ISSN OOn-4995

NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 34

1983

Publi8ked bll
THE NATIONAL INSTITUTE OF GENETICS
Mi8ima, Sizuoka-ken, Japan
1984

Annual Reportof theNational Institute of Genetics
No. 34, 1983

Published by

The National Institute of Genetics, Japan
1984

CONTENTS
General statement..
Staff
,
" .
Council.....
Association for propagation of the knowledge of genetics
Projects of research for 1983
Researches carried out in 1983 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

I.

II.

Molecular Genetics
An improved method for shotgun DNA sequencing. YASUDA,
S., NAKAYAMA, N., JIKUYA, H. and SOEDA, E.
An altered DNA sequence encompassing the ras gene of Harvey
murine sarcoma virus. YASUDA, S., FURUICHI, M. and
SOEDA, E.
The human c-Ha-ras2 is a processed pseudogene inactivated bynumerous base substitutions. SOEDA, E., KAGIMOTO, M.,
M., MIYOSHI, J. and SAKAKI, Y.
DNA rearrangement affecting expression of the BK virus transforming gene. SOEDA, E., WATANABE, S., VCHIDA, S. and
YOSHIIKE, K.

1
3

7
8
9
13

13

14

16

17

Microbial Genetics
The Escherichia coli origin of replication: essential structure forbidirectional replication. TAKANAMI, M., TABATA, S., OKA,
A., SUGIMOTO, K., SASAKI, H., YASUDA, S. and HIROTA, Y.....
Escherichia coli uvrD mutants with thermosensitive DNA-dependent adenosine triphosphatase I (Helicase
RICHET, E.,NISHIMURA, Y., HIROTA, Y. and KOHIYAMA, M..............
Overlapping of the coding regions for a and r components ofpenicillin-binding protein 1 b in Escherichia coli. KATO, J.-I.,SUZUKI, H. and HIROTA, Y.... :.........................
Glycan polymerase with no penicillin-binding activity in Escherichia
coli. HARA, H., VEDA, T. and SUZUKI, H.
A novel glycan polymerase that synthesizes uncross-linked peptidoglycan in Escherichia coli. HARA, H. and SUZUKI, H.. . ..

19

m.

20

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

A Bacillus subtilis gene involved in cell division, sporulation andexoenzyme secretion. SADAIE, Y. and KADA, T.
22
Flagella are formed at the late stage of cell cycle just before theseptum formation in Escherichia coli. NISHIMURA, A. and
HIROTA, Y0
00... 23

III.

Biochemical Genetics and Immunogenetics
Conserved and variable antigenic specificities on the H-2 derived
from a Japanese wild mouse, MOL.SGR. MORIWAKI, K.,
SAGAI, T., MIYASHITA, N. SUZUKI, H. and SHIROISHI, T. .....
Founder effect in the frequency distribution of mouse H-2 observedin the Japanese wild populations. MORIWAKI, K., SAGAI, T.
and MIYASHITA, N
00. 0
0. 00. 0
0. . . . ..
MS-series monoclonal antibodies directed to the H-2 and Ia antigenic determinants of BlO.MOL-SGR mouse. SAGAI, T.,
SHIROISHI, T. and MORIWAKI, K
0............
Effect of H-2 complex on the incidence of urethan-induced mortality and malformation in the mouse embryos. MIYASHITA,
N., NITo, S. and MORIWAKI, K.
Effect of major histocompatibility gene complex (MHC) on thegrowth of embryo-derived teratoma. TAYA, C. and MORIWAKI, K
00
0. 0...........
Restriction fragment length polymorphism of NTS rDNA in molerats. SUZUKI, Ho, MORIWAKI, K., KOMINAMI, R., MURAMATSU,
M. and NEVO, E
00
0.............
Further survery ofT-lymphocyte differentiation antigens in various
mouse subspecies by serological methods. KURIHARA, Y.,
BONHOMME, F. and MORIWAKI, K. '" 0•....... 000" 0.....
Expression of rat class I antigens on the erythrocytes of various
strains. HIRASHIMA, T., NATORI, T. and MORIWAKI, K......
Analysis of mitochondrial DNA in cytoplasms of male-sterile rice.
0..........
NAwA, S., YAMADA, M. A. and FUJII, T

IV.

25

26

28

29

30

31

32
34
35

Developmental Genetics and Somatic Cell Genetics
Electron microscopic studies on the uItrafine structures of Tn vitro
differentiated cells of Drosophila melanogaster. KURODA, Y.
and SHIMADA, Y
0

•••••

0

•

•

•

•

•

•

•

•

•

••

37

CONTENTS

Expression and induction of tissue-specific enzymes in cultured
chicken liver cells. KURODA, Y. and SUGAWARA, E... .... ..
Mutagenic activity of Ames test-negative compounds in cultured
Chinese hamster cells. KURODA, Y., YOKOIYAMA, A. and
KADA, T.
Effects of some growth factors on cultured human diploid cells inclonal culture. KURODA, Y.
Histological analysis of embryonic lethal mutant in Drosophilamelanogaster. MINATO, K.
Mode of action of an inhibitor for stenotele differentiation in
Hydra. FUJISAWA, T.
Molecular cloning of extra organs locus of Dorsophila melanogaster.
YAMAMOTO, M. and MIKLOS, G. L. G.
A novel method for fate mapping of the genital disk: the region
important for the testes elongation and morphogenesis. SAKAIZUMI, M. and YAMAMOTO, M.
Freezing of silkworm ovaries. KUSUDA, J., ONIMARU, K., NOGUCHI, T. and YAMASHITA, O.
V.

iii

38

38
39
40
41
41

42
44

Cytogenetics

Decrease of C-bands and NORs in the black rats from Amami
and Tokunoshima. YOSIDA, T. H.
Location of ribosomal RNA genes in the Amami black rats by insitu hybridization technique. YOSIDA, T. H., SUZUKI, H. and
KANEHISA, T.
Phylogenetic relationship among several karyotype forms of theblack rat and some other related species. BAVERSTOCK, P. B.,
ADAMS, M., MAXSON, L. R. and YOSIDA, T. H.
Frequency of chromosome transolcations in the black and Norway
rats after r-irradiation. YOSIDA, T. H.
A black rat with chromosome alterations born after r-irradiation.
YOSIDA, T. H. .
.
A linkage test on the bared gene to the inversion no. 1. YOSIDA,
T. H
Chromosome translocations occurred in offspring of the Norway
rats after r-irradiation. YOSIDA, T. H.
Translocation between autosome nos. 9 and 18 occurred in a F-344
rat after r-irradiation and its genetics. YOSIDA, T. H.

45

45

46
47
47

48
49
50

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

4/7 translocation occurred in a WM strain rat after r-irradiationand its transmission to offspring. YOSIDA, T. H.
50ll/Y translocation in the NIG-III strain rat occurred after r-irradiation and its genetics. YOSIDA, T. H.
51
1/12 translocation occurred in a WKS-strain rat after r-irradiationand its transmission into offspring. YOSIDA, T. H.
52
A tanuki or Japanese raccoon dog with 40 chromosomes including
two B-chromosomes. YOSIDA, T. H., WADA, M. Y. and
WARD, O. G.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 53
Somatic variation of B-chromosomes in two Japanese raccoon dogs
and Robertsonian fission of no. 8 chromosome. YOSIDA,
T. H., WADA, M., WARD, O. G. and WURSTER-HILL, D. H .. , 53
Robertsonian fission of the no. 6 autosome newly found in a specimen collected in Aichi-ken. YOSIDA, T. H. and WADA,
M.Y
54
A Japanese raccoon dog with three Robertsonian fissions in nos.
5, 8 and II, found newly in Kanagawa-ken. YOSIDA, T. H.
and WADA, M. Y.
. . . . . 55
Variation of B-chromosomes in four youngs from one litter collected in Saitama-ken, YOSIDA, T. H. and WADA, M. Y..... 56
B-chromosomes and Robertsonian fission in 13 Japanese raccoon
dogs, with note on somatic variation of the number of B's.
YOSIDA, T. H. and WADA, M. Y.
57
Karyotype of an Indian spiny mouse with 24 chromosomes.
KUMARI, P., ASWATHANARAYANA, N. V. and YOSIDA, T. H. .. 58
Chromosomes of the Honshu sika deer, Cervus nippon centralis.
WADA, M. Y. and YOSIDA, T. H.
59
A simple technique for chromosome observations from the small
mammals and birds, and karyotypes of some mammals and abird. WADA, M. Y. and YOSIDA, T. H.
60
A simple technique for identification of sex-chromosome (W) inthe bird. WADA, M. Y. and YOSIDA, T. H.
60
Identification of the sex chromosome in a sex reversed peacock.
YOSIDA, T. H., WADA, M. Y., SAKAI, Y. and KONDO, N. .... 61
Karyotypes of four dragonet fish species. MUROFUSHI, M.,
NISHIKAWA, S. and YOSIDA, T. H.
62
Karyotypes of two species in the sandperch fishes. MUROFUSHI,
M., NISHIKAWA, S. and YOSIDA, T. H.
62

CONTENTS

v

Multiple sex chromosome mechanism (XX-Y) newly found in thesnake eel, Muraenichithys gymnotus. MUROFUSHI, M. and
YOSIDA, T. H.
63
Comparative study on two species of snipefishes. MUROFUSHI, M.,
NISHIKAWA, S. and YOSIDA, T. H.
63
Karyotypes of the red swamp crayfish and the Japanese lobster.
MUROFUSHI, M., DEGUCHI, Y. and YOSIDA, T. H.
64
Karyotype of the samll free-living nematode, Caenorhabditis
65
elegans. YOSIDA, T. H., SADAIE, T. and SADAIE, Y.
Inhibition of sister chromatid exchanges in Bloom syndrome cellswith cycloheximide. SHIRAISHI, Y., YOSIDA, T. H. and
SANDBERG, A. A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 65
Chromosome observations on tropical ants from Western Malaysia (2). IMAI, H. T., BROWN, Jr., W. L., KUBOTA, M., YONG,
H.-S. and THO, Y. P.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 66
Modes of inheritance of X-Y dissociation in inter-subspecies hybrids between BALB/c mice and Mus musculus molossinus.
MATSUDA, Y., IMAI, H. T., MORIWAKI, K. and KONDo, K.. . .. 69
Rates of mammalian karyotype evolution by the karyograph method. IMAI, H. T., MARUYAMA, T. and CROZIER, R. H. . . . . . . . 70
Cytogenetic analysis of recombination in males of Drosophila
ananassae. MATSUDA, M., IMAI, H. T. and TOBARI, Y. N. .. 71
Quantitative analysis of karyotype alteration and species differentiation in mammals. IMAI, H. T.
71
Genetic studies on spontaneous sister chromatic exchanges inspermatogenesis of Drosophila melanogaster. YAMAMOTO,
M
72

VI.

Mutation and Mutagenesis in Animals
Induction of specific-locus mutations by high temperature exposures in newly laid eggs of the silkworm. MURAKAMI, A.. .
A new egg colour mutant, light brown egg with black eyes in thesilkworm. MURAKAMI, A. and FUKASE, Y.
Effects of tritium on the induction of recessive visible mutations inprimordial germ-cells of silkworm embryos: the nature of
fi-ray-induced mutants. MURAKAMI, A.
A new type maternal inheritance in the silkworm. MURAKAMI,
A

75
76

78
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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

VII.

Radiation Genetics and Chemical Mutagenesis in Microorganism
and Plants
Induction of prophages in spores of Bacillus subtilis by ultraviolet
irradiation from synchrotoron orbital radiation. SADAIE, Y.
and KADA, T.
82
Detection of natural and environmental bio-antimutagens and82
their mode of actions. KADA, T. and INOUE, T.
Modification of the activities on recA protein by an antimutagenic
metal compound, cobaltous chrolide. INOUE, T. and KADA,
T
83
Restorative effects of the human placenta extracts on gamma-ray84
irradiated mice. KADA, T. and TEZUKA, H.
Effects of DBMA on the gamma-ray mutagenesis in soybean.
FUJII, T..........
85
Absence of mutagenic activity of BP in the soybean mutation test.
FUJII, T. and INOUE, T.
86

VIII. Population Genetics
Diffusion model of intergroup selection, with special reference toevolution of an altruistic character. KIMURA, M.
Rare variant alleles in the light of the neutral theory. KIMURA,
M
Selective constraint in protein polymorphism: study of the effectively neutral mutation model by using an improved pseudosampling method. KIMURA, M. and TAKAHATA, N.
Theoretical sutdy on the accumulation of selfish DNA. OHTA,T
On the evolution of multigene families. OHTA, T.. . . . . . . . . . . . ..
Time until fixation of a mutant belonging to a multigene family.
OHTA, T
Population genetics of multigene families that are dispersed into
two or more chromosomes. OHTA, T. and DOVER, G. A.. ,.
Gene identity and genetic differentiation of populations in thefinite island model. TAKAHATA, N.
Evolutionary dynamics of extranuclear genes. TAKAHATA, N. and
SLATKIN, M.
Linkage disequilibrium of extranuclear genes under neutral mutations and random genetic drift. TAKAHATA, N. . . .... .... ..

88
88

89
90
90
91
91
92
92
93

CONTENTS

vii

Population genetics of extranuclear genomes under the neutral
mutation hypothesis. TAKAHATA, N, 93
A quantitative genetic model of reciprocal altruism: a condition
for kin or group selection to prevail. AOKI, K, 94
Stochastic theory of population genetics. MARUYAMA, T.
95
Analyses of the age of genes and the first arrival times in a finite
95
population. MARUYAMA, T.
Models of evolution of reproductive isolation. MARUYAMA, T... 96
An approach to population and evolutionary genetic theory forgenes in mitochondria and chloroplasts, and some results.
MARUYAMA, T.
97
98
A mathematical model of codon substitution. GOJOBORI, T.

IX.

Human Genetics and Human Cytogenetics
Modern medical practice versus environmental mutagens: their
possible dysgenic impact. MATSUNAGA, E. . . . . . . . . . . . . . . . . 99
Retinoblastoma and ABO blood groups. MATSUNAGA, E. and
MINODA, K.
99
Origin and structure of a minute chromosome in patients withcongenital malformations. NAKAGOME, Y. and NAKAHORI,
Y
100
oning and characterization of a highly Y -specific 3.5 Kb repeatedfragment. NAKAHORI, Y. and NAKAGOME, Y
101/itochondrial DNA polymorphism in Japanese. HORAI, S. and
MATSUNAGA, E.
102:Jifferential enzyme activities in human esterase D phenotypes.
HORAI, S. and MATSUNAGA, E
102
Establishment of a human genomic library with MboI-digested
DNA. NAKAGOME, Y., NAKAHORI, Y. and KUSUDA, J. ..... 103

x.

Behaviour Genetics
The behavioral changes in mice continuously reared under noise
conditions. FUJISHIMA, T
104

XI.

Ecological Genetics and Evolutionary Genetics
Observations on wild and cultivated rices in Thailand for ecologicalgenetic study. MORISHIMA, H., SANO, Y, SATO, Y. and
SHIMAMOTO, T
105

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Invasion of Drosophila simulans into inland area and the effect ofwinter temperature on its frequency. WATANABE, T. K. and
INOUE, Y.
106
Biochemical phylogeny of the Drosophila montium subgroup.
OHNISHI, S. and WATANABE, T. K
107

XII.

