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NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 33

1982

Publiehed bll
THE NATIONAL INSTITUTE OF GENETICS.Hieima, SizuoktJ-ken, JtJJHUI1983

Annual Reportof theNational Institute of Genetics
No. 33, 1982

Published by

The National Institute of Genetics, Japan1983

Photo by Y. Nakagome

CONTENTS
General statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council........... .. ..
.. .. .. . .. . .. .. .. .. . . .
. . ..
Association for propagation of the knowledge of genetics
Projects of research for 1982
Researches carried out in 1982 .. . . .. . . . . . .. .. . . . . . . . . . . . . . . . . . . ..
I.

Molecular Genetics
Genomic information of murine papovavirus K genome. SOEDA,
E., JIKUYA, H. and TAKEMOTO, K. K.
Structure of murine papovavirus K genome: heterogeneity of
nucleotide sequence in the region containing the origin of
DNA replication. SOEDA, E., JIKUYA, H. and TAKEMOTO,

K. K
Automatic DNA sequencer: computer-programmed microchemical
manipulator for Maxam-Gilbert sequencing method. WADA,
A., YAMAMOTO, M. and SOEDA, E.
Cloning of a fibroin gene from wild mulberry silkworm Bombyx
mandarina. KUSUDA, J., ONIMARU, K., NINAKI, 0., SUZUKI,
Y. and TAZIMA, Y.

II.

1

3
6
7
10
12

12

13

14

15

Microbial Genetics
The 245 base-pair oriC sequence of the E. coli chromosome directs
bidirectional replication at an adjacent region. TABATA, S.,
OKA, A., SUGIMOTO, K., TAKANAMI, M., YASUDA, S. and
HIROTA, Y. ..
..
.. .. .
.. .. .. 17
Suppressor genes of a dnaA temperature sensitive mutation in
Escherichia coli. TAKEDA, Y. and HIROTA, Y.
17
The fine architecture and function of the gene coding for PBP-3 ofEscherichia coli. MARUYAMA, 1. N., YAMAMOTO, A., MARUYAMA, T. and HIROTA, Y.
18
N-acetylmuramoyl-L-alanine amidase of Escherichia coli K12:
possible physiological functions. PARQUET, c., FLOURET, B.,
LEDUC, M., HIROTA, Y. and VAN HEIJENOORT, J.
18

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Isolation and characterization of an Escherichia coli mutant lacking
tRNA-guanine transglycosylase function and biosynthesis of
queuosine in tRNA. NOGUCHI, S., NISHIMURA, Y., HIROTA,
Y. and NISHIMURA, S.
19
Synthesis of flagellin and hook subunit protein in flagellar mutantsof Escherichia coli K-12. KOMEDA, Y.
20
Localization of E. coli flagellar gene products using gene fusion
20
technique. KOMEDA, Y.

III.

Biochemical Genetics and Immunogenetics
Urethan-induced lung tumor development in (Susceptible x Resistant)F 1 hybrids ofH-2 congenic strains of mice. MIYASHITA,
N. and MORIWAKI, K.
22
Production of monoclonal antibodies against H-2 complex in
BIO. MOL-SGR mouse strain. SAGAI, T., SHIROISHI, T. and
MORIWAKI, K. .. .. . ..
..
.. .
23
Effect of major histocompatibility gene complex (MHC) on thegrowth of embryo-derived teratoma. TAYA, C. and MORIWAKI, K
24Southern blot analysis of H-2K genes of Japanese wild mice.
SUZUKI, H., SHIROISHI, T., SAGAI, T., GACHELIN, G.,
KANEHISA, T. and MORIWAKI, K.
25Southern blot analysis of ribosomal DNA in Mus musculus.
SUZUKI, H., KOMINAMI, R., MURAMATSU, M., KANEHIsA, T.
and MORIWAKI, K.
26
Serological survey of Thy-l and Lyt-l, 2, 3 antigens in wild mice.
KURIHARA, Y. and MORIWAKI, K.
27

IV.

Developmental Genetics and Somatic Cell Genetics
In vitro formation of adult structures from cells of lethal embryosof Drosophila melanogaster. KURODA, Y.
29
Localization of lectins and their changes in development of chickembryos. KURODA, Y., MATSUTANI, E. and YAMAGATA, T. . 30
Mutagenic activity of environmental aromatic compounds in cultured Chinese hamster cells. KURODA, Y. and ASAKURA, M... 31
Effects of EGF and FGF on growth pattern of cultured human
diploid cells in clonal culture. KURODA, Y.
32

iii

CONTENTS

Hypertrophic growth of neuroblasts in a Notch lethal, Df(l)Notch
8 of Drosophila melanogaster. MINATO, K.
Morphogenetic potentials in chimeric hydra strains. SUGIYAMA,
T., TAKANO, J. and NISHIMIYA, C.
Position dependent nematocyte differentiation in hydra. FUJISAWA,.0••••••

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33
33

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34
A single recessive gene(ter) causing germ cell deficiency and an
extremely high incidence of testicular teratomas in 129jSv-ter
mice. NOGUCHI, To ..
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v. Cytogenetics
Sequentiality of chromosome evolution. YOSIDA, T. H. ..
Parallelism of karyotype evolution. YOSIDA, T. H
Cytogenetical study on a mammary cancer of the domestic cat witha note on an increase of cells with a normal karyotype after
deep freezing. YOSIDA, T. H., and KAWAHARA, K ... 0......
Remarkable difference on frequency of single and twin sister
chromatid exchanges in endoreduplicated Bloom syndrome
cells. SHIRAISHI, Y. and YOSIDA, To Ho
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of
BrdU
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Welsh
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and
D-6
Chinesehamster
Effectcells by treatment with 5-fluorodeoxyuridine. SUZUKI, H.,
SEGAWA, M., YOSIDA, T. Ho and KONDO, K ....
Multiple sex chromosome mechanism (XX-Y) found in two
dragonet fishes. MUROFUSHI, Mo, NISHIKAWA, S. and
YOSIDA, T. H ..
Effect of H-2 complex on the frequency of urethan-induced
chromosome aberrations in the mouse bone marrow cells.
MORIWAKI, K., MIYASHITA, N. and NITo, S.
Implications of the genetic divergence between European wild
mice with Robertsonian translocations from the view pointsof mitochondrial DNA. MORIWAKI, K., YONEKAWA, Ho,
GOTOH, 0., MINEZAWA, Mo, WINKING, H. and GROPP, A. ...
The effects of H-2 complex to the mutagen-induced chromosome
aberrations and micronuclei. NITo, S., MIYASHITA, N. and
MORIWAKI, K.
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37
38

39

40

41

42

43

43

45

iv

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

VI.

Mutation and Mutagenesis in Animals
Genetical behaviour in the silkworm (Bombyx mori L.) carrying
X-autosome translocation. MURAKAMI, A.
The induction of gynogenesis and androgenesis in the silkworm andthe mechanism of their induction. TAMAZAWA, S. and
MURAKAMI, A.
Cytological analysis of mutations produced by super-cooling ofsilkworm eggs at an early stage of development. I. Polyploids. TAMAZAWA, S. and MURAKAMI, A.
Cytological analysis of mutations produced by super-cooling ofsilkworm eggs at an early stage of development. II. Deletionof chromosomes. TAMAZAWA, S. and MURAKAMI, A.
Locus differential sensitivity for the induction of the specific-locus
mutations after irradiation of primordial germ-cells with Xrays in the silkworm. MURAKAMI, A. . . . . . . . . . . . . . . . . . . . ..
A comparison of mutagenic activity of ethylmethanesulphonate(EMS) and methylmethanesulphonate (MMS) in oocytes ofthe silkworm. MURAKAMI, A. . . . . . . . . . . . . . . . . . . . . . . . . . ..

VII.

47

48

50

51

52

53

Radiation Genetics and Chemical Mutagenesis in Microorganism
and Plants
Analysis of DNA damage induced by p-ray of tritiated water.
KADA, T., SADAIE, Y. and INOUE, T.
56
Development of an In vivo germinal mutation detection system inmice. TEZUKA, H., INOUE, T. and KADA, T. . . . . . . . . . . . .. 57
Decrease in r-ray-induced mutations by PLD repair inhibition in
Chinese hamster V79 cells. YOKOIYAMA, A. and KADA, T. .. 58
Adsorption of pyrolysate mutagens by vegetable fibres. KADA,
T., KATO, M. and KIRIYAMA, S.
58
Antimutagens. KADA, T. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. 59
DNA repair in competent cells of Bacillus subtilis. MITA, I.,
SADAIE, Y. and KADA, T.
60
Effects of TPA on the mutagenicity of caffeine and gamma-rays inthe soybean test system. FUJII, T., SHIZAKI, M., FUJIKI, H.
and SUGIMURA, T. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

CONTENTS

VIII.

Population Genetics

The neutral theory as a basis for understanding the mechanism of
evolution and variation at the molecular level. KIMURA, M...
Linkage disequilibrium due to random genetic drift in finite subdivided populations. OHTA, T.
Further study on the genetic correlation between members of amultigene family. OHTA, T.
Allelic and nonallelic homology of a supergene family. OHTA, T...
Linkage disequilibrium, genetic distance and evolutionary distanceunder a general model of linked genes or a part of the genome.
TAKAHATA, N.
Sexual recombination under the joint effects of mutation, selection,and random sampling drift. TAKAHATA, N. .... . . . .. .. . . ..
The disappearance of duplicate gene expression. TAKAHATA,
N
Estimation of evolutionary distance under a mathematical model ofextranuclear DNA molecule. TAKAHATA, N.
A condition for group selection to prevail over counteracting
individual selection. AOKI, K.
Group selection for a polygenic behavioral trait: a differential
proliferation model. CROW, J. F. and AOKI, K.
Additive polygenic formulation of Hamilton's model of kin selection. AOKI, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Polygenic altruism and extinction group selection. AOKI, K. ....

IX.

v

63
64
65
66

66
67
68
69
69
70
71
71

Human Genetics and Human Cytogenetics
Hereditary retinoblastoma: lack of maternal effect. MATSUNAGA,
E
Almost synchronous appearance of bilateral retinoblastomas.
MATSUNAGA, E.
..
Incidence and prevalence of genetic disease (excluding chromosomal
aberrations) in human populations. MATSUNAGA, E.
Distribution of break points in human structural rearrangements.
NAKAGOME, Y., MATSUBARA, T. and FUJITA, H.
High resolution studies in patients with aniridia-Wilms' tumor
association, Wilms' tumor, retinoblastoma and Recklinghausen syndrome. NAKAGOME, Y., SAKURAI, M., ISE, Y.,

72
73
74
75

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

NAKAJO, T., MATSUBARA, T., OKAMOTO, E., SAWAGUCHI, S.,
TSUCHIDA, Y., NAKAHORI, Y., MATSUNAGA, E. and SAITO, S.
75
The loss of kinetochores from chromosomes of aged women (asupplementary note). NAKAGOME, Y., ABE, T., MISAWA, S.,
TAKESHITA, T. and IINUMA, K.
A malformed girl with duplication of major part of 9q. NAKAHORI,
Y., NAKAGOME, Y. and YOKOCHI, T
.

77
77

X. Behaviour Genetics
Selection for the compound learning ability of mice.

FUJISHIMA,

T
The behavioral changes in mice continuously reared under noise
conditions. FUJISHIMA, T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Xl.

79
79

Ecological Genetics
Variation in competitive ability among cohabiting perennial andannual types of Oryza perennis. SANO, Y. and MORIsHIMA, H.
81
Variation in weed tolerance among wild and cultivated rice strains.
MORIsHIMA, H. .
.
Neighbor effects between two co-occurring rice species Oryza
sativa L. and O. glaberrima Steud. SANO, Y., SANO, R. and
MORISHIMA, H. .
.
Association of chromosome and enzyme polymorphisms in naturaland cage populations of Drosophila melanogaster. INOUE, Y.
and WATANABE, T. K
.

XII.

82

83

84

Applied Genetics
Change in herbicide tolerance of barnyard grass in a paddy field.
IYAMA, S.
85
Simulation studies on the factors affecting the pedigree breeding
method. YAP, T. C. and IYAMA, S.
85
Studies on the breeding structure of tree species in the tropical rain
forest. Family clumps and intrapopulation differentiation.
SAKAI, K.-I., ENDO, T., IYAMA, S., MIYAZAKI, Y., HAYASHI, S.,
SHIMAMOTO, Y., GADRINAB, L. U. and JUNIARTI, U.
87

CONTENTS

vii

Effective population number of four hatchery populations of fish.
!YAMA, S. and TANIGUCHI, N, . .. .. .. ..
. .. ..
88
Variation in nitrogen fixing activity among wild and cultivated rice89
strains. OHTA, K., SANO, Y., FUJII, T. and !YAMA, S.
A sterility gene between Oryza sativa and O. glaberrima: its association with photoperiod-sensitivity. SANO, Y. . . . . . . . . . . . . . . . 90
Wx protein in rice. SANO, Y. and MUROFUSHI, A.
91
Diversity of rice cultivars collected in the Asian hilly areas in viewof !ndica-Japonica differentiation. SANO, R. and MORISHIMA,
H
92
94
Books and papers published in 1982 by staff members.
101
Abstracts of diary for 1982
Foreign visitors in 1982
102
Author index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 104

GENERAL STATEMENT
A prospect for switching over the administration category of the institutefrom the present system to that for joint use by the universities has turnedbright. The Ministry of Education, Science and Culture has organized aspecial committee to investigate the reorganization plan of this institute.
The institute was founded in 1949 as one of the national institutes undera jurisdiction of the Ministry. The original idea for characterizing theinstitute as a national institute was to have it independent from the universities. This resulted in much success in selecting qualified researchers
from various universities, but caused several inconveniences to the personal
activities of researchers because of tight service regulations. Even undersuch circumstances innumerable ingenious contributions have been achievedin this institute.
In the meantime, institutes of a new category have been established by thesame Ministry, so as to be suitable especially for the promotion of research.
Those institutes are independent from a specific university but open for
university staffs to carry out cooperative works and are classified into "theinstitute for joint use by the universities." The institute of such category
has several merits, but has to assume, on the other hand, a responsibility
for planning and conducting cooperative researches, in compliance withthe proposal of university researchers. This may add some trouble to thestaff members, but I believe that the new system may provide them with
far more convenience to their research and increased chances to exchange
the views with several experts. Another merit may be the adoption of
guest professor's system. Under the new system a guest professor will beavailable for each group of departments. This new system will be especially
useful for the refreshing the idea and introducing new methodology. Standing at a very crucial turning point of the institute, it is very important that
we must bear in mind not to stray from the right direction.
Regarding the personnel change in the Department of Molecular Genetics, Dr. M. Sugiura, head of the 2nd laboratory, left his position early
September, 1982 and joined with Faculty of Science, Nagoya University,and Dr. K. Miura, head of the Department, will be appointed a professorof the Faculty of Technology of the University of Tokyo next April. Further-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

more, two other staff members of the Department will be transferred to their
new posts. After 13 years since the birth of the Department it will be reorganized comprehensively.
Dr. H. Kihara, who had devoted himself to the development of the institute as the second director for 13 years, attained his 90th birthday on
Octover 21st and still remains hale and hearty. We planned to construct
his relief, congratulating his long life and admiring his great achievementsin the field of genetics. It was engraved by Mr. Shimoyama, a reputed
sculptor, and has been installed on the wall of the landing to the second
floor in the main building. It reminds us of him and encourages us in
the morning and evening.

STAFF
Director

TAZIMA, Yataro, D. Ag.
Members

1. Department of Morphological Genetics
KURODA, Yukiaki, D. Sc., Head of the Department
The 1st Laboratory
MURAKAMI, Akio, D. Ag., D. Sc., Head of the Laboratory
The 2nd Laboratory
KURODA, Yukiaki, D. Sc., Head of the Laboratory
MINATO, Kiyoshi, M. Sc.
2.

Department of Cytogenetics
YOSIDA, Tosihide H., D. Sc., Head of the Department
The 1st Laboratory
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
YAMAMOTO, Masatoshi, Ph. D.
The 2nd Laboratory
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
IMAI, HIROTAMI, D. Sc.

3. Department of Physiological Genetics
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
WATANABE, Takao, K., D. Sc., Head of the Laboratory
The 2nd Laboratory
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Laboratory
4.

Department of Biochemical Genetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory

4

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

YAMADA, Masa-Aki, M. Sc.
The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
ENDO, Toru, D. Ag.
The 3rd Laboratory
SUGIYAMA, Tsutomu, Ph. D., Head of the Laboratory
FUJISAWA, Toshitaka, Ph. D.

5.

Department of Applied Genetics
TAZIMA, Yataro, D. Ag., Head of the Department
The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
FUJISHIMA, Tohru, D. Ag.
The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
The 3rd Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag., Head of the Laboratory

6.

Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department
The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
INOUE, Tadashi, D. Ag.
The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory (the first half year)
SADAIE, Yoshito, D. Sc., Head of the Laboratory (the second halfyear)
TEZUKA, Hideo

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
HORAI, Satoshi, D. Med.
The 2nd Laboratory

STAFF

NAKAGOME, Yasuo, D. Med., Head of the Laboratory

8. Department of Microbial Genetics
HIROTA, Yukinori, D. Sc., Head of the Department
The 1st Laboratory
HIROTA, Yukinori, D. Sc., Head of the Laboratory
NISHIMURA, Yukinobu, D. Sc.
The 2nd Laboratory
YASUDA, Seiichi, D. Sc., Head of the Laboratory
YAMADA, Masao, D. Sc.
9.

Department of Population Genetics
KIMURA, Motoo, Ph. D., D. Sc., Head of the Department
The 1st Laboratory
OHTA, Tomoko, Ph. D., D. Sc., Head of the Laboratory
The 2nd Laboratory
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
TAKAHATA, Naoyuki, D. Sc.
AOKI, Kenichi, Ph. D.

10. Department of Molecular Genetics
MIURA, Kin-ichiro, D. Sc., Head of the Department
The 1st Laboratory
MIURA, Kin-ichiro, D. Sc., Head of the Laboratory
SOEDA, Eiichi, D. Ag.
SHIMOTOHNO, Kunitada, D. Pha.
The 2nd Laboratory
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
SHINOZAKI, Kazuo, D. Sc.
11.

Genetic Stocks Center
YOSIDA, Tosihide H., D. Sc., Head of the Center
Animal Section
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
NOGUCHI, Takehiko, D. Sc.
INOUE, Yutaka, D. Sc.
KUSUDA, Jun, D. Ag.

5

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Plant Section
FUJII, Taro, D. Ag., Head of the Laboratory
SANa, Yoshio, D. Ag.
Microbial Section
SUGIURA, Masahiro, D. Sc., Head of the Laboratory (the first half
year)
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory (the second
half year)
KOMEDA, Yoshibumi, D. Sc.
NISHIMURA, Akiko

12. Experimental Farm
FUJII, Taro, D. Ag., Head of the Farm
MIYAZAWA, Akira
13. Department of Administration
KITAHARA, Kunio, Head of the Department
lORI, Toshiteru, Chief of the General Affairs Section
OOIDE, Yukio, 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
SAKAI, Kan-ichi, D. Ag.
MORIWAKI, Daigoro, D. Sc., Emeritus Professor of Tokyo Metropolitan
University
OSHIMA, Chozo, D. Sc.
aKA, Hiko-Ichi, D. Ag.

COUNCIL
FUJII, Takashi, Chairman, Emeritus Professor of University of TokyoINOUYE, Eiji, Director of Research Institute of Handicap to Growth
IINO, Tetsuo, Professor of University of Tokyo
KONDo, Norio, Professor of Tokyo University of Agriculture

COUNCIL7MISONOO, Keisuke, Member of Atomic Energy Commission
MORIWAKI, Daigoro, Emeritus Professor of Tokyo Metropolitan University
MOROHOSHI, Seijiro, President of Tokyo University of Agriculture andTechnology
NAGAKURA, Saburo, Director of Institute of Molecular Sciences
OOSAWA, Fumio, Professor of Osaka University
SASA, Manabu, President of Toyama Medical and Pharmaceutical University
SHINOZAKI, Nobuo, former Director of Institute of Population Problems
TAKAHASHI, Man-emon, President of Musashi Women's Junior College
UMEZAWA, Hamao, Emeritus Professor of University of Tokyo
YAMAMURA, Yuichi, President of Osaka University
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Emeritus Professor of Tokyo Metropolitan
University
MATSUNAGA, Ei, Managing Director, Head of the Human Genetics Department
YOSIDA, Tosihide H., Managing Director, Head of the Cytogenetics Department
SINOTO, Yosito, Manager
WADA, Bungo, Manager, Emeritus Professor of University of TokyoTAZIMA, Yataro, Manager, Director of the National Institute of Genetics
OSHIMA, Chozo, Manager

PROJECTS OF RESEARCH FOR 1982
Department of Morphological Genetics

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)
Cytogenetics in the silkworm (MURAKAMI)
Studies on recombination in the silkworm (MURAKAMI)
Genetics of the silkworm (MURAKAMI, Fukase and OHNUMA)
Radiation and chemical mutagenesis in the silkworm (TAZIMA and
MURAKAMI)
Department of Cytogenetics

Studies on chromosome evolution and species differentiation in rodents andother small mammals (YOSIDA)
Chromosome study on experimental tumors (YOSIDA)
Experimental breeding and genetics of mice, rats and other wild rodents
YOSIDA and MORIWAKI)
Genetic study on the subspecies differentiation of mouse (MORIWAKI)
Immunogenetical study on the MHC functions in mice (MORIWAKI)
Cytogenetical study of ants (IMAI)
Cytogenetical studies on Drosophila (YAMAMOTO)
Cytogenetical study of fishes (YOSIDA)
Department of Physiological Genetics

Behavior genetics of Drosophila (WATANABE and OSHIMA)
Evolutionary and ecological genetics of Drosophila (WATANABE)
Population genetics of Drosophila (WATANABE)
Theory of population genetics and speciation (MARUYAMA)PROJECTS OF RESEARCH FOR 1982

9

Department of Biochemical Genetics

Studies on transformation in higher organisms (NAWA and YAMADA)
Genetical and biochemical studies of pteridine metabolisms in insects(NAWA)
Analysis of gene action on cell differentiation in higher organisms (NAWA
and YAMADA)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Genetical and biochemical studies of human serum proteins (OGAWA)
Genetical and biochemical studies on Japanese middle size dog (OGAWA)
Genetics of isozymes in plants (ENDO)
Genetic analysis of developmental mechanisms in hydra (SUGIYAMA andFUJISAWA)
Department of Applied Genetics

Behavioral genetic studies in animals (FUJISHIMA)
Theoretical studies on breeding techniques (IYAMA)
Genetic studies of trees in natural forest (IYAMA)
Evolutionary studies on wild and cultivated rice species (MORISHIMA andSANO)
Ecological genetic studies in weed species (MORISHIMA)
Genetic effects of environmental pollution on plant population (IYAMA andMORISHIMA)
Department of Induced Mutation

Molecular mechanisms of radiation- and chemical-induced mutations(KADA, SADAIE, INOUE and TEZUKA)
Environmental mutagens, desmutagens and antimutagens (KADA)
Radiation genetics in mice (TuTIKAwA)
Biochemical factors involved in cellular repair of genetic damage and induced
mutagenesis (INOUE and KADA)
Genetics of Bacillus subtilis (SADAIE and KADA)
Molecular mechanisms of unicellular differentiation III Bacillus subtilis(SADAIE)

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33
Department of Human Genetics

Genetic and cytogenetic studies on retinoblastoma and Wilms' tumor
MATSUNAGA and NAKAGOME)
Clinical cytogenetic studies of congenital abnormalities in man (NAKAGOME)
Molecular cytogenetic studies of human chromosomes (NAKAGOME)
Department of Microbial Genetics

Genetic regulatory mechanisms of DNA replication in E. coli (YASUDA)
Genetic regulatory mechanism of cellular division in E. coli (HIROTA,NISHIMURA and YAMADA)
Molecular genetics on DNA replication (YASUDA and HIROTA)
Genetic studies on bacterial cell envelope (HIROTA and NISHIMURA)
Synthetic bacterial plasmid (YASUDA and NISHIMURA)
DNA replication origin of E. coli (YASUDA, YAMADA and HIROTA)
Department of Population Genetics

Theoretical studies of population genetics (KIMURA, OHTA, TAKAHATA andAOKI)Studies on molecular evolution from the standpoint of population genetics(KIMURA, TAKAHATA and OHTA)
Theoretical studies on the evolution of multigene family (OHTA)
Theoretical studies on the evolution of altruism (AOKI)
Department of Molecular Genetics

Studies on the structure of genome RNA of viruses (SHIMOTOHNO andMIURA)
Studies on the primary structure of DNA (SOEDA)
Studies on structure and function of messenger RNA (MIURA andSHIMOTOHNO)
Cloning of eukaryotic genes and their structural analysis (SUGIURA andSHINOZAKI)
Studies on molecular evolution of chloroplasts (SUGIURA and SHINOZAKI)Transformation of human lung and rat fibroblast cells with viral promoter
DNA (SOEDA)PROJECTS OF RESEARCH FOR 1982

11

Biogenesis of papovaviruses (SOEDA)
Genetic Stocks Center

Studies and conservation of germplasm resources in rice and wheat species(FuJII and SANO)
Specificity of mutagen tolerance in higher plants (FUJII)
Exploitation of genetic ability of nitrogen fixation in Graminae (FuJII, SANO
and IYAMA)
Studies on genetic differentiation in rice (SANO)
Cytogenetic studies of Norway rats and establishment of the chromosomal
mutant stocks (YOSIDA)
Establishment of new experimental animals from wild rodents (YOSIDA andMORIWAKI)
Developmental genetic studies on mouse teratomas (NOGUCHI)
Studies on preservation of rodent embryos by deep freezing (NOGUCHI)
Studies on chromosomal polymorphism in Drosophila (INOUE)
Analysis of fibroin genes of silkworm and its relatives (KUSUDA)
Genetical study of flagellar formation in Escherichia coli K-12 (KOMEDA)
Basic studies on the gene purification and the construction of DNA banks(SUGIURA)
Genetical study on photosynthesis in blue-green algae (SUGIURA)
Coordination of flagellar formation and cell division in E. coli (NISHIMURA,
KOMEDA and HIROTA)Electron microscopic study of cell division in E. coli (NISHIMURA)RESEARCHES CARRIED OUT IN 1982

I.

