,NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 221971

PubliBh«l by

THE NATIONAL INS'ITfUTE OF GENETICS
MUimtJ. Sizuoka-ken. Japan1972

Annual Reportof theI

National Institute of Genetics
No. 22, 1971,
J

Published by

The National Institute of Genetics, Japan
1972

CONTENTS
General statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council....................
Association for propagation of the knowledge of genetics. . . . . . . . . . . .
Projects of research for 1971
Researches carried out in 1971

1
2
5
6
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11

I. Molecular Genetics
Identity of the 3'-terminal sequences in the ten genome segmentsof silkworm cytoplasmic polyhedrosis virus. FURUIcm, Y.
and MIURA, K.
Single-stranded RNA synthesis in vitro by the RNA polymerase
associated with cytoplasmic polyhedrosis virus containing
double-stranded RNA. SmMoToHNO, K. and MIURA, K....

11

II. Microbial Genetics
A non-chemotactic mutant in salmonella. IINO, T. and OGUCHI,
T.
Polymerization of external flagellin at the ends of the hooks
attached to Salmonella cells. IINO, T., SUZUKI, H. and
YAMAGUCHI, S.
Isolation of flagellar hooks from non-flagellate Salmonella mutants.
YAMAGUCHI, S., IINO, T. and KUROIWA, Too... .. .. .. .. . . ..
Ribonucleic acid depndent synthesis of salmonella flagellin bya cell-free system. SUZUKI, H.. . . . . . . . . . . . . . . . . . . . . . . . . .
Phage-induced alterations in Xanthomonas citri. Wu, W. C. andIINO, T.
Transduction by phage PI in Salmonella typhimurium. ENOMOTO, M.and STOCKER, B. A. D.
Ill.

12

13

14
15
17
18
19

Biochemical Gonetics
Amino acid sequence of human IgM immunoglobulins. SmNODA, T., PAUL, C., SHIMIZU, A. and PUTNAM, F. W.. . . . . .

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ii ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Genetic polymorphism of serum transferrin in the field mouse,
Apodemus giliacus. MORIWAKI, K., SADAIE, T. and HAYATA, I.
Electrophoretic pattern of serum protein in indian brown spiny
mouse, Mus platythrix platythrix. TSUCHIYA, K., SADAIE, T.
and MORIWAKI, K.
Androcidin: An exotoxin-like product of SR-spirochetes ofDrosophila that kills male fly zygotes. OISHI, K.... . . . . . .
Migration rate of acid phosphatase isozymes controlled by agene in Oryza sativa and O. perennis. PAl, C. and Endo, T.. .
Effect of foreign DNA on seed formation in egg plants. ENDO,
T....................................................
IV.

V.

DeJlelopmental Genetics
In vitro studies on spermatogenesis of Drosophila melanogaster.
KURODA, Y. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Growth and tissue-specificity of Drosophila embryonic cells in
culture. KURODA, Y.
Differential inhibition by hexosamines of aggregation of normalliver and hepatoma cells in rotation culture. KURODA, Y.. .
Studies on sorting-out mechanism of animal cells in rotation
culture. KURODA, Y.........................
Further studies on radiation effects on sorting-out activity of
HeLa cells. KURODA, Y.
Effects of high molecular fractions of serum protein on growthof mammalian cells in culture. MINATO, K.............
Cytogenetics
Hybridization between several subspecies of black rats (Rattus
rattus). YOSIDA, T. H. and TAKAHASHI, K. . . . .. . . . . . . . . . .
Karyotypes of hybrids between several subspecies of the blackrats (R. rattus). YOSIDA, T. H., SAGAI, T. and TAKAHASHI, K.
A new differential staining technique using sodium dodecyl
sulfate and banding pattern analysis of black rat chromosomes. YOSIDA, T. H. and SAGAI, T.

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24

25

27
28
29
30

31
32

34

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CONTENTS

Similarity of banding pattern of metaphase chromosomes inbalck and Norway rats. YOSIDA, T. H. and SAGAI, T. .....
Banding patterns of Chinese hamster chromosomes revealed
by new techniques. KATO, H. and YOSIDA, T. H.
A new technique to reveal banding pattern of human chromosomes by using urea. SHIRAISHI, Y. and YOSIDA, T. H. ....
Factors involved in the production of banded structures inmammalian chromosomes. KATO, H. and MORIWAKI, K...
Differential responses of several aneusomic cell clones to ultraviolet irradiation. KATO, H. and YOSIDA, T. H.
Induction of sister chromatid exchanges by ultra-violet lightand inhibition by caffeine. KATO, H.
Induction of chromosome aberrations in human leucocytes growing in vitro by treatment with cadmium sulfide. SHIRAISHI,
Y. and YOSIDA, T. H.
Abnormalities of chromosomes found in cultured leucocyte cells
from Itai Itai disease patients. SHIRAISHI, Y. and YOSIDA,
T. H.
Relation between karyotype alteration and aging of tumor stemline cells. YOSIDA, T. H.
Karyological studies in Japanese ants (Hymenoptera, Formicidae)
III. Karyotypes of nine species of Ponerinae, Formicinae, andMyrmicinae. IMAI, H. T. and KUBOTA, M.
A new criterion for classification of mammalian chromosomes.
IMAI, H. T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Karyotypes of four species of bats collected in Japan. TSUCHIVA, K., HARADA, M. and YOSIDA, T. H.
Indian brown spiny mouse, Mus platythrix, having the lowest
fundamental chromosome number. TSUCHIYA, K. and
YOSIDA, T. H.
Possibility of clonal senescence in MSPC-I mouse myeloma
cells during serial transplantation. MORIWAKI, K. andSADAIE, T... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Simplified alkaline phosphate buffer-Giemsa method for inducing banded structure in the mouse chromosomes. MORIWAKI,
K. and SADAIE, T. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iii

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44
46

48
48
50

51

52

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iv ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Callus induction and organ redifferentiation of Triticum, Aegilops
and Agropyron by anther culture. KIMATA, M. and SAKAMOTO, S.

VI.

VII.

Mutation and Mutagenesis in Animals
Effect of protein synthesis inhibitor on repair of X-ray induced
premutational lesions in silkworm spermatids. TAZIMA, Y.
. . .. .. .. . ..
.. .. .. ..
and FUKASE, Y.
Studies on strain differences in radiosensitivity in the silkworm
XIV. Further data on dominant lethals. TAZIMA, Y. and
FUKASE, Y,.
Studies on strain differences in radiosensitivity in the silkworm.
XV. Comparison of recombination values among different
sensitivity strains. TAZIMA, Y. and ONIMARU, K.. . . . . . . . . .
RBE of 14 MeV fast neutrons for visible recessive mutations insilkworm oocytes. MURAKAMI, A.
The nature of specific locus mutations induced by fast neutronsin silkworm spermatocytes. MURAKAMI, A... . . . . . . . . . . . .
Mutagenesis of the silkworm by acridine orange. MURAKAMIA.
Induction of mutations by EMS in female germ-cells of thesilkworm. MURAKAMI, A.
Instability of pM-allelic genes locating on the second chromosome
piece translocated to the W chromosome. TAZIMA, Y. andOINUMA, T.
Radiation Genetics and Chemical Mutagenesis in Microorganisms
and Plants
A cellular factor involved in repair of DNA damage induced byionizing radiation. NOGUTI, T. and KADA, T.. . . . . . . . . . . .
Adenosine triphosphate-dependent deoxyribonuclease activities
in Bacillus subtilis. SADAIE, Y. and KADA, T.. . . . . . . . . . . .
Effect of ultraviolet light on DNA degradation in wild andrecombinationless cells of Bacillus subtilis with or without
toluene-treatment. SADAIE, Y. and KADA, T.
Cell culture of Haplopappus gracilis. AMANO, E.. . . . . . . . . . . . . .

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56

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59
61
62
63
64

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68
70

72
73

CONTENTS

Grafting test of seedling mutants in Capsicum annuum. AMANO,
E.
Radiosensitivity in stored wheat seeds. FUJII, T.............
Fractionation effect of neutrons on mutation frequency in maize.
FUJII, T,. .
.
. . . . .. .. . .

VIII. Population Genetics (Theoretical)
Theoretical foundation of population genetics at the molecularlevel. KIMURA, M.
Linkage disequilibrium between two segregating nucleotide
sites under steady flux of mutations in a finite population.
OHTA, T. and KIMURA, M...............................
Behavior of neutral mutants influenced by associated overdominantloci in finite populations. OHTA, T. and KIMURA, M.. . .. ..
Genetic load due to mutations with very small effects. OHTA, T.
and KIMURA, M.
Associative overdominance caused by linked detrimental mutations. OHTA, T.
Pattern of neutral polymorphism in a geographically structured
population. KIMURA, M. and MARUYAMA, T.. . . . . . . . . . . .
Some methods for treating continuous stochastic processes in
population genetics. MARUYAMA, T. and KIMURA, M.. . . . . .
Time-transformation of a diffusion model of a geographicallystructure population. MARUYAMA, T.
IX.

75
76
77

79

79
80
81
81
82
83
85

Population Genetics (Experimental)
Evidence for the neutral hypothesis of protein polymorphism.
YAMAZAKI, T. and MARUYAMA, T.......................
Detection of single-gene effect by inbreeding. YAMAZAKI, T.. . . .
Deleterious and sterility genes in Japanese and Korean natural
populations of Drosophila melanogaster. OSHIMA, C., and
CHOO, J. K.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

X.

V

Evolutionary Genetics
On the rate of molecular evolution. KIMURA, M. and OHTA, T.. .
On the constancy of the evolutionary rate of cistrons. OHTA, T.
and KIMURA, M.

86
88

90

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vi ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Serological crossreaction of the ascidian liver extract with antirat transferrin rabbit serum. MORIWAKI, K. and SADAIE,
T.

Xl.

93

Human Genetics
An estimate of the effective number of loci for total finger ridgecount. MATSUNAGA, E. . . . . . . . . . . . • • . . . . . . . . . . . . . . . . . . . . . .
Determination of the exchange points in a human ring chromosome. NAKAGOME, Y. and IINUMA, K.
G group chromosomes in satellite associations. NAKAGOME, Y...
Polyploidy in cells cultured from amniotic fluid. NAKAGOME, Y.,
IINUMA, K. and MATSUNAGA, E. . . . . . . . . . . . . . . . . . . . . . . . . .
Y-chromatin in aged males. IINUMA, K. and NAKAGOME, Y.....
Parental age in translocation Down's syndrome. MATSUNAGA, E.
and TONOMURA, A.
Location and neighboring amino acid sequence of inter- andintrachain disulfide bridges of human IgM. SHINODA, T.. .

XII.

95
96
97
98
99
100
101

Behavioral Genetics
Phototaxis and fecundity of Drosophila virilis. OSHIMA, C. and
CHOO, J. K.. . . . . . . .. .. .. . . . . .. . . . . .. . . .. . . .. . . . . .. . . ..
Selection and hybridization analysis on the phototactic behaviourof Drosophila melanogaster. OSHIMA, C. and CHOO, J. K.. .
Circadian rhythm of fecundity of Drosophila virilis under various
light environments. OSHIMA, C. and CHOO, J. K.. . . . . . ..
Bad effect of urbanization and noise environments of a great city
on behaviour of Drosophila. OSHIMA, C. and CHOO, J. K...

XIII.

102
104
105
107

Applied Genetics

Genetic variation in a naturally reproducing sapling-culster in
Thujopsis dolabrata Sieb. et Zucco SAKAI, K. I., IWAGAMI, S.
and MIKAMI, S.
Isoperoxidase variation among island populations of Pinus Thunbergii-densiflora. PARK, Y. G., IYAMA, S. and SAKAI, K. I.. .
Effect of mixing genotypes of barley on the performance of a
population. IYAMA, S. and SAKAI, K. I.. . . . . . . . . . . . .. . . . .

109
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CONTENTS

Genetic changes occurring in wild quails due to "natural selection"
under domestication. KAWAHARA, T.
Multiple ovulation and egg defects in Japanese quail. KAWAHARA, T.
Estimation of heritability from analysis of variance in a population with related parents. FUJISHIMA, T. . . . . . . . . . . . . . . . . .
Estimation of heritability from parent-offspring regression in a
population with related parents. FUJISIDMA, T. . . . . . . . . . . .
Variations in growth pattern among rice varieties. MORISHIMA,
H. and OKA, H. I.
..
The distribution and effects of genes causing F 1 weakness inOryza breviligulata and O. glaberrima. CHU, Y. E. and
OKA, H. I.
Preliminary observations on the establishment of perennial
ryegrass and orchard grass varieties. MORISHIMA, H.. . . . . .
Collection and preliminary observation of cultivated cereals andlegumes in Ethiopia. SAKAMOTO, S. and FUKUI, K.......
Books and papers published in 1971 by research members. . . . . .. . . . .
Abstracts of diary for 1971. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Foreign visitors in 1971. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement... . . . . .. . . .. . . .. . . . . . . . . . . .. . . .. .. . . . . . . .. . .
Author index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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GENERAL STATEMENT
To our deep regret, Dr. Kan Oguma, the founder and the first directorof our institute, died at the age of eighty-six years on September 10, 1971
at Odawara. Indeed Dr. Oguma was the creator of the new institute ofgenetics. The start was made on the occasion of the Twelfth Annual
Meeting of the Genetics Society of Japan in October, 1939. In 1949 hetook office as its first director. Devoted to his duties, in this short time of
six years, he has placed the institute on a stable basis, assembling researchers of ability.
The new library was completed at the end of March of this year as scheduled. The first main floor is now occupied by an office room and a reading
room. Rooms on the second and third floors are for conferences or seminars. The rear half of the building is constructed upon the design of fourstoried bookrooms. On the wall of the third floor Dr. Kihara's portrait
is immortalized in bronze, greeting the visitor with his famous saying,"The History of the Earth is recorded in the Layer of its Crust; The Historyof all Organisms is inscribed in the Chromosomes. Hitoshi Kihara, 1946."
Dr. Mitsuo Tsujita, Head of the Department of Biochemical Genetics,
retired in March, according to the age limit. Devoting himself to his studies
in this institute for twenty-one years since 1950 he has produced many
valuable achievements and contributed his share to the institute prosperity.
This year three members of the staff of the institute traveled abroad as
follows:
Dr. K. I. Sakai, March 1-21, and April 12-June 26, Formosa, for the
purpose of genetic investigations of forest trees in Formosa on one hand,and for giving courses on the other hand at Chung-Hsing University.
Dr. Y. Tazima, June 13-30. U.S.A., to attend the 21st Session of UNSCEAR (the United Nations Scientific Committee on the Effect of Atomic
Radiation) (New York).
Dr. E. Matsunaga, June 24-July 16, Switzerland, West Germany andAustria, to attend the Experts Committee of the WHO (Geneva): Sept.
4-26, France, West Germany and U.S.A., to attned the 4th International
Congress of Human Genetics (Paris).

2 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

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
IMAI, Hirotami, D. Sc.

3. Department of Physiological Genetics
OSHIMA, Chozo, D. Sc., Head of the Department
The 1st Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
WATANABE, Takao K., D. Sc.
OISHI,* Kugao, Ph. D.
CHOO,* long-Kil, M. S.

STAFFThe 2nd Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
SAKAMOTO, Sadao, D. Ag.

4. Department of Biochemical Genetics
TSUJITA, Mitsuo, D. Ag., Head of the Department
The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory
YAMADA, Masa-Aki
The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
Endo, Toru, D. Ag.
The 3rd Laboratory
TSUJITA, Mitsuo, D. Ag., Head of the Laboratory
SAKURAI, Susumu, D. Med.

5. Department of Applied Genetics
SAKAI, Kan-Ichi, D. Ag., Head of the Department
The 1st Laboratory
SAKAI, Kan-Ichi, D. Ag., Head of the Laboratory
KAWAHARA, Takatada, D. Ag.
FUJISHIMA, Tohru, D. Ag.
The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
BAGCHI,* Sujit
PARK,* Y. G.
The 3rd Laboratory
OKA, Hiko-Ichi, D. Ag., 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* Research members under grant from other organization or visiting researchers.

3

4 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

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. Sc., Head of the Laboratory
AMANO, Etsuo, D. Ag.
SADAIE, Yoshito

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
lINO, Tetsuo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
lINO, Tetsuo, Ph. D., D. Sc., Head of the Laboratory
ENOMOTO, Masatoshi, D. Sc.
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

COUNCIL

5

MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Laboratory
YAMAZAKI, Tsuneyuki, Ph. D.

10. Department of Molecular Genetics
MIURA, Kin-ichiro, D. Sc., Head of the Department
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
FUKUI, Teijiro, Chief of the Finance Section
Honorary Members

KIHARA, Hitoshi, D. Sc., Director of the Kihara Institute for BiologicalResearch, Member of Japan Academy, Emeritus Professor of Kyoto
University
KaMAl, 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
TSUJITA, Mitsuo, D. Ag.

COUNCILKIKKAWA, Hideo, Chairman, Professor of Osaka University
Fum, Takashi, Vice Chairman, Emeritus Professor of University of Tokyo* Late6 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

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, Emeritus 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, Emeritus Professor of University of TokyoMARUO, Bunji, Director of Institute of Applied Microbiology, Universityof Tokyo
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 1971
Department of Morphological Genetics

Genetic studies of the silkworm (TAZIMA and ONIMARU)
Repair processes in radiation mutagenesis (TAZIMA and ONIMARU)
Studies on radiosensitivity in the silkworm (TAZIMA and MURAKAMI)
Chemical mutagenesis in the silkworm (TAZIMA and ONIMARU)
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 in Rattus rattus (YOSIDA and SAGAI)
Chromosome studies in rodents (yOSIDA, KATO, TSUCHIYA and SAGAI)
Chromosome alteration and development of tumors (YOSIDA)
Cytogenetical study on monosomic and trisomic cultured mammalian cells(KATO)
Cytogenetical and immunological studies on mouse plasma cell tumor(MORIWAKI, K. and SADAIE, T.)
Biochemical studies on serum transferrin variations in rodents (MORIWAKI,
K., SADAIE, and TSUCHIYA)
Experimental breeding and genetics of mice, rats and other wild rodents(YOSIDA, MORIWAKI, K., TSUCHIYA, SAKAKIBARA and TAKAHASHI)
Cytogenetical study of ants (IMAI)
Department of Physiological Genetics

Population genetics of deleterious genes in natural populations of Drosophilamelanogaster (OSHIMA, WATANABE and CHOO)
Mechanisms of persistence of some lethal 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)

8 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

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)
Genetical and biochemical studies of human serum proteins (OGAWA andODAKI)
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, KAWAHARA and FUJISHIMA)
Genetic studies in wild populations of Japanese quails (KAWAHARA)
Theoretical studies on breeding techniques (SAKAI, !YAMA and FUJISHIMA)
Studies on competition in plants (SAKAI and !YAMA)
Genetic studies in natural stands of forest tree species (SAKAI, !WAGAMI and
PARK)PROJECfS OF RESEARCH FOR 1971

9

Simulation studies on artificial selection (IYAMA)
Evolutionary studies in wild and cultivated rice species (OKA andMORISHIMA)
Analysis of genetic variations in plant type and growth pattern In rice
varieties (MORISHIMA and OKA)
Ecological genetic studies in some grass species (MORISHlMA)
Genetic analysis of isozymes in rice plants (PAl and ENDO)
Department of Induced Mutation

Molecular mechanisms of spontaneous and radiation-induced mutations(KADA, NOGUTI and SADAIE)
Environmental mutagens and carcinogens (KADA, TUTIKAWA, SADAIE andNOGUTI)
Radiation genetics in mice (TUTIKAWA)
Biochemical factors involved in cellular repair of genetic damage (NoGUTI,SADAIE and KADA)
Mutation and differentiation studies of plant tissue culture (FuJII, AMANO
and KADA)RBE and dose rate effects in higher plants (FuJII and AMANO)
Fine structure analysis in maize genetics (AMANO)
Department of Human Genetics

Dermatoglyphics (MATSUNAGA and MATSUDA)
Down's syndrome in Japan (MATSUNAGA and OISHI)
Cytogenetics in man (NAKAGOME, IINUMA and OISHI)
Molecular hybridization studies of human chromosomes (NAKAGOME)
Studies on structure of human immunoglobulins (SHINODA)
Prenatal detection of genetic disorders (NAKAGOME and IINUMA)
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)

10 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Genetics of motility in bacteria (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, MARUYAMA and OHTA)
Studies on molecular evolution from the standpoint of population genetics(KIMURA and OHTA)
Mathematical studies on the genetics of structured populations (MARUYAMA)
Linkage disequilibrium in finite populations (OHTA and KIMURA)
Experimental studies on protein polymorphism in Drosophila (YAMAZAKI)
Simulation studies on linkage disequilibrium in artificial populations (YAMAZAKI)
Department of Molecular Genetics

Studies on the chemical structure of genome of viruses containing doublestanded RNA (MIURA, FURUIcm, SmMoTOHNo and SUZUKI)
RNA polymerase in a virion containing double-stranded RNA (MIURA,SHIMOTOHNO, FURUICHI and KAGAYA)RESEARCHES CARRIED OUT IN 1971
I.

MOLECULAR GENETICS

Identity of the 3'-terminal sequences in the ten genome
segments of silkworm cytoplasmic polyhedrosis virus
Yasuhiro FURUICHI and Kin-ichiro MIURA

Double-stranded RNA preparation isolated from cytoplasmic polyhedrosis
virus (CPV) is a mixture of ten different genome segments as reovirus (1.
Fujii-Kawata, K. Miura and M. Fuke: J. Mol. BioI. 51 (1970) 247). The
3'-terminal nucleosides of double-stranded CPV-RNA segments consistof 50% cytidine and 50% uridine (Y. Furuichi and K. Miura: J. Mol.
BioI. 64 (1972) 619). The distribution of these two kinds of terminal nuc1eosides among the genome segments was investigated. The 3'-terminiof RNA was oxidized and then reduced with 3H-sodium borohydride. The
3'-terminally labelled RNA was separated into three fractions by chromatography on the methylated albumine-Kieselguhr column and into nine
fractions by polyacrylamide gel electrophoresis. Each fraction was hydrolyzed with alkaline or ribonuclease T 2 , which cleaves every internucleotide bondin RNA. The 3H-Iabelled nucleoside trialcohols from the 3'-termini of
RNA was analyzed by paper or thin layer chromatogrpahy. The results
show that every double-stranded RNA segment has an equal amount ofcytosine and uracil as its 3'-terminal base.
When the 3'-terminally labelled CPV-RNA was digested with pancreatic
ribonuclease A, the result was quite analogous to the alkaline or ribonucleaseT 2 digestion, giving equal amounts of C' and U' (trialcohol derivatives ofcytosine and uracil). This suggests that all the penultimate bases of the
3'-termini are pyrimidine. Ribonuclease TJ, which splits polyribonucleotide at guanylic acid residues specifically, gave CpU' and CpC' as 3'-terminally labelled oligonucleotides. This leds to the conclusion that all the penultimate bases of the 3'-termini are cytosine.
From these results, the segmented genome RNA chains in CPV possess
the common 3'-terminal structure as shown below:

12 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22
5' - - - - - - - - - p C p U 3'
3' CpCp5'

It is possible that the transcriptase of the RNA genome may require adefinite and common site for initial binding to the template.

Single-stranded RNA synthesis in vitro by the RNA
polymerase associated with cytoplasmic polyhedrosis
virus containing double-stranded RNA
Kunitada SHiMOTOHNO and Kin-ichiro MIURA

RNA polymerase associated with cytoplasmic polyhedrosis virus, whichcontains double-stranded RNA as a genome, synthesizes single-stranded
RNA in vitro. The optimal conditions for RNA synthesis by this enzyme
were investigated.
The RNA product has the same size distribution as the genome segments,
showing two fractions on glycerol concentration gradient centrifugation.
Each fraction of the synthesized RNA hybridized specifically with thecorresponding size fraction of the denatured genome RNA segments.
Only after transcription has been carried out over the whole length ofeach genome segment does the single-stranded RNA product leave the virus
particle. It appears that the transcription of every segment starts at thesame time, although the transcribed RNA from the shorter segment groupis released from the virion faster than that from the larger group.

RESEARCHES CARRIED OUT IN 1971

II.

13

MICROBIAL GENETICS

A Non-chemotactic Mutant in Salmonella
Tetsuo IINOll and Tomoko OaucHI 21

Among the mutants of Salmonella strain SJ670 (Asakura, S. et al., J.
Mol. BioI. 16: 302, 1966) which fail to swarm on semisolid nutrient plates(NGA), SJ4006 was found to be fully motile in nutrient broth. The mutant
has flagella indistinguishable from the parent strain either in shape or
number. It's growth rate and nutritional requirements were also unchanged
from those of the parent. It was sensitive to flagellotropic phage chi.
Capillary tube method of J. Adler (Nature, 153: 708, 1966) was applied to
examine chemotaxis of the mutant: it failed to make chemotactic bands inbroth or in glucose solution (2.5 X 10-· M). From these characteristics,
the mutant was inferred to be generally non-chemotactic, comparable withthat designated as che- in Escherichia coli (Armstrong, J. B. et al., J. Bacteriol.
93: 390, 1967).
Transduction was carried out with phage P22 from the representative deletion mutants covering various regions of the fla-HI cluster to the nonTable 1. Transductions from various deletion mutantsof the fla-HI region to SJ4006
Donor

Region of deletion

Relative frequencyof recombinant

wild
fla373fla1319
fla1174
fla1139
fla302
fla291
fla234
fla1140
fla1149,none
flaD, B, Q, P, N, A1I1, All, AI
flaAIlI, All, AI, HI, flaL
flaE,K
flaE, K, motA
flaE, K, motA, B
flaE, K, motA, B, flaC
flaC, M
flaC, M
flaC

100.0

98.2
83.6
9.3
4.0
1.6
0.0

0.0
5.9
9.8

11
Present address: Laboratory of Genetics, Faculty of Science, University of Tokyo,
Hongo, Tokyo.
21
Laboratory of Genetics, Faculty of Science, University of Tokyo, Hongo, Tokyo.

14 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

chemotactic mutant. Chemotactic recombinant clones were identified
as swarms on NGA. The results indicated that the mutant site was covered
by deletions of fla291 and fla234, and it was mapped between motB andflaC (Table 1). The trails developing as the result of complementation in
abortive transductants were observed in transductions where recombinantswarms appeared on NGA containing 0.2 % (w/v) agar. Thus it is concluded that SJ4006 is a che- mutant in a cistron homologous either tocheA or cheB of E. coli (Armstrong, J. B. & Adler, J., J. Bacteriol. 97: 156,
1969).
The number of recombinant swarms from the mixture of 109 phage particles and recipient bacteria at m.oj. 3 were scored and converted to thepercentage to the number in transduction from fla+. The order of cistrons
in fla-Hl region is, flaD-B-Q-P-N-AIII-AII-AI-HI-flaL-E-K-motA-B-flaCM.

Polymerization of External FlageIlin at the Ends ofthe Hooks Attached to Salmonella Cells
Tetsuo IINO\}, Hideho SUZUKI and Shigeru YAMAGUCHI" I

Exogenous flagellin monomers can be reconstituted to flagellar filaments
at the tip of flagella attached to the living cells of Salmonella (Iino, T. et al.,1971. Ann. Rep. Nat'l Inst. Genet. 21: 18). The reconstitution experiment
was forwarded with the reaction system in which the cells of a flagellinlessmutant, SJW604, derived from a phase-I stable Salmonella (antigen gt)
were mixed with flagellin monomers of the wild strain TM2 (antigens
i: 1, 2). The mutant strain has a defect in the structural gene of phase 1
flagellin, HI, and consequently produces hooks to which flagellar filaments
are not attached.
To the mixture of 5 x 1010 cells/ml of SJW604 and 8 mg/ml of TM2 flagellin in 0.03 M phosphate buffer (pH adjusted to 6.6 with 1 M KOH), 1/25
volume of 2 M Ka-citrate was added and kept at 26°C. After incubationfor 1 hour, 3.7% of the cells were found to carry reconstituted normal
11
Present address: Laboratory of Genetics, Faculty of Science, University ofTokyo, Hongo, Tokyo.
21
Present address: Department of Biology, School of Education, Waseda University, Totsuka-machi, Shinjuku-ku, Tokyo.

RESEARCHES CARRIED OUT IN 1971

15

flagella; 15 %among them carried two flagella and others only one. Average
length of the reconstituted flagella was 2.2 normal wave units. After twohours of additional incubation, the fraction of flagellated cells increased up
to 6.7 % of the cell population and the average length of their flagella to 2.5
normal wave units. Most of them carried a single flagellum each and 21 %of the flagellated cells carried two flagella. Very rarely cells with three
flagella were detected. The cells incubated in the same condition without
mixing flagellin were entirely nonflagellate as many as 1000 cells being
examined. Thus, the experiment demonstrated that on the mutant cells
flagella are reconstituted from exogenous flagellin without intermediationof ready-made flagellar filaments. When the fla- mutants, which cannot
produce hooks as well as flagella, were used in place of SJW604, reconstitution was not initiated.

