NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 21

1970

PublU1u!d 611

THE NATIONAL INSTITUTE OP GENBnCS
JIiIJbnc. SimoIuJ-keA. Ja:ptIII
1971

Annual Reportof theNational Institute of Genetics
No. 21,1970

Published by

The National Institute of Genetics, Japan
197 1

CONTENTS
General statement
Staff.............
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Council...................
Association for propagation of the knowledge of genetics. . . . . . . . . .
Projects of research for 1970
" ........••............ " . . ..
Researches carried out in 1970
" .. .. . . . . . . .. . . . . .. . . . . . . . . ..

I.

Molecular Genetics
Terminal structure of double-stranded RNA of cytoplasmic
polyhedrosis virus from silkworm (CPV). MIURA, K. and
Furuichi, Y.
RNA polymerase associated with purified virion containing
double-stranded RNA. SHIMOTONO, K. and MIURA, K.. . . .
Double-stranded RNA isolated from the CP virus of pine caterpillar. MIURA, K. and SHIBUYA, A.....................
Structure and function of transfer RNA. FURillCHI, Y. and
MIURA, K.

II. Microbial Genetics
Further mapping of fla and mot cistrons closely linked to HI in
Salmonella abortus-equi and its derivatives. YAMAGUCHI, S.
and IINO, T.
Activity of glutamine dependent carbamyl phosphate synthetasein arginine sensitive mutants of Salmonella typhimurium.
TOSHIMA, H. and ISHIDSU, J.
Activities of enzymes concerning uracil biosynthesis in 6-azauracil
resistant mutants of Salmonella typhimurium. ISHIDSU, J.

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Physiological and enzymological analysis of azide resistantmutants of Salmonella typhimurium. ISHIDSU, J.
Reconstitution of salmonella flagella attached to cell bodies.
IINo, T., SUZUKI, H. and YAMAGUCHI, S.
Possible differential control of flagellin mRNA synthesis in Bacillus
pumilus depending on cell age. SUZUKI, H
.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Phage-induced alteration of colony type in Xanthomonas citri.
Wu, W. C. and IINO, T .....
Genetic analyses of nonmotile double mutants in Salmonella
typhimurium. ENOMOTO, M
A new transducing phage related to P22 of Salmonella typhimurium.
ENOMOTO, Mo and ISHIWA, H .....
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Ill. Biochemical Genetics
Purification and properties of tryptophan pyrrolase in Ephestia.
NAWA, S
Characterization of DNA from eggs of Ephestia. YAMADA, M. A.
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Hereditary change in silworm with DNA. NAWA, So, TSUJITA,
M. and YAMADA, M. A.
Three allelic isozyme variants in rice peroxidase. PAl, C. and
ENDO, T
Lethal effects of a genetic abnromality in the pteridine metabolism
of silkworm larvae. TSUJITA, M. and SAKURAI, S.....
Genetic and biochemical studies of lethal albino larvae of thesilkworm, Bombyx mori. TSUJITA, M. and SAKURAI, S.o ...
The phenotype of double recessive lethal (lem1/lem 1; al/al) larvaeof the silkworm. TSUJITA, M.
Genetic variation observed in membrane proteins isolated frompteridine granules of several oily mutant strains of the silkworm. SAKURAI, S. and TSUJITA, Moo ...
Immunochemical studies of structural membrane proteins ofpteridine granules in hypodermal cells of silkworm larvae.
SAKURAI, S...
Differences in amino acid composition of serum transferrin among
various species of Rattus. MORIWAKI, K., SATO, T. and
TSUCHIYA, K.
Serum transferrin polymorphism in oceanian black rat, Rattus
rattus rattus. MORIWAKI, K. and SATO, Too .....
Serum transferrin patterns of Rattus rattus obtained from Southern
India and Italy. MORIWAKI, K., SATO, T. and TSUCHIYA, K..
Studies on the transferrin of the Mongolian gerbil. MAYEDA, K.
and MORIWAKI, K. . ...
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CONTENTS

Comparative studies of the transferrins of rodents.
K. and MAYEDA, K.

iii

MORIWAKI,

IV. Developmental Genetics
Specific inhibition by glucosamine of aggregate formation of rat
hepatoma cells in rotation culture. KURODA, Y.........
Growth-stimulating effect of peptone on Drosophila ovarian cells in.. .. .
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culture. KURODA, Y.......
Evidence for the restitution of sorting-out activity of HeLa cells
following fractionated X-irradiation. KURODA, Y.......
Further study of relationship between molting and cell cycle ininsect epidermis. MINATO, K... . . . . . . . . . . . . . . .. . . . . . . . .
Effects of culture media on the survival of embryonic quail liverspecific cells. MINATO, K.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Y.

Cytogenetics
Karyotype evolution in Rattus species from black rat, R. rattus.
1. Species with a basic chromosome number, but with structural chromosome difference. YOSIDA, T. H. and TSUCHlYA,K. ..
Karyotype evolution in Rattus species. 2. Species with fewer
chromosome numbers than 2n=42 by Robertsonian chromosome fusion. YOSIDA, T. H. and TSUCHlYA, K... " ....
Karyotype of Indian black rats, Rattus rattus rufesens. YOSIDA,
T. H. and TSUCHlYA, K. .. " ... . .. .. .. .. .. . .. . . . .. .. . . ..
Studies of aneusomic clones isolated from a Chinese hamster cellline following induction of nondisjunction. KATO, H.....
Distribution of two chromosomal types of Japanese wood mouse,
Apodemus speciosus. TSUCHlYA, K. and YOSIDA, T. H... ....
Differences in biochemical characteristics between two sibling
forms of Japanese wood mouse, Apodemus speciosus. TsuCHlYA, K. and MORIWAKI, Koo.........................
Spontaneous sarcomas in black rats, Rattus rattus, bred in laboratory, and chromosome observations. YOSIDA, T. H. and
KATO, H.
Preliminary note on karyotype of guppy and topminnow. YOSIDA, T. H. and HAYASHI, Moo . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Chromosome survey of small mammals in Japan. TSUCHIYA, K.
and YOSIDA, T. H.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Frequency of chromosome polymorphism in pair Nos. 1,3,9 and
13 in black rats, collected in Misima. YOSIDA, T. H... ..
In Vivo proliferation of two sublines of MSPC-I mouse myeloma
in mixed inoculation. MORIWAKI, K. and SATO, T..... "An estimation of the number of definitive spermatogonial cell
divisions in the silkworm. MURAKAMI, A...............

VI.

VII.

Radiation genetics in animals
Effect of the moulting hormone on mutation induction anddevelopment of spermatogenic cells in the silkworm. TAZIMA, Y. and FUKASE, Y.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Studies on strain differences in radiosensitivity in the silkworm.
XI Sensitivity to dominant lethal induction. TAZIMA, Y. and
FUKASE, Y.
Studies on strain differences in radiosensitivity in the silkworm.
XII Different modes of dominance observed in F 1 withregard to radiosensitivity at various levels of biologicalreaction. TAZIMA, Y.
Studies on strain differences in radiosensitivity in the silkworm.
XIII Radiation-induced breakage and rejoining of single
strand DNA. TAZIMA, Y, ONIMARU, K. and KATO, H.....
Relative biological effectiveness of 14 MeV neutrons for recessive
visible mutations in meiotic spermatocytes of the silkworm.
MURAKAMI, A.
Changes in ultraviolet light sensitivity to embryonic killing during
early developing egg of the silkworm. MURAKAMI, A.. . . .
Sex differences in X-ray sensitivity to embryonic killing of dormant
silkworm egg. MURAKAMI, A.. . . . . . . . . . . . . . . . . . . . . . . . . .
Differential nuclease activities in radiation-sensitive and radiation resistant silkworms. SAKURAI, S., KADA, T. and ANDO,
T....................................................

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Radiation genetics and chemical mutagenesis in microorganisms andplantsBiochemical factors involved in the rapair of r-ray-induced

CONTENTS

damage in transforming DNA of Bacillus subtilis. NOGUTI,
T. and KAnA, T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Spores of rec- strains of Bacillus subtilis are sensitive to UV or,-ray. SADAIE, Y. and KADA, T.. . . . . . . . . . . . . . . . . . . . . . . .
Mutagenic action of phloxine. KADA, T., SADAIE, Y. and TUTIKAWA, K.
Host-mediated "rec-test" for chemical mutagens. TUTIKAWA, K.
and KADA, T.
Efficiencies of some mutagens in maize. AMANO, E.. . . . . . . . ..
Mutation experiment in Capsicum annuum. AMANO, E...
Effect of temperature during the split dose interval of gammarays in maize pollen. FUJII, T.........................
Tissue culture from wheat anthers and roots. FUJII, T.......

VIII.

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Population genetics (theoretical)

The length of time required for a selectively neutral mutant toreach fixation through random frequency drift in a finite
population. KIMURA, M.
Probability of fixation of a mutant gene in a finite population
when selective advantage decreases with time. KIMURA,
M. and OHTA, T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetic loads at a polymorphic locus which is maintained byfrequency-dependent selection. KIMURA, M. and OHTA, T.. .
Statistical analysis of the base composition of genes using data on
the amino acid composition of proteins. OHTA, T. and
KIMURA, M.
Development of associative overdominance through linkage disequilibrium in finite populations. OHTA, T. and KIMURA,M.
The rate of decay of genetic variability in a two-dimensionallystructured finite population. MARUYAMA, T...........

IX.

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Population genetics (experimental)
Deleterious genes in natural summer and autumn populations ofDrosophila melanogaster. OSHIMA, c., WATANABE, T. K.
and CHOO, J. K.
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Sterility genes in natural summer and autumn populations of

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

D. melanogaster. OSHIMA, C., WATANABE, T. K. and KAWANISHI, M..............................................
Correlation between productivity and viability of D. melanogaster. WATANA13E, T. K.
Fecundity and longevity of D. melanogaster under constant andfluctuating temperature environments. OSHIMA, C. and
KAWANISHI, M
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Effect of light on oviposition of D. virilis. OSHIMA, C. and
INOUE, K.
Relationship between phototaxis and oviposition of D. virilis.
OSHIMA, C. and INOUE, K.
X.

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Evolutionary genetics
An intergeneric hybrid between Agropyron tsukushiense andElymus mollis. SAKAMOTO, S...........................
Collection of wheats and various other species of the tribe Triticeae
in Iraq, Turkey and Iran. YAMASHITA, K., TANAKA, M.,
SAKAMOTO, S. and ISHII, H.............................
Dynamics of plant domestication: Cultivation experiments with
Oryza perennis and its hybrid with O. sativa. OKA, H. I.
and MORISHIMA, H. '" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Partial self-incompatibility in Oryza perennis. MORISHIMA, H.
and OKA, H. I.
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Human genetics
Inheritance of total finger ridge count. MATSUDA, E. and
MATSUNAGA, E.
Polymorphic red cell enzymes among Japanese. SHINODA, T.. . ..
Structure of human macroglobulin. SHINODA, Too...........
Birth weight in Down's syndrome: Comparison of interchange
trisomies and G trisomies. MATSUNAGA, E... . . . . .. . . .. ..
Parental ages in translocation Down's syndrome. MATSUNAGA,

E.
Distribution of fast renaturining DNA in human chromosomes.
NAKAGOME, Y.
A Cp+mat chromosome found in a malformed boy. OISHI, H.,
MATSUDA, E. and KIKUCHI, Y.

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CONTENTS

vii

Chromosome studies in congenitally abnormal children. NAKAGOME, Y. and MATSUMOTO, H...........................
Clinical conditions of patients with apparently normal chromo0••••.• " •••• " • ••• • . •• •• •• •somes, VII. OISHI, H

XII.

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Ap//ied genetics
Geographic variation on the isoenzyme level of Abies sachalinensis
in Hokkaido. SAKAI, K. I. and MATSUURA, T .. 000• • • • • . . •Genetic differentiation within a natural forest of Cryptomeria
japonica D. Don. SAKAI, K. I. and PARK, Y. Goo 0••• 0.. ••Genetic studies of peroxidase isozymes of the Japanese red pine,
Pinus densiflora Sieb. and Zucco PARK, Y. G. and SAKAI,
K. I. .. 000. 00•.••• 0•• 000. 0•• 000••• 0••.••.• 000• • •An analysis of sexual dimorphism in body size in the Japanesequail. KAWAHARA, Too 00.00 ••• 00000•••• 00 •• 0•• 0•... 0"Monte Carlo studies on the relationship between the system ofreproduction and inbreeding. IYAMA, S 0•. 000•• 0• • • • •Selection in rice: An experiment. IYAMA, Soo
0. 0••• 00•
Growth pattern and its plasticity in wild and cultivated types ofrice. MORISHIMA, H. and OKA, H. 1.. ... 0••. 0... 0. 0•• 0. •Inter-genotypic interaction in rice. HOSHINO, T., MORISHIMA, H.
and OKA, H. I. .... 0. 00..• 000 •• 00.0 •• 00. 000000.000.0..
Evaluation of an electronic instrument for estimation of plantweight. MORISHIMA, H., OKA, H. I. and HOSHINO, T.. 0.. ..
o••••Books and papers published in 1970 by research members .... 0.•. .•Abstracts of diary for 1970 ... 0...........................
Foreign visitors in 197000 •.•• 0.0 .•.• 0•• 00••• 00•••• 00• 0o'Acknowledgement. 000000• 0• 0•• 00 •.•••.• 0• 0• 00• 0•• 00• 00. 0. 0• 00•
Author index
00• 00000.••••...••• 0.••.•. 0•• 00• 00

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GENERAL STATEMENT
A new concrete building, 811 m 2 in floor dimensions, designed for a new
library is now under construction and will be completed in the spring of1971. The library in our institute has been expanding year by year withnew periodicals, monographs and reprints and was in need of a new installation equipped with versatile functions. The first plan to erect a threestoried building had soon to be reduced to two-stories according to the
reduced budget. Fortunately, at this juncture, Dr. H. Kihara stepped in,and the mutilation of our plan was prevented. His Institute for BiologicalResearch offered us a large sum of assistance, allowing us to execute theoriginal plan. We are deeply grateful for the generous help.
In July we have organized for the 3rd time a three day seminar on"Population Genetics and Evolution" as an advanced course in special
fields of genetics. Luckily at that time Dr. J. F. Crow, Professor of Wisconsin University, has been on a visit of three months here as a Visiting
Member. He was one of the speakers at the seminar and gave us an excellent lecture. As another Visiting Researcher, Dr. Kazutoshi Mayeda,
Associate Professor of Wayne State University of Michigan, is staying here
since this summer. He takes part in a US-Japan Cooperative Science
Programme of one year at the Department of Cytogenetics.
Dr. M. Kimura, Head of the Department of Population Genetics, wasgiven an award from the Japanese Society of Human Genetics in November
1970, and presented at the general assembly a prize lecture "Population
genetics and Human genetics."
Three members of our institute, Drs. Tazima, Kada, and Fujii, went to
Evian, France, to attend the 4th International Congress of Radiation
Research. After the Congress, Dr. Tazima, Head of the Department ofMorphological Genetics visited USSR for 10 days. He was invited by
the USSR Academy of Sciences, and was surveying the recent advances ingenetics in that country. After a short time he went again to Europe andattended in Geneva as a Member of National Delegations from 18th to 27th
September the 20th Session of the United Nations Scientific Committee
on the Effects of Atomic Radiation (UNSCEAR). During the summer
session Dr. T. Iino, Head of the Department of Microbial Genetics, gave
lectures at Purdue University, U.S.A., and then attended the 10th International Congress of Microbiology held in Mexico City in August. Dr.
H. 1. aka, Head of the 3rd Laboratory of the Department of Applied Genetics is on leave of two years since November 22, 1970, staying at Central
Luzon State University, Philippines, as a UNESCO Specialist in Crop
Improvement and Seed Production.
Speaking of personnel changes, Mr. T. Oyauchi, who had been Headof the Department of Administration was transferred to Tokyo Educational
University and replaced by Mr. M. Kudo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

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., TSUCHIYA, Kimiyuki
The 2nd Laboratory
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
lMAI, Hirotami, D. Sc.
3.

Department of Physiological Genetics
OSHIMA, Chozo, D. Sc., Head of the Department
The 1st Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
WATANABE, Takao K., D. Sc.
OISHI,* Kugao, Ph. D., CHOO,* Jong-Kil, M. S.
The 2nd Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
SAKAMOTO, Sadao, D. Ag.

STAFF

4.

Department of Biochemical Genetics
TSUJITA, Mitsuo, D. Ago, 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. Ago; FUJISHIMA, Tohru, D. Ag.
The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
BAGCHI,* Sujit; IWAGAMI,* Seiro; PARK,* Y. G.
The 3rd Laboratory
OKA, Hiko-Ichi, D. Ago, Head of the Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag.
HOSHINO,* Tsuguhiro; PAl,* Chiang

6.

Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department
The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
NOGUTI, Takehiko, D. Sc.
The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
FUJII, Taro, D. Ag.
The 3rd Laboratory
KADA, Tsuneo, D. SCo, Head of the Laboratory
AMANo, Etsuo, D. Ag.; SADAIE, Yoshito* Research members under grant from other organization or visiting researchers.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

7. Department of Human Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department
The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
SHINODA, Tomotaka, D. Sc.; MATSUDA, Ei.
The 2nd Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
NAKAGOME, Yasuo, D. Med.; 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
FURUICHI, Yasuhiro, D. Med.

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

12. Department of Administration
KUDO, Masaaki, Head of the Department
MURAMATSU, Masanori, Chief of the General Affairs Section
KATO, Shigeo, Chief of the Finance Section

COUNCIL

5

Honorary 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 of Kyoto University
LILIENFELD, Flora A., Ph. D.
OGUMA, Kan, D. Ag., Emeritus Professor of Hokkaido University
TANAKA, Yoshimaro, D. Ag., D. Sc., Member of Japan Academy, Emeritus
Professor of Kyushu University
Visiting Member

CROW, James E, Ph. D., Member of the National Academy of Science
U. S., Professor of the University of Wisconsin. (June IS ....... Sept. 1)

COUNCILKIKKAWA, Hideo, Chairman, Professor of Osaka University
FUJII, Takashi, Vice Chairman, Professor of Shizuoka University
BABA, Isamu, Director of National Institute of Agricultural Sciences
IZEKI, Shoei, Professor of Gunma University
KAYA, Seiji, Emeritus Professor of University of Tokyo
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
MAKINO, Sajiro, Professor of Hokkaido University
MISONOO, 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
TAKAHASHI, Ryuhei, Professor of Okayama University
TAKEYAMA, Yutaro, Governor of Shizuoka Prefecture
TANAKA, Nobunori, Professor of University of TokyoYANAGIDA, Tomomichi, Director of Institute of Applied Microbiology,
University of Tokyo

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Director of the National Institute of Genetics
TAZIMA, 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

PROJECTS OF RESEARCH FOR 1970
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 culture (KURODA)
Effects of radiation on cells in tissue culture (KURODA)
Department of Cytogenetics

Studies on chromosomal polymorphism of rodents (YOSIDA, 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)PROJECTS OF RESEARCH FOR 1970

7

Cytogenetical and biochemical studies on mouse plasma cell tumor (MoRIWAKI, YAMASHITA and SATO)
Biochemical studies on serum transferrin variations in rodents (MORIWAKI,
SATO, TSUCHIYA and MAYEDA)
Experimental breeding and genetics of mice, rats and other wild rodents(yOSIDA, MORIWAKI, TSUCHIYA, SAKAKIBARA and SONODA)
Cytogenetical study of ants (IMAI)
Department of Physiological Genetics

Population genetics of deterious genes in natural populations of Drosophilamelanogaster (OSHIMA, WATANABE and CHOO)
Mechanisms of peristence of some lethel genes (OSHIMA and WATANABE)
Physiological and population genetics of sterility genes in natural populations of Drosophila melanogaster (OSHIMA and WATANABE)
Analysis of fitness in a fluctuating environment (OSHIMA, WATANABE,
INOUE and CHOO)
Analysis of inversion chromosome in natural populations of Drosophilamelanogaster (OSHIMA and WATANABE)
Cytoplasmic inheritance in Drosophila (OISHI)
Nucleus substitution in wheat and related species (KIHARA, SAKAMOTO
OHTSUKA and YOSHINO)
Cytogenetic studies in the tribe Triticeae (SAKAMOTO)
Studies on ecotypic differentiation in Japanese Agropyron (SAKAMOTO)
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)

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

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 of isozymes in plants (ENDO and PAl)
Effects of exogenous DNA on plant seed formation (ENDO)
Department of Applied 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 and IYAMA)
Genetic studies in natural stands of forest tree species (SAKAI, IWAGAMI
and PARK)
Zymographic studies in forest trees (SAKAI, IWAGAMI and PARK)
Simulation studies on artificial selection (IYAMA)
Evolutionary studies in wild and cultivated rice species (aKA and MORISHIMA)
Analysis of genetic variations in plant type and growth pattern in rice
varieties (MORISHlMA and aKA)
Analysis of sterility genes in Oryza sativa (aKA)
Intergenotypic competition and synergism in rice (HOSHINO and MORISHIMA)
Genic analysis of isozymes in rice plants (PAl and ENDo)
Department of Induced Mutation

Radiation genetics in mice (TUTIKAWA)
RBE and dose rate effects in higher plants (FUJII and AMANO)
Radiation genetics in Arabidopsis (FUJII)
Fine sturcture 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)PROJECTS OF RESEARCH FOR 1970

9

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 NAKAGOME)
DNA replication in human chromosomes (KIKUCHI and OISHI)
Molecular hybridization studies of human chromosomes (NAKAGOME)
Polymorphic red-cell enzymes among Japanese (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 (ENOMOTO)
Transduction mechanism of phage P22 (ENOMOTO and ISHIWA)
Genetics of phytopathogenic bacteria (Wu and IINO)
Department of Population Genetics

Theoretical studies of population genetics (KIMURA)
Studies on molecular evolution from the standpoint of population genetics(KIMURA and OHTA)
Mathematical studies on the genetics of structured populations (MARUYAMA)
Linkage disequilibrium in finite populations (OHTA and KIMURA)
Department of Molecular Genetics

Studies on the chemical structure of genome of viruses containing doublestranded RNA (MIURA, FURUICHI, SHIMOTONO)
RNA polymerase in a virion containing double-stranded RNA (MIURA,FURUICHI, SHIMOTONO)
Structure and function of transfer ribonucleic acid (FURUICHI, MIURA)RESEARCHES CARRIED OUT IN 1970
I.

MOLECULAR GENETICS

Terminal structure of double-stranded RNA of cytoplasmic
polyhedrosis vims from silkworm (CPV)
Kin-ichiro MIURA and Yasuhiro FURUICHI

Genome RNA of CPV is double-stranded and consist of ten segments,
which are detected by electrophoresis on acrylamide gel (I. Fujii-Kawata,
K. Miura, M. Fuke: J. Mol. BioI. 51 (1970) 247). As we have not succeeded to obtain these segments in a preparative scale, mixed segments wereanalyzed. The 5/-termini of the RNA were phosphorylated by polynucleotide kinase using r-32P-Iabeled ATP or GTP only after removal of thepre-existent phosphate group by phosphomonoesterase treatment. Phosphorylation proceeded much faster for the single-stranded state of RNA
than for the double-stranded state. 32P-Iabeled terminal nucleoside wasisolated as 32pNp [nucleoside-5/, 3/-diphosphate] by alkaline (or RNase T 2)
digestion. Preliminary result indicates that purine nucleosides are predominant at 51-termini of CPV-RNA segments.
The 3/-termini of these RNA segments were analysed by 3H-labelingthe terminal ribose moiety with periodate oxidation and successive reduction using tritiated borohydride. None of the 3/-terminus of ten RNA
segments was phosphorylated. All ten RNA segments were labeled in thesame extent. The 3/-terminal nucleosides were determined as 50 % cytidine(ten termini) and 50 % uridine (ten termini) by analysis of nucleoside trialcohols obtained from the ribonuclease T 2 (or alkali) digest of the 3H-IabeledRNA. Since the 3/-terminal of a viral single-stranded RNA was adenosine,or cytosine for a exceptional case so far analyzed, the termini of the doublestranded CPV-RNA segments are unique.
In the course of the latter experiments, a 3H-labeled polysaccharideprotein complex was found in a 3H-labeled RNA preparation.

12 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

RNA polymerase associated with purified virion
containing double-stranded RNA
Kunitada SHIMOTONO and Kin-ichiro MIURA

It is shown that the reovirus contains RNA polymerase activity in apurified virus particle itself. Purified CP virus particle also revealed RNA
polymerase activity in vitro without any pretreatment of the virus preparation. Optimum condition for this RNA polymerase reaction was seeked.
If the product has a specific character, the template must be a doublestranded RNA genome in a virion. The characterization of the reaction
product is now under investigation. Preliminary results show that the
product is a single-stranded RNA and has the large molecular size corresponding to segments of genome RNA.

Double-stranded RNA isolated from the CP
virus of pine caterpillar
Kin-ichiro MIURA and Akiko SHIBUYAAs the size and character of disease of the cytoplasmic polyhedrosis
virus of pine caterpillar is similar to the CP virus of silkworm, the genome
nucleic acid of the former virus was characterized and compared with· thelatter. Midgut of the infected pine caterpillar was gathered by Dr. Katagiri at Asakawa Forestry Experimental Station of the Ministry of Agriculture. The CP virus was isolated as a single pure component from thediseased midgut through purification of polyhedra. It is established frommany kinds of chemical experiments that the nucleic acid prepared byphenol treatment for this virus is RNA and double-stranded. The basecomposition is quite similar to the RNA of CP virus from silkworm. Genome RNA of pine caterpillar CP virus is reproducibly separable into
ten segments as silkworm CP virus by polyacrylamide gel electrophoresis.
Size distribution of these segments of both the virus RNAs is not distinguishable. In the case of reovirus some differences in size distributionof genome RNA segments are detected among different strains. Therefore,
CP virus from pine caterpillar is very similar to silkworm CP virus. Originof both the viruses may be same. Analysis of the size distribution of thesegments of genome nucleic acid would be useful technique for the com-

RESEARCHES CARRIED OUT IN 1970

13

parison of biological species.

Structure and function of transfer RNA
Yasuhiro FURUICHI and Kin-ichiro MIURA(As Miura and Furuichi had engaged in the work on the structure andfunction of transfer RNA in the former laboratories, some works on thisline were also carried out this year.)
Furuichi performed the modification of tyrosine transfer RNA by sulfite
to seek functional site with Prof. Ukita's group in the University of Tokyo.
Uracil residue and isopentenyladenine located in either side of the loopedout anticodon sequence were modified. Although amino acid acceptability
is not changed by the modification, the binding ability of the tryosine
transfer RNA to the messenger RNA-ribosome complex was damaged.
Miura studied the nucleic acid of a living fossil, Brachiopod Lingula,with Dr. N. Shimizu of Nagoya University. DNA of this organism showedsmall GC content (35 %). Several species of RNA molecules were identified. 4s RNA can accept an amino acid by yeast enzyme. This molecule
has a similar structure to transfer RNA of the evolved present organisms,
for example bacteria and rat, in its molecular size, base components andpartial nucleotide sequences.

14 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

II.

MICROBIAL GENETICS

Further Mapping of fla and mot Cistrons Closely Linked to H 1
in Salmonella abortus-equi and Its Derivatives
Shigeru YAMAGUCHI and Tetsuo IINO

Spontaneous non-flagellate or paralyzed mutants were isolated from aphase-l stable strain of Salmonella abortus-equi and its HI-replaced derivatives. Taking trail production on transduction as the criterion, complementation tests were carried out with these mutants. Besides alreadyknown cistrons, that is, flaA, flaB, flaC, flaD, flaE, flaF, flaK, flaL, flaM,
motA, and motB, three cistrons jiaN, jiaP, and jiaQ were recognized, allco-transduced with HI. A group of non-flagellate mutants was shown tobe H 1- or ahi-. FlaA mutants were classified into three subgroups, jiaAI,
jiaAII, and jiaAIII. A group of paralyzed mutants corresponding to
motC was found to belong to the same group as the non-flagellate mutantsof jiaAII, and their average flagellar numbers per bacterium varied by thestrains from almost the same as that of the wild type strain to nearly O.
By deletion mapping supplemented with three-factor cross tests, therelative positions of jia and mot cistrons closely linked to HI were mapped.
They were found to form two clusters. The order of cistrons in one of
the clusters was established to be: -jiaD-jiaB-jiaQ-jiaN-jiaAIII-jiaAIIflaAI-H 1(ahI)-jiaL-, and that of the other cluster was partly determined asfollows: (flaE, jiaK)-motA-mot-B-jiaB-jiaC-jiaM-. (The details were submitted to J. gen. Microbiol.)Activity of glutamine dependent carbamyl phosphate synthetasein arginine sensitive mutants of Salmonella typhimurium
Hiro-o TOSHIMA and Jun-ichi ISHIDSU

It has been reported that the sites of arginine sensitive (ars) and arginineuracil sensitive (aus) mutations in S. typhimurium are closely linked tothe pyrA locus, the structural gene for glutamine dependent carbamyl
phosphate synthetase (carbamyl-P synthetase), by P22 phage mediated transduction (lshidsu, J., 1970. Ann. Rep. Nat'!. lnst. Genet. 20: 95). It has.

