NATIONALINSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 231972

PublitJhMl 611

THE NATIONAL INSTITUTE OF GBNE'l1C8
IIiaimG. 8&uoktl-km, J.,.,.
1973

Annual Reportof theNational Institute of Genetics
No. 23, 1972

I

Published by

The National Institute of Genetics, Japan
1973

CONTENTS
General statement
Staff
Council..........
..
Association for propagation of the knowledge of genetics .. . . . . . . . .
Projects of research for 1972
Researches carried out in 1972 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

Molecular Genetics
The 5'-terminal structure of cytoplasmic polyhedrosis virus(CPV) genome RNA. MIURA, K., SUGIURA, M. and WATANABE, K.
Terminal structure of single-stranded RNA transcribed by thecytoplasmic polyhedrosis (CP) virus. SHIMOTOHNO, K.,
FURUICHI, Y. and MIURA, K.
The 3'-terminal structure of Avian reovirus RNA. FURUICHI,
Y. and MIURA, K.
Nucleotide ratio of the genome segments in a cytoplasmic polyhedrosis virus from silkworm. FURUICHI, Y., RAI, U. and
MIURA, K.

.
II.

Ill.

1
2
5
6
7
11

Microbial Genetics
Absence of messenger ribonucleic acid specific for flagellin in
fla- mutants. SUZUKI, H. and IINO, T.

11

12
13

13

15

Biochemical Genetics
Improved method for separation and identification of rat serumtransferrins: Thin layer acrylamide-gel electrophoresis withacrinol pretreatment. MORIWAKI, K. .. . . . . . . . . . . . . . . . . . .
The second scientific expedition to Southeast, Southwest andCentral Asia for the study of rodents. IV. Electrophoretic
survey of polymorphic transferrin types on the black rat,
Rattus rattus. MORIWAKI, K., TSUCHIYA, K., KATO, H.,
YOSIDA, T. H. and SADAIE, T.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Serum transferrin polymorphism in the red-backed vole, Clethrionomys rufocanus bedfordiae. MORlWAKl, K. and KuwAHATA,T.
Zymograms of serum esterases in the red-backed vole (Clethrionomys rufocanus bedfordiae) separated by starch-gel electrophoresis. KUWAHATA, T. and MORlWAKl, K.
A consideration on the intensity ratio of allelic dimer isozymes
in heterozygotes. ENDO, T.
The genic system controlling acid phosphatase isozymes in Oryza
sativa and O. perennis. PAl, C. and ENDO, T.
Amino acid sequence of human J( chain. SHINODA, T. . . . . . . . . . .
Functional heterogeneity of the variable and constant regions ofhuman Bence-Jones proteins. SHlNODA, T. and TSUZUKIDA,
Y.

IV.

22

23
24
25
26

27

Developmental Genetics
Time-lapse cinemicrographic analyses of embryonic cell behaviorsof Drosophila melanogaster in culture. KURODA, Y.
Inhibition by cyclic AMP and dibutyryl cyclic AMP of aggregation of embryonic quail liver cells in rotation culture. KURODA, Y.
Effects of dextran sulfates on aggregation of embryonic quailliver cells in rotation culture. KURODA, Y.
Differential inhibition by fucoses and concanavalin A of aggregation of normal liver and hepatoma cells in rotation culture.
KURODA, Y.
Colony-forming activity of embryonic human diploid cells in
culture. KURODA, Y.
Effects of 8-azaguanine, EMS and MNNG on colony-forming
activity of embryonic human diploid cells in culture. KuRODA, Y.
Further studies of effects of high molecular serum fractions on
growth of mammalian cells in culture. MlNATO, K.
Genetic analysis of developmental mechanism in hydra. SUGIYAMA, T.

29

30
31
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33
34

36
37
38

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CONTENTS

V.

•

VI.

Cytogenetics
The second scientific expedition for the study of rodents toSoutheast, Southwest and Central Asia, I. Members, aims andschedule of the expedition. YOSIDA, T. H.
The second scientific expedition for the study of rodents to Southeast, Southwest and Central Asia, II. Animal species collected from Southeast, Southwest and Central Asia, andtheir chromosome numbers. YOSIDA, T. H., MORIWAKI, K.,
KATO, H., TSUCHIYA, K. and SAGAI, T.
The second scientific expedition for the study of rodents toSoutheast, Southwest and Central Asia, III. Karyotype
analysis of the black rat, Rattus rattus, collected in Southwest
and Central Asia. YOSIDA, T. R., KATO, H., TSUCHIYA, K.,
MORIWAKI, K. and SAGAI, T.
Giemsa banding patterns of chromosomes in seven Rattus species.
YOSIDA, T. H. and SAGAI, T.
Stable telocentric chromosomes produced by centric fussion in
Chinese hamster cells in vitro. KATO, H., SAGAI, T. and YOSIDA, T. H.
Non-random localization of the centromere In mammalianchromosomes. IMAI, H. T.
Proposal of a new criterion for the classification of mammalianchromosomes. IMAI, H. T.
Comparison between the length method and the weight method
for formulating the centromeric position of mammalianchromosomes. IMAI, H. T.
Studies on chromosomes of the silkworm, Bombyx mori. MuRAKAMI, A. and IMAI, H. T.
The sixth cell population change to occur during serial passages ofthe MSPC-I mouse myeloma. MORIWAKI, K. and SADAIE, T. . .
Ecological genetic studies on the differentiation of Chrysanthemum
species. NAGAMI, S.
Mutation and Mutagenesis in Animals
A new sensitive test system for the detection of weak chemicalmutagens using silkworm oocytes. TAZIMA, Y. and ONIMARU, K.

iii

39

42

43

45

46
46
50

53

54
56
58

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Test for mutagenicity of mercuric and cadmium compounds using
oocyte system of the silkworm. TAZIMA, Y. and FUKASE, Y.

62
Consequences of the selection for mutation frequencies presumably due to replication instability in the descendants ofmitomycin-C treated silkworm. ONIMARU, K. and TAZIMA,Y.
Further studies on the instable pM allele of the silkworm. 1.
Evidence for the occurrence of recombination between
translocated and free second chromosomes in the female.
TAZIMA, Y. and OHNUMA, A.
X-ray-induced crossing-over in oogenesis of the silkworm.
MURAKAMI, A.
A comparison of relative mortalities by 2 MeV neutrons and
X-rays of young and aged dormant silkworm embryos.
MURAKAMI, A. and IWASAKI, T. .
.
Mutagenesis of acridine orange in cleavage nuclei of the silkworm.
MURAKAMI, A.
Mutagenesis of quinacrine compounds in the silkworm. MURAKAMI, A
.

63

64

66

67

69
70

VII. Radiation Genetics and Chemical Mutagenesis in Microorganisms
and Plants
Participation of DNA polymerase I in the repair of r-ray damaged
DNA in permeable Bacillus subtilis cells. NOGUTI, T. and
KADA, T.
Purification of an enzyme specifically involved in repair of gammaray irradiated DNA. NOGUTI, T. and KADA, T.
Mapping of recombination-deficient genes in Bacillus subtilis.
SADAIE, Y. and KADA, T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Efficient procedures for detection of frameshift mutagens. KADA,
T. and SADAIE, Y.
Radiation effect upon sex expression in cucumber. FUJII, T. . . . .
Further studies on the fractionation dose effects with r-rays inmaize pollen. FUJII, T. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Modification of radiation damage in rice. BAG CHI, S. and
FUJII, T.

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73
74

75
76

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CONTENTS

Copper dependency of an induced dwarf rice mutant. SHAMA
RAO, H. K., FUJII, T. and KADA, T.
Radiation and giberellin effects in the induced dwarf rice mutants.
SHAMA RAO, H. K. and KADA, T.
Biological effects of neutrons after post-irradiation storage orwith gamma-ray and neutron combination treatments in rice.
SHAMA RAO, H. K.
Genetic fine structure analysis of wx locus in maize. AMANO,
E.
Mixed pollination for obtaining inbred mutant line in maize.
AMANO, E.
Some experiments with cultured Haplopappus cell lines. AMANO,E.

VIII. Population Genetics (Theoretical)
Population genetics, molecular biometry, and evolution. KIMURA,
M. and OHTA, T. ..•..................................
The effective number of a population with overlapping generations: a correction and further discussion. CROW, J. F.
and KIMURA, M.
Fixation probability of a mutant influenced by random fluctuation of selection intensity. OHTA, T.
Fixation time of overdominant alleles influenced by random
fluctuation of selection intensity. OHTA, T. and KIMURA,
M.
Simulation experiments of multilocus system.
and KIMURA, M.

"

IX.

80
81

82
84
84
85

87

87
88

89
YAMAZAKI, T.

Population Genetics (Experimental)
Spectra of heterozygosities and of evolutionary rates among
different loci. MARUYAMA, T. and YAMAZAKI, T.
Evidence that enzyme polymorphisms are selectively neutral.
YAMAZAKI, T. and MARUYAMA, T.
Further evidence for the neutral hypothesis of protein polymorphism. YAMAZAKI, T. and MARUYAMA, T.
Allelism between lethal genes in Japanese and Korean natural
populations of Drosophila melanogaster. OSHIMA, C.,

89

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

WATANABE, T. K. and CHOO, J. K.
Allelism between sterility genes in Japanese and Korean natural
populations of D. melanogaster. OSHIMA, C., WATANABE,
T. K. and CHOO, J. K.
Effects of density of larvae and temperature on a factor of fitness
in Drosophila melanogaster. OHNISHI, S. and OSHIMA, C. ..

X.

98
100

Evolutionary Genetics
Population size and rate of evolution. OHTA, T.
Evolutionary rate of cistrons and DNA divergence. OHTA, T. . .
On the stochastic model for estimation of mutational distance
between homologous proteins. KIMURA, M. and OHTA, T. ..

Xl.

96

102
102
103

Human Genetics
Effect of changing parental age patterns on chromosomal aber104
rations and mutations. MATSUNAGA, E.
Genetic effect of selective abortion upon the t(2lqDq) transloca105
tion. MATSUNAGA, E.
Evidence for a second structural locus determining tetrazolium
oxidase in man. SHINODA, T., MATSUNAGA, E. and KOSHINAGA, J. .
106
Isozyme variations in man. SHINODA, T., MATSUNAGA, E. and
KOSHINAGA, J.
107
Structural studies on human immunoglobulins. SHINODA, T. .. 108
Prenatal detection of genetic disorders: A program in Shizuoka
prefecture. (A preliminary report). NAKAGOME, Y., Iinuma,
K. and MATSUNAGA, E.
109
G group chromosomes in satellite associations. (A second report).
NAKAGOME, Y.
110
Banding studies of human chromosomes I. Characterization of 3translocations involving C or G group chromosomes and of a
C trisomy mosaic. NAKAGOME, Y., IINUMA, K. and MATSUI,
I.
111
Fluorescence of the Barr body in human amniotic-fluid cells.
IINUMA, K. and NAKAGOME, Y.
111
Polymorphisms of C and Q bands in human chromosomes.
IINUMA, K., MATSUNAGA, E. and NAKAGOME, Y.
112'

CONTENTS

XII.

•

vii

Behavioral Genetics
Genetic analysis of selected populations of Drosophila melanogasterfor positive and negative phototaxis. CHOO, J. K. andOSHIMA, C.
115
Comparison of some quantitative characters between populations
selected for phototaxis in Drosophila virilis. OSHIMA, C. and
CHOO, J. K.
117
Effects of urbanization on the ecological habitat and of noiseand light environments on the development of Drosophila.
OSHIMA, C. and CHOO, J. K.
120
Selection for geotaxis in Drosophila melanogaster. WATANABE, T.
K. and ANDERSON, W.W.
122

XIII.

Applied Genetics

Geographical variation on an isoperoxidase level in populationsof Pinus Thunbergii. SAKAI, K. I. and IYAMA, S.
Effect of population size on selection efficiency of the bulk methodof breeding in autogamous plants. IYAMA, S.
Gamma-ray induced genetic variation in Arabidopsis thaliana.
BAGCHI, S. and IYAMA, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Inbreeding depression in Japanese quail. KAWAHARA, T. ....
A genetic analysis of skeletal characters in Japanese quail. KAWAHARA, T.
A note on the breeding behavior and genetic variations of sunflowers observed in Central Luzon. OKA, H. I.
Performance in Central Luzon of soybean varieties selected in
Taiwan for wide adaptability. OKA, H. I.
Variation in the response to planting density among sunflower
varieties. OKA, H. I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Variations in the growth pattern of Oryza perennis strains. MORISHIMA, H. and OKA, H. I.
Variations in the mortality of perennial rye grass and orchard
grass varieties. MORISHIMA, H. and OKA, H. I.

123
123
124
126
127
128
129
129
130
131

viii ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Books and papers published in 1972 by research members..........
Abstracts of diary for 1972....................................
Foreign visitors in 1972........................................
Acknowledgement
Author index

132
136
137
138
139

..

•

GENERAL STATEMENT•

It is with sadness that I report the deaths of two honorary members of ourinstitute, Dr. Yoshimaro Tanaka and Dr. Taku Komai, one after anotherin July, 1972, and within one year since we were bereaved of our first director,
Dr. Kan Oguma, in September, 1971. We greatly regret that three great
leaders, not only in this institute but in Japanese genetics as a whole passed
away successively in a single year. Drs. Tanaka and Komai, in co-operation
with Dr. Oguma, have made invaluable contributions to the foundation ofthis institute, especially in the training of the young members, and their
devoted efforts have borne rich fruits.
As to international exchange of researchers this year, twelve staff members
traveled abroad on official business to attend international conferences, tomake co-operative studies, to undertake training, and to make an exploration. Among our many foreign visitors, the two months' stay by Prof. J. F.
Crow who, as a Visiting Member, collaborated with Dr. M. Kimura andothers in the Department of Population Genetics, was one which we particularly remember. Dr. Marian R. Goldsmith of Harvard University
stayed about one month in Dr. Tazima's laboratory in the Department ofMorphological Genetics. She made this visit in order to use the facilities
which are available only in this Institute, and was engaged in a study ofdevelopmental genetics, making considerable use of the special mutant
strains of silkworm maintained here.
In March, 1972, Dr. Tetsuo lino, Head of the Department of Microbial
Genetics, was transferred to the University of Tokyo, as Professor of
Genetics. Since he obtained a position here in 1952, he has developed
elaborate microbial genetical studies using Salmonella. For the three
years 1955",1958 he studied abroad at Dr. Lederberg's laboratory at
Wisconsin University. During the past twenty years he has brought about
many remarkable achievements on the cytomorphogenesis of flagella, thegenetic analysis of flagellar antigens, and, recently, the in vitro reconstructionof flagellar fibres from their component flagellin molecules. Dr. Tsutomu
Sugiyama, who came to us from the Central Research Department of the
du Pont Institute, U.S.A., succeeded Dr. M. Tsujita as Head of the Department of Biochemical Genetics in September, 1972.
The older of our two mouseries was reconstructed this year. The newbuilding measures 557 m 2 in floor area, containing 10 breeding rooms
which are conditioned with clean air at 23°C and 60 % humidity and arefurnished with an automatic device to supply water to the animals.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

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

2. Department of Cytogeneics
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.

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

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
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

5. Department of Applied Genetics
SAKAI, Kan-Ichi, D. Ag., Head of the Department
The 1st Laboratory
SAKAI, Kan-Ichi, D. Ag., Head of the Laboratory
KAWAHARA, Takatada, D. Ag.
FUJISHIMA, Tohru, D. Ag.
The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
BAGCHI,* Sujit
KUDO, * Hiromu
The 3rd Laboratory
OKA, Hiko-Ichi, D. Ag., Head of the Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag.
HOSHINO,* Tsuguhiro
PAl,* Chiang

6.

Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department
The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory* Research members under grant from other organization or visiting researchers.

3

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

NOGUTI, Takehiko, D. Sc.
The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
FUJII, Taro, D. Ag.
SHAMA RAO,* H. K.
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
AMANO, Etsuo, D. Ag.
SADAlE, Yoshito#

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

8.IDepartment of Microbial Genetics
The 1st Laboratory
SUZUKI, Hideho, D. Sc.
The 2nd Laboratory
ENOMOTO, Masatoshi, D. Sc., Head of the Laboratory
WU,* Wen Chuan

9.

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

COUNCIL

10.

5Deparment 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

11.

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

12.

Department of Administration
KUDO, Masaaki, Head of the Department
YUHARA, Tokusaburo, Chief of the General Affairs Section
FUKUI, Teijiro, Chief of the Finance Section

Honorary Members

KIHARA, Hitoshi, D. Sc., Director of the Kihara Institute for BiologicalResearch, Member of Japan Academy, Emeritus Professor of Kyoto
University
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, Emeritus Professor of Osaka University
FUJII, Takashi, Vice Chairman, Professor of University of Shizuoka
BABA, Isamu, Director of National Institute of Agricultural Sciences
IKEDA, Yonosuke, Director ofInstitute 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

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

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
SAKATA, Takeo, President of T. Sakata Company
TAKAHASHI, Ryuhei, Professor of Okayama University
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

PROJECTS OF RESEARCH FOR 1972
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 polymorphism in Rattus rattus (YOSIDA and SAGAr)
Chromosome studies in rodents (YOSIDA, KATO, TSUCHIYA and SAGAI)
Chromosome alteration and development of tumors (YOSIDA)
Cytogenetical study on monosomic and trisomic cultured mammalian cells(KATO)
Cytogenetical and immunological studies on mouse plasma cell tumor(MORIWAKI, K. and SADAIE, T.)
Biochemical studies on serum transferrin variations in rodents (MORIWAKI,
K., SADAIE, T. and TSUCHlYA)
Experimental breeding and genetics of mice, rats and other wild rodents(yOSIDA, MORIWAKI, K., TSUCHIYA, SAKAKIBARA and TAKAHASHI)
Cytogetnetical study of ants (lMAI)
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)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

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)
Department of Biochemical Genetics

Studies on transformation in higher organisms (NAWA and YAMADA)
Genetical and biochemical studies ofpteridine 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 and PAl)
Effects of exogenous DNA on plant seed formation (ENDO)
Genetics of fresh water hydra and other lower invertebrates (SUGIYAMA)
Department of Applied Genetics

Quantitative genetic studies in poultry (SAKAI, KAWAHARA and FUJISHIMA)
Genetic studies in wild populations of Japanese quails (KAWAHARA)
Theoretical studies on breeding techniques (SAKAI and IYAMA)
Behavioral genetic studies in mice (FUJISHIMA)
Studies on competition in plants (SAKAI and 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 andMORISHIMA)
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)PROJECTS OF RESEARCH FOR 1972

9

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
and KADA)
Mechanisms of recombination repair (SADAIE and KADA)
Mutation and differentiation studies of plant tissue culture (FuJII, AMANO,SHAMA RAO 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, NAKAGOME and MATSUNAGA)
Structural studies on human immunoglobulins (SHINODA)
Prenatal detection of genetic disorders (NAKAGOME, IINUMA and MATSUNAGA)
Biochemical genetics in man (SHINODA and MATSUNAGA)
Department of Microbial Genetics

Genetics of bacterial flagella (ENOMOTO and SUZUKI)
Genetics of motility in bacteria (ENOMOTO and Wu)
Transduction mechanism of phages (ENOMOTO)
Flagellar synthesis and its regulation in a cell-free system (SUZUKI)
Deparment of Population Genetics

Theoretical studies of population genetics (KIMURA, MARUYAMA and OHTA)
Studies on molecular evolution from the standpoint of population genetics(KIMURA and OHTA)

10 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Mathematical studies on the genetics of structured populations (MARUYAMA)
Linkage disequilibrium in finite populations (OHTA and KIMURA)
Experimental studies on protein polymorphism in Drosophila (YAMAZAKI)
Simulation studies on linkage disequilibrium in artificial populations (YAMAZAKI)
Department of Molecular Genetics

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

RESEARCHES CARRIED OUT IN 1972

I.

MOLECULAR GENETICSThe 5'-Terminal Structure of Cytoplasmic Polyhedrosis
Virus (CPV) Genome RNA
Kin-ichiro MIURA, Masahiro SUGIURA and Kumiko WATANABE

In order to clear the 51-terminal structure of CPV-RNA, the method for
5'-terminal labeling by 32p using polynucleotidekinase was examined.
Satisfactory labeling was achieved after removal of two or three nucleotides
from the complementary 3'-terminaI. It was confirmed that the 51-terminalof every segment RNA were equally labeled without any degradation of amain chain of RNA molecule.
Alkaline digestion and ribonuclease T 2 digestion of the 51-terminal-labeled
CPV-RNA gave 32 pGp and 32 pXpYp in an equal amount each other. As
the ribonuclease T 1 digestion gave the same nucleotides, 32 pXpYp should be
32 pXpGp.
Since the X nucleoside resist to these kinds of digestion, the 2'position of ribose would be blocked. The mononucleotide 32 pX was
obtained from the 51-terminal labeled CPV-RNA by a nuclease from
Penicillium, which was supplied by Dr. A. Kuninaka, or by the successive
treatment of the nuclease Sl from Aspergillus, which was supplied by Dr. T.
Ando, and venom phosphodiesterase. From the behavior in chromatography 32 pX was considered as a derivative of 51-adenylic acid. Hereafter
this will be written as pA*. This is the first time to show the presence ofa strange nucleoside in a viral RNA.
Pancreatic ribonuclease A digestion of the 5'- 32 P-CPV-RNA gave two
trinucleotides. These are identified as 32 pGpGpCp and 32 pA*pGpUp bychromatography.
Ten segments of the 5'-terminal- 32 P-CPV-RNA were separated by gelelectrophoresis. Each segment was extracted with alkaline treatment anddigested by a nuclease from Penicillium. Two labeled mononucleotides,
pG and 32 pA*, were detected equivalently for every segment. Thus, it is12 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

concluded that everyone of ten segments of CPV genome RNA has thesame terminal structure shown as follows:yy
A*GU - - - - - > - - - - CC
U C - - - - < - - - - - - CGG

3'

5'

Terminal Structure of Single-Stranded RNA Transcribed by the
Cytoplasmic Polyhedrosis (CP) Virus
Kunitada SHIMOTOHNO, Yasuhiro FURUICHI and Kin-ichiro MIURA

CP virus contains segmented double-stranded RNA as its genome, andRNA polymerase, which synthesizes single-stranded RNAs carrying lengthof each genome segment.
In order to clear which strand of the double-stranded RNA is transcribedto the single-stranded RNA by the viral RNA polymerase, the 5'-terminal
structure of the single-stranded RNA was analysed by using the specificallylabeled nucleoside triphosphate as a precursor for the 5'-terminal nucleotide.
Nucleoside triphosphates labeled by 32p at f3 (or r) position were prepared
for this purpose. Any f3- (or r-) 32P-labeled GTP or r_32p ATP was not incorporated into the single-stranded RNA product. On the other handf3- 32 P-labeled ATP was incorporated. Upon ultracentrifuging in a glycerol
density gradient, the incorporation of f3-3 2P-labeled ATP into the productRNA was confirmed.
The terminal nucleotide of the single-stranded RNA product was thenanalysed. Aspergillus nuclease S10 which cleaves nucleic acid into 5'nucleotides (Ando, T.: Biochim. Biophys. Acta 114 (1966) 158), gave only
f3-3 2P-labeled ADP (S2ppA). Ribonuclease T 1 yielded 32ppApGp. Therefore, the 5'-terminal sequence of the single-stranded RNA transcribed by
CP virus is identified as ppApGp ------. The RNA carries diphosphateat the 5'-terminal, although the substrate was triphosphate.
The 3'-terminal of this RNA was identified as C by the separate experiments (Annual Meeting of Japanese Biochem. Soc., Nov. 1972). Thus the
single-stranded RNA produced by CP virus has the following structure:
5'3'

ppApGp---------pC

RESEARCHES CARRIED OUT IN 197213

It is concluded that the RNA chain carrying U at the 3'-terminal in the
double-stranded RNA genome segment was used as a template on transcription, and it was completely read out. The fact that the counterpart of
U is a nucleoside derivative of A is noteworthy, since it may participate forthe recognition of the template strand.

The 3'-Terminal Structure of Avian Reovirus RNA
Yasuhiro FURUICHI and Kin-ichiro MIURA

Avian reovirus RNA was oxidized by periodate and reduced by 3H-sodium
borohydride to label the 3'-terminal nucleoside. Since only the 3H-trialcoholof cytosine appeared on alkaline hydrolysis, it is concluded that both the 3'termini of double-stranded RNA is cytosine. Considering the results ofribonuclease digestion of the 3H-Iabeled RNA, the nucleotide sequences
near 3'-termini of every segment RNA are written as follows. The arrangements of three nucleotides from the 3'-termini are common for all the
genome segments as shown in RNA of CPV.
5 ' - - - - - - - -----AGC 3'
3' CUAPy---5'

Nucleotide Ratio of the Genome Segments in a Cytoplasmic
Polyhedrosis Virus from Silkworm
Yasuhiro FURUICHI, Umeko RAI, Kin-ichiro MIURA

CPV-RNA is fractionated into ten bands in a polyacrylamide gel electrophoresis. Each band obtained from the all 32P-Iabeled CPV-RNA
preparation was extracted with 0.3 N KOH. The nucleotide compositionof every RNA segment was analysed by paper chromotography and paper
electrophoresis. As shown in Table I, there are only little differences in
the composition among the segments. The complementarity between purinesand pyrimidines is maintained in all the segments.
Using the 32P-Iabeled CPV-RNA, the cluster of adenylic acid residues was
examined by digestion with pancreatic ribonuclease AI and Takadiastaseribonuclease T 1 which cleave respectively at pyrimidine nucleotides andguanylic acids in RNA. Any longer adenylic acid cluster than hexanucleotide was not detected in the oligonucleotide chromatography in the pre-

14 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

sence of urea. Polyadenylic acid sequence found in a messenger RNA
and a gene of eukaryote cells or some viruses would not exist in CPV-RNA.
Table 1. Nucleotide composition of RNA segment in CPV
Hydrolysis
Segment

Nucleotide composition (%)

Purine&GC
content

Analysis

Ap

Up

Cp

Gp

Pyrimidine

I
II
III
IV
V
VI
VII
VIII
IX
X

A
A
A
A
A
A
A
A
A
A

30.5
28.1
27.9
27.7
28.4
27.4
30.0
28.6
25.5
27.2

31.1
27.6
27.5
29.2
27.9
29.2
30.8
27.4
31.1
29.4

19.0
22.6
21.9
21.3
21.4
20.5
19.5
22.5
19.7
21.0

19.4
21.7
22.7
21.7
22.3
22.9
19.7
21.5
23.7
22.4

0.99
0.99
1.02
0.98
1.02
1.01
0.98
1.00
0.96
0.98

38.4
44.3
44.6
43.3
43.7
43.4
39.2
44.0
43.4
43.4

Total
CPV-RNA

B
C

29.1
27.8

28.4
29.8

21.2
21.1

21.3
20.3

1.02
0.93

42.5
41.4

(%)

A: 0.5 M KOH 37°C 24 hr, Paper electrophoresis

B: 0.3 M KOH 37°C 18 hr, Two dimensional paper chromatography (U. V.
Absorbance)
C: RNase T2 37°C 18 hr, Two dimensional paper chromatography (U. V.
Absorbance)

RESEARCHES CARRIED OUT IN 1972

II.

15

MICROBIAL GENETICS

Absence of Messenger Ribonucleic Acid Specificfor Flagellin in fla- Mutants
Hideho SUZUKI and Tetsuo IINOll

A group of genes called jla is known to control flagellar formation in
Salmonella. It includes as many as twelve (or more) cistrons recognizedby complementation tests. Most of the complementation groups arelocated in clusters close to an Hi gene. The participation of a jla gene inflagella formation is characterized by the lack of specificity for a flagellar
phase in contrast to regulator genes adjacent to an H-gene. Hence a mutation ofjla to jla- results in the loss of ability to produce flagella both in phase
1 and in phase 2.
It interested us to see if jla- mutation accompanies the cessation of transcription of H-genes. We utilized the cell-free system for protein synthesis
to examine the presence or absence of mRNA specific for flagellin in a jlacell. From the jla- strains belonging to one of the following complementation groups,jla-A, -B, -C, -D, -E, -F, -L, -N, -P, and -Q, the representative
one was chosen, and the salt-precipitable RNA was prepared from the jlacells at a middle log phase of growth. The messenger activity of the RNA
for flagellin synthesis was measured by introducing it into in vitro systemfor protein synthesis.
None of the RNAs was found to direct the in vitro synthesis of flagellin,
although sufficient cell-free synthesis of general protein was observed withthese RNAs and a parallel experiment with the RNA of the corresponding
jla+ strain showed the in vitro synthesis of flagellin in a considerable amount.
To examine the complementability among the RNAs in the cell-free system,
the RNA was combined in the following combinations, (A-F), (B-C),(B-E) (C-D), (E-F), (F-L), (N-P) and introduced to the in vitro system forprotein synthesis. With any of these combinations, the synthesis of flagellin
was not observed. The mRNA for flagellin has been lost completely in thecells of the jla- strains.
11

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

16 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

It has been assumed that the site of flagellin synthesis is associated withthe basal structure of a flagellum and at least some of the fla-genes function to produce the components of the basal structure. Any defective
component produced by fla- could give rise to a defective basal structure,
which then would result in the loss of the function to uncover the transcriptional initiation site of an H-gene so that RNA-polymerase may commence synthesis of flagellin mRNA.

RESEARCHES CARRIED OUT IN 1972

III.

17

BIOCHEMICAL GENETICS

Improved Method for Separation and Identification of Rat Serum
Transferrins: Thin Layer Acrylamide-gel Electrophoresis
with Acrinol Pretreatment
Kazuo MORIWAKI

An improvement in electrophoretic method which will give us better
separation and easier identification of rat serum transferrin bands wasdeveloped in the present study. A combination of acrinol precipitation andthin layer acrylamid-gel electrophoresis gives satisfactory results.
For the preparation of the gel, acrylamide was dissolved at the final concentration of 3.8 % in Tris-citrate buffer (pH 7.4) containing 0.064 M Trisaminomethane and 0.01 I M citric acid. 0.3 M borate buffer (pH 8.6)
was used in the electrode vessels. By this discontinuous buffer system
the fast-moving transferrins of Rattus rattus could be separated well, but theslow-moving ones were occasionally overlapped by the slow aI-macroglobulin band. Acrinol pretreatment of the serum, which precipitated manykinds of protein, except transferrin and gamma-globulin, was very usefulFig. 1. Separation and identification of serum transferrins by thin layer
acrylamide-gel electrophoresis accompanied with acrinol pretreatment.

