMAGUCHI, S., 1968. Sensitivity of the g-complex antigenic Salmonella

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strains to M8, a host-range mutant of bacteriophage x. J. gen. Virol. 2: 187190.
YASUDA, N., 1968. An extension of Wahlund's principle to evaluate matingtype frequency. Amer. J. Human Genetics 20: 1-23.
YASUDA, N., 1968. Gene frequency estimation by a counting method. Jap.
J. Hum. Genet. 12: 226-245. (In Japanese)
YASUDA, N., 1968. Distribution of matrimonial distance in the Mishima district. Proc. XII InternatI. Congo Genet. II: 178-179.
YASUDA, N., 1968. Estimation of the inbreeding coefficient from phenotype
frequencies by a method of maximum likelihood scoring. Biometrics 24:
915-935.
YASUDA, N. and M. KIMURA, 1968. A gene-counting method of maximum
likelihood for estimating gene frequencies in ABO and ABO-like systems.
Ann. Human Genet. 31: 409-420.
YOSIDA, T. H., 1968. Relationship between chromosomal alteration and development of tumors in vivo and in vitro. "Cancer Cells in Culture" (Ed.
by H. Katsuta) Univ. of Tokyo Press, Tokyo and Univ. Park Press., Baltimore: 171-194.
YOSIDA, T. H. and L. W. LAW, 1968. Chromosomal alteration and development of tumors, XVII, Chromosomes of the mouse leukemias induced by
Moloney leukemogenic virus (MLV) infection. Cytologia 33: 256-268.
YOSIDA, T. H. and R. A. ROOSA, 1968. Ibid. XVIII. Karyotypes of a 5-fluorodexyuridine resistant subline in the mouse lymphocytic neoplasm, P388,
growing in vitro. Japan. J. Genet. 43: 49-56.
YOSIDA, T. H., H. T. IMAI and M. POTTER, 1968. Ibid. XIX. Chromosome
constitution of tumor cells in 16 plasma cell neoplasms of BALB/c mice. Jour.
Nat. Cancer Inst. 41: 1083-1097.
YOSIDA, T. H. and T. SEKIGUCHI, 1968. Metaphase figures of rat chromosomes
incorporated into mouse cells. Molec. Gen. Genetics 103: 253-257.

ABSTRACTS OF DIARY FOR 1968

106

ABSTRACTS OF DIARY FOR 1968
March

April
May
June

12
15
16
26

17

15
28
19
July
26
September 391120
October18
November 16
December 24

80th Biological Symposium
158th Meeting of Misima Geneticists' Club
81st Biological Symposium
159th Meeting of Misima Geneticists' Club
160th Meeting of Misima Geneticists' Club
28th Meeting of the Board of Councillors
161st Meeting of Misima Geneticists' Club
162nd Meeting of Misima Geneticists' Club
82nd Biological Symposium
83rd Biological Symposium
84th Biological Symposium
85th Biological Symposium
163rd Meeting of Misima Geneticists' Club
164th Meeting of Misima Geneticists' Club
Public Lectures on Genetics (at the National Science
Museum, Ministry of Education, Tokyo)
165th Meeting of Misima Geneticists' ClubFOREIGN VISITORS IN 1968

107

FOREIGN VISITORS IN 1968
Jan.

15 DAss, M., Central Silk Board, Ministry of Commerce, India

Mar.
Mar.
Mar.

16
21

May
June

7
4

Sept.

2-5
2-3

11

2-4
3
3-5
4
4-5
5

5-7
Aug.
13
Aug. 16-19
Aug.
25

28

Aug.
Aug.

29

Aug.