RESEARCHES CARRIED OUT IN 197015

been also known that both activity and repressibility of ornithine transcarbamylase and aspartate transcarbamylase in these strains are quite
normal.
On the basis of the above finidings, the activity of carbamyl-P synthetaseand its regulation pattern were compared between the wild type and thesensitive mutants using crude cell extracts. Unexpectedly, very low activity
was found in ars and aus comparable with that found inpyrA81, a deletionmutant requiring arginine and uracil for growth, even in extracts obtained
from fully derepressed cells grown in minimal medium. This confirms
on one hand the results of transductional analysis which showed that thesemutations have occurred in the pyrA locus, but on the other hand, it remains
to be explained, since these mutants can grow in minimal medium eventhough more slowly than the wild type, how they are able to prepare carbamyl phosphate necessary for biosyntheses both of arginine and uracil.
The answer might be either that even a very low level of carbamyl-P synthetase, if any, is sufficient to support growth, or that the carbamyl-P synthetaseof the mutants is active in intact cells but is easily destroyed or inactivatedduring the procedure of extraction, or that some other pathway is utilized
by the mutants to synthesize this compound. Activity of carbamate kinase,which is also known to exist in microorganisms and to catalyze the reactionof carbamyl phosphate synthesis from NH 4 +, CO 2 and ATP, was found toexist at the wild type level in extracts of mutant cells.
With a wild type strain LT2, the synthesis of carbamyl-P synthetase ispartially repressed by arginine or uracil and their effect is cumulative, The
activity of the enzyme is greatly inhibited by UMP, stimulated by ornithine
and, when UMP and ornithine are given together, the inhibitory effect of
the former is neutralized to quite an extent by the latter. These findings
are in good agreement with the results reported before (Abd-EI-Al, A. and
J. L. Ingraham, 1969. J. BioI. Chem. 244: 4033). Among mutations
occurring in the pyrA locus, several phenotypes have been reported. They
are, in addition to arginine-uracil auxotrophy which is the most frequent
one, arginine sensitivity and arginine-uracil sensitivity described above,arginine auxotrophy, cold sensitive arginine auxotrophy and uracil sensitivity. To explain the regulation pattern of the enzyme activity and theoccurrence of various phenotypes, a model of an allosteric structure of the
enzyme was drawn. Arginine, by binding to the modified ornithine siteon the enzyme, is supposed to interfere with, or contrariwise to stimulate,16 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

the binding of ATP in ars and aus, or in arginine auxotrophic strains, respectively. In aus strains, uracil, after being transformed to UMP, also prevents glutamine from binding to its own site completely because of themodification of UMP site by a mutation. This hindrance is only partial
in the wild type strain. A detailed report is in preparation.

Activities of enzymes concerning uracil biosynthesis in
6-azauracil resistant mutants of Salmonella typhimurium
Jun-ichi ISHIDSU

It has been known that 6-azauracil (6-AU), an analogue of uracil, canact as a co-repressor in place of the latter repressing the pyrimidine synthesizing pathway. However, since it cannot replace uracil or cytosinein active nucleic acid molecules, growth of a wild type strain is inhibited
under the presence of 6-AU unless uracil or cytosine is supplemented inaddition. This means reversely that some, if not all, of the 6-AU resistantmutants can synthesize uracil even in the presence of this drug; that is, they
are consititutive for uracil biosynthesis. Many such mutants, supposed
to have some alteration in genetic regulatory systems of pyrimidine biosynsynthesis, were isolated and their properties were examined.
About 108 washed cells of a wild type strain LT2 of S. typhimurium
were spread over minimal agar medium containing 1.5 mg/ml 6-AU. Afterabout 50 hour incubation at 37°, 100 to 200 large and 10 to 50 minutecolonies were formed on each plate. Thirty-eight small (pyr-1074-pyr1111) and 6 large (pyr-1112 - pyr-1117) colonies were picked up at random
and the level of aspartate transcarbamylase (ATCase) activity was measured
with toluenized cells of each mutant grown under repressed as well as derepressed conditions. The level of the enzyme in small colony formers,
either repressed or derepressed, was found to be as high as 6 to 70 times
the level in the repressed wild type strain. In large colony formers, it wasalso higher than that of the wild type, but the difference was not as significant
as in the case of small colony formers. These evidently synthesize ATCase
constitutively.
The activities of two other enzymes in the pyrimidine biosynthetic pathway, dihydroorotase (DHOase) and dihydroorotic acid dehydrogenase(DHO dehydrogenase), were also examined with toluenized cells. The

RESEARCHES CARRIED OUT IN 197017

synthesis of DHOase was found to be also constitutive in many of the 6-AU
resistant mutants. The derepression effect in the absence of uracil on thesynthesis of DHO dehydrogenase was not apparent, though one of the smallcolony formers, pyr-lOB2, which showed the highest level of ATCase, also
had a very high level of this enzyme even under repressed conditions (x 6.5of the wild type). The regulation of the synthesis of ornithine transcarbamylase (OTCase), which belongs to the arginine synthesizing pathway,
was quite normal in these mutants; namely, its synthesis was not constitutive
and was repressed by arginine. The derepressed level of glutamine dependent
carbamyl phosphate synthetase in pyr-lOB2 was slightly higher than thatof the wild type and the extent of the partial repression by uracil of its synthesis was narrower when compared to the wild type.
Pyr-lOB2, transferred to a glucose minimal medium containing arginine
and uracil from the same medium with no supplementation, showed a transient growth stop when the level of OTCase dropped to the repressed level.
In glycerol medium where generation time is longer, however, such interruption of growth was not observed. Unbalanced growth caused by thedifference in the amount of ATCase and OTCase might be one reason forsuch arrested growth.
Genetical analysis of these mutants yet remains as the next step.

Physiological and enzymological analysis of azide resistantmutants of Salmonella typhimurium
Jun-ichi IsmDsu

As has been reported before (Ishidsu, J., 1970. Ann. Rep. Nat'l. Inst.
Genet. 20: 97), usual isolates of spontaneous sodium axide (NaN3) resistantmutants of S. typhimurium on nutrient agar medium cannot grow on minimal
medium, but require cysteine or some other sulfur containing compounds
in the presence of this drug. In liquid basal medium containing 3 mM
NaN 3, it was found that 20 mM NaSH, 10 mM Na 2S203, 20 mM Na2S03
or 1 mM cysteine could support well the growth of the mutants. Methionine, at the concentration of 1 mM, was less effective. One mM
L-djenkolic acid, which is known to allow the growth of the wild type as asole sulfur source but not to repress the formation of enzymes concerning
the cysteine synthesizing pathway (Dreyfuss, J. and K. J. Monty, 1963.

18 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

J. BioI. Chern. 238: 3781), was much less effective.
Isolation of azide resistant mutants, not dependent on sulfur compounds
under the presence of NaNa, from a wild type strain LT2, pyrA81 and theircysteine dependent azide resistant derivatives, was tried by spreading cellson minimal agar medium containing 4 mM NaNa• PyrA81, a deletion mutant derived from LT2 damaged in the structural gene for glutamine dependent carbamyl phosphate synthetase, was used because the gene maps very
close to one of the known azi loci, aziA (Sanderson, K. E., 1970. Bacteriol.
Rev. 34: 176). In total 7 such mutants were obtained either from azide
sensitive parents directly, or from cysteine dependent azide resistant derivatives, showing that the cysteine dependency is not necessarily a character
accompanying azide resistancy.
Activities of one of the enzymes entering the cysteine biosynthetic pathway, sulfite reductase, were measured with crude extracts. It was found that
NaNa does not repress the formation of this enzyme either in sensitive or incysteine dependent azide resistant strains. Neither does NaNa in reaction
mixtures inhibit the activity of this enzyme. As it has been reported that
NaNa severely inhibits the activity of nitrate reductase (Nason, A. and H. J.
Evans, 1953. J. BioI. Chern. 202: 655), activities of this enzyme in crude
extracts were also examined. NaNa induced the synthesis of the enzyme
quite considerably, especially in young cultures, in any sensitive, cysteine
dependent resistant or independent resistant strains. The last group had atendency to possess more nitrate reductase than the former two groups.
Since NaNa was found to inhibit color yielding up to 90 % or more in thecolor exhibiting reaction mixture also used by the above authors, it was
not possible to elucidate its inhibition of enzyme activity by the method used.
Examination of effects of NaNa on earlier enzymes of the cysteine synthesizing pathway and genetical analysis by an Hfr-F- conjugation experiment are being carried out.

Reconsitituion of Salmonella Flagella Attached to Cell Bodies
Tetsuo IINO, Hideho SUZUKI and Shigeru YAMAGUCHI

Studies in Salmonella of in vitro reconstitution of flagellar filaments fromtheir component flagellin monomers have shown that the flagellar filaments
have structural polarity and that they grow at the end distal to the cell bodyRESEARCHES CARRIED OUT IN 1970

19

(Asakura, S. et al. 1968. J. Mol. BioI. 35: 227). Growth of flagella in vivo
was also demonstrated to take place at their tips (Iino, T. 1969. J. gen. Microbiol. 56: 227). These informations suggested a possibility that even when
the flagella are attached to living cells, reconstitution of flagellar filamentsfrom exogeneous flagellin monomers occurs at their tips under appropriate
conditions. An experiment was carried out to examine this possibility.
The reaction system used was a mixture of cells of a straight flagellar mutant, SJ814, of S. typhimurium and flagellin monomers of the wild strain
TM2 which produced normal flagella. The cells of SJ814 are entirely
non-motile. Failure of their movement was attributed to the loss of spiralstructure of their flagellar filaments (lino, T. and M. Mitani. 1967. J. gen.
Microbiol. 49: 81). The cells of SJ814 starved for 1 hour carried 3.2 flagellaper cell on the average. The length of the flagella ranged up to the size
corresponding to 6 normal wave units. After blending the cells in an electric mixer (Micro-Mixer TM101) for 3 min, most of the flagella longer
than 3 normal wave units were broken to less than 2 normal wave units and
55 % were detached from the cells at or near the base. The cell suspension
was then washed twice with 0.03 M phosphate buffer. To the flagellin solution, which was prepared from the flagella of TM2 by acid dissociation,
0.3 M final concentration of 1 M phosphate buffer (pH 6.9) was added, andmixed with an equal volume of bacterial suspension. Finally 1/25 volumeof 2 M K 3 -citrate was added to the flagellin-cell mixture which was incubatedat 26°C.
During the 4 hours of incubation at the concentration of 5 X 1010 cells
and 10 mg flagellin per ml, neither the number of cells in the mixture nor
the distribution of number and length of straight flagella was significantly
changed. Among the straight flagella longer than a half normal wave unit,
18 % grew normal filaments at their tips and formed heteromorphousfilaments. In addition to these straight and heteromorphous flagella, suchconsisting entirely of normal waves appeared. Their number was muchhigher than that of heteromorphous flagella, and was equivalent to 82 %of the sum of fully detached flagella and such that were broken less than ahalf normal wave unit upon blending. Distribution of the lengths of reconstituted normal flagellar filaments was quite different from that of thenative flagella. Long flagella of more than 7 normal wave units, which
never appear among native flagella, were seen among the reconstituted ones.
The maximum length observed was 20 normal wave units, which is about

20 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

3 times as much as the length of the maximum native flagella. In the mixture after 4 hours, 0.8 % of the cells were wobbling and wriggling. Whenthe cells of the mixture were inoculated into broth and incubated at 37°C,
the fraction of motile cells increased rapidly, reaching 13 % within 20 min
without appreciable increase in cell number. Thereafter, the fraction of
motile cells decreased in inverse ratio with increasing cell number.
These results indicate that exogenous flagellin monomers can be reconstituted to flagellar filaments at the tip of flagella attached to the living
cells, and that the reconstituted flagella on the bacterial body can exert
their locomotive function. (The details were submitted to Nature)

Possible Differential Control of FlageIlin mRNA Synthesis
in Bacillus pumilus Depending on Cell Age
Hideho SUZUKI

The cell-free flagellin synthesizing system, composed of RNA from B.
pumilus and preincubated S-30 from E. coli Q13 was developed, whereby
incorporation of HC-amino acids into the flagellin fraction represented
about 0.2 % of total incorporation (Suzuki, H. and H. Koffler, 1970. Bacteriol. Proceedings 70:23). However, in some reproduced experiments,
the RNA preparation, highly active in stimulating amino acid incorporation
into general protein, was not always active for amino acid incorporation
into the flagellin fraction. Amino acid incorporating activity of an RNA
preparation could be affected by the age of the cells from which the RNA
was extracted. Therefore, incorporation into the flagellin fraction was
examined by using RNA preparations extracted from cells either at an earlyor at a late logarithmic growth phase.
B. pumilus cells were grown in a Penassay broth medium. RNA wasextracted by the phenol method from lysozyme-lysate. Thirty a.D. units/
ml of the 2.5 M sodium acetate-insoluble RNA fraction served as the sourceof mRNA in the reaction mixture of protein synthesis, which contained
2.5 flC/illl of HC-fifteen amino acid mixture. Newly synthesized flagellin
was recovered with carrier flagellin from pH 2 soluble fraction, purified
through reaggregation and chromatographed on a DE-32 (Whatman)column.
The RNA extracted from aged cells stimulated amino acid incorporation

RESEARCHES CARRIED OUT IN 1970

21

into the flagellin fraction at least several times as strongly as that extracted
from young cells, even when incorporation into total TeA-precipitablematerials by the former fell to a low level. This suggests that flagellin
synthesizing ability of the cells varies with their age. Messenger RNA
for flagellin may be produced at a higher rate or readily accumulated upon
aging. Glucose effect might offer another explanation. Glucose contained in the Penassay medium may repress the synthesis of mRNA forflagellin. Upon exhaustion of glucose, cells may produce mRNA forflagellin at a maximum rate. RNA prepared from cells grown in the absenceof glucose exhibited moderately higher activity with respect to incorporation
into the flagellin fraction. However, trials to demonstrate the in vivo
repression of flagellin synthesis by glucose were unsuccessful. Even in anenriched medium, growth of B. pumilus was worse in the absence of glucose
than in its presence. An omission of glucose from the medium may produce
somewhat unfavorable growth condition for the cells, e.g. leading to rapid
aging.
Apart from those explanations, it may be expected that extraction of
RNA from the cells at an appropriate growth phase could produce an RNA
preparation enriched with flagellin mRNA.

Phage-induced Alteration of Colony Type in Xanthomonas citri
Wen C. Wu and Tetsuo IINO

Xanthomonas citri XCJ19, infected with temperate phage PXC7, had its
smooth colonies altered to dwarf colonies. The altered dwarf-colony
clones were lysogenic. After incubation in nutrient broth for 72 hr, eachof them reverted in 0.1-6 % of cells to the production of smooth colonies,
among which 60 % were lysogenic, 25 % resistant, and 15 % sensitive. The
lysogenic dwarf-colony clones could not be recovered to produce smoothcolonies by supplements of amino acids, purines, pyrimidines, vitamins or
nucleic acid bases; they did not change qualitatively in sensitivity to phages
CP1 and CP 2 , fermentation of mannitol and lactose, starch hydrolysis,
catalase activity, and somatic antigens; however, they lysed and liberated
spontaneously phage PXC7 at a considerably higher frequency, and decreased their growth rate and the number of bacterial cells contained in theircolonies, as compared with either their lysogenic smooth revertants or their

22 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

smooth original strain. These results indicate that Xanthomonas citri
XCJl9, when lysogenized with phage PXC7, underwent spontaneous lysis
and liberation of the phage at a considerably high frequency that resulted
in the alteration from smooth to dwarf colonies.

Genetic Analyses of Nonmotile Double Mutants in
Salmonella typhimurium
Masatoshi ENOMOTO

A number of nonmotile-double mutants, mot-fla-, mot-mot- and mot-ahl-,
have been isolated from three parental mot mutants, motA-, motB- andmotC-, of Salmonella typhimurium and used for mapping relative positionsof mot, fla and HI genes. The inferred order is as follows: his-(flaB, flaD)-flaA a-motC-flaA 2-(flaA 2 , flaA1)-(H 1, ahl)-(flaK, flaE, flaJ)-(motA, motB)jlaC-trp. The number of abortive trnasductants issuing from the double
mutants in transduction with the wild-type strain is in inverse proportion
to the distance between two mutations. In this case one mutation should
be located between flaB and ahl (left side) and the other between flaKand flaC (right side), and the number of abortive transductants is very few
as compared with that produced by the double mutants whose two mutations
are together located in either the left side or the right side. This phenomenon
is useful for genetic mapping as a new tool and for studying the mechanism
how the transducing fragments arise from the bacterial chromosome. Details
will be published in Genetics.
A New Transducing Phage Related to P22 ofSalmonella typhimurium
Masatoshi ENOMOTO and Hiromi ISHIWA

Properties of a new general transducing phage, PSA68, isolated from acommercial digestive are studied in comparison with other general transducing phages of Salmonella, L, MG40 and P22. The morphology, the latent
period and average burst size of PSA68 are indistinguishable from thoseof P22. This phage has most resemblance to L serologically and to MG40
in immunity properties; PSA 68 and MG 40 are dominant over L, andP22 is dominant over the other three phages. The buoyant density of PSA68RESEARCHES CARRIED OUT IN 197023

is 1.510 gjcm 3 identical to Land MG40, that of P22 being 1.520 gjcm3 •The relative number of abortive transductants produced by nonmotiledouble mutants are not significantly different among the four phages, though
the transduction frequencies per p.f.u. are different from each other. Thismeans that these transducing phages carry the genetically identical lengthof chromosome fragments. Details will be published in J. gen. Virology.

24 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

III.

BIOCHEMICAL GENETICS

Purification and properties of tryptophan pyrrolase
in Ephestia
Saburo NAWA

The mutant a suffers from the inability to convert tryptophan to formylkynurenine, which is the precursor of ommochromes, the brown eye pigments.
It is known that this conversion is catalyzed by an enzyme, tryptophan
pyrrolase. As reported previously, we obtained individuals of wild-typeeyes in experiments where larvae or eggs, having the recessive genotypea, had been treated with DNA from the wild type. Therefore, it is of interest to examine whether the transformants produce the enzyme normally.
Although tryptophan pyrrolase has been assayed in unfractionated homogenates, purification may be necessary to examine the properties of the
enzyme in detail.
Newly hatched adults of wild type were homogenized in the cold withphosphate buffer. After centrifugation, the protein was precipitated fromthe clear supernatant fraction by the addition of solid ammonium sulfate
to 45 % saturation. The resulting pellet was redissolved, dialyzed, and thentreated with calcium phosphate gel by step-wise elution. The most active
fractions were applied to DEAE-Sephadex column and developed with alinear gradient of NaCl, 0.05 to 0.3 M, in phosphate buffer. Activity wasroutinely eluted at about 0.25 M NaCl. Approximately SO-fold purification was achieved with an over-all yield of about 10 %. The enzyme canbe assayed by measuring kynurenine formation from tryptophan on condition that the added kynurenine formamidase, the enzyme which converts
formylkynurenine to kynurenine, is not rate limiting. The purified enzyme
was unstable, gradually losing activity upon storage at -20°C: about 10%of activity remained after one week and no activity was observed after one
month. In conditions used for the assay, kynurenine formation was shown
to be proportional to time only for one hours at 37°C and two hours at 25°C.
The absolute requirement for methylene blue was observed with the purified
enzyme. Thus, there seems to be a fundamental difference between Ephestia
tryptophan pyrrolase and the enzymes from other organisms such as rat,

RESEARCHES CARRIED OUT IN 1970

25

Drosophila or Pseudomonas which do not require methylene blue. Furthermore, unlike Drosophila tryptophan pyrrolase, no stimulation by ascorbate was observed for the purified enzyme. Tryptophan pyrrolase wasalso purified from transformants. It was observed that the enzymes had
the same properties as the wild-type enzyme.

Characterization of DNA from eggs of Ephestia
Masa-Aki YAMADA

It has been reported that the DNA from eggs at an early developmental
stage was heterogeneous and consisted of a major component and a minor
component (Annual report No. 20). In order to ascertain the localizationof these components, the homogenates of eggs were fractionated into twoparts, one containing the cell and nucleus fraction (CN), and the othercontaining the mitochondria and yolk grain fraction (MY). Eggs ranging
from 0 to 18 hours after they were laid were gently homogenized in 0.03 M
Tris-HCl buffer containing 1 mM EDTA and 0.25 M sucrose, filteredthrough gauze, and centrifuged at 1,500 rpm for 20 minutes. It was foundthat the precipitate (CN fraction) was contaminated with a small amountof mitochondria and yolk grains. MY fraction was precipitated from thesupernatant at 12,000 rpm for 20 minutes. DNA was extracted from eachof those fractions with Tris-EDTA-phenol method, treated with RNase
and a-amylase, followed by deproteinization with chloroform. The
density of DNA was analyzed by using Spinco E.
It was found that the CN fraction contained only one component (p=1.699) having the same density as adult DNA. DNA from MY fraction
consisted of a major (p=1.698) and a minor component (p=1.680). It
seems that the minor component corresponds to that of whole egg DNA,
indicating that it is specifically located in MY fraction. In addition to thosecomponents, two weak bands having densities of 1.677 and 1.668 weredetected in MY fraction. Their properties are at present unknown.
There seems to be a difference between the density of DNA from CN(1.699) and that of the main component from MY (1.698). Though thedifference is quite small, it was always observed in repeated experiments.
Thus, it is probable that those are different DNA s. Moreover, it has
been previously observed that the distribution of the main 1.698 fraction of

26 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

whole egg DNA was asymmetrical in density gradient, indicating that thefraction is heterogeneous. Therefore, it is reasonable to assume that themain fraction of whole egg DNA consists of two components, one fromeN and the other from MY.

Hereditary change in silkworm with DNA
Saburo NAWA, Mitsuo TSUJITA and Masa-Aki YAMADAPrevious communications have reported that treatment of silkworm
larvae of Wl genotype with DNA from wild-type produced wild-type individuals (Nawa, et af. 1971. Genetics 67: 221). In order to know whether
other loci are responding to such treatment, a triple recessive genotype,fem; oc; pe was used for the same experiment. In addition, the use of thismutant was valuable for the examination of a possible contaminant.
Using several samples of wild-type DNA and fem; oc; pe DNA preparedseparately, a series of independent treatments of fem/fem; oc/oc; pe/pe
larvae was carried out. Out of 5,726 larvae injected with wild-type DNA,819 individuals were raised to adults. No eye color mutant was obtained.
From the crosses of the treated adults, either female or male, to untreatedfem; oc; pe, eggs from 187 batches on the whole were examined. No
pigmented egg was obtained. However, when some of the white eggs wereraised to adults, two black-eyed moths were obtained among 3,365 adults(BF l ). They behaved as if they had the genotype fem/fem; oc/oc; pe+/pe,
since they produced fem/fem; oc/oc; pe+/pe and fem/fem; oc/oc; pe/pe in
the proportion of 1:1 when crossed with fem; oc; pe. The appearance ofthe heterozygote in respect of the pe locus may be explained by transformation of the gene by incorporated DNA. The possibility of contamination in this case can be excluded by the fact that they were homozygotes forboth fem and oc loci. If there had been an accidental genetic contamination
produced from the cross of fem/fem; oc/oc; pe/pe to wild type, they would
have had the genotype fem+/fem; oc+/oc; pe+/pe. The line which could
be suspected of being a source of contamination in this case should havebeen fem/fem; oc/oc; pe+/pe+. However, no such line was reared in ourlaboratory.
Some BFl animals, which were pink eyed, were crossed to untreatedlem; oc; pe. In this 2nd backcross generation, one egg batch which contained

RESEARCHES CARRIED OUT IN 197027

two black eggs among 345 white eggs was obtained. This gives a very low
frequency, since a total of 1,418 batches were examined. Although one of
the black eggs did not hatch, another black egg developed into a black-eyed
moth which behaved as if its genotype was lem/lem; oc/oc; pe+/pe. The
white eggs from the same batch developed into pink-eyed moths. However,a cross (9 pairs) between the pink-eyed moths again produced one egg batch
which contained one black egg among 445 white eggs. This may represent
the persistence of introduced pe+ information in unexpressed form in somecell lineages. Similar results to those have been observed in the experiments
using WI. No black egg or black-eyed moth was obtained so far in theexperiments where DNA from lem; oc; pe was injected, though the numberof treatments was rather small. It should be noted that no instance of achange in respect of the genes lem and oc was obtained so far.

Three allelic isozyme variants in rice peroxidase
Chiang PAl and Toru ENDo

Genetic studies on peroxidase isozyme variants have been reported forseveral plants, e.g., morning glory, corn, oats and wild rice, Oryza perennis.
With the exception of O. perennis the genetic behavior of those isozyme
variants has shown the absence of hybrid bands in the heterozygotes. In
the previous papers (Shahi et al. 1969. Japan. J. Genet. 44: 321, Endo 1971.
ibid. 46: 1), two co-dominant alleles, Pe 2A and Pe 4 A at the Pe locus which
specify rice peroxidase isozymes, were reported for two rice strains, W107
and W1294. Also, it was noted that the intensity ratio of the three isozyme
bands deviated from the expected distribution pattern in the lemmas andpaleas of the F I plants and most of the F 2 heterozygous segregants.
Now we have detected a third aIle, Peo c specifying the band OC. The
three parental strains, W593, T65 and WI20-4', each had one major band,
cathodic OC, anodic 2A or 4A, respectively, in the lemmas and paleas.
The three F I plants among them, i.e., T65 X W593, T65 X WI20-4' andW593 X WI20-4'. Each produced not only the bands characteristic forboth parents but also a hybrid band having intermediate mobility. It wasnoted, however, that the intensity ratios of the three bands in those F I plants
almost followed the distribution expected from random association of themonomers, in contrast to that of the F I plant between WI07 and W1294.

28 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

As previously suggested (Endo 1969. this report, 20: 45), it could be assumed to be some factor or factors affecting the alleclic expression of the
Pe locus, resulting in a deviated distribution isozyme pattern in the latterstrains.

Lethal effects of a genetic abnormality in thepteridine metabolism of silworm larvae
Mitsuo TSUillA and Susumu SAKURAI

It has been known that embryos homozygous for the lem! gene produced
from sib-mating individuals having the genotype +!emjlem! develop normallyto young black larvae which hatch by chewing up their egg chorion andgrow normally until the 1st molting. Immediately after the 1st molting
normal larvae become blackish due to melanin biosynthesis in their hypodermal cuticle. However, molted lethal lemon larvae (lem1jlem!) are distinctly yellow due to the presence of a large amount of sepiapterin in their
hypodermal cells. In addition, they can not chew up mulberry leaves and,thus, starve to death. Yellow lethal larvae can also be recognized at the
beginning of the 2nd instar from the cross +lemjlem! X lemjlem l. However,from the cross lemjlem l X +!emjlem l or lemjlem l X lemjlem l, all homozygous
lem!jlem! embryos develop normally, but become partly yellowish and partly
yellowish brown at the stage of body pigmentation. They can not chew
up their egg chorion and die inside the eggs, which show the yellowish
brown dying larvae through the semi-translucent shells.
Based on several biochemical experimental results, the inability to chew
up mulberry leaves or egg chorion has been assumed to be due to incomplete
hardening and imperfect differentiation of the cuticle, especially the mandibular cuticle of the mouth organ, which may be caused by abnormal phenylalanine and tyrosine metabolism.
Since it was found that in the YD 4 strain young homozygous lem!jlem!
larvae can be discriminated from normal (+ lem jlem! and + !emj + lem) larvaeby their body color at the stage immediately after hatching, the activity ofphenol oxidase, i.e., the rate of dopachrome production from dopa and fromtyrosine (Tsujita and Sakurai 1971) was examined.
At the beginning of the 1st instar, normal and homozygous lem!jlem!
larvae showed almost the same enzyme activity. However, in a later

RESEARCHES CARRIED OUT IN 197029

stage of the 1st instar and the stage immeidately after the 1st molting a significantly weaker enzyme activity was detected in lethal lemon larvae thanin normal larvae. Furthermore, it was shown that lethal lemon larvae havea weaker activity of phenylalanine hydroxylation than normal larvae.
It seems that the maternal effect of +1.", gene on sepiapterin reductase
within the homozygous lem/lem eggs laid down by the +I''''/lem l female moth
(Tsujita 1961) has close relation to normal phenylalanine and tryosine metabolism by which homozygous embryos are able to finish their complete
development and a sufficient differentiation of their cuticlular layer.
Genetic and biochemical studies of letahal albino larvaeof the silkworm, Bombyx mor;
Mitsuo TSUJIT A and Susumu SAKURAI

Lethal albino larvae appear immediately after the 1st molt. Since alis a simple recessive gene, from sib-mating of +al/al individuals normal(+al/+al and +al/a/) and lethal albino (ai/a/) larvae segregate in the ratio of
3:1. Doira et al. (1970) showed that the al gene locates on the 5th chrosome (20.5 or 42.9). The phenotype of lethal albino larvae resembles thatof lethal lemon larvae. In both mutants a small amount of melanin is produced in the cuticle layer of the hypodermis and their mandibular cuticle<loes not develop sufficiently due to a defective phenylalanine metabolism,
so that they can not chew up mulberry leaves and starve to death after the
1st molting. These two mutants differ only in body color, namely, the lethal
albino larvae are partly light brown and partly white and their hypodermal-cells contain a large amount of pteridine granules filled with pteridines anduric acid. However, they showed an abnormal pteridine metabolism.
It is well known that in normal larvae uric acid and isoxanthopterin are
accumulated in their hypodermis. However, in lethal albino larvae theamount of uric acid is comparable with that found in normal larvae, while
the amount of isoxanthopterin is much smaller in al/allarvae than in thenormals and they show an accumulation of an unknown fluorescent substance. It is considered from several biochemical experimental resultsthat in lethal albino larvae some step of sepiapterin formation is blockedin the main pteridine metabolism of the silkworm.
A spectrophotometric assay with L-tyrosine or L-dopa as the substrate
revealed that al/allarval extracts have a significantly lower phenol oxidase

30 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

activity than +a!j+a! larval extracts, and the activity of phenylalanine hydroxylase is weaker in aljallarval extracts than in +a!j+a! extracts.
So far as our experimental results thus far obtained are concerned, we
could not show an accurate evidence of direct control of phenol oxidase
production by the +a! gene. Therefore, it seems that in lethal albino larvae
abnormal phenylalanine and tyrosine metabolisms which seriously effectthe differentiation of the hypodermal cuticle are secondary phenomena
caused by the primary defective pteridine metabolism quite like in lethallemon larvae.
The phenotype of double recessive lethal (lem! jlem!; alja/)
larvae of the silkworm
Mitsuo TSUJITA

In order to elucidate the phenotype of double recessive ([em!jlem!; aljal)
homozygotes, segregation from the cross +!emjlem!; +a!j+a!x+!emj+!em;+a!jal was examined in the F 2 , F 3 and F 4 generation. Experimental results
are given in Table 1.
Table 1.
FilialNo.
generation

Segregation from the cross + !em/lem! x +a!/alNo.
hatchedlarvaeHatching
rate

Normal lem!/lem!

al/al

434
460
301
276
373
342
446
417
203
348

97.0
94.3
95.3
95.3
93.8
97.1
98.7
96.9
84.2
85.6

238
225
159
150
200
187
245
210
116
161
1891

98
99
70
63
83
83
110
89
47
66
808

-------

F2
F3

Total
Theoreticalsegregation
ratio

2
18
1
3
15
16
19
3
5
12

Phenotype of larvae

72

69
53
49
63
60
81
66
35
50
598

(al/al+lem!/lem!; al/al)
1855

618

9
9

3
3

824

Total
408
393
282
262
346
330
436
365
198
277
3297
Total
3297

~

1
:
4
X2 =1.66
0.5>P>0.25
3

RESEARCHES CARRIED OUT IN 1970

31

Table 1 shows segregation in normal, lethal lemon and lethal albino larvaein the ratio 9:3 :4. It is clear that the double recessive homozygous larvae
have lethal albino phenotype, indicating the accumulation of an unknown
pteridine compound in their hypodermal cells.
Furthermore, it was confirmed that double recessive (fem/fem; af/af)
larvae which segregated from the cross fem/fem; +al/ +al X +10m/ +10m; +al/al
had also lethal albino phenotype, showing no accumulation of sepiapterinin their hypodermal cells.
These experimental results present evidence in support of the idea thatin lethal albino larvae the formation of sepiapterin is blocked in their main
pteridine metabolic pathway and that they lack the function of tetrahydropteridine quite like in lethal lemon larvae (Fig. 1).
af

fern
fern l-1---> - - > Sepiapterin :'=I~ Dihydrobiopterin -~ Tetrahydrobiopterin- - > 2-amino-4-hydroxypteridine - - >Isoxanthopterin

Fig. 1. A possible main pteridine metabolic pathway from sepiapterin toisoxanthopterin in the silkworm.