18 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

to separate and identify transferrins in the serum. This is demonstrated inFig. 1.
As a routine procedure, 100,u1 serum was mixed with 1 ,ul of 0.6 mM FeC1 3
solution and diluted 4 time with 300 ,ul of 5 mM Tris buffer (pH 8.8). To
that mixture was added 400,u1 of 0.6 % acrinol prepared just before use.
After 30 minutes, coagulated proteins were removed by centrifugation at
3000 rpm for 10 minutes at O°C. An equal volume of cold (-10°C)
ethanol was added to the supernatant. After standing the mixture in an
ice bath for 30 minutes, the protein fraction was spun down at 3000 rpm
for 10 minutes at O°C, after which it was dissolved in 100,u1 of 5 mM Tris
buffer and examined by the acrylamide-gel electrophoresis method mentioned
above.
Using this method one can clearly separate and identify 12 transferrin
types (R, R}, R 2 , N}, N, Ct, C 2 , C, D, E, F and G) in Rattus rattus. That thesebands are transferrin has been confirmed by Fe 59-labelling and autoradiography.

The Second Scientific Expedition to Southeast, Southwest and Central
Asia for the Study of Rodents. IV. Electrophoretic Survey of
Polymorphic Transferrin Type in the Black Rat, Rattus rattus
Kazuo MORIWAKI, Kimiyuki TSUCHIYA, Hatao KATO,
Toshihide YOSIDA and Tamiko SADAIE

Serum transferrin polymorphism in black rats, Rattus rattus, collectedfrom Southeast, Southwest and Central Asia was surveyed using the methodof acrinol precipitation followed by thin layer acrylamide-gel electrophoresisthat was newly developed in our laboratory (For technical details, see
Moriwaki, This Annual Report,). Sera of 270 rats obtained from theseareas were analyzed and 12 transferrin bands were identified as schematically demonstrated in Fig. 1. Among them, Rand N are the same as thosebands already found by us using starch-gel electrophoresis in Japanese populations (Moriwaki et al. Genetics 63: 193, 1969) and C, D, E and Farethe same as those previously reported, also using starch-gel, in PacificIsland population (Malecha and Tamalin, Amer. Nat. 103: 664, 1969).
An additional 5 transferrins, R}, R 2 , N}, C 1 and C 2 , have been found for the
first time by the present expedition. The numbers in the various localities

RESEARCHES CARRIED OUT IN 1972

...

+

Tf

1,1
I I II

0

::0

I'll
, ,I

Gi

z

,,,
I I

19

R
R1
R2
N1
N
C1
C2
C
0

E
F

G

Fig. 1. Schematic demonstration of 12 transferrin bands in Rattus rattus
separated by acrinol precipitation and acrylamide gel electrophoresis.

of each transferrin phenotype observed are summarized in Table 1 and thefrequencies of the transferrin alleles in each locality were computed.
According to our previous data obtained from Southeast Asia andOceania (Moriwaki et al. Annual Report of this Institute No. 20: 44, 1969
and No. 21: 36, 1970), the Tf-C, -D, -E and -F alleles were observed onlyin the Oceanian type R. rattus (2n=38). It is interesting to note thatR. rattus in the Northern parts of India and Pakistan had the Tf-C, -D, -E
and -F alleles, though their chromosomes were of the Asia type, 2n=42.
Furthermore, a Ceylon population having 40 chromosomes also had the
Oceanian type transferrins. Another transferrin allele, Tf c1 , has been
observed in the following 3 populations having different karyotypes: Kalyani(2n=42), Ceylon (2n=40) and West Ghats Mountaines (2n=38).
These findings suggest that the possibility that such polymorphic changes
in transferrin genes as shown above had occurred in the evolutionary history
of R. rattus prior to the karyotypeic differentiation that produced the Asian,
Ceylon and Oceanian types.

tv
0

Table 1.

Distribution of transferrin phenotypes and allelic frequencies in Rattus rattus collected fromvarious localities in Southeast, Southwest and Central Asia-_.-

Localityofcollection

Number Number of indiof
viduals with eachsamples transferrin phenotype R

Asian type (2n=42)
Central Luzon,
Philippine*
Makassar,
Malaysia*
Kuala Lumpur,
Malaysia
Kuala Lumpur,
Malaysia
Kanpur,India
Kalyani,India
New Delhi,
India
W. Ghats Mts.
India
Lahore,Pakistan
Islamabad,
Pakistan
Ceylon type (2n=40)
Kandy,
Sri Lanka---------

Allelic frequencies of transferrins----_._-~--~

Rl- --------

R2

Nl

N

- - - - - . - - ..

Cl

C2-------- - - - - - _ .. _ - - -

.743

.257

~

Z
- - - c::>t'""Remarks-~- -~-

_--

,.

•..

------~--

C

D

E----.--------_--_._._--

F-

G--------------- - ---_.

----

;:0
tTl
"tl

0

;:0
>-l

37

R l(23), R l N(9), N(5)59

R 2 (59)10

8

R(3), RN(2), RD(l), .450
N(l), ND(l), NE(l)R(3), RN(8), RE(I) .500
N(3)C 2 F(l), C(9), CD(l),CF(5)
RCl(2), Nl(l), C l(5) .125

2

C(2)

8

Cl(8)

30

C(4), CE(12), E(14)

.333

.667

14

C(3), CE(7), E(4)

.464

.536

15
16

R. rattus
argentiventer
R. rattus
diardiR. rattus
jalorensis

.100
.300

.100 .050

.467

.033
.031 .750 .031

.125

.188

0

"r1

Z

>
>-l

0
Z

>
t'""

Z

c::
(J1

.750

>-l
>-l

1.00

>-l

Forest

1.00

tTl

0

"r1

0

tTl

Z

tTl

>-l

()

(J1

16

Oceanian type (2n=38)
10Madras,India

Cl(4), ClC(4), ClD(l),ClG(I), CG(I), D(2),DE(3)C(I), CD(l), D(5),DE(2), DF(I).438.156 .250 .094

.063

Z

9

tv

w

.150 .700 .100 .050

Arsikere,**India
Kurnool,**
Nagpur,**India
Hyderabad,**India
Poona,
India
Karachi,Pakistan
ChalusIran.542 .333

.083

.500
.875 .125.500

4ND(l), C(5), CD(l),CF(2), D(3)CF(l)C(3), CD(l)3

CE(3), DE(l), DF(l).167.333 .333 .167

20

C(lO), CD(6), D(4)

.650

.350

3

CE(3)

.500

2

C(2)

.100

12
1

.042

R. rattus
rufescence

it!

--_.

-

-

-

* These amimals were obtained by the First Expedition to Southeast Asia and Oceania in 1968.
The frozen sera were analyzed by the new method.
** Located in Deccan Plateau

.500

tTl
v.>
tTl

g>
tTl
v.>
(j

>
it!
it!

o

o
C

>-l

-Z
I ,C

--.J

N

~

22 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Serum Transferrin Polymorphism in the Red-backed
Vole, Clethrionomys rufocanus befdordiae
Kazuo MORIWAKI and Tsutomu KUWAHATA*

Transferrin polymorphism in the red-backed vole, Clethrionomys rufocanus
bedfordiae, was surveyed by starch gel electrophoresis (For technical details,
see Moriwaki et aI., Genetics 63: 193, 1969). The serum of inbred Long
Evans strain rats, Rattus norvegicus, was used as a standard sample in theelectrophoresis. Serum specimens tested were taken from 148 individuals
collected from 9 populations in Hokkaido: Nopporo-I (32), Nopporo-2(8), Nopporo-3 (18), Teshio (19), Ebetsu (12), Naganuma (8), Daikoku
Island (23), Lake Touya (II), and the laboratory stock obtained from the
N opporo-I population (17). The sizes of the samples are indicated inparentheses. Electrophoretic variants of transferrin were observed only inthe laboratory stock. Out of 17 animals, 2 exhibited a fast moving band
(Tf-A), 13 a slow moving band (Tf-B), and 2 both of them (Tf-AB), as demonstrated in Fig. 1. These bands were identified as transferrin by Fe 59
labelling and autoradiography. The remaining populations all showed the
Tf-B band. The Tf-A allele in the laboratory stock seems to have beenpresent only in a very small number of the original Nopporo-I population.

TtA

TfB
LE

Tt8
TfAB

TfAB
LE
Fig. 1. Three transferring phenotypes, Tf-A, Tf-AB and Tf-B, in the redbacked vole demonstrated by starch gel electrophoresis. Solid triangles indicate
the protein bands labelled with Fe. 59 LE: Serum of inbred Long Evans strain ra
as a standard.
* Forest Experimental Station, The Ministry of Agriculture, Hokkaido.

RESEARCHES CARRIED OUT IN 1972

23

Zymograms of Serum Esterases in the Red-backed Vole
(Clethrionomys rufocanus bedfordiae) Separated by
Starch-gel Electrophoresis
Tsutomu KUWAHATA" and Kazuo MORIWAKI

Serum esterases of the red-backed vole (Clethrionomys rufocanus bedfordiae)
were separated by starch-gel electrophoresis (For technical details, see
+

&,0

D
=

Es-1
Es-2

c=:::J Es-3

c::::::J Es-4

c=:::J Es-S
t:=:::::J Es-6
Es-7
c:::::J Es-8=
Es-2

c:::J c:::::J Es-9

c:::::J Es-10
c:::::J Es-11
Es-3 c:::::J c:::::J Es-12
c:::::J Es-13
Es-4

c::::::J c::::::J Es-14

Es-sD

D

Es-15

D

Es-16

c::::J Es-17

c::::::J Es-18[==:J Es-19

c::::J Es-20
jg
~
I>l
to

Origin
.!!

~

"
>-l
0

Fig. 1. Schematic representation of the esterase zymograms separated by
Starch-gel electrophoresis using the sera of the vole and Long Evans rat.
.. Forest Experimental Station, The Ministry of Agriculture, Hokkaido.

24 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Moriwaki et al., Genetics 63: 193, 1969) and isozyme bands were demonstrated by a histochemical method employing a-naphthylacetate as a substrate.
Sera used in the present study were taken from 148 voles collected from
9 populations which differed in habitat. The esterase zymograms showed
different electrophoretic patterns among the individuals; these are summarized schematically in Fig. 1. Throughout this experiment, the serum of
inbred Long Evans rats (Rattus norvegicus) has been used as a standard.
When the esterease bands were numbered in turn from the fastest, 20
bands in the vole and 5 in the rat were identified on the anodal side (Fig. 1).
No esterase bands were observed on the cathodal side. The 20 esterases
in the vole can be divided into 2 groups; one consists of 11 bands, Es-2,
Es-3, Es-5, Es-7, Es-8, Es-lO, Es-11, Es-13, Es-17, Es-19 and Es-20, which
are rather variable among the individuals and the populations, and the otherconsists of 9 bands, Es-l, Es-4, Es-6, Es-9, Es-12, Es-14, Es-15, Es-16 andEs-18, which are considered to be indispensable to the vole, because theesterese patterns in this animal seem always to be constructed on the basisof these 9 bands.
A comparison of the esterase patterns of the 9 populations from Hokkaido
showed that the Dikoku Island and Teshio populations had exactly thesame patterns, but that the rest of them had obviously different patterns.
When 10- 5 M eserin, 10- 3 M PCMB (p-chloromercuribenzoic acid) and10- 3 M EDTA (ethylendiamine tetraacetic acid) were used as inhibitors tofacilitate the grouping of the esterases, eserin clearly inhibited specific bands,
whereas PCMB and EDTA showed no effect. Estereases inhibited byeserin were Es-5 in the Long Evans rat and Es-9 in the vole; these are bothconsidered to be choline esterase.
A Consideration on the Intensity Ratio of Allelic Dimer
Isozymes in Heterozygotes
Toru ENDO

Allelic dimer isozyme (or dimer allozyme, Prakish et al. 1968, Genetics
61, 841) generally produces in heterozygotes two parental and one hybrid
bands, the latter being intermediate mobility between the parental ones.
These three bands are denoted by FF, FS and SS. Their intensity ratio is

RESEARCHES CARRIED OUT IN 197225

expected to be:
IFF: (1 +a) FS : aSS,when specific activity ratio of FFjSS is Ija. The hybrid band then has thehighest intensity of the three.
If FF and SS have the same specific activity but are produced in different
amounts (1 : m), the intensity ratio is expected to be:
IFF: 2mFS : m 2 SS.
This case is not very rare in the inheritance of dimeric peroxidase in riceorgans (Endo 1971, Japan. J. Genet. 46, 1). Efron (1970, Science 170, 751)
also reported the same case in alcohol dehydrogenase of corn.
It is expected that when the ratios in specific activity and amounts are
1 : a and 1 : m, respectively, the intensity ratio will be:
IFF: m(1+a) FS : m 2 aSS.

The Genic System Controlling Acid Phosphatase Isozymes
in Oryza svtiva and O. perennis
Chiang PAl and Toru ENDo

Since 1967, we have continued analysis of the genic system controllingacid phosphatase isozymes in sativa cultivars and their wild progenitor,
Asian perennis. The Indica and Japonica types of sativa cultivars similarly
show a zymogram composed of three major and three minor bands, but differ
in the mobility of the band group. Five different mobility types of thesame band group have so far been found among perennis strains, two of
which represent the Indica and Japonica zymograms. These isozymes
are commonly observed in the leaf blade, leaf sheath, lemma and palea.
Observations were then concentrated on the leaves. This year, three newmobility types were found in perennis populations of W169 (Thailand),
W573 (Malaya) and W1183 (British Guiana). Accordingly, eight mobility
types of the band group have been identified. Segregation experiments so
far made show that a particular mobility of the band group is controlled bya single genetic unit.
However, a new zymogram characterized by the absence of the band group
was found in two sativa cultivars (1707 and 1708; morphologically con-

26 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

sidered as Indicas and having small seeds). These strains showed in leaf
sheath, a major band with a higher mobility than the above-mentioned bandgroup, which was absent in the leaf blade. It is noted that the band is commonly seen in leaf blade and leaf sheath of other Indicas. Also the twostrains showed a minor band located nearby the start line.
In addition, all sativa and perennis strains have two blur bands with muchhigher mobility than the above-mentioned ones. The highest one wasfound to be beta-naphthylphosphate-specific. The second one as well as
all other bands were alpha-naphthylphosphate-specific.
Furthermore, so far as examined, one major and one minor cathodal
bands were found to be specific to perennis subsp. barthii (African perennis).
All Asian, American and Oceanian perennis strains lack those bands. Thus
the genic system controlling acid phosphatase isozymes in rice seems to
involve at least six gene loci.

Amino Acid Sequence of Human ~ Chain
Tomotaka SHINODA

The primary structure of the variable region of the human type K BenceJones Protein Ni with an Inv (3) genetic marker was determined by sequence
analyses of the tryptic and chymotryptic peptides: the primary structure of
the constant region was deduced by partial sequence analyses of the peptides
and by their homologies. Twenty-five major tryptic peptides were isolated
by chromatography on Dowex I-x 2 and AG 50- X 4 using volatile buffer
systems of increasing pH and ionic strength. The complete sequences of
18 of these peptides were determined. These comprised the entire variable
NH 2-terminal half and 7 peptides from the COOH-terminal half of the protein. Thirty-three chymotryptic peptides, covering all but two of the totalof 21 8 residues, were isolated from the S-carboxymethylated protein. Complete or partial sequence analyses of the peptides covering all but two residuesof the variable region of the molecule were made to obtain overlaps for thetryptic peptides recovered from this region. For the remaining peptides
covering the COOH-terminal half only the partial sequence or the aminoacid compositions and the end groups were determined.
From these results all the tryptic peptides could be arranged in order.
Only one of the tryptic peptides in the NH 2-terminal half of the specimen

RESEARCHES CARRIED OUT IN 197227

had the same sequence as that reported for the corresponding region ofother human fC chains, whereas all the tryptic peptides covering the sequence
from residues 108 (in Ag numbering system) to the COOH-terminus corresponded exactly to the sequences reported for the constant region ofhuman fC chains of Inv (3) allotype. For details, see J. Biochem., 73: 417431 and ibid., 73: 433-446.
Functional Heterogeneity of the Variable and Constant
Regions of Human Bence-Jones Proteins
Tomotaka SHINODA and Yasuharu TsuzuKIDA*

One of the key problems in investigation of the molecular basis of themechanism of the antigen-antibody reaction is the mutual arrangementsof the prosthetic groups which are located on the surface of antibody molecules. As an approach to the problems we determined the relative rate oftrinitrophenylation of amino groups in the molecule of human type K
Bence-Jones proteins of known primary structure, on the basis that morereactive ones are at the surface of the molecules. Lysine-169 of the constant region of the Bence-Jones Protein Ni for which the complete sequence
analysis has been done in our laboratory, appears to be on the surface ofthe molecule since it is the most reactive lysine residue toward the trinitrophenylation reaction under a mild condition. This surface position forlysine-169 seems to be the case in general for type K Bence-Jones protein
since it is also demonstrated to be highly reactive in another type K BenceJones protein (Ka). Lysine-l03 of the variable region is less reactive to thereagent thus appears to be less exposed. Lysine-4lj42 and lysine-183 aremuch less reactive than any of the above mentioned lysine residues. The
NH 2-terminal aspartic acid residue appears to be the least exposed since itis the least reactive among the rapidly reactive amino groups of type K BenceJones protein. The relative reactivity among these amino groups of thespecimen Ni appears in the order lysine-169>lysine-103>lysine-4Ij42;::::;
lysine-I 83 > aspartic acid-I. The above order of the reactivity is also thecase for the specimen Ka. Table I summarizes the results of the sequence
analyses of peptides containing these reactive amino groups. Details are in
the press in J. Biochem.
* Res. Inst. for Atomic Energy, Osaka City Univ.

N

00

Table I.
Specimen

PeptideTNPCnlTNPCn8Ni

TNPCnl9

Summary of amino acid sequences of peptides containing
the reactive amino groups
Sequence

I
4(TNP-)Asp-I1e-Gln-Met
3646(a) Tyr-Gln-Gln-Lys-Pro-(TNP-)Lys-Lys-Ala-Pro-Lys-Leu(b) Tyr-Gln-Gln-Lys-Pro-Lys-(TNP-)Lys-Ala-Pro-Lys-Leu
99
116
Gly-Val-Gly-Ser-(TNP-)Lys-Val-Glu-Ser-Lys-Arg-Thr-Val-Ala-Ala-Pro-Ser-Val-Phe

TNPCn29
TNPCklTNPCk8Ka
TNPCkl9
TNPCk26
TNPCk29

~

t"'"
:;>;l

tTl

<3

El
o'Tl

~

In
Glp-Ser-Gly-Asn-Ser-Gln-Glu-Ser-Val-Thr-Glx-Glx-Asp-Ser-(TNP-)Lys-Asp-Ser-Thr-Tyr
180
186
Thr-Leu-Ser-(TNP-)Lys-Ala-Asp-TyroI
4(TNP-)Asp-I1e-Gln-Met
3646(a) Tyr-G1n-Gln-(TNP-)Lys-Pro-G1y-Lys-Ala-Pro-Lys-Leu(b) Tyr-Gln-Gln-Lys-Pro-Gly-(TNP-)Lys-Ala-Pro-Lys-Leu
99
106
Gly-Gln-Gly-Thr-(TNP)Lys-Val-Asp-Leu
155
173
Glp-Ser-Gly-Asn-Ser-Gln-Glu-Ser-Val-Thr-Glx-Glx-Asp-Ser-(TNP-)Lys-Asp-Ser-Thr-Tyr
180
186
Thr-Leu-Ser-(TNP-)Lys-Ala-Asp-Tyr

~..,

I~

TNPCn26

~

~

t"'"

~

tTl

o'Tl
o

~

Q

z

P

....N

RESEARCHES CARRIED OUT IN 1972

IV.

29

DEVELOPMENTAL GENETICS

Time-Lapse Cinemicrographic Analyses of Embryonic Cell
Behaviors of Drosophila melanogaster in Culture
Yukiaki KURODA

Behavior, locomotion, cell division and changes in shape and size, ofembryonic cells of Drosophila melanogaster were analyzed cinemicrographically under in vitro culture conditions. Tissue fragments from dechorionatedeggs at the stage of sac-like midgut were explanted on cover slips inserted
in T-5 flasks as was described earlier by the author. The culture flasks
were placed in a plastic incubator box in which the termperature was controlled at 28°C, on the stage of a Nikon inverted phase microscope.
The tissue fragments were observed every day and cells coming out fromthe explants were recorded by 16 mm cinefilms. Time-lapse cinemicrography was carried out with a Nikon automatic equipment for interval and exposure and a Bolex camera.
Epithelial-like cells and spindle-shaped cells which were found aroundthe explanted tissues one day after cultivation showed an active migrationand cell divisions. On the contrary muscle cells which became noticeable
after 7 days of cultivation were not exceedingly motile, although they
showed a regular pulsation and pronounced movement of many granules in
the cytoplasm. Most of the conspicuously motile cells were macrophagelike, they appeared a few days after cultivation and migrated very rapidly
at a speed of one cell length an hour (about 30 fl per hour).
. After 8 days of cultivation large cell spheres were prominent.Time-lapse
cinemicrographic studies indicated that the sheath of the spheres which
consisted of monolayer cells expanded and contracted continuously and manycells inside of the spheres were suspended freely in some fluid and active
migration and cell divisions were observed through their transparent sheath.
Some cell spheres were separated into several small compartments by boundary membranes which were similar in appearance to the sheath of spheres.
This suggests that some spheres may fuse into larger spheres. The cellspheres increased in size in further cultivation and retained a healthy appearance for several weeks under in vitro conditions employed. As de-30 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

scribed by earlier worker (Schneider, I. (1972) J. Embryo!. Exp. Morpho!.
27: 353), the maintenance of these cell spheres may be the first step of theestablishment of cell lines from Drosophila embryonic cells.
Inhibition by Cyclic AMP and Dibutyryl Cyclic AMP of
Aggregation of Embryonic Quail Liver Cells
in Rotation Culture
Yukiaki KURODA

Recently it has become known that cyclic 3': 5'-adenosine monophosphate(c-AMP) and dibutyryl cyclic 3': 5'-adenosine monophosphate (DB cAMP) were effective in the inhibition of DNA synthesis and cell growth andalso in inducing morphological changes and enzyme production in a varietyof cultured cells of higher animals. In the present experiment the effects
of c-AMP and DB c-AMP on aggregation of embryonic quail liver cells
were examined in rotation culture.
Dissociated single cells were obtained from 7-day embryonic quail liversby the standard procedure previously described. Three ml of cell suspensions each containing 2 X 10 6 cells in a 25 ml Erlenmeyer flask were rotatedon a gyratory shaker with a constant speed of 70 rpm at 38°C for 24 or 48
hours. C-AMP and DB c-AMP were added to the culture medium at theconcentrations of 0, 0.01, 0.03, 0.1, 0.3 and 0.6 mg/m!. Twenty aggregatesharvested from each flask were photographed, measured for their diameters, and calculated for mean diameters with standard deviations. The
effects of c-AMP on aggregation of embryonic quail liver cells are shownin Table 1.
Table 1. Effects of c-AMP on aggregation of 7-day embryonic
quail liver cells
Concentration(mg/ml)

o
0.01
0.03
0.1
0.3
0.6

Diameter of aggregates (Mean±S.D. Ii)
24 hours

48 hours

202.5±39.7
166.0±26.2
143.5±26.3
122.5±21.9117.5±24.7
101.5±23.3

269.0±47.2
259.0±41.2
238.5±33.9243.5±36.2
157.5±27.7131.0±17.3

RESEARCHES CARRIED OUT IN 1972

31

C-AMP had concentration-dependent inhibitory effects on embryonicliver cells. At the concentration of 0.6 mg/ml aggregates formed after
24 and 48 hours of rotation culture had half the diameter of those obtainedin respective control cultures.
Aggregates formed after 24 hours in media containing c-AMP at various
concentrations were transferred to normal medium in which they wererotated for additional 24 hours. Aggregates which were depressed in sizein the previous cultivation with c-AMP recovered partially their cohesivenessin further cultivation in normal medium, although they did not yet reachthe size of control aggregates.
When DB c-AMP was added to the medium, the sizes of aggregates obtained after 24 or 48 hours of rotation culture are shown in Table 2.
Table 2. Effects of DB c-AMP on aggregation of 7-day
embryonic quail liver cells
Concentration(mg/ml)

o
0.01
0.03
0.1
0.3
0.6

Diameter of aggregates (Mean±S.D. ,,)24 hours

48 hours

231.0±45.8
174.5±37.9
114.5±21.8
89.5±17 .2
92.5±25.7
85.0± 9.2

333.5±67.1
198.0±34.6
110.0±17.6
97.0±15.5
94.0±13.987.5±14.8

DB c-AMP had stronger effects than c-AMP at the corresponding same
concentrations. At the concentrations of more than 0.03 mg/ml no moreincrease was found in size of aggregates for the additional 24 hours ofcultivation. When aggregates obtained after 24 hours in the presence ofmore than 0.03 mg/ml of DB c-AMP were transferred to normal medium,again no recovered increase of aggregate size was found.

Effects of Dextran Sulfates on Aggregation of Embryonic
Quail Liver Cells in Rotation Culture
Yukiaki KURODA

Dextran sulfate is one of polyanionic compounds which bind with cell

32 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

membrane and induce the increase in negativity of cell membrane, resulting
in a disturbance of cohesion among cells. This agent inhibited the metastasis of cancer cells and also decreased the saturation density of tumor cells
in monolayer culture by modifying the surface properties of cell membrane.
In the present experiment the effects of dextran sulfates of various molecularweights on aggregation of embryonic quail liver cells were examined inrotation culture.
Three ml of cell suspensions containing 2 X 106 cells dissociated from 7-day
embryonic quail livers were rotated by the standard procedure at 38°C for
24 or 48 hours. Dextran sulfates of molecular weights of 7,500, 45,000
and 60,000 were added to culture medium at the concentrations of 0, I, 3,
10 and 30 p,gfmI. Twenty aggregates harvested from each flask were
photographed, measured for their diameters and calculated for mean
diameters with standard deviations. The effects of dextran sulfates of various molecular weights on 48-hour aggregation of embryonic quail liver cells
are shown in Table I.
Table 1. Effects of dextran sulfates on 48-hour aggregationof 7-day embryonic quail liver cells
Concentration(pgjml)

o
1
3

10
30

Diameter of aggregates (Mean±S.D. p)
Dextran sulfate
MW=7,500

Dextran sulfate
MW = 45,000

Dextran sulfate
MW=60,000

308.5±30.2
220.5±35.7
145.0±36.9117.5±33.1
95.5±14.0

270.0±26.6
264.0±30.4276.0±23.7
277.5±24.5
275.5±29.7

354.5±39.4
262.5±34.9
175.5±37.3
99.5±14.3
83.0±11.9

Dextran sulfates of molecular weights of 60,000 and 7,500 had pronounced
inhibitory effects on aggregation of embryonic liver cells, whereas that ofmolecular weight of 45,000 had no effect. These results indicate thatdextran sulfates added to culture medium may modify the surface propertiesof cell membrane. The mechanisms by which dextran sulfates induce thedepression of cohesiveness of embryonic liver cells are now under investigation.

RESEARCHES CARRIED OUT IN 197233

Differential Inhibition by Fucoses and Concanavalin A
of Aggregation of Normal Liver and Hepatoma Cells
in Rotation Culture
Yukiaki KURODA

In a series of experiments in which effects of carbohydrate-containing
substances on the mutual cohesiveness of normal and malignant cells were
examined, it had been previously found that hexosamines and their acetyl
derivatives had differential inhibitory effects on normal liver and hepatoma
cells. In the present experiments, effects of D- and L-fucoses and concanavalin A on aggregation of embryonic normal liver cells and DAB-induced
rat hepatoma cells were investigated in rotation culture.
Single cell suspensions were prepared by trypsinization from 7-day embryonic quail livers and monolayer cultures of DAB-induced rat hepatoma
cell lines, dRLN-61, dRLa-74 and dRLh-84, and were rotated for 24 or
48 hours under standard conditions. Hapatoma cell lines, dRLN-61, dRLa74 and dRLh-84, had a low, moderate and high tumor-producing activity,
respectively. D-Fucose and L-fucose were added to culture medium atconcentrations of 0, 1, 3, 10 and 30 mM. Concanavalin A (NBC) wastested at concentrations of 0,0.001,0.003,0.01, 0.03 and 0.1 %. The effects
of fucoses on 24-hour aggregation of normal liver and hepatoma cells areshown in Table 1.
Table 1. Effects of fucoses on 24-hour aggregation of
normal and hepatoma cells..

Inhibition of aggregation of
FucoseConcentration(mM)

D-Fucose

0
1
3
10
30

L-Fucose

0
1
3
10
30

Normalliver cells

HepatomadRLa-74 cells

HepatomadRLh-84 cells

+
+
+
+
+
+

-: 0~30% inhibition, +: 30~70% inhibition, -It: 70~100% inhibition.

+
+
+
-It

34 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

D-Fucose had differential inhibitory effects on normal liver cells andhepatoma cells. At concentrations of more than 3 mM D-fucose affected
the aggregation only of hepatoma dRLa-84, whereas no effects were foundon the aggregation of normal liver cells and hepatoma dRLa-74 cells.
L-Fucose had stronger inhibitory effects than D-fucose. The effects of
L-fucose on aggregation of normal liver cells and hepatoma cells werecorrelated to tumor-producing activities of cells: no effects on normal liver
cells, moderate effects on dRLa-74 cells and strong effects on dRLh-84
cells.
The effects of concanavalin A on aggregation of hepatoma cells having
different tumor-producing activities are shown in Table 2.
Table 2.

Effects of cancanavalin A on 24-hour aggregation of
rat hepatoma cells
Inhibition of aggregation ofConcentration
(%)

0
0.001
0.003
0.01
0.03
0.1

HepatomadRLN-6l cells

HepatomadRLa-74 cells

HepatomadRLh-84 cells

+
+
*
*

For symbols see the footnotes in Table 1.

*
*

+
+

**
*

* indicates flocculation.

