NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 24

1973

PubU.hed bg

THE NATIONAL INSTITUTE OF GENETICS
Mi.ima. Sizuoka·ken, Japan1974

Annual Report:of theNational Institute of Genetics
No. 24, 1973

Photo by Y. Nakagome

Published by

The National Institute of Genetics, Japan
1974

CONTENTS
General statement
Staff................................................................
Council..............................................................
Association for propagation of the knowledge of genetics
Projects of research for 1973
" ....
Researches carried out in 1973

1
3
6
6
7
11

I. Molecular Genetics
The 5' -terminal nucleotide sequences of the double-stranded RNAof human reovirus. MIURA, K., WATANABE, K. and SUGIURA
M
Identification of the abnormal nucleotide located at the 5' -terminusof the genome RNA of cytoplasmic polyhedrosis virus. WATANABE, K., FURUICHI, Y. and MIURA, K.
The 3/-terminal structure of the genome RNA segments of ricedwarf virus. FURUICHI, Y. and MIURA, K.
Methylation related to the initiation of transcription of cytoplasmic
polyhedrosis virus containing double-stranded RNA. FURUICHI,
Y., SHIMOTOHNO, K. and MIURA, K.

II.

11

11
12

13

Microbial Genetics
Studies on control of flagellin mRNA synthesis with fia-ts mutantsof E. coli. SUZUKI, H., ENOMOTO, M. and HIROTA, Y.
14
Expression of Salmonella H2 genes in Escherichia coli K12. ENOMOTO, M..................................................... 15
Isolation of an Hi-repressor (rhi) mutant from the Salmonella-E.
coli hybrid. ENOMOTO M.
16

III.

Biochemical Genetics
The genetic control of tryptophan pyrrolase in Ephestia.
and YAMADA, M.
Structural study of Fc disease protein. SHINODA, T.

NAWA, S.
17
18

VI. Developmental Genetics
In vitro cultivation of developmentally defect cells from deeporange embryos in Drosophila melanogaster. KURODA, Y... 19
Inhibitory effect of cycloheximide on enhanced aggregation of em20
bryonic quail liver cells in dissociation medium. KURODA, Y...

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Mutagenesis to 8-azaguanine resistance in embryonic human diploidcells treated with EMS. KURODA, Y......................... 20
Induction of drug-resistant mutations by furyfuramide in embryonic21
human diploid cells. KURODA, Y.
Studies on serum factors supporting growth of mammlian cells in
culture. MINATO, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 22

V. Cytogenetics
Oceanian type black rats (Rattus rattus) with a subtelocentric M2chromosome obtained from Davis colony. YOSIDA, T. H.
MORIWAKI, K. and SAGAI, T.
Black rat (Rattus rattus) with a single X-chromosome. YOSIDA,
T. H., MORIWAKI, K. and SAGAI, Too.........................
Polymorphism of C-bands of chromosomes in black rats, Rattus
rattus. YOSIDA, T. H. and SAGAI, Too.......................
Induction of sister chromatid exchanges by chemical mutagens andits possible relevance to DNA repair. KATO, H.
Constitutive heterochromatin of Indian muntjac chromosomes revealed by DNase treatment and a C-banding technique. KATO,
H., TSUCHIYA, K. and YOSIDA, T. H.
The seventh cell population change accompanied by a marker
chromosome E in the transplantable mouse myeloma MSPC-l.
MORIWAKI, K.
Synaptonemal complex and male crossing-over in Drosophila
ananassae (abstract). MORIWWAKI, D. and TSUJITA, M. ....
Ecological genetic studies on the pollen germination of Chrysanthemum species. N AGAMI, Soo........... . . . . . . . . . . . . . . . . . . ..

23
23
24
25

26

26
27
28

VI. Mutation and Mutagenesis in Animals
Some aspects of overdominance observed in experiments on radiosensititvity in the silkworm. TAZIMA, Y. . . . . . . . . . . . . . . . . . . . .
Results of mutagenicity testing for some nitrofuran derivatives byapplying a sensitive test system with silkworm oocytes. TAZIMA,Y. and ONIMARU, K.
Production of mosaic eggs at very high frequency with BUdR
treatment in the silkworm. TAZIMA, Y. and ONIMARU, K.....
Analysis of a chromosome-specific genetic instability in the silkworm.
MURAKAMI, A.
Stage sensitivity and dose-response of X-ray-induced recominations
in oogenesis of the silkworm. MURAKAMI, A. . . . . . . . . . . . . . . ..

30

31
32
32
34

CONTENTS

iii

Ultraviolet-light sensitivity of an X-ray sensitive silkworm mutant
rb. MURAKAMI, A. and TAZIMA, Y........................... 35
Induction of recombinants by mitomycin C in oogenesis of thesilkworm. MURAKAMI, A. and ARIGA, 1.
36
Radio-sensitization of cultured mammalian cells by 5-iodouridine.
KURODA, Y., YOKOIYAMA, A. and KADA, T................... 37

VII. Radiation Genetics and Chemical Mutagenesis in Microorganisms and Plants
An early step enzyme participating in the excission type repair of
r-ray damaged DNA in Bacillus subtilis. NOGUTI, T. and
KADA, T
Metabolic activation and Escherichia coli mutagenesis of furylfuramide, a nitrofuran food additive. KADA, T...............
Host-mediated assay of mutagenicity of furylfuramide. TUTIKAWA,
K. and KADA, T.............................................
DNA-damaging products by reaction between sodium nitrite andsorbic acid. KADA, T.
Dose rate effect of gamma-rays in maize pollen. FUJII, T.
Effects of gamma-rays on sex expression in gynoecious cucumber
FUJII, T.
Germination of Podostemaceae seeds. AMANO, E.................
Callus culture of Haplopappus gracilis. AMANO, E.............
Experiments of the grafting of Capsicum mutants. AMANO, E...
Relationship between damage caused by ageing and by irradiationin rice seeds. BAGCHI, S.....................................

38
39
41
43
44
45
46
47
48
50

VIII. Population Genetics (Theoretical)
The age of a neutral mutant persisting in a finite population.
KIMURA, M. and OHTA, T.
51
A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a finite population. OHTA, T.
51
and KIMURA, M.
Effect of linkage on the behavior of mutant genes in finite populations. OHTA, T.
52
IX. Population Genetics (Experimental)
Analysis of heterozygosity in regard to the neutral theory of protein
polymorphism. MARUYAMA, T. and YAMAZAKI, T............. 53
Genes affecting productivity in natural populations of Drosophila

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

melanogaster. WATANABE, T. K.. . . . . . . . . . . . .. .. . . . . . . . . . . . . 54
Genetic changes in natural populations of Drosophila melanogaster.
WATANABE, T.K., WATANABE, T. and OSHIMA, C
56

X. Evolutionary Genetics
Mutation and evolution at the molecular level. KIMURA, M. and
OHTA, T
58
Eukaryote-prokaryote divergence estimated by 5S ribosomal RNA
sequences. KIMURA, M. and OHTA, T.
58
Slightly deleterious mutant substitutions in evolution. OHTA, T.. . 59

XI. Human Genetics
Fingerprints in Ainu. MATSUNAGA, E. and IKEDA, E.............
Sequence analysis of human IgA immunoglobulin. SHINODA, T. ..
Isozyme variations in man. SHINODA, T., MATSUNAGA, E. and
KOSHINAGA, J.
Amplicon model for antibody variability. SHINODA, T.
Frequencies of C and Q variants in human chromosomes. IINUMA
K. and MATSUNAGA, E.......................................

XII.

60
60
61
62
64

Behavioral Genetics

Short-term and long-term memories in different strains of inbred
mice. FUJISHIMA, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

XIII. Applied Genetics
Analysis of genes controlling F 1 sterility in rice by the use of
isogenic lines. OKA, H. 1.. . . . . . . . . . . . . . . . . . .. .. . . .. . . . . . . ....
Breeding of isogenic lines of rice carrying various gene markers
and reciprocal translocations. OKA, H. 1. and MORISHIMA, H...
Character variation and inheritance of sterility in the backcrossprogenies of a species hybrid in rice. OKA, H. 1. and MORISHIMA,H
Phenotypic plasticity and yield stability in rice cultivars. MORISHIMA, H. and OKA, H. 1.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Intervariental variations in growth pattern obtained from the international rice adaption experiment. MORISHIMA, H. and OKA,
H.1.
Variation in weediness among strains of dallisgrass. MORISHIMA,
H
Genic analysis for peroxidase isozymes and their organ specificity

66
66

67
67

68
68

CONTENTSv

in Oryza perennis and O. satiua. PAl, C., ENDO, T. and
OKA, H.1.
69
Intergenotypic competition and cooperation in mix-planting of ricestrains. HOSHINO, T. and MORISHIMA, H..................... 69
Variations in isozymes and other characters among Fa lines derived
from Oryza perennis X O. sativa hybrids. PAl, C. and OKA,
H.1.
70
Further investigation of geographical variation in isoperoxidase in70
populations of Pinus Thunbergii. IYAMA, S.
Gamma-ray induced increase in intra-line variability in Arabidopsis
thaliana. BAGCHI, S. and IyAMA, S. . . . . . . . . . . . . . . . . . . . . . . . . 71
Genetic changes of Japanese wild quails under domestic conditions.
KAWAHARA, T., MITA, A., SAITO, M. and SUGIMOTO, N....... 72
Interstrain variation in gamma-ray induction of hindlimb deformities
in chick embryos. KAWAHARA, T.
72
Serum amylase isozymes in the Japanese quail. KAWAHARA, T.,
WATANABE, S. and YOSHIDA, H.
73
Books and papers published in 1973 by research members
74
Abstracts of diary for 1973 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Foreign visitors in 1973............................. . . . . . .. . . . . . . . . .. 81
Acknowledgement.................................................... 82
Author index........................................................ 83•

GENERAL STATEMENT
In August, the XIII th International Congress of Genetics was held at
Berkeley, California. From this Institute thirteen members attended theCongress: Among them, Dr. Y. Tazima participated in it on behalf of theInternational Genetics Federation as its president; and Dr. M. Kimura andDr. H.1. Oka were invited to the symposia as speakers. In addition, about
ten members of the Institute went abroad to attend other international
conferences, to give lectures, and so on.
We also had many visitors from other countries this year. In April agroup from the U. S. S. R. Academy of Sciences (ten members headed by
Dr. M. A. Markov) visited our institute. Consequent to the establishmentof friendly relations between Japan and the People's Republic of China,
several Chinese scientists visited Japan. From September to November we
received three groups of them: 1) A representative team of botanicalworkers of Academia Sinica (seven members led by Dr. Chen Tsui), 2) A
sericultural mission from the Chinese Association of Agricultural Science
Society (four members led by Dr. Yi-Ling Kao); and 3) A representative
team of Chinese biologists (ten members led by Dr. Ti-Cho Tung). When
we are given the opportunity to welcome scientists from other countries,
we ask them to speak at our "Biological Symposium," which met for the
110 th time at the end of this year. It was started soon after the establishment of the Institute.
Dr. M. Kimura, the head of the Department of Population Genetics, waselected a foreign associate of the National Academy of Sciences of theUnited States of America on the 24th of April. As a Japanese geneticist
Dr. Kimura is the second who has won the honor, following Dr. H. Kihara,
the former director of this Institute.
After Dr. T. lino was appointed professor of the University of Tokyo,
the post of the head of the Department of Microbial Genetics was filled
August 1st by Dr. Yukinori Hirota who was the chief of the research unitof cell division, Division of Molecular Biology, Pasteur Institute.
Dr. Kan-ichi Sakai, the head of the Department of Applied Genetics
retired under the age limit, and has transferred to Kagoshima University
as professor of the Faculty of Agriculture. Dr. Sakai has rendered great
services for 24 years to the Institute since he joined in 1949, the year of
its establishment. In the scientific field he carried forward his original
studies on interspecific competition in plants and genetic variation in forest
trees. He has always played a leading part in this field. Dr. Hiko-ichi

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Oka, the head of the 3rd Laboratory of the same department succeeds
him.
In April Mr. Shin-ichi Tezuka replaced Mr. M. Kudo, the head of the
Department of Administration, who was transferred to the Tenth National
Youth Home as its director.

3

STAFF
Director

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

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

The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
MURAKAMI, Akio, D. Ag., D. Sc.
ONIMARU, Kimiharu

The 2nd Laboratory
KURODA, Yukiaki, D. Sc., Head of the Laboratory
MINATO, Kiyoshi, M. Sc.

2. Department of Cytogenetics
YOSIDA, Tosihide H., D. Sc., Head of the Department

The 1st Laboratory
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
KATO, Hatao, D. Sc.
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.
CHOO,* Jong-Kil, M. S.

The 2nd Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
NAGAMI,* Shuzo, D. Ag.

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

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

4. Department of Biochemical Genetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory
YAMADA, Masa-Aki, M. Sc.
The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
ENDO, Toru, D. Ag.
The 3rd Laboratory
SUGIYAMA, Tsutomu, Ph. D., Head of the Laboratory
FUJISAWA, Toshitaka, Ph. D.
5. Department of Applied Genetics
OKA, Hiko-Ichi, D. Ag., Head of the Department
The 1st Laboratory
OKA, Hiko-Ichi, D. Ag., Head of the Laboratory
KAWAHARA, Takatada, D. Ag.
FUJISHIMA, Tohru, D. Ag.
The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
BAGCHI,* Sujit
The 3rd Laboratory
OKA, Hiko-Ichi, D. Ag., Head of the Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag.
PAI,* Chiang
6. Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department
The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
NOGUTI, Takehiko, D. Sc.
The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
FUJII, Taro, D. Ag.
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
AMANO, Etsuo, D. Ag.
SADAIE, Yoshito, M. Sc.

STAFF
7. Department of Human Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department

The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
SHINODA, Tomotaka, D. Sc.

The 2nd Laboratory
NAKAGOME, Yasuo, D. Med., Head of the Laboratory
IINUMA, Kazuso, B. M.

8. Department of Microbial Genetics
HIROTA, Yukinori, D. Sc., Head of the Department

The 1st Laboratory
HIROTA, Yukinori, D. Sc., Head of the Laboratory
SUZUKI, Hideho, D. Sc.

The 2nd Laboratory
ENOMOTO, Masatoshi, D. Sc., Head of the Laboratory
NISHIMURA, Yukinobu, D. Sc.

9. Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department

The 1st Laboratory
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
OHTA, Tomoko, Ph. D., D. Sc.

The 2nd Laboratory
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Laboratory
YAMAZAKI, Tsuneyuki, Ph. D.

10. Department of Molecular Genetics
MIURA, Kin-ichiro, D. Sc., Head of the Department
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
FURUICHI, Yasuhiro, D. Med.
SHIMOTOHNO, Kunitada, D. Med.

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

12. Department of AdministrationTEZUKA, Asaichi, Head of the Department
YUHARA, Tokusaburo, Chief of the General Affairs Section
FUKUI, Teijiro, Chief of the Finance Section

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24
Honorary Members

KIHARA, Hitoshi, D. Sc., Director of the Kihara Institute for BiologicalResearch, 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.
TSUJITA, Mitsuo, D. Ag.

COUNCILKIKKAWA, Hideo, Chairman, Professor of Hyogo College of Medicine
FUJII, Takashi, Vice Chairman, Emeritus Professor of University of Tokyo
EGAWA, Tomoji, Director of National Institute of Agricultural Sciences
IKEDA, Yonosuke, Director of Institute of Applied Microbiology, Universityof Tokyo
IZEKI, Shoei, Director of National Research Institute of Police Science
KAYA, Seiji, Emeritus Professor of University of Tokyo
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
MAKINO, Sajiro, Emeritus Professor of Hokkaido University
MISONOO, Keisuke, Director of National Institute of Radiological Sciences
OeHI, Yuichi, President of Azabu University of Veterinary Science
SAKAI, Kan-Ichi, Professor of Kagoshima University
SAKATA, Takeo, President of T. Sakata Company
TAKEYAMA, Yutaro, Governor of Shizuoka Prefecture
TANAKA, Nobunori, Professor of Teikyo University
UEDA, Masao, Director of Institute of Population Problems
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

7

PROJECTS OF RESEARCH FOR 1973
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)
Genetics of somatic mammalian cells in culture (KURODA and MINATO)
Department of Cytogenetics

Studies on chromosomal evolution in rodents (YOSIDA, KATO, TSUCHIYA andSAGAI)
Chromosome study on experimental tumors (YOSIDA)
Cytogenetical study on monosomic and trisomic cultured mammalian cells(KATO)
Cytogenetical and immunological studies on mouse plasma cell tumor(MORIWAKI)
Biochemical studies on serum transferrin variations in rodents (MORIWAKI,
and TSUCHIYA)
Experimental breeding and genetics of mice, rats and other wild rodents(YoSIDA, MORIWAKI, 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)
Analysis of inversion chromosome in natural populations of Drosophilamelanogaster (OSHIMA and WATANABE)
Behavior genetics of phototaxis, diurnal rhythmicity of adult emergenceand oviposition in D. melanogaster (OSHIMA and CHOO)
Studies on urbanization and noise environments for the habitat and development in Drosophila (OSHIMA and CHOO)
Ecological genetic studies on the differentiation of Chrysanthemum species(NAGAMI)8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24
Department of Biochemical Genetics

Studies on transformation in higher organisms (NAWA and YAMADA)
Genetical and biochemical studies of pteridine metabolisms in insects (NAWA)
Analysis of gene action on cell differentiation in higher organisms (NAWA
and YAMADA)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Genetical and biochemical studies of human serum proteins (OGAWA andODAKI)
Genetical and biochemical studies on Japanese middle size dog (OGAWA)
Genetics of isozymes in plants (ENDO)
Effects of exogenous DNA on plant seed formation (ENDo)
Genetics of fresh water hydra (SUGIYAMA and FUJISAWA)

Department of Applied Genetics

Quantitative genetic studies in poultry (KAWAHARA and FUJISHIMA)
Genetic studies in wild populations of Japanese quails (KAWAHARA)
Theoretical studies on breeding techniques (IYAMA)
Behavioral genetic studies in mice (FUJISHIMA)
Studies on competition in plants (IYAMA)
Genetic studies in natural stands of forest tree species (SAKAI, IYAMA and
KUDO)
Simulation studies on artificial selection (IYAMA)
Evolutionary studies in wild and cultivated rice species (OKA and MORISHIMA)
Analysis of genetic variations in growth pattern and phenotypic plasticity
in rice (MORISHIMA and OKA)
Ecological genetic studies in some grass species (MORISHIMA)
Genetic analysis of isozymes in rice plants (PAl and ENDO)

Department of Induced Mutation

Molecular mechanisms of spontaneous, chemical- and radiation-induced
mutations (KADA, SADAIE and NOGUTI)
Environmental mutagens and carcinogens (KADA, TUTIKAWA, SADAIE andNOGUTI)
Radiation genetics in mice (TUTIKAWA)
Biochemical factors involved in cellular repair of genetic damage (NOGUTIPROJECTS OF RESEARCH FOR 1973

9

and KADA)
Mechanisms of recombination repair (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)
Genetic fine structure analysis in maize (AMANO)
Department of Human Genetics

Genetic effects of family planning and population planning (MATSUNAGA)
Cytogenetics in man (NAKAGOME and IINUMA)
Molecular hybridization studies of human chromosomes (NAKAGOME)
Studies on human chromosome variants (IINUMA, MATSUNAGA and NAKAGOME)
Structural studies on human immunoglobulins (SHINODA)
Prenatal detection of genetic disorders (NAKAGOME, MATSUNAGA and IINUMA)
Biochemical genetics in man (SHINODA and MATSUNAGA)
Department of Microbial Genetics

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

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

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NQ. 24
Department of Molecular Genetics

Studies on the chemical structure of genome of viruses containing doublestranded RNA (MIURA, SUGIURA, FURUICHI, SHIMOTOHNO, WATANABE,
YAZAKI, MUROFUSHI and KIMURA)
RNA polymerase in a virion containing double-stranded RNA (MIURA,SHIMOTOHNO, FURUICHI and HORI)

RESEARCHES CARRIED OUT IN 1973
I.