MOLECULAR GENETICS

Genomic Information of Murine Papovavirus K Genome
Eiichi SOEDA, Hiroyuki JIKUYA and Kenneth K. TAKEMOTO l )Murine papovavirus K (KV) was first isolated by Kilham in 1953 andhas been classified as a member of papovavirus family. Inoculation ofthis virus to new born mice induces a fetal pneumonia, one of the wellcharacterized pathogenesis, serological studies have indicated that KV
was ubiquitous to a mouse population. KV is different from polyomavirus(PyV) and other papovaviruses in respect to its ability to induce tumour inanimals, but the cultured cells from the mouse lung infected with KV exhibited transformed phenotypes as well. One the other hand, KV sharespermissive host with PyV which can code for middle T antigen. PyV
middle T antigen is thought to be implicated in cellular transformation andtumorigenesis. Gene comparison of PyV with other papovaviruses including simian virus 40 (SV40), human BKV and leV has revealed that PyV
middle T antigen is unique among other papovaviruses, arising a question
how other papovaviruses induce tumours without viral coded middle T
antigen. We are interested in the different pattern of tumorigenicity between PyV and KV and determined the nucleotide sequence of KV DNA
in order to see whether KV genome code for the relevant of PyV middle
T antigen or not.
A total nucleotide sequence of KV DNA has. been deduced by the methodof Maxam and Gilbert. It consists of 4994 uncleotides in length. Examination of the nucleotide sequence indicated that the genomic organization of KV genome was very similar to that of PyV genome except that KV
genome could not code for the relevant of Py middle T antigen. These evidences suggest that papovaviruses have diverged from a common origin
and enforce further that PyV middle T antigen is important for inductionof tumour formation in animals.
1)

Lab. of Viral Diseases, NIAID. NIH, Bethesda, Maryland 20205, USA.

RESEARCHES CARRIED OUT IN 1982

13

Structure of Murine Papovavirus K Genome:
Heterogeneity of Nucleotide Sequence in the Region
Containing the Origin of DNA Replication
Eiichi SOEDA, Hiroyuki JIKUYA and Kenneth K. TAKEMOT01 )Murine papovavirus K (KV) DNA extracted from a viral stock wascloned molecularly in pBR322. Four clones were obtained, of which inserts were different in sizes to each other. Restriction enzyme mapping
indicated that the variation in sizes resides in the Eco RljPst I fragments.
So, these fragments were submitted to sequence DNA and compared withthose of other papovaviruses which were sequenced.
Nucleotide sequences of these fragments were determined by the methodof Maxam and Gilbert. When compared with those of papovaviruses,
GC rich sequences flanked by consecutive AT sequences were found incommon among these fragments and highly homologous to the sequencesof papovaviruses spanning the origin of DNA replication. Downstreamof these regions, one long reading frame starting with ATG initiation coding
remained open to the ends. The predicted amino acid sequences are identicalto that from the N-terminus of VP2j3 of polyomavirus. These evidencesindicated that these fragments span predominantly regulatory region containing the origin of DNA replication and predicted that the early coding
region might be present at the opposite site of the VP-2j-3 coding region.
Heterogeneity of the nucleotide sequences was predominantly present
between the origin of DNA replication and the early coding region which
may be characterized by the presence of tandem repeat sequences.

1)

Lab. of Viral Diseases, NIAID, NIH, Bethesda, Maryland 20205, USA.

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Automatic DNA Sequencer: Computer Programmed
Microchemical Manipulator for Maxam-Gilbert
Sequencing Method
Akiyoshi WADA11, Masahiro YAMAMOT0 2) and Eiichi SOEDA

Recent advances in microprocessors make it possible to provide an automated tool which is able to operate a fairly long series of micro-chemical
Vertically moving cyiinder\.

I

It't.••. I•.•. .• .I• .• .jl&J>•.•>.• •• .•.•

:1

Stock solution

Cap,jInjection nozzle

I
centrifuge rotor

~

Dr er

I ~---_;;]J:~-_--: *~:~),w;"g~ \"""00"

Reaction vessel\

oog'o ''"'"'~po",","bethC:::q:::±==ti==:::::::::::aVertically moving cylinder
IfrmQJ;.LaJtl!Jur<re"-.c.nnJ:mIler..'.

Fig. 1. Block diagram of the system. The system consists of a centrifuge
rotor (radius: 15 cm) which holds reaction vessels. The last two tabes support thesolution manipulation units.
1)
Department of Physics, Faculyt of Science, The University of Tokyo, Hongo, Tokyo,
Japan.
2)
Daini Seikosha Co. Ltd., Takenoshita, Oyama-cho, Sunto-gun, Shizuoka Pref.
410--13, Japan.

RESEARCHES CARRIED OUT IN 1982

15

manipulations for the analysis or the synthesis of biological materials.
Routin and tedious laboratory operations can be replcaed by a button operation for the input of computer commands.
This work is to aim the development of a micro-chemical robot which
carries out a series of chemical reaction steps in DNA sequence analysis
using Maxam and Gilbert's scheme. The performance of the apparatus
includes the quantitative addition of solution, mixing, centrifugation,
draining, drying under vacuum, and cooling and heating of sub-milliliter
solution. Within a two-hour period of operation of this apparatus, one of
the four types of solutions for G, A+C, C+T, or C analysis for a DNA
sample (restriction fragment), is prepared and is ready for subsequent electrophoretic analysis.
In addition to the DNA sequencing, the instrument is applicable treatments for analysis and synthesis of biochemical materials; the steps required
for the reaction sequence are performed one after another, according to acomputer programme that is designed just prior to the operation.
It is proven that the chemical robot operated by a computer commandis capable of speedily accomplishing the fragmentation reaction for DNA
sequencing with accuracy similar to that of a well trained technician.
Cloning of a Fibroin Gene from Wild Mulberry
Silkworm Bomhyx mandarina
Jun KUSUDA, Kimiji ONIMARU, Osamu NINAKI,t Yoshiaki SUZUKI*
and Yataro TAZIMA

Morphological and chromosomal studies showed that Bambyx mandarina
is one of the possible ancestor of domesticated silkworm Bambyx mario
In order to elucidate the phylogenie relationship between two silkworm
species, we attempt to compare the structure of the fibroin gene of B.
mandarina to that of B. mario
The fibroin gene of B. mari was already cloned into E. cali plasmidby Suzuki and the sequence around the 5' end of the gene was determined.
At first, we have isolated the fibroin gene of B. mandarina by cloning techniques using cloned DNA (pFb29) contained the fibroin gene of B. marias a hybridization probe.
* National Institute of Basic Biology.
t Sericultural Experimental Station.

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

The genomic DNA of B. mandarina was digested with Bam HI and
separated by agarose gel electrophoresis. The DNA fragments transfered
to a nitrocellulose filter were hybridized with labeled pFb29. The radioactive probe hybridized to single fragment with molecular weigh of 14.5 Kb.
The fragments recovered from the gel fraction indicated a positive signal
were cloned into Bam HI site of Charon 30.
Twelve positive clones were detected by plaque hybridization to the probe.
Recombinant DNAs prepared from each clones were analyzed on agarosegels after digestion with Bam HI and hybridized to the probe. It was foundthat 7 clones containe 14.5 Kb fragment homologous to the fibroin genesequence of B. mori.

RESEARCHES CARRIED OUT IN 1982

II.

17

MICROBIAL GENETICSThe 245 Base-pair oriC Sequence of the E. coli Chromosome
Directs Bidirectional Replication at an Adjacent Region
Satoshi TABATA*, Atsuhiro OKA*, Kazunori SUGIMOTO*, Mitum TAKANAMI*,
Seiichi YAUDA and Yukinori HIROTA

The replication origin of the E. coli K-12 chromosome has been isolated
as autonomously replicating molecules (oriC plasmid), and the DNA
region essential for replicating function (oriC) has been localized to a sequence of 232-245 base-pairs (bp) by deletion analysis. In this report,
the functional role of oriC was analyzed by using an in vitro replication
system and various OriC+ and OriC- plasmids previously constructed.
The results obtained were summarized as follows: (1) The oriC sequence
contained information enough to direct bidirectional replication. (2) The
actual DNA replication began at a region near, but outside, oriC and progressed bidirectionally. (3) Initiation of DNA synthesis at the specificregion required the dnaA-complementing fraction from cells harboring a
dnaA-carrying plasmid. (For detail, see Nucleic Acids Research, 11, 26172626, 1983).
Suppressor Genes of a dnaA Temperature Sensitive
Mutation in Escherichia coli
Yutaka TAKEDA** and Yukinori HIROTA

Recombinant plasmids were constructed from EcoRI digests of Escherichia
coli chromosomal DNA and pMB9 DNA by selecting for suppression of adnaA-T46 temperature-sensitive mutation. Two types of plasmid capableof suppressing the dnaA mutation were isolated. They did not carry any
genetic markers around dnaA and physical mapping with various restriction enzymes showed that neither of the plasmids contained the dnaA gene.
One plasmid, pYT47, was characterized further and the protein responsible
for the suppression was identified by two-dimensional gel electrophoresis.
* Institute for Chemical Research, Kyoto University, Uji, Kyoto 611.
** Faculty of Agriculture, Nagoya University, Chikusa, Nagoya.

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETlCS NO. 33

The molecular weight of the suppressor protein was about 68 Kdal andthis is clearly different from the dnaA gene product. (For detail, see Mol.
Gen. Genet 187,67-71, 1982).
The Fine Architecture and Function of the Gene Codingfor PBP-3 of Escherichia coli
Ichiro N. MARUYAMA, Akihiko YAMAMOTO, Takeo MARUYAMA and Yukinori HIROTA

A synthetic ColEl plasmid, pLC26-6, found in a gene bank was shownto carry the gene (ltsI or pbpB) coding for PBP-3. Using this DNA fragment, we determined the entire nucleotide sequence of the ftsI, consistingof 1,764 base-pairs which code a polypeptide of 588 amino acid residues
and of a molecular weight 63,850. In this symposium, we present new
findings on the gene's fine structure and mechanism responsible for regulating
the function of PBP-3. (For detail, see "The Target of Penicillin, International FEMS-Symposium on the Murein Sacculus of Bacterial Cell
Walls-Architecture and Growth" (1983). Publ. Walter De Gruyter, BerlinNew-York).
N-Acetylmuramoyl-L-alanine Amidase of Escherichia coli K12:
Possible Physiological Functions
Claudine PARQUET*, Bernard FLOURET*, Mireille LEDUC*, Yukinori HIROTA
and Jean Van HEIJENOORT*

Various experiments were carried out in an attempt to determine the possible physiological function of the N-acetylmuramoyl-L-alanine amidase
purified from Escherichia coli K12 on the basis of its activity on N-acetylmuramoyl- L-alany 1- D- r -glutamyl-meso - diaminopimelic acid [MurNAcLAla-DGlu(msA zpm)]. A Km value of 0.04 mM was determined withthis substrate. Specificity studies revealed that compounds with a MurNAcLAla linkage are the most probable substrates of this enzyme in vivo.
Purified amidase had no effect on purified peptidoglycan and only low levels(1-2.5 %) of cleaved MurNAc-LAla linkages were detected in peptidoglycan
isolated from normally growing cells. However, the action of the amidase
in vivo on peptidoglycan was clearly detectable during autolysis. The
amidase activity of cells treated by osmotic shock, ether or toluene, as*Centre National de la Recherche Scientifique, Institut de Biochimie, Orsay.

RESEARCHES CARRIED OUT IN 198219

well as that of mutants with altered outer membrane compositIon wasinvestigated. Attempts to reveal a transfer reaction catalysed by amidase
were unsuccessful. Furthermore, by its location and specificity, amidase
was clearly not involved in the formation of UDP-MurNAc. The possibility that it might be functioning in vivo as a hydrolase degrading exogeneous
peptidoglycan fragments in the periplasma was substantiated by the factthat MurNAc itself and MurNAc-peptides could sustain growth of E. coli
as sole carbon and nitrogen sources. Finally, out of 200 thermosensitive
mutants examined for altered amidase activity, only two strains had lessthan 50 % of the normal level of activity, whereas ten strains were found topossess more than 50 %. In fact, two of the overproducers encountered
present a 4-5-fold increase in activity. (For detail, see Eur. J. Biochem
133: 371-377, 1983).
Isolation and Characterization of an Escherichia coli
Mutant Lacking tRNA-Guanine Transglycosylase
Function and Biosynthesis of Queuosine in tRNA
Shigeru NOGUCHI*, Yukinobu NISHIMURA, Yukinori HIROTA,
and Susumu NISHIMURA*

An E. coli mutant that lacks tRNA-guanine transglycosylase was isolatedby random screening from a collection of Escherichia coli mutants obtainedwith N-methyl-N'-nitro-N-nitrosoguanidine. The defective gene, named
tgt, was mapped at about 9 min on the E. coli chromosome, and the geneorder was shown to be phoB-tgt-tsx. tgt was transferred to an E. coli
strain with a defined genetic background by PI transduction to investigate
its function. The mutant thus obtained lacked queuosine (2-amino-5[(3, 4R, 5S)-4, 5-dehydroxycyclopent-l- en - 3-ylaminomethyl] -7- ((3 - D- ribofuranosyl)-pyrrolo-[2,3-D]-pyrimidin-4-one) in tRNA, indicating that the
enzyme is actually involved in the biosynthesis of queuosine in tRNA. No
clear biological defect was observed in the mutant, and, in fact, it grewslightly faster than the control isogenic strain. tRNATyr lacking queuosine,
isolated from the mutant, showed no significant biological difference fromnormal queuosine-containing tRNA in amino acid acceptor activity oramino acid transfer in a cell-free protein synthesizing system directed bysynthetic polynucleotide. The only phenotypic change observed in the* The Biology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

mutant thus far is marked reduction of viability when the cells are kept
under unsuitable conditions for growth, suggesting that the presence of
queuosine in tRNA is important to E. coli for survival in the natural environment. (For detail, see The Journal of Biological Chemistry, 257,6544-6550, 1982)
Synthesis of Flagellin and Hook Subunit Protein in
Flagellar Mutants of Escherichia coli K-12
Yoshibumi KOMEDA

I have examined Escherichia coli K-12 flagellar mutants for existence offlagellin and hook subunit protein. Immune precipitation experiments
were employed. Flagellin was detected in mutants defective in the geneflaS, flaT, flaU and flbC. The flaE or flaZ mutants made a little flagellin.
The other mutants did not have flagellin.
The hook subunit protein was found in mutants defective in the genesflaA, flaB, flaC, flaD, flaE, flaG, flaH, flaL, flaM, flaN, flaG, flaP, flaQ,
flaS, flaT, flaU, fla V, fla W, flaX, flaZ, flaZ, flaA, flbC, flbD, and hag. The
flaK,flaI andflbB mutants did not carry the protein. The results conformed
to the prediction by indrect gene fusion study (Komeda 1982).
Localization of E. coli Flagellar Gene Products Using
Gene Fusion Technique
Yoshibumi KOMEDA

It has been known that a fused gene, into which MudII30l (Apr, lac)phage is inserted, codes for a fused enzyme carrying beta-galactosidase as
C-end. The MudII30l (Apr, lac) phage was inserted into each of the E.
coli flagellar genes (fla, flb, hag and mot). The insertion mutants were
expected to carry fused proteins consisted of N-terminal flagellar geneproducts and promoter distal side of beta-galactosidase.
Localization was analyzed in these mutants. Since the beta-galactosidase
is known to be located in cytoplasm, then N-terminal portion is responsible
for the alteration of location if the fused enzyme is located in other thancytoplasm.
At first, motA: : MudII30l (Apr, lac), hag: : MudII30l (Apr, lac), andflaK: : MudII30l (Apr, lac) mutants were studied. These genes code for

RESEARCHES CARRIED OUT IN 1982

21

motility protein, flagellin, and hook subunit protein, respectively. The
products are known to be located in inner membrane, outside, and outside,
respectively. It was found that these fusion products located as follows.

mot producthag productflaK productfusion protein

intact protein

inner membrane
cytoplasm
cytoplasm

inner membrane
outsideoutsideThe motA product was shown to be inner membrane protein by lambda
mot infection study. The fused protein, motA-lacZ product, was also shown
to be located in inner membrane in this study. Therefore, it was expectedthat motA protein has a signal to orient itself into inner membrane. The
hag and flaK products are located outside, but fusion proteins were shown
to be cytoplasmic. This fusion did not have the signal to define locationor original products did not have location signal.
Next, a whole set of flagellar mutants were collected, which carried
MudII301 (Apr, lac) insertions and protein fusion. They were examined
for the location of fused beta-galactosidase activity. Among them, flaU,
jlaA, and flaB fusions were found to be located in membrane fraction.
These gene products were imagined to have signal sequence that alteredcellular locations of the fusions.

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

III.

BIOCHEMICAL GENETICS AND IMMUNOGENETICS
Urethan-Induced Lung Tumor Development in(Susceptible X Resistant)F1 Hybrids of H-2
Congenic Strains of Mice
Nobumoto MIYASHITA and Kazuo MORIWAKIIt has demonstrated that the I-A and I-E regions in the H-2 complex are
related to the regulation of the lung tumor development (Moriwaki andMiyashita, A.R.N.I.G. 31, 38-39, 1981). In the present study, we investigated the urethan-induced lung tumor development in F 1 hybrids between the susceptible strain which is expressing the Ek molecules on the antigen-presenting cell controlled by H-2 a haplotype and the resistant strains
which are not expressing the E molecules. Average number of lung tumor
foci per mouse in (susceptible X resistant)F 1 hybrids were intermediate
between the parental strains of the congenic mice on the A background.
These response patterns are remarkably similar to H-2 linked Ir gene response to lactate dehydrogenase B (LDH-B) and IgG2a myeloma protein.
It can be postulated that the T-cell dependent immunity plays a role in theregulation of the susceptibility to the lung tumor development.
Table 1. Urethan-induced lung tumor development in(susceptible x resistant) F1 mice
Strain
A
A.BY
A.SW
A.CA
(AxA.BY)F1(AxA.SW)F1(AxA.CA)F1():

Alleles of H-2 complex
H-2
haplotype K
Afi A a . Efi Ea D
a
b
s
f
ajb
ajs

ajf

Type ofE molecule

Lung tumor
foci per mouse

EakEfikk k k k db b b b b
s
s
s
s
f
f
f
f
f
kjb kjb kjb kjb kjb djb EakEfik, EakEfib
kjs kjs kjs kjs kjs djs EakEfik, EakEfi8
kjf kjf kjf kjf kjf djf EakEfik, EakEl

30.6±1.3 (24)13.8±O.6 (58)12.3±O.7 (51)17.0±O.6 (58)
23 .9±1. 3 (25)18.9±1.1 (13)21. 7±1.1 (17)

k
b
s
f

Number of mice observed.

RESEARCHES CARRIED OUT IN 1982

23

Production of Monoclonal Antibodies against H-2 Complex
in BIO. MOL-SGR Mouse Strain
Tomoko SAGAI, Toshihiko SHIROISHI and Kazuo MORIWAKI

Previously, we reported that the intra-H-2 recombination frequency of
BIO. MOL-SGR (H_2 wm7) which carries the H-2 complex of Japanese wild
mouse extremely high (about 3 %). In this congenic strain, most of thecrossing over have occured between H-2K and IA region (Nature 300,
370-372). We tried to obtain some hybridomas producing monoclonal
antibodies for H-2 complex of BIO. MOL-SGR, because those antibodies
should be useful for analysis of the high recombination mechanism.
P3UI myeloma cells were fused with spleen cells prepared from three
BIO congenic strains immunized with BIO. MOL-SGR lymphocytes. Activities and specificities of the monoclonal antibodies were roughly analysedby the complement dependent microcytotoxicity test, reported by us preHybridoma cell lines produced against
BIO. MOL-SGR lymphocytes

Table I.
Immunized recipients

Established hybridomas
Hdl, Hd2
HdlO, Hd12, Hd15, Hdl7
Hd24, Hd25, Hd28

BIO
BI0.A
(BI0xBlO.A)Fl
Table 2.

Characterizations of monoclonal antibodies
against BIO. MOL·SGR

H-2 complex
H-2
recombinants
R201
R203
R202
R204
R212
R406
R407
BI0
BI0.BR
BI0.MOL-SGR

K

A

k Iw
k k
k k
w Ik
w w
b Iw
w Ib
b b
k k
w w

E

S

D

w w w
k Iw w
k d Iwk d d
w Id d
w w w
b b b
b b b
k k k
w w w

Type I

Type II

Type III

Type IV

Hd24,25

Hd2, 12, 15
17,28

Hdl

HdlO

+

+
+
+
+
+
+
+

+

+
+
+
+

+
+
+

+
+

+
+
+
+
+

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

viously (Microbiol. Immunol. 25, 1327-1334).
Table 1 summarizes nine hybridomas established. These hybridomas
were classified into four types by the range of reactions (Table 2). Type I
and II produced antibody for H-2K and I regions of BlO. MOL-SGR
respectively. Type III exhibited cytotoxic reaction for H-2 public antigen
on the both K and D regions. It was also crossreactive with BlO. BR.
Type IV antibody was reactive with the I regions, but crossreactive with
BlO.

Effect of Major Histocompatibility Gene Complex (MHC)on the Growth of Embryo-Derived Teratoma
Choji TAYA and Kazuo MORIWAKI

Mouse teratoma can be experimentally induced by the transplantationof early embryos under the kidney capsule. In order to find out the immunological factors governing the in vivo growth of embryo-derived teratoma,
7 day-old embryos of several A. H-2 congenic strains were transplanted
under the kidney capsule of each dault recipients. After 40 days, thegrown tumors were weighed to compare the effect of MHC in the host.
Among five A. H-2 congenic strains, A. TH and A. TL strains weresignificantly greater in the tumor weight than A. BY and A. SW strains.
A strain was the intermediate size of tumor (Table 1). A. TH, A. TL andA strains have d haplotype in H-2D region, but A. BY and A. SW strains
have band s haplotypes respectively. On the other hand, A. TH and A.
SW strains have the same haplotypes in the proximal parts of H-2 comTable 1. Syngeneic transplantation of 7 day-old embryo
under the kidney capsule: Effect of H-2 complex on thegrowth of embryo-derived treatoma
H-2 haplotype
Strains
A.TH
A.TL
A
A. BY
A.SW
BlO. A

K

A

E

D

s
s
k
b
s
k

s
k
k
b
s
k

s
k
k
b
s
k

d
d
d
b
d

No. of miceobserved

Weight ofteratoma

9
23
33
47
23
12

1998.9±476.0 mg
1752. O±3l6. 7
1421.5±219.2
1004.6±167.l
923.6±206.1
606.8±180.0

RESEARCHES CARRIED OUT IN 1982

25

Table 2. Allogeneic transplantation of 7 day-old embryo under thekidney capsule: Effect of H-2 complex on the growth of embryoderived treatoma
StrainsH-2
haplotype

A. BY->BlO
BI0->A. BY
A->BlO. A
BI0. A->A
A. TL->A. TH
A. TH->A. TL
A. SW->A. TH
A. TH->A. SW

b->b
b->b
a->a
a->a
tl->t2t2->tls->t2
t2->sH-2 complex No. of micedifference
observed(Identical)(Identical)(Identical)(Identical)
AE
AE
D
D

11
4
3
2
3
8
2
2

Weight ofteratoma
No tumor
No tumor
No tumor
No tumor
668.0±132.4mg
459.5±84.3
302.0
266.5

plex to H-2D region. Thus, these results suggest that H-2D region or
distal part to H-2D affect the tumor growth in A. H-2 congenic strains.
Teratoma produced in BIO.A strain was smaller than any other A. H-2
congenic strains, suggesting that the genetic background of A strain is favorable to the tumor growth.
In the allogeneic combinations, no tumor was developed from the transplant of A. BY to BIO, BIO to A. BY, A to BIO. A and BIO. A to A, whereas
the definite tumor growth was observed by the introduction of A. TL to
A. TH, A. TH to A. TL, A. SW to A. TH and A. TH to A. SW (Table 2).
These results suggest that the locus/loci controlling the growth of embryonic tissues are located out of MHC as well.
Southern Blot Analysis of H-2K Genes of Japanese Wild Mice
Hitoshi SUZUKI, Toshihiko SHIROISHI, Tomoko SAGAI, Gabriel GACHELIN*,
Takeharu KANEHISA** and Kazuo MORIWAKIThe classical transplanation antigens (H-2 K, D and L) have extensive
polymorphism but the details of their evolutional process remain unknown.
We started the survey of the H-2 gene complex among several subspeciesof Mus Musculus based on restriction enzyme cleavage patterns comparing
to the serological data. Southern blot analysis using class I cDNA probe(pH-2 d-4) revealed the following results.
* Institut Pasteur.
** The Graduate School of Science and Technology, Kobe University.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33(1) BIO H-2 recombinant mice with H-2 haplotype d, k and b exhibited
H-2 K-specific 3.7 Kb, 3.2Kb and 2.5 Kb bands, respectively, in Bgl II
digestion as shown by Xin et al. (EMBO Jour. 4, 467-471, 1982).
(2) DNA from BIO. MOL-SGR showed a H-2Kk like 3.2 Kb band in
Bgl II digestion. Using a recombinant line, R2l4 (KWm7, D d) (see Nature
300. 370-372, 1982), we could map the Bgl II band on K locus. In thisconnection, it may be noteworthy that the antigenicities of H-2K molecules
were relatively similar each other between BIO. BR (H-2 k haplotype) andBIO. MOL-SGR.
(3) BIO. MOL-OKB which carries H-2 K31 (H-2Kd private) antigenic
specificity had a 3.7 Kb band in Bgl II digestion.
These results suggest that H-2K region of each haplotype has its characteristic segment which can be detected by Bgl II digestion of genomic DNA.
During the subspecies differentiation, these haplotype specific Bgl II sites
might have been weB conserved.
Southern Blot Analysis of Ribosomal DNA in Mus Musculus
Hitoshi SUZUKI, Ryo KOMINAMI*, Masami MURAMATSU*,
Takeharu KANEHISA** and Kazuo MORIWAKI

Restriction patterns of ribosomal DNA (rDNA) were examined in fiveinbred strains and several subspecies of Mus Musculus by Southern blot
analysis using two restriction enzymes (Bam HI and Eco RI) and a DNA
probe of 0.7 Kb cloned fragment extending from 3' end of the 28S ribosome
gene to the foB owing spacer region. European subspecies, M. m. domesticus,
M. m. musculus and M. m. brevirostris and Asian subspecies, M. m. bactrianus, M. m. castaneus and M. m. molossinus were examined, each ofwhich showed the characteristic cleavage patterns (Table 1). Intrasubspecific heterogenity in rDNA was found in either of M. m. domesticus,
M. m. musculus, M. m. castaneus or M. m. molossinus. In five inbred strains,
BIO, BALB/c, DBA/I, DBA/2 and CBA, the cleavage patterns of rDNA
in Bam HI and Eco RI were relatively similar to those of M. m. domesticus.