Concanavalin A had stronger inhibitory effects on the aggregation ofhepatoma cells, which had higher tumor-producing activities at concentra. tions up to 0.01 %. At the concentrations of more than 0.03 % of concanavalin A hepatoma cells did not aggregate, but they flocculated.
Colony-Forming Activity of Embryonic Human Diploid
Cells in Culture
Yukiaki KURODA

To elucidate the mechanisms of somatic mutations, phenotypic expression in cultured mammalian cells, and also to establish screening proceduresfor detecting potential mutagens present in human environment, the culture

RESEARCHES CARRIED OUT IN 197235

conditions were examined under which the best colony formation of embryonic human diploid cells was obtained.
Lung cells derived from a 5-month human embryo were cultured inmonolayer in Eagle's BM. Karyotype analysis indicated that more than
86 % of cells had normal diploid female chromosomes. The effects ofvarious inoculation numbers of cells and culture media on colony-forming
activity of cells were tested. Cells dissociated from monolayer cultures atsixth to tenth transfer generations were counted and 103, 3 X 103, 10\ 3 X 104
or 105 cells were inoculated in 60 mm plastic petri dishes in 4 m of Eagle's
BM, Eagle's MEM, Puck's Nl6 or Ham's Fl2 medium each supplemented
with 10% calf serum or in Puck's Nl6CF medium.
After 10 to 16 days of cultivation at 37°C in the atmosphere of 5 % CO 2
and 95 % air, cells in petri dishes were fixed in methyl alcohol and stained in
May-Griinwald-Giemsa solution. The numbers of colonies consisting of
more than 50 cells were counted under a binocular microscope and thecolony-forming activity (plating efficiency) was calculated as follows:
Average number of colonies
Number of cells inoculated
x 100%
Colony-forming activity =The results obtained are shown in Table 1.
Table 1. Comparison of colony-forming activity of embryonichuman diploid cells in various media
Number ofcells
inoculated•

103
3 xl03104
3 X 104
10 5

Colony-forming activity (%)
EageI's BM Eagle'sMEM+10% CS+10% CS

Puck's
N16CF

Puck's N16+10% CF

Ham's F12
+10% CS

0.83
3.70
2.68
1.20
>0.52

0.43
1.09
0.55
0.61
>0.48

0.37
0.77
1.33
0.88
>0.43

10.70
11.24
6.24

1.25
2.66
1.77
1.01

*

*
*

* The number of colonies was not counted because a confluent cell sheet was formed.
Ham's FI2 medium supplemented with 10 % calf serum was the best of
all media tested, followed by Eagle's BM, Eagle's MEM, Puck's NI6CF
and Puck's N16. In Ham's FI2 medium with 10 % calf serum the best colonyforming activity of cells was obtained when 3 X 103 cells were inoculated on
one petri dish.

36 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Effects of 8-Azaguanine, EMS and MNNG on Colony-Forming
Activity of Embryonic Human Diploid Cells
in Culture
Yukiaki KURODA

In the previous investigation it was found that embryonic human diploidcells displayed the best colony-forming activity when cultured in Ham's
F12 medium supplemented with 10 %calf serum. In the present experiments
the effects of some drugs on colony-forming activity of cells were examined.
Drugs employed were an antimetabolite, 8-azaguanine (8AG), a mutagenic
substance, ethyl methan sulfonate (EMS) and a carcinogenic agent, Nmethyl-N'-nitro-N-nitrosoguanidine (MNNG).
104 cells were inoculated in 60 mm petri dishes in media containing
various concentrations of 8AG, EMS or MNNG and were incubated for
14 days at 37°C in the atmosphere of 5 % CO 2 and 95 % air. The cell colonies in harvested cultures were fixed, stained and counted and the colonyforming activity (plating efficiency) was calculated. The results obtained
are shown in Table 1.
Table 1.
Substance

Effects of 8AG, EMS and MNNG on colony-forming
activity of embryonic human diploid cells

Concentration

Average numberof colonies

Colony-forming
activity

Surviving
fraction._-~--~-

o ,ug(ml
1
3
10
30

559.5
234.7
16.7
0
0

5.60
2.35
0.17
0
0

1.00
0.42
0.03
0
0

EMS

OM
10-3
3 x 10- 3
10-2
3 x 10-2

397.0
307.0
44.0
1.0
0

3.97
3.07
0.44
0.01
0

1.00
0.77
0.11
0.002
0

MNNG

OM
10-6
3 x 10-6
10- 5
3 x 10-5
10-4

407.0
311.5
309.0
212.0
33.3
0.3

4.07
3.12
3.09
2.12
0.33
0.003

1.00
0.77
0.76
0.52
0.08
0.0007

8AG

RESEARCHES CARRIED OUT IN 197237

From concentration-survival curve for each drug, Do value was calculated
to be 1.3 ,ugfml for 8AG, 2.2 X 10- 3 M for EMS and 1.7 x 10- 5 M for MNNG.
If cells treated with EMS or MNNG were cultured in selection medium containing 8AG, the effects of EMS or MNNG on mutation frequency from
8AG sensitivity to resistancy may be determined by calculating the colonyforming activity of cells.
Further Studies of Effects of High Molecular Serum
Fractions on Growth of Mammalian Cells
in Culture
Kiyoshi MINATO

To elucidate the effects of high molecular fractions of calf serum on thegrowth of mammalian cells, further analyses of some fractions which could
sustain cell growth were carried out with HeLa S3 cells in monolayer culture.
In Eagle's MEM supplemented with dialyzed calf serum, cells grew almost
at the same rate as that in medium supplemented with whole serum. In
the medium supplemented with calf serum which was salted-out withsaturated ammonium sulfate, the growth rate of cells decreased to 70 80 %of control culture with whole serum. The serum fraction which was
salted-out with 50 100 % saturated ammonium sulfate was effective inproviding 50 60 % of the growth rate of cells in control culture.
With a fraction which was precipitated with 50 % saturated ammonium
sulfate, the initiation of cell growth was delayed for 3 or 4 days and about
one half of cells in culture died. After a lag period, the growth rate of cells
was 50 60 % of control culture. With only one fraction precipitated eitherwith O'V 50 % or 50 'V 100 % saturated ammonium sulfate, no growth rateof cells shown in control culture was obtained.
Some attempts were made to substitute the osmotic pressure of serum
protein by that of other substances. A non-metabolizable disaccharide,
D-cellobiose, had no effects as in culture with no supplementation. A
synthetic macromolecule, polyvinyl pyrrolidone (PVP) k-90, was effective
a little at the concentration of 0.1 %in sustaining survival and growth of cells.
At lower or higher concentrations than 0.1 % PVP was less effective. Linoleic acid and putrescine which were used by Ham (1963, 1964) as substitutes
'V

'V

'V

t

'V

38 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

of albumin and a-globulin of serum were tested with single or combined supplementation. These substances were not effective in promoting cell
growth.

Genetic Analysis of Developmental Mechanisms in Hydra
Tsutomu SUGIYAMA

A new project has been started to study the genetics of fresh water hydra.
The aim of the project is to identify and to analyze the function of genes
which are involved in various developmental processes such as budding,regeneration, transplantation, cell migration and cell reassociation.
Representative species of fresh water hydra native to Japan have beencollected (Hydra parva, H. magnipapillata, H. japonica and Pelmatohydra
robusta) and laboratory conditions for their culture by asexual and sexual
reproduction are being examined.
One temperature sensitive strain of H. magnipapillata was obtained.
This strain shows normal morphology at 18°C but shows twisted filamentous
stem at 23°C. The morphology of the wild type is almost identical atboth temperatures.
Other morphological and conditionally lethal mutants are being sought.

RESEARCHES CARRIED OUT IN 1972

v.

39

CYTOGENETICSThe Second Scientific Expedition for the Study of Rodents to Southeast,
Southwest and Central Asia, I. Members, Aims and
Schedule of the Expedition
Tosihide H. YOSIDAWith support of a grant-in-aid for scientific research from the Ministryof Education we organized the second expedition for the study of rodents
to Southeast, Southwest and Central Asia from September 27 to November
22, 1972. The members of the expedition were as follows:
Tosihide H. Yosida (Leader) )Kazuo Moriwaki
Department of Cytogenetics
Hatao Kato
National Institute of Genetics
Kimiyuki Tsuchiya
The expedition was undertaken with the following aims.
1) Localization of a border line dividing the habitats of Asian- andOceanian-type black rats: The black rat, Rattus rattus, is a peculiar animalin that, as we found previously, it shows a local variation within the same
species in the number and the shape of chromosomes despite the fact that
there are no differences in the external features. This phenomenon isreferred to as a "chromosome polymorphism or geographical variation".
The black rats found in East and Southeast Asia possess 42 chromosomes,
while those in Oceania (Australia, New Zealand and New Guinea) have
only 38 chromosomes, whose morphology also differs markedly from thoseof the former. It has been confirmed by several researchers that the
Oceanian-type black rats are also distributed widely throughout North andSouth America, Europe, and Africa. There is general agreement thatthe black rat first appeared in the Indo-Chinese area, from where it migrated
to Europe through Southwest (India and Pakistan) and Central Asia andfrom Europe they migrated to Oceania, American and Africa, probably
accompanying man. It is reasonable to assume that the transition inchromosome type from the Asian (2n=42) to the Oceanian (2n=38) might
have occurred during the migration period between Asia and Europe, andthat the border-line dividing the distribution of these two types would be40 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

located somewhere in Southwest Asia. One of the aims of the expedition
was to pinpoint such a border region.
2) A search for a transient type of black rat with 40 chromosomes:
In the previous paper we predicted that the karyotype of the black rat evolved
through the following sequence: 2n=42~2n=40~2n=38, and, though a
black rat with 2n=40 has not yet been found in a natural population,
it probably occurs somewhere in the world as a transient type between the
Asian and Oceanian types. We suspected that this type might be foundsomewhere in Southwest Asia.
3) Elucidation of the relationship between the chromosomal changes
and speciation in the genus Rattus: We have examined the chromosomesof 13 species of rats collected in Southeast Asia and Oceania during theprevious expedition, and obtained cytogenetical data implying that all therats belonging to the genus Rattus have been derived from the black rat
through chromosomal rearrangements, and that the polymorphism seen in
the black rat reflects a transitory phase in speciation within the genus
Rattus. Since there have been inadequate karyological studies of rodent
species in these areas, the present expedition aimed to collect and examine
as many rats belonging to the genus Rattus as possible.
4) Comparative survey of the electrophoretic patterns of serum transferrin in the genus Rattus: Diversiforms of electrophoretic patterns ofserum transferrin have been previously demonstrated in the genus Rattus
collected from Japan, Southeast Asia and Oceania by Moriwaki et al.
Recently they have succeeded in comparing these transferrins at the molecular level, that is, in their amino acid compositions. The fourth aim of
the expedition was to pursue a study within the genus Rattus of the possible
parallelism between karyotypic variation mentioned above and molecularvariation in the serum transferrins.
5) Development of a new experimental animal: Recently we havesucceeded in breeding several species of wild rodents collected during thefirst expedition in the laboratory. Some of them are now proliferating. We
planned to bring back to our laboratory various rodents collected during
the second expedition in order to establish new experimental animals.
The schedule of the expedition was as follows:
Tokyo (left Sept. 27, 1972)~Hong Kong~Kuala Lumpur (Malaysia)~
Colombo, Kandy (Sri Lanka)~Madras (India)~Calcutta, Nadia (India)~
Bangalore, Mysore (India)~Poona (India)~New Delhi, Kanpur (India)

RESEARCHES CARRIED OUT IN 1972

Karachi41(Pakistan)~Islamabad (Pakistan)~Lahore (Pakistan)~Teheran(Iran)~Ankara(Turkey)~Istanbul(Turkey)~Europe~Tokyo(arrival Nov.

22, 1972)

Acknowledgements: The members of the expedition wish to express
their gratitude to the following researchers for their kind help in the collection of animals.
Mr. G. W. Chau: Urban Services Dept. Hong Kong.
Mr. B. L. Lim: Institute of Medical Redical Research, Kuala Lumpur,
Malaysia.
Dr. H. F. Fernando: Central Agricultural Research Inst., Peradeniya,
Sri Lanka.
Dr. P. J. Sanzeeva Raj: Department of Zoology, Christian College,
Madras, India.
Dr. G. K. Manna: Department of Zoology, University of Kalyani,
Kalyani, India.
Mr. T. Tachibana: Kasturba Gandhi National Memorial Trust, Asrikere, Mysore, India.
Dr. G. B. Deodika, Dr. M. D. Gadgil: Maharashtra Association for the
Cultivation of Science, Poona, India.
Dr. S. S. Rajan: Indian Agricultural Research Institute, New Delhi,
India.
Dr. A. S. Srivastava: U. P. Institute of Agricultural Sciences, Kanpur,
India.
Dr. Z. Khan: National Health Laboratory, Islamabad,. Pakistan.
Dr. 1. Khan: Pakistan Medical Research Center, Lahore, Pakistan.
Dr. C. M. Tahir: Rice Research Institute, Lahore, Pakistan.
Dr. D. W. Walton: University of Karachi, Karachi, Pakistan.
Dr. P. Mostashfi: Department of Genetics, University of Teheran,Teheran, Iran.

42 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

The Second Scientific Expedition for the Study of Rodents toSoutheast, Southwest and Central Asia, II. AnimalSpecies Collected from Southeast, Southwest andCentral Asia, and Their Chromosome Numbers
Tosihide H. YOSIDA, Kazuo MORIWAKI, Hatao KATO,
Kimiyuki TSUCffiYA and Tomoko SAGAI

Species of rodents and insectivores collected from Southeast, Southwest
and Central Asia by the second expedition are listed in the following table(Table 1).
Table 1. Species and chromosome numbers of rodents and other small
mammals collected in Southeast, Southwest and Central Asia
by the second expedition
Localities
collected

SpeciesNo. of individuals
collected

Chromosome
numbers

Hong Kong

Rattus huang
R. rattus flavipectus

2
10

46
42

Kuala Lumpur(Malaysia)

R. rattus diardii
R. muelleri
R. jalorensis
R. annandalei
R. argentiventer
R. sabanus
R. whiteheadi
R. rajahR. exulans

5

6
7
7
4
8
1
1
1

42
42
42
42
42
42
36
36
42

Kandy
(Sri Lanka)

R. rattus
Bandicota bengalensis
Mus legata

19
2
1

40
42
40

Kalyani(India)R. rattus
Mus musculus
Funambulus palmarum
Suncus murinus

2
1
2
6

42
40
?
40

Madras(India)R. rattus
Mus musculus

23
3

38
40

Mysore(India)R. cutchicus
R. rattus

2
20

36
38, 42(Continued)

RESEARCHES CARRIED OUT IN 1972
Table 1.

43(continued)

Poona(India)R. rattus

20

38

Kanpur(India)R. rattus
Bandicota bengalensis
Mus booduga
Tatera indica

17
2

42
42
40
66

Delhi
(India)R. rattus
Suncus murinus
Bandicota bengalensis

2
3

Rawalpindi(Pakistan)

R. rattus
Bandicota bengalensis
Suncus murinus

143

R. rattus
Bandicota bengalensis
Mus booduga
Tatera indica
Millardia meltada

29
5
I
I
I

42
42
40
66
50

Karachi(Pakistan)

R. rattus
Funambulus pennantiParachinus micropus

4

39

1
1

?
?

Teheran(Iran)

Meriones libycus

3?

Near CaspianSee (Iran)R. rattus

7

38

I

2

42
40
42

I
~

I

--

42
42
40
---

Lahore(Pakistan)

The Second Scientific Expedition for the Study of Rodents toSoutheast, Southwest and Central Asia, III. Karyotype
Analysis of the Black Rat, Rattus rattus, Collected inSouthwest and Central Asia
Tosihide H. YOSIDA, Hatao KATO, Kimiyuki TSUCHIYA,
Kazuo MORIWAKI and Tcmoko SAGAIBlack rats collected in Kalyani, Delhi and Kanpur, located in Northern
India, had 42 chromosomes, that is, the Asian karyotype, but those collectedin Madras and Poona had the Oceanian karyotype (2n=38). Among 12
rats collected in Poona, however, only one rat had Asian type chromosomes(2n=42). Fifteen black rats were collected in Mysore, India. Among44 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

them 8 rats had the Oceanian karyotype (2n=38), but the other 7 rats were
Asian (2n=42). The black rats collected in Rawalpindi and Lahore,
which are located in northern Pakistan, had Asian type chromosomes.
Among 13 rats collected in Lahore, only one had 41 chromosomes as theresult of loss of one acrocentric chromosome. The karyotypes of 3 rats
collected in Karachi, Pakistan, were remarkable in having 39 chromosomes,suggesting the hybrid between the Asian and Oceanian types. All of three
had same karyotype, namely one pair of the large metacentric chromosome(M l ), but only one of the M 2-metacentrics was included in their karyotypes.
All 7 black rats collected near the Caspian Sea in Iran were of the Oceaniantype (2n=38). All 10 black rats collected in Kandy, Sri Lanka (Ceylon)
had 2n=40 chromosomes. This karyotype is characterized by having only
one pair of M 2-metacentric chromosomes and it is suggested that it developedby Robertsonian fusion of pairs No. 11 and 12 in the karyotype of the
Asian rat (Fig. 1). This seemed to be a transient type between Asian(2n=42) and Oceanian types. Through these analyses of the karyotypesof black rats from India, Pakistan, Iran and Ceylon, we could confirm thatIA IP ftA
2

1

alIt,.,.
8

6

t" x
11I

12

'.1

15

14
1

"

•

I

I

~

_"
5

3

Alt
9

1/1I
13

I17I

III

20

Fig. 1. Karyotype of the black rat, Rattus rattus, collected in Kandy, Sri
Lanka (Ceylon).

RESEARCHES CARRIED OUT IN 197245

the Asian type developed first in Southeast Asia, and from there migrated
to Europe through Southwest and Central Asia. The first Robertsonian
fusion of pairs No. II and 12 in the Asian type occurred somewhere in
Southwest Asia, and thus gave rise to the Ceylon type (2n=40). Thereafter
the Oceanian type (2n=38) could have arisen from the Ceylon type by a second fusion of pairs No.4 and 7. The latter type remained isolated on
Ceylon island, while the former became distributed from there to Central
Asia and Europe.
A study of the Ceylon type of the black rat has been published in Jap.
Jour. Genetics 47: 451-454 (1972).

Giemsa Banding Patterns of Chromosomes in Seven
Rattus Species
Tosihide H. YOSIDA and Tomoko SAGAIThe Giemsa banding patterns of the metaphase chromosomes werecompared in seven Rattus species. Four species (R. rattus tanezumi, R.
norvegicus, R. exulans and R. muelleri) all had 2n=42 and their karyotypes
and banding patterns were similar, slight differences were observed. Anothersubspecies (R. rattus rattus) and two other species (R.fuscipes and R. conatus)
had lower chromosome numbers than those above. They had large biarmed
chromosomes developed probably by Robertsonian fusion of the acrocentricchromosomes found in the first four species. The origin of the arms of thebiarmed chromosomes was recognized by their characteristic banding
patterns. The remaining one species, R. sabanus, had 2n=42 chromosomes, but its karyotype differed markedly from the other species describedabove in having two small metacentrics, although those are seven in theothers species. The banding patterns of the chromosomes in this species,
however, were also very similar to those of the others, and therefore theseven small metacentrics, which characterize the karyotypes of many Rattus
species, seem to have originated by pericentric inversion within small acrocentrics, such as those found in the karyotype of R. sabanus. This study
was published in Chromosoma (BerI.), 41: 93-101.

46 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Stable Telocentric Chromosomes Produced by Centric Fission in
Chinese Hamster Cells in vitro
Hatao KATO, Tomoko SAGAI and Tosihide H. YOSIDA

Metaphase examination of pseudodiploid Chinese hamster cells revealed
that spontaneous breaks or fissions occurred rather frequently (2.9 %) at
the centromeric regions of subtelo- or metacentric chromosomes, resulting
in the production of telocentric chromosomes. The centromeric fissions
appeared to occur in every member of the chromosome complement. An
attempt was made to isolate cells possessing such telocentrics from the cell
population and two clonal lines were established which had one and two
telocentric chromosomes, respectively. The banding and labeling patternsof these chromosomes both indicated unequivocally their X chromosome
origin. They were transmitted successively to the daughter cells during a
3-month culture period, showing no tendency to fuse to produce a metacentric chromosome. (Chromosoma 40: 183-192).

Non-Random Localization of the Centromere in
Mammalian Chromosomes
Hirotami T. IMAI

Since Levan et al. (Hereditas 52: 201, 1964) proposed a standard nomenclature for chromosomes in terms of arm ratio, it has been adopted bymany mammalian cytogeneticists in the original form and by several workers
after further modification. Nobody, however, has paid attention to thestatistical distribution of the centromere along the chromosome as a meansof obtaining basic information about the nature of chromosome rearrangements. If the position of the centromere is random, morphologic classification of chromosomes can only be done by using the arbitrary criteria
proposed by Levan et al. But if the pattern of distribution is non-random,
this biological character can be used for the classification of chromosomes.
In order to solve this problem, the present paper examines statistically thefrequency distribution of centromere localization in 16,817 chromosomes
observed in 723 mammals.
The position of the centromere of metaphase chromosomes is expressed
either by the arm ratio (r w ) or by the size of the short arm (Sw) using weight,

RESEARCHES CARRIED OUT IN 197247

according to the photo cut-out technique of Wurster et al. (Cytogenetics
10: 153, 1971). The mean weight of the short arms of a homologus pair ofchromosomes expressed as a percentage of the total weight of the X-containing haploid set is designated as Sw, in the same way that of long arms as
L w, and that of the whole chromosome as Cw, where Cw=Sw+Lw and r w=
LwjSw = (Cw-Sw)jSw. The relative weight of the Y chromosome iscalculated from its percentage of the total weight.
The frequency distribution of the value of r w is shown in Fig. I. The
5. 219 1

4,000

'

..

3.000

lil

~o
~

a::

0 2•000
lL.

o

a::

l!'l

~

::J

Z

1.000

1.0 1.2 1.5 1.9 2.3 3.0 4.0 5.7 9.0
ARM RATIO BY WEIGHT (rw )Fig. 1.

<Xl

Frequency distribution of the arm ratio by weight (r w ).

figure exibits a V-shaped curve, suggesting a non-random localization of
the centromere. In order to know whether the non-random distribution of
rw can be observed in every size of chromosome, the relationship between
frequency distribution of rwand C w was investigated. As shown in Fig. 2,
a V-shaped distribution was observed, which demonstrates the non-random
localization of the centromere more clearly than does the V-shaped patternseen in Fig. 1.

48 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

400

300
In
UJ

~

~

~

is 200
15
a:
~
JE

:::J
Z

100

1.0 1.2 1.5 1.9 2.3 3.0 4.0 5.7 9.0
ARM RATIO BY WEIGHT (rw )00

Fig. 2. Relationship between the frequency distribution pattern of the arm
ratio (rw) and the size of chromosomes (Cw). The arrows attached to the numericalorders indicate the antimode when 2.0::;;Cw<3.0, 3.0::;;Cw<4.0, 4.0::;;Cw<5.0
and 5.0::;; C w< 6.0, respectively.

It is interesting to note that the value of r w at the antimode tends toincrease as value of Cw increases, i.e.; when (1) Cw < 3.0, (2) 3.0 ::;;; C w< 4.0, (3) 4.0::;;; Cw < 5.0, and (4) 5.0::;;; Cw, r w = 2.3, 4.0, 5.7, and 9.0,
respectively (Fig. 2). This evidence is best interpreted by assuming thatthe antimode is fixed for all sizes of chromosomes at a point located distance"k" per cent from the end, where k is a constant fraction of the total weightof the haploid set. According to this assumption, the antimode is expressedby the arm ratio as r w = (Cw - k)/k. k is, then, computed as k = Cw/(rw + 1). When Cw = 2.0 and r w = 2.3, k = 0.6. The same k values,
0.6, were obtained in the cases where Cw = 3.0 and r w = 4.0, Cw = 4.0
and r w = 5.7, and Cw = 6.0 and r w = 9.0. These data suggests that the
antimode is fixed at a point lying about 0.6 per cent distance from the end

RESEARCHES CARRIED OUT IN 197249

of the chromosome, that is, the point where Sw = 0.6.
The frequency distribution of Sw when C w = 1.9, 2.5, 3.4, 4.3, 5.1, and
80

~ 60

::li:

o

III

o

~

540

~

~ 20
~

6

o+-.,.....,,........,.-r.-r..-.....,,......-r--..-"""".....-r-

o

1.0

2.0

3.0

PER CENT WEIGHT OF SHORT ARMS (Sw)

Fig. 3. Frequency distribution of the size of short arms (Sw). The numericalorders show the distribution pattern of Sw when C w = 1.9,2.5,3.4,4.3,5.1, and 6.4,
respectively.
5,422i~....
1.600
1,400

~ 1.200~~ 1.000

~ 800

u

is
~

600

:lZ

~

400
200

O+-,.....,.........................
o
1.0

~

__....,......_ _..........-..-...
2.0

3.0

PER CENT WEIGHT OF SHORT ARMS (SwlFig. 4.

Frequency distribution of the size of short arms (Sw).

50 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

6.4 is shown in Fig. 3. The figure shows an obvious V-shape having anantimode lying at about Sw = 0.6, which strongly supports the previous
conclusion that the antimode is fixed at a point lying at about 0.6 per centof the total length from the end. As the same V-shaped pattern was foundfor all sizes of chromosomes (Fig. 4), the antimode lying at Sw = 0.6 seems
to be an intrinsic characteristic of mammalian chromosomes.
Proposal of a New Criterion for the Classificationof Mammalian Chromosomes
Hirotami T. IMAl

The present author found that the frequency distribution of the size ofshort arms (Sw) is V-shaped with an antimode at Sw = 0.6, which meansin other words that the position of the centromere is non-random in mammalian chromosomes (see this Ann. Rep. 46). Although the biologicalmeaning of this non-random distribution is unknown, the antimode at
Sw = 0.6 seems to be a clear and convenient landmark for classifying thechromosomes of mammals. Based on this intrinsic character, it is possible
to group mammalian chromosomes into two distinct categories, Sw ::;; 0.6
and Sw > 0.6.
In this context, it is interesting to note that 97.4 % of the "acro- or telocentrics" identified by Hsu and Benirschke (Springer-Verlag, 1967, 1968,1969) in their Chromosome Atlases have Sw ::;; 0.6 and 94.4 % of their"meta-, submeta- and/or subtelocentrics" Sw > 0.6 (see Imai, Ann. Rept.
22: 48, 1972). The same phenomenon is found in the various nomenclaturesystems proposed by Baker (1967), Nadler and Lay (1967), Nadler (1968),
Thaeler (1968), Wurster and Benirschke (1968a, b), and Yong (1969) (Table 1).
This evidence suggests that most mammalian cytogeneticists have a priori
discerned this discontinuity in the frequency distribution of short arm size.
The present author proposes to designate the chromosomes with Sw ::;; 0.6"acro- and telocentrics" (A & T) and those with Sw > 0.6 as "meta-, submeta- and subtelocentrics" (M, SM & ST).
This quantitative classification has certain advantages for analysing
karyotype evolution. There is some evidence that chromosome morphologies have often changed from "A & T" to "M, SM & ST" or vice versa by
centric fusion or fission, or by pericentric inversion all three of which must

RESEARCHES CARRIED OUT IN 1972

51

have played an important role in the karyotype evolution of mammals(e.g., Hsu and Arrighi, Cytogenetics 7: 417, 1968; Wurster and Benirschke,
Chromosoma 25: 152, 1968).
As to the chromosomes with Sw > 0.6, it may be convenient to classify
meta-, submeta- and subtelocentric according to the standardized criterion
proposed by Levan et al. (Hereditas 52: 201, 1964). The antimode at
Sw = 0.6 is expressed by the arm ratio in weight (r w ) as rw = (Cw - 0.6)/
0.6, where Cw is the whole size of a chromosome. Then chromosomes canbe defined by means of the arm ratio in weight within the range of r w <(Cw - 0.6)/0.6 as follows; metacentric (median sensu stricto and medianregion) as 1.0::;; rw < 1.7, submetacentric (submedian) as 1.7 ::;; rw < 3.0,
subtelocentric (subterminal) as 3.0 ::;; r w (the terms recommended by Levanet al. were shown in brackets). In the same way, acrocentric (terminal region) is defined as (Cw - 0.6)/0.6::;;r w and telocentric (terminal sensu stricto)
as r w = 00. All the chromosomes with Cw = 1.2 are grouped here as acrocentrics, since their short arms are Sw < 0.6. It is interesting to notethat most of the minute chromosomes identified by Hsu and Benirschke(1967, 1968, 1969) have Cw < 1.2. The present nomenclature is summarized in Fig. 1.
00..----:1: - - - T

ITl------------_

1•
1

A It I•1
minute •S T Is II

1

1
1
1

1
1
1
1

SM {sml

1
1
1
1

1.0

o

1.2

M

2.0

3.0

4.0

5.0

{ml

6.0

IMI,

,

,

,

7.0

8.0

9.0

10.0

PER CENT WEIGHT OF CHROMOSOMES (Cw)

Fig. 1. Nomenclature of mammalian chromosomes. Solid line; rw=(Cw0.6)/0.6. Broken lines; limit lines of arbitrary classification. M; metacentric.
SM; submetacentric. ST; subtelocentric. A; acrocentric. T; telocentric. (M);median sensu stricto. (m); median region. (sm); submedian. (st); subterminal(t); terminal region. (T); terminal sensu stricto. The terms recommended by Levanet al. (1964) are shown in brackets. Minute; chromosomes with C w< 1.2, which area category of acrocentrics, since they have short arms less than 0.6 (Sw < 0.6).

VI

tv

Table 1.

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

Authors

Chromosome
designation
by authors-~~

Correlation between various nomenclature systems andthe new criterion by arm size__ . -~New criterion by the size ofshort arms (Sw)Sw ~ 0.6
Sw > 0.6

ttl

Materials---_._---------------_.--------,-----

References--_ .. - - - - - - - - - -_. -,- ..

A(M, SM & ST)

51 (98.1 %)6(3.1)I ( 1.9%)190 (96. I)

15 species ofbatsSouthwestern
Naturalist 12 (1967)

A(M & S)58 (96.7)
1 ( 0.7)

2 ( 3.3)
140(99.2)

5 species of
Meriones and4 species ofSpermophilus

Z. f. Siiugetierkunde
32 (1967)
Cytogenetics 7 (1968)

ThaelerA(M, SM & ST)

68 (88.3)
14 (10.6)

9(11.7)118 (89.4)

8 species ofThomomys

Chromosoma 25 (1968)

Wurster(A & SA)294 (84.2)

55 (15.8)

Chromosoma 24 (1968)(M & SM)

7 ( 1.4)

485 (98.6)

5 species of
Bovoidea and32 species ofcarnivores
II species ofrats

Chromosoma 27 (1969)

Baker

Nadler & Lay
and Nadler&

Benirschke
Young

A(S & M)-----

167 (96.0)
10( 7.0)

7 ( 4.0)132 (93.0)-------,.,-- - ' ----

(3
~

o"J1

~

o
~
t""

~

..,

::l

c..,
Chromosoma 25 (J 968)

ttl

o"J1
Cl

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

A = acrocentric, M =metacentric, S or SM =submetacentric, ST=subtelocentric, SA=subacrocentric.