MOLECULAR GENETICSThe 5' -Terminal Nucleotide Sequences of the
Double-Stranded RNA of Human Reovirus
Kin-ichiro MIURA. Kumiko WATANABE and Masahiro SUGIURA

The 5' -terminal nucleotide sequences of human ,.reovirus double-stranded
RNA were determined after labeling the RNA with (S2P] phosphate bypolynucleotide kinase. Prior to labeling, it was necessary to perform
sequential oxidation, j3-elimination, and phosphomonoesterase treatment.
This operation seems to remove not only a few nucleotides from the 3'
end but also some material attached to the 5' -terminal nucleotide. The
5/-s2 P-Iabeled RNA was hydrolyzed by alkali or enzymes, and the resultant
mono- and oligo- nucleotides were analyzed chromatographically. The
labeled RNA was separated into the ten constituent genome segments bygel electrophoresis. and the 5' termini of each segment were also analyzed
after alkaline digestion. Each genome segment contained the same two
5' -terminal sequences: GpApUp in one chain and G*pCp in the other. G*p
is a derivative of guanylic acid which renders the 5' -terminal dinucleotide
resistant to hydrolysis by alkali. The results indicate that the transcriptionof reovirus double-stranded RNA starts from the 3' end complementary to
the G*pCp-terminal resulting in the synthesis of single-stranded mRNA
carrying the same 5' sequence as the G*pCp-chain. The presence of amodified nucleotide at the 5' terminus of the strand complementary of themRNA template is a feature common to another double-stranded RNA
virus, cytoplasmic polyhedrosis virus.

Identification of the Abnormal Nucleotide Located at the
5' -Terminus of the Genome RNA of Cytoplasmic
Polyhedrosis Virus. Kumiko WATANABE, Yasuhiro FURUICHI and Kin-ichiro MIURA

Ten segments of double-stranded RNA of the cytoplasmic polyhedrosis
virus (CPV) have the same terminal structure. The 5' -terminal nucleotide
in one strand of the double-stranded RNA is abnormal nucleotide, whichis resistant to alkaline and ribonuclease digestion. After the 5' -termini of

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

the RNA was labeled with [82P] phosphate with polynucleotide kinase, theterminal nucleotide was obtained as 82 pX by Penicillium nuclease, or byvenome phospho-diesterase after treatment with Aspergillus nuclease.
82pX seemed to be a derivative of adenylic acid from the behavior on
chromatography, and to be modified at the 2'-position of ribose moiety,since this nucleotide was resistant to alkali. As a candidate for thisabnormal nucleotide, 2'-O-methyladenosine-5'-phosphate (pAm) was synthesizedchemically, and it was cochromatographed with 82pX on a filter paper using
several kinds of solvent. The radioactive spot of S2pX on a radioautogram
was superimposed on the ultraviolet-absorbing spot of pAm in every solvent
system. Thus the abnormal nucleotide pX is identified as 2'-O-methyladenylic
acid. This is the first report showing the presence of an abnormal nucleotide in a viral RNA molecule.
On transcription of CPV, the same RNA chain as the strand carrying
2'-o-methyladenylic acid at the 5'-end is synthesized. Therefore, thisabnormal nucleoside could be a signal which specifies the starting point oftranscription.

The 3'-Terminal Structure of the Genome
RNA Segments of Rice Dwarf Virus
Yasuhiro FURUICHI and Kin-ichiro MIURA

Rice dwarf virus (RDV) contains twelve genome segments of doublestranded RNA. Total RNA's in RDV particles were extracted by pheonl
treatment and oxidized by periodate. The RNA was then reduced by [8H]
sodium borohydride to label the 3'-terminal nucleoside. On alkaline hydrolysis
the similar amounts of [8H] trialcohols of cytosine and uracil (C' and U')
were detected. The results of ribonuclease digestion of the [8H] labeled
RNA indicate that there are two kinds of the 3'-terminal sequence asfollows: - Py-G-A- U and -G-C-C.
Twelve segments of the 3'-terminal (8H] RDV-RNA were separated into
eight fractions by gel electrophoresis. Two labeled nucleoside-trialcohols,
C' and U', were detected equivalently for every fraction. Thus, it issuggested that every RNA segment of RDV has two kinds of 3',terminal
sequences, that is, one chain carries -Py-G-A-U at the 3'-terminus andthe other chain -G-C-C.

RESEARCHES CARRIED OUT IN 1973

13

Methylation Related to the Initiation of Transcriptionof Cytoplasmic Polyhedrosis Virus Containing
Double-stranded RNA
Yasuhiro FURUICHI, Kunitada SHIMOTOHNO and Kin-ichiro MIURA

Cytoplasmic polyhedrosis virus (CPV) contains a virion-associated transcriptase which, in vitro, can copy one of the two chains of the doublestranded RNA segments. It has been noted that the RNA-polymerising
activity of CPV preparation was relatively low in comparison with theactivity of RNA polymerases obtained from other sources. Since we foundthat the 5'-terminal nucleotide of one chain of double-stranded RNA of
CPV is methylated at the ribose moiety, we thought that methylation
may also take place in the in vivo transcription system and tried adding
a donor of methyl groups for nucleic acid into the reaction mixture forthe in vitro RNA synthesis of CPV. The addition of S-adenosyl-Lmethionine (SAM) to the reaction mixture enhanced RNA synthesis markedly. Synthesis of single-stranded mRNA of CPV proceeded dependingon the presence of SAM.
A methyl residue of SAM was incorporated into an RNA molecule. A
ribose moiety of adenylic acid in the 5'-terminal region of the nascent RNA
was methylated at a very early stage of the trancription. The dependenceof the viral transcription on the presence of SAM and the methylation ofterminal nucleotide suggests that the transcription of CPV is a "methylationcoupled" reaction.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

II.

MICROBIAL GENETICS

Studies on Control of Flagellin mRNA Synthesis
with fla-ts Mutants of E. coli
Hideho SUZUKI, Masatoshi ENOMOTO and Yukinori HIROTA

A number of mutants of E. coli K12, temperature sensitive with respect
to flagella formation, were isolated. They were capable of forming flagellaat 30 0 but not at 41 0 • The ts-characters were mapped in the fia loci.
The mutants could be classified into two categories, type I and type II, by
the mode of flagella generation on temperature shift during the exponential
growth. In the type I mutants a comparatively long lag was required
before acquisition of motility on temperature shift from 41 0 to 30 0 , andelongation of the flagellar filament stopped immediately upon the temperature shift from 30 0 to 41 0 ; while in the type II, flagella were generated
within a short period on shift from 41 0 to 30 0 and filament elongation
continued at least .for 60 min., when the culture was shifted back to 41 0 •m-RNA specific for flagellin in two representative strains belonging toeach type was assayed by flagellin synthesizing activity of the extracted
RNA in the in vitro system. Flagellin synthesizing activity was completely
absent in the RNAs derived from 41 0 cultures of both strains, whereas
the RNAs derived from 30 0 cultures supported synthesis of flagellin equally
well. When the cells grown at 30 0 were transferred to 41 0 , mRNA activity
for flagellin of the type I mutant diminished exponentially with a half-lifeof 7min., while that of the type II was maintained at the same level forat least 60 min. at 41 0 • Administration of 100 r rifampicin/ml at the timeof the temperature shift from 30 0 to 41 0 resulted in an exponential
diminution of flagellin mRNA activity at a half life of 7min. In the typeI mutant, flagellin mRNA synthesis stopped immediately upon temperature
shift to 41 0 • The inactivation of flagellin mRNA synthesis at the hightemperature appears to be irreversible because the type I cells grown at
30 0 and incubated for 70 min. at 41 0 revealed a long lag before regaining
ability to synthesize flagellin mRNA when shifted back to 30 0 • When type
II cells grown at 41 0 were fed with 100 r rifampicin/ml (known as a specificinhibitor for mRNA synthesis) at 10 min. after 30 0 shift, little mRNA
activity for flagellin appeared in the next 10 min. Thus, involvement of aprocessing step in producing active flagellin mRNA is unlikely, and theabsence of flagellin mRNA activity in the mutants at the temperature

RESEARCHES CARRIED OUT IN 197315

non-permissive for flagellation can be ascribed to the failure of flagellinmRNA synthesis, which could be controlled through the jla products.
Expression of Salmonella H2 Genesin Escherichia coli K12
Masatoshi ENOMOTO

PI, the general transducing phage of E. coli, is active on some roughderivatives of the restriction-negative strain of S. typhimurium (e. g.,
galE, rfaH, rfaG and galU) and can be used for transduction between
the two genera. S. typhimurium has two flagellin gene, H1 and H2,
which are separately located on the chromosome and alternately expressedwith a constant rate (phase variation); whereas E. coli has one flagellin
gene, hag, corresponding to Salmonella H1.
An attempt was made to introduce Salmonella H2 genes into E. coli
K12 by PI transduction, because an H2 region includes at least a phasedeterminant and an H1 repressor gene. A nonflagellate K12 recombinant,with H1-i ah1-transduced from S. typhimurium, was isolated and used
as recipient for H2 transduction. The three sorts of the Salmonella H2region, H2-1.2 vh2+, H2-e, n, x vh2+ and H2-e, n, x vh2-, were tried fortransduction into the K12 H1-i ahr derivative (EJ34). The eighteentransductants were obtained at a frequency 1 to 5 X 10- 9 jpfu and all proved
to have the phase-2 antigen of the donor. It is expected that Salmonella
H2 incorporated into the EJ34 chromosome will manifest variation between
active and inactive states, i. e. flagellate and nonflagellate phases. Alltransductants except two did not manifest such variation and their H2
was found to be incorporated into hag locus. The 1.2 and e, n, x gene athag were unable to repress the H1 allele and were susceptible to repression
by a product of the original H2 region. The two exceptional transductants,
EJ38 and EJ44, showed a high rate of variation between flagellate andnonflagellate phase. EJ38 with antigen e, n, x derived from the H2-e, n, x
vh2+ donor and EJ44 with e, n, x derived from the H2-e, n, x vh2- donor
were found to receive the vh2 allele along with H2-e, n, x from each donor.
The vh2- gene which causes fixation of flagellar expression in either phase-lor phase-2 in Salmonella was suppressed in an E. coli background. The
H2 locus of EJ38 and EJ44 was mapped by Hfr crosses at around 50
minutes, between lys and nalA, on the standard chromosome map of E.
coli. H2-e, n, x of EJ44 was cotransducible with tyrA or pheA at afrequency about 3%; whereas with EJ38, contransduction with these two

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

markers was not observed «0.3%). This indicates that the integrationsite of H2 differs between EJ38 and EJ44.
Isolation of an HI-repressor (rhI) Mutant
from a Salmonella-E. coli Hybrid
Masatoshi ENOMOTOA strain, EJ283, showing an extraordinarily high rate of 0- H variation(ca. 1.5xlO- 2 /cell/generation for change from phase-2, e, n, x to nonflagellate phase-I) was isolated in the course of PI transduction of a SalmonellaH2-e, n, x vh2- region into the KI2 Hl-i ahl- derivative (EJ34). EJ287,
made by transduction of Hl-i ahl+ allele into EJ283, was used for isolationof rhr, a mutant of an Hl repressor gene. It is expected that some cellsof the rhl mutant will have flagella with both antigens, phape-I i andphase-2 e, n, x, because the i antigen is always expressed and the e, n, x
antigen is intermittently expressed with a very high rate. When suchmutant cells are present in the parental culture composed by the cells with
antigen i and those with e, n, x, the mutant cells expressing both antigens
will agglutinate with the cells expressing either i or e, n, x by the additionof corresponding anti-flagella serum. The mutant cells will be concentrated
in the agglutinated cells by repeating the procedure with alternate use ofanti-i and anti-e, n, x serum.
The rhl mutant, EJ289, could be isolated as expected by this method.
The mutant did not form swarms on semisolid medium supplemented witheither anti-i or anti-e, n, x serum, though it did on unsupplemented medium.
When PI grown on EJ289 was applied to an Hl-i strain on semisolid
medium with anti-i serum no trails were evoked. This indicates that thetransduced H2-e, n, x region is unable to repress endogenotic Hl-i. Further
Hl-i and H2-e, n, x of EJ289 were separately transduced into the Hl-i
ahr strain.
Expression of the i antigen in the Hl-i aht+ transductants was repressedby the wild H2-e, n, x region. The H2-e,n, x transductants, when used as
donors in transduction to the Hl-i strain, had no ability to repress Hl-i
expression. EJ289 was confirmed to carry the rhl mutation closely linked
to H2-e, n, x and the wild Hl-i allele.

RESEARCHES CARRIED OUT IN 1973

III.

17

BIOCHEMICAL GENETICS

The Genetic Control of Tryptophan Pyrrolase in Ephestia
Saburo NAWA and Masa-Aki YAMADAAs reported previously, we obtained individuals with wild-type eyes inexperiments where the recessive mutant a had been treated with DNA
from the wild type. The mutant a suffers from the inability to convert
tryptophan to formylkynurenine. Although this conversion is catalyzed by
the enzyme tryptophan pyrrolase, it is not clear whether the mutant produces none of the enzyme or directs the production of altered polypetides.
Tryptophan pyrrolase is absent also in the mutant of the vermilion
cistron of Drosophila. It is known that the vermilion mutant produces analtered polypeptide which is in an inactive form in the presence of atyrosyl t-RNA and that RNase treatment of homogenates of the mutant
leads to the activation of the mutant enzyme. In the mutant a of
Ephestia, however, no appearance of tryptophan pyrrolase activity bytreatment with ribonuclease T 1 or pancreatic RNase was observed. On theother hand, the activity of tryptophan pyrrolase in the a+ /a heterozygote
was super-additive, being greater than half the tryptophan pyrrolase
activity in the a+/a+. Moreover, when a/a extract was mixed with a+/a+crude extract, the normal a+ enzyme level was enhanced by 20-40 percent.
The a-material responsible for the super-additivity could be purified froma/a extract by the same procedures as used for tryptophan pyrrolase ofwild type. The material was shown to be heat labile and precipitable withammonium sulfate (40-60% saturation), like a protein. The protein was
fractionated with Sephadex gel filtration and the degree of super-additivityof each fraction was examined. The material showing the enhancement of
tryptophan pyrrolase activity was fractionated in the same place as thatfor tryptophan pyrrolase of wild type. Thus, if the super-additivity resultsfrom a reaction of the a-protein with a+-protein, then the mutant enzyme
exists in the a/a extract, in a form with molecular weight indistinguishable
from the wild type. We propose that the mutant a directs the productionof altered polypeptides, suggesting that the a cistron is the structural genefor tryptophan pyrrolase.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Structural Study of Fe Disease Protein
Tomotaka SHINODAFc disease or heavy chain disease proteins are believed to be identicalto heavy chains of normal immunoglobulins, except that the former haveinternal deletions ranging in size from about 20 to 240 residues. These
smaller products seem to have resulted from abnormalities of the mechanismof gene expression rather than from in vivo degradation of the intact
products. Since the genetic mechanism of these phenomena is not yet wellunderstood and since studies of such proteins are considered to be of greatvalue in connection with the theories concerning the genetic control ofantibody synthesis, a sequence study has been carried out using an Fc
disease protein designating NIG 43 (rI' a+, x+, z-; genetic markers were kindly
determined by Dr. H. Matsumoto).
The protein was purified by a variety of methods and finally crystallized.
Four fragments, FI, FIl, FIll and FIV, were obtained from completely
reduced and alkylated protein by CNBr treatment. Of these FIl, FIll and
FIV were shown to be homogeneous upon amino acid and NH.-terminal
analyses, but FI had a heterogeneous NH 2 -terminal. Sequence analysis
revealed that FI had two NH 2 -terminals, serine and threonine, in nearly
equal amounts, suggesting that the intact Fc protein was a mixture of atleast two molecular species, one beginning with serine and the other withthreonine. FIV, a 17-residue fragment, had an identical sequence except
one with the COOH-terminal portion of the ri heavy chain; thus it wasconsidered that FIV occupied the COOH-terminal of the protein.
Although there have been several reports on sequences of gamma chains
including a partial sequence of heavy chain disease protein, no case has
been shown to have amino acid interchange in the COOH-terminal fragment.
The present case seems to be the first example which shows that aminoacid interchange (i.e. Ala ----> Gly) occurred in the COOH-terminal region.
Whether the interchange is associated with some serological type is notknown. The phenomenon is provisionally referred to as "Chemotype."

RESEARCHES CARRIED OUT IN 1973

IV.

19

DEVELOPMENTAL GENETICS

In Vitro Cultivation of Developmentally Defect Cells
from Deep Orange Embryos in DrosophilamelanogasterYukiaki KURODA
In the previous experiment on the cultivation of embryonic cells fromthe wild-type strain (Oregon-R) of D. melanogaster; muscle cells, epithelial
cells, fibroblastic cells, small cells and nerve cells were found to maintaintheir respective characteristics for a prolonged period of cultivation. (Fordetails, see Development, Growth and Differentiation 16: 55-66.)
In the present experiment cells from genetically lethal embryos, deeporange (1-0.3), were cultured to study when and what types of cells wereaffected by the lethal factors under the same conditions as those used for
cultivation of wild-type embryonic cells. Among 463 eggs collected frommatings of dor/dor females and dor/Y males, 4.1% died before gastrulation,
38.7% died by abnormal gastrulation, 44.0% died after the stage of sac-likemidgut, 13.2% died after segmentation of the body and none were foundto hatch.
When cells from deep orange embryos after gastrulation were cultured,
mesodermal cells such as fibroblastic cells and muscle cells were maintainedin an active state for a few weeks beyond the prospective lethal period of
the deep orange embryos. On the other hand, nerve cells, epithelial cells
and small cells (assumed as imaginal disc cells) scarecely grew. No extension
or poor extension of nerve fibers, and neither maturation nor differentiationof epithelial cells was found. This suggests that the lethal effects of the
dor gene may be manifested specifically in the ectodermal or endodermal
cells of embryos.
When deep orange embryonic cells were cultured in medium containing
an unfertilized wild-type egg extract, they were maintained in an active
state for a longer period, during which the extension of nerve fibers andthe maturation of epithelial cells were observed. The nature and characterization of the effective factors present in the wild-type egg extracts
which may repair the deep orange defect are now under investigation.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Inhibitory Effect of Cyeloheximide on Enhaneed Aggregationof Embryonie Quail Liver Cells in
Dissoeiation Medium
Yukiaki KURODA
The effect of cycloheximide on the aggregation of dissociated liver cells
from 7-day quail embryos in the presence and absence of the medium inwhich the cells were dispersed (dissociation medium), was examined inrotation-mediated cell cultures.
Cycloheximide had a concentration-dependent inhibitory effect on cellaggregation. With 30 pgjml of cycloheximide the aggregates formed after
24 hours were about one third the size of those in control cultures. Its
inhibitory effect appeared after a time lag of about 6 hours and was notcompletely reversible.
When dissociated cells were centrifuged and resuspended in fresh medium
and then rotated without dissociation medium, they formed, after 24 and48 hours, aggregates three fifths and five sixths the size of aggregates
formed in dissociation medium. Resuspension of the above centrifuged
cells in the dissociation medium resulted in restoration of aggregate size,
and the diameters of the aggregates were comparable to those of theaggregates formed when the original cell suspension was cultured directly.
In the presence of cycloheximide, dissociation medium was not effectivein enhancing cell aggregation and the size of aggregates formed wascomparable to that in cultures of resuspended cells without the dissociation

medium.
The size of cell aggregates depended on the period of trypsin treatment,with prolonged treatment resulting in smaller aggregates. This decreasein the size of aggregates was more marked in the presence of cycloheximide.
Mutagenesis to 8-Azaguanine Resistanee in Embryonie
Human Diploid Cells Treated with EMS
Yukiaki KURODA
Human diploid cells derived from a 5-month embryonic lung were treatedwith ethyl methanesulfonate (EMS) and cultured in selection medium containing 8-azaguanine (8AG) to induce mutation to 8AG-resistance. The
effects of factors such as inoculum size and mutation expression time on
the induced mutation frequency of cells were examined.
When cells were treated with 10- 2 M EMS for 2 hours and selected withRESEARCHES CARRIED OUT IN 197321

10 pgjml of 8AG, the induced mutation frequency was highest on inocula-tion of 2.5 X 10~ cells and lower with bigger or smaller inocula. When EMStreated cells were selected with 30 pgjml of 8AG, the highest induced
mutation frequency was obtained on inoculation of 10~ cells. With similar
sizes of inocula, the induced mutation frequencies in cultures selected with
30 pgjml of 8AG were lower than those in cultures selected with 10 pgjml
of 8AG.
The mutation frequency was higher when 10~ cells treated with EMS
were cultured in normal medium for 48 hours (mutation expression time)
before selection with 8AG, than in cultures with longer or shorter mutation
expression time. This mutation expression time of 48 hours corresponds tothe time during which about half the cell population treated with EMS
divides once.
Microscopic examination of the numbers of cells in colonies indicatedthat with or without EMS treatment, human diploid cells do not all growexponentially; but that in some cases after each cell division, only one
daughter cell continued to divide. These results were reported, in part, in
the First International Conference on Environmental Mutagens. (See also
Mutation Research 21: 226.)
Induction of Drug-Resistant Mutations by Furylfuramide
in Embryonic Human Diploid Cells
Yukiaki KURODA

Furylfuramide (FF) is now widely used as an antimicrobial food preservative in Japan. Recently this nitrofuran derivative was found to induce
chromosome aberrations in human lymphocytes and mutations in bacteria,
yeast and silkworms.
In the present experiment the mutagenic effect of FF on 8-azaguanine(8AG) resistant mutations in cultured embryonic human diploid cells was
examined. The effects of various concentrations of FF on cell survival
were tested and the Do values were calculated from the concentrationsurvival curves. The Do values for 2 hour- and 14 day-treatments with
FF were 11 pgjml and 1.9 pgjml, respectively.
When cells were treated with 1 pg(ml of FF for 2 hours, incubated for
48 hours in normal medium, and selected with 30 pg(ml of 8AG, the induced
mutation frequency was 19.2x10-~. When cells were treated with higher
concentrations of FF, the induced mutation frequencies increased gradually.
Thus cells treated with 30 pg(ml of FF gave the mutation frequency of

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

73.3xlO-~.

