NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 201969

Publi8l&ed bll

THE NATIONAL INS'ITI'UTE OF GENETICS
Mitlima. Simoka-ken. Japtm

1970

Annual Reportof theNational Institute of Genetics
No. 20, 1969

Published by

The National Institute of Genetics, Japan
1970

CONTENTS
General statement
Staff.....................................
..
Council............................................................
Association for propagation of the knowledge of genetics... . . . . . . . .
Projects of research for 1969................
Researches carried out in 1969..........................
1.

Cytogenetics
Segregation of three types of the largest No.1 (A-I) chromosome
in Rattus rattus bred in a population room. YOSIDA, T. H. and
TSUCHIYA, K.
Polymorphism of No.9 (A-9) and No. 13 (A-13) autosomes in
Rattus rattus collected in Japan. YOSIDA, T. H. and TSUCHIYA,
K. and MASUJI, H..........................................
Frequency of chromosome polymorphism in Rattus rattus collected in East and Southeast Asia.
YOSIDA, T. H. and
TSUCHIYA, K.
Fl and F2 hybrids between Asian and Oceanian black rats withdifferent karyotypes. YOSIDA, T. H., KATO, H. and SAKAKIBARA,

K....................................................

Karyotypes of black rats, Rattus rattus, obtained from Honolulu,
Hawaii. YOSIDA, T. H. and TSUCHIYA, K.
Local differences in the frequencies of chromosome polymorphismin the house rat, Rattus rattus, in Japan. YOSIDA, T. H.,
TSUCHIYA, K. and SONODA, J.
Karyotypes of Rattus bowersii collected in Malaysia. YOSIDA,
T. H. and TSUCHIYA, K.
Protective effects of protamine sulfate on disintegration of isolated
metaphase chromosomes. KATO, H., SEKIYA, K. and YOSIDA,
T. H.

A preliminary study on the uptake of isolated nuclei by mammalian cells in vitro. SEKIYA, K., KATO, H. and YOSIDA,
T. H.
Nondisjunction of chromosomes in a synchronized cell population
initiated by reversal of colcemid inhibition. KATO, H. and
YOSIDA, T. H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Establishment of several Chinese hamster cell lines with specificchromosomal trisomies. KATO, H. . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Peculiar marker chromosomes in Yoshida rat sarcoma. MATSUSHIMA,
T. and YOSIDA, T. H
" ..............................
Synthesis rate of intracellular gamma·globulin in diploid andtetraploid MSPC·l myeloma cells. MORIWAKI, K. and IMAI,
H. T.
Exchange between cell populations during serial transplantationof MSPC-l mouse myeloma. MORIWAKI, K. and YAMASHITA, J...
Cytological studies of mouse gliomas. YAMASHITA, J.
Aggregate·promoting substance from HeLa cells in rotation culture.
KURODA, Y.................................................
Pachytene pairing in translocation heterozygotes having a hugeknob close to the breakpoint. OHTA, Y.....................

II. Physiological and developmental genetics
An attempt to obtain long·term culture cells from Drosophilamelanogaster. KURODA, Y.
Clonal analysis of embryonic limb cartilage cells in creeper mutant of chicken. KURODA, Y. and SHIBUYA, T.
Effect of molting hormone on the cell cycle in insect epidermis.
MINATO, K. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cytoplasmic male sterility and virus infection in Capsicum annuum
L. OHTA, Y.
A variant found in the progeny from grafting in Capsicum annuum. OHTA, Y.
Induction and inhibition of nucellar embryo development in Citrus.
FURUSATO, K. and OHTA, Y.
III.

Biochemical genetics
Syntheses of drosopterins. NAWA, S. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetic effects of DNA in Bombyx. NAWA, S., YAMADA, M. A. and
TSUJITA, M.
Characterization of incorporated DNA in Ephestia eggs treatedwith DNA. YAMADA, M. A. and NAWA, S.
DNA specific in early development in Ephestia. YAMADA, M. A. . .
Incorporation of 14C-phenylalanine or 14C-tyrosine into thehypodermal cuticle of the silkworm. I. Lethal lemon larvae.
TSUJITA, M. and SAKURAI, S. ..••..........................
Incorporation of 14C·phenylalanine or 14C·tyrosine in to thehypodermal cuticle of the silkworm. II. Lethal albino larvae.
TSUJITA, M. and SAKURAI, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sepiapterin reductase in the silkworm. TSUJITA, M. and SAKURAI, S.

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CONTENTS

Characterization of pteridine granule membrane in larval skincells of the silkworm. SAKURAI, S. and TSUJITA, M.
Immunochemical study of pteridine granule membrane in
hypodermal cells of silkworm larvae. SAKURAI, S. . . . . . . . . .
Aldolase isozyme patterns of brain and brain tumors in mammals.
YAMASHITA, J. and MORIWAKI, K...........................
Biochemical characters of serum transferrin in several Rattus
species collected from South-East Asia and Oceania. MORIWAKI,
K., TSUCHIYA, K. and SAKATA, H.
Organ-specific expression of allelic peroxidase isozymes in heterozygotes of Oryza perennis. ENDO, T.

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IV. Evolutionary genetics
Intraspecific differentiation of genome in AegiloPs caudata.
KIHARA, H. and OHTA, Y.
Comparison of the developmental pattern between cultivated andwild types of rice. MORISHIMA, H. and OKA, H. I.
A survey of genetic variations in the populations of wild Oryza
species and their cultivated relatives. MORISHIMA, H. and
OKA, H. I.

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Y. Mathematical studies on population genetics
The rate of molecular evolution considered from the standpointof population genetics. KIMURA, M.
The number of heterozygous nucleotide sites maintained in a
finite population due to steady flux of mutations. KIMURA,

M..........................................................
The average number of generations until extinction of an individual mutant gene in a finite population. KIMURA, M.
and OHTA, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .
Linkage disequilibrium at steady state determined by random
genetic drift and recurrent mutation. OHTA, T. and KIMURA,
M. .....................•...................................
Some relationships between local and global genetic variabilities.
MARUYAMA, T..............................................
Speed of gene substitution in a geographically structured population. MARUYAMA, T......................................
On the fixation probability of mutant genes in a subdivided
population. MARUYAMA, T.
The variance effective number of human population. YASUDA, N. . .

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

VI.

Experimental studies on population genetics
Characteristics of Japanese natural populations of D. melanogas:er.
OSHIMA, C. and WATANABE, T. K.
Recessive autosomal sterility genes concealed in natural populations. OSHIMA, C. and WATANABE, T. K.
A new mutant gene' tra-b' on the second chromosome. WATANABE,
T. K.
A heterotic inversion on the second chromosome of D. melanogaster.
WATANABE, T. K. and WATANABE, T.......................

VII. Radiation genetics and chemical mutagenesis in animals
Studies on strain differences in radiosensitivity in the silkworm.
VII. Strain differences in sensitivity to germ cell killing.
TAZIMA, Y. and ONUMA, A.
Studies on strain differences in radiosensitivity in the silkworm.
VIII. Strain specificity at various response levels. T AZIMA,Y. and ONIMARU, K.
Studids on strain differences in radiosensitivity in the silkworm.
IX. Relation between mutation induction and cell killing.
TAZIMA, Y. and FUKASE, Y.
Studies on strain differences in radiosensitivity in the silkworm.
X. Spontaneous mutation frequencies observed in different
radiosensitive strains to mutation induction. TAZIMA, Y.
and ONIMARU, K.
A comparison of mutagenicity of 14 MeV fast neutrons on primordial germ cells among different X-ray sensitive silkworm
strains. MURAKAMI, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of ultraviolet light on the oocytes of the silkworm withspecial regard to strain sensitivity. MURAKAMI, A. ..
Comparison of stage sensitivity to X-ray during meiosis in silkworm oocytes. MURAKAMI, A.
Changes in radiosensitivity during the second meiotic stage ofsilkworm oocytes. MURAKAMI, A...........................
Radiation-dependent loss of sorting-out activity of HeLa cells.
KURODA, Y.................................................
VIII. Radiation genetics in microorganisms and plants
Correlations between mutation, transformation efficiency and radiosensitivity in mutants of Bacillus subtilis. SADAIE, Y. and
KADA, T.
Gamma-ray mutagenesis, repair and chemical radiosensitization

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IX.

CONTENTSv

in bacteria. KADA, T......................................
Macromolecular synthesis in bacteria radiosensitized with iodine
compounds. NOGUTI, T. and KADA, T......................
Rejoining inhibition of radiation-induced DNA strand-breaks inradiosensitized bacteria with iodine compounds. NOGUTI, T,
and KADA, T...............................................
Genetic resistance to radiosensitization with iodine compounds.
KADA, T...................................................
Possible toxic effects of halogen atoms in radiosensitization withsome halogenated pyrimidines. KADA, T., SADAIE, Y. and
NOGUTI, T.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Repair systems for UV damage in phage T6. AMANO, E. and
SEKIGUCHI, M.
Wave length dependence of photoreactivation of UV irradiatedmaize pollen. AMANO, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preliminary intragenic mapping of induced wx mutants in maize.
AMANO, E..................................................
Fractionation and dose rate effects of gamma-rays on mutation
frequency in maize. FUJII, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A green revertant obtained from a chlorina mutant in diploid
wheat by gamma-irradiation. FUJII, T. . . . . . . . . . . . . . . . . . . . .
Relative biological effectiveness of monoenergetic fast neutronson oilseeds. RAJAN, S. S.
.........................
Callus formation in wheat anthers. FUJII, T. .. .. .
..

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Microbial genetics
Correlation between the number of flagella and the efficiency of
locomotion in peritrichously flagellate bacteria. IINO, T. ...
Genetical studies of flagellation and motility in Pseudomonas
aeruginosa. IINO, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extraordinary polymerization of Salmonella flagellin in vitro.
HOTANI, H., ASAKURA, S. and IINO, T. . . . . . . . . . . . . . . . . . . . . .
Chlorate-resistant mutants of Salmonella typhimurium. ENOMOTO,
M.

Transductional analysis of the gene controlling arginine sensitivity in Salmonella typhimurium. ISHIDSU, J...............
Some properties of azide resistant mutants of Salmonella typhimurium. ISHIDSU,].
High frequency reversion and modified efficiency of amber suppression induced by A-lysogenization in Escherichia coil K12.
KADA, T...................................................

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

X.

Human genetics
Sexual variation in finger pattern types and ridge counts inGermans. MATSUNAGA, E., MATSUDA, E. and SCHADE, H. ..
Polymorphic hematological traits of the Japanese. SHINODA, T.
Structure analysis of human immunoglobulins. SHINODA, T.....
Partial purification of genetically different phosphoglucomutaseof human red cells. SHINODA, T.
A new variant of /X 2 -globulin fraction in human serum. OGAWA,
Y. and ODAKI, N...........................................
Mutation rates of translocation Down's syndrome. KIKUCHI, Y.,OISHI, H., TONOMURA, A., KURITA, T. and MATSUNAGA, E. . .
Clinical conditions of patients with apparently normal chromosomes, VI. OISHI, H. and KIKUCHI, Y......................
Qualitative relationships between X chromosome and leucocyte
drumstick. OISHI, H. and TONOMURA, A.
Pattern of DNA synthesis in human X chromosomes with structural abnormalities. KIKUCHI, Y. and OISHI, H.............

XI.

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Applied genetics
Further study on competition in sugi-tree, Cryptomeria japonica,
with special reference to the age of the forest. SAKAI, K.
I. and HAYASHI, S.
Genetic variability on enzymatic level in natural forests ofThujopsis dolabrata. SAKAI, K. I., MIYAZAKI, Y. and MATSUURA, T.
Family analysis in a natural forest of Thujopsis dolabrata: A
new method for quantitative genetic studies. SAKAI, K. I.
and MIYAZAKI, Y.
Genetic parameters of organ and body weights in Japanese quail.
KAWAHARA, T. and SAITO, K...............................
Variance and covariance analysis of some traits of Japanese quail.
KAWAHARA, T. and KUSAKA, R.
Early vs. sustained vigor type in growth and their bearing onyielding POtential in rice. OKA, H. I. and MORISHIMA, H...

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Books and papers published in 1969 by research members..........
Abstracts of diary for 1969.........................................
Foreign visitors in 1969 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Author index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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GENERAL STATEMENT
Dr. Hitoshi Kihara retired from directorship in April. He was thesecond in the short sequence of our young institute's directors, succeeding
Dr. Kan Oguma, the first director, in 1955.
During his service of fourteen years, Dr. Kihara spared neither trouble
nor toil in that still difficult time and exerted himself to the utmost
trying to put the new institute on a more stable basis. Through hisconstant endeavors the institute could take long strides toward further
developments. Many achievements were attained, the number of departments was steadily growing, the buildings were set up and fromthe modest beginning an internationally acknowledged National Instituteof Genetics emerged. For his most distinguished services, rendered tothe institute, Dr. Kihara will be always remembered.
All the more, Dr, Kihara has our great admiration for having notrelaxed his pace as an active researcher. At present, he is working on
wheat in his own laboratory in the vicinity of our institute. We all wish
him the best of luck and continued success in his great lifework.
On the 24th of February His Imperial Highness the Crown Prince
visited the institute. This was the second visit of His Highness since1955. The Prince showed great interest in our recent development presented by several staff members.
This year our institute has attained the age of twenty years. During
this period the number of department increased from three to ten. The
tenth department, molecular genetics, started this year, and Dr. Kin-ichiro
Miura has taken office as Head of the department.
Among the facilities created with the help of new budgets the following
two are noteworthy:(1) Experimental farm comprising 7,082 m 2 in our neighborhood,(2) Electron computer, TOSBAC-3400.
Dr. M. Kimura, Head of the Department of Population Genetics stayed
three months, April I-July 6, at Princeton University, giving lecturesand engaging in cooperative researches.
Among the foreign visitors during the current year, were: Dr. Riojun
Kinoshita of City of Hope Medical Center, Los Angeles, Prof. and Mrs.
E. L. Tatum of Rockefeller University, New York, Dr. and Mrs. Sui
Chuan Hsu of Taiwan Agricultural Research Institute, Taipei, and Prof.
L. Tolmach of Washington University, St. Louis.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

STAFF
Director

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

1. Department of Morphological Genetics
TAZIMA, Yataro, D. Ag., Head of the Department

The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
MURAKAMI, Akio, D. Ag.
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
KATO, Hatao, D. Sc.; SEKIYA,* Kunio

The 2nd Laboratory
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
IMAI, Hirotami, D. Sc; YAMASHITA,* Junkoh, D. Med.

Department of Physiological Genetics

3.

OSHIMA, Chozo, D. Sc., Head of the Department

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

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

*

Research members under grant from other organization or visiting researchers.

STAFF

4.

Department of Biochemical Genetics
TSUJITA, Mitsuo, D. Ag., Head of the Department

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, D. Med.

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.

The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
MIYAZAKI,* Yasusada; PARK,* Y. G.

The 3rd Laboratory
aKA, Hiko-Ichi, D. Ag., Head of the Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag.

6.

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

The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
NOGUTI, Takehiko, D. Sc.

The 2nd Laboratory
KADA, TSUNEO, D. Sc., Head of the Department
FUJII, Taro, D. Ag.

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

7.

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

The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Labo:-ato;-y
SHINODA, Tomotaka, D. Sc.; MATSUDA, Ei.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

The 2nd Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
KIKUCHI, Yasumoto, D. Sc.; OISHI, Hidetsune, D. Sc.

8.

Department of Microbial Genetics
IINO, Tetsuo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
IINO, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
ENOMOTO, Masatoshi, D. Sc.; YAMAGUCHI,* Shigeru, D. Sc.; ISHIWA,*
Hiromi
The 2nd Laboratory
IINO, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
SUZUKI, Hideho, D. Sc.; ISHIDsu, Jun-ichi; WU,* Wen Chuan

9.

Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
OHTA, Tomoko, Ph. D.
The 2nd Laboratory
MARUYAMA, Takeo, Ph. D., Head of the Laboratory

10. Department of Molecular Genetics
MIURA, Kin-ichiro, D. Sc., Head of the Department
11.

Experimental Farm
SAKAI, Kan-Ichi, D. Ag., Head of the Farm
MIYAZAWA, Akira

12. Department of Administration
OYAUCHI, Toru, Head of the Department
ANDO, Yoshiichi, Chief of the General Affairs Section
KATO, Shigeo, Chief of the Finance Section
Honorary Members and Visiting Members

KIHARA, Hitoshi, D. Sc., Director of the Kihara Institute for BiologicalResearch, Member of Japan Academy, Emeritus Professor of Kyoto
University
KOMAI, Taku, D. Sc., Member of Japan Academy, Emeritus Professor of
Kyoto University
KUWADA, Yoshinari, D. Sc., Member of Japan Academy, Emeritus Professor

COUNCILof 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

COUNCILOKADA, YO, Chairman, Emeritus Professor of University of Tokyo
WADA, Bungo, Vice Chairman, Emeritus Professor of University of Tokyo
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
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
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
SAKATA, Takeo, President of T. Sakata Company
TACHI, Minoru, Director of Institute of Population Problems
TAKEYAMA, Yutaro, Governor of Shizuoka Prefecture
TANAKA, Nobunori, Professor of University of TokyoYANAGIDA, Tomomichi, Director of Institute of Applied Microbiology,
University of Tokyo
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Director of the National Institute of Genetics
T AZIMA, Yataro, Managing Director, Head of the Morphological Genetics
Department
OSHIMA, Chozo, Managing Director, Head of the Physiological Genetics
Department
KIHARA, Hitoshi, Manager, Emeritus Professor of Kyoto University
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

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

PROJECTS OF RESEARCH FOR 1969
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 (TAZIMA and 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 cultu;-e (KURODA)
Effects of radiation on cells in tissue culture (KURODA)
Department of Cytogenetics

Studies on chromosomal polymorphism of rodents (YO~1DA, TSUCHIYA and
KATO)
Chromosome alteration and development of tumor (YOSIDA and MATSUSHIMA)
Study on incorporation of isolated chromosomes into cultured mammaliancells (KATO, SEKIYA and YOSIDA)
Cytogenetical study on monosomic and trisomic cultured mammalian cells(KATO)
Cytogenetical and biochemical studies on mouse plasma cell tumor (MORIWAKI,YAMASHITA and SAKATA)
Biochemical studies on serum transferrin variations in rodents (MORIWAKI,
SAKATA and TSUCHIYA)
Experimental breeding and genetics of mice, rats and other \viie! rodents(YOSIDA, MORIWAKI, TSUCHIYA, SAKAKIBARA and SONODA)
Department of Physiological Genetics

Population genetics of deleterious genes in natural populations of Drosophila melanogaster (OSHIMA and WATANABE)
Mechanisms of persistence of some lethel genes (OSHIMA and WATANABE)
Physiological and population genetics of sterility genes in natural populationsof Drosophila melanogaster (OSHIMA and WATANABE)
Analysis of fitness in a fluctuating environment (OSHIMA and WATANABE)
Analysis of inversion chromosome in natural populations of Drosophilamelanogaster (OSHlMA and WATANABE)
Nucleus substitution in wheat and related species (KIHARA, SAKAMOTO
and OHTA)PROJECTS OF RESEARCH FOR 1969

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Basic studies on hybrid wheat breeding (KIHARA)
Cytogenetic studies in the tribe Triticeae (SAKAMOTO)
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)
Department of Appl ied Genetics

Quantitative genetic studies in poultry (SAKAI and KAWAHARA)
Genetic studies in wild population of Japanese quails (KAWAHARA)
Theoretical studies on breeding techniques (SAKAI and IYAMA)
Studies on competition in plants (SAKAI, IYAMA and MIYAZAKI)
Genetic studies in natural stands of forest tree species (SAKAI, MIYAZAKI
and PARK)
Zymographic studies in forest trees (SAKAI, MIYAZAKI and PARK)
Simulation studies on artificial selection (IYAMA)
Evolutionary studies in wild and cultivated rice species (OKA and MORISHIMA)
Analysis of genetic variations in plant type and growth pattern in rice
varieties (MORISHIMA and OKA)
Analysis of sterility genes in Oryza sativa (OKA)
Department of Induced Mutation

Radiation genetics in mice (TUTIKAWA)
RBE and dose rate effects in higher plants (FUJII and AMANO)

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Radiation genetics in Arabidopsis (FuJII)
Fine structure analysis in maize (AMANO)
Biological effects of ultraviolet radiation (KADA, FUJII and AMANO)
Radiation-induced and chemical mutagenesis in microorganisms (SADAIE
and KADA)
Studies on biochemical factors involved in induced mutagenesis on thecellular level (NOGUTI, TUTIKAWA and KADA)
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 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)
Studies on structure of human immunoglobulins (SHINODA)
Department of Microbial Genetics

Genetic fine structure analysis on microorganisms (IINO, ISHIDSU and
YAMAGUCHI)
Genetics of cellular regulatory mechanisms (SUZUKI and ISHIDSU)
Genetics of bacterial flagella (IINO, ENOMOTO and SUZUKI)
Genetics of motility in bacteria (ENOMOTO)
Genetics of host range in bacteriophages (lINO, ENOMOTO, YAMAGUCHI and Wu)
Transduction mechanism of phage P 22 (ENOMOTO and ISHIWA)
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)
Mathematical studies on the genetics of structured populations (MARUYAMA)
Linkage disequilibrium in finite populations (OHTA and KIMURA)
Department of Molecular Genetics

Studies on the chemical structure of genome of viruses containing doublestranded RNA (MIURA)RESEARCHES CARRIED OUT IN 1969I.

CYTOGENETICS

Segregation of Three Types of the Largest No.1 (A·I) Chromosome
in Rattu8 rattu8 Bred in a Population Room
Tosihide H. YOSIDA and Kimiyuki TSUCHIYA

In order to ascertain the way of segregation in natural condition of!
three chromosome types (A/A, A/S and S/S)1) of the largest No.1 chromosome pair of the Japanese house rat, Rattus rattus, some animals werebred in a population room. The size of the room was 250x430>,300cm.
Shelters for the nest were partitioned off by board at the side wall of
the room. Food and water were administered every day, but no heat
was supplied in winter. Breeding of the animals in the population room
began in September with one pair having the A/S chromosome type.
After one year all animals bred in the room were captured and weresacrificed for chromosome observation. 84 animals thus obtained segregated into 27(A/A), 50 (A/S) and 7(S/S). This segregation ratio wassignificantly different from that of the expected 21: 42: 21 by x2 test (X 2
=30.95, p<O.OOl). Thus the rats with SIS chromosomes seem to be lessadaptive to the natural conditions of Misima than those with A/A and A/Schromosome types.
As already reported by us (this Annual Report 18:53, 1968), the segregation of three chromosome tyPes in F1 hybrids from A/SxA/S, which werebred in usual animal cages did not deviate significantly from the expectedratio, but SIS animals were somewhat fewer than those with A/A and
A/S. When SIS females were crossed to A/A, A/S and other SIS males,
the litter size of the hybrids was also smaller than that of hybrids between A/S( ~) and S/S( 0). Relation between chromosome types andbody size of the animals bred in the population room was examined forthe above 84 rats. The body length with SIS chromosome pair was for all
under 150mm, while that of those with A/A and A/S pair attained 200
mm. Thus the rats with S /S chromosome pair do not grow as well as
the animals with A/A and A/S pairs in the population room. They seem1)
In the previous papers we used the term for telocentrics (T), but hereafter
we will use for them the term for acrocentrics (A), so A/A and A/S in the present
paper correspond to T /T and T /S of the previous reports.

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

to be less adaptive than those with the other karyotypes, and therefore
their number was smaller than that of the others in the population room.
From the above results, we must consider a random genetic drift, because
the number of animals bred in the room was small. To solve this problem
the above experiment will be repeated.

Polymorphism of No. 9 (A·9) and No. 13 (A·13) Autosomes in
Rattu8 rattu8 Collected in Japan
Tosihide H. YOSIDA, Kimiyuki TSUCHIYA and Hiroshi MASUJIl)

Polymorphism of the largest acrocentric autosomes (A-I) in relation toacrocentrics (or telocentrics) and subtelocentrics was already reported in
the previous communications (Yosida, T. H. et al. 1966. This Annual Report
16: 51-52; 1967. Ibid. 17: 61-63; 1968. Ibid. 18: 51-54). In addition we
found in the Japanese population also polymorphisms of No. 9 (A-9) and
No. 13 (A-l3) autosome pairs which were characterized by acrocentrics
and subtelocentrics. Most of the animals had acrocentric A-9 and A-13
autosomes, but a few ones had heteromorphic pairs consisting of anacrocentric and a subtelocentric member and a homomorphic pair of subtelocentric chromosomes was also found. A study on the frequencies of
A·9 and A-13 polymorphisms in natural population is now in progress.
Due to the polymorphism of three chromosome pairs, the Japanesehouse rats, R. rattus, have very complicated karyotypes. If A-I, A-9
and A-13 chromosomes form homomorphic pairs either acrocentrics or sub·telocentrics, theoretically 8 different karyotypes are expected. Amongthem three are shown in Figs. 1 to 3. In Fig. 1, three chromosome pairs(A-I, A-9 and A·13) are acrocentric, in Fig. 2 A-I and A-9 pairs are subtelocentric, A-13 pair is acrocentric and in Fig. 3 A-I pair is acrocentricand A-9 and A-13 pairs are subtelocentric.

1)

Okayama University Medical School.

RESEARCHES CARRIED OUT IN 1969

••

I•

4

5

9{A/A)

103•• ••.a I"
34

89(5/5}

1\-1(S/5)

2

6

7

11

12

13(A1A)

15

16•• •• •• I . eA

8

•1

I•

M-14

17

19

1:1 tl

11

.1
e..

AA
10

•• •• ••
la

18

20

5

••
19

•• as •• ••
18

J.

a.

20

x y

17

Fig. 2.

Fig. 1.

IIA-1(A1A)

"
a.
6

11

"I

loA
12

IS
M-14
19

Fig. 3.
Figs. 1-3. Three karyotypes of Rattu8 rattu8 collected in Japan. l.
A·I, A·9 and A-I3 pairs are acrocentric. 2. A-I and A-9 pairs are subtelocentric, but A-I3 pair is acrocentric. 3. A-I pair is acrocentric, but A-9
and A-I3 pairs are subtelocentric.

Frequency of Chromosome Polymorphism in Rattu8 rattu8
Collected in East and Southeast Asia
Tosihide H. YOSIDA and Kimiyuki TSUCHIYAIn the previous paper (Yosida et at. 1969, this Annual Report 19: 11)
we had reported only about chromosome numbers and presence or absenceof chromosome polymorphism in Rattus rattus collected in Southeast Asia.

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

In the present contribution the karyotypes and frequency of chromosome
polymorphism of animals collected in East and Southeast Asia are dealt
with. All animals collected in these areas had 42 diploid chromosomes,
although fluctuation from 42 to 44 chromosomes was observed in R. rattus
diardi collected in Kuala Lumpur. As previously reported Rattus rattus
collected in Japan and Korea showed polymorphism of the largest No. 1
chromosomes in respect to telo- or acrocentric (A) and subtelocentric shape(5). In the Japanese population about 75 percent of rats had the A/Apair, about 20 percent the A/S pair and the remaining 5 percent the 5/5
pair.
The animals collected in East Asia (Taiwan and Hong Kong) and thecontinent of Southeast Asia (Bangkok and Kuala Lumpur) showed the A/Apair or they were polymorphic having A/A, A/S and 5/5 pairs. The
number of animals collected and the frequencies of A/A, A/S and 5/5
pairs were different by the locality (Table 1). All rats collected in HongFrequency of chromosome polymorphism (A-I) of

Table 1.

Rattus rattus in East and Southeast Asia_______________

LocaIities

Karyotyp~-~-_________

Japan(19 local.)Seoul,
Korea

Tainan,TaiwanHong
Kong

Bangkok,Thailand

A/A
AIS
SIS

343
90
20

18
17
4

3
0
0

12

27
12

Total

453

29

3

12

42

Kuala
LumpurLuzon,
Philip.

Mindanao,
Philip.

Java,
Indonesia

CelebesIndonesia

1

0
0

0

0

25

0
12

0
15

0
0
20

25

12

15

20

~calities

Karyotype

i

. ~I

AlA
A/S
SIS

i

Total

i

3
3
i~--

7

0
0

3

Kong and Taiwan showed only the A/A pair, but the number of animals
collected was small. In the Bangkok population the frequencies of rats
with A/A, A/S and 5/5 pairs were similar to those of the Japanese population, while in Kuala Lumpur population the frequencies of A/S and 5/5
were very high although the number of animals collected was very small.
This situation is comparable to that found in Hong Kong population. Ac-

13

RESEARCHES CARRlED OUT IN 1969

cording to the opinion of mammalian systematists, the house rat (R. rattus
jlavipectus) in Hong Kong is a different subspecies from that' (R. rattus
diardii) in Malaysia. A difference between the subspecies is seen also in
different frequency of karyotype polymorphism.
On the other hand all rats collected in islands located in Southeast Asia,
such as Luzon and Mindanao of Philippines, Celebes and Java of Indonesia
showed only SIS pairs. Based on the above investigation the rats can beclassified by the karyotypes as follows:
Polymorphic A/A, A/S and SIS
Karyotypes

A/A only

Population

Northern
Japan,
Hong Kong

Few
SIS
South andSoutheast Japan,
Korea,
ThailandSIS only

Few
A/A
Malaysia

Luzon,
Mindanao,
Java
Celebes

F 1 and F 2 Hybrids between Asian and Oceanian Black Rats
with Different Karyotypes
Tosihide H. YOSIDA, Hatao KATO and Katsumi SAKAKIBARA

In a previous paper (Yosida et at 1969, Jap. Jour. Genet. 44: 89) wereported that black rats collected in East and Southeast Asia had 42
chromosomes, while those collected in Australia, New Zealand and New
Guinea had 38 diploid chromosomes. The latter karyotype was characterized by having two pairs of large metacentric chromosomes, one larger
than the other. The other chromosomes were similar to those of the
Asian black rats. Fl hybrids between Australian (2n=38) and Japaneserats (2n=42) were obtained. They had 40 chromosomes consisting of 19
Australian rat and 21 Japanese rat chromosomes. Fl hybrids could beobtained by crossing Japanese females (Cj!) to Australian males «(;) andin reciprocal direction of the cross. Test crosses of Fl and back crosses
to Japanese and Australian rats were carried out. Spermatogenesis in Fl
males seems to be proceeding normally, and normal meiotic figures andspermatozoa were observed. Until now only one F2 animal was obtained.
It was a male and was very healthy. The number of chromosomes in
the F2 male was counted in 73 cells. Among them 70 cells had 39 chromosomes, including 3 large metacentrics, one larger than the other two.

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

The number of chromosomes in F2 rats is expected to vary from 38 to
42. One fourth of F2 rats should have 39 chromosomes. Among them
one half should have one large metacentric and two small metacentricelements. The present F2 rat seems to have that karyotype. From thekaryological analysis it is assumed that one large metacentric chromosome came from either of the two parents, but the two smaller metacentric
members (A-11) were contributed by both parents. The A-9 pair of theF2 rat was heteromorphic, although that of the father rat was an SIS
homomorphic pair. The A-9 pair of the maternal rat should be a heteromorphic pair (A/S), although it was not yet observed.
Hybrid between a black rat of New Guinea with 38 chromosomes andthat of Malaysia with 42 chromosomes, R. rattus diardii, was obtained in
the laboratory. It also had 40 chromosomes like the hybrid between
Australian and Japanese black rats.

Karyotypes of Black Rats, Rattus rattus, Obtained from
Honolulu, Hawaii
Tosihide H. YOSIDA and Kimiyuki TSUCHIYA

It was already reported that samples of black rats, Rattus rattus, collected in East and Southeast Asia had 42 chromosomes, while those collectedin Oceania (Australia, New Zealand and New Guinea) had 38 chromosomes(Yosida, T. H. et al. 1969. Jap. J. Genet. 44: 89-91). Recently we obtained
black rats from Honolulu, Hawaii, by courtesy of Dr. S. R. Malecha of
the University of Hawaii. They had the chromosome constitution of Oceaniantype; namely 38 chromosomes including two large metacentric pairs.
According to Bianchi (1969, Experimentia 25: 1111-1112.), the black rats
in Argentina have 38 chromosomes like the Oceanian rats. Recently we
had two informations about rat karyotypes; one from Texas, U.S.A. (R. J.
Barker, personal information) and the other from Rome, Italy (E. Capanna,
personal information). They also had 38 chromosomes like the Oceanian
black rats. From the above investigations, it may be assumed that theEuropean black rats have 38 chromosomes and had migrated from Europe
to Oceania (Australia, New Zealand, New Guinea), North America (Texas,
Hawaii) and South America (Argentina). The migrated rats seem to haveoccupied the new land and made it their territory, so that Asian rats do
not penetrate the territory of Oceaeian type rats and vice versa. In Japan
we collected 453 black rats in 19 different localities, but none had the
Oceanian karyotype.