***

Tokyo University, School of Medicine.
The Graduate School of Science and Technology, Kobe University.

27

RESEARCHES CARRIED OUT IN 1982
Table 1.

Restriction fragment of rDNA in various mouse subspecies
Restriction enzymes
Bam HI

Length of fragments
(Kb)

2.3

M. m. domesticus

+++
++
+

M. m. musculus
M. m. brevirostris
M. m. bactrianus
M. m. castaneus
M. m. molossinus

±

++

6.6

++
++
+++
+
+++
+++
+++
++

Eco RI
5.5

6.6

7

9

10

+++

+

+++
+++
+++
++

++
+
+

++
++

++
++
+++

DNA probe; cloned 0.7 Kb segments from 3' end of the 28S gene to the following spacer
region.

Serological Survey of Thy-l and Lyt-l,2,3 Antigens
in Wild Mice
Yasuyuki KURIHARA and Kazuo MORIWAKI

Thy-I and Lyt-I,2,3, thymocyte surface antigens of mice, are known as
differentiation antigens. Each of them has two alleles (e.g. Thy-I a andThy-Ib). In Thy-I locus, Thy-P and Thy-Ib alleles determine two antigenic molecules, Thy-I.I and Thy-I.2. Distribution of those antigens inlaboratory mice has already been known. We examined these antigensof wild mice maintained in our laboratory, using the microcytotoxicity test.
Although most laboratory mice express Thy-1.2 and only 15 strains express Thy-I.I, we found that almost all M. m. subspecies except M. m.
domesticus expressed Thy-I.I. Such data agreement with the generally
accepted assumption that the laboratory mice mainly derived from M. m.
domesticus. In survey of Lyt-I and Lyt-2, we could detect only Lyt-1.2
and Lyt-2.l, but not Lyt-l.l and Lyt-2.2 (Table 1).
Thus, thymocyte antigens such as Thy-I, Lyt-I and Lyt-2 seem to be
monomorphic within each subspecies.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33
Table I. Survey of differentiation antigens in Mus musculus subspecies
and some laboratory mice
Strains
Wild mice
M. m. domesticus
molossinus
musculus
castaneus
bactrianus
M. m. subspecies
BJN III
SHHI
JYGI
LZHII
Inbred mice
C57BL/6
BALB/c
129
DBA/2
AKR

Thy-l

Lyt-l

Lyt-2

Lyt-3

2
1
1
1
1

2
2
2
2
2

1
1
1
ND
ND

2
1
1
ND
ND

2
ND
2
2
2
2
2
2
1

2
2
2
1
2

1
1
ND
ND
2
2
2
1
1

2
2
2
2
I

ND:

not detected.

RESEARCHES CARRIED OUT IN 1982

IV.

29

DEVELOPMENTAL GENETICS AND SOMATIC
CELL GENETICS
In Vitro Formation of Adult Structures from Cells of
Lethal Embryos of Drosophila melanogasterYukiaki KURODA

When cells from post-gastrula embryos of Drosophila melanogaster are
cultured in ecdysterone-free medium, they differentiate into larval epithelial
cells, muscle cells, cellular spheres and nerve cells having their respective
characters and functions. On the other hand, when these embryonic cells
are cultured in ecdysterone-containing medium, they form adult structures
such as the leg, the wing and the compound eye. This indicates that larvalor adult tissues and cells can be selectively differentiated from embryoniccells by adding or removing of ecdysterone in the medium.
Homozygous and hemizygous embryos of a sex-linked mutant, deeporange (dor, 1-0.3; salivary gland chromosome map, IFI-2A2) died aroundthe stages of gastrulation and mid-gut formation during embryogenesis.
When cells from post-gastrula embryos of the dor mutant were cultured
in ecdysterone-free medium, epithelial cells, muscle cells and nerve cells
differentiated normally. However, dor cells had defects in the differentiation and function of some specific types of cells. The syncytium formationof muscle cells, the formation of cellular spheres and the droplet secretionon the nerve fibers were not observed in these cultures.
When embryonic dor cells were cultured in medium containing 1-10
Itg/ml of ecdysterone, some structures of the adult legs developed. Thissuggests that the dor lethal gene may express in some specific types of cells
which show defects in normal differentiation and function, but some othertypes of cells differentiate normally and form not only larval tissues andcells but also adult structures in culture.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Localization of Lectins and Their Changes in Developmentof Chick Embryos
Yukiaki KURODA, Etsuya MATSUTANI and Tatsuya YAMAGATA 1 )Mesenchyme cells dissociated from the limb buds of chick embryos differentiate into cartilage cells under in vitro culture conditions. We havepreviously reported that lectins had stimulating effects on the in vitro
chondrogenesis of these mesenchyme cells (Develop. BioI., 89: 521-526,
1982).
In the present experiment, the localization of endogenous lectins andtheir quantitative changes in the limb bud tissue of developing chick embryos were examined by using the immunofluorescent antibody technique,to find the role of lectins in normal development of chick embryos. The
localization of galactose which binds specifically to chick lectins was also
examined by using a FITC-Iabeled plant lectin, RCA.
No lectins were detected in the limb bud of chick embryos before thestage 24. They appeared in muscle tissue at the stage 25 and increased inamount as the developmental stages proceeded. Lectins were also foundin the prospective cartilage region at the center of the limb bud and increased further in the process of osteogenesis. In the muscle tissue, lectins
were present in the cytoplasm of cells, whereas in cartilage tissue lectins
were detected in the intercellular matrix.
No galactose was detected in muscle tissue, whereas it was detected in
cartilage tissue after the stage 24. Mesenchyme cells in the limb bud arestabilized to express the differentiated character of the cartilage cells andobtain the autonomous differentiating activity after the stage 24. The timeof quantitative changes in endogenous lectins and galactose in the limb
bud coincided with the time when mesenchyme cells are stabilized theirdifferentiating activity. The intimate relationship between these two events
may be suggested. This was supported by an experiment in which cultured
mesenchyme cells were stimulated to differentiate into cartilage cells byadding embryonic chick lectin to medium.

1)

Mitsubishi-kasei Inst. of Life Science, Machida, Tokyo.

RESEARCHES CARRIED OUT IN 1982

31

Mutagenic Activity of Environmental Aromatic Compounds
in Cultured Chinese Hamster Cells
Yukiaki KURODA and Masumi ASAKURA

The mutagenic activity of most aromatic compounds has not been detected in microbial assay systems. Recently, however, some aromaticcompounds like aniline were reported to be carcinogenic in experimental
animals. In the previous report, it has been found that aniline had a strong
mutagenic activity in cultured Chinese hamster V79 cells.
There have been no report on the mutagenic activity of other aromaticcompounds such as chloroaniline, nitrobenzene and hexachlorobenzene.
These compounds are produced in large amounts as the materials for dyestuff, mordants and medicines by the manufacturing industry and frequently
found in natural water and substratum in our environment. In the present
experiment, the activity of these compounds to induce 8-azaguanine (8AG)and ouabain (OUA)-resistant mutations in V79 cells was examined.
0-, m- and p-chloroaniline had stronger cytotoxic effects than aniline on
V79 cells. The values of LD 50 for 4 hour treatment of these three compounds were 0.5-0.6 mg/m/. The frequency of 8AG-resistant mutations
induced by these compounds were 1.3-2.5 x 10- 5 which were lower thanthat of aniline. No detectable OUA-resistant mutations were induced bythese compounds.
Nitrobenzene showed almost the same cytotoxic effect as that of chloroaniline. The frequency of 8AG-resistant mutations induced by nitrobenzene
was as low as 1.6 x 10- 5 • The induced mutation frequency increased to
5.6 x 10- 5 , by adding of S-9 Mix, showing a metabolic activation of nitrobenzene.
Hexachlorobenzene had a strongest cytotoxic effects among all aromaticcompounds tested. Its LD 50 value for 4 hour treatment was 0.04 mg/m/.
However, hexachlorobenzene was weakly mutagenic for induction of 8AGresistant mutations (1 X 10- 5). No OUA-resistnt mutations were detectedby treatment with this compound. The tumor-forming activity of thesearomatic compounds are unknown, but it is necessary to examine theircarcinogenicity in experimental animals.

1)

Japan Bioassay Laboratory, Hadano, Kanagawa.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Effects of EGF and FGF on Growth Pattern of Cultured
Human Diploid Cells in Clonal Culture
Yukiaki KURODA

Normal human diploid cells are extensively used as a model system ofcell aging, since they have a definite life span of about 50 population doublings(PD). In the present study the mode of proliferation of individual cells
was analyzed by clonal cultures of young cells (6PDL) and aged cells (18PDL). The effects of epidermal growth factor (EGF) and fibroblast growth
factor (FGF) on the mode of prliferation of both cells were also examined.
The mode of proliferation was analyzed by scoring the number of cells
per colony in replicate cultures in many petri dishes which were inoculatedwith 3 x 103 single cells in 60 mm dishes and fixed every 24 hours in cultivation. In cultures of young cells, beside some synchronously dividing cells
which increased in number as 2n , other asynchronously dividing cells wreeobserved. In the latter cells, after one cell division one daughter cell proceeded to the next division, but another daughter cell did not proceed to thenext division. Some cells remained as single cells in whole culture periodof 7 days.
In cultures of aged cells, the mode of proliferation was almost the sameas that of young cells. But the rate of proliferation was prolonged inaged cells. During cultivation for 72 hours a most actively dividing cell
produced 42 cells after more than 5 divisions in young cell cultures, whereasa most actively dividing cell in aged cell cultures produced only 10 cells
for the same culture period.
When EGF or FGF was added to medium, in young cell cultures theproliferation rate was markedly stimulated by these growth factors, although the mode of proliferation remained unchanged. The number ofcells which remained as single cells started to divide again and the numberof single cells decreased. This indicates that these growth factors had
stimulatory effects on the proliferation of both dividing and non-dividing
cells in young cell population.
On the contrary, the proliferation of aged cells were not or slightly affectedby EGF and FGF. This suggests that human diploid cells may be reducedin their responsibility to these growth factors as their culture age increased.

RESEARCHES CARRIED OUT IN 198233

Hypertrophic Growth of Neuroblasts in a Notch Lethal,Df (1) Notch 8 of Drosophila melanogaster
Kiyoshi MINATO

The lethal alleles at the Notch locus of Drosophila melanogaster are known
to be lethal in embryogenesis, producing an extensive hypertrophy of
nervous tissue. To know whether this hypertrophy is due to either theformation of too many neuroblasts segregated from the ectodermal layer
in the early period of embryonic development or an increased rate ofcell divisions of neuroblasts, one of Notch lethals, DI(I) Notch 8 wasanalyzed for the growth of nervous tissue in the histological preparations,by using a recently developed technique with more efficient fixation(Zalokar, 1976) and embedding in resins which made an easy orientation of
fixed materials.
At the final stage of nerve differentiation 10 or 12 hours after fertilization,
the fully condensed central nervous system was seen in normal embryos.
At this stage, there were about twice as many ganglionic cells, thoughloosely condensed, in lethal embryos as those in normal embryos. At
the earlier stage of 5 or 6 hours, neuroblasts or ascendants of ganglioniccells became firstly distinguishable from ectodermal cells. At this stage,
there were already about twice as many neuroblasts in lethal embryos as
those in normal embryos. Thereafter the mitotic activity of neuroblastsin lethal embryos was not different from that in normal embryos.
These results indicate that the hypertrophy of nervous tissue in this mutant
embryo may be due to only the hypertrophy of neuroblasts at the earlier
stage of embryogenesis.

Morphogenetic Potentials in Chimeric Hydra Strains
Tsutomu SUGIYAMA, Jun TAKANO and Chiemi NISHIMIYA

In order to determine which cell types in hydra are responsible for morphogenetic potentials, we have constructed chimeric hydra from the standard
wild type (lOS) and the regeration-deficient strain (reg-l 6) or the slow buddingstrain (L4). Reg-16 has a greatly reduced head regenerative capacity andL4 has a very low budding rate and is large in size. Both strains have avery low head-activation potential and a high head-inhibition potential
comparing to 105.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Hydra tissue consists of three self-renewing cell lineages: the ectodermal
epithelial cell lineage (referred to as Ect), the endodermal epithelial celllineage (referred to as End) and the interstitial cell lineage (referred to as I)
which includes interstitial stem cells and their differentiation products,
nerve cells and nematocytes. By combining these cell lineages between
two strains it is possible to construct chimeric strains (Marcum and Campbell, 1978; Wanek, 1981). For example, from 105 and reg-16 the following
6 chimeric strains can be constructed: Ectlo5/End105/1reg'16' Ectreg'16/Endreg'16/
1105 , Ect105/Endreg'16/1105', Ectlo5/Endreg -16/ I reg -16' Ectreg'16/Endl05/1l05' Ectreg_16/Endl05/1reg'16'
The head regenerative capacity of chimeric strains constructed from
105 and reg-16 and the budding rate and the size of chimeric strains constructed from 105 and L4 were examined in detail and compared to thoseof their respective parental strains.
The regenerative capacity was greatly reduced when the reg-16 epithelial
cell lineages are present in chimeras, although it was slightly higher thanthat of reg-16. The influence of the reg-16 endodermal epithelial cells
was larger than that of the reg-16 ectodermal epithelial cells on head regeneration. The influence of the reg-16 interstitial cells on head regeneration was slight but significant. These results indicate that the regenerative
defect of reg-16 resides mainly in its epithelial cells and that the endodermal
epithelial cells contribute more to head regeneration than the ectodermal
epithelial cells.
The budding rates and the sizes of chimeras constructed from 105 andL4 resembled to their donor strain of the ectodermal epithelial cell lineage.
The endodermal epithelial cells and the interstitial cells showed no influenceon these traits. These results indicate that the ectodermal epithelial cells
are the determinant of the budding rate and the size of hydra.
Position Dependent Nematocyte Differentiation in Hydra
Toshitaka FUJISAWA

Nematocytes are hydra's stinging cells used for capturing preys and forprotection. There are 4 types of nematocytes (stenotele, desmoneme,
holotrichous isorhiza and atrichous isorhiza), all of which are differentiatedfrom multipotent interstitial stem cells.
The spatial distribution of the differentiating nematocytes along the bodyRESEARCHES CARRIED OUT IN 1982

35

column is very characteristic. For example, stenoteles are differentiatedpredominantly in the lower budding region and their number decreases
gradually in the more distal regions, while desmonemes and isorhizas aredifferentiated mainly in the distal half of the body column.
In order to know the mechanism by which the position dependent patternof nematocyte differentiation is formed, stenotele differentiation was analysed
in detail.
Among possibilities the following two are most likely for the position
dependency of stenotele differentiation: (1) The inhibitor for stenotele differentiation is produced in the head and its concentration decreases in the
more proximal regions. More stenoteles are differentiated in the regions
where the inhibitor concentrations are lower. (2) The stimulator for
stenotele differentiation is produced in the foot and its concentration decreases in the more distal regions. More stenoteles are differentiated in
the regions where the stimulator concentrations are higher. The following
two experiments were carried out to differentiate these possibilities.
1) The head tissue was grafted laterally into the lower budding region
to see if the grafted tissue inhibits stenotele differentiation. The foottissue was grafted laterally into the distal region to see if the grafted tissue
induces stenotele differentiation. The results showed that only the headtissue inhibited stenotele differentiation.
2) The inhibitor for stenotele differentiation has been found in the hydra
tissue. To examine if this factor is most abundant in the head and its concentration decreases gradually in the more proximal regions, the body columnof hydra was divided into 5 regions (head, upper body region, middle body
region, lower body region and foot) and the concentration of the factor
was determined in each region. The results showed that the inhibitor
concentration was the highest in the head and it gradually decreased in the
more proximal regions.
These results favor the first possibility.
A Single Recessive Gene(ter) Causing Germ Cell Deficiency
and an Extremely High Incidence of Testicular Teratomas in
129JSv-ter Mice
Takehiko NOGUCHIThe genetic basis of a germ cell deficiency which is accompanied by an

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

extremely high incidence of testicular teratomas, and which is peculiar to
the 129/Sv-ter strain of mice (J. Nat!. Cancer Inst. 69: 907-913, 1982) was
studied.
In the males, germ cell deficiency became more severe as age advanced.
The males having germ cell deficient testes were sterile whether they had
bilateral teratomas or not. Of 147 males with abnormal testes 110 (74.9 %)had tumors bilateraly, 28 (19.1 %) had them unilateraly, and only 9 (6.1 %)were tumor free. In the females, however, the deficiency did not progress,and most of the females were fertile.
Germ cell deficient males exhibited smaller testes except when the testes
had teratomas. The detection of the abnormal males was possible byobserving the appearance of the seminiferous tubules even when they had
teratomas. In the females, the germ cell deficiency correlated with smaller
ovaries. The establishment of the method of detecting the abnormal individuals made it possible to study the genetic basis of the abnormality.
One hypothesis is that the germ cell deficiency may stem from the homozygous state of a recessive mutant gene(ter).
Matings between females with small ovaries and males with normal testes,
but without the genetic factor, produced no FI offspring having germ cell
deficient gonads. However if these FI offspring were mated together,individuals having germ cell deficient gonads appeared again in the F2generation. This strongly suggests that the genetic factor is recessive.
When females having small ovaries were mated with fertile males having
the genetic factor, half of the Fl offspring (146/296) had abnormal gonads.
If the FI offspring with normal gonads were mated together, F2 animals
with abnormal gonads appeared at a frequency very close to 1/4. These
results strongly suggest that the genetic factor is a single recessive gene.
This hypothesis implies that this mice colony is composed of animals withthree different genotypes, +/+, +/ter and ter/ter.
Teratoma incidences estimated for the males with different genotypes
were 1.3% for +/+, 22% for +/ter, and 94% for ter/ter.

RESEARCHES CARRIED OUT IN 1982

v.

37

CYTOGENETICS

Sequentiality of Chromosome Evolution
Tosihide H. YOSIDA

When we observe the karyotypes in some rodent species, the chromosome
evolution seems to have occurred sequentially from one to others. For
example, the chromosome polymorphism in pair nos. 1, 9 and 13 found in
Asian type black rat (2n=42) should have occurred by the sequential inversion of these acrocentrics. The Ceylonese and Oceanian type black rats
with 40 and 38 chromosomes also seemed to have occurred in southern
India by sequential Robertsonian translocation. Another sequential
karyotype evolution was observed in the black rats distributed in the
Mauritius Island. This island was found by Portuguese in the 16th century.
In the Oceanian type black rats migrated to this island the centric fission
should have occurred sequentially in metacentric pair nos. 14 and 18 during
a short time after the rats landed on this island. Possibilities of species
differentiation through the sequential centric fusions were found in theother Rattus species, such as R. fuscipes (2n=38), R. leucopus (2n=34)
and R. conatus (2n=32) distributed in Australia and New Guinea. Sequential centric fusions and fissions were also observed in Rattus villosissimus with 50 chromosomes in Australia.
Karyotype of the tabacco mouse, Mus poschiavinus, with 26 chromosomes
has been derived from centric fusion of 14 acrocentric pairs (Gropp et al.
1970). A similar karyotype change by the centric fusion has been observedin Mus (Leggada) minutoides indigenous to Africa (Matthey 1963). Thesechromosome variations seemed to have occurred by sequential events ofthe centric fusion. The Indian spiny mouse, Mus platythrix, was characterized by having 26 acrocentric chromosomes due to sequential tandem fusionof each two in several acrocentric pairs (Yosida 1980a). Basic karyotypeof the house shrew, Suncus murinus, had 40 chromosomes, but those inhaviting in southern India and Sri Lanka had 2n=32 and 30 (Satya Parakash
and Aswathanarayana 1976; Yosida 1982). Karyotypes of the latter specimens have been resulted from the sequential centric fusion of 5 and 6 acrocentric paris, respectively.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

A good evidence of the sequential karyotype change was provided in the
LEW-strain rat, Rattus norvegicus, breeding in the laboratory. In one
female a translocation between pair nos. 1 and 12 was primarily observed(Yosida 1980b). In the next generation inversion of pair no. 1 was detected.
This was explained that the 1/12 translocation should have spontaneously
occurred and in the next generation inversion might have sequentially occurred in the normal chromosome partner in the early embryogenic stage.
A new hairless mutant (ba) appeared after a few inbreeding generations in
the inversion stock of this animal (Yosida 1981). The above findings provide
a good evidence for the sequentiality of chromosomal and/or gene mutation.
Parallelism of Karyotype Evolution
Tosihide H. YOSIDA

Phenomena of parallelism of the karyotype evolution due to the pericentric inversion, Robertsonian fusion and fission has been found frequently
in genus Rattus. Some examples of the parallelism will be described in
Rattus and some other mammals and discussed on a mode of the karyotype
evolution.
1) Examples in genus Rattus: Basic karyotype of the black rat (R. rattus)
with 2n=42 was characterized by 13 acrocentric (nos. I to 13) and 7 metacentric autosome pairs (nos. 14 to 20) and acrocentric X and Y (the Asiantype). Acrocentric and subtelocentric polymorphism due to pericentric
inversion was observed in pair nos. 1, 9 and 13 of this animal. Some other
Rattus species, such as R. annandalei, R. muelleli, R. exulans, R. losea andR. norvegicus had 2n=42 and their karyotype were similar to the blackrat, but pair nos. I, 9 and 13 were either one of the acrocentric or subtelocentric. It seems to have occurred by parallelism of karyotype evolution
in these Rattus species.
Parallelism of the karyotype evolution by the Robertsonian fusion was
found in genus Rattus. Both Ceylonese type black rat, R. rattus kandianus,
and R. bowersii had 2n=40 with one large metacentric pair derived fromthe Robertsonian fusion between acrocentric nos. 11 and 12. A similar
karyotype to the Oceanian type black rat with (2n=38) having two large
metacentric pairs due to the Robertsonian fusion was found in R. fuscipes
living in Australia. Karyotype evolution by the other Robertsonian fusions
was observed in R. leucopus (2n=34) and R. conatus (2n=32). They should

RESEARCHES CARRIED OUT IN 1982

39

have developed by parallel karyotype evolution due to Robertsonian fusion.
Mauritius type black rats were characterized by having 4 small acrocentric
pairs due to the Robertsonian fission of two small metacentric pairs, nos.
14 and 18. A parallel karyotype evolution to the Mauritius black rat was
found in R. villosissimus (2n=50), which was remarkable by having extra
12 small acrocentric pairs derived from the Robertsonian fission of 6 small
metacentric pairs, nos. 14 to 19.
2) Examples in the other mammals: Evolution by inversion of chromosomes was often reported in the other genus in Rodentia, such as Peromyscus,
Neotoma, Thomomys, Ctenomys and some others. A typical case of theparallel karyotype evolution through the Robertsonian fusion was foundin two different species belong to genus Mus, Mus minutoides distributedin Africa and the tabacco mouse (Mus poschiavinus) distributed in Europe.
Parallel karyotype evolution through the Robertsonian fusion was reported
in the other taxon, such as Bovidae, Equidae, Felidae and Primates.
Karyotypes in mammals would have evolved through such parallelism andsequentiality described in the previous report.
Cytogenetical Study on a Mammary Cancer of the Domestic Catwith a Note on an Increase of Cells with a Normal
Karyotype after Deep Freezing
Tosihide H. YOSIDA and Kyoko KAWAHARA

Karyotypes of a mammary cancer spontaneously developed in the domestic cat were analysed in comparison with those of normal somatic
cells (2n=38). Normal karyotype of the domestic cat was characterizedby having 2n=38, consisting of eighteen autosome pairs classified into A
to F groups. Chromosome number in 100 cancer cells growing in vitroof the mammary cancer ranged from 26 to 110. Interesting is the factthat this tumor was characterized by having the bimodal distribution ofchromosome numbers; hypodiploid and hypotetraploid. In hypodiploidcells several chromosomes were monosomic consisting of one mode at 31
chromosomes. Normal diploid chromosome number (2n=38), however,
was found only in two cells (2 %). Such chromosome constitution wasobserved for about 10 culture generations, so far the cultivation was continued by use of the same technique.
The primary culture of the tumor cells was stored into the deep freezer

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

at -80°C by use of 10% DMSO for 7 days. The cells thus frozen for 7
days were cultured again in the same culture median as above. Shape ofcultured cells growing in vitro after deep freezing was not different fromthose before freezing. Chromosome numbers in tumor cells in one batch(A) were counted in 180 cells in the first culture generation after freezing.
Among them 159 cells (88.3 %) had a normal diploid number of 38 chromosomes. In the culture from the other batch (B), 50 cells were counted, among
them 46 cells (92.0 %) had 38 chromosomes. The karyotypes of all cellswith 38 chromosomes obtained from these batches were normal. Th~secomplements were maintained for about 10 generations so far the cells
cultured in vitro. It could not be examined by us whether the cells withthe diploid karyotype in the feline tumor developed after deep freezing
have still malignant characteristics or not, because the diploid cells could
not be transplanted to the same host. If the tumor malignancy has reduced
by increase of diploid cells after deep freezing, the evidence presented may
be available for seeking any clue for medical treatment of the tumor.
Remarkable Difference on Frequency of Single and Twin
Sister Chromatid Exchanges in EndoreduplicatedBlood Syndrome Cells
Yukimasa SHiRAISHl1 ) and Tosihide H. YOSIDA

The increase rate of sister chromatid exchanges (SCEs) in Bloom syndrome(BS) cells has been known, but its mechanism is unknown. Shiraishi et al.
(1981) showed that when BS cells labeled with bromodeoxyuridine (BUdR)
for one round of DNA replication were fused with nonlabeled normal
cells, the hybrid cells had a normal level of SCE at the first mitosis after
fusion. On the other hand, single and twin SCEs in endoreduplicatedcells have been shown that an equal number of SCEs occurred in each of
the two cell cycles. On the basis of these data, a 50 % reduction in BS
chromosome SCE frequency could theoretically be expected to be observed
one cell cycle after fusion. But we observed a clearcut normalized levelof SCE. Therefore, the theoretical half reduction is clearly inconsistent
with that of our exact data (Shiraishi et al. 1981). In order to know, thefrequency of SCE in the first and second cell cycles the frequencies of single
and twin SCE were analysed in endoreduplicated cells induced by treatment
1)

Kochi Medical College, Nangoku, Kochi-ken.