When the mutation expression times were varied, the mutation frequency
was higher in cultures with 48 hours of mutation expression time than incultures with longer or shorter mutation expression times. During thismutation expression time about half the cell population treated with FF
divided once.

Studies on Serum Factors Supporting Growth ofMammalian Cells in Culture
Kiyoshi MINATO

The nature and functions of serum factors which support growth ofcultured mammalian cells were analyzed using HeLa S3 cells in monolayer
culture.
HeLa S3 cells were cultured in TD-15 flasks in Eagle's MEM supplemented with calf serum. Cell Growth was determined from the changes
in the average numbers of cells per unit area of the culture flasks. In
the previous experiments, the growth rate in the logarithmic phase of cell
growth was calculated to determine quantitatively the growth-supporting
activity of serum or its fractions. This method was based on the factthat at lower concentrations of serum lower growth rates were obtained.
However, in the later experiments, it was found that the relation ofserum concentrations to the growth rate of cells varied depending on thelots of serum. Even in this case, the cumulative number of cells whichwere maintained on each day was found to depend on the concentration ofserum supplemented; at low concentrations cells ceased to grow in lower
cell density, although the growth rate was not changed.
When dialyzed serum was used, the cumulative number of cells was also
dependent on the concentration of dialyzed serum. It was lower than thatin control cultures with whole serum, and corresponded to that in cultures
supplemented with serum at one fourth concentration, although the growthrate was the same as that in control cultures. Since this dialyzed serum
was considered to be deficient in low molecular nutrients such as vitamines
and amino acids which were contained in Eagle's MEM, it is likely thatthe difference in the cumulative number of cells, which was dependent on
the concentration of serum, was not due to such vitamins and amino acids.
These results indicate that other factors in the serum which were partiallylost in the process of dialysis were responsible for the gowth of HeLa S3cells.

RESEARCHES CARRIED OUT IN 1973

V.

23

CYTOGENETICS

Oceanian Type Black Rats (Rattus rattus) with a
Subtelocentric M 2 Chromosome obtained
from Davis Colony
Tosihide H. YOSIDA, Kazuo MORIWAKI and Tomoko SAGAIThe black rats (Rattus rattus) used in the present study were kindly
supplied by Dr. Rex Marsh and Mr. Ray Record in the Department of
Animal Physiology, University of California at Davis, when we visited
there in August, 1973. According to Dr. Marsh's correspondence, threepairs of black rats were collected in San Lorenzo, California in 1970, andsince then they have been bred in an outdoor population cage. Ten rats
(5 females and 5 males) randomly caught from the breeding colony were
given to us. Chromosomes were observed in short-term cultured cells fromtheir tail tips.
All 10 black rats obtained from the Davis colony were characterized as
the Oceanian type having 38 chromosomes, except one which had 37 chromosomes. All of them had two large biarmed autosome pairs (M! and M 2 ).
Among them the larger one (M!) was the same metacentric pairs as thosefound in the other Oceanian type black rats. On the other hand, thesmaller one (M2 ) in 5 rats was a metacentric homomorphic pair similar to
those found in the other Oceanian type black rats, but the remaining 5
rats were remarkable by having a metacentric and a subtelocentric heteromorphic M 2 pair. The morphology of the subtelocentric M 2 chromosome
was similar among the 5 rats examined. The metacentric M 2 and thesubtelocentric M 2 chromosomes were almost the same size. This suggeststhat the subtelocentric M 2 was derived from pericentric inversion of themetacentric M 2 • The comparison of G-banding patterns between metacentricand subtelocentric M 2 chromosomes strongly suggests that the latter wasderived from the pericentric inversion of the former.

Black Rat (Rattus rattus) with a Single X-Chromosome
Tosihide H. YOSIDA, Kazuo MORIWAKI and Tomoko SAGAI

Nine of the ten black rats obtained from the Davis colony showed 38
chromosomes as the Oceanian type, but the remaining one showed 37
chromosomes. Chromosome numbers were counted in 6 rats to examine

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

the distribution. In 5 rats (4 females and one male) the majority of cells
showed 38 chromosomes, although a few aneuploid cells were observed.
Only one rat among them showed 37 modal chromosome number, althougha few aneuploid cells were also observed.
The karyotype of the rat with 37 chromosomes is remarkable by having
a single X chromosome. From the G-banding pattern analysis, the single
chromosome is clearly shown by its characteristic X band. All autosome
pairs are easily distinguished from each other by their characteristic Gbands, but the X chromosome having two dark bands in the middle regionof the long arm is unpaired. The external and internal features of theblack rat were apparently female. From these observations this rat ischaracterized by the sex chromosome abnormality with X -0.
Polymorphism of C-bands of Chromosomes in Black Rats,

Rattu8 rattu8
Tosihide H. YOSIDA and TOMOKO SAGAr

It has already been reported that the Asian type black rats, Rattus
rattus with 2n=42 chromosomes, are characterized by chromosome polymorphism with respect to acrocentrics and subtelocentrics. The Giemsa
band, however, was quite uniform in all subspecies of the black rats, butalso in the related species of genus Rattus. The C-band of the animals,
however, showed high polymorphism in regard to positive and negative
staining, especially in the Japanese black rat, Rattus rattus tanezumi.
The rats were characterized by 13 autosome pairs (No.1 to 13) of acrocentrics or subtelocentrics; and 7 autosome pairs (No. 14 to 20) of metacentrics and acrocentric X and Y chromosomes. In Japanese black rats,
pairs No.1, 4, 7, 9, 11 and 13 showed polymorphism in regard to positive
and negative C-bands. Pairs No.2, 5, 6, 8 and 10 have negative bands,
while pairs No.3 and 12 have positive bands. It is interesting that thepair No. 1 chromosomes showing acrocentric and subtelocentric polymorphism were C-band negative in subtelocentrics, but positive in acrocentrics.
In pair No.9 which is also characterized by acrocentric and subtelocentric
polymorphism, however, the correlation between the C-band and the chromosome type could not be observed. The above study was carried out in27 rats obtained from our breeding colony, but in the natural populationof Japan the polymorphism was also observed, although the polymorphic
chromosome was slightly different from the above rats. The C-band polymorphism was very rare in the Philippine black rat, R. rattus mindanensis,

RESEARCHES CARRIED OUT IN 197325

the Hong Kong black rat, R. r. flavipectu8, and the Malayan black rat,
R. rattu8 diardii. In the latter species, however, pair No.1 was polymorphic similar to Japanese black rats.
In the Oceanian type black rats with 2n=38 chromosomes, the C-bandpolymorphism has not been observed. A peculiar feature of the C-band inthese animals is that the bands in metacentric pairs No. 11 to 20 weregenerally smaller than those in Asian type rats. The C-band character in
Ceylon type black rat (2n=40) was almost similar to that of the Oceanian
type.
Induction of Sister Chromatid Exchanges by Chemical
Mutagens and Its Possible Relevance to DNA Repair
Hatao RATO

Several chemical mutagens were tested for their ability to induce sister
chromatid exchanges in Chinese hamster chromosomes. Among them, theeffects of 4-nitroquinoline-1-oxide (4NQO) and mitomycin C (MMC) werevery similar to those of ultraviolet light in that the exchange frequency
increased with increasing dose of chemicals and decreased markedly in thepresence of 1 mM caffeine during a post-treatment period. The frequency
of proflavin-induced sister chromatid exchanges was also found to be dose
dependent, but it was insensitive to caffeine post-treatment. On the other
hand, no appreciable increase was detected in the incidence of sister
chromatid exchanges in cells treated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) over a 100-fold range of variation in chemical dose. Caffeine
by itself raised the exchange frequency only slightly over the control level.
It was found that 4NQO and MMC exerted remarkable delayed effects on
the exchange induction, whereas proflavin did not. This seems to suggestthat the lesions caused by the former mutagens would be long-lived andrepeatedly provoke sister chromatid exchanges.
These data imply that there are several possible ways in which theinitial DNA lesions ultimately lead to the formation of sister chromatid
exchanges, and that at least UV-, 4NQO- and MMC-induced sister chromatid
exchanges have evolved through a caffeine sensitive repair process, probably
related to a post-replication repair of DNA damage. (For details, seeExptl. Cell Res. 85: 239-247, 1974.)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Constitutive Heterochromatin of Indian Muntjac
Chromosomes Revealed by DNase Treatment
and a C-Banding Technique
H. KATO, K. TSUCHIYA and T. H. YOSIDA

A karyotype of a female Indian muntjac, Muntiacus muntjak vaginalis,
was studied. The karyotype was unique in that No.1 and No.3 homologous pairs were heteromorphic with respect to the size of their secondary
constrictions. In these pairs, one of the homologs always had a longer
secondary constriction than that of the corresponding homolog. Heterochromatin in the secondary constriction region was visualized with difficulty
by a C-banding technique, but was demonstrated clearly by a DNase treatment followed by Giemsa staining, which also revealed the size differenceof the secondary constriction. Centromeric constitutive heterochromatinof No.1 chromosome was also found to differ in size between the homologs.
On the basis of the heteromorphic character of No.3 chromosome, oran X-autosome complex, it was possible to confirm autoradiographicallythat X-inactivation had occurred in random fashion in this animal.

The Seventh Cell Population Change Accompanied by a
. Marker Chromosome E in the Transplantable
Mouse Myeloma MSPC-l
Kazuo MORIWAKIThe mouse myeloma MSPC-l has been serially transplanted in BALB/cmice since 1966. During the eight years, the major cell population has
had at least six changes which could be detected either by the successive
occurrence of marker chromosomes, A, B, C and D, or by the loss ofcapability to produce the specific gamma-A globulin. The sixth cell population NP38-ABCD subline, was established in September 1972 at transplant
generation 123. It was successfully propagated in the solid form until
generation 163 in March 1974, when it was spontaneously converted to theascites form with tetraploidy. This phenomenon was considered to becertain kind of clonal senescence (Moriwaki and Sadaie, This Annual Report
22: 52, 1972). Our previous data on the successive karyotypic changes inthis tumor line (Moriwaki et al. J. Nat. Cancer Inst. 47: 623, 1971) suggested that the variant cells with newly emerged marker chromosome canescape from the clonal senescence and actively proliferate to constitute themajor cell population in the subsequent transplant generations.

RESEARCHES CARRIED OUT IN 1973

27

In the case of NP38-ABCD subline, we have maintained two series:
One of them changed to the ascties form at generation 163 as stated above,
whereas the other one which was once stored in a deep freezer (-80°C)
exhibited cell population shift from NP38-ABCD to NP38-ABCDE at generation 140 in March 1974. The new marker chromosome E seems to be
produced by a pericentric inversion of the smallest chromosome, No. 19,
in the NP38-ABCD subline cell. This was confirmed by either G-band or
C-band analysis. The C-banding pattern, especially, has clearly demonstrated
that the D-marker chromosome was translocated with the centromericheterochromatin at the terminal region of the C-marker chromosome andthat pericentric inversion near the centromeric region of No. 19 chromosome
induced the E marker chromosome. The newly established NP38-ABCDE
subline has been propagated until now as a stable solid form.
Synaptonemal Complex and Male Crossing-over in

Drosophila ananassae
Daigoro MORIWAKI and Mitsuo TSUJITA

The synaptonemal complex has been known to be intimately related topairing of homologous chromosomes and meiotic exchange, being normally
restricted to the nucleus of meiocytes.
Although it has been known that crossing-over does not take place inthe male of Drosophila species, D. ananassae is a notable exception inwhich spontaneous crossing-over occurs frequently in the males. Individuals
from T 15 - 4 strain, one of the inbred lines derived from the Tonga strain
which had shown the highest recombination value in males, were crossedwith double recessives, b se, and crossing-over in the F 1 males was counted
between band se. Crossovers appeared in their progeny with an averagefrequency of 8.9%, which is 18% of 48.7%, the recombination value between
band se in females.
Testes were dissected from the third instar larvae of sibs of the F 1
males (b se/+T 15 - 4) and examined by electron microscopy to determine
whether the synaptonemal complex is formed in pro-spermatocytes. The
synaptonemal complex with typical tripartite structure which was seen in
pro-oocytes could not be found by examining a number of pro-spermatocytes.
According to Grell, Bank and Gassner (1972 Nature 240: 165-167), no structure was found which could be identified as a synaptonemal complex, inspite of their observation of a number of pro-spermatocytes from the testes
of D. ananassae by electron microscopy. They interpreted this as follows:

28

ANNUAL REPORT 0F NATIONAL INSTITUTE OF GENETICS NO. 24"demonstration of high levels of crossing-over in the apparent absence ofthe complex implies that it is not an inseparable feature of meiotic exchange."
In the present case, however, we were able to find imperfectly developed
synaptonemal complexes, though they were not typical. In leptotene andearly zygotene cells, the axial filament which makes up the center of eachchromosome was clearly observed. Furthermore, in zygotene cells, homologous chromosomes formed incomplete synapses with the aid of an imperfectly developed synaptonemal complex. It is considered, therefore, thatmale crossing-over takes place between these imperfectly synapsed homologues, although its frequency is much lower than that of females.
Ecological Genetic Studies on the Pollen Germinationof Chrysanthemum Species
Shuzo NAGAMI 1)

Pollen grains of 16 Chrysanthemum species were cultivated artificiallyon the medium A and B (NAGAMI, 1972). The results are shown in Table
1. The conclusions are as follows: (1) In general, medium B gives better
pollen germination than medium A. (2) There is no, remarkable relationTable 1. Species name (H: Hagi strain, Y: Yuhara str., T: Tsubakidomaristr., A: Awasima str., M: Manazuru str., K: Kamakura str.), subsection name (SN. D: Dendranthema, L: Leucanthemum, N: Nipponicae),
distribution (d. CJ: Central Japan, NJ: North Japan, SJ: South Japan,
c: coast species, f: field sp.), chromosome number (cn. X=9), and poIIen
germination rate (grA: rate by medium A, grB: rate by medium B)
of chrysanthemums.
SN

d

cn

grA

grB

2.
3.

C. Makinoi
C. boreale
C. nipponicum

D
D
N

CJ-f
CJ-f
NJ-c

2X
2X
2X

7.5%
0
5.3

6.1%
0
15.3

4.

C. indicum

D

CJ-f

4X

0

5.0

D
D
D

CJ-c
CJ-\"
CJ-c

6X
6X
6X

0
12.0
10.3

6.0
22.4
35.0

species namel.

5. C. indicum var. hexaploid (H)6. C. indicum var. hexaploid (Y)7. C. japonense

1)
Professor, Department of Biology, Faculty of Education, Yakohama National
University.

RESEARCHES CARRIED OUT IN 1973

29

(continued from page 28)
species name

SN

d

cn

grA

grB

(T)
8. C. Shiwogiku(A)
9. C. Shiwogiku
10. C. Shiwogiku var. kinokuniense
11. C. Weyrichii
12. C. japonense var. octoploid

D
D
D

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

8X
8X
8X
8X
8X

1.0
0
0.7
6.5
0

13.5
0
0.6
7.7
7.1

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

lOX
lOX
lOX
lOX

0
6.2
12.5
20.0

5.6
17.5
32.0
11.2

13.
14.
15.
16.

C. japonense var. crassum
C. arcticum ssp. Maekawanum(M)
C. pacijicum(K)
C. pacijicum

L

D
D
L

D
D

ship between taxonomical conditions and pollen germination rate, or between
distributional conditions and pollen germination rate. (3) In proportion to
the increase of Chrysanthemum polyploidy, a gradual increase is found in
the size of pollen grains, but no increase is observed in the pollen germination rate. (4) Some species, such as C. indicum var. hexaploid, C.
Shiwogiku and C. pacificum, show clear intra-specific differences of pollen
germination rate.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

VI.

MUTATION AND MUTAGENESIS IN ANIMALS

Some Aspects of Overdominance Observed in Experimentson Radiosenstitivity in the Silkworm*
Y. TAZIMA

Overdominance is regarded at times as the cause and at other times as
the effect of a hybrid vigour. In this paper the relation between ordinary
dominance and apparent overdominance is discussed. The argument is basedon the results of our experiments with the silkworm in which radiosensitivities at several biological response levels were used as criteria.
By raising two sensitive and one resistant parental strains and theirhybrids simultaneously, the radiosensitivity of Fl's was compared with thoseof parental strains. The modes of dominance contrasted markedly between
different reaction levels: Overdominance was always observed with regard
to the sensitivity to embryo killing (whole individual level) ; whereas withregard to mutation induction, sensitivity in F l was invariably intermediate
between both parental strains (gene level). Between those two reactionlevels, with respect to cell killing, either overdominance or the intermediate
sensitivity was observed, depending on the combination of the cross (celllevel).
These findings indicate that as far as radiosensitivity is concerned, theinherent expression of dominance is of the intermediate type (as in thecases of mutation induction) ; but may be modified toward the overdominant
type because in each strain there is an upper limit for survival of radiationinduced lesions, and this limit may be shifted to a higher level in F linduviduals than in both parental strains, presumably due to a hybrid
vigour. In this regard, the overdominance observed for embryo killing was
not a real one, Qut a modified form of ordinary type of dominance. The
overdominance observed for drug resistance, pest resistance, and so on may
belong to the latter type.

* Presented at the Second General Congress of SABRAO, held on Feb. 22-28,
1973. A full paper is due to appear in Ind. J. Genet. and Plant Breed.

RESEARCHES CARRIED OUT IN 1973

31

Results of Mutagenicity Testing for some Nitrofuran
Derivatives by Applying a Sensitive Test
System with Silkworm Oocytes*
Yataro TAZIMA and Kimiharu ONIMARU

A highly sensitive test system which is applicable for the screening of
weakly mutagenic chemicals, has been established in the silkworm using
oocytes. It is essentially a kind of specific loci method, using egg color
mutants as markers, characterized by an administration of chemicals to wild
type females at a mid-pupal stage, so that the chemicals are incorporated
into developing oocytes. The method proved to be effective for the detection of a weakly mutagenic compound Panfuran, a derivative of nitrofuran(Tazima, 1973). Using this system we are continuing the mutagenicity
testing of other nitrofuran derivatives. The results reported here are forFurylfuramide [2-(2-furyl)-3-(5-nitro-2-furyl) acrylic acid amide], and Furazolidone [3-(5-nitrofurfurylidenamino)-2-oxazolidone].
Both compounds have low solubility in physiological saline solution andinjection of 0.025 ml of saturated solution, administered doses per head of
3.1,ug for Furylfuramide and 7.2,ug for Furazolidon, did not produce asignificant increase in mutation frequency. However, at higher doses,
which were prepared by dissolving the chemicals in a mixture of one partacetone and 4 parts of physiological saline solution, Furylfuramide gave
clearly positive results. Induced frequencies were 98.0x10-~ at +pe locus
and 54.5x10\~ at +re after injection of 19.2,ugfhead and 70.4xlO-~ at +pe
and 41.7x10-~ at +re after injection of 9.6,ugfhead. In contrast, Furazolidone did not iricrease the mutation frequency within dose range of 12.5,ug
and 25.0,ug per head.
Since Furylfuramide is used widely in Japan as a food preservative, atest for mutagenicity of this compound with a mammalian system isurgently needed.