Genetic variation observed in membrane proteins isolated frompteridine granules of several oily mutant strainsof the silkworm
Susumu SAKURAI and Mitsuo TSUJITA

W-C, Cl24 and E-Iemon were used as normal strains with non-transparent
larval skin, and wa, wb , wo l , wo a, wo h , oa and oc were used as oily mutant strains
with semi-transparent or transparent larval skin. Pteridine granules wereisolated from the hypodermal cells of normal strains and oily mutants on
the 4th day of 5th instar. The cytoplasm of the hypodermal cells whichcontains the granules were homogenized with teflon-glass homogenizer in
8.56% sucrose containing 1 mM NaHCOs and 5 mM EDTA, pH 7.5(NaHCOs-EDTA-sucrose). After homogenization in NaHCOs-EDTAsucrose, unbroken cells and floating cell components of the hypodermis were
removed by filtration through a nylon cloth. The resultant filtrate waslayered over discontinuous density gradients consisting of 20, 25, 30, 35,
40, 45, 50, and 80% (w/v) sucrose and was centrifuged in the Tominaga
No.7 S W rotor at 3,000 rpm for 2 hrs at Q-4 0 c.

32 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Using the above method, the normal pteridine granules formed a compact
zone at the 50 %/80 % interface, while the abnormal ones of wo l and wo"oily mutants did the same at the 40 %/45 % interface. On the other handin the case of w" oily mutant the specimen was collected from the upper layerof 8.56 % sucrose. After washing once with 0.01 M sodium phosphatebuffer, pH 7.0, containing 5 mM EDTA and 5 mM 2-mercaptoethanol,
the pellet was resuspended in a solution containing 0.8 % sodium dodecyl
sulfate (SDS), 0.025 M Tris, 0.3 M NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 8.5. The suspension was incubated at 37°C for 2 hrs tosolubilize the membranes. Insoluble material was then removed by centifugation. The soluble sample was dialyzed overnight at room temperature
against 0.1 % SDS, 0.1 M NaCl, 1 mM 2-mercaptoethanol (SDS-NaClEDTA-2-mercaptoethanol) pH 8.5 and the dialysed solution was chromatographed at room temperature through a 2.4 X 50 cm column of Sephadex
G 75 equilibrated with SDS-NaCI-EDTA-2-mercaptoethanoI. The protein fraction eluted at the void volume was then applied to a 4.0 x 55 cm
column of Sephadex G 200 equilibrated with same buffer at room temperature. The effluent protein peak was collected and dialysed overnight
against 0.025 M Tris, 0.1 M NaC!, 1 mM EDTA, 1 mM 2-mercaptoethanol,
pH 8.5. The pH of dialysed solution was lowered to 5.5-5.7 by adding 1 %acetic acid and the resulting precipitate after standing overnight at 4°C
was collected by centrifugation at 9,000 x g, freeze-dried and stored at -20°e.
The membrane protein fraction of pteridine granules consisted of equalshares of lipid and protein. Protein, lipid and carbohydrate content aswell as its amino acid composition of the delipidated membrane proteinsobtained from normal pteridine granules are very similar to those obtained
from the abnormal pteridine granules of oily mutants. Freeze-dried membrane protein obtained from a normal strain was dissolved in 1 % SDS
in 0.05 M Tris-O.38 M glycine buffer containing 8 M urea, 10 mM EDTA
and 10 mM 2-mercaptoethanol, pH 8.8 and incubated at 37°C for 1 hr.
The solution was analyzed by 7.5 % polyacrylamide gel in 0.05 M Tris0.38 M glycine buffer containing 0.1 % SDS, 5 M urea, 1 mM EDTA,
1 mM 2-mercaptoethanol, pH 8.8 (SDS-urea polyacrylamide).
Electrophoresis was carried out at 5 rna/gel at 5-10°e. A single band
was observed, indicating that the membrane protein of normal pteridine
granules was composed of an electrophoretically homogeneous protein
component. However, it was revealed by SDS-urea polyacrylamide gel

RESEARCHES CARRIED OUT IN 1970

33

electrophoresis that the SDS-urea treated pteridine granule membraneproteins of oily mutants were dissociated into multiple protein bands. Twoor seven separate bands were observed by the following multiple gel analysis.
These results show that the electrophoretic patterns of proteins obtained
from several oily mutant larvae not only differ from that of the protein
obtained from the strain with non-transparent skin, but also differ fromeach other among oily mutant strains studied. It has been known (Tsujita
and Sakurai 1967) that the size and shape of pteridine granules produced
in hypodermal cells of oily mutant larvae are abnormal, and the amountas well as the density of the mature granules are much smaller than in thenormal strain. Thereby, it is considered that the transparency of larvalskin of wS multi-allelic oily mutants and the complexity of the electrophoretic
pattern of the membrane protein obtained from these oily mutants are bothdue to an alteration of structural protein components resulting in an abnormal
structure or assembly of those pteridine granule membranes.

Immunochemical studies of structural membrane proteins of pteridine
granules in hypodermal cells of silkworm larvae
Susumu SAKURAI

Oily mutant strains, wS, w", wb , wO" and oc with semi-transparent ortransparent larval skin and normal larval strains, W-C, Cl24 and E-lemonwith normal granules and non-transparent skin were used. It has been
known that the mutated loci of these strains, w", wb and wO" belonging to
WS multiple allelic series are located on chromosome 10, and that of oa
on chromosome 14.
Pteridine granules were isolated from the hypodermal cells of normal
strains and oily mutants using discontinuous sucrose density gradients as
described in the preceding report. The pellet was dissolved in a solution
with PH 9.0, containing 1 % sodium deoxycholate, O.oI M NaHCO s , 0.3 M
NaCl, 1 mM EDTA, I mM 2-mercaptoethanol and 0.01 M ammonium
formate and allowed to stand at 37°C for 2 hrs to solubilize the membranes.
The protein fraction of the membrane was purified by several procedures
such as precipitation at isoelectric point and gel filtration in Sephadex.
Antisera against the membrane proteins obtained from normal and abnormal pteridine granules were prepared by immunizing rabbits with them.

34 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

The immunization was done by intraperitoneal and intravenous injectionof the antigens. Ouchteriony double diffusion test was performed in Petridishes with 0.8 % agar in 0.01 M ammonium formate buffer, PH 7.3, containing 0.01 M NaHCOi, 0.9% NaCl, 1 mM EDTA and 1 mM 2-mercaptoethanol, added with 0.01 % sodium ethylmercurithiosalicylate for preservation. At immunodiffusion, the antiserum to E-lemon membraneprotein produced a single precipitin band with normal membrane proteinsobtained from C124 and W-C as well as with the homologous antigen.
The membrane proteins of E-lemon and oa oily mutant gave a complete
identical precipitin line with normal membrane antiserum. Antiserum to
wa abnormal membrane protein reacted with two precipitin bands against
the homologous antigen, one of which was revealed to be partially in common with the band between anti-E-lemon serum and E-lemon protein. A
similar common line was also seen at immunodiffusion between antiserumto E-lemon membrane protein and the membrane proteins of w3 , wb , andwoa • Anti-w a membrane protein antiserum reacted against abnormal
membrane proteins of w3 , wa and wb all with two precipitin bands but ofslightly different pattern from one another.
It is therefore proposed that the +W3 gene located on chromosome 10
is the structural gene for a membrane protein subunit. The results ofimmunodiffusion test suggest that immunological differences of membraneproteins of w3 , wa and wb are due to a defect of the primary structure ofmembrane protein subunits.

Differences in Amino Acid Composition of Serum Transferrin
among various Species of Rattus
Kazuo MORIWAKI, Tamiko SATO and Kimiyuki TSUCHIYA

Amino acid composition of serum transferrin was analyzed in various species of Rattus obtained from South East Asia and Oceania. Transferrin
was purified through several steps; acrinol treatment of the serum followedby DEAE-cellulose column chromatography of the soluble fraction, starch
block electrophoresis of DEAE-separated j3-g10bulin fraction and finallygel-filtration through Sephadex G-200 column. Purified transferrin washydrolyzed and analyzed as to the amino acid composition using automatic
amino acid analyzer (JEOL Model JLC-5AH and Hitachi Model KLA-3B).

RESEARCHES CARRIED OUT IN 1970

35

Residue number of each amino acid per mole was computed by assumingthat the molecular weight of transferrin is approximately 67,000. Differencein the number of each amino acid residue among various species is summarized in Table 1. These differences may represent, to a considerable
extent, the differences in amino acid sequence among these species. In
order to support this notion, composition difference and sequence difference
in a single species of protein were compared among various kinds of higherorganisms. If we use the data on cytochrome-c as a typical example (Dayhoff, M. 0.: Atlas of protein sequence and structure, vol. 4, 1969), almost
a linear relationship between composition difference and sequence difference
can be observed in the range less than 40 amino acid differences. This rpobably indicates that differences in amino acid composition of certain specific
Table 1. Total difference in the number of each amino acidresidue of serum transferrin among various species of Rattus

~...

Q

'-~'"

t<>

~

R. rattus argentiventer (M)R. muelleri
R. rattus argentiventer (C)R. norvegicus
R. rattus tanezumi TfN
R. rattus mindanensis
R. bowersi
R. rattus diardii
R. rattus tanezumi TfR
R. conatus
R.legatus
R. rattus rattus (N.G.)
R. exulans

Total difference

.;;;:
~

~

:::
'"

~

~

...

1: .::J::::'".:;'"'E::: ::::'"
i:~~'t<>" .;=:::'" ~''"" ·SE<::s~g ...:>.c~ §'" ~ 'Ec'" ~1:: :::: 1:: ~ "" ~
::::

:>.

<::s

z

...

~

l::'"

E

~

~

~

~

""'~"

~

~

,-..,

'-

c::i

E-<

~

'E:::

'"

''""

~

~

'" ~<::s::::

~

~

~

c

<>

~

'"<::s

~
~

~

~

~

~

~

...

'"

::::
:::

-S!

~

~

29 17 20 24 22 30 24 30 34 44 54 44
29
24 24 26 24 20 28 34 30 42 50 62
17 24
22 30 22 32 32 38 28 44 49 59
22 24 28 32 32 44 48 60 42
20 24 22
24 26 30 22
30 20 34 18 58 44 60 48
22 24 22 24 30
28 34 30 29 52 54 56
28 34 48 48 56 56
30 20 32 28 20 28
24 28 32 32 34 34 28
38 42 52 60 37
53 36 62 62
30 34 38 32 18 30 34 38
34 30 28 44 48 29 48 42 53
36 46 76
44 42 44 48 44 52 48 52 36 36
38 84
54 50 49 60 60 54 56 60 62 46 38
88
44 62 59 42 48 56 46 37 62 76 84 88
372 392 397 398 404 405 418 441 467 514 568 677 704

Abbreviation. (M): Malaysia, (C): Celebes, (N.G.): New Guinea, TfR: R-typetransferrin, TfN: N-type transferrin.

36 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

protein between two species can be used as a substantially meaningful
tool for indicating distance index.

Serum Transferrin Polymorphism in Oceanian Black Rat,

Rattus rattus rattus
Kazuo MORIWAKI and Tamiko SATO

Starch gel electrophoretic survey was carried out on serum specimens of
62 black rats collected from 4 localities in Oceania. Transferrin bands onthe gel was identified by Fe~9 labelling and autoradiography. Detailed
analytical procedure has been reported by us (Moriwaki, K. et al., Genetics
63: 193, 1969). Naming of transferrin phenotypes followed Malecha andTamalin (Amer. Nat. 103: 664, 1969). Table 1 demonstrates the distribution of transferrin phenotypes in various localities of Oceania. That Oceanian rattus is markedly different from the Asian rattus in the karyotype
has already been reported (Yosida, T. H. et al., Jap. J. Genet. 44: 89, 1969):
the former has 38 diploid chromosome numbers and the latter 42. In
the present study, difference between both rattus was found also in thetransferrin alleles. Oceanian rattus have Tfc, TfD, TfE and TfF, whereas
Asian rattus have TfR and TfN.
Frequencies of each allele in Oceanian materials studied were as follows.
In Brisbane Tfc=0.33, Tf D=0.60, TfE=0.03 and TfF=O.03. In Cairns
Table 1. Distribution of transferrin phenotypes in Oceanian
Rattus rattus.
Transferrin
phenotype
C
CD
CE
CF
D
DE
DF
EF
Total

BrisbaneinAustralia
2
4Localities
CairnsinAustralia2
51

Wellington
in
New Zealand
21
2

2
8
1
1
2
18

Pt. Moresbyin
New Guinea
3
2

8

8

23

13

RESEARCHES CARRIED OUT IN 1970

37

TfC=0.83, TfD=0.08 and TfE=0.08. In Wellington Tfc=0.96 and TfD=
0.04. In Port Moresby Tfc =0.46 and TfE =0.54.

Serum Transferrin Patterns of Rattus rattus obtained
from Southern India and Italy
Kazuo MORIWAKI, Tamiko SATO and Kimiyuki TSUCHlYA

Serum transferrin patterns of black rat, Rattus rattus, obtained from
Mysore in southern India and from Rome, Italy were analyzed by starchgel electrophoresis. Identification of transferrin bands on the gel was
carried out by Fe~9 labelling and autoradiography. Detailed operational
processes have already been reported (Moriwaki, K. et al., Genetics 63:
193, 1969). In five R. rattus, kindly sent to us by Dr. T. Tachibana in
India, two were Tf-C type and the remainder Tf-CD type. The nomenclature for the transferrin types is as described by Dr. Malecha for Hawaiian
R. rattus (Malecha, S. R. and R. H. Tamalin, Amer. Nat. 103: 664, 1969).
Two R. rattus obtained from Dr. Capanna in Italy contained one Tf-C
and one Tf-CD types. These transferrin alleles, Tfc and TfD, were also
observed in the Oceanian R. rattus (Moriwaki, K. et al., this Annual Report).
Thirty eight diploid chromosomes were observed in these Indian R. rattus(yosida, T. H. and K. Tsuchiya, this Annual Report), and also in the Italian
R. rattus (Capanna, E. et al., Experientia, in press). The biochemical dataobtained above seem to coincide with these cytogenetical findings in suggesting that European type R. rattus (Tf=C, D, E and/or F, 2n=38) aredistributed in southern India and Italy.

Studies on the transferrin of the Mongolian gerbil
Kazutoshi MAYEDA1) and Kazuo MORIWAKIThe two dimensional electrophoresis of serum proteins of the Mongolian
gerbil, Meriones unguiculatus, showed seven evenly spaced beta-globulin
bands. In order to confirm that all seven bands were transferrin, autoradiographic study was carried out using radioactive FeCIs• After tagging
11 Supported by (N. S. F.) U. S.-Japan Cooperative Science Program; on leave fromthe Dept. of Biology, Wayne State Univ., Detroit, Michigan.

38 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

the transferrin with this substance, two dimensional electrophoresis showed
all seven bands to be radioactive, indicating that they are transferrin.
Subsequently, the transferrin was purified by means of preparativeelectrophoresis and column chromatography. Physical characterizationof the purified transferrin showed that the gerbil transferrin has a molecularweight of approximately 67,000, which corresponds well with other rodent
transferrins such as the rat and the mouse.
The purified transferrin still maintained seven distinct bands upon starchgel electrophoresis and agar gel electrophoresis. In order to determine
the effect of sialic acid on the electrophoretic mobilities, the purified gerbil
transferrin was incubated in purified neuraminidase (Sigma Chemical Co.)for varying length of time. Subsequent electrophoresis showed that themobility of all the bands were retarded, relatively, after such treatment. We
were unable to obtain a single electrophoretic banding pattern for thisprotein even after prolonged incubation in neuraminidase.
In order to further characterize the observed seven bands, anti-serum
to the purified gerbil transferrin was made by injecting it into rabbits. Subsequently, the purified gerbil transferrin was analyzed by the immuno-electrophoresis technique in agar gel. The precipitation pattern showedthat all bands were antigenically identical.
Amino acid analysis of the purified transferrin was then carried out.
As reported in the following article, the amino acid content of the gerbil
transferrin does not seem to differ greatly when compared with transferrinsof other rodents. The greatest difference seems to occur when the aminoacid content of the gerbil transferrin is compared with those of the golden
hamster. The least difference occurs between the gerbil transferrin andthe mouse transferrin.

Comparative studies of the transferrins of rodents
Kazuo MORIWAKI and Kazutoshi MAYEDA 1 )In a continuing study of the genetics of rodents, we have investigated thetransferrin proteins obtained from several species of rodents. In order tomake a thorough comparison, amino acid contents of the transferrins
1)
Supported by NSF U. S.-Japan Cooperative Science Program; on leave from theDept. of Biology, Wayne State Univ., Detroit, Michigan.

RESEARCHES CARRIED OUT IN 1970

39

obtained from several different species of rodents were determined.
The molecular weight of the transferrins of all the rodents studied wasapproximately 70,000 by the technique of thin layer gel filtration analysis.
This corresponds well with the published results of Gordon and Louis(Biochem. J. 88: 409, 1963) who reported that the molecular weight of the
rat transferrin is 67,000. The method of purification of the transferrins is
described in a previous article (Moriwaki, K. et al., this Annual Report).
Such purification process resulted in transferrins contaminated by otherserum proteins by less than 5 %. Amino acid analysis was carried out on
Hitachi Model KLA 3B and JEOL Model JLC-5AH amino acid analyzers.
The results of the amino acid analyses are presented in Table 1. Generally
speaking, there seems to be no great difference in the amino acid content of
the transferrins from different rodents studied. In order to better visualize
the extent to which differences exist, we have rearranged the data in Table 1
by making a pair-wise comparison as shown in Table 2. For example, comparing mouse with rat, the absolute number of amino acid difference between these two species is 20. Similarly, the number of amino acid difference
between the transferrins of the golden hamster and gerbil is 74.
Table 1. Amino Acid Content of Various Transferrins
Amino Acid

Rat

Mouse

Apodemus

Peromyscus

GerbilHamster

Lys
His

41
14
21
60
27
36
52
41
49
46
6
34
5
18
45
20
26

46
16
21
58
28
37
50
41
50
46

37
12
22
56
28
37
45
39
52
50

38
21
55
30
36
54
37
47
51

49
19
18
56
26
39
51
39
52
51

34
5
17
49
26
25

37
6
19
46
27
25

38
2
20
47
18
22

48
12
21
58
25
35
48
15
45
62
9
36
7
21
45
27
28

Arg

Asp
Thr
Ser
Glu
Pro
Gly
Ala
Cys
Val
Met
lieu
Leu
Tyr
Phe

* Not determined

12

-*
36
3
18
46
16
28

40 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Because of the technical errors involved with amino acid analyses, totalamino acid difference of 20 is probably not significant. That is to say, thetransferrins of the mouse and rat are probably identical. The mouse transferrin is relatively close to the transferrins ofgerbil, peromyscus and apodemus
but significantly different from the golden hamster transferrin.
Table 2. Number of Amino Acid Difference Between Species
Rat
Rat
Mouse
Apodemus
Peromyscus
GerbilHamster

20
40
36
40
78

Total

214

Mouse

Apodemus

Peromyscus

Gerbil

20

40
44

36
52
26

40
32
44
52

44
52
32
72

26
44
80

52
60

74

220

234

226

242

Hamster
78
72

80
60
74
364

RESEARCHES CARRIED OUT IN 1970

IV.

41

DEVELOPMENTAL GENETICS

Specific Inhibition by Glucosamine of Aggregate Formation of
Rat Hepatoma Cells in Rotation Culture
Yukiaki KURODA

It is well known that malignant transformation of cells is accompanied
by many biochemical, immunological, biological and morphological alterations of the surface cell membrane such as cellular adhesiveness, contact
inhibition of movement and growth, electrophoretic cell mobility, also
antigens and chemical compositions of the surface membrane. Recently
some quantitative and qualitative changes in glycolipids or glycoproteinsin the surface membrane were detected following malignant transformation.
It has been previously found by the author that aggregate formation of dissociated cells was enhanced by the supernatant of the medium in which cells
were cultured for a few hours and this supernatant contained proteins andhexosamines.
In the present experiment the effects of some hexosamines and their acetyl
compounds on aggregation of DAB-induced rat hepatoma cells were examined in a rotation culture. Three rat hepatoma cell lines were used:dRLN-61, dRLa-74 and dRLh-84, which had low, moderate and hightumor-producing activity, respectively. The cell suspensions containing
3 X 105 cells in 3 ml culture medium were rotated under standard conditionsin the presence of various hexosamines and their acetyl compounds at theconcentrations of 1, 3, 10 and 30 mM.
Among the hexosamines tested D-glucosamine was most effective in reducing the diameter of cell aggregates from all three hepatoma lines. Dgalactosamine and D-mannosamine had not any or a slight effect even at
the concentration of 30 mM on the aggregate formation of hepatoma cells.
Acetyl-D-glucosamine, acetyl-D-galactosamine and acetyl-D-mannosamine
were also ineffective at the concentrations tested up to 30 mM. No differential effects were found of these hexosamines and their acetyl compounds
on hepatoma cells having various tumor-producing activities.
Phytohemagglutinin Concanavalin A, isolated from the jack bean andknown to agglutinate malignant cells, was also tested for affecting aggrega-42 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

tion of rat hepatoma cells. No effect of Cancanavalin A was found atconcentrations less than 0.1 mg/mI. At higher concentrations than 0.3 mg/
ml Concanavalin A prevented completely aggregate formation of rat hepatoma cells in a rotation culture.

Growth-Stimulating Effect of Peptone on Drosophila
Ovarian Cells in Culture
Yukiaki KURODA

In the series of experiments planned on a long-term cultivation of Drosophila cells, it was found that peptone had a pronounced growth-stimulating effect on Drosophila ovarian cells. Ovaries obtained from 48-hourpupae of the Oregon-R strain of D. melanogaster, which were reared understerile conditions, were cut into several fragments and cultured in T-5
flasks with 0.8 ml of Kuroda's Drosophila medium K-6' (Kuroda, Y. 1969.
Japan. J. Genetics 44, SuppI. 1: 42; 1970. Exp. Cell Res. 59: 429), which
was slightly modified and supplemented with 0.1 mg/ml fetuin and 15 %fetal bovine serum.
Peptone (Wako Pure Chern. Ind. Ltd.; Pancreatic digest of casein) wastested at various concentrations for its growth-stimulating effect on ovariancells. It was found that 5 mg/ml of peptone enhanced the outgrowth offibroblastic cells from explanted ovarian fragments. At higher concentrations
than 10 mg/ml, peptone had a slightly toxic effect on those cells.
In the presence of 5 mg/ml peptone fibroblastic cells initially issued fromthe cut end of ovarian fragments, stretched their cytoplasm on the surfaceof culture flasks, and formed a monolayer sheet around the original explants.
Mitotic figures were frequently observed. Under a phase microscope somesmall granules were observed in the cytoplasm and one or two nuclei were
found in the nucleus. From their morphology and behavior, these fibroblastic cells seem to be derived from the lumen cells in the ovariolar cavity.
Some insect cell lines established by Grace (1962. Nature 195: 788; 1966.
Nature 216: 613) were also derived from ovarian tissues of Antheraea
eucalipti and Bombyx mori. Although it is uncertain that Grace's cell lines
originated from the same type of cells in the ovary as the fibroblastic cells
observed in the present study, these ovarian cells may have some advantage
in growing under in vitro culture conditions.

RESEARCHES CARRIED OUT IN 1970

43

Evidence for the Restitution of Sorting-Out Activity of
HeLa Cells Following Fractionated X-Irradiation
Yukiaki KURODA

HeLa cells have a characteristic sorting-out activity when intermixed
with embryonic quail liver cells in a rotation culture. It has been previously
found that the sorting-out activity of HeLa cells was quantitatively expressedby the number of quail liver cells intermingled in HeLa cell aggregates andthat of HeLa cells in quail liver cell aggregates. These percentages ofintermingled cells increased with gradually increasing doses of X-ray given
to HeLa cells.
In the present experiment the presence of a restitution process in the lossof sorting-out activity of HeLa cells following X-irradiation appeared
when HeLa cells were exposed to fractionated X-ray and the percentages ofintermingled cells in aggregates of each type of cells were examined.
HeLa cells which were exposed to the first dose of 1,000 R X-ray wereincubated for 2 or 4 hours, then exposed to a second dose of 1,000 R. Thesecells were intermixed with 7-day embryonic quail liver cells and allowed
to form aggregates in a culture rotated for 24 or 48 hours. The harvested
aggregates were examined for the number of both types of cells in the histological preparations. The results of such experiments are shown in Tables
1 and 2.
In the above tables it is indicated that the percentage of intermingled
quail liver cells in HeLa cell aggregates and that of intermingled HeLa
cells in quail liver cell aggregates markedly decreased when the irradiated
Table 1. Effect of fractionated X-irradiation expressed as thenumber of quail liver cells intermingled in 24-hour
aggregates of irradiated HeLa cells
Dose

No. of total cells
in aggregates·

No. of quail liver
cells in aggregates·

Per cent of
quail liver cells

OR
1,000 R
2,000 R
1,000 R+l,OOO R(Interval, 2 hr)
1,000 R+l,ooO R(Interval, 4 hr)

52.3
77.0
.50.0
54.7

4.3
13.3
17.3
13.3

8.2
17.3
34.6
24.3

56.7

16.0

28.2

44 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21
Table 2. Effect of fractionated X-irradiation expressed as thenumber of irradiated HeLa cells intermingled in 24-hour
aggregates of quail liver cells
DoseOR
1,000 R
2,000 R
1,000 R+l,ooo R(Interval, 2 hr)
1,000 R+l,ooo R(Interval, 4 hr)

No. of total cells
in aggregates·

No. of HeLa cells
in aggregates·

Per cent of
HeLa cells

156.3
147.3
145.3
150.7

1.7
5.0
14.0
7.3

1.1
3.4

133.3

8.7

9.6
4.8
6.5

• The average numbers of total cells and intermingled cells in the largest sections
through three different aggregates were calculated.

HeLa cells were incubated for 2 hours before the second X-ray exposure,
followed by a slight increase when the cells were incubated for 4 hours.