Applied Genetics
Correlated selection responses in characters and allozymes of rice.
MORISHIMA, H.
109
The genetic basis of hybrid chlorosis found in a cross between two
Japanese rice cultivars. SATO, Y. and MATSUURA, S.
110
Chromosomal location of Pgi-2 locus in rice. SANO, R. and
MORISHIMA, H.
III
Differential regulation of waxy gene expression in rice endosperm.
SANO, YIII
Temperature effects on the Wx protein level and amylose content
in rice endosperm. SANO, Y., MAEKAWA, M. and KIKUCHI,
H
112
Genetic variation of nitrogen fixing activity in the progeny population of rice hybrid. IYAMA, S. and VAN HINTUM, T. J. L. .... II3

Books and papers published in 1983 by staff members
Abstracts of diary for 1983
Foreign visitors in 1983
Author index
,
"

II5
124
125
128

GENERAL STATEMENT
In introducing the Annual Report of the institute that covers 1 year
from January to December 1983, I should like to begin by paying tribute
to my predecessor, Dr. Yataro Tazima, who directed the institute fromMarch 1, 1975 to September 30, 1983. During that period Dr. Tazima
had exerted himself for the development of the institute in many ways.
In particular, in order to switch over the administrative category of theinstitute from the present system to that for joint use by universities, hemade tenacious endeavor for the negotiation with the authorities concerned. As a consequence, the Ministry of Education, Science and Culture
set up in April an ad hoc committee to investigate the reorganization planof the institute (Chairman: Prof. Y. Yamamura, President of Osaka University). According to the report of the committee, this institute will haveprospect to be reorganized into the one for joint use by universities fromthe next fiscal year. The institute conferred the title of Honorary Member
on Dr. Tazima.
Having assumed, since October 1, heavy responsibility as director, I will
do my best for further development of the institute so as to contribute moreto the promotion of studies in genetics and related fields in this country.
Since the foundation in 1949, the staffs of the institute have been cultivating
the tradition to work assiduously in friendly rivalry with each other by
breaking down academical cliquism or sectarianism. I was advised by Dr.
Takashi Fujii, Chairman of the Board of Councillors of the institute, thatthe institute should carryon such tradition and independence. I will
always keep his advice in mind.
Some of the events deserving special mention here are as follows. Dr.
Tosihide H. Yosida, head of the Department of Cytogenetics, was awardeda Purple Ribbon Medal from the government for his long scientific contributions, including studies on the karyotypical evolution in Rodents.
By the way, the same medal was awarded to Dr. Kan Oguma, who was
the first director of the institute and teacher of Dr. Yosida. An Encouragement Award of the Association for the Promotion of Genetic Studies wasgiven to Dr. Tomoko Ohta, head of the laboratory, Department of Population Genetics, for her theoretical studies on evolution and variation in

2

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

multigene families. Dr. Motoo Kimura, head of the Department of Population Genetics, published a book entitled "The Neutral Theory of Molecular
Evolution" (Cambridge University Press, 1983). This theory has provokeda great deal of controversy among geneticists in the world since he first
proposed it in 1968. Through 15 years ordeal, evident proofs supporting
his theory are appearing one after another.
On April 23, the institute was opened to the general public. In addition
to the exhibition of each department and moviefilms, a lecture was given
by Dr. Yosida on "Chromosomes: From virus to humans." In the campus
cherry blossoms of double-flowers species were in full bloom and thevisitors numbered about 2500. On November 5, public lectures were given
at the National Science Museum in Tokyo by Dr. Yukiaki Kuroda on "Cellgenetics in vitro" and Dr. Yosida on "Chromosomes and evolution oforganisms." More than 200 people listened to the lectures in spite of theweekend afternoon.
In regard to personnel change, Dr. Kin-ichiro Miura, head of the Department of Molecular Genetics, was appointed to professor of the Faculty ofTechnology, University of Tokyo in April; Dr. Kunitada Shimotohno,
researcher of the same department, to laboratory head of the National
Cancer Center Research Institute in March; and Dr. Kazuo Shinozaki,
researcher of the department, to assistant of Faculty of Science, Nagoya
University in April. Furthermore, Dr. Yoshibumi Komeda, researcher,
Microbial Section, Genetic Stocks Center, was promoted to lecturer ofFaculty of Science, University of Tokyo in November. For 15 years Dr.
Miura had assumed leadership of the Department of Molecular Genetics
and produced many achievements which added luster to the institute's
reputation, for instance, he identified the cap structure of the 5' end ofmessenger RNAs that is peculiar to eukaryote cells. Lastly, to our greatsorrow, Dr. Takehiko Noguchi, researcher, Genetic Stocks Center, died ofheart attack on November 24 at the age of 41. Dr. Noguchi had been
studying on developmental genetics of mouse, especially on mutant strains
with high frequency of testicular teratomas. Besides, he had been engaging
in the work of practical use of keeping mouse stocks by freezing fertilized
eggs. The sudden death of Dr. Noguchi, who had been expected muchby the colleagues in and out of the institute, cannot be enough deplored.
We had many visitors from abroad as the last year, with whom informations and views on recent studies were exchanged actively. Among them,

GENERAL STATEMENT

3

Dr. Ick-dong Yoo (working in Dept. of Microbial Genetics) from Korean
Ministry of Agriculture, Forestry and Fishery, Mr. T. J. L. van Hintum(Dept. of Applied Genetics) from Agricultural University in Wageningen,and Mr. Nai-Kai Zhu (Dept. of Induced Mutation) from Institute of Environmental Chemistry of Academia Sinica, were staying rather long fortechnical training and cooperative work.

STAFF
Director

MATSUNAGA, Ei, D. Med., D. Sc.
Members1.

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
GOJOBORI, Takashi, Sc.

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

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

5.

Department of Applied Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department
The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., 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
SATO-HIRAOKA, Yoichiro, M. Ag.

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
SADAIE, Yoshito, D. Sc., Head of the Laboratory
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
NAKAGOME, Yasuo, D. Med., Head of the Laboratory

5

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

8. Department of Microhial 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
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Laboratory
SOEDA, Eiichi, D. Ag.
The 2nd Laboratory
MIURA, Kin-ichiro, D. Sc., Head of the Laboratory
11.

Genetic Stocks Center
YOSIDA, Tosihide, H., D. Sc., Head of the Center
Animal Section
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
INOUE, Yutaka, D. Sc.
KUSUDA, Jun, D. Ag.
Plant Section
FUJII, Taro, D. Ag., Head of the Laboratory
SANO, Yoshio, D. Ag.
Microbial Section
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
NISHIMURA, Akiko

COUNCIL

7

12. Experimental Farm
FUJII, Taro, D. Ag., Head of the Farm
MIYAZAWA, Akira
13. Department of Administration
AKATSUKA, Takao, 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.
TAZIMA, Yataro, D. Ag.

COUNCIL
FUJII, Takashi, Chairman, Emeritus Professor of University of TokyoINOUYE, Eiji, Director of Institute of Developmental Research
IINO, Tetsuo, Professor of University of Tokyo
KONDO, Norio, Professor of Tokyo University of Agriculture
MISONOO, 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
NAKAJIMA, Tetsuo, Professor of University of Tokyo
OKAZAKI, Yoichi, Director of Intitute of Population Problems
OOSAWA, Fumio, Professor of Osaka University
SASA, Manabu, President of Toyama Medical and Pharmaceutical University

8

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

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, Director of National Institute of
Genetics
YOSIDA, Tosihide H., Managing Director, Head of the Cytogenetics Department
SINOTO, Yosito, Manager
WADA, Bungo, Manager, Emeritus Professor of University of TokyoTAZIMA, Yataro, Manager
OSHIMA, Chozo, Manager

PROJECTS OF RESEARCH FOR 1983
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)
Cytogenetics in the silk worm (MURAKAMI)
Pheno-genetics of the silkworm (MURAKAMI)
Radiation- and chemical-induced mutagenesis in the silkworm (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)
Department of Biochemical Genetics

Studies on transformation in higher organisms (NAWA and YAMADA)
Genetical and biochemical studies of pteridine metabolisms in insects(NAWA)

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

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 and
SATO)
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)
Enviromnental 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 in Bacillus subtilis(SADAIE)
Department of Human Genetics

Genetic and cytogenetic studies on retinoblastoma and Wilms' tumor(MATSUNAGA, NAKAGOME and HORAI)
Clinical cytogenetic studies of congenital abnormalities in man (NAKAGOME)PROJECTS OF RESEARCH FOR 1983

11

Molecular cytogenetic studies of human chromosomes (NAKAGOME)
Studies on DNA polymorphisms in human populations (HORAI and MATSUNAGA)
Department of Microbial Genetics

Genetic regulaotry 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 primary structure of DNA (SOEDA)
Transformation of human lung and rat fibroblast cells with viral promoter
DNA (SOEDA)
Biogenesis of papovaviruses (SOEDA)
Genetic Stocks Center

Studies and conservation of germplasm resources in rice and wheat species(FUJII and SANO)
Specificity of mutagen to tolerance in higher plants (FUJII)
Exploitation of genetic ability of nitrogen fixation in Graminae (FUJII, SANO
and !YAMA)
Studies on genetic differentiation in rice (SANO)12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Cytogenetic studies of Norway rats and establishment of the chromosomal
mutant stocks (YOSIDA)
Establishment of new experimental animals from wild rodents (YoSIDA andMORIWAKI)
Studies on chromosomal polymorphism in Drosophila (INOUE)
Analysis of fibroin genes of silkworm and its relatives (KUSUDA)
Coordination of flagellar formation and cell division in E. coli (NISHIMURA,
and HIROTA)
Synthetic ColE! plasmids carrying genes for cell division in Escherichia
coli (NISHIMURA)RESEARCHES CARRIED OUT IN 1983

I.

MOLECULAR GENETICSAn Improved Method for Shotgun DNA Sequencing
Shuhei YASUDA, Naohiro NAKAYAMA, Hiroyuki JIKUYA
and Eucm SOEDA

Shotgun DNA sequencing is one of the most rapid and easiest methodsof determining the sequence of a large DNA molecule (Deininger, P. L.,
Anal. Biochem., 129, 216-223, 1983). It involves choosing clones randomly
from a shotgun library of DNA fragments cloned into a Ml3 phage vector,
sequencing them by the "dideoxy" method and, finally building up to thecomplete sequence by overlapping the subfragment sequences. The optimum library should contain recombinants with inserts which span thewhole region of the DNA of interest, share short pieces of overlapping
sequences with each other and are present in roughly equal proportions in
the library. Difficulty occurs in cloning DNA larger than 1 kb (kilo base)in length into M13 vectors, and sequences smaller than 0.4 kb should be
avoided since this is the maximum length that can be read from a polyacrylamide sequencing gel. Thus, in order to construct such a library, it isimportant to dissect DNA randomly so as to fall within this size range andprocess them for blunt-end ligation to M13 vectors. Probably, the combination of physical shearing by sonication and repair by T4 DNA polymerase is one of the best procedures to achieve it. However, the cloning
efficiency by this method was rather low as compared with that obtainedwith use of restriction fragments.
In order to promote shotgun DNA sequencing further, the method forconstruction of M 13 phage clone libraries was modified, including theshearing of DNA into appropriate sizes by sonication and the processingof the sheared DNA for blunt-end ligation to the phage vectors.
By changing the volume of DNA solution applied as well as time and amplitude of sonication, the DNA was dissected randomly to generate fragments predominantly 0.4-1 kilo bases in length, this size range being suitable

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34
Table 1. Effect of the processings of sonicated DNA on cloning efficiency
Sonicated ADNA (20 ng)

White
White PlaqueBlue PlaqueNuclease PI

T4pol.

Nuclease

ADNA/AluI(20 ng)

9
9
111

30
30
87

PI & T4 pol.
113
113
130

251
251
51

The sonicated DNA was processed to generate blunt-ends by T4 DNA polymerase,
nuclease PI and both, respectively, and cloned. The cloning efficiency is represented by
the number of white plaques. Phage ADNA/AluI fragments bearing flush-ends are used
as a positive control.

for shotgun DNA sequencing. Furthermore, by introducing nuclease PI
treatment prior to the repair of the sheared DNA by T4 DNA polymerase,the cloning efficiency increased three fold compared to when T4 DNApolymerase was used alone (Table 1).
An Altered DNA Sequence Encompassing the ras Geneof Harvey Murine Sarcoma Virus
Shuhei YASUDA, Masato FURUIcm and Eiichi SOEDA

Harvey murine sarcoma virus (Ha-MuSV) was originally isolated fromtumours induced by inoculation of Moloney murine leukemia virus (MoMuLV) into rats. This virus was able to transform fibroblast cells in culture and induce sarcomas in susceptible mice. In the virus-infected cells,
a transformation specific protein with a molecular weight of 21 kilo daltons,
p2l, was produced. The closed circular DNA intermediates of the viralgenome have been isolated from the infected cells and cloned molecularly
and physically characterized. Studies on transforming ability of the subgenomic DNA permitted its localization within the SmaI-PstI fragment in
the proximal half of the viral genome.
The DNA fragment encompassing the ras gene of Harvey murine sarcoma virus was sequenced and the coding region of a transforming protein,p2l, was assigned to the sequence (Figure). Examination of uncleotide
sequence, taken together with the result of analysis of the ras mRNAs,
has revealed that p2l is encoded from a continuous coding region startingwith the 5' proximal initiation codon but is not a processed protein. How-

15

RESEARCHES CARRIED CUT IN 1983
AGACCCCGCTCTAGTGGCAGTGTGTTGGTTGATAGCCAAAGTTAATTTTTAAAA

54nCATAGTGTTTTGGGGGTTGGGGATTTAGCTCAGTGATAGAGCTCTTGCCTAGCAAGCGCA

114

AGGCCCTGGGTTCGGTCCC~GCTCTGAAAAAAAGGAAAGAGAAACAAAACAAAAACATA174T

TAGTGTTTTATCTGTGCTTATGCCCGCAGCCCGAGCCGCACCCGCCGCGGACGGAGCCCA
T A
T
A
T
T
TGCGCGGGCCCAGTCGGCGCCCGTCCGCGCCCCCGCCCTGCCCCGGCCCCGGCCCCGGGG

294

GCAGTCGCGCCAGCAAGCGGTGGGGCAAGAGCTCCTGGTTTGGCAGCCCCTGTAGAAGCG354

ATG ACA GAA TAC AAG CTT GTG GTG GTG GGC GCT AGA GGC GTG GGA
Met- Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly

399
15

MG AGT GCC CTG ACC ATC CAG CTG ATC CAG AAC CAT TTT GTG GAC
Lys Ser Ala Leu Thr I Ie Gin Leu I Ie Gln Asn His Phe Val Asp

44430

GAG TAT GAT CCC ACT ATA GAG GAC TCC TAC CGG MA CAG GTA GTC
Glu Tyr Asp Pro Thr lie Glu Asp Ser Tyr Arg Lys Gin Val Val

489
45

ATT GAT GGG GAG ACG TGT TTA CTG GAC ATC TTA GAC ACA ACA GGTI Ie Asp Gly Glu Thr CyS Leu Leu Asp 11 e Leu Asp Thr Thr Gly

534
60

CM GM GAG TAT AGT GCC ATG CGG GAC CAG TAC ATG CGC ACA GGG
Gin Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly579
75

GAG GGC TTC CTC TGT GTA TTT GCC ATC MC MC ACC MG TCC TTTGlu Gly Phe Leu CyS Val Phe Ala I Ie Asn Asn Thr Lys Ser Phe

624
90

GAA GAC ATC CAT CAG TAC AGG GAG CAG ATC AAG CGG GTG AAA GATGlu Asp 11 e His Gin Tyr Arg Glu Gin Ile Lys Arg Val Lys Asp

669
105

TCA GAT GAT GTG CCA ATG GTG CTG GTG GGC AAC MG TGT GAC CTG
Ser Asp Asp Val Pro Met Val Leu Val Gly Asn Lys Cys Asp Leu
T G
GCC GCT CGC ACT GTT GAG TCT CGG CAG GCC CAG GAC CTT GCT CGC
Ala Ala Arg Thr Val Glu Ser Arg Gin Ala Gln Asp Leu Ala Arg
Gly
AGe TAT GGC ATC CCC TAC ATT GAA ACA TCA GCC AAG ACC CGG CAG
Ser Tyr Giy 11 e Pro Tyr 11 e Glu Thr Ser Ala Lys Thr Arg Gin

714
120
759
135

GGT GTA GAG GAT GCC ·TTC TAC ACA CTA GTA CGT GAG ATT CGG CAG
Gly Val Glu Asp Ala Phe Tyr Thr Leu Val Arg Glu I 1e Arg Gln

849
165

C.AT AM CTG CGG AAA CTG MC CCG CCT GAT GAG AGT GGC CCT GGC
His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu Ser Gly Pro Gly

894
180

TGC ATG AGC TGC MG TGT GTG CTG TCC TGA CACCAGGTGAGGCAGGGACCCyS Met Ser Cys Lys Cys Val Leu Ser Ter

944
189

234

804
150

AGCAAGACATCTGGGGCAGTGGCCTCAGCTAGCCAGATGAACTTCATATCCACTTTGATG 1004
G CG
TCCCTGCTCCCCCAATTCTGCCAATCCCCCTGCCTGCA
1042

Fig. 1. The nucleotide sequence of the fragment encompassing H-ras geneof Ha-MuSV. The nucleotide sequence of the AccI-PstI fragment was determinedby the methods of Maxam and Gilbert and of Sanger et aI., and compared with thesequence reported earlier. The differences in nucleotide sequences from the published one are indicated above the sequence line. Arrows (T) indicate the positions
where nucleotides were added to the original sequence. The deletion of dinucleotide
CC occurred between position 134 and 135 shown in the box. The predicted aminoacid sequence of p21 is also shown below the sequence line with one replacementof glycine by alanine at position 122.

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

ever, there were found several differences between the sequence publishedby Dhar et al. (Science, 217, 934-937, 1982) and ours, including 9 deletions, 7 substitutions and 2 insertions of nucleotides in the published sequence of 997 nucleotides in length. Among these, one of the substitutions occurring in the coding region resulted in amino acid replacementof glycine by alanine at position 122 of p21. The latter suggests that p2l
is encoded continuously from the 5' proximal ATG triplet in the single
open reading frame.
This work was published in Nucleic Acids Research, 12, 5583-5588 (1984).

The Human c-Ha-ras2 Is a Processed Pseudogene
Inactivated by Numerous Base Substitutions
Eiichi SOEDA, Masaki KAGIMOTO,1) Jun MIYOSHI2 )and Y oshiyuki SAKAKI 2 )Normal human DNA contains sequences related to retroviral oncogenes,
and activation of these proto-oncogenes is considered to be involved in
expression of the transformed phenotype in naturally occurring tumors.
The transfection assay of NIH 3T3 cells with tumor DNA disclosed activated versions of ras-related proto-oncogenes, comprised of at least threemembers (c-Ha-ras, c-Ki-ras and N-ras), in human solid tumors, cell lines
of epithelial origin and certain types of hematopoietic malignancies. Basedon hybridization analysis, the human genome was found to contain additional cellular homologues of transforming genes of the Harvey andKirsten murine sarcoma viruses. As characterization of normal rasrelated sequences and determination of their exact numbers might be ofclinical significance in the diagnosis and prevention of cancer, we attempted
to identify homologous sequences by screening a human genomic DNA
library using the v-Ha-ras probe at various hybridization stringency. Amongthese clones, the c-Ha-ras2 gene, is reportedly located on the X-chromosome
and has lost introns. There has heretofore been no information on its
precise gene structure and oncogenic potential. We have determined thenucleotide sequence of the c-Ha-ras2 and demonstrate that it is a processed
1)
First Department of Internal Medicine, Kyushu University, Faculty uf Medicine,
Fukuoka 812.
2)
Research Laboratory for Genetic Information, Kyushu University, Faculty of
Medicine, Fukuoka 812.