~

(j

CI.l

~

....tvRESEARCHES CARRIED OUT IN 1972

53

Comparison between the Length Method and the Weight Method for
Formulating the Centromeric Position of
Mammalian Chromosomes
Hirotami T. IMAIThe location of the centromere has been considered to be the most usefullandmark for the morphological classification of metaphase chromosomes.
Nevertheless, if we want to understand chromosome rearrangements quantitatively, it is desirable to observe the position of the centromere in interphase chromosomes, since the chromosome rearrangements which haveplayed an important role in karyotype evolution would have occurred on
the stretched DNA strands during interphase. However, the position of
the centromere of interphase chromosomes can only be computed indirectlyfrom two-dimensional images of metaphase chromosomes.
Assuming a uniform thickness and a uniform width for long and shortarms, the per cent length of each arm against total length of the haploidset (S1 and L 1) is correlated with the per cent volume of each arm against
total volume of the haploid set. If the DNA strands are uniformly foldedin the metaphase chromosomes, the S1 and L 1 values should roughly correspond to the DNA content. Recent electron micrographs of the metaphase chromosomes seem to support this assumption (Comings and Okada,
Cytogenetics 9: 436, 1970; Stubblefield and Wray, Chromosoma 32: 262,
1971). Furthermore, a linear relationship between the DNA content of
metaphase chromosomes and their length has been demonstrated by Radley(Exp. Cell Res. 41: 217, 1966).
However, the assumption that the widths of the short and long arms areapproximately equal in every size of chromosomes has seldom been examined. To study this, the relationship between the length of the short arms
(S1) and the ratio of arm widths (mean width of short arm/mean width oflong arm) was examined using the chromosomes of Folios no. 1-25 obtained
from the Chromosome Atlas by Hsu and Benirschke (Springer-Verlag, 1967).
It was discovered that the ratio of arm widths was about 1.0 in chromosomeswith S1 > 1.0, but that it was reduced remarkably in those with S1::S;; 1.0(Fig. 1). The use of the length method for the determination of the short
arm size with S1 ::s;; 1.0 seems to result in overestimation and error. The
sizes of minute short arms can be determined more accurately by the weight54 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

method than the length method. Assuming a uniform thickness of themetaphase chromosomes on a glass slide as well as of the photographic
paper used, the Sw values correspond to DNA content in the short arm (seethis Ann. Rep. 46).

---------I-----1------I------L----- i

III

I

I
o

1.0

2.0

3.0

4.0

5.0

PER CENT LENGTH OF SHORT ARMS (5,)

Fig. I. Comparison of widths of short and long arms. Broken line; expectedline assuming that the widths of both arms of a chromosome are qual. Solid circle;
the observed ratio of arm widths.

Studies on Chromosomes of the Silkworm, Bombyx mori
Akio MURAKAMI and Hirotami T. IMAI

It has been established that the diploid and haploid numbers of silkworm
chromosomes are, 56 and 28, respectively. To reach this final figure, however, it has taked about four decades after the first observation by Henking(1892). This would be due to a technical difficulty to estimate the exact
chromosome number by the paraffine-sectioning method. During the lasttwo decades, techniques of chromosome preparation have developed remarkably in mammals and others. Recently, Imai (Chromosoma, 37(1972): 193-200), one of the present authors, demonstrated exact chromosome numbers and clear cut metaphase figures in ants by employing theimproved squash method.
This preliminary communication will report on the result of observations
on the silkworm chromosomes according to the colchicine hypotonic squash
method developed by Imai (1972).

RESEARCHES CARRIED OUT IN 1972

r
•• •
.. ••
• ..
•
.", .
.1

..

,

." ,

-..

•

•
3

~

,

2

I

•

,

..,.

•

•ttl-

..

•
.-.
. ..,
,.,. • ••I-.• • •

• •

• •
•
••

•

-

fit . '••

, .,...
•

55

•

4

• "• •

5

Fig. I.
FIGURE LEGENDSI) Mitotic metaphase of spermatogonial cells (2n=56).
2) First meiotic metaphase having one minute fragment (arrow).
3) First meiotic metaphase (n=28).
4) & 5) First meiotic metaphase and diakinesis with one chain quadrivalentori~inated from reciprocal translocation (arror).

56 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Testes obtained from 3rd or 5th instar larvae were dipped in a colchicinehypotonic solution (0.01 % colchicine in 0.45 % sodium citrate solution) for
30-45 minutes at room temperature. After this treatment, the testis wastransfered on a slide slightly tipped to remove the hypotonic solution completely. The testis teared into pieces in a drop of 60 % acetic-ethanol (I: 3)
fixative was squashed gently by finger pressure. The squashed preparation
was frozen on a block of dry ice for a couple of minutes to take off thecover glass using a razor. After the ice was melted by blowing air over it
gently with a hair-dryer, the slide was immediately dipped in absolute glacialacetic acid for 30 seconds to reduce cytoplasmic staining, and was dried
again with the hair-dryer. The preparation was stained for about 10 minutes
in Giemsa solution (Merck) diluted 30 times in Sorensen's phosphate buffer(pH 6.8).
Typical mitotic metaphase figure of spermatogonial cells (2n=56) andthe haploid chromosome (n=28) at meiotic metaphase of spermatocytes
are shown in Figs. 1 and 3, respectively. These observations coincide withthe standard number of chromosomes of the silkworm reported by authors
in the past. It is worthwhile to note that the colchicine-hypotonic squash
method is a simple procedure for providing exact and reproducible preparations. Thus, it seems to be certain that the method is useful for detecting cytological abnormalities of the silkworm chromosome. In fact, someobservations were carried out on minute fragment chromosomes (Fig. 2)
and a translocated chromosome (Figs. 4 and 5), which have been drived
from X-rayed silkworms.
However, it can be learned from these figures that silkworm chromosomes
could not be characterized each other by the location of centromers. Results obtained in the present experiment support the observations by Friedlander and Wahrman (1. Cell Sci., 7 (1970): 65-89) who indicated that thesilkworm chromosome was one of diffused centromer type or holokinetics
as often observed in other Lepidoptera.

The Sixth Cell Population Change to Occur During
Serial Passages of the MSPC-l Mouse Myeloma
Kazuo MORIWAKI and Tamiko SADAIE

The mouse myeloma MSPC-I has exhibited cell population changes at

RESEARCHES CARRIED OUT IN 197257

least 5 times during serial passages since 1966. These were detected bysingle non-disjunction, the appearance of marker chromosomes (A, BandC) and loss of the ability to produce gamma-globulin. In the middle of1972, a sixth cell population change was detected by the appearance of the
D-marker chromosome. This new cell population has grown rapidly, andafter 9 generations the former subline having 39 chromosomes and theA, Band C markers had changed completely to the new one having 38
chromosomes and the A, B, C and 0 markers (see Fig. I).

,

•

,"

,

'C

<~D

-...
;.

C

1#

4

\

Fig. I. Banded karyotype of a NP-38-ABCD subline cell in MSPC-l mouse
myelom,l. D-marker chromosome is translocated on C-marker.

The cell population shift that is described above could be repeated byusing frozen tumor cells of subline NP-39-ABC which were stored at-80°C 5 generations before the D-marker was observed. In this case thecell population shift was very fast. For instance, generation 114 consistedof 100 % NP-39-ABC cells, generation 117 75 % NP-39-ABC and 25 %NP-38-ABCD and generation 119 100% NP-38-ABCD.
The rate of cell population shift seems to be affected by the inoculum
size of the tumor cells. If a sample of 10 5 cells containing equal numbersof NP-39-ABC cells and NP-38-ABCD cells was transplanted subcutaneously, the resulting tumors contained almost equal numbers of both subline

58 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

cells. But if 10 3 cells were tested in the same manner, the NP-38-ABCD
cells tended to proliferate more rapidly than the NP-39-ABC cells.
Ecological Genetic Studies on the Differentiation ofChrysanthemum Species
Shuzo NAGAMIli

About 40 wild Chrysanthemum species are reported in Japan (Kitamura,1940). In 1972, 22 species among them were sampled from their respective
natural populations, and were transplanted in the garden of National
Institute of Genetics in Misima for studies on the pattern of species differentiation. The results of pollen investigations are summarized as follows:
I. Ultramicro-structures of pollen grains were observed by using ascanning electron microscope. The pollen grains of all these species were
spherical and had many small spines on the surface. In proportion to theincrease of Chrysanthemum polyploidy, gradual increase was observed in thesize of pollen grains, but no increase was found in the number of pollen spines.
Clear differences among the 22 species were frequently seen in the shape ofpollen spines.
2. Chrysanthemum pollen grains did not germinate well on culture media
so far devised. But two suitable media were found recently, whose components were as follows: (A) sucrose 25 % and agar 1.5 %, and (B) sucrose
25 %, agar 1.5 %, boric acid 250 ppm and malonic acid 10 ppm. In general,the latter gave better pollen germination than the former. Germination
rate varied from 0 % to 35 %. Pollen longevity of Chr. pacificum (2n=90)
was about 6 hours after emission at room temperatures, but it was extended
to 72 hours when stored at -15°C with silica gel (Nagami, Akizawa andIwanami, 1972).
3. Isozymes in the pollen grains of Chr. Shiwogiku var. kinokuniense(2n=90) were examined by starch gel electrophoresis. The results wereas follows: (1) bands of acid phosphatase, esterase, asparate aminotransferase, leucine aminopeptidase, alcohol dehydrogenase and malate dehydrogenase (+ nicotinamide-adeninedinucleotidephosphate) were clearly identified, (2) weak bands were observed for malate dehydrogenase (+ nicotinamideadeninedinucleotide) and isocitrate dehydrogenase (+ nicotinami~-- -Professor of Department of Biology, Faculty of Education, Yokohama National
University.
1)

RESEARCHES CARRIED OUT IN 197259

deadeninedinucleotide), but (3) no isozyme band was observed for peroxidase, succinate dehydrogenase, glutamate dehydrogenase (+ nicotinamideadeninedinucleotide) and a-glycerophosphatedehydrogenase).

60 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

VI.

MUTATION AND MUTAGENESIS IN ANIMALSA New Sensitive Test System for the Detection of
Weak Chemical Mutagens Using Silkworm Oocytes
Yataro TAZIMA and Kimiharu ONIMARU

With a purpose of exploiting a sensitive system for the detection of weakchemical mutagens, researches have been carried out with silkworm in two
directions: one was screening of sensitive strains and the other was inquiring
into the most sensitive cell stages for the chemical treatment. Seven representative strains for differential radiosensitivity have been compared in
respect to their sensitivity to chemical mutagens by injecting a same dose ofphysiological saline solution of mitomycin-C to mature male larvae. The
difference thus observed among strains was far less than anticipated, showing only four fold difference between the most and the least sensitive strains.
Furthermore, induced mutation frequencies fluctuated from experiment toexperiment. In contrast, differences in mutability among different cellstages were far larger than those between strains in both sexes.
It has been known in the silkworm that oocytes were extraordinarily
sensitive to the mutagenic action of radiation around the stage of meiotic
metaphase I and II. We, therefore, focussed our study to the treatment ofcells at those stages with chemical mutagens. Since meiotic metaphasetakes place immediately after oviposition in this insect, we first tried toadminister mitomycin-C to ova in the ovariole by injecting it into the body
cavity of female pupae one day before emergence. The chemical were injected into wild type female pupae in several doses, and mutation frequencies
were measered by specific loci method. Linear relation was obtained whenexperimental data were plotted on logarithmic scale between administereddoses and mutation frequencies, indicating that the injected chemical
penetrated the chorion and induced the mutations. However, the mutation
frequencies were far lower than expected.
Next, injection was performed at earlier pupal stages: i.e., 3 or 5 days
before emergence. Injected dose was 5 ,ug/head. The results are partly
shown in Table 1.
The results clearly indicated that injection three days before emergence

RESEARCHES CARRIED OUT IN 1972

61

Table 1. Changes in mutation frequency according to the stage of
injection into female pupae. (Treated + + ~ x pe re J, 723 rb)*---------------------

Injected
stage
Cont.
1 day
before3 daysbefore5 daysbefore----No. ofeggs
observed

Mutation frequency (10- 5)pe

pe mosrere mos

26682
19509

11.2
20.5

15.0
0

3.7
15.4

14122

226.6

191.2

5910

1895.11844.3others**Induced
total

Mut. freq.

3.7
15.4

33.6
51.3

1.8

177.0

49.6

644.4

61.1

1336.7930.66006.7

597.3

50.7

- - - - _ ~ _ -* Injection: MMC 5 Ilg in 0.025 ml physiological saline per head.
** Induced mutation frequency: x 10-5jllgjlocus.

was 10 times and that five days before was more than hundred times as effective as the injection one day before. The effectiveness seemed to be inversely
correlated with the formation of a chorion, which is completed one day
before the emergence. The amount of chemicals to be incorporated into theegg plasm might be very limited after the completion of the chorion but it
might be fairly large before the completion of the chorion.
These findings seemed to permit us to develop a very sensitive test
system for the detection of weakly mutagenic chemicals. This was confirmedby the examination of the mutagenicity of a weak mutagen, Panfuran. Thischemical is a derivative of nitrofuran and widely used in Japan as clinicallyuseful topical anticeptics. The weak mutagenicity of this compound to
the silkworm has already been reported by us (Tazima and Fukase, 1966).
The effect was so indistinct that we could hardly conclude the positiveeffect. In the present experiment, the chemical was injected into female
pupae four days before emergence in doses 4.5 and 17.5 ,ugjhead. The
results were diffnitely positive.
Thus, the method was concluded to be a very sensitive test system which
could be utlized for the detection of weakly mutagenic chemicals.

62 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Test for Mutagenicity of Mercuric and Cadmium Compounds
Using Oocyte System of the Silkworm
Yataro TAZlMA and Yosoji FUKASE

Ever since the causality of organic mercuries and cadmium compounds
was recognized with regard to serious human injuries, a suspicion has been
aroused upon the mutagenic action of those compounds. In order to get ananswer to this question we carried out experiments with use of silkworm
oocyte system soon after we noticed its high sensitivity. The compounds
we tested were Methylmercury chloride, Mercuric phenylacetate, Cadmium
carbonate and Cadmium hydroxide. Those compounds, disolved in aphysiological saline solution, were injected into wild type female pupae fourdays before emergence, whereby enough quantity of chemicals could havebeen incorporated into egg plasm. Doses given were 0, 0.1, 1, 10 and
100 fig per head in cases of mercuric compounds and saturated concentration and its 1/4 in cases of cadmium compounds. Injection volume was
0.025 ml/head. Mutations were measured by specific loci method using pe
and re marker genes. Results for Methylmercury chloride are shown in
Table 1.
Table I.
Dose
,ug/head
0
0.1
1
10
100

Results of mutagenicity test for Methylmercury chloride.
(Treated + + <f x pe re c5, 723 C108). _ - - . _ - , , - - -.. _ - - - - _ .
5)Mutation frequency (10-No. of eggs
observed
30365
39992
33275
31158
3947

pe

pemos

re

re mos

total

9.9
20.0
27.0
35.3
25.3

23.1
45.0
57.1
22.5
50.6

13.2
10.0
15.0
9.6
0

6.6
10.0
6.0
12.8
0

52.8
85.0
105.1
80.2
75.9
----""._-As seen from the table, the compound seemed to exert toxic effects to growing oocytes at higher doses but almost no mutagenic effects upon the silkworm germ plasm. Likewise, Mercuric phnylacetate, Cadmium carbonate
and Cadmium hydroxide failed to be detected of their mutagenicity by thistest system.
Since the method used in this experiment is very sensitive one, it can beconcluded that those chemicals are not mutagenic in the silkworm.

RESEARCHES CARRIED OUT IN 1972

63

Consequences of the Selection for Mutation Frequencies Presumably
Due to Replication Instability in the Descendants ofMitomycin-C Treated Silkworm
Kimihraru ONIMARU and Yataro TAZIMA

We reported earlier (Onimaru and Tazima, 1969, this Report No. 19)that in the descendants of EMS and/or Mitomycin-C treated silkworm,
a mutation from + to re had continued to occur generation aftergeneration. In that experiment wild type males were treated and crossed
to pe re females. In the F 1 (hereinafter represented by C 1) several mutants,
whole types and mosaics for pe and re, appeared in addition to expectedwild type. Those wild type females were back crossed to pe re males andC 2 were obtained. The expected phenotypes in this generation were 1 +:1 pe, because pe is epistatic to re and recombination does not occur in thefemale. It was observed in C 2 that, in addition to those expected phenotypes, re mutants appeared in some batches (29 betches among 418 tested)
and the frequency of re within a batch was 4.8 % on an average. Wild typefemales from those re producing batches were back crossed again to pe remales and thus the strains were maintained (C g , C 4 ••• ). The occurrenceof the re mutation continued to later generations.
The frequency of re producing batches to total observed was 29/418 in
C 2 , 77/221 in C g and reached 100 % in C 4 • The frequency of the appearanceof re within a batch varied remarkably among batches and in high re producing batches the ratio of + to pe became lower than unity. Wild typefemales from non-re producing batches in C g were tested in a similar way,by sampling 50 eggs from each of 20 batches randomly, but no re producing
batches were discovered in C (0/198). Those observation strongly suggested that the observed case could be regarded as due to the replication instability as already reported for Schizosaccharomyces and Drosophila.
Since the above experiment was carried out on mixed batch basis, next
experiment was undertaken on separate batch basis, so as to investigate
more exactly the change in the frequency of re producing batches. The
experiment started from three batches of C 1 • The frequency of re producing
batches in C 2 were 5/86, 5/100 and 4/113, in which the numerator denotes anumber of re producing batches and the denominator a total number of observed batches. In C g the frequency reached 100 % in some families but
was fairly low in others, showing large variation among families. In C 4

64 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

all batches became re producing. However, the effect of selection was notclear with regard to the frequency of re eggs within a batch, which is shownin parenthesis in the Figure I.
Fig. 1. Procedure and consequences of the selection of mutation frequency
in the descendants of mitomycin-C treated individuals.
Cl
C2
C3
C4
0.621---> 4/7 (0.7)
0.6---->5/86 11 (6.9)31

0.7--->5/100(4.7)

0.8 ---->4/II2 (5.5)~ --->47/47 (12.9) [i~:~=:~~;~~ Hg:1~

5.7 ---> 7/7 (17.0)[ 11.2 ---> 2/15 (1.3)
14.1 --->IO/IO (14.0)
11.6 --->40/41 ( 7.4)
10.8 --->13/13 (10.8)
0.9--->[ 0.2 ---> 0/29 ( 0.0)
0.2 ---> 1/48 ( 0.3)

[b:~=:jg;jgH:6~[I~:~ =: 8/8 (5.3)

0.2 ---> 2/7 (0.2)
0.2 ---> 2/69 ( 0.01)

Note:I) 5/86 denotes 5 re producing egg batches among 86 tested.
2) Per cent of re eggs within each re producing egg batches.
3) Average percentage of the re eggs within re producing egg batches.

It should be noted that the occurrence of re mutation in all batches was
attained as early as in C 3 in this experiment, one generation earlier than theformer experiment which was carried out on mixed batch basis. Whenmutation frequency was reasonably high, the appearance of re producing
batches could be expected to reach 100 % in C3 • This was actually observed
for about half number of families. These results suggested that in addition
to a instable primer at least a co-factor is necessary for the production of
mutation in this strain.
Further Studies on the Instable pM Allele of the Silkworm
I. Evidence for the Occurrence of Recombination Between
Translocated and Free Second Chromosomes in the Female
Yataro TAZIMA and Akio OHNUMAIn the previous report (this Annual Report No. 22) we described an

RESEARCHES CARRIED OUT IN 1972

65

instable pM allele locating on the second chromosome segment of a W.II
translocation. Although very seldom mottled females with regard to pM
marking appear and when crossed to p males, they produce daughters ofseveral phenotypes, belonging to the same multiple allelic group: pH, pM,
pSa, + P, and p. In contrast, their sons comprised only one phenotype, p.
The newly arisen phenotypes were transmitted to their female offspring as
if each of them were a complete mutation that had occurred at pM locus on
the translocated segment. For the interpretation of the appearance ofseveral phenotypes belonging to the same allelic group, an assumption wasnecessary that mutation had occurred several times at the same locus during
very early cleavage stages in the pM mottled female. Such instability ofpM locus can be interpreted by assuming, as was the case for variegated position effects in Drosophila, that an euchromatic region involving pM locus
and a heterochromatic region of W chromosome were brought in a close
proximity due to the translocation. It is only hypothetical and does not
explain why existence of two kinds of chromosome region in close proximity cause instability of pM locus.
In order to see if the two free second chromosomes behave freely fromthe translocated segment of the second chromosome, W· pM/ Z, p Yjpy females
were crossed to pyjpy males. The results were surprising. It was revealed
that recombination could occur between translocated and a free chromosome
even in the female.
Among seven families tested six behaved just as expected. Whereas,
one family showed quite abnormal segregation; i.e., pMy~ 61, pMy~ 63,
pSay~ 100, py~ 1, pY(JI 68, and py(JI 179.
The newly arisen pSa Y females were transmitted per se to the next generation, when they were back crossed to py males. These results could only
be interpreted by assuming the occurrence of recombination between translocated and a free second chromosome during a very early cleavage stage,
giving rise to a mosaic comprising two kinds of genetic constitution; W.pM jZ,
pyjp Y and W.pSa Y/Z, pyjpy. The former genetic constitution accounts
for the appearance of pM Y ~ 6I, pMY ~ 63, p Y c3' 68, pyc3' 68, and the latter
may account for the appearance of pSa Y ~ 100 and pyc3' 11 I.
These evidences suggest that the observed instability at pM locus is not
only due to mutations occurring at pM locus but also due to an unknown
mechanism of chromosome recombination.

66 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

X-ray-Induced Crossing-Over in Oogenesisof the Silkworm
Akio MURAKAMI

Physical and chemical agents have been known to induce or enhance thegenetic recombinations or corssing-overs in both meiotic and mitotic cells.
Recently, the repair mechanism in recombination for radiation-induced
DNA lisions has attracted special attention.
Tanaka (1913) found that in the silkworm, crossing-overs do not normallyoccur in oogenesis. Later, many investigators have attempted to produce
artificial induction of crossing-overs by means of X-irradiation to oogenesisof the silkworm. They found a number of exceptionals which might haveresulted from the induced crossing-overs. Most of the experiments, however, was lacking in sufficient progeny tests except for a few cases as criticizedby Tanaka (1953) and dealt with the middle or late growth stage oocytes inpupae although the most likely cell stage for induction of crossing-overs
may be an early growth stage oocytes. In the present study an investigation
was carried out by using egg-color (pe and re) markers to know whether
X-rays could induce crossing-overs in oogenesis of the silkworm.
Early stage oocytes in fourth instar larvae carrying the heterozygous
genotype (;:~ ~) were irradiated with 180 kVP X-rays (1000 and 2000 R
at a dose-rate of 300 R/min) and they were then crossed to male moths having the homozygous genotype for pe and re genes. Spontaneous crossingovers were also checked in unirradiated control. By this procedure, any
crossing-overs could be detected by finding exceptional black and yellowishwhite eggs among normal yellowish-white and red eggs. Since exceptional
yellowish-white eggs could not be distinguished from normal ones, a progeny test has been done for black exceptional eggs. In view of the matingprocedure, however, the crossing-over type black eggs also could not be
distinguished from the polyploid type black eggs. To make clear thisconfusion, the moths developed from the exceptional black eggs were backcrossed again to moths carrying the homozygous for pe and re genes. Since
it was well-known that tetraploid (4n) on triploid (3n) type eggs weregenerally much larger or smaller in size than crossing-over 2n type eggs,
difference in egg size was useful for distinguishing each other. In addition, by comparing egg-color segregation ratios in their hybrids, it was pos-

RESEARCHES CARRIED OUT IN 197267

sible to classify them into crossing-over and polyploid types: female
moths which produce yellowish-white and black eggs in the ratio of I to I
and male moths which produce yellowish-white, red and black eggs invarious ratios could be regarded as the crossing-over type. While, female
moths which produce yellowish-white, red and black eggs in various ratios
could be regarded as the incidence of polyploid type. A lack of this classmale moth would be interpreted to be due to retardation of XO type malecells in the process of gametogenesis.
The results indicated that the frequency of exceptional black eggs increased
with doses of X-rays. One crossing-over type moth was found out of fiveexceptionals examined in the control experiment. Thus, the spontaneous
frequency of the crossing-over in silkworm females was estimated to be
3 X 10- 6 • This suggests that the crossing-over in oogenesis of the silkworm
does not completely lacked, but is very strongly inhibited. Three females(17 X 10- 5) and two males (11 X 10- 5) crossing-over types were found out often exceptionals in the 1000 R series and one female (12 X 10- 6) out of twelve
exceptionals in the 2000 R series. Although this experimental scale was notenough, one can be said that there were less frequently induce crossing-overs
in the 2000 R series than in the 1000 R series in contrast to polyploid.
This suggests that the polyploid, which may be due to inhibition of celIdivision, would be much more frequently induced with the high doses
than with the low doses. No crossing-over was observed in pupal oocytes.
In any case, it may be given a conclusion that X-rays could enhance thecrossing-over in early growth stage oocytes of the silkworm.
A Comparison of Relative Mortalities by 2 MeV Neutrons
and X-rays of Young and Aged Dormant
Silkworm Embryos
Akio MURAKAMI and Tamiko IWASAKI'"To establish a relationship between the radiosensitivity and the aging
is one of important and fascinating subjects in radiobiology. It has generally been thought that there are various biological and technical limitations in
insects as compared to mammals to investigate such a biological event.
The silkworm has a biological advantage that the duration of dormant em'" Division of Biology, National Institute of Radiological Sciences, Chiba.

68 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

bryonic stages can be regulated by the temperature with a slight change
in the physiological activity of embryos. The present experiment carriedout to make clear a relationship between the radiosensitivity and the age ofembryos was investigated by comparing the sensitivity of embryos at two
different stages which were exposed to radiations having two different ion
densities.
Embryos of the CI08 silkworm strain at two different dormant stages,
prehibernated (3 month-old) and post-hibernated (10 month-old) stages,
were exposed to either 2 MeV neutrons (at a dose-rate of 100 rads/min) or
180 kVp X-rays (at a dose-rate of 300 R/min) and embryonic mortalities
were determined by the reduction of hatchability. The neutron beam was
obtained from a Van de Graaff accelerator in the National Institute of Radiological Sciences, Chiba and gamma contamination was less than 10
per cent.
It was clearly observed that there was a larger shoulder in the dose-response curve for young dormant embryos than in that for aged ones regardlessof the radiation quality. This was more pronounced with neutrons thanwith X-rays. Practically, no shoulder was observed in the curve for aged
dormant embryos exposed to 2 MeV neutrons. LD 50 of X-rays and 2 MeV
neutrons for young emrbyos was about 1400 and 350 rads, respectively,
while that for aged embryos was about 600 and 100 rads, respectively. Thisfinding indicates that aged embryos were much more sensitive in embryonicmortality than young ones. Comparing the LD 60 value of 2 MeV neutrons
with that of X-rays, a relative effectiveness (or RBE) of 2 MeV neutrons forthe radiation-induced embryonic mortality can be estimated. Thus, RBE
values for young and aged dormant embryos were about 4 and 6, respectively, suggesting that the RBE would be increased with the age of embryos.
A possible reason for changes in radiosensitivity and RBE in the processof embryonic development may be due to either the increase in radiosensitivity or the decrease in repair mechanism in accordance with the processof senescence of embryos.
In comparing the RBE values for the embryonic mortality in the silkworm
of 2 MeV neutrons with that of 14 MeV neutrons in the previous report(Murakami and Kondo, this Report, No. 14 (1964): 106-107), the former
is 1.2 to 1.8 times more effective than the latter, indicating that the RBE
for the embryonic mortality clearly depends on the LET. This observation
would be interpreted as due to the chromosomal damage as revealed cytolo-RESEARCHES CARRIED OUT IN 197269

gically in Artemia which was exposed to 2 MeV neutrons (Iwasaki et a/.,Radiation Res., 45 (1971): 288-298).

Mutagenesis of Acridine Orange in Cleavage Nucleiof the Silkworm
Akio MURAKAMIIn the previous paper (Murakami, 1972), it was shown that acridine orange(AO) was mutagenic for germ-cells in female silkworm pupae, but not for
male pupae. The explanation for these findings was that AO, which hadtaken into ooplasm of oocytes (or eggs), would affect metabolic and noncrystallized DNA in either meiotic oocytes or cleavage nuclei by intercalating with DNA bases. In yeast cells, however, it was observed that 5-aminoacridine was mutagenic for meiotic cells, but antimutagenic for mitotic cells(Magni et a/., 1964). The present study was carried out to check whether
AO is mutagenic for mitotic cleavage nuclei in the silkworm.
Mutation frequencies were estimated by the specific-locus method using
egg-color genes, pe and reo For selectively testing the mutagenic action of
AO for mitotic cleavage nuclei, AO was injected into seven day-old female
pupae with double markers. After emergence they were then mated to
ClO8 wild type male moths. The germ-cells of female pupae at the stageof injection were prophase I oocytes. Oviposited F 1 eggs were scored on
the visible phenotype with complete or mosaic mutations.
A positive mutagenic effect of AO was clearly observed on chromosomes
originated from the wild type male. The majority of mutants recovered was
mosaics which had small mutated areas on the egg, while complete mutations detected was remained on a level of the control. It is of interest
to note that the mutant area in mosaics produced by treated pupae withdouble markers was less than that in mosaics produced by treated wild type
pupae which were mated to marker male moths. If mutational eventsoccurred in oocytes, mutated areas may become a large size, on the contrary, mutational events occurred in somatic cells at the stage of cleavage
divisions, the areas may become small size in the process of egg development.
Thus, it may be concluded that most of mutants detected in the present
experiment would be originated from mutational events in the stage of
mitotic cleavage divisions.

70 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Although in a small scale experiment, it was observed that AO wasmutagenic for meiotic spermatocytes and that the ratio of complete to mosaic
mutations was an equal. It was also observed that the mutants detectedin the treated wild type female pupae (Murakami, 1972) would be originatedfrom mutational events in both mitotic cleavage nuclei and meiotic oocytes.
These findings in the silkworm seem to be incompatible with observations
in yeast cells (Magni et al., 1964) and in E. coli (Streisinger et al., 1966).
One of possible interpretations for this contradiction among different biological materials would be the difference in biophysical conformation of their
DNA molecules.