30

ULLAL, S. R., Central Silk Board, Ministry of Commerce,
India
SHAH, S., Central Silk Board, Ministry of Commerce, India
SOBELS, F. H., State Univ. of Leiden, Netherlands
LATARJET, R., Radium Institute, France
ANOS, A., Center of Corn Breeding, National Institute of
Research in Agronomy, Spain
RUPER, E. A., The Rockefeller Foundation, U.S.A.
DAss, H., Research Branch, Canada Dept. of Agriculture,
CanadaBARIGOZZI, C., Universita degli Studi di Milan, Italy
PETRAS, M., University of Windsor, Canada
FOSTER, M., University of Michigan, U.S.A.
AUERBACH, C., University of Edinburgh, U.K.
RIZKI, T. M., University of Michigan, U.S.A.
SUZUKI, D. T., Univ. of British Columbia, Canada
KIMBALL, R. F., Oak Ridge National Laboratory, U.S.A.
SOBELS, F. H., State Univ. of Leiden, Netherlands
DONN, S. J., University of Toronto, Canada
FISZEARLE, M., University of Toronto, Canada
NIDDER, R., University of Toronto, Canada
WOLF, B. E., Freiden UniversWit, Germany
OFTEDAL, P., Norsk Hydros Institute for Cancer Research,
NorwaySENGBUSCH, P. V., Max-Planck-Institut, Germany
CHOWDHURY, S. N., Sericultural Experiment Station, India
CROW, J. F., University of Wisconsin, U.S.A.
FELSENSTEIN, J., Institute of Animal Genetics, Scotland
SEARS, E. R., University of Missouri, U.S.A.
SMITH, H. H., Brookhaven National Laboratory, U.S.A.
BURDICK, A. B., Adelphi University, U.S.A.
ARSENIEVA, Institute of Developmental Biology, U.S.S.R.
WILLIAMSON, D., Philadelphia University, U.S.A.
POULSON, D. F., Yale University, U.S.A.
PAIK, Y. K., Yonsei University, Korea
CORDEIRO, A. R., Univ. do Rio Grande do SuI, Brazil
LEE, W. J., Seoul University, Korea

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

Aug.
Aug.

BERNSTROM, P. A., Hilleshog Sugar Beet Breeding Institute,
SwedenBOROJEVIC, S., University of Novi Sad, Yugoslavia
BROWN, D. F., Bishop's University, Canada
DALY, K. R., San Fernando Valley State College, U.S.A.
DUBOVSKY, J., Komensky University, Czechoslovakia
ERIKSSON, T.R., University of Uppsala, Sweden
FRYDENBERG, 0., University of Aarhus, Denmark
GAMBLE, E. E., Ontario Agricultural College, Univ. of
Guelph, Canada
GARDNER, C.O., University of Nebraska, U.S.A.
GUTZ, H. K. W., Graduate Research Center, Southwest,
U.S.A.
HANKS, G. D., University of Utah, U.S.A.
HOROVITZ, S., Universidad Central de Venezuela, Venezuela
JORDAAN, J. P., University of Stellenbosch, Republic of South
AfricaKASHA, K. J., University of Guelph, Canada
KHOSHOO, T. N., National Botanic Garden, U.P., India
LINDQVIST, K. G., Hilleshog Sugar Beet Breeding Institute,
SwedenMARIE, R. A., Ecole Nationale Superieure, Agronomique,FranceMATOUSEK, J., Laboratory of Physiology and Genetics ofAnimals, Czechoslovakia
MAYO, C. M. E., University of Adelaide, Australia
RAJASEKARSETTY, M. R., University of Mysore, India
Rasmuson, S. B., University of Umea, Sweden
SOSNA, M., Institute of Experimental Botany, Czechoslovakia
SPRAGUE, L. M., The Rockefeller Foundation, U.S.A.
STEVENSON, H. Q., Southern Connecticut State College,
U.S.A.
TOSSELL, W. E., Ontario Agricultural College, Universityof Guelph, Canada
31-1 BAND, H. T., Michigan State University, U.S.A.
31 EHLING, U. H., Gesellschaft fUr Strahlenforschung, Germany
KOOPMAN, A., University of Groningen, Netherlands
KRANZ, A. R., University of Frankfurt, Germany
KUNKEL, H. A., UniversWit Hamburg, Germany
LACROUTE, F. C., Institut de Botanique, France
OWEN, R. D., California Institute of Technology, U.S.A.