RESEARCHES CARRIED OUT IN 1982

41

with colcemid. Each SCE that arised during S1 appearred as a twin SCE
at the second mitosis (M2), and an exchange that occurred during S2 gave
rise to a single SCE in only one of the pair of daughter chromosomes at
M2. In the present study we demonstrated that though a manyfold increaseof single SCEs was detected, twin SCEs were very rare. Namely 141 single
SCEs on the average were counted in endoreduplicated BS metaphase, while
only 5.4 twin SCEs were countable. This finding strongly suggests thatmost of BS SCE occur during second cell cycle when BrdU-containing
DNA is used as a template for replication.
Effect of BrdU on SCE in Welsh Onion and D-6 Chinese
Hamster Cells by Treatment with 5-Fluorodeoxyuridine
Hitoshi SUZUKI, Michiharu SEGAWA,1) Tosihide H. YOSIDA and
Katsuhiro KOND0 1 )5-Fluorodeoxyuridine, an inhibitor of thymidilate synthetase catalyzed
conversion of dUMP to dTMP, induced sister chromatid exchanges depending on the concentration of BrdU used for chromosome label during
the two cell cycles to detect SCEs in D-6 Chinese hamster in vitro as well as
Welsh onion in vivo. On the contrary, mitomicin C did not produce, at
any concentration, any dependent-effect of 5-Bromodeoxyuridine. In D-6
cells, when BrdU label was carried out only during the first cell cycle andFdU was added to the culture medium in the absence of labelling BrdU
during the second cell cycle, the SCE induction caused by FdU was completely suppressed by a synchronous addition of thymidine or BrdU, while
the induction was not suppressed by deoxycytidine or uridine. Based onthese studies, it was suggested that SCE induction by FdU is depended on
the thymidylate starvation in the process of DNA synthesis.

1)

Faculty of Integrated Arts and Science, Hiroshima University.

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Multiple Sex Chromosome Mechanism (XX-Y) Found in
Two dragonet Fishes
Makoto MUROFUSHI,* Shohei NISHIKAWA** and Tosihide H. YOSIDA

Although it has been considered that the sex-chromosomes are notdifferentiated in fishes, XX-Y mechanism has been found by us in filefish(Murofushi et al. 1980). Very similar case of the multiple-sex-chromosome
mechanism has been found to occur in two dragonet fish species, Callionymus
beniteguri and C. ornatipinnis collected from the near coast of Yamaguchi
and Shizuoka Prefecture. The diploid chromosome-number of the female
specimens of these two dragonet species was thirty-eight (2n=38), all elements being subtelocentric. In contrast, the diploid chromosome-numberof the male ones was thirty-seven (2n=37), among which one large metacentric chromosome was conspicuous in their karyotypes. The metacentricelement occurring only in the male specimens was the Y chromosome. Basedon the different chromosome-numbers in the male and the female in these
species it is suggested that the male has the X X 2-Y, while the female has
the X X 2-X X 2 • To confirm the above findings the secondary spermatocytes were investigated in the male specimens of these two species.
There were two cell types, one of them being n= 19, whereas the other n= 18.
In the former cells, one metacentric chromosome was always present,
whereas in the latter, no such element was found. The above findings haveshown that the sex-chromosome mechanism of these two species is maleheterogametic with an X X 2-Y mechanism.
The metacentric Y chromosome in the multiple sex-chromosome
mechanism occurring in these fishes should have been derived from theRobertsonian fusion between an ordinarily acrocentric Y and one memberof an autosome pair. These findings suggest that the Robertsonian fusion
between the Y chromosome and an autosome is to be one of the mechanisms
in morphological differentiation of the sex-chromosome in fishes.
j

j

j

j

* Mishima Junior College, Nihon University, Mishima.
** Shimonoseki University of Fisheries, Shimonoseki.

RESEARCHES CARRIED OUT IN 1982

43

Effect of H-2 Complex on the Frequency of Urethan-induced
Chromosome Aberrations in the Mouse Bone Marrow Cells
Kazuo MORIWAKI, Nobumoto MIYASHITA and Shinji NITO*

We reported that the susceptibility to urethan induced lung tumor is
genetically controlled by H-2 complex, in addition to ptr gene. To pursue
the mechanism of genetic regulation by H-2 complex for the induction oflung tumor, the frequency of chromosome aberrations after urethan treatment was surveyed in various H-2 congenic strains. To 6 weeks mice, 10 %urethan was given at 0.5 mg/g B.W.. After 24 hrs, valid chromosome
breaks were scored in the bone marrow cells. Percentage of aberrant cells
was 22.3 % in A strain (H-2 a ), whereas 11.0 % in A. BY (H-2 b ), 10.5 % in
A. CA (H-2 f ) and 6.8 % in A. SW (H-2
This tendency was confirmedin B10 H-2 congenic mice as well. Micronucleus test done in the samecombinations of the congenic mice exhibited parallel results with thosein chromosome breaks. Further cytogenetical surveys in H-2 recombinantstrains, A. TH and A. TL, suggested the possible relevance of a locus to
the right of H-2IE region.
S).

Implications of the Genetic Divergence between European
Wild Mice with Robertsonian Translocations from the View
Points of Mitochondrial DNA
Kazuo MORIWAKI, Hiromichi YONEKAWA**, Osamu GOTOH**,
Mitsuru MINEZAWA***, Heinz WINKING**** and Alfred GROPP****

Genetic divergences between the wild mouse populations with various
Robertsonian translocations from the Poschiavo Valley, Yougoslavia,
Milan and the Apennines, were estimated based on the mitochondrial (mt)DNAs. The mtDNA isolated from the liver were analysed by agaroseslab-gel electrophoresis after digestion with eight kinds of restriction endonucleases; BamHI, EcoRI, HindU, HindUI, Pst!, HpaI, Hpall and BglL
These preparations were further used to make restriction maps. From whichapproximate divergence time between each Rb variation was calculated
to be in the order of 105 years (Table 1). These data appear to be in con* Tanabe Seiyaku Co.
** Saitama Cancer Center Research Institute, Saitama.
*** Primate Research Center, Inuyama.
**** Medizinische Hochschule Lubeck, Lubeck.

t:
:>
Z
Z

Table 1. Effect of H-2 complex on the urethan-induced chromosome
aberrations and micronuclei
Chromosome aberration test
Strainc:::>

Micronucleus test

t""

~

tT'l

Number ofobserved cell

Breaks percell (%)

Percentage ofaberrant cell

Number ofobserved cell

800
700
400
600
300
800
300
300
700
400
400
700
500
400
400
400
400
600

19.1±4.0
39.3±4.937.0±9.1
30.7±4.431.3±1.535.9±8.922.7±1.2
37.7±1l.1
20.0±6.742.3±4.2
46.0±6.731.1±9.0
42.0±6.6
19.5±1.0
6.8±2.6
13.8±1.3

14.3±2.3
22.9±3.4
22.3±3.6
21.2±1.5
20.0± 1.0
20.4±3.3
15.7±4.0
25.7±5.8
14.3±4.4
27.3±1.5
27.0±2.2
22.3±2.1
22.2±1.6
17.0±0.8
6.8±1.3

4000
4000
4000
4000
4000
4000

ID.5±1.0

10.5±1.3
11.0±1.4

Micronucleus
per cell (%0)

"t:I

0

~

.-)

BID
BID. A
BID. BR

BlO. D2
BID. S(BI0xBI0. A)Fl
BID. A (2R)
BID. A (3R)
BID. A (4R)
BID. A (5R)
BID. S (9R)
AjWy

A.AL
A.TL
A.SW
A.TH
A.CA
A.BY

13.3±3.7

6000
6000
6000
4000
6000
4000
4000

ID.5±1.3

6000
4000

27.5±6.6
38.5±6.0
21.0±4.2
22.8±6.0
28.5±6.2
38.0±4.4
Not Tested
40.8±4.2
29.2±4.6
40.0±.5.1
Not Tested
30.3±5.4
23.8±4.0
25.3±5.918.8±9.3
Not Tested
30.3±4.2
28.0±4.9

0

'Tl

Z

:>
.-)

0
Z

:>
t""

.....

Z
.....

CZl
.-)

.-)

c::.-)
tT'l

0

'Tl

Cl
tT'l

Z

tT'l

.-)

.....
(')

CZl

Z

9
w
w

RESEARCHES CARRIED OUT IN 1982

45

Table 1. Sequence divergence and time of divergence estimated from
mtDNA among five variant Robertsonian populations and six
subspecies of Mus musculus
Robertsonian variations

mus
(D)

mus
(B)

mol

cas

bac

sequence divergence (%)
1.1
0.8 0.0
1.8
1.8
1.4 1.5 2.4
0.4
1.5 0.8
1.0
1.0
1.4 1.5 0.3
1.1
1.1
0.7

4.0
4.9
4.8
4.5
4.2

3.4
4.0
4.2
4.0
3.6

6.1
6.1
4.9
4.6
4.3

7.5
7.4
6.1
5.9
5.5

Time of divergence (x 106 years)
0.53 0.28 0.53 0.39
0.28 0.0 0.63
0.77 0.74 0.63
0.49 0.53 0.84
0.52 0.28 0.35
0.49 0.14
0.35
0.49 0.53 0.11
0.39 0.39 0.25

1.40
1.72
1.68
1.58
1.47

1.19
1.40
1.47
1.40
1.26

2.13
2.13
1.72
1.61
1.51

2.63
2.59
2.14
2.06
1.93

Zadar Mil-II CD

Pos
Zadar
Mil-II
CD
CB
Pos
Zadar
Mil-IICD
CB

M. m. subspecies

1.5

0.8
2.2

1.5
2.1
1.4

CB

dom

brv

flict with the present concept that the Rb variations occurred during thelast several thousand years. Both, however, might be reconciled by assuming genetic introgression of the founder with a small number of Rb
translocations into other subspecies populations genetically remote by theorder of 106 years, and the subsequent rapid accumulation of Rb translocations unique to each population due to a hybrid dysgenesis-like mechanism. Occurrence of a new Rb (9, 15) translocation in the intersubspecies hybrid between M. m. molossinus and M. m. domesticus on the Ogasawara Islands in Japan may support this notion.
The Effects of H-2 Complex to the Mutagen-Induced
Chromosome Aberrations and Micronuclei
Shinji NITo*, Nobumoto MIYASHITA and Kazuo MORIWAKI

Moriwaki et af. (1983) have recently reported that the H-2 complex affects
the frequency of urethan-induced chromosome aberrations.
In the present study, the effects of H-2 complex was examined with the
6 chemical mutagens (cyclophosphamide: CP, mitomycin C: MMC, adria* Tanabe Seiyaku Co.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

mycin: AD, potasium chromate, colchicin and colcemid) and r-ray.
We used 6-weeks male mice of the BlO and BlO. A congenic strains, andthe sensitivity to the mutagens (in terms of chromosome aberrations andmicronuclei) was compaired between the two strains, in which the haplotype of the BlO and B10. A is H-2 b and H-2" respectively, and the latteris sensitive to urethan.
The results are summarized in Table 1. The table clearly indicates thatthe BlO. A strain is more sensitive than the B10 strain to most of mutagens,except colchicin and colcemid which do not attack DNA molecule. Almost
the same tendency was obtained by the two methods employed (i.e., chromosome aberration and micronucleus test).
The high sensitivity of the BIO. A strain may be due to the DNA repair
capacity, and is controled by genes linked to the H-2 complex.
Table 1.

Comparison of effects of H-2 complex to the mutagen-induced
chromosome aberrations and micronuclei between the
BIO and BIO. A cngenic strains

CompoundDose(mg/kg)Number of breaks
per cell (~;;)
BIO (H_2 b )

CP
MMC
AD
PotasiumcromateColchicin
Colcemid
r-ray
N.B. Mean±SD.
marrow.

Number of micronuclei(%r)-----_._----

BIO. A(H_2 a )BIO (H-2 b )BlO. A(H-2 a )25
1.55

22.5±10.6 50.0±4.2*
18.0±0.0 40.5±7.8*
15.5±2.1
19.0±2.825.7±4.344.5±3.2
29 .O±3.6

34.3±5.4*54.7±6.0*
36.5±5.9*

50
1
10
200R
200R

NT
NT
NT
48.5± 7.8
69.0±0.O

5.0±0.628.3±5.0
1O.7±2.6
38.0±3.912.1±2.1*
24.2±1.6
2.7±2.433.5±4.6

NT: Not tested.

NT
NT
NT
70.5±5.0*
85.5±0.7** . P<O.05.

(+)

( +)

( +): Gross destruction of bone

RESEARCHES CARRIED OUT IN 1982

VI.

47

MUTATION AND MUTAGENESIS IN ANIMALS

Genetical Behaviour in the Silkworm (Bomhyx Mori L.)
Carrying an X-Autosome Translocation
Akio MURAKAMI

Translocations of the silkworm Y (previously W) to other chromosomes
including X(Z), could be easily detected. Several cases of such type translocations have been published and utilized in agricultural industry to date.
While no translocation of X to autosome has yet been published. Recently
we found two X-autosome translocations in this insect which had been treatedpupal oocytes with chemical mutagens, MNNG and Sterigmatocystine(STC). These two lines have a translocation between X chromosome andan autosome no. 5 linkage group. The present communication describes
genetical behaviours of the firstly obtained X-5 translocation which has
been derived from an MNNG-induced fractional-body mutant expressedboth wild-type black (+) and yellowish-white (pe) serosa membrane cells
in the egg. According to the genetical analysis of this line, a proximal
piece of the 5th linkage group chromosome which contains the wild-typeallele of the pe (pink eye and pink egg, 0.0), but not the ok+ locus (5: 4.7),-.,
is translocated onto the X chromosome or xpe+. This translocation line
is designated to be as the T(X: 5)1. Another X-5 translocation [T(X: 5)2] isoriginated in STC-induced fractional-body mutant and is also not included,-.,
at the ok+ region, Xpe+.
,-.,
When the females having the translocated X chromosome Xpe+ and,-.,heterozygous for egg-colour genes + -+- /pere or Xpe+ /Y: ++ /pere werecrossed with the males homozygous for the normal X chromosome andthe egg-colour genes, X/X:pere/pere, female black (X/Y: ++/pere) andyellowish-white (X/Y: pere/pere) eggs in their progeny would have been
free from the translocated chromosome. In male progeny, however, both
,-.,
,-.,
black (xpe+ IX: ++/pere) and red (xpe+ IX: pere/pere) eggs should be thetranslocation heterozygous for X chromosome. Consequently, the numberof black and red eggs should be expected in a ratio of one to one. In otherwords, the number of black, yellow, and red eggs per batch should besegregated in a ratio of 2: 1: 1. For reference, as is well known, egg-colour

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

for the pe and re gene is due to the colour in somatic (serosa-membrane)cells and the pe gene is epistatic to the re gene, so that eggs homozygous
for pe and re genes turn yellowish-white in their serosa cells.
Whereas the number of black eggs was clearly less than the expectation.
In opposition, yellowish-white eggs distinctly exceeded the expectation andred eggs came short of the expectation. In addition to these unusual segregations, a significant number of fleckered eggs which are composed of blackand yellowish-white serosa cells. The decrease of black or red eggs andthe increased of yellowish-white eggs might be interpreted on the assumptionthat the pe+ segment on the X chromosome did not at least partly affect on
the pe gene on the autosome no. S in the heterogametic male. The selec,.-.
tion for cells having the Xpe+ chromosome might have been influenced on
the decrease of the black eggs in males. It is also not reasonable to fully
exclude a possibility of a loss of the pe+ segment on the X chromosome.
The occurrence of the fleckered eggs might be caused by an incomplete
the way,
affection of the pe+ to the pe gene on the autosome no. S. By,.-.
the cross between females having chromosome constitution, x/ype+: ++/pere and males having X/X: pere/pere, it could not observed the unusual
phenomenon. The abnormal observation in the T(X/S)! line seems to beanalogous to findings in mice (Lyon, 1961; Russell et al., 1961; etc.) interpreted as the X chromosome inactivation hypothesis. It is possible tosuppose that mechanisms responsible for the X chromosome inactivation
appear to be a universal phenomenon regardless of the heterogametic female
or male. In any case, it required much more information to get a final
conclusion whether or not the present finding with the T(X: S)1 line in thesilkworm is due to the X chromosome inactivation.
The Induction of Gynogenesis and Androgenesis in the
Silkworm and the Mechanism of Their Induction
Susumu TAMAZAWA* and Akio MURAKAMI

Gynogenesis and androgenesis are known to be induced in the silkworm
relatively easily by low temperature treatment of the egg, but the mechanismof the induction has not been elucidated. Whether the ontogenesis is derived
from fusion of the egg nucleus and polocyte, fusion of two sperm nuclei
or from a single female or male nucleus can be theoretically assumed from* Faculty of Agriculture, Hokkaido University, Sapporo.

RESEARCHES CARRIED OUT IN 198249

the growth stage of the female and male nuclei in the eggs at the start of thetreatment. Therefore, we analyzed ontogenetically the relationship between the mechanism of gynogenesis or androgenesis and the developmental
stage of the sperm and egg nuclei. For the detection of androgenesis,
we used the hybrid: WZ +/w 2 +; ch+/ch+; pS/ps';f.XW2/W 2 ; ch+/ch; P+/p+e!
and for the detection of gynogenesis: W2 /W 2 ; ch+/ch; p+/p';f.XW 2 +/W 2 +;ch+ /ch+; pS/pse!. Thirty minutes was allowed for egg laying, and theeggs collected between 0 and 2lOth minute after being laid were kept at 0°or lOoC for 24 hours to induce abnormal ontogenesis. To identify theorigin of the individuals thus obtained, they were backcrossed with a line
having W 2 , ch and p genes homozygously. Then, the segregation of thevarious characters in the BF 1 were obtained.
In the BF 1 of the individuals induced from the eggs laid during 0-60
minutes, in the experiment to induce androgenesis, ch+: ch and p+ : p weresegregated at the ratio of I: I which confirms that they had been derived
from the fusion of 2 sperm nuclei. On the other hand, in the BF 1 of theindividuals induced 60-120 minutes in the same experiment, only ch+ andp+ were segregated in most, which confirms that they had been derived froma single sperm nucleus. The mosaic individuals with ch+ and ch detected
simultaneously were presumed to have derived from 2 sperm nuclei withdifferent genetic types.
In the experiment to induce gynogenesis, in contrast to the male nucleus
ontogenesis, both male and female were induced from gynogenetic individuals. The females all showed dominant larval characters (ch+; p+),and the male individuals had either dominant (ch+ ;p+) or recessive (ch;p)character. In the BF 1 of the female individuals hatched from normal
eggs, ch+; ch, p+ ; P and ';f.: e! segregated at the ratio of I: I, which confirms that they had been derived from the fusion of egg nuclei and polar
bodies with different genetic types. In the BF 1 of the male individuals,
only the dominant characters (ch+ or p+) were segregated, and they had
normal ability to fertilize. Thus, the males were confirmed to be 2n andhad derived from the egg nucleus with X chromosomes. Simultaneously,
triploids and tetraploids were frequently induced, but the mechanism of
induction of these polyploids is under examination.
In the present experiments, it has been genetically confirmed that androgenesis and gynogenesis can be induced by low temperature treatment of
the egg, and that as has been suggested previously, besides the individuals

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

derived from the fusion of 2 sperms or egg nucleus and polocyte individuals
produced by androgenesis and gynogenesis derived from single gamates,i.e., monogamy, can develop normally and they are capable of fertilization.
Low temperature treatment was suggested to have some effect on the ploidyof chromosomes (DNA) of female and male (pro)nucleus and to promote
the development of the individual just as if it was fertilized normally.
Cytological Analysis of Mutations Produced by Super-coolingof Silkworm Eggs at an Early Stage of Development
I. Polyploids
Susumu TAMAZAWA* and Akio MURAKAMI

Polyploids of silkworms are relatively easily induced by various physicaland chemical treatments of the egg. The polyploids frequently induced
are triploids. Although tetraploids were indirectly obtained by crossing
6n ¥ X 2nd', there were few cases of tetraploids induced directly by physicalor chemical treatment. However, when the oocytes obtained by crossing
between the stocks labeled by visible genes (p/p; re+/re+, W2/W 2, ch/chx
pS/ps, re/re, W2+ /W 2+, ch+ /ch+) were exposed to -lOoe for 24 hours at thestage just after to 90 minutes after the egg was laid, i.e., at first meiosis, andthen incubated at 25°e for 20 hours, hatched and reared by standard methods,
along with triploids which are presumed to have been produced by conjugation of a diploid synkaryon of oocytes produced by low temperature
treatment and one sperm as has been reported, tetraploid females were
found to be produced by syngamy of a triploid synkaryon produced during
the meiosis of autosome constitution (p/p/p/ps, w2 /w Z/W Z/w 2 +, ch/ch/ch/ch+)and one sperm. But no tetraploid males have been detected to date. The
autosome constitution of the egg given low temperature treatment during
the period beginning at the 90th minute after being laid until the 210th
minute (the first cleavage stage) via syngamy of egg (sperm)-pronucleus
stage, wasps/psp/p, W2 +/W2 +W 2 /W 2 ' re+/re, ch+/ch+ch/ch, and the sex chromosome constitution of the female and male tetraploids X/X Y/Y and X/X X/X
were obtained at the same frequency. The explanation given is that although normal chromosome duplication of the normally fertilized nucleus
can proceed at the first egg cleavage stage, the spindle fiber is inactivatedby the low temperature treatment. Thus the two daughter nuclei cannot* Faculty of Agriculture, Hokkaido University, Sapporo.