Further Study of Relationship between Molting andCell Cycle in Insect Epidermis
Kiyoshi MINATO

It has been previously found that the epidermal cells of larvae of Philosamia cynthia ricini may have a peculiar growth cycle: they do not divide
after DNA synthesis in the same larval perid. DNA synthesis takes placesynchronously prior to the period of the pause for molting of each larval
instar, followed by cell division in the next larval instar after passing therelatively long G 2 period.
A previous autoradiographic analysis has ascertained the possibility ofsuch a process. To confirm it further, a similar experiment was arranged.
Fourth instar larvae were continuously labeled with H3-thymidine and theincorporation into mitotic figures in various tissues was survayed during
the same instar period.
As the result, mitotic figures of epidermal cells were scarcely labeled(1/115, 0.9 %). On the contrary, the incorporation of HS-thymidine into
mitotic figures in the fat body, haemocytes, and trachea was frequently
observed at the ratio of 85/226 (37.6%), 61/97 (63.0%) and 11/30 (36.7%),
respectively.

RESEARCHES CARRIED OUT IN 1970

45

These facts indicate that epidermal cells in mitotic stage were not labeledwith HB-thymidine when injected during the same instar period, although
the incorporation of H 3-thymidine was observed in mitotic figures in otherlarval tissues. This result confirms the possibility that DNA synthesis
takes place at the stage preceding the larval stage in which the cell divisionoccurs.

Effects of Culture Media on the Survival of Embryonic
Quail Liver-Specific Cells
Kiyoshi MINATO

It has been previously reported that tissue-specific cell colonies and nonspecific colonies were isolated in the primary cultures of embryonic chickchondrocytes, retinal pigment cells and myogenic cells (Cahn, R. D. andCahn, M. B. 1966. Proc. N. A. S. 55: 106; Coon, H. G. 1966. Proc. N. A. S.
55: 66). To isolate tissue-specific cells selectively, the following attempts
were made.
Liver cell suspensions were prepared from 7-day or lO-day quail embryos
by trypsinization and cultured in monolayer in TD-flasks or plastic petri
dishes. Eagle's basal medium, minimum essential medium, Parker's TC199 medium, and Ham's F-12 medium were tested with the supplementof 10 % calf serum. Although small differences were found among themedia tested, Eagle's minimum essential medium seemed to be most suitable
for maintaining liver parenchymal cells. In this medium liver parenchymal
cells became flattened after 1 or 2 days of cultivation and formed an epithelial cell layer. Giant cells with relatively transparent cytoplasm were
frequently observed. No difference was found among the sera obtained
from various manufactory sources. The adhesiveness of cells to glass orplastic surface was better in fetal bovine serum than in calf serum. In
the former serum the parenchymal cells were not epithelial in shape anddegenerated after 3 or 4 days of cultivation, although non-specific fibroblastic cells grew rapidly. For the maintenance of liver parenchymal cells
calf serum seemed to be better than fetal bovine serum. Further investigations are carried out to find better culture conditions for the cells.

46 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

V.

CYTOGENETICS

Karyotype evolution in Rattus species from black rat, R. rattus.
1. Species with a basic chromosome number,
but with structural chromosome difference
Tosihide H. YOSIDA and Kimiyuki TSUCHIYABlack rats, R. rattus, inhabiting wide area of East and Southeast Asia
have 2n=42 chromosomes, among which 13 pairs (No.1 to 13) being acrocentrics of various sizes, 7 pairs being small metacentrics, and the rest
pair being a large and a small acrocentric X and Y chromosomes. Amongacrocentric group, No.1, 9 and 13 pairs were found to be remarkably
polymorphic, occurring in three chromosome types, namely an acrocentric
homomorphic pair (AI A), a subtelocentric homomorphic pair (SIS) andan acrocentric and a subtelocentric heteromorphic pair (A/S). Basedon the population survey of the frequency of chromosome polymorphismin the rat distributed in East and Southeast Asia, it is suggested that theacrocentric chromosomes in the three pairs are the basic karyotype, and thatthe subtelocentric chromosomes arose from the acrocentric ones by pericentric inversion. The following four species, namely the Norway rat,
Rattus norvegicus, Rattus exulans, Rattus annandalei and Rattus muelleri
have the same chromosome number (2n=42) and similar karyotype as theblack rat. They have 13 acrocentric or subtelocentric autosome pairs and7 small metacentric autosome pairs. External morphology of the Norway
rat, R. norvegicus, is very similar to that of the black rat, and are distributed
throughout the world. These animals are characterized by having No.1,
No. 9 and No. 13 subtelocentric homomorphic pairs. All R. exulans
collected in Philippine, Indonesia, Thailand and Hawaii have 2n=42 andsimilar karyotype to the black rat, but they are remarkable by having No.1
and 9 subtelocentric pairs. R. annandalei collected in Malaysia is also characterized by having a similar karyotype to the black rat, but only No.9 pair
was subtelocentric. All 3 species have acrocentric X and Y chromosomes,
but R. muelleri collected in Malaysia had a large metacentric X chromosome.
This species had a basic Rattus karyotype with 2n=42, but No.1 and No.9
pairs are usually subtelocentric. Based on the above comparative karyo-RESEARCHES CARRIED OUT IN 1970

47

type analysis in the four Rattus species, it is strongly suggested that the
karyotypes in the above four species have arisen from chromosome polymorphism in the Asian black rat with 2n=42.

Karyotype evolution in Rattus species, 2. Species withfewer chromosome numbers than 2n=42 byRobertsonian chromosome fusion
Tosihide H. YOSIDA and Kimiyuki TSUCHIYA

It has been reported that R. rattus is classified into two geographicalraces (or subspecies) by chromosome numbers, 2n=42 and 38, and further
suggested that the latter evolved from the former by Robertsonian type ofchromosome change. Those rats with 42 chromosomes are distributedin East and Southeast Asia, but those with 38 are found in Oceania, Europe
and America. Rattus fuscipes which was collected in Brisbane, Australia,
had 2n=38 and the idiogram is very similar to that of R. rattus with 2n=38.
It is characterized by having two large metacentrics as found in Oceanian
R. rattus. These chromosomes are suggested to have arisen by Robertsonian fusion of No.4 and No.7 and No. 11 and No. 12 acrocentrics numbered according to the basic karyotype with 2n=42. Among six rats collected at Brisbane, two had 2n=39 chromosomes which was remarkableby having one extra chromosome belonging to small metacentrics.
Five Rattus sp. collected in a jungle close to a suburb of Port Moresby,
New Guinea, had 2n=34 chromosomes. Four large metacentrics orsubmetacentrics which are suggested to have developed by Robertsonian
fusion of Nos. 2 and 12, Nos. 3 and 6, Nos. 4 and 7, and Nos. 6 and 10
chromosomes observed in the basic karyotype of 2n=42 are included.
No. 9 pair is acrocentrics. Seven small metacentrics characterizing thebasic karyotype of the black rat are observed in this species too. Four
Rattus conatus collected in Cairns, Australia, had 2n=32 chromosomes.
They had five large metacentric or submetacentric elements. Biarms of
those chromosomes seem to have arisen by Robertsonian fusion of Nos. 2
and 5, Nos. 3 and 6, Nos. 4 and 7, Nos. 8 and II and Nos. 10 and 12. No.9
pair is submetacentrics. Seven small metacentric pairs quite similar to
those of black rat are observed in these animals.
Both species, R. fuscipes and R. rattus rattus, have the same chromosome

48 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

number and similar karyotypes, but the former is native to Australia, thelatter from Europe. They should have differentiated independently fromthe basic karyotype with 2n=42 by the Robertsonian type of change. R.
eona/us and Rattus sp. are also suggested to have differentiated independently
in Australia and New Guinea, because the constitution of the large biarmed
chromosomes in the former is different from that of the latter. Interestingis the fact that No.1 chromosomes which are characterized by acrocentricand subtelocentric polymorphism in the Asian black rats are usually subteleocentric in the above three species, but No. 9 pair is different dependingon the species, namely, R. fuscipes and Rattus sp. showed acrocentric, but
R. conatus subetlocentric.

Karyotype of Indian black rats, Rattus rattus rufesens
Tosihide H. YOSIDA and Kimiyuki TSUCHIYABlack rats are roughly classified into two groups by chromosome constitution, namely Asian type with 2n=42 and Oceanian type with 2n=38.
(Yosida et al., 1969, Jap. J. Genet. 44: 89-91, Chromosoma 33: 3~O,
1971). The latter type R. rattus rattus is distributed in Oceania (Australia,
New Zealand, New Guinea), North America, South America, Europe andEgypt. We suggested from data obtained by population survey and karyotype analyses that the black rats with 2n=38 are derived from the Asian
black rat with 2n=42 by Robertsonian fusion of four acrocentric chromosomes. We also suggested that the black rats with the Oceanian typedeveloped in the course of migration from Asia to Europe. Karyotypes ofthe black rats in India which is on the route of migration from Southeast
Asia to Europe should be interesting to determine the karyotype evolutionof this animal.
Three black rats were obtained by courtesy of Mr. Toshio Tachibana in
Mysore of India. Chromosomes of these rats, named Rattus rattus rufesens,
are clearly 2n=38 chromosomes. Among them two pairs are large metacentrics as observed in Oceanian type rats. According to Ray-Chaudhudi
and Pathak (1971) the animals collected in Varanasi located in Northern
India had 2n=42, but our material collected in Mysore located in Southern
India had 2n=38. From these observations, it is suggested that karyotypechange in black rats occurred somewhere in the Indian district.

RESEARCHES CARRIED OUT IN 1970

49

Studies of aneusomic clones isolated from a Chinese hamster
cell line following induction of nondisjunction
Hatao KATO

A brief exposure of Chinese hamster cells to colcemid induced chromosomal
nondisjunction. Although nondisjunction occurred randomly in every
member of the chromosome complement, the viability of progeny cells
differed markedly. Karyotype analyses of cells sampled at definite timeintervals after induction of nondisjunction suggested strongly that cellswith aneusomies of large chromosomes would die off or undergo karyotype
remodeling by the time of the 3rd or 4th mitosis. The elimination ofcells with unfavorable chromosome constitution appeared to begin as early
as in the first cell cycle following the occurrence of nondisjunction, because
we found no cell to lack the X chromosome in contrast to the frequent
detection of those with two X's in 20-hour sample. On the other hand, cellswith aneusomies of small chromosomes such as No.9 or No. 10 (or No.
11) appeared to proliferate steadily.
Making use of this behavior, several trisomic as well as monosomic cellclones were isolated at a relatively high frequency from colcemid-treatedcell population. Aneusomic lines now in our possession are as follows:
No.9 monosomy (one line); No. 10-11 monosomy (one line); No.9 trisomy(2 lines); No. 10-11 trosomy(5 lines); No. 7-8 trisomy (2 lines); No. 4 trisomy(one line); No. 10-11 trisomy with two Y's (one line); No.9 and No. 7-8
double trisomies lacking Y (one line).
Studies of differences in phenotypic expressions of these clonal lines are
now in progress. Preliminary studies demonstrated a significant variationsof UV sensitivity among clones with different doses of a specific chromosome
type. (Details of this report will be published in Cytogenetics).

Distribution of two chromosomal types of Japanesewood mouse, Apodemus speciosus
Kimiyuki TsucffiYA and Tosihide H. YOSIDA

Japanese wood mouse, Apodemus speciosus, which has a similar bodysize as the house mouse, is widely distributed in Japan. They are classified
into two sibling forms by chromosome numbers, i.e., 2n=46 and 48 (Shimba
et al., 1969). The change is due to Robertsonian fusion of chromosomes.

50 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

In order to determine the distribution of these two forms, we collected
347 specimens in 66 different localities in Japan. The distributions of thetwo chromosomal types are strictly divided into two parts of northern andsouthern Japan. The borderline of the distribution is on a line connecting
Toyama and Hamamatsu. Northern population from this borderline
had 2n=48, while southern population showed 2n=46. Four animals
collected in the Hamamatsu region, however, had 2n=47 chromosomes
which was determined to be a hybrid type between the two sibling forms.
The animals with 2n=46 can not be distinguished from those with 2n=
48 by external morphology, blood transferrin pattern or other biochemical
examinations, but the two forms are separated strictly into two geographic
regions. To determine the ecology of the animals, we tried to breed themin captibity in laboratory cages. Although the animals survive in thecages for one or two years when fed commercial rat food, offspring could
not be obtained. In a natural population room set up in the field, we
succeeded in obtaining F 1 hybrids. They showed hybrid karyotypes with
2n=47 chromosomes, similar to those obtained in Hamamatsu region.

Differences in Biochemical Characteristics between Two Sibling
Forms of Japanese Wood Mouse, Apodemus speciosus
Kimiyuki TSUCHIYA and Kazuo MORIWAKIAs stated in a previous article in this Annual Report (Tsuchiya, K. and
T. H. Yosida), two sibling forms of Japanese wood mouse, Apodemus speciosus, 2n=46 and 48, are found in Japan. The former is distributed in thesouthern parts and the latter in the northern parts, both of which are indistinguishable from each other in morphological characters. In thepresent study we attempted to determine differences in biochemical characters
between the two sibling forms, if any.
First, optimal temperature ofliver acid phosphatase was compared. Whenthe enzyme activity was assayed at pH 4.6 using para-nitrophenol phosphate
as a substrate, the animals collected from 3 localities, Misima (2n=48, 6),
Tsushima (2n=46, 6) and Kumamoto (2n=46, ~) exhibited almost identical optimal temperature, 45°C.
Secondly, the whole serum proteins of the materials obtained from
Kyoto (2n=46, 6), Misima (2n=48, 6) and Nopporo (2n=48, ~) wereRESEARCHES CARRIED OUT IN 1970

5J

compared by agar-gel immunoelectrophoresis using rabbit anti-wood mousewhole serum (2n=48) antibody. All of the animals equally showed atleast 11 precipitation arcs on the gel and no visible difference was detectedin the corresponding arcs among them.
Thirdly, comparison of isozyme patterns of serum a-esterase was madebetween the northern populations (2n=48, 17 individuals) and the southern
populations (2n=46, 32 individuals). Though no electrophoretic patterns
definitely characteristic of either population have been found, an apparent
difference in the average number of esterase bands was observed as follows;
northern populations=4.1 bands, southern populations=3.7 bands.
Bottom half of starch-gel, upper half of which was used for demonstration of esterase isozymes mentioned above, was stained with amido blacklOB for detection of serum proteins. Polymorphic features were observed inslow-a2-macroglobulin and in certain ,a-globulin which was not iron (Fe59 )binding protein (transferrin), but these polymorphisms were not associatedwith either of the two kinds of Apodemus populations.
Lastly, difference in erthrocytic antigens between the two groups waschecked by determining agglutination titer of erythrocytes obtained fromgroups. Anti-serum was prepared in rabbits by immunizing with erythrocytes of one type, 2n=48 in this case. Four batches of northern group(2n=48) gave an equal titer of 1/24 and 4 batches of southern group (2n=46) 1/12, equally. This difference, however, does not seem to be significant.
Recently it has been revealed that another Apodemus species, Apodemus
miyakensis, collected from Miyake island has similar 48 chromosomes as
those of the northern type. These animals showed different electrophoretic
mobilities in seruin transferrins and albumin. Immunoelectrophoretic
analyses of the whole serum using anti "48" rabbit serum demonstrated
that one of the post-albumin proteins which can be detected in Apodemus
speciosus is missing in this species.

Spontaneous sarcomas in black rats, Rattus rattus, bred inlaboratory, and chromosome observations
Tosihide H. YOSIDA and Hatao KATO

In black rats, Rattus rattus, bred in the laboratory, three sarcomas, two
fibrosarcomas and one hemangiosarcoma developed spontaneously. One

52 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21of the fibrosarcomas developed in one female (RRJ-497a) in the fourth
generation of sister-brother mating in the Japanese black rats, and theother fibrosarcomas developed in a male (RA-2A) of the F 1 hybrids obtained by mating an Australian female (A-6) and a Japanese male rat(RRJ-373A). In the above female (RRJ-497a), the tumor developed in theperitoneal cavity as a solid tumor, and in the male, developed in the hypoderm at the left ventral site as a large tumor (about 35 x 66 x 77 mm).
The Japanese rat (RRJ-497a) had 2n=42 and No.1 A/A chromosomes,
but the F 1 hybrid rat showed 2n=40, being a hybrid karyotype between
Japanese (2n=42) and Australian rat (2n=38). Chromosomes could beobserved in bone marrow cells of the rats, but could not be successfully
studied in the tumor tissues.
One hemangiosarcoma developed in the hypoderm at dorsal site in one
female (RRT-519) in the sixth generation of sister-brother mating of theblack rats collected in Korea. This rat had 2n=42 and No.1 A/A chromosomes. The chromosomes in the tumor tissue were successfully observedin situ and in cultivated cells. Remarkable feature was that most tumor
cells had hypotetraploid chromosome numbers. The chromosome numberof 101 cells observed were distributed widely from 42 to 167, but about
80 percent of the cells were at the hypotetraploid level (74 to 83). Amongthe 101 cells, those with 83 chromosomes showed the highest frequency(22 cells), cells with 80 chromosomes ranked second (14 cells) and thosewith 82 chromosomes ranked third (10 cells). Only five tetraploid (2n=84) and one hypertetraploid cells (85) were observed. Remaining sevencells were hyperdiploid (42 to 44), three cells were near triploid (63,68 and69) and two cells were hypooctoploid (149 and 167).

Preliminary note on karyotype of guppy and topminnow
Tosihide H. YOSIDA and Makoto HAYASm11

This paper is a preliminary note on the chromosome constitution of guppy,
Lepistes reticulatus and topminnow, Gambusia holbrook. These two species
belong to Cyprinodontidae, Teleostomi. Chromosomes of the guppy havebeen reported by Winge (1923) and Iriki (1932). According to these reports,
11
Department of Bioloby, Faculty of Science, Kwansei Gakuin University, Nishinomiya.

RESEARCHES CARRIED OUT IN 197053

the diploid chromosome number in the fish is 2n=46. Our observations
carried out in female somatic cells agree with those observed by them.
All 46 chromosomes are acrocentrics and the sex chromosomes are notrecognizable in the somatic cells (Fig. I).
Chromosomes of topminnow have been reported by Geiser (1924) who
described that the fish has 2n=36 chromosomes. The results of the present
observation, however, differ markedly from that obtained by Geiser. Our
observations revealed that the chromosome number in this fish is 2n=48
in female somatic cells. Among 48 chromosomes, one large metacentricchromosome is remarkable in the female cell. The metacentric chromosome
seems to be an X element (Fig. 2). Based on the above observations, it
is suggested that the sex determining mechanism in the topminnow is afemale heterogamety (XY). The sex chromosome mechanisms in theseshould be determined in more detail by further observations of male andfemale somatic cells.

"."
. ...
., .:..

•

t.

.,

I

I'

• ·"-,,,6"",fIt·••':';. • tt~.t!JI'& .~<.~.

1

_r'

"..

Figs. 1 and 2. Metaphase chromosomes in female somatic cells.
1) Guppy, Lepistes reticulatus.
2) Topminnow, Gambusia holbrook. Arrow
indicates the X-chromosome.

54 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Chromosome survey of small mammals in Japan
Kimiyuki TSUCHlYA and Tosihide H. YOSIDA

Chromosomes in small mammals belonging to Insectivora, Chiroptera,
Lagamorpha, Rodentia and Carnivora living in Japan were studied. The
names of the species, diploid chromosome numbers, and the karyotypes
are shown in the following table.
- - - - _.. _-Karyotype*2nSpecies

M SM ST-

Insectivora
TalpidaeDymecodon pilirostris
Urotrichus talpoides hondonis
-

-

-

34
34

22
22

--

4
4

62
58

Lagamorpha
Leporidae
Lepus timidus ainu
L. brachyurus angustidens

48
48
-

--- - -

--

--

M
M

?
m

60
52

ST
SM

SM
?

36

SM

A

_--

2

6
-

--

6
6

4

46

(Y)

(X)

A
-

- - - - - - _ ..

-Chiroptera
Rhinolophidae
Rhinolophus comutus comutus
R. ferrumequinum mikado
Vespertilionidae
Miniopterus schreibersi fuliginosus

Rodentia
Sciuridae
Petaurista leukogenys nikkonis
Muridae
Clethrionomys rutilus mikado
C. rufocanus bedfordiae
C. rexAschizomys niigatae
Eothenomys kageus
E. smithi smithi
Microtus montebelli montebelli
M. m. brevicorpus
Ondatra zibethicus
Apodemus miyakensis
A. argenteus
A. giliacus
Micromys minutus hondonis--------------------

10
10

6
6

-

-

38

24

56
56
56
56
56
56
30
30
54
48
46
55
68

2
2
2
2
2
2
12
12
2
4
4
4
6

10
10

---- -

20
20-- -- -- -

12

12
12

2
2

4

4

M
M

38

52
52
52
52
52
52
2
2
50
38
40
48
18

A
A
- - ---

SM

A

A
A
A
ST
ST
A
SM
SM
A
A
ST
A
A

M
A
ST
M
SM
SM
A
A
A
A
A
A
A

55

RESEARCHES CARRIED OUT IN 1970
M. m. japonicus
M. m. aokii
Mus musculus molossinus
M. m. yonakuniensis
Tokudaia osimensis muenninki
Rattus legatus
Carnivora
Canidae
Nyceterutes procyonides viverrinus
Mustelidae
Mustela sibirica itatsi

2
2

18
18
38
38
4 20
34

A
A
A
A
M
?

A
A
A
A
ST
?

42

14 12

14

A

ST

38

10

12

SM

A

68
68
40
40
42
44

6
6

4
4

14
8

6

38
38

8

* M=metacentrics, SM=submetacentrics, ST=subtelocentrics, and A=acrocentrics.
Frequency of chromosome polymorphism in pair Nos. 1, 3, 9 and
13 in black rats, collected in Misima
Tosihide H. YOSIDA

The apearance of chromosome polymorphism in pair Nos. 1, 9 and 13
in balck rats, Rattus rattus, has been reported (Yosida et al. 1965, 1970,1971). Among the chromosomes, frequency of No.1 chromosome polymorphism has been reported in the rats collected in various localities inJapan. In the rats, polymorphism of pair No.3 was newly found which
has not been reported to date. This pair showed acrocentric and subtelocentric polymorphism as seen in the other chromosome polymorphism ofNos. 1, 9 and 13. In the present paper, frequencies of the acrocentric
homomorphic pair (AlA), acrocentric and subtelocentric heteromorphic
pair (A/S) and subtelocentric homomorphic pair (SIS) in the above fourchromosomes in 16 animals collected in Misima are presented as a preliminary report. The frequencies are as shown in the following table (Table
1).
Table 1. Frequencies of three chromosome types in pairNos. 1, 3, 9 and 13 in 16 animals collected in Misima
Pair No.
Pair typ; _________

AlA
A/S
SIS

No.1

No.3

No.9

No. 13

8
7

9
5
2

5

10
1

1

3
8

o

56 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

As seen in the table, frequencies of three chromosome types (A/A, A/S
and SIS) in the rats differ for the different chromosomes, but those of pairNos. 3 and 13 are somewhat similar. Ratio of subtelocentric to acrocentricchromosomes in the three pairs (Nos. 1,3 and 13) is 0.4, 0.4 and 0.2, respectively. In pair No.9, however, the frequency of subtelocentric chromosome
is markedly higher, and the ratio of A/S is 1.5. The frequency of acrocentricand subtelocentric chromosomes varies by colony of rats collected. For
instance, among 16 black rats, 4 were collected in one house (colony A)
and 8 rats collected in another house (colony B) which is about 5 km
distant from colony A. The frequencies of subtelocentrics to acrocentrics
in the four chromosome pairs are shown in the next table (Table 2).
Table 2. Frequency of subtelocentric to acrocentric in fourchromosome pairs in two different colony~

Pair No.
------------,

No.1

No.3

No.9

No. 13

No .of animals

B

1.6
0.1

0.3
0.3

0.3
4.3

0
0.8

4
8

Colony ~A

The above table shows that the difference of frequency of chromosome
polymorphism seems to be affected by random drift of the animals.

In Vivo Proliferation of Two Sublines of MSPC-l Mouse
Myeloma in Mixed Inoculation
Kazuo MORIWAKI and Tamiko SATO

Two sbulines of MSPC-1 mouse myeloma, P-40 and NP-39, were tested
for their proliferating ability with the mixed or separated subcutaneous
inoculation. P-40 subline derived from an early transplant generationof MSPC-1 tumor is a producer of myeloma protein and has 40 chromosomes
without any marker. NP-39 subline derived from a later generation isa non-producer and has 39 chromosomes with 3 markers, A, B, and C.
When 106 P-40 cells was subcutaneously inoculated into the left abdomen
and 5 x 103 NP-39 cells into the right abdomen separately, the population
size of the former cells became 4.0 X 108 and that of the latter 1.4 X 108 after
2 weeks. In the mixed inoculation with the same cell doses as employed
above, however, the resulting tumor, after 2 weeks, consisted of 1.5 x 108

RESEARCHES CARRIED OUT IN 197057

P-40 cells and 4.8 x 108 NP-39 cells. The cell numbers for such experiments can be computed from the total number of cells in the grown tumor,
the ratio of P-40jNP-39 in the metaphase figures and the mitotic index
in each subline of tumors inoculated separately. The results, summarizedin Table 1, clearly demonstrate that in the mixed inoculation, growth of
P-40 cells is markedly retarded and growth of NP-39 cells is considerablyenhanced. These interactions between the two sublines seem to occur only
when they are in close contact with each other.
In order to determine the possibility of "allogeneic inhibition" likeinteraction between these two sublines of cells, intact P-40 cells were subcutaneously inoculated by mixing with NP-39 cells heavily irradiated with
Cesium 139 (5000 r), treated with cold glycerine for one week or treatedwith cold acetone. No significant inhibition of the prolieferation of P-40
cells by the NP-39 cells treated in such manner has been observed thus far.
Possibility of other kinds of immune mechanisms involved is now underinvestigation.
Table 1. Interaction between two sublines of MSPC-l tumor
in their in vivo proliferation.
Total cell number

Inoculation

Tumor line
Initial

Final

Separated

P--40
NP-39
Total

106
5 x 1034.0xlOS
1.4 X 1085.4xlOS

Mixed

P--40
NP-39
Total

106
5x103

1.5 x lOS
4.8 x 1086.3xlOS

Expected

4.7xlOS
1.6x lOS
6.3xlOSAn estimation of the number of definitive spermatogonial
cell divisions in the silkworm
Akio MURAKAMI

Since the first report of Kawaguchi (1928), there have been few papers.describing the number of definitive (or secondary) spermatogonial divisions in the silkworm. According to Kawaguchi, six-definitive spermatogonial divisions occur resulting in the production of 64 primary spermatocy-

58 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

tes. In some insect orders, however, it has been shown that the number ofsuccessive definitive spermatogonial mitosis is not strictly constant, but
fluctuates to some extent even within the some testes. To better understandthe mechanism of spermatogenesis, it is worth while to study the number ofspermatogonial divisions and other related subjects.
For these reasons, the present experiment was conducted to estimate
the number of definitive spermatogonial mitosis using our usual experimental material, the CI08 silkworm strain. The testes used for observations
were obtained either from the 4th or the 5th instrar larvae. The preparations were sampled by the squash method and were observed directly, without
staining, under a phasecontrast microscope. If the total spermatocytes andspermatids within the cyst are counted, it would be easy to assume fromwhat cell type of the definitive spermatogonial period the cyst arose before
passing into the primary spermatocyte period.
As reported by other authors, synchronous cell divisions within the cyst
either for the secondary spermatogonia or the meiotic spermatocytes wasobserved. The size of the cyst is proportional to the total number of cells
within it, even in the rare event when the total number of cells vary from its
theoretical number. It was also observed that there are at least two different
types of spermatids per cyst: one of which is the type with 256 cells per cyst
as reported by Kawaguchi and another is the type with 128 cells. These
correspond to the six and five definitive spermatogonial divisions, respectively. According to a rough estimate of the frequency of these two types
within an individual, the former type was found greater than 75 per centand the latter type was observed less than 25 per cent. Although withlow frequency, the type with 64 cells was also observed. In any case, it
was concluded that the type with six definitive spermatogonial divisions is
the main process in this material.
To confirm the findings, calculation of the number of primary spermatocytes Gust prior to the first metaphase) per cyst was also made by using
the same material in parallel. From this observation, the number ofprimary spermatocytes per cyst could be classified into three different types,
among which the type with 64 cells which correspond to six definitive spermatogonial divisions was more frequently found than those for 32 and 16
cells which correspond, respectively, to five and four divisions. Thisfinding is not inconsistent with that obtained from the estimate for thenumber of spermatids per cyst.

.'

RESEARCHES CARRIED OUT IN 1970

59

Thus it can be concluded that, in the silkworm, the main type in thenumber of synchronous definitive spermatogonial divisions is six, but there
are relatively low frequencies of other types. However, the question still
remains as to which type takes part in the final fertilization.

60 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

VI.