Isolation of Flagellar Hooks from Non-flagellate
Salmonella Mutants
Shigeru YAMAGUCHI!), Tetsuo IIN02 ) and Tsuneyoshi KUROIWA 3 )A bacterial flagellum is composed of a filament, a hook, and a basal
body. The filament is the distal helical part of the flagellum and is apolymer of flagellin. The basal body is the proximal structure, boundto the cell wall and membrane (DePamphilis, M. L. and Adler, J., 1971.
J. Bacteriol. 105: 384). The hook connects the filament with the basal
body. Both filaments and hooks can be detached from flagellated cellsby mechanical shaking (Abram, D. et al., 1970. J. Bacteriol. 101: 250).
In Salmonella, the non-flagellate phenotype results from mutation ineither the H or the fla genes. The H genes, HI and H2, are the structuralgenes for phase-! and phase-2 flagellins, respectively. The precise functionsof fla genes are not known. Fourteen fla complementation groups havebeen recognized. So-called non-flagellate mutants lack the filament, but
it is not known if they possess other parts of the flagellum. In the present
study the possession of hooks by non-flagellate mutants, including both11
Present address: Department of Biology, School of Education, Waseda University, Totsuka-machi, Shinjuku-ku, Tokyo.
2)
Present address: Laboratory of Genetics, Faculty of Science, University of Tokyo,
Hongo, Tokyo.
3)
Tokyo Metropolitan Isotope Research Centre, Setagaya, Tokyo.

16 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

those representative of all the known fla complementation groups (flaAI,
All, All, B, C, D, E, F, K, L, M, N, P, and Q) and the HI mutants derived
from phase-l stable strains of Salmonella abortus-equi SL23, was examinedby electron microscope observation of material mechanically detachedfrom bacteria.
The procedures for the isolation of hooks were as follows. A suspensionof bacteria in saline was shaken at 750 strokes/min and centrifuged at
10,000 x g. The supernatant was further centrifuged at 98,000 x g for 1 hr
and the pellet was resuspended in 0.1 M Tris-hydrochloride (pH 7.8). Thissuspension was then given two cycles of differential centrifugation first at
10,000 x g for 10 min to sediment the bacterial debris and then at 98,000 x g
for 1 hr to sediment the hooks. The final pellet was resuspended in a small
volume of 0.1 M Tris-hydrochloride (pH 7.8), to which lysozyme (0.02 %)and Brij-58 (1 %) were added to lyse the contaminating fragments of cellwall and membrane. After incubation at 30 D e for 30 min, the suspension
was centrifuged at 98,000 x g for 1 hr. The resulting pellet was resuspended
in distilled water and observed with an electron microscope.
Hooks were found in all preparations obtained from HI mutants but
none in those from fla mutants. Hooks obtained from HI mutants wereof almost uniform length (72±8 nm) and diameter (18 nm). Their sizeand shape were indistinguishable from those of the wild strain. One
end of those hooks appeared to be frayed or split and the other end to be
blunt. Surface patterns of hooks suggest a structure composed of several
fine coils and twisted to form a short helix of relatively small pitch.
Hook and filament are not only distinguishable morphologically but
they also differ in serological specificity (Lawn, A. M., 1967. Nature,
London 214: 1151) and solubility in acid and/or alcohol (Abram, D. et al.,1970. J. Bacteriol. 101: 250). However, it is still possible that both arecomposed of flagellin molecules in different conformational states. But
the presence of hooks in HI mutants clearly shows that hook formation is
not under the control of the flagellin structural gene and that hooks are
therefore composed of subunits other than flagellin.
It has been shown that flagellar filaments have a structural polarity andare formed by sequential polymerization of flagellin molecules at the distal
end of each filament (Asakura, S. et al., 1968. J. Mol. BioI. 35: 227; lino,
T. 1969. J. gen. Microbiol. 56: 227; Emerson, S. V. et al., 1970. Science
169: 192). The structural similarity of the distal frayed end of the hook to

RESEARCHES CARRIED OUT IN 1971

17

that of the filament may indicate the formation of the hook by sequential
polymerization of subunit molecules. Hooks are about the same length
irrespective of the presence or absence of the filaments. Therefore the possibility of the addition of flagellin to those distal ends being the factor limiting their maximum length is excluded. (The details were submitted toJ. gen. Microbiol.)Ribonucleic Acid Dependent Synthesis of Salmonella
Flagellin by a Cell-Free System
Hideho SUZUKI

It has been known from serological and genetic studies that two structural genes, HI and H 2 , concerned with flagellar protein of Salmonella donot simultaneously express their genetic information, namely only either ofthem is active at one time (phase variation).
To develop an assay which can determine the biochemical process(es)causing the regulated expression of these genes, the cell-free flagellin synthesizing system was constructed with preincubated S-30 of E. coli andRNA of Salmonella, after the method used for synthesis of B. pumilus
flagellin (H. Suzuki and H. Koffler, 1970).
RNA was extracted by phenol method from the Brij 58-lysate of EDTAlysozyme-treated Salmonella cells. Most part of activity to stimulate
flagellin synthesis was attached to the RNA fraction insoluble in 2.5 M
sodium acetate. Therefore, the sodium acetate-insoluble fraction was used
as a source of an informational RNA. Newly synthesized flagellin wasrecovered with carrier flagellin from acid-soluble (pH 2.3) fraction, purified
through reaggregation with ammonium sulfate and chromatographed on
DEAE-cellulose column. Chromatography of flagellin at pH 8 on DEAEcellulose could separate at least four serologically-distinct kinds of flagellin:a, enx, i and 1.2, eluting them in this order.
In vitro synthesized flagellin, as identified by chromatography, corresponded to the serological type of the cells from which the informational
RNA derived. When RNA of phase I monophasic strain and that ofphase 2 monophasic strain were introduced simultaneously to the cellfree protein synthesizing system, both phase I and phase 2 type of flagellin
were synthesized. RNA extracted from the cells of a diphasic strain direct-

18 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

ed synthesis of both phase 1 and phase 2 flagellin, in such a ratio as expected
if the culture was at equilibrium with respect to phase variation. The cellsof the diphasic strain were propagated from a single colony expressing either
phase I or phase 2. RNA prepared from these cells stimulated in vitro
synthesis of flagellin predominantly in the same phase as expressed by theoriginal colony.
These results indicate non-existence of a regulatory activity causative ofphase variation at a translational level, and support the notion that activityof the genes concerning phase variation is regulated by control of phasespecific mRNA production.

Phage-induced Alterations in Xanthomonas citr;
Wen Chuan Wu and Tetsuo IINOli

Temperate phage PXC7 was isolated from lysogenic strain XCJl8 andpropagated on sensitive strain XCJl9 of the citrus canker bacterium Xanthomonas citri. When it lysogenized XCJl9, the tenetic characteristics ofthis bacterium were modified by an alteration from smooth to dwarf colony
types accompanied with the changes in some of bacterial cells occurring in
chains and response to virulent phage CP 2 from sensitive to resistant. The
lysogenic dwarf convertants, incubated in nutrient broth, reverted in somecells to the production of smooth colonies, among which some consistedof lysogenic cells and others of non-lysogenic cells, either resistant or sensitive. Both lysogenic and resistant smooth revertants failed to adsorb
phage PXC7 and gained resistance to CP 2 • Moreover, they had also grownsome of their cells in the form of chains. All of these changes, however,were not found with the sensitive smooth revertant.
The lysogenic dwarf convertant, after it arose from smooth strain XCJl9,
continued to liberate phage PXC7 spontaneously at the frequency considerably higher than its lysogenic smooth revertant. The resistant smooth
revertant did not liberate the phage. They did so even after having entered
their susceptible host. As a result, the lysogenic dwarf convertant could not
grow sufficiently to developing canker lesions as its lysogenic, resistant, andsensitive smooth revertants and smooth original strain. In addition, the11
Present address:
Hongo, Tokyo.

Laboratory of Genetics, Faculty of Science, University of Tokyo,

RESEARCHES CARRIED OUT IN 1971

19

lysogenic dwarf convertant also reverted in the host tissue in some cells to
produce smooth colonies, which consisted either of lysogenic or sensitive
cells. Smooth revertants arisen in such a way have the advantage of survival.
The lysogenic smooth revertant protects istelf from phage infections. The
lysogenic and sensitive smooth revertants may grow sufficiently to achieve
normal canker development, if they enter again their susceptible host. On
the other hand, the sensitive smooth revertant may also be relysogenized.
If this does occur, the phage-induced alterations in Xanthomonas citri showing in culture media might also take place under natural conditions.
The details were published in Annals of the Phytopathological Societyof Japan 38 (2,4), 1972.

Transduction by Phage PI in Salmonella typhimurium
Masatoshi ENOMOTO and Bruce A. D. STOCKER

The general transducing phage PI, active on E. coli and Shigella, does
not attack smooth S. typhimurium lines, which fail to adsorb it. However,phage PI (grown on S. typhimurium, to circumvent DNA-restriction) plateswith high efficiency on S. typhimurium LT2 mutants with some sorts oflipopolysaccharide core defect: classes rfaH and rfaG, deficient of galactosyl- and of glucosyl-LPS transferases, class gal U, lacking UDP-glucose
pyrophosphorylase, and class galE, lacking UDP-galactose epimerase.
Transduction of nutritional characters by phage Plkc, at frequencies of
10- 5 to 10- 6 per plaque-forming unit, was obtained to LT2 recipients ofthese classes.
When motility was transduced by phage P I from a motile LT2 galEdonor to non-motile mutants of an LT2 gal E line the rate of cotransductionof HI (phase-I flagellar antigen gene) was 50-90% for allfla and mot locitested (except for the unlinked flaF); the corresponding rates for transduction by phage P22 vary from 0.01 % to 38 %. flaD was co-transduced withhis by phage PI. When phage PI grown on a motile LT2 donor wasapplied to various mot A fla double mutants the number of trails (abortivetransductants) produced was about the same as when the recipient was
motA- but fla+; when phage P22 was used the number of trails obtained
from some double mutants was less than I % of the number obtained fromthe motA- fla+ recipient. These observations indicate that the chromosome

20 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

fragments in PI transducing particles are longer than those in P22 transducing particles, as is expected from the known DNA contents of the phages.
Phage PI grown on E. coli K12 or Shigella dysenteriae 16 was applied to
mot and jla derivatives of the LT2 galE restriction-negative line. Swarms,
i.e., complete transductants, were very infrequent. The appearance oftrails indicated that E. coli K12 has (at least) genes functionally corresponding
to jlaA +, jlaC+, jlaD+, jlaK+, jlaF+, motA+, and motB+ of S. typhimurium
but Shigella has only jlaF+ or a part of it.

RESEARCHES CARRIED OUT IN 1971

III.

21

BIOCHEMICAL GENETICS

Amino acid sequence of human IgM immunoglobulins
Tomotaka SHINODA, Claudine PAUL, Akira SHIMIZU,
and Frank W. PUTNAM

Amino acid sequence analysis of one IgM globulin and comparative
sequence analysis data on other IgM globulins have given new evidence on
the evolutionary relationships and genetic control of immunoglobulins.
To assemble the sequence of the fl heavy chain, Fab and Fc fragments wereprepared by tryptic cleavage at high temperature, 11 different fragmentsby CNBr cleavage, and more than 300 different peptides by digestion ofthese fragments or the whole fl chain and the whole IgM by trypsin, chymotrypsin, thermolysin and pepsin. Sequence analysis was done by conventional methods and by use of the automatic protein sequenator for
CNBr and Fc fragments and large peptides. The results have permitted
the ordering of the fragments and the peptides and the location of five
intrachain disulfide bridges, four interchain bridges, and five glycopeptides.
The effect of the disulfide bridges and of carbohydrates on IgM conformation has been studied by molecular model building. Comparative structural
study of several fl chains supports the concept of the identity of the constantregion (Cu) of different fl chains and the division of the N-terminal region(VH ) into variable-sequence subgroups shared by fl, lX, and r chains. (Science
173: 629 (1971); Prog. Immunol., 1: 291 (1971); Am. N. Y. Acad. Sci., 190:
83 (1971)). The entire work was carried out at Zoology Department, Indiana
University.

Genetic Polymorphism of Serum Transferrin in the
Field Mouse, Apodemus giliacus
Kazuo MORIWAKI, Tamiko SADAIE and Isamu HAYATA*

Remarkable chromosomal polymorphism in the field mouse, Apodemus
giliacus, collected in Hokkaido has been preliminarily reported by Hayata
et af. (Proc. lap. Acad. 46: 567, 1970). Starch gel-electrophoretic survey* Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo.

22 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22of serum in these mice revealed an apparent transferrin polymorphism;
namely fast moving band designated TfA, slow moving band TfB and the
mixture of them TfAB. The identity of transferrin was proved by analysing
the electrophoretic pattern of Fe 59-tagged serum by autoradiography.
Both of them have moved between haptologin and slow-az-macroglobulin
on the gel. Typical electrophoretic pattern of TfAB type-serum is shownin Figure I.

~

+

~

~

TfB TfA
Fig. I.
giliacus.

Alb

Starch gel electrophoretic pattern of TfAB type serum of Apodemus

Table 1.

Parents

Progeny test of serum transferrin variants in
Apodemus giliacus

Number ofLitters

Progenies--------~

TfA--------------

TfAxTfA
TfAxTfAB
TfABxTfAB

TfAB

TfB

Total

~-----------~

3
2
I

18
6
1

0
3
2

0
0
2

18
9
5

Among 5 mice collected in the field, 3 were TfA and 2 were TfAB. Laboratory crosses of these mice partly succeeded and demonstrated segregationof the transferrin types in the progenies as shown in Table 1. These results
might suggest that the mode of inheritance is based on a pair of codominant
alleles, Tf A and Tf B . There seems to be no linkage relationship between
the biarmed chromosomes characteristic in this species and those transferrin
types.

Electrophoretic Pattern of Serum Protein in Indian Brown
Spiny Mouse, Mus platythrix platythrix
Kimiyuki TSUCHIYA, Tamiko SADAIE and Kazuo MORIWAKI

Starch-gel electrophoresis of serum protein of Indian brown spiny mouse,
Mus plathythrix platythrix, kindly sent by Mr. Tachibana in Mysore, India,

RESEARCHES CARRIED OUT IN 197123

was carried out by the routine procedure in our laboratory (Moriwaki, K.
et al. Genetics 63: 193, 1969). Figure 1 demonstrates the electrophoretic
pattern where the sera of Mus muse/us (BALBjc mouse) and of Rattus
rattus rattus (Oceanian type black rat, 2n=38) were run in parallel as references. Identification of transferrin among those electrophoretic bands1]:;,t'~I:I'-"']1i!1~

AB_]>1]~:

C

+

~.:

AlbuminFig. 1. Starch gel electrophoretic pattern of serum protein in Indian brown
spiny mouse. Mus platythrix playthrix. A: Mus muse/us (BALBjc mouse bearing
rA-myeloma), B: Mus platythrix platythrix, C: Rattus rattus rattus (Oceaniantype black rat, 2n = 38). White dots indicate transferrin identified by Fe 59 -1abeling.

was conducted by Fe59 -labeling of the protein on the starch-gel plate followedby autoradiography with X-ray film. It seems to be interesting from theview point of species differentiation that· the electrophoretic mobility ofalbumin in Mus platythrix platythrix was very similar to that in Mus musculus,
whereas that of transferrin in the former species was rather close ot that in
Rattus rattus rattus.

Androcidin:

An exotoxin-like product of SR-spirochetes ofDrosophila that kills male fly zygotes?
Kugao OISHIIi

The SR-condition in Drosophila by SR-spirochetes is a phenomenon characterized by an absence of male progeny. SR fly strains with transovariallytransmitted SR-spirochetes are maintained by mating them with males
from normal strains. Infection with SR-spirochetes results in the selective
death of male zygotes.
It has been proposed that SR-spirochetes produce an exotoxin-like substance, androcidin, which kills male zygotes (Oishi, 1971). Thus, if one
11

Postdoctoral fellow supported by the Japan Society for the Promotion of Science.

24 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

eliminates SR-spirochetes from hemolymph of the SR flies and introduces
the spirochete-free hemolymph into normal flies, it may be expected thatthe injected normal flies will produce only female progenies after a shortlag as long as the androcidin activity remains high, and then progenies ina I; I sex-ratio as the androcidin activity is diluted out.
ORNSR flies (Oregon-R strain of D. melanogaster carrying NSR strainof SR-spirochetes) were first injected with spv-2 (a virus strain normallyassociated with WSR strain of SR-spirochetes, this virus multiplies in andlyses NSR spirochetes) to eliminate NSR spirochetes from the fly. Normal
Ore-R females (1-5 days old) were injected with the NSR-free hemolymph
thus obtained, mated, and reared individually at 25°C. Flies were transferred into new culture bottles every two days and progenies examined forthe sex-ratio according to the brood.
In a recent experiment, out of 34 flies injected II produced enough progenies and of which 7 produced progenies in an expected a manner.
The results strongly support the working hypothesis mentioned above.
Refinement of the procedures and techniques is being made.

Migration rate of acid phosphatase isozymes controlled
by a gene in Oryza sativa and O. perennis
Chiang PAl and Toru ENDo

In our previous papers (Shahi et al. 1969, Jap. J. Genet. 44: Endo et al.
1971, Ibid. 46), variations in leaf acid phosphatase were examined in sativa
cultivars including the Indica and the Japonica types. The both types eachshowed a specific zymogram consisting of three major bands accompanied
by at least three minor bands. The difference between the two types wasin the migration rate of the set of three major bands. Extensive examination of zymographic variations was continued with hybrid materials between
strains of O. sativa and O. perennis. Five different zymograms on starch
gel were so far detected in O. perennis. Two of them were identical to thoseof the Indica and Japonica types of sativa. The other three also had threemajor bands accompanied by at least three minor bands, and these bands
appeared to migrate as a group at different rates according to genotype.
The lowest migration rate was found in the Indica type of sativa and the second lowest in Afl07 (0. perennis subsp. barthii from Africa). The migra-

RESEARCHES CARRIED OUT IN 1971

25

tion rate was higher in the order of W628 (Asian perennis), Japonica types
and W1426 (barthii). The F 1 hybrids of these strains always produced threehybrid bands and had nine major bands in total unless some of them overlapped one another. The F 1 populations examined produced only threedifferent (two parental and F 1 ) zymograms. Although the action of genes
controlling these isozymes remains unknown, yet the results obtained suggest
the presence of a locus specifying the migration rate of a set of three major
bands accompanied by minor bands.

Effect of foreign DNA on seed formationin egg plants
Toru ENDO

Repeated grafting experiments among different pepper strains strongly
suggested the possibility of creating of so-called grafthybrids. Although
the frequency was still very low, some characters of the strains used as stock
appeared in the offspring of the scion or in the G 1 generation. Sexual F 1
hybrids of those strains were morphologically uniform, whereas the G 1
plants were variable and showed certain fruit characters which were notobserved in the F 1 hybrids. Though the mechanism is not yet elucidated,graft-hybridization may be due to the translocation of decomposed nuclear
fragments from the xylem tissue of the stock to the germ cells of the scion.
In order to evaluate this hypothesis, the following experiment was madeon the stem 60 to 70 mm below the flower petiole of each plant by using agimlet. The hole (2 mm) was covered with vinyl tube (10 mm diameter),rubber stopper, a mixture of lanolin-celite and aluminum foil. Foreign
DNA solution (0.2 to 0.5 mg/ml SSC) was poured into the tube and was
kept for 24 or 48 hours. The solution was then substituted with the sameamount of SSe.
After this treatment, 5 to 10 days old fruits obtained from self-pollination
were examined. The control (SSC only) produced normal fruits with 150
to 200 seeds. Treatments for 48 hrs with 0.5 mg animal DNA (calf thymus
and salmon sperm) resulted in fruit abscission. Treatments with 0.2 to
0.5 mg animal DNA for 24 hr often interrupted fruit browth but seldomgave fruits half or one-third in size of the normal fruiss. They had 3 to 8
seeds only, but plants from the seeds appeared to be normal. Corn DNA

26 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22(isolated from 16 days old endosperm) was also tested. A preliminary
experiment with corn DNA showed a comparatively weak retarding effecton fruit development. A continuous treatment with corn DNA (I mg/ml
ssq, however, resulted in the death of the upper part of the plant which
extended toward the of the treatment. An additional treatment with plant
hormone (6-benzyladenin and/or indole-3-acetic acid, 1 ppm) was ineffective.

RESEARCHES CARRIED OUT IN 1971

IV.

27

DEVELOPMENTAL GENETICS

In Vitro Studies on Spermatogenesis of
Drosophila melanogasterYukiaki KURODA

Testes obtained from pupae of the Oregon-R strain of D. melanogasterwere cultured by a previously described procedure (Kuroda, Y. (1970) Exp.
Cell Res. 59: 429). Those aseptically obtained from 48-hour pupae whichwere grown under sterile conditions were cut into several fragments in
Drosophila physiological salt solution. They were found to be filled with
germ cells at various maturation stages. The anterior fragments of the testes
contained spermatogonia, about 5 f.l in diameter. The middle fragments
contained germ cells at more advanced stages of spermatogenesis, spermatocytes and spermatides, about 15-20 f.l in diameter. In the posteriorfragments spermatides at early stages of spermiogenesis were found.
These fragments of testes were cultured at 28°C in T-5 flasks with 0.8 ml
of Medium K-17, which was slightly modified from Medium K-6' and supplemented with 0.1 mg/ml fetuin, 5 mg/ml peptone and 15 % fetal bovine
serum.
After 24 hours of cultivation, none or only slight detectable changes wereobserved in anterior or middle fragments which contained spermatogonia,
spermatocytes or spermatides, although the germ cells increased slightly insize and testicular sheath cells grew and extended on the glass surface of theculture flasks.
On the other hand, the spermatides in the posterior fragments expanded
markedly toward both ends along the long axis of the sperm bundles, indicating that they attained more advanced stages of spermiogenesis. This
result indicates that under culture conditions employed the process of spermiogenesis could be continuously traced under a phase microscope, whereas
it is difficult to examine the early process of spermatogenesis.

28 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Growth and Tissue-Specificity of Drosophila Embryonic
Cells in Culture
Yukiaki KURODA

Embryonic cells of Drosophila melanogaster were cultured in a chemicallydefined medium supplemented with fetuin, peptone and fetal bovine serum.
Dechorionated eggs at the stage of dorsal extension after gastrulation (4.5hours after egg laying) and at the stage of sac-like midgut (12 hours after
egg laying) were surface-sterilized, torn into small fragments and explantedon the glass surface of T-5 flasks. More active migration and division ofcells were observed when tissues from the latter stage of embryos were used
for cultivation.
At the early period of cultivation, fibroblastic cells and spindle-shaped
cells came out from the cut end of tissue fragments, and stretched theircytoplasm on the surface of culture flasks. The fibroblastic cells strongly
resembled in their morphology observed in the cultures of pupal ovaries.
The slender spindle-shaped cells were specific for embryonic tissues inculture and have not been found in the cultivations of pupal ovaries andtestes. Both types of cells increased gradually in number in further cultivation and formed cell sheets around the original fragments after 48 hours.
In addition to the above types of cells some nerve fibers were found toextend from fragments of nervous tissue. They were fine thread-like andbranched off at their distal endings. The deposition of some small droplets
was found on the nerve fibers which increased in size in further cultivation.
After 7 days of cultivation large muscle cells became noticeable. They
were motile, made contact with each other and fused to form syncytial complexes, some containing more than ten nuclei. These muscle cells pulsatedsynchronously with other muscle cells in contact with them. Their pulsation continued for more than forty days in culture when the culture medium
was changed every week.
When tissue fragments of v embryos were explanted in a medium whichcontained 0.1 mg/ml tryptophan, almost the same types of cells as thoseobserved in cultures of wild type embryos came out from the explants andgrew actively under the employed culture conditions.
An attempt was made to culture embryonic tissues from the SR strainof D. melanogaster. About one half of the embryos in this strain ceased
their development about 4 hours after egg laying. They were assumed to29

RESEARCHES CARRIED OUT IN 1971

be male. When tissue fragments from presumed male embryos were
cultured, a conspicuous difference was found in the types of cells that cameout from the tissues. Neither growth of fibroblastic cells and spindle-shaped
cells nor extension of nerve fibers were observed. After 9 days of cultivation, however, muscle cells grew abundantly and their active pulsations wereobserved.

Differential Inhibition by HexosamiDes of Aggregationof Normal Liver and Hepatoma Cells in
Rotation Culture
Yukiaki KURODA

Recently some quantitative and qualitative changes in glycolipids or
glycoproteins in the surface membrane of cells have been detected following
malignant transformation. In the present experiment the effects of somehexosamines and their derivatives on aggregation of embryonic normalliver cells and DAB-induced rat hepatoma cells were examined in rotation
culture.
Single cell suspensions were obtained by trypsinization from 7-day
embryonic quail livers and monolayer cultures of DAB-induced rat hepatoma
cell lines, dRLa-74 and dRLh-84, and were rotated for 24 or 48 hours understandard conditions in the presence of hexosamines and their acetyl compounds at the concentrations of I, 3, to and 30 mM.
The effects of D-glucosamine on aggregation of normal embryonic liver
cells and rat hepatoma cells are shown in Table I.
D-Glucosamine had inhibitory effects on normal liver cells at the conTable 1.

Concentration of
D-glucosamine

OmM
ImM
3mM
lOmM
30mM

Effects of D-glucosamine on aggregation of normalliver and hepatoma cells
Inhibition of 24 hr-aggregation ofNormalHepatoma
Hepatoma
liver cells
dRLa-74dRLh-84

++
++
++
++

+
+

++

++

30 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

centration of 1 mM and which increased gradually at higher concentrations.
On the other hand o-glucosamine had no effect on aggregation of hepatoma
cells at concentrations to 3 mM.
o-Galactosamine had more differential inhibitory effects on normal liver
cells and hepatoma cells. At the concentration of 10 mM o-galactosamine
affected strongly the aggregation of normal liver cells, whereas no effect
was found on the aggregation of hepatoma cells.
N-acetyl-o-glucosamine was ineffective or slightly effective for aggregation of both normal liver cells and hepatoma cells at the concentrations
tested up to 30 mM. Very interesting were the effects of N-acetyl-ogalactosamine. It had an inhibitory effect on aggregation only of heatoma
dRLh-84 cells, which had a high tumor-producing activity, whereas noeffects were found on the aggregation of normal liver cells and hepatoma
dRLa-74 cells which had a low tumor-producing activity (Table 2).
Table 2. Effects of N-acetyl-D-galactosamine on aggregationof normal liver and hepatoma cells
Concentration of
N-acetyl-Dgalactosamine

Inhibition of 24 hr-aggregation ofNormalHepatoma
Hepatoma
liver cells
dRLa-74dRLh-84

OmM
ImM
3mM
lOmM
30mM

++
++
-------- - - - - - - - - - - - - - - - - - - - - - - These results suggest that changes in some amino sugar composition of
the surface membrane may be produced following malignant transformation.

Studies on Sorting-Out Mechanism of Animal Cells
in Rotation Culture
Yukiaki KURODA

In the process of topographical heterogenization in homogeneous cell
populations and formation of the highly specialized architectures in histogenesis and organogenesis of higher animals, the mutual cohesiveness andRESEARCHES CARRIED OUT IN 197131

sorting-out mechanism of constituent cells may play some extremely important role.
For analyzing the sorting-out mechanism of animal cells the time-lapse
examination of the sorting-out process of HeLa cells and 7-day embryonic
quail liver cells was carried out and the correlation between the sorting-out
activity and aggregate-forming activity of both types of cells was examinedin respect to their responses to some inhibitors for RNA and protein syntheses and to glucosamine.
When mixed cell suspension of HeLa and quail liver cells was rotationcultured, quail liver cells started to aggregate at first, and 3 hours later HeLa
cells in the same suspension started to cohere. Aggregates of two types ofcells independently increased in size by cohesion of the same type of cells orcell clusters. This supports the hypothesis of "differential adhesiveness",and it is suggested that the substances responsible for the aggregate-forming
activity and sorting-out activity may be qualitatively different among a varietyof tissues.
Both the sorting-out activity and aggregate-forming activity of cells were
inhibited by puromycin and actinomycin D. The inhibition by the antibiotics of the former activity was accompanied by that of the latter activity,
suggesting that the substances responsible for both activities may be identicalwith or closely related to each other and produced through the processes
susceptible to the inhibitors of DNA-dependent RNA synthesis and protein
synthesis.
Glucosamine also affected the sorting-out activity and aggregate-forming
activity of cells. Its inhibitory effects were concentration-dependent, andnot reversible after the treated cells were transferred to normal medium. It
was suggested that glucosamine may be substituted for N-acetyl-glucosamine
present on the cell surface and that this process may be produced throughsome metabolic processes.