RESEARCHES CARRIED OUT IN 1982

51

divide and male and female tetraploids with 112 chromosomes are induced.
Cytological Analysis of Mutations Produced by Super-Coolingof Silkworm Eggs at an Early Stage of Development
II. Deletion of Chromosomes
Susumu TAMAZAWA and Akio MURAKAMI

We previously reported that many polyploids, mosaics including gynandromorphs, abnormally developing individuals derived from mono (or di)
sperm(s), and individuals induced by fertilization of single egg nucleus oregg nucleus and polocyte were produced when the eggs obtained by theabove mentioned cross or its reverse cross were treated with low temperature, -SoC (4 days)~-lOOC (1 day), during the early stage of development, that is, between the 90th minute after being laid which was immediately
after completion of meiosis and the 210th minute after being laid. Besides
these, many mottled silkworms with different degrees of deletion of fragmental chromosomes which include the pS locus on chromosome 2, weredetected. Mottled silkworm have been reported to be induced by ionizing
radiation. In such cases it has been considered that ionizing radiation causes
chromosome breakage and in the cells from which the chromosome fragment containing the dominant gene pS factors has been released, the recessive character (p) is expressed, and thus both recessive and dominant characters are expressed in a complicated pattern. This suggests that chromosomal fragmentation is' also induced by low temperature treatment, andthe fragmented chromosome is of the diffuse kinetochore type. Among
the F, of the treated individuals, normal (p) silkworms were frequently
detected besides the expected type, black stripes (pSjp). Genetical analysisof several of such exceptional silkworms showed that the spots of the larval
character were detected only in p and not in pS silkworms. This showsthat mutants were induced in pS (2-0.0), but this has been explained to be
due to deficiency of chromosomes caused by chromosome breakage near pS
rather than to the induction of gene mutations by the low-temperature treatment. Furthermore, many chocolate newly hatched larvae (ch) were detectedamong the F, besides the expected black newly hatched larvae (ch+ jch), andthe chocolate newly hatched larvae were also considered to be due to deletionat the ch+ locus of the allele of ch (13-9.6) because none of the BF, examined
for their phenotypic expression had the ch+ factor. Besides these mutants,

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

kidney shaped eggs (ki) and grey eggs (gr) were also found. By elucidating
the genetic characters of these mutants, a conclusive decision can be madeon whether the above mentioned exceptional silkworms were produced bybreakage of chromosomes or by gene mutations.
Locus Differential Sensitivity for the Induction of theSpecific-Locus Mutations after Irradiation of
Primordial Germ-Cells with X-rays
Akio MURAKAMIIn the silkworm, various systems for measuring the mutagenicity of internal .a-emitters at low dose-rates have been devised. Among them theexperiments have extensively been carried out with the use of primordial
germ-cells in diapausing eggs (or embryos). However, this system islacking in several data on the mutagenic response of the primordial germcells to radiations, among other things, the dose-mutation frequency relationships. The present communication reports the results of experiments
for the dose-response kinetics in the stage germ-cells induced with external
X-irradiation by the egg-colour specific-locus method. Diapausing eggs
of wild-type (ClOB X Aojuku)F 1 hybrids were irradiated with six different
doses (125, 250, 500, 750, 1000, and l250R) of 180 kVp X-rays (25 mA,
1.0 mm Al filter and a dose-rate at 1Rjmin). Non-treated materials were
set up for the control. After completion of diapause, both treated andnon-treated eggs were incubated at 25°C and hatched larvae were bred in
the usual way. The resultant moths were mated with marker stocks homozygous for egg-colour genes, pe and re, on the 5th chromosome for detecting
the recessive visible mutations.
The dose-mutation frequency curves obtained were non-linear (or multihit) fashion regardless of sex and clearly varied with the locus. Mutagenicity of males was significantly higher than that of females as well as otherstage germ-cells. The mechanism responsible for this sex-differential
sensitivity is not yet clear, but it may be caused by different repairability
and germinal selection for the cells having lesion between the sexes. Also,
the difference in mitotic division times and in time for the duration of one
cell cycle in primordial germ-cells (and gonial cells) may have influenceon the sex differential radiosensitivity.
The dose-response curves for both loci showed the same fashion to 750R,

RESEARCHES CARRIED OUT IN 1982

53

but mutagenicity of the pe+ locus was higher than that of the re+ locus.
The curve at the pe+ locus was exponentially increased up to the highest
dose at 1250R. At the re+ locus the curve was slightly increased from 750R
to 1000 R and declined to some extent at 1250 R. This difference in the doseresponse curve between the loci might be due to a gene site (locus) on thechromosome: the pe+ gene locus is locating on the proximal site (5: 0.0),
whereas the re+ locus on the middle portion (5: 31.7). According to an
well accepted opinion, most of the specific-locus mutations in higher organisms are considered to be either small deletion or deficiency type rather
than gene mutations at the molecular level. If this view is correct, thesystem used in this experiment, a single-break event at the pe+ locus, would
be sufficient to induce the deficiency and two-break events at the re+ locus
required for induction of the deletion. Such the difference in mutational
events between the loci would be affected a viability of mutants detected.
Actually, the viability of mutants at the pe+ locus was clearly higher thanthat at the re+ locus. The cluster size for the pe+ locus mutants per batchbasis was also significantly larger than that for the re+ locus.
It should be noted that hatchability for the mutants after treatment ofprimordial germ-cells with X-rays was markedly lower (less than 10 %)when compared with other stage germ-cells irrespective of locus. In addition, almost of all larvae hatched were lost before progeny tests. The
marked reduction of hatchability in F, and of viability in F 2 appeared to beanalogous to either dominant or inherited lethality, suggesting that X-rays
could induce a fairly large number of chromosomal rearrengements includingtranslocations and many other things in the primordial germ-cells of silkworms.
A Comparison of Mutagenic Activity of Ethylmethanesulphonate(EMS) and Methylmethanesulphonate (MMS) in Oocytesof the Silkworm
Akio MURAKAMIIn the silkworm, EMS is more highly mutagenic to pupal spermatozoa by
the egg-colour specific-locus method than MMS. According to the recent
studies on the dose-mutation frequency relationship in the spermatozoa
with EMS and MMS, it showed a linear fashion at a dose down to a noneffective (or threshold) dose. Based on these data, the non-effective dose

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

of EMS was calculated to be I x 10- 3 and that of MMS was 4.2 X 10- 3 p,//pupa. Similarly, diethylsulphate (DES) is more mutagenic to the spermatozoa than dimethylsulphate (DMS). Such the tendency is extended tonitrosoguanidines, ENNG and MNNG. These findings suggest that in
the silkworm, ethylating agents are generally potent mutagens to the low
metabolic spermatozoa as compared with methylating ones. The mechanism responsible for this differential sensitivity of the alkylating agents inthe spermatozoa is not clear. To understand the mechanism, we intended
to study the mutagenic activity of EMS and MMS in highly metabolic premeiotic oocytes at the mid-stage of pupae.
Freshly prepared EMS and MMS solution (1 X 10- 3 to I X 10- 1 pi/pupa)
in 0.85 % NaCl were administered by the injection method into the bodycavity of mid-stage wild-type F 1 (C108xAojuku) female pupae. Controls
were applied with 0.85 % NaCl solution alone. After emergence, they weremated with the marker stock homozygous for pe and re gene loci to detect
the recessive visible mutations.
The dose-response kinetics in the premeiotic oocytes with EMS showeda sigmoidal fashion regardless of loci. The frequency of mutations in thisgerm-cells treated with a nearly killing dose of MMS was just increased
above the control level. This situation was almost similar to dialkylsulphates, DMS. The mutagenic activity of these chemicals in oocytes at thelate stage of pupae had also been made by the specific-locus method. In
the EMS treatment group, a marked increase of the mutational incidence
was found in this stage oocytes as compared with the oocytes at the early or
mid stage pupa. While a very low frequency of the mutations was detectedfollowing the MMS treatment in the oocytes at the mid-stage pupa, theincidence of mutations was slightly increased in the late stage oocytes.
The present data and others clearly indicate that the ethylating agent
treatment, which has proven to be highly mutagenic at inducing the specificlocus mutations in matured spermatozoa, was slightly mutagenic in thisrespect in the oocytes. This situation was effectively pronounced in thetreatment with the methylating agents. The differential sensitivity between
oocytes and spermatozoa to the alkylating agents may be interpreted being
due to a different repair activity for the premutational damage in theirrepair system. The weak mutagenicity following exposure of the alkylating
agents to oocytes may be dependent on the effective repairability for the
premutational damage. Especially, the damage with the methylating agents

RESEARCHES CARRIED OUT IN 198255

seems to be more susceptible to the maternal repair system in ooplasma
as compared with the ethylating ones. The differential sensitivity of germcell types to any form of genetic damage by the kind of alkylating agentswould be also likely to be one of the interpretations. Another possibility
may be still considered that the methylating compounds were more effectively
subjected to inactivation in oocytes (or pupae) before the compound (orits metabolite(s» reaches the target cells. In any case, further studies are
required to check these possibilities to elucidate the differential mutagenic
activity between methylating and ethylating agents in a various type ofsilkworm germ-cells.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

VII.

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICROORGANISMS AND PLANTS
Analysis of DNA Damage Induced by .a-Rays of Tritiated Water
Tsuneo KADA, Yoshito SADAIE and Tadashi INOUE

We previously reported that the efficiency of inactivation of DNA by tritiated water (HTO, upto 100 p,Ci/m/) increases by lowering the HTO concentration and that such an increase in efficiency of inactivation leveled
off at concentrations of 0.01-0.001 p,Ci/ml (Sadaie et al., J. Rad. Res. 22:
387, 1981; Proc. 7th Inter. Congr. Rad. Res., Bl-18, 1983). It is probablethat the efficiency of damage production in DNA might be reduced at highconcentrations of the isotopes due to the "ineffective" recombination ofradicals. It is also possible that certain primary potential damages in DNA
might be efficiently progressed into fixed damages which would lead to theinactivation of molecules.
In order to obtain more information about the nature of DNA damages
involved in the above dose-rate effects of tritium p-irraidation, several
types of experiments were carried out.
First, strains carrying DNA-repair deficiencies such as uvr-19 and/or
ree-43 were constructed and used as recipients of irradiated transforming
DNA. UV-irradiation on transforming DNA using recipients carrying asimple repair deficiency (uvr or ree), moderately reduced the yield of transformants. This was in contrast to the results obtained using wild recirecipients. The double deficient strain (uvr ree) produced markedly reduced transformants. No such difference was found when transforming
DNA was irradiated to 3H-p-rays or 32 p-p-rays. It is suggested that tritium
irradiation caused DNA damage which can not be repaired by cellular repairfunctions working in the UV-repair.
HC-labelled covalently close circular (ccc) DNA of colicin £, was prepared and kept at O°C in a 100 mM NaCl-lO mM potassium phosphatebuffer (pH 7.0) containing 439 p,Ci/ml of HTO (p-irradiation at a dose rateof 128 rad/day). Since ccc DNA changes its sedimentation coefficient bya single nick in one of the DNA strand, we carried out neutral SDG analysis
every 2 days on the irradiated samples with or without a following treat-

RESEARCHES CARRIED OUT IN 1982

57

ment of AP-endonuclease. Results of the above analysis indicated thatthe dose of j3-irradiation producing one AP-endonuclease-sensitive site is
0.24 kR. When similar experiments were carried out with r-irradiation of
137Cs, it required 0.19 kR to produce one AP-endonuclease-sensitive site.
Therefore, RBE for production of apurinic or a pyrimidinic is 0.79 (=0.19/0.24).
One of the marked effects of tritium-irradiation concerns an in vitro
repair susceptibility. We previously reported that an appropriately diluted
crude homogenate of human placenta can repair r-irradiated transforming
DNA of Bacillus subtilis. No such repair was observed with 3H-j3-rays.
Development of an In Vivo Germinal Mutation Detection
System in Mice
Hideo TEZUKA, Tadashi INOUE and Tsuneo KADA

We are trying to examine the mutation induction with beta-rays fromtritiated water in mice using the Malling's method (Ansari et al., Proc. Natl.
Acad. Sci. 77: 7352-7356, 1980). Alteration of Lactate dehydrogenase-X(LDH-X; LDH-C4) from the original mouse-type to rat-type can be detected by an immunofluorescent antibody technique which enables to
examine one mutant sperm among 10 6 sperms on a microscopic slide.
There is a discrete species-difference with LDH-X but no strain difference
is detected so far.
LDH-X was purified from testes of matured male young rats (2.2 mg
of LDH-X from 39 animals) according to the Ansari's methods (Ansari,
Biochem. J. 199: 75-79, 1981). Rabbits were immunized with several
injections of rat LDH-X. Rabbit antiserum against rat LDH-X was
prepared (titer X 128 to X 256) and absorbed extensively with mouse sperms.
The resulting preparation was specific for rat LDH-X judged by the fluorescent antibody technique for artificial mixture of mouse and rat sperms.
We are now examining the mutation frequency in mice treated with procarbazine or ethylnitorosourea.
Using this method, 10 mice per experimental groups give reliable data
and therefore 50 animals for one experiment may be enough for evaluationof the mutagenicity due to an agent.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Decrease in r-Ray-Induced Mutations by PLD Repair
Inhibition in Chinese Hamster V79 Cells
Akiko YOKOIYAMA and Tsuneo KADA

Since the first demonstration of the repair of potentially lethal damage(PLD) in irradiated mammalian cells in culture (Phillips and Tolmach,1966), a number of studies have been carried out on factors and conditions
which modify repair efficiencies. Recently Nakatsugawa and Sugahara(1980 and 1982) found that purine analogues such as 3'-deoxyadenosine(3'-dA) significantly inhibited the repair of PLD, suggesting promisinguses of these chemicals to sensitize tumors in radiation therapy.
We studied the effect of 3'-dA on the frequency of radiation-induced
6-TG resistant mutations. The plateau phase cells of Chinese hamster
V79 exposed to r-rays from a 137Cs source were incubated for 0, 3 or 6hours in Hanks' BSS with or without 3'-dA, then diluted and inoculated toestimate the PLD repair as well as the induced mutation frequency. The
expression time of 5 days was adopted for 1000 rad irradiated cells. The
presence of 3'-dA in the course of post-irradiation repair incubation markedly reduced the capacity of repair in irradiated cells. The mutation frequency
decreased gradually in the course of PLD repair taking place in the cells
in Hanks' BSS. In the presence of 5 x 10- 6 M and 1 x 10- 4 M of 3'-dA, amarked decrease in the frequency of mutations was found, though there
were certain variations of mutation frequencies which could be caused byinactivation of 3'-dA by the cellular deaminase.
Adsorption of Pyrolysate Mutagens by Vegetable Fibres
Tsuneo KADA, Masayuki KATO!) and Shuhachi KIRIYAMA2 )It has been recently shown that pyrolysates of amino acids and proteins
contain highly mutagenic products (for review, see Sugimura, Cancer 49:
1970, 1982). We have previoysly shown that a number of vegetables suppressed mutabilities of certain pyrolysate mutagens (for review, see Kada,
Proc. 3rd ICEM, 355, 11982). A number of vegetables have also been shown
to possess a desmutagenic effect on the mutagenic activities of the crude
products obtained from broiled fish (Yoshikawa et al., 3rd ICEM 3P19,
1)
2)

Japan Biological Chemistry Centre, Gifu-ken.
Dept. of Agriculture, Hokkaido University, Sapporo.

RESEARCHES CARRIED OUT IN 1982

591981). Later we found that one of the desmutagenic principles in cabbage
was a hemo-protein exhibiting peroxidase activity (Inoue et al., Agric. BioI.
Chem. 45: 345, 1981). From a group of vegetables such as burdock whichcontain heat-resistant factors we isolated a comples compound having amolecular weight higher than 300,000 (Morita et al., 3rd ICEM, 3P20, 1981).
We summerize in this communication our results showing adsorption ofpyrolysate mutagens by vegetable fibres.
Fibres of vegetables were prepared according the procedures describedby Kiriyama et al. (The Jour. of Nutrition 109: 388,1979). To each aqueous
solution of Trp-P-I, Trp-P-2 or Glu-P-I, vegetable fibres were added andkept at room temperature for several hours. The mutagenic activities
before and after the addition of fibres were determined using the Ames'
strain of Salmonella TA98 with S9 activation. The fibres of following
vegetables reduced markedly the mutabilities of the above three pyrolysate
mutagens: cabbage, burdock, radish, banboo shoot, Japanese onion, carrot,
peaman, spinach and soybean sprout. Results of different studies showedthat the mutagens were adsorbed irreversibly by the fibres.
Antimutagens
Tsuneo KADAIn the past ten years, a number of mutagens have been detected as aresult of mutagenicity assays using microorganisms. They include not
only synthetic chemical compounds but also many natural compounds.
On the other hand, little information has been available about antimutagenic
factors in the environment. We carried our systematic screening of antimutagens in our environment and found a number of new natural antimutagens. They include certain biometal (cobalt and germanium) compounds, ingredients of certain spices and food such an cinnamon and green
tea, crude homogenates of mammalian placental tissues, etc. Chemical
characterization was done on some active principles such as protoanemoninof Ranunculus and Anemone (Minakata et al., Mutation Res. 116: 317,
1983).
We previously showed that cobaltous chloride was effective in reducing
mutations of the base-change type. However, our recent studies revealed
that this metal compound worked as an efficient antimutagen in certain
frame-shift mutagenesis (Mochizuki and Kada, Mutation Res. 95: 145,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

1982).
We collaborated with Dr. T. Ohta et al. (Institute of Environmental
Toxicology, Tokyo) in studies on cinnamaldehyde, an active principle ofcinnamon. Cinnamaldehyde markedly suppressed mutations induced by
4NQO or AF2 in Escherichia coli WP2 uvrA- trpE-. Mutations induced
by MMS or EMS were slightly inhibited. However, cinnamaldehyde was
not effective on MNNG-induced mutagenesis of MNNG. Different aspects
in the mode of action of cinnamaldehyde suggest that this antimutagen mightact by reducing errors of an inducible error-prone DNA repair pathway(Ohta et al., Mutation Res. 107: 219,198; Ibid. 117: 135, 1983).
DNA Repair in Competent Cells of Bacillus subtilis
Izumi MITA, Yoshito SADAIE and Tsuneo KADA

Two main pathways, excision repair and postreplication repair, are known
to operate in DNA repair in bacterial cells in the dark. Usually, thesepathways have been studied with rapidly growing vegetative cells. Generally, the former process functions without chromosomal replication, andthe latter process requires chromosomal replication. The culture of Bacillus
subtilis contains, in addition to vegetative cells, competent cells which takeup exogenous DNA and appear to be derived from early sporulating cells.
Competent cells appear to be metabolically less active and nondividing, andhave resting chromosomes. Therefore, DNA damage in competent cells
might be repaired by an excision repair process but not by a postreplication
repair process. Further, DNA repair in these specific cells might have aunique feature related to cellular differentiation, because competent cells
seem to be derived from sporulating cells.
To investigate whether or not excision repair and postreplication repair
can function in competent cells of Bacillus subtilis, a series of isogenic transformable strains of Bacillus subtilis carrying the uvr-19 or rec-43 mutation
or both were constructed. Both mutations are assumed to block excision
repair and postreplication repair respectively. The number of competentcells was scored as that of Trp+ transformants.
The mutations uvr-19 and rec-43 made both competent and noncompetent cells highly sensitive to UV irradiation and their effects were additivein a doubly deficient strain. We also examined the effects of uvr-19 andrec-43 mutations on the repair of UV-irradiated exogenous transforming

RESEARCHES CARRIED OUT IN 1982

61

DNA in competent cells. Transforming DNA was UV irradiated andused to transform the competent cells of repair-proficient or deficient strains.
The uvr-19 and rec-43 mutations reduced the repair capacity of the wildtype strain to a certain extent, whereas competent cells with two mutations
lost the capacity to repair transforming DNA to a greater extent. Therefore,
UV-induced damage in transforming DNA seemed to be repaired efficiently
and independently by processes requiring uvr-19+ and rec-43+ gene products.
The strains used in this study carry a long deletion (about 2 X 107 daltons)
fdpAI, which blocks the utilization of gluconate or inositol as a carbon source.
The transformation from Fdp- to Fdp+ was very sensitive to UV irradiation because of a large target size. The uvr-19 mutation did not affect the
UV sensitivity of Fdp+ transformation, whereas the rec-43 mutation did.
These results indicate that the uvr-19+ -dependent repair process could notfunction on transforming DNA (possibly distinct by having been converted
to a single strand and looped out at the site of the fdpAI mutation of thehost chromosome), whereas the rec-43 +-dependent repair process could
function. (J. Bacteriol. 155, 933, 1983 and 153, 813, 1983).
Effects of TPA on the Mutagenicity of Caffeine and Gamma-raysin the Soybean Test System
Taro FUJII, Masumi SHIZAKI, Hirota FUJIKI and Takashi SUGIMURA

12-0-tetradecanoylphorbol-13-acetate (TPA), a well known tumor promoter in mouse skin carcinogenesis, was tested for its effects on mutagenesis,
using a soybean test system which was indicated by the appearance of variously colored spots on leaves. The mutagenic activity of TPA was tested
at concentrations of between 2 and 20 p,g/ml. No differences in the average
number of spots per leaf were observed. When seeds were treated with
0.01 %caffeine solution, averages of 3.7 mutant spots per leaf were observed.
These spots were classified into 3 types, yellow (forward mutation), darkgreen (backward mutation) and double (somatic crossing over) spots.
Treatment with TPA at concentrations of 2 and 5 11g/ml reduced the mutagenicity of caffeine, giving averages of 2.5 and 2.2 spots, respectively. Whenseeds were treated with 0.02 % caffeine solution, the total number of mutant
spots increased to about 10 spots per leaf. Treatment with TPA at a concentration of 5 p,g/ml reduced the total number of spots to 4.4 per leaf.
However, no reduction of spots was observed at a concentration of TPA

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

of 2 flgfml. Treatment with 0.03 % caffeine solution, treatment with TPA
at a concentration of 20 flgfml significantly decreased the total number ofspots from 15.8 to 8.9. However, no decrease in spots was observed whenseeds were treated with 0.05 % caffeine solution simultaneously with 10 and
20 flgfml of TPA. The remarkable finding was that the concentration of
TPA modified the mutation frequency greatly, depending of the concentration of caffeine. Interestingly, TPA did not reduced the mutagenicity of
0.05 % caffeine solution.
To examine the TPA action on radiation mutagenesis, combined treatment of TPA and gamma-rays were studied. TPA post-treatment did notaffected the gamma-ray mutagenesis (500 R). Spotting frequency of 11.3
and 11.9 per leaf observed in 10 and 20 flgfml TPA post-treatment, respectively, were similar to that obtained in water post-treatment plot. Sincesome chemicals have a potentiating effect of radiation induced damage
when they were present in the material, gamma-ray post-treatment werecarried out to seeds pre-soaked in water or TPA. Mutant spots increased
considerably with gamma-ray treatments, but there were no differences
between radiation treatments singly and in combination with TPA.
Because of the difficulties in the handling of the present material, soybean
seeds were treated with solutions of TPA plus caffeine. Reduction of mutant spots was actually observed on treatment with these solution. It is
unknown how TPA reduced the mutagenicity of caffeine, TPA suppresses
non-repair-associated DNA fragmentation in murine and human cells,
and also inhibits the appearance of this DNA fragmentation after X-irradiation. Although it is still unknown whether this suppressive effect of TPA
is related to reduction of the mutagenic activity of caffeine, the promoter
had no effect on the gamma-ray-induced mutation frequency. Therefore,
TPA presumably reduced the concentration of active caffeine molecules
inside the embryo cells of the seed. The observation that TPA had noeffect at the highest concentration of caffeine used is in accord with such amechanism of action.

RESEARCHES CARRIED OUT IN 1982

VIII.

63

POPULATION GENETICS (THEORETICAL)The Neutral Theory as a Basis for Understanding
the Mechanism of Evolution and Variation
at the Molecular Level
Motoo KIMURAThe neutral theory (or more precisely, the neutral mutation-random
drift hypothesis) asserts that the great majority of evolutionary changes
at the molecular level are caused by the random fixation of selectively neutralor nearly neutral mutants in the species, rather than by positive Darwinian
selection. It also claims that much of the intraspecific genetic variability
at the molecular level, such as is demonstrated by protein polymorphisms,
is selectively neutral so that polymorphic alleles are maintained in the species
by mutational input and random extinction. Thus the neutral theory
regards protein and DNA polymorphisms as a transient phase of molecular
evolution. The essential aspect of the neutral theory is not that the alleles
involved are selectively neutral in the strict sense. Rather, the emphasis
is on mutation and random drift as explanatory factors in molecular evolution because the selection intensity involved is exceedingly small. The
theory does not deny the occurrence of deleterious mutations. In fact,
selective constraint due to such negative selection is a very important partof the neutralist explanation of some important features of molecular evolution. The neutral theory does not deny the possibility that some changes
are adaptive. Thus it is by no means antagonistic to the Darwinian theoryof evolution by natural selection. However, because of its emphasis on
mutation and random drift, and also because of its concern with negative
selection rather than positive Darwinian selection, the theory clearly differs
in its theoretical framework from the traditional neo-Darwinian or "synthetic" theory of evolution.
The neutral theory is accompanied by a well-developed mathematicaltheory based on the stochastic theory of population genetics. The latterenables us to treat evolution and variation quantitatively, and therefore
to check the theory by observations and experiments. The neutral theory
in the above sense does not rule out the possibility that the mutant alleles

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

involved have different effects on form and function. These mutants canbecome selected under a special circumstance, even if they are selectively
neutral (i.e., selectively equivalent) under the prevailing environmental
conditions. This means that "neutral" variations can be the raw material
for adaptive evolution. Some important topics relating to the neutral
theory are; (i) how it explains the main features of molecular evolution;(ii) what kind of mathematical models are employed to treat the mechanism
for the maintenance of protein polymorphisms; (iii) how it differs from rival"selectionist" hypotheses in the face of hard facts; and (iv) why neutral
mutations are so prevalent at the molecular level, and how they relate tonatural selection at the phenotypic level. For details, see Kimura (1982)
In Molecular evolution, protein polymorphism and the neutral theory (ed.
Kimura, M.), pp. 3-56. Japan Scientific Societies Press, Tokyo and SpringerVerlag, Berlin Heidelberg New York.
Linkage Disequilibrium Due to Random Genetic
Drift in Finite Subdivided Populations
Tomoko OHTA

Linkage disequilibrium between two linked loci was studied for a finite
population with a subdivided population structure. Wright's island model
was used; extinction and replacement of colonies were also incorporated.
Two alleles with symmetric mutation rates were assumed at each locus(AI and A z at the first locus, and B 1 and B 2 at the second locus) and equilibrium properties of linkage disequilibrium coefficients were analyzed. In
terms of the analogy with the subdivision of inbreeding coefficient, thevariance of linkage disequilibrium is divided into several components:
Dis (variance of within-colony disequilibrium), D~T (variance of correlationof Al and B) on different gametes from the same colony relative to that of
the total population), and DJT (total variance of disequilibrium). Alternatively, the total variance can be subdivided into Dl~ (variance of correlationof Al and B 1 on one gamete from a colony relative to that of the average
gamete of the population) and D~zT (variance of the ordinary disequilibriumof the whole population). When migration is limited, the variance becomes large if the correlation of Al and B 1 of one colony is taken relative
to that of the whole population (D'i,r and Dl~)' Also, when the rate ofextinction-replacement of colonies is high, the whole-population disequili-

RESEARCHES CARRIED OUT IN 1982

65

brium coefficient (D~2T) can become fairly large. The analyses were extended
to the infinite aIlele model, and it was found that the disequilibrium components may get very large when migration is limited if the correlation ofalleles at the two loci within a colony is taken relative to that of the entire
population. In other words, with limited migration, random genetic driftof gamete types prevails in each colony. A possible test in which thevariance components of disequilibrium are compared is suggested; the test
discriminates between epistatic natural selection and limited migration,
showing the latter to be the main cause of various observed linkage disequilibria. In particular, observed linkage disequilibria, such as thoseamong markers in the major histocompatibility complex of man and mouse,
may well be explained by limited migration, without assuming epistatic
natural selection. For details, see Proc. Natl. Acad. Sci. USA 79, 19401944, and Genetics 101,139-155.
Further Study on the Genetic Correlation
between Members of a Multigene Family
Tomoko OHTA

The extent of genetic similarity (in terms of identity coefficients) wasinvestigated among members of a multigene family that is evolving undermutation, unequal crossing-over and random genetic drift. The method of
Kimura and Ohta (1979) was used, but the possibility was incorporated
that the length of the shift (in terms of the number of genes) involved inunequal crossing-over can be more than one gene unit. Extensive numericalanalyses show that, at equilibrium, the identity coefficients between twogene members are practicaIly independent of their distance apart on thechromosome when the mean length of the shift at unequal crossing over ismore than 10 % of the total size of the family. In that case, the approximate treatment by Ohta (1980) is shown to be valid, but when the mean
length of the shift is less than 10%, the average identity coefficient at equilibrium is underestimated. In order to clarify the effect of natural selection
responsible for keeping the family size stable, Monte Carlo simulation studies
were performed. The results indicate that the identity coefficients are
not greatly influenced by natural selection on gene family size, particularly
when the number of genes shifted is smaIl compared to the family size. In
addition, with sufficiently strong selection on family size, unequal cross-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

overs with a large shift (such as with maximum shift of 90 % of the familysize) contribute very little to evolution and their net effect is almost indistinguishable from those with a small shift. For details, see Genetics
99, 555-571.