* Presented at the Second Annual Meeting of the Environmental Mutagen
Society of Japan held at Misima, Sept. 22, 1973.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Production of Mosaic Eggs at Very High Frequency
with BUdR Treatment in the Silkworm
Yataro TAZIMA and Kimiharu ONIMARU

It was reported in late 1950 that transition type mutations had been
induced in bacteriophage when base analogues of DNA, as 5-bromouracil
and/or 2-aminopurine, were incorporated by mistake into DNA during theprocess of its multiplication. However, our experiments in with silkworm
spermatogonia were used as materials, failed to demonstrate a similar
mechanism in this insect. Recently, we have been able to establish a very
sensitive test system of chemical mutagens using developing oocytes in thesilkworm. We again tested the mutagenicity of BUdR to the silkworm
using this system. The results were surprising. The incidence of mosaic
eggs were observed at strikingly high frequencies.
Wild type female pupae were injected with BUdR at a dose of 50 pg/headin physiological saline solution. Injection was administered 4-5 days before
the emergence of the moth. After emergence, injected females were matedwith males of a double recessive marker strain, pe reo In F 1 the incidenceof mosaic eggs having tiny red cell patches among normal (dark brown)
serosa cells was observed at a frequency of 1.6x10- 2 • Mosaic eggs werealso observed with regard to pink and normal cells. More frequently
observed were mosaics that had either small or large opaque patches on
their transparent chorion. Similar treatment with FUdR, BU and/or AP
did not produce such aberrations.
Since the chorion is the product of follicle cells of the mother moth, theappearance of opaque patches seems to reflect the abnormal function of
those follicle cells, and red or pink patches in serosa cells indicates thatthe abnormality developed during early segmentation of the zygote. The
two phenomena may have some common processes. Presumably, both might
have been caused by an error in the process of edcoding genetic information
during transcription or translation.

Analysis of a Chromosome-Specific Genetic Instability
in the Silkworm
Akio MURAKAMI

In an experiment on radiation-induced mutagenesis carried out in 1971,
in which mature larvae of the C108 wild type strain silkworm male were
irradiated with 180 kVp X-rays (5 kR: dose-rate of 100 R/min) and crossed

RESEARCHES CARRIED OUT IN 197333

to the marker strain female moths having a recessive visible egg-color
mutant gene, re (red egg: V-31. 7), six mosaic type mutant eggs were
found out of 110,000 F 1 eggs. The moths developed from the mosaic
mutant eggs were backcrossed again to moths homozygous for re gene inorder to test their transmissibilities. Among six moths tested, five produced
red and black eggs in various ratios as expected, whereas the remaining
one produced a large number of unexpected mosaic eggs in addition to
expected red and black eggs. Furthermore, when all these mosaic eggs
which appeared in the BF1 were crossed to moths homozygous for re gene,they again produced a number of unexpected mosaic eggs (BF2 ) besides
expected red and black eggs as observed in the BF1 eggs. By repeatingprocedure, a similar tendency has been observed in the production of BFa
eggs.
From the finding in the single re locus tested so far, it seems likely thatthe above instable (or mutable) line, tentatively MVINSTA, may be classified
as the line having locus-specific genetic instability. Also, it can be said
that this MVINSTA line behaves as a semidominant one.
In the foregoing description, the relevant event occurred at the re locus
on chromosome 5. It is of interest to analyze whether or not the MVINSTA
line has an effect upon another gene located on chromosome 5, pe (pinkegg: V-0.0). It was observed that the chromosome in the MVINSTA line
was affected at the pe locus as well as at the re locus. This was also
confirmed in studies on the other two gene loci locating on the chromosome
5, ok (kinskiryu translucent, V-4.5) and oc (chinese translucent, V-40.8).
Both genes control a larval oily skin character which is due to lack of uricacid in epidermal tissues.
It is of further interest to know whether the MVINSTA line has an effectupon chromosomes other than chromosome 5. In this connection, experiments were carried out using several gene loci, os (sex-linked translucent,chromosome 1-0.0), lem (lemon, chromosome 3-0.0), W2 (white egg-2,
chromosome 10-3.4) and ck (chocolate, chromosome 12-9.6). No genetic
effect of the MVINSTA line on these loci and/or chromosomes was found.
These findings may lead to a conclusion that the MVINSTA line shows achromosome (5)-specific instability.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Stage Sensitivity and Dose-Response of X-ray-Induced
Recombinations in Oogenesis of the Silkworm
Akio MURAKAMI
The object of the present study was to investigate the stage sensitivity
and the dose-response relation in X-ray-induced recombinations in silkworm
female gametogenesis as well as in Drosophila male gametogenesis, in which
the crossing-over does not occur normally.
Silkworm females from early instar larval stage to the late stage pupal
stage, having pe (V-0.0) and re (V-31. 7) mutations in trans type heterozygotes were exposed to 1000 R of X-rays and mated to males homozygouSfor pe and reo In this test system, recombinants occur as exceptional
black(+pe re+) and pink (pepe re)eggs in addition to normal pink (pe +)
re
pe re

+re) eggs. Since the recombinant pink eggs can not be
and red (pe redistinguished from the normal ones, a progeny test has been done only forthe black eggs. In view of the mating scheme, however, the recombinant
black eggs cannot be distinguished from other exceptional types, such as
polyploid eggs, which may come from apparent non-disjunctional events.
Thus, by backcrossing the exceptional black eggs with marker strain moths
homozygous for pe and re, they can be identified as recombinant eggs andfurther classified into either homologous exchange (crossover) or nonhomologous interchange type.
The results of the experiments indicated that some recombinants were
induced by X-rays at any stage in female germ cells. A considerable
number of recombinants was induced by X-rays in 4th or 5th instar larvae,
suggesting that a peak of the sensitivity to X-ray-induced recombinations
occurs at the early stage of prophase I oocytes, which in general, is themost likely stage for the crossing-over to take place. At other developmental stages, however, the incidence of recombinations was almost equal
to or somewhat less than that of the untreated control series. It is ofinterest to note that no cluster effect of induced recombinants was observed
even at the early larval stage of 1st to 2nd inster larvae, suggesting that
gonial cells do not respond to the induction of recombinations by treatment
with X-rays. Among 122 recombinants recovered in X-irradiated groups
in the present experimental series, the frequency of homologous exchange
type recombinants was about three times as high as that of non-homologous
interchange types.

RESEARCHES CARRIED OUT IN 1973

35

When recombinants induced by different doses of X-rays were analyzed,
it was found that the yield in the mature larval oocytes which showed apeak sensitivity to induction of recombinations increased curvelinearly up
to a dose of 2500 R. The yield of recombinants in oocytes of 4th instar
larvae increased also curvilinearly up to 1000 R and then decreased, probably
due to cell-killing. In pupal oocytes, no increase in recombinants wasobserved up to 2500 R, while a markedly large number of polyploid type
exceptionals were found.

Ultraviolet-Light Sensitivity of an X-ray Sensitive
Silkworm Mutant rb
Akio MURAKAMI and Yataro TAZIMA

A silkworm mutant rb (red blood) is one of X-ray sensitive stocks for
either mutation induction or killing effect (Tazima, 1956). In order toinvestigate the mechanism underlying the biological effects of ionizingradiations, the UV sensitivity of rb mutant was examined and comparedwith that of some other UV sensitive stocks. The stocks Kansen andKojiki are resistant to UV light; whereas the stock Aojuku is sensitive.
Also, as reported already, the stocks Kansen and Aojuku is of the X-ray
resistant class; whereas the stock Kojiki is of the class relatively sensitive
to X-rays (Tazima and Murakami, 1971). After mature oocytes and/or eggs
at early stages of cleavage from the above stocks were irradiated with
UV, their sensitivities to killing effects and to mutation induction were
determined by measuring embryonic mortality and by scoring pe: re eggcolor specific locus mutatins, respectively.
As to the killing effect of UV-light on mature oocytes and/or eggs ofthe mutant rb, a large shoulder was found for dose-embryonic hatchability
curve in semilogarithmic plot with UV resistant stocks, whereas a slightshoulder and an almost linear dose-response relation were found for UV
sensitive stocks. This finding suggests that the rb stock may be one of the
UV resistant stocks.
As to the mutagenic effect, the mutant rb showed almost the same rateof induced-mutation as those of UV resistant stocks (Kansen and Kojilci).
As has been reported elsewhere, the stock Aojuku was UV sensitive formutation production. Thus, it was suggested that stocks which are UV
sensitive for killing effect are also UV sensitive for mutation-production
and vice versa.
These experiment indicated that an X-ray sensitive stock rb was not

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

UV sensitive, but UV resistant. Furthermore, there may be no positive
correlation between UV and X-ray sensitivity of oocytes and/or eggs toboth biological effects; and the mechanism(s) involved in killing andmutagenic effects of UV-light may be, at least in part, different fromthose of ionizing-radiations.
Induction of Recombinants by Mitomycin C in Oogenesisof the Silkworm
Akio MURAKAMI and Isao ARIGA ' )Although no crossing-over occurs normally in silkworm females, it has
been reported that a significant number of recombinants were clearly
detected in prophase I oocytes by treatment with X-rays. The present
communication reports our findings on the induction of recombinations in
oogenesis of the silkworm by treatment with mitomycin C (MC) which is
known to effectively break DNA strands.
For detection of recombinants, the egg-color method was employed. The
detailed principle for the method was described in the preceding report.
Females from the first instar larval stage to the middle pupal stage having
two recessive egg-color mutant genes, pe (pink egg: V-0.0) and re (V-31. 7)in the state of trans type heterozygotes, were treated with either feedingor injection of MC dissolved in 0.85% NaCI solution, and crossed to male
moths homozygous for both mutant genes.
The results indicated that the presence of recombinants as well as polyploid type eggs was detected in any stage of germ cells after treatment
with MC. The stage showing a peak sensitivity to MC-induced recombinations was the last (mature) larval stage, probably corresponding to the
nuclear phases from zygotene to diplotene which is the most likely stage
for crossing-over to take place. At the peak sensitive stage the frequencyof homologous exchange (crossover) type recombinantions was 4.12 X 10- 5(8/193,984) and that of non-homologous interchange types was 6.70 X 10- 5(13/193,984). This ratio of the frequencies of homologous exchange to nonhomologous interchange type recombinations is contary to that of experimental results with X-rays, suggesting that mechanisms related to the
induction of recombinations by treatment with MC may be different fromthose with X-rays. It should be noted, however, that the present finding
in the silkworm appears to agree with the result of experiments on
Drosophila treated with MC (Schewe et al., 1971).
1)

Kanagawa-Ken Sericultural Research Center, Ebina-Shi, Kanagawa-Ken.

RESEARCHES CARRIED OUT IN 1973

37

In the control series, seven eggs out of 189,007 F i eggs so far tested
were products of recombinations. Six of them were classified as thehomologous exchange (crossover) type and the remaining one was an interchange type recombinant. Thus, the spontaneous frequency of homologous
exchange type recombinations was calculated to be 3.17xl0- 5 • This finding
and others suggested that crossing-over is not completely absent in thesilkworm female, but it may be suppressed in some ways.
Radio-Sensitization of Cultured Mammalian Cellsby 5-Iodouridine
Yukiaki KURODA, Akiko YOKOIYAMA and Tsuneo KADA

It is well known that cells are more sensitive to ionizing radiation or
ultraviolet light when some chemical agents are present at non-toxic low
concentrations during irradiation. Such enhancement of the radiosensitivityof cells by halogenated thymidines such as 5-bromodeoxyuridine or 5iododeoxyuridine was thought to be due to the replacement of thymidine
in DNA with thymidine analogues (Zamenhof et al., Nature 181: 827, 1961;
Kaplan et al., Radiat. Res. 16: 98, 1962; Erikson and Szybalski, Cancer
Res. 23: 122, 1963). On the other hand, our previous studies have shownthat radiosensitization with some iodine compounds such as 5-iodouracil, 5iododeoxyuridine or 6-chloropurine took place by r-irradiation at O°C without
drug incorporation into bacterial cells, but may involve radiation-provokedradicals containing iodine atoms which might attack certain cellular sites(Kada et al., Int. J. Radiat. BioI. 18: 281, 1970).
When Chinese hamster cells at acidic pH's were exposed to r-rays andthe number of colonies was scored after 6 to 11 days of incubation, many
more cells were inactivated in the presence of the drug than in its absence.
5-iodouridine was markedly effective at concentrations more than 1.0 mM,
whereas 6-chloropurine had no such radio-sensitizing effect even at a concentration of 3.0 mM.
This radio-sensitizing effect of 5-iodouridine may be due to radiationinduced toxic products from the reagent, because the cells were inactivatedefficiently only by contact with the previously irradiated drug solution.
The toxicity of the irradiated drug solution increased markedly when the
pH was shifted to the acidic side. Such a toxicity was not detected with
6-chloropurine. It is concluded that the radio-sensitization with 5-iodouridine
does not require its incorporation into cellular DNA, at least under theconditions employed in the present studies.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

VII.

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICROORGANISMS AND PLANTS
An Early Step Enzyme Participating in the Excission Type
Repair of r-ray Damaged DNA in Bacillus subtilis
Takehiko NOGUTI and Tsuneo KADA

Ionizing radiation produces complex DNA damage including lesions in
nucleoside moieties as well as simple chain scission in phosphodiester bonds.
Most complex lesions are assumed to be modified and converted to single
strand interruptions with 3'OH termini prior to subsequent excision-type
repair process, because this terminus structure is essential for the initiationof the DNA polymerase I action involved in it (this Annual Report, No.
23: 72-73, 1972). In this connection we have shown that a protein factor
exists and enhances the priming activity of r-ray impaired DNA. Because
Table 1. Effect of the primer activating enzyme (Phosphocellulose
fraction) on DNA's subjected to different treatments
Treatment

PA enzyme
treatment*

Control+
r-ray (0.5 kR)+
Control+Heat (70°C, pH 5.0, 30min)+
Control+
MMS (25 mM, 37°C, 30 min)UV (15 erg/mm 2 , 1 min)UV (15erg/mm 2 , 4min)+++Priming activity
for DNA polymerase I
(%)

100
105
42
167
100
100
111
196
100
116
82
344
78
93
63
78

* 0.64 pg enzyme protein was mixed with 60pg T7 DNA and incuba ted for
30 minutes at 37°C.

RESEARCHES CARRIED OUT IN 1973

39

this enzyme was expected to play a role in an early step of the excision
repair, we purified the factor from Bacillus subtilis cells by means of
DEAE cellulose and phosphocellulose chromatography. One of the two
peaks which appeared in phosphocellulose fractions was first examined as
to its specificity. As shown in Table 1, this enzyme enhanced the priming
activity of heat or MMS treated DNA like r-ray irradiated DNA. On theother hand, the enzyme had little effect on the priming activity of intact
DNA or UV irradiated DNA. The exonuclease activity of the enzyme wasvery low. Endonuclease activity was examined by sedimentation analysis
in sucrose density gradient. When T7 DNA samples which had been previously denatured with alkali or formamide were analysed in parallel inneutral sucrose density gradient containing formaline, a significant numberof nicks were introduced by subsequent action of the enzyme at alkali
labile lesions produced by ionizing radiation. This nicking action on thealkali labile region of DNA was more clearly demonstrated if DNA washeated to 70°C for 30 minutes at pH 5.0. It is very probable that the
DNA lesion susceptible te the above enzyme may be apurinic or apyrimidinicsites because it is known that such lesions are alkali labile and considered
to be formed in general by heating, MMS treatment and r-ray irradiationof DNA. Our primer activating enzyme may be analogous to the apurinic
site-specific enzyme reported by Verly et al. (Nature New Biology 244:
67, 1973).
Metabolic Activation and Escherichia coli Mutagenesisof Furylfuramide, a Nitrofuran Food Additive
Tsuneo KADA

The author has formerly reported that 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) shows a potent DNA-damaging capacity as shownby the rec-assay utilizing Bacillus subtilis strains, and very efficiently
induces reversions in Escherichia coli strains B/r WP2 try and WP2 try
hcr (Jap. J. Genetics 48: 301, 1973). Similar observations were also madeby S. Kondo and H. Ichikawa-RYo (Jap. J. Genetics 48: 295, 1973). Our
tentative experiments have shown recently that the genetic capacity oftransforming DNA extracted from Bacillus subtilis strain 168T was notinactivated by treatment with furylfuramide in phosphate buffer (pH 7.0).
This result in addition to the positive in vivo activity of furylfu:r:amide
would indicate that the chemical may be metabolically converted to an
active form in the cell and react with cellular DNA to produce mutations.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Similar activations must take place also in cultured human cells to induce
chromosome aberrations and DNA repair synthesis (Tonomura and Sasaki,
Jap. J. Genetics 48: 291, 1973); and in silkworms to induce visible colour
mutations (Tazima, Y., 1973).
The above metabolic activation hypothesis of mutation inductions by
furylfuramide indicates that mutations would not be induced in cells lacking
this activation capacity. It is also supposed that these cells may beresistant to the lethal action of the drug because the supposed activatedform should be involved in the cell killing through production of DNA
damage. We therefore tried to isolate such strains from E. coli WP2 try.
In a typical one isolated, reverse-mutations were hardly induced by furylfuramide and supplementation of rat liver extract was required for mutationinduction.
Cells of WP2 try were grown overnight in broth and spread densely on
solid broth agar containing furylfuramide (10 pg/ml). A number of substrains were selected and purified from drug-resistant colonies which appeared.
A typical strain called C801 was used in the following experiments: Cells
in broth were collected during exponential growth and washed once by
151M phosphate buffer (pH 7.0); they were resuspended in the same buffer
at a concentration of about 2x10 B cells per ml. Furylfuramide was dissolved
in distilled water at a concentration 100 pg/ml. Liver homogenate wasprepared as described by B. N. Ames et al. (Proc. Nat. Acad. Sci. 70: 782,
1973). This fraction contained NADP and glucose-6-phosphate as supplements and was sterilized by filtration with a Millipore membrane. For
experiments of mutation induction, two ml of bacterial suspension, one mlof furylfuramide solution and one ml of S9 were mixed at O°C and kept
at 37°C for 10 minutes; the cells were then collected and washed by centrifugation with 151M phosphate buffer in cold (O°C) and finally resuspended
in 0.5 ml of the same buffer. The number of viable cells and the frequencyof revertants were determined by spreading appropriately diluted cellularsuspensions on broth-supplemented minimal agar (MB agar; T. Kada et al.
Mutation Res. 16: 165, 1972). For the controls without drug and/or S9,corresponding volumes of buffer were sqpplemented and incubated similarly.
Results obtained are shown in Table 1. The following points are clear:
1) Reversions were hardly induced by furylfuramide (25 pg/ml, at 37°C
for 10 minutes) in the strain C801 derived from WP2 try. In the caseof the original drug-sensitive strain, it was found that a similar treatment yielded mutations that were more than 200 Try+ cells per 107
survivals.
2) When supplemented with S9 fraction the drug produced a significant

RESEARCHES CARRIED OUT IN 1973

41

Table 1. Activation by rat liver homogenate of furylfuramide-induced
reversions in a drug-resistant derivative C801 of E. coli WP2 try.
Treatment of the cells was done at 37°C for 10min.
(See the text for details of experiments.)