RESEARCHES CARRIED OUT IN 1983

17

pseudogene surrounded by several direct repeats and contains numerous
base substitutions as well as a notable mutation (AGT at codon 12 of thep2l protein) responsible for oncogenic conversion of the known ras genes.
(For detail, see Nucleic Acids Research, 12, 1821-1828, 1984)
DNA Rearrangement Affecting Expression of the BK Virus
Transforming Gene
Eiichi SOEDA, Sumie WATANABE, 1) Seijiro UCHIDA2 ) and Kunito YOSHIIKE1 )The BK virus (BKV) mutant, designated pm-522, has a number of characteristics distinct from those of the wild-type (WT) BKV. On humanembryonic kidney (HEK) cell cultures, pm-522 forms turbid plaques smaller
than clear plagues of WT BKV. This mutant is approximately five times
as tumorigenic in hamsters as WT BKV and can induce hamster insulinomas,
which so far have not been induced by the plague isolates of WT BKV.
Despite its somewhat inefficient growth in HEK cells, BKV pm-522 produces
many more foci of permanently transformed cells in hamster or rat cell
cultures than WT BKV does. Apparently, pm-522 is a host range mutant
that can express its transforming gene in nonpermissive cells more efficiently
than WT BKV.
The high transforming capacity of pm-522, compared with that of WT
BKV, is probably ascrible to the capacity of unintegrated pm-522 genomes
to express continuously their early functions in the nonpermissive cells andto transform them transiently before stable transformation is established.
Rat cells infected with either pm-522 or WT BKV contain unintegrated
BKV DNA in their nuclei during the first 2-week period, before foci oftransformed cells become detectable. The free viral genomes continue
to be transcribed to direct synthesis of T antigen, which renders the cells
transiently transformed, in pm-522 infection. In WT infection, however,transcription of T antigen gene occurring, initially seems to be repressedsoon after infection and T antigen production cease. Apparently, WT
BKV is susceptible to some repressive mechanism, which pm-522 can escape,in rat cells.
BKV pm-522 has a deletion at approximately map unit 0.72 in the nonDepartment of Enteroviruses, National Institute of Health, Tokyo 141.
Departmefft of Viral Infection, Institute of Medical Science, University of Tokyo,
Tokyo 108.
1)

2)

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

coding region (topographically, the putative control region for early transcription) near the origin of DNA replication, within the HindIlI C segments.
Construcion and characterization of recombinant viruses between pm-522(HindIlI C fragment) and wt-501 (HindIII ABD fragment) has shown that
HindIII-C (450 base pairs around the origin of replication) of pm-522 contains the mutation responsible for both the altered plaque morphology andthe high transforming capacity. Therefore, sequencing the HindIlI C
segment is expected to correlate the change in nucleotide sequence tobiological functions of BKV.
In the present study, we compared the nucleotide sequence of wt-50l
HindIlI C segment with that of pm-522 HindIII-C, which contains the mutation responsible for the altered plaque type and trasforming capacity. The
difference between the two BK viruses was the local DNA rearrangement(deletions and duplications) that had occurred in the putative control region
for early transcription in pm-522 DNA. Whereas wt-50l had three setsof 68-base pair repeats (the central set had a deletion of 18 base pairs) inthis region, pm-522 had one set of 68-base pair unit and two sets of shorter
37 base pair repeats. Three BK virus mutants, forming clear large plagues
like those of wt-50l but capable of tranforming rat cells, were derived fromthe recombinant virus earring the HindIII C segment of pm-522. Thesemutants had further duplications of shorter segments originating from the
pm-522 sequence in the putative early control region. (For detail, see J.
Virol., 51, 1-6, 1984)

RESEARCHES CARRIED OUT IN 1983

II.

19

MICROBIAL GENETICSThe Escherichia coli Origin of Replication: Essential
Structure for Bidirectional Replication
Mituru TAKANAMI, Satoshi TABATA, Atsuhiro OKA, Kazunori SUGIMOTO,
Hitoshi SASAKI, Seiichi YASUDA and Yukinori HIROTA

The 245 base-pair oriC sequence of the E. coli K-12 chromosome wassubjected to localized mutagenesis in vitro which uses sodium bisulfite, anda large number of mutants carrying a single to multiple GC to AT changes
were isolated. The replicating ability of these mutants was assayed byintroducing into cells, and the correlation between the position of mutation and replicating function was analysed. Combining the results obtained
from this and previous studies together, we concluded that oriC provides
multiple interaction sites precisely separated by spacer sequences, and initiation factors cooperatively interact on these sites to form an initiation
complex.
The functional role of the 245 bp oriC sequence was investigated by using
an in vitro replication system and various Ori+ and Ori- plasmids. The
result of analysis clearly indicated that DNA replication started at a region
near, but outside, the 245 bp oriC sequence, and proceeded bidirectionally.
The replicating function was reduced by mutations introduced only in
oriC, but not by sequence replacements in the flanking regions. The result
indicates that the 245 bp oriC sequence contains information enough fordirecting bidirectional replication in the vicinity of oriC. It was also notedthat initiation of DNA synthesis at the specific region required the dnaAcomplementing fraction from cells harboring a dnaA-carrying plasmid.
Based on the above observations, a possible model for bidirectional replication directed by oriC is discussed. (For detail, see "Mechanisms of
DNA Replication and Recombination", pp. 257-273, Alan R. Liss Inc.,New York, 1983)

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Escherichia coli uvrD Mutants with Thermosensitive
DNA-dependent Adenosine Triphosphatase I(Helicase II)
Evelyne RICHET, Yukinobu NISillMURA, Yukinori HIROTA
and Masamichi KOillYAMA

Three mutants producing thermosensitive DNA-dependent Adenosine
triphosphatase (ATPase) I were screened from a collection of temperaturesensitive mutants of Escherichia coli K12. ATPase I purified to near
homogeneity from one of the mutants (JEI0l00) possesses both thermosensitive DNA-dependent ATPase and DNA helicase activities. We
have shown that ATPase I is encoded by the uvrD gene as first suggested
by Oeda et al. (1982): (i) the thermosensitive ATPase I mutation present
in JEI1040 lies in or very close to the urrD gene, (ii) ATPase I activity isabsent in uvrD21O, uvrDl56, and uvrD252 mutants. Thus the thermosensitive mutations correspond to new uvrD mutations. However, the mutation present in JEl1040 confers neither UV sensitivity nor mutator phenotype at high temperature. Evidence is presented that the mutant ATPase
I is stabilized in vivo at 42°C. (For detail, see Mol. Gen. Genet. 192, 378385, 1983)
Overlapping of the Coding Regions for a and r Components
of Penicillin-Binding Protein 1 b in Escherichia coli
Jun-ichi KATO, Hideho SUZUKI and Yukinori HIROTA

The mode of biosynthesis of penicillin-binding protein (PBP)-1 b in
Escherichia coli was investigated by use of the plasmid carrying the ponB

(PBP-lb) gene region. Analyses of the products synthesized in minicells
and in vitro showed that PBP-l b was synthesized as two molecular species
corresponding to the a and r components of PBP-l b. The coding regions
for the a and r components were located within the ca. 3.7 kb MluI-HincII
fragment and transcribed in the direction from the HincII to the Mlul site.
The capacity for producing the a component was abolished by a deletion extending to the MluI site ca. 0.7 kb inward from the HincII end of the ca. 3.7 kb
fragment; the remaining 3.0 kb region with the MluI site at both ends directed
the production of the r component alone. The production of the r component was enough to correct all the known defects caused by a ponB mutation.
In addition to these results, the analyses for cross-reacting materials produced

RESEARCHES CARRIED OUT IN 198321

in correspondence to the various deletions indicated that the coding regions
for the a and r components overlapped and that the N-terminal portion was
responsible for the difference between the two components. The distal
region about 0.7 kb long inward from the MluI end of the MluI-HincIl
fragment was dispensable for producing the functional PBP-l b, although
the PBP-l b produced was curtailed. By a larger distal deletion reachingalmost to the middle of the MluI-HincII fragment, the polypeptide producedfor PBP-lb lost the ability to bind penicillin and still retained a low but
significant activity for glycan synthesis. We suggest, therefore, that thepolypeptide portion required for transglycosylase activity resides on the
N-terminal half of PBP-lb, followed by the middle portion necessary forpenicillin-binding and the C-terminal part dispensable for the function of
PBP-lb. (For detail, see Mol. Gen. Genet. 196,449-457, 1984)
Glycan Polymerase with No Penicillin-binding Activity
in Escherichia coli
Hiroshi HARA, Taro UEDA and Hideho SUZUKI

The enzymes involved in the last stage of peptidoglycan synthesis in
Escherichia coli have been found among penicillin-binding proteins (PBPs):
first PBP-lb and subsequently PBP-la and PBP-3 were identified as peptidoglycan synthetases that carry out both transglycosylation and transpeptidation. PBP-l b appeared to be a major peptidoglycan synthetase,bacause the membrane fractions of mutants defective in PBP-lb were
almost unable to support in vitro peptidoglycan synthesis. We found,however, that the activity for peptidoglycan synthesis was detectable even in
the membrane fractions defective in PBP-l b on addition of non-ionic polar
substances like glycerol to the reaction mixture. The investigation of theglycerol-stimulated activity suggested that two or more glycan polymerases
other than PBP-lb were responsible for the activity and that a glycan polymerase with no penicillin-binding ability may exist in E. coli. This glycan
polymerase with no penicillin-binding ability has no transpeptidase activity and consequently should require cooperation of a cross-linking enzyme to serve sacculus synthesis. (For detail, see "The Target of Penicillin,
Proceedings of International FEMS Symposium on the Murein Sacculus of
Bacterial Cell Walls" Eds. R. Hakenbeck et al., pp. 583-588, Walter deGruyter, Berlin, 1983)

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

A Novel Glycan Polymerase that Synthesizes Uncross-Linked
Peptidoglycan in Escherichia coli
Hiroshi HARA and Hideho SUZUKI

A simple and efficient procedure to assay peptidoglycan synthesis in vitro
was established. By this procedure, a novel activity for glycan polymerization in Escherichia coli was found in the fraction containing no detectablepenicillin-binding protein (PBP). This polymerase activity was relativery
insensitive to moenomycin, showed requirement for Ca2+ or Mn2+ but not
for Mg2+, and led to production of uncress-linked glycan chains. These
properties distinguished the glycan polymerase from the activities shwon by
the fractions containing PBPs. The glycan polymerase catalyzing polymerization of glycan units from lipid intermediates was purified and identified as a protein of 34 kdal. (For detail, see FEBS Letters 168, 155-160,
1984)

A Bacillus suhtilis Gene Involved in Cell Division,
Sporulation and Exoenzyme Secretion
Yoshito SADAIE and Tsuneo KADA

To elucidate the divisional control of sporulation in Bacillus subtilis, we
examined the effect of temperature sensitive septum initiation mutations on
sporulation and competent cell formation and found that the div-341 mutation (Miyakawa et al. Mol. Gen. Genet. 181: 207) resulted in the early
spoO phenotype at an intermediate permissive temperature (37°C) where
vegetative growth was largely intact. Sporulation, exoenzyme production
and competent cell formation were reduced at 37°C in the mutant. The
div-341 mutation was found closely linked to the sacU h 32 mutation (Kunst
et al. Biochimie 56: 1481) by transformation which results in pleiotropic
phenotype: hyperproduction of exoenzymes and derepressed sporulation in
the presence of excess nutrients. A double mutant (div-341 sacU h 32) retained the sacU h 32 property of hyperproducer of exoenzymes even at 37°C
and showed the sacU h 32 property of sporulation at 30°C and an intermediate property at 37°C. The filamentous growth at higher temperature(40°C) of the div-341 strain could not be recovered by the introduction of
the sacU h 32 mutation into the strain.
From the opposing properties of both mutations and their close linkage

RESEARCHES CARRIED OUT IN 198323

we conclude that the sacU gene might be a regulator gene for the div-341
structural gene and that derepression of the div-341 gene, which is causedby the sacU k 32 mutation, might be responsible for the high production of
exoenzymes and high incidence of sporulation in the presence of excess
nutrients. The div-341 gene product might be involved in a step required
for the excretion of some exoenzymes and cell surface proteins. Uponstarvation for nutrients, the div-341 gene might be derepressed to carry out
early sporulation events. (Mol Gen. Genet. 190: 176).
Flagella Are Formed at the Late Stage of Cell Cycle Just
before the Septum Formation in Escherichia coli.
Akiko NISHIMURA and Yukinori HIROTA

We discovered a phenomenon in E. coli that the formation of flagella iscoupled with cell division (Nishimura and Hirota, Ann. Rept. Natl. Inst.
Genet. 32 (1981), 31-35). In this paper, we report that flagella in E. coli
are formed at the late stage of the cell cycle, just before septum formation.
Formation of flagella in wild type cells, PA3092, was studied as follows.
PA3092 was exponentially grown in broth at 30°. The cells were negatively
stained with phosphotungstic acid (pH 7.0) and electron micrographs weretaken. On the EM photographs, the number of flagella per cell (N) and thecell length (I) was measured. The total number of cells measured was 372.
These cells in random population were classified into 12 groups by cell
length (It). The average number of flagella per cell length, D t (=Nt/l t),
was calculated for each cell length group (It). The cell length (It) was used
as one of the factors which possibly indicate the stage in the cell cycle,
because rod cells like E. coli grow lengthwise without changing thickness.
If flagella are formed continuously abreast of increase in cell length, D twould be constant irrespective of cell length. On the contrary, if flagella
are only formed at a special stage of the cell cycle, D t would decrease ininverse proportion to the increase in cell length.
The results are as follows. The cells in the smallest class (Io= 1.56 ,urn),
i.e. the class of newborn cells, were found to have the highest value of D t(D o=5.23/,um). The larger the cell size (It) became, the lower the value of
D t became in inverse proportion to cell length. D t increased again after
the cell size reached 1.7 times of the cell size of newborn cells. A septum
was observed in a portion of the cells in this class. The cells which have a

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

20
.c

+-'

en
c _

c

~o

.Q
n.

- ,
~o
,-

8.10

Qj

+-'

'"::J

'"

01

'"

4=
'"
o

+-'

.8

~

Q)

..0

E

o 1 ---"----------;!:2--=~
1-

cell
(A)

::J

C

5

05 1-~~--'---'-~~-:::2~-W...."""-';31cell lengHl (it/iol(B)

Fig. 1. Length (It) distribution and the number of flagella per cell length (D t)of E. coli PA3092. (A) length measurements of exponentially growing wild typecells at 30°. The total number of cells measured was 372. The average length of thecells was 2.59 ,urn. The hatched area represents the distribution of dividing (constricting) cells (5.15 % of total); the average length was 3.53,um. The average
length of the newborn cell (7.00% of total) was 1.56,um. The length distribution
sloping on right side indicates the exponential increase of cell length. (B) logarithmicplot of the relative number of flagella per cell length (D t ) versus relative cell length(It) in each length class. The average number of flagella per cell length of newborn
cells (Do) was 5.23 ,urn.

septum between the two daughters had almost the same value of D t as
newborn cells.
From these results, we suggest that, during the cell cycle, the flagella areformed at the stage of septum formation. After the completion of cell
division, formation of flagella is suppressed.

RESEARCHES CARRIED OUT IN 1983

III.