Allelic and Nonallelic Homology of a Supergene Family
Tomoko OHTA

A model to explain the high degree of polymorphism at the major histocompatibility complex (MHC) is described. The model incorporates
domain transfer between the different loci in a supergene family by either
gene conversion or double unequal crossing-over. Population genetics
theory is used to formulate changes in the probabilities of allelic and nonallelic gene identities and equilibrium values are obtained. The observed
degree of allelic and nonallelic homology in the complex can be explainedby assuming that a domain is converted at a rate of 10- 5 to 10- 6 per generation and reasonable values of other parameters. This rate of domain transfer
is compatible with the observed high mutation rate at marker loci in the
major histocompatibility complex. For details, see Proc. Nat!. Acad.
Sci. USA 79,3251-3254.

Linkage Disequilibrium, Genetic Distance and Evolutionary Distanceunder a General Model of Linked Genes or a Part of the Genome
Naoyuki TAKAHATA

A general model of linked genes or a part of a genome is proposed whichassumes multiple states at each of linked multiple sites (loci) and whichenables us to study various problems in molecular population genetics ina unified way. Based on the diffusion approximation method, several
formulas with special reference to linkage disequilibrium and genetic distanceare derived for neutral mutations in finite populations. In particular,when the r sites are completely linked and the mutation rate v is independentof site, the mutation scheme at each site following the so-called K allele
model, the equilibrium identity probability of the r sites within a populationof the effective size N. is given by

RESEARCHES CARRIED OUT IN 1982

67

1
+
rf)
r= TtKrf)(1+Krf)(1+K(r-1)f)P

r(r-1)f)2
r-l

n (r-j)f)r
Il (1 + K(r-j)f)
;=0

+ ;:~

(1)

where f)=4N.v/(K -1). Note in (1) that the first term on the right-handside is equivalent to the homozygosity expected on the K allele model.
Clearly, Pr-:::=.l/(1 +Krf) when f) ~ 1. On the other hand, the identity probability between two isolated populations of the divergence time t generations
is given byJr(t)= lr

Eo [r Cp { ~o pCi -1)qKP-qJp_iO)}e-2PKvt/(K-1I]

where rCp is the binomial coefficient.

(2)

Formula (2) reduces to

Ji t )={ ~ +(I_+)e-ZKvt/(K-llrfor Jp(O) = I (p=l, 2, "', r) and it also reduces toJr(t)=Jr(O)e-Zrvt

(3)

(4)

for K=oo. Formulas (3) and (4) are closely related to Jukes and Cantor's
evolutionary distance and Nei's genetic distance, respectively. It is argued
that the present model and formulas are potentially useful to analyze DNA
sequence data from the standpoint of population genetics. (Genet. Res.
1983,39: 63-77).

Sexual Recombination under the Joint Effects of Mutation,
Selection, and Random Sampling Drift
Naoyuki TAKAHATA

The advantage or disadvantage of sexual reproduction or recombination
for the accumulation of mutant genes in a panmictic population is studied
under the joint effects of mutation, selection, and random sampling drift.
Three different methods are used; numerical integration of Kolmogorov
backward equations, simulation of stochastic difference equations, and68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Monte Carlo experiments. Comparison of the results between sexual andasexual populations shows that the effect of recombination depends on initial
linkage disequilibrium, mutation rate v, selection intensity s, and population size N e • The mode of selection is also an important factor and thelarge effect of recombination is observed when mutant genes are individually deleterious (advantageous) but collectively favorable (harmful).
Under a given model of selection, the large effect of recombination is expected when genetic variation is produced not by mutation but by recombination. Extreme values of Nev and Nes make the effect insignificant.
The long-term benefit of recombination in a panmictic population is expected
only under rather stringent conditions; thus, the present results do not support the classical view that the widespread occurrence of sexuality in theliving world may be accounted for recombinational advantage for selection.
(Theoretical Population Biology, 1982,22: 258-277).
The Disappearance of Duplicate Gene Expression
Naoyuki TAKAHATA

Based on a numerical method for integrating Kolmogorov backward
equations and a simulation method for solving stochastic differential equations, I studied the process of fixation of null (non-functional) alleles at
duplicate loci. Assuming that normal alleles irreversibly mutate to nullalleles, and that the population is finite and panmictic, I obtained the mean
and median times until fixation measured in unit of generation, the amountof heterozygosity at specified times and the trajectories of null allele frequencies under various types of selection. It is shown that duplicate genes
may rapidly return to the diploid condition when only double homozygotes
for null alleles are selected against and linkage is tight. On the other hand,a prolonged retention of duplicate gene expression is expected if the heterozygous effect on fitness is pronounced. We have related the results of theseanalyses to recent observations on amounts of polymorphism, on loss of
duplicate gene expression in tetraploid organisms, and on pseudo genes inmammals. Evolutionary implications of duplicate genes are discussed
quantitatively from the viewpoint of population genetics. (In Molecular
Evolution, Protein Polymorphism and the Neutral Theory (1982) (ed. M.
Kimura), pp. 169-190. Japan Scientific Societies Press, TokyojSpringerVerlag, Berlin).

RESEARCHES CARRIED OUT IN 1982

69

Estimation of Evolutionary Distance under a Mathematical
Model of Extranuclear DNA Molecule
Naoyuki TAKAHATA

The evolutionary distance between two related populations is studied
based on a mathematical model of transmission genetics of extranuclear
genomes. Several formulas are derived assuming neutral mutations, whichallow us to estimate the distance in the presence of not only intrapopulationalvariation but also within-cell variation. Disregard of back and parallel
mutations in distant populations underestimates the distance while neglectof intrapopulational and within-cell variations in closely related populations overestimates the distance. The formulas take into account thecomplete linkage between nucleotide sites in question so that they are potentially useful to analyze data obtained by the use of restriction enzymes.
In the light of the present study, we can rigorously examine the situations
under which the use of several conventional formulas may cause serious
bias in estimating the distance. (RIMS, Kokyuroku 457, 141-156, 1982).
A Condition for Group Selection to Prevailover Counteracting Individual Selection
Kenichi AOKIThe dynamics of a deleterious gene favored by group selection (a gene foraltruism) are studied, based on a one-locus two-allele model similar to themodels of Levins (1970), Eshel (1972), and Levin and Kilmer (1974). The
genetic system is assumed to be haploid, but the main theoretical result
can be generalized to the diploid case. Let N be the effective group size,m the migration rate, s the selection coefficient against the gene, and a(p)the non-extinction probability of a group with fraction p of the gene. Given
the distribution of p among the (infinite number of) groups with mean p
and variance a p 2, let b and a be the regression of a(p) on p and the mean ofa(p), respectively, with k=.bla. Then if e is the maximum of the positive
quantities liN, s, and k, and if we can ignore terms of 0(e 2 ) (0 indicatessame or smaller order), we have as the condition for the increase of p,a 2Sf='p(T~p» s+k .
If we assume further that e= liN and that recolonization is of the propagule

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

pool type (Slatkin, 1977) or a~ 1 so that the effect of recolonization on a/
can be safely neglected, and if, as numerical results indicate,jtakes transient
values that are within 10 % of the equilibrium value in the absence of individual and group selection, this condition is in the simplest casekNm<- 2s
given k/s-O(1). Thus if, for example, individual and group selection areof the same intensity, then the number of "immigrant genes" per groupper generation must be less than 1/2, which indicates a fairly strong isolation.
In order to check the validity and the range of applicability of the above
formulae, and to observe the behavior of the model where selection is strong,
the recurrence equations in the probability distribution of Np were iterated
numerically, for group sizes of 10 and 100. Monte Carlo experiments werealso conducted as a further check and to observe the effect of finite group
number. For details, see Evolution 36, 832-842.
Group Selection for a Polygenic Behavioral Trait:
A Differential Proliferation ModelJames F. CROW and Kenichi AOKIConditions for natural selection to increase a polygenic behavioral trait
are derived for a model in which the population is divided into a very large
number of partially isolated groups of variable and varying size. Specifically, we consider an altruistic trait that is deleterious to the individual
but raises the mean fitness of the group. We assume, for each generation,that all groups have the same proportion of males, k, at the time of migration and that each group contributes M f females and M m males to a poolof migrants, from which M f females and M m males are randomly parceledout to each group. This assumption ensures that, at equilibrium between
random drift and a low level of migration and neglecting the small per locus
effect of selection, each group has the same expected value of Wright's fixation index, FST=F. At equilibrium, this is approximately 1/(1 +4M.),
where M.=2kMf +2(1-k)Mm • The trait will increase when (b-c)/c>(1- F)/2F=2M., where b is the expected benefit to the group and c is the
expected cost of a unit change in the mean value of the altruistic trait. In
particular, the group selection analogue of Hamilton's inequality, c/b<r,
where r is the coefficient of relationship, is obtained. The effect of isolation

RESEARCHES CARRIED OUT IN 1982

71

is enhanced if migration is mainly between adjacent groups and if groupsplitting is along family lines, as data on population structure of primates
seem to indicate. For details, see Proc. Natl. Acad. Sci. USA 79, 26282631.
Additive Polygenic Formulation ofHamilton's Model of Kin Selection
Kenichi AOKI

Hamilton's condition for the evolution of altruism by kin selection isrederived on an additive polygenic model. The approach uses intraclasscorrelation. It is then shown that the mean inclusive fitness is non-decreasing. A common ground for the comparison of kin and group selection
is provided, permitting a discussion of the differences between and therelative efficacy of kin and group selection. For details, see Heredity, 49,
163-169.
Polygenic Altruism and Extinction Group Selection
Kenichi AOKIConditions are derived for altruism to evolve by the differential extinctionof groups. The altruism is assumed to be determined by many loosely
linked genes, acting additively within and between loci, and an independent
environmental component. The results confirm those obtained on a monogenic model by a different method in a previous paper (Evolution 36, 832842). For details, see Jpn. J. Genet. 57, 297-300.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

IX.

HUMAN GENETICS AND HUMAN CYTOGENETICS
Hereditary Retinoblastoma: Lack of Maternal Effect
Ei MATSUNAGA

The question of possible maternal effect on variable penetrance and expressivity in hereditary retinoblastoma is of interest for two reasons. First,
because a significant proportion of patients with retinoblastoma are diagnosed shortly after birth, tumor formation can be initiated in the intrauterine stage, so that transplacental maternal factors may modify the degree of expressivity. Second, retinoblastoma-like tumors can be producedin rodents and baboons by the injection of human adenovirus 12. Although
the transmission pattern of retinoblastoma in certain kindreds showing
affected collateral relatives who are connected via unaffected persons could
be explained by a chromosomal mechanism as well as by successive selection of host resistance genes, the possibility of delayed mutation whichassumes a labile premutation cannot be excluded. Such a premutation
may involve a vertically transmitting tumor virus that finally becomesintegrated into the host genome in the germ cell line. If this is the case,
we may expect more instances of transmission through maternal than
through paternal lines, at least until the supposed agent is intergrated into
the host genome.
Analysis of two-generation data from published pedigrees with familial
cases of retinoblastoma showed that, while the proportion of bilateralcases among affected children varied consistently with expressivity of theparents who transmitted the gene, the proportion did not differ with thesex of the parents. These findings support the view that inherited hostresistance to the development of hereditary retinoblastoma is determinedby modifying genes, and not influenced by transplacental maternal factors,
and that the resistance is mainly directed to the induction (promotion)phase. As a by-product of this study, it was found that there was a significant deficit of bilaterally affected mothers relative to bilaterally affected
fathers, which could not be accounted for by ascertainment bias or by differential survival or fertility between sexes. It is suggested that female
patients who survived bilateral retinoblastoma had much less chance ofRESEARCHES CARRIED OUT IN 1982

73

marriage as compared to male survivors. Examination of pedigrees withmultiple cases ofretinoblastoma, transmitted via unaffected carriers, revealed
no sign of cytoplasmic inheritance associated with a vertically transmitting
virus. For details, see Hum. Genet. 62: 124-128, 1982.
Almost Synchronous Appearance of Bilateral Retinoblastomas
Ei MATSUNAGA

Reexamination was made of previously published data (Matsunaga, E.
1981: Am. J. Med. Genet. 8: 375-387) concerning distribution of 139
bilateral cases of retinoblastoma by Reese group as ascertained when the
diagnosis was first made in either eye. In 124 (89.2 %) of the 139, thedisease was already bilateral when the diagnosis in one eye was made,although in such cases the tumor in the first eye with the diagnosed disease
had usually progressed more than had the tumor in the second eye, resulting
in a negative correlation between eyes with respect to the Reese group oftumors. However, there was a high positive correlation between ages ofthe patients at diagnosis in the right and left eyes, even after removal of
the bias due to the fact that the diagnosis for each eye was never independent.
Furthermore, the proportion of simultaneously bilateral cases with tumorsof the same group in both eyes (20/139= 14.4 %) was significantly higher
than the expected (6.1 %) assuming independent diagnosis for each eye.
These two findings seem to contradict the two-mutation model whichassumes that in the gene carriers the eyes acquire tumors independently.
One may argue that if tumors in the gene carriers were initiated by asomatic mutation at a very early stage of development, the simultaneous
occurrence of bilateral tumors in both eyes would be explained. If thiswere the case, then assuming equal rate of tumor growth, the age at diagnosis of the patients with tumors of the same Reese group in both eyes
should tend to be much lower as compared to the patients with sequentiallybilateral cases or with tumors of different Reese group in each eye. Contrary to this expectation, the mean age of the 20 patients at diagnosis (andstandard deviation) was 11.7 months (6.4), which was significantly (P<O.OI)
higher than the mean of 9.0 months (8.5) for the remaining 119 patients.
This difference is probably due to bias in ascertainment; in the latter groupof patients, clinical signs of the eye with the advanced tumor could havebeen contrasted by the second eye with or without a growing tumor and74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

so the parents brought their children earlier to a physician than in the caseof the former group. For details, see Am. J. Med. Genet. 11: 485-487,
1982.
Incidence and Prevalence of Genetic Disease (Excluding
Chromosomal Aberrations) in Human Populations
Ei MATSUNAGA

Detailed knowledge of the birth frequency or the cumulative incidence
over all ages of genetic diseases in human populations is a prerequisite forassessing the magnitude of possible genetic hazards caused by environmental mutagens. However, both theoretical and practical difficulties
are involved in precisely measuring the total frequency of these diseases.
Two sets of data from large-scale population studies, one from Northern
Ireland and the other from British Columbia, are compared with eachother and with the results from ad hoc surveys for individual monogenic
disorders. With due allowance for differences in approach, examination
indicates that the data from the large-scale population studies are inadequate.
However, it could provide a crude estimate of the total frequency of genetic
diseases and a fairly reliable estimate of the individual frequency of certain
genetic disorders with early onset that are familiar and readily diagnosed.
In addition to environmental mutagens, there are a number of factors
associated with current human activity that may change the incidence ofgenetic diseases. In order to monitor the human population for environmental mutagens, the change in frequency of sporadic cases of those genetic
diseases that arose from fresh mutation and that can be easily detected as
early as possible should be followed closely. The mechanism of datacollection currently being employed in some countries for childhood cancers,
certain congenital malformations, and inborn errors of metabolism could
be extended to include the so-called sentinel phenotypes. The rationale
and feasibility of using retinoblastoma and Wilms' tumor (nephroblastoma)
as examples of such population monitoring are described. For details,
see Mutation Res. 99: 95-128, 1982.

RESEARCHES CARRIED OUT IN 1981

75

Distribution of Break Points in Human Structural Rearrangements
Yasuo NAKAGOME, Takako MATSUBARA and Hiroko FUJITA·

In 1976, Nakagome and Chiyo (1976) reviewed 117 reported unrelatedcases with two-break rearrangements and found that among 220 breakage
points, breakages were preferentially located within G-light bands. Similar
observations have been made by others (Nielsen & Rasmussen 1976; Yu
et al. 1978; Evans et al. 1978). On the other hand, there have been reports
in which the breakages were located at a junction (interface) between adjacent G-dark and G-light bands (Dutrillaux et al. 1977; Aurias et al. 1978;
Brogger 1978).
In the present study, we attempted to resolve the issue on the location of
breakages. Three types of structural rearrangements (inverted duplications,isodicentrics and rings) were studied as it was realized recently that they
were capable of providing information not obtainable from other types ofrearrangements. We found that break points are primarily located within
the G-light bands; a small number of breaks are located in G-dark bands.
Breakages at the interface were exceedingly rare (For further details: see
Nakagome et al. Amer. J. Hum. Genet. 35: 288, 1983).

High Resolution Studies in Patients with Aniridia-Wilms'
Tumor Association, Wilms' Tumor, Retinoblastoma and
Recklinghausen Syndrome
Yasuo NAKAGOME, Minoru SAKURAIl), Yasushi ISE2 ), Toshio NAKAJO S),
Takako MATSUBARA, Eizo OKAMOT0 4 ), Shigenori SAWAGUCHI~),
Yoshiaki TSUCHIDA'), Yutaka NAKAHORI, Ei MATSUNAGA andSumio SAITO')

Since the first cytogenetic report by Francke et al., there have been several
confirmatory observations in which an interstitial deletion of the short armof chromosome II was detected in patients with aniridia-Wilms' tumor* Department of Child Health, Osaka City University, Osaka.
1)

2)
3)
4)

~)
6)

Dept. Pediatrics, Mie Univ., School of Med., Tsu.
Dept. Pediatrics, National Cancer Center Hosp., Tokyo.
Dept. Surgery, National Children's Hospital, Tokyo.
Dept. Surgery, Hyogo College of Med., Hyogo.
Dept. Surgery, Univ. Tsukuba, Ibaraki.
Dept. Pediatric Surgery, Univ. Tokyo, Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

association. In the present study, high-resolution chromosome analysis
was carried out in patients with aniridia-Wilms' tumor association (AWTA)
as well as Wilms' tumor, retinoblastoma and Recklinghausen syndrome.
As to AWTA, both of two possible hypotheses that the lip deletion causes
transposition of an oncogene next to a promotor in the proximal II p or thedeletion of a specific site within IIp13 band in itself predisposes the tumor,
were evaluated based on the distribution of break points. It is also attempted to determine whether every case of AWTA or any of following
groups of patients show II p deletion: cases of Wilms' tumor without aniridia,
tumor itself in a case of Wilms' tumor without constitutional lip deletionand cases of aniridia or hemihypertrophy not associated with Wilms' tumor.
Chromosome studies were carried out by the standard leukocyte-culture
as well as the acridine-organge high-resolution banding technique (Nakagome & Matsubara, this report No. 31: 103, 1981; Matsubara & Nakagome,
Cytogenet. Cell Gent. 35: 148, 1983). Small pieces of tumors were cultured
for 10 to 14 days. Six cases of aniridia-Wilms' tumor association, 4 casesof Wilms' tumor associated with various congenital abnormalities, 16 casesof Wilms' tumor (in 5 of them cultured tumors were also studied), one caseeach of aniridia and hemihypertrophy, one case of retinoblastoma (cultured
tumor) and 4 cases of Recklinghausen syndrome were analysed. In allcases of AWTA, II P deletion was detected. Points of breakages were:del(lI) (plI. 2pI3), del (11) (pI 1. 2pI4.3), del (11) (p1302p p1403), del(1l)(pl1. 2pI3), normal/del (11) (p13 p13) and del (11) (p12 p14.3). In a caseof Wilms' tumor with microcephalus and a few minor anomalies, a 46,
XX/47, XX, +8 mosaicism was detected. In a case of retinoblastoma(bilat.), blood culture revealed a normal karyotype, however, culture of thetumor revealed a normalfdel(13) (q14 q34) mosaicism. All other cases
revealed a normal karyotype at 850 to 400 band level.
As to cases of AWTA, proximal break points scattered between p 11.2
and pl302 and the distal ones between p13 and p14.3. The finding is inconsistent with the promotor-oncogene hypothesis. The middle part of the
p13 band is the common segment of deletion in all 6 cases. Both the proximal 20% and the most distal part of the p13 band was excluded from thecommon deletion segment. (Blood samples of some cases of Wilms' tumor,
with or without aniridia, were kindly made available by Dr. S. Nagahara,
Osaka Children's Medical Cneter; by Dr. Y. Takada, Kure National
Hospital and Dr. Y. asawa, Niigata Univ. Further details: Nakagome

RESEARCHES CARRIED OUT IN 198277

et al. submitted).
The Loss of Kinetochores from Chromosomes ofAged Women (A Supplementary Note)
Yasuo NAKAGOME, Tatsuo ABE'), Shinichi MISAWAl),
Tatsuya TAKESHITA2 ) and Kazuso IINUMA 3)

In the Last issue of this report we described that in aged women the frequency of chromosomes that had inactivated kinetochore and lost Cd-bandpositive material was significantly increased. Later it was pointed outthat our control mostly consisted of infants and children and the difference
between the aged and the control group might have been due to developmental effect rather than postpubertal aging.
We added sexually mature women in the control group and tabulated
individual control data were prepared. Of 6474 scored chromosomesfrom aged women, 62 were Cd-negative; this was the case in 12 of 3849
chromosomes from the controls. The difference was highly significant(p<O.OOl). There was no difference within the control group, i.e., between
sexually mature women and infants plus children.
We trust that our finding is relevant to the etiology of Down's syndrome
and other numerical chromosome abnormalities with either maternal or
paternal age effects (for further details: see Nakagome et al. Amer. J.
Hum. Genet, in press).
A Malformed Girl with Duplication of Major Part of 9q
Yutaka NAKAHORI, Yasuo NAKAGOME and Tsunehiro YOKOCHI

Chromosome abnormalities involving the short arm of a no. 9 chromosome have been repeatedly described. On the other hand, those involving
its long arm, especially middle part of it, is very unusual. In the present
report, an 8-moth-old girl with almost full 9q trisomy is described.
The patient was born to a 33-year-old mother. A 3-year-old sister wasmentally and physically normal. The birth weight was 2,300 g (37 weeks).
She was referred to us when she was 8 months of age. She was able to
1)

2)
3)

Dept. of Medicine, Kyoto Prefectural Univ., Kyoto.
Dept. of Health Sciences, Medical Univ. of Yamanashi, Yamanashi.
Dept. of Pediatrics, Ohta Hospital, Tokyo.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

control her head but she couldn't sit by herself. Physical examinations
revealed growth and metnal retardation, her weight being 5,565 g (-3SD),
length 62.5 cm (-3SD) and head circumference 37.5 cm. Other features
included white and pale eczematous skin, microdolicocephaly, narrow
face, deep orbits, horizontal palpebral fissures, hypotelorism, bilateral innerepicanthal folds, beaked nose, thin upper lip, low set ears, congenital dislocation of the left hip joint, and small hand with relatively long tapering
fingers. Both thumbs were held in the flexed position and pressed firmly
against the palm. Dermatoglyphic study revealed bilateral simian creases,
low axial triradii, and following finger-tip pattern: WL W W W U (r) WLU
W WL and L (1).
Chromosome studies were done by the standard leukocyte-culture technique. One of the No.9 chromosomes was unusually long. Chromosomes
were further examined by the C, high-resolution band, and DistamycinDAPI staining. It was concluded that the abnormal no. 9 chromosome
had the duplication of the major part of the long arm of it; dir dup (9) (q12q32). Both parent had a normal karyotype. (Nakahori et al. in preparation).

RESEARCHES CARRIED OUT IN 1982

X.

79

BEHAVIORAL GENETICS

Selection for the Compound Learning Ability of Mice
Tohru FUJISHIMA

A selection has been made bidirectionally for the discriminated avoidance
performance (DAR, %) of mice (60-70 days of age) in the second of two
daily training sessions, each comprising fifty learning trials. The fundamental stock was constructed from crossing of four inbred mouse strains(C3HjHeMs, SWMjMs, C57LjJMs and D103jMs), each having different
characteristics of DAR measured with an automated Y-type maze apparatus(Experimental Animals, 29(4), 383-390, 1980). The selection has been
made on the basis of the within-family level, and the crossing on the circularmating system. Careful attention has been paid to keep the same inbreeding
coefficient in both selected lines.
The data for the ninth selective generation were obtained this year. They
indicated that the cumulative genetic gain for the average of males andfemales was 12.9, being statistically significant. As for the component
abilities, the difference in avoidance between the DAR High line and the
DNA Low line (High line minus Low line) was 23.8, while the difference
in discrimination was -3.2 although the difference for the parental population was positive.
These results suggested that there was negative genetic correlation between avoidance and discriminatory abilities, and the selection for hihger
DAR resulted in the higher avoidance ability of mice rather than the higher
discriminatory ability.
The Behavioral Changes in Mice Continuously Reared under
Noise Conditions
Tohru FUJISHIMA

It was found in the previous experiments that continuous rearing undernoise conditions caused emotional change as well as decrease of body weightin mice. This year, the experiment was carried out to investigate the effective period of noise to the behavioral change in mice. Mice continuously

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

reared under non-noise conditions were kept under noise condition fromtheir birth to weaning (21 days old) and then returned to the non-noise
condition. Their behavioral performances were compared with those of
the control. The mice of the two groups were originally derived from thesame full sib families. They were kept at 25°C with 12 hour light and 12
hour darkness. The mice in the noise treated group were exposed six times
to noise (pink, 100 phon) for one hour each at one hour interval during thedark period (6 p.m. to 6 a.m.). The avoidance and discriminatory learning
performances of mice approximately 70 days old were measured with anautomated Y-type maze apparatus. Their activity and body weight werealso recorded.
The results showed that no behavioral change occurred by the exposure
during the post natal period. This suggested that the behavioral changes
having been observed in the noise treated group so far might have resulted
from the effect of noise given to mice before birth.