15

RESEARCHES CARRIED OUT IN 1969

Local Differences in the Frequencies of Chromosome Polymorphismin the House Rat, Rattus rattus, in Japan
Tosihide H. YOSIDA, Kimiyuki TSUCHIYA and Jun SONODA

Local differences in the frequencies of the polymorphic largest No. 1(A-1) chromosome pair which was characterized by consisting of an acrocentric (A) and a subtelocentric (S) chromosomes were already reported(Yosida 1968, this Annual Report 18: 51.). We have newly obtained 86
animals from 7 localities of Japan, namely Sendai, Mizukaido (Ibaragi),
Yokohama, Mikurajima (Izu islands), Okayama, Tsushima (Kyushu) andOkinawa. The frequencies of the three chromosome types of the A-I
pair are given in Table 1. As the table shows, rats collected in Sendai
Table 1.

Karyotype

Frequency of three chromosome types of the largest No. 1
pair in Rattus rattus newly collected in Japan
Localities

Sendai Mizukaido Yokohama Mikurajima Okayama Tsushima Okinawa

A/A
A/S
SIS

11

Total

11

2

2
1
0

6
3
2

23
5
1

7

3

11

29

10
8
1

1
2
3

4

1

19

6

were all of A/A type, while those collected in Mizukaido, Yokohama and
Mikurajima located in Kanto district showed a higher frequency of
A/S and SIS types. The Okinawan population was rather similar to
those of some localities of Kyushu (Okinoerabu and Kusudomari), but in
the Tsushima population the frequency of A/S and SIS types was higher
than in the other Kyushu populations, and was rather similar to that ofSeoul population in Korea.
In total 453 rats were collected until now in 19 localities of Japan.
Among them 343 (75.5%) had the A/A, 90 (19.9%) the A/S and the remaining 20 (4.4%) the SIS pair. All 146 animals collected in northern (Sapporo,
Sendai) and northwestern Japan (Niitsu, Obama and Tottori) had the A/Atype, while those collected in the other localities had A/A, A/S and SIS
types. This group was also roughly divided into 3 populations by thefrequency of the subtelocentrics; eastern population (79 rats) in Kanto
district (Mizukaido, Ooizumi, Yokohama and Mikurajima) showed a higher
frequency of A/S (46%) and SIS types (17%), while the southeastern population (98 rats) collected in Tokai (Hiratsuka, Misima, Hamamatsu) and16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Sanyo (Okayama and Ube) showed lower A/S (31%) and SIS (3%) percentage.
In the southern population (100 rats) collected in Kyushu district (Kusudomari, Okinoerabu and Okinawa) the lowest frequency of A/S (18%) andSIS (1%) types was observed.
From the study of those local differences it is assumed that rats with
A/A pair are the original type which was the first to propagate widelyin Japan, and those with SIS pair migrated later secondarily to Kanto
district (Tokyo, Yokohama), and are propagating from there to the otherparts. This assumption seems to be supported by observations made inSoutheastern Asia, and by our crossing experiment in the laboratory as
well as a breeding experiment in a population room.

Karyotypes of Rattus bowersii Collected in Malaysia
Tosihide H. YOSIDA and Kimiyuki TSUCHIYA

Rattus bowersii collected in Kuala Lumpur, Malaysia, had 40 diploid
chromosomes. Their karotypes are remarkable by having one large metacentric chromosome pair. Rattus rattus collected in Asia had 42 chromosomes, but those collected in Oceania had 38 chromosomes (Yosida et at.
1969, Jap. J. Genet. 44: 89-91.). The latter karyotypes are characterizedby containing two large metacentric pairs, one larger than the other.
From a comparison of idiograms of these two forms it was assumed that
two arms of the large metacentric element might have developed byreciprocal translocation of acrocentric pairs No.4 and 7, and the smaller
one by reciprocal translocation of acrocentric pairs No. 11 and 12. Rattus
fuscipes collected in Australia had 2n-c-cc 38, and their karyotypes were very
similar to those of the black rats in Oceania.
The karyotypes of R. bowersii had one smaller metacentric pair, whose
size corresponded to the smaller metacentric pair of the Oceanian black
rats. This chromosome seems to have originated from the fusion of No.
11 and 12 acrocentric pairs of R. rattus. The other chromosomes of thebowersii rat are essentially similar to those of R. rattus in Asia; namely
7 small metacentric pairs, 11 acro- or subtelocentric autosome pairs, andan acrocentric sex chromosome pair. The karyotype of R. bowersii seems
from appearance to be intermediate between that of Oceanian black rats
or R. fuscipes and that of Asian black rats. Subtelocentrics of No.1 andNo. 9 pairs are similar to those of the other Rattus species, but subtelocentric No. 2 and No. 3 pairs are remarkable in this species. From theabove investigation it is assumed that the bowersii karyotype developed

REARCHESES CARRIED OUT IN 196917

from that of the Asian black rats by fusion of Robertsonian type ofacrocentric No. 11 and 12 pairs, and also a centromeric inversion of No.
2 and 3 pairs.

Protective Effects of Protamine Sulfate on Disintegrationof Isolated Metaphase Chromosomes
Hatao KATO, Kunio SEKIYA and Tosihide H. YOSIDA

It has been demonstrated that isolated metaphase chromosomes werereadily phagocytized by mammalian cells in vitro. Recently Yosida andSekiguchi (Molec. Gen. Genetics, 103: 253, 1969) presented an evidence
for the appearance of some of the incorporated chromosomes in harmony
with the host genome. In the present study, we intended to obtainfurther information on the behavior of the incorporated chromosomes at
an early phase of the phagocytic phenomenon and to find appropriate
conditions which would protect the engulfed chromosomes from the digestive
action of cellular enzymes in the hope to increase the survining rate ofincorporated chromosomes.
Metaphase chromosomes were isolated from mouse lymphoma L5178Y
cells. The isolated chromosomes were first exposed in vitro to DNase
dissolved in phosphate buffered saline. A disintegration of chromosomes
occurred within 5 min after exposure to the enzyme at 37°C. On theother hand, when protamine sulfate was added to the incubation mixture,
chromosome morphology as well as stainability by Feulgen reaction remained intact even 90 min after incubation with DNase. This protective
effect of protamine sulfate was constantly manifested at concentrations
higher than 10 ,ltg/ml.
When the isolated chromosomes were added to a monolayer culture ofChinese hamster cells, they were phagocytized by the recipient cells. The
frequency of cells taking up the exogenous chromosomes reached a peak
4 hours after their addition, and then fell rapidly. This decline appeared
to be due to degradation of incorporated chromosomes within the cytoplasm of recipient cells. When chromosomes were labeled with 3H-thymidine
prior to isolation and then added to the culture, a uniform labelingof the nuclei of recipient cells was detected 4 hours after their addition,suggesting reutilization by the recipient cells of labeled DNA precursors
derived from the degraded chromosomes. The incidence of labeled nuclei
increased steadily up to 10.2% by the 14th hour.
Intracellular disintegration of incorporated chromosomes was also

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

prevented by their pretreatment with protamine sulfate. The incorporation
frequency showed a steady rise till 6 hours after the addition of chromosomes and reached a plateau. The percentage of labeled nuclei of recipient
cells remained very low until 12 hours (1.3%) after the addition of protamine-treated chromosomes.

A Preliminary Study on the Uptake of Isolated Nuclei
by Mammalian Cells in vitro
Kunio SEKIYA, Hatao KATO and Tosihide H. YOSIDA

The present report deals with cytological observations of the uptake ofisolated nuclei by cultured mammalian cells, and their fate in the recipient
cells during an early phase of cultivation. The nuclei were isolated frommouse FM3A cells, which lacked in hypoxanthine-guanine phosphoribosyltransferase activity, and were added to the culture of mouse 3T6-8T
cells which were deficient in thymidine kinase activity (TK-). The recipient
cells were grown in plastic dishes as monolayers in Eagle's minimum
essential medium supplemented with 10% calf serum.
At various time intervals after incubation with the isolated nuclei, thecells in each dish were fixed in methanol-acetic acid (20: 1) after a brief
wash with phosphate buffered saline, and stained with Giemsa solution.
Phagocytic activity of the recipient cells was indicated by a gradualincrease in the frequency of binucleate cells after the addition of isolated
nuclei. In untreated control cultures, the incidence of binucleate cells
was only 0.1%, whereas in cultures that had been exposed to isolated
nuclei, the frequency increased up to 3.1% by the 4th hour. In thesebinucleate cells, one of the nuclei was apparently smaller and stained
more densely than the other, indicating that it was an engulfed donor
nucleus.
Another characteristic feature of the recipient cells was the appearance
in their cytoplasm of vacuoles, which sometimes contained densely-stained
donor nuclei. The frequency of cells possessing cytoplasmic vacuoles
increased steadily after treatment; from 0.8% in untreated cells to 2.4%,
5.0% and 6.5%, after 2, 4, and 12 hour-incubation with the isolated nuclei,
respectively.
In autoradiographic studies employing tritium-labeled nuclei, the symptom of disintegration of incorporated donor nuclei, indicated by dispersion of silver grains over the cytoplasm of recipient cells, was first detected
18 hours after the addition of nuclei.

RESEARCHES CARRIED OUT IN 196919

In parallel with these cytological observations, the 3T6-8T cells (TK-)
were cultured in selective medium (HAT) following exposure to nuclei
isolated from FM3A cells (TK+) in the hope of obtaining cells which
acquired thymidine kinase activity. So far, no surviving colony has beenobtained.

..
Nondisjunction of Chromosomes in a Synchronized Cell Population
Initiated by Reversal of Colcemid Inhibition
Hatao KATO and Tosihide YOSIDA

Stubblefield and Klevecz (Exptl. Cell Res., 40: 660, 1965) reported amethod of initiating a synchronized cell population by collecting andreplacing colcemid-arrested metaphase cells in fresh medium. Theirmethod appeared to have the definite advantage of obtaining a large yieldof mitotic cells as compared with the original selective detachment method
developed by Terasima and Tolmach (Exptl. Cell Res., 30: 344, 1963).
However, this modified method seems to require a further sure proof thatthe cells are completely free from lasting effects of colcemid arrest,
especially in a long term culture. In the present study, we found thatchromosomal nondisjunction occurred frequently in colcemid-reversed cells.
Chinese hamster cells (Don) were grown in 100-mm plastic (Falcon)
petri dishes. When the cultures were growing exponentially, cells weretreated with colcemicl at a concentration of 0.05 pg/ml. The metaphasecells were collected 30 min, 1, 2 and 4 hours after the addition of thedrug, respectively, and placed in 60-mm plastic dishes containing prewarmed normal medium. Some of the cultures were fixed at the firstmitotic stage following a release from the colcemid block. Chromosomal
examination revealed that nondisjunction had occurred at various frequencies depending upon the duration of colcemid arrest; the percentage
of cells possessing the modal chromosome number decreased with increasing
duration of the colcemid block (e.g., 88.3% in the untreated control culture, 76.6%, 61.5%, 52.0%, and 29.8% in cultures exposed to colcemid for
30 min, 1, 2 and 4 hours, respectively). Chromosomal monosomies andtrisomies were observed frequently. Tetrasomies were also observed.
These results appear to suggest that in a certain proportion of colcemidreversed cells the damages of the spindle fibers and their contact withthe centromeres were not completely repaired.
Further cultivation of the remaining cultures and their karyologicalexamination carried out at definite time intervals revealed that cells with

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

monosomies disappeared rapidly from the cell population. The colonies
formed by cells exposed to colcemid for 4 hours were apparently smaller
than the colonies derived from those submitted to 2-hour-arrest. The
plating efficiencies determined on the 7th day of incubation were 73.0%
in the untreated control cultures, 53.3% in those submitted to 2-hourcolcemid arrest, and 44.3% in those that had recovered from 4-hour-arrestby colcemid.
Establishment of Several Chinese Hamster Cell Lines with
Specific Chromosomal Trisomies
Hatao KATO

The method of estimating gene dosage effect in trisomic individuals has
been used by various workers for the localization of genes in mammalian
autosomal chromosomes. Such studies would be much easier if one could
induce nondisjunction in cultured cells and isolate a variety of cell clones
with chromosomal trisomies.
According to our previous report (Kato and Yosida, 1970. Exptl. CellRes., 60: 459-464.) nondisjunction can be readily induced by a brief exposure of cells to colcemid. Making use of this technique, seven celllines with trisomic chromosomes were successfully isolated from cultured
Chinese hamster cells.
Chinese hamster cells (Don) have 22 chromosomes. They were treatedwith colcemid (0.05pg/ml) for 2 or 3 hours, and the metaphase cells wereselectively detached from the culture dishes. They were then washedfree from colcemid and placed at a concentration of 100 cells pe,' dish in
100-mm plastic culture dishes containing pre-warmed fresh medium. Colonies formed by these colcemid-reversed cells were isolated from the dishby cylinder technique. In the first experiment, twenty five colonies
were randomly picked out and chromosome counts were performed. Fourteencolonies were found to have chromosome numbers in diploid range, whereas
the other eleven colonies possessed chromosome numbers at tetraploidlevel. Out of the diploid colonies, three were found to have trisomic
chromosomes; trisomy of no. 9, of no. 10, and double trisomies of no. 7
and no. 10 chromosomes. The rest of the diploid colonies showed a karyotype identical to that of the parental cells.
Although the incidence of tetraploid clones was very high among thecolonies isolated in the first experiment, they were easily distinguishedin the living culture from the diploid ones on the basis of difference insize of metaphase cells. Therefore, in the next experiment, colonies withRESEARCHES CARRIED OUT IN 196921

small metaphase cells were picked out, all turning out to have chromosome numbers in diploid range. Out of 35 colonies isolated, four had
trisomic chromosomes (no. 9 trisomy, no. 9 trisomy accompanied by an
extra Y, no. 10 trisomy, and no. 11 trisomy) and three had 21 chromosomes. All other colonies had the same karyotype as the original Don
cells. Karyotype analysis of clones with 21 chromosomes revealed thatthey were not genuine monosomic clones. The reduction of the chromosome number in these clones was due either to translocation of a part of
the Y to other chromosomes or to centric fusion of acrocentric homologues.
Chromosomes involved in these structural rearrangements were no. 7, MI,
no. 10 and the Y. Clones possessing trisomies of large chromosomes (e.g.,
no. 1, 2, 4, and 5) have so far not been isolated.
Seven trisomic clonal lines have been maintained for approximately twomonths since they had been isolated without losing their specific chromosome configurations.

Peculiar Marker Chromosomes in Yoshida Rat Sarcoma
Toshiharu MATSUSHIMA and Tosihide H. YOSIDAIn Yoshida rat sarcoma two peculiar marker chromosomes in metaphasecells were recently observed. These two elements, one a large subtelocentric, LS-marker, and the other a small submetacentric chromosome,the SS-marker, were characterized by an abnormally elongated part resembling a long satellite. The SS-marker was more remarkable than theLS-marker. The length of the quasi-satellite was different by the cell.
In some cells with the SS-marker it had almost the same length as along ordinary arm but in some other cells it was shorter. In many cells
it showed two parallel daughter threads, but in few cells they formed aring by joining the ends of the separated daughter chromatids of thesatellite.
The occurrence of the two marker elements in the tumor sto:.:ks maintained in five different institutes (this Institute, Sasaki Medical Institutein Tokyo, Medical School in Gifu University, Takeda Biological Institutein Osaka, and Medical School in Tohoku University, Sendai) was examined.
Almost similar LS- and SS-markers were found in stocks of this Institute,
Sasaki Medical Institute, Gifu University and Tohoku University, but theLS-markers in a stock of Takeda Institute had a different shape, as if asmall chromosome was united with the satellite by translocation. Frequencyof LS- and SS-markers was markedly different by the stock. Higher

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

frequency of the markers was observed in stocks of this Institute, Sasaki
and Takeda, slightly lower in Gifu and a very low in Tohoku.
The SS- and LS-markers had sometimes the appearance of a satellite
association. From the figures the long threads seem to have a relation
to nucleolar organization. Late replication of DNA in the thread regionof LS-markers was observed by labeling with H8-thymidine, but was notfound in that region of SS-markers.

Synthesis Rate of Intracellular Gamma-Globulin in Dipliod
and Tetraploid MSPC-l Myeloma Cells!)
Kazuo MORlWAKl and Hirotami T. IMAl

It was previously reported that no significant difference was found in
the rate of gamma-globulin synthesis between diploid and tetraploid MSPC1 myeloma cells when it was determined in the excreted globulin fraction(Moriwaki, K. et al. Symp. Cell Chern. 20: 195-·204 1969). To exclude a
possibility that this phenomenon was caused by a difference between diand tetraploid cells in the ability to excrete the protein, the rate of
intracellular myeloma protein synthesis was assayed in both sublines of
the tumor.
The tumor cell suspension was incubated for 2 hours at 37°C with
C14-leu. The soluble fraction was extracted from sonicated cells followedby precipitation at pH 5. Proteins soluble at the acidic pH were chromatographed on DEAE-cellulose column to separate the gamma-globulin
fraction to which anti-MSPC-l myeloma protein rabbit serum was added
for the precipitation of the specific protein. Radioactivity in the antiserumprecipitated fraction represented the rate of intracellular myeloma protein
synthesis. No significant difference was observed between the synthesis
rates of cells of both sublines (Table 1), suggesting the possibility thatan "allelic exclusion" mechanism functioned in the tetraploid myeloma
cells producing gene dosage compensation, which did not take place inthe total protein synthesis of these tumor cells (Moriwaki, K. and H. T.
Imai. 1969. This Annual Report 19: 15-16).
In order to determine the synthesis rate of light and heavy chainsseparately, the myeloma protein precipitate was further reduced andalkylated by mercaptoethanol and monoiodo-acetic acid, followed by separation in Sephadex G-lOO column with 1 N acetic acid. The ratio of light1)
Supported by Public Health Service grant CA-07798-06 from the National Cancer
Institute, U.S.A. (T. H. Yosida).

RESEARCHES CARRIED OUT IN 196923

Table 1. Rate of myeloma protein synthesis in diploid and tetraploid
MSPC·1 myeloma cells
C14·leu incorporated (cpm/10 6 viable cells/120 min.)Fractions
Total protein
Myeloma protein
Light/Whole ratio

Diploids

Tetraploids

2634 (5)190 (5)0.294552 (5)216 (5)0.29

Figures in parentheses indicate the number of tumor-bearing mice used for theassays.

chain to whole molecule in the synthesis rate of the diploids was almost
the same as that of the tetraploids (Table 1). This finding indicates that
inhibition of myeloma pl'otein synthesis in tetraploid cells is not restricted
either to light or to heavy chains.

Exchange between Cell Populations during Serial Transplantationof MSPC·l Mouse Myeloma!'Kazuo MORIWAKI and Junkoh YAMASHITA

During a 2-year serial transplantation of MSPC·1 mouse myeloma themodal chromosome number has shifted from 40 in the early generations
to 39 with 3 marker chromosomes, A, B, and C, in the late generations.
The present study was undertaken to reveal a possible mechanism of suchan exange between two tumor cell populations. The tumor cells withmodal number 40 in early transplant generations designated as subline
P-40 have been stored in liquid nitrogen for several months and usedsimultaneously with another subline, NP·39 ABC, of a later transplant
generation, for mixed inoculation experiments. As the growth rate of NP39 ABC subline was considerably higher than that of P·40 subline, 10 3
cells of the former and 106 cells of the latter developed tumors of thesame size in two weeks, when they were inoculated separately on bothsides of abdomen of a host mouse. On the other hand, when cells ofboth sublines were inoculated as a mixture of 10 3 NP-39 ABC cells and
106 P·40 cells, more than 98% of the tumor cells developed after two
weeks consisted of NP-39 ABC cells (Table 1). In order to know what
cellular function plays a role in the inhibition of P-40 cells, NP-39 ABC
cells irradiated by 15,0001' gamma-rays which had completely blocked!)
Supported by a Grant in Aid (Nos. 9151 and 4051) for Fundamental Scientific
Research from the Ministry of Education in Japan.

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Table 1. "Allogeneic inhibition "-like phenomenon between two syngeneic tumors,namely P-40 and NP-39 ABC sublines of MSPC-l myeloma
Chromosome constitution
Subcutaneous
inoculation

Inoculum

Grown tumor(2 weeks old)

Separately
Left abdomen, 106
Right abdomen, 103

40
39 ABC

40>98% (10/10)
39 ABC>98% (10/10)

106
Mixed

40

+

+

103

39 ABC

39 ABC>98%(10/10)

Figures in parentheses represent the number of mice developing tumors, in
relation to the total number inoculated.

proliferation of the cells, were inoculated mixed with viable P-40 cells.
Comparing the control which contained a mixture of heavily irradiatedand untreated P-40 cells, the growth of the tested tumor seemed to be
fairly retarded.
Those findings suggested that an " allogeneic inhibition"-like mechanism
may exist in the inhibitory action of NP-39 ABC cells against P-40 cells,
though they are syngeneic to each other in histocompatibility.

Cytological Studies of Mouse Gliomas
Junkoh YAMASHITA

The techniques of studying metaphase chromosomes have been wellestablished in the study of ascites tumors and in vitro cultivated cells, but
one has not been always successful in obtaining reliable chromosome
preparations directly from solid tumors without an eventually selective
action of tissue culture. We found that a direct drying method following
enzymatic digestion with extremely diluted trypsin (0.02%) is satisfactory
for the karyological study of glial tumors in mice.
Three transplantable methyl-cholanthrene-induced mouse gliomas werestudied.
1) 203 glioma (obtained through the kindness of Dr. Y. Ishida, Department of Pathology, Gumma University). A wide distribution of chromosome numbers ranging from 63 to 132 was observed at the 23rd transplant
generation. Among 100 cells analysed, the frequency of cells with 67
chromosomes was the highest (30 cells). Four kinds of marker chromosomes

RESEARCHES CARRIED OUT IN 196925

were observed; large metacentrics (Ml), small metacentrics (M2), median
submetacentrics (SM) and minutes (m).
2) No.4 glioma (originally induced). Chromosome numbers were distributed from 39 to 82 at the second transplant generation. The highest
frequency (52 among 100 cells) had cells with 40 chromosomes. No marker
chromosome was observed.
3) No.5 glioma (originally induced). Among 100 cells examined at thefirst transplant generation, chromosome numbers ranged from 37 to 84
with the highest frequency at 39 (71 cells). No marker chromosome wasobserved.
In younger No. 4 and No. 5 gliomas with small number of transplantpassage, chromosomal abnormalities were less frequent and the proportionof cells in the diploid range was higher than in the older 203 glioma. It
was reasonably assumed that the chromosomal aberration was not anessential factor in carcinogenesis but a secondary product of stepwise
genetic changes during tumor progression. These results were consistent
with the documented fact on most murine tumors that they have a common tendency to tetraploidy after a period in the diploid range. Nevertheless, it should be emphasized that the chromosome analysis will be ofgreat importance as a visible indicator of invisible genetic changes intumors.

Aggregate-Promoting Substance from BeLa Cells
in Rotation Culture
Yukiaki KURODA

It has been previously found by the author that in mouse spontaneous
mammary gland tumors, mouse plasma cell tumors and chick Rous sarcoma
characteristic changes in aggregate-forming activity of dissociated cells
were exhibited in the process of carcinogenesis (Kuroda, 1968. Gann 59:281; Kuroda, 1969. Ann. Rep. NatI. Inst. Genet. Japan 18: 11). In
embryonic chick liver and retina cells some cell-free materials showing
aggregate-forming activity were obtained from a culture medium in which
the cells were cultured for a few hours (Kuroda, 1968. ExptI. Cell Res. 49:
626; Lilien, 1968. Develop. BioI. 17: 657). These findings suggest that thecharacteristic alteration of aggregate-forming activity of neoplastic cells
may depend upon the qualitative or quantitative changes in their aggregatepromoting substance(s).
In the present experiment the author attempted to isolate cell-free

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

materials showing aggregate-forming activity from HeLa cells in comparison with those from normal human diploid cells. When HeLa S3 cells
were cultured in Eagle's basal medium supplemented with 10% calf
serum at 20°C at 70 rpm on a gyratory shaker for 24 hours, the characteristic aggregates which were formed at 38°C under otherwise identicalconditions were not detected. It was found that a cell-free supernatantobtained from culture medium in which HeLa cells were previously rotated
at 38°C for 2 hours showed aggregate-forming activity on newly liberated
HeLa cells at 20°C in rotation culture.
The supernatant which was obtained from 2-hour rotation cultures of
3 x 106 cells in 3 ml culture medium was diluted with the culture mudium
into various concentrations and tested for their effect on the aggregationof HeLa cells at 20°C. Culture medium containing fifty per cent supernatant had the highest activity among various concentrations examined.
Isolation of the corresponding supernatants from normal human diploidcells and comparison of the activity between those from HeLa cells andnormal cells are now under investigation.

Pachytene Pairing in Translocation Heterozygotes Having
a Huge Knob Close to the Breakpoint
Yasuo OHTA

An exceptionally huge knob (3L111) was found by the present author on
the long arm of chromosome 3 (position .58) in an inbred line, N. C. Inb.
No. 12, derived from the variety Jarvis Golden Prolific (Ohta 1965).
To examine the effect of this huge mass of heterochromatin on thesynapsis during meiotic prophase, two translocation heterozygotes having
the breakpoint close enough, one proximal and the other distal, to theknob position, were selected for hybridization to the inbred line. Strains
T3-8h, the breakpoint at .53, and T3-9f, at .63, were obtained from Dr.
E. B. Patterson, for which the author is grateful to him. They werecrossed to the inbred line, 3L111, and the Fl hybrids were examined
cytologically.
Pachytene pairing in a translocation heterozygote, 3L111/T3-9f, was analysed. Three major types were found:1) complete asynapsis or asynapsis in each of the four arms of thecross-shaped configuration in segments adjacent to the breakpoint, wasobserved in about 50 per cent of the cells analysed although the lengths
of asynaptic arms varied, 2) asynapsis in two or three arms of the

RESEARCHES CARRIED OUT IN 196927

cross-shaped configuration was observed in about 35 per cent of the cells
and, 3) complete homologous pairing throughout the cross-shaped configuration in the rest of the cells, in which two cases existed: either thehuge knob paired with the knobless homologue or it folded down itself
and V-shaped (partly non-homologous pairing in a strict sense). In themost extreme case of 1) the asynaptic segment was as long as about
two-fifths of the length of the long arm of chromosome 3, but no case of
asynapsis beyond the centromere was observed.
Pachytene pairing in another translocation heterozygote, 3U l1 /T3-8h,
was also analysed. Three major types were found:1) complete asynapsis or asynapsis in each arm of the cross-shaped
configuration in the segments adjacent to the breakpoint, 2) complete
homologous pairing throughout the cross-shaped configuration and, 3) partly
homologous pairing and partly asynapsis. The observed frequency ofeach of the three types was about 1: 1: 1.
From the above observation the following conclusion is drawn: Heterochromatin when exists in huge amount heterozygously does prevent pairingof homologous chromomeres or ordinary chromosome segments in the region
adjacent to it, but it does not for homologous centromeres, indicating thatdifferent mechanisms are involved between synapsis of chromomeres andthat of centromeres during meiotic prophase.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

II.

PHYSIOLOGICAL AND DEVELOPMENTAL
GENETICSAn Attempt to Obtain Long-Term Culture Cells from
Drosophila melanogasterYukiaki KURODA

To obtain some long-term culture cells from D. melanogaster, the author
has been searching for the most suitable conditions under which the cellsof this insect can be maintained in luxuriant growth. Embryonic tissues
and larval imaginal discs, gonads, intestines, Malpighian tubes, salivary
glands and ganglions were cultured in various chemically defined media
with or without supplementation of some macromolecular substances.
For the cultivation of embryonic tissues, it was found that embryos at
the time of gastrulation (4 hours after egg laying) were a better sourceof materials for cultivation than those at earlier or later stages. Attachment of explanted tissues to the glass surface of the culture bottles
was an essential factor for the maintenance of cells in active living state.
It was found that the preincubation of explants in physiological salt solution for 60 minutes resulted in an enhancement of their attachment to
the glass surface.
Various chemically defined media which have been previously devisedfor cultivation of insect tissues by many investigators were tested fortheir suitability in culturing Drosophila tissues. None of them were foundto be more efficient in sustaining cell growth than medium K-6 and its
modified medium K-6' which was devised by the author for organ cultureof Drosophila imaginal discs.
Supplementation to medium K-6' of 10% calf serum was found to be
the best among sera from various sources and at various concentrations
examined. The addition to this supplement of heat-treated blood collectedfrom fifth instar larvae or pupae of silkworm exhibited no growth improvement in the culture of embryonic Drosophila tissues. Among ecdysone
analogues tested ecdysterone at the concentration of 0.01 pg! pI had a slight
growth-promoting effect, whereas inokosterone at as low concentration as
0.0001 pg!pl had an inhibitory effect.
Dodecyl methyl ether, a substance acting as juvenile hormone, wastested at various concentrations for its growth-promoting effect on embryonic Drosophila tissues. The result is shown in Table 1.
It was found that 0.1 pg! pI dodecyl methyl ether stimulated the growth

29

RESEARCHES CARRIED OUT IN 1969
Table 1.

Effects of dodecyl methyl ether (DME) on the growthof embryonic Drosophila tissues in culture

Concentrationof DME (pg( pI)

No. of explants
testedNo. of explants inwhich growth wasobserved

Per cent
growthControl
10.0
1.0
0.1
0.01

16
13
2
8
22

12
2
1
7
18

75
15
20
88
82

of tissues, whereas with higher concentrations than 1.0 flgf fll an inhibitory
effect was observed as compared with the control cultures without it.
Fetuin, a fraction of fetal calf serum (Grand Island BioI. Co.) and phytohemagglutinin P (Difco) were also tested for their growth-promoting
activity. The results are shown in Table 2.
Table 2.

Effects of fetuin and phytohemagglutinin (PHA) on the growth ofembryonic Drosophila tissues in culture

Substance

No. of explants
testedNo. of explants inwhich growth wasobserved

Per cent
growthControlFetuin, 0.1 pg(plPHA, 10 pg(pI
PHA, 100 pg(pI

34
24
14
31

28
18
14
23

82
90
100
74

A marked stimulatory effect on the growth of explanted tissues wasfound with 0.1 flgf fll fetuin or 10 flgf fll phytohemagglutinin. For obtaining
cell strains throughout a long-term cultivation of Drosophila tissues further
improvements of culture conditions and attempts to subculture the primary explants are now under investigation.