25

BIOCHEMICAL GENETICS AND IMMUNOGENETICS
Conserved and Variable Antigenic Specificities on the H-2
Derived from a Japanese Wild Moose, MOL.SGR
Kazuo MORIWAKI, Tomoko SAGAI, Nobumoto MIYASffiTA,
Hitoshi SUZUKI and Toshihiko SHIROISffi

Eight monoclonal antibodies of mMS series have recently been preparedusing BlO.MOL-SGR strain (This Report, No. 33 1983). We surveyed the presence of reactivities with these mMS antibodies in various mouse subspecies
in the world and also in another species, Mus speciligus. As demonstrated
in Table I, an H-2 antigen detected by mMS 38 seems to be most conservative in the present survey. It was able to react with all the Mus musculus
subspecies examined at the higher phenotypic frequency and moreover
reacted with the other species, Mus speciligus. K33 antigenic specificity
detected by monospecific conventional antibody was also widely observed
not only in Mus musculus species but in M. speciligus (This Report, p. 00).
Southern blot analysis using a H-2K DNA probe demonstrated that regardless of their subspecies, all mice having K33 determinant commonly
shared 2.5 Kb DNA fragment digested with BgIII restriction enzyme.
The antigens detected by mMS 1 and 37 were also relatively conservative.
They were distributed within Mus musculus species at the higher frequency,
Table 1. Frequency distribution of MOL-SGR H-2 specificities detectedwith mMS series monoclonal antibodies in various wild mice
Phenotypic frequencies
Name ofspecies orsubspecies
M. m. molossinus
M. m. subsp.
CHN
M. m. castaneus
M. m. musculus
M. m. domesticus
M. speciligus

No. ofSampleMonoclonal antibodies (mMS)

cAb

1

37

38

24

25

42

39

40

36
15

.89
.23

.36
.37

.69
.67

.06
.00

.06
.00

.28
.00

.17
13

.64

.36

.20

.13

19
8
17
13

.79
.88
.23
.00

.74 1.00
.63
.63
.59
.35
.00 .31

.00
.00
.06
.00

.00
.00
.12
0.0

.42
.25
.00
.00

.47
.63
.00
.00

.42
.25
.00
.00

502

.34

.13
.18
.00

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

but not in M. speciligus. The most variable determinant examined wasthat detected by either mMS24 or mMS25. It reacted only with the donor
strain and DOM.PGN strain which derived from Canadian wild populationin the Ontario State. These two mouse strains are evolutionarily remote eachother by around one million years (Yonekawa et al. 1980). The antigenic
determinant on H-2 molecule detected by mMS 24-25 is considered to behighly variable. But, presumably it could remain unchanged by chance inthe both lineages of wild mice, even though they have been separated longtime ago.
Founder Effect in the Frequency Distribution of Mouse H-2
Observed in the Japanese Wild Populations
Kazuo MORIWAKI, Tomoko SAGAI and Nobumoto MIYASHITA

Extensive polymorphism of H-2 class I antigens has already been demonstrated both in the laboratory mouse strains and the European wild
populations. We have surveyed the wild populations of Asian mice,
mostly Japanese ones, by using monospecific antisera directed to both of
the well-known H-2 private specificities and the newly identified ones inJapanese mice.
As shown in Table 1, the Japanese wild populations exhibited relatively
higher frequencies of several H-2 specificities such as K26, D32, K20,
KD501, KD507, K509 and K51O. Phenotypic frequencies of all these
specificities were more than 10 % which was apparently higher than thosein Chinese wild population. On the other hand, the number of H-2 specificities detected was less in China mainland than in Japan. Probably theJapanese populations were originated from a small fraction of the Chinese
populations. This has been also suggested from the cytogenetical surveyof chromosome C band patterns both in Japanese wild mice and Chinese
ones. No such differences in frequency distribution of H-2 were observed
between the European population and their Canadian derivatives which
we surveyed in this study. Presumably the founder of the Canadian population had a size large enough to eliminate founder effect, when they weretransfered from Europe.

Table 1. Frequency distribution of H-2 antigenic specificities in various wild mice
Phenotypic frequencies H-2 haplotype and antigenic specificities
Name ofspecies &subspecies

M. musculus
molossinus
subsp. CHN
castaneus
domesticus*
domesticus**
M. speciligus

No. ofsample

b

k

q

u

~
~

(j

K
33
115
41
28
49
320
14

f

d

~

tTl

CI.l

D

2

.00 .00.12 .05.18 .04.12 .06.08 .03.88 .00* Collected from Ontario, Canada.
** Gotze et al. 1980

K
31

D

4

K D
26 9

K D
23 32

K D
17 30

K KDKDKDKD K K
20 501 502 503 507 509 510.00 .00.05 .00.00 .04.20 .04.15 .16.00 .00.26 .10.24 .07.29 .36.10 .12.07 .02.00 .00.00 .13.00 .05.14 .39.00 .02.06 .05.00 .00.00 .01.00 .05.00 .04.00 .04.02 .06.00 .00.16 .43 .08 .02 .11 .16 .40.15 .27 .12 .00 .00 .05 .15.21 .42 .00 .07 .04 .11 .21.36 .16 .02 .00 .00? ?
.01.00 .00 .00 .00 .00 .00 .00

:I:

tTl

CI.l

~

~

tTl

tJ
0
C

>-l

Z
.....

\0
00

w

~

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

MS-Series Monoclonal Antibodies Directed to the H-2 and Ia
Antigenic Determinants of BIO.MOL-SGR Mouse
Tomoko SAGAI, Toshihiko SHIROISHI and KAzuo MORIWAKI

B10.MOL-SGR (H_2wm7) carries the H-2 complex of Japanese wild
mouse and its intra-H-2 recombination frequency is extremely high (About
3 %. See Nature 300: 370-372, 1983). As previously reported, we haveobtained several hybridoma cell lines secreting monoclonal antibodies to
H-2 gene products of BIO.MOL-SGR (This Report No. 33, 1983). Recently we have established total 19 hybridoma clones and analysed strain
distribution of their specificities. The intra-H-2 subregions corresponding
to each of those specificities were decided using BlO.MOL-SGR recombinants. As shown in Table I, 8 clones are secreting antibodies directed
to class I antigen and the others those to class II antigens. Among theTable 1. Characterization of monoclonal antibidoes against BIO.MOL-SGR
Hybridomadesignation

Class IClass II

Recipient

Tentative
antigenic
determinantSpecificity

Cross reactive
haplotyped, k, f, r, v, pk, q, r, v
k, q, S, r, v, p, u,
(d)
none
none
f, v, p(f, v, p)k, f, r, j, p, (b, d)

MSI
MS37
MS38

BIO
BIO
BIO

msl
ms2
ms3

Kwm7Dwm7
Kwm7Dwm7
Kwm7Dwm7

MS24
MS25
MS42
MS39
MS40

BlOxBlO.A
BIOxBlO.A
BIO
R212
R212

ms4
ms4
ms5
ms6
ms7

Kwm7
Kwm7
Kwm7
Dwm7
Dwm7

MS34
MS2
MSl5
MS43
MSl7
MS28
MS33
MSIO
MSl2
MS35

BlO.D2
BIO
BlO.A
BIO
BIO.A
BIOxBIO.A
BIO.D2BIO.A
BIO.A
BIO.D2

msl
ms2
ms2
ms2
ms3
ms3
ms3
ms4
ms5
ms6

MS36

BIO.D2

ms7

I_Awm7
or
I_Ewm7

none
v
v
v
q,v,p
q, v, pq, v,pb, d, q, v, Pf, q, v, jb, k, q, s, r, v,
j, p, ub, f, q, s, r, v,
j, p

RESEARCHES CARRIED OUT IN 1983

29

former antibodies, MS-24 and 25 (H-2K region specific) did not react withany inbred and BIO.MOL congenic strains tested. They seem to be highly
specific to the H-2 molecule of BIO.MOL-SGR, while MS-I directed toboth H-2 K and D region was crossreactive with all the BIO.MOL congenic
strains tested. The latter antibodies for class II were classified to 7 groups
by their strain distribution pattern. MS-34 monoclonal antibody was
specific to BIO.MOL-SGR and the other reacted with BIO.SM, BIO.MOLTEN2 and BIO.MOL-NSB, each of which has different class I antigens.
Those antibodies should be useful for studying the mechanism of highrecombination in BIO.MOL-SGR and surveying H-2 complex in the wild
mice as well.
Effect of H-2 Complex on the Incidence of Urethan-Induced
Mortality and Malformation in the Mouse Embryos
Nobumoto MIYASHlTA, Shinji NITo· and Kazuo MORIWAKI

Teratogenecity of carcinogens and the similarity in the mechanism ofcarcinogenesis and teratogenesis have been studied by many investigators.
Genes, however, which regulate the susceptibility to carcinogen-induced
teratogenesis have not been well determined. We studied the effect of
H-2 gene complex on the urethan-induced malformation in mouse embryos
by using H-2 congenic strains. Urethan (ethyl carbamate) was used as ateratogenic agent because it can penetrate the placental barrier and reachthe embryos at any stage of pregnancy.
Six to eight week-old virgin females of BIO.A and BIO strain were used.
These mice were weighed and mated from 4 p.m. to 12 p.m. with male miceand their vaginal plugs were checked to determine Day 0 gestation. On
Day 10, they were again weighed and received a single s.c. injection ofurethan, 1.00 mg/g or 0.75 mg/g body weight. The pregnant mice weresacrificed on Day 18 and the implants, deaths and survivors of the fetuses
were checked. Subsequently, the fetuses were taken out and their externalmalformation were examined. As shown in Table I, BIO.A mice had significantly higher rate of mortality than BIO mice at the dose of 1.00 mg/g ofurethan. As for the external malformations (Table 2), BIO.A also hadhigher frequency than BIO at the dose of 0.75 mg/g of urethan. These
results indicate that the H-2 gene complex or its closely linked gene(s) are•

Tanabe Seiyaku Co.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34
Table 1. Effect of H-2 complex on the mortality of mouse embryo
following transplacental administration of urethan

Strain
BlO
BI0.A
BIO
BI0.A
BI0
BI0.A

No. offemale micewithimplantation

Dose(mg/g)
1.00
0.75
Control

12
11
13
11
14
19

No. of implants
Av.
9.4±1.98.5±1.99.2±1.1
11.0±1.3
9.4±2.5
10.3±2.0

Deaths%Total No.
113
93
119
121
132
195

10
31
17
17
16
31

8.8
33.3*
14.3
14.0
12.1
15.9

Living fetuses
No.
%
103
62
102
104
116
164

91.2
66.7*
85.7
86.0
87.9
84.1

• P<O.OOITable 2. Effect of H-2 complex on the external malformations
by urethan in mouse embryo
Strain
BIO
BI0.A
BIO
BI0.A
BI0
BI0.A
•

Dose
(mg/g)
1.00

0.75
Control

No. of living fetuses
Total
Male Female
103
62
102
104
116
164

52
31
52
51
65
87

51
31
50
53
51
77

Fetuses with malformation
No.

%

25
23
2
11
0
2

24.2
37.1
2.0
10.6
0.0
1.2

X 2 test (p)
N.S.·
<0.02
N.S.

N.S., not significant.

involved in determining susceptibility to the mortality and malformationinduced by urethan.
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 transplantation ofearly embryo under the kidney capsule. Previously we reported that thedifference in the weight of tumors which were derived from the syngeneictransfer of 7-day-old embryo was observed among five A.H-2 congenic
strains 40 days after transplantation. H-2D locus or its neighboring regions
were suggested to affect the growth of the embryo-derived tumors. It has

RESEARCHES CARRIED OUT IN 198331

not been clear yet whether this difference in the tumor growth is related tothe degree of further malignant change in those induced teratoma or not.
In the present report, the tumors induced in a susceptible strain, A.TL,
and in a resistant one, A.SW, were examined histologically. The ratio ofmalignant teratomas to all the developed teratomas was 18/28 in A.TL
strain and 15/31 in A.SW strain. Though the former was a little higher
than the latter, there was no significant difference between them. Even inin the smaller teratomas, the foci of malignant teratoma cells had been
observed. These results suggest that a putative H-2linked gene controlling
the teratoma growth is not related to the rate of malignant transformation,
instead to the degree of nutrient supply through the introduction of blood
capillaries into the developing teratoma, because the larger teratomas were
found to have a plenty of blood vessels introduced from the recipient.
Restriction Fragment Length Polymorphism of NTS rDNA
in Mole Rats
Hitoshi SUZUKI, Kazuo MORIWAKI, Ryo KOMINAMI*,
Masami MURAMATSU* and Eviator NEVO"

Mole rats in Israel (Spa/ax ehrenbergi) have chromosome number polymorphism such as 2n=52, 54, 58 and 60. We attempted to estimate thegenetic diversity in DNA level among those mole rat populations. Southernblot analysis with ribosomal DNA probe prepared from mouse and two
restriction enzymes, EcoRI and BamHI, revealed that each individual had
various sizes of major restriction fragments both digested by EcoRI and
BamHI. Since the probe specificically hybridizes to an area of 3'-end of
28S rRNA gene in mice and probably in mole rat as well which is considered
to be highly conserved, this length polymorphism seems to depend on
sequence polymorphism of non-transcribed spacer (NTS) DNA. This
fact was also confirmed by the construction of restriction maps by Southernblot analysis with several restriction enzymes. In the genomes of molerats collected from 13 different localities in Islael, we could characterize
the major types of NTS of rDNA repeating units based on the EcoRI and
BamHI fragment lengths as shown in Table 1. Generally, mole rats carrying
the same chromosome number had a similarly characteristic combination***

Tokyo University.
University of Haifa, Israel.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34
Table 1. Restriction flagment length polymorphism of rONA in mole rats

Chromosomal
species
52

54

58

60

60

TypeofNTS
Type of population

No.

Near 52/58 hybrid zone
CentralMarginal
Near 54/58 hybrid zone
CentralMarginal
Near 58/52 hybrid zone
CentralNear 58/54 hybrid zone
Near 60/58 hybrid zone
CentralMarginal
Isolate

4
4
4
4
4
4
4
4
4
4
4
4
2

A

B

+

+t
+t

+
+
+
+

+t

+

C

o

E

F

G

+t

+t

+

+t

+

+t

+
+

+

+t

ttt
ttt
+t

ttt
ttt

+

Each restriction NTS DNA types is tentatively designated by the letters.
Approximate restriction fragment sizes (kb) introduced either by BeoRI or BamHI are
as follws in the parentheses: A (6.6-7.4, 8-9), B (8.6-9, 10-10.5), C (8, 10), 0 (7.5-8.2,
8.5-9), E (12.5-13, 9.5-10), F (12-14,16-18), and G (16-17,10-11).

of the types of NTS each other, suggesting that the specificities of the NTS
of rDNA had been generated after the chromosomal rearrangement. Somemole rats living in near 58/54 hybrid zone, however, had neighbor's type ofthe combination. This phenomena is possibly explained by a gene flowfrom 54 population to 58.
Further Survey of T-Iymphocyte Differentiation Antigens
in VariOllS Mouse Subspecies by Serological Methods
Yasuyuki KURIHARA, Fran<;ois BONHOMME* and Kazuo MORIWAKl

We have surveyed geographical distributions of Thy-I, Lyt-I and Lyt-2
alleles in wild mice using serological methods. European subspecies, Mus
musculus domesticus and M. m. brevirostris, exhibited Thy-I.2 antigen, but
Asian ones mostly Thy-I.I. Lyt-2.2 was unique to some European mice,and Lyt-2.I was widely distributed both in Asian and European mice.
Mus musculus subspecies from wild populations examined so far expressed* Institut des Science de I'Evolution, Montpellier, France.

RESEARCHES CARRIED OUT IN 1983

33

Table 1. Allelic distribution of T-lymphocyte differentiation antigens
in various mouse subspecies
Name of species
or subspecies
Mus musculus
M. m. molossinus

M. m. castaneus
M. m. musculus
M. m. domesticus

M. m. bactrianus
M. m. brevirostris
M. m. subspecies
Seychells

China

Mus spretus
Mus spicilegus

Allele of differentiation antigens
Thy-l

Lyt-l

Lyt-2

No. of miceobserved

1
1
1
1
1
1
1
1
2
2
2
1
NT
2

2
2
2
2w
2w
2w
2
2
2
2
2
2
2
2

1
lw
NT
1
NT
lw
lw
1
1
2
1,2

39
1
6

1
1
1
1
1
1
1

2
2
2

8
3
20
6

2
2
1

1
2
2

1

NT
2

1

1
10
3
28
49
2
6
1
1
5

1

w:

showed weak reactivity
no reaction
NT: not tested

only Lyt-1.2. We also surveyed two closely related species, Mus spretus
and M. spicilegus. Though their lymphocytic phenotypes were similar to
those of Asian mice, part of M. spicilegus expressed Lyt-1.1 that could not
be found in the survey of M. musculus (Table 1).
Certain Asian mice related to M. m. castaneus had weak reactivities toanti-Lyt-1.2 and anti-Lyt-2.1 monoclonal antibodies by complementmediated cytotoxicity test, which was confirmed by absorption test as well.
At present, we can not decide whether the weak reactivities resulted from

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

the less amount of expression or from the little change of the antigenic
molecules on cell surface. Further experiments to solve this problem arecurrently in progress.
Expression of Rat Class I Antigens on the Erythrocytesof Various Strains
Tsukasa HIRASHIMA*, Takashi NATORI** and Kazuo MORIWAKIIn BI rat strain (haplotype i) RTl.E antigens can not be expressed on
the erythrocytes (Heing, W. et al. Journal of Immunology, 128: 402, 1982).
We have surveyed this trait among 10 inbred strains of rat by using either
hemagglutination method (Natori et al. Transplant. Proc. 13: 1563, 1979)
or flat plate micro-cytotoxicity method (Shiroishi et al. Microbiol. Immunol. 25: 1327, 1981).
Each antigenic specificity of RTl was detected by the following antisera.
"1": WKAH anti-F344 absorbed with ACI. "2": F344 anti-TO. "3":
K(DA) anti-BN. "4": WKAH anti-ACI (monoclonal antiserum).
As shown in Table 1, RTI specificity "1" can not be expressed on theerythrocytes in several strains such as W jMs, BNjfMai, ACIjMs and
NIGIIIjMs. Similar differential expression of RTl on erythrocytes andlymphocytes has also been observed in the specificities "2" and "4". SpeciTable 1.
StrainReactivity of four alloantisera with erythrocytes and lymphocytes
Hemagglutination activity
RTl antigenic specificities
2

F344/Ms
BUF/Ms
LEJ/Ms
W/Ms
WKA/Ms
BN/fMai
ACI/Ms
ALB/Ms
WKA/Hok
HlGIII/Ms++• Otsuka Seiyaku Co.

•• Hokkaido University.