Further Studies on Radiation Effects on Sorting-out
Activity of HeLa Cells
Yukiaki KURODA

In the previous studies the presence of a restitution process in the lossof sorting-out activity of HeLa cells following X-irradiation was revealed32 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

when HeLa cells were exposed to fractionated X-rays. The most noticeable
restitution was found when the time of interval between fractionated X-rays
was 2 hours (For details, see Kuroda Y. (1971) Radial. Res. 48: 565).
In the present experiments the metabolic process of substance(s) whichwere relevant to the sorting-out activity of HeLa cells and susceptible to
X-irradiation was investigated. HeLa cells which were irradiated with2,000 R X-rays were intermixed with 7-day embryonic quail liver cells.
The mixed cell suspensions were rotated by the standard procedure andsorting-out process of both cells was examined at an interval of 3 hours.
The sorting-out activity of HeLa cells for quail liver cells was retained forthe initial six hours of rotation culture, then it was gradually lost in further
cultivation.
When mixed suspensions of non-irradiated HeLa cells and quail liver cells
were rotated in a medium containing 0.5 ,ug!ml actinomycin D, the essentially identical result was obtained: the sorting-out phenomenon persisted
for the initial six hours, then it disappeared in further cultivation. When
10 ,ug!ml puromycin or 1 ,ug!ml cycloheximide was added to the culture
medium, the sorting-out activity of HeLa cells for quail liver cells was inhibited immediately after addition of the antibiotics.
As a result of these experiments it was suggested that the substance(s)relevant to the sorting-out activity of the cells may be produced through theprocess susceptible to the inhibitors of DNA-dependent RNA synthesis
and protein synthesis and that the damage in the sorting-out activity producedby X-rays may be in the process of DNA-dependent RNA synthesis (transcription).

Effects of High Molecular Fractions of Serum Protein
on Growth of Mammalian Cells in Culture
K. MINATO

Supplementation of serum is necessary for the growth of most mammaliancells cultured in chemically defined media. It has been found in many
earlier works that a-globulin and albumin fractions of serum were effective
in promoting the growth of cells. It has not been known, however, whether
the effect of these high molecular substances was due to their protein molecule, as carriers of some other low molecular substances such as vitamins

RESEARCHES CARRIED OUT IN 197133

and hormones, or as nutrient sources for cell growth.
In the present experiment effective substances contained in the serum andtheir funct.ional mechanism were analyzed in monolayer cultures of HeLa
cells by adding some test substances to Eagle's (1959) chemically defined
medium.
As for the concentrations of serum, 5 % was the most effective for thegrowth of cells among concentrations tested in the range of 0.65-10 %. At
the concentrations lower than 5 % the growth rate decreased in proportion
to the logarithm of serum concentration. As for the density of cells at theinoculation, the lower the density was, the more delayed was the initiationof the logarithmic cell growth. At the cell density more than 1 X 10' cells
per ml no difference was found in the growth of cells.
Effects of some fractions of serum protein on cell growth was assayed.
The dialysed serum was almost as effective as whole serum, although the dialysate had little growth promoting activity. When serum was fractionatedby precipitation with various concentrations of ammonium sulfate, a fraction from which low molecular substances were removed by saturated
ammonium sulfate delayed the initiation of cell growth, with no detectableeffect on the growth rate. A seurm fraction precipitated with 52 % ammonium sulfate was ineffective in promoting cell growth.
These results suggest that the growth promoting substances in the serum
may be in a fraction precipitated with ammonium sulfate at the concentrations between 52 % and saturation. More detailed analysis is now underinvestigation.

34 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

V.

CYTOGENETICS

Hybridization between several subspecies of blackrats (Rattus rattus)
Tosihide H. YOSIDA and Koroku TAKAHASHIThe black rat (Rattus rattus) is divided into many subspecies, but theopinions of the taxnonomists are inconsistent. Some have claimed thatsome subspecies should be classified as independent species of the black
Table 1. Results of crossing experiments among several
subspecies of R. rattus
Parents

Fertility

R. r. tanezumix
R. r. diardii

Breeding generation

Very good

R. r. tanezumix
R. r. mindanensis

Very goodF2

R. r. tanezumix
R. r. Thailand

Very goodF2

R. r. tanezumix
R. r. rattus

Good

F2 (Fl was semisteril)

R. r. mindanensis

x

Good

R. r. rattus
R. r. diardii
x
R. r. rattus

Good(R. r. tanezumi x R. r. diardii) Flx(R. r. tanezumi x R. r. rattus) Fl------ - ---------

Good----- - - - - - - - - - - -(R. r. tanezumi x R. r. diardii) Flx(R. r. diardii x R. r. rattus) F 1

GoodSecond generation------------------First generation

RESEARCHES CARRIED OUT IN 1971

3S

rat. For instance, subspecies R. rattus diardii in Malaya is preferred to beconsidered to represent species R. diardii by Yong (1969). To classify properly the animal groups it is important to know the result of matings between
subspecies of the black rats. Therefore we collected several subspecies ofthem, bred them and then mated them in the laboratory.
The following subspecies were used in crossing experiment: Japanese
black rats, R. rattus tanezumi; Malayan black rat, R. rattus diardii; Australian, New Guinean, and Hawaiian black rats, R. rattus rattus; Philippine
black rat, R. rattus mindanensis; and Thailand black rat, R. rattus Thailand.
The results ofcrossing experiments are given in the following table (Table 1).
Based on the above experiments it can be said that subspecies of blackrats in Asia, namely R. r. tanezumi, R. r. diardii and R. r. mindanensis, andR. r. Thailand, which are all characterized by 42 chromosomes in diploid,
are very similar and hybrids between them breed easily in the laboratory.
Therefore, it is not necessary to classify them any independent species.
Black rats in Oceania which are remarkable by having 38 chromosomes
could be classified into separate subspecies, because the F 1 hybrids between
the rats and Asian rats are semisterile.
Reference
YONG, H. S.:

Chromosoma (BerI.) 27: 245-267 (1969).

Karyotypes of hybrids between several subspeciesof the black rats (R. rattus)
Tosihide H. YOSIDA, Tomoko SAGAI and Koroku TAKAHASHI

It was already reported in the previous papers (Yosida et al. 1965, 1969,
I97Ia) that the Asian black rats had 2n=42 chromosomes, but pairs No.1,
No. 9 and No. 13 are remarkable by acrocentric and subtelocentric polymorphism. Pair No. 1 in almost all Philippine black rats (R. rattus mindanensis) was subtelocentrics, but most of the pair No. 1 in Thailand (R.
rattus Thailand) and Japanese black rat (R. rattus tanezumi) were acrocentrics(Yosida et al. 1971b). Segregation of the acrocentric and subtelocentricchromosomes was just theoretical as was in the case of matings within theJapanese population. In the previous paper (Yosida et al. I97Ib), the present
author suggested that polymorphic chromosomes found in Japanese black36 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

rats chould be caused by natural mating between black rats with acrocentric
pair No. I originally had lived in Japan and those in islands of Southeast
Asia characterized by subtelocentric pair No. 1. Based on the results ofthe present maing experiments the above suggestion seems to be correct.
Hybrids between Asian (2n=42) and Oceanian black rats (2n=38) arealso easily produced in the laboratory and they had 40 chromosomes, but
the F 1 hybrids were semisterile and only one F 2 hybrid was obtained fromcrossing between F 1 hybrids (Yosida et al. 197Ic). The F 2 rat had 39
chromosomes which were characterized by M-I/A4, A-7, and M-2/
M-2. Acrocentric No.4, No.7, No. II and No. 12 pairs in the Asian
black rat are shown by A-4, A-7, A-ll and A-12, respectively. The
metacentrics arose due to Robertsonian fusion of No.4 and No. 7 and
No. I I and No. 12 are denoted by M-I and M-2, respectively. Three rats
with the same karyotype as the above mentioned were collected in
Eniwetok island, South Pacific, which seemed to be produced by natural
hybridization of the rats with 2n=38 and 42 chromosomes. One offspring obtained in the laboratory by mating between the rats with 2n=39
which were collected in Eniwetok had also 2n=39 and the same karyotype
as the above.
In the laboratory it is difficult to obtain many F 2 rats, and therefore
back crosses of the F 1 rats with 2n=40 to Asian black rats with 2n=42
Table 1. Chromosome constitution in 17 rats obtained byback cross (Fl x Japanese rats)
Type

Chromosome constitution

No. of rats obtained

I
II
III
IV

M-l/A-4, A-7, M-2/A-ll, A-12
M-l/A-4, A-7, A-ll/A-ll, A-12/A-12
A-4/A-4, A-7/A-7, M-2/A-ll, A-12
A-4/A-4, A-7/A-7, A-ll/A-ll, A-12/A-12

3
8
4
2

were performed. From back crosses 17 rats were obtained and their chromosome types were analysed (Table I). Production of rats with 16 karyotypes
was theoretically expected from the above back crosses, but only four types
were obtained. Frequencies of rats with the four karyotypes were also
theoretically the same, but that of type II was the highest observed (50 %).
Explanation why frequency of the type II does occur so more often higher
than those of the other types is difficult at the present time. It is important

RESEARCHES CARRIED OUT IN 197137

to know that the above four types have the same number of chromosome
arms, but the rats with a higher or a lower number of chromosome arms
should degenerate in the embryonal stage.
References
YOSIDA, T. H., NAKAMURA, A. and FUKAYA, T.: Chromosoma (Ber!.) 16: 70-78 (1965).
YOSIDA, T. H., TSUCHlYA, K., IMAI, H. and MORIWAKI, K.: Jap. J. Genet. 44: 89-91(1969).
YOSIDA, T. H., TSUCHlYA, K. and MORIWAKI, K.: Chromosoma (Ber!.) 33: 30-40 (1971a).
YOSIDA, T. H., TSUCHIYA,K. and MORIWAKI,K.: Chromosoma (Ber!.) 33: 252-267 (1971b).
YOSIDA, T. H., KATO, H., TSUCHlYA, K. and MORIWAKI, K.: Chromosoma (Ber!.) 34:
40--50 (1971c).

A new differential staining technique using sodium dodecyl
sulfate and banding pattern analysis ofblack rat chromosomes
Tosihide H. YOSIDA and Tomoko SAGAI

Differential staining techniques revealing the banding patterns in metaphase chromosomes were reported by several investigators. A new techniquewhich uses sodium dodecyl sulfate has been developed by us. In thepresent paper the procedure of the technique and the banding patternanalyses of the black rat chromosomes are reported.
Technique: The slides obtained by the conventional air drying procedure
were immersed in a mixture of 2X sse (0.3 M NaC!, 0.03 M sodium citrate,
pH 6.8) and 0.1 % (wjv) SDS (sodium dodecyl sulfate) for a few seconds
at room temperature. They were then washed with running tap water andstained for about 10 min. in Geimsa solution dilluted in Sorensen's buffer(pH 6.8).
Banding pattern: By use of the above technique the banding pattern of
metaphase chromosomes of black rat was revealed. The karyotype of
the Japanese black rat (Rattus rattus tanezumi) consists of 42 chromosomes(21 pairs), among which 13 autosome pairs are acrocentrics, 7 autosome
pairs are metacentrics and X and Yare acrocentrics. In all chromosome
pairs, a characteristic banding pattern was produced by the above technique.
The number of segmental bands in pair No.1 chromosomes was 7, that ofpairs No.4, 7, 9, 11 and 12 was two, and of pair No. 11 was three. Even

38 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

though the number of segmental bands is the same in pairs No.4, 7, 9, 11
and 12, the pattern is different by each chromosome pair. Pair No. 1 isalso characterized by having two constrictions, one near the centromere
and the other near the distal end, and pair No.2 by one constriction near
the distal end.
Pairs No.1 and 9 in the Japanese black rats are polymorphic with respect
to acrocentric and subtelocentric form. As to the origin of the subtelocentrics the present author considered to have arisen from a pericentric inversion in the acrocentrics (Yosida et al. 1965). From the banding patternanalysis of these chromosomes it is strongly suggested that the breakage
and the subsequent pericentric inversion have occurred near the centromereof pairs No.1 and 9 and thereby subtelocentrics have developed. Oceaniantype black rats (R. rattus rattus) are characterized by having 38 chromosomes, among which two pairs are large biarmed elements, which are assumed to have derived by Robertsonian fusion of pairs No.4 and 7, and pairs
No. II and 12 present in Asian type black rat (Yosida et al. 1969, 1971).
From the comparative analysis of banding patterns of these related chromosomes, our assumption on the origin of the large biarmed elements in the
Oceanian type black rats seems to be proved.
References
YOSIDA, T. H., NAKAMURA, A. and FUKAYA, T.: Chromosoma (Berl.) 16: 70-78 (1965).
YOSIDA, T. H., TSUCillYA, K., IMAI, H. and MORIWAKI, K.: Jap. J. Genet. 44: 89-91(1969).
YOSIDA, T. H., TSUCHlYA, K. and MORIWAKI, K.: Chromosoma (Ber!.) 33: 252-267(1971).

Similarity of banding pattern of metaphase chromosomesin black and Norway rats
Tosihide H. YOSIDA and Tomoko SAGAI

Chromosome numbers in Asian black rat (Rattus rattus) and Norway rat(R. norvegicus) are 2n=42, and their karyotypes are also very similar inboth species. In the black rats pairs No.1, 9 and 13 are characterized byhaving acrocentric and subtelocentric polymorphic pairs. Based on thesimilarity of karyotypes in both species it was suggested that the black rat
is closely related to the Norway rats (Yosida 1971).

RESEARCHES CARRIED OUT IN 1971

39

Banding patterns of chromosomes in black and Norway rats, which wererevealed by Yosida-Sagai method, were compared (Fig. I). As seen in the
figure, banding pattern in both species is very similar. Subte10centric
No. I chromosomes in the Norway rat had two constrictions as similar to

Fig. 1. Banding pattern of metaphase chromosomes in black rat (R. rattus)
and Norway rat (R. norvegicus). In each pair, left two chromosomes zre of R. r.
rattus (R. r. in the figure) and right two R. norvegicus (R. n. in the figure).

the subtelocentric homomorphic pair in the black rat, and their banding
pattern is also similar. No.2 acrocentric chromosomes in the Norway rat
showed one constriction near distal end as seen in the black rat. Banding
pattern of the chromosomes in the former species is also similar to that in
the latter. No.9 chromosomes are usually subtelocentrics in the Norway
rat and the banding pattern is similar to subtelocentric homomorphic pairin the black rats.
Reference
YOSIDA, T. H.:

This Ann. Rep. 21: 46-47 (1971).

40 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Banding Patterns of Chinese Hamster Chromosomes
Revealed by New Techniques
Hatao KATO and Tosihide H. YOSIDA

Two kinds of technique were newly developed to reveal banding patternsof the Chinese hamster chromosomes. Both techniques were essentiallythe same as those used for the extraction of proteins. The techniques consisted of incubation of air-dried metaphase preparations in a solution
composed of I) 2 M NaCI and 5 M urea for 20 min at 37°C, or dipping theslides for 2 seconds in 2) phosphate buffer (pH 8.0) containing 0.07 % 2mercaptoethanol, 2 M urea and 0.05 % SDS, and staining them in a Giemsa
solution. Banding patterns produced by these techniques appeared to be
identical to those induced by the methods reported by previous workers,
requiring post-fixation incubation of slides in warm saline.
The banding pattern was typical for each chromosome pair, permitting
unequivocal identification of several pairs which were hardly distinguishedby the conventional staining procedures. It was confirmed that these patterns had been well preserved in the chromosomes of the cultured cell line.
Details are presented in Chromosoma 36: 272 (1972).

A new technique to reveal banding pattern of humanchromosomes by using urea
Yukimasa SHIRAISHI and Tosihide H. YOSIDA

The present report deals with a new technique to reveal the banding
pattern of human chromosomes by the use of urea. Slides prepared byroutine air drying technique were treated with urea-Sorensen buffer solution(three volume of 8 M urea and one volume of Sorensen buffer solution)
for ten minutes at pH 6.8 and 37°C. Afterwards, throughly washed intap water to avoid the precipitation of urea, and then they were stainedwith Giemsa solution diluted about 50-70 times with Sorensen buffer (pH6.8) for 5-20 minutes at room temperature.
By this technique, a specific banding pattern of each pair of homologous
chromosomes was revealed, permitting easy identification of all human
metaphase chromosomes. The appearance and location of darkly stained
bands are consistent and specific in each metaphase chromosome. How-RESEARCHES CARRIED OUT IN 1971

41

ever, they were not always constant as to cell stage. At prometaphase,many finer bands could be distinguished alpng the chromosome lengths,
but supercontracted metaphase chromosomes are also disadvantageous forthe observation of banding pattern, because the bands lie too close. The
chromosome to be used for analysis of banding pattern should have a suitable
degree of contraction. A suitable condition of the chromosome is when
the length of pair No. I is contracted to about 10 fl. To find such suitably
contracted metaphases in specimens prepared by the present technique isquite easy. The banding pattern revealed by urea-Sorensen buffer incubation showed that the patterns appear to be almost identical to those given
by previous workers.

Factors Involved in the Production of Banded Structuresin Mammalian Chromosomes
Hataa KATO and Kazua MORIWAKIIt has so far been believed that the band formation in chromosomesreflects a denaturation-reassociation process of repetitious nucleotides
and that a post-fixation incubation of slides for a considerably long period
is indispensable. As described in the previous report, we found that the
banded structure was inducible within a few seconds when freshly preparedslides were properly treated prior to staining with a Giemsa solution.
This finding seemed to be imcompatible with the postulation mentioned
above, and prompted us to investigate to what degree factors other than therepetition of DNA sequence can be involved in this phenomenon.
Over 70 reagents were tested for their ability to produce bands in the
Chinese hamster chromosomes by incubating air-dried slides in aqueous
solutions of these reagents for a definite period prior to the Giemsa staining.
Acids were found to be without effect on the band production. Many ofsalts were able to induce bands if their pH was alkaline. Strong bases werealso found to be potent band-inducing reagents. They produced bands
only in a few seconds. Protein denaturants such as urea, guanidine-HCI
and several surface active compounds were also effective in band production.
In the light of these results, it seems very likely that the solubilization or
extraction of chromosomal proteins, probably of acid nature, would be the42 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

primary cause of the appearance of the banded structure in chromosome arms.
Details of this work are presented in Chromosoma 38: 105-120 (1972).

Differential Responses of Several Aneusomic Cell Clones
to Ultra-Violet Irradiation
Hatao KATO and Tosihide H. YOSIDAWith the use of a colcemid-reversal method (Kato, H. and Yosida, T. H.
1970. ExptI. Cell Res. 60: 459), we have succeeded in inducing chromosomal
nondisjunction in cloned Chinese hamster cells and so far obtained clonal
lines with various types of trisomic and monosomic chromosomes (Kato,
H. and Yosida, T. H. 1971. Cytogenetics 10: 392). The present reportdeals with the sensitivity of some of these aneusomic clones to ultra-violet
light in terms of the colony forming ability after irradiation.
Exponentially growing cells were trypsinized, plated at desired concentrations into 60 mm plastic dishes and exposed to UV light, 2537 A,
emitted from a 15 W germicidal lamp at an incident dose-rate of 10 ergs/
mm2 /sec. The cultures were then incubated for 7 days. Colonies with morethan 25 cells were counted as viable.
Clones 19a and 67b were trisomic and a clone 67f was monosomic forNo. 10 chromosome, respectively. All of them showed a significant difference in the sensitivity from the parental control line, D-6. Do values were57.5 ergs/mm 2 for the D-6, 57.5 ergs/mm 2 for the 19a and 67b and 40 ergs/
mm2 for the 67f. The heightening of the UV sensitivity was also detectedin another type of trisomic clones. No. 9 trisomy clones, lie and 54e,
showed almost the same Do values as that of the No. 10 trisomy. A disomic
clone was isolated from a long term culture of the monosomic clone. This
clone was found to be more resistant than the monomosic one, while apparently more sensitive than the control disomic line. It appears that thedisomic has arisen through duplication of a monosomic chromosome in amonosomic cell by nondisjunction, thus possessing two identical chromosomes.
There was no statistically significant difference among these clones in theability of unscheduled DNA synthesis following UV-irradiation.
It is very likely that the increased UV-sensitivity of these aneusomic
clones is attributed to their altered chromosomal constitutions. The

RESEARCHES CARRIED OUT IN 197143

addition or loss of a chromosome to or from the genome, irrespective of thetype of the chromosome, may force cells to modify their genic expression,
resulting in the alteration in the activity of various enzymes including
those involved in the DNA repair process. Details of this work arepresented in Exptl. Cell Res. 74: 15-20 (1972).

Induction of Sister Chromatid Exchanges by Ultra-Violet
Light and Inhibition by Caffeine
Hatao KATO

Sister chromatid exchanges on the Chinese hamster chromosomes werestudied by pulse-labeling cells with 3H-thymidine at various concentrations
and by fixing them at the 2nd post-labeling mitosis. There was no significant change in the exchange frequency regardless of a 40-fold range ofvariation in the tritium dose (0.76-0.96 exchanges per No.1 chromosome),
whereas both chromatid and isochromatid deletions increased linearly withthe tritium dose. This suggest that the sister chrematid exchanges are
spontaneous events, not largely affected by endogenous radiation fromincorporated tritium.
When cells were submitted to synchronization treatments utilizing either
excess thymidine, 5-fluorodeoxyuridine or hydroxyurea, the frequency ofthe sister chromatid exchanges increased twice as high as the control level.
Ultra-violet irradiation on cells treated with excess thymidine induced an
additional rise in the exchange frequency (4.20 per No. 1 chromosome at
40 ergsjmm 2), but only in those irradiated at the early S phase. The postirradiation treatment with caffeine caused a significant decrease in theexchange frequency, while a striking increase in aberrant chromosomes,
mostly of deletion type. Further studies are in progress on the basis of aworking hypothesis that the Chinese hamster cells have a repair mechanism
similar to the recombinational post-replicational repair process which is
known in microorganisms, its occurrence being visualized as the sister chromatid exchange at metaphase.

44 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Induction of chromosome aberrations in human leucocytes growing
in vitro by treatment with cadmium sulfide
Yukimasa SHIRAISHI and Tosihide H. YOSIDA

Chromosome aberration in cultured human leucocytes are induced bytreatment with cadmium sulfide. Human leucocytes from a normal female
were cultured according to the routine phytohemagglutinin method. After
72 hours incubation at 37°C, cadmium sulfide was added to the culture
medium at the final concentration of 6.2 x 10- 2 {tgjml, and then cultured
for 4 and 8 hours in the medium. By treatment with the cadmium sulfide,
several chromosome aberrations, such as chromatid breaks and translocation were observed. In the untreated control, no chromosome aberrations
were noted in the observation of 50 cells. On the other hand, in the treatedcells chromatid breaks were observed at 38 % in 4 hour treatment and 42 %in 8 hours treatment. Translocations were observed at 12 %in only 8 hourtreatment. Dicentric like chromosomes were often observed in 4 and 8hours treatment, but it is difficult to recognize whether they are real dicentric
or only a twist occuring in two doughter chromatids.
Since chromatid aberrations were found so frequently in culured cells
treated for 4 and 8 hours before harvest, these aberrations should haveoccurred in the late Sand G 2-periods. Based on the above investigations
it seems highly possible that cadmium sulfide has some mutagenic action
on genetic material as seen in some other radiomimetic chemicals.

Abnormalities of chromosomes found in cultured leucocyte
cells from Itai Itai disease patients
Yukimasa SHIRAISHI and Tosihide H. YOSIDAIn the previous paper of this report we found that cadmium sulfide produced a marked increase in the frequencies of chromosomal aberrations.
Itai Itai disease, which is a kind of diseases caused by environmental pollution, seems to be caused by cadmium. However, it was discussed by several
scientists whether this compound is really related to the cause of the disease.
To give an information on the cause of this disease, we examined the chromosomes of the patients.
Peripheral leucocytes obtained from 10 patients (female) and six normal

RESEARCHES CARRIED OUT IN 197145

females as contral were cultured according to the routine procedures. All
cultures were incubated for 72 hours at 37°C, and slides were prepared byroutine air-drying methods.
The frequency of the cells with the chromosome abnormalities was assessed in 100 to 200 cells in each of the patients and normal subjects. In
normal humans, the chromatid breaks were observed at 0 to 2.0 %. In
7 among 10 patients, however, chromosome abnormalities such as translocation, chromatid breaks, dicentric or dicentric like chromosomes and acentric
fragments, were observed at about 10 to 50 %. Among the abnormalities,
chromatid breaks were observed as a common type in these patients. In
addition, cells with chromosome translocations were found more frequently
than the dicentric or acentric fragments. On the other hand, three patients
showed a lower frequency of chromosome abnormalities (2 to 3 %). Thesefrequencies are not sifinificantly different from those of control subjects.
Bloods were taken three different times from the patients and chromosomes
were observed sequencially. The frequencies of the chromosome abnormalities in 7 patients were always higher, but slightly different by the time
obtained the blood was taken. There is a question that why 7 patients had
so high frequencies of chromosome abnormalities. Should not the otherthree patients without higher chromosome abnormalities be a real Itai Itaidisease? Another question is that the state of chromosome abnormalities
are similar to the Fanconi's syndorome as observed by Bloom et al. (1966)
and Dosil et al. (1970). Therefore, we have to examine whether the chromosome abnormalities found in the 7 patients were not caused by theother disease. It is remainsed as a further important problem to solve therelation between chromosome abnormalities and Itai Itai disease.
References
BLOOM, G. E., WARNER, S., GARALD,

P. S. and DIAMOND, L. K.: New Eng!. J. Med.
271: 8-14 (1966).
DOSIL, H., Hsu, L. Y., TODARO, G. J., LEE, S. L., HIRSCHHORN, K., SELlRIO, E. S. and
ALTER, A. A.: Blood 36: 341-352 (1970).

46 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Relation between karyotype alteration and aging oftumor stemline cells
Tosihide H. YOSIDA

Based on the extensive cytogenetical studies of the Yoshida rat sarcoma,
Makino (1952, 1957) has established a concept of tumor stemline cells forthe transplantable tumors. According to him, the most important characterof tumor stemline cells is the constancy of stemline idiogram through manytransfer generations. The constancy, however, is not always absolute as
Makino (1957) later has recognized it himself. Change of tumor stemline
karyotypes has been studied by many investigators in several tumors.
Based on the cytogenetical studies of several experimental tumors the present
author (Yosida 1966, 1968) explained the karyotype alteration of tumor
stemline cells to be depending on a sequential event of mutation and selection of tumor cells. Here is a question why transplantable tumors have astemline each consisting of a certain karyotype through many generations,in spite of the continuous and random occurrence of mutations in the cell
population.
To consolidate the different opinion on the behaviour of tumor stemline
cells, constancy and variability, the idea of the "aging" of the tumor cells
will be proposed. In human diploid cell strain, the diploid karyologicalcharacter is limited to about 50 serial passages (Hayflick and Moorhead 1961).
The diploid cells are the first stemline of the cell population. In this casethe first 50 passages seems to be the age of the diploid stem cells. By proliferation of aneuploid cells occurred after diploid cells have degenerated thesecond stemline cells developed. The second stemline cells are also limited
by "aging" like as the first diploid stem cells, and then the third and fourth
stemline cells develop sequencially.
In karyotype alteration in the tumor stemline cell could be explaned by
the same mechanism as above. As examples of the karyotype alterationin tumor cells four cases will be described here. In the Yoshida rat sarcoma
the karyotype change was observed sometimes during the tumor transplantations (Yosida 1959, Matsushima and Yosida 1971). In the MY mousesarcoma drastic change of karyotype from diploidy to tetraploidy was observed about the 90th transplant generation, which corresponds to about
6 years (Yosida et al. 1960). In the case of the Shay's rat chloroma the
karyotype alteration occurred sequentially with a period of about 2 years

47

RESEARCHES CARRIED OUT IN 1971

(Yosida and Sakai, unpublished). A rapid karyotype alteration has been
observed in mouse plasma cell tumors. In this case MSPC-I mouse plasma
cell tumor which established by us the karyotype change from diploidy totetraploidy occurred within one to two transplant generations (Yosida 1968,0'"0"0" 0 .. 0" (~)
~

aged

o

~

' - - - - - "..." .-----.j, ''-----.".-----'''''----Stemline·A

Stemline·B

Stemline·C

Fig. 1. Scheme of karyotype alteration in tumor stemline cel1s by aging.
A, Band C show tumor cel1s in each stemline. A', A", B' and B" show mutant
cel1s which are not developed as a new stemline. Broken circle denotes the cel1
degeneration by aging.

Yosida et al. 1970). Thereafter the stemline karyotypes of the tumor
changed sequentially one to another in the period of about several transfer
generations (Moriwaki et al. 1971). Karyotype alterations in the tumor
stemline cells will be explained by the "aging" of the tumor cells (Fig. I).
Stemline karyotype "A" degenerates after a certain transfer generations byage, and the new cells with karyotype "B", which occurred as result of
mutation and selection from the stemline "A", increase in the cell population, and the tumor is occupied by the new stemline "B". The stemline"B" is replaced by stemline "C" by aging after a certain number of transfer
generations.
The concept of aging in tumor stemline cells has been reported in 1971
Annual Meeting of the Society of Chromosome Research in Japan, by
Matsushima and Yosida (1971), and by Yosida (1972).
References
HAYFLICK, L. and MOORHEAD, P. S.:

Exp. Cel1 Res. 25: 585-621 (1961).