Clonal Analysis of Embryonic Limb Cartilage Cells in Creeper
Mutant of Chicken
Yukiaki KURODA and Toru SHIBUYA

It is known that the Creeper (CP) gene is a dominant gene on the C
chromosome of chicken. Embryos homozygous for Cp (CP/CP) are lethal
at an early stage of embryonic development, whereas embryos heterozygous

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

for Cp (CP/+) continue to live for a long time after hatching showing
deteriorating growth of their hind limbs. Among offsprings from a crossbetween CP/+ and +/+ parents, CP/+ embryos can be morphologicallydistinguished from +/+ embryos on the eighth day of incubation.
In the present experiment chondrocytes were dissociated by trypsinization of hind limbs of CP/+ and +/+ embryos at various stages of development, and the growth rate, identification of colony-forming cells andtissue-reconstructing activity of dissociated chondrocytes from both strains
were investigated.
The growth rate of chondrocytes from 8-day or lO-day old + / + embryos
was 1.3"-'1.6 times higher than that from CP/+ embryos at the corresPOnding stages. Two morphologically distinguishable kinds of colonies wereobserved when dissociated chondrocytes from embryos of both genotypes
were inoculated on 60 mm plastic Petri dishes: cartilage-making cell colonies(CMC) and fibroblastic cell colonies (FC). Percentages of each type tototal colonies formed from single cells of both genotypes are given in
Table 1.
Table l. Percentages of cartilage-making cell colonies (CMC) and fibroblastic
cell colonies (FC) formed from dissociated limb chondrocytes of Cp/ +and +/+ embryos
Stage ofembryos

Genotype

No. of colonies
countedNo. ofCMC

No. ofFC

Per cent
CMC

8-day

Cp/+

575
424

366
257

209
167

63.7
60.6

525
401

265
178

260
223

50.5
44.4

344
257

146
96

198
161

42.4
37.0

+/+
10·dayCp/++/+
12-dayCp/++/+

Percentages of CMC to total colonies formed from dissociated cells of

CP/+ embryos were significantly higher than the corresponding values of+/+ embryos regardless of age of embryos from which the cells wereobtained. In reconstructed tissues from dissociated Cp/+ cells after rotation
culture, a well differentiated cartilage matrix which was metachromaticallystained with toluidine blue was found, whereas reconstructed tissues from
dissociated +/+ cells exhibited less differentiation of cartilage matrix
which was faintly stained with toluidine blue.
From the above findings it is suggested that limb chondrocytes of Cp/+
have differentiated precociously as was indicated by their retarded growth

RESEARCHES CARRIED OUT IN 1969

31

rate, higher percentage of CMC, and more differentiated cartilage matrix
formation in comparison with the chondrocytes of +/+ embryos.

Effect of Molting Hormone on the Cell Cycle in Insect Epidermis
Kiyoshi MINATO.,

In a series of experiments dealing with the analysis of cell kinetics
during the formation of larval cuticle at the molting stage of Philosamia
cynthia ricini, it has been previously found that there was a long Gzphase (about 48 hours) between the DNA synthetic phase and the following
mitotic phase through the molting. To elucidate further the correlation
between the characteristic duration of the long Gz phase and the phenomenon of molting taking place during this phase, an experimental
induction of larval molting was attempted by injecting ecdysterone, asubstance having molting hormone activity.
When ecdysterone at the concentration of 20 pg/g body weight was
injected into larvae at the initial stage (6~7 hours after molting) or an
early stage (12 hours after molting) of the fourth instar which seemed to
be under no influence of molting hormone, the larvae entered precociously
into the pause prior to molting 12~17 hours after injection. However,most of them died before the molting was completed.
In the same way when larvae at the middle stage (26 hours after molting)of the fourth instar were injected with ecdysterone, a precocious pause
prior to molting was also induced 12~17 hours after injection, in contrast
to the non-injected control larvae in which no pause was detected at thistime. In this case most of the injected larvae proceeded normally into
the fifth instar through molting. The time between the injecting of
ecdysterone and the initiation of molting was 12~17 hours, indicatingthat it was constant regardless of the age at which the larvae were
injected. The relation between the concentrations of ecdysterone used
for injection and the time length following injection before molting remains
to be examined. The effect of these experimental inductions of molting oneach phase of the cell cycle of larval epidermis is now under investigation.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Cytoplasmic Male Sterility and Virus Infection
in Capsicum annuum L.
Yasuo OHTA

A phenotypical similarity is known to exist between cytoplasmic andvirus-induced male sterility in certain plants. In some cases it is probablethat viruses behave as cytoplasmic agents or in some way react withthem.
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).
The first and second inoculation experiments have been described in
the previous papers (Ohta 1968, 1969), so that only the results are now
given in Table 1. The inocula were broad-bean wilt virus (BWV), threestrains of cucumber mosaic virus, i.e. ordinary (CMVo), leguminous (CMVl)
and necrotic spot (CMVn), ordinary and yellow strains of tobacco mosaic
virus (TMVo and TMVy) and a mottle strain of potato virus X (PVXm).
With these previous results only, the cause of reduced pollen fertility
could not be determined. Therefore, the third and the fourth experiments were performed. They are listed also in Table 1. The inoculated
plants were (1) 5-Fresno Chile x Fushimiamanaga FI, (2) Fresno Chile x
Fushimiamanaga FI, (3) Fushimiamanaga x Fresno Chile FI and (4) Fushimiamanaga. The above three FI hybrids possessed an identical genotype(Rfrf) and the same genetic background, the only difference being in
the cytoplasm between the first (5) and the second (N) or the third (N),and practically no difference in the influence of cytoplasm between thesecond and the third. As inocula a hydrangea isolate of CMV(CMVh), a
tomato strain of TMV (TMVt) and a tomato ringspot virus (TMRV}
were also employed.
Markedly reduced pollen fertility appeared only in plants having the(5) cytoplasm, indicating that this specific cytoplasm was responsible forthe reduction in pollen fertility by inoculation with certain viruses.
Progeny tests have provided more evidence indicating that pollen fertility
in plants having (5) cytoplasm and Rfrf genotype, and should be pollen
fertile, was reduced following infection with specific viruses.
It is concluded that a certain interaction between specific viruses anda particular cytoplasm (5) is responsible for the reduced pollen fertility.
In other words, the specific cytoplasm (5) modifies the action of Rf-geneby interacting with specific viruses.

•

Table 1. Pollen fertility (%) of virus-inoculated Capsicum annuum
Exp.
no*

Inoculated plants

Inoculated with+_ _ _ :-:I
Cont. ----~------------~BWV CMVo CMVI CMVh CMVn TMVo TMVy TMVt TMRV AMV PVXm B5
t!j

1st (1) S-F.C.xFu. (8)Rfrf(4) Fushimiama. (N)RfRf

92.7
83.4

93.5 64.2
93.9 86.7

68.0
93.8

2nd (1) S-F.C. X Fu. (8) Rfrf 91.2 75.3
(4) Fushimiama. (N) Rf Rf 92.6 88.0

72.5
90.8

77.6
84.9
69.2
80.7
80.4
81.8

3rd (1) S-F.C. X Fu.
(2) N-F.C. X Fu.
(3) Fu.xN-F.C.
(4) Fushimiama.

(8) Rfrf(N)Rfrf(N)Rfrf(N) RfRf

88.0
90.8
90.5
84.3

65.2
85.1
77.0
78.7

64.1
82.1
71.6
87.4

4th (1) S-F.C. X Fu.
(2) N-F.C. X Fu.
(3) Fu. xN-F.C.
(4) Fushimiama.

(8) Rfrf(N)Rfrf(N)Rfrf(N) RfRf

91.6
94.7
90.9
92.0

70.9
86.4
85.5
86.3

63.2
89.9
89.9
90.5

~

92.7
94.3

:-:I

()

:::c:

89.3
88.3

86.9
91.0
74.6
88.8
84.7
84.5

63.9
89.3
90.0
90.4

87.3
93.6

t!j

(fl

()

~

85.6
80.1
83.6
88.2

:-:I
:-:I

~
t:1
0

75.2
85.7
86.9
90.2

c:::

>-l

Z
<J:)

0>

<J:)

* 1st: 1967. 2nd: 1968 3rd: 1969a. 4th: 1969b.
+ See text for the abbreviation of virus name.

t3

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Since it is evident that a cytoplasmic entity reacts in this way withspecific viruses, its nature and origin were discussed, and the following
speculation was proposed. A cytoplasmic entity for male sterility in
Capsicum could have originated from an exogeneous virus in the courseof evolution followed by the loss of infectivity. Then it may be supposed
to have arrived at a state of a kind of plasmon or plasmid of RNA nature.
(The details were submitted to Japan. J. Genetics Vol. 45.)

A Variant Found in the Progeny from Graftingin Capsicum annuum
Yasuo OHTA

If a hereditary trait could be altered by grafting, how could such an
event take place. One may speculate that the mechanism would be akind of transformation or transduction. There are evidences which indicatethat transformation does take place in insects such as Bombyx (Tsujita
and Nawa 1969) and Ephestia (Nawa and Yamada 1967, 1968). Recently
alteration of certain Mendelian traits by grafting was reported in red
peppers (Capsicum annuum L.) (Kasahara 1967, 1968). My curiosity was
aroused and I repeated Kasahara's experiment.
My grafts were performed between two cultivars of Capsicum annuum
L. provided by Dr. Kasahara: Tochigisantaka (abbrev. T) and Kiiro (K).
The material has been maintained by selfing for more than five generations. Three fruit characters were chosen for analysis: erect vs. pendent,
fasciculate vs. non-fasciculate fruiting, and yellow vs. red ripe pericarp.
Cultivar T is erect, fasciculate, and red (uP, fa, y+Cl+); and K is pendent,
non-fasciculate, and yellow (Up+, fa+, YC1). Crossing experiments madefor control revealed that three characters are Mendelian traits, the pericarp
color being inherited as dihybrid, and that the four genes concerned are
inherited independently.
Grafting was made by usual cleft-graft method in reciprocal combinations.
Young plants, two to three months old, having 20 to 30 leaves and alreadyinitiating flower buds, were used as stocks, while about three weeks oldseedlings served as scions. To be sure that the scions depended mainlyon the metabolism of the stocks, leaves of the scions were removed twicea week during their growth except for two or three leaves at the top.
Several flowers of each scion and stock were artificially self-pollinated.
Seeds thus obtained were sown and grown into the Gl generation. Several
plants of each family were randomly selected for selfing and their seeds

RESEARCHES CARRIED OUT IN 196935

gave rise to the G2 generation.
No particular phenomenon was observed either in the course of grafting(Go) or in the Gl generation. As to the G2 generation, in the progeny ofa T scion grafted onto K, a single variant was found whose fruits were
red and erect but not fasciculate, indicating a change from recessive fa
to dominant fa+. The possibility of a mistake by contamination is refutedby the fact that the plant still possesses another recessive character,namely erect. To obtain more evidences about this variant, whether wasit originated by a spontaneous mutation or by grafting, for instance,
selfing as well as backcrossing with both parents were made. Also thesister plants were selfed for further investigation.

Induction and Inhibition of Nucellar Embryo Development
in Citrus
K. FURUSATO aud Y. OHTA

From the viewpoint of citrus breeding it is important to obtain hybrid
seedlings from polyembryonic seeds which usually contain nucellar embryos. However, true hybrids can be obtained when the nucellar embryos
are eliminated by certain treatments. The present paper deals withexperiments aiming at inducing artificial development inhibition of nucellar
embryos in the Rutaceae genera, Citrus, Fortunella and Poncirus.
Neither legitimate nor nucellar embryos developed when no pollination
was carried out after emasculation in the parthenocarpic and polyembryonic C. unshiu. To examine the direct effect of pollination on the numberof embryos in a seed, the following crosses were made: polyembryonic(poly-) x poly-species, poly- x monoembryonic (mono-) species and monoX poly-species. In the case of C. unshiu as the female parent, the numberof embryos in a seed seemed to be slightly higher when pollen of monoembryonic species was applied than when that of polyembryonic species
was used.
Small amounts of 0.01% solution of a-naphthalene acetic acid (aNAA)
were applied to the stigmata of 100 emasculated flowers of C. unshiu. Of
15 fruits obtained, none had set seeds. When injected into young fruitsof both mono- and polyembryonic species, no change was observed in thenumber of embryos in a seed. In the case of C. aurea, a monoembryonicspecies, young seeds were excised from treated fruits and examined microscopically. No nucellar embryos were found. Maleic hydrazide (MH)solution was either injected into young fruits or sprayed on the leaves of

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

fruit bearing branches. Generally speaking, the mean embryo numberin the treated group decreased slightly in C. natsudaidai and F. japonica,
whereas it showed an increase in C. unshiu. In the remaining three species
no change was seen.
It was attempted to prevent transportation of nutrients in branches
bearing young fruits. Bark ringing produced no change in the numberof embryos. Incisions on one side of the branches produced a s,lightly
higher embryo number in C. unshiu, C. sinensis (Valencia) and C. pseudoparadisi, whereas it produced a lower embryo number in C. leiocarpa
and F. japonica, while no change was seen in C. grandis and C. natsudaidai.
Effect of fertilizer and soil was examined with potted three-year-old F.
japonica. When lime superphosphate was used, the mean embryo number
was slightly higher than in the control. This is interesting since it iswell known that much phosphate is required for seed formation. Anotherexperiment was carried out to ascertain the effect of fertilizer. 1.0%
urea solution was sprayed on the leaves. The embryo number was notaffected by the treatment.
Potted plants of F. japonica were placed either in a phytotron kept at
30°C constantly day and night or in a glasshouse without heating. The
experiment lasted from the end of September, when the plants were in
full bloom, to the end of December. During that period the temperaturein the glasshouse was below 30°C in daytime and much lower at night.
The embryo number was the same at the temperatures used. (The details
were published in Seiken ZiM No. 21. 1969)

RESEARCHS CARRIED OUT IN 1969

III.

37

BIOCHEMICAL GENETICS
Syntheses of Drosopterins
Saburo NAWA..

The possibility of syntheses of Drosophila eye pigments, drosopterins, on
the reaction with reduced pteridine and anion from a-keto acid has been
already suggested by Nawa and Forrest (this Annual Report, No. 13). In
co-operation with Sugiura and Goto of University of Gakushuin, a large
quantity of two kinds of red pigments sufficient for various kinds ofanalyses were obtained in the reaction between reduced pteridine andd, l-,B-hydroxy-a-keto-butyric acid. Combustion analyses, UV-spectra, ORDcurves, Rf-values and electrophoretic mobilities of the synthetic pigments
were identical with those of drosopterin and isodrosopterin respectively.
The incubation of 7, 8-dihydro-2-amino-4-hydroxypteridine with d, l-ahydroxyacetoacetic acid gave also the same products. Thus, synthesisof drosopterins in this manner was completely proved to be achieved.
Furthermore, these synthetic procedures suggest strongly a possibility
that in vivo drosopterins are derived from the reaction between reduced
pteridines and keto-acids. (For details, see Tetrahedron Letters No. 34,
1969).

Genetic Effects of DNA in Bombyx
Saburo NAWA. Masa-Aki YAMADA and Mitsuo TSUJITA

It has been shown that treatment of Ephestia larvae or eggs with arecessive eye-color genotype with DNA from wild-type produced wildtype individuals in the treated generation and in subsequent generations(Nawa and Yamada, 1968. Genetics 58: 573). In co-operation with Sakaguchi,
the experiment has been designed for inducing DNA-mediated genetictransformation in silkworm. There are many advantages in the use ofsilkworms in this kind of experiment. For example, the egg color ofwhite-l is determined not only by its own genotype but reflects that ofits mother. Of most important is that eggs are deposited on a paper bya female placed in a separate box (moth funnel) within a short time,making contamination of an egg absolutely impossible.
When larvae of w-l/w-l (white eyes and white egg color) were treated

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

with DNA prepared from wild-type larvae, black-eyed moths were obtainedin the treated generation and in the progeny by backcross to untreated
w-l/lO-l moths. The change of w-l to w-l+ was detected in eggs in somecases by their color, but there were cases when the change was detected
only after raising to adults. Several unexpected phenomena were observed in subsequent backcross generations of these mutants. 1) Morethan half of the black-eyed moths behaved as if their genotype was
w-l+/zv-l+. 2) A few behaved as if their genotype was w-l+/w-l, butsome of them produced significantly higher number of black-eyed adults
than would be expected, when crossed to untreated w-l/w-I. 3) Egg
color produced by some of these mutants was unusual and the abnormality
was also found even in later generations. 4) Sometimes, repeated appearance of mutations in later generations of outcrossing of mutants wasobserved. Mechanisms for these unusual feature were considered interms of replication of the effect of incorporated DNA. (The details were
submitted to Genetics).
A triple recessive genotype, fern; oc; pe was also treated with + DNA.
Both in the progeny by cross of the treated animals to untreated fern; oc;
pe and in the 2nd backcross generation, several individuals having the
genotype fern/fern; oc/oc; pe+/pe were obtained. It was observed that thegene pe+ has persisted for several generations and segregated like anormal allele at the pe locus. So far no instance for the change of fem-+
fem+ or oc-+oc+ has been obtained.

Characterization of Incorporated DNA in Ephestia Eggs
Treated with DNA
Masa-Aki YAMADA and Saburo NAWA

Evidences of DNA uptake by Ephestia eggs have been reported previously (Annual Report No. 18). DNA was extracted from eggs treatedwith 3H-DNA of Salmonella for 4 hours using SDS-phenol method. Preparative cesium chloride density gradient centrifugation of the DNA
solution was carried out in SW 39 rotor of Spinco L centrifuge. The
patterns of DNA fractionation showed that the peaks of radioactivity were
found at the positions of double stranded and single stranded DNA of
Salmonella. This was verified by analyzing the mixture of native anddenatured Salmonella DNA. When DNA extracted from the eggs immediately after the treatment with labelled DNA was fractionated, single
stranded DNA was detected as a shoulder of the double stranded peak.

RESEARCHES CARRIED OUT IN 1969

but when eggs were left standing for 15 hours after treatment with DNA
single stranded DNA was obtained in a separate peak higher than thatof the double strand. These results indicate that DNA was incorporated
into the eggs at double stranded state and was denatured gradually into
the single strand.
DNA Specific in Early Development in Ephestia
Masa-Aki YAMADADNA extracted from eggs at an early development stage was found tobe heterogeneous. Eggs were collected for 0-18 hours after spontaneous
laying in plastic boxes. DNA was extracted from the eggs with TrisSDS-phenol method, treated with RNase and deproteinized with chloroform.
The density of DNA was analyzed by using Spinco E analytical centrifuge.
Preparative cesium chloride density gradient centrifugation of the DNA
was performed in an angle rotor ;f 40 of Spinco L centrifuge. It was
found that the eggs at the early stage of development contained at least
two DNA fractions which were sensitive to DNase. The values of theirbuoyant densities were 1.696 (the same as that of adult DNA) and 1.672.
The distribution of the main 1.696 fraction in density gradient was asymmetrical, indicating that the fraction was heterogeneous. When this DNA
was denatured at 100 for 20 minutes, two bands with density of 1.723
and 1.702 were obtained, but in the adult DNA denatured at 100 only
one band with density 1.723 was detected. Therefore the 1.696 fraction
may have consisted of two components. The minor component with 1.672
density was specific at the early development stage, since it was notfound in late stage eggs. Further study of these components is in progress.
00

Incorporation of HC-phenylalanine or HC-tyrosine into the Hypodermal
Cuticle of the Silkworm. I. Lethal Lemon Larvae
Mitsuo TSUJITA and Susumu SAKURAI

Incorporation experiments with HC-phenylalanine or HC-tyrosine as metabolites into the hypodermal cuticle of normal, lemon and lethal lemon
larvae were carried out. It was found from the experimental results thatin lethal lemon larvae the radioactivity of alkali-insoluble protein containing
as principal component of hard cuticle protein, sclerotin, is conspicuously
weaker in both head and body than in normal larvae.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Incorporation of HC-phenylalanine or He-tyrosine into the Hypodermal
Cuticle of the Silkworm. II. Lethal Albino Larvae
Mitsuo TSUJITA and Susumu SAKURAI

Incorporation experiments with HC-phenylalanine or HC-tyrosine as metabolites into the hypodermal cuticle of normal and lethal albino larvae
were carried out. It was found that in lethal albino larvae also the alkaliinsoluble protein containing as the principal component of hard cuticle
protein, sclerotin, is conspicuously weaker in both head and body parts
than in normal larvae.

Sepiapterin Reductase in the Silkworm
Mitsuo TSUJITA and Susumu SAKURAI

Tile occurrence of two different reductases, sepiapterin reductase anddihydrofolate reductase, in the silkworm was demonstrated (Matsubara
et al. 1963. Nature 199: 808-809 and Katoh et al. 1969. Handbook of IVthIntern. Congo Pteridine: 32-33). The former reductase catalyses sepiapterin
to dihydrobiopterin and the latter catalyses dihydrofolate to tetrahydrofolate.
In our previous paper (Tsujita 1961. Jap. J. Genet. 36: 337-346) the
enzyme reducing sepiapterin in the reaction solution containing crude
enzyme solution prepared from normal larvae, NADPH and phosphatebuffer at pH 6.2-7.0 was called pterin reductase. For the catalytic reaction by this enzyme, the requirement for NADPH as a cofactor is quite
specific and the optimum rate of the reaction occurred at about pH 6.8.
The enzyme activity in the reaction solution is not inhibited by aminopterin. The appearance and increase of dihydrobiopterin in the reaction
solution was detected.
lem
lem l

t"'--=:Dihydrobiopterin
I

Sepiapterin

Sepiapterin
reductasei(NADPH)
Fig. I.

~ ~ Tetrahydrobiopterin

Dihydrofolate
reductasei(NADPH)

Main pteridine metabolic reaction in the silkworm.

It may be safely said from these experimental results that the enzyme
reducing sepiapterin in our experiments is really sepiapterin reductase.
This enzyme activity is strong in normal larvae, but in lemon larvae a

RESEARCHES CARRIED OUT IN 196941

much lower activity is detected than in normal larvae. Furthermore, ill
lethal lemon larvae the enzyme activity is almost lacking.
Each crude enzyme solution prepared from normal, lemon or lethallemon larvae showed almost the same activity of dihydrofolate reductase.
It is considered from these experimental results that the main pteridine
metabolic reaction as shown in Figure 1 was taking place by the two kindsof reductase.
Characterization of Pteridine Granule Membrane in Larval
Skin Cells of the Silkworm
Susumu SAKURAI and Mitsuo TSUJITA

Using pteridine granules isolated from the larval hypodermis of anormal silkworm strain and various abnormal oily mutants the following
chemical analysis was carried out.
Isolated pteridine granules were solubilized by adding 1% sodium deoxycholate in 0.01 M NaHCOs-O.01 M ammonium formate buffer (pH 9) atroom temperature. Dialysis was carried against 0.01 M NaHCOs-1 mM
EDTA-1 mM 2-mercaptoethanol-0.01 M ammonium formate buffer (pH 9)
in a cold room at 4°C. The dialysed sample was chromatographed on a
4 x 55 cm column of Sephadex G 200 equilibrated with 0.01 M NaHCOs-1 mM
EDTA-1 mM 2-mercaptoethanol-0.01 M ammonium formate buffer (pH 9).
The protein eluted at the void volume was dialyzed overnight at 4°C
against 0.01 M NaHCOs-EDTA-mercaptoethanol-ammonium formate buffer(pH 9) and the sample was precipitated between pH 4.0 and 5.6, indicating
a low isoelectric point typical of acidic protein.
The lipid content by weight of the chloroform-methanol soluble fraction
amounted to 35-40% of dry weight. Protein content was 50-60% of membrane dry weight. Carbohydrates seemed to be low accounting for 0.31.0% of dry weight. These results show that pteridine granule membraneprotein was composed almost entirely of protein and lipid.
The hydrolysates by 6 N HCI at 110 0 of lipid-free membrane proteinsobtained from oily mutants and normal strains were analyzed by anautomatic amino acid analyzer. The results indicated that membraneproteins were replete with hydrophobic amino acids, such as alanine,
leucines, phenylalanine, methionine and valine. Cysteine content was
the lowest and that of acidic amino acids, aspartic acid, glutamic acidwas high in comparison with the basic amino acid content. The hydrophobic and acidic amino acid composition of the membrane proteins of
w b oily mutant and C-124 normal strain were very similar. On the

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

contrary, lysine content of normal membrane protein was somewhat low incomparison with wb oily mutant.

Immunochemical Study of Pteridine Granule Membrane in
Hypodermal Cells of Silkworm Larvae
Susumu SAKURAI

As a part of investigations planned to clarify the relationship between
normal and abnormal proteins, an immunological analysis of membraneproteins was carried out with the use of Ouchterlony double-diffusion
reaction.
C-124, E tern and W-C were used as normal strains with non-transparent
larval skin, and wa , wb , wo h (Aojyukuhakuran oily) and oa were used as
mutant strains with semi-transparent or transparent larval skin. It has
been known that among those mutants wa , wb and wo h belong to Wa multiple.allelic series located on chromosome 10, but that oa is located on chromosome 14.
Immunization was done in rabbits by eight intravenous injections of 80
mg membrane protein, each in 1% aluminium sulfate solution over a
period of one month. Precipitin lines were allowed to develop at room
temperature. Using antiserum to normal membrane protein or using
antiserum to abnormal membrane proteins obtained from oily mutants,
several membrane preparations from different oily mutants were comparedin double diffusion analysis.
On immunodiffusion, anti-E tern normal membrane protein antibody gavea single precipitin band with its antigen, normal membrane protein of
E tern. Anti-woh abnormal membrane protein antibody reacted against
its antigen, and abnormal membrane protein of wo h oily mutant displayed
several precipitin bands. On the other hand, membrane protein of oaoily mutant gave a precipitin line with anti-E tern membrane antibody,indicating an identity pattern with membrane protein of E tern. Whenmembrane proteins of E tern and w a oily mutant were allowed to reactwith anti-E tern membrane antibody a reaction showing a complete difference
occurred. Similar results were seen on immunodiffusion when anti-E tern
membrane antibody reacted with membrane protein of E tern and membraneproteins of wo h and w b oily mutants. These facts suggest that transparencyof larval skin of Wa multi-allelic oily mutants is due to a defect of membrane proteins themselves.

RESEARCHES CARRIED OUT IN 1966

43

Aldolase Isozyme Patterns of Brain and Brain Tumors in Mammals
Junkoh YAMASHITA and Kazuo MORIWAKIIt has already been reported that there were three molecular speciesof aldolase in mammalian tissues; aldolase A in muscle, aldolase B in liver
and aldolase C in brain, and that the aldolase B found in normal rat
liver disappeared in fast-growing rat hepatomas and instead aldolase A
was produced.
1) The brain of the Japanese monkey (Macaca fuscata), was studied
to ascertain whether or not aldolase patterns have topographic characteristics within the brain. The sample was homogenized with one volumeof 20 mM tris-HCI buffer (pH 7.4) containing 0.15 M KCI and 5 mM EDTA.
The aldolase of the supernatant fraction obtained after centrifugation at
105,000 g for 60 min was separated electrophoretically on a cellulose acetate
membrane and stained as described by Matsushimaetal. (Biochem. Biophys.
Res. Comm. 30: 565-570, 1978). All parts of the brain showed five distinct
bands of A-C type. There were aldolase C(C4) on the cathodal side,
aldolase A(A4) on the anodal side and three hybrid bands (A3Cl, A2C2
and AlC3) between them. The patterns in the cerebral cortex, corpus
callosum, hippocampus, thalamus, midbrain, pons and medulla were similarand A3Cl activity was the most pronounced among the five bands, whileboth in the cerebellar vermis and hemispheres the most pronounced was
C4 band. No gross difference was detected between the gray and thewhite matter. The peripheral nerve obtained from the sciatic nerve also
showed five bands of A-C type, among which A4 was predominant, although
the overall activities were weaker than in the brain.
2) The aldolase patterns of three transplantable mouse gliomas were
investigated. No. 4 and No. 5 gliomas showed five bands of A-C type,with more pronounced activities at AlC3 and A2C2 instead of A4 and C4of normal mouse whole brain. 203 glioma, on the other hand, presented
four bands lacking in C4, but definitely carried C components such as
AlC3, A2C2 and A3CI. In contrast to No. 4 and No. 5 gliomas whichwere recently induced, 203 glioma had been serially transplanted through23 generations. The findings indicated that all three tumors had their
origins in the brain, although the present aldolase patterns somehow
deviated from the original form.
3) One case of human brain tumor (glioblastoma) obtained as a surginal
specimen was studied in similar manner. Comparison was made with
the normal cerebral cortex which had to be sacrificed in the surgicalprocedures. This tumor showed five bands of A-C type, but C4 activity

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

was extremely weak.
These results coincided with observations madel in hepatomas, namelythat the aldolase isozyme patterns have a common trend to become closer
to the muscular (A) type during tumor progression. It is very likely thatthe aldolase isozyme patterns of brain tumors might be new diagnostic
tools helping to evaluate their origin and the degree of malignancy,
histological diagnoses being equivocal.

Biochemical Characters of Serum Transferrin in Several Rattus
Species Collected from South-East Asia and Oceania
Kazuo MORIWAKI, Kimiyuki TSUCHIYA and Harumi SAKATAElectrophoretic pattern and molecular weight of serum transferrin of
several wild Rattus species collected from South-East Asia and Oceania
were compared. Identification of transferrin bands in starch gel electrophoresis was carried out by labelling serum transferrin with Fe 59 and byautoradiography on non-screen type X-ray film. Approximate molecularweight of transferrin was determined by thin layer gel-filtration on Sephadex
G-200 plates. Fe 59 labelled serum was applied on these plates, followed
by 20 hours elution. Separated serum proteins were taken up on a filter
paper on which the mobility of transferrin, that is its approximate molecular weight, was determined by autoradiography. The data thus obtained
are summarized in Table 1.
Table 1. Electrophoretic pattern and molecular weight of serum transferrin
in several Rattus species collected from South-East Asia and Oceania
Electrophoretic
bands of Tf
Species

Place of collection

Betweenoriginand SU2

Rattus rattus

Okinawa, Japan
Tainan, Taiwan
Khao Yai, Thailand
Luzon, Philippines
Bogor, Java
Makasar, Celebes
Kuala Lumpur, Malaysia
Kuala Lumpur, Malaysia

0
0
0
0
0
0
0
0

H
H
H
H
H

R. Bowersii
R. sabanus

Approx. Chromolecu- mosome
Betweenlar
number(2n)11Sa2 and weight
Hp.
2
2
0
2
2
2
1
1

70,000
H
H
H
H
H
H
H

42
42
42
42
42
42
40*
42

45

RESEARCHES CARRIED OUT IN 1969
Table 1 (continued)
Electrophoretic
bands of Tf

Species

Place of collection

R. jalorensis
R. argentiventer
R. exulans
Bandicota indica

Kuala Lumpur, Malaysia
Kuala Lumpur, Malaysia
Makasar, Celebes
Tainan, Taiwan

R. rattus rattus
H

H

H

Approx. Chromolecu- mosome
larnumber
Between Between(2n)1I
originSa2 and weightHp.
and Sa2H

32
38(39)
42
42(43,44)

2

2

H

40

g
Brisbane, AustraliagWellington, New Zealand g
Port Moresby, New Guinea gCairns, Australia

Cairns, Australia
R. Conatus
Brisbane, Australia
R. !uscipes
Kuala Lumpur, Malaysia
R. muelleri
Kuala Lumpur, Malaysia
R.diardi
R. rattus (Japan) }F

R. rattus rattus

70,000

1
2
1
2

2
2
2
2
0
1
0
1
0
1
0
1
1
0
0
2

0
0
0
0

i

H

42
42
42
44

H

38

H

38

H

38

H
H

/I

/I
/I

H

38

1) Yosida, T. H. et at. 1969. Mam. Chromo News. 10: 217-219.
This data will be reported in this Annual Report, 1970. Abbreviations: Tf;
transferrin, Sa2: Slowa2-macroglobulin, Hp; Haptoglobin.

*

Organ-Specific Expression of Allelic Peroxidase Isozymes in
Heterozygotes of Oryza perennis
Toru ENDO

Two allelic peroxidase isozymes, 2A and 4A, were zymographicallydetected on starch gels in an annual strain W107 (Pi) and a perennial
strain W1294 (P2) of a wild rice, Oryza perennis Moench, respectively.
Both isozymes were specified by codominant alleles, Peu and Pe u at the
Pe locus (Shahi et at. 1969. Japan. J. Genent. 44: 321). The heterozygote
Pe2A fPe u produced a hybrid isozyme 3A together with both parental isozymes in some plant organs, i.e, leaf blade and leaf sheath as well as inlemma and palea. An organ-specific difference in the intensity ratio of
isozyme bands was observed. In the leaf blade and leaf sheath, the46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

intensity ratio seemed to follow the distribution expected from random
association of the monomer components of 2A and 4A isozymes. However, the ratio of the three bands in lemma and palea deviated fromexpectation as the intensity of 2A was very weak and that of 3A did notcorrespond to the sum of the intensities of 2A and 4A bands. The same
fact was also found in 24 out of 25 heterozygous F2 segregants.
The zymograms of 60 F2 plants were classified into PI, Fl and P2 types.
The ratio was considered to be 1:2:1, under the assumption that it was
distorted due to gametic selection whose coefficient was calculated to be
0.857. One particular segregant was found which had an Fl type zymogram
but the intensity ratio of the three bands corresponded to the expecteddistribution from association of the monomers, i.e., the intensity of 3A
was approximately the sum of those of 2A and 4A bands. In leaf sheath
zymograms, however, no difference in the intensity ratio was detectedamong the 25 F2 heterozygotes, including that particular plant. Such anorgan-specific profile of the allelic isozymes in the heterozygotes wastentatively explained by a competitive activation of the alleles due to two
kinds of inducing systems each carried by the parental strains. In theparticular plant, one of the inducing systems might have been deleted
or inactivated by mutation, resulting in the activation of both alleles at
a similar level.