FOREIGN VISITORS IN 1968

109

PASSARGE, E., Institut fUr Humangenetik, Germany
PFITZER, P., UniversWit Dusseldorf, Germany
ROBBELEN, G. P., University of Gottingen, Germany
SCHMIEGER, H., Gesellschaft fUr Strahlenforschung, Institut
Mikrobiologie, Germany
BOGDANOV, Y. F., Institute of Molecular Biology, U.S.S.R.
BORzHKOVSKAYA, G. D., Institute of Cytology & Genetics,
U.S.S.R.
DUBININA, L. G., Institute of General Genetics, U.S.S.R.
GEODAKIAN, V. A., Institute of General Genetics, U.S.S.R.
KIKNADZE, I. I., Institute of Cytology & Genetics, U.S.S.R.
KOROCHKIN, L. I., Institute of Cytology & Genetics, U.S.S.R.
KOZHIN, S. A., Leningrad State University, U.S.S.R.
KVITKO, K. V., Leningrad State University, U.S.S.R.
LEBEDEvA, L. I., Institute of Cytology & Genetics, U.S.S.R.
LUCHNIK, N. V., Institute of Medical Radiology, U.S.S.R.
LUCHNIKOVA, E. M., Leningrad State University, U.S.S.R.
OGANESIAN, M. G.,
Institute of Experimental Biology,
U.S.S.R.
PETROV, R. V., Institute of Biophysics, U.S.S.R.
POMERANTSEVA, M. D., Institute of Genetics, U.S.S.R.
PRIJLINN, O. ]., Institute of Experimental Biology, U.S.S.R.
RATNER, V. A., Institute of Cytology, & Genetics, U.S.S.R.
RONICHEVSKAYA, G. M., Institute of Cytology & Genetics,
U.S.S.R.
RUBIKAS, I. P., Institute of Biochemistry, U.S.S.R.
SHAKHBAZOV, V. G., Kharkov State University, U.S.S.R.
SHEREDEKO, L. N., Ukrainian Research Institute of Agriculture, U.S.S.R.
SKAVRONSKAYA, M. G., Academy of Sciences of the USSR,
U.S.S.R.
TODuA, V. A., All Union Institute of Tobacco & Makhorka,
U.S.S.R.
Sept.
9 VOGEL, F., Universitat Heidelberg, Germany
SCHLEIERMACHER, E., Universitat Heidelberg, Germany
Sept.
10 MUKAI, T., North Carolina State University, U.S.A.
Sept.
11 SCHADE, H., University of Dusseldorf, Germany
Nov.
12 Hadders, G., Institute for Forest Improvement, Sweden
13-26 PETRAS, M., University of Windsor, Canada

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 19

ACKNOWLEDGEMENT
The editor wishes to express his sincere gratitude to Dr. F. A. LILIENfor her kindness in reading the original manuscripts. (Kazuo

FELD

MORIWAKI)AUTHOR INDEX111

AUTHOR INDEXAMANO. E
Y.E
ENDO. T
ENOMOTO, M
FUJII. T
HAYASHI. M
HAYASHI. S
IiNO. T
1MAI. H. T
ISHIDSU, J
IYAMA, S
KADA. T
KAWAHARA. T
KIHARA. H
KIKUCHI. Y
KIMURA. M. .
KUMAGAI. M
KURODA. Y
MARUYAMA. T
MATSUNAGA. E
MINATO. K
MITA. AMIYAZAKI, Y
MORISHIMA. H
MORIWAKI. K. .
MURAKAMI. A
NAMIKI. M
NAWA. SNISHIDA. T.. . . . . . .. :

CHU.

75
.41.4234
81
76. 77. 78
71
9580
10. 1582
96
71. 73. 74. 83
97. 9939
86. 87. 89.44. 4587
18. 21. 22. 66
47. 49, 84
84, 8623
97
93, 94
41. 42
15. 4064. 6573
27. 2893

OGAWA. Y
91, 92
OHBA. K93.44. 45
OHTA. T
OHTA. Y19. 24. 39
86, 87. 89
OISHI. H
OKA. H.I.
19, 34, 41. 42.100
ONIMARU. K
61, 62. 63
OSHIMA, C
53. 54. 55. 57
SADAIE. Y
71
SAKAI. K. I.
93. 94. 95. 99
SAKATA. H
40
SAKAMOTO. S. .
39
SAKURAI, S
29, 30. 31, 33
15
SEKIGUCHI. T. .
SEKIYA. K
1634, 42, 100
SHAHI. B. B
87
SHIBATA. K. .
SHINODA. T
35, 36, 89
59, 60, 61. 62, 63
TAZIMA. Y
TONOMURA. A
86
TSUCHIYA. K
10, 11. 12, 13, 40
29, 30. 31, 33
TSUJITA. M
67, 69TUTIKAWA. K
53. 54. 55. 57, 58
WATANABE. T. K
YAMADA. M. A
27, 28YAMAGUCHI. S. .
81
YASUDA, N. ..
51
YONEDA. Y. ..
25, 37YOSIDA, T. H
9. 10. 11, 12, 13, 15

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