48 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22
MAKINO, S.: Chromosoma (Ber!.) 4: 649-674 (1952).
MAKINO, S.: Intern. Rev. Cyto!. 6: 26-84 (1957).
MATSusIDMA, T. and YOSIDA, T. H.: Gann 62: 389-394 (1971).
MORIWAKI, K., IMAI, H. T., YAMASIDTA, J. and YOSIDA, T. H.: J. Nat. Cancer Inst. 47:
624-637 (1971).
YOSIDA, T. H.: Z. Krebsforsch. 63: 209-214 (1959).
YOSIDA, T. H.: Jap. J. Genet. 41: 439-451 (1966).
YOSIDA, T. H.: In "Cancer Cells in Culture" ed. by H. Katsuta. Univ. Tokyo Press (1968).
YOSIDA, T. H., ISIDHARA, T. and HAMADA, T.: Gann 51: 55-56 (1960).
YOSIDA, T. H., IMAI, H. T. and MORIWAKI, K.: J. Nat. Cancer Inst. 45: 411-418 (1970).
YOSIDA, T. H.: Proc. Japan Acad. 48 (No.4): 1-8 (1972).

Karyological studies in Japanese ants (Hymenoptera, Formicidae)
Karyotypes of nine species of Ponerinae,
Formicinae, and Myrmicinaem.

Hirotami T. IMAI and Masao KUBOTA l }The karyotypes of nine Japanese ants in three subfamilies (Ponerinae,
Formicinae, and Myrmicinae) were successfully analysed by the improved
squash technique. Three ponerine species had 2n=7 and n=4 (Ponera
scabra), 2n=22 (Brachyponera sinensis), and 2n=28 and n=14 (Cryptopone
sauteri). Four formicine species had 2n=18 and n=9 (Camponotus sp. and
C. tokioensis), 2n=26 and n= 13 (Camponotus japonicus), and 2n=30 and
n=15 (Lasius niger). Two myrmicine species had 2n=18 and n=9 (Leptothorax congruus), and 2n=37, 38, 39, and n=17, 18, 19,20 (Pheidole nodus).
It was found that the variation of chromosome number observed in P. nodus
was caused by Robertsonian type polymorphism. For details, see Chromosoma (Ber!.) 37: 193-200 (1972).

A new criterion for classification of mammalian chromosomes 2 }Hirotami T. IMAI

Using the Chromosome Atlases by Hsu and Benirschke (1967, 1968, 1969),
a search was made for a quantitative criterion for discriminating the "acro13 Nakasone, Odawara.
The author indebted to Drs. E. Matsunaga and K. Moriwaki for their valuableadvice and encouragement.
l}2}

RESEARCHES CARRIED OUT IN 197149

or telocentrics" from the "meta-, submeta- and/or subtelocentrics". Examination of some measurements for each chromosome with reference tothe classification made by these authors showed that, although they hadtaken into account the arm ratio and/or the size of the short arm, there was

f:

fI')

W

~

500

I:
I:I:

I'

~

I'

0

~::c 400

I:

,:

u

LL

0

':I:

300

,:

c:
w
CD

~

I~

,',
I '••

200

I'

Z

~I ....' ,.'

,

100

0
0

1.0
2.0
3.0
PER CENT WEIGHT OF THE SHORT ARMS (Sw)

4.0

Fig. 1. Frequency distribution of chromosomes according to the size of theshort arms (Sw). Open circle and broken line; the "acro- or telocentrics" identified
by Hsu and Benirschke. Solid circle and solid line; the "meta-, submeta- and/or
subtelocentrics" identified by these authors, where the chromosomes showing Sw
4.0 were omitted. Dot line; the total chromosomes examined.

no consistent rule in their classification that could be defined quantitatively.
On the other hand, the frequency distribution of the size of the short arm(measured in wieght relative to the X-containing haploid set) of all the chromosomes revealed an apparent bimodality (Fig. 1), which made it possible
to classify the chromosomes into two distinct categories, one with the short
arm smaller than 0.6 (acro- or telocentrics) and the other with the short
arm larger than 0.6 (meta-, submeta- and subtelocentrics). This simple

50 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

rule would give betwetr agreement with the Hsu and Benirschke's classification as compared with the arm ratio system; it would further enable us toscore the fundamental number (NF) more objectively than before.

Karyotypes of four species of bats collected in Japan
Kimiyuki TSUCHIYA, Masashi HARADA and Tosihide H. YOSIDA

Karyotypes of greater horseshoe bat (Rhinolophus ferrumequinum mikado),
Hosono's whiskered bat (Myotis hosonoi), Japanese pipistrelle (Pipistrellus
abramus) and large noctule (Nyctalus lasiopterus aviator) belonging all to
the Chiroptera were analysed.
Five greater horseshoe bats were collected from Numazu in Shizuoka
Pref. (l female), Takine in Fukushima Pref. (2 males) and Akiyoshi in Yamaguchi Pref. (l female and I male), a male Hosono's whiskered bat from Mt.
Fuji, 2 male Japanese pipisterelles from Takasaki in Gunma Pref. and afemale large noctule from Misima in Shizuoka Pref. The karyotypes wereanalyzed in the femoral and fumerus bone mallow cells using a rutine airdrying technique and staining with acetic orcein.
Table 1.

Karyotype formulae of 4 Chiropteran species

Species2nRhinolophus ferrumequinum mikado
Myotis hosonoi
Pipistrellus abramus
Nyctalus lasiopterus aviator

58
44
26
42

Karyotype formulae
M
4
10
12
8

SM

8
2

ST

A

X

Y

52
32
4
30

SM
SM
A
M

A
A
m

The diploid chromosome number in greater horsehoe bat, Hosono's
whiskered bat, Japanese pipistrelle and large noctule were 58, 44, 26 and 42,
respectively. Karyotypic formulae of these species are listed in Table I.
Karyotype of the greater horseshoe bat (R. f mikado) was identifiable fromthat of R. f ferrumequinum already observed by Capanna and Civitelli(Caryologia, 17: 361, 1964). Karyotype of Hosono's whiskered bat (M.
hosonoi) was almost indistinguishable from that of M. macrodactylus observed by Sasaki (Mamm, Chromo Newsletter, 11: 22, Figs. 8, 1970). The
present data on the karyotype of Japanese pipistrelle (P. aburamus) have

RESEARCHES CARRIED OUT IN 1971

51

confirmed the previous observation by Takayama (lap. l. Genet., 34: 107,
1959). The karyotype of N. I. aviator was similar to that of N. noctula
reported by Dulic et al. (Experientia, 23: 945, 1967).

Indian brown spiny mouse, Mus platythrix, having the lowest
fundamental chromosome number
Kimiyuki TSUCHIYA and Tosihide H. YOSIDA

Ellerman has reported that the Indian brown spiny mouse, Mus platythrix
platythrix Bennett, is distributed in the Deccan district of India (The Fauna
of India, 3: 771, 1961). One male was sent from Mysore, South India, by

Xy
lOr I

II:,IIJI
181ft
Fig. 1. Karyotype (A) and banding pattern, (B) revealed by NaOH treatment of a male Indian brown spiny mouse.

courtesy of Mr. Tachibana in Octobber, 1971. The external measurementsof this specimen were as follows: Length of head and body 120.0 mm,
hind foot without claw 17.5, ear from notch 17.0, and body weight 59.5 g.
Chromosomes were observed in bone marrow cells by routine air-drying
technique. Diploid chromosome number was 26 in 20 cells, and all autosome pairs and the X and Y chromosomes were acrocentrics (Fig. 1).

52 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

According to Gropp et al. (Experientia, 25: 875, 1969) the tobacco mouse,
Mus poschiavinus, has 26 diploid chromosomes, but the karyotype is obviously
different from that of Mus platythrix platythrix, namely the former has 7
pairs of metacentrics and the fundamental number (FN) 40, whereas FNof the latter is 26. Diploid chromosome number in the African pigmy mouse,
Mus minutoides, was 18, but all chromosomes were metacentrics, and FN
was 36 (Matthey, Rev. suisse Zoo\., 73: 585, 1966). Fundamental numberin the Indian brown spiny mouse is the lowest for the genus Mus, so far as
the present knowledge of their karyotypes is concerned.

Possibility of Clonal Senescence in MSPC-l Mouse Myeloma
Cells during Serial Transplantation
Kazuo MORIWAKI and Tamiko SADAIE

The present author had already made his opinion known of the possibilityof cell population change in MSPC-l mouse myeloma due to the senescenceof a cell clone followed by the proliferation of a new variant cell clone during
serial transplantation (Moriwaki, K. and H. T. Imai, Acta Haem. lap. 33:
67, 1970; Moriwaki, K. et al. l. Nat. Cancer Inst. 47: 623, 1971). For supporting further this hypothesis, quantitative comparison of the growth rate
between 2 cell populations obtained from an earlier transplant generationand from the later one, was undertaken in this study.
MSPC-l myeloma has exhibited at least 5 times of cell population change
during about 50 in vivo serial passages. Three of them were detected by
the appearance of the marker chromosomes, A, B, and C, probably due toisochromosome formation, and one of the remaining was found to be a lossof the capability for rA-globulin production. Fifth change was a single
non-disjunction, which seems to have occurred close to one of the isochromosome formations mentioned above. It has been visualized by the
banding pattern analysis of chromosomes (On the analytical procedure, see
this Annual Report p. 53). These changes are summarized in the following flow chart.
i
n dj
i
n p
i
P-40~->P-4o-A-)P-39-A-->P-39-AB-->NP-39-AB-->NP-39-ABC" dj ',.P-41(P: rA producer; NP: rA non-producer; Number: modal chromosomes;

RESEARCHES CARRIED OUT IN 1971

53

A.B and C; marker chromosomes; i: isochromosome formation; n dj:
non-disjunction; n p: loss of an ability of rA production)
In order to reveal the mechanism of those cell population changes, thequantitative comparison of growth rates between P-41 subline instead of
P-4~A in this case from the earlier transplant generation and NP-39-ABC
subline from the later generation, from each 104 viable cells were inoculated subcutaneously. After 2 weeks the total cell numbers of the individual
tumors were counted; P-41 was 0.8x 108 and NP-39-ABC 2Ax 108 • In
vivo growth rate of NP-39-ABC seems to have become larger than that of
P-4I by 3 times after 4 changes in the tumor cell population. This may
imply that the ratio of increase in in vivo growth rate per single population
change is approximately 1.32, because 1.324 =3.0. Provided that one candetect new variant cells by karyotype analysis when the minor population
exceeds approximately 2 % of total cells and that their growth rate is larger
than that of the major cell population surrounding them by 1.32 times,
further 14 transplantations are needed before the tumor cell population is
occupied mostly by the variant cells, because 1.3214 =50. In fact, however,
the tumor cell population changes have been completed more rapidly. For
instance, at generation 21 the tumor cell population consisted of more than
98 % P-4~A cells, whereas at generation 27 more than 98 % P-39-AB
cells occupied that tumor. Such rapid population change has to be explained not simply by the larger growth rate of the new variant cells but also by
possible senescence of aged cell clones. So far nobody considered the concept of clonal senescnce in transplantable tumor cells based on the quantitatively valid evidence which can exclude the possibility of cell population
shifts by the selection of fast growing variant cells. The present study seems
to have successfully demonstrated the possible clonal senescence as such.

Simplified Alkaline Phosphate Buffer-Giemsa Method forInducing Banded Structure in the Mouse Chromosomes
Kazua MORIWAKI and Tamika SADAIE

The recent advance in cytogenetical methods for producing banding
patterns in the metaphase chromosomes has made it possible to identify
clearly each pair of mouse chromosomes which was almost impossible fora long time. The present study attempted to modify those procedures re-

54 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

commended by several workers (Sumner, A. T. et al. Nature New Biology
232: 31, 1971; Schnedl, W. Chromosoma 35: 111, 1971: Miller, O. J. et al.
Proc. Nat. Acad. Sci. 68: 1530, 1971) to more simiplified forms. We usedbone marrow cells of normal BALB/c mouse and MSPC-l mouse myeloma
cells as materials.
Table 1.

NaOH
pretreatment

+
+
+
+

Effects of alkaline pretreatment and solvent for Giemsa
solution on the stained features of metaphase
chromosomes MSPC-I mouse myeloma cells
Solvent for
Giemsa solution

Features of staining
Whole chromosome

Distilled water+o.25 M Sucrose+

0.35% NaCI+MilS Sorensen buffer

+

Dots

Bands

+
+
+
+

The chromosome preparations of those cells have been made following
routine procedures for fixation and air-drying. Those preparations weretreated with 0.005 N sodium hydroxide for 1-2 seconds at room temperature
followed by staining with Giemsa solution for 5 minutes diluted 25 times
by M/15 Sorensen phosphate buffer (pH 6.8). This simplified procedure
was good enough to induce clear banded structure on the metaphase chromosomes of those cells. In order to simplify this method further, Giemsa
solution was diluted with distilled water or 0.25 M sucrose but neither ofwhich could induce definite bands. Giemsa solution diluted with 0.35 %sodium chloride could give a reliable bnading pattern. The data of thoseexperiments are summarized in Table 1. The details of further survey ofsuitable conditions for inducing band patterns was reported elsewhere (Kato,
H. and K. Moriwaki, Chromosoma 38: 105, 1972).

RESEARCHES CARRIED OUT IN 1971

55

Callus induction and organ redifferentiation of Triticum,
Aegilops and Agropyron by anther culture
Mikio KIMATA and Sadao SAKAMOTO

Callus induction and organ redifferentiation were studied by the antherculture of 17 Triticum, 11 Aegilops and 8 Agropyron species. Out of threedifferent culture media used for callus induction, the best result was obtainedby the Miller's medium supplemented with 2.21 mg/l 2, 4-D. Callus formation from pollen grains was observed in four Triticum, two Aegilops
and two Agropyron species. On the contrary, a high frequency of callus
formation from anther filaments was obtained in emmer wheats. In order
to induce organ redifferentiation, calluses were transferred to the same
medium as mentioned above omitting 2,4-D. Root formation was observedin several species. Sprouting appeared only from calluses derived from pollen
grains of C C Cu Cu, an artificially synthesized amphiploid (2n = 28) between
diploid Ae. caudata and Ae. umbellulata, and many plantlets took shape.
However, all were haploid albino plants (2n= 14). 117 days after transplanting an albino plant with seven leaves flowered. However, the spikeof this plant included only a single spikelet without pistil and stamens. The
details were published in Japan. J. Palynology 8: 1-7 and Japan. J. Genetics
47: 61-63.

56 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

VI.

MUTATION AND MUTAGENESIS IN ANIMALS

Effect of protein synthesis inhibitor on repair of X-ray induced
premutational lesions in silkworm spermatids
Y. TAZIMA and Y. FUKASE

Our previous experiments (this Report No. 16: 100(102) showed that postirradiation treatment of silkworm spermatogonia with many metabolic inhibitors increased mutation frequency to a considerable extent. Although
it was surmised that the treatment with those chemicals suppressed therepair of premutational lesions, it was impossible at that time to exclude
another possibility that those treatment might intensify the blocking of thecell cycle progression caused by the X-ray irradiation thus increasing themutation frequency. Since DNA synthetic cycle does no longer exist in the
spermatids, the latter possibility could be rejected only if the irradiation
experiment had been carried out with these cells. Furthermore, repair ofradiation induced premutational lesions has since been demonstrated byirradiating the spermatids with two split doses at an appropriate timeinterval (Tazima, 1959, Jap. J. Genet. 44 Suppl 1: 123-130).
An experiment has, therefore, been carried out in which a treatment
with a protein synthesis inhibitor was combined with fractionated irradiation
given to spermatids.
Wild type males of strain C108 were irradiated at mature larval stage
with two split doses of 500 R each given three hours apart. Immediately
before the initial 500 R exposure, chloramphenicol and/or puromycinin physiological salt solution were injected interperitonealy. The injection
was 0.02 ml per capita with various concentrations, i.e., 0.01 % (2r) and
0.1 %(20r) for chloramphenicol and 0.005 %(I r) and 0.02 %(4 r) for puromycin. Control group was also irradiated but was injected with physiologicalsaline only. Both treated and control males were crossed to pe re females
for the measurement of induced mutation frequency. The results are given
in Table 1.
The results showed that those chloramphenicol and puromycin treatedmales exhibited significantly higher mutation frequencies than saline treatedcontrol. Since in this case the possibility of blocking the progression of cellRESEARCHES CARRIED OUT IN 197157

cycle was excluded, it seemed more likely that inhibition of protein synthesis
increased the mutation frequency. That is to say, the process of protein
synthesis seems to be necessary for the repair of premutational lesions.
Table 1. Mutation frequencies obtained after fractionated X-ray
irradiation of silkworm spermatids with or without aprotein synthesis inhibitor
Treatment
Saline only
CMPO.Ol%
CMPO.l%Purom 0.005 %PuromO.02%

nnumber

Mutation frequency (x 10-6)
Number of mutantspe pe, + re re, + pe pe, + re re, +total

18.24217.72617.21718.26019.82479911
3Observed

22
21
33
32
39

3
3
4
4
8

4
8
6
7
8

38
51
52
60
15

121
119
192
175
197

16
17
23
22
40

22
45
35
38
40

197
232
302
295
292

Studies on strain differences in radiosensitivity in the silkworm
XIV. Further data on dominant lethals
Yataro TAZIMA and Yosoji FUKASE

In the previous issue of this Report (No. 21: 61-62), we have reported
that mature sperm were rather insensitive to radiation in the inductionof dominant lethals and that the differences in sensitivity was not clear
among strains at this cell stage but became marked when spermatogonia
has been irrdiated.
Since only two strains, rb as a sensitive and Kansen as a resistant one,and their hybrid were used in the previous experiment, the irradiation experiment was continued using another sensitive strain Sekko and a less sensitive strain Kojiki in addition to rb. F 1 hybrids between rb and either
Sekko or Kojiki were also irradiated. Those hybrids were fairly resistant toradiation. In contrast, the parental strains were much more sensitive andbecame sterile when irradiation doses were higher than 2000 R for Sekko
and 3000-4000 R for Kojiki and rb, thus invalidating the irradiation withappreciably high doses that ensure a reasonable yield of dominant lethals.
The data thus obtained sometimes did not represent good regression curves
for parental strains, although the difference became more or less marked
among strains. The radiosensitivity in F 1 again seemed to fall between
both parents.

S8 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

With the purpose of avoiding so far as possible the disturbance due to thesterilizing effect, comparisons have been made by irradiating spermatids
in mature larvae. Strains used were again both sensitive and less sensitive
strains, i.e., rb, Sekko, Kojiki, Aojuku and Kansen, together with their
Table 1. Frequencies of dominant lethals induced in malegerm cells of the silkworm
Irrad.
cell
stage

Exptl
seasonDose(kR)

Hatchability
decline (%)

Freq. of dom.
lethals per
kR(%)

703
711
711
703
711
703
711
711

4
4
1
2
2
4
4
4

2.5
4.3
3.3
10.0
15.5
5.0
24.0
7.3

0.62
1.10
3.3
5.0
7.7
1.25
6.00
1.82

AoxKa
ubxSe
Sexrb
rbxKo
Koxrb

713
712
713
712
713
712
713
713
712
713
712
713

4
3
4
4.5
4
4
4
4
4
4
4.5
4

4.6
3.0
5.0
10.0
10.0
10.0
15.0
7.0
5.0
8.0
8.0
15.0

1.15
1.00
1.25
2.20
2.50
2.50
3.75
1.75
1.25
2.00
1. 77
3.75

KansenAojuku
Sekko
rb

701
701
701
701

10
10
10
10

10.0
10.0
10.0
10.0

1.00
1.00
1.00
1.00

StrainKansenKojikiSekko
rb

S-gonia
rbxKa
Sexrb
Koxrb
Kansen
Sekko
AojukuKojikirb
S-tidsSpermhybrids, rb X Kojiki, rb x Sekko and Aojuku x Kansen. Selection at thisstage permitted to deliver radiation doses up to 4000 R in all parental strains
and to 6000 R in all hybrid combinations guaranteeing more reliable data
than irradiation to spermatogonia. The induction rates per unit dose werereduced. The differences among strains also became smaller but seeemed

RESEARCHES CARRIED OUT IN 197159

to represent more reliable data showing good regression curves.
The data so far obtained are summarized in Table 1. As is shown in thetable, induction rates of dominant lethals are very low in the silkworm.
After the irradiation of mature sperm the rate was about two figures lower
than for the mouse and about one figure lower than for Drosophila.
It was also confirmed in these experiments that with regard to the induction of dominant lethals the sensitivity in F 1 was intermediate between
both parents, as has been already observed for recessive visible mutations
at marked loci.

Studies on strain differences in radiosensitivity in the silkworm
XV. Comparison of recombination values among
different sensitivity strains.
Yataro TAZIMA and Kimiharu ONIMARU

It has been known for several microorganisms that many radiation sensitive mutants not only lack the capacity to repair their damaged DNA but
are also uanble to undergo genetic recombination. Those mutants were
mostly discovered as UV-sensitive but some were known to be sensitive also
to X-rays. Conversely, in some other cases recombination deficient mutants
were observed to be more sensitive to radiation than the wild type. It istherefore of great interest to examine if there is in the silkworm any close
relationship between radiosensitivity and recombination properties. In
this work a comparison of recombination values has been made amonggroups with different genetic background in F 1 and BF1 of crosses withstrains of different radiosensitivity.
For the measurement of recombination values the section between +pe
and +Te of the Vth linkage group was chosen. Strains utilized for thecomparison were Aojuku, Kansen, Kojiki, Sekko and rb. All of them had+pe+re/+pe+re genotype. Females of each strain were mated to double
recessive pe re males. F 1 males were then back-crossed to pe re females
and more than 250 batches of each cross were subjected to the examinationof recombination values in F 1 males (Cross I). F 1 females were crossed
again with males of their parental strain of wild type, i.e., Aojuku, Kansen
etc. Those BF1 males were then crossed to pe re females. About one halfof the batches thus obtained comprised wild type eggs only. Another half

60 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

were segregating three types of egg color, whose male parents were assumed
to be heterozygous pe ref ++. They were also assumed to comprise bothhetero- and homo-zygotes with respect to the major sensitivity gene(s) and/or
their modifiers, if any were present. Those bactches were subjected to theexamination of recombination values in BFI males (Cross II).
40
30
20

702

D

Ao;luku:296

x : 25.59

10

".2:

19.76

D:

247

0
30
20

x : 26.88

10

".2:

15.60

n:

297

0
30
>.
u

20

., 10

<::

:>

.,c-

o

~

30

....

20

Xansen

10
0
30

rb

x : 29.43

20f1"7.:

10
0
16.5

20.5

24.5

28.5

32.5

36.5

14.25

40.5

44.5

Recombination value (%)

Fig. 1

The results of Cross I are given in Fig. I. As is seen from the figure,difference was clearly observed among strains, being the lowest 25.59 %for Aojuku and the highest 29.43 % for rb. The differences were significant
by t-test analysis among strains except for that between Kansen and rb.
In Cross II more distinct differences were observed among strains, although
the tendency was almost similar to that of Cross I. The lowest recombinaIRESEARCHES CARRIED OUT IN 1971

61

tion frequency was 25.03 for Aojuku and the highest was 30.30 for rb. The
array of recombination values among strains was almost in parallel withthat of radiosensitivity with exception of Kansen, but in the reverse order.
The result that the most sensitive strain rb showed highest recombination value was indeed against our expectations from the findings in microorganisms. The phenomenon might perhaps be related to the activity ofa relevant enzyme(s) existing in those silkworm strains. Kada and his collaborators recently revealed that activity of A type DNase in rb strain is
ten times as high as in Aojuku.
The finding that recombination values were clearly different among thegroups even in F 1 indicates that the modifying factor(s) for recombination
properties are almost dominant.

RBE of 14 MeV fast neutrons for visible recessive
mutations in silkworm oocytes
Akio MURAKAMIThis communication shows some preliminary data on the relative mutagenic
effects of 14 MeV neutrons as compared with 137Cs gamma-rays for prophase
I oocytes of the silkworm. Prophase I oocytes of the wild type strain C108
were irradiated either with fast neutrons or gamma-rays in 13 day-old pupae.
The pupae were exposed to neutrons of 250, 540, and 1,000 rads (at doserate of 2-7 rads/min) and gamma-rays of 500, 1,000, 1,500, and 2,000 R (atdose-rate of 100 R/min). The gamma-ray of the neutrons was negligible.
Mutation frequencies were estimated by the specific locus method using eggcolor genes, pe and reo
The dose-response relations for both radiations slightly deviated fromthe linear relation so that the RBE varied according to the dose comapred:at lower doses the RBE was about 2.3 and at higher doses it was slightly
reduced to about 2.0. This may indicate that there is no drastic change in
the RBE value for the cell before and during meiosis, as found in male silkworm. It may also indicate that the RBE of 14 MeV neutrons for the specificlocus mutation in silkworm oocytes is lower as compared with that of 2.5
MeV neutrons, which had a higher vaerage LET (Machida and Nakao, 1969).
In addition to the study mentioned above, the RBE of 14 MeV neutrons
to gamma-rays has been compared in oocytes among four different X-ray

62 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

sensitive strains (Kojiki and Sekko as representing the sensitive strain andAojuku and Kansen as representing the resistant strain). These four strains
were irradiated with a single dose of neturons (400 rads) and l37es gammarays (1,000 R) in late pupal stages. In this experiment, the ratio of mutation
rate per rad of the neutrons to that of gamma-rays was roughly regarded as
the RBE of the neutrons. The fast neutrons have shown RBEs of about
2.3 and 2.4 at pe and re loci, respectively. But no appreciable difference
was found in RBE among four strains regardless of their sensitivities.

The nature of specific locus mutations induced by fast neutronsin silkworm spermatocytes
Akio MURAKAMI

The analysis of the nature of fast neutron-induced mutations may provide
useful information for the understanding of the change in RBE (relative
biological effectiveness) with the progression of gametogenesis. From thisview point, mutants at egg-color specific loci which were obtained in the
study on the relative genetic effectiveness of 14 MeV neutrons have been
investigated with regard to their lethality in homozygous condition.
Whole-body mutants for either pe or re locus were picked up from the F 1
eggs. To discard the non-irradiated pe: re chromosome, mutants, pe andre, were crossed to, +re/+re and pe+/pe+, respectively. Thus 22 mutantsat re locus and 6 at pe locus were analyzed as to their segregation ratios in
the F 2 and lehtality in homozygous condition for the muation bearing
chromosome. The majority of them showed segregation into three different
phenotypes, expected wild and marker phenotypes as well as a newly induced
mutant phenotype. The segregation ratio in the F; was very close to 2: 1: 1
throughout for all cases.
Among 28 mutants analyzed, mutants associated with embryonic lethals
were 21 and the remaining 7 mutants were viable. This figure does notseem to be lower in the frequency of viable mutants than that for low LET
radiations reported by Tazima and Onimaru (this Report, No. 10 (1960):
115-117).
From these results, it could be said that specific locus mutations obtained
after fast neutrons-irradiation of spermatocytes are in a broad sense of apoint mutation, including small deficiency or deletion. But it should be

RESEARCHES CARRIED OUT IN 197163

noted that mutations associated with gross chromosome aberrations would
frequently be subjected to selection during the course of spermatogenesis.

Mutagenesis of the silkworm by acridine orange
Aldo MURAKAMI

It has been known that certain acridine compounds are mutagenic in
microorganisms when supplemented to the culture media. As a plausible
interpretation it was assumed that (1) the reaction between acridine dye
and DNA would result in a frameshift mutation as due to the intercalationof the chemical between DNA bases and (2) the complex of DNA whichstacked with acridine dye could be unstabilized in their molecular state underlight conditions leading to mutations such as copy errors.
In male Drosophila, there are many reports that monofunctional quinacrine mustard ICR-170 which has two main reactive parts, one being an
acridine dye and another an alkylating mustard, is mutagenic for the postmeiotic cells, while one of acridine dyes, proflavine, which is a powerful
mutagen for T 4 phages, is not mutagenic for mature sperm (Carlson et al.,1967). Each reactive portion in the ICR-170 seems to have the mutagenic
action on germ-cells. Accordingly, it seems unlikely to exclude a possibilityof mutagenesis of the ICR-170 as an alkylating agent.
Such being the case, it would seem to open the question whether acridine
dyes have mutagenic action on higher organisms as well as microorganisms.
The present communication reports a positive mutagenic action of acridine
orange (AO) on silkworm oocytes, but no such action in mature sperm.
Eight-day old silkworm wild-type, C108 strain, pupae were injected with
0.5 mg per pupa in 0.85 % saline. Germ-cells of this stage pupae are
mature sperm and prophase I oocytes, respecively, for male and female.
They were mated after emergence of moths from pupae to marker strain
moths having recessive egg-color genes, pe and reo The F 1 eggs were scoredon the visible phenotype as either whole or mosaic (fractional) mutant.
Results indicated that the AO is not an effective mutagen for mature sperm,
while the AO it induces significantly high mutational incidences for prophase I oocytes. In addition, the recovered mutants were overwhelmingly

64 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

mosaic types. A test for transmissibility and gonadal mosaicism is inprogress.
The mechanism by which the AO induces mutations in the silkworm is
not yet known, but the mechanism proposed for microorganisms could be,in part, extended to the present finding. The preponderance of mosaic
mutants would be induced when the AO in egg-plasm were intercalated between DNA bases during meiotic and cleavage stages and the final mutational event were fixed by delayed effect within the first few divisions of thezygotes after syngamy. But it could not exclude the possibility when DNA
which stacked with the AO in newly deposited oocytes (or eggs) was received
sufficient amount of visible light to induce mutations as the photodynamic
effect.
No increase in mutation frequency of pupal mature sperm was observedwith the treatment of the AO. It is well known that the DNA moleculein the sperm is crystallized and its structure may remarkably differ fromthat of meiotic and cleaving nuclei. Thus it could be said that the AO
may be mainly incorporated into non-crystallized DNA, but not crystallized
DNA. This thought seems to be compatible with the idea proposed by
Streisinger et al. (1964) that the acridine ring insertion would be observed
during DNA replication.
We are carrying out further studies and we hope to test which hypothesis
is applicable to the present finding by using other acridine dyes either underlight or dark condition.