+
+
+

3

4

2

+
+
+
+

+

Cytotoxic activity

+
+
+
+

+
+
+
+
+
+

3

+

4

+
+
+
+

RESEARCHES CARRIED OUT IN 1983

35

ficity "3" was detected only in BN/fMai strain both on erythrocytes andlymphocytes. Whether each of those antigenic specificity is coded for by
RT1.A or RT1.E is not yet clear.
Meantime, it should be desirable to adopt the RTl specificity on lymphocytes as a genetic profile of inbred strain.
Analysis of Mitochondrial DNA in Cytoplasms ofMale-Sterile Rice
Saburo NAWA, Masa-aki YAMADA, Yoshio SANO and Taro FUJII

A number of cytoplasmic variations conferring male sterility in maize
have been recognized and classified into several groups depending on theability of nuclear genotypes in tester stocks to restore male fertility. On
the other hand, several discrete low molecular weight DNAs in mitochondria
were found in all lines (including normal cytoplasm) with corresponding
different banding patterns. These patterns were shown to be correlatedwith several type cytoplasms defined by nuclear fertility restorer genes.
Among these extrachromosomal DNAs present in mitochondria, the candidates for the male sterility were S-l and S-2, linear episomes of 6.2 and5.2 kb, respectively, which were present only in the S-cytoplasms. Certain
cms-S strains have undergone spontaneous reversion to male fertility at ahigh frequency. It has been known that concomitant with the reversion of
the male sterility to the fertile condition was the disappearance of themitochondrial plasmid-like DNAs (S-l and S-2) and changes in the mitochondrial chromosomal DNA.
In rice, cytoplasmic male sterility was obtained by Shinjo and Omura.
Shinjo found that the cytoplasmic male sterility was controlled by a nuclear
restoration gene Rf and produced the isogenic BT lines having genetic
background of Taichung 65 by repeated back crossing. Yamaguchi andKakiuchi detected two small mitochondrial DNAs, B-1 (1.5 kb) and B-2(1.2 kb), in BT cytoplasm of male sterile rice, whereas none in normal fertile
cytoplasm. Although no spontaneous reversions have been reported fromBT lines of rice, Fujii obtained two revertants to male fertility by treating
BT lines with EMS. We examined mt-DNAs from the revertants, wheresome changes of B-t and B-2 would be expected if the small mt-DNA are
the molecular components responsible for these reversions. Mitochondrial
DNAs were prepared from rice lines with normal cytoplasm (n; rfrf), with

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

BT source of male-sterile cytoplasm (ems; RfRf) and with the revertantsof fertile cytoplasm (rev; rfrf). Agarose gel electrophoresis of these preparations revealed a main high-molecular-weight DNA band. However, no
additional low-molecular-weight DNA bands were detected in all cytoplasms,
when young shoots were used as the source of mitochondria fraction. It
was found that preparations from calluses of the male sterile cytoplasm
contained two fast-migrating bands at the positions corresponding to approximate molecular weights of 1.5 and 1.1 kb, respectively. However,it became evident that these bands were detected in mitochondrial preparations from normal fertile cytoplasm and from the revertants. Both of
the bands were sensitive to ribonuclease and resistant to deoxyribonuclease.
The evidence that the two bands migrated at the same positions as 28 S
and 18 S ribosomal RNA, respectively, suggests that they were derived fromribosomes as contaminants. After RNase treatment, additional electrophoretic bands that might be DNAs of molecular weights of B-1 and B-2
were not detected in all cytoplasms. We used the male sterile cytoplasm
with the nuclear restorer gene (ems; RfRf) as the source of mt-DNA to
get many seeds, on the basis of the fact in maize that the fertility elements
are present in the S-cytoplasm groups regardless of nuclear background,even in the cytoplasm with the nuclear restorer gene. This may not fit
the case of BT lines of rice. If so, our data suggest that concomitant withthe reversion of the male sterility in BT rice to fertile condition, either by
the nuclear restorer gene or by treatment with EMS, is the disappearanceof the mitochondrial plasmid-like DNAs, B-1 and B-2. Further analysis
of mt-DNAs of ems; rfrf, however, is necessary.

RESEARCHES CARRIED OUT IN 1983

IV.

37

DEVELOPMENTAL GENETICS AND SOMATIC
CELL GENETICS

Electron Microscopic Studies on the Ultrafine Structuresof In Vitro Differentiated Cells of DrosophilamelanogasterYukiaki KURODA and Yutaka SHIMADA*In the series of tissue culture studies of Drosophila embryonic cells, thetissue specificity and time specificity of the lethal gene actions have been
investigated by examining the defects in cultured cells from some sexlinked recessive lethal embryos. Thus, the one-to-one correspondence
has been analyzed between a differentiated character or function and a
specific gene on a specific locus on the chromosome.
When undifferentiated cells dissociated from post-gastrula embryos of
the wild-type strain were cultured in medium K-17 supplemented with 15 %fetal calf serum and 0.1 p,gjml fetuin, they differentiated into various embryonic or larval cells such as muscle cells, epithelial cells, cellular spheres
and nerve cells. Their specific morphology and function were easily characterized under a light microscope.
In the present experiment, the ultrafine structures of these differentiatedcells were examined under an electron microscope. Cells dissociated frompost-gastrula embryos of the Oregon-R strain of D. melanogaster were
cultured for various days. Muscle cells, epithelial cells and nerve cells
which differentiated in vitro were fixed with 2.5 % glutaraldehyde and 1 %osmic acid solution. Then ultrathin sections were prepared, stained withuranyl acetate and lead citrate, and observed under an electron microscope.
In muscle cells, the formation of typical myofibrils was observed. In
syncytium of fused muscle cells, the characteristic M and Z lines in the A
and I bands of sarcomeres were clearly found. In nerve cells, many neurofibrils were parallel running in nerve fibers which extended and branchedduring cultivation. In epithelial cells, the formation of interdigitations,gap junctions and desmosomes were observed between the membranes ofadjacent cells.

* Department of Anatomy, School of Medicine, Chiba University, Chiba.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

The results suggest that the ultrafine structures of these differentiatedcells may be useful for analyzing the more detailed characteristic action of
the specific genes during normal development.
Expression and Induction of Tissue-Specific Enzymes in
Cultured Chicken Liver Cells
Yukiaki KURODA and Etsuko SUGAWARA'"To examine the effect of various chemicals on the function of livingorganisms, the induction of tissue-specific enzymes was used as markers.
The treatment with 0.05 % trypsin solution was found to give a good result
to dissociate cells from livers of 6-day chickens. Collagen (Vitrogen 100)
was effective in adhesion of liver cells on the surface of petri dishes. An
attempt was made to induce the activity of tyrosine aminotrasferase (TAT)in these liver cells.
Dissociated liver cells were cultured in medium 199 supplemented with
10 % fetal calf serum. After cultivation for 22 hours, IO,uM hydrocortisone was added to culture medium and the activity of TAT was determined.
The enzyme activity rose up about three times 24 hours after addition ofhydrocortisone. The addition of insulin before treatment with hydrocortisone had no effect on the induction of the enzyme activity.
The effective concentrations of hydrocortisone for induction of the TAT
activity were 1-30,uM and no difference in effectiveness was found withinthese concentrations. The addition of 10,uM dexamethasone was also
effective in induction of the TAT activity. These results indicate that thissystem may be used for examining the effect of various chemicals on thefunction of living organisms.
Mutagenic Activity of Ames Test-Negative Compounds in
Cultured Chinese Hamster Cells
Yukiaki KURODA, Akiko YOKOIYAMA and Tsuneo KADAIn the last decade, various mutagens in the environment has been effectively detected by the microbial systems including the Ames tests in thepresence of the microsome fraction of mammalian livers. On the other
hand, not so few chemicals showing no mutagenicity in microbial systems'" Department of Research, Chubu-Rosai Hospital, Nagoya.

RESEARCHES CARRIED OUT IN 198339

were found to have the carcinogenic activity in experimental animals.
In the present experiment, several carcinogens which had no mutagenicity
in the Ames test and was proposed to be tested for their mutagenicity in
the International Programme on Chemical Safety (IPCS) of the World
Health Organization (WHO), were examined in their mutagenicity incultured Chinese hamster V79 cells.
Chemicals tested were hexamethylphosphoramide (HMPA), o-toluidine,benzene, safrole and phenobarbital as carcinogens and caprolactam andbenzoin as non-carcinogens. Cells were treated with these chemicals at
various concentrations for 3 hours, and their cytotoxic effects were examined by the colony-forming activity of cells. HMPA, o-toluidine,benzene and caprolactam had no or weak cytotoxic effects at concentrations up to 500 pg/mI. Safrole, phenobarbital and benzoin had moderate
or strong cytotoxic effects on V79 cells.
Next, the activity to induce 6-thioguanine (6TG) resistant mutations ofthese chemicals in V79 cells was examined. Safrole and o-toluidine induced mutations at 200 pg/ml or 400 pg/ml without addition of S-9 Mix.
HMPA and phenobarbital also induced mutations at concentration of
500 pg/ml. The mutagenicity of benzene was not or slightly detected in
V79 cells, both with and without S-9 Mix. On the other hand, caprolactam
and benzoin had no mutation-inducing activity without S-9 Mix.
These results indicate that four carcinogens except for benzene induced
6TG-resistant mutations, whereas two non-carcinogens had no mutagenic
effect in Chinese hamster V79 cells. Thus, the correlation has been foundbetween the tumor-producing activity in animals and the activity to induce
6TG-resistant mutations in Chinese hamster V79 cells.
Effect of Some Growth Factors on Cultured Human Diploid
Cells in Clonal Culture
Yukiaki KURODA

In the previous study, the effects of epidermal growth factor (EGF) andfibroblast growth factor (FEF) on the mode of proliferation of human
diploid cells have been analyzed in clonal culture. It has been found thatboth EGF and FGF had growth-stimulating effects on both dividing andnon-dividing cells in population.
In the present experiment, effects of platelet-derived growth factor

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34(PDGF), fibronection and hydrocortisone on the growth pattern of human
diploid cells at the sixth population doubling level were examined in clonal
culture.
PDGF had a growth-stimulating effect on cells at a concentration of
0.1 unit/ml, but an inhibitory effect at higher concentrations. Fibronectin
also showed a growth-stimulating effect on only dividing cells at a concentration of 10 ng/ml. Higher concentrations of fibronectin showed adecrease in its stimulating effect on the growth of cells.
Hydrocortisone was the most effective among these three growth factors.
At a concentration of 0.05 pg/ml, it specifically stimulated the growth of
only dividing cells in population, but no effect on non-dividing cells, resulting in 10 % increase in the average cell number per colony, compared withthat in control cultures. These results indicate that the action of hydrocortisone was different from that of EGF or FGF. The former was effective on only dividing cells, and the latter was effective on both dividing andnon-dividing cells in population. The difference in the action of growth
factors may be related to the control action of growth factors for the senescence of human diploid cells.
Histological Analysis of Embryonic Lethal Mutant in

Drosophila melanogaster
Kiyoshi MINATO

The process of nervous hypertrophy in an embryonic lethal mutant,Df(l) Notch-B, of Drosophila melanogaster has been analyzed histologically.

The hypertrophy of nervous tissue in this mutant appeared to be caused bymore segregated neuroblast cells than those in normal embryos in theearlier period of embryonic development.
In this year, a further improvement of fixative technique was successfully
made to make more accurate analysis possible. Moreover, a new incubation method of embryos was devised, in which each embryo was incubatedin many column-drops of liquid paraffin made in the space of 450 pm
depth between the cover-slip and the slide glass. This method makes it
possible to observe very easily under the microscope the change of theexternal morphology of many embryos in a better orientation, without
developmental arrest by lack of oxygen and, hence, to analyze quantitatively
even the mutants having various lethal times and lethal features.

RESEARCHES CARRIED OUT IN 1983

41

Using this method, the external morphology of Notch-8 embryos in theprocess of neuroblast segregation was microscopically observed in ventral
view for ventrally-segregating neuroblasts, but no significant difference
could be detected between mutant and normal embryos. With the same
method, the analyses of other embryonic-lethal mutants are now in progress.
Mode of Action of an Inhibitor for Stenotele Differentiationin Hydra
Toshitaka FUJISAWA

An inhibitor for stenotele differentiation has been found in Hydra tissue.
It was examined at which stage during stenotele differentiation the factor
exerts its action.
Stenotele is one of 4 nematocytes (sting cells) in Hydra, all of which
differentiate from interstitial stem cells after 2 to 5 rounds of cell division.
Commitment of a particular type of nematocyte occurs near the SjG2
boundary in the terminal cell cycle just prior to cytodifferentiation.
The tactics to determine the timing of the inhibitor action during stenotele
differentiation is the following. The precursor cells are exposed to theinhibitor for certain time periods and allowed to differentiate into stenoteles.
Since the cell cycle time of precursor cells (18h), the stenotele differentiation time (72h) and the maturation time of stenotele capsule (24h) are
known, it is possible to reconstruct the differentiation pathway of cells which
respond to the inhibitor and to detect the timing of the inhibitor action.
By using the above method it was determined that the inhibitor exerts
its action for about 6h period after the SjG2 boundary in the terminal cell
cycle. The inhibitor applied before the SjG2 boundary or after first 6h of
G2 phase had no effect. These results appear to suggest that commitment
is a process extending over about for 6h during which the process is vulnerable to the external signals and can be changeable but after which the process
is no longer reversible.
Molecular Cloning of extra organs Locus of Drosophila melanogaster
Masatoshi YAMAMOTO and George L. G. MIKLOSWe tried to clone the DNA of extra organs gene which plays an im-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

portant role in the morphogenesis especially at the process of imaginal
disk development. Since the gene is known to localize on the 20AI-2
bands in the salivary chromosome, we first looked for a cloned DNA fragment which is from the very intimate bands of the Drosophila genome. We
planned to use the DNA fragment as a probe and walk into the eo locus.
Among already published DNA fragments, collagen-like DNA was reported
by Monson et al., to hybridize in situ at the region 19E-19F. The gift DNA
from Monson was then fine mapped to determine the gene locus of collagen.
In the proximal region of the X chromosome in Drosophila there are abundant chromosomal rearrangements and thus it is possible to make up ahemizygous deficiency or a duplication at the level of one band genetically.
We made up a series of deficiencies in the region by appropriate chromosomal manipulations. By Southern hybridization it is possible to determine
on which band the DNA fragment used as a probe localizes in the X. If aprobe DNA comes from a band deleted as a hemizygous deficiency theintensity of hybridization of the DNA is about a half strength of the one
hybridized with diploid amount of autosomal DNA which is considered as
an internal control. From this series of experiments the collagen-likeDNA was determined to locate at 19F6 which is one band distal from 20Al.
We then used this collagen-like DNA as a probe for the walking and cloned
28 kb long DNA to the proximal and 24 kb to the distal. The DNA fragment of the most proximal was not in the chromosomal fragment of
T(X; Y)Bl54R , but within y+ Ymal 126 chromosome. In genetical examinattion, eo phenotype appears in 5 % of the individuals whose genotype is
Df(l)A7/y+ Ymal 126 , but it significantly increases up to 85-90 % in the fliesof Df(I)A7/ T(X; Y)Bl54 R • This indicates at least a part of eo gene islocated between the breakpoints of T(X; Y)Bl54 and y+ Ymal 126 • We
have thus almost reached to one end portion of the eo gene. Weare now
trying to clone the DNA up to the breakpoint of T(X; Y)Bl54.
A Novel Method for Fate Mapping of the Genital Disk: The Region
Important for the Testes Elongation and Morphogenesis
Mitsuru SAKAIZUMI and Masatoshi YAMAMOTO

The adult testes of Drosophila melanogaster is composed of a pair ofcoiled tubes approximately 2 mm in length and 0.1 mm in diameter. The
adult reproductive system is derived from the gonads and the genital disk.

RESEARCHES CARRIED OUT IN 1983

43

The somatic gonadal cells develop independently from the germ cells andperform the morphohenesis of the adult testes. The rest of reproductive
system is contributed by the development of the genital disk. At the larval
life however the gonads and the imaginal disk are not connected.
At 1940's, C. Stern demonstrated from the studies of organ culture that
it is the pupal stage when these tissues are connected and then the larval
spherical gonads start elongation and coiling. Since then no study on thetestes morphogenesis has been done and it remains totally as a dark boxof the Drosophila development.
By using the mutant extra organs(eo) which causes a deletion or a duplication in a region of genital disk as well as other imaginal disks, we havestudied correlations between the failure of testes elongation and the anomalies in the male genitalia.
It is obvious that the testes elongation is totally dependent on the imaginal
disk development because the male of complete absence from the outergenitalia (derived from genital disk) retains under-developed sphericalgonads equivalent to that of 72 hrs after puparium formation. Furthermore, if the half of the outer-genitalia is deleted one side of the paired internal genitalia is also missing and one of the gonads remains as undeveloped. The examination of about 450 eo individuals led us to conclude
as follows;
1. There is a hierarchy in the appearance of abnormalities within the
disk. This suggests the process of genital disk development namely thecell lineage. The higher abnormalities to the lower in the outer-genitalia
goes posterior lateral plate=posterior clasper ~ anterior clasper ~ anterior
lateral plate ~ penis ~ genital arch --+ anal plate. When the anal plates
are missing the whole male genitalia is without a failure completely deleted.
On the other hand there are many males having deletions in the posterior
region of lateral plates and claspers but normal in the anal plates.
2. The region which is important for induction of testes elongationand coiling can be fate mapped at the overlapping area between the region
for the posterior lobe of lateral plate and the posterior portion of theclasper. The region may well become the testicular duct of mesodermal
origin and function in the induction of testes elongation.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Freezing of Silkworm Ovaries
Jun KUSUDA, Kimiharu ONIMARU, Takehico NOGUCHI
and Okitugu YAMASHITA·

Since the successfull preservation of mouse embryos at -196°C, thefreezing technique has been applied to store the oocytes and embryos ofseveral mammalian species for the purpose of the maintenance of their
genetic strains economically and safely. However, such a subzero temperature preservation is not reported in insect. Here, we show that silkworm
ovaries preserved in liquid nitrogen produce the mature eggs retaining thecapacity of embryogenesis and post-embryonic development.
Ovaries were taken out from 2 day old fifth instar larvae of a hybrid of
Nl06 and Cambodge which carried a dominant marker gene. They weresuspended in successive concentrations (0.5 M, 1.0 M, 1.5 M) of glycerolin Grace's medium containing 15% fetal calf serum. The organs were
packed into plastic semenstraws and cooled to -7°C. Straws were seededby holding with forceps precooled in liquid nitrogen to induce iceformation.
They were gradually cooled to -35°C at a rate of 1°C/min using a program
freezer and the straws were finally plunged into liquid nitrogen. Afterfreezing for 48 hr, the ovaries were thawed rapidly by agitating in a 40°C
water bath (at a rate of 500°Cfmin) and glycerol was washed out. The
treated ovaries were transplanted into the haemocael of 4 day fifth instar
larvae carrying no marker genes. The larvae grew to adults normally.
Since the planted ovaries were not connected with the host oviduct,
mature eggs were dissected out from adults and parthenogenetically activated by incubation at 46°C for 18 min. The activated eggs were thentreated with HCl solution (specific gravity, 1.075 at 15°C) at 46°C for 5 min
to prevent diapause. About 10 days after the acid treatment, larvae hatchedfrom eggs. They grew to fifth instar and exhibited the mark of marker
gene on their integuments. Thus, the silkworm ovaries can survive infreezing temperature retaining the capacity of further development.