RESEARCHES CARRIED OUT IN 1969

IV.

47

EVOLUTIONARY GENETICS

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

Aegilops caudata L. includes two varieties, typica and polyathera Boiss.
These two varieties are characterized by awnedness at the lateral spikelets. The following three strains were used in our experiment:
Variety
OriginCharacters
No.1 polyathera
Turkeyawned, waxy, earlyNo.2 typica
Volos, Greece
awnless, non-waxy, late
No.3 typica
Hama, Syria
awnless, non-waxy, early
Reciprocal hybrids between any two of the three strains were easily
obtained. The chromosome behavior in the Fl's was normal throughout
meiosis, but the hybrids were almost completely pollen sterile. For the
combination of no. 1 with no. 2, for instance, the reciprocal hybrids backcrossed successively in great numbers to both parents as pollen providers
gave rise to two substitution backcross lines (SB) and two restoration
backcross lines (RB) as follows:/ x (no. 1 o)n .... ·RB n(no. 1 ~ Xno. 2 0) Fl",
X(no. 2 o)nooSB n/x(no. 1 0 )n
·SB n(no. 2 ~ Xno. 1 0) Fl",X (no. 2 0 )n ... ooRB n
In the same way the other two combinations were carried out so that inall six SB lines and six RB lines were obtained.
In the combination of no. 1 with no. 2, both pollen fertility and seedsetting began to increase from Bl and normal fertility was recovered by
B2 in both SB and RB lines when backcrossed to no. 2 as the male parent(Figure 1). On the contrary, when no. 1 was the male parent, pollen
and seed fertility remained almost at zero until B2, then began to increase from Ba and normal fertility was recovered by Bs in both SB andRB lines. Such delay in the repair of fertility by successive backcrosses
was termed "delayed effect" by Kihara (Kihara, H. 1968. Proc. III Internatl. Wheat. Genet. Symp.: 125-134). The delayed effect was observedin the SB line with cytoplasm of no. 2 and in the RB line with cytoplasm{)f no. 1. The latter case is most interesting since the delayed effectappeared in no. 1's own cytoplasm.

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 2()
100

.

.

I

80

I
/

/

§

/
/

>.

a
.......
....

Q)

c::
Q)

/

60

.

/

0/
/

//
//

40

o //
/

;:g
P-.

o

/

/

/.

/

20

/

/
/

/

/

0

- - -- - -() --- - - ..!---.,....-----,----.--F,

8,

82

83

85

Fig. 1. Pollen fertility in successive backcrosses in therestoration and substitution lines of reciprocal hy·
brids, Aegilops caudata no. 1 X no. 2 (1964-1969).
0: RB to no. 1.
.: SB to no. 1.
6.: RB to no. 2.
&: SB to no. 2.

In the combination of no. 2 and no. 3, only the B2 backcross genera
tion is available until now but essentially the same tendency may bealready noticed concerning the recovery of fertility. While in the com·bination of no. 1 and no. 3, pollen fertility was about 10 and 13 per centin the reciprocal F1's, it was about 69 and 58 per cent in B1, about 70
and 65 per cent in B2 backcrossed with pollen of no. 3 and no. 1, respectively. Distinct delayed effect was not observed in this combination but
no. 1 was slightly inferior to no. 3 as pollen parent in recovering fertility.
The real cause of the delayed effect is still unknown. From our investigations, it may be concluded i) that a delayed effect was observed
between no. 1 and no. 2 and also between no. 2 and no. 3 even in RB
lines having their own cytoplasms, but not between no. 1 and no. 3, ii)that no. 2 recovered fertility quicker than no. 1 or no. 3 in the order of
no. 2:J;>no. 3>no. 1,
indicating that the three strains of Ae. caudata are slightly different intheir genomes but, probably not in their cytoplasms. The situation is alittle different from our previous expectation.

RESEARCHES CARRIED OUT IN 1969

49

Comparison of the Developmental Pattern between Cultivatedand Wild Types of Rice
Hiroko MORISHIMA and H,I. OKA

To look into difference in the developmental patterns between wild andcultivated types and their variation due to growing condition, three sativa,
two perennis and one glaberrima strains were grown in gravel culture(using Matsushima's formula) and in paddy, and were measured for dry
matter weight, length of various organs and other yield characters.
Measurements were taken three times during the growing period and at
maturity. The results may be summarized as follows: 1) The wild
type has a lower growth rate in the early stage than the cultivated type.
2) Its growth rate rapidly increases at a certain stage before heading,but also rapidly declines after the maximum is attained. In contrast,
improved cultivated varieties were found to keep up a relatively low
growth rate for a long period. 3) In cultivated types the panicle andthe third internode (from top) elongate simultaneously about ten days
before heading, and the elongation of first and second internodes resultsin heading. In the wild type, the elongation of the third internode results in heading, and the first and second internodes elongate after heading. Probably an amount of nutrients may be consumed in this process
reducing the deposit of carbohydrates in the endosperms. 4) Examining
the variation in size of various organs due to growing conditions, wild
plants appeared to be phenotypically more plastic than cultivated plants.
They showed a large variability in the length of panicle and internodes
which were much less variable in cultivated types.

A Survey of Genetic Variations in the Populations ofWild Oryza Species and their Cultivated Relatives
Hiroko MORISHIMA and H,I. OKA

The first-generation plants raised from population seed samples belonging to Oryza perennis, O. sativa, O. breviligulata and O. glaberrima, eachcollected from its natural habitat or a farmer's field, were observed forvarious metric characters, and their intrapopulational genetic variability
was estimated for each character and also in terms of generalized variance.
In O. perennis, perennial and allogamous forms generally had in their
populations more genetic variations than the annual and autogamous
forms, while the latter were more diversified from population to population.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

The level of heterozygosity was estimated from the ratio of withinline to between-line component of genetic variance. Perennial and allogamous forms were more heterozygous than annual and autogamous ones.
Diallel analysis of a perennis population showed appreciably large meansquares due to dominance for certain characters. Further, illtrapopulational variability in spikelet size was observed using the original seeds
collected from various sites. Though cultivated forms (sativa and glaberrima) were largely homozygous, their populations in primitive agricultural conditions contained as much genetic variations as wild forms.
This was considered to be due to introgression and disruptive selectionin the environment.

RESEARCHES CARRIED OUT IN 1969

V.

51

MATHEMATICAL AND STATISTICAL STUDIESON POPULATION GENETICS
The Rate of Molecular Evolution Considered from theStandpoint of Population Genetics
Motoo KIMURA

The rate of amino acid substitutions was computed for hemoglobins invarious lines of vertebrate evolution. The method of calculation was asfollows. Let n aa be the total number of amino acids in each of two
homologous polypeptide chains being compared with each other and let
d aa be the number of sites in which they are different. Then the mean
number (Kaa) of substitutions per amino acid site over the whole evolutionary period that separated these two polypeptides may be estimatedfrom
Kaa= -2.30 loglO(l- Pa) ,where p'l=daa/naa is the fraction of differing sites.
of this estimate is

The standard error

Ih=VPa/{(l-Pa)naa} .

The rate of substitution per amino acid site per year may then be computed fromkaa=Kaa/(2T) ,where T is the number of years that have elapsed since divergence froma common ancestor.
The estimated rates of amino acid substitutions based on comparisonsof the alpha hemoglobin chains of various mammals with that of the
carp are about the same as those based on comparisons of the carp alphaand mammalian beta or the alpha and beta chains in mammals. All thesecomparisons give roughly 10- 9 for k aa • In addition, comparison of humanbeta hemoglobin with lamprey globin gives a similar value.
These remarkable uniformities in the rate of amino acid substitutions
in evolution support the hypothesis that a large fraction of amino acidsubstitutions that occurred in these proteins are result of random fixationof selectively neutral or nearly neutral mutations.
This leads to the conclusion that random genetic drift is playing
an important role in determining the genetic structure of biological

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

populations and the prediction that genes in "living fossils" will haveundergone as many DNA base substitutions as corresponding genes inmore rapidly evolving species.
A new term pauling was proposed for a unit of evolutionary rate at
the molecular level, defined as the rate of substitution of 10- 9 per aminoacid site per year.
For details, see Kimura (1969. PNAS 63:1181-1188).

The Number of Heterozygous Nucleotide Sites
Maintained in a Finite Population Due to
Steady Flux of Mutations
Motoo KIMURAIn the present study the following model was assumed: The numberof nucleotide sites making up the haploid genome is so large, while themutation rate per site is so low, that whenever a mutation occurs it represents a mutation at a previously homallelic site, that is, a site inwhich no mutant forms are segregating in the population.
Since a mutant that appears in a finite population is either lost fromthe population or fixed in it within a finite length of time, under continued production of new mutants over many generations, a statisticalequilibrium will be reached between the production of new mutants andtheir random extinction or fixation. In such an equilibrium, a stable
distribution will be realized with respect to frequencies of mutants among
different sites, if we restrict our consideration only to those sites inwhich mutant forms are currently segregating.
We will denote by rp(P, x) the frequency distribution at equilibrium suchthat rp(P, x)dx is the expected number of sites in which the frequencies
of mutants lie in the range (x, x+dx), and p is the frequency at thetime of the first occurrence of the mutant at each site.
Let l.'m be the number of sites in which new mutants appear in the
population each generation, and let

be the expection of an arbitrary function f(x) with respect to this distribution. Integration in the right hand side of the above equation isover the open interval (0,1). This is an approximation to the summation over all frequency classes from x=1/(2N) to x=1-l/(2N). It can

RESEARCHES CARRIED OUT IN 196953

then be shown that It is given byIt(p) = {l-u(P)} ~: ¢ f(~)U(~) d~ +u(P)L f(~){l-u(~)}d';
¢

,

where

in which u(P) is the probability of eventual fixation, u'(~)=du(;),d;, andVaz is the variance in the rate of change in mutant frequency x per
generation.
The number of heterozygous nucleotide sites per individual at equilibrium (denoted by H(P) is given by If(P) with f=2x(1-x). In thespecial case of neutral mutations, u(P)=p and the above formula for It
reduces to
H(P)=4Neli m p(l-p) ,where N e is the effective population number.
In a mammalian species having an effective population number of 10,000,
if the majority of molecular mutations due to base substitutions are almost
neutral with respect to natural selection, and if they occur at the rateof 2 per gamete per generation, the average number of heterozygous
nucleotide sites per individual becomes about 8 x 104 • Mutation is usedhere to designate any change in the nuclear DNA whether it codes protein or not.
For details, see Kimura (1969. Genetics 61:893-903).

The Average Number of Generations until Extinction of
an Individual Mutant Gene in a Finite Population
Motoo KIMURA and Tomoko OHTA

Since a majority of mutant genes are lost from the population by chance
within a small number of generations, it is desirable to know the average number of generations until extinction of an individual mutant genein a finite population, excluding those cases where it is eventually fixed.
Using the method of diffusion equations, we have obtained approximation formulae for the cases of selectively neutral, semidominant deleterious, and recessive deleterious mutations.
Let to be the mean number of generations until extinction of an individual mutant gene (excluding the cases of eventual fixation) in a

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

population with "variance" effective number N. and actual population
number N. When the mutant is selectively neutral, we haveto2~110g.(2N)= 2~; log.(2N) .

(1)

When the mutant is deleterious and semidominant with selection coefficients s' and s'/2 against the mutant homo- and heterozygotes respectively,

to = 2Ze {log.(2N) -log.(2N.s') + 1-r} ,(2)where r is Euler's constant, 0.577, and 2N.s' is assumed to be muchlarger than 1.
When the mutant is recessive with selection coefficient s' against the
mutant homozygotes,
1
to = 2N.
N { log. (2N)-Zlog.
(2N.s') +e

1-1-.. }(3 )Monte Carlo experiments were performed to check these three formulae.
The agreement between the theoretical and experimental results was
satisfactory.
For details, see Kimura and Ohta (1969. Genetics 63:701-709).

Linkage Disequilibrium at Steady State Determined byRandom Genetic Drift and Recurrent Mutation
Tomoko OHTA and Motoo KIMURA

Linkage disequilibrium caused by random genetic drift in a finite
population was investigated using diffusion models, assuming that a steadystate has been reached under recurrent mutation and random samplingof gametes.
We will assume that a pair of alleles Ai and A z are segregating in thefirst locus and alleles B l and B z are segregating in the second locus.
Let gl, gz, ga and g4 be the frequencies of the 4 chromosome types A1Bl,
A1Bz, AzBl and AzBz in the population. Then, the coefficient of linkage
disequilibrium is D=glg4-gz{Ja, the frequency of Ai is P=gl+UZ and thefrequency of Bl, q=gl+ga. By solving the equations for the moments ofthe distribution the expected value of DZ was obtained in terms ofN.e and N.k, where N. is the effective population number, e is the

RESEARCHES CARRIED OUT IN 196955

recombination fraction between the two loci and k is the sUm of themutation rates in all directions.
Since the value of D depends heavily on gene frequencies, a morestable measure of linkage disequilibrium, a~=E{D2}/E{pq(1-P)(1-q)} hasbeen used, and ad was termed the standard linkage deviation. It wasthen shown that, at equilibrium, we havea~=1!{3+4Ne(c+k)-4/(5+2Nec+4Nek)}.

The standard linkage deviation, ad, is approximately equal to the correlation of gene frequencies between the two loci. For a large Nec, a~ becomes about 1!(4NeC) , and this also holds for the case of steady decay
without mutation.
As long as all the segregating loci are selectively neutral, linkage disequilibrium has no effect on the behavior of individual genes. However,when selection occurs, the effect of linkage disequilibrium may become
important.
Apparent overdominance may arise at an intrinsically neutral locus by
the presence of overdominant or ordinarily dominant loci tightly linked
to it. The magnitude of such overdominance is roughly proportional to
the sum of D2 between the neutral and the remaining selected loci.
:!.<'or details, see Ohta and Kimura (1969. Genetics 63:229-238).

Some Relationships between Local andGlobal Genetic Variabilities
Takeo MARUYAMA

Consider a typical natural population occupying a geographically structured habitat in which migration occurs. Let fo be the probability thatthe homologous genes in actually mated gametes are identical by descent,and let j be the same probability for two homologous genes chosenrandomly from the whole population. Then for neutral genes at equilibrium, the following relationship holds,-(1- fo)(1-2u)

f=

4Nu

where It is the mutation rate and N is the total population size.
If u=O, we have transient processes in which fo and j approach zero
as time increases. The rate at which these quantities approach zero isof genetical interest. Let t indicate the generation, h(t)=1-jltl and56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

h o(t)=l-!oltJ.

Then the following relationships hold,'1(t+ 1) = 1- ho(t)h(t)
2Nh(t)(1)andho(t) = - 2N d~~t) .

Formula (1) due to Robertson (1964).

Speed of Gene Substitution
in a Geographically Structured Population
Takeo MARUYAMA

In evolutionary theory, the number of generations (T) required for a
new gene arisen by mutation to become fixed in the population plays animportant role. This problem was first solved by Kimura and Ohta (1969)
for a random mating population of finite size. Later Kimura (1970) obtained the distribution for this quantity. Many natural populations, if
not all, have a geographical structure and matings in a population dependon the distance between the two individuals. Thus as a whole the population may not be a random mating unit and this fact should be taken
into account in the calculation. Using the one- and two-dimensional stepping stone models, this problem was investigated. The result can be
expressed as follows,mean of T=J:....
A

second moment of T=!l.
,{Z
in which~r (for large N) for one-dimensional habitat,21Vn
1m} . = - - (for large n), } . = - - (for large N) for two-dimensional square
2Nn4nhabitat,
10m}.=-z- (for large

4n

n),

}.=

where n=number of colonies, N=colony size and m=migration rate. It
was also shown that the distribution of T from simulation experiments
fits well with Kimura's distribution obtained for a random mating

RESEARCHES CARRIED OUT IN 1969

population.

57

This is illustrated in Table 1.

Table 1. Frequency distribution of T/E[T] where E indicates themean value of T. The parameters are n=10, N=1 andm=O.1. The number of the simulations from which
the table was constructed was 100
T/E[T]

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.5<

Simulation .01
Kimura's .01
formula.06 .06 .11 .12 .13 .05 .07 .04 .05 .04 .04 .02 .20.04 .07 .10 .11 .10 .09 .08 .07 .06 .05 .04 .03 .15

On the Fixation Probability of Mutant Genes in a Subdivided
Population
Takeo MARUYAMA

Since all natural populations are geographically distributed, it is an
important problem to consider the ultimate fixation probability for mutant
genes in a population with geographical structure. Although populations
may be usually continuously distributed, the two-dimensional steppingstone models is often a good approximation to their structure. Here wewill consider a population consisting of a finite number of partially isolated colonies and obtain an approximation formula for the fixation probability for one locus with two alleles Al and A 2 which are additive infitness.
Let Ni be the size of colony i, Y/tJ be the number of A 2 alleles incolony i at the t-th generation and y(tl be the total number of A 2 allelesat the t-th generation, i.e. y(tJ= ~ Y/tJ and N= ~ Ni. Assume that theiisampling of gametes from one generation to the next is done independently
in each colony. Then we have,
E [exp (~2SYlltJ)] ·E[(exp (-2SY2(tl)] . ... =E[exp (-2sY(tl)] ,(1)

where E stands for the expected value. Now suppose that selection occurs independently in each colony. Then
E[exp (-2sYi(tJ)]=E[exp (-2sYi (t+!J)] ,for all i.

(2 )

Thus

E[exp (-2sYI (t+1I)] ·E[exp (-2sY2(t+II)] . ... =E[exp (-2s( ~ Yi 1t + II ))];follows from (1) and (2).

=E[exp (-2sY(t+II)]=E [exp( -2sY(tl)] ,Therefore E[exp(-2sYW)] is invariant over

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

population subdivision and selection within colonies. The invariance stillholds over migration of individuals between colonies since this does notchange the total number of Az alleles. If each colony can be reachedfrom any other colony by migration in one generation or several generations either Al or Az will be eventually lost from the whole population.
For such a situation, as above, we haveexp (-2syIO»)=U (P) exp (-2s2N)+(1- U (P)) ,in which U (jj) is the fixation probability of A z in the population with theinitial frequency,

where Pi is the initial frequency in colony i. Therefore we have

U(P)= l-exp (-4NsP) .
l-exp (-4Ns)(3)

This is equal to Kimura's fixation probability in a panmictic populationof size N= ~ N i • It should be noted that, in the derivation of (3),ineither a fixed migration pattern nor fixed colony size is assumed, though
the total population size is assumed to be constant.

The Variance Effective Number of Human Population
Norikazu YASUDA

A method is devised for estimating the variance effective number ofa population based on gene frequency data. Let us suppose that the genefrequencies of two alleles Al and A z in the parental population are P and(j, respectively. The frequency of gene A 1 in the filial generation (x)will be x=i/2N, where 2N is the number of gametes which contribute to
the filial generation and which are considered as a random sample froman infinite number in the gamete pool produced by the parents, and i isthe number of Al genes in the filial population. The mean x and variance
V~ of x will be respectively x=p and V~=pq/2N.
The change of gene frequency between two generations, op=x-P, has
an expectation of zero and a variance pq/2N; i.e. ap=o and Vo p =pq/2N.
The variance effective number (or size) of population is thus defined by

N.=pq/2Vo p •RESEARCHES CARRIED OUT IN 196959

By applying an angular transformation, the variance will be independentof the gene frequency of the parental generation: that is, p=sin 2 8 gives
Vae= l(8N, where 08=8'-8 and the prime indicates the filial generation.
In practice the variance of 08 may be computed fromn Vae = .I: ~(8)2 ,in which n is the number of loci studied.
For a multiple allelic locus, 08i can be measured for each allele separately(considering the others being pooled). In this case, however, the contribution of each gene to Vae will be (m-I)(08)2/ m where m is the numberof alleles. Thus the variance in general will be

nV8e =....!.. .I: {(081)2+c082)2} + 2 .I: {(08 1)2+ (08 2)2 + (08 s)2}+ ...
2 k3
3 ks
m-l
+ - - .I: {(081)2+ ... +(08 m)2} ,mkm

where n(=k2+2ks+'" +(m-l)km) is the number of independent genes
examined, and ki the number of loci with i alleles. The variance effective
Table 1.
Population
Japan1)
YashiroKurodaniH-group2)A

LI.ut! ~5l.ut
Brazil3)g

Variance effective number in some human populations

N

N'

N.

N./N'

fr

n

---

71

18
10

8
9

0.44
0.90

0.06477
0.06166

6
4

93
128
83
94
78
86
51
92
2,136

107
192
134
80
301
93
168
117
4,834

52
60
35
24
69
29
25
19
540

0.49
0.31
0.26
0.30
0.23
0.31
0.15
0.16
0.11

0.00966
0.008370.01465
0.021400.007310.017400.02061
0.026410.00093

8
8
8
8
8
8
8
8
32

48

N=sample size of parental population.
N'=sample size of filial population.
N.=estimated variance effective number.
fr=the inbreeding coefficient due to random genetic drift.
1) Fujiki et al. (1968), 2) Steinberg et al. (1967), 3) Morton (1964).

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

number is thus obtained fromN e =1/8Vso.

The rate of fixation or the change of inbreeding coefficient (fr) due torandom gene frequency drift is then 1/2Ne =4Vso.
Application of the method is made to some human populations andpertinent figures are summarized in the table. Although the numbers
obtained (N,) are underestimates due to migration and other possible causesof fluctuation in gene frequencies between two generations, the results
clearly show that: the smaller the size of population (N'), the more random
drift of gene frequency takes place as indicated by small N e • The ratiosof effective to actual numbers are 0.11"'0.90. It is of interest that isolated populations give smaller ratios than a relatively large population.
For details, see Yasuda, N. (1969. Jap. J. Hum. Genet. 14(1): 10-16).

RESEARCHES CARRIED OUT IN 1969

VI.

61

EXPERIMENTAL STUDIES ON
POPULATION GENETICS

Characteristics of Japanese Natural Populationsof D. melanogaster
Chozo OSHIMA and Takao K. WATANABE

The mean total frequencies of lethal and semilethal chromosomes were21.5 per cent in coadapted genetic background and 36 per cent inSamarkand isogenic background. The isogenic background decreased theviability of homozygotes for semilethal and subvital chromosomes and thecoadapted background, on the contrary, increased it. A clear negative
correlation between the frequency of deleterious chromosomes and relative
viability of quasinormal homozygotes was ascertained.
Complete allelism tests were carried out between lethal genes extracted
from the same or from different populations. The frequencies of allelism
between lethals extracted simultaneously from a population were fluctuating, but the average rate was 3.03 per cent. The rate of elimination(lQ2) of lethal genes was estimated to be 0.0014 which was about two
times higher than in the American populations.
On the other hand, allelism tests were performed between lethal genes
extracted in successive two or three years, and a fairly large proportionof all lethal genes was found to have been maintained for a long time inthe same population. About 40 per cent of all lethaIs were persistent(one year) and about 22 per cent were long-term lethals (persistent forover two years). Consequently, about 60 per cent lethals have newly
arisen. The mean viability of persistent lethal heterozygotes was estimated to be equal to or rather better than that of normal heterozygotes
with coaclapted genetic background.

Recessive Autosomal Sterility Genes Concealed
in Natural Populations
Chozo OSHIMA and Takao K. WATANABE

The frequency of sterile chromosomes among non-lethal chromosomes
extracted simultaneously from a natural population was reported to be
12.6 per cent in the previous Annual Report (No. 19). Thereafter 36 male

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

sterile chromosomes and 19 female sterile chromosomes have been maintained by Cy-balanced system. The frequencies of allelism between maleand female sterility genes were determined by half diallel crosses between Cy-sterility lines. Among 630 crosses, double heterozygotes fortwo male sterility genes found in 95 crosses were sterile when they werecrossed with virgin Oregon-R females. The frequency of allelism of malesterility genes was 15.1 per cent and that of female sterility genes was
10.5 per cent. Such high frequencies were due to a frequent appearanceof a few kinds of sterility genes, for example, 13 male sterile chromosomes, 6 male sterile chromosomes and 6 female sterile chromosomes were
found to carry allelic genes as shown in Table 1.
Table 1. Frequency of sterile chromosomes extracted from a natural
population in Katsunuma and allelism between sterily genes
No. of second chromosomes tested

475

Sex
No. of sterile chromosomes
Frequency of sterile chromosomes (Q)No. of chromosomes tested for allelism
No. of crosses
No. of allelic crosses
Frequency of allelism (l)No. of different loci of sterility genes
Frequency of appearance
1
2
3
6

13

Male

Female

44

25
0.053

0.093
36

19

171

630
95
0.151

0.105

17

12

13

10

18

2

1*

1**
1

1

* Allelic group A.
** Allelic group B represented in Table 2.
From the observation of salivary gland chromosomes, those male sterile
chromosomes were found to have a heterotic inversion C on the right armand one of them had additionally a heterotic inversion B on left arm, as
shown in Table 2.
The loci of those male and female sterility genes could not be exactly
determined because they were located on inversion C and were assumed
to be different. Although most of the sterile chromosomes manifested
semilethality in homozygous state, they were assumed to exist frequently
and persistently in the natural population. Such as assumption could be
supported by linkage with heterotic inversion C. However, it remains to

RESEARCHES CARRIED OUT IN 1969
Table 2.

63

Linkage relationship between frequent sterility genes
and inversions

Numerical symbolof chromosome

Group A
Male

Group B
Female

372
594
597
27
277
549

st.
st.
st.
st.
st.
st.

+
+
+

Inversion
2R

Viability ofhomozygoteBsemilethal
semilethal
subvitalsemilethal
semilethal
semilethal

2£

+
+
+

st.
st.
st.

+.

+

C
C
C
C
C
C

be proved whether sterility and semilethality are the result of pleiotropic
actions of one gene or are individual actions of two different genes involved.
Frequency of sterile flies collected from natural populations was assumed to be about three per cent in both sexes as shown in Table 3.
Table 3.

Frequency of sterile flies in natural populations in Kofu
and Katsunuma locality

Sex

Population

Month of No. of flies
collection
testedMale

Katsunuma
Kofu
Katsunuma

Oct. '68
Oct. '69
Dec. '69

Total
FemaleKatsunuma
Katsunuma
Katsunuma
Total

Oct. '69
Nov. '69
Dec. '69

No. of sterileflies

Frequency

604
226
42

18
6
1

3.0
2.7
2.4

872

25

2.9

236
242
83

10
4
2

4.2
1.7
2.4

561

16

2.9

(%)

On the other hand, the rate of elimination of male sterility genes byhomozygosis in a population could be calculated by using the values of I
and Q of Table 1. If both second and third chromosomes were assumed
to have the same values for I and Q, the rate of elimination of malesterility genes by homozygosis could be calculated to be 0.151 x 0.171=
0.00258. The rate of elimination of female sterility genes by homozygosis
could be calculated similarly to be 0.0006. From the results, about one
tenth of sterile male flies and one fiftieth of sterile female flies werecaused by autosomal recessive sterility genes and most of them could be
attributable to some physiological or environmental factors.

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

A New Mutant Gene' tra-b' on the Second Chromosome
Takao K. WATANABE

When the viabilities of homozygotes for many second chromosomes
extracted from a cage population were estimated by Cy-method, a chromosome carrying a mutant gene, termed 'transformer-b' was found.
In the Fl offspring of Cy/tra-b heterozygous flies, Cy/tra-b heterozygous
flies and tra-b/tra-b homozygous flies emerged as expected in the ratio of
2 : 1. However, all tra-b homozygous flies were male. Female flies, whose
genotype was X/X; tra-b/tra-b, were evidently transformed into phenotypic male flies and their mating ability was normal, but they werecompletely sterile duE' to their reduced testes.
A. H. Sturtevant (1945. Genetics 30: 297-299) reported a transformer
gene (tra: 3-45) on the third chromosome. The functions of both tra andtra-b gene were very similar, but male flies, whose genotype was X/Y;
tra/tra, were fertile.
The locus of tra-b gene was assumed to be close the Lobe gene (2-72.0)
on the right arm and it remains to be proved whether the sterility of
tra-b homozygous male flies is due to the pleiotropic action of that gene ornot.
A Heterotic Inversion on the Second Chromosomeof D. melanogaster
Takao K. WATANABE and Taishu WATANABE

A second chromosome inversion, In(2L)B, has been maintained in thenatural populations of Kofu and Katsunuma at the frequency of about 30
per cent during the past 7 years. Two cage populations, containing theheterotic inversion at 25 per cent, were started in the laboratory. One
has been kept in a constant 25°C environment and the other in anenvironment fluctuating 20-30°C two t~mes a day. After about 15 months,
200 flies were sampled from each population, and their salivary chromosomes were examined. From the results, the equilibrium frequency of
the inversion was assumed to be about 20 per cent in the constant andabout 11 per cent in the fluctuating environment.
The viability of random heterozygotes was estimated by using Cymethod, and the random heterozygous female and male productivities
were estimated by the number of their offspring. There relative values(mean) of normal and inversion B heterozygotes and inversion homozygotes
are given in Table 1.

RESEARCHES CARRIED OUT IN 1969
Table 1.

Environment

Constant

Fluctuating**

65

Relative viability. female and male productivity of In(2L)B
heterozygotes.
Productivity

Genotype

No. of crosses

Viability

9

0

+1+
+IB
BIB
+1+
tlB
BIB

125
81
8

1.00
0.99
0.98

1.00
1.11**
0.96

1.00
0.96
1.02

152
38
3

1.00
0.93
1.03

1.00
1. 21**
0.71

1.00
1. 21**
1.02

Highly significant

Although the viabilities in both environments were not statisticallydifferent, the productivity of inversion female and male heterozygotes
showed a significant superiority over normal heterozygotes. This result
reveals one of the mechanisms by which the inversion, In(2L)B, has been
maintainecl in natural and cage populations.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

VII.

RADIATION GENETICS AND C~EMICALMUTAGENESIS IN AN IMALSStudies on Strain Differences in Radiosensitivity in the Silkworm.
VII. Strain Differences in Sensitivity to Germ Cell Killing
Yataro T AZIMA and AKIO ONUMA

Strain differences in radiosensitivity to cell killing have been studied
with regard to spermatogenic cells using fertility decline as a ctiterion.
The materials used were seven representative strains which had been
selected for embryonic killing; two resistant, two intermediate and three
sensitive.
It has been known that spermatogenic cells are extremely sensitive tokilling by radiation during the larval period from late fourth instar toearly fifth instar, when most of the germ cells are just before the firstmeiotic metaphase. Hence, r-irradiation was administered to the larvae
at fifth instar day one (V-I) with several different doses. The killing
effect on spermatogenic cells was expressed per batch basis as an index
of the average number of fertilized eggs relative to control, when irradiated males had been crossed to non-irradiated females. From dosereaction curves LD50 values were calculated.
The results of our first experiment showed remarkable differencesamong the strains with the following LD50 values: Aojuku, 2,359 R; Kojiki,
1,650R; Kansen, I,OOOR; and Sekko, 300R. The maximum difference insensitivity among strains were about eight fold.
Surprisingly enough, the sensitivity to germ cell killing did not run inparallel with that to embryonic killing. Sekko, which belongs to theintermediate group with regard to embryonic killing showed extremely
high sensitivity to cell killing. In contrast, Kojiki, which was classified
as most sensitive to embryonic killing was found to be fairly resistantto cell killing. A further experiment revealed that strain rb was moresensitive than strain Sekko with respect to cell killing, although LD50
values became a little higher for both strains than in the first experiment.
In conclusion, there was no parallelism between the sensitivity to germ
cell killing and the sensitivity to embryonic killing.