RADIATION GENETICS IN ANIMALS

Effect of the moulting hormone on mutation induction anddevelopment of spermatogenic cells in the silkworm
Y. TAZIMA and Y. FUKAsE

It has been reported by Morohoshi et al. (1967, Jap. J. Genet. 42: 425)
that the moulting hormone promotes pupation as well as moulting in thesilkworm depending on the quantity of the co-existing juvenile hormone.
These findings prompted us to carry out an experiment to find out whether
the same hormone promotes the development of spermatogenic cells. We
found some years ago that the frequency pattern of mosaic and whole bodymutants was changing remarkably with the progress of spermiogenesis(Tazima and Onimaru, 1969, Mutation Res. 8: 177). Namely, until fifth
instar day 4.5 relatively more completes than mosaics were produced,
around V-4.5 the ratio of mosaics to completes reaching unity, and fromthis stage onward relatively more completes than mosaics were produced.
By using this relation as an index we may assess the development of germ
cells. The present experiment has, therefore, been attempted with two
moulting hormones, ecdysterone and inokosterone, which were kindly
provided by Prof. T. Takemoto of Tohoku University.
The hormones, dissolved in physiological saline solution, were injected
into the male larvae of CI08 at about the middle stage of the fifth instar.
The injected doses per capita were 15 r, 5 rand 0 r in 0.05 ml injection.
The hormone-injected larvae attained maturity one or two days earlier
than the non-injected control. .The injected group with saline matured
a half day earlier. All groups were irradiated simultaneously with 2000
R r-rays when the hormone-injected groups had attained maturity. They
were mated, after emergence, to pe re females for the detection of induced
mutants and the proportion of mosaics to total number of mutants wascalculated.
The results thus obtained were: in Expt. I 0.73±0.05 (ecdy. 15 r), 0.70±
0.05 (ecdy. 5 r) and 0.67±0.05 (saline only) and in Expt. II 0.62±0.07(ecdy. 15 r) and 0.61±0.05 (ecdy. 5 r) and 0.55±0.07 (saline only). The
difference between the hormone injected group and the control was not sta-

RESEARCHES CARRIED OUT IN 1970

61

tistically significant, but the regressions of the incidence of mosaics against
the injected dose were in good agreement between two experimental series.
From those results it can be concluded that ecdysterone has a promoting
effect on the development of spermatogenic cells of the silkworm.
We have also examined whether the moulting hormone increases ordecreases radiation induced mutation frequencies. Both ecdysterone andinokosterone were injected to male larvae at fifth instar days 5 and 6. Injected per capita were 5 r ecdysterone, 5 r and lOr inokosterone and no
hormone in 0.05 ml injection. Six hours after injection the larvae were
irradiated with 2000 R r-rays. Injection of ecdysterone 5 r increased
dearly the mutation frequencies over the saline control, whereas inoko-sterone failed to show its effectiveness in increasing mutation frequency.

Studies on strain differences in radiosensitivity in the silkworm
XI Sensitivity to dominant lethal induction
Y. TAZIMA and Y. FUKASE

Sensitivity to dominant lethal induction was compared among silkworm
strains having different radiosensitivity to embryonic killing, by irradiating
male germ cells at two developmental stages; mature sperm and young
spermatogonia.
For the irradiation of mature sperm, the strains used were Kansen andAojuku as the less sensitive and Sekko and rb as the highly sensitive strains.
X-rays were administered to male moths of each strain in four dose groups,'0, 5, 10 and 15 kR. In the experiment with young spermatogonia Kansen,
rb and their hybrid were used as materials. Doses administered were 0, 2
and 4 kR for both parental strains and additional 6 and 8 kR doses for the
hybrid.
Irradiated males as well as males of the non-irradiated control were-crossed to non-irradiated F I females of a hybrid between two commercial
breeds, FxB x 95. The use of such hybrid females ensured the deposition of non-defective eggs so that the dying embryos due to dominantlethals were easily determined.
Results: After the irradiation of mature sperm, no clear difference wasobserved among the four strains. In contrast, irradiation of young spermatogonia revealed a clear diffeernce between strains, exhibiting higher

62 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

incidence of dominant lethals in the rb strain. In the F) the incidence ofdominant lethals fell between the two parental strains. The results werein agreement with those obtained for recessive visible mutations, although
the difference between the strains was much reduced for dominant lethals.

Studies on strain differences in radiosensitivity in the silkworm.
XII Different modes of dominance observed in F) with regard toradiosensitivity at various levels of biological reaction
Y. TAZIMA

Using several radiosensitive and radioresistant strains of the silkworm,
which were selected with regard to embryonic killing, we have carried out
a comparative study in radiosensitivity at various reaction levels, i.e. sensitivity to embryonic killing (individual specimen level), cell killing (celllevel), dominant lethals (chromosome level) and mutation induction atmarked loci (gene level).
Among several findings obtained so far the most instructive was the contrasting mode of dominance observed between mutation induction andkilling of embryos by radiation in F) hybrids produced between two sensitive
strains. Namely, when F) hybrids, Kojiki X rb and Sekko X rb, were compared with the parental strains, both showed intermediate sensitivity withrespect to mutation induction, but overdominance with respect to embryonic killing even in the same hybrid combination.
Incidentally, as to germ cell killing either type of intermediate sensitivity
or overdominance was observed according to the combination of the hybrid. Kojiki X rb showed themselves to be intermediate, while Sekko X rb
belonged to the overdominant type. Although the above two hybrids have
not yet been examined, the sensitivity to dominant lethal induction wasseen to be intermediate in the case of F 1 between Kansen and rb.
These results could roughly be interpreted as showing that radiosensitivity
in F 1 represents the intermediate type at the gene level, the overdominant
type at the individual specimen level, and a transitional type at the cellularlevel.
The intermediate type of dominance observed for mutation induction seems
to suggest that a modification of gene action, responsible for pigment
formation in this case, by several intracellular and/or extracellular con-

RESEARCHES CARRIED OUT IN 197063

ditions, if any, is rather slight. Whereas, the possibility of such modification is fairly large in the case of individual specimen killing because thesame F I crosses exhibited overdominance with regard to embryonic killing.
The increased vigour of the F I for instance, seems to relieve the radiation
lesions by an appreciable degree leading to an increased survival.
It can be concluded from the above results that overdominance is theoutcome of the combined effect of several relevant factors even when eachcomponent allele acts additionally.

Studies on strain differences in radiosensitivity in the silkworm.
xm Radiation-induced breakage and rejoining of
single strand DNA
Y. TAZIMA, K. ONIMARU and H. KATO

In order to have a better insight into strain differences in radiosensitivity,
radiation-induced breakage and rejoining of single strand DNA have been
compared among different sensitivity strains.
For the sedimentation analysis the alkaline sucrose density gradientmethod was used with a slight modification of the procedure as proposedby McGrath and Williams (1966) following Terashima and Tsuboi (1967).
The cells used were spermatogonia and/or spermatocytes of one or twodays old third instar larvae. Labelling of DNA was performed by in vitro
culture of the testes in K6 medium containing 3H-thymidine with a specificactivity of 20 flc/ml for 5-6 hours.
The labelled testes were divided into three groups, each comprising 15,
in cold K6 medium: two groups were for X-irradiation (10 kR) and one
group for non-irradiated control. One of the two former groups was
incubated after X-ray irradiation in a fresh K6 medium for 30 or 45 minutes
at 25°C, so as to allow the rejoining of broken DNA strands. After appropriate treatment the testes in each group were torn to pieces in a hollow
glass and drop of culture medium was added. The mixture was then subjected to filtration through tissue paper. The cell suspension (0.1 m!)
was loaded gently on 0.2 ml of 2 % solution of sodium dodecylsulfate, which
was overlaid on 5-20 % sucrose gradient at pH12 in 5 ml cellulose nitratetubes. Gradients were centrifuged at 22000 rpm for 90 min. at room temperature in a swinging bucket rotor of a "Tominaga 88-500" centrifuge.

64 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

The contents were collected in about 30 fractions, to which cold 5 % trichloroacetic acid was added. The precipitate was washed on a millipore
disc with ethanol, and acetone-dried for liquid scintillation counting.
It was anticipated that the rejoining capacity of DNA strands might
differ among the strains reflecting the different radiosensitivities. The
results obtained so far are, however, contrary to this expectation. Eitherwith respect to strand breakage or to rejoining, no clear difference wasobserved among the seven strains. Even such most sensitive strains as
rb and Sekko have been found to have the capacity for rejoining single
strand breaks in DNA as was observed for the resistant strain Kansen.
Those results indicate that the differential sensitivity among silkworm
strains is not due to a differential capacity for rejoining single strand breaks
in DNA.

Relative biological effectivenss of 14 MeV neutrons
for recessive visible mutations in meiotic
spermatocytes of the silkworm
Akio MURAKAMI

Meiosis is a biologically important cell process in the course of gametogenesis. However, very little information is available as to radiationinduced mutagenesis in meiotically dividing cells, although there are numerous
published reports for either pre- or post-meiotic germ cell in various organisms. This situation is more pronounced with densely ionizing (or highlinear energy transfer: LET) radiations than with less densely ionizing (lowLET) radiation. Such being the case, there are few reports as to the genetic
RBE of fast neutrons in meiotic cells.
This communication presents the result of experiments on the RBE of
14 MeV fast neutrons as compared to mes ,-rays for recessive visible mutations in the early first meiotic cells at the first day of fifth instar larvae inthe silkworm. The ,-ray doses given were three different doses given
were three different doeses: 250, 500 and 1,000 R. For 14 MeV fast neutrons, irradiation was given at three different doses: 150, 250 and 540 rads.
Mutations at both the pe and re loci were detected by the egg-color-specificlocus method.
The cell at this stage showed extremely high radiosensitivity to cell de-

RESEARCHES CARRIED OUT IN 197065

generation which was not found in post meiotic cells. With 1,000 R of
r-rays and 540 rads of neutrons, more than ninety-five per cent of the cellin meiosis resulted in sterility. However, these effects were negligible withother lower doses either for neutrons or r-rays. Thus data presented
come from the remaining two low doses for both radiations. The average
mutation rate obtained on the two loci in this study was 10.9 X 10-7 for
r-rays. This rate was somewhat less than that of spermatids which havethe highest rate throughout spermatogenesis as observed by Tazima (1961).
The frequency of mutations increased non-linearly with dose for both radiations. Hence RBE was estimated tentatively by comparing the estimateddoses for each radiation at an arbitary mutation frequency level. The
RBE obtained in this manner is 1.8. This value is about one-third that inin post-meiotic cells or mature sperm and is the same as those in primordial
germ cells and in spermatogonia. This finding could be explained reasonably as due to an interaction between effects of germinal selection and sensitivity on irradiated cells.

Changes in ultraviolet light sensitivity to embryonickilling during early developing egg of the silkworm
Akio MURAKAMI

To analyse the relationship between UV sensitivity and embryonic development, the present experiment was carried out using silkworm synchronizedcell population during early embryogenesis. Embryos (or eggs) wereobtained from crosses between Cl08 strain wild type females and pe andre marker strain males. This mating procedure has the advantage of permitting the estimation of both cell killing in terms of lowered hatchability
and the measurement of the mutational incidence of the egg-color-specificloci of pe and reo The UV source was a germicidal lamp and the visible
light as the photoreactivating source was obtained from the fluorescencelight. These eggs were subjected to UV treatment with relevant timeintervals from immediately after oviposition up to 16 hr old eggs.
A marked change of UV sensitivity was observed with the progress ofembryonic development. Sensitivity to UV was relatively low in the first
10 hr after oviposition, and then it gradually increased with egg ages. Since
10 hr-old eggs, an extremely high sensitivity was observed. In the silkworm,

66 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

there is a well known fact that cleavage nuclei migrate somewhat into theinner part of the egg and those in the blastula stage move to the upper surfaceof the egg. Thus it can be concluded that these high sensitive stages correspond to the stage of blastula, while low sensitive stages correspond to
the age of eggs from meiotic oocytes until the blastula stage.
It is of interest to note that photoreactivation to UV damage was more
effective in eggs for UV sensitive stages than in those for resistant stages.
In addition, no cyclic change of UV sensitivity in eggs during early cleavage
stages (from 200 to 400 min after oviposition) was observed in accordancewith the cleavage division cycle. But the cyclical change of sensitivity toionizing radiation in this cleavage stage of the silkworm has been observed(Murakami, 1969). This suggests that the nuclei in the egg stage are notresponsive to UV killing because the nucleus is shielded by cytoplasm.
Although the cytoplasm was not so sensitive to UV, the death of cleaving
egg may be attributable, in part, to cytoplasmic injury following exposure
to UV. This finding is suggestive to the observation that the cytoplasm isable to repair the damage induced by ionizing radiation (Murakami, 1969).
In any case, the present study indicated that the effect of UV to embryonickilling is related to the event occurring within or governed by both nucleus
and cytoplasm in the silkworm egg.

Sex differences in X-ray sensitivity to embryonickilling of dormant silkworm egg
AIda MURAKAMI

Since 1965, we have been studying the radiosensitivity to embryonickilling of the silkworm and the results of these experiments have been reported
in various communications. On the occasion of the XII I.e.G., Dr. Oster
asked whether there is a difference in the sensitivity of embryonic killing toradiation between sexes. At that time, we did not have detailed data toanswer the question. However, it was empirically known that females
are more sensitive to radiation killing than males as well as other biologicaldamage in general. To answer this important question, experiments presented here were carried out, using X-irradiated dormant embryos of the
Cl08 silkworm strain. As this strain lacks the relevant sexual trait for
sexing the egg stage, the sensitivity could not be estimated separately inRESEARCHES CARRIED OUT IN 1970

67

males and females on the day of egg hatching. However, by the fifth instar,
the larvae could be sexed in the usual way by studying the ventral sex organs.
Such being the case, the sensitivity in irradiated embryos was estimated,on the first day of the fifth instar, as being due to the reduction of their survival fractions as compared with the control.
The dose-response relations for the egg-hatchability decreased exponentiallywith dose as reported in earlier papers. Surviving larvae were kept until
the fifth instar stage and their sexes determined to estimate the radiosensitivity in males and females. It was observed from this experiment thatthe female embryo is significantly more sensitive to X-ray killing than themale embryo. This difference in radiosensitivity may be interpreted as
being due to 1) difference in the sex-chromosome constitution: the femalesof this insect have ZW heterozygous chromosome constitution, while themale has ZZ homozygous, 2) difference in the direct and/or indirect physiological function of the sex chromosome and 3) difference in whether
recombination occurs or not as one of repair mechanisms in somatic cells
as well as in germ cells: recombination in the female silkworm is absent in
the germ cell; on the other hand this phenomenon is present in the male.
Fortunately, in the silkworm, there have been created various translocation strains for the purpose of establishing male-rearing method. Mostof these strains have translocations between the W chromosome which
has female determing factors and autosomal chromosomes which have
either egg or body color mutant genes. By using these strains, it would bepossible to estimate correctly the radiosensitivity between male and femalein egg and/or early larval stages. We have carried out experiments withthese strains. Similar but more pronounced difference in sensitivity between
sexes to that found in the C108 strain was observed in these strains. It
can be said from this finding that strains with translocations are moresensitive to X-rays than those free of translocated chromosomes.
Differential Nuclease Activities in Radiation-sensitive
and Radiation Resistant Silkworms
Susumu SAKURAI, Tsuneo KAnA and Tadahiko ANDo*Recent studies on recombinationless mutants of microorganisms suggestthat they are lacking in certain nucleases. Because the increased radiation* Institute of Physical and Chemical Research.

68 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

sensitivities of these mutants are due to the absence of recombination repair
mechanisms involving functions of specific nucleases, it appears to beinteresting to examine similar problems in higher organisms possessing different sensitivities to radiations. It is known that strains of silkworm
remarkably differ as to their radiation sensitivities (Tazima and Murakami,.
Gamm-Field Symp. 8: 53, 1970). Therefore the activities of some typicalstrains were analyzed.
Ten g of 3rd instar larvae were homogenized with 10 ml of 0.01 M TrisHcl buffer (pH 7.0) containing 0.01 M 2-mercaptoethanol and the supernantant solution after centrifugation at 8000 g for 10 minutes was collected.
Ten ml of crude extract were combined with 50 ml of 0.01 M Tris-HCI+
0.01 M 2-mercaptoethanol (THM buffer, pH 7.0), then 12 m1 of 5% streptomycin sulfate solution were added in the course of 30 minutes at cold.
Into the supernantant solution obtained by centrifugation (8000 G, 10
minutes), 27 g of powdered ammonium sulfate were added slowly in thecourse of 30 minutes at cold. The supernatant obtained by centrifugation(8000 G, 10 minutes) was treated again with 21 g of ammonium sulfate.
The precipitate obtained by centrifugation (8000 G, 10 minutes) was dissolved in 5 ml ofTHM buffer, and dialyzed overnight against 21 of the same
buffer at 4°C. The dialyzed enzyme solution was then charged on DEAEcellulose column (2.5 g, equilibrated in THM buffer, 1.5 cm X 20 cm) andeluted with 300 ml of THM buffer and 300 ml of THM buffer containing
0.4 M NaCI by gradient solution. Nuclease activities of 5ml fractions
were determined in transformation experiments with Bacillus subtilis.
0.1 ml fractions (previously diluted 10 times with phosphate buffer) weremixed with 0.1 ml solutions of transforming DNA (arg+) containing 6 mM
MgS0 4 and kept at 37°C for 10 minutes. The transforming activity (Arg+)
in 0.1 ml of each reaction mixture was determined using 0.9 ml of competentbacteria (HI7, arg- try-).
Two typical strains, Aojuku and rb were studied in detail. The former
is radiation-resistant while the latter is remarkably sensitive. Results obtained by fractionation experiments indicated that the strain Aojuku contained
two nucleases (tentatively named A and B) respectively at 0.02 Mat 0.10 M
NaCI region of elutes, on the other hand, the strain rb contained a nuclease
A' at 0.02 M NaCI region. Because it is supposed that A and A' may besimilar enzymes, the strain rb lacks in B nuclease. Further studies arein progress to elucidate the characters and specificities of these enzymes.

RESEARCHES CARRIED OUT IN 1970

VII.

69

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICROORGANISMS AND PLANTS
Biochemical Factors involved in the Repair of r-ray-induced
Damage in Transforming DNA of Bacillus suhtilis
Takehiko NOGUTI and Tsuneo KAnA

In an approach to understand the biochemical mechanisms involved in
the repair of DNA damage induced by ionizing radiations, we have carriedout experiments using toluene-treated cells of B. subtilis. Media, conditionsof irradiation and technics of transformation experiments had been previously described (Kada, Noguti and Namiki, Int. J. Radiat. BioI. 17: 407).
Cells of strain Hl7A (arg+ try-) were harvested at their growing phasein TF medium and were exposed to r-ray (30 kR) of mcs. Immediately
after irradiation, the bacteria were shaken in 1 % toluene-phosphate buffer(pH 7.4, 0.05 M) at O°C for 10 minutes and were collected by centrifugation.
The toluene-treated cells were incubated in phosphate buffer containing
various supplements at 37°C for repair of DNA damage. The complete
mixture contained 40 pM of each of four deoxynuc1eosides triphosphates(dXTP), 150 pM of NAD and 5 mM of MgCI 2 • Cell lysates containing
DNA were prepared as follows: Cells were first incubated at O°C for 30
minutes in a lysozyme solution (1 mg/ml in 0.5 M NaCI-O.02 M EDTA0.02 M Tris-CI-25 % sucrose), then incubated in the same solution supplemented with equal volume of Brij-58 in 0.01 M Tris-Cl (pH 7.2) at 37°C
for 20 minutes; the cellular suspensions were mixed with two volumes of
1 % SDS-O.I M Tris-Cl (pH 9.0); the resulting lysates were heated to 60°C
for 10 minutes and dialysed with a millipore filter (0.05 p of pore size) chamber against 0.15 M NaCI-O.OI5 M sodium citrate, then against the above
medium supplemented with 2 M NaCI for 2 hours. The dialysates werediluted and their transforming activities (Arg+) were determined using
HI7 (arg- try-) as recipient.
The transforming capacity in the lysate was inactivated to approximately
I/lO by irradiation but rose higher more than several fold in the course of the
37°C incubation in the complete mixture. Such an increase was not foundby removing anyone of the components (Table 1).

70 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Because 4dXTP are substrates for DNA synthesis by DNA-polymerase
and NAD is a co-factor required for DNA-ligase function in B. subtilis, theabove results strongly indicate that DNA-polymease (probably I) andDNA-ligase may be involved in the repair of r-ray induced damage of DNA
in B. subtilis.
Table 1. Requirements for repair of r-ray-induced damage of transforming
DNA in toluene-treated cells of B. subtilis. The complete mixture
contained in phosphate buffer 40 pg of each of 4dXTP,
150 pM of NAD and 5 mM of MgCb
r-ray
dose
(kR)

Incubation
mediumIncubationin minutes

Transformants
per ml ofcompetent
culture

0
0
0
0
30
30
30
30
30
30
30
30

Complete
Complete-4dXTP-4dXTP
Complete
Complete-4dXTP·4dXTP
-NAD
-NAD
-Mg++
-Mg++

0
10
0
10
0
10
0
10
0
10
0
10

11460
7320
9820
6300
1100
6440
1270
740
1170
1370
1360
980

Spores of rec- Strains of Bacillus subtilis
are Sensitive to UV or r-ray
Yoshito SADAIE and Tsuneo KADA

Since recombination repair is an efficient mechanism securing cellularresistance to irradiations, it may be interesting to ascertain, whether thiskind of repair mechanism is also operating in the high radiation resistanceof spores in which the conformation of DNA is modified so that they tolerate
well radiations.
A number of r-ray-sensitive mutants were isolated by treatment with
N-methyl-nitroso-N'-nitrosoguanidine from a derivative named H17(try- arg-) of Bacillus subtilis Marburg possessing wild radiation sensitivity.

RESEARCHES CARRIED OUT IN 1970

71

Two strains showing stable sensitivities to irradiations in the course ofexperiments over two years were named respectively M45 and L43. Cellsof M45 were exposed to transforming DNA extracted from strain 168
possessing wild radiosensitivity and bacteria showing intermediate sensitivities between those of M45 and HI? were isolated. One of them wasnamed RIO. All these mutants carry the same growth requirements (argtry-) as HI?
Bacteria grown exponentially in TF medium were harvested and theirradiosensitivities were determined. Spores were prepared in Schaeffer's
medium. r-irradiations were carried out with r-ray of mcs.
Inactivation studies with i"-ray showed that strains M45, L43 and RIO
were more radio-sensitive than their parental HI? (Table 1). Increased
sensitivities of mutants were also found with UV. That these mutants havegenetic recombination deficiencies was concluded on the basis of following
additional experiments. First, they all possessed Hcr+ capacities on UVexposed phage M2. Secondly, frequencies of transformation and transduction (with phage PBSl) were reduced in comparison with those found in
the wild strain.
We found that spores of rec- strains were sensitive to both r-ray and UV
when compared to HI? which is rec+ (Table 1).
The results given above show that the recombination repair is involved
in DNA damage of spores. It is noted here that Munakata and Ikeda(Biochem. Biophy. Res. Comm. 33: 3, 1968) have shown that spores of an
UV-sensitive strain, not involving ree character, were sensitive to UV but
not to r-irradiation.
Table 1.

Radiation sensitivities of rec- mutants of B. subtilis----~ ~----- - - -

---~

~-

---

"-

% of survivals
Strains

Vegetative cells

r-ray_ _ _ _ _--'-=(1=3c.:.3 kR)
H17
11.4
L43
0.118
M45
0.038
RIO
1.23

UV
(32 ergs·mm- 2)
49.9
0.240
0.182
26.9

Sporesr-ray(160 kR)
16.7
0.158
0.171
0.576

UV
(1776 ergs·mm- 2)
4.53
0.240
0.065
1.39

72 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Mutagenic Action of Phloxine
Tsuneo KADA, Y oshito SADAIE and Kiyoshi TUTIKAw A

Current hypothesis on radiation-induced mutagenesis suggests that mutagenic errors may be produced by recombination repair of DNA damage
induced by irradiations. If this hypothesis is true, it is supposed that any
chemical agent producing DNA damage subjected to recombination repair
may be mutagenic. It is expected that the lethal action of such a chemical
may be stronger in mutants deficient in genetic recomination capacity thanin wild strains. We have recently isolated a number of rec- mutants ofBacillus subtilis Marburg. A typical set of rec+ and rec- strains, 17A (arg+try-) and 45T (arg- try+) were used in these experiments.
An easy method of testing lethal actions of different chemicals was devised.
Papaer disks of 5 mm diameter were prepared by punching thick filter papers.
A disk was soaked in a drug solution and then placed on the surface of abroth agar plate. The plate was stood overnight at room temperature.
Cultures of strains 17A and 45T grown exponentially in TF medium werediluted 10 times and streaked separately from the edge of the disk. By
incubation for 24 hours at 37°C, grown bacteria became visible except the
inhibition zone in dependence on the strain and the drug used.
Studying a number of dyes, we found that phloxine, a red dye, inhibits
more the growth of 45T (rec-) than 17A (rec+). Further studies werecarried out using E. coli BJr WP2 try- to examine if pholoxine is reallymutagenic. Cells of the strain WP2 grown exponentially in broth were
washed, resuspended in phosphate buffer (0.067 M, pH 7.0) containing
phloxine at different concentrations and kept at 37°C for 20 minutes. Afterwashing out the dye by centrifugations, the cells were plated, after appropriate dilutions, on synthetic minimal agar supplemented with 1 %liquid broth to determine survivals and induced mutations. In a typicalexperiment, the number of viable cells per ml decreased from 4.8 X 108 to
1.3 X 108 by treatment with 30 pgJml of phloxine. The absolute number ofrevertants approximately doubled; their frequency per 108 viable cells was
5.0 for the non-treated control and 33.3 for the treated sample. By repeating similar experiments, we concluded that the dye is an active mutagen.

RESEARCHES CARRIED OUT IN 1970

73

Host-mediated "rec-test" for Chemical Mutagens
Kiyoshi TUTIKAWA and Tsuneo KAoA

Because different chemicals may be modified by in vivo metabolisms, it
is important to assess their mutagenecity in consideration of such modifications. Gabridge and Legator (Proc. Soc. ExptI. BioI. Med. 130: 831,
1969) first deviced the host-mediated assay of mutagenes using strains ofSalmonella. They injected intraperitoneally bacteria into mice that previously had received drugs through intramuscular injections and showedthat bacteria taken out from the peritoneum were mutated by certain drug
metabolities.
When experiments on mutation induction are carried out, contamination with other microorganisms must be avoided. Much more skill isrequired in a host-mediated assay of mutagenecity. On the other hand,studies on cellular sensitivities to chemicals are easy. In the previous report,
we have shown that chemicals possessing mutagenic capacities have increased lethal actions on rec- (strain 45T) over rec+ (strain 17A) cells ofBacillus subtilis. This relationship was adopted to the host-mediated assay
of potential mutagens.
A mixture (0.2 ml in volume) of rec+ and rec- bacteria were injected intraperitoneally into mice which had received a test drug through injection
into a leg muscle. After 60 minutes, the peritoneum was washed with 3 ml
portions of phosphate buffer and filled up to 10 mI. The number of survivals in this bacterial suspension was determined, by plating on basal medium
containing tryptophane (for 17A, arg+ try-) or arginine (for 45T, arg- try+).
Though the bacteria of both types were inactivated gradually even in thecontrol mice without drug injection, the existence of chemical activity
Table 1. Inactivations of rec+ and rec- strains of Bacillus subtilis
in the peritoneum of mice that had received muscle
injections with drugs% of survivals

Dose of drug
(mg/kg of mouse)

17A (rec+)45T (rec-)Phloxine

0
167

23.2
20.4

13.1
10.1

Butter yellow

0
100

32
6.2

38
10.8

Drug

74 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

producing damage subjected to recombination repair could be detected by
the difference of survival levels of rec+ and rec- cells.
Results obtained with phloxine and butter yellow are shown in Table 1.
Phloxine is a red dye whose in vitro mutagenecity on bacteria has recently
been confirmed by us. Butter yellow is a famous carcinogen. It is shownthat phloxine inactivated ree and rec+ cells to similar extents in mice, suggesting that this drug may not be an active mutagen in vivo at least at thehigh concentration examined. Butter yellow is inactive in vivo at leastas to production of DNA damage subjected to recombination repair.

F.fficiencies of some mutagens in maize
Etsuo AMANO

The starch character, wx in maize is expressed in the pollen grain andwild type recombinant pollen grains between independently induced wx
mutants are detectable, if present, by differential staining with iodine. Bysuch pollen analysis it has been shown that most of the ethyl methanesulfonate (EMS) induced mutants produced wild type recombinants whencrossed to standard wx stocks. The presence of many such recombinant
producers, good transmission and high ratio of leaky mutants suggest thatthe chemical mutagen, EMS, is very likely to induce base exchange mutations in maize. To compare the mutagenic effects and the genetic alterations induced with those of EMS, ionizing and ultraviolet (UV) radiations
were applied to maize as mutagen.
Since isolation of mutants as stocks were a primary requirement in thisanalytical study, experimental methods were modified to suit each mutagen.
Ionizing radiations were applied to dry seeds of a homozygous multiple
dominant marker stock (Yg 2 C I Sh Bz Wx, Y), and test crosses were madewith a recessive tester (Yg 2 C sh bz wx, y) to detect recessive mutation inendosperm characters. EMS soaking treatments were given to water presoaked seeds and the rest of the process was the same as in ionizing radiations. In both cases, treatment dosages were as high as to give 80 % survival in the treated generation. In case of UV treatment, pollen of the dominant stock were irradiated in a single layer by germicidal lamps with doseof 1 x 104 ergsjmm2 which was enough to decrease seed fertility to 50 % orless under the dark condition used. The irradiated pollen were used toRESEARCHES CARRIED OUT IN 197075

pollinate the same dominant stock (sib pollination), and self pollinatedears of the following generation were examined for the presence of mutationin the endosperm or other mutation detectable in the seed. Thus in thiscase the recessive lethal mutation could be tested besides the above
mentioned markers.
Since the experimental procedures were different among the mutagens
used, results summarized in Table 1 cannot be compared directly witheach other. Even with such reservations and also those on treatment dosage,
EMS seems to induce more mutations than other mutagens. It has beenreported (Amano and Smith, Mutation Res. 2 (1965) 344-351) that fission
fast neutrons effectively induced mutations in loci C, sh and wx. However,recent endeavors to induce mutations in the wx locus with radiations were
not successful except only one wx kernel found so far in a r-ray treatmentgroup. An other finding to be noted was the high incidence of recessive
lethals induced by UV treatment. As the UV treatment and other procedures were carried out under dark condition (yellow sodium lamp in a
darkroom and dim dry cell head lamp for night pollination in the field),photorepairable damage might be fixed as mutation. Photo-repairabilityof this mutation should be further investigated.
Table 1.