•

Department of Agriculture, Nagoya University.

RESEARCHES CARRIED OUT IN 1983

v.

45

CYTOGENETICS

Decrease of C-Bands and NORs in the Black Rats from
Amami and Toknnoshima
Tosohide H. YOSIDA

Large and clear heterochromatic C-bands are generally observed near
the centromere of all chromosomes in the black rats (Rattus rattus), but
the Japanese black rats (R. rattus tanezumi) were remarkable by showing adecrease of C-bands in some chromosome pairs (Yosida and Sagai 1975,
Chromosoma 50: 283-300). The animals collected from the plain area of
Amagi and Tokunoshima islands located in southern Japan, were characterized by a remarkable decrease of C-bands in their karyotype. They
had C-bands in only two of 13 acrocentric autosome pairs, and complete
decrease of C-bands occurred in the other II acrocentric autosome pairs.
On the other hand, the black rats collected from the high land of the islands
showed the usual C-band type. The nucleolar organizer regions (NORs)of the black rats are generally observed in three chromosome pairs (nos.
3, 8 and 12) (Yosida 1979, Proc. Jap. Acad. 55B: 481-586), but in the animals having decrease of C-bands collected from the island, the defficiencyof the NORs was observed in pair no. 3. The rats with usual C-bands
and NORs collected from the high land of Amami were suggested highly
to be an ancestral type of the Japanese black rats which were migrated
primarily from the continent of Asia, but those with the decrease of Cbands and NORs would be derived from the former by the diminishingmutation of the inner chromosomal organizations such as C-bands andNORs (Proc. Japan Acad. 59B: 211-214).
Location of Ribosomal RNA Genes in the Amami Black Rats
by in situ Hybridization Technique
Tosihide H. YOSIDA, Hitoshi SUZUKI and Takeharu KANEillSA1)

In the black rats, the nucleolar organizer regions (Ag-NORs) of chromosomes have been observed in chromosome pairs 3, 8, and 13 in all sub1)

Faculty of Science, Kobe University, Kobe.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

species belonging to several geographical type (Yosida 1980, Cytogenetcsof the Black Rat, Univ. Tokyo Press), but those collected from Amami had
only two NORs in chromosome pairs 8 and 13 described in the previous
report. To know whether the NOR is really lost in the chromosome pairno. 3 in this sepcimen, we investigated the site of NOR genes in the Amami
black rat by means of in situ molecular hybridization technique by use of
the radioactive complementary r-RNA. The result obtained was as follows;in the Amami black rats the location of ribosomal 18S+28S RNA genes
was found in the centromeric regions of the pair nos. 8 and 13, but in thechromosome no. 3, small silver cluster was rerely observed. This meansthat the copy number of r-RNA genes would be extreamely fewer in pairno. 3 than those of pair no. 8 and 13 (Proc. Japan Acad. 59B: 215-218).
Phylogenetic Relationship among Several Karyotype formsof the Black Rat and some Other Related Species
P. B. BAVERSTOCK1 >, M. ADAMS 1), L. R. MAXSON 1 ) and T. H. YOSIDA
The black rat, Rattus rattus, consists of the Asian (2n=42), Ceylonese(2n=40), Oceanian (2n=38) and Mauritius (2n=42 by fission) types. The
Asian type Was divided into two types; high and low C-bandings. As therelated species to the R. rattus, R. norvegicus, R. losea and R. exulans wereused. We used isozyme electrophoresis and microcomplement fixation to
elucidate the genetic distance and phylogenetic relationship among each of
the various karyotypic forms of R. rattus and other related species. The
results showed that (1) the Mauritius type black rat was genetically very
similar to the Oceanian form, suggesting that this island population has
undergone very rapid chromosomal evolution; (2) the Ceylonese type was
genetically distinct from the Oceanian type; the genetic difference wasprobably insufficient, however, to prevent future introgression; (3) the levelof genetic differentiation occurring between the Asian type on the one hand
and Oceanian, Ceylonese and Mauritius types on the other hand was suggested that these two groups were either full species or incipient species;(4) in contrast to data from amino acid composion of transferrin and fromrestriction endonuclease digests of mtDNA, the present data suggested
that the various karyotype froms of R. rattus were phylogenetically more
1)
Division of Vetarinary Science, Department of Agriculture, Adelaide, South Australia, Australia.

RESEARCHES CARRIED OUT IN 1983

47

closely related to each other than any is to R. norvegicus and others, andthat they are related by a series the Asian, Ceylonese and Oceanian types;(5) the R. rattusjR. norvegicus divergence would have occurred 2", 8 million
years ago, whereas various chromosome types of R. rattus diverged overthe last 4 million years (Genetics 105: 965-983).
.
Frequency of Chromosome Translocations in the Blackand Norway rats after r-Irradiation
Toshihide H. YOSIDA

Polymorphism or geographical variations due to chromosome mutations
were often observed in the natural population of the black rat (Rattus
rattus), while in the Norway rat (R. norvegicus) it was very few. On the
difference of the chromosomal divergence in these two species, the present
author (Yosida 1981, Environmental Mutagen and Carcinogen, Univ.
Tokyo Press) has suggested that it should be dependent on circumstancesin which both species were encountered during the migration from theirnative land to the other parts. Radioactivity exposed from the earth orcosmic rays seemed to be one of important facters in the circumstances.
Another explanation to the above event is that the black rat is more sensitive
to mutagens than the Norway rat. To solve this problem a rate of chromosomal mutation in the black and Norway rats was examined by irradiation with r-ray. The 400 to 750R r-rays from C S137 were exposured tomales of the black and Norway rats and they were mated to the non-irradiated females. The rate of chromosome mutations in offspring thus obtained was just the same as 0.02 in both species. This result suggests strongly
that the black rat with a higher cocurrence of chromosome mutation in thenatural population is due to the heavier exposure from the natural radiation during migtation than in the Norway rat (Proc. Jap. Acad. 59B: 263266).
A Black Rat with Chromosome Alterations Born afterr-Irradiations
Toshihide H. YOSIDAIn the previous paper the same frequency of the chromosome alterationsin the black and Norway rats in the F 1 generation born after r-irradiation

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

has been reported. In that experiment we found that only one Ceylonesetype black rat (Rattus rattus kandianus) among 50 F 1 offspring thus obtained
showed chromosomal alterations. The karyotype of the normal Ceylonesetype black rat was characterized by having 40 chromosomes (20 pairs),
among which 11 autosome pairs were acro- or subtelocentrics, one autosome
pair (M 2) was a large metacentric which was resulted from the translocation between acrocentric pair nos. 11 and 12. The remaining 7 autosome
pairs were small metacentrics and the X and Y were long and short acrocentrics. One male black rat born after mating between irradiated maleand non-irradiated female had the following abnormalities; a deletion atabout one fourth of long arm of pair no. 1 was found, and the broken end
was translocated to the terminal side of the no. 5 chromosome. A pericentric inversion had been taken in one of the pair no. 9. The anothercomplicated chromosome alteration was found to occur between M2 (11/12)
and no. 13. Namely, the Robertsonian fission occurred in the metacentric
M 2 , and no. 12 chromosome thus formed was translocated again to the no.
13 chromosome. The male black rat with such chromosome alterations
appeared to be healthy and grew into the adult stage. The external sexualorgans of the male seemd to be normal, but it was sterile (Proc. Japan
Acad. 59B: 308-311).
A Linkage Test on the Bared Gene to the Inversion no. 1
Tosihide H. YOSIDAAs previously reported (Yosida 1980) an LEM strain rat with an inversion
no. 1 was established from the spontaneous chromosome mutants occuured
in an LEW strain rat. In the fifth inbreeding generation of the LEM strain
rat, a bared (ba) mutant appeared spontaneously (Yosida 1981). Basedon the mating between the mutant and the normal haired LEM strain rats,
the bared character was shown to be due to a simple and recessive mutant
gene. To know whether or not the ba gene is located on the inversion
no. 1 chromosome, the segregation ratio in the F 2 generation from hybrids
between the bared rat with inversion no. 1 and the normal Wistar strain
rat (WM) without inversion was investigated. All F 1 rats thus obtained
had the normal hair coat, but their karyotypes showed without exception an
inversion/normal heterzygous pair no. 1 as expected. Among 55 F 2 offspring obtained from matings between the F 1 hybrids described above, 16

RESEARCHES CARRIED OUT IN 1983

49

rats were bared, while the remaining 29 rats were normally haired. The
segregation ratio was not significantly different from the expected value asa simple and recessive mutant gene. A relation between the segregation
ratio of karyotypes and that of the bared rats was examined in 21 F 2 offspring. Three among them were bared, but the remaining 18 rats were normal.
The ratio of the bared to the normal rats was not significantly different
from the expected one. The karyotype of the bared rats was all characterized by having the inversion and normal heteromorphic pair no. 1. On
this basis it was strongly suggested that the bared gene might not be linked
to the inversion pair no. 1, but to any other chromosome pairs (Proc.
Japan Acad. 59B: 531-354).
Chromosome Translocations Occurred in Offspring of the
Norway Rats after r-Irradiation
Tosihide H. YOSIDA

It is previously reported that the frequrncy of the Norway rats with thechromosome translocation in the offspring born after mating between
irradiated male and non-irradiated females was 0.02, which was just thesame as that obtained in the black rats. In that experiment 400 to 750R
of r-rays from CS137 were irradiated to the males of Norway rat and they
were mated to the non-irradiated females. By such matings 273 offspringfrom 55 litters were obtained. Among them 6 individuals had chromosometranslocations. For the present study ACI/N, ALB, BUF, F-344, LET, NIGIII, WM and WKS strains were used. On the induction of translocations,
there was no strain difference of the rat. Ten autosome pairs, such as nos.
1, 3,4, 7, 8, 9, 11, 12, 14, and 18 and the Y chromosome were involved in
the translocation. The frequency was calculated from the chromosome
length and found that it was not different significantly from the differenceof the chromosome length or type. It occurred ar random through thelength of chromosomes. The translocatoon of the chromosomes occurredin the offspring in the rat after r-irradiation seemed to be always reciprocal(Proc. Jap. Acad. 60B: 46-49).

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Translocation between Autosome nos. 9 and 18 Occurred in a
F-344 Rat after r-Irradiation and its Genetics
Tosihide H. YOSIDAIn the previous report it was described that 6 rats born after r-irradiation
had various types of translocation between autosomes and also the autosome and sex chromosome. Among them the translocation between
autosomes 9 and 18 was found in a F-344 male. In the normal karyotypeof the rat no. 9 was characterized by acrocentric homologous pair and no.
18 by metacentric homologous pair. In the one male rat born after rirradiation, however, one of the no. 9 chromosomes was remarkable by thelarge submetacentric, and one of no. 18 chromosomes by small acrocentric.
This karyotype was developed by following two events; the fission of thepair no. 18 at the centromeric region, and translocation of the one arm of
the no. 18 to the centromeric side of pair no. 9 was taken place. This relation was clearly shown by G-banding staining. The male rat with 9/18
translocation was mated to normal F-344 females and 38 F, offspring wereobtained. They were segregated to 19 normal and 19 translocation heterozygotes, just we expected. To obtain the F 2 offspring, the male andfemale translocation heterozygotes were mated and 63 F 2 offspring wereobtained. They were segregated only to two types, such as 13 normal rats
and 50 translocation heterozygotes. The rats with the translocation homozygous pair were never obtained in the F 2 and thereafter. The 9/18 translocation homozygotes were suggested to be lethal (Proc. Japan Acad. 60B:
50-53).

4/7 Translocation Occurred in a WM Strain Rat afterr-Irradiation and its Transmission to Offspring
Tosihide H. YOSIDA

Among several translocations occurred in the Norwwy rats born aftermating between r-irradiated males and a non-irradiated females, a translocation between pair nos. 4 and 7 was found in a male WM strain rat. About
two third in the distal part of one no. 7 chromosome were deleted and thebroken end was translocated to the terminal region of one of the pair no.
4. By such a translocation, one of the pair no. 4 was of an extraordinary
long acrocentric, while one of the pair no. 7 was like a small dot.

RESEARCHES CARRIED OUT IN 1983

51

By mating between the male with translocation and the 5 normal femalesof the WM strain rats, 14 offspring were obtained. The mean litter size
was 2.8± 1.3 which were smaller than the normal litter size in this strain.
Among 14 offspring, 7 rats (3Sj!: 40') were the hetrrozygotes having the 4/7translocation and the normal partner, but the remaining 7(2Sj! : 50') showed
the normal pairs of nos. 4 and 7. Among the 5 males, however, 4 were thenormal XY-sex chromosome mechanism, but the remaining one was characterized by having 43 chromosomes including XYY-sex chromosomes.
This male showed the normal external feature, but it is unknown at present
whether or not, the male is a fertile. Such an abnormal sex chromosome
should be due to the non-disjunction of the Y chromosome in the maleparent with 4/7 translocation (Proc. Japan Acad. 60B: 88-91).

ll/Y Translocation in the NIG-III Strain Rat Occurred afterr-Irradiation and its Genetics
Tosihide H. YOSIDA

A remarkable case of the translocation between the autosome no. 11
and the Y chromosome was found in a male of the NIG-III strain rats bornafter mating between the ,-ray irradiated male and the non-irradiated female.
A large part of the Y chromosome remaining the centromere was translocated to the short arm of the no. 11 chromosome and the remaining Y
with the centromere was deformed as an extremely small element like adot. By mating between the lIlY translocation male and the normal female,
133 rats (73Sj! : 600') were obtained in the F 1 to F 2 generations. Among 73
females, 62 were of the normal female karyotype with 42 chromosomes
including the normal pair no. 11 and two Xs. Ten females, however, had
41 chromosomes, among which one had the normal pair of no. 11, but asingle X, and the remaining 9 females showed the translocation heterozygous no. 11 and a single X. Remaining one female had 42 chromosomes,
but its karyotype was conspicuous by having a translocation heterozygous
pair no. 11 and two Xs. Eventhouth these 10 had the large part of the Y
element by the transloction, their sex characters were always of femalenessbecause the dot-Y element was not included.
Among 60 males thus obtained, 46 had the translocation heterozygous
pair no. 11 and X and dot-Y sex elements similar to the male parent. The
other 13 males, however, had 43 chromosomes, among which 40 were normal

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

chromosomes including normal pair no. 11, but the sex chromosomes wereremarkable by having two Xs and one dot-Yo The occurrence of the XO
female and XX/dot-Y males was considerably high in their frequencies andthey were found at almost identical number (10~ : 138') (Pore. Japan Acad.
6OB: 125-128).
1/12 Translocation Occurred in a WKS-Strain Rat afterr-Irradiation and its Transmission into Offspring
Tosihide H. YOSIDA

A new translocation between pair nos. 1 and 12 occurred in one male ofthe WKS-strain rats born after mating between the irradiated male and nonirradiated females. The translocation between these two chromosomes
occurred spontaneously in the Lewis strain rat has been reported by the
preswnt author (Yosida 1980). In that case, the long arm of the no. 12
was broken and the broken end was translocated to the short arm of thepair no. 1. In the present case, however, the long arm of the pair no. 1
was broken and the broken end was translocated to the short arm of thepair no. 12. Thus, one of the pair no. 1 became shorter than the normal
partner, while one of the pair no. 12 deformed to meta- or submetacentrics.
Although the reciplocal translocation between these two chromosomes wasdifficult to demonstrate by the conventional or G-banding staining, it was
clearly shown by the tranlocation of the nucleolar organizer region (NOR).
The translocation male was mated to the normal females of the samestrain rats. The 23 F 1 hybrids (10~: 138') from 4 litters thus obtained
were segregated to 8 normal homozygotes (5~: 38') and 15 translocation
heterozygotes (5~ : 108'). The segregation ratio was not significantly different from the expected one. The 32 offspring (17 ~ : 158') in total 9 litterswere obtained by mating between female and male heterozygotes. They
were segregated to 12 (4~: 88') normals, 18 translocation heterozygotes(12~ : 60') and 2 tranlocation homozygotes (1 ~ : 18'). The segregation
ratio was significantly different from the expected one (Proc. Japan Acad.
60B: 227-230).