67

RESEARCHES CARRIED OUT IN 1969

Studies on Strain Differences in Radiosensitivity in the Silkworm.
VIII. Strain Specificity at Various Response Levels
Yataro T AZIMA and Kimiharu ONIMARU

A comparative study of various kinds of radiosensitivity among silkworm
strains has yielded several informations. Above all it has been madeclear that each strain has its own specific response level in radiosensitivity.
First, using embryonic killing as a criterion for selection, we have extracted several radiosensitive and radioresistant strains from the worldwide collection of silkworm strains maintained at the Sericultural
Experiment Station.
Second, using those strains and an already established radiosensitive
strain rb and a standard type strain C108, we have carried out a comparative study of sensitivity to cell killing and sensitivity to mutationinduction by radiation. The results are summarized in Table 1.
Table 1.

Radiosensitivity at various levels for seven representative strains

StrainEmbryonic
killingGerm cellkilling

KansenAojuku

+
+

*

CIOB
Sekko

**

Kojiki
Ascoli
rb

#tt
#tt
#tt

Mutation induction in
Spontaneous
Spermato- Spermatids Spermmutation
gonia

+

+

#tt

*
*

+

±

*
*+

#tt

#tt

*

-tit

-tit

+
+
+
+
+
+

+

*

*

*

In this table the strains are classified into three groups, sensitive,
intermediate and resistant, according to the sensitivity to embryonickilling. As pointed out previously, sensitivity to embryonic killing, sensitivity to germ cell killing and sensitivity to mutation production do not
run parallel to another among the strains except for the hyPersensitiverb, which showed always the highest sensitivity. The most noteworthy
is Sekko. Although it is placed in the intermediate group with regard
to embryonic killing, it showed extreme sensitivity to germ cell killing
and to mutation induction in spermatogonia. Another extreme case is
Aojuku. It was highly resistent to germ cell killing, but intermediatewith regard to mutation induction.

68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Regarding mutation response a marked difference is observed among
strains when spermatogonia were irradiated. The difference. however,diminishes with advancing germ cell stage.
The above results seem to indicate that sensitivity to radiation is not
only strain specific but also developmental phase specific.

Studies on Strain Differences in Radiosensitivity in the Silkworm.
IX. Relation between Mutation Induction and Cell Killing
Yataro TAZIMA and Yosoji FUKASE

A contrasting mode of dominance has been discovered betv,-een mutationinduction and germ cell killing by radiation in Fl hybrids produced between sensitive strains.
Strains used for this study were rb and Sekko. It has been alreadyreported with respect to embryonic killing that the Fl between t\VO sensitive strains, Kojiki and Ascoli, became resistant showing far higher LD50
values than both parental lines. A similar phenomenon of overdominance
was observed with respect to germ cell killing, when Fl hybrids between
rb and Sekko were compared with the parental strains. Contrarily, thesame Fl hybrids exhibited intermediate sensitivity with respect to mutation induction. The intermediate sensitivity in Fl regarding mutationinduction was also confirmed in another cross produced between rb andKojiki.
In this study the sensitivity to mutation was estimated in terms of themutation frequency at the specific loci, +pe and ..:.. re, after irradiation ofearly spermatogonia and sensitivity to cell killing was studied by irradiating spermatocytes using fertility decline as a criterion. Since visible
mutations at the specific loci represent chromosoJlal events incurred in
the germ cell and cell killing is estimated from the fertility decline
produced in the same cell line, both results could be reasonably compared.
The intermediate type of dominance observed in Fl with regard to
mutation induction suggests that a modification of chromosomal lesions byseveral intracellular and/or extracellular factors is rather slight. Whereas,
the overdominance exhibited in the same F 1 cross with regard to cellkilling indicates that the possibility to such modification is fairly large
in the case of cell killing. Increased vigor in Fl, for instance, seems torelieve radiation lesions to an appreciable extent leading to increased
survival. It is concluded from these results that factors other than chromosomal lesions are also concerned with cell death.

RESEARCHES CARRIED OUT IN 1969

69

Studies on Strain Differences in Radiosensitivity in the Silkworm.
X. Spontaneous Mutation Frequencies Observed in Different
Radiosensitive Strains to Mutation Induction
Yataro TAZIMA and Kimiharu ONIMARU

Since remarkable differences in radiosensitivity were discovered amongsilkworm strains with regard to mutation induction, we have examined
whether such differences could be detected with respect to spontaneous
mutation frequency.
Seven representative strains for differential sensitivity were used forthis purpose; i.e. rb and Sekko as the most sensitive, Aojuku, C108 andAscoli as intermediate and Kansen and Kojiki as the least sensitive.
Among them the best analysed were rb, Sekko and Kansen. Mutation
frequencies were examined only for the male. In order to minimize environmental variables influencing mutation frequency, all strains wereraised simultaneously. The mutation frequency was assesed by utilizing
specific loci method.
Spontaneous mutations can occur at any time of germ cell development.
Therefore, it is impossible to guess exactly the stage of their occurrence.
However, whether they had occurred at pre-meiotic stage or post-meiotic
stage could be surmised from their appearance. Should a mutation occur
in germ cells at premeiotic stage, it could be detected as whole typemutation in the next generation. Whereas, would it occur after themeiosis in one of the DNA double strands, it might be manifested as amosaic mutant in the next generation. Furthermore, mutations arisingat pre-meiotic stage are sometimes characterized by their occurrence inclusters.
The data obtained for the past three years are given in Fig. 1. The
difference is clear among strains at +pe locus. Mutation frequencies werehigher in strains rb and Sekko than in Kansen. The difference was,however, less clear at +re locus, owing to lower mutation responses. The
highest peak which appeared in the score for 692 was due to the clusterlike appearance of the mutants. When more than two mutants appearing
in the same batch were counted as one mutation, the peak fell c:own toapproximately average level. Nevertheless, the relative order in mutation
frequency among strains remained unchanged.
These results suggest that strains sensitive to mutation induction byradiation show hig~1er mutation frequency even under non-irradiatedcondition.
As to the incidence of mosaics no clear difference was observed among

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20
x 10- 5

Spontaneous

mutation frequencies

40+

Kansen

pe

locus

rb

Sekko
30

>0
c

•tr
20
.::•
~

,/"'

-

/"'

10

671

~

/"'

,"""

/

,

"" "/
"-

,

/

/\
\

/

\
\

\

\

\

672

673

681

682

V

683

691

692

693

Rearing season
Fig. 1. Spontaneous mutation frequencies observed for three silkworm
strains during past three years.

strains, suggesting that the occurrence of spontaneous mutations at
postmeiotic stages does not differ appreciably among strains.

A Comparison of Mutagenicity of 14 MeV Fast Neutrons
on Primordial Germ Cells among Different X-ray
Sensitive Silkworm Strains
Akio MURAKAMI

A comparison of the mutagenicity of 14 MeV neutrons among strains
having different radiosensitivities may give a better insight intot he factors
affecting relative biological effectiveness CRBE) values of the two kinds ofradiation. The present study has been carried out with primordial
germ cells of the silkworm in order to compare the mutation rates ofneutrons to those of X-rays among five different strains. Treatments
were given to newly hatched larvae. Mutation rates were estimated by
the egg-color-specific-locus method using the pe and re genes.

RESEARCHES CARRIED OUT IN 1969

71

For X·rays, there was a remarkable difference in mutation frequencies
between the most and the least sensitive strain. The difference was,however, less pronounced for neutrons. Comparing the mutation rates of
the two radiations (or RBE of neutrons to X·rays), it has been shownthat the RBE values of resistant strains are higher than those of sensitive
strains. The result suggests that the efficiency of neutrons in inducing
mutations varies according to radiosensitivity perhaps due to interaction
between germinal selection and reparability of biological damage.

Effects of Ultraviolet Light on the Oocytes of the Silkworm
with Special Regard to Strain Sensitivity
Akio MURAKAMI

Sensitivity to ultraviolet light was studied in order to compare strain
sensitivity of different X·ray sensitive strains with regard to killing andmutagenic effects.
After a UV irradiation was delivered to mature oocytes and/or eggs in
the early cleavage stages of the silkworm, their sensitivities to killingand mutagenic effects were measured in terms of embryonic mortality
and scored by the pe:re egg·color-specific·locus method, respectively.
The results showed the occurrence of distinct differences for bothbiological effects on oocytes and eggs: for the killing effect there wasfound a difference of 4-5 times or more and for mutagenicity a differenceof 10 times or more between the most and the least resistant strain. The
results further indicated that strains sensitive to UV radiation killing arealso sensitive to mutation·induction and vice versa. There was often nOpositive correlation betw'een UV and X·ray sensitivities for both biologicaleffects. In the treated oocytes many more fractional than whole·bodymutations were induced with UV, while X·rays gave the opposite result.

Comparison of Stage Sensitivity to X-rays during Meiosis
in Silkworm Oocytes
Akio MURAKAMI

Meiosis comprises two different types of division, a reductional and an
equational division. Comparison of the radiosensitivity of cells these twokinds of divisions may provide some clews for a better understanding of
the mechanisms underlying the effects of X·ray in the higher organism.

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Such consideration prompted the present experiment.
The radiosensitivity was determined by LDso value from the dosehatchability curves. The strain used was wild type C108. Six doses of
X-rays (180 k Vp, 25 rnA, 1.0 mm Al filter and a dose-rate at 1,000 R/min)were used: 1,000, 2,000, 3,000, 4,000, 5,000 and 6,000 R. For the irradiation of prophase I oocytes the X-rays were administered to eggs in theovaries of late stage pupae or moths which were mated after irradiatedwith male moths having pe and re markers. This sC:1eme alh,';ed for asimultaneous check for induced mutations and cell killing. For the irradiation of post-oviposition eggs (oocytes at metaphase I through the timeof syngamy), eggs were used of wile! type moths mated with the marker
strain. Samples were collected every 10 min. X-irradiations \vere applied
to eggs at 10 min intervals from immediately after oviposition until firstmitotic stage.
The results indicated that cells in the dividing phase were mO;'e sensitive to X-rays than those in any other phases for both first and second
meiosis, and that cells in the first meiosis were more resistant than those in
the second meiosis. Furthermore the sensitivity of the second meiotic
cells was almost similar to tha t of the early mitotic cells. The differences
in sensitivity may be interpreted as being due to (1) whether cells areassociated with DNA synthesis or not ane! (2) the difference in chromosomal structure at the time of irradiation.

Changes in Radiosensitivity during the Second Meiotic
Stage of Silkworm Oocytes
Akio MURAKAMIIn the previous report, we have demonstrated that in cleavage cells theradiosensitivity to cell killing and to mutation varied with the progressof the division cycle (this Annual Report, No. 16, 1967). The most sensitive
phase to cell killing was found to be the dividing phase, whereas that tomutation production was late dividing phase and/or early interphase.
The present work was undertaken in order to extend the observation
to the second meiotic oocytes.
Mutation frequencies were estimated by the specific-locus metho::l using
the pe and re genes. Radiosensitivities for cell killing were measured by
the reduction of hatchability in comparison with the control. Materials
were oocytes and/or eggs laid by the wild type female moths matedwith the marker male moths. Samples were collected every 10 min at

RESEARCHES CARRIED OUT IN 196973

25°C. They were irradiated with 2,000 R X-rays (180 kVp, 25 rnA, 1.0 mm
Al filter and a dose-rate of 1,000 R/min) at 10 min intervals from their ageof 60 until 140 minutes. They corresponded to eggs from meiotic divisionof oocytes to the stage of syngamy.
Mutations most frequently found in this work were fractional-bodymutations and whole-body mutations were scarce. The peak stage forcell killing was observed to be at 110 min age corresponding to the dividing
phase of the second meiotic oocytes, whereas for mutation production to
be at 120 min age corresponding to the end of the second meiotic division
or early pro-nuclear stage. These findings indicate that the sensitive
phases for both biological events do not coincide, suggesting that themechanisms involved in both events are different. The mutation rate
observed for the oocytes around the end of the second meiosis was almost
similar to that observed for the early cleavage stage cells showing approximately 10 times higher value than the other stages of oogenesis.
The above results could be interpreted, at least in part, on the assumption that cells around the end of the second meiotic division stage are in
the course of DNA synthesis or chromosomal duplication.

Radiation-Dependent Loss of Sorting-Out Activityof BeLa Cells
Yukiaki KURODA

It has been previously found that HeLa cells has a characteristic
sorting-out property when intermixed with embryonic chick and quailliver cells in rotation culture. In the present experiment changes in thissorting-out activity of HeLa cells were quantitatively examined after Xirradiatio:1 of HeLa cells. HeLa 53 cells were irradiated with various
doses of X-ray (175 KVp, 25 rnA, distance 40 em, filter 1.0 mm AI, dose
rate 300 R min). After irradiation 3 X 10 5 HeLa cells were intermixed with
6 x 10 5 liver cells dissociated from 7-day quail embryos. Mixed cell suspensions were rotated at 37°C at 70 rpm on a gyratory shaker for 24 or 48
hours, then the formed aggregates were fixed, sectioned, stained andhistologically examined.
Non-irradiated HeLa cells which were intermixed with quail liver cells
formed aggregates in which a small number of liver cells was randomly
engulfed by overwhelming HeLa cells. HeLa cells which were irradiatedwith various doses of X-ray formed aggregates in which quail cells engulfed
by HeLa cells gradually increased in number with increasing X-ray doses.

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Changes in the number of quail cells in aggregates of HeLa cells after
24 hours of rotation culture are shown in Table 1.
Table 1.

Dose (R)

o
400
1,200
1,600
2,000

The number of quail liver cells engulfed in aggregatesof X-irradiated HeLa cells

No. of total
cells inaggregate

No. of HeLa
cells inaggregate

No. of quail
cells inaggregate

Per centof
quail cells

112
143
131
113
139

102
125
109
78
90

10
18
22
35
49

8.9
12.6
16.8
31.0
35.3

It was also found that the number of X-irradiated HeLa cells engulfed
in aggregates of quail liver cells increased with gradually increasing
doses of X-ray. The result of counts for each type of cells in theseaggregates formed after 24 hours of rotation culture is shown in Table 2.
Table 2.

The number of X-irradiated HeLa cells engulfed in aggregatesof quail liver cells

Dose (R)No. of total
cells inaggregate

No. of quail
cells inaggregate

No. of HeLa
cells inaggregate

Per centof
HeLa cells

0
400
1,200
1,600
2,000

475
155
416
346
220

467
147
384
317
195

8

1.7
5.2
7.7
8.4
11.4

8
32
29
25

The figures given in Table 2 show that the percentage of HeLa cells
engulfed in quail cell aggregates was relatively lower than the percentageof the latter engulfed in the aggregates of the former. The differencein these percentages suggest that the formation of discrete aggregatesof each type of cells which were brought about by the mutual sorting-out
property of both types of cells may depend upon a chasing out activity·of the aggregated cells on the intermingled unlike cells and escaping
activity of intermingled cells from aggregates of unlike cells.

RESEARCHES CARRIED OUT IN 1969

VIII.

75

RADIATION GENETICS IN MICROORGANISMS
AND PLANTS

Correlations between Mutation, Transformation Efficiency andRadiosensitivity in Mutants of Bacillus subtilis
Yoshito SADAIE and Tsuneo KADA

Bacillus subtilis strain H-I7 (try arg) \yas treated with N-methyl-N'nitro-N-nitrosoguanidine and surviving cells were screened for the increasein radio-sensitivity. Among mutants isolated and purified, seven weresensitive to both gamma-rays and UV.
All the mutant strains had reduced rates of gamma-induced resistanceto Kanamycin (1 fig/ml) in comparison with the original strain H-I7. Whilestrain H-I7 is genetically stable as an efficient recipient for transforming
DNA carrying arginine and tryptophane markers, the efficiency of transformation was remarkably low in radiosensitive mutants. It is evident
that a recombinational event between extracellular DNA and the recipient
chromosome is required for marker incorporation in transformation.
It has been supposed by different authors that simultaneous occurrenceof enhanced r-ray- and UV-sensitivities may be due to genetic lack inrepair involving the recombinational process· Thus the reduction of
induced mutations in our radiosensitive mutants would posseses a similarmolecular basis.

Gamma-Ray Mutagenesis, Repair and ChemicalRadiosensitization in Bacteria
Tsuneo KADA

One of the possible mechanisms involved in the radiosensitization withcertain chemical reagents may be an inhibition of repair of radiation
damages on the level of cellular DNA. If it were so, it is possible thatradiation-induced mutagenesis might be modified in some way. Reversions to Prototrophy in Escherichia coli B/r try WP2 were studied under
mild sensitizing conditions using KIOa as a radiosensitizer (Kada, T. 1969.
Int. ]. Radiat. BioI. 15: 271).
When bacteria were irradiated at O°C with gamma-rays from 137CS in
phosphate buffer (O.067M) containing a non-toxic level of KIOa, many
more cells were inactivated as compared to the control without the re-

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

agent. To observe the effect of the reagent at the time of irradiationon the yield of mutations, cellular suspensions (~5XlOB/ml) containing
increasing concentrations (l~5 mM) of KIO a were exposed to gamma-rays(l~5 kR) at O°C, then the cells were washed twice with cold phosphatebuffer and resuspended in the same solution of 1/10 volume. Plating was
carried out after appropriate dilutions to determine the number of viablebacteria on minimal glucose agar enriched with 0.2% Difco liquid brothand revertant cells on the same minimal agar with or without broth enrichment. All plates were incubated at 37°C for 3 days.
Results of some typical experiments are shown in Table 1. It is clear
Table 1. Effect of KIO a (PI) addition during gamma-irradiation onradiation-induced reversions of Escherichia coli B/r WP2 try
Number of viable
cells per ml
Dose ofr-rays
(kRI

0
1.4
2.7
4.0

-PI

Frequency of induced mutants
per lOB survivals
Minimal Med.

Broth (0.2%)
enriched MM

-PI

+PI

-PI

+PI

0
1.9
2.9
3.5

0
0.9
0.7
0.9

0
3.4
4.0
7.1

0
4.4
3.0
3.1

+PI

2.4x lOB

2.4x lOB

1.7xlOB
1.5x10B
1.3xlOB

1.7x10B
1.2x1OB
6.9x10'that, while some decrease of viable cells was observed under above
sensitizing conditions, the frequency of reversions decreased also markedly.
Recent studies carried out in this laboratory on macromolecular synthesis in bacteria that had been irradiated with gamma-rays in the presence of KI03 have shown that de novo DNA synthesis as well as rejoining of radiation-induced DNA strand-breaks were prevented in the courseof post-irradiation incubation. It is thus supposed that potential mutant
cells might be inactivated more efficiently than the non-mutant population because of lacking in rejoining repair or that some specific mutagenic
lesions might be more preferentially repaired than lethal lesions in theabsence of DNA synthesis.

•

77

RESEARCHES CARRIED OUT IN 1969

Macromolecular Synthesis in Bacteria Radiosensitizedwith Iodine Compounds
Takehiko NOGUTI and Tsuneo KADA

In order to elucidate the biochemical mechanisms underlying increaseof cellular death by radiosensitization with iodine compounds such as
iodoacetic acid (I· CH2' COOH) , potassium iodide (KI) or potassium iodate(KIOa), DNA. and protein synthesis were studied in cells of Bacillus
subtilis strain H-17.
Bacteria exponentially grown in TF medium were irradiated with 7kR
gamma-rays from mcs in the presence of 0.8 mM KI, 0.25 mM KIOa or
0.2 mM I· CH2' COOH, washed in cold and reincubated in the same medium.
Cellular incorporations of aH-thymidine or HC-amino acids into acid-insoluble
materials were measured in parallel cultures. We observed in repeatedexperiments that, while irradiation or drug treatment alone had little
effect on the macromolecular synthetic pattern as compared to that of thecontrol culture, the incorporation of radioactive precursors into DNA was
more inhibited than into protein (Table 1).
Above results indicated a close correlation between increased cellular
inactivation and inhibition of DNA synthesis in radiosensitized cells.
Similar relationships are well known concerning the effect of ultraviolet
Table 1. Effect of the presence of iodine compounds during gammairradiation on incorporation of radioactive precursors into DNA orprotein in cells of Bacillus subtilis. Treated cells were washed and reincubated for 40 minutes. Radioactivities were measured at this stage by
precipitating and washing cells by 5% TCA and expressed in percentage
as comparedto the control.

r-ray
(kR)

Drug (mM)

Survival
fraction (%)(DNA)

14C-amino
acids(Protein)

0
7

None
None

0

[ KI
(0.8 )
KIO a(0.25)
I·CH 2 ·COOH (0.2 )

100
53
100
100
100

7

[ KI
(0.8 )
KIO a(0.25)
I·CH 2 ·COOH (0.2 )

100
133
88
108
85
3
0.2
1

100
104
84
96
62
16
6
2

aH-thymidine

Treatment of cells

7

0.03
5

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

irradiation of bacteria. However, the radiosensitization with iodine compounds was not due to the increase of in situ radiation damage incellular DNA (Kada, T., T. Noguti and M. Namiki. 1970. Int. J. Radiat.
BioI. 17: 40,7), unlike the case with ultraviolet light that directly modifies
DNA molecules. We suppose that some transient products containing
iodine atoms might be produced by irradiation and that might reactwith certain enzymes involved in DNA metabolisms or modify the cellular
membrane site thus affecting preferentially DNA synthesis.

Rejoining Inhibition of Radiation-Induced DNA Strand-Breaksin Radiosensitized Bacteria with Iodine Compounds
Takehiko NOGUTI and Tsuneo KADA

Evidences have been accumulated recently in various studies on radiation-induced cellular inactivation indicating that the radiation-induced
lethality and its repair are closely related to the production of strandbreaks in cellular DNA molecules and their rejoinings in post-irradiation
growth. Radiosensitization by cellular incorporation of 5-bromouracil into
DNA was correlated to the inability of strand-break rejoinings (Kaplan,
H. S. 1966. Proc. Nat. Acad. Sci. 55: 1442). Extensive studies were carried out to see if such a mechanism might be also involved in the caseof radiosensitization by extracellular presence of iodine compounds suchas potassium iodate (KI08), potassium iodide (KI) or iodoacetic acid (I· CHz'
COOH) at the time of gamma-irradiation.
Cells of Bacillus subtilis strain H-17 were labeled with 8H-thymidine
for several generations during growth in TF-medium. Exponential bacteria were washed, and exposed to gamma-rays (usually 10 kR) from mcs
in suspension in cold phosphate buffer (pH 7.0) containing increasing concentrations of reagent. Cells were washed immediately after treatment
and inoculated in TF-medium. Sedimentation analYsis in alkali-sucrose
gradint of cellular DNA was carried out by techniques already described(Kada, T., T. Noguti and M. Namiki. 1970. Int. J. Radiat. BioI. 17: 407)
on samples collected 0 minutes and 40 minutes after aerobic incubation
under shaking at 37°C.
Results obtained have shown following points (Table 1). (a) Irradiation
provoked in vivo single-strand breaks in DNA molecules of smaller sizes.
The presence of drug did not affect the number of breaks producedin situ by irradiation. (b) Cellular incubation followed by irradiation
without reagent allowed broken molecules to rejoin. However, such aR

79

RESEARCHES CARRIED OUT IN 1969
Table 1. Effect of the presence of iodine compounds during gammairradiation on production of strand-breaks in cellular DNA and theirrejoining. Treated cells were washed, lysed and analysed by sedimentation (38,000 rpm for 80min) in alkali-sucrose gradient (5-20%). In thistable, the position of main peak of DNA is shown as distance (cm) from thebottom. Total length of the gradient was 4.0 cm.
Treatment

NoneKI(1 mM)KIOg
I·CHz·COOH(0.2mM)(0.3mM)

Not irradiated.
Contact withdrug for 30 minutes at O°C.

1.1

1.1

1.1

1.1

Irradiated (20 kR at O°C, 30 minutes required) in the presenceof drug. No incubation.

2.4

2.3

2.3

2.3

Irradiated (20 kR) in the presenceof drug and incubated for 40
minutes.

1.2

2.5

2.4

event was strongly inhibited or totally prevented in cells irradiated in
the presence of the reagent.
These results favor the view that rejoining inhibition of radiationinduced DNA strand-breaks may be rosponsible at least for part of theenhanced cellular death under mild radiosensitization conditions.

Genetic Resistance to Radiosensitization with Iodine Compounds
Tsuneo KADA

A question could be posed if inactivation of a specific cellular metabolism could be involved in radiosensitization. One way to answer it
may be by isolating mutant strains possessing modified responses to radiosensitization and by determing their genetic characters. Previous studies(Kada, T., T. Noguti and M. Namiki. 1970. Int. J. Radiat. BioI. 17: 407)
have indicated that the sensitization might not be due to the increase ofradiation-induced in situ damage in cellular DNA. Therefore it is possible to suppose that some metabolisms may be involved in the repair ofradiation damages in cellular DNA in the radiosensitization process, inaddition to other possible target metabolisms.
A F- strain PA309 of Escherichia coli K12 was treated with N-methylN'-nitro-N-nitrosoguanidine, and the cells were grown overnight. The

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

bacteria were then exposed to 1 mM solution of iodoacetic acid, pH 5.4,
that had previously been exposed to 6.7 kR of gamma-rays from l31Cs,
and the surviving cells were screened for genetic resistence.
Non-mutant bacteria are usually killed very efficiently at O°C in thepresence of persistent toxic products from the reagent induced by irradiation. However, the above isolated mutant strains were fairly resistantto radiation-induced toxicity from iodoacetic acid. They were also resistant to radiation-induced toxic products from potassium iodide or potassium iodate, but not to unirradiated iodoacetic acid or iodine dissolved inpotassium iodide solution. We observed that typical three mutant strains
resistant to irradiated iodine compounds were also more resistant toradio-sensitization with these compounds, that is, simultaneous treatment
with gamma-irradiation and the reagent in neutral buffer. Genetic analysis is under way in order to determine the location of genes responsible for the resistance
These observations indicate: (a) Important part of the enhancementin radiation lethality by irradiation of cells in the presence of iodine
compounds may be due to the action of toxic transient products whoselife may be pH-dependent. (b) The cellular sites responsible for radiosensitization were similar in all experiments with the three iodine sensitizers
studied and/or similar radiochemical products were involved in the radiosensitization with these iodine compounds (details reported in Int. Radiat.
BioI. 1970. 17: 419).

Possible Toxic Effects of Halogen Atoms in Radiosensitization
with Some Halogenated pyrimidines
Tsuneo KADA, Yoshito SADAIE and Takehiko NOGUTIIt has been well established that cells or viruses containing 5-bromouracil
or 5-iodouracil in their DNA are more sensitive to ionizing radiation or
ultraviolet light (Zamenhof, S., R. De Giovani and S. Greer. 1958. Nature
181: 827; Opera-Kubinska, Z., Z. Lorkiewicz and W. Szybalski. 1961.
Biochem. Biophys. Res. Comm. 4: 288; Stahl, F. W., J. M. Grasemann,
L. Okun, E. Fox and C. Laird. 1961. Virology 13: 98). These observations offered a very important argument for the assumption that primarylesions for radiation-induced cellular lethality may reside in DNA molecules.
Radiation chemical studies on DNA possessing 5-bromouracil or 5-iodouracil
substituents strongly suggest that dehalogenation may occur by both

RESEARCHES CARRIED OVT IN 196981

iOlllzmg and ultraviolet radiations of brominated or iodinated bases in{;ellular DNA (Rupp, W. D. and W. H. Prusoff. 1965. Biochem. Biophys.
Res. Comm. 18: 158; Zimbrick, J. D., J. F. Ward and L. S. Myers, Jr.
1969. Int. J. Radiat. BioI 16: 525). It is very likely that such an event willproduce abnormal characters in DNA molecules and might weaken the
DNA backbone structure or cause irreparable strand-scissions (Beer, J. Z.,
J. T. Lett and P. Alexander. 1963. Nature 199: 193; Aoki, S., R. P. Boyceand P. Howard-Flanders. 1966. Nature 209: 686; Kaplan, H. S. 1966. Proc.
Nat. Acad. Sci. 55: 1442).
We have been carrying on studies on mechanisms of radio-sensitization
with iodine compounds such as potassium iodide, potassium iodate or iodoacetic acid (Kada, T. 1969. Int. J. Radiat. BioI. 15: 271; Kada, T.,
T. Noguti and M. Namiki. 1970. Int. J. Radiat. BioI. 17: 407; Kada, T.
1970. Int. J. Radiat. BioI. 17: 419). Because all these agents produced byirradiation at pH 5.4 persistent toxic products inactivating bacteria efficiently at O°C and that mutant strains of Escherichia coli K12 resistant
Table 1. Radiosensitization with 5-iodouracil (IV) and toxic effect of r-irradiateddrug solution in different microbial strains. Bacteria grown exponentiallyin broth were washed and resuspended in phosphate buffer (pH 7.0 or 5.4).
(a) They were irradiated at O°C with r-rays from mcs, with or without
supplementation by 1 mM IV. (b) Phosphate buffer solution containing
1 mM IV was irradiated at O°C ,then combined with bacteria. The mixture was then kept at O°C for 30 minutes.
Survivals were determined by plating on broth agar.
Number of colony forming
cells per ml

Conditions of treatment

Irradiation

pH

mM
of
IV

Minutesofcontact

Salmonella
Escherichia typhimurium
coli B
TM2

Bacillus
subtilis
H-17

8.3x105
9.0x15 5l.lx 106l.Ox 106

8.0x1057.8x105

4.7x1()49.0x 1033.2x105
2.0x1052.2x1055.1x103

None

7.0
7.0

0
1

Simultaneous
treatment withr-rays (10 kR).and IV

7.0
7.0

0
1

None

5.4
5.4

0
1

30

8.0x 105
7.5x105l.3x 106l.Ox 106

8.1x1058.0x 105

Treatment by
r-irradiated(10 kR) IV
solution

5.4
5.4

0
1

30
35

8.1x105<1

9.3x 105<1

7.5x10 5
<1

30

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

to the above products were also resistant to radiosensitization, that is.
simultaneous treatment with gamma-rays and the reagent in neutral buffer,
it is supposed that an important part of cellular death under sensitizing
conditions may be due to the above transient toxic products very probably
containing iodine atoms.
Studies were carried out to examine if halogenated purines or pyrimidines might produce toxic products. Table 1 indicates that 5-iodouracil
produces by gamma-irradiation at pH 5.4 highly toxic persistent toxicities
that killed very efficiently at aoc different kinds of microorganisms
such as Escherichia coli, Salmonella typhimurium as well as Bacillus subtilis.
The enhancement of radiation lethality was also observed under usual
radiosensitizing conditions, that is, when they were irradiated in neutral
phosphate buffer solution containing the reagents.
The above results strongly suggest the possibility that toxic products
could be also produced from cellular DNA possessing iodouracil substituents. If this were the case, the toxic effects of halogen atoms produced
under irradiation might playa certain role in radiosensitization by cellularincorporation of iodinated bases. (Related results were reported, Int. JRadiat. BioI. 18: 281. 1970).

Repair Systems for UV Damage in Phage T6
Etsuo AMANO and Mutsuo SEKIGUCHIll

Bacteriophage T4 is resistant to ultraviolet (UV) irradiation in comparison with T6. UV resistance of T4 may be mainly due to a repair
system or systems active in the dark. It has been domonstrated that an
extract of T4-infected cells contains an enzyme whose action excises
pyrimidine dimers from UV irradiated DNA (Sekiguchi, M. et al. 1970.
J. Mol. BioI. 47: 231-242). Lack of this specific excision activity in strains
of UV-sensitive phages such as T4vl and T2 supports the view that it isinstrumental in the repair of T4 (Yasuda, S. and M. Sekiguchi. 1970. J.
Mol. BioI. 47: 243-255). In the present experiments, similar biochemical
methods were used to investigate repair activities in T6 and T5 phages.
To prepare the HC-thymine labeled DNA, cells of E. coli strain B3(thy-) were infected with T4 or T6, and phage DNA was extracted byphenol treatment from purified particles. Cell-free extracts were prepared by means of sonication from phage-infected cells of E. coli strain
11

Dept. of Biology, Fac. Sci., Kyushu Univ.