Induction of mutations by EMS in female germ-cellsof the silkworm
Akio MURAKAMI

Ethyl methanesulfonate (EMS) has widely been used as a mutagen for
intragenic changes in phages as well as in higher organisms. There aremany reports as to the EMS mutagenesis in the male silkworm. Little is
known, however, about the mutagenesis of this chemical for the female
silkworm. The present experiment was intended to obtain the informationof EMS mutagenesis for female germ-cells of this insect. In the female
silkworm, the post-meiotic stage begins about 2 hours after oviposition andlasts only 10 min. The syngamy takes place about 30 min thereafter.

RESEARCHES CARRIED OUT IN 1971

65

EMS in 0.85 % NaCl solution was injected into the ventral abdomen of
the Cl08 wild-type female silkworm in 13 day-old pupae (or 1-2 days before
emergence). The germ-cell at this stage is in prophase I oocytes. For
the detection of the mutants the egg-color specific locus method using pe
and re genes was employed. After emergence of moths from the treatedfemale pupae, they were crossed with the double recessive marker male moths.
F 1 eggs were scored for egg-color mutations and classified as whole (complete) or fractional (mosaic) mutants.
Results indicated that a high mutation induction was observed for thelate stage pupae following treatment of the EMS. Almost all mutants werefractionals containing both mutant and non-mutant cells, while whole mutants were very rare, as was found for mature sperm in pupae and moths. In
the present study, it is of much interest that some complex mosaic eggs were
found possessing three different phenotypes, +, pe, and re, in one individual.
Fractionals may originate from the alteration by EMS in one of double
stranded DNA by the production of two different cell lines of wild andmutant phenotypes in the mosaic egg. Explanation for rarely observed
whole body mutants is difficult since a single-hit event on double-stranded
DNA in one chromosome may be more probable than a simultaneous doublehit event. A tentative interpretation is that two independent mutational
events on both strands in oocytes might have occurred by the reason of ahigh frequency of fractional-type mutations. However, it would seem unlikely to exclude the possibility that the fractional mutation changes into
the whole mutations prior to syngamy because DNA replication may takeplace in pronuclear cells.
It has been known that in the male silkworm EMS is less mutagenic forpre-moiotic germ-cells than for post-meiotic germ-cells (Tazima and his
associates, this Report, No. 18 (1968): 87-88). The present finding indicated, however, that EMS is a powerful mutagen for pre-meiotic oocytes inpupae. For reference, no remarkable increase in mutation frequency oflarval oocytes was observed with the treatment of EMS. If EMS, which
had been incorporated into egg-plasm, were mutagenic for post-meiotic andcleavage nuclei in eggs, alteration on DNA (or chromosome) would be
produced during these cell stages. This thought seems to applicable to the
production of complex mosaic mutants.
Analysis for transmissibility of mosaic eggs and gonadal mosaicism of

66 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

the moths obtained from the mutant eggs are in progress by mating thesemutants again to the marker strain. The data obtained so far indicatedthat mutant characters of about one-third of mosaic eggs were transmitted
to the F 1 generation either as wild or mutant eggs. It should be noted,
however, that a secondary mosaic egg was rarely observed among somedesendants of the primary mosaics. The origin of such type mosaics may beinterpreted as due to the replicating instability as observed in fission yeast(e.g., Nasim, Mutation Res. 4 (1965): 753-763) and in the male silkworm(Onimaru and Tazima, this Report No. 19 (1968): 63).

Instability of pM-allelic genes locating on the second chromosome
piece translocated to the W chromosome
Yataro TAZIMA and Toshio OINUMA'"..-

W.IIM is a stable translocation involving p.\{ gene on the second chromosome piece and the female determining gene(s) on the W chromosome
part. Although very seldom a detachment of the translocated piece occurrs
giving rise to pM males, p+ females and pM-mottled females.
The pM-mottled females, when crossed to p males, gave birth to daughtersof four kinds of phenotype belonging to the same multiple allelic group,i.e., pB, pM, p+ and p in addition to mottlings for those traits. In contrast,
their sons comprised only one phenotype, p. The newly arisen four phenotypes were all transmitted to their descendant as if each of them were acomplete mutation that had occurred on the translocated chromosome.
Namely, when they were mated to p males, pB females produced pB daughtersand p sons and p+ females yielded p+ daughters and p sons showing about
1: 1 sex ratio. These findings suggested that those newly arisen seggregates..were not the products of detachment of the W.II M translocation, but kindof genetic change, perhaps a mutation, had occurred in chromosome whose..transmission was confined to the female line. Since W.II M was the only
chromosome that satisfies such requirement, it was assumed that all thosesegregates were the products of mutation that occurred at the pM locus.

..-

The W.IIM translocation was discovered in 1949 as a mosaic of larval

..-

..-

marking among W.IIP bearing individuals, which had W.p+ y oa constitu'" The Silk Science Research Institute.

RESEARCHES CARRIED OUT IN 1971

67

tion, presumably due to a mutation that occurred spontaneously from p+
to poll. Accordingly, it follows that the mutation occurred repeatedly atthis locus, first from p+ to p.'ff and then from p.ll to pH or p+.
Mottlings were also transmitted per ce from generation to generation
mostly through the female line. When the male was a carrier, though it
occurred less frequently, their mode of transmission was quite unstable.
Mottlings have been obtained with regard to three mutant chracters,i.e., pH, pM and p+. They always produced more than two kinds of allelicphenotype among their daughters in addition to a few mottled individuals.
As mentioned above, complete type mutants never showed such kind ofgenetic behavior in their descendants. They differ greatly in this respectfrom the mottlings. When mutation occurred, it was only to one direction
from dominant to recessive, except for the case from p.ll to pH, when the discrimination between two phenotypes was sometimes ambiguous. Since
the genetic change occurred so fequently in this case, it could be regarded
as a kind of ever-sporting gene. Although the mechanism is not yet known,
it seems almost certain that the instability is caused by some sort of chromosome rearrangements which leads to misreplication of the p-Iocus geneas well as to mechanical breakage of the chromosome.

68 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

VII.

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICROORGANISM AND PLANTS

Cellular factor involved in repair of DNA damage
induced by ionizing radiation
Takehiko NOGUTl and Tsuneo KADA

We have recently shown, for Bacillus subtilis cells irradiated with i-ray
and made permeable by toluene-treatment, that rejoining of DNA singlestrand breaks as well as recovery of transforming activity was taking placewhen the permeable cells were subsequently incubated in the presence ofprecursors and cofactors for DNA-polymerase and DNA-ligase (Noguti, T.
and Kada, T., 1972, J. Mol. BioI. 67: 507). Because 3'-OH chain termini
are required for the function of the Kornberg-type DNA-polymerase and3'-OH end groups of chain breaks of DNA irradiated with X- or i-ray may
be complicated due to damage of deoxyribose residues (Kapp, D. S. andSmith, K. C., 1970, Rad. Res. 42: 34; Bopp, D. S. and Hagen, u., 1970,
Biochim. Biophys. Acta, 209: 320), certain "cleaning factor" might have worked prior to the repair process involving DNA-polymerase and DNA-ligase.
Existence of such a factor is suggested in the present studies.
When cells of Bacillus subtilis strain 17A were irradiated with 30 KR of
i-ray, treated with I % toluene at O°C and incubated at 37°C in potassium
phosphate buffer containing 4dXTP, MgH and 3H-TTP, incorporation of
the label in the DNA fraction increased remarkably during the incubation
period. Such an increase was very mild if irradiation was not given. Addition of N-ethylmaleimide (1.5 mM), an inhibitor for semi-conservative
DNA synthesis in toluene-treated E. coli cells, had no effect on the result.
When DNA was extracted from i-ray irradiated and toluene-treated cells
and was used as primer for purified Micrococcus DNA-polymerase (Zimmerman, B. K., 1966, J. BioI. Chern., 241: 2035) for in vitro DNA synthesis,
we found that the incorporation rate of the radio-active precursor was
several fold greater with DNA extracted from cells irradiated with 30 KR
r-ray than with that from unirradiated cells.
Because the priming activity of cellular DNA was enhanced by i-rayirradiation of the cells, the situation is completely reversed from the case

69

RESEARCHES CARRIED OUT IN 1971of in vitro irradiation as shown by Harrington, H. (1964, Proc. Nat. Acad.
Sci., U.S., 51: 59; 1966, J. Mol. BioI., 15: 152). It is supposed that DNA
chain termini required for DNA-polymerase function may have been produced enzymatically in the cell. In order to examine the presence of sucha factor in cellular proteins, following experiments were performed. Toluenized cells were treated with lysozyme and Brij-58 and centrifuged at 12,000 g
for 20 minutes; the supernatant fraction was used as "cellular extract".
Table 1. Effect of Pronase treatment and heating on the "cleaning"
activity of cellular extract. Pronase treatment was done at aconcentration of 3 mg/ml for 20 minutes at 3rc.
Heating of cellular extract was done in boilingwater for 10 minutes
r-ray
dose
(R)

Minutes of
incubation
periodIncorporation
rate(cpm/60 min./tube)

Brij-lysozyme solution

0
1000

40
40

498
164

Cellular extract

0
0
1000
1000

0
40
0
40

452
918
210
2140

Pronase-treated cellular extract

0
1000

40
40

461
181

Heated cellular extract

0
1000

40
40

316
180

Treatment

DNA was extracted from phage T7, divided into three parts and treatedin following different ways. In the first, the priming activity for Micrococcus DNA-polymerase was determined directly on the DNA sample. The
second portion was combined with the cellular extract of B. subtilis, incubated and then the DNA extraction procedure was followed. The third portion, which served as a control for the second, was combined with the Brijlysozyme solution containing no cellular extract, and the DNA extraction
procedure was performed exactly as was done for the second portion. The
rate of in vitro DNA synthesis with Micrococcus DNA-polymerase was reduced in the first or third treatment, as radiat.ion dose increased, while it wasenhanced by the second treatment where the irradiated DNA samples were

70 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

combined with the cellular extract.
Adjoining experiments were performed using T7 DNA to determine
whether the above cellular factor is an enzymatic protein or not. Results
given in Table 1 show the following points: (1) In vitro irradiation of T7
DNA with 1000 R r-ray reduced considerably its priming activity for Micrococcus DNA-polymerase. A similar radiation effect was obtained even
if the irradiated DNA was incubated with the Brij-lysozyme extract notcontaining the cell and the DNA isolation procedure was followed as usual.
(2) The priming activity increased remarkably compaired to the controls by
incubating the irradiated DNA with the cellular extract, but only slightly
if the DNA was not irradiated. (3) Such an increase was not found when
the cellular extract was treated with Pronase or heated before incubation
with the irradiated DNA.
From the above results it is clear that the cellular factor enhancing thepriming activity of DNA following r-ray-irradiation is a protein. We
therefore suppose that this factor may "clean" radio-lesions in DNA andproduce 3'-OH chain-termini required for DNA-polymerase function.
Further purification studies of the above factor are in progress.

Adenosine triphosphate-dependent deoxyribonucleaseactivities in Bacillus subtilis
Yoshito SADAIE and Tsuneo KADA

Deoxyribonucleases requiring ATP for their function have been detected
in wild strains of Escherichia coli and several other microbial species but werelacking in rec Band rec C mutant strains of E. coli, and its possible role ingenetic recmbination has been discussed (Tsuda, T. and Strauss, B. S.,
1964, Biochemistry 3: 1678; Winder, F. G. and Coughlan, M. P., 1967,
Biochim. Biophys. Acta, 134: 215; Buttin, G. and Wright, M., 1968, Cold
Spring Harbor Symp. Quant. BioI., 33: 259; Oishi, M., 1969, Proc. Nat.
Acad. Sci., U.S., 64: 1292; Anai, H., Hirahashi, T., Yamada, M. andTakagi, Y., 1970, J. BioI. Chern., 245: 775; Barbour, S. D. and Clark, A. J.,
1970, Proc. Nat. Acad. Sci., U.S., 65: 955; Goldmark, P. J. and Linn, S.,
1970, Proc. Nat. Acad. Sci., U.S., 67: 434). Recently, Vovis, G. F. andButtin, G. (1970, Biochim. Biophys. Acta, 224: 42) showed that a recombination-deficient mutant strains of Diplococcus pneumoniae lacked in the ATP-RESEARCHES CARRIED OUT IN 1971

71

dependent deoxyribonuclease activity. We found these enzyme activities
in wild and recombinationless strains of Bacillus subtilis.
The deoxyribonuclease activity in the cell lysate was assayed by measuring
the radioactivity in the acid-soluble product released from native or heatdenatured 3H-DNA. Cells of Bacillus subtilis 168 thy were labelled with
SH-thymidine. DNA was extracted and purified by the phenol method.
Table 1. ATP-dependent and non-dependent nucleaseactivities of Bacillus subtilis
Min. ofincubationforreactiono30
60

c.p.m. in acid soluble material---Native DNA
Denatured DNA
-ATP
+ATP
-ATP
+ATP

- - - -

50.8
114.5
101.0

----------

79.4
793.4
1274.2110.6
748.6
1306.042.6
837.6
1545.6Heat-denaturation of DNA was performed by heating the SSC solution(0.15 M NaCl-O.oI5 M sodium citrate) of 3H-DNA at 100°C for 10 min.
and cooling rapidly in ice-water. Preparation of Brij-58 cell lysate wasbased on the method described by De Lucia and Cains [Nature 224: 1164
(1969)]. Cells of B. subtilis Hl7 grown in 100 ml TF-medium supplemented
with 10 ,ug/ml arginine and 10 ,ug/ml tryptophane were collected by centrifugation at their late log phase and were resuspended in 5 ml of 10 % sucrose0.1 M Tris-HCl (pH 8.3). After addition of lysozyme and EDTA (finally50,ug/ml and 5 mM respectively), the mixture was kept at 37°C for 30 min.
Further lysis of cells was carried out by supplementing Brij-58 and MgS0 4(finally 0.5 % and 0.05 M respectively) and incubating at 37°C for 5 min.
The lysate was centrifuged at 19,500 g for 30 min. and the supernatant wascollected and used immediately for the enzyme assay. The reaction mixture for measurement of ATP-dependent deoxyribonuclease activity contained, in 0.5 ml of Tris-HCl buffer (62 mM, pH 8.7), 0.1 ml of the cell
extract, 20,ug 3H-DNA and 0.1 mM ATP (neutralized previously with
NaOH). This mixture contains some amount of Mg++ derived from thecell extract. The mixture was incubated at 37°C and the reaction was
stopped, at indicated time, by cooling to O°C and adding 2 drops of 0.2 %bovine albumin and 2 ml of 10% TCA. The sample was finally centrifuged
at 12,500 g for 15 min. and the supernating 0.2 ml fraction was used for72 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

counting the radioactivity of acid-soluble material with Beckman Scintillation Counter Model 225.
Results obtained with cells of strain HI? possessing wild recombination
capacity are shown in the Table. It is shown that the strain has two kindsof deoxyribonuclease activities: the one requiring ATP and acting on native
DNA; the other specific to denatured DNA and independent of ATP. These
enzyme activities were also found in several typical recombinationlessmutant strains of Baeillus subtilis so far studied.

Effect of ultraviolet light on DNA degradation in wild andrecombinationIess cells of Bacillus subtilis
with or without toluene-treatment
Yoshito SADAIE and Tsuneo KADA

In some typical mutants of E. eoli Kl2 carrying single or multiple ree
genes, positive correlations have been pointed out between cellular possession of ATP-dependent nuclease and degree of DNA-degradation following UV-irradiation. Strains carrying a ree A gene alone exhibit "reckless"DNA-degradation, on the other hand those carrying a single ree B or ree C
gene, or both, in addition to the ree A show "cautious" DNA-degradation
which is the character shown by ree B or ree C mutant strains (Willetts,
N. S. and Clark, A. J., 1969, J. Bacteriol., 100: 231). The ree B+ or ree C+
gene determines ATP-dependent DNase which, therefore, seems to be engaged in the degradation of DNA after UV-irradiation (Buttin, G. andWright, M., 1968, Cold Spring Harbor Symp. Quant. BioI., 33: 259).
To examine these relationships in B. subtilis recombinationless derivatives
which we isolated, cells of different strains were labelled with 3H-thymidine
and the release of acid-soluble radioactive materials was measured following
UV-irradiation. We found that the cellular degradation of DNA wasinitiated by UV-irradiation and that, in spite of high UV-sensitive characters,
the extent of DNA degradation of two ree strains (M45 and L43) was slightly
higher, but not "reckless", than that of the wild strain (HI?). The situation
is not therefore comparable to the case with E. eoli strains.
Toluene-treatment makes the cellular membrane permeable and accessible to small molecules such as co-factors for cellular nucleases. The
treatment at aoc was effective in Bacillus subtilis (Noguti, T; and Kada, T.,

RESEARCHES CARRIED OUT IN 1971

73

1972, J. Mol. BioI., 67: 507). Cells labelled with 3H-thymidine were exposed
to UV of different doses, toluenized and washed at O°C. Cells were thenincubated in potassium phosphate buffer with various supplements such as
ATP, Mg++ or 4XTP, and radioactivities in TCA soluble materials were
determined.
Following were main results obtained.
(1) When toluene-treated cells were incubated in phosphate buffer without supplements at 37°C, no detectable degradation of cellular DNA wasobserved. However, if the incubation was done with Mg++, a considerable
DNA-degradation took place. Similar results were obtained using thewild strain (HI7) as well as the recombinationless strains (M45 and L43).
(2) In these wild and recombinationless cells of strains H 17, M45 and L43,
the presence of ATP promoted further the above Mg++-stimulated degradation. However, ATP alone without Mg++ was not effective enough toprovoke the DNA degradation. This degradation may be independent of
DNA synthesis in the cell, because addition of 4dXTP (together with ATP
and Mg++) to the incubation buffer did not modify the degradation pattern.
(3) UV-irradiation of the cells before toluene-treatment did not promote
the DNA-degradation described above.
This observation was similar in both the wild (HI7) and the two recombinationless strains (M45 and L43) studied.
These observations lead us to assume that, either in wild or recombinationless cells, toluene-treatment impaired the integrity of cellular membrane
which may be required for manifestation of UV-induced DNA-degradationand made cells susceptible to certain endogenous nucleases including theATP-dependent one.

Cell culture of Haplopappus gracilis
Etsuo AMANO

A single cell suspension culture of a plant could be useful in UV andother mutagenesis studies. For this purpose, Haplopappus gracilis (2n=4)
seemed to be promising as the callus tissues derived from it is soft and fastgrowing. Small chromosome number would make the cytological analysis
easier and might help to stabilize the ploidy of the cell.

74 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

The surface of the seeds was disinfected with 1: 1 mixture of 95 % ethanol
and 3 % H 20 2 for 10 min. After rinsing the seeds twice with distilled water
they were sown on a mineral supplemented agar medium in glass capped
test tubes, and were placed under continuous illumination by a whitefluorescent lamp and kept at 25°C. From the germfree seedlings the tissues
were dissected and placed on a slant medium, which contained inorganic
salts (T. Eriksson (1965) Physiol. Plant. 18: 976-993) and organic supplements including 2.4.0 (2 ppm), Vitamines, yeast extract (0.3 %) andsucrose (2 %). The medium was adjusted to pH 5.8 by 1 N - NaOH andwas used either as liquid or solidified by agar (0.8 %). For inductionof callus, 10 ppm of 2.4.0 was effective, but standard concentration of
2 ppm for callus culture also worked well.
Three callus lines were derived from leaf (HL), stem (HS) and root (HR).
A line of callus (HSe) was induced directly from a seed placed on 2.4.0
containing medium. Another line (HNC) was rather spontaneous in origin. A cutting of stem, placed on mineral supplemented agar medium
without 2.4.0 ,produced fine powdery cells from the stem surface near thebase. These cells were transferred to slant medium and became a callus
line. All of the five callus lines were soft and grew well and fast. But
some differences among them were observed.
1) Hs tended to show a granular organization, especially at low (0.5ppm) 2.4.0 concentration, but the others did not. 2) HSe, HS and HR
turned to dark brown by aging but HNC and HL kept their fresh light color
longer. This coloration was remarkable by its progressive widening of thecolored area when developed in a culture on an agar plate in a Petri dish.
3) Microscopical observation revealed rather homogeneous cell populationsin HNC and HL in contrast to the mixture of variously shaped cells in HSe.
Liquid culure could be made in 100 ml Erlenmeyer flasks on a rotating
stand (2 rpm). The suspension of cells, however, was not fine enough andvisible aggregates were present. We were not yet successful in developing
good size colonies from a filtered cell suspension. Cytological observationby aceto-carmine staining of the liquid cultured materials (HSe) showed apair of meta-centric and a pair of submeta-centric chromosomes. No
poly- or aneuploid cells were observed.

75

RESEARCHES CARRIED OUT IN 1971

Grafting test of seedling mutants in Caspicum annuum
Etsuo AMANO

Modification of genetic traits of scions by grafting has been known in
Solanaceae. To study the presence and, if present, the characteristics ofthe phenomenon, with comparable genetic background, seedling mutants
have been induced in Capsicum annuum var. Sapporo Wase. Among the
mutants induced by ethyl methanesulfonate, seven chlorophyll mutants
were tested for survival by grafting.
Table 1. Results of grafting of seedling color mutants of Capsicum annuum,
var. Sapporo Wase. Scions were grafted on normal ( +/+) plants,
at the stage when the cotyledons were opening
Scion
alAl
alA2
ygAl
ygA2
IgAl
IgA2
IgA 3
*.

Normal plants segregatedMutantViability

Grafting

Survival*

Grafting

Survival*lethal

10

0
0
0
0
8
2
7

13
2

13
2

4
4

4
4

5

17
weak

26
9
2
9

Survival after two weeks.

Two albinos and two yellow mutants among the seven mutants tested,
were lethal and died before producing the first leaf. These lethal mutants
could not grow even on a sucrose supplemented nutrient agar medium. As
shown in the table, these lethal mutants could not survive on normal green(+/+) stocks, although the sister green plants segregated from the same fruit
i.e. mutant/ + and +/+) could be grafted successfully.
The remaining three mutants, yellow- or light-green, were all weak but
viable. Most of the grafts obtained from these viable mutants were successful and the scions grew further. Although all of the three mutants could
be distinguished from the normal green sisters segregated in seedling stage,
they got greener and in one mutant mottling on the new leaves developed,
so that somatic reverse mutation sector would be difficult to score.
Graftings of very distinct color mutants, albino and yellow, were notsuccessful. But their heterozygotes will be useful for a somatic mutation

76 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

test of mutagenes, as the inactivation of normal gene in mutant/ + heterozygous tissue can be scored as mutant colored sectors. Experiments ofdouble mutagenesis in search of a reverse mutation sector in a mutated tissue
are being tried using the yellow mutants.

Radiosensitivity in stored wheat seeds
Taro FumIn this study, an attempt was made to obtain some information about therelationship between storage and radiosensitivity of Triticum monococcum
var. f/avescens. Seeds harvested in July 1970 were stored in a low temperature storage room (l2°C with 40% humidity) until the sowing time which
was October 27, 1971. One lot of stored seeds was irradiated with l31Cs
source of 6 kCi strength, during January 1971 and again stored under similar
conditions until sowing time. A second lot of stored seeds as well as freshly
harvested seeds were irradiated with the same source on October 27, 1971.
All three lots were sown simultaneously on October 27, 1971. One hundred
seeds were used for each treatment and the doses administered were 0, 3,
5, 7, 10, 15 and 20 kR.
Germination percentage and seedling height of 20 days old seedlings wereused as an index of radiation injury. In general, germination rate of stored
seeds was lower than that of fresh seeds. There was no dose effect ongermination in the fresh and non-post irradiation stored seeds. Drastic
effect of radiation was observed in the 9 months post-irradiation storage
series beyond 15 kR treatment.
Radiation damage in relation to seedling height was noticed at 20 kR
in fresh seeds while with post and non-post irradiation storage seeds it
was at 7 kR. The seedling height reduction was however more drastic in
post-irradiation storage as compared to non-post irradiation storage seeds,
the percentage seedling height at 20 kR being 5.3 and 36.2, respectively.
At low doses viz., 3 and 5 kR, stimulation effect was noticed in fresh as well
as stored seeds. From the above data it may be inferred that post-irradiation storage has a definite radiation damage effect unlike irradiated seeds sown
immediately.

77

RESEARCHES CARRIED OUT IN 1971

Fractionation effect of neutrons on mutation frequency in miaze
Taro FUJII

Recovery of premutational damage or decrease of mutation frequency
in relation to Bz-gene, after maize pollen grains irradiated by gamm-rays,
fractionated at 2 h interval, was already reported (Jap. J. Genet. 46, 243251). To obtain further information about the mutational response at
the same locus with high energy fractionation radiation treatments, two
Table 1.

Relationship between mutation frequency
and fractionation treatments

Dose(rads)First experimentSingle treatment
250
389
641
Fractionation treatment (2 h)148+167
205+193271+250
Second experimentSingle treatment
323
432
680
Fractionation treatment (3 h)154+158
198+240316+330

Numberof seeds

Mutationfrequency

5951
5615
6353

2.07
2.87
5.10

6375
6494
6411

2.31
3.11
4.35

2723
2447
1732

1.29
2.98
4.50

5107
3553
5613

1.94
2.56
3.53

(%)

experiments were conducted with maize pollen grains, using the 14 MeV
neutron source. The fractionation time intervals in these experiments were
2 and 3 hours. Three dosages ranging from 250 rads (minimum) to 640
rads (maximum) were selected in the first experiment. In the second experiment, the three doses ranged between 323 and 680 rads. The results
are presented in Table 1.
The differences observed between the total dose in single and fractionation

78 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

treatments are due to the differential output of energy emitted by the neutron
source at different dates of treatments. Mutation frequency increased
linearly in relation to dose both in single and in 2 h fractionation treatments.
Results of the second experiment with 3 h interval treatment, on the other
hand, showed some differences in mutation frequency between single andfractionation series. Although there was a dose relationship response inboth single and 3 h fractionation treatments the mutation frequency in general was less in the fractionation treatment.
From the above results it may be assumed that the mutational effect offast neutrons could be repairable but the extent of recovery is smaller thanin the case of gamma-ray treatments, as indicated in the earlier studies wherea significant recovery or lowering of mutation frequency was observed.
Further, the environmental conditions which are known to modify greatly
the effect of gamma-rays, however do not appear to do so in the case ofneutron treatments.

RESEARCHES CARRIED OUT IN 1971

VIII.

79

POPULATION GENETICS (THEORETICAL)
Theoretical foundation of population genetics
at the molecular level
Motoo KIMURA

A theoretical framework based on the diffusion equation method wasused to treat some problems of population genetics at the molecular level.
To represent the mode of production of molecular mutants, two models
were considered. One is the "model of infinite sites" and the other is the"model of infinite alleles."In the model of infinite sites, it is assumed that the number of nucleotide
sites making up the genome is so large while the mutation rate per site isso low that whenever a mutant appears it represents a mutation at a new site.
This was used to investigate such problems as the behavior of mutants ina finite population, the rate of mutant substitution in evolution, and theamount of heterozygosity and linkage disequilibrium under steady flux ofmutations.
In the model of infinite alleles, it is assumed that the number of possible
allelic states of a cistron is so large that whenever a mutant appears it
represents a new, not preexisting allele. This was used to investigate thenumber of alleles and probability of polymorphism in a finite population.
A hypothesis that mutants are selectively neutral at the majority of sites
but are selected at a relatively small number of sites was examined, and theroles of associative overdorninance and subdivided population structure
in maintaining genetic variability were considered. For details, see Theor.
Population Biology 2: 174-208.

Linkage disequilibrium between two segregating nucleotide
sites under steady flux of mutations
in a finite population
Tomoko OHTA and Motoo KIMURA

Linkage disequilibrium or nonrandom association of mutant forms
between two segregating nucleotide sites in a finite population was studied80 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

using diffusion models, assuming that the number of nucleotide sites making
up the genome is so large while the mutation rate per site is so low thatwhenever a mutant appears, it represents a mutation at a homoallelic site,i.e., a site in which no mutant forms are currently segregating in a population. It was shown that under steady flux of molecular mutations in a
finite population, if we measure the amount of linkage disequilibrium
between two segregating sites by
a~ = E{D 2 }/E{x(l -x)y(l - y)},where D is the ordinary coefficient of linkage disequilibrium, and x and y
are the frequencies of mutants at the two sites, then we havea~ ~ (5+ 2R)/(l1 + 26R+8R

2),

where R=N.c in which N. is the effective size of the population and c is
the recombination fraction between the two sites. It was pointed out that
if multiple alleles in a random mating population are generated throughsegregation at two or more nucleotide sites within a cistron, a strong linkage
disequilibrium is usually expected between those sites, even in the absenceof selection. For details, see Genetics 68: 571-580.