Mutagenesis of Quinacrine Compounds
in the Silkworm
Akio MURAKAMI

The acridine compounds are commonly used in medicine as antibacterial,
carcinostatic and antimalarial drugs. They are also used as dyestuffs in
the textile industry. Quinacrine hydrochloride (QH) or atebrine, andquinacrine mustard (QM) are acridine derivatives that have been used as
antimalarial and carcinostatic agents, respectively.
In the silkowrm, acridine orange, acridine yellow and proflavine arehighly mutagenic for female germ-cells (or prophase I oocytes) in pupae
after injection regardless of the lighting condition (Murakami, 1972). In
contrast, these acridines are not mutagenic for mature sperm. Therefore,
female germ-cells in silkworm pupae seem to be a simple and relevant mutational assay system for acridines and related compounds.
This communication reports a positive mutagenic effect of QH and QM
on silkworm oocytes as well as on cleavage nuclei, but not on mature sperm.
QH (0.25 and 0.50 mg/capita) and QM (0.04 mg/capita) in saline were
injected into the ventral abdomen of eight day-old wild type C108 silkworm
pupae. The germ-cells at this stage pupa are in prophase I oocytes andmature sperm in female and male, respectively. They were then mated after
emergence to marker strain moths carrying double recessive egg-color genes,
pe and reo F I eggs were examined for their egg color to determine the occurrence of mutations.
The results indicated that mutagenicity of QH was low for pupal oocytes

RESEARCHES CARRIED OUT IN 1972

71

treated, while that of QM was markedly high and about ten times more
effective than QH. No significant mutagenic effect of both compounds wasobserved on mature sperm. This sex difference in mutagenic activity ofthe quinacrine compounds for pupal silkworms may be caused by thedifferential DNA conformation in germ-cells, either female meiotic and mitotic cells or mature sperm.
In comparing the mutagenicity of other acridine compounds, acridine
orange, acridine yellow and proflavine, for pupal silkworm oocytes, QH wasthe least effective among them. It is known that QH did not bind lessstrongly to DNA than did proflavine (Drummond et al., 1965). Hence it
can be said that intercalation of acridines and related compounds between
DNA bases would be a necessary condition to induce mutation by thesecompounds, but not a sufficient one.
The majority of mutants recovered in the present experiment was mosaic
types as observed in the experiments with acridine orange. This may beinterpreted as being due to preferential insertion of quinacrine compounds
into one of double-stranded DNA molecules. If so, a possible mechanism
how complete mutants appear in meiotic oocytes would be an induction ofrecombination-like events between paired homologous DNA molecules if
one of them (temporarily) contains an intercalated compound.

72 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

VII.

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICROORGANISMS AND PLANTS
Participation of DNA Polymerase I in the Repair of
r-ray Damaged DNA in Permeable Bacillus
subtilis Cells
Takehiko NOGUTI and Tsuneo KADA

Gamma-ray irradiation reduces biological activity and molecular sizeof cellular DNA. We have recently shown that the DNA damage was
repaired in B. subtilis cells made permeable by toluene treatment when they
are supplied with substrates and cofactors for DNA polymerase and ligase(Noguti, T. and Kada, T., J. Mol. BioI. 67: 507,1972). Mutant cells lacking
DNA polymerase I failed to repair the DNA damage in the above system(Table 1). Gamma-ray irradiation and toluene-treatment of wild cells showed remarkable high level of DNA synthesis compared to the non-irradiatedcontrol. On the other hand, the pol- cells showed a very reduced level ofDNA synthesis upon r-ray irradiation. This r-ray stimulated DNA synthesis seemed to be an essential process of the repair.
Table 1.

Repair of r-ray damaged DNA in toluene-treatedpo/+ and po/- cells---~._._--

Strain

HA 101 (po/+)

r-ray
dose
0
0
0
30
30
30

Incubation
medium(kR)
buffercompletebuffercomplete

Incubation
period(min.)% transformants

0
10
10
0
10
10

100
59
41
4.8
3.2
33
---._._---- - - - - -HA 101 (59) F(po/-)

0
0
0
30
30
30

buffercompletebuffercomplete

0
10
10
0
10
10

100
73
39
8.1
4.1
5.5

RESEARCHES CARRIED OUT IN 197273

HPUra(6-(p-hydroxyphenylazo)-uracil) has been reported to be a specificinhibitor for semiconservative DNA replication of B. subtilis cells (Brown,
N. C., J. Mol. BioI. 59: I, 1971), and more recently to inhibit action of pol
III (Mackenzie, J. M. et al., Proc. Nat. Acad. Sci. U. S., 70: 512, 1973).
The r-ray stimulated DNA synthesis was refractory to this inhibitor with adose sufficient enough to inhibit ATP dependent DNA synthesis to less than
one tenth.
These results led to conclude that the type I DNA polymerase is actually
involved in the above repair.

Purification of an Enzyme Specifically Involved in
Repair of Gamma-ray Irradiated DNA
Takehiko NOGUTI and Tsuneo KAnA

The involvement of a third enzyme in addition to DNA polymerase I
and DNA ligase relevant to the r-ray irradiated DNA was shown lastyear and described in the previous annual report (Noguti, T. and Kada, T.,
No. 22: 68, 1972). Gamma-ray irradiation in vitro usually reduces priming
activity of DNA for DNA polymerase I. However, the priming activity of
DNA extracted from irradiated cells was rather more enhanced than thatwithout irradiation. It was further shown that treatment of irradiated DNA
with cellular extract brought about remarkable enhancement of the priming
activity. If the cellular extract was heated at wooe or treated with pronase,the above effect was disappeared. The SDS or phenol treatment also caused
inactivation of the activity. Because such an effect was incubation dependent,
we assume that our extract contains an specific enzyme. This cellularenzyme was expected to play a role in the repair by converting damaged
DNA into a better primer for DNA polymerase I.
We made efforts to purify this cellular enzyme. The assay method of its
activity consists of measuring the amount of radioactive precursors incorporated into the primer DNA which had been irradiated with r-rays,
treated with the enzyme and purified again. In the present study, Mycrococcus Iysodeikticus DNA polymerase (type I) was prepared and used.
The cellular extract (F-I) containing less than 2 % of cellular DNA wasprepared from B. subtilis cells grown in e+ medium by treating with Brij-58
and lysozyme at low temperature (at 7-8°C). Subsequently, the extracted

74 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

protein was condenced by ammonium sulfate precipitation (F-II). Specificity of this enzyme fraction toward r-ray damaged DNA is shown in
Table I. After dialysis, the F-II was adsorbed on DEAE cellulose column
and eluted by a linear gradient with NaC!. Most of active fractions werecollected and condensed by dialysis against solid sucrose (F-II1). After
dialysis, the F-III was adsorbed on phosphocellulose column and eluted bya linear gradient with (NH 4)2S04. Two peaks appeared between 0.05 M
and 0.2 M (NH4)2S04 concentrations. The mode of action of these active
fractions is now under investigation.
Table 1. Specific enhancement of priming activity of gamma-ray
irradiated DNA after treatment with F-II fraction
Gamm-irradiation
on T7 DNA

Incubation

Priming activity(kR)(min.)

(cpm)

o
4

o

430

60

466

o

188
1750
125
2060

60
8

o
60

Mapping of rec Genes in Bacillus subtilis
Yoshito SADAIE and Tsuneo KADA

Past isolation and characterization studies on bacterial mutants defectivein genetic recombination have been carried out mainly in E. coli, revealing
the roles of different rec genes in processes of recombination, repair andmutation as well as in other DNA metabolisms. Recombination-deficientstrains of Bacillus subtilis were also isolated. Three of rec mutations
are actually mapped (Hoch, J. A. and Anagnostopoulos, C., J. Bacterol.
103: 295, 1970; Sinha, R. P. and Iyer, V. N., J. Mol. BioI. 72: 711, 1972).
Studies on the recombination in B. subtilis have unique features; chase oftransforming DNA in the course of integration into host chromosome andbiochemical studies of DNA molecules in mutants with various genetic defects in the recombination capacity.
Through isolations of r-ray sensitive strains of B. subtilis, we charac-

RESEARCHES CARRIED OUT IN 197275

terized several recombination-deficient strains. Some of our studies are
already described (Kada, T., Tutikawa, K. and Sadaie, Y, Mutation
Res. 16: 165, 1972; Sadaie, Y. and Kada, T., Mutation Res. 17: 138, 1973).
The chromosomal location of the ree mutations was first studied in a strain
named M45 which showed very reduced frequencies both in genetic transformation and PBSI transduction. One class of revertants of M45 having
intermediate sensitivities to radiations and mitomycin between the M45
and wild strains possessed a mutation in the reeA gene. This mutation
was linked to ura-l and causes reduction in the frequency of transformation
but not that of PBSI transduction and therefore designated as reeA45.
PBSI phages grown on the M45 or on their revertants, did not give any
Rec+ transductant among Ura+ transductants in recipient cells of GSY
1037 (ura-l reeAl). When DNA isolated from the revertants of M45
was used for transformation of the M45, no Rec+ transformant was obtained
and most transformatns were of reeA45 type. DNA from the wild strain
could transform the M45 to Rec+ and a few reeA45 type transformants weredetectable. These results with another observation indicate that the strain
M45 carries a reeA45 mutation and an additional ree mutation linked
closely to the reeA. We tentatively name this new ree gene ree45.

Efficient Procedures for Detection of
Frameshift Mutagens
Tsuneo KADA and Yoshito SADAIE

There are increasing numbers of reports indicating that many carcinogens
have mutagenic capacity after metabolisation in mammals (Maher, V. M.,
Miller, E. C., Miller, J. A. and Szybalski, W., 1968, Mol. Pharmacol. 4: 411;
Fahmy, O. G. and Fahmy, M. J., 1972, Int. J. Cancer 9: 284; Cookson,
M. J., Sims, P. and Grover, P. L., 1971, Nature, New Biology 234: 186;
Huberman, E., et aI., 1971, Proc. Nat. Acad. Sci. U. S. 68: 3195). Important carcinogens such as polycyclic aromatic hydrocarbons or acetylaminofluorens are really frameshift mutagens in vivo (Ames, B. N., Sims, P. andGrover, P. L., 1972, Science 176: 47; Ames, B. N., Gurney, E. G., Miller,
J. A. and Bartsch, H., 1972, Proc. Nat. Acad. Sci. U.S. 69: 3129). Because
the frameshift mutagenesis may involve some specific interactions between
mutagens and DNA, it is usually required to exame a sample with plural

76 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

testing strains having different sites for mutation detections.
We deviced a simple method named "rec-assay" for screening new chemicalmutagens by comparing cellular sensitivities of wild and recombinationlessstrains against different chemicals (Kada, T., Tutikawa, K. and Sadaie, Y.,
1972, Mutation Res. 16: 165). Because diverse chemical damages in DNA
are repaired through cellular recombination functions in wild cells but not
in recombination-deficient cells, most agents showing increased lethal
activity on rec- over rec+ cells may have damaged cellular DNA. It is highly
probable that such DNA-damage-provoking. agents are mutagenic.
We have isolated a number of rec- strains on which genetic and biochemical
studies were carried out (Sadaie, Y. and Kada, T., 1973, Mutation Res. 17:
138). It was recently found that certain rec- strains were remarkably sensitive to typical frameshift mutagens compared to wild strains. Among otherrepair-deficient mutant strains studied, three pol- strains as well as five hcrstrains showed cellular sensitivities which are similar to those of wild strains
against typical frameshift mutagens. Strains carrying either recB or rec43(this carried by the strain L43 isolated by us) showed remarkably increased
sensitivities to frameshift mutagens compared to wild cells. Furthermore,
the strain M45 carrying a recA gene (recA45) and a rec45 gene (tentatively
named) showed the highest sensitivity. The above findings indicated thatcomparative studies on cellular sensitivities of wild and M45 strains may beefficiently applied to the detection of frameshift mutagens.
Actually we tested, in collaboration with Dr. Y. Shirasu, a number ofpesticides and found at least two potent frameshift mutagens: 2,4-Dinitrophenylthiocyanate and Sodium p-dimethylaminobenzenediazosulfonate.

Radiation Effect upon Sex Expression in Cucumber
Taro FUJIIIn a monoecious plant like cucumber, the sex expression could be changed
with environmental conditions, such as temperature, day length or hormones
like ethylene or kinin etc. in spite of genetic background controlling the sex
factors. It is well known that ionizing radiations modify certain enzymaticor hormonal system in addition to the genetic effect. Present experiments
were conducted to examine the effect of r-rays on the sex expression in
cucumber.

RESEARCHES CARRIED OUT IN 197277

Dry seeds of commercial variety "Kaga-Fushinari" were exposed to 10
and 20 kR of 137Cs r-rays at a dose rate of 40 kR/h. Twenty seeds wereused in each lot and the seeds irradiated in Feb. 28, 1972 were sown in soil
inside the greenhouse. The development of female flowers were examined
during the successive internodal growth of the plants. In most cases, thefirst female flower was noticed on the 6th node but in some instances on the
7th. or 8th node. Two or mor~ female flowers clustering on the same node
were also observed and there was no male flower on such nodes. Most of
the plants continued their growth exceeding 60 nodes or leaves but thepresent examinations were terminated at the 50th node, since some plants
did not survive beyond this stage. Data on the effect of r-rays on the sex
expression is presented in Table 1. The average number of nodes that developed female flowers decreased from 9.1 in the non irradiated control to
7.5 or 6.5 with 10 or 20 kR treatments. The total number of female flowers
in the control was 221, but it decreased considerably after the radiation exposure. A reduction over than 50 % of female flowers was noticed by a
20 kR treatment. We conclude that the r-ray exposure to cucumber seeds
alters the sex expression by reducing the number of female flowers. Thoughmechanisms involved are not clear, one possible explanation may be achange in the rate of auxin synthesis, since this hormone is reported to beradiosensitive (Brown, G. N. and F. G. Tayler Jr., Rad. Bot. 7: 67, 1967).
Differential radiosensitivities of growth hormones could perhaps account foralterations in the normal balance of sex expression and development.
Examination of hereditary alterations, if any, is now under way.
Table 1. Average number of female flowers developedin r-ray irradiated cucumber~-----

Dose ofr-raysNode number(kR)

1-10 11-20 21-30 31-40 41-50
2.7
1.2
3.5
3.8
5.5
1.6
1.3
1.6
3.0
3.8
0.6
1.2
2.2
0.6
1.7

0
10
20

~

Average numberof nodesdeveloped female
flowers

Total
number of
femaleflowers

9.1
7.5
6.5

221
145
101

78 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Further Studies on the Fractionation Dose Effects withr-rays in Maize Pollen
Taro FUJII

Recovery of premutational damage or decrease of mutation frequency inmaize pollen grains by fractionation dose treatments with r-rays has alreadybeen reported (Fujii, T., Japan. J. Genet. 46: 243, 1971). To obtain further
information about the extent of recovery, maize pollen grains having a Bz
gene were irradiated with 2 or 3 kR r-rays as well as with these doses splitted
into two equal parts by administering them in two fractionations with 2
and 3 hours intervals. Subsequently, the irradiated pollen grains were used
to pollinate the recessive stock. Mutation from Bz to bz can be observed in
the F 1 seeds which have bronzy aleuron color.
A single dose of 2 or 3 kR showed a mutation frequency of 6.76 or 9.11 %(Table 1). In the fractionation treatments, there was a decrease in mutation frequency but the frequency did not vary much between the two interval
periods. The frequency of whole type mutations was markedly lower inthe fractionation lots than in the single irradiation lots, while the frequencyof partial type mutations did not show any significant difference between the
single and fractionated treatments. Although the nature of mutationalcourse or origin in these two mutant types are not clear, the present results
confirm the earlier findings that up to 2 hours interval period there enhanced
Table 1.
Treatmentwith r-rays(kR)

Mutation from Bz to bz by single and fractionatedirradiations with r-rays in maize
NumberofseedsSingle treatment
2
5534
5575
3
Fractionated (2 hours interval)
5937
1 +1
4881
1.5+1.5
Fractionated (3 hours interval)
5612
I +1
5257
1.5+1.5
Not irradiated7646

Mutation frequency (%)..

_- ----_.,--_.__ ..

Whole (a)Partial (b)(a)+(b)

4.37
6.13

2.39
2.98

6.76
9.11

2.53
4.13

1.58
2.29

4.11
6.45

2.51
4.20
0.04

2.07
2.21
0.09

4.58
6.41
0.13

RESEARCHES CARRIED OUT IN 1972

79

recovery, and no further recovery was observed beyond this interval. In
the present material, the 2 hours interval period appears to bring about
the maximum recovery and the rate in recovery of premutational damage
induced by ,-rays seems to be approximately 30 per cent.

Modification of Radiation Damage in Rice
Sujit BAGCHI and Taro Fum

Ten cultivated varieties of rice were subjected to 10-40 kR of ,-rays with adose rate of 10 kR/hr from a 137CS source. Considering the germiability
at 40 kR as a criteria for selection, four varieties were taken into account in
the present experiments. Among these four varieties two (Nos. 451, 647)
showed higher germiability and were presumed to be radiation resistant andthe other two (Nos. 545, 564) showed considerably low germiabilities andassumed to be radiation sentitive. In the next part of these experiments,
the four varieties were subjected to irradiation under different conditions,such as dry-seed irradiation, dry-seed irradiation combined with pre- or posttreatment of temperature at 60°C for 30 min. as well as wet-seed (24 hr soaked
in water) irradiation. These experiments were conducted primarily to observe differential radio-tolerant capacities in the above four vatieties whichwere selected as radio-resistant and sensitive ones. Two quantitative
characters such as germiability and seedling height were taken into account. It was found that the radiation tolerance capacity of germination
is not always reflected by the seedling height, thus showing non-dependencywith each other.
In the next part of these experiments all the four varieties were subjected
to fractionated dose treatments both in dryas well as in wet-seed conditions,
i.e., each treatmental dose was divided into two equals leaving a gap of timein between. In the dry-seed fractionated dose treatment, the time intervals
were 24-96 hrs for applied doses of 10-50 kR. In the case of wet-seedtreatments, the time intervals were 6-24 hrs for applied doses of 2.5-20 kR.
It was found that, in the dry-seed treatment at 50 kR dose with 96 hr interval,
the germiability and seedling height were very low when compared with thevalue at 50 kR dose with 48 hr intervals. This lag in the germiability and in
the seedling height has been thought to be due to lack of metabolic-repair(Traut R., Proc. IAEA, 67-78, 1966). In the wet-seed treatment, the ger-80 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

miability did not show any marked decrease except in one sub-treatment,
however when the mean seedling height was considered, a pronounced decrement could be observed except in three sub-treatments. The detrimental
effects of irradiation in seedling height of wet-seed treatments is presumed tobe due to the damage occurred at the metabolic-pathway, thus hindering themetabolism proper. Though any definite conclusion could be drawn fromthe above experiments, this sort of damage was more pronounced in thetreatment using wet-seeds than dry-seeds.

Copper Dependency of an Induced Dwarf Rice Mutant
H. K. SHAMA RAo, Taro FUJII and Tsuneo KADA

Seeds of eleven dwarf rice mutants and parent Norin-8 obtained fromDr. T. Kawai were cultured in the modified Murashige and Skoog's medium
containing copper, cobalt and molybdenum or in the modified White's
lacking them, in order to ascertain the physiological or biochemical differences, if any. Interestingly one of the mutants, MGS-95, was found todevelop into normal seedlings in the modified Murashige and Skoog's
medium but not in the White one. This observation made us to conceive theMurashige and Skoog's medium to contain an element (or elements) which
may be a specific requirement for this mutant.
Since the modified White's medium lacked Cu, Co or Mo, it was supplemented individually with these minor inorganic elements for culturing
MGS-95 and Norin-8. Observations made at 5, 10, 20 and 30 days after
culturing revealed, in the presence of Cu, 100 per cent callus initiation around
10 days in the mutant, while the parent required 30 days to attain this stage.
In the absence of Cu, 80 % callus initiation occurred around 20 days in
Norin-8 and only 25 % in MGS-95. The presence of molybdenum did nothave any effect on early callus initiation in both the mutant and parent.
On the other hand about 50 to 60 % calluses were observed around 10 daysin the presence of cobalt.
Optimum Cu requirement in MGS-95 was ascertained by culturing in themodified white's medium supplemented with 0, 0.025, 0.05, 0.1 and 1.0 mg/
I of Cu, respectively. Besides early callus initiation in the mutant, there
was also a shift from shoot to root callus formation which was maximum(76 %) at 0.05 mg/I Cu concentration. Still higher concentrations inhibited

RESEARCHES CARRIED OUT IN 197281

root callus development.
The differences in the concentration of 2,4-D in the White's (20 mg/l)
and the Murashige and Skoog's media (2.2 mg/l) does not appear to haveinfluenced the nature and frequency of callus development. It was confirmedthat even at 20 mg/l concentration of 2,4-D early callus initiation as well as
the shift from shoot to root callus development appeared only in the presence of various Cu concnetrations in the supplemented White's medium.
These results have clearly demonstrated an induced differential response
for copper requirement of a mutant. Since the strain MGS-95 was produced as a result of mutation, it is probable that a genetic change is exercising on the level of a protein to exhibit the copper dependent character involved in the regulation of callus formation. It is required in future to
elucidate what type of gene expression is involved in the copper effect.

Radiation and Giberellin Effects in the
Induced Dwarf Rice Mutants
H. K. SHAMA RAO and Tsuneo KADA

Seeds of eight dwarf rice mutants and parent Norin-8, kindly supplied
by Dr. T. Kawai, National Institute of Agricultural Sciences, were usedin this study. Of these 8 mutants, one was derived from r-rays (MGS-28),
3 from X-rays (MGS-46, 50 & 95), 2 from pile neutrons (MGS-54 & 55)
and 2 from ethylene imine (MGS-130 & 131) treatments.
Moisture stabilized (12.5 %) dry seeds were exposed to 0, 9.5, 28.5 and47.5 Krads of l37Cs r-rays and 0, 1.5, 2.6 and 4.4 krads of 14.1 MeV fastneutrons. There were 4 replications. The neutron doses administered were
determined by the seed elements composition analysis and sulfur activation.
Seedlings were raised under constant temperature (±28°C) and continuous
illumination (2800 lux) inside a phytotron. The extent of radiation damage
was measured based on height recorded on 14 days old seeldings.
To study the GAs effect on the induced dwarf rice mutants and parent,
well germinated seeds were first sterilized in equal volume of ethyl alcoholand 30 % vol. HaO a for 10 min., and subsequently transplanted asepticallyon 0.5 % agar media inside 500 m!. beakers. After the establishment ofroots, 4 ml of 20 ftg/l GAs was applied inside each beaker. Growth differentiation was recorded after 14 days growth of these seedlings raised under82 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

constant temperature (±28°q and continuous illumination inside the growthroom.
MGS-46, 55 and 95 were radiosensitive with significantly pronounced
effect at higher doses of both r-rays and 14.1 MeV fast neutrons. For e.g.
with 28.5 krads of r-rays, the percentage seedling height were 38.3, 31.4
and 37.5 respectively compared to 58.1 in the parent Norin-8. With 2.6
krads of fast neutrons, the seedling height percentages were 65.3, 55.9,
43.9 and 84.6 for MGS-46, 55, 95 and Norin-8 respectively.
On the other hand in MGS-130 and 131, the seedling height percentages
were significantly higher than in Norin-8, i.e. 63.1 and 61.4 %with 28.5 kradsof r-rays and 87.2 and 88.2 % with 2.6 krads of fast neutrons and thus revealed radioresistance. The other 3 mutants MGS-28, 50 and 54 were likethe parent Norin-8. RBE's amongst the mutants and parent however varied
only between 7.3 and 8.8. Interestingly only the mutants derived after
ethylene imine treatment were radioresistant; of the 3 radiosensitive mutants,
one had been induced with pile neutrons and the other 3 with X-rays.
Growth promotion with GA 3 was observed to a similar extent in theparent and the radiation induced mutants (125 to 150 % over control)
except for the absence of such an effect in MGS-95 (93.7 % over control)
which was found to be copper dependent for callus differentiation.
In MGS-130 and 131 the growth was rather inhibited i.e. 84.8 and 61.9%
of control, respectively. Since these 2 mutants are now relatively lesssensitive to radiations, a certain target site or function responsible for radiation sensitivity might have been modified as a result of mutation. Further,
the absence of GA 3 effect may be the consequence of genetic alterationin the above presumed biological site or function. In the case of MGS-95,
it may be interesting to examine in future if the GA-dependence of growth isalso related to copper concentrations.

Biological Effects of Neutrons after Post-irradiation
Storage or with Gamma-ray and Neutron
Combination Treatments in Rice
H. K. SHAMA RAO

The present experiments were conducted to find out the influence of postirradiation storage as well as pre/post gamma-ray and neutron combination

RESEARCHES CARRIED OUT IN 1972

83

treatments on the biological effects in rice. Moisture stabilized dry seedsof commercial variety Norin-8 were used. The seedlings were raised underconstant temperature condition. Seedling heights were recorded 14 days
after sowing and the extent of radiation damage was measured in relation
to percentage seedling heights over respective controls.
In the storage experiment, the 14.1 MeV fast neutron doses administered
were 1.5 and 2.6 krads and the 137CS gamma-ray doses were 19.0 and 38.0
krads, respectively. Post-irradiation storage treatment consisted of storingfor 10, 20 and 30 days at 30°C. It may be noted that enhanced radiation
damage occurred up to 20 days storage with some recovery at 30 days, inboth radiation treatments. For, e.g., with 2.6 krads of neutrons, theseedling height percentages were around 94,84 and 89 %at 10, 20 and 30 days
storage periods, respectively. The damage was much greater with 38 krads
gamma-rays, the seedling height percentages being around 67, 47 and 69 %for similar periods of storage, respectively. At 20 days storage where thedamage was maximum, it was nearly 50 % more with gamma-rays comparedto neutrons. Even the fast neutrons were found to enhance the radiation
damage by nearly 16 % with 20 days post-irradiation storage as compared
to about 50 % damage with gamma-rays.
In the pre/post neutron and gamma-ray combination experiment, thedoses administered were 0.7 and 9.5 krads of neutrons, and 9.5 and 19.0
krads of gamma-rays. Although both the treatments showed dose dependence for seedling heights reduction, the extent of radiation damage differedin the combination procedures. For e.g., neutrons when administered
immediately after the gamma-ray treatment (9.5 krad + 1.3 krad) revealed
seedling height percentage to be 64 %, whereas for a similar total dose when
gamma-rays were used as a post-treatment the seedling height was around
79 % of control. Similar trends were observed even with lower doses ofneutrons (0.7 krad), the gamma-ray dose being constant (9.5 krad). The
seedling height recovery as a result to post-irradiation gamma-ray treatment
could be explained on the repair processes known to occur with such low
LET treatments. On the other hand, the densely ionizing fast neutrons whenadministered as a post-treatment after gamma-rays could block the recovery processes. The extent of recovery could therefore be much less thanwhen gamma-rays are used as a post treatment.

84 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Genetic Fine Structure Analysis of wx Locus in Maize
Etsuo AMANO

Since the wx character of starch is expressed in pollen grains, infrequent
intracistronic recombination are detectable relatively easily for a higher plant.
When independently induced wx mutants were crossed with standard wx
testers and if any non-wx (Wx) recombinant pollen were produced, they
could be scored as blue black grain among light-brown colored pollen ofparental character by appropriate iodine staining. The frequency of blue
black grains might reflect the map distance between the tester wx site and thetested wx. Preliminary mapping data have been reported (Ann. Rep. Nat.
Inst. Genetics, 20: 85, 1969).
Further experiments using other wx testers such as wx R (presumably
intracistronic deletion) indicated different distribution of the wx sites fromthe detailed work of Nelson (Genetics 60: 507, 1968). According to orthodox linkage experiments, he mapped marker loci and wx sites as bZ-WX H21 WX C-wx 90-v.
However, his pollen analysis data themselves suggested adifferent order which would also give a good interpretation for the present
experiments. Using the same standard wx stocks to determine the EMS
induced wx sites, a preliminary map which was constructed refering to two
wx stocks, wx c and WX H21 required only a little modification in the present
experiments. If the WX H21 site locates between wx c and wx 90 as suggested
in the present experiments and Nelson's pollen data, the distribution of thewx mutant sites will be relatively even, i.e. two mutant sites locating outside
wx c , three between wx c and wx H2 \ seven between WX H21 and wx 90 , and eightoutside wx 90 • If the WX H21 must be placed outside WXC -wx90 region, distribution of wx mutant sites may become very uneven, since most of the mutants
induced by EMS should be placed outside WX H21 • The order of the standard
wx stocks should be determined by orthodox linkage test with larger population and compared with the pollen data, while further diallele cross experiments with more wx mutants are required.

Mixed Pollination for Obtaining Inbred Mutant Line in Maize
Etsuo AMANO

In maize, a number of conveninent marker stocks have been available

RESEARCHES CARRIED OUT IN 197285

for mutation studies. Mutagen treated dominant homozygous plants canbe pollinated with an endosperm marker tester (C sh r bz wx, y) to detect
recessive mutations in these loci on the cob. However, since the induced
mutant genes could only be detected as heteroallelic hybrids with the testermarker, there are some difficulties in obtaining established mutant lines.
Genetic back-ground may interfere with the results of analytical studies suchas genetic fine structure mapping in wx locus, and progenies of hybrids inmaize often show unstable growth. Moreover, identification of segregatednewly induced mutant genes would be possible only for special cases likeleaky wx mutants, or after laborious tests of recombination with the tester.
To avoid these difficulties, pollination of the mutagen-treated plant wasmade with mixed pollen of untreated parental line and the tester line.
Pollen of the two lines were collected from tassels cut in the field on a previous day and kept separately in vases. Roughly equal volumes of pollenof a stock 6311R (untreated CI Sh 1 Bz Wx, Y) and a stock 639 (C sh 1 bz wx,y) were mixed in a Petri dish. Pollinations were made using small brushes.
Test pollination to 639 resulted in good fertility and expected segregation
ratio, suggesting absence of disturbance like certation. When the mixed
pollen were pollinated to 6311R, inbred kernels could be separated fromhybrid ones. This classification can be tested further, since the latter willshow hybrid vigor when planted in the field. If a mutant could be detected
after seed treatment with a mutagen on a cob as hybrid kernels, presumably
half of the 6311 R kernels on the same cob may carry a newly induced mutant
gene. Inbred homozygous mutant will be segregated in the next generationfrom some of the inbred kernels. A few mutant cobs have been obtainedin this way by preliminary experiments.