RESEARCHES CARRIED OUT IN 1969

83

1100, an endonuclease I-less mutant of K12. A germicidal lamp was used
as UV source. UV-irradiated radioactive DNA was incubated with thecell extract, and the specific excision activity of the cell extract wasmeasured using Dowex I ion-exchange column chromatography. T4-infected
cell extract showed specific excision of thymine dimers from UV irradiated T4 or T6 DNA. Cell extract from T6-infected cells showed none at
all 0:- only very weak activity in excising dimers when irradiated T4 or
T6 DNA was used as substrate. Lack of the specific excision activity
may be the reason for increased UV sensitivity of phage T6 in comparison
to T4.
In T6, photoreactivation of UV damage was observed when appropriate
host bacteria were used. Under photoreactivation conditions, the survival
rate of UV-irradiated T6 phages was comparable to that of T4. In addition to these two repair systems tested, a liquid holding effect was observed in the course of incubation of phage-infected cells in the presenceof chloramphenicol. Some preliminary experiments indicated that thiseffect may be independent of UV sensitivity, photoreactivation ability or
host cell reactivation of bacteria, although the effect seemed to dependon host cell strains used.
In T5 phage, none of the above three repair systems was detected.
However, some technical difficulties in phage adsorption did not allow adefinite conclusion.
This study was carried out at Dept. of Biology, Fac. Sci., Kyushu
Univ., while one of the authors (E. A.) received a support from Kagaku
Gijutsu-cho (Science and Technology Agency).

Wave Length Dependence of Photoreactivation of
UV Irradiated Maize Pollen
Etsuo AMANO

The mutagenic effect of ultraviolet light (UV) on the linked endosperm
marker genes (C sh bz wz) in maize, and its photoreactivation was studied.
Within the range tested the mutagenic effect was found only at 250 mp
and no significant effect was noted at 306 mp or more. Photoreactivation
could be observed from the decrease of mutant frequency after postirradiation with light of longer wave length. The same methods as in aprevious experiment (Amano, E. 1968. this Annual Report 19: 75) wereused in the present work which deals with a wider wave length range
and a larger number of maize kernels. Since mutant spectra in terms

84

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

of entirely vs. fractionally mutated kernels, or stable type os. B. F. B.
mosaics, did not differ from single locus mutation vs. multiple loci, theresults are represented in Table 1 according to their classification. Kernels
which were affected by mutation in more than 1/8 of surface area werescored as mutant kernels. Multiple loci mutations which include B. F. B.
mosaics may be brought about by chromosome breakage taking place onsome point of the mutation process.
To produce damages by UV, four germicidal lamps set in a plane wereused as UV light source, and irradiations were made at an intensity of
100 erg/mm 2 /sec for 60 sec. As a standard PR condition, postirradiation
by visible light from a high presure mercury fluorescent lamp at 6,000
lux for 30 min was used and the treatment reduced the mutation frequencies very effectively. Immediate pollination after UV irradiation in an
open field on a sunny afternoon without aluminium foil light shielding
also reduced the frequencies. Postirradiation by monochromatic lights,
with a constant dose of 3 x 10 5 erg/mm 2 , revealed that the m03t effective
wave length was around 400 m,u producing a decrease in both mutation
categories, single locus or multiple loci. Severe damage at 290 m,ll andTable 1. Photoreactivation of UV irradiated maize pollen. Kernels with 1/8
surface area or more affected by mutant characters were scored as mutated. Multiple loci mutants include B.F.B. mosaics.
UV
PR
irradiation condition*2(erg/mm )

0
0
6,000

"
"

6,000

"
"
"
"
"
"

*

No. ofobservedkernelsdark
6,000 lux
dark
6,000 lux
field PR
290 mp313 mp334mp
365 mp
405mp
436mp
546mp

For details see text.

14,548
5,202
3,393
1,467
3,300
0
1,522
1,995
4,614
2,893
3,625
2,227

No. ofFrequency of mutant kernels (%)
kernels per
pollinatedSingleMultiple
Total
ear
locus
loci
175.28
80.03
54.73
20.96
44.59
0.0
19.27
28.10
57.68
36.62
51.06
29.69

0.00
0.00
0.47150.06810.09090.20620.05774.27350.06811.60600.20620.05774.74500.1363
1.69690.93550.6516
0.34670.38010.27570.98786.04463.30822.73082.86893.03444.66997.0302
3.95983.07753.2492
3.31035.6578RESEARCHES CARRIED OUT IN 196985

very high mutation frequencies at 313 mp might be due to the results ofvery high dose used at these wave lengths or to the effects of contaminating light of shorter wave length. 540 mp light (yellow green) was
ineffective in reducing the UV damages. Photoreactivation was observedin both single locus and multiple loci mutations, and this may suggestthat UV damages may be reparable in the early state regardless of theirconsequences, point mutation or chromosome breakage.

Preliminary Intragenic Mapping of Induced
wx Mutants in Maize
Etsuo AMANa

The structural relationship among wx mutant genes in maize can beanalyzed by scoring Wx recombinant pollen produced in Fl. Normal Wx
Table 1. Recombination frequencies between standard wxs and wx
mutants of maize induced by ethyl methanesulfonatewx

mutant164A-)

94
92
84
82
89
5
76
93
3
90
74
67
4
79
wx-639WX 1l2l

wx 90

Wx pollen frequency (%)
leakymutant

+

-j-

+
+

Cross with

Cross with

U'X H2l

wx-6390.03760.04200.00670.00990.00180.00190.01950.01270.01820.02230.02160.02400.03220.03360.0258<0.0006
0.0152

0.025
0.005
0.00
0.03
0.035
0.04
0.04
0.075
0.14
0.06
0.055
0.065
0.06
0.075
<0.0003
0.03110.050086

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

pollen grains observed in Fl might be mostly the results of intragenic
recombination and their frequencies might depend on the distance between the mutant sites tested (Nelson, O. E. 1962. Genetics 47: 737-742).
14 lUX mutants independently induced by EMS treatments were studied.
All F 1 endosperms expressed waxy phenotype, and the cross with wx-639
showed that they are all in the same cistron. In all tests, more than 1.8
x 10 5 pollen grains were surveyed in eac:1 cross, and Wx pollen frequencies in homozygous standard stocks were much lower than those usually
founc; in Fl. In the present tests, only WX H21 was used as standard lUX
and the results are shown in Table 1. Previous results with wx-639 are
also included in the table. Since the tests with WX H21 and wx-639 weremade in different years, the results may not represent precise three point
tests, but may by comparison give an idea on the locations of the mutated sites. Of the 14 EMS induced wx genes, two might be located outside of LVx-639 , two between lOx-639 and lOX H21 and ten outside of lOXH21 •Artificial induction of lOX mutation by radiation treatments have been
tried but only a few established wx mutants were obtained. Very low
survival frequencies of the three fast neutron induced wx mutants andother evidences of deletion type mutations by radiation suggest that thisdifficulty may mostly be due to structural damages by radiation.

Fractionation and Dose Rate Effects of Gamma-rays
on Mutation Frequency in Maize
Taro FUJI

Mutation frequency at 1,400 rad exposure to gamma-rays by two fractions with 2 hr interval is lower than that delivered by single dose, and
it is imaginable that some of premutational damage induced by gammarays could be repaired during this interval (Fujii, T. 1968. Ann. Rep.
Nat!. Inst. Genet. 18: 78-79). Under this assumption, the relation between mutation frequency and dosage by fractionation was examined andthe dose rate effect was also investigated.
Pollen grains with Bz-gene were irradiated by gamma-rays in a single
treatment or in two fractions with the intensity of 40 kR/h, and low dose
rate with 2 kR/h. Irradiated pollen grains, when used on the recessive
stock, allow to observe mutation from Bz to bz in Fl seeds with bronzy
aleuron co~or. Two types of mutations, whole and partial, could be recognized by this method. Table 1 shows that total mutation frequency(sum of whole and partial) increased with the increasing dosage from

RESEARCHES CARRIED OUT IN 196987

500 to 2,000 R, both in single and fractionation series, but it was lower infractional than in single treatments. When the mutation frequency between the single and fractionation series was compared, the frequencyof partial type mutation did not show any significant difference between the two series, while the frequency of whole type mutation at
total dosage of 1,000 and 2,000 R lots was significantly lONe:" in fractionation lots than in single lots. Similarly, lower mutation frequencies inlow dose rate lots in comparison with those of acute lots also depended
on the frequency of whole type only, those of partial type showing a nonsignificant difference between the acute and the chronic series.
These results suggest that the repair of gamma-ray induced premutational event under fractionated or chronic irradiation occurred merely
for whole mutation. Although similar kinds of mutation could also be
induced by ultraviolet treatment, the trend of recovery was incompatible
with that of gamma-rays; almost equal extent of recovery was observedin those two types when photoreactivation treatment was applied after
Table 1.

Relation between mutation frequency and dosage or dose rate

Treatment(R)

Numberof seeds
examined

Mutation frequency (%)~~~~~~~~~~~-----

Total

Whole

Single (acute) irradiation (40 kR/h)
0.66
0.35
500
6,994
2.55
1.42
1,000
9,611
2,000
7,391
6.21
3.72
Fractionated (acute) irradiation (40 kR/h, 2 hr interval)
250+ 250
4,459
0.70
0.27
500+ 500
5,914
1.82
0.81**
1,000+1,000
7,934
5.47
2.79**
Chronic irradiation (2 kR/h)
1,000
7,489
1.92
1.03*
2,000
7,611
3.89
1.72**
Control
8,890
0.04
0.01
*, **

Partial

0.31
1.13
2.49
0.43
1.01
2.68
0.89
2.17
0.03

Significant at 5 and 1% levels, respectively.

UV-exposure. Photoreactivation occurred in neither of these two typesof gamma-ray induced mutations (Fujii, T. 1969. Rad. Bot. 9: 115-123).
Namely, the kind of premutationa1 event and the fixation of the event
must differ according to various mutagens, since reparability is thus

88

ANNUAL REPORT -OF NATIONAL INSTITUTE OF GENETICS NO. 20

different while gamma-ray and UV-induced mutation is phenotypically thesame.

A Green Revertant Obtained from a Chrorina Mutant

in Diploid Wheat by Gamma-Irradiation
Taro FUJII

A recessive chlorina mutant induced by X-rays in Triticum mOllococcum
flavescens has been maintained for a number of years in homozygous
condition and has never shown any kind of spontaneous mutation. Itspigmentation was uniform and it remained Yellowish green from seedlingstage to maturity (Fujii, T. 1955. Proc. Japan Acad. 31: 88-92). Dry dormant seeds of this chlorina mutant were irradiated by 5 and 10 kR ofgamma-rays derived from 137es source. Germination and survival rates,
as well as seed sterility in the irradiated lots showed no difference fromthe non-irradiated control lot. All plants of the control and 10 kR lots
had the same chlorina coloration. However, one plant out of 185 survivals
in the 5 kR lot gave revertant tillers, namely among 72 tillers 61 showednormal green color. Ten chlorina tillers and ten green were selfed.
Average seed fertility was 75% for the chlorina tillers and 9296' for thegreen ones, their fertility being similar to that of the original chlorina
and green strains.
The seeds were sown and the offspring in the next generation were
examined. All seedlings derived from 10 selfed and 1 open-pollinatedchlorina tiller had the original chlorina coloration, and all seedlings (234seedlings from 10 selfed and 288 seedlings from 7 open-pollinated tillers)
derived from the green tiller had normal green coloration. As it is known
that the cells for tiller formation are differentiated already in the embryo of the dormant seed, a mutational event which takes place in response to a mutagen among the innumerable cells of a ripe embryo is
usually of chimeric kind. Some of these cells have further developed
to initials of other tillers. It was at first assumed that the normal green
tillers which arose due to irradiation of the chlorina plant are back mutations of one of the chlorina alleles of the homozygote. If so, segregationof chlorina seedlings (at least about 130 on the basis of 3 : 1) was expected in the seedling population derived from normal green tillers. This
unexpected result remained of a somewhat similar investigation by Tullen
et al. (1968. Genetics 59: 45-55) who assume a mutation of a suppressor
in which the phenotypic effects of a given mutation are modified toward

RESEARCHES CARRIED OUT IN 196989

the wile] type by the presence of a second, non-allelic mutation. But
there is an essential difference between the two cases. In their case thechlorina ~hows a tendency to greening and the newly arisen green plants
have a ~omewhat weaker pigmentation than the normal greens. Therefore, further experiments are necessary before deciding how to interpret
my finelings.

Relative Biological Effectiveness of Monoenergetic
Fast Neutrons on Oilseeds
S. S. RAJAN!lIt is wen known that seeds which are rich in vegetable oils are relatively highly resistant to damage by ionizing radiations when compared
to seeds not so rich, as for instance, the cereals. In an attempt to throw
more light on this radio resistance property of oilseeds, the EBR of monoenergetic fast neutrons (14.1 MeV from a Toshiba NT 200-25 neutron
generator employing D-T reaction) were estimated with reference togamma-rays (l37Cs-6 kCi, at the rate of 70 kR/h), with germination, cotyledonaly abnormalities and seedling height as the biological end points.
The seed material chosen for the study were, one variety of Brassica
campestris, and two of Sesamum indicum. These were chosen since theirchromosome numbers (2n=20 and 26 respectively) as also their nuclear
volumes are comparable while their oil contents were different, viz., ca.
40% in Brassica and ca. 50% in the Sesamum material. Further, the oil inthe Brassica seeds contain a high percentage of erucic acid, an unsaturated
C22 compound and a relatively higher content of unsaturated fatty acids
viz., 94"'; as compared to 85% in Sesamum. The neutron doses were firstmeasured by the sulphur activation method but later estimated as thefirst collision dose using the chemical analyses (courtesy Kyushu University) of the elements H, C, Nand 0, in the seed. Treated seeds alongwith the controls were raised in the pots in the glass house and observations were recorded on germination, cotyledonary abnormalities and seedling height on the 10th, 20th and 30th day after sowing.
The LDso estimates for neutrons and gamma-rays and the RBE value
for the neutrons are given in the Table 1 for germination as well as forseedling heights at three stages of growth. The LDso values for both the1)
Present address: Division of Genetics, Indian Agricultural Research Institute,
New Delhi. India.

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

radiations were higher in the case of Brassica seeds than for Sesamum.
This was so for all the four parameters studied. This is contrary to expectations based on their relative contents of oil. The RBE values in thecase of the Brassica were in the neighbourhood of 10, while they wereless for Sesamum. Thus, while the neutrons were found to be ten times
as effective as gamma rays, for the Brassica, they were only 5 to 8 times
as effective for Sesamum. This indicates the Sesamum to be more radio
resistant of the two with reference to neutron irradiation. Another pointof interest was that while in the case of Brassica, the RBE values were
almost the same for germination as well as for seedling heig:1ts at all thestages studied, (10.00 to 10.40), those for Sesamum showed a wider variation (4.57 to 9.21). In the latter the RBE for seedling height was almost
half of that for germination particularly in the variety 45 BON. This
points to the necessity of specifying the stage and nature of measurementin the estimation of RBE values.
It is fairly well established that organic peroxides play an importantrole in the radiation induced biological damage. In seeds with a highdegree of unsaturated fatty acids the oxidation products are mainly from
Table 1. The LD ao estimates for fast neutrons and gamma-rays and theRBE values for fast neutrons for Brassica and Sesamum seeds

Biologicalend point

LD ao
RBE
R a d'la- I Dose.
.
In
Brassica Sesamum Sesamum!Brassica Sesamum Sesamum
tlOn I krads campes- indicum indicum Icampes- indicum indicumI
tris
45 BON 219-3QA
tris
45 BON
219-3QA
I
I

Germination

Fast
3
I
neutrons 10
Gamma104
rays

12.6

7.7

9.5

13.1

6.7

8.8

11.2

8.4

9.3

11.2

4.6

6.5

103

11.7

9.3

7.5

104

11.9

4.2

6.8

103

12.5

6.8

8.2

104

13.0

3.6

6.7

10.33

8.90

9.21

10.00

5.41

6.89

10.17

4.57

9.06

10.40

5.36

8.18

Seedlingheight10daysFast
I103
neutrons
Gamma-I 104
rays

Fast
I
neutrons
20 days Gamm:l
rays
Fast
neutrons
30days Gammarays

RESEARCHES CARRIED OUT IN 196991

the unsaturated fatty acids. The extent of unsaturated fatty acids in
Brassica campestris is 94% of the total oil content as compared to 85% inSesamum indicum. But in terms of unsaturated carbon atoms, 5.5% and7.1% of carbon atoms respectively are unsaturated. If the extent of unsaturation of the carbon atoms determine the semitivity to radiations,
the Sesamum material should be more sensitive to radiations than the
Brassica seeds. While the LD so values lend support to this, the RBE values
do not. In both the seeds the fatty acids are stored in the cotyledonsand very little is found in the embryo. If we score the cotyledonary abnormalities like, early senescence, cleft cotyledons and tricotyledons, thesewere met with only in the Brassica material and not in the Sesamum.
This is again contrary to expectations on the degree of unsaturated carbon atoms these seeds contain. It thus appears that neither the total oil
content nor the degree of unsaturation of carbon atoms of the fatty acids
is related to radio resistance of oilseeds and one should look for this property in other components of the seed. It is probable that native antioxidants like tocopherols in the seed play an important role in this system. (The author is grateful to the Japan Society for the Promotion of
Science for sponsorship under their Visiting Professorship Programme.)

Callus Formation in Wheat Anthers
Taro FUJII

Anthers containing pollen grains at tetrad stage in diploid, tetraploidand hexaploid wheats were used as the materials. They were planted
on modified White's medium supplemented with 20 mg/1 of 2, 4-D andsolidified with 0.9% agar in a test tube. They were incubated underdark condition and constant temperature of 25°C. The pH of medium
was adjusted to 5.8. Callus formations started about 4 weeks after planting. Differences in the production of callus among the species were observed and the results are given in Table 1. Among diploid species, about
3% of explants formed callus in the wild T. aegilopoides, while the tendency to callus formation was very low in the cultivated T. mOJ1ococcum.
A similar tendency was also observed in tetraploid species, namely, wild
T. dicoccoides showed a relatively high callus production of about 18%,
and no callus at all was found in about 600 explants of cultivated T. durum.
It is interesting that no callus was observed in two cultivated hexaploid
wheats, T. aestivum and T. spelta, so far examined. Callus tissues obtained from the above diploid and tetraploid species could be successfully

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20
Table 1.

Ability to form callus in di-, tetra· and hexaploid wheats

StrainNumber of
explants

Number of antherswith callus (%)

698

23 (3.3)

Diploid wheats

T. aegilopoides
T. monococcum(5030)*(5031)*(5074)*(5075)*

2,410
979
1,733
509
1,001

o
o
2 ?

o

o

Tetraploid wheats

T. dicoccoides
T. durum492
592

86 (17.5)

638

o
o

o

Hexaploid wheats

T. spelta
T. aestivum

631* Homozygotic strain of X-ray induced recessive mutant.
subcultured with the same or other kinds of synthetic medium and havebeen maintained over several passages up to present. Although the ability
to form callus is lower in diploid than in tetraploid species, its growth
was better in the former. The use of haploid or polyhaploid callus cells
for mutation experiments is under way. In spite of the very slow growth,
modified White's medium supplemented with 2,4-D was the best for wheat
anthers, among three types of medium, namely, Erickson's, and Murashige
and Skoog's.
Callus induction was also tried in roots of diploid wheats, maize andArabidopsis which are our materials for mutation experiments. Calli wereformed easily in these materials, and their growth was more vigorous
than of those obtained from anthers. Roots were formed from Arabidopsis
callus on modified White's medium while wheat and maize did not formroots on the same medium. Requirements for callus formation and differentiation must be different by different materials. (The guidance in
culture techniques of Dr. Hiroo Niizeki is gratefully acknowledged.)

RESEARCHES CARRIED OUT IN 1969

IX.

93

MICROBIAL GENETICS

Correlation between the Number of Flagella and the Efficiency
of Locomotion in Peritrichously Flagellate Bacteria
Tetsuo lIND

In peritrichously flagellate bacteria such as Salmonella, several flagellaappear from all over the surface of a cell. When a bacterium moves,they are bundled winding around each other. In order to estimate thecorrelation between the number of flagella per bacterium and the efficiency
of bacterial locomotion, the distribution of moving speed was compared
between two bacterial populations differing in flagellar number per bacterium. The experiment was carried out on a wild strain of S. typhimurium, TM2, and its leaky fla mutant, 5J772, which produces flagellaabout half the number of flagella per cell generation as compared withthe wild strain. The speed was measured by the motility track methodof Vaituzis & Doetsch (1969. Appl. Microbiol. 17: 584). The result indicatedthat the decrease in the number of flagella is paralleled to the decreasein the speed of bacterial locomotion. It was also shown that the fractionof cells showing no translational movement exceeds the cell fraction having
less than three flagella in both strain. Of the cells showing no translational movement, one third to one fourth of them wobbled. These results
may mean that a flagellar bundle must be composed from more than three
flagella for a peritrichously flagellate cell to move and that the increasein the number of flagellar filaments in a bundle enhances the speed ofbacterial movement. When the number of flagella per bacterium is lessthan four, rotation exerted by the flagellar bundle may not be strong
enough to drive forward the cell body. It may be worth noting herethat in Pseudomonas and the related genera, which are characterized
by the presence of a single or a small number of polar flagella, can movetranslationally as efficiently as the movement of peritrichously multiflagellate cells of Salmonella in liquid media. However, the movementsof the former fall off more steeply with the increase of viscosity of themedia than the movements of the latter. (The detail submitted to Recent
Progress in Microbiology, 1970)

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Genetical Studies of Flagellation and Motility
in Pseudomonas aeruginosa
Tetsuo IINO

Fifteen mutants which fail to spread on semisolid media were isolated
from a motile strain, MlO, of Pseudomonas aeruginosa. They were classified into three groups as regards flagellar characteristics and cellular
motility. Group-l comprised ten mutants, group-2 four mutants and group3 a single mutant. The mutants in group-l (fia-) are non-flagellate.
The mutants in group-2 (spr-) produce flagella both morphologically andantigenically indistinguishable from those of the parental strain, and they
are motile in liquid media. Differing from chemotactic mutants (che-) ,they show positive chemotaxis to oxygen and glucose. The mutant ingroup-3 produces straight flagella, consequently, it is non-motile.
Transductions were carried out with bacteriophage F1l6 between thesemutants and P. aeruginosa P133, which produces flagella antigenicallydistinct from MlO. As the results, mutant sites of fia- and spr- wereclassified into three and one complementation groups respectively. The
mutant site of straight flagella was mapped in H, i.e. the structural geneof flagellin. It was further shown that H, fia and spr disclosed in thisstudy cluster on a chromosome and they are capable to be transduced
simultaneously by F1l6. By mating experiments between P13 (FP+ adefia-) and P138 (FP- trp- his-), this gene cluster was mapped between leu
and ade.

Extraordinary Polymerization of Salmonella Flagellin in vitro
Hirokazu HOTANl1), Sho ASAKURA1) and Tetsuo IrNO

A cellular component which promotes polymerization of Salmonella
flagellin under physiological environmental conditions was fractionatedfrom an autolysate of the deflagellated bacterial cells by differential centrifugation. This component is membranous in nature and probably originating from the cell wall and/or cytoplasmic membrane. Filaments producedin this polymerization are straight and are thinner than flagella. They
are highly stable against heat (boiling for 5 min), acid (0.1 M He!) andalkali (0.1 M NaOH) and never depolymerize under the environmental
conditions where flagella completely depolymerize. These filaments are
named P-filaments. Serological comparisons between P-filaments and flagella1) Institute of Molecular Biology, Nagoya University.

RESEARCHES CARRIED OUT IN 196995

showed that the two types of filaments have common specific antigenicities(The detail published in Biochim. Biophys. Acta, 1969).

Chlorate-Resistant Mutants of Salmonella typhimurium
Masatoshi ENOMOTO

Chlorate-resistant (chI) mutants were isolated on nutrient medium supplemented with KCIO a lO.2% w/v) and glucose (0.2% w/v) in the prolonged
incubation under anaerobic condition. A frequency of mutations wasapproximately one per 106 cells. The chi mutants were classified into
three groups (chIA, chIB, and chIC) by transductions with phage P 22 andmatings with an Hfr strain, SW 1391. The order of chi and other loci usedin the experiment was inferred to be his-trp-chIC-chIB-chiA-bio-gai.
The chIC mutants showed no growth on minimal medium, which wasrestored by the addition of NAD (nicotinamide-adenine dinucleotide) or acommercial vitamin complex, Panvitan (Takeda). The chi mutants lostthe activity of nitrate reductase, because of the defect of an enzyme
system capable of using both chlorate and nitrate as a common substrate.
Specific activity of nitrate reductase in the wild-type strain, TM2, was
148 (m,umole NOz/mg protein/min) in a soluble extract and 1045 in an insoluble One and that of the chi mutants was less than 0.7% of TM2. Details
will be published elsewhere.

Transductional Analysis of the Gene Controlling Arginine
Sensitivity in Salmonella typhimurium
Jun-ichi ISHIDSU

It has been reported that the site of an arginine sensitive mutation ins. typhimurium, ars-l, is linked to pyrA locus by P 22 phage mediatedtransduction at the co-transduction frequency of 30 to 60% (Eisenstark,A. 1967. Nature 213: 1263). Linkage relationship between various ars
and aus (sensitivity to both arginine and uracil) mutants and mutants
whose map position scatter around all over the range of the pyrA locus
was analyzed. pyrA mutants were used as recipients and P 22 L4 phagegrown on ars or aus mutants were employed as donors. Washed cell suspensions of 4 to 5 x 109/ml were mixed with phage suspensions in broth(lOlO/ml) at the ratio of 1 to 1, and 0.1 ml aliquots of each mixture wereplated on minimal agar plates. After about 48 hour incubation at 37°C,

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

transductant colonies were scored. There found two types of colonies
with respect to their sizes, namely large and small. After their characteristics were examined by transferring each colony onto minimal andminimal with 10- 3 M arginine plates using toothpicks, it can be said that
small colonies are consist of transductants which have received ars or aus
allele from the donor, while cells forming large colonies have not received
such allele. By this indication, co-transduction frequency was calculated
for each combination. They are listed in Table 1.
Table 1.

Co-transduction frequency C%) in crosses between ars or aus
and pyrA mutants~Recipient*1
Donor .~
ars-1
ars-2
ars-3
aus-1
aus-2
aus-3
aus-4
aus-5

pyrA 39 pyrA127 pyrA102 pyrA 78 pyrA 81 pyrA103 pyrA109

75.1
77.3
73.8
34.8
41.4
47.4
43.0
49.6

82.9
87.4
87.2
11.8
30.8
33.8
37.1
39.1

86.4
86.4
89.5
59.7
66.3
72.2
62.3
72.5

89.0
91.9
93.5
58.8
75.1
83.9
69.2
84.8

89.8
87.7
90.6
50.0
71.1
81.1
61.9
74.2

91.5
91.2
91.9
65.3
73.8
84.4
73.1
80.6

98.7
94.4
95.5
79.8
88.2
89.9
83.5
93.8

* Arranged in accordance with the results of genetical analysis made by Yan
and Demerec CYan, Y. and M. Demerec. 1965. Genetics 56: 643).

From these results, eight ars or aus mutants are arranged in the ordershown below, taking each pyrA mutant as standard.

pyrA 39-ars-2-ars-l-ars-3-aus-5-aus-3-aus-4-aus-2-aus-l
pyrA127-ars-2-ars-3-ars-l-aus-5-aus-4-aus-3-aus-2-aus-1
pyrAI02-ars-3-ars-2-ars-l-aus-5-aus-3-aus-2-aus-4-aus-1
pyrA 78-ars-3-ars-2-ars-l-aus-5-aus-3-aus-2-aus-4-aus-l
pyrA 81-ars-3-ars-l-ars-2-aus-3-aus-5 -aus-2-aus-4-aus-l
pyrAI03-ars-3-ars·l-ars-2-aus-3-aus-5-aus-2-aus-4-aus-1
pyrAI09-ars-l-ars-3-ars-2-aus-5-aus-3-aus-2-aus-4-aus-l
Although some discrepancies still remain in detail, it is clear that bothars and aus are linked to pyrA by transduction, that their order is ~pyr Aars-aus- and that they are located on the right hand side of pyrARESEARCHES CARRIED OUT IN 1969

97

between pyrA and ara because pyrA39 is known to map farmost andpyrAl09 nearmost from ara gene (Yan, Y. and M. Demerec. 1965. Genetics 52: 643) and it is evident from Table 1 that both ars and aus arelinked to pyrAl09 much closer than to pyrA39.

Some Properties of Azide Resistant Mutants ofSalmonella typhimurium
Jun-ichi ISHIDSU

Sixty-eight spontaneous sodium azide (NaN a) resistant mutants (aziwere isolated from S. tYPhimurium wild type strain LT2 andsome mutants derived from LT2 (pyrA8l, ars-l and aus-l) grown on nutrient agar plates containing 4 x lO- a M NaN a. Thirty-nine of them formed
large colonies and the rest formed minute colonies. Surprisingly, neitherof them could grow on minimal medium containing the same concentration of NaNa, even when requirements were supplemented in case theparent was an auxotroph. By auxanography, it was found that, when
NaNa is present, they all require methionine or cysteine and in addition
minute colony formers grow only anaerobically. In either case methionine
was a better supplement. Many sulfur containing compounds were tested
for their ability to support growth of the mutants under the presence of
NaN a. NaSH (about 2x 10- 2 M) was found to support the growth of bothtypes quite effectively and Na2S0a the growth of large colony formers.
Physiological and genetical analyses of these mutants are being continued.
501~568)

High Frequency Reversion and Modified Efficiency of Amber
Suppression Induced by A-lysogenization in
Escherichia coli K12
Tsuneo KADAHigh frequency spontaneous reversions in a threonineless strain ofEscherichia coli K12 have been previously reported (Kada, T. 1970. Mutation Res. 10: 91 and 103). This frequency was increased more than 104
fold by introduction of a gene named mod+ linked to the leu locus andthe site of frequent mutations resided at a locus named Jgr locating between gal and try. It has been supposed that the presence of mod+permitted to detect frequent genetic changes taking place at Jgr locus,
bringing about suppressor functions on the level of ribosomes.

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Frequent reversions also took place at many auxotrophic loci by
A-lysogenization of E. coli K12 S-1014 (F-, thr-1014 leu modfgr try his arg
thi str-r). Two categories of strains S-1014 (J.) were obtained: category
I remained apparently unchanged except for the possession of J.; lysogenic
strains of category II had the frequency of reversions promoted 103~1O.
fold at all auxotrophic loci studied (thr, leu, try, his and arg) and became
sensitive to streptomycin (this Annual Report 18: 118. 1967).
In order to examine if the phage J. might have introduced a geneworking similarly to mod+ into S-1014 having the mutable fgr locus, strain
S-1014 was lysogenized using phage particles liberated from non-mutable
lysogenic strains of category I. No transfer of the hypothetic gene was
expected in these experiments. However some of isolated S-1014 (A) strains
were mutable. These observations are against the supposition that J.
transduced specifically a gene that makes bacteria mutable.
In other experiments, mutable lysogenic strains were cured of J. bymeans of superinfection with phages ¢170 kindly provided by Dr. Iijima(Osaka). It was found that some bacteria remained mutable even after
curing and were sensitive to streptomycin. These results rather indicatethat a physical integration event may be responsible for the genetic
change responsible for high mutability.
A remarkable genetic modification other than mutability and streptomycin sensitivity accompanied with lysogenization was the change concerning the efficiency of amber suppression. The parental strain S-1014
has an amber suppressor gene (su Jl+) and permits the growth of mutantsof T4 phages possessing amber nonsence codons. It was observed thatthe lysogenic mutable strains (category II) was not permissive at all(Table 1). On the other hand, the parental S-1014 and all the lysogenic
derivatives were lacking completely a permissive capacity for an ochre
Table 1.

Plating efficiencies of mutants of phage T4 possessing nonsensecodons on different F- strains of Escherichia coli K12
T4 mutants
Strains

Amber
Am 52

S-1014 (,1-, str-r)
I-A (,1+ catogory I, str-r)
II-B (,1+ category II, str-s)
BI t + 4 (Permissive indicator)r ry
for ochre 427

B 17

N 69

Ochre
427

1.0
1.0
1.0
6.3x10- 1
1.2
0.97
1.4
5.0x10- 1
3.4xlO- 5 4.4x10- 1 4.2x10- 6 7.5x10- 6
1.0

RESEARCHES CARRIED OUT IN 196999

T4 mutant.
Ribosomal mutations affecting efficiency of amber suppression wererecently reported by Strigini and Gorini (J. Mol. BioI. 1970. 47: 517). To
interpret our observations we may suppose that the chromosomal integration of A phage might cause mutations at a mutator or specific suppressor
gene residing at the integration site.