Supplements fortreatment of bacteria

Number of
viable cells
per ml insuspensions oftreated andwashed cells

Number of revertant
Try+ colonies foundon MB agar; 0.1 ml
portions were spread
and a mean of
triplicated plates
was obtained

Frequency of
induced Try+revertants
per 107 viable
cells

Furylfuramide and S9Furylfuramide only
S9 onlyNone

1.3x10'1.5x10'1.6x10'1.6x10'

337
24
32
20*

24±0.5less than 1.0
less than 1.0(0)* These colonies appeared during residual growth of auxotrophic cells inminimal agar supplemented with a small amount of broth.
induction of reversions, 24 times more than that found without S9. Sucha mutation induction was not observed with supplementation of S9 without
drug.
The above results indicate very clearly that mutation induction by fury1furamide requires a metabolic activation by liver homogenate. It is supposedthat a similar metabolism may take place in the cells of E. coli. Further
studies are required to determine whether our drug-resistant cells are reallylacking the supposed activation enzymatic capacity.

Host-Mediated Assay of Mutagenicity of Furylfuramide
Kiyosi TUTIKA W A and Tsuneo KADA
Host-mediated assays are usually devised to detect the mutagenic activityof metabolites of chemical agents in experimental animals (M. C. Cabridge
and M. Legator, Proc. Soc. ExptI. BioI. Med. 130: 831, 1969). In thepresent example, studies were carried out to evaluate the genetic toxicity
in the peritoneum of mice to which furylfuramide had been administeredorally.
The experimental procedures are based on the host-mediated rec-assay
formerly described (T. Kada, K. Tutikawa and Y. Sadaie, Mutation Res.
16: 165, 1972). Seven to nine weeks female or male of closed colony ddY-

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

SLC were used in the experiments. The chemical substances furylfuramide(FF, 2-(2-Furyl)-3-(5-nitro-2-furyl)-acrylamide), was suspended in 0.5% sodium carboxymethyl cellulose solution and administered orally with doses
ranging from 190.6 to 4.7 mg per Kg of body weight. One hour afteradministration of the drug, a 0.5 ml portion of bacterial suspension consisting of 17A (Rec+ arg+ try- str-r) and 45T (Rec- arg- try+ str-r) cells ofBacillus subtilis in a modified Vogel Bonner medium without glucose (MM)was injected into the peritoneal cavity of each mouse. One hour after
injection of the cellular suspension, the mouse was sacrificed by cervicaldislocation and was injected with 1 ml of MM into the peritoneum.
Immediately the abdomen was kneaded gently in order to promote suspenTable 1. Determination of the DNA-damaging capacity in theperitoneal cavity of mice (ddY-SLC) orally administered
FF by means of the host-mediated rec-assay
Experimental
number(sex of miceused)

Number of viable cells of B.
subtilis strains per ml ofbacterial suspension. Meansof 3 mice were obtained

FF (mg/Kg)
given toeach mousl (males)

0.0
47.7
II (females)

0.0
47.7
III (males)

0.0
9.0

17A Rec+

45T Ree-

Administrated
to mice before
drug treatment
Recoveredfrom miceIbid.

7.9X10 6

2.1X106

2.3x106(100%)
2.4x106(104%)

9.2x10'(100%)
2.5x10 4(2.7%)

Administrated
to mice before
drug treatment
Recoveredfrom mice
Ibid.

8.0x10'7.5x10 4

1.6x10·(100%)
2.6x10 4(16.3%)

6.5x10 4(100%)
0.5x10 2(0.08%)

2.7x106

2.9x10'7.6x10·(100%)
6.5x10·(85.5%)

2.8x10·(100%)
9.7x10 4(34.6%)

Administrated
to mice before
drug treatment
Recoveredfrom mice
Ibid.

RESEARCHES CARRIED OUT IN 1973

43

sion of the bacterial cells in the injected fluid. Then the fluid was withdrawn directly with an injection syringe. About 1 ml of fluid could thus
be recovered from an animal and the exudate was filled up to 10 ml with
MM. The recovered bacterial suspension was diluted appropriately with MMand plated on two kinds of agar media: MM+Arg-Try and MM-Arg+
Try to count the number of viable cells of the original and recovered
suspensions.
The results obtained are summarized in Table 1. With an administrationof 190.6mgjKg, the toxic activity found in the peritonial cavity of female
mice was very high and both the rec+ and rec- cells were killed completely.
Similar activity, though less enhanced, was observed with 95.3mgjKg.
With a dose of 47.7mgjKg (about 1.5mg per mouse), some DNA damaging
activity appeared in the peritoneal cavity 60 minutes after administration,
as suggested by the fact that many more cells of 45T Rec- were killedthan 17A Rec+. In one series of experiments, male animals were administereda dose of 47.7mgjKg. The genetic toxicity found seemed to be more thanthat observed in female mice. However, we avoid concluding anything
about the sex difference because these experiments are not yet repeatedenough. With a dose of 9 mgjkg, the toxic activity decreased remarkably
compared to that observed with higher doses, but some activity was stilldetected.
In order to estimate quantitatively the level of DNA damaging activity
in vivo, model experiments were carried out in vitro. Mixture of Rec+and Rec- bacteria were kept at 37°C in MM containing different amounts offurylfuramide, and survival curves were obtained for two kinds of cells. The
toxicity level obtained in one series of experiments (Table 1, b) was thus
found to be between 0.015 ppm and 0.15 ppm of furylfuramide tested invitro. The activities found may be due to either FF which was notmetabolized or certain metabolized compounds which permeated into theperitoneal cavity through the intestinal wall or veins (presented at the
1973 Technicon Symposium, Tokyo).

DNA-damaging Products from Reaction between Sodium
Nitrite and Sorbic Acid
Tsuneo KADA

Compared to toxicological studies of single substances, little attention
has ever been given to products derived through reactions taking place
among chemicals in our environment. Sodium nitrite and sorbic acid often

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

co-exist in food materials as food additives. We found that they produce"rec-assay positive" substances under moderate or natural conditions. The
rec-assay procedures were useful for easy detection of DNA-damaging
chemicals or potential mutagens by comparing growth sensitivities of wild
and recombination repair-deficient strains of Bacillus subtilis against test
chemicals (Mutation Res. 16: 165, 1972; Jap. J. Genetics 48: 301, 1973;
Mutation Res. 26: 243, 1974).
Sodium nitrite was weakly positive in the rec-assay at high concentrations
but the effect was not detectable at low concentrations. Sorbic acid was
negative in the assay. When sodium salt sorbic acid and sodium nitrite
were dissolved in distilled water to obtain one molar concentration foreach chemical, the solution slightly coloured at O°C or at room temperature
indicating some reactions taking place. The reaction seemed to be further
enhanced by heating at 100°C. When the reaction mixture was rec-assayed,some positive results were observed compared to those obtained in thecontrol experiments in which each single chemical was assayed. The positiveeffect showed its maximum value at fifth minuts in the course of heating
at 100°C, then decreased.
Dr. H. Hayatsu and his collaborators (University' of Tokyo) have carriedout chromatography analysis of the reaction products and obtained several
fractions possessing rec-assay positive characters. Their preliminary chemical
analysis suggested formation of some nitroso-compounds. Studies are under
way to find mutation specificity of the above DNA-damaging products
from reaction between sodium nitrite and sorbic acid.

Dose Rate Effect of Gamma-rays in Maize Pollen
Taro FUJII
Recovery of premutational damage or mutation frequency decrement inmaize pollen grains treated with ionizing radiations has been studied withfractionation dose treatment with gamma-rays or fast neutrons, and we
concluded that the premutational damage could be reparable in the case of
gamma-rays if a 2-hr interval period was adopted (Ann. Rep. 23: 78-79).
In order to get further information about such a reparability, the dose
rate effect of gamma-rays was studied using 1 or 40 kR/h intensities of 3
different doses. Two different dose rates were used in these experiments
at different distances from a 6 kCi 187Cs-source. A 40 kR/h intensity wasmost commonly used with our facility. Materials and method used in the

RESEARCHES CARRIED OUT IN 197345

mutation detection were similar to those used in our previous experiments.
In the 40 kR/h series, 1, 2 and 3 kR lots showed mutation frequencies of
2.18, 4.60 and 5.92% in "total" (sum of whole and partial type) mutations.
In the 1 kR/h series, however, a clear decrease in mutation frequency wasobserved, showing 1.30, 3.07 and 3.90% in the 3 dose lots. The mutation
frequency decrement in the low dose rate series was about 30% of the highdose rate series. From these results, we can say that some efficient recovery
of premutational damage might have occurred with the 1 kR/h intensity.
The frequency of whole type mutations was markedly lower in low dose
rate lots than in the high dose rate lots, while the frequency of partial
type mutations did not show any significant difference in the low and highdose rate treatments.
Though the actual nature of whole and partial mutations is not clear,
the above results are in accord with those of our previous experiments inwhich single and fractionation dose were done. Thus, recovery of about
30% of premutational damage induced by gamma-ray occurred with fractionation dose treatment over a 2-hr interval or by low dose rate treatment
with 1 kR/h. However, such a recovery seems to be concerned only withthe whole type mutation.

Effects of Gamma·Rays on Sex Expressionin Gynoecious Cucumber
Taro FUJIIIn monoecious plants, the sex expression is influenced by genetic, environmental and hormonal factors. Reduction in the number of pistillate (female)
flowers in monoecious cucumber by gamma-ray treatment has already beenreported (Ann. Rep. 23: 76-77). Gynoecious strain (MSU) in cucumber
normally produces only female flowers under genetic control, and in thegibberellin treatment at an early seedling stage is required to develop
staminate (male) flowers or to obtain homozygous seeds. Dry seeds ofMSU-713-5, kindly supplied by Dr. S. Komochi of Hokkaido Agric. Exp.
Station, were exposed to 10, 20 and 40 kR of gamma-rays, and the growthhabit and flower development examined up to the 50 th nodal stage. Twenty
seeds were used in each lot. All plants in 10 and 20 kR lots showed nodifference in growth with good germination percentages like in the nonirradiated control lot. One among 18 plants of 40 kR lot had developed 88
male flowers in total over 18 nodes between 4th to 35th nodes, in addition

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

to female flowers. In this monoecious-like plant (MSU-713-5 M), twofruits were obtained by selfing, but no ripened seed was observed.
Other male flowers were crossed to the original MSU-strain and 15 fruits
with ripe seeds were obtained. Fertility and germination percentages in
the F 1 seeds examined so far was about 50%. Most of the F 1 plants
showed normal gynoecious type, but 4, 1 and 2 plants among 33, 5 and 14
plants from 3 examined fruits again developed male flowers. The numberof male flowers in these plants was merely 2 or 3 on one or two basal
nodes in each plant, while the male parent had developed many male flowers.
Although we can not say at present that the male flower development in
the gynoecious strain might be due to mutation, it may be possible thatsome transmissible alteration of the sex expression might have been induced
by gamma-ray treatment. Selfing and back- or test-crosses using male
flowers developed in F 1 plants are now underway to check whether or not
the altered sex expression is introduced by heritable change. Modificationof the sex expression, either genetic or non-genetic, might be useful instudies of the sex differentiation in plants.
Germination of Podostemaceae Seeds
Etsuo AMANO

Podostemaceae plants are found mostly in tropical streams or rapids andhave very strange flowering habits. When the water level of the riverdecreases and the plants are exposed, they produce flowers and seeds fora short period. There have been some reports on the taxonomy andevolution of this family, as well as some notes on their strange physiologyof self maceration when the tissues are taken out of water. However, there
is no detailed report on the life cycle or on the genetic relationships among
species. Even the chromosome number is not yet known. In cooperation
with the Kihara Institute for Biological Research, the author succeededin germinating the seeds which had been collected by a member of the Kihara
Institute from the tropical rivers in Surinam, South America.
Among the media tried, the best result was obtained with 1/10 diluted
salts mixture of Murashige and Skoog's medium. Other conditions adopted
were constant temperature (25°C) and continuous illumination with Biolux
fluorescent lamps. Of the four kinds of seed material tested, Mourera
jluviatilis, Apinagia longijolia and Apinagia sp. successfully germinated.
However, Apinagia sp. having the smallest capsules and seeds failed togerminate. The germination took place on the surface of agar medium,RESEARCHES CARRIED OUT IN 197347

but the seeds had to be submerged in liquid medium for further growth.
The seeds were generally very small. The length of-the largest seeds (M.
fiuviatilis) was about 1/4mm, and that of the smallest (A. sp.) was about
1/6mm. The seeds seemed to have some gluing materials which werereleased by wetting. Thus they could be attached to slide glass or groundglass surfaces by wetting the seeds with distilled water and redrying them.
These slides with adhering seeds could then be surface-disinfected by dipping
into equal mixture of 3% H 20 2 and ethanol for two to five minutes. Afterrinsing twice with sterilized distilled water, they were put into a deep
Petri dish containing liquid medium.
Most seeds germinated on the third day after sowing. The fertility ofthe seeds was usually more than 80% and the germination rate was over
80% of the fertile seeds. The seedling that emerged from a seed coatwith the root part first was green and had two cotyledon-like leaves, which
may really be cotyledons. There was no significant root organization except
fine root hairs that grow directly from the round bottom of the seedling.
The cotyledons opened flat, and had a thick disk-shaped meristematic tissue
between them. Two long, narrow, flat leaves emerged from the meristematic disk at right angles to the cotyledons, forming a flat" cross". The
third and fourth leaves came out in a similar manner, but, instead of
forming a radial rosette, they came out on one side of the previous leaves,suggesting shift of the apical meristem toward one of the cotyledons.
After the fourth or fifth leaf appeared from the shifted meristem, theleaves had branches, one on each side of the leaf. The leaves had several
remarkable spots which glistened in incident light. These spots looked
like some kind of condensed crystal. Since the seedlings are easily damagedby algae, they have been kept under aseptic culture conditions.
Callus Culture of Haplopappus gracilis
Etsuo AMANO
Cultured cells of a Compositae plant, Haplopappus gracilis (Nutt) Gray,
have a low chromosome number (2n=4) and grow relatively fast on modified
Murashige and Skoog's agar medium containing 2, 4 D (2 ppm), yeast extract(0.3%), vitamins and sucrose (2%). Even after two years' culture with
about 50 transfers, the chromosome number has remained stable at 2n=4.
To obtain haploid cell lines, flower bud cultures were tried. Youngflower buds were taken from plants grown in the green house about two
to four days prior to opening of the ligulate flower. Florets of the central

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

part of the flower were picked out and placed on the same agar slant as
used for the callus culture (2 ppm of 2, 4D). The anthers were too small
to inoculate separately from another part of the floret. No special disinfection procedure was used for the materials, but nearly half of the slants
were free of contaminating microorganisms. The floret grew on the agar
medium and bloomed some days later. About a month later, callus tissues
were found on the floret. They were then transferred to new medium androughly grouped according to the part of the floret where the callus
originated. After five transfers on the agar medium, chromosome numbersof the cells were examined. Of 15 lines examined, all carried the twopairs of chromosomes that are expected for normal diploid somatic cells.
There were some morphological differences among these callus lines, butthere seems no relation to the original tissue. A very soft callus has been
selected and maintained as HB line for future experiments.
The Haplopappus callus cells grew so fast at 25°C and under dark
conditions, that transfers were necessary every ten days. However, we
had some difficulties with experiments in liquid culture. Conditions forthe stable suspension culture were examined and the following points
suggested: (1) Sufficient shaking is necessary during incubation to disperse
cells in the culture medium. (2) Better results were obtained with 1/2
concentration of the medium than full strength or 1/10 dilution with thesame 2, 4D concentration. (3) Fast and good growth was observed at theinitial concentration of 2 mg wet weight per ml. Condition (3) seems to
be important because, if the initial concentration was too low, growth isvery slow until the cells reach this concentration. Under favorable conditions, the maximum rate of the growth was about 7mg wet weight/ml/day.
Experiments on the Grafting of Capsicum Mutants
Etsuo AMANO
Graftings of leaf color mutants onto the normal plants of parental line
were done to test modification of the genetic traits by grafting. The
phenomenon has been reported in Solanaceae plants, but it is necessary totest it in a genetically purified experimental system in order to study thegenetic aspects or even to confirm the phenomenon itself. For this purpose,some usable leaf color mutants have been induced in Capsicum annuum
L. var. Sapporo Wase, by ethyl methanesulfonate (EMS). The characters
IgA3 and yg2 could be grafted on normal green homozygotes of parentalline above the 2nd leaf. Both mutants segregated in a ratio of one mutant

RESEARCHES CARRIED OUT IN 197349

to three normal plants, suggesting that they are simple recessive mutants.
If a normal dominant factor (gene?) can be transferred to the scion or to
the seed, normal green tissues or seedlings may be expected.
The grafts were cultured in the greenhouse. The IgA3 character canbe observed best in very young leaves; they recover normal green color as
they mature. A yellow green or light green mutant, yg2, is a stable anddistinct mutant of the leaf color. Leaves of some 17 yg2 scions were
examined but no significant color patch of normal green color was foundin these leaves. To test the progeny of the grafts, each flower but of thescions was covered by a small paper bag to ensure self pollination.
Seeds thus obtained were sown onto steam-sterilized soil in wooden flatsin the green house. The color character IgA3 can be examined on the
cotyledon, but better on the first and second young leaves. Out of 34
selfed fruits, 780 seedlings were examined and all of them were mutants.
The yg2 mutant can be examined on the cotyledon. Out of 50 selfed fruits,1271 seedlings were examined and all of them showed yg2 phenotype. Sincc
leaves of the scions were not removed during the culture of the grafted
plants, nutritional dependence of the scion on the stock might be small.
Another leaf color mutant, y1, may be a good material for such a nutritional dependence on the stock. The character y1 is a recessive yellow
color mutant at the seedling stage. It is very weak or lethal unlessgrafted on the normal green stock. When it was grafted on the parental
normal green plant, growth was nearly normal. The tissues near nodes
had some chlorophyll but leaves were pale yellow or creamy white. The
leaves had a relatively short life and were replaced by new leaves. On
these pale yellow leaves, a few small green patches could be observed.
The shape of the green patches was such that the mutational events might
have taken place in the leaf primodia. The frequency of the green patches,
however, was not high, and only three patches were found among 216 y1
leaves. Furthermore, the patches were about 1mm wide and 4mm longat the largest. Rough calculation using the number and the area of leaves
indicated that the frequency of mutational events might be as low as
1.4xlO- 4 • This frequency is much lower than that reported as around 10-:.
In the case of yg2 scions, no normal green patches were found in 374
leaves from 17 graftings.
In the all present experiments, no distinct graft mutation phenomenon
was observed. The genetic markers induced by EMS were not significantly
modified by the stock as reported by different authors. It is likely thatthe so-called graft mutations might involve physiological modifications or
cytoplasmic influences, or some effects of infection with the stock used.

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Relationship between Damage Caused by Ageing and by
Irradiation in Rice Seeds
Sujit BAGCHIIn order to look into the relationship between the damages caused byageing and irradiation in rice plants, seeds of fifty-one rice varieties whichwere kept in a desiccator at a room temperature for five years and thenin a 0-1°C room for seven years were tested for their germinabilities andseedling heights. Among them, two varieties with high (451, Surjamkhi
from India and 647, Padi Ase Banda from Indonesia) and two with low(545, Shinriki and 564, Nakamura both from Japan) germination percentages
were selected. The fresh seeds of those selected varieties were irradiatedby gamma-rays with doses of 10 kR, 30 kR and 50 kR. The germinationand seedling height of the treated as well as unirradiated control plots
were measured.
It was found that varieties with high and low germinabilities in aged
seeds also showed high and low resistances to irradiation for both germination and seedling height, when their freshly harvested seeds were irradiatedby gamma-rays. It was suggested from the result that there may besimilar biological effects between damages due to ageing and irradiation.

RESEARCHES CARRIED OUT IN 1973

VIII.

51

POPULATION GENETICS (THEORETICAL)The Age of a Neutral Mutant Persistingin a Finite Population
Motoo KIMURA and Tomoko OHTA

Formulae for the mean and the mean square age of a neutral allele
which is segregating with frequency x in a population of effective size N.
have been obtained using the diffusion equation method, for the case of
4N. v<1 where v is the mutation rate. It has been shown that the averageages of neutral alleles, even if their frequencies are relatively low, arequite high. For example, a neutral mutant whose current frequency is
10% has an expected age roughly equal to the effective population size N.
and a standard deviation 1.4 N. (in generations), assuming that this mutant
has increased by random drift from a very low frequency. Also, formulae
for the mean" first arrival time" of a neutral mutant to a certain frequency
x have been presented. In addition, a new, approximate method has beendeveloped which enables us to obtain the condition under which frequencies
of "rare" polymorphic alleles among local populations are expected to beuniform if the alleles are selectively neutral. It was concluded that exchangeof only a few individuals on the average between adjacent colonies per
generation is enough to bring about such a uniformity of frequencies. For
details see Genetics 75: 199-212.