Some Experiments with Cultured Haplopappus Cell Lines
Etsuo AMANO

Five callus lines of Haplopappus gracilis (2n=4) have been induced andmaintained in our laboratory since 1971. Origins and morphologicalcharacteristics were described previously (Ann. Rep. Nat. Inst. Genetics
22: 73,1972). These lines have been kept on agar slant containing inorganic
salts (Eriksson, T, Physiol. Plant. 18: 976, 1965), vitamines, yeast
extract (0.3 %), 2.4 D (2 ppm) agar (0.8 %) and saccharose (2 %). Among

86 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

the lines, HL (induced from leaf), and HNC (spontaneous callus appearedon the stem surface) seemed to possess better characters for suspension
culture than those of HSe (from seed), HR (from root) or HS (from stem).
Liquid suspension culture was made in small Erlenmeyer flasks kept on arotating stand (2 or 6 rpm). Component of the liquid medium used was
the same as that of slant medium shown above except agar.
At 28°C, the fresh weight of HL cells in 20 ml of liquid medium increased
linearly for a week and resulted in ten-fold increase of weight. The cellvolume, measured either by natural sedimentation for 60 min. or by centrifugation for 1 min. at 2000 rpm, continued to increase linearly for 20
days. The results were comparable except that the centrifugation method
gave packed cell volumes which were almost half of those obtained by the
sedimentation method. The linear growth in initial phase might suggest
a low mitotic activity in some of daughter cells. Rough estimate of themitotic cell cycle was about 35 hours according to the increase of fresh weight.
The chromosome number of the five cell lines was studied on samples
kept for more than one year on agar slant. The cell lines were cultured inliquid suspension of 20 ml for a week. After 20 hours of cold treatment,
cells were collected by centrifugation (2000 rpm, 1 min.) and fixed in 1:3
acetic alcohol fixative. The chromosome number was determined withacetocarmine squash preparations. Among the five cell lines, HS contained
tetraploid cells (10%) and an aneuploid cell (one with three chromosomes in
64 metaphases). One cell in the 88 HR cells examined had only two chromosomes. Other cells and three cell lines (HL, HNC and HSe) showed either
normal four metaphase chromosomes or eight anaphase chromosomes. To
test effect of the suspension culture, small samples were taken from fresh
slant cultures and stained by acetocarmine stain without pretreatments.
Only a small number of metaphases could be examined in this way, but thechromosome number was the same as that of the suspension culture.
By a simple sedimentation procedure, a fine cell suspension, free of large
cell aggregates, could be obtained from a cell line, HNC. This cell sample
could incorporate sH-TTP into acid-insoluble fraction when incubated fortwo hours with appropriate precursors and energy sources. Addition of
5 % Brij-58 which can increase permeability in yeast cell, was not successful
to promote sH-TTP incorporation in the present material.

RESEARCHES CARRIED OUT IN 1972

VIII.

87

POPULATION GENETICS (THEORETICAL)

Population Genetics, Molecular Biometry, and Evolution
Motoo KIMURA and Tomoko OHTA

Amino acid substitutions in protein evolution and protein polymorphisms
in natural populations are not separate phenomena; they represent two aspects of a single phenomenon, i.e., the spreading of neutral or nearly neutral
mutations, caused by random genetic drift.
The behavior of the neutral mutants within a finite population was analysedmathematically. In particular, the constancy of the evolutionary rate was
explained in terms of constant occurrence of neutral mutations.
The most rapidly evolving molecules known so far are the fibrinopeptides
A and B. It has been found that the middle segment of the proinsulin
molecule shows nearly the same evolutionary rate as fibrinopeptides A and
B. Since these molecules have no known function after they are removed
from the rest of the molecules, so selective constraint must be minute for them,and hence, almost all mutations (amino acid replacements) are likely to
be selectively neutral. Thus, the rate of amino acid substitutions in evolution is approximately equal to the mutation rate for them.
From the standpoint of the neutral mutation-random drift theory, theaverage amino acid composition of proteins represents the equilibrium
condition under mutational substitutions of nucleotides, as can be predictedfrom the code table. This prediction is in fact turned out to be in good
agreement with the actual observations. For details, see Proc. 6th Berkeley
Symp. of Mathematical Statistics and Probability Vol. 5: 43-68.
The Effective Number of a Population with Overlapping
Generations: a Correction and Further Discussion
James F. CROW and Motoo KIMURA

An approximate formula is proposed for the effective number, N., of a
population such as the human population where generations overlap andreproduction occurs at various ages. When y is the age (e.g., in years), thevariance-effective number is given by

88 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

N ev = [ [NyVydy = [V,where Ny is the number of age y and vy is Fisher's reproductive value at agey; V is the total reproductive value of the population, and [ is the weighted
mean probability of surviving into the reproductive period,[=rl~bye-my dy .

The formula can also be written
N ev = NoIr,
where 'r is the mean age of reproduction. These are also a good approximation to the inbreeding-effective number.
These formulas all assume that the population has attained a stable age
ratio and that the age-specific birth and mortality rates are constant.
Modifications when these conditions are not met are discussed. We also
retract an earlier, incorrect formula. For details, see Amer. J. Hum. Genet.
24: 1-10.
Fixation Probability of a Mutant Inftuenced by Random
ftuctuation of Selection Intensity
Tomoko OHTA

The probability of fixation of a mutant gene in a finite population wasinvestigated by taking into account the effect of random fluctuation of
selection intensity. It was assumed that the change of gene frequency per
generation by selection is given by sx(l - x) where s is the selective advantage and x is the gene frequency. It was shown that not only the productof the effective population number and average selection coefficient (Nes)is important, but also the ratio of the mean and the variance of selection
coefficient (sIV.) has an important influence on fixation probability. In
particular, when this ratio is small, a mutant gene, even if selected against,
becomes fixed in the population like a selectively neutral mutant. In
general, when random fluctuation of selection intensity is not negligible it
may be convenient to use 'effective selection coefficient'. The bearing of
the present findings on the neutral mutation-random drift theory of evolution
and variation at the molecular level was discussed. For details, see Genetical Research 19: 33-38.

RESEARCHES CARRIED OUT IN 197289

Fixation Time of Overdominant Alleles Influenced byRandom Fluctuation of Selection Intensity
Tomoko OHTA and Motoo KIMURA

It was demonstrated that the number of generations until fixation orloss of an overdominant alleles is influenced by random fluctuation of
selection coefficients. When 2s < Vs, where s is the mean selection coefficient against either homozygote and Vs is the between-generation varianceof the selection coefficient, overdominance generally accelerates rather thanretards fixation of segregating alleles. This finding should have important
bearing on our consideration of the behaviour of polymorphic variants which
are nearly neutral but have very slight overdominance. When the population size (N.) is extremely large, not only N.s but also s/Vs have to beconsidered in discussing the effectiveness of overdominance. For details,
see Genetical Research 20: 1-7.

Simulation Experiments of Multilocus System
Tsuneyuki YAMAZAKI and Motoo KIMURA

In order to obtain information on what type of chromosomes are established by selection when many loci are tightly linked, we performed simulation
experiments under the following assumptions: (1) Population size-100
diploid individuals, (2) The number of linked loci on a chromosome-88,(3) Recombination value between adjacent 10ci-O.002 without interference,
and, (4) Individual fitness-determined by the multiplication of fitness
values among loci.
The results are summarized as follows: I) When there are two overdominant alleles at each locus and the selection coefficient against either
homozygote is 0.1, almost complete linkage disequilibrium is establishedamong segregating loci after approximately 200 generations. Then, thepopulation consists of two complementary chromosome types, and fixation (or loss) of alleles seldom takes place. 2) When more than two alleles
exist at each locus with 10 % selection coefficient (against homozygotes)
complementary chromosome types are established similar to the two alleliccase. The maximum number of complementary chromosomes is equal to
the number of alleles per locus. 3) When the selection coefficient (s) of90 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

overdominant loci is small, the rate of fixation or loss of alleles is slower
with linkage than without linkage. However, when selection coefficient islarger than a certain value, linkage tends to accelerate fixation rather thanretard it. Moreover, the most efficient selection coefficient to maintain
polymorphisms in a population is neither a large s nor a small s, but the
optimum is an intermediate value of s, though it is a function of recombination fraction. 4) When frequency-dependent selection of minority-advantage
type is operating, two complementary chromosome types are also establishedby selection; namely, one with majority alleles in most loci, and the otherwith minority alleles.

RESEARCHES CARRIED OUT IN 1972

IX.

91

POPULATION GENETICS (EXPERIMENTAL)
Spectra of Heterozygosities and of Evolutionary
Rates among Different Loci
Takeo MARUYAMA and Tsuneyuki YAMAZAKI

Kimura and Ohta suggest that protein polymorphism and protein evolution should be regarded not as separate phenomena, but as two aspects ofa single process. They further argue that most of naturally occurring polymorphisms are due to random frequency drift of selectively neutral mutantsin finite populations, (Kimura and Ohta, 1971, Nature 229: 467). On theother hand, Kimura, King and Jukes have argued that most of molecular
evolutionary changes are due to fixation in species of the neutral mutants,(Kimura, 1968, Nature 217: 624; King and Jukes, 1969, Science 164: 788).
Under the hypotheses of neutrality, Kimura and Crow showed that the"effective number" of allele at a locus is I + 4 Nu, where N is the population
size and u is mutation rate per gene per generation, and Kimura proved thatthe evolutionary rate of amino acid substitution is exactly equal to mutation
rate per site per year and is independent of other factors. Both of thesequantities are observable and are known to vary greatly among loci. A
considerable amount of data for heterozygosity is available in publishedliteratures, and Dayhoff's Atlas of Protein Sequence and Structure provides
the evolutionary rates for a number of proteins. Since both of thesequantities are proportional to mutation rate, if they are really two different
aspects of a single process, their spectra ought to be similar to each other.
Namely, the distributions of the values of 4 Nu and of the evolutionary
substitution rates should have an identical pattern. The purposes of thisnote are to obtain the spectra from available data and to examine whether
or not they appears to be identical.
The spectrum of the values of 4 Nu among different loci is constructedfrom data of the Drosophila species. From the literatures were collectedgene frequency data of some 140 loci and the value of 4 Nu was calculated
for each locus by first computing the reciprocal of the homozygote probability
and then substructing one from it. The whole range of the values weredivided into ten classes of equal interval. The distribution of the values with

92 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23(0) Heterozygosityo5
Relative magnitude

10(b) Evolutionary rate

o

5

10

Relative magnitudeFig. 1. The spectra of the evolutionary rates (nv) and of the heterozygosities(4Nu) among different loci.

respect to the classes is the spectrum and is presented in Fig. I (a). On theother hand, the amino acid substitution rate, which is denoted by v in thisnote, given in Dayhoff's Atlas (page 50) was multiplied by the number (n)of amino acid residues of that protein. This value (nv) should be themutation rate per locus per year because of Kimura's principle that the evolutionary rate is equal to mutation rate. Since the rates in Dayhoff's Atlas
were calculated from mammalian data and mammals have approximately
same length of one generation time, the value of nv should be proportional tomutation rate per gene per generation which is therefore comparable to thevalue of 4 Nu. The values of nv among different proteins were also divided
into ten classes of equal interval and the distribution is presented in Fig. I(b). The two spectra presented in the Figure appear to be quite similar, andtherefore the result is consistent with the hypothesis of Kimura and Ohta
and with the neutral hypothesis of molecular evolution.

RESEARCHES CARRIED OUT IN 197293

Evidence that Enzyme Polymorphisms are Selectively Neutral
Tsuneyuki YAMAZAKI and Takeo MARUYAMA

In a recent paper, Johnson, G. B. (Nature New BioI. 237: 170, 1972) has
examined published data with special reference to the neutral hypothesisof protein polymorphisms and concluded that they appear to be contrary
to the following theoretical expectation based on the neutral hypothesis.
Here we denote by n. the inverse of the homozygosity (the effective number

20

•
15

•
•
t:J

10

c:

•

•
•

5

o 0-----L.-.---l-.....L-.....L.--J
0.4 0.6"fie/no0.8

1.0

Fig. 1. The relationship of the actual to the effective number of aIleles in
the combined data, (all the data based on a sample of size greater than 100 genes
are included). The curves indicate the theoretical expectations of the relationship.
Each dot indicates the average coordinate of the analysed data which belong to agiven value of na. The number of protein loci used for this figure is 255.

94 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

of alleles) and by n a the actual number of alleles. In these notations, Johnson's prediction is that, if all the alleles at loci in question are selectively
neutral, the ratio, n./n ao should decrease as n.. increases. He has presented
three different sets of data, all showing the opposite to the above prediction,and has argued that the finding is against the truth of the neutral hypothesis.
In this note, we have extended the same examination as Johnson to moredata including a number of different organisms and also provided theexact theoretical expectation of ratio n./fi.. as a function of fi. and sample
size (Fig. 1). (A bar indicates the expectation of the quantity under consideration.) On the contrary to Johnson's claim, we find that the observed
data are in good accord with the theoretical expectations, while our theoretical expectation turned out to be the same as his prediction.
Since we could provide the precise theoretical expectation as a functionof fi.. and the sample size, we have included all the alleles reported in theliteratures used for the analysis, while Johnson had excluded those whose
frequency is less than 0.01. It is possible that the exclusion ofless frequent
alleles had lead Johnson to the false conclusion. It is certain that quantitatively more precise theoretical expectation, than Johnson's qualitative prediction merely on the direction of the slope, had been necessary to draw any
conclusion from the kind of analysis attempted by him. The present paper
provides both the exact expectation and the analysis based on more dataof some two hundred fifty loci. The latter is particularly important, for thecontroversy is not concerned with a set of any selected loci, but it is concernedwith average fate of all loci.
From Fig. 1 it is clear that the data is consistent with the theoretical expectation based on the neutral hypothesis. We therefore present this result
as evidence for the truth of the neutral theory.

Further Evidence for the Neutral Hypothesis ofProtein Polymorphism
Tsuneyuki YAMAZAKI and Takeo MARUYAMA

In our previous papers, published polymorphism data were used inconjunction with an invariant principle to conclude that the available data
are consistent with the "neutral theory" of Kimura and Ohta, (T. Yamazaki
and T. Maruyama, Science 178: 56-58 and this Report No. 22: 86-87.)

RESEARCHES CARRIED OUT IN 1972

95

The principle is that when we assess the amount of heterozygosity, given thatthe gene frequency is specified, the total amount is independent of the
population structure. The data then available were some 400 proteins.
Since then more data were published and therefore we made the same survey
0.20

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0

0.2

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Gene frequency (y)j; 0.40
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Gene frequency (YlFig. 1 and 2. Distribution patterns of heterozygosity. The curves indicate thetheoretical expectations: (1) neutral; (2) advantageous (Ns=lO); (3) deleterious(Ns=-10); (4) Overdominance (Ns=10).

96 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

of all available data which amount to 1045 proteins. The present result
turned out to confirm the previous ones, and therefore our claim was greatly
reinforced. The result is presented in Fig. 1.
We have applied the same analysis to polymorphism data of humanblood groups alone. Although the amount of available data is undoubtedly
insufficient to draw any strong conclusion, the result indicates that the distribution of heterozygosity deviates from that of neutrality. This is probably
due to some sort of balance selection. The result is presented in Fig. 2.

Allelism between Lethal Genes in Japanese and Korean
Natural Populations of Drosophila melanogaster
Chozo OSHIMA, Takao K. WATANABE and Jong-Kil CHOOll

Many flies of D. melanogaster were collected simultaneously in September
1971 at two sites of Anyang, the suburbs of Seoul, Korea and in November
at Katsunuma and Shikishima in Yamanashi prefecture, Japan. The
frequency of lethal second chromosomes extracted from each male fly wasreported in the previous Ann. Rep. No. 22: 90.
The frequencies of allelism between lethal genes concealed in the same
population and these different populations were determined by half and full
diallel crosses. The results were shown in Table 1.
Although the distances between two sites of Anyang and between twosites of Yamanashi were about 8 km and 20 km respectively, no remarkabledifference was found between frequencies of allelism within and between
populations. The map distance between Anyang and Yamanashi wasmeasured to be about 1000 km, but the frequency of allelism between lethal
genes in both populations was higher than that expected between geneticallyunrelated populations.
Among many individual lethal genes, 8 lethal genes (a - h), locating atdifferent loci, were confirmed to appear on several chromosomes as shownin Table 2.
Seven lethal genes, except h, were found to be located with another lethal
genes on a chromosome and on the other hand, seven lethal genes, except
C, were found to be concealed in both Japanese and Korean populations
1)

Visiting researcher from Department of Biology, Chung-Ang University, Seoul, Korea.

Table 1. Results of allelism between lethal genes
Between populations

Within population
Population

testedchromoNumber ofcrosses

allelic
crossesFrequencyofallelismtestedchromoNumber ofcrosses

allelic
crosses

(%)

(%)

--

Katsunuma
Shikishima

40
40

780
780

12
11

FrequencY:;tlof
ITlallelism
gj

1.54
1.41

40
40

1600

23

1 44
.

>
:;tI

g

ITl
(Zl

Between populations---- -------- -Population

Number oftested crosses allelicchromocrosses----Frequencyofallelism

Population(%)

Between populations-------------

Number oftested crosses allelicchromocrosses--------------------

Frequencyofallelism(%)

Katsunuma
Anyang 1

40
19

760

3

0.39

Katsunuma
Anyang 2

40
24

960

11

1.15

Shikishima
Anyang I

40
19

760

0

0.00

Shikishima
Anyang 2

40
24

960

4

0.42

Total

80
43

3440

_____

Japan
Korea

18

(")

>
:;tI

c:

t!l
v

0

~
....
:

~

N

0.52

\0
-...J

98 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23
Table 2. Allelismic relationship between lethal genes found commonly
in Japanese and Korean populations
Chromosome
SymbolPopulation

040 A*
O4OB
013
024
061 '"
091
045 A*
045 B
005 A*
oo5B
043 A, B
071 A,B,C
063 *
023
035
037
055

Katsunuma
Katsunuma
Anyang 1
Anyang 2
Katsunuma
Shikishima
Katsunuma
Katsunuma
Anyang 2
Anyang 2
Katsunuma
Shikishima
Katsunuma
Anyang 2
Anyang 2
Anyang 2
Katsunuma

Lethal gene (tentative symbol)

a
a
a

b

b
b
b

d

c
c

d

d
d

e
e
e
f
f

g
g
h
h

*; double lethal chromosome.

respectively. Such lethal genes would be persisted for a long time inthese natural population, but a migration of insect by a seasonal wind from
Korea to Japan is unknown.

Allelism between Sterility Genes in Japanese and Korean
Natural Populations of D. melanogaster
Chozo OSHIMA, Takao K. WATANABE and Jong-Kil CHaO

The frequency of sterility second chromosomes among semilethal andquasinormal chromosomes extracted from Katsunuma, Shikishima andAnyang 1, 2 natural populations was reported in the previous Ann. Rep.
No. 22: 91.
The frequencies of allelism between sterility genes concealed in these
populations were determined by the fertility test crosses of hybrid flies between different sterility lines. The results are shown in Table 3.

Results of allelism between sterility genes

Table 3.

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

Within population---

Between populations- - - - - - - - - - - - - - - - - - ---- -_.. _----- - - - - - - - - -Population

testedchromoNumber ofcrosses

allelic
crossesFrequencyofallelism

Number of-tested- -·crosse-schromo__.

._~-------

alleliccrosses

Frequencyofallelism(%)(%)

Katsunuma!? Shikishima(l)

Katsunuma
Shikishima
Anyang 1

17
12

136
66

32
23

496
253

14

12

204

3

1.47

15
32

3.02
12.65

32
23

736

40

5.43

V1

17.58
8.89

14
10

140
140

10
10

7.14
7.14

g

0.00
3.57

6
8

48

o

0.00

!? Anyang 2

91
45

16

10

Anyang 1
Anyang 2

6
8

15
28

o

(l)

17

0.74
1.52

4
1

tTl

testedchromoNumber ofcrosses alleliccrossesFrequencyofallelism

Population

Number of
Frequency
tested---crosses -ailafc
ofchromocrosses
allelism(%)!? Katsunuma

17
14

238

6

2.52

Katsunuma
Anyang 2

Shikishima

12

14

168

o

0.00

Katsunuma
Anyang I

29

174

o

Shikishima
Anyang 1

22
6

132

29
24

696

!? Anyang 1
(l)
(l)

(%)

Anyang 1!? Total

Japan
Korea

6

17
10

170

o

0.00

Shikishima
Anyang 2

12
10

120

o

0.00

0.00

Katsunuma
Anyang 2

29

232

o

0.00

o

0.00

Shikishima
Anyang 2

22
8

176

o

0.00

6

0.86

51
14

714

o

0.00

(l)

Total

Japan
Korea

8

V1

~

:;.:l
:;.:l

Between populations

Between populations
Population

~
~
:;.:l

~

o
..,c

Z

....
'D
-..l

IV

'D
IQ

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

It was confirmed by the allelism test that three female sterility genes locating at different loci respectively, existed in both Japanese and Korean populations. One of them, named Is 302, was found in one chromosome extracted from Katsunuma population, and it appeared in 4 chromosomes
among 14 chromosomes extracted from Anyang 1 population. This fact
could be hardly considered only by the occurrence of common mutation
at each locus.

Effects of Density of Larvae and Temperature on a Factor
of Fitness in Drosophila melanogaster
Seido OHNISHI and Chozo OSHIMA

Fitness of organisms is greatly influenced by environmental factors such astemperature, nutritions and population density. Developmental time fromegg to adult, one of the component of fitness, was examined under several
different temperatures and densities. The fertilized eggs of Oregon-R
strain were collected for two hours and placed on the medium in small vials(20 X 90 mm) and thereafter they were kept under constant (25°C) andfluctuating (20-30°C, one cycle a day) temperatures respectively. Threekinds of densities (20 60 and 180 eggs per vial) and five replications foreach were adopted simultaneously under both temperature conditions. Allflies emerged were counted every eight hours and then the developmental
time and the viability (ratio of numbers of adult to egg) in every vials wereobtained as shown in Table 4.
The developmental time was prolonged significantly by the density IllTable 4. Mean developmental time (hours from egg to adult) andviability (ratio of number dault to egg) in three densities underconstant (25°e) and fluctuating (20- 30 D e) temperatures
Developmental time

Viability

Density·
20
60
180

const.

fluet.

eonst.

fluet.

201.25±0.40
203.99±1.08
210.71±0.73

217.17±1.09
218.70±0.54224.52±1.50

0.890±0.037
0.923±0.026
0.823±0.018

0.930±0.034
0.817±0.037
0.777±0.01O• Number of eggs per vial.

RESEARCHES CARRIED OUT IN 1972101

creased under both temperature conditions and also more prolonged by thefluctuating temperature.
On the other hand, the viability decreased proportionately with thedensity increased under the fluctuating temperature, while such a result
could not be found under the constant temperature. From the results,
an optimum density was presumed to be about 60 eggs per vial. It is evident
that the density of larvae seems to control the viabiLty, and its pattern is
influenced by the condition of temperature. The coefficient of correlation
between the developmental time and the viability was estimated to be-0.5797 for the constant and -0.6335 for the fluctuating temperature.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

x.

EVOLUTIONARY GENETICS

Population Size and Rate of Evolution
Tomoko OHTA

It is suggested that in evolution there is much substitution of nearly
neutral mutations, for which the selection intensity varies from time to timeor from region to region. Since the variance among the selection coefficients of new mutants decreases when the environment becomes uniform,
the probability of a mutant being advantageous to the species as a wholeincreases in more uniform environment. Therefore the rate of gene substitution increases in smaller populations, as smaller populations are likely
to be distributed over less varied environments.
The adequacy of the model was discussed in relation with the following
facts or plausible postulates. 1. A large number of amino acid substitutions during a period corresponding to the formation of new species.
2. Rapid evolution at the phenotypic level of populations having a small
size. 3. Many extinctions and expansions of the species in the past. For
details, see Jour. Molecular Evolution 1: 305-314.

Evolutionary Rate of Cistrons andDNA Divergence
Tomoko OHTA

The ratio of DNA divergence to cistron divergence was estimated forvarious comparisons of Drosophila and mammalian species. It was shownthat a negative correlation exists between this ratio and the length of one·
generation. It was proposed that the majority of the amino acid substitutions in evolution are the result of random fixation of nearly neutral mutations for which selection intensity fluctuates considerably but the average
selection coefficient is very slightly negative. On the other hand, theevolutionary rate of DNA divergence is negatively correlated with thegeneration time if the majority of mutations are completely neutral and if
mutation rate at the nucleotide level is mildly dependent on the generation
time. For details, see Jour. Molecular Evolution 1: 150-157.

RESEARCHES CARRIED OUT IN 1972

103

On the Stochastic Model for Estimation of Mutational
Distance between Homologous Proteins
Motoo KIMURA and Tomoko OHTA

A set of simple equations is derived which gives the relationship between
the observed fraction of amino acid differences per codon and the "evolutionary distance" per codon. Holmquist's stochastic model is used in which
the mutation is assumed to occur at random among the four bases (A, T, G,
C) in the variable part of the molecule. Let P d be the observed fraction ofamino acid differences per codon and let DE be the evolutionary distance(in terms of the number of nucleotide substitutions) per codon. Then,P d = 1 - (1 - 1y (1 - 1/4)

DE =

-

(9/4) log. (1 - 41/3)

where 1 represents the fraction of nucleotide sites for which the two cistrons
differ from each other (0 :::;; 1 :::;; 3/4). For details, see Jour Molecular
Evolution 2, 87-90.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

XI.

HUMAN GENETICS

Effect of Changing Parental Age Patterns on
Chromosomal Aberrations and Mutations
Ei MATSUNAGA

Among a number of possible genetic consequences of family planning(Matsunaga, E. (1966) JAMA 198: 533), the effect of changing parental age
patterns upon recurring mutations, both chromosomal and genic, is the one
which we can appreciate as of obvious "eugenic" relevance. Here, theargument is based on deduction from two empirical facts. First, demographic data in most industrialized countries show that there has been over
several decades, as a result of family p'anning practiced by more and more
people, a gradual concentration of childbearing at younger ages than informer times. Second, there are strong, positive associations of parental
ages with the production of certain chromosomal aberrations and genemutations. Whatever biological mechanisms are involved in such associations, the genetic consequence of younger parenthood is beneficial because it
lessens the additional genetic burden that would have arisen if the parents
had borne children at advancing ages.
Using demographic data in Japan, it was shown that the incidence of
certain trisomies (2I-trisomy, XXV and XXX, 13-trisomy and 18-trisomy)
must have decreased by 30--40% during the past 20 years since 1948, simply
because of the changing parental age patterns caused by family planning.
In addition, it was deduced that certain dominant conditions (chondrodystrophy, Apert's syndrome and Marfan's syndrome) must have decreased
by about 20 % during the same period. Since they include relatively
common disorders like Down's syndrome, a reduction in incidence by thisamount through family planning would represent a major saving in medicalcare and in human suffering. (For details, see Social Biology 20: 82, 1973).

RESEARCHES CARRIED OUT IN 1972

105

Genetic Effect of Selective Abortion Upon thet(21q Dq) Translocation
Ei MATSUNAGA

Among a variety of chromosomal aberrations, a balanced heterozygote
carrying the t(2IqDq) translocation is probably the most important indication for prenatal diagnosis. The incidence at birth of carriers of this type oftranslocation has been estimated as 3/21,996 = 1.4 X 10- 4 (D. N. (1972)
Ionizing Radiation II), and the risk of having a child with Down's syndrome
is 10% for female carriers while it is reduced to about 2% for male carriers(Hamerton, J. L. (1968) Cytogenet. 7: 260). If prenatal diagnosis followed
by selective abortion of unbalanced heterozygotes could be performed inevery carrier parent after one affected child is born, the reduction in theincidence at birth of inherited cases of Down's syndrome would be only afew per cent. However, if carriers could be diagnosed by premarital counseling with reference to his or her relatives being affected with translocation
Down's syndrome, the reduction would be much greater. On the other
hand, if reproductive compensation took place, the prevalence of carriers
would increase. This point may he outlined as follows.
Assume that premarital diagnosis of all carriers could be made in orderto monitor each of the pregnancies, and full reproductive compensation by
the births of two phenotypically normal children were introduced. Thenthe prevalence of carriers in the nth generation, y", would be such thaty" = (1/2 + k) y"-l + 2 m,

where k is the relative proportion of balanced heterozygotes among phenotypically normal progeny of male carriers, and m is the mutation rate giving
rise to a balanced heterozygote of the t(21qDq) translocation. While thevalue of m has been estimated as 0.6-0.7 X 10- 6 (Polani, P. E. et al. (1965)
Cytoget. 4: 193; Kikuchi, Y. et al. (1969) Jap. J. Human Genet. 14: 93),
it is still an issue whether or not k is significantly greater than 0.5 (Hamerton,
J. L. ibid.; Dutrillaux, B. and Lejeune, J. (1969) Ann. Genet. 12: 77; Jacobs,
P. A. et al. (1970) Ann. Human Genet. 34: 119). With k equal to 1/2,
the prevalence of carriers would increase from the present level of 1/7,000
to 1/5,000 in four generations, and to 1/4,000 in eight generations. However, if k is 0.6, as is suggested by the family data given by Hamerton,

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

the prevalence would increase to 1/3,700 in four generations, and to 1/2,200
in eight generations. It is to be noted that, if carriers were diagnosed only
retrospectively, then the rate of increase in their prevalence would becomeslightly slower.
Needless to say, if, based on prenatal diagnosis, not only fetuses that are
unbalanced heterozygotes, but also those that are balanced heterozygotes,
could be aborted, the eugenic effect would be as great as in the caseof dominant conditions. This raises of course an ethical issue. The
above calculation implies that, even if we go without sacrificing fetuses thatare balanced heterozygotes, the extent of the possible consequent dysgenic
effect need not be of serious concern, at least within several generations.

Evidence for a Second Structural Locus Determining
Tetrazolium Oxidase in Man
Tomotaka SHINODA, Ei MATSUNAGA and Jushiro KOSffiNAGA*

Upon electrophoresis of tissue extracts in starch gels and stained fortetrazolium oxidase, two different zones of the activity are demonstrated,
one of these corresponds to that of hemolyzates and the other to a distinct
form absent in hemolyzates. The former, having a greater mobility, isprovisionally referred to as soluble or cytoplasmic form (s- TOX), and thelatter as mitochondrial or bound form (m- TOX). The both forms of the
enzyme appear to have common substrate specificities and physicochemical
characteristics, as far as the data obtained from our preliminary experiments are concered.
An inherited variation in tetrazolium oxidase has recently been reported
by Shinoda (Jap. J. Hum. Gent., 15: 144-152) for a Japanese family which
was followed up to three generations. The variant has been referred to as
TOX 2-1, heterozygous for TOXI and TOX 2 alleles at TOX locus. We now
suggest an existence of a second structural locus determining this enzyme inman, based on the results of isozyme typing for various organs obtained byauthopsy.
During the course of the survey for multiple molecular forms of enzymes
in man, we encountered a rare case in which the enzyme in m-TOX fraction
exhibited an altered electrophoretic pattern. The electrophoretic pattern* Medical Examinar's Office, Tokyo.