RESEARCHES CARRIED OUT IN 1982

XI.

81

ECOLOGICAL GENETICS

Variation in Competitive Ability
Among Cohabiting Perennial and Annual Typesof Oryza perennis
Yoshio SANO and Hiroko MORISHIMA

Variation in competitive ability among strains of a common wild rice,
Oryza perennis, is a complex showing various patterns depending on thecompetitors. When tested with a perennial or cultivated strain, perennial
strains were stronger in competition than annual strains, while annual strains
were stronger than perennial strains when tested with an annual strain(Ann. Rep. 31). In the present experiment, two populations in which
the plants were differentiated into the perennial and annual types (Ann.
Rep. 32) were studied to look into within-populational variation in competitive ability. Competitive ability was evaluated by the difference in plantweight between pure and mixed stands as before.
The Bhubaneswar (India) population was found in a depression, about
30 m in diameter and deeper than 50 cm in the center. The periphery ofthe depression was covered by an annual type, while the central part was
occupied by a perennial type. The perennial and annual types each wasrepresented by a seed bulk from each of 25 individuals. They were grownin pure and mixed stands in the two experimental plots. In one plot, toexamine the effect due to water depth, the ground was inclined making
difference in water depth ranging from 5 cm to 40 cm. In this plot, theperennial type was a strong competitor against the annual type irrespectiveof water depth. In the other plot (5 cm deep), to simulate habitat disturbance by men and animals, the plants in the half of the plot were cut at aheight of 5 cm and tramped down one month after transplanting, remaining
the other half undisturbed. The perennial types showed a marked reduction in plant weight in both pure and mixed stands under disturbed conditions in comparison with the annual type.
The Chiengrai (Thailand) population was found in a strip of roadsidedepression. It seemed to contain the plants ranging from perennial to annualtypes growing in clumps. Fourteen progeny lines each representing a single

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

plant in the original population were grown in pure and mixed stands
using annual (W107) and perennial (W120) strains as the associate. Although a range of variation was observed in competitive ability, no significant difference was detected between the perennial and annual types. The
same lines were also tested regarding weed tolerance. Comparisons of
their performances between weeded and non-weeded conditions showedthat perennial strains were generally tolerant to weed.
In general, the perennial and annual types are allopatric. The results
obtained from the sympatric populations suggest that the two types aremutually exclusive rather than co-operative, however, to what extent competition between them is important for their differentiation remains unknown.
Variation in Weed Tolerance Among Wild andCultivated Rice Strains
Hiroko MORISHIMA

A series of experiments to study variation in weed tolerance and theeffect of weed-mediated natural selection in rice plants are carried out.
In 1981 and 1982, 170 cultivated varieties (Oryza sativa) and 37 wild ricestrains (0. perennis) were grown in a weeded plot (W), a barnyard grass sownplot (E; Echinochloa crus-galli), and a sedge sown plot (C; Cyperus difformis
and other miscellaneous weeds). Weed tolerance of each strain was evaluated by the difference in log-transformed plant weight of rice between
weed-free and weed-sown plots (E-W, C-W). Cultivated strains showeda range of variation in weed tolerance, though no significant difference wasfound between the Indica and Japonica types. In wild strains which showa perennial-annual continuum, the perennial type was generally more tolerant
than the annual type. This suggests that the perennial type is adapted toecologically saturated habitats having strong competitive ability against
coexisting species. Sedge tolerance and barnyard grass tolerance werepositively correlated (r=0.52** in O. sativa, r=0.57**, in O. perennis).
It was found further that weed tolerance was negatively correlated withperformance in weed-free plot suggesting a trade-off relationship (r=-0.37** '" -0.52**). These associations might be the products of natural
selection, since they were not observed among hybrid progenies which werereared under weed-free condition and tested under the same conditions

RESEARCHES CARRIED OUT IN 1982

83

mentioned above (Ann. Rep. No. 32).
Neighbor Effects between Two Co-Occurring Rice Species
Oryza sativa L. and O. glaberrima Steud.
Yoshio SANO, Reiko SANO and Hiroko MORISHIMA

Two cultivated rice species, Oryza sativa L. and O. glaberrima Steud.,are reproductively isolated each other but morphologically very similar.
In West Africa, these two species are often grown mixed unconsciously infarmers' fields. To study the selective effects of species coexistence in thehistory of a crop community on the interaction between neighboring plants,
two sets of co-occurring sativa and glaberrima derived from seed samples
collected from two fields in Nigeria, one was upland and the other was
rainfed lowland, were examined. Neighbor effects between these four
strains, two sativa and two glaberrima, were evaluated by a mixture-diallel(reported in Ann. Rep. No. 30) and a substitutive experiments.
In the substitutive experiment (a replacement series), these four strains
were grown under wet and dry conditions in pure stands and in species mixtures with three different proportions keeping plant density constant. Number of seeds produced per plant was recorded. In the mixture of sympatricstrains collected from the upland field, sativa was a strong competitor under
wet condition, but a weak competitor under dry condition against glaberrima,suggesting that competitive relationship may change according to water
regime. This could be a mechanism to allow coexistence of two species
with different adaptability in a farmland with heterogeneous environments
prevailing in West Africa. In the mixture of sympatric strains collectedfrom the lowland filed, on the other hand, sativa was a strong competitor
against glaberrima irrespective of water regime. With this combination,
however, total yield of mixed plot was higher than that expected from yieldsof their pure plots. Such cooperative interaction can be explained as the
product of natural selection working in the long history of cohabitation.
The above results agreed well with those obtained from a mixture-diallel
experiment. Further, a trend of frequency dependency that minor component took advantage over major component in reproduction was observed.
This can be also a mechanism to help species coexistence. Together withthe result from a mixture-diallel experiment, the above result is to be published in J. Appl. Ecology (Vol. 42, in press).

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Association of Chromosome and Enzyme Polymorphisms
in Natural and Cage Populations of Drosophila
MelanogasterYutaka INOUE and Takao K. WATANABE

The frequencies of a polymorphic inversion, In(2L)t, and of Adh anda-Gpdh alleles were analyzed in three natural populations of Drosophilamelanogaster from Japan. Significant positive correlations between thefrequencies of In(2L)t and Adh s or a-Gpdh F were detected due to tight linkage. An analysis of correlation with latitude showed that the negativecline of Adh s frequency could be explained entirely by its linkage withIn(2L)t; the frequency of Adh s on the standard chromosome did not show alatitudinal cline. To the contrary, the cline of a-Gpdh F frequency itself
was positive, and its linkage with In(2L)t makes the positive cline unclear.
These results suggest that the two allozymes themselves respond to latitudinal
natural selection in different ways. When these populations were transferred to laboratory cages and maintained for a long time, they lost thechromosomal polymorphism but retained stable enzyme polymorphisms,
although allele frequencies in the cage were not the same as in nature. The
frequencies of Adh and a-Gpdh alleles were close to those in earlier cage
populations of the same geographical origin.

RESEARCHES CARRIED OUT IN 1982

XII.

85

APPLIED GENETICS

Change in Herbicide Tolerance of Barnyard Grassin a Paddy Field
Shin-ya IYAMA

A natural selection experiment of barnyard grass in a rice paddy fieldhas been conducted since 1975 in the experimental plots which consist of
the herbicide treatments with (i) propanil and (ii) propani1+MO, and (iii)
control without treatment. Plants obtained from each plot in 1981 weregrown and harvested individually and then their seeds were used for examination of tolerance to propanil in 1982. Tolerance was measured by
the survival frequency of the seedlings treated with a given amount of propanil solution at two weeks old. Average survival frequency of the plantsfrom the treated plots was higher than that of the plants from control, suggesting that the consecutive application of herbicide changed the genetic
structure of barnyard grass population regarding the tolerance to propanil(Table 1).
Table 1. Viability of propanil treated seedlings of barnyard grassstrains obtained from the paddy field under the application ofherbicide since 1975.

•

Treated with

No. of strains

Viability

Propanil
Propanil+MO
Control1615150.40± .04
0.44±.04*
0.31±.04

Significantly different from control at the 5 % level.

Simulation Studies on the Factors Affecting
the Pedigree Breeding Method
T. C. YAp· and Shin-ya IYAMA

Computer simulation of the pedigree breeding method was carried out
on five different sizes of F 2 populations (5000, 1000, 500, 200 and 100), four*

University of Agriculture, Malaysia.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

levels of environmental variance so as to give heritability values in the F 2
as 0.05, 0.2, 0.5 and 0.9 in six dominance effects (complete and partial recessive, no dominance, partial and complete dominance and overdominance)
and three recombination values between adjacent loci (1/2, 1/8 and 1/32)
in order to study the effects of these factors in relation to genetic gains of
the selection. Twenty loci of equal effects were assumed to control thecharacter. The number of plants selected and progenies produced per
plant for each generation of selection are given in Table 1.
Table 1. Number of lines and number of plants grown per line at eachgeneration after Fa in the pedigree selection scheme

No. of lines
No. of plants
grown/line

Fa

F4

F,

Fe

Fr

F8

F9

FlO

100
60*
10

40

20

12

6

5

3

2

20

30

50

100

100

100

100

Fll

F 2 population sizes used: 100,200,500,1,000,5,000.
* In the case of F 2 population size being 100, 60 Fa lines were selected, other cases
100 Fa lines selected.
Table 2. Mean genotypic values after 10 generations of selection for no
dominance case with respect to different population sizes andheritability values (Possible maximum value=20 and genotypicvalue of F l = 10)
Recombination
value

h2

1/2

F 2 population size
5000

1000

500

200

100

0.05
0.2
0.5
0.9

16.2
17.6
18.5
19.3

16.0
17.2
17.8
18.8

16.6
17.3
18.0
18.6

15.9
16.9
17.5
18.2

15.7
16.4
17.2
18.0

1/8

0.05
0.2
0.5
0.9

16.4
17.5
18.3
18.7

16.0
16.9
17.9
18.4

16.3
17.0
17.7
18.3

15.8
16.8
17.9
17.8

15.7
16.7
17.3
17.5

1/32

0.05
0.2
0.5
0.9

15.4
16.8
16.9
17.0

15.1
16.1
16.5
17 .1

15.0
16.6
16.3
16.8

15.1
15.9
16.2
16.8

15.3
15.5
16.2
16.5

RESEARCHES CARRIED OUT IN 1982

87

Based on the results of 10 runs, it was found that there was a steady increase of genotypic means in response to selection for complete recessive
to partial dominance cases and the genotypes developed were homozygous
for all loci. For complete dominance and overdominance cases, the response to selection was erratic and genotypes developed were still heterozygous even after 10 generations of selting. Results of the response to selection for various degrees of dominance were quite similar and as expected,
high heritability values gave relatively high genotypic values after 10 generations of selection or vice versa for all the population sizes studied.
With respect to the population size, if the aim is to look for the recombinants which could recover 80 % to 85 % of the desirable genes from the F 2
generation, perhaps population size of 100 to 200 F 2 plants may be large
enough to serve the purpose, judging from that genotypes developed fromsmaller populations (l00 to 200) were not very much inferior to those developed from large population (1000 to 5000) for no dominance case which
is of more concern in breeding self-pollinated crop species (Table 2).
Studies on The Breeding Structure of Tree Species in
The Tropical Rain Forest. Family Clusters andIntrapopulation Differentiation
Kan-Ichi SAKAI, Toru ENDo, Shin-ya IYAMA, Yasusada MIYAZAKI*,
Shigesuke HAYASHI*, Yoshiya SHIMAMOTO*, Lilian U. GADRINAB**
and Ulfah JUNIARTI**

Breeding structures of two tropical rain forest tree species, Altingia excelsa
in Java and Agathis borneensis in Kalimantan were investigated. All trees
with a diameter at breast height exceeding 6 cm growing in a 50 X 50 m
quadrat set in a forest were recorded for their position. In all 38 among
148 trees recorded were Altingia excelsa in Java and 21 among 230 were
Agathis borneensis in Kalimantan. Twigs with mature leaves were collectedfrom each tree for isozyme study as well as morphological measurements.
Peroxidase isozyme pattern of each tree was determined by electrophoretic
analysis and "disagreement counts" between trees in the population were
calculated based on the zymogram patterns. Assuming that similarity in* University of Kyushu, University of Kagoshima and University of Hokkaido,
respectively.
** BIOTROP, Bogor, Indonesia.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

the assortment pattern of the isoperoxidase bands tells genetic relationshipbetween trees, on the one hand, and that inbreeding increases the incidenceof smaller values of the disagreement counts, on the other, it has been concluded that inbreeding occurs considerably in Altingia excelsa and to someextent in Agathis borneensis. Finding that trees showing very low disagreement counts are located close to each other, they were grouped as afamily. It was found that different families were quite dissimilar in respectof iso-peroxidase constitution and in several leaf characters as well. The
approximate distance between two trees at which they can mate is estimatedto be 16.5 meters and the area one family occupies 200 to 250 m2, assuming that a family cluster can be a single breeding unit in Altingia
excelsa, within which trees mate at random. Some families were mixedly
distributed with each other within the mating distance, but they were foundstill genetically differentiated from each other. The reason for this reproductive isolation among families might be interpreted to be due to genetic differences between families in flowering time.
In Agathis borneensis, there was no indication of family cluster formation.
Related trees may have been distributed widely scattering in the forest, andthe inbreeding of the species may be due, not to outcrossing between relatives, but to self-fertilization of individual trees.
Effective Population Number of Four Hatchery Populations of Fish
Shin-ya IYAMA and Nobuhiko TANIGucHl*

Based on the amount of genetic drift, the effective population number ofhatchery population of black seabream, Acanthopagrus schlegeli was estimated. Four hatchery populations, two of which were F i and othertwo were F 2 progenies were established from the fish captured in the natural
waters. From the data of 15 polymorphic isozyme loci in the fish populations, the variance of genetic drift (Va) was calculated by using the standardized deviation from the original frequency in each locus. Since theoriginal allele frequencies were not equal from locus to locus, each deviation
was standardized before calculation: di=(Xi-Pi)j';Pi(l-Pi). When theallelic frequency in F i population was estimated by N s samples, the effective
number of parents (N.) was calculated by N.=(l-lj2Ns )j2(Vd -lj2N.).
For the data obtained from the second generation, N. was estimated from* Kochi University.

RESEARCHES CARRIED OUT IN 198289

Table I. Estimated effective population number (N.) and range ofconfidence at 95 % probability. n: No. of original parents used ineach hatchery
Hatchery

n

Vd

N

Range

Kochi
Okayama
Kagawa
Osaka

102
102
71

.031881.0543956.047833.052427

25.48
15.70
27.70
24.48

15.46-100.85
5.32- 42.99
9.42- 72.36
8.47- 61.06

44

the following relationship: Vd = 1/2N.+(I-l/2N.)(I-(l-l/2N.)2), assuming that the effective number in F 1 and F 2 are equal.
Range of confidence at 95 % probability for N was calculated using theconfidence intervals of estimated variance Vd ,I: d//X 2 .m< Vd<I: d//X 2 .915'
assuming that the distribution of d is normal.
The result revealed that the effective population number was smaller than
the number of parents used in any of the four populations. This may haveresulted from far fewer parents actually contributing to the next generation,or unequal number of male and female parents.
Variation in Nitrogen Fixing Activity among Wild andCultivated Rice Strains
Kouki OHTA, Yoshio SANO, Taro FUJII and Shin-ya IYAMA

The acetylene reducing activity (ARA) of Oryza species including two
cultivated species (156 strains) and ten wild species (76 strains) was investigated. A wide range of variation in ARA was observed in wild species
and tropical cultivars, especially in the Sativa group. The three other species
belonging to Sativa group, O. glaberrima, O. perennis and O. breviligulata
also showed similar variations. As far as the average ARA of each species
was concerned, much higher ARA was observed in a cultivated species, O.
sativa than in its ancestral wild species, O. perennis. On the other hand,the species belonging to Officinalis and other groups were at the lower levelof ARA compared with the species in Sativa group so far examined. The
difference between the Sativa and the other groups was significant statistically. The ARA of four tetraploid species, O. minuta, O. latifolia, O.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

eichingeri and O. ridleyi, was lower than that of other diploid species. The
nitrogen fixing activity in rice species appeared to have no particular relationship with ploidy and origin.
ARA was generally lower in Japanese cultivars than that of wild andtropical strains. Analysis of variance revealed that the variation in the
ARA found among the varieties used was statistically significant in eachexperiment. Correlations among ARAjplant, ARAjg dry root and dryroot weightjplant were calculated in the Japanese cultivars. Positive correlation was found either between ARAjplant and root weight" or between
ARAjplant and ARAjg dry root weight. But no significant correlation was
found between ARAjg root weight and root weight. High correlations
observed between dry root weightjplant and ARAjplant, and between
ARAjplant and ARAj g dry root weight indicated that both the amountof root and the efficiency of root playa role for nitrogen fixing activity.
However, insignificant correlation observed between root weightjplant andARA/g dry root weight indicated that the two characters are independent.
This may suggest the possibility of improving nitrogen fixing activity ofrice by increasing the amount of root as well as improving the nature ofroot.
A Sterility Gene between Oryza sativa and O. glaberrima:
Its Association with Photoperiod-Sensitivity
Yoshio SANO

The interspecific F i hybrid between the two cultivated rice species, Oryza
sativa L. and O. glaberrima Steud., is male sterile but female fertile. In
order to extract and examine elements causing F i pollen sterility, a backcross experiment was carried out. The sativa (Taichung 65) and glaberrima(W025) parents were considered to have SaG/SaG and Sa/Sa, respectively.
The Sa locus had no deleterious effect on the development of microsporein the homozygote but induced abortion of pollen not carrying it in theheterozygote Sa/SaG. There appears to be no adverse effect of Sa on megaspores or somatic cells, which is different in genic action from those of S1and S2 reported previously. The SaG locus was tightly linked with la (lazyhabit) on the 9th chromosome. In addition, plants homozygous for Sa
proved to be photoperiod sensitive and showed the heading date about
10 days later than those having Sa/SsG or SaG/SGa in summer seasons.

RESEARCHES CARRIED OUT IN 198291

As to F 1 sterility barriers in rice, the genes examined so far were of gametophytic type although those of sporophytic type were also possible to be
extracted. It is considered that vast numbers of haploid genomes can bescreened by intensified gametophytic selection, and adaptive changes could
be achieved at relatively little cost. Frequently observed sterility genes ofgametophytic type might suggest that gametic selection is favorable for their
fixation of genes controlling F 1 hybrid sterility otherwise they are deemed
to be quickly extinct (J. Hered. 74, Nov.-Dec., 1983, in press).

Wx Protein in Rice
Yoshio SAND and Akemi MURDFUSHI

The Wx locus on the first linkage group in rice controls the productionof amylose in the endosperm. Starch produced in the endosperm thatcarries the Wx allele is composed to 15-23 % amylose, but homozygous
Wx endosperm lacks amylose and contains only amylopectin. Reported
evidences in maize show that the Wx gene product is almost certainly the
starch-granule-bound NDP sugar-starch glucosyl transferase. We report
here a subunit with 60,000 dalton specific to Wx strains in rice. First, seedpolypeptides from glutinous (wx) and non-glutinous (Wx) strains werecompared by means of a two-dimensional polyacrylamide gel electrophoresis
according to O'Farrell. Comparisons between Taichung 65 and an isogenic
line of Taichung 65 carrying wx gene revealed that only a difference detectedin spot patterns is the presence of a subunit in Taichung 65 (non-glutinous,
Wx). The molecular weight of the subunit was approximately 60,000.
Secondly, SDS extracts of starch granules from dried endosperm contain
only three proteins as visualized on polyacrylamide gels. There was asingle major protein with 60,000 dalton which is completely missing in 15 wx
strains examined. More than 100 non-glutinous strains showed the major
protein called Wx protein in maize. In addition, variation in the quantitative levels of the Wx gene product was detected among strains. We intend
to ask if regulatory changes of the Wx gene is related to differentiation in
rice.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

Diversity of Rice CuItivars Collected in the Asian Hilly Areas
in View of Indica-Japonica Differentiation
Reiko SANO and Hiroko MORISHIMA

Asian rice cultivars tend to differentiate into two varietal groups, theIndica and Japonica types, which can be recongized on the basis of character
association pattern. They could be effectively classified into the above
two types with few intermediates, using a discriminant function combining
four characters, KCI0 3 resistance, low temperature resistance, apiculus
hair length and phenol reaction (Morishima and aka, 1981). These twovarietal groups were found to differ markedly in gene frequencies at six
isozyme loci (Px-2, Acp-l, Cat-A, Pgi-A, Pgi-B, Est-E) indicating nonrandom
allelic association among loci. In 101 varieties randomly chosen from alarge number of primitive cultivars collected in various Asian countries,
the varieties classified as the Indica type showed 18 different isozyme genotypes in the combination of the above six loci, while those classified as the
Japonica type showed only two genotypes which differed from any of thosefound in the Indica type. Thus, the Indica and Japonica types definedby character association pattern have specific isozyme genotypes, respectively.
The Himalayan foothill is known as a region in which a large amountof variability in rice is found. One hundred fifty land races collected in
Nepal, Assam, Shikkim and Northern Thailand were studied in the hopeof looking into the incipient stage leading to the Indica-Japonica differentiation. When examined by the discriminant function mentioned above,
they showed a continuous variation between the Indica and Japonica types
with many intergrades, though the Indica type dominated in number inthe present collection. In total, 35 different isozyme genotypes (in the
combination of six loci) were found among them. Nonrandomness ofallelic association among isozyme loci as well as specific association between
character set and isozyme genotypes clearly observed in the samples fromall Asia were not distinct among these hill rices. Further, intra-populational variations were also surveyed in eight populations. Though mostof them were highly polymorphic in characters as well as in isozymes, bothvariations were not associated.
It was concluded that primitive cultivars grown in the hilly areas containa large amount of genetic variability between and within populations, but

BOOKS AND PAPERS PUBLJSHED IN 1982 BY STAFF MEMBERS

93

that differentiation into the Indica and Japonica types has not well developed
as in the other areas in Asia. In general, the accumulation and maintenanceof variations in a crop species would be due to various factors, such as antiquity of domestication, dissemination, hybridization, isolation followed by
selection under different climatic and ethnological conditions. Most
probably, varietal diversity and whin-populational heterogeneity found in
the hilly areas of Asian continent are a function of long-continued primitiveness in agricultural society.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