100

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

X.

I-IUMAN GENETICS

Sexual Variation in Finger Pattern Types and Ridge
Counts in Germans
Ei MATSUNAGA, Ei MATSUDA and Heinrich SCHADEl I

The dermal ridge patterns on finger tips vary in many respects between males and females. Among various racial samples hitherto investigated, males have generally more whorls and radial loops than females,
while the females have more ulnar loops and arches than males. Thereis also a sexual variation in the pattern size as measured by ridge count.
The total ridge count, i. e., the sum of the ridge counts for each of ten
fingers of a given individual, is on average larger in males than infemales. Relevant data from a Japanese sample consisting of 200 males
and 200 females were reported previously (Ann. Rep. Nat. Inst. Genet.
18: 120, 1967). An interesting finding in our study revealed a significant
sexual difference in the mean ridge count per digit for a \vhorl but not
for a loop. This was rather unexpected because the larger total ridge
count had been thought to imply a larger pattern size in males than infemales irrespective of the pattern type, whorl or loop.
In order to see whether the above finding may have a general significance, we examined finger prints of a German sample consisting of 243
males and 220 females, which had been collected by one of us (H. S.).
Table 1 shows the total ridge counts as well as the percentages of four
major pattern types according to sex. As compared with the Japanese,
the Germans have on average in both sexes smaller total ridge counts, less
Table 1.

No.

Total ridge counts and percentages of different patterntypes among Germans
Total ridge
count

Pattern types (%)

Sex
examined
M
F

243
220

Diff. (M-F)

Mean

S.D.

Arch

142.4
130.8

51.00
53.32

11.6*

Loop
Ulnar

5.2
7.5

5.6
3.4

57.8
61. 7

31.4
27.5

-2.3

2.2

-3.9

3.9

* P<0.02.
1)

Whorl

RadialInstitute for Human Genetics, University of Dusseldorf,Germany.

101

RESEARCHES CARRIED OUT IN 1969

whorls and more loops and arches. However, the sexual variations in thefrequency of pattern types and the total ridge count are in the same
direction as those found among Japanese.
Table 2 represents the mean ridge count per digit according to sex
Table 2.

Mean ridge count of a whorl and a loop, based on 243 males
and 220 females in Germany
All digits

Sex

Whorl

Ulnar
loop

Radial
loop

II

III

IV

V

18.3
19.0

19.8
18.5

17.7
16.8

19.2
18.5

M
F

21.3
20.2

16.9
16.9

Diff. (M-F)

1.1

0

-0.7

1.3

0.9

0.7*

M
F

15.5
13.9

10.1
9.8

11. 7
11. 7

14.4
14.2

13.4
12.1

13.2
12.4

Diff. (M-F)

1.6

0.3

0

0.2

1.3

---------------_ ..

0.8**

M
F

9.4
9.0

9.6
8.4

Diff. (M-F)

0.4

1.2

* P<0.02, ** P<O.OOl.

and pattern types for the Germans. Here, the males have on averagelarger ridge count than females for all pattern types, although there are
more or less variations according to digits which may be due to chance
fluctuation. This finding apparently differs from the one obtained from
our Japanese sample for which the sexual difference was seen only for awhorl. Further studies are needed to see whether the discrepancy is due
to a real ethnic difference or to some sampling error.

Polymorphic Hematological Traits of the JapaneseTomotaka SHINODA

Polymorphic genetic systems, particularly of red cell enzymes recognizedin man have revealed striking differences between the populations, notonly in specific gene frequencies but also in the presence or absence ofparticular genes.
In order to estimate gene frequencies of £everal red cell enzymes andto make a survey of regional variations, if any, in the distribution of

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

various phenotypes, an extensive investigation has been carried out using
blood samples collected from three different areas, Tokyo, Mishima andNagoya. Parts of this work were already published in Jap. J. Hum.
Genet., 14: 316-323, 1970 and Jap. ]. Genet. 45: 147-152, 1970.
Acid phosphatase: Of 405 samples, collected in Tokyo and examined,
18 (4.4%) were of type A, 129 (31.9%) of type AB and 258 (63.7%) of type
B. Accordingly, the frequencies of the genes p' and pb may be estimatedas 0.204 and 0.796, respectively. In Mishima, 685 samples were so far
analyzed. Of those 28 (4.1%) were of type A, 231 (33.7%) of tyPe AB and
426 (62.2%) of type B. The frequencies of the genes p' and p b may be
estimated as 0.209 and 0.791, respectively.
Adenosine deaminase: A total of 1,499 samples, collected in Tokyo,
Mishima and Nagoya were analyzed for ADA phenotypes. In our samples
ADA 1 type was the most common and occurred in roughly 94% of the
populations. ADA 2 type was rare and was found only once in each
population. Table 1 summarizes the results.
Table 1. Distribution of ADA phenotypes and estimates of ADAI
and ADA2 gene frequencies among unrelated Japanese
Area
Tokyo
Mishima
NagoyaTotal

No.
examined
286
(%)

645
(%)

586
(%)

1,517
(%)

ADA phenotypes

Gene frequency

1

2-1

2

ADAI

ADA2

268
(93.7)
606(94.0)
549(93.7)

18
(6.3)
38
(5.9)
36
(6.1)

0

0.969

0.031

1
(0.1)
1
(0.2)

0.969

0.031

0.968

0.032

1,423
(93.8)

92
(6.1)

2
(0.1)

0.969

0.031

As far as the frequencies of the genes ADAI and ADA2 are concerned,
no marked differences were found among the populations.
Phosphoglucose isomerase: Analysis of 933 blood samples was made for
PGI types. So far, three cases of type 3-1, 2 of type 4-1 and one rare
type, tentatively designated as PGI 6-1 J, were found. Further survey
for the enzyme system is under progress on blood samples collected fromdifferent areas.
Phosphoglucomutase: Nearly 2,900 samples were collected during thisyear in two different areas. Of those 2,742 samples were so far analyzed.
Table 2 summarizes the result. The variation found in zymograms was

RESEARCHES CARRIED OUT IN 1969
Table 2.
Area

Shizuoka
Total

Distribution of PGM 1 phenotypes among unrelated JapaneseN?
exammed

Tokyo

103

PGM1 phenotypes
1

585(60.6)
1,677 1,001(%) (59.7)
965

(%)

2,642
(%)

1,586
(60.0)

2-1

2

320(33.2)
576(34.4)

54
(5.6)
89
(5.3)

1
(0.06)

896(33.9)

143
(5.4)

1
(0.04)

3-1

3-2

7-1

8-1

unidentified

1
(0.1)

3
(0.3)
5
(0.3)

1
(0.1)
1
(0.06)

1(0.1)
4(0.24)

8(0.3)

2(0.08)

5(0.2)

1(0.04)

concerned in every instance with PGMl but not with PGM2 pl1enotypes.
In addition to three common phenotypes, 19 cases in all of rare variants
were observed, some of them were similar to PGMl types 3-1, 3-2, 7-1,
7-2 and 8-1. On the basis of the incidence among 2,642 unrelated persons,the frequencies of genes PGMll, PGM12, PGM13, PGM17, and PGM18 havebeen estimated to be 0.7720, 0.2239, 0.0004, 0.0015 and 0.0004 respectively,
with no difference between the two areas.
6-Phosphogluconate dehydrogenase: Three phenotypes designated as
types A, AC and C were found throughout the survey. Type A was themost common in our samples and occurred in roughly 87% of the population with no difference among the areas investigated. So far 1,078 samples
were analyzed for PGD types. Of those 936 (86.8%) were of type A, 137(12.796) of type AC and 5 (0.5%) of type C, respectively. Accordingly,
the frequencies of the genes PGDA and PGDe were estimated to be 0.933
and 0.067, respectively.
Fumarase: As to variation in fumarase, 600 samples were tested, but
all samples except one case were of the same type, designating as FUM
1. The enzyme exhibited two isozyme components, one major with faster
mobility and one minor with slower mobility. The only variant consistedof two equally intense isozyme bands, one occupying the same position
as the major component of tyPe 1, the other migrating at the same rate
as the minor component of type 1. The variant was tentatively designated as FUM 2-1.
Nucleoside Phosphorylase: Red cell nucleoside phosphorylase exists in
two isozyme bands, one major with faster mobility and a minor withslower mobility. The enzyme was stable for months, if stored at -22°C.
Of 960 samples tested, all but two were of the same type, designated as
NP 1. The two variants showed two equally intense isozyme bands, oneof which had the same migration rate as the major component of NP 1

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

and the other was its minor component. The variants are tentatively
designated as NP 2-1. Further analysis of larger samples is under progress.
Besides the enzymes mentioned above, several other enzymes such as
adenylate kinase. citrate dehydrogenase, glycerokinase, pyruvate kinase
and enolase are under investigation.

Structure Analysis of Human Immunoglobulins
Tomotaka SHINODA

Bence-Jones proteins, now generally recognized as a light chain of
immunoglobulins carrying either Inv or Oz serological factor, were isolated
from urine of patients with multiple myeloma and were purified byseveral procedures such as (NH4)zS04 precipitation, chromatography on
DEAE-cellulose column and gel filtration in Sephadex. By these procedures
specimens (referred to as HBJ-l and HBJ-2) were found to be pure, notonly in starch gel electrophoresis but also in amino-terminal analyses by
DNP-method. They were of /(-type, but were not tested for the Inv
serological factor. Molecular weight determination revealed the figure
22,000-23,500 for either specimen. An aspartic acid residue was found tobe the amino-terminal for both proteins. Pepsin digests of intact HBJ-land HBJ-2 were prepared in 2% by weight of pepsin using 0.01 N HCI at
37°C for 8 hours. After lyophilization, the digest was separatd on afilter paper (60x80 cm) by electrophoresis at pH 6.5 and 2,500 volts for
90 min. The "diagonal" maps, prepared in a similar way for the purifi·
cation step, were compared for both samples. Of four pairs of cysteic
acid containing peptides, three were found to be common to two samples,but the remaining pair was different in mobility in electrophoresis at
pH 6.5. The common peptides may have derived from the constant partand the carboxyl-terminal cystine of Bence-Jones protein, whereas theremaining pair derived from the variable part of the protein.
Sequence analysis for the variable part is under progress.
Partial Purification of Genetically Different Phosphoglucomutaseof Human Red Cells
Tomotaka SHINODA

In order to investigate the relationship between phenotypic vanatIOn
and biochemical nature of human red cell enzymes, partial purification

RESEARCHES CARRIED OUT IN 1969105

has already been carried out of acid phosphatase in the preceding year
(Shinocla, T. 1968. this Annual Report 19:35). In this year, attempts weremade to purify genetically different types of phosphoglucomutase (PGM)[a -D-giucose-1, 6-diphosphate: a -D-giucose-phosphate phosphotransferase
EC. 2.7. :'i. 1] isolated from normal human red cells. As vvell as on starch gelelectrophoresis heterogeneity was observed on column chromatography
made v,ith CM- or DEAE-cellulose and NaCI gradient. The partiallypurified enzyme appeared to show essentiallY the same electrophoretic
pattern on starch gel as the hemolyzate, except that faster cathodal minor
components disappeared from the purified material. Heat stability of PGM
was carriec1 out at various temperatures, and was found to be different
according to the phenotype of the enzyme, the order of their stability at
50°C being PGM 2> PGM 1 PGM 2-1. A large scale purification wasmade using bottles of blood purchased from a Blood Center. By combination of several procedures, such as (NH4)2S04 precipitation, DEAE- andCM-cellulose column chromatography and gel filtration with Sephadex,
specific activities of 35-140 units/mg for PGM 1 and of 22-135 units/mg
for PGM 2-1 were attained. Blood samples of PGM 2 type were notavailable in large quantity, since the incidence of PGM 2 was less than
8% among Japanese.
When purified material and stroma free hemolyzate were submitted togel filtration in Sephadex, the enzyme, regardless of its type, was eluted
as a single, and nearly symmetrical peak. In contrast to starch gelelectrophoresis, no clear-cut separation among the isozymes of PGM wasobserved on gel filtration, presumably because they are similar in molecular sizes.
Recombination experiment was carried out without success using theintact enzyme and a partially trinitrophenylated material.

=

A New Variant of a 2-globulin Fraction in Human Serum
Yoshito OGAWA and Norio ODAKI

A new polymorphism of subfraction of a2-globulin in human serum wasfound by two-dimensional electrophoretic analysis (Cellulose acetate - Disc)and immunochemical examination.
Three autosomal codominant alleles, designated by symbols a2'F, a2'M andal s are responsible for the observed five phenotypes (alM/al M, alF/al M,
a2'F/ a2'", alF/al F and alM/al S ) on the basis of the family and population
data. Occurrence of phenotypes associated with a2's, gene, seems to be106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

very rare in the Japanese population.

No phenotype corresponding toa/Sjaz'S was therefore found in one thousand and eleven cases.

Details of this work had been published in Physico-Chemical Biology
Vol. 14 (1969).

Mutation Rates of Translocation Down's Syndrome
Yasumoto KIKUCHI, Hidetsune OISHI, Akira TONOMURA!I,
Takehiko KURITAzl and Ei MATSUNAGA

It is now known that G trisomy is responsible for the overwhelming.
majority of the cases with Down's syndrome, while translocation andmosaicism account for a very small number. The purpose of the present
study was to estimate the frequency of patients with chromosome translocation within the general population as well as the rate of particularchromosomal mutations leading to translocation Down's syndrome.
Since the last report of 1966, we have examined chromosomes of further
673 cases of Down's syndrome. Whenever a patient was found to have
46 chromosomes which included a presumptive translocation the chromosomes of the parents and other close relatives were also examined. For
estimating the frequency of translocation, other data reported in Japan
were combined with the present one.
Among the total of 902 patients with Down's syndrome, 41 or 4.5%
were of translocation type. Among them 23 or 56.1% were of t(DqGq)
and 18 or 43.9% were of t(GqGq) type. These two groups of 23 and 18 cases
contained 3 and 4 families respectively, where neither parent was studied.
If we apportion sporadic and inherited classes according to the proportions
worked out for the families where both parents were studied chromosomally, then the number of sporadic and inherited cases among thet(DqGq) patients was 13.8 (60%) and 9.2 (40%), respectively. The corresponding figures for the t(GqGq) patients were 15.4 (86%) and 2.6 (14%),respectively.
The incidence of Down's syndrome among newborns in Japan has
recently been estimated to be about 0.134%. Using this figure, the frequency of translocation Down's syndrome among all live births may be
estimated on the assumption that they have the same mortality rate as theprimary trisomies, to be 6.03 X 10- 5 or 1 in 16,600 live births, as compared
1) Department of Cytogenetics, Tokyo Medical and Dental University.
2) Pediatric Clinic, Kohnodai National Hospital.

RESEARCHES CARRIED OUT IN 1969107

with the incidence of Down's syndrome of 1 in 750 live births.
From the above estimate and the proportion of sporadic cases, the figureof 2.15 X 10- 5 was obtained for the ill'ltation rate of gamete with t(DqGq)or t(GqGq) in unbalanced form, resulting in sporadic cases of translocation
Down's syndrome in each generation. However, this represents an underestimate, probably by a factor of 4, if early embryonic deaths due tochromosome mutation are taken into account. Upon the consideration of
the proportion of inherited cases, the mutation rate for the production oftranslocation heterozygotes carrying t(DqGq) or t(GqGq) was estimated tobe 0.87xlO- 5 • It was noted that our figures are remarkably close to thosereported by European workers. The details of this work were publishedin the Jap. J. Human Genet. 14: 93-106, 1969.

Clinical Conditions of Patients with Apparently Normal
Chromosomes, VI
Hidetsune OISHI and Yasumoto KIKUCHI

Based on chromosome analysis performed on cultured leucocytes,
patients with various pathological conditions were found to have apparently
normal chromosome assortments of 46 chromosomes. Cases of some interest
are listed below.

Name

Age Legal sex

Clinical conditionsa) Multiple deformations with mental retardation
105 H. W. 2 years
M
Arachnodactyly; transverse palmar creases;
external rotation of lower extremity; hypertonia
106 T.K. 3 years
M
Hypertelorism; high-arched palate; micrognathia; transverse palmar creases; logopathy
107 D.T. 3 years
M
Strabismus; high-arched palate; epilepsy;
logopathy
108 M.L
6 years
F
Arachnodactyly; congenital cataract; nystagmus
109 M.L 4 months F
Congenital heart disease; hypertelorism;high-arched palate; small deformed lobes ofthe ear; low-set ears; short neck; cubitus
valgus; genu varum; pes abductus; alopecia; hemangioma

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

110 M.F.

111

4 months

M

M.Y. 10 months

F

112 K.H.

3 monthsF

113 N.ll.

2 months

M

114 M.L3 months

Mb) Sex anomalies
115 K.S. 1 yearF

116 K.N. 3 years
F
117 H.G. 29 years
M
118 K.A. 2 years
Mc) Hereditary diseases
119 K.K. 6 years
F
120 1. Y. 16 years
M
121 M.N. 4 years
M

Low-set ears; hypertelorism; epicanthus;
high-arched palate; short neck; brachycephaly
Microcephaly; pseudohydrocephalus; higharched palate; hemiatrophy; incurved fifth
fingers
Microcephaly; craniostenosis; exophthalmos;
high-arched palate; low-set ears; micrognathia; polycystic kidney
Microcephaly; trigonocephalus; narrow andoblique palpebral fissures; exophthalmos;
cleft palate; short neck; umbilical hernia
Microcephaly; high-arched palate; micrognathia; retentio testis abdominalis
Male pseudohermaphroditism; short neck;shield chest; cubitus valgus; vaginal atresia
Female pseudohermaphroditism
True hermaphroditism
Female pseudohermaphroditism
Madan's syndrome
Pachydermoperiostosis
Familial vertebral dysplasia

Qualitative Relationships between X Chromosome
and Leucocyte Drumstick
Hidetsune OISHI and Akira TONOMURAli

It is generally considered that the so-called drumstick found in polymorphonuclear leucocytes of normal females is an expression of one of
the X chromosomes being inactivated. This interpretation may be extended to subjects with structurally abnormal X chromosomes, although
the qualitative correlation between the X chromosome and the drumstickis still obscure.
Comparison of the sizes of drumsticks has been made for four normal
females and four patients with abnormalities of the X chromosome. Of
1) Department of Cytogenetics, Tokyo Medical and Dental University.

RESEARCHES CARRIED OUT IN 1969109

the four patients three showed a sex-chromosome constitution of an X and
an isochromosome for the long arm of the X (46,XXqi) and the remaining
one had a deletion of the short arm in one of the two X chromosomes(46,XXp-). The mean value of the drumstick/nucleus (D/N) ratio estimated from normal female cells in terms of chromatin mass showed
3.00±0.39%, whereas in cells with 46,XXqi and with 46,XXp- the meanvalues were 4.22±0.49% and 2.22±0.20%, respectively. These results
strongly suggest that the proportional changes involved in the amount of
X chromosome material are reflected in the size of the drumsticks, since
the mean value of the D/N ratio in cells with 46,XXp- was comparable
to half the value in cells with 46,XXqi. Furthermore, if we assume thatin normal female cells one of the two X chromosomes is totally heterochromatic and forms a drumstick, a part of the drumstick manifested by
the chromatin of the short arm may be expressed as:
drumstick (X) - drumstick (Xp-) or 1/2 drumstick (Xqi) .
The estimation from the above data gives 0.78 or 0.89% for this value.
Based on this assumption, the ratio of the short arm to the long arm of
the X chromosome can be calculated as, 0.35 or 0.42. However, according
to a note on length measurements of human chromosomes in Penrose's
report (1964), the arm ratio of the X chromosome is represented as 0.58.
Since the estimates of the arm ratios in terms of area measurements areclosely related to those based on length measurements (Oishi and Tonomura,1965), the difference may indicate that a part of the X chromosome (about
10.1 or 14.6%) is euchromatic and remains within the nucleus.

Pattern of DNA Synthesis in Human X Chromosomeswith Structural Abnormalities
Yasumoto KIKUCHI and Hidetsune OISHI

In normal female cells one of the X chromosomes synthesizes most of
its DNA at the end of DNA synthesis period (S period). This late replicating X chromosome is most easily identified by autoradiographic
terminal labeling of the chromosome complement. Recently, internal
asynchrony of this late replicating X chromosome has been observed. In
this study we examined the pattern of DNA synthesis of structurally
abnormal X chromosomes as compared to that of a normal X chromosome.
The studies were performed on cultured leucocytes obtained from thefollowing two female patients with Turner's syndrome; one with 46,XXp-,

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

and the other with 45,X/46,XXqi. DNA synthesis in the chromosomes
at the end of the S period was analysed by means of 3H-thymidine autoradiography.
An Xp- chromosome was morphologically similar to the members ofgroup D except that its short arm and the satellite region were missing.
Therefore the cells with 46,XXp- had 7 large acrocentrics. Afterdeveloping the autoradiographic film, 43 labeled metaphase plates wereobserved. In most cells one of the 7 large acrocentrics was heavily
labeled, being presumably an Xp-.
Sixty-five metaphases with 46, XXqi were formed to be labeled. An
Xqi resembling in size and arm ratio the autosome no. 3 was the mostheavely labeled chromosome of the entire complement during the end of
the S period. No such late replicating chromosome was observed in thecells with 45,X.
Internal asynchrony of DNA synthesis was observed frequently in those
two abnormal X chromosomes. In some Xqi chromosomes the labeling
patterns on their both arms seemed to be asymmetrical. These findings
indicate that in the Xp- and Xqi chromosomes DNA synthesis is apparently
completed first in the centromeric region, followed by the distal part of
the arm, and the proximal part of the arm is the last to complete
synthesis.
The results may indicate that in those patients with one normal andone abnormal X, the structurally abnormal X chromosome was always
inactivated in preference to the structurally normal X chromosome in thecells. Evidences from the labeling pattern of the Xp- and Xqi chromosomes suggest that both behave in a fashion similar to the long arm ofa normal X chromosome.
Details of this study have been published in the Jap. J. Human Genet.
15: 114-123, 1970.

RESEARCHES CARRIED OUT IN 1969

XI.

111

APPLIED GENETICS

Further Study on Competition in Sugi-Tree, Crgptomeria japonica,
with Special Reference to the Age of the Forest
Kan-Ichi SAKAI and Shigesuke HAYASHIA part of results of this study has been reported in the last issue of
this Annual Report (No. 19, pp. 95-96). Further data are added in thepresent report. Investigated were two forests, both raised from seedlings.
Two hundred disks from one, and one hundred disks from the other forest
were collected, and stem growth was measured for yearly ring width in-eight directions in order to estimate the mean annual growth. Bothforests were about 50 years old, though in one of the two it was impossible
to measure growth older than the age of 25 years, because the disks were-collected from the bottom of stumps remaining on the cleared area andswelling by roots of yearly rings disturbed correct measurements of growtholder than 25 years. Correlation of stem growth between paird adjacenttrees was calculated. When the correlation was positive and high, effectof intertree competition was understood to be none or slight, whereas
low and positive, zero or negative correlation was assumed to show theoccurrence of competition.
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 during thefirst six or seven years correlations were positive and high, and no competition appeared to occur in both forests. The value of correlations,however, decreased thereafter, indicating that competition started among
the trees. The intensity of competition was increasing year after year
probably up to a certain stage of maturity, so far as neither spontaneous
nor artificial thinning was done. When thinning was performed, competition appeared to weaken for several years, though it was again
recovered later. It appeared that the start of competition was associatedwith the start of vigorous growth of trees. Results of this study will bereported in a journal of forestry.

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. ::0

Genetic Variability on Enzymatic Level in Natural Forestsof Thujopsis dolabrata
Kan-Ichi SAKAI, Yasusada MIYAZAKI and Takashi MATSUURA

Two natural forests of Thujopsis dolabrata growing in two promontories
in Aomori prefecture were investigated to find out whether they were
genetically different. The two promontories are isolated from each otherby about 10 kilometer wide straits. Needle leaves were collected fromthe thickest bough of each tree of both forests for the study of leaf
characters on one hand, and electrophoretic analysis on the other. The
measurements concerned length and width of main leaf, width of compound leaf, length and vertical length of lateral leaf and leaf thickness.
The electrophoretic study was performed by the technique developed bySmithies (1955) and Endo (1968).
In an early stage of our study of electrophoretic peroxidase variability.
53 bands were counted as the maximum number. However, after a statistical treatment of the material, the number was reduced to 42. With those
42 isoperoxidase bands, two comparisons were made between the twoforests. The first was a comparison of the average number of bands exhibited per tree. In one of the two forests, called Masukawa, the average number was 16.29±2.17, while in the other called Ohata, it was 15.61±2.76. The t-test failed to reveal a statistical significance of the difference between the two forests. The second was a comparison of incidences of various bands in the two forests. It was found by statisticalcomparison that among 42 bands, there were 15 pairs which were statistically significant. This amounted to 36% of 42 pairs, which was far
more than 5% expected by chance only. In other words, the two populations were considered to be significantly different so far as isoperoxidase
distribution is concerned.
As to the measurements concerned with leaf characters, it was foundthat two of them, namely vertical length of lateral leaf and leaf thickness, were significantly different between the two forests. The verticallength of lateral leaf in Masukawa forest was 4.51±O.52mm while it was
4.12±O.50 mm in Ohata. The leaf thickness was in the former 1.25±O.24mm and 1.42±O.30 mm in the latter. It was thus concluded that the
two natural forests of Thujopsis dolabrata which were ecologically notmuch different but geographically isolated by straits were geneticallydifferentiated from each other.

RESEARCHES CARRIED OUT IN 1969

113

Family Analysis in a Natural Forest of Thujopsis dolabrata:
A New Method for Quantitative Genetic Studies
Kan-Ichi SAKAI and Yasusada MIYAZAKI

The aim of our study was to develop a method for genetic investigation of quantitative or physiological traits of forest trees in a natural
stand where individuals of different age are growing at various densities.
For this study, it was necessary to detect sib-trees constituting families
in the forest. The detection of sib-trees was made possible by a comparative study among trees with respect to their isoenzyme patterns ofperoxidase. The approach started first with accurately mapping on asection paper the numbered individual trees of the forest. Then a few
measurements concerned with growth as well as leaf characters of those
trees were taken. Leaf samples from each tree were investigated forelectrophoretic variation of peroxidase isozymes.
Two natural forests of Thujopsis dolabrata, one with 45 and the otherwith 83 trees, were investigated. The electrophoretic study was conducted by the technique developed by Smithies (1955) and Endo (1968).
The number of isozyme bands was 42 in total. Comparison of isozyme
bands among different trees within the same forest was made by measuring the number of azygous bands or the "disagreement count". During
this comparative study of peroxidase among trees, we noticed that there
was every indication that the disagreement counts between trees growing in the neighborhood were often very small in comparison with thosebetween trees growing far apart in the same forest. The lower disagreement count or higher similarity in the isozyme pattern among trees
growing in the neighborhood might suggest that genetically related individuals are standing rather in proximity than being scattered.
It has been considered that trees which differed from each other by a
low disagreement count might, in some way or other, be sib-trees. In
order to examine if this hypothesis is correct, variance of a few vegetative characters within each group of trees classified according to low aswell as high disagreement count was investigated. It was then foundthat the within group variance of those vegetative characters were verysmall when the disagreement count among trees was so low as 0, 1, 2
or 3. In other words, trees which are comparatively similar in isozyme
patterns resemble each other also in vegetative characters, and they may
most probably be genetically related members of the same familY·
Sometimes, trees having higher disagreement count than 4 might besibs, but we have conservatively concluded that disagreement count 0

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

would indicate vegetative propagules while the disagreement counts 1,
2 and 3, seed-propagated sib-trees. We have been thus able to make agenetic study in a natural forest of Thujopsis dolabrata on the basis offamily analysis as outlined above. Details of this study will be publishedin a forestry journal.

Genetic Parameters of Organ and
Body Weights in Japanese Quail
Takatada KAWAHARA and Katsumi SAITOGenetic and phenotypic correlations among whole body weight andthose of various organs and their heritability were analyzed. The weights
recorded were those of total body (XI), bones (X2), heart (Xs), lungs (X~),
liver (Xs), gizzard (Xa), intestines (Xl), pancreas (Xa), spleen (Xg), kidneys(XlO), ovary or testes (Xu), oviduct (XI2) and eviscerated body (XIS) collected from 25 week old birds. Sinistral and dextral sides of lungs, kidneys and testes were pooled and weighed. Data were collected from 584
progenies (305 females and 279 males) originated from 68 pair matingseach of one sire with one dam. The estimates of heritability and thecoefficients of genetic and phenotypic correlations among various traits
are given in Tables 1 and 2. It is found from the two tables that theheritabilities of body weight and eviscerated body weight of females are
low in comparison with the males. Comparison between male and female
with regard to genetic and environmental variances of eviscerated
body weight showed that the environmental variance was approximately
the same between both sexes, whereas the genetic variance was different.
The same seemed to hold true for total body weight, though it was lessapparent than in eviscerated body weight. This fact may suggest adosage effect of sex-chromosomes on body weight, particularly on muscle
weight.

Tahle 1.

Heritnhility of and genetic and p.henotypic correl:1.tions among \":lrious tt"ails in m:lles 2S weeks after hatching.
--- ---X,
X,
X,
X,.,\'13
X.
Xo
X;
X IO
X l1
X"
..

X,

ii'
--

_~~_._._-_.

--~---------'-~--'------'._-- - - - - - - - - - - - - _ . _ . _ - .. ---_._-----_._-~._-------~-------_._-------------

X,

0.693

0.186**

0.440**
I

0.097

0.6H**

o. :~S{1**O.:11[)**

0.367**

O.I1R

0.459**

0.992**

0.027

X,

0.17·1

0.777

0.123

0.265**0.023

IJ.OJO

1J.013

1J.0270.001

1J.081

1J.1050.154'

X"

1J.6V0-0.025

1J.3090.553

0.231**

1J.272**0.1:;8*0.306-0.130*

0.084
0.024

0.061

0.332

0.210**-0.0:170.354**

0.832

0.192**-0.0160.190**

0.171**

0.412**
0.069

0.650

-0.2290.301-0.1840.404**

0.451**

0.411**

0.142*

0.425**-0.0950.529*""
0.294**

X.
X,-------

0.044--0.-3413-----------

X,

0.399

-0.3270.249-0.286

x-0.,,96-0.461

0.406

X,
X,
XIO,

O.fi2~

0.210

0.lR1

-0.628- 0.220-0.049

0.010

0.26R

0.270

~::

I

0.261
0.401

0.648
0.260

0.267**
0.174

0.261**

0.151*

0.306*'

0.373**'

0.11R

0.296**-0.175"-0.0800.3R5-- 0.390

0.126

0.690

0.282

0.123

0.327**

0.032

0.312**

0.OR7

0.040

0.103

0.515

0.065

0.022

0.100

0.086

0.416**

0.77R

0.194

0.6RO

-0.236

0.549

0.5R2

- 0.156

0.379

0.129

0.02R

0.177

0.453
.- 0.215

0.2R3-0.056

0.213

0.233

0.313

0.006

-0.031

0.998

0.161

0.674

-0.026

0.695

0.399

0.610

0.537

- 0.0370.781!

0.244**--~-"""'--'--'-I_(),73~_1 -0.081-0.105

I 0.693

Genetic correlation coefficients*. **:X,

X2

X,

O.:lOO
0.399
0.082
0.089
0.463
-0.165
0.388
0.583
0.088
0.205
0.606
0.260
0.900

0.:18:1**
0.748.----~-----~--~-----

X3

X.
- ----------

X.

X'I
X,
X8

i

X,I
X

IO I

X" I

X121

XI3

8

I .~"

'"

I, 'E...
0

u
u

:::0

l:'
a

;l>

'no
I~~

.c
.,.,'"

h'.

~

t"1

:::0

(")

:I:

~

O.liOI

0.371
0.084
0.022
0.536
0.193
-0.173
0.292
0.104
0.169
0.209

;l>

:::0
:::0
t"1

Heritability of nnd genetic and phenotypic correlations among v;uious traits in females 25 weeks after hatching.

h'

X,

"

(")

----------

X,

'"
I '"
'u
I IE
'-'

Exceed the So,; and 10 ;; significance h~velst respectively.