Results of mutagen treatments in maize.
for several years.

Summing up of experiments-----

MutagenX-, r-rays
Thermal neutrons
UV
EMS

*.
**.

Fertilecobs
examined

Mutations
C

sh

wx

multipleothersfrequency (%)

2203
453
690
2270

0
0
0
19

1
0
2
8

1
0
0
20

0
0
0
1

-t-*
(53)**

0.045
0.00
0.00
0.925

wx mutation

Abnormally shaped kernels were often found.
Out of 479 ears of 1970 harvest. Many of them were recessive lethals.

Mutation Experiment in Capsicum annuum
Etsuo AMANO

Transfer or alteration of a genetic factor in a scion by grafting wasoften observed in Solanaceae. Recent studies on transformation inhigher plants seem to give further support to these observations. To

76 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

study the basis of mutations obtained by grafting, mutants were newly
induced in Capsicum annuum var. Sapporo Wase. Use of chlorophyll ormorphologically mutated seedlings within a single variety should makeeasy the progeny test on a simple genetic background. Since structural
alterations in such genetic markers may reveal the efficiency of graft mutation, two mutagens with different effects, r-ray and ethyl methanesulfonate(EMS) were used. The chemical mutagen, EMS is a very effective mutagen
and is known to induce point mutations in higher plants.
Seeds were presoaked in distilled and deionized water for 24 hours before
soaking them in EMS solution (up to 0.1 M) for 5 hours. After soaking
in water for 24 hours at the same 27°C as in other treatments, seeds weresown in sterilized soil in wooden flats. The seedlings were kept in the flatsso that their growth was suppressed. Each plant gave only one fruit.
To induce larger structural alterations, r-ray irradiated samples were used.
Fruits were collected from plants irradiated in a r-greenhouse with a 137Cs
source. Chronic irradiation was used in expectation of accumulation of
mutations.
Mutant seedlings were surveyed in wooden flats in the greenhouse. Eachfruit was considered to represent a single mutational unit. Two morphological, four lethal and three viable chlorophyll mutations were foundamong 746 fruits from EMS treatments. Out of 531 fruits from r-rayedmaterials tested, 210 gave more than four seedlings but none segregated the
expected mutant. Chronic r-ray doses used were more than 5 kR as meassured by glass dosimeters.
Some preliminary graftings were tried on normal green plants. Graftings of two green seedlings (control) and three scions of each stock (selfgrafting) were successful but none of the four lethal chlorophyll mutants(12 graftings in total) gave a result.

Effect of Temperature during the Split Dose Interval ofGamma-Rays in Maize Pollen
Taro Fum

Recovery of premutational damage in maize pollen grains by fractionatedgamma-ray treatments with 2 hours interval have been already reported(Ann. Rep. Natl. Inst. Genet. 20, 86-88). In order to get more information

RESEARCHES CARRIED OUT IN 197077

about the apparent loss of premutational damage in both whole and partial
mutations, the effect of temperature in the interval period was investigatedin the present experiment. Material was the same as in the previous experiment, namely, mutation from Bz to bz was examined. The interval
time was settled as 2 hours since a significant decrease in mutation frequency
was usually observed under this condition, and three different dosages,
0.5, 1 and 2kR in total, were applied to 9heck the relation dose versus mutation frequency. Irradiated pollen grains in three single dosage lots weredivided into two parts. Prior to pollination on the female side, one halfof the irradiated pollen grains was kept under a low temperature (about
5°C) for 2 hours and the other part was kept under room temperature (about
28°C) for the same duration. Similar treatment was also applied to the fractionated lots, namely one half of pollen grains irradiated with one half ofsingle dosage were kept under room temperature for a given interval, while
the other half of the pollen grains were stored under low temperature before
the second irradiation. Pollination was carried out after those treatments.
The total mutation frequency increased by increasing the dose from
0.5 to 2 kR, both in single and fractionation series, but it was always
lower in fractional than in single treatment. Furthermore, the storage
temperature of pollen grains did not affect considerably the mutation
frequency; similar mutation frequencies were observed in room and low
temperature lots of each dosage for single or fractionation series as wellas in non-irradiated control lots. The results suggest that the reductionof gamma-ray induced mutation under fractionation conditions occurs
merely for the whole mutation and that low temperature treatment does
not prevent recovery. However it is very difficult to imagine at present
that the supposed recovery would usually involve the enzymatic processus
because no temperature dependency was observed at least in the rangeof 5-30°C.

Tissue Culture from Wheat Anthers and Roots
Taro FUJII

Callus induction from anthers was observed in di-, tetra- and hexa-polid
species. Anthers containing pollen grains at tetrad stage were planted in

78 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

a test tube on modified White's medium supplemented with 20 mgll 2,4-D
and solidified with 9 gil agar. Casein dehydrogenase, one of the known
efficient substances for callus growth, was added to the medium at concentration of 1 gil. Callus formation from the anthers of two wild species,
Triticum aegilopoides (diploid) and T. dicoccoides (tetraploid), was significantly higher on this medium than when no casein was added. Furthermore, callus formation was observed in anthers of T. durum, while cultivatedtetraploid species did not form any callus without casein. No callus was
yet observed, so far examined, in three cultivated species, T. monococcum(diploid), T. spelta and T. vulgare (hexaploid) like in the previous experiment (Ann. Rep. Natl. Inst. Genet. 20, 91-92). The result suggests thatthe callus may be induced with certain chemicals specifically required.
To obtain an information on radiation sensitivity of cultured cells, calluses induced from dipolid wheat roots were irradiated with 0.5, 1, 2 and5 kR of gamma-rays, because callus growth induced from pollen grains isvery slow and is not produced in quantities is large enough for irradiation
experiments. Irradiated calluses were inoculated again on the same medium(modified White's with 2,4-D), and the growth rate was measured by theincrease of wet weight 70 days after the treatment. In the control lot, weightincrease was about twice as large as the initial one, but about five times aslarge increase was observed in 0.5 and 1 kR lots. Growth rate in 2 kR
lot was almost the same as that of the control lot, and 5 kR irradiation
diminished growth rate amounted to 1.5 times of the initial weight (datafrom two repetitions). Although the growing plants or wet tissues arerather more sensitive to radiation than dry seeds, for instance LD 50 of diploid
wheat seedlings is less than 1 kR, stimulation of callus growth was observedin lower dose lots. Similar growth rate as that of the control lot was observed at 2 kR irradiation, a dose beyond LD loo for seedlings. Such resistance in cultured callus cells was reported for tobacco (Venketeswaran
and Partanen 1966, Radiat. Bot. 6, 13-20). High radio-resistance in callus
cells might be partly due to that the undifferentiated cell population which
can grow by the growing point had substituted the undamaged cell for thedamaged cell, whereas in the seedlings the growing point was already differentiated.

RESEARCHES CARRIED OUT IN 1970

VIII.

79

POPULATION GENETICS (THEORETICAL)

The length of time required for a selectively neutral mutant
to reach fixation through random frequency drift
in a finite population
Motoo KIMURA

Frequency distribution of the length of time until fixation (excluding
the cases of eventual loss) of a selectively neutral mutant in a finite population was obtained based on the solution of Kimura (1955) on the processof random genetic drift. With 4Ne generations (N., effective populationsize) as the unit length of time, the distribution has mean ,111 = 1, standard
deviation a=0.538, skewness 1.11 = 1.67 and kurtosis 1.12=4.51 (Fig. 1). For
details, see Kimura (1970), Genet. Res. Camb. 15: 131-133.

1.0

y
0.5

0.0 L.L_~_ _~_ _~_ _~""'============
0.5
1.0
1.5
2.0
2.5
3.0
3.5

t/(4N e )Fig. 1.

Probability of fixation of a mutant gene ina finite population when selective advantage
decreases with time
Motoo KIMURA and Tomoko OHTA

A theory was developed which enables us, for the first time, to obtain
the probability of fixation of a mutant in a finite population when its selective advantage decreases at a constant rate with time. The theory is based

80 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

on diffusion models and it can be extended to cover a more general casein which the selective advantage can be expressed as a function of the exponential function of time.
Let u(P) be the probability of ultimate fixation of a mutant allele withinitial frequency p and having selective advantage se- kt at time t. Then,assuming that p is small, the fixation probability in a population of effective
size N e is given approximately byu(p)={ Y/(l-e- Y )} p ,where Y is the solution of the differential equation
dY
dS

I(S- Y)(l_-----:~~~)_
KS[l-(l+ Y)e-

Y],

in which S=4Ne s and K=4Ne k. This equation was solved numericallyand the values of Yare tabulated for various combinations of Sand K.
When Sand K are large, the following approximation formulae were founduseful to estimate Y: (i) K<f-.S, we have Y-;::::,S-K, while (ii) if S~2K, wehave Y/(l-e- Y )-;::::,e S /(K+2).
In order to check the validity of the theory, an extensive computation
was carried out by using the method of multiplying the transition probability
matrices, and the result turned out to be satisfactory.
As an application of the present theory, the probability of establishing a
new inversion initially containing an exceptionally small number of deleterious genes was worked out. For details, see Kimura and Ohta (1970),
Genetics 65: 525-534.
Genetic loads at a polymorphic locus which is maintainedby frequency-dependent selection
Motoo KIMURA and Tomoko OHTA

If a polymorphic locus is maintained in finite populations by frequencydependent selection with selective neutrality at equilibrium, it is generallyaccompanied by two genetic loads, that is, the dysmetric and the drift
loads. The former arises because the fitness of the population may not
be at a maximum at the equilibrium gene frequency and the latter because
genetic drift in small populations displaces the gene frequency from its
equilibrium value.

RESEARCHES CARRIED OUT IN 197081

In some simple models of frequency-dependent selection considered, thedrift load is independent of selection coefficients and is approximately equalto (n-l)/(2Ne), where n is the number of alleles and N. is the effective
population size.
For details, see Kimura and Ohta (1970), Genet. Res. Camb. 16: 145-150.

Statistical analysis of the base compositionof genes using data on the amino acidcomposition of proteins
Tomoko OHTA and Motoo KIMURA

A statistical method was developed to estimate base composItIOn ofgenes (cistrons) from amino acid composition of proteins. The method
enables us to obtain base frequencies in the first and second positions ofcodons through an iterative process with a computer. However, withrespect to the second position of codons, the G-C (guanine and cytosine)content can be estimated directly and without error by a simple formula.
Using this method, 17 vertebrate proteins were analyzed to obtain the
distribution of base frequencies among genes in the vertebrate genome.
The average G-C content turned out to be about 52 % for the first position
and 43 % for the second position of codons. Their variances are higher
than expected. It was also noticed that adenine content is almost always
higher than uracil content, indicating different base composition between
two complementary strands of vertebrate DNA. The problem of randomness in base arrangement in relation to molecular evolution by random
drift and selection was discussed.
For details, see Ohta and Kimura (1970), Genetics 64: 387-395.

Development of associative overdominance through linkage
disequilibrium in finite populations
Tomoko OHTA and Motoo KIMURA

Associative overdominance arises at an intrinsically neutral locus throughits non-random association with overdominant loci. In finite populations,even if fitness is additive between loci, non-random association will becreated by random genetic drift.

82 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

The magnitude of such associative overdominance is roughly proportional
to the sum of ai's between the neutral and the surrounding overdominantloci, where ad 2 is the squared standard linkage deviation, defined between
any two loci by the relation
ai=E(D 2 )IE{p(l-p)q(1-q)} ,in which p and l-p are frequencies of alleles Al and A 2 in the first locus,
q and I-q are frequencies of alleles B I and B 2 in the second locus, and D

is the coefficient of linkage disequilibrium. A theory was developed basedon diffusion models which enables us to obtain formulae for ai under various
conditions, and Monte Carlo experiments were performed to check thevalidity of those formulae.
It was shown that if Al and A 2 are strongly overdominant while B I and
B 2 are selectively neutral, we have approximately

ai= 1/(4Nec) ,provided that 4Nec~ I, where N e is the effective population size and c is therecombination fraction between the two loci. This approximation formulais also valid between two strongly overdominant as well as weakly overdominant loci, if 4Nec~ 1.
The significance of associative overdominance for the maintenance ofgenetic variability in natural populations was discussed, and it was shownthat Nes', that is, the product between effective population size and the coefficient of associative overdominance, remains constant with varying Nco
if the total segregational (overdominant) load is kept constant.
The amount of linkage disequilibrium expected due to random drift inexperimental populations was also discussed, and it was shown thatai= l/(n-l) in the first generation, if it is produced by extracting nchromosomes from a large parental population in which D=O.
For details, see Ohta and Kimura (1970), Genet. Res. Camb. 16: 165177.
The rate of decay of genetic variability in a two-dimensionallystructured finite population
Takeo MARUYAMA

The rate at which a population approaches homozygosity is an importantnumber in population genetics. In particular, the asymptotic rate is related

RESEARCHES CARRIED OUT IN 197083

to the amount of genetic variability maintained in a population and to the
speed of gene substitution. Previously, I have studied the rate for a population linearly subdivided into colonies.
Populations occupying two-dimensional habitats however are biologicallymore interesting than linear cases. In this note I want to investigate suchcases of torus-like lattice structure. Considering the direct product of two
circular lattices with nl points and with n2 points, assume that a populationconsists of nln2 colonies and that each colony occupies a grid point of thelattice. Assume furthermore that there are N diploid individuals in eachcolony.
Let m be the rate at which a colony receives immigrants from its fourneighboring colonies in one generation. It receives immigrants from eachat a rate of mj4 per generation. Let fW be the probability that two randomly chosen homologous genes, one each from colonies which are i-steps
apart in one dimension andj-steps apart in the other, are identical by descent.
Letng l be the probability of identity by descent when the two genes are
sampled from a single colony without replacement. If we assume that the
mutation rate is u and every mutant is new to the population, it can beshown that the average value of f~g lover a long period time is given by
I' JO

l'

1(Tfltld _S
00
t~ 2NT +S= ;~-TJo

where

with

~kl=[I--i-(1 - cos 2: k)J[I- ~ (1 - cos 2~I)J
1

and N T=Nnln2'

Now lethU =I-fW·
1

Assuming that reintroduction of lost alleles into the population by
mutation or migration does not occur or occurs at a negligibly small rate,
we are interested in determining the asymptotic rate at which the hU I approaches zero. If we let H(t) be the matrix [hW]"lX"2' we want to determine a con stant A such that

84 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

for large t.

According to Robertson (1964), we have

l_A=_I~ lim J-[o.:=-.!--lim
1-/0
2NT u I0 1-f
2N T u I0 1-} -/0 .

(1)

4N T uapproximately. Several examples of the value of I-A calculated by (1)and the exact value obtained by an iteration method are compared in Table 1.
Table 1. Numberical comparison of the eigenvalues
obtained by formula (1) and the exact value
nl Xnz

20x20
50x50
50x50
50x50
90x90
90x90
190 x 190

N
5
5
5
25
5
10
5

mN
0.1
0.1
1.0
5.0
1.0
4.0
2.0

Eigenvalue (1-,l)lim 1-/;/1-/
ulO

0.163
0.131
0.612
0.889
0.578
0.852
0.710

from (1)exact value

2.039 x 10- 5

2.040x 10-5
2.616 X 10-6
1.222x 10- 5
3.554xl0-6
3.570 X 10-6
2.632 X 10-6
8.920 X 10-7

2.610x 10- 6
1.223 x 10- 5
3.556 x 10-6
3.566 x 10- 6
2.631 x 10- 6
8.931 x 10- 7

The values by the two different methods agree remarkably well.
Let j be the probability that randomly chosen two homologous genes
from the entire population are identical by descent. Then the value ofratio (1-/0)/(1- j) is of considerable interest, because it tells us whether
or not the population behaves like a panmictic one. I have calculated alarge number of numerical examples and have found a fact that when thenumber of colonies is sufficiently large, the value of ratio depends only on
the value of mN (number of migrating individuals), and it is almost independent of number of colonies. Furthermore when mN~ 1, the value of(1-/0)/(1- j) is approximately equal to unity. Therefore if a population
is of two-dimensional and mN ~ I, it behaves very much like a randommating population, even if the population consists of large number ofcolonies. It is interesting that in a one-dimensional population, the ratio
depends on both mN (local property) and the number of colonies (global
property), but in a two-dimensional case it depends only on mN (local pro-RESEARCHES CARRIED OUT IN 197085

perty). Several examples of one-dimensional and of two-dimensional
cases are listed in Table 2.
Table 2.
nl Xn2

Examples of numerical values of lim 1-1011 -I
ujO

N

mN

lim 1-/0/1-/

10
50
10
50
5
5
5
5
10
5
5
50
50
20
50
20
100
100
200
100
5
5
5
50
500
5000

0.01
0.01
0.01
0.03
0.05
0.1
0.1
0.1
0.2
0.2
0.2
1.0
1.0
5.0
5.0
5.0
10.0
10.0
50.0
50.0
0.5
0.5
0.5
0.5
50.0
50.0

0.01270.01140.01110.03290.0496
0.11450.09550.08110.20540.17550.15880.56380.50360.86030.83300.82420.90900.90080.98630.98090.10110.05010.02080.10070.92450.9237

ujO--~-----

100 X 100
200 X 200
500 X 500
200 X 200
500 X 500
loox 100
300 x 300
1000 x 1000
100 x 100
300 x 300
1000 x 1000
loox 100
500 x 500
200 x 200
500 x 500
1000 x 1000
500 x 500
1000 x 1000
100x 100
1000 x 1000
100 x 1200 x I
500xl
100 x I
100 x Iloox 1In the case of one-dimensional circular population studied, it was shownthat if the number of colonies is large, the dominant eigenvalue can be approximated as the smaller of mrr 2/n 2 or 1/2Nn, where n=number of colonies.
An analogous feature seems to exist in the two-dimensional case studied
here. Here we assume that the number of colonies along both axes are thesame, i.e., nl =n2 == n. If mN < 1 the value of 1-A is approximately equal
to m/n2 and if mN> 1 it is approximately equal to 1/2Nn 2, i.e.,86 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21mnl-A~-2( more precisely l-A< 2m)n2
if mN<l1
l-A~2Nn2( more precisely l-A<2~n2)
if mN>l.

This relationship is illustrated in Figure 1.
I

Nn l "2

.. ..

--<
(a)

. ..

"I = "2 = 200

I

N = 100
10-a

10-9
0.01

10

0.\

100

mN
10- 6

m
"I "2

10- 7

(b)

--<
I
-

/.,<i'~"I = "2= 200
10-8m = 0.01

I09L..-...J....

......J

0.01

0.1

I
mN

..l-

...,;

10

100

Fig. 1. The relationship between I-A and mN. The lines indicate thevalues of the approximations discussed in the text. In (a) the value of N is fixed
and the value of m is varied, while in (b) m is fixed but N is varied.

RESEARCHES CARRIED OUT IN 1970

87

Let us now assume that a population is divided into partially isolated ncolonies of equal size N. Then the inequality 1-). ~ 2mjn holds for a moregeneral situation. The equality is atained if hU I <hljl and hU I zhlij, foreveryi*j and i'*j'. This seems to be the case in a two-dimensional population with very small amount of migration, i.e., mN < 1.

88 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

IX.

POPULATION GENETICS (EXPERIMENTAL)
Deleterious genes in natural summer and autumn
populations of Drosophila meianogaster 1J

Chozo OSHIMA, Takao K. WATANABE and Jong-Kil CH00 2 1

Many flies were collected at three sites in Katsunuma locality on July
31st and October 12th of 1970. The summer population was not verylarge, but the number of flies increased remarkably since September andthen the autumn population became very large.
Many second chromosomes, extracted from each male fly, were analyzed
by Cy-Pm method and the results are given in Table 1.
Table 1. Frequencies of deleterious and normal second chromosomes
isolated from summer and autumn populations of D. melanogaster~~~lethalsemilethal

subvital

normalNo. ofchromosomes
tested14(32.6)
21 (42.9)
35 (32.7)

4 ( 9.3)
3 ( 6.1)
10 ( 9.3)

4 ( 9.3)
2 ( 4.1)
7 ( 6.5)

21 (48.8)23(46.9)55 (51.4)

43
49
107

70 (35.2)

17 ( 8.5)

13 ( 6.5)

99 (49.7)

199

Frequency (%)
SeasonSite-----------------------

Marufuji
Summer Koshiien
Minaminoro

--~-

Total

--

Marufuji
Autumn Koshiien
Maruki21 (21.0)
15 (25.9)
19 (34.5)

12 (12.0)
10 (17.2)
8 (14.5)

10 (10.0)
5 ( 8.6)
2 ( 3.6)

57 (57.0)
28 (48.3)
26 (47.3)

100
58
55

Total

55 (25.8)30(14.1)

17 ( 8.0)

111 (52.1)

213

The frequency of lethal chromosomes in the summer population was muchhigher and also that in the autumn population was higher than the meanfrequency in the autumn populations in the previous years.
The frequencies of allelism were determined by half diallel crosses between
65 lethal strains isolated from male flies of the summer population and 89
lethal strains isolated from male and female flies of the autumn population.
The results are given in Table 2.
112)This work was supported by a grant (0801) from the Ministry of Education.
Visting researcher, Department of biology, Chung-Ang University, Seoul, Korea.

RESEARCHES CARRIED OUT IN 1970
Table 2.

89

Results of allelism between lethal strains
Summer population

Autumn population

65
2,080
171
8.22

89
4,005
133
3.32

------------------------------------------_~_--

Summer population
Allelic group
No. of lethal genes
I
II

III

IV
V

VI
Single letahl gene

Autumn population
Allelic group
No. of lethal genesI8
14 (2*)II
7III
5
11 (8*)IV
7 (7*)V
4
VI
7
11 (3*) - - - - - V I I
10
VIII
5
7
4
{I~
2
XI
3
2
XII
2
2
XIII
2
18 (27.7 %)Single lethal gene
24 (27.0%)------*:

visible mutant rbl geneallelic relationshipThe high frequency of allelism decreased with the frequency of lethals in
the course of about two months. The frequency of single lethal genes,
which had no allelic genes, was about 27 per cent in both populations.
Other clustering lethal genes could be divided into 6 and 13 groups in thesummer and autumn populations, respectively.
The representative 10 lethal strains of the summer populations were crossed
diallelly with all 89 lethal strains of the autumn population and the frequencyof allelism was 6.62 per cent. It was recognized that many lehtal genes ofallelic groups I, II, III and IV of the summer population were allelic respectively with those of allelic groups II, IV, VII and X, XI of the autumn population. These lethal genes have persisted for about two months and were
distributed over about 106 square meters in Katsunuma locality.
Most chromosomes of allelic group II in the summer population and all
chromosomes of allelic group IV in the autumn population had not only
an allelic lethal gene, but also a visible mutant gene for reduced bristle (rbl).

90 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

A few chromosomes of group II in the summer population were proved tohave at least three deleterious genes.

Sterility genes in natural summer and autumn populations of
D. melanogaster 1 )Chozo OSHIMA, Takao K. WATANABE and Masaoki KAWANISHI

Frequencies of sterile male and female flies among several hundred flies
collected simultaneously from the summer and autumn populations of1970 in Katsunuma locality were found to be 4.3, 4.8 per cent and 3.4,
7.8 per cent respectively.
Among 342 second chromosomes extracted from each male fly in summer
and autumn populations of 1970, 69 chromosomes were found to be sterility
chromosomes. The results are shown in Table 3.
Table 3.

Frequency of sterility chromosomes and frequ~ncies of male,
female and both sexual sterility chromosomes1970 (July, October)Collection time
Class of viability

Semilethal

Subvital

Normal

Total

46
13
(28.3)

4112(29.3)

255
44(17.3)

342

No. of tested chromosomes
No. of sterility chromosomes
Frequency (%)
No. of male sterility chromosomes (%)
No. of female sterility chromosomes (%)
No. of both sexual sterility chromosomes (%)

69(20.2)

32 (46.4)
27 (39.1)
10 (14.5)

The frequency of sterility chromosomes was higher than 12.6 % in 1968.
By half diallel crosses between sterility lines, which have been maintainedby the Cy-sterility balanced system, the frequency of allelism was determinedin the summer and autumn populations as Table 4 shows.
The sterility strains, which were extracted from a natural populationin Katsunuma locality in 1968 and have been maintained in our laboratory,
were crossed diallelly with new sterility strains extracted from the summer
populations of 1970. On the other hand, diallel crosses between the sterility
strains of the summer and autumn populations were performed.
1)This work was supported by a grant (84097) from the Ministry of Education.

RESEARCHES CARRIED OUT IN 1970
Table 4.

91

Results of allelism between the strility genes
July 31

Collection timeSex
No. of sterility chromosomes
No. of crosses
No. of allelic crosses
Frequency of allelism (%)
Frequency of finding
1
2
3
5
7

October 12

Si?-

(l)

Si?-

(l)

II
55
10
(18.2)

19
171
1
(0.6)

24
276
14
(5.1)

23
253
26

6

No. of sterility genes
17
14
1

1
1

(10.3)

10
2**

1*
1

. persistent sterility gene.

*, **.

One male sterility gene (ms 801) and one female sterility gene (fs 801)
have persisted for two years. These male sterility genes (ms 202, B, C)
in the autumn populations of 1970 were linked with different female sterility
genes (fs 206, fs 217). Three different male sterility genes in the summer
population of 1970 were found to be combined in double sterility chromosomes(mss 203 A-C). On the other hand, a female sterility gene (fs 102) has increased in the autumn population. The results are shown in Table 5.
The breeding pattern of D. melanogaster in Katsunuma locality has been
scarcely known, but the places of hibernation and the dispersion of flies
were probably localized in the district.
Table 5. Persistent and allelic sterility genes during two years
in Katsunuma locality
Collection date

Oct. 2, 1968

July 31, 1970

Oct. 12, 1970

Female sterility gene

Is801 A-F - - - - I s 102 - - - - - / s 201 A-E *(6 chromosomes)(1 chromosome)(5 chromosomes)

Male sterility gene

ms 801 A-M(13 chromosomes)

allelic relationshipms 202 A **I--ms 202 B·Is 206
ms 202 C·Is 217(3 chromosomes)
ms 114I
mss 203 A
ms 103=\_ _\--mss 203 B
ms 102--mss 203 C

ms 113(1 chromosome)*, **.. persistent sterility genes.

I_92 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Correlation between productivity and viability of
D. melanogaster
Takao K. WATANABE

A total of 224 second chromosomes were extracted from a cage population,
which has been maintained at constant 25°C for about 30 generations.
Viabilities and productivities of homozygotes and heterozygotes for thesechromosomes were estimated.
The mean viability of homozygotes for female sterility chromosomes
was remarkably lower than that of other homozygotes and they reduced
significantly the productivity of male flies, but on the contrary the homozygotes for male sterility chromosomes did not show such an effect on theviability and the productivity of female flies.
The correlations between productivities of both sexes, being homozygous
and heterozygous for all chromosomes, were positive, and the correlationin the homozygotes was significant at 5 per cent level.
The correlations between productivity and viability of male and female
heterozygotes and female homozygotes for all chromosomes or fertility
chromosomes were not significant, but the correlations between productivity
and viability of male homozygotes for all chromosomes or fertility chromosomes were positive and significant at 5 per cent level and 1 per cent level
respectively.
Sterility genes of both sexes might be slightly deleterious in heterozygous
condition for productivity of the same sex, but they should be neutral on
viability.
Fecundity and longevity of D. melanogaster under constant andfluctuating temperature environments
Chozo OsmMA and Masaoki KAWANISm

Two cage populations were set up with about 1,000 F 2 flies from 192
female flies collected in October of 1966 from natural populations in Kofu
and Katsunuma localities.
One cage population (F) has been maintained in an incubator, whose temperature fluctuated two times a day between 20 and 30°C by a program
controller. Another cage population (C) has been maintained in theculture room, whose temperature was kept constant at 25°C.