RESEARCHES CARRIED OUT IN 1972107

of s-TOX fraction of this subject was found to be identical with that of theother individuals. This unusual pattern was constant in starch gel electrophoresis of extracts obtained from various organ, such as skeletal muscle,lung, heart, brain, pancreas, spleen, and kidney. The specimens taken fromthe individual were analyzed for other enzymes of over 20 different kinds,
and all of them were found to belong to one of the common types or one ofthe phenotypes when the enzyme systems were polymorphic. These datastrongly suggest that the altered form in m-TOX fraction of the individual is
not the result of secondary modifications, but a true structural alteration
resulting from a mutation at the m-TOX locus of the individual. Based onthese evidences we proposed the second structural locus for tetrazolium
oxidase in man.
Isozyme Variations in Man
Tomotaka SHINODA, Ei MATSUNAGA and Jushiro KOSHINAGA*

Isozyme variations in red cells and tissues obtained by autopsy have been
investigated for the total of 16 different enzyme systems. As to isozymes
in red cells, acid phosphatase adenosine deaminase, phosphogulcomutase,6-phosphogluconate dehydrogenase and s-glutamate-pyruvate transaminase
were found to be polymorphic, with the practecally the same frequencies
in the previous results. Estimated frequencies for respective enzyme
Eystems were as follows; AP : pa = 0.20, p b = 0.80; ADA: ADAl = 0.97,
ADA 2 = 0.03; POM l : PGM ll = 0.76, PGM l2 = 0.24; POD: PGDA=
0.91; PGDc = 0.09; s-OPT: GPP = 0.60, GPP = 0.40. Other variants
found in red cells were POM 7-1 0/340), PHI 3-1 0/340), PHI 4-1 (3/340), s-OOT 2-1 (3/340), s-OOT 3-1 0/340), and s-ICD 2-1 0/340), respectively. No variants were found in red cell samples with respect to otherenzyme systems.
As to tissue specimens, the following enzyme systems were found to be inmultiple forms: they were adenosine deaminase (ADA 2-1, 3; ADA 1, 61;
total 64), alcohol dehydrogenase (ADH a 1,37; ADH a 2-1, 47; ADH a 2,14;
total 98), 6-phosphogluconate dehydrogenase (POD A, 57; POD AC, 7; total64), phosphoglucomutase POM 1, 39; POM 2-1, 21; POM 2, 4; total 64),
phosphohexose isomerase (PHIl, 63; PHI 4-1,1; total 64), m-glutamate* Medical Examinar's Office, Tokyo.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

oxaloacetatetransaminase (GOT 1,53; GOT2-1, II; total 64), and s-glutamate
pyruvate transaminase (GPT I, 20; GPT 2-1, 24; GPT 2, 6; total 64).
In addition to these enzyme systems an isocitrate dehydrogenase wasfound to be highly heterogeneous, not only in s-fractions but also in m-fraction. There are three types of s-ICD; types I and 2 exibit only one major
band either the most cathodal in the former or the most anodal in thelatter, whereas the third one consists of three major bands, two of these are
identical with those of either type I or type 2, respectively. The remaining
band has an intermediate mobility between the bands I and 2. This figuresuggests that the third type is likely to be heterozygous form for types I
and 2. At present we are not very certain whether these phenomena areunder genetic control.

Structural Studies on Human Immunoglobulins
Tomotaka SHINODA

Amino acid sequence analyses were carried out using a fragment obtainedby specific scission at Asn-Gly bond with a chemical agent from purified
human type K IgA myeloma protein (Mu). Peptides were obtained bytryptic and chymotryptic digestions of the fragment following column
chromatography on Chromobeads-P in volatil buffer systems. After purification by high voltage paper electrophoresis at pH 3.7 or 6.5, peptides weresequenced by manual Edman-dansyl technique. The fragment contained
the first 65 residues of the variable region of the a chain.
When the sequence of the fragment is compared with those reported forother human heavy chains, there are several noteworthy aspects: a) the
sequence greatly resembles that reported for VHIII heavy chain subgroup;b) there are three regions in which the sequences appear to be less variable;i.e., positions 2 though 10, 17 through 29, and 36 through 43; c) the halfcystine residue at position 22 and two tryptophan residues at positions 36
and 47 are invariant; d) this region seems to have no class specificity insequence but is rather subgroup specific. The evidence strongly suggeststhat the variable region of the a heavy cha n has the characteristic structural feature common to other heavy chains and this will give further support
to the genetic hypothesis that each immunoglobulin chain is coded by morethan one gene, probably two, one for the variable region and the otherRESEARCHES CARRIED OUT IN 1972

for the constant region.
Res. Commun.

Details are in the press in Biochem.

109

B:ophys.

Prenatal Detection of Genetic Disorders: A program in Shizuoka
prefecture. (A preliminary report)
Yasuo NAKAGOME, Kazuso IINUMA and Ei MATSUNAGA

In 1972 a program was set out to monitor "high-risk" pregnancies in the
Shizuoka prefecture. It was a joint effort of both the Department of
Human Genetics, National Institute of Genetics, Mishima and the Shizuoka
branch of the "Nichibo", a nation-wide organization of obstetricians andgynecologists. There were about 330 members in the Shizuoka branch,most of them being in private practice. The total number of births in thisarea was about 60,000 per year and most, if not all, of them werecovered by the branch members. The prefecture has no medical college.
Indications of amniocentesis included pregnancies of chromosomal translocation carriers, women who had had a chromosomally abnormal child,
women of 40 years or older, carriers of severe sex-linked diseases and carriers of a few metabolic diseases. Amniocentesis was carried out by thebranch members and the amniotic-fluid samples were transferred to our laboratory.
Sixty-three cases have been examined so far. Twenty-six of them wereby various reasons other than genetic diseases (e.g., hydramnion, historyof irradiation in early pregnacy). Feasibility of various culture techniques,effects of storage prior to the start of cultures and the incidence of polyploidcells in cultures were studied using these samples (Nakagome et al., Lancet
2: 387, 1972 and unpublished data). Examinations of pregnancies of two
different translocation carriers, t(6q+; 18q-) and t(Cp-; Dq+), revealed
a balanced carrier in the former and an unbalanced Dq + foetus in the latter.
The banding study on the latter family is in progress. There were 23
pregnancies that were examined because of a previous delivery of a childwith Down's syndrome (regular trisomy). In one of them, who had had
two miscarriages and was 42 years old, the foetus was 47, XXY. The details
will be published elsewhere (Iinuma et aI., Human Hered., in press). Pregnancies of two known carriers of haemophilia revealed a female and amale foetus respectively. All other cases, including a pregnancy with the110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

history of previous delivery of cat-cry syndrome and 8 cases with maternalages over 40, showed a normal karyotype.
G Group Chromosomes in Satellite Associations.
(A second report)
Yasuo NAKAGOME

In the last issue of this organ a slight excess of the involvement of no. 21
chromosomes in satellite associations was described (Nakagome (1972)
Ann. Rep. Nat. Inst. Genet. 22: 97). A total of 152 G group chromosomes
in satellite associations were then identified by the technique of G-staining.
Eighty-seven of them were no. 21s and 65 were 22s. The difference was not
s:gnificant.
Additional 385 G chromosomes have been scored. The details of the techniques used are described elsewhere (see next page). The results are shownin the table. Out of a total of 537 (152 plus 385) G group chromosomes,
297 were no. 21s and 240 were 22s. There seems to be an excess entry of no.
21 chromosomes into satellite associations (x 2 = 6.05, d.f. = 1, p < 0.02).
The details will be published elsewhere (Nakagome, Cytogenet. Cell Genet.,in press).
Table 1. No. of G group chromosomes involved in satellite associations_._-_.
No. of G's associatedIndividuals-----------. _ - -..

1
2
3
4
5
6
7
8
9
Combined

x2

Sex
F
M
M
F
F
M
F
F
M

Total

i21

i22

Expected

60
18
134
59
33
18
15
42
158
537

30
10

30
8
63
27
15
5
5
19
68
240

30
9
67
29.5
16.5
9
7.5
21
79
268.5

71

32
18
13
10

23
90
297

---------~-* x 2 for combined data, d.f. = 1, 0.02> p.
Heterogeneity x 2 =4.0, d.f.=8, p>0.5.

0.22
0.48
0.42
0.27
3.56
1.67
0.38
3.06
6.05*
-

----

----

Sum of x 2s=10.06, d.f.=9, p>0.25.

RESEARCHES CARRIED OUT IN 1972

111

Banding Studies of Human Chromosomes. 1. Characterization of
3 Translocations Involving C or G Group
Chromosomes and of a C Trisomy Mosaic
Yasuo NAKAGOME, Kazuso IINUMA and lehiro MATSUI

Individual chromosomes in the C or G group cannot be distinguished withcertainty either by conventional karyotype analysis or autoradiography.
However, the techniques of Giemsa banding and quinacrine fluorescencehave made it possible to recognize every individual chromosome pair by its
characteristic pattern of bands. In the present report, chromosomes involved in 3 unusual translocations, including two Cs and a G, were identified. Further, the points of exchanges were determined within narrow
limits. A C group chromosome in a case of mosaic C trisomy was also
identified. The techniques of Giemsa staining and quinacrine fluorescenceare described elsewhere (Nakagome et al. (1973) J. Med. Genet. 10: 174).
A family with 2 cases of 18 trisomy had a t(Cq +; Eq-) translocation
through 3 generations. II). every case examined, translocation was identified
as rcp (6; 18) (q2; q1). In a pregnancy of a carrier woman, the embryo was
identified to be a translocation carrier by amniocentesis.
In a girl with t(Cq-; Dq+) translocation the chromosomes involved were
11 and 14. The karyotype was identified as 46, XX, rcp (11; 14) (q12 or 13;
q32 ?). Both of her parents showed a normal karyotype. In the third translocation, routine chromosome analysis revealed a 45, XX, +C, - E, -G
karyotype. One of the C group chromosome was mediancentric andabout the size of a no. 12. The karyotype was identified as 45, XX, -17,
- 22, + der (17), t(17; 22) (p 12 or 13; q11 ?). An extra chromosome in acase of mosaic C trisomy was identified as no. 10. The correlation between
phenotype and these chromosomal changes are being studied.

Fluorescence of the Barr Body in Human
Amniotic-8uid Cells
Kazuso lINUMA and Yasuo NAKAGOME

Fluorescent staining technique has been proved to be a reliable tool forprenatal identification of fetal sex, as it ensures the presence of the Y chromosome in interphase nuclei of male cells from human amniotic fluid.

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Moreover, the sex-chromatin or Barr body when stained with quinacrine
mustard has been observed to fluoresce in certain cell types under certain
conditions, such as cultured fibroblasts. However, no recognizable fluorescence of the Barr body has been observed in the nuclei of uncultured humanamniotic-fluid cells from female fetuses. Our report describes the fluorescence of the Barr body in freshly obtained amniotic-fluid cells.
Approximately 3 ml of fresh amniotic fluid was centrifuged, and thecell pellet was fixed in acetic-methanol (1: 3) for 15 minutes. After centrifugation, one drop of cell suspension was placed on a glass slide, gently
blown, and flame-dried. Our fluorescent staining technique was described
elsewhere (Iinuma and Nakagome, (1972) Jap. J. Hum. Genet. 17: 57).
The fluorescent Barr body could be readily identified by its size, intensityof staining, and its usual position next to the nuclear membrane. Restaining with carbolfuchsin confirmed its correspondence with the sex-chromatin.
The incidence of nuclei with fluorescent Barr bodies varied from 2 % to 16 %in a total of 10 cases with the conception of a female fetus. In one case, bothV-chromatin and fluorescent Barr body were observed in the same nucleus:
its karyotype from cultured fibroblasts was 47, XXV. For details, see
Lancet 1: 436 (1973).

Polymorpbisms of C and Q bands in Human Chromosomes
Kazuso IINUMA, Ei MATSUNAGA, and Yasuo NAKAGOME

Using the new techniques of staining C and Q bands of human chromosomes described by Arrighi & Hsu (Cytogenetics, 10: 81, 1971) and byCaspersson et al. (ExptI. Cell Res. 60: 315, 1970), respectively, we havestudied a total of 29 unrelated persons, consisting of 13 males and 16 females,
without any detectable abnormality in the karyotype. Polymorphism
involving constitutive heterochromatin (C-band variants) was found to occur
at a high rate in chromosomes Nos. 1,9 and 16, where the length of densely
stained heterochromatic regions of the secondary constriction was used asa marker. The results are summarized in Table 1, in which "cc" stands
for the homozygous form with two "normal" chromosomes and "Cc"for the heterozygous form with one normal and one (C) with increased lengthof densely stained heterochromatin in the region of secondary constriction.
In our sample there was no case with a homozygous "CC" form. It is

RESEARCHES CARRIED OUT IN 1972
Table l.

113

Summary of C-band variants
--_

..

~

Chromosome No.

CC

Cc*

cc

Total

1
9
16

0
0
0

9
10
1

10
9
18

19
19
19

..

_-

----------

X2**-------

Frequency of C

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

1.83
2.42
0.01

0.24
0.26
0.03

-----_._--"------* C indicates the variant with increased length of densely stained heterochromatinin the region of secondary constriction.

** Calculated on the basis of the Hardy-Weinberg's equilibrium.
Table 2.
____~_______

Chromosome No.
3
4
13
14
15
21
22

Summary of Q-band variants' , _ 0 _ _ _ _ . •_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ • _ _ _ _ _ _ , _ _ _ _ _ _

FF

Ff*

ii

5
0
5
0
0
0
0

16
14
14
6
1
7
5

5
12
7
20
25
19
21

Total
- - - ----

X2**------------ .

Frequency of F----_._-_._._._----_.,._~-----

26
26
26
26
26
26
26

0.18
3.53
0.18
0.44
0.02
0.10
0.08

* F indicates the variant with strongly fluorescing paracentric region.

0.50
0.27
0.46
0.12
0.02
0.13
0.09

-------** Calculated on the basis of the Hardy-Weinberg's equilibrium.

not certain whether the chromosomes designated either as C or c are homogeneous among different subjects with respect to the quantity of the heterochromatin but the values ofX 2 calculated on the basis of the Hardy-Weinberg's
equilibrium showed a good agreement between the observation and the,expectation. The frequencies of the variants are also shown in the table.
Polymorphism involving Q-bands was observed to occur in chromosomes
Nos. 3,4, 13, 14, 15, 21 and 22, each of them showing either strongly (F) or
weakly (f) fluorescing paracentric regions or satellites. Again, assumingthat there are only two types of chromosomes involved in each of the polymorphisms concerned, the results are summarized in Table 2. The X2
gives a larger but insignificant value (3.53 for 1 d. f.) for the polymorphism ofchromosome No.4; it is not clear whether this is due to chance alone or toother causes, although it is sometimes difficult to ~iscriminate the fluorescentmarker in this particular chromosome. In addition, the length of intensively
fluorescing distal part of the long arm of Y chromosome was considerably
variable from person to person. The length of the largest Y chromosome
was equal to that of D group chromosomes, while the smallest one corresponded to two thirds of No. 21 chromosome.

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Further, metaphases from 6 pairs of identical twins, one pair of fraternal
twins, one pair of siblings and their parents were analysed with C and Q
banding techniques. A combination of respective markers was characteristic
in each individual, and comparison of these combinations within a family
revealed a pattern of regular inheritance from parents to children. Particularly, a complete concordance in the combinations within pair of identical twins, whose zygosity was determined independently by the conventional
methods using blood types, serum types, and isozyme patterns of red blood
cells, was noticeable, in contrast to the discordance between fraternal twinsor siblings.
A part of this study was reported in the 17th annual meeting of the Japan
Society of Human Genetics (1972).

RESEARCHES CARRIED OUT IN 1972

XII.

115

BEHAVIORAL GENETICS

Genetic Analysis of Selected Populations of Drosophila melanogasterfor Positive and Negative Phototaxis.I )Jong-Kil CHOO and Chozo OSHIMA

The results of selection of D. melanogaster (Okinawa strain) for bothphotopositive and photonegative directions for 25 generations were reported in the previous Ann. Rep., No. 22: 104-105. Such selections wererepeated for more 10 generations, and mean photoscores and variances ofboth photo-positive and negative populations for two periods were shownin Table 1. Realized heritability was calculated by average ratio of theselection response to the selection differential (Falconer 1960). The regression slops, plotted against the cumulated selection differential, were calculatTable 1. Results of selection: Mean photoscores and variances of original
photopositive and negative populations, and of reversely anddisruptively selected populations_Q!:iginal selection

Generationof
selection

X

26-30
31-35

3.21
3.35

Negative

Positive

X

q2

7.95
8.40

7.86
7.48

q2

5.43
6.52

Reverse selection
Posi.--->Nega.

28-30
31-33
34-35

Nega.--->Posi.

X

q2

X

q2

4.39
6.59
7.52

8.97
9.82
10.40

7.01
6.79
6.03

12.27
9.93
12.04

Disruptive selection
Nega. ~ x Posi. is
Posi. ~ x Nega. is

27-29
30-32
33-35
1)

X
5.47
6.41
6.66

q2

X

q2

9.60
12.30
9.86

5.88
6.47
6.59

9.18
10.62
10.40

This work was supported by a grant (4050) from the Ministry of Education.

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

ed for early 20, late 15 and total 35 generations. Combined female andmale realized heritabilities of both photo-positive and negative populations
for total 35 generations were 0.0160 and 0.0245 respectively. The responses
to both directions of selection were not symmetrical. The realized heritability of photo-positive population for the late 15 generations was verylow.
At 27th generation, reverse selections for both photo-positive and negative
populations were started. Fifteen pairs of flies showing the most negative
phototaxis in the photopositive population were crossed, and such reversed
selection was repeated for the following 8 generations. On the other hand,such reverse selection was performed for the photonegative population.
Mean photoscores and variances of both populations in the period ofreverse selection were shown in Table 1. The effect of reverse selection frompositive to negative was rapid and the photoscore decreased to the neutrallevel by only four generations, but the opposite reverse selection did notshow such rapid effect.
At 26th generation, disruptive selections were started. Fifteen females
and males, showing the most positive and the most negative phototaxis fromphoto-positive and negative populations respectively, were crossed to makea hybrid population. Such procedures were repeated for the following
9 generations. The results were presented in Table 1. Mean photoscoresof these populations accepting reciprocal disruptive selections for three
periods were similar with each other. However, these values seemed toincline gradually to photonegative side.
Hybridization analysis between photo-positive and negative populations
was carried out at 26th, 30th and 34th generation, and the results wereshown in Table 2. Photoscores of reciprocal hybrid flies at 26th generation
Table 2. Photoscores and variances of hybrid flies between photo-positive
and negative populations at 26th, 30th and 34th generation
Gen. 27

Gen. 31

X

<1 2

X

Photopositive popul.
Photonegative popul.
Average

3.01
7.86
5.43

4.34
8.47

Posi. ~ x Nega. rJ
Nega. ~ x Posi. rJ

5.62
5.88

9.50
8.62

Population

Gen. 35
2

X

2.78
8.07
5.62

5.95
9.42

3.49
8.42
5.95

8.30
6.86

6.95
6.84

10.17
9.51

7.16
7.08

9.94
10.11

<1

<1

2

RESEARCHES CARRIED OUT IN 1972117

was close to the mean of both parental populations, however, those of
30th and 34th generations were slightly biased to photonegative side. From
the results described above, some photonegative polygenes manifestingnegative phototaxis would be partially dominant or epistatic over thephotopositive polygenes.

Comparison of Some Quantitative Characters between Populations
Selected for Phototaxis in Drosophila virilis 1 )Chozo OSHIMA and long-Kil CHOO

Selections to make photo-positive and negative populations in D. virilis(Tokyo strain) were carried out for 40 generations. The results of thefirst 30 generations were reported in the previous Ann. Rep., No. 22: 103104. The experimental method using a maze apparatus was quite the sameas that of D. melanogaster. In this case, body weight and body length(from the 3rd segment of antenna to the end of abdomen) of flies, sampledfrom both photo-positive and negative populations at 34th, 36th and 40th
generations, were measured by Mettler's physical balance and the magnifying projector. Wing length (from the humeral crossvein to the end of 3rd
longitudinal vein) was measured at the same time. Each generation wasmeasured with 30 pairs of flies (about 7 days-old), taken at random from thetotal number of flies of each population. The results of these measurements
were shown in Table 3. The body weight of photopositive female and male
was definitly lighter than that of photonegative female and male flies respectively. The same relationship was deservedly noticed in the body length,
but no difference was observed in the wing length. The ratio of body weightover body length of photonegative flies was greater than that of photopositive flies.
Walking speed of flies to a light way was measured by a long tube, of whichinside diameter was 1.5 cm and length was about 1.3 m. The tube wasdivided into 8 regions (18 cm) by septum having a hole and these regions
were numbered from 5 (light) to -2 (dark). These tubes were connected
in a straight. A glow lamp (60W) was placed at 30 cm above the 5 region
and the -2 region was covered with a dark box. About 40 female and male:flies were introduced separately into the 0 region, and they were allowed to1)This work was supported by a grant (92715) from the Ministry of Education.

........
00

I
~

o
~

Table 3. Body weight, body length and wing length of photo-positive
and negative flies
Generation

Body weight

Body length

Wing length

Population

Photopositive
population

Photonegative
population

Male

FemaleMale

FemaleMaleo'Tl
Ratio ofbody weight/body length
FemaleMaleof selection

FemaleGen. 34
Gen. 36
Gen. 40
Average

1. 75
1.90
1.95
2.16
1. 76
1.68
1.89±.041.84±.03

3.22
3.29
3.44
3.23
3.45
3.16
3. 39±.03 3.20±.02

2.91
2.75
2.70
2.59
2.57
2.45
2. 73±.02 2.60±.02

0.53
0.63
0.51
0.56

0.59
0.60
0.53
0.58

Gen. 34
Gen. 36
Gen. 40
Average

2.21
2.69
2.76
2.19
2.26
2.80
2. 75±.04 2.22±.04

3.78
3.39
3.37
3.82
3.74
3.43
3. 78± .02 3.40± .02

2.97
2.81
2.51
2.69
2.70
2.61
2. 79±.02 2.64±.02

0.71
0.72
0.75
0.73

0.65
0.64
0.66
0.65

~

o

~

Z

VJ

~
tri

c::

o'Tl
o
~

gj

()
til

~

N

w

~

tI1

Table 4. Walking speed of photo-positive and negative flies
in a test tube for 5 minutes
Population

Photopositive

Generationof
selection
Gen. 38 ~Gen. 40 ~Gen. 38 isGen. 40 is-----Gen. 38 ~Photonegative

Gen. 40 ~Gen. 38 isGen. 40 is

V2

~
~

Percentage of flies distributed in regions of a test tube

5
61.65
82.96
60.82
78.51
10.00
6.51
5.75
11.03

4
6.77
3.70
5.26
8.26
6.47
4.73
7.47
5.15

3
8.27
2.96
11.70
4.96
5.88
10.06
7.47
13.97

2
4.51
2.96
8.77
1.65
11.76
13.02
5.17
7.35

1
8.27
2.22
4.09
2.48
14.71
15.98
12.64
11.76

0
3.76
3.70
3.51
3.31
29.41
26.63
36.21
21.32

-1
5.26
1.40
5.68
0.83
14.11
14.79
16.67
16.91

Total flies

-2
1.50
0
1.17
0
7.65
8.28
8.62
12.50

@
tI1

V2

133
135
171
121
170
169
174
136

~
~
~
....

gj
0
C

>-l

....
Z
....

'"......
N

....
....

'"

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

walk to either light or dark ways and after 5 minutes, the number of flies
distributed at each region was counted. Such experiments were performed
four times with different flies. From the results represented in Table 4,
the walking speed of both photo-positive and negative flies could be compared with each other. About 80 percent of photopositive flies walked
fastly to the light way, but about a quarter of l'lhotonegative flies did not
move and about 20 percent of them prefered the dark way. The behaviorof both photo-positive and negative flies could be recognized by such asimple method.

Effects of Urbanization on the Ecological Habitat andof Noise and Light Environments on theDevelopment of Drosophila. 1 )Chozo OSHIMA and Jong-Kil CHOO

In November 1972, Drosophila species were collected by sweeping at
two places: National Park for Nature Study in Tokyo city and Tamazawa
in the suburbs of Misima city. Names of genera, and numbers of species
and specimens, collected at two places, were summarized in Table 5.
The montium species group (D. auraria, D. rufa and D. lutea) was dominant
species in autumn at two places, but about fifty percent of specimens collected at Tamazawa were D. immigrans. The similar result was observed in1971 as reported in Ann. Rep., No. 22: 107-108. Furthermore, many
Table 5.

Drosophilid flies collected at two places
in November 1972
Number of species (No. of specimens)

Genus

National Park for Nature Study(Tokyo city)

Tamazawa(Misima city)
3 ( 25)

Leucophenga
Mycodrosophila
Scaptomyza
Drosophila

3 ( 33)16 (730)~~~~~-~---

22 (788)

Total~-~--- ----~~~~

1)

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

RESEARCHES CARRIED OUT IN 1972

121

flies of a new species, D. (S.) oshimai (named by Choo and Nakamura:
Kontyii, 1973), were collected at Tamazawa and Shizuoka in autumn andwinter seasons.
The effects of noise and light environments on the development (fromegg to adult) of D. melanogaster (Oregon-R strain) were examined. A
sound level of 100 phone, 2000 cycle/sec. was produced by a machine,
Audio Signal Generator (SNR-ll), which set in an incubator with program
controllers of photo and temperature conditions. Many eggs laid by several
hundreds of female flies for one hour were collected, and each of 50 eggs
was transferred to a small vial containing cornmeal, ebios and sugar medium.
Two small sheets of gauze were used as a cap of vial in noise environment,
and a sponge plug was used in light environment. A group was composedof ten vials having fifty eggs. One group was kept in constant noise and light(2500 lux) environment, and another group was kept in constant quiet andlight environment. The temperature in these environments was kept at
25°C. In other cases, light environment was changed to constant dark
(0 lux) environment and also to periodical light and dark (Ll2; D12) environment. Periodical noise and quiet environment (NI2; Q12) was also made.
The results were presented in Table 6.
Table 6. Time of development (egg to adult) under noise and quiet,
light and dark environments----

Environment

Mean time ofdevelopment

Variance

Eclosion rate

Const. Land Q
Const. Land N
Const. D and Q
Const. D and N
Peri. L12 : D12, Const. Q
Peri. N12 : Q12, Const. L

207 hr 35 min
197 hr 35 min
221 hr 20 min
210hr 49 min
212 hr 18 min
190hr 40 min

6 hr 38 min
5 hr 49 min
6tv 45 min
7hr14min
4 hr 45 min
6 hr 15 min

88.45
80.40
81.47
86.00
90.80
89.60

(%)

L, Light environment; D, Dark environment; Q, Quiet environment; N, Noise environment.

The light environment accelerated the development about 5 percent fasterthan the dark environment, and the noise environment accelerated it about
10 percent faster than the quiet and dark environment. However, nosignificant differences among the rates of eclosion in these environments
was observed.

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Selection for Geotaxis in Drosophila melanogaster
Takao K. WATANABE and Wyatt W. ANDERSON

Geotactic selection and hybridization studies were carried out with a natural population of D. melanogaster. Behavior was measured by using Hirsch
classification maze. The initial population was near neutral to gravity,
yet it responded to both positive (downward) and to negative (upward)
selections with a realized heritability of about 0.14. A stabilizing selection
toward neutral gravity was also carried out simultaneously. At generationsof 6, 9 and 10 all possible hybrid crosses among the selected populations
were examined. Each geotactic value of the hybrid population was notdifferent from the mid-parent value. But, average value of the hybrids
was slightly deviated to downward. Frequencies of polymorphic inversionsof the initial population generally reduced in every population. During
the selection of ten generations, neutral population seemed to maintain more
inversions than the negative and positive populations. Negative population eliminated all second chromosome inversions, while positive population
decreased the frequencies of the third chromosome inversions. There wasno significant depression in productivity (number of progeny) of eachselected population during the selection for geotaxis.

RESEARCHES CARRIED OUT IN 1972

XIII.

123

APPLIED GENETICS

Geographical Variation on an lsoperoxidase Level in
Populations of Pinus Thunhegii
Kan-Ichi SAKAI and Shinya !YAMA

Collection of needle-leaves on an individual tree basis was performed infive districts along the sea coast of Japan proper. Two of them are facing
Japan Sea and designated for the sake of convenience JE (Niigata-Ken)and JW (Tottori-Ken), while the remaining three on the side of Pacific
Ocean, being designated PE (Miyagi-Ken), PC (Shizuoka-Ken) and PW(Hiroshima-Ken). From each of two to five populations in each district,
twenty to seventy trees were sampled for their needle-leaves. The squeezed
sap of those needle-leaves was investigated for the isoperoxidase variationby the aid of the starch-gel electrophoresis. It has been found from thisstudy that there was a distinct variation among populations in respect ofincidence of several isoperoxidase bands. It is of interest to find that the
populations growing in western parts of Japan proper had higher incidencesin general than those from eastern parts. For example, incidence of Q
band in populations from PW and JW was 88 and 52 %, respectively, while
it was 14, 20 and 13 % in the remaining three districts, i.e. PC, PE and JE.
The same tendency was also found for other bands. In addition to this,it was observed that the staining intensity of isoperoxidase bands in samples
from central and eastern districts looked apparently weaker than that inwestern districts. Further investigations are under way.
Effect of Population Size on Selection Efficiency of the Bulk
Method of Breeding in Autogamous Plants
Shinya !YAMA

The bulk method of breeding is widely used for selection of quantitative
traits in autogamous crop plants. The principle of the method is as follows:
After crossing between two parental stocks, the progeny populations arepropagated for several generations without selection. During this period,
the plants become homozygous and desirable recombinations can take place.

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Then, selection is applied to the hybrid population. In practice, the sizeof population which one can maintain is small as compared with the numberof possible gene combinations.
A computer simulation experiment was conducted to find how the sizeof population in each generation influences on the probability of recovery
of desirable recombinants. After crossing AAbb X aaBB, the probability of
AABB genotype in the F u generation was estimated under various propagation schemes (Table 1). The probability of occurrence of AABB on the
average for 1,000 runs did not differ among the different schemes if the twoloci had the same strength of linkage. However, the frequency of runs inwhich AABB was ultimately lost distinctly differed according to the schemes.
It was concluded from the results that a small population size in early
generations (F 2 and Fa) definitely resulted in the loss of desired genotypes.
An example of simulation is presented in Table 1.
Table 1. Frequency of runs losing AABB before Fu under different
propagation schemes. (No. of runs = 1,000)
Receombination value between A and B

Propagation schemenl* n2 na n4 n6 •.• nlO

0.1

0.2

0.25

0.3

0.5

0.4

---~----

~

-

_

.

_

(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)

4
8
8
16
6
6
12
24

4
4
8
4
4
6
3
3

4
4
2
2
3
2
2
2

.. __ . _ . _ - -

4
2
2
2
2
2
2
1

1. ... 1
1. ... 1
1. ... 1
1. ... 1
1. ... 1
1. ... 1
1. ... 1
1. ... 1

121
14
18
4
75
52
18
4

86
10
3
2
45
28
2
0

63
3
5
0
32
19
1
0

57
11
4
0
29
16
1
0

58
7
3
0
13
22
0
0

79
5
5
0
18
14
0
0

*) ni =number of descending plants from each parental plant at i-th generation.
Population size at F6 was 256 for (1) to (4) and 144 for (5) to (8).