Table 2.

X,

I-

0.288*"
0.236*'
0.227**
0.125*
0.232
0.206**
0.651
0.184-0.0770.069-0.007-0.084
0.375
0.480
0.198
0.448
0.191
---0.083
0.487-0.09,9
0.124
0.381
0.362
0.272
-0.1800.024

X,-------

.----

X,

Xu

X,

XH

0.170"
O.OSS
0.02·1

0.3Rl*'O.163*"
O.I64*'

O.:n7'*--------~

0.47:;"
0.074
0.135*--._-------~--.-

XIO

Xu

X

-----~~-~-

O.IU'
0.H5*
_0.469 . ! 0.076-0.176
I 0.400~ 0.23:;0.458--0.079
0.438
0.347
0.207
0.463
0.506
0.333
0.428
0.335
0.069
0.318
0.234

0.145*

0.321**
0.048
0.175 __ I
0.164
-0.274
0.989
0.046
0.152
0.234

0.109
O.1!l2't-t
0.031
O.222~:*
0.070
0.193""
0.048
0.279**
0.140*
0.104
0.149**
0.157** -0.043
0.426 i O.OGO
0.023
0.501
0.IR3
0.08·1
0.338
0.G17
0.676
0.61:\
0.34G
0.138

-1-

tl

.

c::

-~-~-

0.261**
0.934*O.399**'
0.378'*
0.163**
0.147*
0.081
0.405**
0.201**
0.072
0.223**
0.196**
0.034
0.158**
0.178**
0.115*
0.280*"
0.395**
0.327**
0.303**
0.002
0.051
0.099
0.007
0.220**
0.165**
0.294*'
0.230**
0.186**
0.280**
0.229**
0.324*'
0.08:;
0.075
0.102
0.180**
0.294 I 0.223**
0.245**
0.232**
0.300
0.413**
0.264**
0.239**
0.480 -----o:71il-. 0.54-2
0.304~().129~I-0,'348--0.210-o.4SS-1

0

XIS

12

-I

l-j

'"

"'"

iE
~

a

u

Z

.....

$

:3"

]'a"
u
u

'5,
~
0

"
'"Po.
~

.....
.....

(Il

I
*. **:

Genetic correlation coefficients

Exceed the 5% and 1% significance levels, respectively.

h'116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Variance and Covariance Analysis ofSome Traits of Japanese Quail
Takatada KAWAHARA and Ryo KUSAKA

Genetic and phenotypic correlations among various traits and theirheritability were investigated in a random bred population of Japanesequail. Measurements on birds were taken at several developmental stages.
Traits examined at sexual maturity were body weight (X9) , egg weight(XlO ) and age (Xu). Hen-day egg production rate was recorded up to
120 days after the first egg lX12). Eight traits were measured in 23 week
old birds. They were egg weight (Xl), albumen weight (X2) , yolk weight(XS) , shell weight (Xf) , shell thickness (X5 ), egg length (X6 ), egg width(X7) and body weight (Xe). The birds were killed at the age of 25 weeks
in order to take weight of oviducts (XiS) and ovaries (Xlf). Data on eggs
were obtained from measuring three eggs per quail, they were collectedfrom 305 female descendants originated from 68 pair matings each ofone male with one female. The ovary data were collected from 289
birds, since the remaining 16 females had no egg material in their uteri.
The estimates of heritability and the coefficients of genetic and phenotypic correlation among various traits are given in Table 1. It is foundfrom Table 1 that the genetic correlations between age at sexual maturity
on one hand and ovary and oviduct weights on the other were -0.448
and -0.211, respectively. The genetic correlation between egg production rate and weight of those two sexual organs was close to -zero. This
may indicate that egg laying performance is genetically independent fromthe size of sexual organs. Sexual maturity and size of sexual organs
were, however, found to be genetically associated. In the present study,
it was found that the genetic correlation between egg number and egg
size in quails was as low as -0.053 or zero. This is of interest in viewof the widely accepted opinion that the same correlation in chickens israther high in negative direction. It should be noticed here that yolk
weight and egg production rate were negatively correlated either genetically or phenotypically.

\-

h'

Xl

Xl

0.452
0.982
0.666
0.712
0.040
0.705
0.985

X,

X,
X,
X,
X.
X,
X,
X.

XlO
..Yl1
X12

Xu
Xu

I

Table 1.

lIeritahiltty of anti gClletic illld phenotypic currelatioll':> among variuu':> trait:::; in quails.

..\"2

Xa

X4

0.907**

0.762**
0.524**
0.355

0.627**
0.530**
0.459**

~I
0.390
0.628
0.041
0.582
0.853

X~

XI;

X7

0.035
0.702**- 0.009
0.649**- 0.025
0.603**~I~I 0.444**
0.493**-0.149 '~I---0:484- 0.087
0.550
0.582 '~!-O.535 0.560
0.538
0.095 -~--

I

o.m

0..

O.~

o.m

O.~

o.~

0.373
0.635
-0.212-0.053
0.909
0.366

0.287
0.580
-0.186
0.030
0.816
0.184

0.386
0.444
-0.115-0.239
0.634
0.551

0.196
0.315
-0.231
0.006
0.545
0.425

0.037
0.109
-0.1060.286
0.071-O.I:,!I

0.235
0.401
-0.127- 0.074
0.695
0.28!)x,X.

Exceed the 5% and l!3o significance levels, respectively.

X"

X"

X"

X"

-0.054- 0.081--0.001-0.0480.021--0.170**0.439**
0.495**
0.209**

0.347**
0.247**
0.424**-0.0060.045-0.0210.010
0.147*-0.016

0.341**
0.027
0.326**--0.075-0.0810.334**

0.145*

0.372**

0.370**

0.267**
0.310'*

0.114*-0.0950.235**
0.265**

0.202**-0.0120.861**

0.478**

0.292**

0.371**

0.831**

0.405**

0.280**

0.397**

0.709**

0.388**

0.233**

0.232**

0.498**
0.037
0.427**
0.538

0.351**
0.050
O. 41 ~~**
0.360**

0.230**
0.026
0.288**
0.335**
Q.2J-ii--0.170**
0.172 ~~O-.~I 0.419**
0.226 -0.567"- -----0.-5].10.592
0.100
0.152
0.6l!!
O.fl6:1

0.200**-0.0330.237**
0.226**
0.370~
0.521**

O.15!!
0.218
0.412
0.·16:1

0.125
0.290
0.3:14O.:1l6(;cnclic corn~lation (~ucnicients*. **:

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118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

Early vs. Sustained Vigor Type in Growth and their Bearing
on Yielding Potential in Rice
H.I. OKA and Hiroko MORISHIMATo examine genetic variation in the growth curve parameters and theircontribution to factors determining grain yield, 40 randomly chosen F1lines of a cross between two contrasting rice varieties were measured
for dry matter weight and for other yielding characters during the growing period. From the dry weight data, growth curves were computed
using linear and quadratic <-Pearl-Reed) logistic equations, and various
values derived from them were compared among lines. The results
showed that: 1) The growth curves of the lines varied genetically between the "early-vigor" and "sustained-vigor" types. 2) Total panicle
length per unit area, repre3enting the potentiality for carbohydrates tobe produced in grains, seemed to depend on the growth rate at floral
initiation, and panicle weight per unit length, representing the rate ofcarbohydrate supply to the panicles, seemed to depend on the growthrate at heading. These two values were negatively correlated. 3) "Earlyvigor" types tended to produce more panicles but had a low rate of carbohydrate supply than "sustained vigor" types. Either "potentiality" or"supply" was a limiting factor in grain production, depending upon thepattern of growth being either "early vigor" or "sustained vigor" type.
This experiment was carried out in the International Rice Research Institute by Dr. T.T. Chang and Mr. Oscar Tagumpay, and the authors
were engaged in data processing. This paper will be published in"Theoretical and Applied Genetics" 40: 50-55, 1970.

RESEARCHES CARRIED OUT IN 1969

119

BOOKS AND PAPERS PUBLlSI..II:D IN 1969
BY STAFF MEMBERS( 1) CHU, Y. E., H. MORISHIMA and H. 1. OKA 1969. Reproductive barriers distributedin cultivated rice species and their wild relatives. Jap. J. Genet. 44: 207223.
( 2) CHU, Y. E., H. MORISHIMA and H. 1. OKA 1969. Partial self-incompatibility
found in Oryza perennis subsp. barthii. Jap. J. Genet. 44: 225-229.
( 3) CROW, J. F. and M. KIMURA 1969. Evolution in sexual and asexual populations:
A reply. Amer. Nat. 103: 89-91.
( 4) DOHI, Y., T. AMANO and T. !rNO 1969. Finding of a g-determinant in theflagella of Salmonella healsingborg. Biken Journal 12: 137-139.
( 5) ENDO, T. 1969. Hybrid enzymes (in Japanese). Heredity (Tokyo) 7: 15-23.
( 6) ENOMOTO, M. and S. YAMAGUCHI 1969. Different frequencies of cotransduction of mote and H1 in Salmonella. Genet. Res., Camb. 14: 45-52.
( 7) ENOMOTO, M. 1969. A method for isolating paralysed (mot-) mutants fromnon-flagellated cells of Salmonella. Genet. Res., Camb. 14: 89-92.
( 8) FUJII, T. 1969. Photoreactivation of mutations induced by ultraviolet radiationof maize pollen. Radiation Bot. 9: 115-123.
( 9) FUJII, T. 1969. Relative biological effectiveness of high LET radiations inhigher plants. Jap. J. Genet. 44(Suppl. 1): 431-442.
(10) FURUSATO, K. and Y. OHTA 1969. Induction and inhibition of nucellar embryo
development in Citrus. Seiken Zih6 21: 45-54.
( 11) HASHIGUCHI, S. and H. MORISHIMA 1969. Estimation of genetic contribution ofprincipal components to individual variates concerned. Biometrics 22: 9-15.
(12) HOTANI, H., S. ASAKURA and T. IINO 1969. Extraordinary polymerization of
Salmonella flagellin. Biochim. Biophys. Acta 194: 572-583.
(13) IINO, T. 1969. Polarity of flagellar growth in Salmonella. J. gen. Microbiol.
56: 227-239.
( 14) IINO, T. 1969. Genetics and chemistry of bacterial flagella. Bacteriol. Rev.
33: 454-475.
(15) IINO, T. 1969. Regulation of self-assembly in morphogenesis (in Japanese).
Kagaku 39: 489-495.
( 16) KADA, T. 1969. Radiosensitization by potassium iodate and related compounds.
Int. J. Radiat. BioI. 15: 271-274.
( 17) KADA, T. 1969. Selective growth of radiation sensitive mutants in Escherichia
coli K12. Jap. J. Genet. 44(Suppl. 2): 33.
(18) KADA, T. 1969. Repair events and radiation-induced mutagenesis. NIRS
Report: 9-13.
(19) KIKUCHI, Y., H. OISHI, A. TONOMURAand T. KURITA 1969. Incidence oftranslocation Down's syndrome (in Japanese). Saishin-Igaku 24: 309-312.

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20(20) KIKUCHI, Y. 1969. Human chromosomes and its abnormalities (in Japanese).
World Obst. and Gynecol. 21: 244-253.
(21) KIKUCHI, Y., H. OISHI, A. TONOMURA, K. KURITA and E. MATSUNAGA 1969.
Translocation Down's syndrome in Japan: Its frequency, mutation rate oftranslocation and parental age. Jap. J. Human Genet. 14: 93-105.
( 22 ) KIMURA, M. 1969. The number of heterozygous nucleotide sites maintainedin a finite population due to steady flux of mutations. Genetics 61: 893-903.
(23) KIMURA, M. 1969. The rate of molecular evolution considered from thestandpoint of population genetics. Proc. Nat. Acad. Sci., Wash. 63: 11811188.
(24) KIMURA, M. and J. F. Crow 1969. Natural selection and gene su':Jstitution.
Genet. Res. 13: 127-141.
(25) KIMURA, M. and T. MARUYAMA 1969. The substitutional load in a finite
population. Heredity 24: 101-114.
(26) KIMURA, M. and T. OHTA 1969. The average number of generations until
fixation of a mutant gene in a finite population. Genetics 61: 763-771.
(27) KIMURA, M. and T. OHTA 1969. The average number of generations until
extinction of an individual mutant gene in a finite population. Genetics 63:
701-709.
(28) KIMURA, M. 1969. Population Genetics by W. J. Ewens (book review). Amer.
Scientist 57(4): 356A.
(29) KOSUDA, K., O. KITAGAWA and D. MORIWAKI 1969. A seasonal survey of thegenetic structure in natural populations of Drosophila melanogastgr. Jap. J.
Genet. 44: 247-258.
(30) KURITA, Y., H. SHISA, M. MATSUYAMA, Y. NISHIZAWA, R. TSURUTA, and T. H.
YOSIDA 1969. Carcinogen-induced chromosome aberrations in hematopoietic
cells of mice. Gann 60: 91-95.
(31) KURODA, Y. 1969. Differentiation of the cell-free materials with aggregateforming activity from embryonic chick liver cells. Nucleic Acid Metab?lism,
Cell Differentiation and Cancer Grow~h (eds. E. V. Cowdrey and S. Seno)
pp. 277-285. Pergamon Press, Oxford and New York.
(32) KURODA, Y. 1969. Dissociation and reconstruction of tissues (in Japanese).
Methods of Cell Biophysics S!udy (ed. Soc. Biophys.) pp. 279-293. YoshikaShoten, Tokyo.
(33) KURODA, Y. 1969. Technique of animal cell culture. VII. (in Japanese).
Heredity (Tokyo). 23(1): 74-79.
(34) KURODA, Y. 1969. Technique of animal cell culture. VIII. (in Japanese).
Heredity (Tokyo). 23(2): 75-79.
(35) KURODA, Y. 1969. Technique of animal cell culture. IX. (in Japanese).
Heredity (Tokyo). 23(3): 74-78.
(36) KURODA, Y. 1969. Technique of animal cell culture. X. (in Japanese).
Heredity (Tokyo). 23(4): 74-79.
(37) KURODA, Y. 1969. Reconstruction of tissues (in Japanese). Seitai-No-Kagaku(Tokyo). 20(3): 106-122.

RESEARCHES CARRIED OUT IN 1969
(38)
(39)
(40)
(41)

(42)
(43)
(44)

(45)
(46)
(47)
(48)

(49)
(50)
(51)

( 52)
(53)
( 54)

(55)
(56)
(57)

121

KURGDA, Y. 1969. Tissue reconstitution and its control mechanisms (inJapanese). Igaku-No-Ayumi (Tokyo). 71(9): 454-461.
KURODA, Y. 1969. Differentiation of ommatidium·forming cells of Drosophilamelanogaster in culture (in Japanese). Jap. J. Develop. BioI. 23: 74-76.
KURODA, Y. 1969. Growth and differentiation of insect cells in culture (inJapanese). Jap. J. Develop. BioI. 23: 144-147.
KVRODA, Y. 1969. Hybridization at cellular level (in Japanese).
KagakuAsahi 29(13): 51-56.
KVRODA, Y. 1969. Growth and differentiation of embryonic cells of Drosophilamelanogaster in vitro. Jap. J. Genet. 44 (Suppl. 1): 42-50.
MARUYAMA, T. 1969. Genetic correlation in the stepping stone model with
non-symmetrical migration rates. J. Appl. Prob. 6: 463-477.
MATSUXAGA, E. and T. MARUYAMA 1969. Human sexual behaviour, delayed
fertilization and Down's syndrome. Nature 221: 642-644.
MATSUNAGA, E. 1969. Polymorphism of ear-wax and its biological implications(in Japanese). Kagaku 39: 482-488.
MATSUNAGA, E. 1969. Genetic epidemiology of chromosome aberrations (inJapanese). J. Japan Med. Ass. 62: 661-672.
MATSU:-IAGA, E. 1969. Some reflections on the biological consequences offamily planning. J. Med. Educ. 44: 87-90.
MATSUNAGA, E., E. MATSUDA and K. ASHIZAWA 1969. Sexual variation in
finger pattern size and pattern types. VIIlth Congress of Anthropologicaland Ethnological Sciences: 224-225.
MATSUNAGA, E. 1969. Some comments on the genetics of schizophrenia (inJapanese). Psychiat. Neurol. Jap. 71: 1294-1297.
MIKI, C., S. YEE, N. YASUDA and N. E. MORTON 1969. Alltype. A Genetic
Program Library (ed. N. E. Morton): 24-27. Univ. of Hawaii Press.
MIYAZAKI, Y. and K. 1. SAKAI 1969. Use of zymography for identification of
a clone in Cryptomeria japonica D. Don. J. Jap. Forestry Soc. 51(9): 235239.
MORISHlMA, H. 1969. Differentiation of pathogenic races of Piricularia oryzae
into two groups, Indica and Japonica. SABRAO Newsletter 1: 81-94.
MORISHIMA, H. 1969. Variations in breeding system and numerical estimationof phylogeny in Oryza perennis. Jap. J. Genet. 44 (Suppl. 1): 317-324.
MORISHIMA, H. 1969. Phenetic similarity and phylogenetic relationships among
strains of Oryza perennis, estimated by methods of numerical taxonomy.
Evolution 23: 429-443.
MORIWAKI, D. and S. ITO 1969. Studies on puffing in the salivary gland chromosomes of Drosophila ananassae. Jap. J. Genet. 44: 129-138.
MORIWAKI, K., K. TSUCHIYA and T. H. YOSIDA 1969. Genetic polymorphismin the serum transferrin of Rattus rattus. Genetics 63: 193-199.
MORIWAKI, K., H. T. IMAI and T. H. YOSIDA 1969. Effect of ploidy changes
on the rate of protein synthesis in the mouse plasma cell tumor, MSPC-l(in Japanese). Symposia Cell. Chern. 20: 195-204.

122 ANNUAL REPORT OF NATIONAL INSTINUTE OF GENETICS NO. 20(58) MORIWAKI, K., H. T. IMAI and T. H. YOSIDA 1969. Polyploidization and protein synthesis in mammalian tumor cells. Jap. J. Genet. 44 (Suppl. 1): 71-83.
( 59 ) MURAKAMI, A. 1969. Comparison of radiosensitivity among different silkworm
strains with respect to the killing effect on the embryos. Mutation Research8(2): 343-352.
(60) MURAKAMI, A., and T. ITO 1969. Co-mutagenesis: An interpretation of theeffect of post-irradiation treatment with base analogues in the silkworm.
Mutation Research 7(3): 479-481.
(61) MURAKAMI, A., and P. TEULADE 1969. Variation of radiosensitivity in synchronized cell populations of silkworm embryos exposed to X-rays. Int. J.
Radiation BioI. 15(4): 315-322.
( 62) MURAKAMI, A. 1969. Comparison of sensitivity to ultraviolet light among
different X-ray sensitive silkworm strains. Radiation Res. 39: 462-463.
(63) NAWA, S. 1969. Hybrids on the molecular level (in Japanese). Kagaku Asahi
29(13): 42-48.
(64) OGAWA, Y. 1969. Comparison of analytical results of serum protein fractions
on paper and cellulose acetate strips by Disc electrophoresis (in Japanese).
Physico-Chemical BioI. 14(1): 52-55.
(65) OGAWA, Y. 1969. Serometrics and Sartorius, new two cellulose acetate strips
for electrophoresis (in Japanese). Physico-Chemical BioI. 14(1): 55-56.
( 66) OGAWA, Y. 1969. On a new polymorphism of human serum found in Japanese
by using two-dimentional electrophoresis (Cellulose acetate-Disc) (in Japanese).
Physico-Chemical BioI. 14(3): 259-260.
(67) OGAWA, Y. 1969. Selecta, a new German cellulose acetate strip for electrophoresis (in Japanese). Physico-Chemical BioI. 14(3): 284.
(68) OHTA, T. and M. KIMURA 1969. Linkage disequilibrium due to random
genetic drift. Genet. Res. 13: 47-55.
(69) OHTA, T. and M. KIMURA. Linkage disequilibrium at steady state determinedby random genetic drift and recurrent mutation. Genetics 63: 229-238.
(70) OHTSUKI Y., and A. MURAKAMI 1969. Cytological observations on the nuclear
division in the early embryonic development of the silkworm (in Japanesewith English resume). ZooI. Magazine (Tokyo) 77(12): 383-387.
(71) OISHI, H., Y. KIKUCHI, E. MATSUDA, and T. KURITA 1969. A case of 'cri
du chat' syndrome. Jap. J. Human Genet. 14: 151-159.
( 72) OISHI, H. 1969. Human chromosomes (in Japanese). The Cell 1: 12-18.
( 73) OKA, H. I. 1969. A note on the design of germplasm preservation work ingrain crops. SABRAO Newsletter 1: 127-134.
( 74) OSHIMA, C. 1969. Persistence of some recessive lethal genes in natural populations of Drosophila melanogaster. Jap. J. Genet. 44(Suppl. 1): 209-216.
(75) OSHIMA, C. and M. KAWANISHI 1969. Adaptation of Drosophila melanogaster
under the constant and fluctuating temperatures (in Japanese). Environ.
Control in BioI. 71: 21-29.
( 76) SAKAI, K. I. 1969. The future of the breeding science. SABRAO Newsletter
1(2): 61-68.

RESEARCHES CARRIED OliT IN 1969123

( 77 ) SAKAI, K. I. 1969. Breeding science in future and forest tree breeding (inJapanese). Forest-tree Breeding 55(5): 1-2.
( 78) SAKAI, K. I. 1969. Problems in the breeding plan by elite-tree selection of
Cryptomeria (in Japanese). Ringyo-Gijutsu 324: 8-13.
( 79 ) SAKAMOTO, S. 1969. Interspecific hybrid between the two species of thegenus Taeniatherum of the tribe Triticeae. Wheat Information Service 28:
26-27.
(80) SHAH I. B. B., H. MORISHIMA and H. I. OKA 1969. A survey of variations inperoxidase, acid phosphatase and esterase isozymes of wild and cultivatedOryza species. Jap. J. Genet. 44: 303-319.
(81) SHAHI, B. B., Y. E. CHU and H. I. OKA 1969. Analysis of genes controlling
peroxidase isozymes in Oryza sativa and O. perennis. Jap. J. Genet. 44:
321-328.
(82) SHINODA, T.1969. Molecular aspect of immunoglobulin formation (in Japanese).
Internal Medicine 23: 624-632.
(83) SHINODA, T. 1969. Molecular structure of immunoglobulin (in Japanese).
Heredity (Tokyo) 23(5): 26-33.
(84) SHINODA, T. 1969. A note on the frequency of red cell acid phosphatasetypes in Japan. Jap. J. Human Genet. 13: 249-255.
(85) SUGIURA, K., M. GOTO and S. NAwA 1969. Die Synthese der Drosopterine.
Tetrahedron Letters No. 34: 2963-2965.
(86) TAZIMA, Y. 1969. The records of the XIIth International Congress of
Genetics. Proc. XIIth Internat. Congo Genet. 3: 25-46.
(87) TAZIMA, Y. and K. ONIMARU 1969. Frequency pattern of mosaic and wholebody mutants induced by ionizing radiations in post-meiotic cells of themale silkworm. Mutation Research 8: 177-190.
(88) TAZIMA, Y. 1969. Analysis of radiation sensitivity of silkworm spermatogonia and its implications in the study of mutation. Comparative Cellularand Species Radiosensitivity (ed. by V. P. Bond and T. Sugahara): pp.
164-172. Igaku Shoin Ltd., Tokyo.
(89) TAZIMA, Y. 1969. Repair in mutation process studied in low and high radiosensitivity strains of the silkworm. lap. J. Genet. 44 (Supp!. 1): 123-130.
(90) TAZIMA, Y. 1969. Comparison of mutagenic effects of 14 MeV neutrons, Trays and some chemical mutagens upon silkworm spermatogenic cells. Jap.
l. Genet. 44(Supp!. 2): 71-72.
(91) TITANI, K., T. SHINODA and F. W. PUTNAM 1969. The amino acid sequenceof a /C-type Bence-Jones protein. J. Bio!. Chern. 244: 3550-3560.
(92) TSUJITA, M. and S. SAKURAI 1969. Lethal effects of a genetic abnormality
in pteridine metabolism of silkworm larvae. IVth International Congress
on Pteridines at Toba, Hand book: 30-31.
(93) TSUJITA, M. and S. SAKURAI 1969. Incorporation of 14C-phenylalanine or
14C-tyrosine into the hypodermal cuticle of the silkworm. 1. Lethal lemon
larvae. Proc. Japan. Acad. 45(10): 943-949.
(94) TSUJITA, M. and S. SAKURAI 1969. Incorporation of 14C-phenYlalanine or 14C-

124 ANNUAL REPORT OF NATIONAL INSTINUTE OF GENETICS NO. 20

(95)
(96)

(97)

(98)
(99)

(100)

(101)
(102)
(103)

(104)

(105)

(106)

tyrosine into the hypodermal cuticle of the silkworm. 2. Lethal albino
larvae. Proc. Japan. Acad. 45(10): 949-954.
WATA~ABE, T. K. 1969. Persistence of a visible mutant in natural populations of Drosophila melanogaster. Jap. J. Genet. 44: 15-22.
WATANABE, T. K. 1969. Frequency of deleterious chromosomes and allelism
between lethal genes in Japanese natural populations of Drosophila melanogaster. Jap. J. Genet 44: 171-187.
WIKLER, M., T. SHINODA, H. KOHLER and F. W. PUTNAM 1969. Macroglobulin
structure: Homology of mu and gamma heavy chains of human immunoglobulin.
Science 163: 75-78.
YAMAGUCHI, Sand T. IINO 1969. Genetic determination of the antigenic
specificity of flagellar protein in Salmonella. J. gen. Microbiol. 55: 59-74.
YAMASHITA, K., S. SAKAMOTO and K. FUKUI 1969. A preliminary report ofthe Botanical Team of the Kyoto University Scientific Expedition to the
Sahara and the Surrounding Areas, December 1967-March 1968. Wheat Information Service 28: 27-31.
YASUDA, N. 1969. Estimation of the inbreeding coefficient from mating type
frequency and gene frequency. Computer Application in Genetics (ed. N. E.
Morton): 87-96. Univ. of Hawaii Press.
YASUDA, N. 1969. The estimation of the variance effective number basedon gene frequency. lap. J. Human Genet. 14: 10-16.
YASUDA, N. 1969. The inbreeding coefficient in northeastern Brazil. Human
Heredity 19: 444-456.
YONEDA, Y. and T. ENDO 1969. Effect of low concentration of hydrogen
peroxide on indoleacetate oxidase zymogram in Pharbitis nil. Plant andCell Physio\. 10: 235-237.
YOSIDA, T. H., K. TSUCHIYA, H. T. IMAI and K. MORIWAKI 1969. New chromosome types of the black rat, Rattus rattus, collected in Oceania and F 1
hybrids between Japanese and Australian rats. Jap. J. Genet. 44: 89-91.
YOSIDA, T. H., K. TSUCHIYA, H. T. IMAI, K. MORIWAKI and T. UDAGAWA
1969. Chromosome numbers of rodent species in Southeast Asia and Oceania.
Mamm. Chromo Newslet. 10: 217-219.
YOSIDA, T. H., K. MORIWAKI and H. T. IMAI 1969. Chromosome alterationand gene function in tumor cells (in Japanese). Nihon-Rinsho 27 (Suppl.):142-151.

RESEARCHES CARRIED OUT IN 1969

125

ABSTRACTS OF DIARY FOR 1969•

January
30 166th Meeting of Misima Geneticists' Club
February 28 167th Meeting of Misima Geneticists' ClubMay
6 168th Meeting of Misima Geneticists' Club26 169th Meeting of Misima Geneticists' Club27 170th Meeting of Misima Geneticists' Club
June
July
14 86th Biological Symposium
26 87th Biological Symposium
September 19 171st Meeting of Misima Geneticists' Club
October
30 172nd Meeting of Misima Geneticists' Club
November 5 173rd Meeting of Misima Geneticists' Club
6 88th Biological Symposium
15 Public Lectures on Genetics (at the National Science
Museum, Ministry of Education, Tokyo)
21 174th Meeting of Misima Geneticists' Club
December 19 175th Meeting of Misima Geneticists' Club126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

FOREIGN VISITORS IN 1969
April
June

7-9 LONG, T. Swansea University, Great Britain
19 MESHCHEROV, M.
20 STUKALIN, B. Scientific journalist, USSR
KOLSTOVOI, B.
111111
BESLOV, S.
11
11
11
BRITANS, Y.
11
11
8
GOLOVACHEV, V.
8811
KULIKOV, F.
11
11
PEKELIS, V.
1188
TOLSTIK, A.
11118
KHANG, K.
11
11..
TATUM, E.L. The Rockefeller University, U.S.A.
MATTE, R. University of Montreal, Canada
VALENZUELA, R. G. WHO, Geneve, Switzerland
SHALIN, Y. P.
CHUMARK, A. G.
MARKARJYANTS, S. B.
DASS, M. Member of the Parliament, Vice-chairman, Central
Silk Board, India
SONI, K.L. Director of Sericulture, Jammu & Kashmir,
India
SHARMA, R. Director of Sericulture, Assam, India
MAHADEVAPPA, D. Assistant Director of Sericulture, Mysore,
India
TOMACH, 1. J. Washington University, U.S.A.
SZABOLCSI, G. Institute of Biochemistry, Hungarian Academyof Sciences, Hungary
SESHADRI, V. S. Indian Agricultural Research Institute,
India
8

July
AugustOctober

14
26
7

29

30

November 6
13
15

RESEARCHES CARRIED OUT IN 1969127

ACKNOWLEDGEMENT
The editor wishes to express his sincere gratitude to Dr. F. A. LILIENFELD
for her kindness in reading the original manuscripts. (S. IY AMA)

r

•

•

4.

128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 20

AUTHOR INDE:X
Amano, E
82, 83, 85
Asakura, S
94
Endo, T
.45
Enomoto, M. ..
95
Fujii, T'"
86, 88, 91
Fukase, Y
68
Furusato, K. .
35
Hayashi, S. .
111
Hotani, H. .
94lino, T93, 94, 94
Imai, H. T
22
Ishidsu, J
65, 97
Kada, T. .
75, 75, 77, 78, 79, 80, 97
Kato, H13, 17, 18, 19, 20
Kawahara, T
114, 116
Kihara, H. .
.47
Kikuchi, Y
106, 107, 109
Kimura, M
51, 52, 53, 54
Kurita, T
106
Kuroda, Y
25, 28, 29, 73
Kusaka, R
116
Maruyama, T
55, 56, 57
Masuji, H. .
10
Matsuda, E. .
100
Matsunaga, E
100
Matsushima, T
21
Matsuura, T
112
Minato, K
31
Miyazaki, Y
112, 113
Morishima, H.49, 49, 118
Moriwaki, K. .
22, 23, 43, 44
Murakami, A. .
70, 71, 71, 72
Nawa, S. .
37, 37, 38
Noguti,T
77,78, 80

Odaki, N
105
Ogawa, Y
105
Ohta, T. ..
53, 54
Ohta, Y26, 32, 34, 35, 47
Oishi, H
106, 107, 108, 109
Oka, H.1.
49, 49, 118
Onimaru, Koo67, 69Onuma, A
66
Oshima, C
61,61
Rajan, S. S
89
Sadaie, Y
75, 80
Saito, K
114
Sakai, K.1.
101, 112, 113
Sakakibara, K. .
13
Sakata, H. .
44
Sakurai, S
39, 40, 40, 41, 42
Schade, H
100
Sekiguchi, M
82
Sekiya, K
17, 18
Shibuya, T
2!'Shinoda, T
101, 104, 104
Sonoda, J
15
Tazima, Yoo66, 67, 68. 69
Tonomura, A
106, 108
Tsuchiya, Koo. 9, 10, 11, 14, 15, 16, 44
Tsujita, Moo
37, 39, 40, 40. 41
Watanabe, T
64
Watanabe, T. K
61, 61, 64, 64
Yamada, M. A
37, 38, 39
Yamashita, J
23, 24. 43
Yasuda, N
58
Yosida, T. H. .
9, 10, 11, 13, 14,
15, 16, 17, 18, 19, 2L

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