BOOKS AND PAPERS PUBLISHED IN 1982
BY STAFF MEMBERS
AOKI, K. 1982: Polygenic altruism and extinction group selection. Jap. J. Genet. 57:
297-300.
AOKI,1982: A condition for group selection to prevail over counteracting individual
selection. Evolution 36: 832-842.
AOKI, K. 1982: Additive polygenic formulation of Hamilton's model of kin selection.
Heredity 49: 163-169.
CROW, J. F.* and AOKJ, K. 1982: Group selection for a polygenic behavioral trait: A
differential proliferation model. Proc. Natl. Acad. Sci. USA 79: 2628-2631.
IHARA, M.* and T. ENDo 1981: Genetic control of alcohol dehydrogenase in the Japanese
species of Trillium. Jap. J. Genet. 56: 397-407.
ENDo, T. 1981: Differential regulation of peroxidase isozymes coded by Px-l locus inrice. Jpn. J. Genet. 56: 175-183.
SUN, C. R.,* T. ENDO, M. KUSUDA and M. SUGJURA 1982: Molecular cloning of thegenes for ribosomal RNAs from broad bean chloroplast DNA. Jpn. J. Genet. 57:
397-402.
FUJII, T. 1982: Mutagenicity testing of chemical mutagens in higher plants. In "Environmental Mutagens and Carcinogens" (Eds. T. Sugimura & others), 399-410,
Univ. of Tokyo Press and Alan R. Liss (New York).
FUJISAWA, T. and C. N. DAvm* 1982: Commitment during stenotele differentiation in
Hydra is localized near the S/G 2 boundary in the terminal cell cycle. Develop. BioI.
93: 226-230.
ICHIKAWA, T.* and T. FUJISHIMA 1982: Effects of 6.5 and 17.5 hour milking intervals
on the yield and udder health in dairy cows. Jap. J. Zootech. Sci. 53: 355-358.
TAKEDA, Y.* and Y. HIROTA 1982: Suppressor genes of a dnaA temperature sensitive
mutation in Escherichia coli. Mol. Gen. Genet. 187: 67-71.
ASADA, K.,* K. SUGIMOTO,* A. OKA,* M. TAKANAMI* and Y. HIROTA 1982: Structureof replication origin of the Escherichia coli K-12 chromosome: the presence of spacer
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IMAI, H. T. and K. MORIWAKI 1982: A re-examination of chiasma terminalization andchiasma frequency in male mice. Chromosoma (Berl.) 85: 439-452.
MATSUDA, Y.,* H. T. IMAl, K. MORIWAKI, K. KONDO* and F. BONHOMME* 1982: X-Y
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INOUE, T., M. SASAKI,* A. YOKOIYAMA* and T. KADA 1982: Primer activating enzyme
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B. A. Bridges and D. G. Harnden), 305-326, John Wiley and Sons Ltd.
KADA, T. 1981: Mutagenicity of selected chemicals in the rec-assay in Bacillus subtilis.
In "Comparative Chemical Mutagenesis" (Eds. F. J. de Serres and M. D. Shelby),
19-26, Plenum PubI. Corp.
KADA, T. 1982: Mechanisms and genetic implications of environmental antimutagens.
In "Environmental Mutagens and Carcinogens" (Eds. T. Sugimura and others),
355-359, Univ. of Tokyo Press and Alan R. Liss (New York).
KADA, T., T. INOUE and M. NAMIKI* 1982: Environmental desmutagens and antimutangens. In "Environmental Mutagenesis, Carcinogenesis, and Plant Biology"(Ed. D. J. Klekowski, Jr.), Vol. 1, 135-151, Praeger (New York).
HIRANO, K.,* HAGIWARA, T.,* OHTA, Y.,* MATSUMOTO, H.* and KADA, T. 1982: recAssay with spores of Bacillus subtilis with and without metabolic activation. Mutation Res. 97: 339-347.
MOCHIZUKI, H.* and T. KADA 1982: Antimutagenic effect of Ge-132 on r-ray-induced
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MOCHIZUKI, H.* and T. KADA 1982: Antimutagenic action of cobaltous chloride on
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MOCHIZUKI, H.* and T. KADA 1982: Antimutagenic action of mammalian placental extracts on mutations induced in Escherichia coli by UV radiation, r-rays and N-methylN-methyl-N'-nitro-N-nitrosoguanidine. Mutation Res. 95: 457--474.
MOCHIZUKI, H.* and T. TADA 1982: Restorative effects of human placenta extract in
X-ray-irradiated mice. J. Radiat. Res. 23: 403-410.
MORIMOTO, 1.,* T. WATANABE,* T. OSAWA,* T. OKITSU,* and T. KADA 1982: Mutagenicity screening of crude drugs with Bacillus subtilis rec-assay and Salmonella/
microsome reversion assay. Mutation Res. 97: 81-102.
YOSHINO, K.,* T. NOHMI,* R. MIYATA,* M. ISHIDATE,* N. OZAWA,* M. IsOBE,* T.
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KIMURA, M. (ed.) 1982: Molecular Evolution, Protein Polymorphism and the Neutral
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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

and others), 91-94, The Society for Insect Cells in Japan.
KURODA, Y. 1982: Totipotency of animal cells cultured in vitro. (in Japanese) Tissue
Culture 8: 335-341.
MATSUTANI, E.* and Y. KURODA 1982: Effect of lectins on chondrogenesis of cultured
quail limb bud cells. Develop. Bio!. 89: 521-526.
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MATSUNAGA, E. 1982: Almost synchronous appearance of bilateral retinoblastomas.
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1982: Japanese wild mouse, Mus musculus molossinus, has duplicated immunoglobulin r 2a genes. Nature 298: 82-84.
KATAOKA, T.*, T. NIKAIDO*, T. MIYATA*, K. MORIWAKI and T. HONJO* 1982: The
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leukemia virus-related sequences in various redent species. Proc. Japan Acad. 58:
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SHIROISHI, T.*, K. MORIWAKI and T. SAGAI* 1982: A wild-derived new H-2 haplotype
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YONEKAWA, H.*, K. MORIWAKI, O. GOTOH*, N. MIYASHITA*, S. MIGITA, F. BONHOMME,*
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Hum. Genet. 34: 182-194.
NAKAGOME, Y., T. YOKOCHI*, T. MATSUBARA* and F. FUKUDA* 1982: Preservation ofwhole blood for chromosome analysis. Cytogenet. Cell Genet. 33: 254-255.
MATSUBARA, T.*, Y. NAKAGOME, N. OGASAWARA*, S. OKA* and T. YOKOCHI* 1982:
Maternally transmitted extra ring (21) chromosome in a boy with Down's syndrome.
Hum. Genet. 60: 78-79.
NOGUCHI, S.*, Y. NISHIMURA, Y. HIROTA and S. NISHIMURA* 1982: Isolation and characterization of an Escherichia coli mutant lacking tRNA-guanine transglycosylase. J.
BioI. Chern. 257: 6544-6550.
NOGUCHI, T. and L. C. STEVENS* 1982: Primordial germ cell proliferation in fetal testes
in mouse strains with high and low incidences of congenital testicular teratomas.
J. Nat. Cancer Inst. 69: 907-913.
NOGUCHI, M.*, T. NOGUCHI, M. WATANABE and T. MURAMATSU* 1982: Localization ofreceptors for Dolichos bi/forus agglutinin in early post implantation embryos in mice.
J. EmbryoI. expo Morph., 72: 39-52.
NOGUCHI, T., C. TAYA* and K. MORIWAKI 1982: Search for transplantation method
facilitating establishment of normal diploid teratocarcinomas from "resistant"
C57BL mice. In "Genetic Approach to Developmental Neurobilology"; 101-110,
Tokyo Univ. Press.
NOGUCHI, T., C. TAYA*, T. SHIROISHI*, M. NOGUCHI, Y. NISHIMUNE*, Y. OGIso,* A.
MATSUSHIRO* and K. MORIWAKI 1982: Normal diploid teratocarcinomas derived
from BIO H-2 congenic mice. In "Teratocarcinomas and Embryonic Cell Interactions" (Eds. T. Muramatsu and others), 41-56, Japan Scientific Societies Press(Tokyo) and Academic Press.
OHTA, T. 1982: Some evolutionary problems of multigene families considered from the98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

standpoint of population genetics. In "Molecular Evolution, Protein Polymorphism and the Neutral Theory" (Ed. M. Kimura), 59-77, Japan Scientific Soc. Press(Tokyo) and Springer-Verlag (Berlin).
OHTA, T. 1982: Population genetics of multigene families. Adv. Biophysics 15: 173199.
OHTA, T. 1982: Further study on the genetic correlation between members of a multigene family. Genetics 99: 555-571.
OHTA, T. 1982: Linkage disequilibrium due to random genetic drift in finite subdivided
populations. Proc. Natl. Acad. Sci. USA 79: 1940-1944.
OHTA, T. 1982: Allelic and nonallelic homology of a supergene family. Proc. Natl.
Acad. Sci. USA 79: 3251-3254.
OHTA, T. 1982: Linkage disequilibrium with the island mode!. Genetics 101: 139-155.
OKA, H. I. and H. MORISHIMA 1982: Ecological genetics and the evolution of weeds. In"Biology and Ecology of Weeds" (Eds. W. Holzner and N. Numata), 73-89, Dr.
W. Junk Pub!. (The Hague).
OKA, H. I. and H. MORISHIMA 1982: Phylogenetic differentiation of cultivated rice, XXIII.
Potentiality of wild progenitors to evolve the Indica and Japonica types of ricecultivars. Euphytica 31: 41-50.
L SATO, S.*, S. MATSUOKA* and Y. SANO 1982: Reconstruction of a linkage group corresponding to the Nishimura's second chromosome in rice, Oryza sativa L. Jap. J.
Breed. 32: 232-238.
SASAHARA, T.*, T. SENGOKU* and Y. SANO 1982: CO 2 and water vapour exchange as
related to shade tolerance of Oryza punctata Kotschy. Photosynthetica 16: 365-361.
SANO, Y. and H. MORISHIMA 1982: Variation in resource allocation and adaptive
strategy of a wild rice, Oryzaperennis Moench. Bot. Gaz. 143: 518-523.
SHIMOTOHNO, K. and H. M. TEMIN* 1982: Spontaneous variation and synthesis in U 3
region of the long terminal repeat of an avian retrovirus. J. Virol. 41: 163-171.
SHIMOTOHNO, K. and H. M. TEMIN* 1982: Loss of intervening sequence in genomic mouse
J-globin DNA inserted in an infectious retrovirus vector. Nature 299: 265-268.
SHINOZAKI, K. and M. SUGIURA 1982: Sequence of the intercistronic region between the
ribulose-l,5-bisphosphate carboxylase/oxygenase large subunit and the coupling
factor J subunit genes. Nucleic Acids Research 10: 4923-4934.
SHINOZAKI, K. and M. SUGIURA 1982: The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-I, 5-bisphosphate carboxylase/oxygenase.
Gene 20: 91-102.
SHINOZAKI, K., Y. SASAKI*, T. SASKIHAMA*, and T. KAMIKUBO* 1982: Coordinate
light-induction of two mRNAs, encoded in nuclei and chloroplasts, of ribulose 1.5bisphosphate carboxylase/oxygenase. FEBS Lett. 144: 73-76.
SHINOZAKI, K., N. TOMIOKA*, C. YAMADA* and M. SUGIURA 1982: Cloning and characterization of a plasmid DNA from Anacystis nidulans 6301. Gene 19: 221-224.
DENo H.*, A. KATO, K. SHINOZAKI and M. SUGIURA 1982: Nucleotide sequences oftobacco chloroplast genes for elongation tRNAMet and tRNAVa': the tRNAti~cgene contains a long intron. Nucleic Acids Research 10: 7511-7520.
SOEDA, E. and T. MARUYAMA 1982: Molecular evolution in papova viruses and in

BOOKS AND PAPERS PUBLISHED IN 1982 BY STAFF MEMBERS

99

bacteriophages. Advances in Biophysics 16: 1-16.
TODOH, N.* and M. SUGIURA 1982: The complete nucleotide sequence of a 16S ribosomal
RNA gene from tobacco chloroplast. Gene 17: 213-218.
TAKAIWA, F.* and M. SUGIURA 1982: The complete nucleotide sequence of a 23S ribosomal gene from tobacco chloroplasts. Eur. J. Biochem. 124: 13-19.
TAKAIWA, F.* and M. SUGIURA 1982: Nucleotide sequence of the 16S-23S spacer region
in an rRNA gene cluster from tobacco chloroplast DNA. Nucleic Acids Research
10: 2665-2676.
UCHIMIYA, H.*, H. KATO*, T. OHGAWARA*, H. HARADA* and M. SUGIURA 1982: Sequence-specific methylation of ribosomal RNA genes contained in the nuclear DNA
of tobacco. Plant & Cell Physiol. 23: 1129-1131.
TAKAIWA, F. * and M. SUGIURA 1982: The nucleotide sequence of chloroplast 5S ribosomal
RNA from a fern, Dryopteris acuminata. Nucleic Acids Research 10: 5369-5373.
TAKAIWA, F.*, M. KUSUDA, N. SAGA*, and M. SUGIURA 1982: The nucleotide sequenceof 5S rRNA from a red alga, Porphyra yezoensis. Nucleic Acids Research. 10: 60376040.
TAKAIWA, F.*, M. KUSUDA and M. SUGIURA 1982: The nucleotide sequence of chloroplast 4.5S rRNS from a fern, Dryopteris acuminata. Nucleic Acids Research 10:
2257-2260.
SUGIYAMA, T. 1982: Roles of head-activation and head-inhibition potentials in patternformation of Hydra: Analysis of a multi-headed mutant strain. Amer. Zool. 22:
27-34.
TAKAHATA, N. 1982: Linkage disequilibrium, genetic distance and evolutionary distance under a general model of linked genes or a part of the genome. Genet. Res.
Camb. 39: 63-77.
TAKAHATA, N. 1982: Sexual recombination under the joint effects of mutation, selection and random sampling drift. Theor. Pop. BioI. 22: 258-277.
TAKAHATA, N. 1982: Estimation of evolutionary distance under a mathematical modelof extranuclear DNA molecule. RIMS, Kokyuroku 457: 141-156.
TAKAHATA, N. 1982: The disappearance of duplicate gene expression. In "Molecular
Evolution, Protein Polymorphism and the Neutral Theory" (Ed. M. Kimura), 169190, Japan Scientific Soc. Press (Tokyo) and Springer-Verlag (Berlin).
TAZIMA, Y. 1980: Mutagens, comutagens and antimutagens of natural origin. In"Well-Being of Mankind and Genetics" Proc. XIV Intern. Congo Genetics, Moscow,
1(1): 207-214, USSR Acad. Sci.
TAZIMA, Y. 1982: Mutagenic and carcinogenic mycotoxins. In "Environmental Mutagenesis, Carcinogenesis and Plant Biology" (Ed. E. J. Klekowski, Jr.) Vol. 1, 66-95,
Praeger (New York).
TAZIMA, Y. 1982: A brief sketch of environmental mutagen studies in Japan. In "Environmental Mutagens and Carcinogens" (Eds. T. Sugimura and others), 91-100,
Univ. of Tokyo Press and Alan R. Liss (New York).
TAZIMA, Y. 1982: Apparent threshold and its significance in the assessment of risks
due to chemical mutagens. In "Environmental Mutagens and Carcinogens", Proc.
3rd Intern. Confer. on Env. Mutagens, 728-735, Univ. of Tokyo Press (Tokyo).

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33
SHIBUYA, T.*, T. MUROTA and K. TUTIKAWA 1982: Mouse spot tests with alkylnitrosoureas. Mutation Res. 104: 311-315.
MUROTA, T.*, T. SHIBUYA* and K. TUTIKAWA 1982: Genetic analysis of an N-ethylNnitrosourea-induced mutation at the hemoglobin .a-chain locus in mice. Mutation
Res. 104: 317-321.
TOMITA, 1.*, K. NAKAMURA*, Y. YAm* and K. TUTIKAWA 1982: Teratogenicity/fetotoxicity of DEHP in mice. Environ. Health Perspect. 45: 71-75.
WATANABE, T., M. MATSUDA*, S. OHNISHI* and F. HIBARA* 1982: Notes on the systematics of Drosophilajambulina. Jap. J. genet. 57: 561-567.
YAMADA, M. S. NAWA and T. WATANABE 1982: A mutant of SR organism (SRO) in
Drosophila that does not kill the host males. Jap. J. Genet. 57: 301-305.
YAMADA, M. and Y. HIROTA 1982: Hfr-mediated conjugative transfer of pBR322 vector
carrying the chromosomal DNA of Escherichia coli. Gene 20: 471-475.
YAMADA, M., Y. TAKEDA*, K. OKAMOTO* and Y. HIROTA 1982: Physical map of thenrdA-nrdB-ftsB-glpT region of the chromosomal DNA of Escherichia coli. Gene
18: 309-318.
YOSIDA, T. H. 1981: Population cytogenetics of the black rat, Rattus rattus. Proc.
XIV. Intern. Congo Genet. II: 96-117.
YOSIDA, T. H. 1982: Environmental mutagens and karyotype evolution in mammals.
Proc. III. Intern. Conf. Environ. Mut. (1981): 445-561.
YOSIDA, T. H. 1982: Notes on the karyotype of a male hybrid between the domestic
cat and the Leopard cat. Proc. Japan Acad. 58(B): 5-8.
YOSIDA, T. H. 1982: Chromosomal alteration and the development of tumors, XXV.
Quantitative changes of nucleolar organizer regions (NORs) in the Indian spiny mousetumors developed by 3-methylcholanthrene. Proc. Japan Acad. 58(B): 169-172.
YOSIDA, T. H. 1982: Cytogenetical studies on insectivora, II. Geographical variationof chromosomes in the house shrew, Suncus murinus (Soricidae), in East, Southeast
Asia, with a note on the karyotype evolution and distribution. Jap. Jour. Genet. 57:
101-111.
YOSIDA, T. H. 1982: A preliminary note on preferential selection of eudiploid tumor
cells after deep freezing. Proc. Japan Acad. 58(B): 299-302.
SHIRAISHI, Y.*, T. H. YOSIDA, and A. A. SANDBERG* 1982: Single and twin sister chromatid exchanges in endoreduplicated Bloom syndrome cells, Proc. Japan Acad.
58(B): 260-264.
SHIRAISHI, Y.*, T. H. YOSIDA and A. A. SANDBERG* 1982: Analysis of single and twinsister chromatid exchanges in endoreduplicated normal and Bloom syndrome Blymphoid cells. Chromosoma (Berl.) 87: 1-8.
HARADA, M.*, T. UCHIDA,*T. H. YOSIDA and S. TAKADA 1982: Karyological studies of
two Japanese noctule bats (Chiroptera). Caryologia 35: 1-9.
SUZUKI, H.*, M. SEGAWA*, T. H. YOSIDA and K. KONDO* 1982: Dependent effects
5-bromodeoxyuridine and uridine on frequency of sister chromatid exchanges inchromosomes of Welsh onion and D-6 Chinese hamster induced by 5-fluorodeoxyuridine. La Kramosomo 11-27-28: 817-825.
* Author not onN.I.G. staff.

ABSTRACT OF DIARY FOR 1982

ABSTRACTS OF DIARY FOR 1982
January
February
March
May
June

AugustSeptember
OctoberNovember
December

18
18
4
10
7
12
15
21
27
20
27
25
8
29
3
13
20

278th Meeting of Misima Geneticists' Club
279th Meeting of Misima Geneticists' Club
189th Biological Symposium
190th Biological Symposium
191st Biological Symposium
192nd Biological Symposium
193rd Biological Symposium
280th Meeting of Misima Geneticists' Club
194th Biological Symposium
195th Biological Symposium
196th Biological Symposium
197th Biological Symposium
281st Meeting of Misima Geneticists' Club
198th Biological Symposium
199th Biological Symposium
200th Biological Symposium
282nd Meeting of Misima Geneticists' Club

101

102

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

FOREIGN VISITORS IN 1982
May 1, '80-Apr. 30, '82
Feb. 16, '81-Sept. 30, '82
January
12-18
16-17
February

26-Mar. 7

March

24-25
25-26

April

1-3
6-0ct.4
12-0ct. 11
13-16

May

IO
20-21
28

June

6-7
11-13
11-13
14-15

ACHERMANN, Josef, UniversiHit Zurich,
Switzerland
SUN, C. R., Fudan University, China
HOTTA, Y., University of California, U.S.A.
OHTSUBO, E., State University of New York,U.S.A.
CROW, James F., University of Wisconsin,
U.S.A.
RAJAN, S. S., F·A·O Expert, United Nations, Iraq.
SZYBALSKI, W., University of Wisconsin,
U.S.A.
WERNEK, Mass., University of New York,U.S.A.
GONI, Beatriz, University of Uruguay,
Uruguay.
YAP, Thoo Chai, University of Agriculture,
Malaysia.
JEFFERSON, Roland M., U.S. National Arboretum, U.S.A.
RAETZ, Cristian, University of Wisconsin,
U.S.A.
GHYSEN, J. M., Universite de Liege, Belgium.
HOPPE, Petter c., The Jackson Laboratory,
U.S.A.
SUEOKA, N., University of Colorado, U.S.A.
WEISBLUM, Bernard, University of Wisconsin,
U.S.A.
KADO, C. 1., University of California, U.S.A.
WATSON, John M., Australian National
University, Australia.

FOREIGN VISITORS IN 1982

July

17-20

August22-23
23

September

2Q-Dec.20
20

October

4-12
26

November26
29-30

December

13-14

103

MULLER, Andreas J., Akademie der Wissenschafter, Germany
EIGEN, Manfred, Max-Planck-Institut, Germany.
BROCK, Richard D., S.C.I.R.O., Division of
Plant Industry, Australia.
YASSUDA, Yatiyo Y., University of Sao
Paulo, Brazil.
KING, Robert C, Northwestern University,
U.S.A.
BARADJANEGARA, A. A. A., Pusut Penelition
Teknik Nuklir, Indonesia.
Naomani, M. K. R., Benchamin, K. V.,
Venugopala Pillai, S., Chandrashekharaiah.,Central Sericultural Research and Training
Institute, India.
KOKKE, Robert, United Nations University,
Tokyo.
KassEL, Hans, UniversiUit Freiburg, Germany.
OHNO, S., City of Hope Research Institute,
U.S.A.

104

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 33

AUTHOR INDEX
ABE, TAOIU, K77
69, 70, 71
AsAKURA, M. .
31
CRow, J. F
70
ENDo, T •••••..............•........ 87
FLouRET, B
18
FUJII, T
61,89
FUJIKI, H. . •••....••••..•.••.•.•••.. 61
FUJISAWA, T •...•...••..••....••.... 34
FUJISHIMA, T
79
FUJITA, H •....•.........••.••.••.... 75
GACHELIN, G •..........•.•...•...... 25
GADRINAB, L. U •..••......••..••••... 87
GOTOH, O •.....••....••••......... .43
GROPP, A . • . . . . . . . . • • • . . . . . . . . . . . . . . 43
HAYASHI, S •.•.......•...•••.....•.. 87
HIROTA, Y
17,18,19lINUMA, K
77
INOUE, T •............•.....••.•.. 56, 57
INOUE, Y
84
ISE, Y75
IYAMA, S .........•...•••.. 85, 87, 88, 89
JIKUYA, H •••••........••..•.•••• 12,13
JUNIARTI, U
87
KAoA, T56,57,58,59,60
KANEHISA, T •.....•.•.•....•••••. 25, 26
KATO, M ...••......•.••••.•........ 58
KAWAHARA, K •......•..•••..•...•.. 39
KIMURA, M •.•..••.•••.•......•...•.. 63
KIRIYAMA, S •••...........••.•.•.... 58
KOMEDA, Y"
20
KOMINAMI, R •••.••.•.•.•••.•••••.•.. 26
KONDo, K •........•..••..•.•••..•••• 41
KURIHARA, Y. .
27
KURODA, Y
29,30,31,52,32
KUSUDA, J
15
LEDUC, M •••••......•.••••••....... 18
MARUYAMA, I. N. • ..••••••••••.•.•.. 18
MARUYAMA, T •••.•..••••••••••.•••.• 18

MATSUBARA, T

75

MATSUNAGA, E •....••••.••• 72,73, 74, 75
MATSUTANI, E •••.•..••••••..••••••.• 30
MINATO, K.

• •••..••••.•..•••••..•.. 33

MINEZAWA, M •••.••.•••••..•.••... .43
MISAWA, S ••••...•••.•••••••••.••••. 77
MITA, I.

60MIYASHITA, N •..•......•••.•.. 22, 43, 45
MIYAZAKI, Y

87

MORISHIMA, H •...•••••..••••.. 81, 82, 84
MORIWAKI, K.

22,23,24,25,26,27,43,45

MURAKAMI,A•.•.••. .47, 48, 50, 51, 52, 53
MURAMATSU, M •.•...••..••.••...... 26
MUROFUSHI, A

91
42
NAKAGOME, Y
75, 77
NAKAHORI, Y
75, 77NAKAJo, T • • . . • . . . . . . . . . . . . . . . . . . . . . 75
NINAKI, 0
15
NISHIKAWA, S . . . . • . . . . . • . • . . • . . . . . . .42
NISHIMlYA, C • . . . . . . . . . . . . . • • . • . . . . . . 33
NISHIMURA, S ••••....••.•.•.....•.... 19
NISHIMURA, Y
19
NITo, S •.........••.•••.......•. .43, 45
NOGucHI, S. • ••......•..•.•.••.•.... 19
NOGucHI, T •...•...•.•...•.•••...... 35
OHTA, K •..•••..•.••.•.....•..••.... 89
OHTA, T •..•.••..•••••...•.•.. 64,65,66
OKA, A •.......•••.•...••.•.•••• 26,17
OKAMOTO, E. . •.•..••••••......•.••. 75
ONIMARU, K. .
15
PARQUET, C
18
SADAIE, Y
56, 60SAGAI, T •.•••.•.....•••••..••..•• 23, 25
SAITO, S .........••••••.....•••••.•. 75
SAKAI, K. I.
87
SAKURAI, M
75
SANO, R
83
SANO, Y81,83,89,90,91
MUROFUSHI, M

AUTHOR INDEX

105

SAWAGUCHI, S .•••.•..............•.. 75

TAMAZAWA, S .......•••.•..... 48, 50, 51

SEGAWA, Moo ..•..•.....••..•....... .41

TANIGUCm, N

SHIMAMOTO, Y • • . . . . . . . • . . . . . . . . . . . . . 87

TAYA, Coo .••...•.........•..•.•..•.. 24

SHllVUBHI, Y ••....•....•............. 40

TAZIMA, Yoo ..•.•••.••.•..••.....•... 15

SmROIsHI, T •.•.•......•.......... 23, 25

TEZUKA, H

88

57. • • . . . . . . . . . . . . . . . . . . . . . 61

TSUCHIDA, Y .•.....••...••••.•.•.•.•. 75

12, 13, 14
17
SUGIMURA, T. . . . . . . . . • . . • . . . . . . . . . . . 61
SUGIYAMA, T • . . . . • . . . . . . . . . • . . . • • . . . 33
SUZUKI, H
25, 26, 41
SUZUKI, Y • . . . . . . . . . . • . . . . . . . . . . . . . . 15
TABATA, S . • . . . . . . • . . . . . . . . . . . . . . . . . 17
TAKAHATA, N
66, 67, 68, 69
TAKANAMI, M • . • . . . . . . . . . . . . . . . . . . . . 17
TAKANO, J. .
33
TAKEDA, Y • • . . . . • • . . . . . . . . . . . . . . . . . . 17
TAKEMOTO, K. K. "12,13
TAKESHITA, T • . . . . . . . . . . . . . . . . . . . . . . . 77

18
14
WATANABE, T. K
84
WINKING, H. • • . . . . • . . . . . . . . . . . • . . . . 43
YAMAGATA, T ••..•....•..••••.••.... 30
YAMAMOTO, A. .
18
YAMAMOTO, M • • • . . . . . . . . . . . . . . . . . . . . 14
YAP, T. C
85
YASUDA, S
17
YOKOCHI, T • . . . . . . . . . . . . . . . . . . . . . . . . 77
YOKOIYAMA, A
58
YONEKAWA, H ••..........•.•....•... 43
YOSHIDA, T. H •...... 37,38,39,40,41,42

SHIZAKI, M.
SOEDA, E

SUGIMOTO, K

VAN HEIJENOORT, J

WADA, A

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