RESEARCHES CARRIED OUT IN 1970

93

After about two years, flies were sampled and used in the present experiment. Flies sampled from C population were divided into two groups
and one group was exposed to constant temperature (C-c group) while
another group was exposed to fluctuated temperature (C-f group). Flies
sampled from the F population were similarly exposed to fluctuated temperature (F-f group) and to constant temperature (F-c group).
Individual longevity and fecundity of many female flies and hatchabilityof eggs laid in each vial were estimated under these constant and fluctuating
temperature environments. The results for the four groups were compared.
The mean longevity of female flies in the F-f group was 31.3 days and thatin the C-c group was 29.0 days. The longevity in constant temperature
was generally shorter than in fluctuating temperature, but the mean longevityof female flies in the F-c group was remarkably reduced being 25.7 days.
The fecundity and the longevity of female flies may be closely related quantitative characters. Young female flies in the C-c group laid significantly
more eggs until they were 10 days old than in the C-f group. However,
the total number of eggs laid for 40 days in the F-c group having the shortest
mean longevity was smaller than in other groups.
The hatchability of eggs laid by young female flies (1-10 days old) in allgroups was higher than 90 per cent and no significant difference was found.
However, the hatchability of the C-c group decreased faster with the ageof female flies and that of old female flies (20 days old) was 67 per cent.
'On the other hand, that of old female flies (20 days old) of the F-f group
was 81 per cent.
Longevity, fecundity and hatchability may be polygenic characters.
When their variances of all groups were calculated and compared witheach other, homeostatic gene systems of flies in these F and C populations
could be expected. The degree of the system in the former population
was recognized to be higher than in the latter population, but the charactersin the former population varied in manifestion under constant temperature·environment. (Published in Environ. Cotrol in BioI. 7:21-29, 1969).

94 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Effect of light on oviposition of D. virilis
Chozo OSHIMA and Koji INOUE!)

Many flies of D. virilis were collected from a warehouse of a brewerycompany in Tokyo in the spring of 1970. A cage population was set up
with them and has been maintained in the culture room, whose temperature was kept at 25°C. After three months, 12 lines were established withthe offspring of different female flies extracted from the population. Ninepairs, isolated from each line, were divided into three groups. When they
were six days old, they were treated with three kinds of light conditions;
1. constant light (2500 Lux), 2. dark (I5-minute light/day), and 3. periodical
light and dark (9 hours 2500 Lux, 9 hours 0 Lux, 3 hours 2500~0 Lux).
The first two groups were cultured under constant light and dark conditions, and the third group was cultured in an incubator, whose light
condition was controlled periodically by a program controller. The experiment was performed at constant 25°C.
A pair of male and female flies were placed in a creamer and transferred
every day to a new one. Eggs laid on a culture medium were scored. The
experimental results for ten days are given in Tables 6 and 7.
The character of oviposition was remarkably affected by light conditions
and it was observed that the mean number of eggs per fly increased linearlyin the order of constant light, periodical light and dark, and dark
(I5-minute light/day) conditions. The phenotypic variances under those
three conditions were significantly different and the variance under theperiodical light and dark condition was the smallest while that underdark(I5-minute light/day) condition was the largest.
Daily fluctuations of oviposition under these three conditions were comTable 6.

Mean number of eggs per day per fly under threekinds of light conditions

Condition

Mean numberof eggs- - - - - _ . _ - _ . _ - - _ _--.
..

Constant light
Periodical light and dark
Dark (IS-minute light/day)Range__. _ - - - - - - - - -

50.91
54.16
63.86

11
Temporary researcher, present address; Kurume Branch of Horticultural Research
Station, M. A. F. Kurume, Fukuoka-ken.

RESEARCHES CARRIED OUT IN 1970
Table 7.

95

Result of analysis of variance for total number of eggs
laid by three female flies of each line for 10 days

Source ofvariation

D.F.

S.S.

M.S.

F

Condition
CL
CQ
Line
CxL
Error

2
1
1
11
22

326,977.02
301,994.01
24,983.01
1,574,402.77
1,055,494.32
1,536,645.33

163,488.51
301,994.01
24,983.01
143,127.52
47,977.01
21,342.30

3.408
6.295**

72

6.706**
2.248**

107

pared by the run test method. The fluctuation was recognized only under
the periodical light and dark condition. The total number of eggs laid in
the first five days was higher than in the next five days under both constantlight and dark (IS-minute light/day) conditions.

Relationship between phototaxis and oviposition of
D. viri/is
Chozo OSHIMA and Koji INOUE

One cage population was set up with flies collected from a warehouseof a brewery company in Tokyo and has been maintained in the cultureroom. After three months, about three hundred flies were extracted, andmale and female flies were separated. They were introduced into a mazeapparatus having eleven choice points in order to select positive and negativeflies with respect to phototaxis. About 30 pairs of photopositive flies andon the other hand, about 30 pairs of photonegative flies were crossed invials and their offspring were subjective to selection. After several generations, the degrees of positive and negative phototaxis of flies seemed toreach the equilibrium state. The regression coefficient of selection curves
was calculated to be 0.475.
Eleven lines were established with flies extracted from the 7th generationof photopositive population and twelve lines were established with flies
extracted from the 9th generation of photonegative population. Ninepairs were isolated from each line and their oviposition under three kindsof light condition were examined as described in the previous experiment.

96 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

The mean number of eggs per day per fly increased in the order of constantlight, periodical light and dark, and dark (IS-minute light/day) conditions
as shown in Table 8, but such a result could be observed more clearly inphotopositive flies.
Table 8. Mean daily number of eggs laid by photopositive andphotonegative flies under three kinds of light condition
Photopositive flies

Photonegative flies

ConditionFirst
10 daysSecond10 daysFirst
10 daysSecond10 days

Constant light
Periodical light and dark
Dark (is-minute light/day)

40.04
44.90
51.53

21.09
30.47
34.15

42.46
40.86
50.24

33.63
36.76
40.79

Flies laid more eggs in the first 10 days than in the second 10 days, andthis pattern of oviposition was recognized clearly in photopositive flies.
The difference between numbers of eggs laid for both first and second ten
day periods by photonegative flies under the periodical light and dark condition was the smallest. The rhythm of oviposion may be most efficiently
maintained by the same flies.
The running speed of photopositive flies through the apparatus was
faster than that of photonegative flies.

RESEARCHES CARRIED OUT IN 1970

X.

97

EVOLUTION GENETICS

An intergeneric hybrid between Agropyron tsukushienseand Elymus mollis
Sadao SAKAMOTO

El. mollis Trin. is distributed widely from Korea to Japan, Kamchatka
and Alaska, northeastern U.S.S.R., and Northern and Eastern Canada,
Greenland and Iceland. This species is tetraploid (2n=28) and includes
three subspecies. A strain collected at the seashore of Abashiri, Hokkaido,
has been maintained in Misima as a clone. It usually does not head in
Misima but two spikes were produced in 1969; their pollen grains were
dusted on emasculated florets of Agropyron tsukushiense (Honda) Ohwi
var. transiens Ohwi (2n=42) collected in Misima.
From this cross 22 F l plants were produced in 1970. Crossability was
30.6 % calculating from the number of hybrid plants produced and thenumber of florets pollinated. Individual variation in growth behaviorof F l plants was observed. Out of 16 F l plants observed only seven were
heading. The shape of the F l spikes was of Agropyron type but the empty
glumes were hairy as those of the Elymus parent. One of characteristics
distinguishing Elymus from Agropyron is the production of two spikelets
at almost all nodes of the rachis. This character was not expressed in Fl.
Thus, this behavior was quite different from that observed in F l hybrids
between El. dahuricus or El. sibiricus and Ag. tsukushiense where 1/3-1/4of all nodes of a rachis had two spikelets (Sakamoto 1965). All pollen
grains of F l plants were completely abortive.
Average chromosome pairing at MI of PMCs of the F l amounted to 2.0
bivalents and 30.9 univalents. Almost all bivalents ranging from one toseven were elongated and connected loosely by terminal chiasma. These
results indicate a lack of genomic homology between the three genomes of
Ag. tsukushiense and the two genomes of El. mollis.

98 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Collection of wheats and various other species of thetribe Triticeae in Iraq, Tnrkey and Iran
Kosuke YAMASHITA11, Masatake TANAKA1 1, Sadao SAKAMOTO and Hirotoyo ISHIl1 1

Northern Iraq, Southeastern Turkey and Southwestern Iran, or briefly,
the eastern part of the so-called Fertile Crescent, was regarded from older
findings to be the most important area which could provide interestingmaterials in respect of origin, differentiation and domestication of tetraploid
wheats. For instance, a number of reports were known of massive standsof Triticum dicoccoides together with its putative progenitors, Tr. boeoticum
and Aegilops speltoides. But only a few studies became known of theircytogenetic relations. The finding alone of Tr. dicoccoides in these parts
was a powerful enough incentive to organize a new expedition and to collecta wealth of material of those plants which would at last allow a thoroughstudy of their cytogenetic affinities to Palestinian dicoccoides Tr. timopheevi
and Tr. araraticum.
Also, a thorough exploration of archaeological excavation of such age
as 7,000 years B. C. promised to be highly interesting in view of the domestication problems in these parts.
Therefore, a botanical expedition of Kyoto University to the NorthernHighlands of Mesopotamia was sent to Iraq, Turkey and Iran from May
Table 1.

Specimens of Triticeae collected in Iraq, Turkey
and Iran during the expedition

Genus name

Aegilops
Agropyron
Crithopsis
Eremopyrum
Henrardia
Heteranthelium
Hordeum
Psathyrostachys
SecaleTaeniatherum
Triticum1)

Kyoto University.

No. of species

No. of samples

15
7
1
5
1
1
8
1
3
2
5

486
18
13
81
5
55
179
3

20
70
221

RESEARCHES CARRIED OUT IN 197099

10th to July 25th, 1970, for the collection of wild as well as cultivated wheats
and other Triticeae.
Along the expedition routes many large samples of Triticum, Aegilops
and other taxa belonging to the tribe Triticeae were collected. The collected items are briefly summarized in Table 1. At the same time wild
Gramineae indigenous to the area were also collected. The whole collection made by the expedition contains about 450 herbarium specimens of
Triticum, Aegilops and their relatives. The specimens were placed on fileat the Royal Botanic Gardens, Kew, the Smithsonian Institution, Washington, D. c., the National Science Museum, Tokyo and the Department of
Botany, Kyoto University, Kyoto.

Dynamics of Plant Domestication: Cultivation Experimentswith Oryza perennis and Its Hybrid with O. sativa
Hiko-Ichi OKA and Hiroko MORISHIMA

To look into the dynamics of domestication in rice, three population samples of the Asian form of Oryza perennis (the wild progenitor of O. sativa)
collected from natural habitats, and seven hybrid populations derived fromsativa X perennis crosses were grown for several generations at Taichung,Taiwan. They were observed for characters distinguishing wild from cultivated types. To investigate the influence of propagating methods, thehybrid populations were grown both in bulk and in pedigrees.
The results clearly indicated that when populations containing sativa
genes were grown in the field, the frequency of alleles characterizing thecultivated type increased in response to "cultivation pressure". This
genotypic change toward the cultivated type was more rapid in bulk populations than in pedigrees. Studies of character correlation showed that thepedigree populations gradually restored parental character associations in
response to natural selection, but the same trend early realized in bulk
populations could be masked by the elimination of wild types. It was also
found that plants showing cultivated characters were more homozygous
than those with wild characters, suggesting that natural selection for cultivated characters resulted in an increase of selfing rate. Comparing naturally
sown populaions with those seeded by hand, seeding was found to be aneffective agent causing "cultivation pressure". The details will be published100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

in EVOLUTION 25: 356-364.

Partial Self-incompatibility in Oryza perennis
Hiroko MORISHIMA and Hiko-Ichi OKA

The African form of Oryza perennis Moench (subsp. barthii) is partly
self-incompatible (Chu et at. 1969. Japan Jour. Genet. 44: 225-229). To
examine whether or not other geographical races of this species also showa tendency to self-incompatibility, four Asian, two American, and two
Oceanian strains each represented by several plants were grown, and selfand cross-pollinations were made by hand. In so far as the materials used
are concerned, self- and cross-pollination brought about the same amountof seed set indicating that they were self-compatible, though seed fertility
differed among strains.
Plants belonging to several African strains (barthii) were also tested. Mostof them consistently showed partial self-incomaptibility, but one strain,
ML3-2, was found to be self-compatible. It has some different morphological characteristics from typical barthii plants. The F 1 hybrid between
this strain and other barthii plants showed partial self-incompatibility.
Different barthii strains appeared to differ in the degree of self-incompatibility.

RESEARCHES CARRIED OUT IN 1970

XI.

101

HUMAN GENETICS

Inheritance of Total Finger Ridge CountEi MATSUDA and Ei MATSUNAGA

The distribution of total ridge count has been examined using finger
prints from unrelated Japanese, i.e. 313 males and 313 females. The mean
total ridge counts were found to be 151.28±46.09 for the males and 142.20±
44.81 for the females, the difference being significant. The values of gl(skewness) and g2 (kurtosis) obtained for the male distribution are
gl=-0.150±0.138, g2=0.016±0.275.

For the female distribution the corresponding values are respectively;
gl=-0.286±0.138, g2=-0.OO9±0.275.

Based on family data, correlation coefficients for total ridge count between
various types of relatives have been estimated, and the results are shownin Table 1.
Table 1. Correlations for total ridge count between relatives

Parent-child
Midparent-child
Mother-child
Father-child
Parent-parent

No. of pairs

Correlation coefficient

1,048
470
533
515
195

0.436±0.028
0.598±0.037
0.390±0.040
0.480±0.039
0.015±0.072

Using data on 187 sibships, the sib correlation was calculated, after
correction for sex difference, to be 0.463±0.038. Finally, the regression
coefficient of total ridge count of offspring on midparent value was estimatedto be 0.819. Although these estimated values are slightly lower than thoseobtained by Holt for the British population, (Ann. Eugen. 20: 270-281,
1956), it appears that they are consistent with the hypothesis that total
ridge count is controlled by a number of autosomal genes with almost additive effect.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Polymorphic Red Cell Enzymes among JapaneseTomotaka SmNODA

Polymorphisms of several enzyme systems in red cells have been investigated in 2,423 unrelated individuals, which were taken in three different
areas. Among them, acid phosphatase, adenosine deaminase, 6-phosphogluconate dehydrogenase and phosphoglucomutase were found to
be polymorphic, with practically the same frequencies in different populationgroups except for a few variants. Estimated gene frequencies for respective
enzyme systems were as follows; AP: pa=0.20, pb=0.80; ADA: ADA 1 =0.97, ADA2=0.03; PGD: PGD A =0.93, PGDc=0.07; PGM1 : PGM11 =0.77,
PGM1 2=0.22. As to rare PGM variants, there were four types; three ofthem were identified to be PGM 1 5-1, 7-1 and 8-1, but the remaining type
was not yet identified with certainty. In phosphoglucose isomerase about
99 % were of type PGI 1, while the remaining 1 % were accounted for byfour different variants, namely PGI 3-1, 4-1, 5-1 and 6-1 J, their incidencesin percent being respectively 0.2, 0.8, 0.1 and 0.05. No electrophoretic
heterogeneity was found in the samples with respect to adenylate kinase
type, in agreement with the previous result.
Additional enzyme systems, such as citrate dehydrogenase, hexokinase,malate dehydrogenase, succinate dehydrogenase, nucleoside phosphorylase
and fumarase were examined in some of the samples, but no electrophoretic
polymorphism was found by the current technique employed. One rarevariant in tetrazolium oxidase was found among the samples; the phenotype
was tentatively designated as TOX 2-1, and its mode of inheritance wasdetermined by family studies.
Details of the above results were published in Jap. J. Human Genet. 15:
133-143 and 159-165, 1970.
Structure of Human Macroglobulin
Tomotaka SmNODA

The Fc fragment prepared from whole Ig M by trypsin treatment at hightemperature was subjected to a selective cleavage at methionyl residues bycyanogen bromide. By combination of several procedures, at least fivefragments were isolated in a pure form, and they were further characterized.
Of these, a peptide with 11 amino acids having a carbohydrate branch was

RESEARCHES CARRIED OUT IN 1970

103

located at the NH 2-terminal and an octapeptide was located at the COOHterminal of the heavy chain. The order of the remaining cyanogen bromide
fragments was not yet fully established. In addition to isolation and characterization of cyanogen bromide fragments from Fe, an extensive work has
been carried out to isolate and locate disulfide bridges, both inter and intra
chains in the whole macroglobulin. At present, amino acid sequences
around five disulfide bridges were established. Of these, one was proved
to be located in VH region, while the remaining four were located in the
Fe region. Exact positions of each bridge in the Fe fragment were notyet established. Further attempts to isolate disulfide bridges are now going on.

Birth Weight in Down's Syndrome:
Comparison of interchange trisomies and G trisomies
Ei MATSUNAGA

From cytogenetical evidence it is assumed that the amount of excess
chromatin mass in interchange trisomies with t(DqGq) must be slightly
less than in regular G trisomies. As far as physical signs of Down's syndrome are concerned, however, no difference has been noted between
patients with interchange trisomy and G trisomy types. The author
examined birth weights of 58 pairs of interchange trisomics/G trisomies,
matched for sex, maternal age and birth order. The mean difference inbirth weight within a pair (interchange trisomic minus G trisomic) was
calculated to be only -8.2g, with standard deviation of 86.3 g. Thisfinding indicates that the two types of chromosome aberration have quantitatively a comparable effect upon intrauterine development of the affectedfetus.

Parental Ages in Translocation Down's Syndrome
Ei MATSUNAGA

Parental ages were analyzed for 49 cases of DIG and 38 cases of GIG
translocation Down s syndrome. These cases were collected from morethan 1,700 patients with this syndrome karyotyped in various laboratories

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

in Japan. With respect to parental karyotypes, they were classified into
inherited (19 DIG and 4 GIG), sporadic (18 DIG and 21 GIG) and unknownclasses (12 DIG and 13 GIG). When the data were combined for sporadic
and unknown classes, the mean paternal ages in years were 30.9 for DIG
and 31.4 for GIG, both being close to the population average (30.9), while
the mean maternal ages were 26.1 for DIG (1.6 years below the population
average) and 29.1 for GIG (1.5 years above the population average), thedifference between these two figures being significant. This finding suggeststhat maternal age may have some selective effect upon interchange of theacrocentric chromosomes.

Distribution of Fast Renaturing DNA in Human Chromosomes
Yasuo NAKAGOME

The distribution of fast renaturing (repetitive) DNA in human mitotic
chromosomes was studied by in situ denaturation-renaturation technique(Arrighi, F. E. et al. 1970. Chromosoma 32: 224). After the denaturation
with NaOH, chromosome preparations were allowed to renature in situ
in either 2 or 6 X SSC solution at 66°C for 15 hours. The renatured segments of chromosomes were identified as darkly stained bands along thechromatids in Giemsa stained preparations. Every band observed was located at the centromeric region of a chromosome (centromeric heterochromatin).
The same procedure, however, was found to be not satisfactory in studying the nuclei of certain kinds of cells. Polymorphnuclear leukocytes andsperms were among the hard-to-analyse cells. To overcome this difficultyin identifying renatured regions, cells were stained with acridine orange
instead of Giemsa solution. Experiences with viruses revealed that doublestrand DNA showed yellow-green fluorescence but RNA and single-strand
DNA showed flame-red fluorescence under certain conditions (Mayor,
H. D. 1961. Proc. Soc. Exper. BioI. Med. 108: 103). Any segments ofchromosomes or cells that show yellow-green fluorescence, presumably,
contain renatured DNA. On the other hand, the flame-red regions(RNAase
treatment required) should contain denatured DNA. Both the sensitivity
and the specificity of this method in identifying renatured segments ofchromosomes are expected to be much better than the usual Giemsa staining
procedure. This particular experiment is still in progress.

RESEARCHES CARRIED OUT IN 1970

105

A Cp+mat Chromosome Found in a Malformed Boy
Hidetsune OISID, Ei MATSUDA and Yasumoto KIKUCHI

During our current survey of chromosomes in patients with some clinicaldisorders, a case with a Cp+mat chromosome was observed.
The patient was a 27 months old boy who was the first-liveborn child ofa 31 years old mother and a 30 years old father. The gestation lasted
normally for 40 weeks and the birth weight was 3,100 g. The parents were
healthy and not consanguineous. The mother had previously experienced
one stillbirth.
Main clinical features found in the patient were: epicanthal folds, higharched palate, micrognathia, undescended testes, polydactyly of toes andprotuberant heels. The results of dermatoglyphic analysis of the patientwere as follows, a transverse palmar crease was noted on his left hand:
Right hand.-Palmar formula, 11.9.7.1-tt"-P.0.0.L.0. atd angle=55°,
123.5°; digits=U,A,W,W,W. Left hand.-Palmar formula, 3.3.3.1-t-P.
0.0.0.0, atd angle=54°; digits = U,U,W,W,W. Total ridge count=86.
Right toes=A,A,A,A,A. Left toes=A"A,A,A,A.
Chromosome analyses of the patient revealed that a missing C-groupchromosome was substituted by a metacentric chromosome resembling a
no. 3. On the other hand, the karyotype of the mother was characterized
by the presence of two extra chromosomes, one in no. 3 and the other ingroup G, in accordance with the missing two chromosomes one from group
C and the other from group D. Since the mother was phenotypically normal, the apparently balanced karyotype was interpreted as the result of areciprocal translocation between a C- and a D-group chromosome. Autoradiography disclosed the consistent presence of a normal, late replicating
X chromosome in mother's cells and indicated that the extra large chromosome was not a no. 3. In addition, the D-group chromosome involved
was identified with D 1 by means of autoradiography, though the exact
numbering of the C-group chromosome involved was unknown. Therefore, the karyotype of the mother could be written as 46, XX, t(Cp+; Dq-).
The Cp+ chromosome found in the patient was transmitted from the mother,and he was considered to represent trisomy for the distal half of the long armof a D 1 chromosome. The father had a normal male karyotype.

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Chromosome Studies in Congenitally Abnormal Children
Yasuo NAKAGOME and Hidetsugu MATSUMOTO

Among 20 cases studied, 2 showed rare chromosome disorders.
A fourteen year-old girl was referred to us because of short stature(128.8 cm) and sexual infantilism. On physical examinations a few additional minor anomalies such as shield-like chest, short and wide neck andshort fourth metacarpal bones were observed. Dermatoglyphic studies
revealed no unusual findings except for a very high total ridge count (226).
Buccal smear studies revealed very small as well as scanty sex chromatin
spots. No drumstick was observed in 2,000 polymorphnuclear leukocytes.
The karyotype, based on the standard leukocyte-culture technique, showed
two cell lines. One of them had a 45, X complement and the other showeda 46, XX, Cq- karyotype. The Cq- chromosome was smaller than No. 12
but larger than No. 16 and was median centric. Its chromatids were always
attached to each other and looked fuzzy. Autoradiography revealed that
the Cq- chromosome replicated itself at the end of the S-period, i.e. it
represented, in fact, an X chromosome with the deletion of a part of thelong arm. In other words, her karyotype was 45, Xj46, XXq-. The
size of the deletion was estimated to be 45 % of the long arm. This caserepresents one of the mildest deletions of the long arm of the X chromosome
so far reported.
The second case was a three month-old boy with the typical clinical featuresof Down's syndrome. Fourty-seven chromosomes were observed by thestandard leukocyte-culture technique. However, there were only five Ggroup chromosomes. None of them showed the characteristic appearanceof the Y chromosome. There was an extra acrocentric chromosome in
the D group. In most metaphases the extra chromosome was identified
by its peculiar shape. The both chromatids of the long arm were usuallyclose together and looked fuzzy. It was not satellited and was slightly
smaller than the other members of the D group. Based on these findings,
the patient's karyotype was interpreted as 47, XYq+, G+. The patternof quinacrine mustard fluorescence in this unusually long Y chromosome
is under study.

RESEARCHES CARRIED OUT IN 1970

lOT

Clinical Conditions of Patients with Apparently
Normal Chromosomes, vn
Hidetsune OISIUBased on leucocyte cultures of peripheral blood, patients with various
pathological conditions were found to have apparently normal chromosome
assortments of 46 chromosomes. Cases of some interest are listed below.

Case no.
Age
Legal sex
Clinical conditionsa) Multiple deformations with metal retardation
122 M.N. 10 days
Microcephaly; narrow temples; slopingM
forehead; hypertelorism; coloboma chorioideae; cleft palate; short neck; hemangioma
Hypertelorism; antimongoloid slant eyes;
123 T.U. 3 yearsM
a transverse palmar crease (left hand);hemangioma
Microcephaly; antimongoloid slant eyes;
124 Y.T.
5 yearsM
large ears; spasticity
Dubois' sign; incurved fifth fingers; large
125 U.M. 4 years
Fears; logopathy
Microcephaly; micrognathia; hypotonia
126 M.A. 1 year
F
Microcephaly; high-arched palate; ptosis;
1 year127 S.H.
M
malformed and low-set ears; micrognathia; polydactyly; syndactyly; cryptorchidismus; hypertonia
Congenital heart disease; anophthalmia
128 R.T. 2 months F(right); coloboma chorioideae (left); hydrocephalus
Open anterior fontanelle; antimongoloid
129 Y.N. 3 years
M
slant eyes; high-arched palate; a transverse palmar crease (left hand)b) Sex anomalies
Webbed neck (Turner's syndrome?, 46,
130 RO. 5 years
FXX)

131 S.N.

14 years

F

Dwarfism; cubitus valgus (Turner's syndrome?,46,XX)

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

132 Y.K. & M.K.
(sibling)
22 & 26 years
133 C.Y. 15 years

F
F

Testicular feminization (46,XY)
Short stature; shield chest; cubitus valgus;
small uterus (Turner's syndrome?, 46,
XX)

c)Hereditary diseases
134 K.K. 6 months
135 M.S. 3 years

M
F

Familial ectodermal dysplasia
Familial chronic nephritis (Alport's syndrome)

RESEARCHES CARRIED OUT IN 1970

XII.

109

APPLIED GENETICS

Geographic Variation on the Isoenzyme Level
of Abies sachalinensis in Hokkaido
Kan-Ichi SAKAI and Takashi MATSUURA

Abies sachalinensis is one of the most important conifer species in
Hokkaido, growing wild in hilly as well as in mountainous regions. For
the purpose of a provenance test, seedlings from different localities havebeen grown in a hilly land at the outskirts of Sapporo. The present study
deals with the electrophoretic study of needle-leaves collected from 25
trees from each of six strains. The locations from where the six strains
had originated are designated from the east to the west by N (Nemuro),
S (Saroma), U (Urakawa), P (Piuka), K (Kotanbetsu) and H (Higashisetana). The trees were six years of age. Examined were the peroxidase
isoenzymes in the leaf sap. It has been found from the study that theirmaximum number was 27. On the basis of those isoenzymes, the following<comparisons were made among the six populations. The first comparison-dealt with counting the average number of isoperoxidases per tree. It was
found that the mean number as well as its standard deviation was variable.among the populations. The second comparison was made from thefrequency of each of the 27 isoenzymes in the six populations. It was foundthat some populations were slightly different from each other, whereas in-others the difference was very significant. The third comparison was madeby measuring the number of azygous isozyme bands or the "disagreement-count" among trees within and between populations. It was found thatthe disagreement count among some populations was very small, whereas
others showed high values. Combining the results of such comparisons,it was concluded that Abies sachalinensis may have been originally propagated in the eastern to the north-eastern district of Hokkaido. The
trees may have first migrated to the central parts and finally to the westernand south-western parts of the island. The details of this study will bereported in some other journal.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Genetic Differentiation within a Natural Forest of
Cryptomeria japonica D. Don
Kan-Ichi SAKAI and Young-Goo PARK

The present study aimed at detecting intra-population genetic differentiation in a natural forest of Cryptomeria japonica growing in Kochi Prefectureon the Shikoku island. At first, three sites were allocated in the forest.
They were located at the apices of an isosceles triangle, the straight line
distances among the three subpopulations being 760 m, 1300 m and 1300 m,
respectively. In each of these subpopulations approximately 50 trees wereselected at random and their needle-leaves were collected on individual
tree basis. An electrophoretic analysis was conducted with those leaves,in order to measure the variability of isozyme peroxidase patterns. The
total number of isozyme bands of the peroxidase counted and identified in
the present species was 36, though the average number of isozyme bands
actually exhibited per tree in the three subpopulations was 11.68±0.28,
13.73±0.35 and 11.35±0.29, respectively.
The three subpopulations were comparatively examined by twos forthe incidence of each of the 36 bands. It was found that the number ofstatistically significant differences between subpopulations was 10, 12
and 15 out of 36 comparisons. It shows that they were far more numerous
than expected if due only to chance. Thus, it is concluded that a naturalforest of Cryptomeria japonica is not genetically homogeneous but very
heterogeneous, most probably involving numberless subpopulations which
are more or less genetically different from one another. It is suggested thata random genetic drift followed by effective isolation due to a higher occurrence of inbreeding than expected on the basis of wind-pollination may
have occurred in a natural forest of Cryptomeria japonica.

Genetic Studies of Peroxidase Isozymes of the Japanese
Red Pine, Pinus densijlora Sieb. & Zucco
Young-Goo PARK and Kan-Ichi SAKAI

It has been proved recently that isoenzymes are of great use in the studyof genetic variation of forest trees, especially in natural forests of Cryptomeria japonica, Thujopsis dolabrata, and Abies sachalinensis.

RESEARCHES CARRIED OUT IN 1970

111

The present paper describes the results of a study enquiring into thegenetic basis of peroxidase isozyme formation in Japanese red pine, Pinus
densiflora Sieb. et Zucco Materials for this study were selfed progenies
and F I hybrids of a diallel cross among 15 parental trees collected fromdifferent regions of Tohoku district of Japan Proper. The laborious work
'Of collection of mother-trees and the subsequent cross-hybridization havebeen performed by Dr. Y. Takayama and his collaborators in Kitakami
Silviculture Station of Jujo Paper Manufacturing Co. Ltd., due to theirkind help we have been able to make use of the valuable materials for thepresent study.
The electrophoretic analysis has shown that the total number of peroxidase isozymes in the present species was thirteen. Of those thirteen,
four, i.e. H, K, M and N have been investigated. Inferences from comparison among the phenotypes of parents, selfed progenies and F I hybrids
allowed the authors to assume that independent pairs of genes, Ph+ vs.
Ph-, Pk+ vs. Pk- together with the repressor gene, Rk vs. rk, Pm+ vs. Pm-,and Pn+ vs. Pn-, were responsible for the formation of H, K, M and N
bands, respectively. There was no indication of multiple allelism among
those alleles. It was found that H behaved independently against either
K, M or N, whereas K was negatively correlated with M and N, while M
and N were positively correlated with each other though the correlation
coefficients were rather low.