A Model of Mutation Appropriate to Estimate the Number
of Electrophoretically Detectable Alleles
in a Finite Population
Tomoko OHTA and Motoo KIMURA

A new model of mutational production of alleles was proposed which
may be appropriate to estimate the number of electrophoretically detectablealleles maintained in a finite population. The model assumes that the entire
allelic states are expressed by integers ("', A_ l , A o, A l , ••• ) and that if
an allele changes state by mutation the change occurs in such a way thatit moves either one step in the positive direction or one step in the negative
direction. It was shown that for this model the 'effective' number of
selectively neutral alleles maintained in a population of the effective size

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

N. under mutation rate v per generation is given by

n.=v1+8N.v.
When 4N.v is small, this differs little from the conventional formula by
Kimura and Crow, i. e. n.=1+4N.v, but it gives a much smaller estimate
than this when 4N.v is large. For details see Genet. Res. Camb. 22: 201204.
Effect of Linkage on the Behavior of Mutant Genesin finite Populations
Tomoko OHTA

The effectiveness of the apparent or "associated" overdominance caused
by linked selected loci for maintaining genetic variability was investigatedusing diffusion models. It was shown that when a population is subdivided,
the amount of linkage disequilibrium is determined by the local population
size if the recombination fraction is much larger than the migration rates.
Also, it was shown that, although the associative overdominance temporarily
creates a sort of inertia against perturbation in gene frequencies, it cannot
determine any definite equilibrium values. Hence, it has no long-term
effect on the rate of mutant substitution in evolution. For details, see
Theoret. Pop. BioI. 4: 145-162.

RESEARCHES CARRIED OUT IN 1973

IX.

53

POPULATION GENETICS (EXPERIMENTAL)

Analysis of Heterozygosity in Regard to the Neutral
Theory of Protein Polymorphism
Takeo MARUYAMA and Tsuneyuki YAMAZAKI

In conjunction with a theoretical calculation, a distribution of heterozygosity (more precisely, 2x(1-x), where x is the allele frequency) was examinedwith special reference to the neutral hypothesis of protein polymorphisms.
Data of some 400 polymorphic proteins collectively are consistent with thetheoretical expectation based on the neutral hypothesis, although otherpossibilities are not ruled out.
The distribution of neutral alleles for randomly-mating populations is
known (Kimura and Crow, 1964), and from it we can calculate the distribution of the amount of heterozygosity. If we let N be the population
size and u be the mutation rate, the number of alleles whose frequency isin the interval x±i3x/2 is proportional to x- 1 (1_x)4NU-l. Therefore, if we
multiply this formula by 2x(1-x), we get the amount of heterozygosity.
Namely,2(1-x)4NU ,(1)

which is proportional to the sum of the heterozygotes whose gene frequency
is in the interval x±i3x/2.
From the same literature used in our previous paper (Science 183: 1091,
1974), we have collected 1053 alleles (over 400 loci), involving many generaand species. All alleles were classified according to their frequency into
twenty equally spaced classes: 0-0.05, 0.05-0.1, "', 0.95-1. Then theheterozygosity was calculated from the allele frequency, and the values
were summed. Each sum represents the value of the heterozygosity ofthat class.
To determine the theoretical expectation, we need to know the value of
4Nu in formula (1). It can be shown that if f is the probability that two
randomly chosen genes are identical by descent, 1/f=1+4Nu. The valueof f is rather invariant over a wide range of organisms, but it varies
among different loci. The estimated values of 4 Nu for some loci are close
to zero, while those for other loci are as high as 0.5, which is about thehighest we found. Therefore, we present in Figure 1 the curves given by(1) for two extreme values of the parameter, 4Nu=0.5 and 0 together
with observations. The data appear to be consistent with the theoretical

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

1.0
>.

• •

.1=
en
0

g:
N
0

...
<l>

•

+4)

:I:

.....
0.5
0

...

~4Nu=0

•
•

c:

0

•

•

•

::J

••

E

«

•

<l>

>

+=
0

•

4j

a::

0
0

0.5

1.0

Gene Frequency x
Fig. 1. The curves indicate the theoretical expectations based on
the neutral hypothesis. The dots indicate the observed results.
The amount of heterozygosity is normalized so that the total area
under each curve and the dots is one.

expectation. Expectations based on other assumptions have different patterns. For example, that based on the assumption that the majority ofpolymorphisms are maintained by some sort of balancing selection withequilibrium frequency uniformly distributed has a high peak at the neighborhood of x=1jn, where n is the number of alleles maintained in the
population. Therefore, this result supports the neutral hypothesis thatgenetic drift plays the main role in the maintenance of protein polymorphisms
in natural populations.
Genes Affecting Productivity in Natural Populations ofDrosophila melanogaster
Takao K. WATANABE

Two hundred second chromosomes were extracted from the Katsunuma
population in October of 19.72, and the viabilities and productivities ofhomozygotes and heterozrgotes were examined. Viability was measured by

RESEARCHES CARRIED OUT IN 197355

the Cy-method and the productivity by the number of progeny produced
per female. The frequency of lethal-carrying chromosomes was 0.315.
When the average heterozygote viability was standardized as 1.000, theaverage homozygote viability was 0.595 including the lethal lines, and 0.866
excluding them. The frequency of recessive sterile chromosomes among 131
non-lethal lines was 0.092 in females and 0.183 in males. There were two
instances in which homozygosis for the second chromosome caused sterility
in both sexes, which was close to the number expected (2.2) from a randombasis of 0.092xO.183x131. When the average heterozygote productivityof 200 lines was standardized as 1.000, the average homozygote productivity
was 0.532 including femals steriles, and 0.584 excluding them. The ratio
of detrimental load to lethal load was 0.383, while the ratio of partial
sterile load to complete sterile load was 5.767. The average viability oflethal heterozygotes was slightly, but not significantly, lower than that oflethal-free heterozygotes; while the average productivity of lethal heterozygotes was significantly lower than that of lethal-free heterozygotes.
There were no statistically significant differences in viability or productivity
between sterile heterozygotes and non-sterile heterozygotes. The heterozygous effects of viability and productivity polygenes were examined byregressions of the heterozygotes on the sum of corresponding homozygotes.
The regression coefficients were consistently positive for both viability andproductivity if lethal and sterile chromosomes were excluded. The correlation between viability and longevity was significantly positive in homozygotes; the correlation between viability and productivity in homozygotes
was significantly positive when sterile chromosomes were included, but thesignificance disappeared when the sterile chromosomes were excluded. In
the heterozygotes there were no detectable correlations among viability,
longevity and productivity. The homozygous sterile chromosomes in both
sexes had significantly lower viability in homozygotes.
Tabel 1. Average viability and productivity of lethal heterozygotes.
Genotype

No. of crosses

Viability±s. e.

Productivity±s. e.

Non-Iethal/Non-Iethal'
Lethal/Non-lethal
Lethal/Lethal'

90
94
16

1. 0044 ± .0048.9972±.0055.9873 ± .0113

1.0302±.0129.9804 ± .0147*.9462± .0210*** Statistical significance at the 5% level.
** Statistical significance at the 1% level.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Genetic Changes in Natural Populations ofDrosophila melanogaster
Takao K. WATANABE, Taishu WATANABE and Chozo OSHIMA

Annual censuses of genetic variation on the second chromosome havebeen taken in a Japanese natural population for more than ten years; thefrequencies of both recessive lethals and polymorphic inversions have been
examined every fall in the population at Katsunuma. The lethal ferquency
was about 15% in the 1964-1968 period; it increased from 1969 to 1970 to
30%. The frequency doubled in the two years, and the population has
maintained this new lethal frequency in recent years (1970-1973). The
allelism frequency of the lethals did not change so much, showing no
significant correlation with the increased frequency of lethals. The accumulation of mutations other than lethals was also measured by the homozygous genetic load, i. e. the detrimental load (non-Iethals) and the lethalload (lethals). The ratio of detrimental load to lethal load (D/L) was slightly
but not significantly decreased as the lethal frequency increased.
Another sudden change observed in the population was in the frequencies
of chromosomal arrangements. Two polymorphic inversions, In(2L)B and
In(2R)C, had been maintained at high levels of about 35% and 25% during
the early sixties. In 1969 and 1970 their frequencies began to decrease,
until they were reduced by half in the early seventies. Thus, the frequency
Table 1. Frequencies of second chromosome lethals, their allelism
and inversions, and the ratio of detrimentalload to lethal load (D/L).
Year

No. of % of No. of % ofchr.
crosses allelism InB
tested Ie.

% of

In C

ST

1963
1964
1965
1966
1967
1968
1969
1970
1971
1972
1973

668
826
905
323
974
571
201
412
410
351
204

32.0
27.0
35.0
40.5

21.0
25.5
25.5
30.0

53.7
54.4
48.4
41.7

29.0
26.5
15.5
17.0
16.5
13.0

20.5
14.5
14.0
9.0
9.5
12.5

56.4
62.1
71.4
75.1
75.1
74.0

20.2
15.7
15.5
12.4
15.0
15.2
23.1
30.8
29.5
34.8
31.9

6441
3403
8646
1830
6216
3741

2.64
4.20
1.95
2.02
6.44
3.80

6085
3160
1994
666

4.35
1.46
2.96
5.71

D

L

D/L

.128

.167

.767

.121

.166

.729

.232
.256
.221
.213

.366
.349
.428
.385

.634
.734
.516
.553

RESEARCHES CARRIED OUT IN 197357

of inversion-free chromosomes (ST: standard arrangements) increased in the
population as the homozygous load increased. Why this should happen is
unknown. Additional data collected from other Japanese populations(Yamagata, Okinawa) and U. S. populations (Raleigh, Texas) fit very well
the linear regression of lethal frequency on standard arrangement frequency
which was obtained for the Katsunuma population.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

X.

EVOLUTIONARY GENETICS

Mutation and Evolution at the Molecular Level
Motoo KIMURA and Tomoko OHTA

Some consequences of the neutral mutation-random drift hypothesis ofmolecular evolution and polymorphism were worked out with special reference
to the relationships among evolutionary rates, mutation rates, protein
electrophoretic mobilities, functional constraints and molecular structure.
A graphical method was devised to show the pattern of extinction, multiplication and mutational steps involved for selectively neutral mutants ina finite population in the course of evolution. Using the observed mammalian evolutionary rate of protein-encoding cistrons and average heterozygosity for human isozyme polymorphisms, and assuming that the latterare transient states of neutral evolution, it is inferred that, very roughly,
a new allele is substituted in each cistron every 10 million years, eachindividual substitution taking an average of 2 million years. The upper
limit of the evolutionary rate is the mutation rate. Using data on theincidence of rare hemoglobin variants in Japanese populations, it wasestimated that the mutation rate per amino acid site is about 9x10- 8 per
generation. It was shown that in hemoglobins, the rate of amino' acidsubstitutions at the surface parts of the molecules is about 10 times thatin the heme pocket in the course of mammalian evolution, although there
is no indication that mutation rates differ at different parts of the molecule.
For details see Genetics (Supplement) 73: 19-35.

Eukaryote-Prokaryote Divergence Estimated by
5S Ribosomal RNA Sequences
Motoo KIMURA and Tomoko OHTA

In order to estimate the evolutionary distances among 5S ribosomal RNA
sequences, we made the alignment using published data on human, yeast
and bacterial (Escherichia coli and Pseudomonas jluorescens) sequences,and counted the numbers of base differences by comparing them to eachother. From the fraction of different sites between sequences, the average
number of nucleotide substitutions per site that separate the sequences
was estimated. The number turned out to be 0.817 between the eukaryotes
and prokaryotes, whereas it was 0.561 between the human and yeast. The

RESEARCHES CARRIED OUT IN 197359

remoteness of the eukaryotes-prokaryote divergence relative to the humanyeast divergence can be estimated by the ratio 0.817/0.561, which is approximately 1.5. From comparative studies of cytochrome c sequences, the timeof the human-yeast divergence was estimated to be about 1.2x109 years.
Therefore the divergence between eukaryotes and prokaryotes is estimatedto go back to some 1.8X10 9 years. For details see Nature New Biology
243: 199-200.

Slightly Deleterious Mutant Substitutions in Evolution
Tomoko OHTA

The concept of neutral mutant substitution in a population by random
genetic drift can be extended to include random fixation of very slightly
deleterious mutations. In fact, there are a few observed facts whichsuggest that this process is important in molecular evolution. In particular,
Fitch's concept of covarion (concomitantly variable codons) can be understoodreadily by considering the secondary or tertiary structure of the molecule:
If the first mutant disturbs very slightly the normal functioning of themolecule, there are probably many ways of compensating for it and hence
the first mutant provides a new possibility for evolutionary change. A
simple example is coupled base substitutions in the helical region of transfer
RNA. It was pointed out that many facts can be explained by assumingthat the class of mutations with very slight negative effects are important.
For details, see Nature 246: 96-98.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

XI.

HUMAN GENETICS
Fingerprints in Ainu

Ei MATSUNAGA and Ei IKEDA

As part of anthropological studies of the Ainu in Hokkaido, fingerprints were taken from pupils of three junior high schools located in
Niikappu, Urakawa and Piratori townships, in the District of Hidaka.
The material yielded a sample of 923 unrelated subjects, of which 173 wereindentified as Ainu in origin through genealogical investigations carried out
by other members of the team. The rate of admixture with the surrounding Wajin, i. e. the ordinary Japanese in the same environment as the
Ainu, was estimated to be about 30-40%.
It was found that the Ainu had more ulnar loops and less whorls thanthe Wajin, and that they had a significantly larger pattern size of ulnar
loops. In both Wajin and Ainu groups, however, the mean ridge-count ofwhorls per digit was larger in boys than in girls, while no such difference
was found for loops; the result has confirmed the one previously obtainedby us for Honshu Japanese. The distribution of total finger ridge-countin the Ainu showed a marked variation from locality to locality, probably
because of random drift. Details of the studies will be published elsewhere.

Sequence Analysis of Human IgA Immunoglubulin
Tomotaka SHINODA

Amino acid sequence analyses were performed using fragments obtainedby CNBr cleavage at methionyl bond from a completely reduced andalkylated heavy chain of human IgA myeloma globulin. Following thecleavage, four fragments, FI, FIl, FIll and FIV, were separated by column
chromatography in a urea-propionate system. Of these, FIl and FIV were
further purified by ion exchange column chromatographY in phosphateurea-EDTA buffer. FIV had an identical sequence with that of the COOHterminal portion of the alpha chain. FIl was further digested with proteolytic enzyme, and the smaller peptides were separated with a columnof AG 50- x2, AG 1- x2 and by paper electrophoresis at pH 3.7. FI wasa large fragment and contained the total carbohyrates of the intact alphaheavy chain; therefore it was concluded that FI covered almost the entire

RESEARCHES CARRIED OUT IN 1973

61

Fdc-Fc region. Several CMC-containing peptides were isolated from FI
after tryptic digestion. Among them was a proline-rich peptide, which
was later identified to be the hinge peptide. Several other CMC-peptides
were also completely or partially sequenced but the order of each peptide
in FI has not been determined. The results are summarized as follows:
FII, Glu-Val-Gln-Leu-Val-Glu-Ser-Gly-Gly-Gly-Leu - Val- Lys - Pro -Gly -GlySer -Leu - Arg -Leu - Ser - Cys - Ala - Ala-Ser-Gly-Phe-Thr-Phe-Thr-Arg-Gly-GlyLeu-Glu-Trp-Val-Arg-Gln-Ala-Pro-Gly-Lys- Ala - Leu -Glx -Trp- Val- Leu - ValPhe- Ser - Val-Thr -Gly- Asx -Asx-Lys-Phe-Tyr-Thr-Glu-Ser-Leu-Asn-Gly-ArgPhe-Thr-Ile-Ser-Arg-Asx-Asx-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Glx-Met ; FIV,
Ala-Glx-Val-Asx-Gly-Thr-Cys-Tyr; hinge, His-Pro-Thr-Asn-Pro-Ser-Glu-AspVal (Thr, Val, Pro, Cys, Pro, Val, Pro, Ser, Thr, Pro, Pro, Thr, Pro, Ser,fu,~,T~,fu,fu,T~,Pro,~,Pro,~,C~,~,~,Pro,CH~­

Arg; FIT1, Leu-Ser-Leu (His, Arg. Pro, Ala, Leu, Glx, Asx, Leu, Leu, Leu,
Gly, Ser, Glx, Ala, Asx, Leu, Thr, Cys, Thr, Leu, Thr, Gly, CHO) LeuArg; FIT2, Asx-Phe (Pro, Pro, Ser, Glx, Asx, Ala, Ser, Gly, Asx, Leu,
Tyr, Thr, Thr, Ser, Ser, Glx, Leu, Thr, Leu, Pro, Ala, Thr, Glx, Cys, Leu,
Ala, Gly, Ser) Lys; FIT3, Asx-Leu-Cys-Gly-Cys (Tyr, Ser, Val, Ser, Ser,
Val, Leu, Pro, Gly, Cys, Ala, Glx, Pro, His, Gly, Asx) Ly~; FIT4, GlyAsx-Thr-Phe-SerJCys-Met; FIT5, Ser-Val-Thr (Cys, His, Val) Lys.
In addition to these peptides there were a few CMC-peptides, but they
were not pure enough for sequence study.

Isozyme Variations in Man
Tomotaka SHINODA, Ei MATSUNAGA and Jushiro KOSHINAGA*
Recent developments in biochemical analyses have made it possible to assessthe degree of genetic variation of individuals to a much more precise level
than a decade ago. Using tissue extracts, we have undertaken a survey
on a variety of enzymes by means of gel electrophoresis and specific staining.
Last year we analyzed 16 different enzyme systems on 64 samples, andfound that 6 loci were polymorphic. This time we have tested 25 different
loci on a total of 56 tissue samples collected in Tokyo. Samples wererelatively fresh and there existed hardly any problems of secondary isozymes
due to in vivo modifications.
Of 25 different loci tested, 7 were found to be polymorphic with practically the same frequencies as in the previous study. They are: adenosine* Medical Examiner's Office, Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

deaminase (ADA 1, 54; ADA 2-1, 2; total 56), alcohol dehydrogenase (ADH s
1, 20; ADH s 2-1, 27; ADH s 2, 9; total a6), 6-phosphogluconate dehydrogenase (PGD A, 49; PGD AC, 7; total 56), phosphoglucomutase-l (PGM 1
1, 35; PGM 1 2-1, 19; PGM 1 2, 2; total 56), phosphoglucomutase-3 (PGM s
1, 36; PGM 2-1, 18; PGM s 2, 2; total 56), s-glutamate-pyruvate transaminase (GPT 1, 22; GPT 2-1, 25; GPT 2, 9; total 56), m-glutamateoxaloacetate transminase (GOT 1, 49; GOT 2-1, 7; total 56).
In addition to these enzyme systems, a mitochondrial tetrazolium oxidase
was found to exist in multiple forms (m-TOX 1, 55; m-TOX 2-1, 1; total
56). NADP-dependent isocitrate dehydrogenase, aldehyde dehydrogenase
and xanthine dehydrogenase were also found to be in multiple forms. Thesephenomena, however, are not likely to exist in genetic origin but in secondary
modifications caused by, for example, allosteric effect due to unbinding/binding with coenzyme.