RESEARCHES CARRIED OUT IN 1983

53

A Tanuki or Japanese Raccoon Dog with 40 Chromosomes
Including Two B-Chromosomes
Tosihide H. YOSIDA, Masayasu Y. WADA1) and Oskar G. WARD2 )The chromosome number of the tanuki or Japanese raccoon dog(Nyctereutes procyonoides vivverinus) has been described by several investigators to be 2n=42. Recently one specimen was caught in Amagi,
Shizuoka-ken, Japan, and its karyotype was analysed by conventional, Gand C-banding stainings. By use of such techniques, we found that thisspecimen was remarkable by having 2n=40, including two supernumeraryor B-chromosomes. The karyotype of this specimen was consisted of
13 meta- or submetacentric autosome pairs (nos. 1 to 13), 5 acrocentricautosome pairs (nos. 14 to 18) and a metacentric X and a small Y. In
addition to the above chromosomes, two acrocentric B-chromosomes were
included in its karyotype. They were dissimilar in the shape and stained
heavily through their whole length by the C-band staining. All autosome
pairs and the X were differentiated from each other by their G-banding
characteristics, but B's were distinguishable from the other autosomes byabsence of the clear G-bandings. Thus, the karyotype of this raccoon dog
was determined to consist of 36 autosomes, two B-chromosomes and an
XY complement. The previous reports that the raccoon dog had 42 chromosomes thus would be due to failure in identification of B-chromosomes
among autosomes (Proc. Japan Acad. 59B: 267-270).

Somatic Variation of B-Chromosomes in Two Japanese Raccoon
Dogs and Robertsonian Fission of no. 8 Chromosome
Tosihide H. YOSIDA, Masayasu WADA1), Oskar G. WARD 2)
and Doris H. WURSTER-HILLS)

The karyotype of one Japanese raccoon dog (Nyctereutes procyonoides
viverrinus) with two B-chromosomes has been reported in the previous
report. This specimen (RD-8301) captured in Amagi, Shizuoka-ken,Amagi Wild Boar Park, Amagi, Shizuoka-ken.
Department of Ecology and Evolutional Biology, Faculty of Science, Universityof Arizona, Arizona, U.S.A.
8)
Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire,
U.S.A.
I)

2)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Japan, was characterized by having 40 chromosomes, among which 36 (18pairs) were autosomes, two were X and Y chromosomes and the remaining
two were B-chromosomes. Recently, we analysed cytogenetically on theother two raccoon dogs (RD-8302 and RD-8303) newly captured from thesame place as the above, which were remarkable by having a wide somatic
variation of the number of B-chromosomes. We also found a Robertsonian
fission in one of the autosome no. 8 in these two specimens.
The basic karyotype of the RD-8302 was the same as the previous one(RD-8301), but the centromeric fission was observed in one of the chromosome pair no. 8. By such fission, an increase of two new acrocentrics
and decrease of one metacentric no. 8 occurred in this specimen. The
another remarkable karyotype feature of this specimen was the presence ofwide numerical variation of the B's in somatic cells. Among 100 somatic
cells analysed 44 % cells had 41 chromosomes including 2 B's, 21 % cells
had 44 chromosomes having 5 B's. Each with 43 (4 B's) and 42 (3 B's)
were at 18 % and 12 % respectively. Those with 1B and 6B's were alsocounted at 4 and I %, respectively. The karyotype of the RD-8303 wasquite similar to the above specimen due to having the centromeric fission of
one of pair no. 8. This animal, however, was remarkable in having only
two cell types due to the variation of B's. Among 96 cells counted, 79
cells (82 %) had 43 chromosomes including 4B's, but the remaining 17 cells(18 %) had 42 chromosomes including 3B's. Cells with the other variation
did not be observed. Distribution of B's was examined in a specimen,
RD-8301, previosuly reported. Among 88 cells examined, 85 cells (96.5 %)showed exactly 40 chromosomes including 2B's, just as descrived previously,
but one cell had one B and the remaining two cells had 3B's (Proc. Japan
Acad. 6OB: 17-20).
Robertsonian Fission of the no. 6 Autosome Newly Foundin a Japanese Raccoon Dog Collected in Aichi-Ken
Tosihide H. YOSIDA and Masayasu Y. WADA 1)

As described in the previous paper, among three Japanese raccoon dogs,
Nyctereutes procyonoides viverrinus, collected in Shizuoka-ken, Japan, one
did not have any Robertsonian fission, but the other two were characterizedby having the fission of no. 8 chromosome. In one specimen (RD-8409)
1)

Amagi Wild Boar Park, Amagi, Shizuoka-ken.

RESEARCHES CARRIED OUT IN 1983

55

collected from Aichi-ken, Japan, we found a new Robertsonian fission occurred in the no. 6 chromosome. This individual also showed a bimodal
distribution in two and three B-chromosomes in the somatic cells. The
Robertsonian fission of pair no. 6 was clearly demonstrated by G-banding
technique. Namely one partner of the pair no. 6 was normal submetacentric, but the other one was broken at the centromeric region and two
new acrocentrics were formed. The no. 8 chromosomes of this individual
were normal submetacentrics. The two acrocentrics formed by the fissionof no. 6 had clear C-bands like the other normal metacentrics.
In the present specimen, two and three B-chromosomes were observed inmost cells. Among 122 cells counted, 64 (52.4 %) cells had two B's, 56(45.9 %) cells had three B's, and the remaining only two cells had one Belement. Thus, the specimen, RD-8409, was mosaic in the somatic cells
due to the different number of the B-chromosomes (Proc. Japan Acad.
60B: 289-292).

A Japanese Raccoon Dog with Three Robertsonian Fissions
in nos. 5, 8 and 11, Found Newly in Kanagawa-Ken
Tosihide H. YOSIDA and Masayasu, Y. WADA1 )Two types of the Robertsonian fission occurring in no. 6 and no. 8 chromosomes in the Japanese raccoon dog, Nyctereutes Procynoides viverrinus,
have been described in the previous reports. A specimen (RD-8410)
collected from the suburb of Yokohama, Kanagawa-ken, Japan, however,was characterized by having the Robertsonian fission in three chromosomes,
nos. 5, 8 and 11. This specimen had also three and four B-chromosomes
in somatic cells.
The Robertsonian fission of the no. 8 chromosome was similar to the otherspecimens described previously. In the present specimens, however, othernew Robertsonian fission was found in pair nos. 5 and 11. Each one ofthese metacentrics were broken at the centromeric region and the six acrocentrics appeared newly, while three metacentrics decreased from the basic
karyotype. By such events the total chromosome numbers except B's
were increased to 41. All 119 cells so far examined had the same three
Robertsonian fissions as described above. It is interested that all the1)

Amagi Wild Boar Park, Amagi, Shizuoka-ken.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

acrocentrics newly formed by fission of metacentrics had a clear and large
C-bands similar to the other metacentrics.
The present material was also characterized by somatic variation of Bchromosomes. Among 119 cells so far analysed, 71 cells (59.6%) had
three B's, 43 cells (36.1 %) had four B's, and the remaining 5 cells (4.3 %)had two B's, All B-chromosomes found in this specimens were acrocentrics
as found in the other Japanese raccoon dogs, and all these B's were heavily
stained through their whole length by the C-band staining (Proc. Japan
Acad. 60B: 293-296).

Variation of B-Chromosomes in Four Youngs from One Litter
Collected in Saitama-Ken
Tosihide H. YOSIDA and Masayasu Y. WADA1 )The karyotypes of 5 specimens of the Japanese raccoon dog, Nyctereutes
procyonoides viverrinus, collected from several places of Japan, were remarkable by having several numbers of B-chromosomes and also various
types of the Robertsonian fission. These specimens, however, had not
any relation from each other. We newly obtained four young specimensfrom one litter of the Japanese raccoon dog from Saitama-ken. All four
specimens, RD-8404, RD-8405, RD-8406 and RD-8407, had the same
basic chromosome number and karyotype; 2n=38 consisting of 36 autosomes and XX- or XV-sex chromosomes. The Robertsonian fission of
autosomes was not observed in the all four specimens.
All four specimens had the B-chromosomes as similar to the other Japanese raccoon dogs observed previously. Interesting in the present materials,
however, was that among four spcimens, two (RD-8404 and RD-8406) showeda modal distribution in 2B's, but the other two (RD-8405 and 8407) in 3B's.
In RD-8404, 2B's were found in 97 (95.1 %) among 102 cells counted.
Only 4 (3.9 %) and 2 (1.9 %) cells had one B and three B's, respectively. In
the case of RD-8406, 2B's were observed in 84 (80.0 %) among 105 cells
counted, but cells with 3B's were observed at higher frequency (18.1 %)than in the above specimen. In the case of specimens with 3B's, the RD8405 had the 3B's at 85.3 %, 2B's at 12.7% and one B at 2.0 %. In the
RD-8407, the variation was slightly different from the above specimen.
1)

Amagi Wild Boar Park, Agagi, Shizuoka-ken.

RESEARCHES CARRIED OUT IN 1983

57

Among 110 cells counted, 3B's were observed in 64.5%, and 4B's wereat 30.0 %. The cells with 2B's and one B were at 4.5 and 1.0 %, respectively. Based on the above investigations, it is suggested that the differeceof the number of B's in four youngs born from the same parents seems tobe due to the random segregation of B's in the meiosis of the parents. On
the somatic variation of the B's it is suggested to be derived from the nondisjunction in somatic cells in the course of the ontogeny (Proc. Japan
Acad. 60B: 297-300).

B-Chromosomes and Robertsonian Fission in 13 Japanese
Raccoon Dogs, with a Note on Somatic Variationof the Number of B's
Tosihide H. YOSIDA and Masayasu Y. WADA1)

In the previous reports the karyotypes of 9 Japanese raccoon dogs
(Nyctereutes procyonoides viverrinus) were described. All these specimens
had B-chromosomes in their karyotypes ranging from 2 to 4 showing somatic
variations. On the other hand, the Robertsonian fission of some metacentric autosomes has been found in 4 specimens among them. In adition
to these 9 animals, we analysed 4 specimens newly collected from Shizuokaken and Aichi-ken. Based on these 13 Japanese raccoon dogs, the frequency of the B-chromosomes and the Robertsonian fission, with special
interest to the somatic distribution of B's in 1,732 cells.
Among 4 specimens newly collected, 3 (RD-8401, RD-8402 and RD8403) were collected from Shizuoka-ken and one from Aichi-ken. Amongthese 3 specimens, the Robertsonian fission was found in only one specimens (RD-8402), but in the other 3 the basic karyotype was observed.
These all specimens had the B-chromosomes showing some somatic variation. Model occurrence of 3B's was observed in two specimens (RD-8402
and RD-8408) and the other two (RD-8401 and RD-8403) had 4B's.
Among 13 specimens collected in Japan, 5 had the Robertsonian fission;
three specimens in no. 8, one in no. 6 and the remaining one in three chromosomes, nos, 5, 8 and 11. Based on these results, it can be said that about
38.5 % of the Japanese raccoon dogs have the Robertsonian fission. All
these specimens so far examined had the B-chromosomes of two to four,
1)

Amagi Wild Boar Park, Amagi, Yugashima, Shizuoka-ken.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

although some somatic variation was observed in all of them. Among 13
specimens, model occurrence of 2B's were observed in 5 specimens, 3B'sin the other 5 specimens, and the remaining 3 specimens had 4B's. From
the above 13 specimens the somatic variation was examined in 1,372 cells
in total. Among them, cells with 2, 3 and 4B's were frequently observed
at 31.6, 36.2 and 23.3 %, respectively. The other cells with 1, 5, 6 and7B's were extreamly fewer, such as 2.0, 5.0, 0.7 and 1.2 %, respectively.
The cells without B's and those with more than 8B's were not observed.
In the Japanese raccoon dogs, the individuals with few B's would be preferentially selected than those without B's and also more number of B's(Proc. Japan Acad. 60B: 301-305).
Karyotype of an Indian Spiny Mouse with 24 Chromosomes
Parasana KUMARI,l) Nimmakayalu V. ASWATHANARAYANA1 )and Tosihide H. YOSIDAThe Indian spiny mouse, Mus platythrix, has a characteristic karyotype
consisting of 26 acrocentric chromosomes as described by several investigators. Yosida (1979, 1980), one of the present authors, has describedthat the karyotype of this species was derived from that of the house mouse,
M. musculus, by tandem fusion of several pairs of the autosomes. He
suggested that the chromosome evolution might have occurred sequentiallyby the tandem fusion (Yosida 1983). He also suggested that specimens
with fewer chromosome numbers than 2n=26 would be found somewhere
in India by sequential fusion of chromosomes in this species. As predictedby him, we discoverd a male specimen with a reduced chromosome numberof 24, characterized by all acrocentrics.
Among 27 specimens collected in Mysore, 26 showed the usual diploid
number (2n=26), with all acrocentric chromosomes. One male, howefer,had 24 chromosomes. Among 12 autosome pairs (no. 1 to 12) designatedin the original karyotype, one of the smaller pairs, probablly no. 11 appeared to be missing in the present material. This pair might have translocated tandemly to anyone of the other autosome pairs as in the otherlarge acrocentrics, although it was not identified. Interesting is the factthat the Y chromosome in this specimen was considerably smaller than theother specimens with 2n=26. It seem that in the course of development
1)

Department of Zoology, University of Mysore, Mysore, India.

RESEARCHES CARRIED OUT IN 198359

of 24 chromosomes, the deletion in the Y element would have been takenplace (Proc. Japan Acad. 60B: 85-87).
Chromosomes of the Honshu Sika Deer, Cervus nippon centra/is
Masayasu Y. WADA1) and Tosihide H. YOSIDA

Karyotypes of the Ezo sika deer (Cervus nippon yesoensis), Kuyshu sika
deer (c. nippon nippon) and Manchurian sika deer (C. nippon nortulorum)
have been described some investigators (Makino and Muramoto 1966,
Gustavsson and Sundt 1968, 1969, Miyake et al. 1982). On the chromosome complement of the Honshu skia deer it has not yet been reported sofar. The present report deals with the karyotyp of Giemsa, G- and Cband stainings of the Honshu sika deer. Chromosome number of this subspecies was 2n=68 consisting of 32 acrocentric and one metacentric autosome pairs, and an acrocentric X and a submetacentric small Y. They
were identified by their G-banding chractristics. All acrocentric autosomes
and the X chromosome had clear C-bands at their centromeric regions,but not in the matacentic autosome. Small satellites were observed at
the distal ends of pair nos. 1 and 2. Karyotype of the Honshu sika deer
was compared with those of Ezo, Kyushu and Manchurian sika deer whichwere classified as the different subspecies from the Honshu sika deer. The
karyotypes of the Ezo, Kyushu and Manchurian sika deer were similar tothe present material by having 68 chromosomes. Although the small
satellite was observed in the pair nos. 1 and 2 in the Honshu sika deer, it
was not shown in the Ezo and Kyushu sika deer. In the Manchurian sika
deer it was observed only in the autosome pair no. 1. One of the present
authors (Yosida 1980) has described that the differentiation of the nucleolarorganizer is important to the differentiation of species. It is well known
that the nucleolar organizer is located at the satellite region. The sika deer
would be interesting materials to know the above problems (La Kromosome
II, 35-36: 1117-1121).

1)

Amagi Wild Boar Park, Amagi, Shizuoka-ken.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

A Simple Technique for Chromosome Observations from the
Small Mammals and Birds, and Karyotypes of Some
Mammals and a Bird
Masayasu Y. WADA1) and Tosihide H. YOSIDA

The present paper deals with a simple and easy technique for observationof chromosomes in small mammals and a bird and their karyotypes. A
small amount of blood was collected by the claw-cutting method, and thewhole blood was cultured by our routine method for the chromosome observation. Two small mammals, the masked palm civet (Paguma lorvata)and the common palm civet (Paradoxurus hermaphroditus) and domestic
fowl (Gallus gallus domesticus) were used in the present study. The blood(0.2-0.3 ml) of these animals was collected by cutting the base of the claw,
and the whole blood was cultured in vitro by use of the culture medium(MEM) with PHA. Sixty six hours after culture the slides were made forchromosome observation. Chromosome number of the masked palm
civet was 2n=44 and the karyotype was consisted of 9 subtelocentric autosome pairs, 8 acrocentric autosome pairs and 4 small meta or bsumetacentric autosome pairs, and a large metacentric X and the smallest Y.
The common palm civet had 2n=42, consisted of 10 subtelocentric autosome
pairs, 5 acrocentric autosome pairs, 5 meta or submetacentric autosome
pairs and the X (large metacentric) and Y (small dot like) elements. The
chromosome number of the domestic fowl was 78, among which no. 1, 2,
8 and 9 autosome pairs were submetacentrics, no. 3, 6, 7, and 10 to 39
autosome pairs were acrocentrics. Sex chromosome (ZjZ) was shown to
be no. 5 metacentrics (La Kromosomo II-32: 971-976).

A Simple Technique for Identification of Sex-Chromosome (W)in the Bird
Masayasu Y. WADA 1) and Tosihide H. YOSIDA

A simple and applicable method for the identification of sex in the bird
was developed by use of direct air drying preparation. The chromosome
preparations were made from the feather pulp by the following technique.
(1) Mitotic arrest. In young domestic fowl, 0.025 % solution of demecolcine(or colchicine) was injected into the peritoneal cavity (0.2 ml per one in1)

Amagi Wild Boar Park, Amagi, Shizuoka-ken.