Behavior of neutral mutants influenced by associatedoverdominant loci in finite populations
Tomoko OHTA and Motoo KIMURA

The behavior of isoallelic mutants in finite populations was investigatedwith special reference to nonrandom association of neutral isoallelic mutants
to overdominant loci by random genetic drift. The overdominant loci are
assumed to be sparsely distributed along the whole chromosomes and theneutral loci are distributed more densely among them. The behavior ofneutral mutants is influenced by the surrounding overdominant loci and theapparent selective force takes the form of "associative overdominance".
This was treated theoretically using a model assuming that the overdominantloci are equally spaced and have equal selection coefficients. For this model
the approximate magnitude of associative overdominance was estimated.
Mont Carlo experiments proved the validity of the theoretical prediction,although the estimated degree of associative overdominance is not as effec-

RESEARCHES CARRIED OUT IN 197181

tive as true overdominance. For the set of parameters used in the experiments, (effective population size 100 or 200, 211inked overdominant lociwith heterozygote advantage of 0.01 in chromosome segments of 0-0.66
recombination fraction) it was about 1/4 as effective as true overdominancein retarding gene fixation. The associative overdominance at intrinsicallyneutral loci, will contribute, at least partly, to bring about a constant
distribution of neutral alleles, by preventing fixation of these alleles in local
populations. For details, see Genetics 69: 247-260.

Genetic load due to mutations with very small effects
Tomoko OHTA and Motoo KIMURA

A unified treatment of substitutional and mutational loads was presented assuming a steady flux of molecular mutations in a finite population.
The case of genic selection for diploid populations ("no dominance") was
analysed in detail. We restrict our consideration to segregating loci,disregarding the part of the load coming from fixed deleterious mutants~
An important point which emerged from the analysis is that mutations
with very small effects (having selection coefficients of the order of the reciprocal of the effective population number) can create considerable genetic
load. This suggests that slightly detrimental mutations constitute a real
threat to eventual extinction for a species having small population number.
If N. is the effective population size and s is the selective disadvantage ofthe mutant, the mutational load as a function of N.s may be used to definethe "cline" between neutral and deleterious mutations. The load depends
on IN.sl rather than lsi alone. The importance of mutations with verysmall effects for variation and evolution of the species was discussed. For
details, see Jap. J. Genet. 46: 393-401.

Associative overdominance caused by linked
detrimental mutations
Tomoko OHTA

Associative overdominance due to linked detrimental mutations wasinvestigated using the method of moment equations based on diffusion mo-

82 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

dels. The expectation of the apparent selective value at the marker (neutral)locus has been evaluated. Assume two linked loci, at one of which thesteady flux equilibrium is reached under constant mutational input of deleterious mutations (with rate v) having disadvantages hs in heterozygote ands in homozygotes. At another locus, the neutral alleles are segregatingwith frequencies near 0.5. Let N. be the effective size of the populationand c be the recombination fraction between the two loci. Then the coefficient of associative overdominance at the neutral locus can be obtained bytaking the expectation with respect to chromosome frequencies at steady
flux equilibrium. It becomes approximatelys , ::::;

2N.(cL1 - L ohs)
cJ2hs'++

where (L 1 -Lo) is the inbreeding depression caused by deleterious mutations under complete inbreeding, and N.hs/? 1 and hs/?v are assumed.
More generally, if the inbreeding depression of a chromosome segmentwith a length of recombination fraction Cis (L 1 -L o) then s' at the neutral
marker at the edge of the segment is,s::::;

L 1 - L o I C + hs
2N.C oghs 'where hs is the average heterozygote disadvantage of detrimentals.
The significance of the associative overdominance is discussed in relation to actual observations. It is proposed that the most of the observed
heterozygote superiority including inversion chromosomes of Drosophila,
isozyme alleles in Avena and ABO blood group genes in man could be explained by the associated detrimentals. For details, see Genet. Res.,
Camb. 18: 277-286.

Pattern of neutral polymorphism in a geographicallystructured population
Motoo KIMURA and Takeo MARUYAMA

In a two-dimensional stepping-stone model of finite size, if a pair ofalleles happen to segregate in the whole population, marked local differentiation of gene frequencies can occur only if migration between colonies issufficiently rare so that Nm < I, where N is the effective size of each colony

RESEARCHES CARRIED OUT IN 197183

amd m is the rate at which each colony exchanges individuals with four
surrounding colonies each generation. On the other hand, if Nm~4, thewhole population behaves as if it were panmictic and the allelic frequencies
become uniform over the entire distribution range unless mutation isunusually high. Tendency toward local differentiation is much weaker in
two-dimensional than in one-dimensional habitats. For details, see Genet.
Res. Camb. 18: 125-131.

Some methods for treating continuous stochastic
processes in population genetics
Takeo MARUYAMA and Motoo KIMURA

The purpose of this note is to give methods of calculation of quantities
associated with the genetics of natural populations. The methods are basedon diffusion approximation.
Consider a random mating population of constant effective size and alocus at which two alleles, say Al and A 2 , are segregating. Then the change
in the frequency of Al follows a Markov chain. Let us denote by x(w, t)
the gene frequency of Al in sample path w at time t, with p=x(w, 0). Letf(x) be an arbitrary function defined on the open interval (0, 1). We define
the following quantityFin)(p) == U(P)E{[[W'f(X(W, ~»d~

JIX(w, r(w» I}=

where E{ } indicates the expectation with respect to the paths, r(w) is the
fixation time, and u(p) is the fixation probability of AI' Then Fin,(p) represents the fixation probability times the expectation of the n-th momentof the sum of the quantity given by f(x) from time until fixation, given thatthe initial frequency is p. Now let°

Then ¢()"p) satisfies the conditions lim ¢()"p)=u(p) and.~oL¢()', p)

where+ ),¢()" p)f(p) = 0,(1)

84 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

L = Vq1!-~~_ +M ~
2

dp 2

ap dp

and where Map and Yap are respectively the mean and variance of the change
of gene frequency per generation.
The above treatment gives the sum of the quantity, but it is also desirable
to know the expectation of the quantity at a specified time. This can bedone by solving for the transition probability explicitly. In doing this weneed to solve a partial differential equation. An alternative way is to use
the Laplace transform. Let
gI(t, p) == u(p)E{f(x(w, t» I x(w, ,(w» = I}

and letThen G(A, p) satisfies the ordinary differential equation
LG(A, p) - AG(A, p) =-f(p)u(p).

Here the mutation in one or both directions is allowed.
Example. We shall calculate the expectation of the gene frequency of aneutral gene (AI) in the course of fixation. For mathematical simplicity,
instead of calculating the frequency of A lo we calculate the frequency of A 2 •Then we need to solve

P04~ p) d2~~2' p) - AG(A, p) + pO - p) = O.
The appropriate solution is G(A, p) = (p(l-p»)jCA+Oj2N) and its inverse
Laplace transform istgI(t,P) = p(1 - p)e- 2N •Therefore the mean gene frequency of the successful gene (AI) is given byt

I - (1 - p)e-2ft .

Similar analogues of the above treatments for calculations including bothcases of fixation and loss of Al can be constructed easily.

RESEARCHES CARRIED OUT IN 197185

Time-transformation of a diffusion model ofa geographically structure population
Takeo MARUYAMA

Consider a population consisting of a series of colonies and consider
a locus at which two aIIeIes (A l and A 2) are segregating. Let X""t,i be thegene frequency of A l at time t in sample path w, and let Nt,i be the size ofcolony i at time t. Let N = I: Nt,i= a constant, and X t = liN I: X""t,i.
ii°

Let the relative fitnesses of AlA h A l A 2 and A 2 A 2 be 2s, sand respectively.
We assume that mating and selection occur independently in each colony
and that only migration connects the colonies. Let

and let P(T, X, Y) be the probability that the overaII gene frequency of Al
is X when T",=O and is Ywhen T",=T. Then irrespective of the geographicalstructure of the population, P(T, X, Y) satisfies the foIIowing Kolmogorov
backward euqation:

oP

1

02p

s

oP

~ = 8N oX2 + '2 oX'
and therefore we have the solution for the transition probability:

where S = 4Ns.
The process governed by P(T, X, Y) is a standard Brownian motion over
(0, I) with absorbing states at Y =0 and Y = 1.

86 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

IX.

POPULATION GENETICS (EXPERIMENTAL)

Evidence for the neutral hypothesis of protein polymorphism
Tsuneyuki YAMAZAKI and Takeo MARUYAMAA question that is currently much discussed is the mechanism for maintenance of polymorphisms in natural populations. There are two main
hypotheses: One is that the polymorphism is mainly due to neutral,favored, or harmful alleles that are in the process of increasing to fixationin the population or are being lost by drift. The other hypothesis is thatthese are maintained by recurrent mutation of alleles that are maintained bybalanced selective forces, such as heterosis or frequency dependent selection.
The purpose of this note is provide evidence to distinguish the two possibilities. Consider an arbitrary number of polymorphic loci in a Mendelian
population of arbitrary geographical structure. Then classify the polymorphisms into equally spaced classes according to their gene frequencies in theentire population (not local gene frequency), e.g. ten classes of 0.1 interval.
Say genes (polymorphisms) All' A 12 , ••• , AI" belong to the first class, A 21 ,A 2 , ••• , A 2 to the second class and so on. Let H(A i .) be the actual number
2"
3of heterozygotes formed between gene Ai.3 and any other in the entire population. Then if all the alleles are neutral, the sum of H(A i 3.) over all jis equal for all i, i.e.
(I) n = L; H(A I .) = H 2 = L; H(A 2 .) = H a = L; H(A a .) = ... = H".
j
3
j
3
j
3
This is true regardless of whether the population is divided into wholly or
partially isolated subgroups or is geographically continuous with isolation
by distance.
We should note here that the number of polymorphisms which belong to aparticular class is influenced by the population structure and it can not beknown unless we know the details of the structure. The same is true forthe distribution of gene frequencies of polymorphisms. Therefore it isquite remarkable that when we assess the amount of heterozygotes, giventhat the gene frequencies are specified, the total amount is invariant under
the population structure. This invariance holds true for the selective caseof additive effect. When there is dominance between alleles, the invariant
property itself breaks down. However the sum of heterozygotes, the Hi,

RESEARCHES CARRIED OUT IN 197187

can be calculated for a random mating population under otherwise the same
assumptions.
The absolute values of the Hi are not so much informative for they depend
on mutation rate and population size. But the relative values of Hi are of
our interest. Four different situations, in which all the mutant genes are1) neutral, 2) additively advantageous, 3) additively deleterious and 4)
overdominant, are illustrated by the curves in Figure 1. The cases 1) and 2)
assume somewhat similar pattern while the other two are entirely different.
o.

°O.!:--*-==~~~o.l:'3-~o.4~-:0.5·
Gene frequency (Y)Fig. 1. Distribution patterns of heterozygosity. The curves indicate thetheoretical expectations: (1) neutral; (2) advantageous (Ns=lO); (3) deleterious(Ns=-lO); (4) overdominance (Ns=10). The dots indicate the observed results.
(The total area under each curve and the dots is unity.) From Yamazaki and Maruyama (1972).

We note that when we wish to compare the pattern of distribution of
the Hi' we can combine data, from different Mendelian populations andeven from different species. We have done this by collecting data for some
400 polymorphisms in Drosophila, mice, sparrow and man. The resultis shown by the dots in Figure 1, (Yamazaki and Maruyama, 1972, Science
178: 56-58).

88 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Detection of single-gene effect by inbreeding
Tsuneyuki YAMAZAKI

The technique of inbreeding is often used in detecting the effect of singlegene. However, it is rather difficult to interpret the results of experiments
correctly unless the experiments are carefully planned. For example, in a
recent issue of Nature, C. Wills and L. Nichols (Nature 233: 123 (1971))
published a short article claiming evidence for single gene heterosis at theoctanol dehydrogenase (ODH) locus of Drosophila pseudoobscura. In
brief their methods and results are as follows; In each generation, pairwise
brother-sister matings were made using only heterozygotes for the locus.
The expected ratio of progeny from this mating is 1: 2: 1. An excess ofheterozygotes was observed under the stressed media containing octanol,
the substrate of ODH, after the 12 generations of inbreeding, although nodeviation from Hardy-Weinberg proportion was observed under the same
medium in outbred flies. Thus, they concluded that the heterosis was pronounced under the stressed media when the background genes were madehomozygous by inbreeding and that this heterosis was attributed to theeffect of ODH locus alone.
However, there is one serious shortcoming in their method which makes
this conclusion unwarranted. Namely, the rate of increase of inbreeding
coefficient by this inbreeding method is much quicker in the homozygotes
than in the heterozygotes. To clarify this point, I have performed simulation experiments, assuming the length of the chromosome to be either 50,
or 100 morgans. The method of inbreeding was exactly the same as thatused by Wills and Nichols. The rates of the decrease of heterozygosity inboth genotypes are shown in Figure 1. Even after 12 generations ofinbreeding, approximately 28 % of linked genes remain heterozygous in theheterozygotes with respect to the marker locus when the chromosome lengthis 50 morgans. On the other hand, only 3 % of linked genes remain heterozygous in the homozygotes with respect to the marker. (17 % in theheterozygotes, and 4 % in the homozygotes in case of 100 morgans.) The
excess of the heterozygotes will naturally arise due to the difference in theamount of heterozygosity in the background of the two genotypes, considering a large amount of genetic variability concealed in chromosomes.
Nevertheless, there is tendency that an excess of heterozygotes were more
pronounced under the octanol medium than under the KCI medium, at

RESEARCHES CARRIED OUT IN 197189

each locus. It has been found from our experiment that octanol medium
is more harmful to the survival of Drosophila than KCI medium. Therefore, the results obtained by Wills and Nichols can be interpreted as follows:(1) an excess of heterozygote at the marker locus was observed after inbreeding, because a sufficient difference in the amount of heterozygosity at the100

0.01 a10

20 &> 40 50 60 70

80

90 100Generation
Fig. I. Rate of the decrease of heterozygosity of the linked genes by inbreeding, in both heterozygote and homozygote with respect to the marker gene. The
values are the average of 10 experiments.

genes linked to the marker was produced between the two genotypes in thecourse of inbreeding. (2) An excess of heterozygote was observed under
the octanol medium but not under the KCl medium because octanol is more
harmful to the survival of flies than KCI and that the homozygotes werepreferentially killd in the octanol medium.

90 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Deleterious and sterility genes in Japanese and Korean natural
populations of Drosophila melanogaster 1 )Chozo OSHIMA and Jong-Kil CHOO·)

Many flies of Drosophila melanogaster were collected simultaneously inthe middle of September, 1971 at two sites of Anyang, the suburbs of Seoul,in Korea, and on the other hand, many flies of the same species were collected
simultaneously in November at Katsunuma and Shikishima in Yamanashi
prefecture. The distances between two sites of Anyang and between twosites of Yamanashi were about 8 km and 20 km respectively.
Many second chromsomes, extracted from each male fly, were analyzed
by Cy jPm method, and the results are shown in Table 1.
Table 1. Frequencies of deletrious and normal second chromosomes
isolated from Korean and Japanese natural populations of
D. melanogaster
Frequency (%)

No. of tested
chromosomes

lethalsemilethal

subvital

normal80107

19 (21.6)
24 (22.4)5( 5.7)6 ( 5.6)

11 (12.5)
12 (11.2)

53 (60.2)
65 (60.7)

Total

195

43 (22.1)

11 ( 5.6)

23 (11. 8)118(60.5)

Katsunuma
Shikishima

208
202

53 (25.5)
68 (33.7)

35 (16.8)
16 ( 7.9)

27 (13.0)22(10.9)

93 (44.7)
96 (47.5)

410

121 (29.5)

51 (12.4)

49 (12.0)

189 (46.1)

Country

Population

Korea

Anyang- I
Anyang-I1

Japan

Total

The total frequency of lethal and semi-lethal chromosomes in Japanese
populations was about 42 percent, and that in Korean populations was
about 28 percent. The frequencies of delterious chromosomes in Kofu andKatsunuma natural populations during several years (1965-1968) havebeen maintained to be a about 22 percent, but the frequency of deleterious
chromosomes was found to be about 40 percent in both summer and autumn
populations in 1970. The cause of such an increase in the last two years
remains unexplained.
The mean viabilities of homozygotes for all second chromosomes andThis work was supported by a grant (5080l) from the Ministry of Education.
Visiting researcher from Department of Biology, Chung-Ang University, Seoul,
Korea.
1)

2)

RESEARCHES CARRIED OUT IN 197191

also for quasinormal chromosomes were represented in Table 2. The
mean viability of all homozygotes of Japanese populations was lower significantly than that of Korean populations.
Table 2. Mean viabiliies of homozygotes for all second chromosomes
and also quasinorrnal chromosomes of D. melanogaster
Country

Population

Korea

Japan

All homozygotes

Anyang- I
Anyang-II

No. of tested
chromosomes
88
107

Total

Quasinormal heterozygotes

viability

No. of tested
chromosomes

viability

22.46±1.4522.18±1.33647730.35±0.5630.38±0.50

19522.31±0.98

14130.36±0.37

Katsunuma
Shikishima

208
202

18.35±0.96
18.09±1.03

120
118

29.37±0.4429.84±0.45

Total

410

18.22±0.66**

238

29.66±0.32** Highly significant at 1 % level.
Table 3.

Frequencies of sterility second chromosomes in Korean andJapanese natural populations of D. melanogaster
Frequency (%)

Country

Population

No. of tested
chromosomes

Female

Male

Korea

Anyang- I
Anyang-II67
81

15 (22.4)
8 ( 9.9)6( 9.0)6 ( 7.4)

Japan

Both sexualo2 ( 2.5)

Total
21 (31. 3)
16 (19.8)

Total

148

23 (15.5)12( 8.1)

2 ( 1.4) 37 (25.0)

Katsunuma
Shikishima

152
128

13 ( 8.6)
10 ( 7.8)

28 (18.4)
21 (16.4)

4 ( 2.6)
2 ( 1. 6)

45 (29.6)
33 (25.8)

Total

280

23 ( 8.2) 49 (17.5)

6 ( 2.1)

78 (27.9)

A total of 428 second chromosomes, excluding lethal chromosomes, wasexamined to carry recessive sterility gene. The results were shown in Table 3.
Total frequencies of sterility second chromosomes in natural populationsof both countries were not so different, but the frequency of male sterility
chromosomes in Japanese populations was much higher than that of female
sterility chromosomes, and the frequency of female sterility chromosomesin Anyang-l population was especially high. The frequency of sterility
second chromosomes in Katsunuma locality increased in the last two years
as deleterious chromosomes.

92 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22x.

EVOLUTIONARY GENETICSOn the Rate of Molecular Evolution
Motoo KIMURA and Tomoko OHTA

There are at least two outstanding features that characterize the rate ofevolution at the molecular level as compared with that at the phenotypiclevel. They are; (1) remarkable uniformity for each molecule, and (2) very
high overall rate when extrapolated to the whole DNA content.
The population dynamics for the rate of mutant substitution was developed, and it was shown that if mutant substitutions in the population are
carried out mainly by natural selection, the rate of substitution is given byk = 4N.51V,

where N. is the effective population number, 51 is the selective advantage ofthe mutants, and v is the mutation rate per gamete for such advantageousmutants (assuming that 4N.51~ 1). On the other hand, if the substitutions
are mainly carried out by random fixation of selectively neutral or nearly
neutral mutants, we havek = v,

where v is the mutation rate per gamete for such mutants.
Reasons were presented for the view that evolutionary change of aminoacids in proteins has been mainly caused by random fixation of neutral
mutants rather than by natural selection.
It was concluded that if this view is correct, we should expect that genesof "living fossils" have undergone almost as many DNA base replacements
as the corresponding genes of more rapidly evolving species. For details,
see J. Molecular Evolution 1: 1-17.

On the constancy of the evolutionary rate of cistrons
Tomoko OHTA and Motoo KIMURA

The variations of evolutionary rates in hemoglobins and cytochrome camong various lines of vertebrates are analysed by estimating the variance.
The observed variances appear to be larger than expected purely by chance.

RESEARCHES CARRIED OUT IN 197193

If the amino acid substitutions in evolution are the result of random
fixation of selectively neutral or nearly neutral mutations, the evolutionaryrate of cistrons can be represented by the integral of the product of mutation rate v(s) and fixation probability u(s) in terms of selection coefficients around the neutral point. Namely,k = 2N

~

2/N,u(s)v(s)ds,-2/N,

where Nand N, are respectively the actual and effective sizes of the population. This integral is called the effective neutral mutation rate.
The influence of effective population number and generation time on theeffective neutral mutation rate is discussed. It is concluded that the uniformity of the rate of amino acid substitutions over diverse lines is compatible with random fixation of neutral or very slightly deleterious mutations which have some chance of being selected against during the course of
substitution. On the other hand, definitely advantageous mutations will
introduce significant variation in the substitution rate among lines. Approximately 10 % of the amino acid substitutions of average cistrons might
be adaptive and create slight but significant variations in evolutionary rate
among vertebrate lines, although the uniformity of evolutionary rate isstill valid as a first approximation. For details, see J. Molecular Evolution 1: 18-25.

Serological Crossreaction of the Ascidian Liver Extract with
Anti-Rat Transferrin Rabbit Serum
Kazuo MORIWAKI and Tamiko SADAIE

There have been numerous reports on the existence of iron-binding protein, transferrin, in the sera of various classes of vertebrates as extensively
reviewed by Lush (The Biochemical Genetics of Vertebrates Except Man,
North-Holland, 1966). For the purpose of surveying the phylogenical stepin which the transferrin molecule had emerged, phylum protocordata was
firstly investigated in this study.
Liver of the ascidian, Halocynthia roretzi collected at Kesennuma in Miyagi Pref., was homogenized with 3 volumes of 5 mM Tris-buffer (pH 8.6)
followed by centrifuging at 10,000 rpm for 30 min at O°c. As the crude94 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

extract indicated a detectable crossreaction with anti-black rate transferrin
rabbit serum in the Ouchterlony test, further purification of the transferrinlike substance has been undertaken employing DEAE-cellulose and Sephadex G-150 column chromatography. Elution from DEAE-column following gradual increase of sodium chloride concentration up to 0.1 M in 5 mM
Tris-buffer (pH 8.6) introduced 2 peaks, one of which designated peak 2
contrained a certain substance reacting with the anti-transferrin serum.
Though an ability of binding iron was demonstrated by Fe59 -radioimmunoassay in the crude extract above obtained, it was shown to be involved inpeak 1.
anti X5563 MP
anti Rattus Tf R\

anti Wister Tf""- \0 1 o~-"_,~ 0 ~ 0'-'"..II M51'C:::'.!~~,ll..",peak 2w 0

anti X5563 MP

Fig. 1. Cross-reaction of the ascidian liver extract with anti-transferrin andanti-globulin rabbit sera in the Ouchterlony test.

The peak 2 was re-chromotographed on Sephadex G-150 using 5 mM
Tris-buffer and 2 protein peaks, approximate molecular weight 40,000 and
150,000, have come out. Both fractions, however, equally had positive
cross-reactions with various rabbit antisera; anti-black rat transferrin,
anti-norway rat (Wistar rat) transferrin, anti-mongolian gerbil transferrin,
anti-mouse r-chain (H-chain of X 5563 rG myeloma globulin). Whereas
they had negative reaction with anti-mouse a-chain (H-chain of MSPC-lrA myeloma globulin). These data are demonstrated in Figure 1. It isof great interest that the ascidian liver extract seems to share a certain
antigenic structure not only with serum transferrins of black rat, norway
rat and mongolian gerbil but also with rG class heavy chain of mouse immunoglobulin.

RESEARCHES CARRIED OUT IN 1971

XI.

95

HUMAN GENETICS

An estimate of the effective number of loci fortotal finger ridge-count
Ei MATSUNAGA

In the previous report (Matsuda and Matsunaga, 1971, Ann. Rep. Nat.
Inst. Genet. 21: 101) it was noted that the frequency distributions of total
finger ridge-count from an unrelated sample of 313 males and 313 females
were more or less negatively skewed in both sexes, with the mean and thevariance 151 and 2,124 for males and 142 and 2,008 for females, respectively.
The parent-child correlation estimated from 1,048 pairs was 0.44±0.03
and the sib-sib correlation from data on 188 sibships was 0.46±0.05, while
the parent-parent correlation was 0.02±0.07. The regression of child on
midparent was 0.82±0.05, and a linearity test gave no sign of dominance.
These results indicate that the great majority of variation in total ridgecount in the Japanese are to be attributed to genes with additive effect.
From the non-normality of the frequency distribution of total ridgecount in population, Holt (Ann. Human Genet. 20: 159, 1955) conjectured
that only a small number of genes having appreciable effect is involved,
since a large number of additive genes would give a nearly normal distribution. A simple method is suggested here to give a rough estimate of theeffective number of loci for this character.
It is assumed that 1) the variation in total ridge-count is determined byn pairs of alleles with perfectly additive effect, 2) there are only two alleles,
A and a, at each locus with frequencies p and q (p+q= I) respectively, 3)
the effect in single dose of A is a and that of a is zero in terms of ridge-count,and 4) the genes are segregating freely in the population. Then x=2nap+ fl, Vg =2na 2pq and d=2na, where x is the population mean, fl the basevalue assumed by the genotype homozygous for the gene a at all loci, Vg
the genetic variance and d the maximum range of the frequency distribution.
In the case of total ridge-count, the value of fl is obviously zero since individuals with zero total ridge-count have actually been observed. It follows that

96 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

x

p=([,
a = _t!!,g_ ---x(d - x)andx(d - x)n =-iv -.
9

Vg can be estimated from the observed variance mutiplied by the heritability, or twice the parent-child correlation, which corresponds to about
0.9 in our data. The value of d cannot be estimated with certainty. The
observed range of frequency distribution in our sample was about 260
in both sexes, so that d must be greater than this. On the other hand,because the distribution is negatively skewed, the value of p must be larger
than 1/2, and from p=x/d, d cannot exceed twice the value of x. Assuming d=290 and applying the observed values for the mean and the variancein our data, we obtain the following estimates for males: p=0.52, a=26.4
and n=5.5. The corresponding values for females can be obtained in thesame way, but in this case, allowing for the sex difference in the mean, thesame value for x as in males was used. The estimates are essentially thesame as for males: p=0.52 a=25.0 and n=5.8. If d=280, this would bring
only a slight reduction of the estimate of n.
It is to be noted that, if the value of a differs from locus to locus, theestimate of n gives its lowest limit. Therefore, the minimum number ofloci may roughly be estimated as about six, which must be taken as a first
approximation because of the many assumptions involved.

Determination of the exchange points in a human ring
chromosome
Yasuo NAKAGOME and Kazuso IINUMA

Ring chromosomes have been described in all 7 groups of human chromosomes. In none of these rings so far reported, the breakage points were
determined. The only example of determination of exchange points instructural abnormality was that reported by Bobrow and Pearson «(1971)
J. Med. Genet. 8: 240). They determined the exchange points in a translocation involving no. 4 and no. 18.

RESEARCHES CARRIED OUT IN 1971

97

A ring chromosome was observed in a 4 month-old boy with cri-du-chatsyndrome. His karyotype was 46,XYq+, 5r, based on either of the quinacrine fluorescence and the Giemsa-banding techniques. The ring seemed
to be very stable. Out of 184 cells karyotyped, rings were observed in 181
cells, 2 of them being dicentric and 179 monocentric.
The points of breakages in the process of ring formation were determinedby the comparison of G-banding patterns of the ring with those of the normal
no. 5 chromosome. It was shown that the normal no. 5 chromosome had
a narrow light band at the end of each arm, followed by a dark band proximal
to it. The light and the dark bands were designated as p 15 and p 14 bands
for the short arm, and q 35 and q 34 bands for the long arm. The lengths ofthe two light bands, amounted to 11.4 and 8.5 % of the length of the normal no. 5 chromosome (L) respectively. In every ring, where individual
bands were identified, either of the p 14 and the q 34 bands could be recognized. Therefore, two points of breakages in the formation of the ring must
be within either of the p 15 and the q 35 bands. The length of a light band
between the two dark bands, p 14 and q 34, in the ring was 14.4 % of the L.
The size of deletion in the process of ring formation was estimated to be
5.5 % of the L. The fluorescence technique seemed to be less efficient thanthe Giemsa-banding technique used here as far as the pattern in a ring isconcerned. (For details, see Nakagome et al. Cytogenetics, in press)

G group chromosomes in satellite associations
Yasuo NAKAGOME

Either of satellite associations and separation difficulties of chromosomes
at anaphases are likely to result from persistence of nucleoli (Heneen andNichols, (1966) J. Cell BioI. 31: 543). It was established that 3 pairs of
D group chromosomes were involved in satellite associations randomly(Nakagome, (1969) Cytogenetics 8: 296). However, in some of the caseswith translocation involving D group chromosomes, non-random association pattern was demonstrated (Nakagome and Bloom, (1970) J. Med.
Genet. 7: 371).
The present study dealt with two pairs of G group chromosomes, nos. 21
and 22, in satellite associations. Metaphases were derived from normal
individuals. The Giemsa banding technique was used for the purpose of

98 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

identifying each pair. A total of 152 G group chromosomes in satellite
associations were identified. Eighty seven of them were no. 21s and 65
were no. 22s. There was a slight excess of 21s, however, the difference was
not significant (x 2 = 3.18 d.f. = 1). The study is stilI in progress and additional cells are being scored to elucidate the difference, if any, between 2
pairs of G group chromosomes.