Amplieon Model for Antibody Variability
Tomotaka SHINODA

Among the most fascinating and challenging problems in mammalian
genetics, the nature of the genetic control of antibody variability is yet tobe solved. Vertebrate organisms are believed to be endowed with theability to synthesize thousands of different species of antibodies, eachpresumably encoded by a different antibody gene. So far two major linesof hypotheses have been proposed concerning the mechanism of antibody
variability: the germ line and somatic theories. The somatic theory
postulates that antibody genes arise by appropriate mutational events
during somatic differentation, whereas the germ line theory claims thateach organism has a separate germ line for each antibody polypeptide
chain. Both theories appear to have paradoxes within themselves fromthe standpoint of basic biological economics.
To escape the paradox, an alternate model referred to as "Ampliconmodel" is proposed for antibody variability, based on our own and other'ssequence data on immunoglobulins. The basic idea of the model is asfollows:
Since the total number of germ line genes in haploid mammalian species
may not, on the average, exceed "-'106 , and since the organisms can hardly
spare genes only to code antibody molecules, the maximum number of thegenes which code for antibodies should be far less than the above figure.
If we follow the assumption that each antibody polypeptide chain, both

RESEARCHES CARRIED OUT IN 1973

63

PSEUDOCISTRON
VkI
VkII
VkIlI
Ck
S ' 1 - -- - X 's' 1- - -- y1-- - - Z •S '---------'T S!
~1I1!~lIi!~!I!11 ~i212~lI22pm4 ~. f" +-1 I
~

's'

DNA

-A~

DNA
COPIESn COPIESPARALLEL GENEAMPLIFICATIONMAYGIVE RISETO'" 10 2 COPIES( AMPLICON )Fig. 1. Amplicon model for antibody variability.

light and heavy, is encoded by a set of separate genes-V and C genes-thenat least 14 different genes may be assumed to be in the constant regionof antibodies. As for the variable region, it has become increasingly clearthat the first 110 residues of both light and heavy chains can be classified
into subgroups according to their sequence similarity. If it is assumed
that each V-region subgroup is encoded by a certain number of germ line
genes, ~ 10 for example, then the total number of the germ line genes
for antibodies may be on the order of ~102. This figure corresponds tono more than 1/1000 of the total informational part of the genetic materialof mammalian genome, if we follow an assumption that only a few percent of the genetic material is informationally active in mammalian organisms. The idea is illustrated in Fig. 1, where stacks of a certain numberof repeated DNA segments are oriented in the order within each subcistron
which constitutes a "pseudocistron" together with other subcistrons ofdifferent subgroups. Each subcistron consists of a set of different but
similar DNA segments of about 400 nucleotide pairs. As for light chains,the model postulates that each subcistron can produce a number of copies
called" amplicon" which later undergoes genetic fusion with the C gene(s)within the same pseudocistron. In the case of the heavy chains, the model
requires at least 5 sets of the C genes, and these genes can fuse randomly
with anyone of the genes of any subgroups to form a complete stretchof antibody gene.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

Frequencies of C and Q Variants in Human Chromosomes
Kazuso IINUMA and Ei MATSUNAGA

Unrelated individuals with normal karyotypes, a total of 27 males and
28 females, have been studied with C and Q banding methods. Their
karyotypes have further been characterized by distinctive patterns of C
and Q polymorphisms in those regions defined as variable in the Paris
Conference (1972). After C banding treatments, two sizes of heterochromatin blocks in secondary constrictions were observed. The large one,
appearing as the most frequent type, was designated as 'C', and theother infrequent one as 'c '. Frequencies of 'c' of #1, 9 and 16 chromosomes were 0.18, 0.19 and 0.07, respectively. Fluorescence intensity of theparacentric regions was determined of #3, 4, 13, 14, 15, 21 and 22 chromosomes. Strongly fluorescing ones were designated as 'F', and weakly
fluorescing ones as 'f'. Frequencies of 'F' were 0.58, 0.31, 0.48, 0.14,
0.02, 0.12 and 0.20, in orders of the chromosomes mentioned above. For
each of these chromosomes, the expected incidences of 'CC' or 'FF',, Cc' or 'Ff' and 'cc' or 'ff', were calculated from the frequencies, andthe values obtained were compared with those observed in our samples.
No significant discrepancy (p<0.05) was seen in any chromosome except# 4 and 13. Q variants of # 4 are in fact difficult to distinguish from eachother because both of them have a strongly fluorescing band adjacent tothe very variable region near the centromere. However, Q variants of # 13
are much more easily identified, because of the absence of any confusing
segment around the region, and of a clear-cut appearance of ' F' in contrast
to 'f'. The implication of this discrepancy observed in #13 is not clear.
In addition, 5 patients with regular 21 trisomy and their normal parents
were studied of their Q variants of #21 chromosomes. In one family, it
could be safely denied that the non-disjunction had occured in the second
stage of maternal meiosis.
A part of this study was reported in the 18 th annual meeting of theJapan Society of Human Genetics (1973).

RESEARCHES CARRIED OUT IN 1973

XII.

65

BEHAVIORAL GENETICS

Short-Term and Long-Term Memories in Different
Strains of Inbred Mice
Tohru FUJISHIMAThis experiment was designed to obtain basic information for a selection
experiment on the learning ability of mice. Adult mice of 15 inbred
strains, each comprising 10 males and 10 females, were measured for rightleft discrimination ability, conditioned avoidance and escape responses, andactivity using a Y-maze apparatus in which lamp and buzzer were used asconditioned stimulus and electric shock as unconditioned stimulus. Twosessions, each consisting of 50 trials, were carried out at 24 hour intervals,
and the short-term and long-term memories were measured in percentage.
The long-term memory was calculated as the difference between the performance in the first session (denoting short-term memory within the sameday) and that in the second session.
The results indicated that there were significant differences in all charactersamong the strains. As to the short-term and long-term memories in discrimination ability, respectively, C3H/He and C57BL/6 was high and high,D103 low and low, C57L, C57BR, DBA/2 and DBAf/Lw low and high, andSWM and RF high and low. In general, related strains showed similar
performances in learning ability.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

XIII.

APPLIED GENETICS

Analysis of Genes Controlling F 1 Sterility in Riceby the Use of Isogenic Lines
Hiko-Ichi OKA

In order to look into the genetic basis of intervarietal F , sterility inrice (Oryza 8ativa L.), a series of backcrosses (up to B ,S ) was carried out
using Taichung 65 (Japonica type) as the recurrent parent and several
Indica varieties as donor parents. A number of "isogenic F , -sterile lines"
were isolated by test-crossing fertile F 2 plants obtained from the selfingof partly pollen-sterile backcross segregants. Crossing experiments with
the isogenic lines confirmed the author's previous hypothesis that there
are sets of duplicate gametic lethals (8 genes) and gametes carrying adouble recessive combination (8 , 8 2 ) of them deteriorate during development:
though in the present hypothesis the genes are considered to affect thedevelopment of microspores only. Assuming that Taichung 65 has 8,181 +2/+2
and a donor parent as well as an isogenic F , -sterile line derived from it
have +,1+182/82, pollen grains with +,82 have shown in the genetic background of Taichung 65 a higher fertilizing capacity than those with 8 , + 2 ,while those with +, +2 have a lower fertilizing capacity. This certational
advantage of alien genes was considered to be an internal mechanism that
helped the development of F , sterility relationships among rice varieties.
The isogenic F , -sterile lines derived from different donor parents each had
a set of 8 genes at different loci. Linkage relations were detected between
the 8 loci and three gene markers. (Genetics, in press)

Breeding of Isogenic Lines of Rice Carrying Various Gene
Markers and Reciprocal Translocations
Hiko-Ichi OKA and Hiroko MORISHIMAThis work has been continued for 10 years though not reported in previous
issuse of the Annual Report. Isogenic marker lines were isolated from theselfing of heterozygotes backcrossed to the recurrent parent 7 to 16 times.
Donor parents carrying different markers were obtained through the kindnessof Dr. M. Takahashi of the Hokkaido University, and Taichung 65 (T65,
a Japonica type from Taiwan) was used as the recurrent parent throughout

RESEARCHES CARRIED OUT IN 197367

the experiments. The marker genes so far isolated in isogenic lines are:wx (glutinous endosperm, 1st chromosome), d 2 (Ebisu dwarf, 2nd), 19 (liguleless, 2nd), Ph (phenol reaction, 2nd), Rd (red pericarp, 3rd), Re (brown
pericarp, 4th), d 1 (Daikoku dwarf, 6th), la (lazy habit, 8th), ne (bract
development, 9th), be (brittle culm, 11th), gb (glabrous leaf, 12th), etc.
The pleiotropic effects of these genes are under observation.
Further, 30 isogenic lines of T65 carrying different reciprocal translocations were obtained from the progeny of radiation-induced semi-sterile
plants after recurrent back-crossing (3 times) and test-crossing. Whichchromosomes are involved in each of them is still under observation. As
these lines will be of some use for rice geneticists, their seeds are offeredto the workers who need them.
Character Variation and Inheritance of Sterility in the
Backcross Progenies of a Species Hybrid in Rice
Hiko-Ichi OKA and Hiroko MORISHIMAThe F 1 hybrid between two cultivated rice species, Oryza sativa L. andO. glaberrima Steud. (highly pollen sterile though meiotic chromosome
pairing is normal), was backcrossed by the parental strains, and the characters distinguishing between the parental species and pollen fertility wererecorded for BnF1 and B nF 2 plants. This experiment has been continued
since 1966, and the B 8 F 2 plants were observed this year. The pattern ofcharacter variations as shown by correlation coefficients differed according
to the genetic background of the plants estimated by the coefficient ofrelationship to one of the parents. As to the genetic basis of sterility, it
was suggested that some unfavorable interaction between the sporophytic
and gametophytic effects of particular genes could be the major factor
causing the deterioration of microspores. Further experiments on thegenetic basis of this hybrid sterility are under way.
Phenotypic Plasticity and Yield Stability in Rice Cultivars
Hiroko MORISHIMA and Hiko-Ichi OKA

Our part as coworkers of the International Rice Adaptation Experiment(IBP/UM) was to look into varietal variations in the plasticities of grain
yield and yield-component characters. The data studied C1l-me from testsof 10 rice varieties of different origins at seven experimental stations

68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

distributed in Japan, Taiwan, India, and U. S. A. which were continued for
two years (1971 and 1972; Phase B). Though the details will be publishedelsewhere, one point suggesting the nature of genotypic difference in yieldstability is presented here. An examination of interrelationships between
the standard deviations of various agronomic characters indicated thatplasticity in a developmental stage could be associated with stability inthe subs9quent' stage. Thus, yield stability appeared to depend on a certain
degree of plasticity in the tillering stage and non-plasticity in later stages.
It was also pointed out that successive selection for better yield of hybridmaterials raised in different cropping seasons, called "disruptive seasonal
selection", was an effective method for improving yield stability.

Intervarietal Variations in Growth Pattern Obtained fromthe International Rice Adaptation Experiment
Hiroko MORISHIMA and Hiko-Ichi OKA

Variations in logistic growth curve, among rice varieties were investigatedusing dry-matter weight data from the International Rice Adaptation
Experiment (IBP/UM). Two major trends of variation were detected, one
represented by the developmental stage at which growth rate becomesmaximum (variation between" early- and late-vigor" types), and the otherrepresented by the durability of a high growth rate (between "sustainedand dash-vigor" types). These variations were found to be controlled byvarietal genotypes. Early vigor contributes to the production of many
spikelets, and late vigor to filling the spikelets with grain. Sustained-vigor
types gave more grain yield than dash-vigor types. The magnitude ofchange in these vigor types due to fertilizer application also appeared to
be a varietal characteristic.

Variation in Weediness Among Strains of Dallisgrass
Hiroko MORISHIMA

Seeds were collected from several natural populations of dallisgrass(Pasupalum dilatatum) at different sites in Kyushu and in the Misima

area, and the plants from the seeds were examined with the hope of
demonstrating ecological characters supporting the weediness of this species.
Observations were made in an experimental field for variations in metriccharacters, the response to walking-over by man, density response, andRESEARCHES CARRIED OUT IN 197369

competition with a closely related native species, P. thunghergii. Dallisgrass produces its seeds mainly by apomixis. As expected from this breeding behavior, strains from different populations did not differ much incharacters though the differences were partly significant statistically. The
response to walking-over by man significantly differed among strains.
When mix-grown with P. thunghergii, dallisgrass was advantageous at alow planting density but was disadvantageous at a higher density. The
weediness of dallisgrass as compared with other weed species is still underobservation.

Genic Analysis for Peroxidase Isozymes and Their Organ
Specificity in Oryza perennis and O. sativa
Chiang PAl, Toru ENDO and Hiko-Ichi OKA

Peroxidase zymograms of the leaf blade, leaf sheath and lemma-palea
were observed in progeny of crosses between strains of Oryza perennis
Moench and O. sativa L. Bands OC, 2A, and 4A were found to be specified by codominant alleles, Px?C, PX;A, and Pxi A, respectively. These
alleles produced hybrid bands lA and 3A in their heterozygotes. Regarding
band 4C specified by PxjC (independent of PXl), it was found that aperennis strain (WI20-4, derived from an Indian natural population) had
the band in the leaf sheath only, while another (WI20-5, from the same
population) showed it in the leaf blade only. Observations of their selfed
progenies and hybrids suggested that there were two independent dominant
genes repressing band 4C, one (R L B 4C) acting in leaf blade and the other(R LS 4C) acting in leaf sheath. On this basis, the organ-specific intensity
variations of the 4C band observed in four different crosses could also be
explained. These genes were considered to be "regulatory genes" repressing the activity of Pxi c •(Published in Can. J. Genet. Cytol. 15: 845-853, 1973)

Intergenotypic Competition and Cooperation in
Mix-Planting of Rice Strains
Tsuguhiro HOSHlNO and Hiroko MORlSHlMA

A series of mix-planting experiments was conducted with various rice
varieties and hybrid-derived lines to study intergenotypic competition andcooperation in mix-planting of rice strains. Three different models given

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

by P. Jacquard and J. Caputa (1970, Ann. Amelior. Plantes 20: 115-158)
were employed. They were: 1) evaluation of aggressiveness and resistanceto aggression, 2) analysis of "domination matrix", and 3) estimation ofregression coefficient on mean values for various combinations of mixplanting. In addition to the strong effect of competition, cooperation
effect as shown by a gain or a loss in both mix-planted genotypes wasdemonstrated in certain combinations of strains. The detail will be publishedelsewhere.

Variations in Isozymes and Other Characters Among Fa Lines
Derived from Oryza perennis X O. sativa Hybrids
Chiang PAl and Hiko-Ichi OKA

The Asian strains of Oryza perennis Moench, the wild progenitor of
O. sativa L., carry a number of different alleles for peroxidase and acidphosphatase variations. However, only a small part of them is foundamong cultivars of O. sativa. Why were many isozyme alleles lost when
the wild plants were domesticated? In order to look into this problem,
142 Fa lines from 6 perennis-sativa crosses which had different isozyme
alleles were observed for three characters distinguishing between wild andcultivated forms of rice, i. e., seed shedding, rachis angle, and awn length.
No particular linkage relations were found, however, between genes for
those characters and isozymes. The problem should now be consideredfrom a different angle.

Further Investigations of Geographical Variationin Isoperoxidase in Populations ofPinus Thunbergii
Shin-ya IYAMA

Variation in isoperoxidase of Pinus Thunbergii populations was investigated in the area along the Japan Sea coast. Needle leaves were collectedfrom about 40 trees randomly selected from each of six local populations
in Toyama, Ishikawa and Fukui prefectures. Sap squeezed from the needle
leaf was used for electrophoretic study of isoperoxidase variation. Incidencesof several bands of peroxidase isozyme were investigated. Comparing theresult with those obtained previously for the populations in Eastern andWestern Japan, the populations used for the present study were similar toRESEARCHES CARRIED OUT IN 1973

71

those from Eastern Japan and had lower frequencies of the bands thanthose in Western Japan. For example, the average frequency of band Q
in the present populations was 8.8%, while those in Shizuoka and Niigata
prefectures (Eastern Japan) were 24% and 21%, and those in Hiroshima
and Tottori prefectures (Western Japan) were 89% and 62%, respectively.

Gamma-ray Induced Increase in Intra-Line Variability in
Arabidopsis thaliana
Sujit BAGCHI and Shin-ya !YAMA

We reported before (Ann. Rep. N. 1. G. No. 23) an increase in intra-line
variability in M, generation due to gamma-ray irradiation. To clarify if
this increase was due to developmental instability or merely genetic segregation within line, further investigations were conducted in the M r and M sgenerations which were obtained by the "one plant one offspring" scheme.
For the present investigation, about 100 plants were selected at random
from each of the M s populations (Control, 20 kR and 80 kR; each consistingof about 1000 plants), and M r lines each consisting of approximately 20
plants were observed. Measurements were taken on days to first floweringand plant height at 40 th day, on a single plant basis. The results ofanalysis of variance showed that (1) radiation induced little change in
population means except for days to first flowering in 20 kR populations;(2) inter-line variation was increased by irradiation, and the irradiatedpopulations had larger genetic variances than the control; (3) intra-line
variability in both characters increased in the irradiated populations, particularly in the 80 kR population; (4) no significant correlation was foundbetween the line mean and within-line variability in 80 kR population. Since
the parental M 6 plants were assumed to be almost homozygous on accountof self-pollination, increased intra-line variability was considered to be due
to induced mutations that increased developmental instability. To reconfirm
Table 1. Estimated mean, genetic and environmental variances for
two quantitative characters of Arabidopsis thaliana
Plant height (em)Days to flowering
Population
Control
20kR
80kR

Mean

ag 2

ae

28.53
24.02
29.89

0.0196
0.07140.03701.12851.50932.3501

2

Mean

ag 2

ae 2

24.00
26.82
22.33

0.07130.07392.36611.6193
1.53662.8499

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

this, a selection experiment was conducted. M 7 lines showing high andlow intra-line variability for plant height were selected from 80 kR population, and the intra-line standard deviation of their progeny lines wasinvestigated. Selection was effective and estimated heritability (0.34) was
found to be significant. It may be concluded that irradiation has induced
genetic changes not only in quantitative characters but also in their developmental stability.

Genetic Changes of Japanese Wild Quails Under
Domestic Conditions
Taka tada KAWAHARA, Akihiko MITA, Masami SAITO
and Norio SUGIMOTOThis experiment was carried out to find the genetic changes in Japanesewild quails reared for several generations under domestic conditions.
Examinations were made on several characters of the quails which had
been reared without any artificial selection for five generations since they
were captured in the field.
The results indicated that the most striking changes of the birds toward
the domestic types occurred in time of sexual maturity (which was earlier),
and in fertility, hatchability, survival rate, egg production rate, bodyweight and fitness index (which all increased). A decrease in the variabilityof characters was also observed in progressive generations.

Interstrain Variation in Gamma-ray Induction of Hindlimb
Deformities in Chick Embryos
Takatada KAWAHARA

Gamma irradiation of eggs at a very early stage of embryonic development produced facial anomalies, and at 48 to 60hr of incubation induced
deformities in the hind- and forelimbs. Strains differed in the frequency anddegree of deformities in hindlimbs. Barred Plymouth Rock showed the lowest
frequency, and White Leghorn the highest of the three breeds tested. The
reciprocal F 1 hybrids between the two breeds, when irradiated, showedthat the incidence of deformities could be maternally determined. In the
backcrosses, however, the paternal genotype had a strong influence on theincidence. The Nagoya breed showed an incidence intermediate between
the above two breeds. Its reciprocal F 1 hybrids with White Leghorn

RESEARCHES CARRIED OUT IN 197373

showed transgressive matrocliny, and a maternal effect was apparent in
the backcrosses. T}1e heritability estimate for the frequency of hindlimb
deformities was 0.303 on the average. The incidence and the magnitudeof hindlimb deformities were highly correlated. The incidence was negatively correlated with the number of somites at 48 hr of incubation, but was
uncorrelated with shell thickness.
(Published in Radiation Research 57, 332-341, 1974)
Serum Amylase Isozymes in the Japanese Quail
Takatada KAWAHARA, Seiki WATANABE and Haruhiro YOSHIDA

The sera from a domestic and a wild strain of Japanese quails, andtheir F 1 hybrids, were examined for amylase isozymes by the use ofpolyacrylamide gel electrophoresis.
Seven anodal bands were detected and were classified into three groups:
Regions I, II and III. Observing 641 birds of the three strains to determine
the gene frequencies for two bands appearing in Region II, there were
three phenotypes, AA (a fast band), AB and BB (a slow band) which were
controlled by autosomal codominant alleles called Amy-1 A and Amy-p.
The frequency of Amy-p was generally higher than that of Amy-P. The
frequencies of Amy-P were 7.7% in the wild and 21.7% in the domestic
strain.