Gamma-ray Induced Genetic Variation in Arabidopsis tlla/iana
Sujit BAGCHI and Shinya IYAMAIn order to look into the pattern of induced genetic variations in quantitative characters, a pure line, Landberg, of Arabidopsis thaliana was irradiated with gamma-rays. The experiment started with treating the seedsof the plants with doses of 20 and 80 kR. The subsequent propagation was

RESEARCHES CARRIED OUT IN 1972

125

conducted according to the "one plant one offspring" scheme (Sakai and
Suzuki, 1964) with population size of about 1,000 in each treatment. At
the M s generation 20 plants were selected at random from each of the
20 kR and 80 kR bulk populations in addition to the 14 plants from thecontrol population. In the M 4 generation, lines each consisting of approximately 20 plants were grown from the selected plants. Measurements
were taken on days to first flowering, plant height and number of leaves
on a single plant basis.
The data were subjected to analysis of variance, and genetic and environmental variances were estimated based on the components of between- andwithin-line mean squares as shown in Table 1. It was found from theresults that radiation had induced little change in the mean values of the
population except for days to flowering and plant height in the 20 kR treatment. Of noteworthy, however, was an increase in the inter-line variability
due to irradiation and it was of interest to find that this trend was very
distinct for all three measured characters in the 80 kR treatment. The
estimated values of genetic variances were always larger in irradiatedpopulations than in the control.
Unexpected, however, was an increase of the environmental variances in
irradiated populations. In order to reconfirm whether or not this is a generaltrend, intra-line variability was investigated in the irradiated and control
line groups. It was found that some lines from the irradiated populations
were highly variable within themselves while no control line showed such
Table 1. Estimated mean (x), genetic variance (u~) and environmental
variance (u:) for three metric characters.
------

Treatment

Days toflowering---._---"'----

Control

u 2g
u e2

x
20kR

u 2g
u e2
X

80kR

Number ofleaves

24.61±2.441.3521
7.378627.63±3.12
3.980511.9730
25.78±4.1315.7701
19.84239.52±0.98
0.01891.1262
9.61 ±1.27
1.00871.797810.97±1.491.85142.7285- - - - - - - - _..

x

u g2
u e2

28.25±1.440.69592.491224.27±2.186.2231
6.044829.28±2.54
13.0520
5.8811._------------

Plant
height..

- - _ . _ - ~ . __-126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23
Table 2.

Character Treatment

Frequency distribution of standard deviations forwithin-line variation in M4 generation< 1.0

1.0 1.8 2.6 3.4 4.2 5.0 5.8 6.6
1
1
1
1
1
1
1
1
1.8 2.6 3.4 4.2 5.0 5.8 6.6 7.0

No. oflines
observed..

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

Days
tofloweringControl
20kR
80 kR

3
1

8
8
6

3
6
6

2
7

1
1

2
---- -----------_.~---~._--~~~----~~---_.-

Plant
heightControl
20kR
80kR

No. ofleavesControl
20kR
80kR

3
1
11

4
4

3
14
12

5
8
2
2
2

4
6
4

1

3
8

1

2

2

2

-- .

14
20
20

__ .... _---14
20
20
14
20
20

variability (Table 2). This high variability might be due to intra-line
segregation or developmental instability induced by irradiation. Recent
investigations on this point, though not completed yet, suggest that increased
developmental instability could be the major factor in this phenomenon.

Inbreeding Depression in Japanese Quail
Takatada KAWAHARA

Birds from semi-domesticated wild strain of Japanese quail, Coturnix
coturnix japonica, those from a domestic strain, and their reciprocal F lhybrids were investigated with regard to the tolerance against inbreeding
depression. The total number of matings with which the experiment did
start was 467 of which 191 was from the wild, 85 from the domestic, 83 fromdomestic (~) X wild (t), and 108 from wild (~) X domestic (t). The
birds were propagated by successive full-sib mating. Birds at the age of 12
to 20 weeks were used for mating, and the eggs were hatched twice per
generation at a two-week interval to raise the progeny from eggs stored lessthan two weeks. As the control, the domestic strain was propagated bycircular pair mating system in order to avoid inbreeding. No conscious selection for performance traits was practiced. Traits examined were fertility,
hatchability, viability (up to 20 weeks after hatching), survival index (fertility
X hatchability X viability), egg production rate up to 60 days after the first

RESEARCHES CARRIED OUT IN 1972

127

egg, fitness index (survival index X egg production rate), age at sexual maturity, egg weight (average for first three eggs at sexual maturity) and bodyweight at 4, 8, 12, 16,20 weeks of age and at sexual maturity.
The results of this investigation was summarized as follows:
1) The wild and two reciprocal F l hybrid strains were generally lesstolerant to inbreeding than domestic strain. These three strains died out inthe third or fourth generation of inbreeding, whereas the domestic strain
survived until the fifth generation. No significant difference was foundbetween the two reciprocal hybrids in this respect.
2) The degree of inbreeding depression was measured by the regressionof a given trait on inbreeding coefficients (F). The mean amount of changes
in the traits for a 10% increase in Fwas -9.10% in fertility, -11.34% in
hatchability, -11.63% in viability, -10.64% in survival index, -7.39%
in egg production rate, - 8.99 % in fitness index, + 3.6 days in sexual maturity and -0.03 g in egg weight. In a similar manner, the body weightdecreased with inbreeding, the decrease per 10% increase in F being -4.19 g
for females and -2.99 g for males.
3) The lethal equivalents as described by Morton et al. (1956) were
estimated. The total load (A + B) of fitness index in terms of lethal equivalent was between 6.5 and 9.0 per gamete or 13 and 18 per zygote. Thus,
the estimates of Band B/A statistics in Japanese quail were found to be higher
than those reported by Sittmann et al. (1966) using domestic quails.

A Genetic Analysis of Skeletal
Characters in Japanese Quail
Takatada KAWAHARA

Genetic and phenotypic correlations among traits and heritability valuesof skeletal characters were estimated in a random-bred population of domestic
Japanese quail. Investigated were total body weight, eviscerated bodyweight, total bone weight, keel length, keel depth, lumbosacral bone lengthand skull width. In addition to them, length and weight were measured
in the following nine bone characters, i.e., coracoid, scapula, humerus,
ulna, carpometacarpus, femur, tibiotarsus, tarsometatarsus and thoracic
vertebrae. Data were collected from 584 progenies (305 female and 279
male birds) originated from 69 pair matings. The birds were killed at the128 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

age of 25 weeks and autopsied.
The results of this study are summarized as follows:
1) The female birds tended in many characters to be significantly larger
than the males except for keel length and depth, skull width, coracoid weightand eviscerated body weight, in which the males were significantly larger
than the females. Weight of scapula and carpometacarpal bones did not
significantly differ between sexes.
2) Heritability values were estimated from the analysis of variance ofthe data for various bone characters. In the males, bone length generally
had higher heritabilities than bone weight. Females showed lower heritability values than males and in females, the values for bone length were
not higher than those for bone weight. When the data for females andmales are pooled, the heritability for the hind-limb bone complex including
femur, tibiotarsus and tarsometatarsus, was 0.819 for length and 0.789 forweight; the fore-limb bone complex consisting of humerus, ulna andcarpometacarpus, had somewhat lower values, e.g., 0.726 for length and0.637 for weight. Lower heritability values were found in lumbosacralbone length (about 0.35 for both male and female) and in skull width(0.378 for male and 0.479 for female).
3) Genetic and phenotypic correlations among various traits weregenerally higher in the male than in female. The phenotypic correlation
between length and weight of the same bone estimated among nine different
bones was 0.431 in male and 0.266 in female. The genetic correlation also
showed a similar trend, namely, 0.595 in male and 0.273 in female. Correlations of body weight or eviscerated body weight with bone characters werehigher in male than in female. The body weights were strongly correlatedwith bone length but weakly correlated with bone weight. Bones in thesame body part were strongly inter-correlated, but those in different bodyparts showed lower correlations.

A Note on the Breeding Behavior and Genetic Variations ofSunflowers Observed in Central Luzon
Hiko-Ichi OKA

Two sunflower (Helianthus annuus L.) varieties, Arrowhead and Armaveric,RESEARCHES CARRIED OUT IN 1972129

were used for determining self-fertility, outcrossing rate and other genetic
parameters. Isolation of single plants by a distance reduced seed fertility
to about 20 per cent indicating their tendency to self-incompatibility.
Outcrossing rate was estimated to be 75 per cent from the frequency ofplants with striped seed (a dominant character) in the progeny of Armaveric(with black seed) raised together with Arrowhead (with striped seed). The
heritability values estimated from parent-offspring regression were 52 %for number of days to flowering, 68 % for plant height, and 22 % for head
diameter. The number of days to flowering and plant height were geneticallycorrelated, but head diameter was not correlated with the former two characters.

Performance in Central Luzon of Soybean Varieties
Selected in Taiwan for Wide Adaptability
Hiko-Ichi OKA

Ten soybean varieties from Taiwan and two local ones were tested in threedifferent seasons at the Central Luzon State University, Munoz (l5°N),
Philippines. Of the ten Taiwan varieties, eight had been selected for yieldstability following the breeding scheme of "disruptive seasonal selection".
The results proved that, if planted at a proper density, at least two Taiwan
varieties (CH-2 and Fall-17) could have a high yielding potential (about
2.5 ton/ha) and a small yield variance among the six seasons compared(spring, summer and fall in Taichung and rainy, early dry, and late dry at
Munoz). Generally, varieties adapted to the fall cropping in Taiwan appeared to be adaptive in the tropical conditions in Central Luzon. Correlation studies among yield characters indicated varietal differences in thephysiological responses to seasonal conditions, yet yield stability was recognized to be a genotypic character established in certain Taiwan varieties.
This may serve as an evidence for the usefulness of disruptive seasonal
selection for obtaining wide adaptability. (Published in SABRAO Newsletter 5: 29-38, 1973).

130 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

Variation in the Response to Planting Density among
Sun80wer Varieties
Hiko-Ichi OKA

Ten sunflower (Helianthus annuus) varieties were tested at four different
planting densities. They generally responded to close spacing by reducing
their height and organ size, but differed in the magnitude of their responses.
Logistic functions were computed from the data of plant height to estimate
the number of days to half-size time and maximum growth rate. Further,
regressions of these growth parameters and other character values on planting density (linear and quadratic) were computed in each variety. The results indicated significant varietal differences in the mode of developmental
plasticity. This work was carried out at the Central Luzon State University, Philippines, in the 1971/72 dry season.

Variations in the Growth Pattern of Oryza Perennis Strains
Hiroko MORISHIMA and Hiko-Ichi OKA

As previously reported (1969 and 1970), we have continued investigations
on the growth pattern of wild (Oryza perennis) and cultivated (0. sativa)
rice strains for six years to look into its role in adaptation. Six wild andthree cultivated strains were tested in an experimental field, automatic
shortday plots (latitudinally controlled by using astrodials), and in gravel
culture. In each condition, a strain was represented by about 20 plants
and four to five plants were sampled several times in the period from floral
initiation to maturity for recording plant weight and length of certain organs.
A part of plant weight data were, however, obtained by the use of an electronic "grass meter". The growth pattern in dry matter weight and elongation patterns of individual organs were represented by logistic equations tocompute certain parameter values, i.e., days from maximum growth toheading (early vs. late vigor) and to the time when growth rate declines tohalf of the maximum (growth persistence), and growth rates 25 days before
heading, at heading, and 10 days after heading; as to the elongation patternsof panicle and first to fourth internodes (from the top), the number ofdays from half-size time (t 1/ 2) to heading was computed for each organ.

RESEARCHES CARRIED OUT IN 1972

131

Variance analysis of the data proved that most of these values were genotypically controlled.
In cultivated as well as perennial wild strains, the elongation of thirdinternode and that of panicle were synchronous (t 1/ 2 being about one week
before heading) and the following elongation of first and second internodes
resulted heading. In contrast, in wild strains of annual habit, the panicle
elongated earlier than the third internode, and the elongation of the latter
resulted in heading. Then, the first and second internodes elongated after
heading to lift up the panicles high. This behavior seems to help the plants
to disperse seeds.
From the growth curves of dry matter weight, wild strains were generally
found to have lower growth rate than cultivated ones. The annual wild
strains showed high vigor around the heading time and declined rapidly,
particularly under shortdays, while the strains of perennial habit continued
growth longer and were characterized by vegetative growth after heading.
It was found further that wild strains generally had higher developmental
plasticity as to organ size than cultivated ones, particularly in the lengthof panicle, third internode and leaf blade. Among the wild strains, perennial
ones were more plastic than annuals. This suggests that the wild plants ofperennial habit can survive varying adverse environments, as they are
resistant to drought and submersion. In contrast, annual plants may beforestallers.

Variations in the Mortality of Perennial Rye Grass andOrchard Grass Varieties
Hiroko MORISHIMA and Hiko-Ichi OKA

An observation of differential survival in different environmental conditions
was initiated in 1970 using three varieties of perennial rye grass (Lolium
perenne) and also three of orchard grass (Dactylis glomerata). As reported
last year, 400 viable seeds per plot (1 m X 1 m) were sown in 1970 September
in pure stand (6 varieties) and species mixture (1: 1,9 varietal combinations).
The 15 populations were tested in an experimental field (4 replications differently managed later) and on road side (2 replications).
In general, many seedlings died in two months after germination. Then,the established plants gradually decreased in number. The infant mortality

132 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

was higher on road side than in field, and higher in orcharad grass than inperennial rye grass. However, the mortality of established plants tended
to be higher in the field than on the road side, and higher in perennial rye
grass than in orchard grass. Variance analysis of the survival data (afterarc-sine transformation of percentages) showed that not only the difference
between the two species but also those among varieties of the same species
were significant. This indicates that mortality under a certain conditionis genetically determined. The heritability estimates for the within-species
differences in survival were about 15 % for infant mortaility and about 8 %after establishment. Negative correlations between infant and adult mortalities were found within perennial rye grass varieties. This seems tosuggest an adaptive role of the death of young plants.
In the summer of second year, most of the experimental plants on roadside were driven out by weeds which were better adapted to the warm climate in Misima. In the experimental field, the effects of cutting and herbicide application were tested. Contrarily to expectation, these environmental stresses tended to increase the survival rate possibly because of
reduction of mutual shading of the plants. This tendency was more pronounced in orchard grass than in perennial rye grass.

RESEARCHES CARRIED OUT IN 1972

133

BOOKS AND PAPERS PUBLISHED IN 1972
BY STAFF MEMBERS( 1) AMANO, E. 1972. Genetic fine structure analysis of mutants induced by ethylmethanesulfonate. Gamma Field Simposia No. 11 (in press).
(2) CHU, Y. E. and H. I. OKA 1972. The distribution and effects of genes causing Fl
weekness in Oryza breviligulata and O. glaberrima. Genetics. 70: 163-173.
( 3) CROW, J. F. and M. KIMURA 1972. The effective number of a population withoverlapping generations: A correction and further discussion. Amer. J. Human
Genetics. 24: 1-10.
( 4) ENDO, T. 1972. Application of the Nadi reaction to rice peroxidase stain. Bot.
Mag. Tokyo. 85: 147-151.
(5) ENOMOTO, M. and H. ISHIWA 1972. A new transducing phage related to P22 ofSalmonella tiphimurium. J. Gen. Virology 14: 157-164.
(6) ENOMOTO, M. 1972. Genetic studies of chlorate-resistant mutants in Salmonella
typhimurium. Jap. J. Genet. 47: 227-235.
( 7 ) FUJISHIMA, T. and H. T. FREDEEN 1972. General formulae for estimating heritability in a population with related parents. Can. J. Genet. & Cytol. 14: 549-557.
(8) FURUICHI, Y. and K. MIURA 1972. The 3'-termini of the genome RNA segmentsof silkworm cytoplasmic polyhedrosis virus. J. Mol. BioI. 64: 619-632.
(9) HAYASHI, S. and K. I. SAKAI 1972. Competition in relation to growth of trees in
Cryptomeria forests. (In Japanese) J. Jap. Forest. Soc. 54: 218-225.
(10) HUNG, A. C. F., H. IMAI, and M. KUBOTA 1972. The chromosomes of nine ant
Soc. species (Hymenoptera: Formicidae) from Taiwan, Republic of China. Ann.
Ent. Soc. America 65: 1023-1025.
(11) IINO, T., H. SUZUKI and S. YAMAGUCHI 1972. Reconstitution of Salmonella flagellaattached to cell bodies. Nature, New BioI. 237: 238-240.
(12) IINUMA, K. and Y. NAKAGOME 1972. Y-chromatin in aged males. Jap. J. Human
Genet. 17: 57-61.
(13) IMAI, H. and M. KUBOTA 1972. Karyological studies of Japanese ants (Hymenoptera, Formicidae) III. Karyotpes of nine species in Ponerinae, Formicinae, andMyrmicinae. Chromosoma (Berl.). 37: 193-200.
(14) KADA, T. 1972. Mechanisms of chemical radiosensitization. (In Japanese) Jap.
J. Cancer Clinics 18: 600--604.
(15) KADA, T. 1972. Screening of chemical mutagens by microbial method. (InJapanese) Iden 26: 46-52.
(16) KADA, T. 1972. Chemical and biochemical characteristics in the recovery ofcellular damage induced by ionizing radiation in the presence of iodine compounds.
(In Japanese) KURRI-Report, TR-I05, 26-30.
(17) KADA, T., K. TUTIKAWA and Y. SADAIE 1972. In vitro and host-mediated "rec-134 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

(18)
(19)
(20)
(21)
(22)

(23)

(24)

(25)
(26)
(27)
(28)
(29)

(30)

(31)

(32)

(33)

(34)

assay" procedures for screening chemical mutagens; and phloxine, a mutagenic
red dye detected. Mutation Res. 16: 165-174.
KATO, H. and T. H. YOSIDA 1972. Banding patterns of Chinese hamster chromosomes revealed by new techniques. Chromosoma 36: 272-280.
KATO, H. and K. MORIWAKII972. Factors involved in the production of banded
structure in mammalian chromosomes. Chromosoma 38: 105-120.
KATO, H. and T. H. YOSIDA 1972. Differential responses of several aneusomic cellclones to ultraviolet irradiation. Exptl. Cell Res. 74: 15-20.
KIMURA, M. and T. OKrA 1972. On the stochastic model for estimation of mutational distance between homologous proteins. J. Molec. Evolution. 2: 87-90.
KIMURA, M. and T. OKrA 1972. Population genetics, molecular biometry, andevolution. Proceedings of the Sixth Berkeley Symposium on Mathematical Stat.
and Probability. 5: 43-68.
KURODA, Y. 1972. Analysis of sorting-out mechanism of animal cells in rotationmediated cell culture. Aspects of Cellular & Molecular Physiology (ed. by K.
Hamaguchi), Univ. Tokyo Press, 237-257.
KURODA, Y. 1972. Interaction between isotypic and heterotypic cells. (In Japanese) Saibogaku-Taikei I. General Aspects & Cell Membrane (ed. by K. OGAWA
et af.), Asakura-Shoten, 341-369.
KURODA, Y.1972. Studies on the sorting-out mechanism of animal cells in rotationmediated cell culture. Proc. IV Internal. Congr. Histochem. Cytochem. 59-60.
MAEDA, K. and K. MORIWAKI 1972. Comparative studies of the transferrins ofrodents. Genetics 71: s 38.
MARUYAMA, T. 1972. Rate of decrease of genetic variability in a two-dimensional
continuous population of finite size. Genetics 70: 639-651.
MARUYAMA, T. 1972. The rate of decay of genetic variability in a geographicallystructured finite population. Mathematical Biosciences. 14: 325-335.
MARUYAMA, T. 1972. The average number and the variance of generation atparticular gene frequency in the course of fixation of a mutant gene in a finite
population. Genet. Res. Camb. 19: 109-114.
MARUYAMA, T. 1972. Some invariant properties of a geographically structured
finite population: distribution of heterozygotes under irreversible mutation. Genet.
Res. Camb. 20: 141-149.
MARUYAMA, T. 1972. Distribution of gene frequencies in a geographically structured
finite population I. Distribution of neutral genes and of genes with small effect.
Ann. Human Genet. Lond. 35: 411-423.
MARUYAMA, T. 1972. Distribution ofgene frequencies in a geographically structured
population II. Distribution of deleterious genes and of lethal genes. Ann. Human
Genet. Lond. 35: 425-432.
MARUYAMA, T. 1972. Distribution of gene frequencies in a geographically structured population III. Distribution of deleterious genes and genetic correlation
between different localities. Ann. Human. Genet. Lond. 36: 99-108.
MARUYAMA, T. 1972. A note on the hypothesis: Protein polymorphism as a phaseof molecular evolution. J. Molecular Evolution. 1: 368-370.

BOOKS AND PAPERS PUBLISHED IN 1972

135(35) MARUYAMA, T. 1972. Population genetics (a review). Jap. J. Appl. Stat. 2: 1-29.
(36) MATSUNAGA, E. 1972. Eugenics: its role in future family planning in Asia. Second
Asian Population Conference. Paper no. POP/APC. 2/IP/32.
(37) MATSUNAGA, E. 1971. Genetics of congenital malformations. (In Japanese)
Proc. 18th General Assembly of Jap. Med. Assoc., 808-812.
(38) MATSUNAGA, E. 1972. Ecology of the earth, with special reference to population
problems. (In Japanese) Suri Kagaku 103: 25-30.
(39) MATSUNAGA, E. 1972. Genetic effects of environmental pollution. (In Japanese)
Pullution-Environmental Sciences (ed. by M. Tachi et at.), Mainichi News Papers(Tokyo), 341-359.
(40) MATSUNAGA, E. 1972. Prenatal diagnosis of hereditary diseases. (In Japanese)
J. Jap. Med. Assoc. 68: 1100-1105.
(41) MATSUNAGA, E. and A. TONOMURA 1972. Parental age and birth weight in translocation Down's syndrome. Ann. Human Genet. 36: 209-219.
(42) MORIWAKI, K. and T. SADAIE 1972. Method of the karyotype observation and thebanding pattern analysis in bone marrow cells and myeloma cells of mouse. (InJapanese) Experimental Methods in Immunology (ed. Immunol. Soc. Japan) 2:
343-349.
(43) MURAKAMI, A. 1972. Acridine orange mutagenesis in silkworm. Jap. J. Genet.
47: 331-334.
(44) MURAKAMI, A. 1972. Effects of ultraviolet light on the early embryos of the silkworm and its biological significance. (In Japanese) Zool. Mag. (Tokyo) 81: 41-48.
(45) MURAKAMI, A. and M. MIKI 1972. Age-dependent changes of radiosensitivity inembryo of Bombyx mori. J. Radiat. Res. 13: 183-192.
(46) NAKAGOME, Y. 1972. Cytogenetic aspects of leukemia. (In Japanese) Shoni Igaku
5: 149-164.
(47) NAKAGOME, Y. and K. IINUMA 1972. Prenatal diagnosis. (In Japanese) Iden 26(4):7-12.
(48) NAKAGOME, Y. and K. IINUMA 1972. Prenatal diagnosis of congenital defects.
(In Japanese) Sanfujinka no Sekai 24: 725-736.
(49) NAKAGOME, Y., K. IINUMA and E. MATSUNAGA 1972. Polyploidy in cultured amniotic fluid cells. Lancet. 2: 387.
(50) NAWA, S. 1972. Molecular genetics in insects-transformation in higher organisms.
(In Japanese) Different Slices of Biochemistry (ed. by S. Suzuki, M. Ishimoto &M. Kageyama), 427-443, Kodansha (Tokyo).
(51) NOGUTI, T. and T. KADA 1972. Semi-in vitro repair of radiation-induced damage intransforming DNA of Bacillus subti/is. J. Mol. BioI. 67: 507-512.
(52) OHTA, T. 1972. Evolutionary rate of cistrons and DNA divergence. J. Molec.
Evolution. 1: 150-157.
(53) OHTA, T. 1972. Fixation probability of a mutant influenced by random fluctuationof selection intensity. Genet. Res. Camb. 19: 33-38.
(54) OHTA, T. 1972. Population size and rate of evolution. J. Molec. Evolution. 1:
305-314.

136

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23(55)

OHTA, T. and M. KIMURA 1972. Fixation time of overdominant alleles influenced
by random fluctuation of selection intensity. Genet. Res. Camb. 20: 1-7.
OSIDMA, C., K. INOUE and S. ISHIWA 1972. Influence of light and dark environmentson the oviposition of Drosophila virilis. (In Japanese) Environ. Control in BioI.
10: 187-191.
OSIDMA, c., K. INOUE and J. K. CHOO 1972. The selection for both photopositiveand photonegative directions in D. virilis. (In Japanese) Environ. Control in BioI.
10: 192-197.
PUTNAM, F. W., A. SHIMIZU, T. SHINODA and C. PAUL 1972. Variation and homology in immunoglobulin heavy chains. Fed. Proc. 31: 193-205.
SHINODA, T., A. SHIMIZU and F. W. PUTNAM 1972. The primary structure of thehuman IgM heavy chain. (In Japanese) Symp. on Protein Struct. 23: 41-44.
SHINODA, T., C. PAUL, A. SHIMIZU & F. W. PUTNAM 1972. Amino acid sequence ofhuman IgM immunoglobulin. Fed. Proc. 31: 445.
SHIRAISHI, Y., H. KURAHASHI and T. H. YOSIDA 1972. Chromosomal aberrations
in cultured human leucocytes induced by cadmium sulfide. Proc. Jap. Acad. 48:
133-137.
SHIRAISHI, Y. and T. H. YOSIDA 1972. Chromosomal alterations in cultured leucocyte cells from Itai itai disease patients. Proc. Jap. Acad. 48: 248-251.
SHIRAISHI, Y. and T. H. YOSIDA 1972. Banding pattern analysis of human chromosomes by use of a urea treatment technique. Chromosoma (BerI.). 37: 75-83.
TAZIMA, Y. 1972. Recovery from genetic radiation damage in higher forms. (InJapanese) Symposia Cell BioI. 23: 19-24.
YAMAZAKI, T. 1972. Detection of single-gene effect by inbreeding. Nature, New
Biology. 240: 53-54.
YAMAZAKI, T. and T. MARUYAMA 1972. Evidence for the neutral hypothesis ofprotein polymorphism. Science 178: 56-58.
YOSIDA, T. H. 1972. Is aging of tumor cells related to the alteration of stemline
karyotype? Proc. Jap. Acad. 48: 268-273.
YOSIDA, T. H. and T. SAGAI 1972. Banding pattern analysis of polymorphic
karyotypes in the black rat by a new differential staining technique. Chromosoma(BerI.) 37: 387-394.
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: 451454.

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BOOK AND PAPERS PUBLISHED IN 1972

ABSTRACTS OF DIARY FOR 1972
March
May
June
July
September
OctoberNovember
December

17
31
16
29
2
7
22
27
14
1
15

195th Meeting of Misima Geneticists' Club
196th Meeting of Misima Geneticists' Club
99th Biological Symposium
197th Meeting of Misima Geneticists' Club
198th Meeting of Misima Geneticists' Club
199th Meeting of Misima Geneticists' Club
200th Meeting of Misima Geneticists' Club
201st Meeting of Misima Genefcists' ClublOOth Biological Symposium
202nd Meeting of Misima Geneticists' Club
203rd Meeting of Misima Geneticists' Club137

138 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

FOREIGN VISITORS IN 1972

16 DOl, Roy H., University of California, U.S.A.
12 SCHEIN, M. W., West Virginia University, U.S.A.
22 de SERRES, F. J., NIEHS-NIH, U.S.A.
FREESE, E., N.I.H., U.S.A.
NICHOLS, W. W., Institue for Med. Research, Camden,
U.S.A.
October
3 BOUNHIL, J. J., Biological Laboratory 0' the University,
Bordeaux, France
December 19 HERNANDEZ-ARAGON, L., Centro de Investigacions Agricolas de Sinaloa, Mexico

May
July
August,•ACKNOWLEDGEMENT139

ACKNOWLEDGEMENT

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

..

.

-

140

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 23

AUTHOR INDEXAMANO, E

84

ANDERSON, W. W • . . . . . . . . . . . . . . . . , .122

79, 12496, 98,115,117,120
CROW, J. F
87
ENDo, T24, 25
FUJII, T • . . . . . . . . . . . . . . . . . . . . . 76, 78, 79
FUKASE, Y.
.
62
FURUICHI, Y
12, 13
IINO, T
15
IINUMA, K
109, 111
IMAI, H. T
46, 50, 53, 54
IWASAKI, T
67
IYAMA, S
123
KADA, T72, 73, 74, 75, 80, 81
KATO, H
18,42,43,46
KAWAHARA, T. .
126, 127
KIMURA, M
87,89,103
KOSHINAGA, J.
106, 107
KURODA, Y
29, 30,31, 33, 34, 36
KUWAHATA, T. .
22, 23MARUYAMA, T
91,93,94
MATSUI, I.
111
MATSUNAGA, E. .. 104,105,106,107,109,112
MINATO, K
37
MIURA, K
11,12,13
MORISHIMA, H. .
130, 131MORIWAKI, K
17, 18,22,23,42,43,56
MURAKAMI, A . • . . . . . . . . 54, 66, 67, 69, 70
BAGCHI, S

CHOO, J. K

58
109, 110, 111
NoaUTI, T. . . . . . . . . . . . . . . . . . . . • . 72, 73
OHNISHI, S
100
OHNUMA, A. . . . . . . . . . . . . . . . . . . • . . . • . 64
OHTA, T87,88,89,102,103
OKA, H. I.
128, 129, 130, 131
ONIMARU, K. .
60, 63
OHIMA, C.
96,98,100,115,117,120
PAl, C
25
RAI, U . . . . . . . . . . . . . . . . . . . . . . • . . • . . . 13
SADAIE, T. . . . . . . . . . . . . . . . . . . . • . . 18, 56
SADAIE, Y
74,75
SAGAI, T.42, 43, 45, 46
SAKAI, K. I.
123
SHAMA RAO, H. K
80,81,82
SHIMOTOHNO, K. . . . . . . . . . . . . • . . . • . . . 12
SHiNODA, T
26, 27, 106, 107.108
SUGIURA, M . . . . . . . . . . . . . . . . . . . . . • • . . 11
SUGIYAMA, T . . . . . . . . . . . . . • . . . • . . . • • . 38
SUZUKI, H
15
TAZIMA, Y
60, 62, 63, 64
TSUCHlYA, K
18, 42, 43
TSUZUKIDA, Y . . . . . . . . . . . . . . . . • . . . . . . 27
WATANABE, K. .
11
WATANABE, T. K
96, 98,122
YAMAZAKI, T
89, 91, 93, 94
YOSIDA, T. H
38, 39,42,43,45,46
NAGAMI, S. •NAKAGOME, Y

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