Polyploidy in cells cultured from amniotic fluid
Yasuo NAKAGOME, Kazuso IINUMA and Ei MATSUNAGA

Up to 100% polyploidy has been described in amniotic fluid cultures ofpregnancies with normal diploid embryos (Walker et al. (1970) Lancet 2:1137 and Milunsky et al. (1971) J. Pediat. 79: 303). If in fact this discrepancy frequently happens, the prenatal diagnosis of polyploid embryos would
be impossible. It was reported that prolonged culture of amniotic fluid
cells increased the incidence of polyploid cells.
The present report describes a technique by which polyploidy was successfully kept below a few percent. In addition, abundant mitoses wereobtained within a short period of time. One and a half to 2.0 ml of amniotic fluid was placed in each of a few Falcon Petri dishes (35 mm diameter)
and incubated overnight in a 5 % CO 2 atmosphere. Next morning thesupernatant was replaced with 1.5 ml of fresh medium (MEM supplemented
with 20 % fetal calf serum). At this time a few fibroblasts were observedin most of the dishes. Cultures were fed every other day. Some dishes
were ready for harvest on 4th day of culture. Usually several countablemetaphases were then obtained from each dish when harvested. In thepresent series, cultures were harvested between 10th and 14th day of culture
to ensure the presence of abundant mitoses. Before harvesting, usuallyin the evening, the culture medium was replaced with fresh medium. Next
morning, colcemid (final concentration 0.06 r/ml) was added. After 4 to 5
hours, cultures were trypsinized and harvested. Special care was taken to
avoid to lose cells in the process of harvest.
A total of 482 metaphases were obtained from 6 different cultures. There
were 11 tetraploid and 471 diploid metaphases, the incidence of tetraploid
being 2.3 %. In 3 cultures, some of the dishes were cultured further, up to
54 days and then harvested. Cells in mitosis were few in these cultures.

RESEARCHES CARRIED OUT IN 197199

All 76 metaphases observed were diploid.
The number of cultures and cells observed after prolonged period of culture is still limited. However, the present study suggests that the overwhelming increase of polyploid cells may be avoided by improving of culture
technique.

Y-chromatin in aged males
Kazuso IrNUMA and Yasuo NAKAGOME

Oral smears from 102 males between the ages from 8 months to 87 years
were examined in order to clarify whether ageing has any effect on theincidence of Y-chromatin, a highly fluorescing segment of Y chromosome
stained with quinacrine derivatives, in the epithelial cells. Slides wereprepared by the conventional technqiue including 20 minute staining in
0.005 % quinacrine mustard dihydrochloride in McIlvaine's buffer at pH
7.0~ One hundred well preserved cells per person were examined and scoredfor Y-chromatin positive cells under a Nikon-FL fluorescence microscope.
Cells with Y-chromatins were further divided into three classes: (a) single
body, showing a single distinct fluorescing spot, (b) duplex body, showing
two similar spots adjacent to each other and (c) multiple bodies, multiple
spots other than those scored as duplex.
The scores for total Y-chromatin positive cells (a+b+c) ranged from 43
to 99 % with an average of 79.1 ± 11.1 % (Standard deviation). The incidences of cells with duplex and multiple spots were 11.5 % and 2.9 % respectively. The regression equation upon age based upon individual records was given by Y= -0.056X+81.85 for total Y-chromatin positive
cells. There seems to be a slight and insignificant decrease with advancing
age (t=1.17, d.f.=loo, 0.3>p>0.2). When cases were divided into twogroups, one containing 42 subjects over 65 years of age and the other containing 60 subjects of younger age, then the mean scores were 77.6 and80.1 %, respectively, the difference between the two groups was small but
significant (x 2 =9.24, p<0.005). It is noteworthy that the incidence ofY-chromatin positive cells above 75 years of age exhibited the lowest score,
and that this was the group contributed to the decrease of Y-chromatin
positive cells in the older males. Of 102 subjects, one case with normal

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

phenotype had an unusually large Y-chromatin, suggesting the presence ofa large Y chromosome. For details, see Iinuma and Nakagome (1972), Jap.
J. Hum. Genet. 17: 1.

Parental age in translocation Down's syndrome
Ei MATSUNAGA and Akira TONOMURA·

In continuation from the preceding year's work, data on the parental
age at the birth of patients with translocation Down's syndrome have beencollected from various cytogenetic centers in Japan. The number of cases
available for analysis amounts to 102. Their distribution by the type andthe origin of translocation is as follows: 23 t(DqGq), 29 t(GqGq) and one
G/G tandem translocation of sporadic origin; 20 t(DqGq) and 4 t(GqGq)
with maternal inheritance, and 6 t(DqGq) with paternal inheritance; and9 t(DqGq) and 10 t(GqGq) of unknown origin. Examination of the datausing vital statistics as controls revealed no evidence for paternal age effect,
while maternal age was shown to be of importance in the etiology of thesporadic t(DqGq) or t(GqGq) trisomy. The mean maternal age wasdecreased for the t(DqGq) (25.5 years) but increased for the t(GqGq)(28.5 years), both decrease and increase being significant when comparedwith the control (27.1 years); the mean difference between paternal andmaternal age was significantly greater in the former (4.4 years) than in thelatter group (2.3 years). When the data are combined for the sporadic andunknown origin, the relative risk increases with advancing maternal age forthe t(GqGq) while it decreases for the t(DqGq). These findings, together
with the greater variance in maternal age for the t(GqGq) than for the t(DqGq), suggest that the majority of the sporadic t(GqGq) are either t(2Iq21q)
or 2lqi and the rest t(2Iq22q), and that non-disjunction, isochromosome
formation and centric fusion between non-homologous acrocentrics may all
be correlated, in the first two instances positively and in the last one negatively, with maternal age. On the other hand, no alteration of maternal
age was found for the t(DqGq) inherited from the mother. Detailed data
will be published elsewhere.

•

Dept. of Cytogenetics, Tokyo Medical and Dental University.

RESEARCHES CARRIED OUT IN 1971

101

Location and neighboring amino acid sequence of interand intrachain disulfide bridges of human IgMTomotaka SHINODA

To determine the amino acid sequence and position of the disulfide bridges
in IgM obtained from patients with macroglobulinemia, purified material
was digested with pepsin and the peptides were separated by high voltage
paper electrophoresis. Peptides containing cysteine were also isolated aftertryptic, peptic, or thermolysin digestion of the p heavy chain of IgM and of
Fab and Fc fragments. The bridge peptides were sequenced after oxidationor alkylation followed by purification by paper electrophoresis or column
chromatography. The results showed that human IgM has five intrachain
disulfide bridges, and four interchain brideges. Two of the interchain
bridges link p heavy chains together, one interchain bridge joined each lightchain to a heavy chain, and one bridge holds five monomeric units together
to form a pentamer of one million molecular weight. The sequence and thelocation of the bridges in the p heavy chain were as follows: Thr-CysThr-Phe-Ser-GlyjTyr-Cys-Ala-Arg-Val-Val-Asn, Fd variable; Gly-Glu-Cysj
Val-Ser-Cys-Glx-Asx-Ser, L-H interchain; Val-Gly-Cys-LeujVal-Cys-Lys(Glx, His, Asx, Asx, Gly, Pro, Lys, Glx), Fd constant; Ile-Cys (Glx, Ala, Thr,
Gly, Phe, Ser, Pro, Arg) GlnjThr-Cys (Arg, Val, Asx, His, Arg, Gly, Leu,
Thr) Phe, Fd-Hinge; Cys-Val-Pro-Asx-Glx-Asx-Thr-Ala-Ile-Arg, Fc p;
Thr-Cys-LeujThr-Cys-Thr (Val, Thr, His, Thr, Asx, Pro, Ser, Leu, Pro, Leu)
Lys-Gln, Fc p; Ile-Thr-Cys-LeujTyr-Thr-Cys-Val-Val-Ala, Fc p; Ser-AspThr-Ala-Gly-Thr-Cys-Tyr, C-terminal.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

XII.

BEHAVIORAL GENETICS

Phototaxis and fecundity of Drosophila virilis. l )Chozo OSHIMA and Jong-Kil CHOO

Many flies of Drosophila virilis were collected from the warehouse of abrewery company in Tokyo, in April 1970, and a cage population was set
up with them. Approximately after three generations, about 200 females
and 200 males were sampled randomly from the population. The sexes
were run separately through the maze apparatus and they chose either lightor dark pathway eleven times when they came to the fork of a way. About
twenty pairs of flies having the similar extreme photoscores, positive or negative, were mated, and their offspring were subjective to the same selection. These both photo-positive and negative populations were inbred
separately in a milk bottle containing culture medium at 25°C. Suchselections were repeated over for 30 generations. The mean photoscores andvariances for every ten generations were shown in Table 1.
The mean photoscores and variances of the photo-positive population
have not changed remarkably throughout the experiment, but the mean
photoscores of the photo-negative populations increased gradually andon the contrary its variances have reduced.
The regression coefficient of differences in photoscores between thephoto-positive and negative populations was calculated to be 0.2382 for theformer 15 generations and to be 0.3542 for the latter 15 generations of
selection.
The fecundity of flies in these phototactic populations at the 20th and30th generations was examined. Six pairs of F l flies of each four isoline
extracted from these populations respectively were cultured separately in asmall vial and transferred everyday to a new vial under the three kinds oflight condition: 1) constant light, 2) constant dark, 3) periodical light anddark (9: L, 3: L~D, 9: D, 3: D~L). These eggs laid by each female on
the cultural medium were scored everyday for ten consecutive days. The
experimental results are shown in Table 2.
The mean number of eggs laid by photo-negative flies was about 20 percentl}This work was supported by a grant (4050) from the Ministry of Education.

Table 1.

Mean photoscores and variances for every ten generation of
Drosophila virilis populations--~---_._---

Generationof
selection

Mean photoscore and variance of both
sexes of the photo-positive population
FemaleMale
Both sexes-- - - - - - - - - - - - - - - ---Mean photoscore and variance of both
sexes of the photo-negative population
FemaleMale
Both sexes
---

-

..

~

_._~

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

X

4.15
4.28
4.37

0-10
11-20
21-30

------,----_.~

(12

X

(12

X

(12

9.94 4.67 11.37 4.51
10.31 4.56 10.04 4.43
10.74 4.44 10.53 4.41

10.61
10.19
10.71

X

(12

X

(12

X

(12

6.22 11.51 6.16 10.91 6.20 11.35
7.09 9.04 7.15 9.41 7.10 9.29
6.52 8.73 6.25 8.62 6.36
8.51

~

tTl

en

!i1~

g
tTl

en

~
~

Table 2. Mean number of eggs per day per female for 10 consecutive days under threekinds of light conditions, which is Fl fly of four isoline extracted from thephoto-positive and negative populations at 20th and 30th generation

~

oo
C

>-l

PopulationLight
condition

Gen. 20

Number of eggs/day/female
Gen. 30

Photo-positive

Constant light
Constant dark
Periodical L: D

45.21±3.5235.23±2.28
48.21±2.91

38.87±2.72
32.62±3.2635.52±1.9339.54±3.1233.92±2.77
41.87±2.42

Photo-negative

Constant light
Constant dark
Periodical L: D

67. 67±4.4956.13±4.1756.30±2.4748.31±2.1951.26±2.56
49.55±2.37

57.99±3.3453.70±3.3752.93±2.42

Combind

--Z
\0

.....

ow

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

more than that laid by photo-positive flies, but no significant difference wasobserved among these different light conditions within each population.
The mean number of eggs per day per female and also their standard errors
showed a tendency to decrease for ten generations.

Selection and hybridization analysis on the phototactic
behaviour of Drosophila melanogaster
Chozo OSHIMA and long-Kit CHOO

From female flies of Drosophila melanogaster collected at Nago-cho,
Okinawa in April 1970, eleven iso-female lines were established and total
452 females and 365 males of F 1 fles were collected from these iso-femalelines and introduced into a population cage. The procedure of selection
by using the maze apparatus in this experiment was similar to that in theexperiment of Drosophila virilis. The effects of selection in both photopositive and photo-negative directions are summarized in Table 3.
Table 3. Mean photoscores and variances of every five
selected generation of both photo-phostive and negative
populations of D. melanogaster
Generationof
selection

Photo-positive population---------------------

Photo-negative population--~-~---~--~- --~------

Score

Variance

Score

Variance

o

5.04

1- 5

4.37
4.32
4.14
3.22
3.44

12.17
8.94
9.11

5.04
5.50
6.29
6.62
7.27
7.66

12.17
9.84
10.87
10.99
10.72
8.72

6-10
11-15
16-20
21-25

8.00
6.47
6.37

The photoscore of the foundation population was 5.04, and it has diverged
gradually and fairly by the repeated selection for 15 generations and thephotoscores of both photo-positive and photo-negative populations would
reach to the equilibrium states respectively at around 20th generation. On
the other hand, the variances of the photo-positive population have decreased gradually, but those of the photo-negative population have not sochanged.
The heritability W) could be calculated by the following formular:

105 1RESEARCHES CARRIED OUT IN 1971h2=(0'~-0':)/0'~ where O'~ was the wild type variance of the foundation popu-0':

was the isogenic variance of the selected population. (N. M.
lation andHadler, 1964, Genetics 50: 1269-1277). After 25 generations of selection,
the heritabilities of the photo-positive and negative populations were obtained to be 0.597 and 0.368 respectively, and the combined heritability ofboth populations was 0.483.
The photoscore and variance of hybrid populations between the photopositive and negative populations at 21st generation were examined and theresults were shown in Table 4.
Table 4. Photoscores and variances of hybrid populations between
photo-positive and negative populations at 21st generation
Generation
21

Male

FemalePhoto-positive
Photo-negative

Photoscore
3.19
7.93

Average

5.56

F 1 (posi x nega)
E (nega x posi)

5.40
6.10

Average

5.75

6.37
9.66

Photoscore
3.73
7.97

11.14
10.18

5.67
5.47

Variance

Combined

Variance
7.28
8.03

Photoscore
3.48
7.95

Variance
6.92
8.80

5.72

5.85
8.96
11.11

5.57

5.53
5.81

10.06
10.61

5.67

The photoscores of the hybrid populations were close to the average onesof these photo-positive and negative populations. As expected, the variances of the hybrid populations were larger than those of all parental populations. The phototactic behaviour was assumed to be dependent a polygenic
system.

Circadian rhythm of fecundity of Drosophila viri/is undervarious light environments1 )Chozo OSHIMA and Jong-Kil CHOO

A cage population of Drosophila virilis was set up with 123 females and
137 males which were collected from the warehouse of a brewery company
in Tokyo on April 7, 1971. The population has been kept in an incubator,
1)

This 'York was supported by a grant (91265) from the Ministry of Education.

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

whose temperature fluctuated diurnally between 20 and 30 0 e by a program
controller. After two months, 20 pairs randomly sampled from the population were mated separately in small vials and they were allowed to lay eggs
for 24 hours on culture medium and they were transferred into a new vialeveryday. F 1 flies of four strains among twenty parental strains were usedin this experiment. Twelve pairs (F l ) isolated from each strain were divided
into three groups and the number of eggs laid everyday by these pairs werescored as maintained under three kinds of light environments at constant
25°e. These light environments were as follows: I) constant light: 2000
lux by fluorescent lamp, 2) Periodical light and dark: 9 hours 2000 lux,
9 hours 0 lux and 3 hours changing from light to dark reciprocally by
program controller, 3) constant dark: 0 lux, in this case, the flies were transferred under a red lamp (3 lux). The experiment continued for twenty
days.
Table 5.

Linear regression coefficients for the fecundity curves of four strainsof D. virilis from 3rd to 20th days under three kinds of lightenvironments and the results of analysis of variance

StrainConstant
light

Periodical
light & dark

Constant
dark

AverageAD-2.772-0.905-4.761-4.913-2.041
0.565-0.230-0.896-3.292-0.531-2.776-1.090-2.702-0.290-2.589-2.299Average-3.338-0.651-1.922

B

C

d.f.

S.S.

M.S.

F

Environ.
StrainEn. x St.
Error

2
3
6
36

57.8333
46.1699
32.3224
222.1121

28.9166
15.38995.38706.1697

5.69**
2.49*

Total

47

358.4374

The mean numbers of eggs per day per female were 62.5 under the constantlight, 61.4 under the periodical light and dark and 62.4 under the constantdark environemtnt. No significant difference was detected among thetotal eggs poroduced under the three kinds of light environments.
Linear regression coefficients for the fecundity curves of four strains from

RESEARCHES CARRIED OUT IN 1971

107

3rd to 20th days were calculated and the average values of four strains
under the constant light, constant dark and periodical light and dark
environments were -3.338, -1.922 and -0.651 respectively. The analysis
of variance for these regression coefficients was performed and the difference
among three light environments was highly significant at 1 percent level andalso significant difference among four strains was found at 5 percent level.
The results were presented in Table 5.
The circadian rhythm of fecundity was assumed to be markedly influenced
by the light environment and the rhythm could be recognized to be maintained under rhythmically fluctuated light environment. However, B strain
showed the similar responses for these light environment.

Bad effects of urbanization and noise environments of a great city
on behaviour of Drosophila!)
Chozo OSHIMA and Jong-Kil CHOO

Drosophila flies and relative species were collected simultaneously by asweeping net at two places in November 1971. One place was a rural district, Tamazawa in Misima city and another place was the National Park
for Nature Study in Tokyo city. Collected flies were classified, and thenumber of genera, species and specimens at two places were compared asshown in Table 6.
Table 6.

Drosophila flies collected at two places in
November 1971

National Park for Nature Study (Tokyo city)
Tamazawa (Misima city)

GeneraSpecies7

31Specimens

287

4

20

316

Any bad urbanization effect of Tokyo could not be recognized on the
result. The montium species group (D. auraria and D. rufa) and the immigrans species group (D. immigrans) were dominant species at Tamazawa,
and on the other hand, Scaptomyza pallida was found to occupy about
60 percent in total specimens collected at National Park for Nature Study.
It was examined whether the oviposition of two strains of D. melanogaster
II

This work was supported by a grant from the Ministry of Environmental Agency.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22(Anyang and Katsunuma strains) and of one strain of D. virilis (Azumabashi
strain collected in Tokyo) would be affected under the noise environment or
not. The noise, 3000 cycle/sec. and 100 phone, was produced by a speakerof the Audio Signal Generator (SNR-ll) in an incubator KOITOTRON
having program controller of photo and temperature conditions.
Fourty sib-pairs of newly emerged flies from five lines of each strain weredivided into two groups. The one group was subjected to the noise environment and the other group was cultured under the quiet environment. Eachpair of flies was put in a small vial which was covered with a thin paper cap
and contained the cornmeal-agar-medium. They were transferred to a
new vial everyday. After transferring flies, eggs laid on medium, werescored. Such procedures were continued for 12 or 16 days. The photocondition was a constant light (2000 lux) and the temperature was kept
at 24°C constantly. The results are shown in Table 7.
Table 7. Mean number of eggs per female per day during the first
period (6 or 8 days) and the second period (6 or 8 days)
under the noise and quiet environments
Environment

Period

Quiet

Noise

D. melanogaster

~~--_

..

__ . - - - - . - - - - - -D. virilis--------

Anyang strain

Katsunuma st.

Azumabashi st.

1st
2nd

n.19±8.67
61.54±2.77

62. 73±7. 73
68.65±1.69

67.69±3.01
62.84±0.98

Total

66.87±4.61

65.69±3.89

65.27±1.65

1st
2nd

35.59±6.31
55.22±4.46

34.55±5.86
69.08±2.1152.66±2.4546.95±1.51

Total45.41±4.5155.15±4.87

49.81±1.57

The state of oviposition of D. melanogaster was remarkably depressed
during the first period, but such depressions were relieved at the second
period. On the other hand, the state of oviposition of D. virilis under thenoise environment was about 20 percent depressed consistently during thewhole period.

RESEARCHES CARRIED OUT IN 1971

XIII.

109

APPLIED GENETICS

Genetic variation in a naturally reproducing sapling-cluster
in Thujopsis dolabrata Sieb. et Zucco
K. I. SAKAI, S. IWAGAMI and S. MIKAMI

Reproduction in natural forests is one of the most interesting problems
in silviculture and has been investigated ecologically as well as physiologically.
Little attention, however, has been paid from the standpoint of genetics.
The present study deals with genetic analysis of sapling clusters of Thujopsis
dolabrata spontaneously growing in a cut-over area of a natural forest of
Cryptomeria japonica D. Don. The sapling cluster is of an echinus form
with a diameter of a few meters, each consisting of more than several "individuals" which were terrestrially saparated from the others. Saplingclusters form a stretch of a crowd on the area. From each of eight clusters
selected at random, samples of needle-leaves were collected from five"individuals". Juice squeezed from collected leaves was investigated forisoperoxidase variation in starch-gel electrophoresis. Isoperoxidases werecompared among saplings within each cluster in order to find if they werevegetatively propagated or from seed. Needless to say, propagules fromthe same clone will exhibit a very similar, not necessarily the same due totechnical variation, pattern of isoenzyme bands. Isoenzyme similarity
between saplings was estimated by measuring the disagreement count between
azygous bands or by difference in staining intensity between paired bands.
It was found from this study that, of eight clusters, two were found to begroups of vegetative propagules from single clones, five mixtures of clones
and seedlings, while the remaining one was a mixure of seedlings. Investigation by analysis of variance of needle characters gave additional evidenceof the genetic constitution of sapling clusters. It is expected in future that
only one or two trees within each cluster would grow to maturity at the costof the other. What selection would be responsible for the differential
survival among saplings constituting a cluster is an interesting problem.

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

Isoperoxidase variation among island populations ofPinus Thunbergii-densijlora
Y. G. PARK, S. IYAMA and K. I. SAKAIThe Seto inland sea surrounded by Japan Proper, Shikoku and Kyushu
islands is dotted with numberless islets, on which the Japanese pine species,
Pinus Thunbergii and P. densiflora are naturally growing. These two pine
species, though they have been defined taxonomically as separate species,
are often hard to discriminate one from another. Thus, in the present
study, we treat them collectively as a single species, Pinus Thunbergiidensiflora.
We have collected needle-leaves from approximately 30 trees in each of
20 populations growing on eleven islets. They were investigated electrophoretically for isoperoxidase variation. The maximum number of isoperoxidase bands detected so far in Pinus Thunbergii-densiflora was thirteen,
but observations were made on four of them: H, K, M and N. The averagefrequencies of these four bands were 21, 17, 77 and 2 %, respectively. Bystatistical comparison among populations of frequencies of these four bands,
it was found that some populations were significantly different from othersin the frequencies of one or two isozymes. On the basis of such a comparison, populations not significantly different from each other were collected
in one group, taking also into consideration their locations. Thus, twenty
populations investigated in the present study were classified into six groups.
It is interesting to find that some groups consisted of only one population
while other involved six populations which were distributed on different islets.
How do such groups of populations come into existence on separate islets
will be left for further enquiry.

Effect of mixing genotypes of barley on the performance of a
population
Shinya IYAMA and Kan-Ichi SAKAIThis experimental study has been carried out in the past three years andis still under way. The experiment consisted of 12 cultivars of barley grownin single stands and 60 mixtures of four cultivars selected at random fromthe twelve. Attention was paid to make the entry of each cultivar in mix-

RESEARCHES CARRIED OUT IN 1971

111

tures equal. It was found from the experiment conducted in 1968-1969
that some mixtures yielded better than that expected on the basis of performances of component cultivars, or were overcompensatory, while otheryielded similarly to the means of their components (compensatory) or lessthan that (undercompensatory). Selection was conducted for and against
overcompensatory effect of mixtures and six overcompensating and six
undercompensating mixtures were grown in 1969-1970. The result of thisstudy was, however, contrary to our expectation: the overcompensatory
as well as undercompensatory effect of mixtures was not always recurring.
It was then suspected if some or other environmental conditions including
the year effect might have played a role in the overcompensating mechanismof the mixtures. Thus, another experiment was conducted in 1970-1971
with the same mixtures but grown on variable fertilizer levels. Then, it
was found that poor fertilizer application caused the overcompensatory effector the cooperative effect of component cultivars, while higher level offertilizers showed negative cooperation or undercompensation. Further
experimental study is now in progress.

Genetic changes occurring in wild quails due to"natural selection" under domestication
Takatada KAWAHARA

One of the most interesting problems in the breeding of avian species
would be to enquire into the biological changes in birds which take place
during domestication. For such an investigation, Japanese quails are useful.
It should be used because they are being domesticated in Japan since thefirst decade of this century, on the one hand, and their wild relatives still
propagate and migrate between the northern districts and the central partsof Japan proper.
The present paper reports the result of comparative observations on somephysiological changes occurring in wild Coturnix coturnix japonica rearedin cages for three generations. The materials for this study have originatedfrom 268 wild birds captured in the field during the past four years. Wild
birds are hardly distinguishable from the domestic ones in their appearance,
but are distinctly different in some physiological aspects, for instance, occurrence of non-layers in cages at least up to 20 weeks after hatching, in112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

number of days to first egg or sexual maturity under continuous light,
egg production during 60 days after first egg and body weight at maturity(20 weeks of age).
The present observations have revealed that the frequency of non-layers
in wild birds in a cage for the three successive generations, F h F 2 and Fa,of propagation within the flock was 50.0,30.9, and 23.4 percent, respectively,
while in the domestic strain none was a non-layer. In the wild strain, there
was a great individual variation among birds, which laid egg before the ageof 20 weeks, in respect to the number of days required for attaining sexual
maturity. The average age at sexual maturity in the first generation offspring of wild birds was 11 0.0±2.7 days in contrast to 50.1 ±0.3 days of domestic
ones. The second and third generation of the wild strains showed an
improvement toward earlier maturity and their mean ages were 82.8±2.3
days and 81.1 ± 1.5 days, respectively.
The hen-day egg production rate was 44.5±3.7 % in the first generation
offspring of the wild strain, whereas it was 88.3±0.5 %in the domestic strain.
The second and third generations of the wild birds showed a slight improvement of the rate, i.e., 49.6±2.2 % in the second and 52.8± 1.8 % in the third
generation.
When wild brids were captured in the field, body weight of the female
was 97.6±1.0 g., and the first generation offspring weighed 95.3±1.3 g.
The second and third generation offspring of the wild strain, however, gained
weight during domestication, their body weight being l00.9±0.8 g. and104.0±0.8 g. The body weight of the domestic female bird was 133.5±
0.5 g., on the average.
It is thus concluded that wild strains can show a rapid improvement ingrowth as well as in egg production as a result of natural selection in domestic
environment.

Multiple ovulation and egg defects
in Japanese quail
Takatada KAWAHARA

In a domestic strain of the Japanese quail, it was noticed that some families
tended to produce more defective eggs than the other. Among the defective
eggs observed were double-yolked eggs, shell-less (membrane) eggs and soft-

RESEARCHES CARRIED OUT IN 1971

113

shelled eggs. Progeny test showed that, of the three defects mentioned above,
the occurrence of double-yolked eggs was significantly different among fullsib families. The heritability value calculated from the analysis of variance
was 0.49. Investigation indicated that the correlation between doubleyolk and egg production was -0.14 when measured on individual bird basis,
while it was -0.29 when measured on family basis. Birds which laid
double-yolked eggs tended to show earlier sexual maturity, the correlation
coefficients being -0.12 and -0.14 on individual bird and family basis.
The same correlation coefficients between double-yolk and ovary weight oroviduct weight were 0.33 for individual birds and 0.11 for families or 0.19
and 0.25, respectively. It is of interest to find that the egg productivity andweight of the sexual organs, however, were not correlated. In order tofind out the mechanism for such an unusual behavior, a post-mortem examination was conducted with female birds immediately after ovipositionof a double-yolked egg. It was then found that double-yolk was caused bysimultaneous release of ova instead of successive occurrence of single ovulations.

Estimation of heritability from analysis of variancein a population with related parents
Tohru FUnslllMA

Conventional method for estimating heritability, i.e. h2 = 4a:/(a: + a~
+ a:) etc., where a:, a~ and a: are the components of variance for sire, damand full-sib in the analysis of variance, bases on the assumption that no
genetic relationship exists among any of the parents. However, such an
idealized population where there is no relationship among parents is seldom
encountered in practice.
The purpose of this paper is to present general formulae for estimating
heritabilities based on analysis of variance in a population where genetic
relationships may exist among sires, among dams and/or between sires anddams, including mates.
Heritabilities based on sire families, dam families and sire and dam families
can be estimated as follows;114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

h2d =

(W

lJ

- -

+ lJ + S)(rl - r2) + lJr3~--~~-~~-~

~--

and
h;+d =

S+lJ(W+lJ+S)rl- Wr3 'where r1, r2 and r3 are Wrigtht's coefficents of relationship between full-sibs,
between half-sibs and between non-sibs, and S, lJ and Ware the components
for sire, dam and full-sib in analysis of variance, respectively.
Only when there is no relationship among parents, rl = 1/2, r2= 1/4 andr3 =0, the equations reduce to the conventional one.
It was found that heritability estimates must be corrected according to
the genetic relationship among members in the population and that theconventional method, ignoring the genetic relationship, may lead to bothover- and under-estimation of the heritability depending on the parental
relationships.