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

BOOKS AND PAPERS PUBLISHED IN 1973
BY STAFF MEMBERS(1) CHOO, J. K. and K. NAKAMURA 1973. On a new species of Drosophila(8ophophora) from Japan, (Diptera). Kontyu 41: 305-306.
( 2 ) ENDO, T. 1973. Isozyme loci and a strategy of differentiation in plants.
Seiken Zih6 24: 89-104.
(3) FURUICHI, Y. and K. MIURA 1973. Identity of the 3f -terminal sequences inten genome segments of silkworm cytoplasmic polyhedrosis virus. Virology
55: 418-425.
(4)* GEFTER, M. L., T. KORNBERG, I. J. MOLINEUX, H. G. KHORANA, L. MINDICH
and Y. HIROTA 1973. Studies on DNA polymerases II and III of E. coli.
"DNA synthesis in vitro" (ed. by Wells, R. D. and R. D. Inman), Univ.
Park Press; 71-82.
(5)* HIROTA, Y. 1973. Analysis of cell division and DNA replication in E. coliby a genetic method. Folia Microbiol. 18: 269.
(6) IINUMA, K. and Y. NAKAGOME 1973. Fluorescence of Barr body in humanamniotic-fluid cells. Lancet. 1: 436-437.
(7) IINUMA, K., Y. NAKAGOME and I. MATSUI 1973. 21 trisomy and prenatallydiagnosed XXY in two consecutive pregnancies. Human Hered. 23: 467469.
(8) KADA, T. 1973. Escherichia coli mutagenicity of furylfuramide. Jap. J.
Genet. 48: 301-305.
(9) KADA, T. 1973. Mutagenicity testing of chemicals in microbial systems.
(In Japanese) "New Methods in Environmental Chemistry and Toxicology",
Int. Acad. Print. Co., Tokyo: 127-133.
(10) KADA, T. 1973. Cellular metabolization of chemical mutagens. (In Japanese)Adv. in Med. Sci. (Igaku-no-Ayumi) 84: 776-782.
(11) KADA, T. 1973. DNA repair of damage induced by ionizing radiation. (InJapanese) Adv. in Med. Sci. (Igaku-no-Ayumi) 87: 613-614.
(12) KADA, T. 1973. Recent remarks on the detection of potential carcinogens.
(In Japanese) Adv. in Med. Sci. (Igaku-no-Ayumi) 85: 492-498.
(13) KADA, T., Y. SADAIE and Y. SHIRASU 1973. Detection of frameshift mutagens
in pesticides by "rec-assay" procedures (abstract). Mutation Res. 21: 207.
(14) KATO, H. 1973. Induction of sister chromatid exchanges by UV light andits inhibition by caffeine. Exp. Cell Res. 82: 383-390.
(15) KATO, H., T. SAGAI and T. H. YOSIDA 1973. Stable telocentric chromosomes
produced by centric fission in Chinese hamster cells in vitro. Chromosoma(Berl.) 40: 183-192.

* Contributions from other institutes.

BOOKS AND PAPERS PUBLISHED IN 197375(16) KAWAHARA, T. 1973. Comparative study of quantitative traits between
wild and domestic Japanese quail (Coturnix coturnix japonica). Exp.
Animals 22: Suppl. 139-150.
(17) KAWAHARA, T. 1973. Wild species and Iivestocks-especially on domestic fowls.
(In Japanese) Iden 27: 16-20.
(18) KIMURA, M. and T. OHTA 1973. Mutation and evolution at the molecularlevel. Genetics. 73: Suppl. 19-35.
(19) KIMURA, M. and T. OHTA 1973. Eukaryotes-prokaryotes divergence estimatedby 5S ribosomal RNA sequences. Nature New BioI. 243: 199-200.
(20) KIMURA, M. and T. OHTA 1973. The age of a neutral mutant persisting ina finite population. Genetics. 75: 199-212.
(21) KUMAGAI, M., Y. KIKUCHI, H. OISHI, E. MATSUDA and J. KATO 1973. A casewith short arm deletion of chromosome 18 (18 p- syndrome). Jap. J. Human
Genet. 18: 24-36.
(22) KURODA, Y. 1973. Macromolecular requirements of embryonic Drosophila
cells in culture. Proc. 3rd Int. Colloq. on Invertebrate Tissue Culture (ed.
by J. REHACEK et al.); 187-194, Publ. House of the Slovak Acad. Sci.
(Bratislava).
(23) KURODA, Y. 1973. Differential inhibition of histoformative aggregation of
rat hepatoma cells in culture by concanavalin-A. Gann. 64: 555-561.
(24) KURODA, Y. 1973. Growth and phenotypic expression of embryonic cells
from Drosophila melanogaster in cell culture. (abstract). Genetics 74: s147148.
(25) KURODA, Y. 1973. Mutagenesis to 8-azaguanine resistance in cultured human
diploid cells. (abstract). Mutation Res. 21: 226.
(26) KURODA, Y. 1973. Effects of amino sugars on the adhesiveness of rat
hepatoma cells in rotation culture. (In Japanese) Symp. Cell. BioI. 24: 29-38.
(27) KURODA, Y. 1973. Cytoplasm and evolution. (In Japanese) "Heredity of
Nucleus and Cytoplasm" (ed. by C. OSHIMA et al.): 163-194, Asakura Shoten(Tokyo).
(28) KURODA, Y. and M. HORIKAWA 1973. Mutations in cultured mammalian cells.
(In Japanese) "Procedures for Detecting Mutations Induced by Chemicals"(ed. by Y. TAZIMA et al.): 97-117, Kodan-sha Scientific (Tokyo).
(29) MARUYAMA, T. 1973. Isolation by distance, genetic variability, the timerequired for a gene substitution and local differentiation in a finite, geographically structured population. "Population Genetics Monographs" (ed.
by N. E. MORTON) vol. 3: 80-81, Univ. Press of Hawaii (Hawaii).
(30) MARUYAMA, T. 1973. Diffusion model for geographically structured populations. "Population Genetics Monographs" (ed. by N. E. MORTON) vol. 3: 4044, Univ. Press of Hawaii (Hawaii).
(31) MARUYAMA, T. 1973. The variance of the number of loci having a given
gene frequency. Genetics 73: 361-366.

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

MARUYAMA, T. 1973. The substitutional load and mutational load in a finite
population. Evolution 27: 95-99.
(33) MARUYAMA, T. 1973. Diffusion models and Brownian motion in population
i'enetics. Jap. J. Genet. 48: 231-234.
(34) MARUYAMA, T. 1973. Some theoretical aspects of isozyme polymorphism
viewed from the standpoint of population genetics. Seiken Ziho 24: 75-88.
(35) MATSUDA, E. 1973. Genetic studies on total finger ridge-count among Japanese. Jap. J. Hum. Genet. 17: 293-318.
(36) MATSUNAAGA, E. 1973. Introduction to the genetics of cardiovascular diseases. Jap. Circul. J. 37: 33-34.
(37) MATSUNAGA, E. 1973. Effect of changing parental age patterns on chromosomal aberrations and mutations. Social BioI. 20: 82-88.
(38) MATSUNAGA, E. 1973. Heredity and marriage of mental defectives. (InJapanese) Seishin-Hakujakuji-Kenkyu 172: 18-24.
(39) MATSUNAGA, E. 1973. Gene mutation and birth defects. (In Japanese) Igakuno-Ayumi 84: 748-752.
(40) MATSUNAGA, E. 1973. Biological consequences of a stationary population.
(In Japanese) Sanfujinka-no-Sekai 25: 935-939.
(41) MATSUNAGA, E. 1973. Genetic effects of environmental pollution. (In Japanese) Popular Medicine, Suppl. 4: 209-313.
(42) MORIWAKI, D. and Y. TOBARI 1973. Spontaneous male crossing-over of frequent occurrence in Drosophila ananassae from Southeast Asian populations.
Jap. J. Genet. 48: 167-173.
(43) MORIWAKI, K. 1973. Serum transferrin polymorphism in black rats collectedfrom Asia and Oceania. (abstract). Genetics 74: s 185-186.
(44) MORIWAKI, K., K. TSUCHIYA and T. H. YOSIDA 1973. Breeding and geneticsof rat, Rattus rattu8. Exp. Animals 22: Suppl. 211-220.
(45) MURAKAMI, A. 1973. A comparison of mutation induction by 14 MeV fastneutrons and mcs r-rays in meiotic spermatocytes in the silkworm. Mutation Res. 17: 73-80.
(46) MURAKAMI, A. 1973. Mutagenesis of acridine orange in mitotic cleavage
nuclei of the silkworm, Bombyx mori. Mutation Res. 20: 67-70.
(47) NAKAGOME, Y. 1973. G-group chromosomes in satellite associations. Cytogenet. Cell. Genet. 12: 336-341.
(48) NAKAGOME, Y., K. IINUMA and K. TANIGUCHI 1973. Points of exchange ina human no. 5 ring chromosome. Cytogenet. Cell. Genet. 12: 35-39.
(49) NAKAGOME, Y., K. IINUMA and I. MATSUI 1973. Three translocations involving C-or G-group chromosomes. J. Med. Genet. 10: 174-176.
(50) NAKAGOME, Y., K. IINUMA and I. MATSUI 1973. Trisomy 10 with mosaicism.
A clinical and cytogenetic entity. Jap. J. Human Genet. 18: 216-219.
(51) NOGUTI, T., Y. SADAIE and T. KADA 1973. Biochemical mechanisms of repair
of EAN damage induced by ionizing radiation (abstract). Genetic'S 74: Suppl.
s 19"8.

(32)

BOOKS AND PAPERS PUBLISHED IN 1973

77(52) OHTA, T. 1973. Slightly deleterious mutant substitutions in evolution. Nature
246: 96-98.
(53) OHTA, T. 1973. Effect of linkage on behavior of mutant genes in finite
populations. Theor. Pop. BioI. 4: 145-162.
(54) OHTA, T. and M. KIMURA 1973. A model of mutation appropriate to estimate
the number of electrophoretically detectable alleles in a finite population.
Genet. Res. Camb. 22: 201-204.
(55) OKA, H. I. 1973. Performance in Central Luzon of soybean varieties selected
in Taiwan for wide adaptability. SABRAO Newsletter. 5(1): 29-38.
(56) OSHIMA, C. 1973. Noise and light environments accelerating the developmentof Drosophila flies. (In Japanese) Sci. Amer. (Japanese edition) 3(6): 47-49.
(57) OSHIMA, C. 1973. The role of the insectron and its future prospects. (InJapanese) Bioteck 4(5): 389-395.
(58) OSHIMA, C. and T. K. WATANABE 1973. Fertility genes in natural populationsof Drosophila melanogaster. I. Frequency, allelism and persistence of sterility
genes. Genetics 74: 351-361.
(59) PAl, C., T. ENDO and H. I. OKA 1973: Genic analysis for peroxidase isozymes
and their organ specificity in Oryza perennis and O. sativa. Can. J. Genet.
& Cytol. 15: 845-853.
(60)* PUTNAM, F. W., G. FLORENT, C. PAUL, T. SHINODA and A. SHIMIZU 1973.
Complete amino acid sequence of the mu heavy chain of human IgM
immunoglobulin. Science. 182: 287-291.
(61)* PUTNAM, F. W., T. SHINODA, A. SHIMIZU, C. PAUL, G. FLORENT and E. RAFF
1973. Salient structural features of human IgM immunoglobulins. 3rd. Int.
Convoc. Immunol. 40-59.
(62)* RICARD, M. and Y. HIROTA 1973. Effect des sels et autres composes sur Ie
phenotype de mutants thermosenibles d' Escherichia coli. Ann. Microbiol.
(Inst. Pasteur). 124 A: 29-43.
(63)* RICARD, M. and Y. HIROTA 1973. Process of cellular division in E. coli:
physiological study on thermosensitive mutants defective in cell division.
J. Bacteriol. 116: 314-322.
(64)* RYTER, A., Y. HIROTA and U. SCHWARZ 1973. Process of cellular division in
E. coli. Growth pattern of E. coli. murein. J. Mol. BioI. 78: 185-196.
(65) SADAIE, Y. and T. KADA 1973. Radiation inactivation and recombination
repair in Bacillus subtilis spores. Mutation Res. 17: 138-141.
(66) SHAMA RAO, H. K. and T. FUJII 1973. Biological effect of high LET radiations after post-irradiation storage or with low and high LET combination
treatments in rice. Jap. J. Breed. 23: 121-124.
(67) SHIBUYA, T. and Y. KURODA 1973. Studies on growth and differentiation of
cartilage cells from Creeper chick embryos in culture. Jap. J. Genet. 48:
197-205.
(68) SHIMOTOHNO, K. and K. MIURA 1973. Single-stranded RNA synthesis in vitro
by the RNA polymerase associated with cytoplasmic polyhedrosis virus con-

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

taining double-stranded RNA. J. Biochem. 74: 117-125.
(69) SHIMOTOHNO, K. and K. MIURA 1973. Transcription of double-stranded RNAin cytoplasmic polyhedrosis virus in vitro. Virology. 53: 283-286.
(70) SHINODA, T. 1973. Amino acid sequence of a human kappa type Benec-Jones
protein I. J. Biochem. 73: 417-431.
(71) SHINODA, T. 1973. Amino acid sequence of a human kappa type Benec-Jones
protein II. J. Biochem. 73: 433-446.
(72) SHINODA, T. 1973. Amino acid sequence of the first 65 residues of IgA
myeloma protein. Biochem. Biophys. Res. Comm. 52: 1246-1251.
(73) SHINODA, T. 1973. Structural study of human IgA myeloma protein (abstruct).
Genetics 74: s 525.
(74) SHINOHARA, H. and T. MARUYAMA 1973. Evolution of glycoproteins as judged
by the frequency of occurrence of the tripeptides Asn-X-Ser and Asn-X-Thr
in proteins. J. Molec. Evol. 2: 117-122.
(75) SHIRAISHI, Y. and T. H. YOSIDA 1973. Comparative banding pattern analysis
in human chromosome induced by urea and trypsin treatments. Jap. J.
Genet. 48: 11-17.
(76) SUZUKI, H. and T. !INO 1973. In vitro synthesis of phase-specific flagellinof Salmonella. J. Mol. BioI. 81: 57-70.
(77) TAZIMA, T. and K. ONIMARU 1973. An unstable allele of Bombyx mori locatedon the second chromosome piece of the W· II translocation (abstract).
Genetics 74: s 273.
(78) TAZIMA, Y. 1973. Naturally occurring mutagens. (abstract). Mutation. Res.
21: 239-240.
(79) TSUCHIYA K., K. MORIWAKI and T. H. YOSIDA 1973. Cytogenetical survey inwild population of Japanese wood mouse (Apodemus speciosus) and its
breeding. Exp. Animals 22: Suppl. 221-230.
(80) WATANABE, T. K. and C. OSHIMA 1973. Fertility genes in natural populationsof Drosophila melanogaster. II. Correlation between productivity andviability. Jap. J. Genet. 48: 337-347.
(81) WATANABE, T. K. and T. WATANABE 1973. Fertility genes in natural populations of Drosophila melanogaster. III. Superiority of inversion heterozygotes. Evolution 27: 468-475.
(82) YAMAZAKI, T. and T. MARUYAMA 1973. Evidence that enzyme polymorphisms
are selectively neutral. Nature New BioI. 245: 140-141.
(83) YAMAZAKI T. and P. E. THOMPSON 1973. The dependence of segregationdistortion on chromosome pairing in Drosophila melanogaster. Jap. J.
Genet. 48: 217-229.
(84) YASUDA, N. and M. KIMURA 1973. A study of human migration in theMishima district. Ann. Human Genet. Lond. 36: 313-322.
(85) YOSIDA, T. H. 1973. Evolution of karyotypes and differentiation in 13 Rattus
species. Chromosoma (Berl.) 40: 285-297.
(86) YOSIDA, T. H. and T. SAGAI 1973. Similarity of Giemsa banding patterns

BOOKS AND PAPERS PUBLISHED IN 1973

79

of chromosomes in several species of the genus Rattu8. Chromosoma (BerI.)
41: 93-101.
(87) YOSIDA, T. H., K. MORIWAKI and K. TSUCHIYA 1973. Collection, breeding andgenetics of Rattu8 species in Asia and Oceania. Exp. Animals 22: Suppl.
201-210.
(88) YOSIDA, T. H., H. KATO, K. TSUCHIYA, T. SAGAI and K. MORIWAKI 1972.
Ceylon population of black rats with 40 diploid chromosomes. Jap. J. Genet.
47: 451-454.

80

ABSTRACTS OF DIARY FOR 1973
January
February
March

April
May

June

AugustSeptember
OctoberNovember

December

26 204 th Meeting of Misima Geneticists' Club23 205 th Meeting of Misima Geneticists' Club26 101 st Biological Symposium
9 206 th Meeting of Misima Geneticists' Club13 102 nd Biological Symposium
22 207 th Meeting of Misima Geneticists' Club27 208 th Meeting of Misima Geneticists' Club
1 103 rd Biological Symposium
10 104 th Biological Symposium
14 209 th Meeting of Misima Geneticists' Club15 210 th Meeting of Misima Geneticists' Club23 211 th Meeting of Misima Geneticists' Club23 105 th Biological Symposium
8 106 th Biological Symposium
10 107 th Biological Symposium
1 212 th Meeting of Misima Geneticists' Club26 213 th Meeting of Misima Geneticists' Club2 214 th Meeting of Misima Geneticists' Club16 108 th Biological Symposium
22 109 th Biological Symposium
22 215 th Meeting of Misima Geneticists' Club11 216 th Meeting of Misima Geneticists' Club22 217 th Meeting of Misima Geneticists' ClubJ

81

FOREIGN VISITORS IN 1973
February 26-27
March
16-17
April
6
May
1
July
22-24
November
9

HAMKALO, B. A., Oak Ridge Natl. Lab., U. S. A.
ADLER, H. I., Oak Ridge Nat!. Lab., U. S. A.
MARKOV, M. A. & others, Academy of Sciences, U. S. S. R.
BEALE, G. H., University of Edinburgh, Great Britain
SUZUKI, D. T., University of British Columbia, Canada
TUNG, T. C., & others, A representative team of Chinese
biologists, People's Republic of China
16 EIGEN, M., Max-Planck-Institut, Germany
22 IRVING, C. C., Veterans Administration Hospital, U. S. A.

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 24

ACKNOWLEDGEMENTS
The editor wishes to express his sincere gratitude to Dr. M. Slakin,Mrs. S. Slatkin and Dr. J. F. Crow for their kindness in reading the original
manuscripts. (Y. Nakagome)

83

AUTHOR INDEXAMANO, E
46, 47, 48
ARIGA, 1.
36
BAGbHI, S. .
50, 71
ENDO, T
69
ENOMOTO, M
14, 15, 16
FUJII, T. .
.44, 45
FUJISHIMA, T
65
FURUICHI, Y
11, 12, 13
HIROTA, Y;
14
HOSHINO, T
69
IINUMA, K
64
IKEDA, E
60
IYAMA, S
70, 71
KADA, T37, 38, 39, 41, 43
KATO, H. .
25, 26
KAWAHARA, T
72,73
KIMURA, M
51, 58KOSHINAGA, Joo
61
KURODA, YO'
19,20,21,37
MARUYAMA, T. .
53
MATSUNAGA, E
60, 61, 64
MINATO, K
22
MITA, A72
MIURA, K
11, 12, 13
MORISHIMA, H •.......... 66, 67, 68, 69
MORIWAKI, K
23, 26MORIWAKI, D
27
MURAKAMI, A
32, 34, 35, 36

NAGAMI, S. .
28
NAWA, S
17
NOGUCHI. T
38
OSHIMA, C
56
OHTA, T51, 52, 58, 59
aKA. H
66,67,68,69,70
ONIMARU. K. .
31. 32
PAl. C. ..
69, 70SAGAI, T. .
23, 24
SAITO, M
72
SHIMOTOHNO, K
13
SHINODA. T. .
18.60,61,62
SUGIMOTO. N
72
SUGIURA. MOo
11
SUZUKI, H. .
14
TAZIMA, Y
30, 31, 32, 35
TUTIKAWA. K
41
TSUCHIYA, K
26
TSUJITA, M
27
WATANABE. S
73
WATANABE, K. .
11
WATANABE, Too
56
WATANABE, T.K
54,56
YAMADA, M•........................ 17
YAMAZAKI, T
53
YOKOIYAMA, A
37
YOSHIDA, Hoo73
YOSIDA, T. H. ..
23, 24, 26

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