An Analysis of Sexual Dimorphism in Body Sizein the Japanese Quail
Takatada KAWAHARA

In the Japanese quail, the sex difference in body weight is pronounced,
the female being heavier than the male. The difference becomes apparent
at the age of 2 or 3 weeks after hatching, and it continues throughout whole
life. The main purpose of the present investigation was to elucidate thecause of the sex difference in the body weight. Data have been collectedfrom three stages at the age of 2, 4 and 25 weeks. Three hundred andeighty-two birds were used in total. The number of birds examined was42, 36 and 304 birds at 2, 4 and 25 weeks after hatching, respectively. They
consisted of the same number of males and females collected from a number

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

of full-sib families. The total body weight was partitioned into the following components, i.e. bones, heart, lungs, liver, gizzard, intestines, pancreas~
spleen, kidneys, gonads, oviduct, egg materials in oviduct and eviscerated
body weight.
Results of this study are summarized as follows:
1) Whole body weight in the female was significantly heavier thanthat of the male at each of the three developmental stages. Almost all
female organs showed a tendency to show heavier weight than the maleorgans at younger stages (2 and 4 weeks of age), though the difference
was not statistically significant, except for a few organs such as digestive
and the related organs and eviscerated body weights. Sexually matured
birds (25 weeks of age), however, exhibited statistically significant difference
between the sexes in all organ weights, except heart weight. Organs whichshowed the greatest difference between the sexes were sexual organs,liverin and testines.
2) Of interest is the eviscerated body weight in relation to the developmental stages of the bird. Total body weight in the three stages was always
larger in females than in males. The eviscerated body weights were heavier
in female than in males in the early two stages, but that at mature stage
showed apparently reversion, that is, the male was heavier than the female.
The difference between sexes or female minus male in the eviscerated
body weight was +2.0 g. at 2 weeks, +4.1 g. at 4 weeks, but -11.6 g. at
25 weeks of age, respectively.
3) Comparison between male and female with regard to genetic andenvironmental variances of eviscerated body weight at 25 weeks of age
showed that the environmental variance was approximately the same
between the sexes, whereas the genetic variance in males was larger thanin the female. Such a sexual difference in genetic variance was not foundin other organs. Further investigation into the genetic mechamism of thissexual dimorphism in the quail is under progress.
Monte Carlo Studies on the Relationship between the System
of Reproduction and Inbreeding
Shin-ya IYAMA

To maintain a population of wild organisms or breeding material underartificial control, it is required to find an appropriate structure of breedingRESEARCHES CARRIED OUT IN 1970

113

population to protect it from inbreeding depression. The author has
undertaken two Monte Carlo experiments to investigate the effect of control of the reproductive system on the progress of inbreeding in a population. The following assumptions were made in these experiments: 1) nonoverlapping generations, 2) constant population size, 3) no polygamy allowed;
mating performed by twos between a male and female pair, 4) sex ratio 1:1,
and 5) no relationship among members of the initial population.
Wright's coefficient of relationship for all possible combinations among
the members was computed and recorded for each generation. Inbreeding
coefficient of the progeny population derived from given matings wascalculated on the basis of the above record up to 100 generations. Effective
population number (N.) was estimated by fitting the formula known for adioecious population, 1,,=(1/2N.)+(I-(I/N.))/"_1 +(1/2N.)/,,_2, to theresults, where I" stands for the inbreeding coefficient at the n-th generation.
Experiment 1: In a random mating population, two types of propagation were considered. One was that the number of progeny from each mating to the next generation was variable. Since the population size wasconstant, the number of male or female progeny per mating was assumed
to follow the Poisson distribution with mean of unity. The other was thateach mating pair contributes always one male and one female to the next
generation. Monte Carlo experiment was carried out for various population sizes (N) and the effective population number (N.) was estimated(Table 1). In the case of variable progeny number, N. was found consistent with N as expected. N. for the case of constant progeny number
agreed well with 2N-l which has been suggested by Wright (1938) theoretically. It is concluded that the control of the reproduction to produce
an equal number of progeny from each mating pair makes the effective
population size almost as large as double of the case with variable progeny
number which is probable in uncontrolled population.
Experiment 2: When the population consists of more than two subpopulations, circular group mating was proposed by Kimura and Crow
(1963) to keep inbreeding as low as possible. Practically it might be,
however, troublesome since it is necessary to be done for every generation.
Then,effectiveness of mixing the subpopulations with certain generationinterval was examined as an alternative. In this experiment, equal numberof progeny was assumed. Random mating was performed within subpopulations but they were mixed together and redistributed at random into

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21
Table 1. Estimated effective population number for the matings
producing variable and equal number of progeny
Actual population
number (N)Variable numberof progeny

Equal numberof progeny

10
20
30
40
50
60
100
150
200

10.0
19.7
28.3
37.2
48.9
55.8
104.2
150.8
197.9

17.9
38.0
57.8
77.5
96.9
117.0
195.7
294.4
392.9

Table 2. Estimated effective population number for the populationconsisting of more than two subpopulations
Total
population
numberNo. ofsubpopulations

Size ofsubpopulation

60

2

30

j

60

3

20

j

60

5

12

60

6

10

10

6

Effective
population
numbernone
10
5
circularnone
105
circularnone
105
circularnone
105
circular
none

57.8
110.2
113.8
118.1
38.0
104.1
110.9
118.6
22.0
92.6
104.5
115.9
18.0
85.9
99.5
114.5
10.0
68.1
87.5
98.5

1
1

60

Mixing
type

j

10

5
circularsubpopulations at every 10th or 5th generation. Results are shown in
Table 2 in comparison with those of non-mixing and circular group mating.

RESEARCHES CARRIED OUT IN 1970

115

N. in circular group mating was nearly equal to the effective number of
total population considered as an undivided one. Mixing effect improved
the situation largely towards that of the circular group mating, frequent
mixing making it still better. Furthermore, it was shown by the investigation of inbreeding coefficient at each generation that the more frequently
mixing occurs, the smaller was the progress of inbreeding in the period during the mating was performed within each subpopulation.

Selection in Rice: An Experiment
Shin-ya IYAMAThe experiment was arranged in order to investigate what changes would
occur when selection was made for low-yielding genotypes. The mainmaterial used in this study is represented by five populations, all derived
from a cross between two or three japonica varieties and kept unselecteduntil the fifth or sixth generation. They were:
CR141 F o Kinmaze X San-in No. 63
CR143 F o Hoyokux36-1
CR157 F 5 SachikazexYamabiko
CR161 F5 Shizukei Mochi No. 5xYamabiko
CRl64 F 5 Norin No. 29 X (Hokushin Shuho X Chushin)
Throughout the experiment, the material was cultivated by single plant
per hill planting with 25 cm X 15 cm spacing. The characters examined
were panicle yield, plant weight, plant height and panicle number per plant.
In 1968, selection for panicle yield was performed on an individual plantbasis for each population. Each 50 plants were selected towards low andhigh yield from about 2,000 plants. In 1969, lines derived from the selected
plants were cultivated with randomized block design with three replications,each plot consisting of 20 plants. Characters were measured on plot basis
except panicle length which was obtained from ten samples chosen at
random from each plot, and then converted into plant basis.
Analysis of variance of data showed that the difference between high andlow selected groups in panicle y.ield was significant for all populations,indicating that the selection was effective. Heritability values obtained
from the ratio of realized gain against selection differential were 0.07 to
0.17. Selection of panicle yield showed a statistically significant effect on

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

plant weight, plant height, panicle length and panicle weight, except plantweight and panicle length in CR141 and panicle length in CR157. It wassuggested that the attained change in minus direction in panicle yeild byselection might be due rather to the change in weight per panicle than that
panicle number per plant.

Growth Pattern and Its Plasticity in Wild andCultivated Types of Rice
Hiroko MORISHIMA and Hiko-Ichi OKA

To compare the growth pattern and its plasticity between wild andcultivated types of rice, four cultivated, two wild and five intermediate(hybrid derivative) strains were grown in paddy and in gravel culture at two
fertilizer levels from 1967 to 1970. Measurements for the length of variousorgans and dry-matter weight were taken several times during the growing
period and at maturity. Yielding characters were also recorded at maturity.
From the dry-weight data, logistic growth curves were computed to obtainvarious growth parameters.
The results may be summarized as follows: 1) In the cultivated type,the panicle and third internode elongate simultaneously and the following
elongation of the first and second internode results heading. In somewild types, however, elongation of the third internode resulted in heading
and the first and second internodes elongated after heading. 2) The wild
type differs from the cultivated type in that the increase in dry weight after
heading is partly due to vegetative growth and does not necessarily imply
grain production. 3) Modern cultivars showed late vigor and sustained
growth, while primitive ones showed early vigor and rapid decline. 4)
Cultivated types responded to fertilizers mainly by increasing panicle number
and grain yield, the size of organs remaining relatively constant, while wild
types tended to be plastic as to organ size in response to fertilizers. 5)
Annual fluctuation in characters was more pronounced in wild than incultivated type. It may be concluded from the above experimental resultsthat the wild type is highly responsive to natural environmental conditions,while the cultivated types are responsive to man-controlled conditions.

RESEARCHES CARRIED OUT IN 1970

117

Inter-genotypic Interaction in Rice
Tsuguhiro HOSHINO, Hiroko MORISHIMA and Hiko-Ichi OKA

A mix-planting experiment with rice was conducted to investigate intergenotypic competitive interaction. Ten F 11 lines taken from pedigrees
of a cross between two varieties, Taichung 65 (Japonica) X Pai-ku (Indica),
were grown in pure stands and in mixture with each parental strain withthree replications. They were observed for heading date and various yieldcharacters at maturity. During the growing period, dry-matter weight wasmeasured three times, and logistic growth curves were computed to obtaiJ;lgrowth parameters. The effect of mix-planting on each genotype wasestimated by subtracting the value of pure stand from that of mixed stand,and was designated as a for tested lines (resistance to aggression) and f3
for tester strains (aggressivity of a tested line). The values of a+f3 anda-f3 were considered to show synergism and competition between two component genotypes in mixed plot, respectively.
Main conclusions obtained may be summarized as follows: 1) Analysis
of variance revealed that not only a and f3 but also the effects of synergism
and competition significantly differed between the tested lines. 2) a and f3
were negatively correlated in most characters, though the correlation coefficients were low. 3) Principal component analysis of character correlations
in a and f3 showed that, in addition to a general factor, a differential factor
is involved, namely, variation between the lines which are plastic as topanicle size but stable as to panicle number and those having the opposite
trends. 4) The larger is the difference in growth pattern between two component genotypes, the more synergistic they are in grain production.

Evaluation of an Electronic Instrument for Estimation of Plant Weight
Hiroko MORISHIMA, Hiko-Ichi OKA and Tsuguhiro HOSHINO

To estimate the weight of standing plants, an electronic instrument called"Herbage meter" was developed in collaboration with the Central ResearchInstitute of Electric Power Industry. The instrument is essentially anelectric capacitance meter. When the measuring head is placed over the
standing plants, a change in the electrical capacitance of the system iscaused which is closely related to the amount of herbage. Plant weight
can be estimated from meter reading by calibration.

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

Using ten rice strains, reading of the herbage meter and actual dry-matterweight of sampled plants were compared every week during the growing
period. Before heading, they were highly correlated with each other,though the regression coefficient in the prediction equation for cultivatedstrains significantly differed from that for a wild strain having a quite different plant type. Coefficients of determination (r 2) were 0.92 in cultivatedstrains and 0.96 in a wild strain. When the data for wild and cultivatedstrains were pooled, the coefficient of determination was 0.88, and wasraised up to 0.89 by adding plant height to meter reading in the form ofmultiple regression. After heading, however, meter reading gradually
decreased, while dry-matter weight increased with carbohydrate accumulation in the grains. This might be due to a change in electric resistance ofplants after heading. Meter reading may indicate fresh weight rather than
dry weight. This technique, therefore, seems to be applicable when thewater content remains within a certain range. It was concluded that sequential record of vegetative growth can be taken by this technique more
efficiently than by the ordinary sample-cutting method which reqires muchlabor and is often unreliable owing to a large inter-plant variation in dry
weight.

RESEARCHES CARRIED OUT IN 1970

119

BOOKS AND PAPERS PUBLISHED IN 1970
BY S1AFF MEMBERS(1) BLOOM, A. D., Y. NAKAGOME, A. AWA and S. NERDSHI 1970. Chromosome aberrations and malignant disease among A-bomb survivors. Amer. J. Public Health.
60: 641-644.
(2) CHU, Y. E. and H. I. OKA 1970. The genetic basis of crossing barriers between
Oryza perennis subsp. barthii and its related taxa. Evolution. 24: 135-144.
(3) CHU, Y. E. and H. I. OKA 1970. Introgression across isolating barriers in wild
and cultivated Oryza species. Evolution. 24: 344-355.
(4) CROW, J. F. and M. KIMuRA 1970. An Introduction to Population Genetics
Theory. xiv+591. Harper and Row (New York).
(5) FUll-KAWATA, I., K. MIURA and M. FUKE 1970. Segments of genome of viruses
containing double-stranded ribonucleic acid. J. Mol. BioI. 51(2): 247-253.
(6) FUKATAMI, A. and D. MORIWAKI 1970. Selection for sexual isolation in Drosophilamelanogaster by a modification of Koopman's method. Japan. J. Genetics. 45:
193-204.
(7) FURUICHI, Y., Y. WATAYA, H. HAYATSU and T. UKITA 1970. Chemical modification of yeast tyrosine tRNA with bisulfite. A new method to modify isopentenyladenosine residue. Biochem. Biophys. Res. Commun. 41(5):1185-1191.
(8) HOTANI, H., T. Om, H. KAGAWA, S. ASAKURA and S. YAMAGUCHI 1970. Biochemical evidence for identical structure of p-filament and flagellin. Biochim. Biophys.
Acta. 214: 207-215.
(9) KADA, T. 1970. Studies on the mutability of Escherichia coli K12. I. Suppression and high spontaneous mutation in a threonine auxotroph. Mutation Res. 10:
91-102.
(10) KAoA, T. 1970. Studies on the mutability of Escherichia coli K12. II. Modification of radiation sensitivity by reversions in a threonine auxotroph. Mutation
Res. 10: 103-109.
(11) KAoA, T. 1970. Radio-sensitization with iodine compounds. II. Studies on mutant
strains of Escherichia coli K12 resistant to radiation-induced toxic products fromiodoacetic acid, potassium iodide or potassium iodate. Int. J. Radiat. BioI. 17:419--430.
(12) KAoA, T., T. NOGun and M. NAMIKI 1970. Radio-sensitization with iodine compounds. I. Examination of damage in deoxyribonucleic acid with Bacillus subtilistransformation system by irradiation in the presence of potassium iodide. Int. J.
Radiat. BioI. 17: 407--418.
(13) KAoA, T., Y. SADAIE and T. NOGun 1970. Radio-sensitization with extracellular
halogenated purines and pyrimidines and related compounds. Int. J. Radiat. BioI.
18: 281-285.
(14) KAro H. and T.H. YOSIDA 1970. Nondisjunction of chromosomes in a synchronized120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21
cell population initiated by reversal of colcemid inhibition. Exptl. cell Res.
60: 459--464.
(15) KIKUCHI, Y. and H. OISHI 1970. Internal asynchrony in late replication of human
X chromosomes with structural abnormalities. Japan J. Human Genet. 15: 114123.
(16) KIMuRA, M. 1970. Stochastic processes in population genetics, with special reference to distribution of gene frequencies and probability of gene fixation. Biomathematics Vol. 1" Mathematical topics in population genetics (ed. K. KOJIMA).
178-209 Springer-Verlag (Berlin).
(17) KIMuRA, M. 1970. The length of time required for a selectively neutral mutant
to reach fixation through random frequency drift in a finite population. Genet.
Res. Camb. 15: 131-133.
(18) KIMURA, M. and T. OHTA 1970. Probability of fixation of a mutant gene in a finite
population when selective advantage decreases with time. Genetics 65: 525-534.
(19) KIMURA, M. and T. OHTA 1970. Genetic loads at a polymorphic loci which ismaintained by frequency-dependent selection. Genet. Res. Camb. 16: 145-150.
(20) KURODA, Y. 1970. Differentiation of ommatidium-forming cells of Drosophilamelanogaster in organ culture. Exptl. Cell Res. 53: 429--439.
(21) KURODA, Y. 1970. Cytodifferentiation of Drosophila melanogaster in organ culture(in Japanese with English summary). Nucleus and Cytoplasm 14: 49-55.
(22) KURODA, Y. 1970. Effects of X-irradiation on sorting-out property of animalcells in rotation culture (in Japanese). Japan. J. Develop. BioI. 24: 69-70.
(23) KURODA, Y. 1970. Biology of cultured animal cells (in Japanese). The Heredity(Tokyo) 24(5): 2-9.
(24) MARUYAMA, T. 1970. Rate of decrease of genetic variability in a subdivided population. Biometrika 57: 299-311.
(25) MARUYAMA, T. 1970. Analysis of population structure. I. One-dimensional
stepping-stone models of finite length. Annals of Human Genetics 34: 201-219.
(26) MARUYAMA, T. 1970. On the rate of decrease of heterozygosity in circular steppingstone models of populations. Theoretical Population Biology 1: 101-119.
(27) MARUYAMA, T. 1970. Effective number of alleles in a subdivided population.
Theoretical Population Biology 1: 273-306.
(28) MARUYAMA, T. 1970. Stepping stone models of finite length. Advances in Applied Probability 2: 229-258.
(29) MARUYAMA, T. 1970. On the fixation probability of mutant genes in a subdivided
population. Genet. Res. Camb. 15: 221-225.
(30) MARUYAMA, T. and N. YASUDA 1970. Use of graph theory in computation of inbreeding and kinship coefficients. Biometrics 26: 209-219.
(31) MATSUNAGA, E. 1970. Chromosomal aberrations and maternal age (in Japanese)
Rinsho-Kagaku 6: 648-655.
((2) MATSUNAGA, E. 1970. Report of the study group on medico-legal determinationof disputed paternity (in Japanese). Japan J. Legal Med. 24: 193-230.
(33) MATSUNAGA, E. 1970. Future of the world population (in Japanese). The
Heredity 24(11): 2-5.

RESEARCHES CARRIED OUT IN 1970

121(34) MATSUNAGA, E., K. OMOTO, T. SffiNODA, H. OISHI and E. MATSUDA 1970. A
further study on the family with anomalous inheritance of haptoglobin types. JapanJ. Human Genet. 15: 166-175.
(35) MIURA, K. 1970. Nucleic acid chemistry (in Japanese). The Nucleic Acids. (eds.
Y. Takagi and Y. Miura) 1-35 Asakura-shoten (Tokyo).
(36) MWRA, K. 1970. Structure and function of the tRNA (in Japanese). Biochemical
Genetics (eds. Y. Kajiro et at.) 123-141 Igakushoin (Tokyo).
(37) MWRA, K. and H. HAYASffi 1970. On the transfer RNA (in Japanese). Jyohokagaku-koza (Information Sciences) B·7·2. Genetic Information II (ed. A. Tsugita). 27-62 Kyoritsu-shuppan (Tokyo).
(38) MORISmMA, H. and H. I. OKA 1970. A survey of genetic variations in the populations of wild Oryza species and their cultivated relatives. Japan J. Genetics 45:
371-385.
(39) MORIWAKI, D., Y. N. TOBAR! and Y. OGUMA 1970. Spontaneous crossing-over inthe male of Drosophila ananassae. Japan. J. Genetics 45: 411-420.
(40) MORIWAKI, K. and H. T. IMAI 1970. The mechanism and significance of periodic
ploidy alterations in the mouse myeloma, MSPC-l. Acta. Haem. Japon 33: 67-78.
(41) MURAKAMI, A. 1970. A comparison of mutagenicity of 14 MeV fast neutrons on
primordial germ cells among five different X-ray sensitive silkworm strains. Int.
J. Radiat. BioI. 17: 479-482.
(42) MURAKAMI, A. 1970. Strain sensitivity of silkworm eggs to killing and mutation
by ultraviolet light. Radiat. Res. 44: 146-153.
(43) NAKAGOME, Y. and A. D. BLOOM 1970. The satellite associations of the D groupchromosomes in translocation carriers. J. Med. Genet. 7: 371-376.
(44) OHTA, T. and M. KIMURA 1970. Statistical analysis of the base composition of genes
using data on the amino acid composition of proteins. Genetics 64: 387-395.
(45) OHTA, T. and M. KIMURA 1970. Development of associative overdominance throughlinkage disequilibrium in finite populations. Genet. Res. Camb. 16: 165-177.
(46) OKA, H. I., H. MORISHIMA, T. T. CHANG and O. TAGUMPAY 1970. Analysis ofgenetic variations in plant type of rice. V. Early vs. sustained vigor types in growth
and their bearing on yielding potential. Theoretical and Applied Genetics 40:
50-55.
(47) SAKAI, K. I., S. HAYASHI and K. TOMITA 1970. Clone analysis and genetical studyof quantitative characters in a natural forest of Cryptomeria japonica. Silvae
Genetica 19(4): 124-128.
(48) SHINODA, T. 1970. Polymorphism of red cell adenosine deaminase in the Japanese
population. Japan J. Genetics 45: 147-152.
(49) SHINODA, T. 1970. Inherited variation in tetrazolium oxidase in human red cells
Japan J. Human Genetics 15: 144-152.
(50) SHINODA, T. 1970. Inherited variations in red cell phosphoglucose isomerase among
Japanese. Japan J. Human Genetics 15: 159-165.
(51) SHINODA, T. and E. MATSUNAGA 1970. Polymorphism of red cell phosphoglucomutase among Japanese. Japan J. Human Genetics 14: 316-323.
(52) SHINODA, T. and E. MATSUNAGA 1970. Studies on polymorphic types of several

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21
red cell enzymes in a Japanese population. Japan J. Human Genetics 15: 133-143.
(53) SHINODA, T., K. TITAN! and F. W. PurNAM 1970. Amino acid sequence of human,'l chains. I. Tryptic peptides of protein Ha. J. BioI. Chem. 245: 4463-4474.
(54) SHINODA, T., K. TITAN! and F. W. PuTNAM 1970. Amino acid sequence of human,'l chains. II. Chymotryptic peptides and sequence of protein Ha. J. BioI. Chem.
245: 4475-4487.
(55) TAZIMA, Y. 1970. Studies on radiation genetics of the silkworm and their application to sericulture. Ann. Rept. of Nippon Agr. Res. Inst. 1969 pp. 36-51.
(56) TAZIMA, Y., and A. MURAKAMI 1970. Analysis of strain differences in radiosensitivity of the silkworm. Gamma-Field Symposium 8: 53-66.
(57) TSAI, K. H., Y. C. Lu and H. I. OKA 1970. Studies on soybean breeding in Taiwan.
4. Adaptability to fall cropping explored by disruptive seasonal selection of hybrid
populations. SABRAO Newsletter 2: 91-102.
(58) TSAI, K. H. and H. I. OKA 1970. Genetic studies of yielding capacity and adaptability in crop plants. 4. Effects of an earliness gene, mh, in the genetic background of
a rice variety, Taichung 65. Botanical Bulletin of Academia Sinica 11: 16-26.
(59) TSUJITA, M. and S. SAKURAI 1970. Lethal effects of a genetic abnormality in thepteridine metabolism of the silkworm larvae. In "Chemistry and Biology of
Pteridines." Proc. 4th Intern Symp. Pteridines. The Chemistry and Biology.
Intern. Print. Co. Ltd., Tokyo pp. 425-434.
(60) WATANABE, T. K. and C. OSHIMA 1970. Persistence of lethal genes in Japanesenatural populations of Drosophila melanogaster. Genetics 64: 93-106.
(61) YOSIDA, T. H. 1970. Chromosomes in cancer cells (in Japanese). Clinic All-Round
19(5): 832-838.
(62) YOSIDA, T. H., H. T. IMAI and K. MORIWAKI 1970. Chromosomal alteration anddevelopment of tumors, XXI. Cytogenetic studies of primary plasma cell neoplasms
induced in BALB/c mice. J. Nat. Cancer Inst. 45: 411-418.
(63) YOSIDA, T. H., T. KUROKI, H. MASUR and H. SATO 1970. Chromosomal alterationand the development of tumors, XX. Chromosome change in the course of malignant transformation in vitro of hamster embryonic cells by 4-nitroquinoline I-oxide
and its derivative, 4-hydroxyaminoquinoline I-oxide. Gann 61: 131-143.

RESEARCHES CARRIED OUT IN 1970

ABSTRACTS OF DIARY FOR 1970
January
February
March
April
May

June
July

23
27
26
23
1
4

22
25
13-15
31

AugustOctober28
1
12

November 20
24l76th Meeting of Misima Geneticists' Club
l77th Meeting of Misima Geneticists' Clubl78th Meeting of Misima Geneticists' Clubl79th Meeting of Misima Geneticists' Club
90th Biological Symposium
9lst Biological Symposiuml80th Meeting of Misima Geneticists' Club
l8lst Meeting of Misima Geneticists' Club
Summer Seminor of the Institutel82nd Meeting of Misima Geneticists' Clubl83rd Meeting of Misima Geneticists' Clubl84th Meeting of Misima Geneticists' Club
92nd Biological Symposiuml85th Meeting of Misima Geneticists' Club
93rd Biological Symposium

123124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

FOREIGN VISITORS IN 1970
March
April

May

1-4 STICH, H. E, University of British Columbia, Canada
4 WILSON, W.O., University of California, U.S.A.
15 LEVY, S., National Institute of Health, U.S.A.
17 SAMS, J. R., Indian River Poultry Farms, U.S.A.
17 ARVIDSON, R. B., Hy-Line Poultry Farms, U.S.A.
1-2 KING, R. C., North Western University, U.S.A.
4 GILLOIS, M., French Nat' I Organisation for Agricultural
Research, France

June 15September 1
October
7
November 3
924

CROW, J. F., University of Wisconsin, U.S.A.
GUTTMAN, R., Hebrew University of Jerusalem, Israel
KIMBER, G., University of Missouri, U.S.A.
COLE, R. K., Cornell University, U.S.A.
SCHAEFFER, P., Institute de Microbiologie, Universite deParis, France
27 LAW, G. R. J., Hy-Line Poultry Farms, U.S.A.
December 8 MENGESHA, N. M., Agricultural College of the Haile
Sellassie University, Ethiopia

RESEARCHES CARRIED OUT IN 1970125

ACKNOWLEDGEMENT

..

",

'I

.

The editor wishes to express his sincere gratitude to Dr. F. A. LILIENFELD
for her kindness in reading the original manuscripts. (T. MARUYAMA)

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 21

AUTHOR INDEX
Amano, E
Ando, T
Choo, J. K
Endo, T
Enomoto, M
Fujii, T. .
Fukase Y
Furuichi, Y
Hayashi, M
Hoshino, T
Iino, T
Inoue, K. .
Ishidsu, J
Ishii, H
Ishiwa, H
Iyama, S. ..
Kada, T
Kato, H
Kawahara, T
Kawanishi, M. .
Kikuchi, Y
Kimura, M
Kuroda, Y
Maruyama, T
Matsuda E
Matsumoto, H. .
Matsunaga, E
Matsuura, T. .
Mayeda, K
Minato, K
Miura, K
Morishima, H
Moriwaki, K

74, 756788
27
2276, 7760,61
11, 1352
117
14, 18, 21
94, 9514, 16, 1798
22112, 115
67, 69, 70, 72, 73.49, 51, 63
111
90, 92
105
79, 80, 81.41, 42, 43
82
101, 105
106
101, 103
109
37, 3844, 45
11, 12, 13
99,100,116,117
34,36,37,38,50,56

Murakami, A
57, 65, 65, 66
Nakagome, W
104, 106
Nawa, S. .
24, 26
Noguti, T. .
69
Ohta, T. .
79, 80, 81
Oishi, H
105, 107
Oka, H. I.
99,100,116, 117
Onimaru, K. .
63
Oshima, C. .
88, 90, 92, 94, 95
Pai, C
27
Park, Y. G
110
Sadaie, Y
70, 72
Sakai, K. I.
109, 110
Sakamoto, S
97,98
Sakurai, S
28,29,31,33,67
Sato, T34, 36, 37, 56
Shibuya, A. .
12
Shimotono, K
12
Shinoda, T
102
Suzuki, H
18, 2098
Tanaka, M. .
Tazima, Y
60, 61, 62, 63
Toshima, H
14Tsuchiya,K34,37,46,47,48,49, 50, 54
Tsujita, M
26, 28, 29, 30, 31
Tutikawa, K.
72, 73
Watanabe, T. K.
88, 90, 92
Wu, W.C
21
Yamada, M. A
25, 2614, 18
Yamaguchi, S. .
Yamashita, K. .
98
Yosida, T.H... .46,47,48,49,51,52,54,55

r

.
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