Estimation of heritability from parent-offspring regression
in a population with related parents
Tohru FUJISHIMA

The conventional method for estimating heritability from the parentoffspring regression is strictly applicable only to a population where there
are no genetic relationships among the parents, but populations commonly
encountered in animal breeding are seldom free of such a genetic relationship.
The purpose of this paper is to present the procedure for estimating heritability applicable to the population where genetic relationships exist among
the parents.
1) Heritability estimated from intra-sire dam-offspring regression:
The heritability can be estimated as;

h=
2

where

2b 1
I - (I - 2bl)m~ ,b1 = the coefficient of regression of offspring on dam within sirefamily,RESEARCHES CARRIED OUT IN 1971

115

m~= Wright's coefficient of relationship between dams within sirefamily.
It is clear from the equation that h2=2b 1 only when m~=O.
2) Heritability estimated from mid-parent - offspring regression:

h2 =

2b 2
2 + [2(mf - ml) - m2 - mal (I - b2) ,where

b2 =the coefficient of regression of offspring on the average ofparents' phenotypic values in a pair-mating population,
mf, mh m2 and ma=Wright's coefficients of relationship between
mates, between sires and dams mated to different
sires, between dams mated to different sires,and between sires, respectively.
It should be noted that h2=b2 only when mf=ml=m2=ma=0.
Variations in growth pattern among rice varieties
Hiroko MORISHIMA and Hiko-Ichi OKA

Variations in growth pattern among rice varieties were studied for better
understanding of their "general adaptability". As a part of "International
Rice Adaptation Experiment" (under UMjIBP), about 50 varieties fromvarious temperate countries were evaluated at two different fertilizer levels
for 3 years (1968-1970) in the Sizuoka Agricultural Experiment Station.
From the dry weight data taken during the growing period (3 times) and at
maturity, logistic growth curves were computed for each variety in each plot
to obtain parameter values showing growth pattern and yielding capacity:maximum growth rate, days from maximum growth to heading, days frommaximum growth to the time when growth rate is reduced to half of the
maximum (growth persistence), growth rates at 10 different stages, total
spikelet number per unit area, grain yield, percentages of grain yield attributable to the growth before and after heading, etc. Studies of variations in these parameter values indicated the following:I) Analysis of variance showed that the varieties significantly differedin most parameter values; annual variations were significant in growth
persistence, total spikelet number and grain yield; fertilizer application affected maximum growth rate, days from maximum growth to heading, grain

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

yield, etc.; and genotype-environmental interactions were significant inmany parameters.
2) To examine the pattern of sequential change in growth rate, correlation coefficients were computed among the estimates of growth rate at 10
different stages and were subjected to principal component analysis. The
results showed that the changing pattern in growth rate would be oscillatory.
However, varieties with smaller amplitude tended to give higher yield.
3) Major variation trends found among the varieties were: i) early vs.
late vigor type (maximum growth around the time of floral initiation or atlater stages), and ii) short-period rapid growth vs. sustained slow growth
types. These two variations were independent of each other. The later
the maturity of a variety, the more it tended to early vigor and sustained slow
growth type. Such varieties appeared to have a high yielding capacity.
The variations in growth type caused by fertilizers were similar to those due
to varietal genotypes.

The distribution and effects of genes causing F I weakness inOryza brevi/igu/ata and O. g/aberrima
Yaw-En CHU and Hiko-Ichi OKA

Weakness of F I plants is frequently found in hybrids between strains of
Oryza breviligulata (wild) and O. glabberrima (cultivated rice) endemic toWest Africa. A set of two complementary dominant weakness genes,
WI and W 2 , was found to control the observed F I weakness. Many breviligulata strains had W lo while most of the glaberrima and semi-wild strains
had W 2 or were free of both. In the weak F I plants, tissue differentiation inadventitious roots seemed to be disturbed. Modifier genes affecting theexpression of the weakness genes appear to be also present. (Published in
Genetics 70: 163-173)
Preliminary observations on the establishment of perennial ryegrassand orchard grass varieties
Hiroko MORISHIMA

In order to investigate differential survival of two grass species under
different conditions, an experiment was conducted using three varieties of

RESEARCHES CARRIED OUT IN 1971

117

perennial ryegrass (Lolium perenne) and also three of orchard grass (Dactylis
glomerata). After a germination test, 300 viable seeds per plot (1 m X 1 m)in size) were sown in 1970 in September in pure stand (6 varieties) and species
mixture (1: 1, 9 varietal combinations). The 15 populations were tested
in two environments: i) road side (2 replications, with and without whiteclover respectively) and ii) experimental field (fertilized and weeded, 4replications differently managed in 1971 for another observation). The
number of surviving plants was counted two months after seeding and thenevery three months.
In the road side plots, the number of surviving plants in pure stand twomonths after seeding significantly differed between species and among thevarieties. It ranged from 20 % to 28 % of viable seeds in perennial ryegrass
and from 5 % to 16 % in orchard grass, and the differences among species
and varieties persisted untill next year. In the experimental field, thesurviving rates two months after seeding were about 40 % in all plots andwere reduced to 10-15 %next spring showing no significant differences among
species and varieties. In the rate of establishment, competition between
species was not apparent at least in the first several months. Perennial
ryegrass had a relative frequency of about 75 % in road side and about 60 %in experimental field, and the relative frequencies did not change much for
one year. The data suggested that the rate of establishment in an environment could be genotypically controlled.

Collection and preliminary observation of cultivatedcereals and legumes in Ethiopia
Sadao SAKAMOTO and Katsuyoshi FUKUI!)

Observations and collections of various cultivated plants in Ethiopia
were carried out, from December, 1967 to March, 1968, by the Botany
Team of the Kyoto University Scientific Expedition to the Sahara and thesurrounding areas. About 1,000 samples from the field and 750 samples
from 21 local markets were collected during the expedition. Several random
samplings from the fields were also made.
Preliminary observation and analysis of variations found in the collected
samples of the following cereals and legumes were carried out.
11

Institute for the Study of Languages and Cultures of Asia and Africa.

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

cereals: wheat, barley, teff, sorghum, finger millet and maize
legumes: field pea, chick pea, horse bean, lentil, Vigna, Phaseolus andgrass pea
An abundant variation in various characters, such as morphology andcoloration of spikes and seeds, was observed in the samples of wheat, barley,sorghum, field pea, chick pea, Vigna and Phaseolus. It was speciallycharacteristic that the occurrence of colored forms was observed commonlyin the present collections of the most cereals and legumes. A close association of weedy Lolium and Avena with wheat and barley cultivation in Ethiopia
was clearly demonstrated. Importance of Ethiopia as one of the most important world centers for the origin and differentiation of cultivated plants
was again confirmed from the present study. The details were published in
Kyoto University African Studies 7: 181-225.

BOOKS AND PAPERS PUBLISHED IN 1971

119

BOOKS AND PAPERS PUBLISHED IN 1971
BY STAFF MEMBERS(1) CROW, J. F. and T. MARUYAMA 1971. The number of neutral alleles maintained in a
finite, geographically structured population. Theor. Popui. BioI. 2: 437-453.
(2) ENDO, T. 1971. Expression of allelic peroxidase isozymes in heterozygotes of
Oryza perennis. Jap. J. Genet. 46: 1-5.
( 3) ENDO, T., B. B. SHAHI and C. PAl 1971. Genetic convergence of the specific acidphosphatase zymograms in Oryza sativa. Jap. J. Genetics 46: 147-152.
( 3) ENOMOTO, M. 1971. Genetic analysis of nonmotile double mutants in Salmonella
typhimurium: a new mapping method by abortive transduction. Genetics 69:
145-161.
( 5) FUJII, T. 1971. Effect of fractionation treatment with gamma-rays on the mutation
frequency in maize. Jap. J. Genetics 46: 243-251.
.
( 6 ) HASHIGUCHI, S. and H. MORISHIMA 1971. Principal component analysis for multivariates which are not directly measurable and its application to genetic data.
(In Japanese). Oyo Tokei 1(2): 89-95.
(7) HIGURASHI, M., Y. NAKAGOME, I. MATSUI and M. NAGANUMA 1971. Cytogenetic
observations in children with leukemia. Paediat. Univ. Tokyo 18: 36-40.
(8) HIRATSUKA, N., K. SHIMODA and Y. OGAWA 1971. New clearing procedure of cellulose acetate strips after electrophoresis. (in Japanese). The Physico Chemical
Biology. 15: 276-277.
( 9) HOSHINO, T., H. MORISHIMA and H. I. OKA 1971. Evaluation of an electronic
instrument for estimating plant weight. (In Japanese). Proc. Crop Science Soc.
Japan 40: 545-556.
(10) IINO, T. and M. ENOMOTO 1971. Motility. Methods in Microbiology. 5 A (ed.
J. R. NORRIS and D. W. RIBBONS). 145-153. Academic Press (London).
(11) KATO, H. and T. H. YOSIDA 1971. Uptake of isolated chromosomes by mammaliancells and protective effect of protamine sulfate. Exp. Cell Res. 65: 454-462.
(12) KATo, H. and T.H. YOSIDA 1971. Isolation ofaneusomicclones from Chinese hamster
cell line following induction of nondisjunction. Cytogenetics 10: 392-403.
(13) KIMATA, M. and S. SAKAMOTO 1971. Callus induction and organ redifferentiationof Triticum, Aegilops and Agropyron by anther culture (in Japanese). Jap. J.
Palynology 8: 1-7.
(14) KIMURA, M. 1971. Theoretical foundation of population genetics at the molecularlevel. Theoretical Population Biology 2: 174-208.
(15) KIMURA, M. 1971. Population Genetics and Human Genetics, With Special
Reference to Variation and Evolution at the Molecular Level (Japanese with English
summary). Jap. J. Human Genet. 16(1): 1-14.
(16) KIMURA, M. and T. OHTA 1971. Theoretical Aspects of Population Genetics.
ix+219 Princeton Univ. Press (Princeton).

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22
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KIMURA, M. and T. OHTA 1971. Protein polymorphism as a phase of molecular
evolution. Nature 229: 467-469.
KIMURA, M. and T. OHTA 1971. On the rate of molecular evolution. J. Molecular
Evolution 1: 1-17.
KIMURA, M. and T. MARUYAMA 1971. Pattern of neutral polymorphism in a geographically structured population. Genet. Res. Camb. 18: 125-131.
KOSUDA, K. and D. MORIWAKI 1971. Increase of genetic variability throughrecombination in Drosophila melanogaster. Genetics 67: 287-304.
KURODA, Y. 1971. Effects of X-irradiation on tissue formative activity and sortingout activity of HeLa cells in rotation culture. Radiation Res. 48: 565-577.
KURODA, Y. 1971. Changes in tissue-reconstituting activity of cells in carcinogenesis.
(in Japanese). Proc. Symp. 29th Japan Cancer Assoc. 34-41.
KURODA, Y. 1971. Methods in medical chemistry by using tissue culture techniques(in Japanese). Metabolism and Enzyme I, Methods in Medical Chemistry 2A(ed. H. Wada) 351-378 Nakayama-shoten (Tokyo).
KURODA, Y. 1971. Sorting-out mechanism of animal cells in culture. (in Japanese).
The Cell (Tokyo) 3(5): 9-21.
KURODA, Y. 1971. Aging in proliferating cell system. (in Japanese). The Heredity(Tokyo). 25(11): 40-47.
KURODA, Y. 1971. Studies on the sorting-out mechanism in tissue reconstitutionof animal cells in vitro. (in Japanese). Jap. J. Develop. BioI., 25: 31-32.
MARUYAMA, T. 1971. Speed of gene substitution in a geographically structured
population. Amer. Nat. 105: 253-265.
MARUYAMA, T. 1971. Analysis of population structure II. Two-dimensional
stepping stone models of finite length and other geographically structured populations. Ann. Hum. Genet., Lond. 35: 179-196.
MARUYAMA, T. 1971. The rate of decrease of heterozygosity in a population
occuping a circular or a linear habitat. Genetics 67: 437-454.
MARUYAMA, T. 1971. An invariant property of a structured population. Genet.
Res., Camb. 18: 81-84.
MARUYAMA, T. 1971. A stochastic problem in population genetics (in Japanese).
Proceedings of Tokei-Suri Kenkyusho Symposium no. 3, part 2 Markov processes,
116-125.
MARUYAMA, T. and M. KIMURA 1971. Some methods for treating continuous
stochastic processes in population genetics. Jap. J. Genet. 46: 407-410.
MATSUNAGA, E. 1971. Future of world population from the ecological viewpoint.
(in Japanese). Jyoshi-Dai-Tsushin (News from the Japan Women's Univ.) 271:
9-24.
MATSUNAGA, E. 1971. A testimony in the Thalidomide trial. in ThalidomideTestimonies of Savants. (in Japanese) 85-124 Tokyo Univ. Press.
MATSUNAGA, E. 1971. Parental ages and brith weight in translocation Down's
syndrome. Excerpta Medica Intern. Congress Series No. 233, 4th Intern. Congo
Human Genet. 119.

BOOKS AND PAPERS PUBLISHED IN 1971
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121

MATSUSHIMA, T. and T.H. YOSIDA 1971. Change of stemline karyotypes in Yoshidasarcoma by appearance of peculiar marker chromosomes (Chromosomal alterationand development of tumors, XXII). Gann 62: 339-394.
MORIWAKI, K., H. T. IMAI, S. YAMASHITA and T. H. YOSIDA 1971. Ploidy fluctuations of mouse plasma cell neoplasm MSPC-l during serial transplatation. J. Nat.
Cancer Inst. 47: 623-637.
MUKAI, T. and T. MARUYAMA 1971. The genetic structure of natural populations ofDrosoph./a me/anogaster IX: A prediction of genetic equilibrium. Genetics.
68: 105-125.
MUKAI, T. and T. YAMAZAKI 1971. The genetic structure of natural populations ofDrosophila me/anogaster. X. Developmental time and viability. Genetics. 69: 385398.
MURAKAMI, A. 1971. Comparison of the stage sensitivity to X-rays during meiosis
in the egg of the silkworm, Bombyx mori. Int. J. Radiat. BioI. 19: 167-176.
MURAKAMI, A. 1971. Relative biological effectiveness of 14 MeV neutrons for
inducing dominant lethals in mature sperm of the silkworm: A comparison of the
RBE for dominant lethals and specific-locus mutations. Jap. J. Genet. 46: 6774.
MURAKAMI, A. 1971. Radiation-induced recessive visible mutations of oocytes
during meiosis in the silkworm. Genetics. 67: 109-120.
MURAKAMI, A. 1971. Further studies on the radiation-induced recessive visible
mutations in oocytes during meiosis in the silkworm. Proc. Japan Acad. 47: 631634.
MURAKAMI, A. 1971. The relative biological effectiveness of neutrons in inducing
visible recessive mutations in the silkworm, Bombyx mori L.-A comparison of
RBE between recessive visible and lethal mutations induced by neutrons among
different organisms (in Japanese). Kyoto University Research Reactor InstituteTechnical Report (KURRI-TR) No. 87: 3Q-40.
MURAKAMI, A. 1971. Possibility of the use of reactor radiations to entomology(in Japanese). Report of Task Group for the Use of Atomic Piles in Medicine andBi%gy (Kyoto Univ. Research Reactor Institute, Osaka-fu). 66-72.
NAWA, S., B. SAKAGUCHI, M. YAMADA and M. TSUJITA 1971. Hereditary change
in Bombyx after treatment with DNA. Genetics. 67: 221-234.
NOGUTI, T., Y. SADAIE and T. KADA 1971. Radiosensitization with iodine compounds III. Macromolecular synthesis and repair in Bacillus subtilis irradiated in
the presence of iodoacetic acid, potassium iodide or potassium iodate. Int. J.
Radiat. BioI. 19: 305-322.
NAKAGOME, Y. 1971. Prenatal diagnosis and genetic counselling (in Japanese).
Handbook of Pediatrics (ed. M. Enjoji et a/.). 1-19 Nakayama Shoten (Tokyo).
NAKAGOME, Y. 1971. Prenatal diagnosis of genetic diseases (in Japanese). Iden
25(9): 61.
OGAWA, Y. 1971. The apparatus of electrophoretic analysis (in Japanese). Medical
Apparatus Culture 12: 55-64.

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22
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OGAWA, Y. and N. OOAKI 1971. Comparision of disc electrophoretic analyses ofhuman serum protein frations on various cellulose acetate strips. (in Japanese).
The Physico Chemical Biology 15: 185.
OGAWA, Y. and N. OOAKI 1971. Disc electrophoresis of the preserved humansera (in Japanese). Medicine and Biology 82: 77-80.
OGAWA, Y. and N. OOAKI 1971. The clearing procedure of cellulose acetate strips.
(in Japanese). Medicine and Biology 82: 127-130.
OHTA, T. 1971. Linkage disequilibrium and associative overdominance due torandom genetic drift. Jap. J. Genet. 46: 195-206.
OHTA, T. 1971. Associative overdominance caused by linked detrimental mutations. Genet. Res. Camb. 18: 277-286.
OHTA, T. 1971. Studies on molecular evolution from the standpoint of population
genetics. (in Japanese). Kagaku, 41: 642-650.
OHTA, T. and M. KIMURA 1971. Functional organization of genetic materials as a
product of molecular evolution. Nature 233: 118-119.
OHTA, T. and M. KIMURA 1971. Amino acid composition of proteins as a productof molecular evolution. Science 174: 150-153.
OHTA, T. and M. KIMURA 1971. On the constancy of the evolutionary rate ofcistrons. J. Molecular Evolution 1: 18-25.
OHTA, T. and M. KIMURA 1971. Linkage disequilibrium between two segregating
nucleotide sites under the steady flux of mutations in a finite population. Genetics
68: 571-580.
OHTA, T. and M. KIMURA 1971. Behavior of neutral mutants influenced by associated overdominant loci in finite populations. Genetics 69: 247-260.
OHTA, T. and M. KIMURA 1971. Genetic load due to mutations with very small
effects. Jap. J. Genet. 46: 393--401.
OKA, H. and H. MORISHIMA 1971. The dynamics of plant domestication: Cultivation experiments with Oryza perennis and its hybrid with O. sativa. Evolution.
25: 356-364.
PuTNAM, F. W., A. SHIMIZU, C. PAUL and T. SHINOOA 1971. Tentative structure ofhuman IgM immunoglobulin. Prog. Immunoll: 291: 307.
PUTNAM, F. W., A. SHIMIZU, C. PAUL, T. SHINOOA and KOHLER, H. 1971. The
amino acid sequence of human macroglobulins. Ann. N.Y. Acad. Sci., 190: 33103.
SAKAI, K. I. 1971. Genetic differentiation in a population of forest tree species.
SABRAO Newsletter 3(1): 71-72.
SAKAI, K. I. 1971. Domain and development of the breeding science. (in Japanese).
Recent Advances in Breeding Sciences No. 12: 25-33.
SAKAI, K. I., IWAGAMI, S. and S. MIKAMI 1971. Genetic variation in naturally
reproducing sapling-clusters in Thujopsis dolabrata. (in Japanese). J. Jap. For.
Soc. 53(8): 256-259.
SAKAI, K. I., MIYAZAKI, Y. and MATSUURA 1971. Genetic studies in natural populations of forest trees. I. Genetic variability on the enzymatic level in natural

BOOKS AND PAPERS PUBLISHED IN 1971

123

forests of Thujopsis dolabrata. Silvae Genetica 20(5-6): 168-173.
SAKAI, K. I. and Y. G. PARK 1971. Ibd. III. Genetic differentiation within a forestof Cryptomeriajaponica. Theor. and Appl. Genet. 41(1): 13-17.
(70) SHIMIZU, A. C. PAUL, T. SHINODA, H. KOHLER and F. W. PUTNAM 1971. Variationand homology in the Mu and Gamma chains of human immunoglobulins. Science
173: 629-633.
(71) SHIMIZU, N. and K. MIURA 1971. Studies on nucleic acids of living fossils. I. Isolation and characterization of DNA and some RNA components from the Brachiopod
Lingula. Biochim. Biophys. Acta. 232: 271-277.
(72) SHIMIZU, N. and K. MIURA 1971. Studies on nucleic acids of living fossils. II.
Transfer RNA from the Brachiopod Lingula. Biochim. Biophys. Acta. 232: 278288.
(73) SHINODA, T. and E. MATSUNAGA 1971. Studies on polymorphic types of several
red cell enzymes among Japanese. Excerpta Medica Intern. Congress Series No.
233, 4th Intern. Congo Human Genet. 164.
(74) SHIRAISHI, Y. and T. H. YOSIDA 1971. Differential staning of human chromosomesby treatment with urea. Proc. Japan Acad. 47: 729-731.
(75) TAZIMA, Y. 1971. Radiation mutagenesis. (in Japanese). Effect of Radiation(ed. Y. HIYAMA). Univ. Press, Tokyo pp. 293-311.
(76) TAZIMA, Y. 1971. Molecular mechanism of mutagenesis. (in Japanese). Insect
Biochemistry (Essays to Commemorate the Retirement of Professor R. AKUNE).
Kyushu Univ. Press, Fukuoka pp. 65-72.
(77) TAZIMA, Y. 1971. Problems of protection against the genetic effect of radiation.
Biological Aspects of Radiation Protection (eds. Sugahara and Hug). Igaku ShoinLtd., Tokyo pp. 5-15.
(78) TAZIMA, Y. 1971. On the relative biological effectiveness of neutrons to r-rays ininducing mutations in the mouse (a review, in Japanese). Kyoto University Research
Reactor Institute-Technical Report (KURRI-TR) No. 87: 22-29.
(79) TAZIMA, Y. 1971. Problems in the evaluation of medical substances from the view
point of mutation induction (in Japanese). Gendai No Rinsho (Tokyo) vol. 5:
320-325.
(80) TSUCHIYA, K. 1971. Chromosomes of two insectivorous bat species from Japan(CHIROPETRA). J. Mammalogical Soc. Japan 5: 114-116.
(81) TSUJITA, M. and S. SAKURAI 1971. The mecanism of lethality of al/al silkworm
larvae (in Japanese). Symposia Cell BioI. 22: 19-26.
(82) WADA, A., I. KAWATA and K. MIURA 1971. Flow-dichroic spectra of doublestranded RNA. Biopolymers 10: 1153-1157.
(83) YAMAGUCHI, O. and D. MORIWAKII971. Chromosomal variation in natural populations of Drosophila bifasciata. Jap. J. Genet. 46: 383-391.
(84) YAMAZAKI, T. 1971. Measurement of fitness at the esterase-5 locus in Drosophila
pseudoobscura Genetics. 67: 579-603.
(85) YOSIDA, T. H., K. TSUCHIYA and K. MORIWAKI 1971. Frequency of chromosome
polymorphism in Rattus rattus collected in Japan. Chromosoma (Berl.) 33: 30-40.
(69).

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22(86) YOSIDA, T. H., K. TSUCHIYA and K. MORIWAKI 1971. Karyotype differences of
black rat, Rattus rattus, collected in various localities of East and Southeast Asiaand Oceania. Chromosoma (Berl.) 33: 252-267.
(87) YOSIDA, T. H., H. KATO, K. TSUCHIYA and K. MORIWAKI 1971. Karyotypes andserum transferrin patterns of hybrids between Asian and Oceanian black rats,
Rattus rattus. Chromosoma (Berl.) 34: 40-50.

DIARY FOR 1971

ABSTRACTS OF DIARY FOR 1971
January
March
April
May
June

July
September
October

December

20
21
5
22
7
4
25
25
16
23
13
15
28
17

186th Meeting of Misima Geneticists' Club
94th Biological Symposium
187th Meeting of Misima Geneticists' Club
95th Biological Symposium
188th Meeting of Misima Geneticists' Club
189th Meeting of Misima Geneticists' Club
96th Biological Symposium
190th Meeting of Misima Geneticists' Club
191st Meeting of Misima Geneticists' Club
192nd Meeting of Misima Geneticists' Club
193rd Meeting of Misima Geneticists' Club
97th Biological Symposium
98th Biological Symposium
194th Meeting of Misima Geneticists' Club125

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

FOREIGN VISITORS IN 1971
January

20-21

February

8

July
July
October10134October

28

OctoberNovember
December

30
20
7

LEIGHTON, J., Univ. of Pittsburgh, School of Medicine,
U.S.A.
BIRNBAUM, H. National Science Foundation, Tokyo
Office, U.S.A.
KRISHNASWAMI, S., Indian Sericulture Institute, India
GRAHAM, K. M., Univ. of Malaya, Malaysia
COOK, R. C. National Parks and Conservation Association, U.S.A.
CANTAVIEJA, M. H., Agricultural Supervisor, The
Philippines
WALLACE, J., Hy-Line Poultry Comp., U.S.A.
DEVARAJURS, D., Central Silk Board, India
DUNHAM, C. L., NAS-NRC, U.S.A.
EDINGTON, C. W., USAEC, U.S.A.
NEEL, J. V., Univ. of Michigan, U.S.A.

ACKNOWLEDGEMENT127

ACKNOWLEDGEMENT,

The editor wishes to express his sincere gratitude to Dr. F. A. LILIENFELD
for her kindness in reading the original manuscripts. (K. MIURA)128

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 22

AUTHOR INDEXAMANO, E. . .............•.•...•. 73, 75

NAKAGOME, Y . • . . . . . . . . . . 96, 97, 98, 99

J

CHOO, J. K

NOGUTI, T ••..•••..••......•••••.•••. 68~1

90, 102, 104, 105, 107
CHU, Y. E
116
ENDO, T
24,25
ENOMOTO, M . • . . . . . . . • . . . . . . . . . . . . . . 19
FUJII, T . . . . . . . . • . . . . . . . . . . . . . . . . 76, 77
FUJISHIMA, T . . . . • . . . . . . . . . . . . . . 113, 114
FUKASE, Y
56, 57FUKUI, K. .
117
FURmcm, Y . • . . . . . . . . . . . . . . . . . . . . . . 11
HAYATA, I.
21
HARADA, M. . ..•.................... 50
IINO, T. • .......•........ 13, 14, 15, 18
IINUMA, K.
96, 98, 99
IMAI, H. T. •.48
IWAGAMI, S • • . . . . . . . . . . • . . . . . . . . . . . 109
IYAMA, S •......•.....•............. 110
KADA, T. •68, 70, 72
KATO, H • . . . . • . . . . . . . . . . .40, 41, 42, 43
KAWAHARA, T . . . . . • . . . . . . . . . . . 111, 112
KiMATA, M • . . . . . . • . . . . . • . . . . . . . . . . . 55
KiMURA, M. . ..... 79, 80, 81, 82, 83, 92
KUBOTA, M .........•.......•••••... .48
KURODA, Y • . . . . . . . . . . 27, 28, 29, 30, 31
KUROIWA, T. • ..............•....... 15
MARUYAMA, T . • . . . . . . . . . . 82, 83, 85, 86
MATSUNAGA, E •.•........... 95, 98, 100
MIKAMI, S. . .......•.....•••....... 109
MINATO, K •............•............ 32
MIURA, K
11, 12MORISHIMA, H
115, 116MORIWAKI, K. • .•• 21, 22, 41, 52, 53, 93
MURAKAMI, A •••••••••••. 61, 62, 63, 64

79,80,81,92

OHTA, TOINUMA, T.

• ...•.....•.....•...•••. 66

OISHI, K •...•...................•••. 23
OGUCHI, T •••.•.....•••......••.•.•• 13
OKA, H. I. • ...•..............• 115, 116
ONIMARU, K.

• ..•...•.•••...•..••••• 59

OsmMA, C ••....•. 90,

102, 104, 105, 107

PAl, C .•........••.•....•....•••••.• 24
PARK, Y. G ••..........•.......•... 110
PAUL, C ••......••..•.....••.•...... 21

21
21, 22, 52, 53, 93
SADAIE, Y ••....••...•.•.•...•..• 70, 72
SAGAI, T
35, 37, 38
SAKAI, K. I.
109, 110
SAKAMOTO, S. ..
55, 117
SmMlzu, A ........•...•.•....... , ••. 21
SHIMOTOHNO, K. . .........••.•..•... 12
SHINODA, T ••..•.....•.......••• 21, 101
SHiRAIsm, Y •..•.•...•.......... .40, 44
STOCKER, B. A. D
19
SUZUKI, H ••...•.....•...•....... 14, 17
T AKAHAsm, K. • .....•..........• 34, 35
TAZIMA, Y
56, 57, 59, 66
TSUCHIYA, K.
22, 50, 51
TONOMURA, A
100
Wu, W.C
18
YAMAGUCHI, S •............•••..• 14, 15
YAMAZAKI, T ••....•.•........... 86, 88
PUTNAM, F. W

SADAIE, T

YOSIDA, T. H.

. . .. 34, 35, 37, 38, 40, 42, 44, 46, 50, 51

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