RESEARCHES CARRIED OUT IN 1983

61

dividual of the 7 day young chick). The injection was made 1 hr before
harvesting the feather. The feather was plicked for chromosome observations. (2) Slide preparation. The base of feather was cut in the petri
dish into about 1 mm3 , and immediately transferred to 0.075 M KCI solutopn for 50 min at room temperature. Two or three pieces of the cut pulp
were removed from the hypotonic solution and placed on a clearn slide.
Then, the materials were minced into small pieces and fixed on the slide by
applying the method of Imai et al. (1977; Chromosoma 59: 341-393). The
slide was air-dried for one day. (3) Staining. The completely air-driedslides were stained with 3 % Giemsa solution for 10 min. For C-bandingsof the slide, the Giemsa stain was removed from the slide by immersion into
50% acetic acid. Then, the trypsin C-banding method was applied (Wangand Shoffner 1974; Chromosoma 47: 61-69). By the above thechniquethe metaphase chromosomes became generally vaguely by the long treatment with trypsine, but only the W-chromosome was stained darkly andconspicously. In the brid the individual with the dark stained chromosome
was determined to be female (WZ-type) (Proc. Japan Acad. 59B: 223-226).
Identification of the Sex Chromosome in a Sex Reversed PeacockTosihide H. YOSIDA, Masayasu Y. WADA, Yohei SAKAI
and Norio KONDO

The peacock is a typical sexual dimorphism bird. The upper tail covers
are beautiful and long in the mail, while they are not in the female. We,
however, encountered an intereting case that a peacock, which was recognized primarily as a female in the secondary sexual characteristics, haschanged into a male character after full-grown and the upper tail covers havebegun to grow just as those of the male. Due to such a change of thesecondary sexual feature, it was difficult to determine whether or not, thispeacock was a real female. It has been well known that the bird is characterized by having a sex determining mechanism as the female dimorphismor the ZW-type.
Sex chromosome (W) of the bird can easily be demonstrated by our simple
technique by use of the feather pulp described in the previous paper. By
applying this method, the W-chromosome of the sex reversed peacock could
cleary be demonstrated. By use of this technique, we found that the sex
reversed bird was primarily a female, because the W-chromosome was

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

exactly included. The chromosoe constitution of the bird was also analysedin cultured cells from the biopsy skin by applying the serial conventional
Giemsa, G- and C-band staining methods, and confermed that the sex reversed peacock had 32 autosome pairs and ZW-sex chromosome complement (Proc. Japan Acad. 59B: 304-307).
Karyotypes of Four Dragonet Fish Species
Makoto MUROFUSHI 1), Shohei NISHIKAWA2 ) and Tosihide H. YOSIDAThe karyotypes of four dragonet fish species, Callionymus punctatus, C.
beniteguri, C. ornatipinnis and C. doryssus, were examined. The chromosome number in the male and the female of C. punctatus was 2n=38, allof which were acrocentrics. The sex chromosome in this species was anusual X-Y complement, although it was not identified from the other autosomes. The chromosome numbers of C. beniteguri and C. ornatipinnis
were 2n=38 in the female and 2n=37 in the male. The sex chromosome
mechanism of these two species was XX-Yo All autosomes and X-chromosomes were subtelocentrics, but the Y-chromosome was large metacentric. On the other hand, the chromosome number of C. doryssus was
2n=32 in the male, among which one pair was large metacentric and theremaining fifteen pairs were acrocentric. The sex chromosome was not
identified in this species, because the female specimens were not examined.
The secondary constriction was found in one chromosome pair of all four
species; in the pair no. 1 of C. beniteguri, in the pair no. 2 of C. doryssus,
and in the pair no. 10 of C. ornatipinnis and C. punctatus. Position of thesecondary constriction of the pair no. 10 in the latter two was different
from each other (La Kromosome II-34: 1079-1084).
Karyotypes of Two Species in the Sandperch Fishes
Makoto MUROFUSHI 1 ), Shohei NISHIKAWA2) and Tosihide H. YOSIDAThe karyotypes of the species in the sandperch fishes (Parapercis pulchella
and P. sexfasciata) were examined. The diploid chromosome number in
P. pulchella was 2n=42, while P. sexfasciata, 2n=26. The karyotype ofthe formaer species was consisted of 4 metacentric pairs (nos. 1 to 4) and1)

2)

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

RESEARCHES CARRIED OUT IN 1983

63

17 acrocentric pairs (nos. 5 to 21), but in the latter species 11 metacentric
pairs (nos. 1 to 11), one submetacentric pair (no. 12) and one acrocentric
pair (no. 13). The number of chromosome arms in these two species was
50. The secondary constriction was found in pair no. 6 in P. pulchella
and pair no. 4 in P. sexfasciata. One heteromorphic pair (no. 12) andone small metacentric pair (no. 11) like as a minute was found in the latter
species. The sex chromosome could not be identified in both species.
(La kromosome II-35: 1122-1125).

Multiple Sex Chromosome Mechanism (XX-Y) Newly Foundin the Snake Eel, Muraenichithys gymnotus
Makoto MURoFusm1) and Tosihide H. YOSIDA

The multiple sex chromosomes (XX-Y) in the fish have been found in asingle filefish species (Murofushi et al. 1980) and two dragonet fish species(Murofushi et al. 198ft). A similar case of the multiple sex-chromosome
has newly been found in one species of the snake eel, Muraenichthys
gymnotus. Six specimens including 3 males and 3 females of the snake
eel were used in this study. They were collected from two-hundred meter
depth of the Suruga Bay, Shizuoka-ken, Japan.
The chromosome number of the female snake eels was 2n=48, consistingof subtelocentric pairs and 22 acrocentric pairs, while in the males it was
2n=47. In the male cells one large metacentric was always observed.
Judging from the multiple sex-chromosome mechanism in the other fish
species found in the filefish and dragonet fish, the large metacentric element
was suggested to be the Y-chromosome. Thus, the sex-chromosome
mechanism in this species is inferred to be the multiple X 1 X 2 - Y. (Proc.
Japan. Acad 60B: 21-23).

Comparative Study on Two Species of Snipefishes
Makoto MURoFusm1J, Shohei NrsmKAwA2) and Tosihide H. YOSIDAThe karyotypes of two species in the snipefish, Macrorhamphosus sagifueand M. japonicus, were comparatively examined. Two specimens in the1)2)Mishima Junior College, Nihon University, Mishima.
Shimonoseki University of Fisheries, Shimonoseki.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

former species and four specimens in the latter species were collected fromtwo hundred meter depth of the Suruga Bay, Shizuoka-ken, Japan. The
diploid chromosome number of these two species of the snipefishes wasforty-eight (2n=48), all of which were acrocentrics ranging from large tosmall in the size. The secondary constriction was found near the centromere of the pair no. 9 in the both species. The idiograms of these two
species and the length of each chromosome pair were very similar fromeach other.
These two snipefish species were separated by the difference of ratio inbody size and length of spine, but the karyotypes of M. sagifue could not
be distinguished from that of M. japonicus. Okada and Suzuki (1951)
asserted that these two of the snipefishes should be integrated to one species
as M. scolopax. Based on the present karyological data, the opinion of
the latter authors seems to be reasonable (Jap. Jour. Genet. 59: 155-158).
Karyotypes of the Red Swamp Crayfish and the Japanese Lobster
Makoto MUROFUSHl1 ), Yosihaki DEGUCHl2 ) and Tosihide H. YOSIDAThe karyological studies on Crustacea are insufficient and most of thestudies have been carried out by the classical technique by several investigators. The chromosomes of the red swamp crayfish, Procambarus clarkii,
and the Japanese lobster, Nephrops japonicus, have also been observed by
Niiyama (1934, 1939) by that technique. We obtained the red swamp
crayfish from the paddy field in Mishima, Shizuoka-ken and the Japanese
lobster from Suruga bay, Shizuoka-ken. The chromosomes were observedby an air-drying method modified from our routine technique for fishes(Murofushi and Yosida 1979).
The diploid chromosome number in male and female specimens of the
red swamp crayfish was 2n= 188, consisting of 42 meta- or submetacentricand 52 subtelo- or acrocentric pairs. The diploid number of male andfemale specimens of the Japanese lobster was 2n= 136, among which 46
pairs were of meta- or submetacentrics and 22 pairs were of acro- or subte1ocentrics. Niiyama (1934 and 1939) has reported that the chromosome
number of the former spesies was 2n= 192 and that of the latter species,
2n= 164. The difference seen in these two studies are probably due to the1)2)Mishima Junior College, Nihon University, Mishima, Shizuoka-ken.
College of Agriculture and Veterinary Mediciffe, Nihon University, Setagaya, Tokyo.

RESEARCHES CARRIED OUT IN 198365

difference of the method utilized in the karyotype analysis (Proc. Japan
Acad. 60B: 306-309).
Karyotype of the Small Free-Living Nematode,
Caenorhabditis elegans
Tosihide H. YOSIDA, Tamiko SADAIE and Yoshito SADAIE

The small free-living nematode, Caenorhabditis elegans, has recently
been widely utilized as an attractive organism in molecular biology. In
the adult hermaphrodites, only about 800 somatic cells are included in one
individual and their cell lineage can easily be recognized in the body. The
haploid DNA of this worm was to be only about 20 times as large as thatof the genome of E. coli. It is also known to have six linkage groups.
The karyotype of this worm by the light microscope, however, has not yet
been demonstrated so far. By use of the drying technique, we succeeded
to observe the chromosomes of this small nematode in somatic and germ
cells. The number of the diploid chromosomes was determined to be twelve
and they were classified into six homologous pairs by G-band staining.
Six chromosome pairs were similar in its size. The smallest chromosome
was about 0.7 time as large as the largest one. In the present comunication we could not assign the number of the linkage group and also recognize
the X-chromosome from the autosomes. A series of the first meiotic chromosomes was also successfully demonstrated. All of these cells showed
to habe six tetrads (Proc. Japan Acad. 60B: 54-57).
Inhibition of Sister Chromatid Exchanges in Bloom
Syndrome Cells with Cycloheximide
Yukimasa SHIRAISHI1), Tosihide H. YOSIDA and Avery A. SANDBERG2 )Effects of cycloheximide (CH) and deoxycytidine (dC) on the frequencyof sister chromatid exchanges (SCEs) in normal and Bloom syndrome (BC)cells labeled with BrdU during first, second and third cell cycles were evaluated, using endomitotic and three-way differentiation analyses. WhenCH at 0.2 and 2.0 flgfml was added to normal and BS cultures of BrdU
labeled endomitoses, the rate of single SCEs was significantly decreased in
1)

2)

Department of Anatomy, Kochi Medical School, Nankoku, KochL
Roswell Park Memorial Institute, Buffalo, New York, U.S.A.

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

BS cells, though the rate of reduction in single SCE was slight in normal
cells. No significant change was detected in the twin SCE rate. In BS
cells, treatment with CH at 0.2 and 2.0 flgfml produced significant reductions in SCE in both the second (SCE 2) and third (SCE 3) cell cycles-sometimes reaching normal level. Treatment with dC at 13 and 26 flgfml resulted in almost no significant changes in rates of SCE during first, second
and third cell cycles. When CH was added to BrdU labeled normal andBS cell cultures, the cell growth rates improved from 35 to 70 per cent overthe control level in the BS cells, though in normal cells CH resulted in a lower
cell growth rate in a dose dependent manner. Deoxycytidine did not noticeably affect the cell growth rates in BrdU labeled normal and BS cultures.
The finding that the reduction of BrdU-induced SCE in BS is paralleled bycell growth improvement is of special interest (Cancer Genet. & Cytogenet.
in press).

Chromosome Observations on Tropical Ants from Western
Malaysia. II
Hirotami T. IMAI, William L. BROWN Jr. 1 ), Masao KUBOTA2 ),Hoi-Sen YON03 ) and Yow Pong TH04 )Imai and Kubota went to West Malaysia in 1982 and 1983 for a karyological survey of tropical ants. The project was performed in cooperation
with H. S. Yong and Y. P. Tho. The identification of species was madeby M. Kubota and W. L. Brown, though it remains unsettled yet in someant groups, e.g., Pheidale and Campanatus. We already described chromosomes of 73 ant species collected in 1982 (Ann. Rep. Natl. Inst. Genetics,
32: 71-73). The present paper reports chromosomes of 86 species (39genera, 6 subfamilies) collected in 1983. A total of 119 colonies, labelled
as HI83-(I-119), were collected from the following localities: campus ofForest Research Institute (HI83-1-42), Jeram Toi (HI83-43-46, 49), Pasoh
Forest (HI83-47-74, 103), Gombak Station of the University of Malaya(HI83-76-83, 96, 109-115), Genting Highlands (HI83-84-95), VIu Gombak
1)

2)
3)

4)

Department of Entomology, Cornell University, Ithaca, New York, 14853, U.S.A.
Nakasone 13, Odawara, Kanagawa-ken, 250, Japan.
Department of Genetics and Cellular Biology, University of Malaya, Kuala Lumpur,
Malayasia.
Forest Research Institute, Kepong, Malaysia.

RESEARCHES CARRIED OUT IN 1983

67

Table 1. Chromosome numbers of Malaysian ants. II
Taxa (Colony number)

Chromonumber(n) 2nPONERINAE( 9) 18
Platythyrea quadridenta(HI83-22, 50)
P. tricuspidata (HI83-116)
96
Gnamptogenys menadensis*
42(HI83-6l)
G. binghami (HI83-21)(22)
Leptogenys diminuta=sp. 1*
38(HI38-14, 15, 86)
L. myops=sp, 2* (HI83-113)(24)
L. sp. 3 (HI83-109)
48
L. iridescens (HI83-47, 65)
46
L. borneensis (HI83-66)
46
Diacamma sp. 1*
44(HI83-16, 99, 106)
Amblyopone reclinata
38(HI83-20)
Pachycondyla "n. sp. A of Brown"=Mesoponera sp. 1(HI83-63, 71)(18) 36( 8) 16
P. leeuwenhoeki
P. rubra*=Mesoponera sp. 2
38, 40(HI83-3, 11, 12)
Robertsonian polymorphism
P. tridentata (HI83-51)
28
Ponera japonica (HI83-94)
12(12) 24
Hypoponera pruinosa(HI83-11O)
H. sp. 2 (HI83-35)
38
H. sp. 3 (HI83-45)
36
Cryptopone testacea( 9) 18(HI83-54)
Odontomachus simillimus
44=sp. 1* (HI83-17, 18)(15) 30
O. rixosus=sp. 2* (HI83-38)(15)
O. latidens (HI83-25)
PSEUDOMYRMECINAE
Tetraponera sp. 2 (HI83-97)

42

Taxa (Colony number)
DORYLINAE
Aenictus laeviceps? (HI83-103)

Chromonumber(n) 2n22

MYRMICINAE
Aphaenogaster beccarii*
30(HI83-55)
Pheidole sp. 1* (HI83-101)(10)
P. sp. 7 (HI83-7)
16
P. sp. 11 (HI83-53)
22
P. sp. 12 (HI83-44)(10) 20
P. sp. 13 (HI83-102)
38
P. sp. 14 (HI83-32)
20
P. sp. 15 (HI83-6)
20
P. sp. 16 (HI83-104)( 9) 18
P. sp. 17 (HI83-27)
20,21Translocation polymorphism
P. sp. 18 (HI83-8, 88)
20
P. sp. 19 (HI83-9, 90)
20
P. sp. 20 (HI83-58, 60, 108)
(18) 36
P. megacephala (HI83-82)
20
Crematogaster sp. 1*
26(HI83-59)
C. sp. 4* (HI83-5, 49)
24
C. sp. 5 (HI83-4)
26
C. sp. 6 (HI83-72)
26
Monomorium sp. 2*
22(HI83-85)
M. sp. 3 (HI83-96)(11) 22
M. sp. 4 (HI83-105)(11)
Lophomyrmex bedoti
38(HI83-73,75)
49,50
Vollenhovia sp. 1(HI83-46, 69)
Robertsonian polymorphism
V. sp. 2 (HI83-93, 95)
33,34
Robertsonian polymorphism(11) 22
Acanthomyrmex sp. 3(HI83-10, 98,114)
Pristomyrmex sp. 2 (HI83-77) (14)68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34
Table 1. Continued
Taxa (Colony number)

Chromonumber(n) 2nLordomyrma sp. 1 (HI83-37)(11) 22
Myrmicaria sp. 2* (HI83-87)
44(23) 46
M. sp. 4 (HI83-43)
Tetramorium pnyxis (HI83-56) 20
T. e/eates (HI83-30)
28
T. seneb ? (HI83-29)
20(12) 24
Smithistruma taipingensis*(HI83-11l)
Strumigenys godeffroyi
40(HI83-31)
16
Dacetinops concinna(HI83-115)
Eurhopa/othrix n. sp.
18(procera group)
Brown (in press)
DOLICHODERINAE
Do/ichoderus bitubercu/atus*(HI83-83) B-chromosome
polymorphism
Iridomyrmex cordatus(HI83-74)
Technomyrmex sp. 1*(HI83-118)
FORMICINAE
Anop/o/epis /ongipes*(HI83-1, 2, 117)
Acropyga acutiventris*(HI83-28, 119)
Robertsonian polymorphism

30,31,
32,33
16
30

34
28, 29Taxa (Colony number)
P/agio/epis sp. 1 (HI83-62)
Pseudo/asius sp. 1(HI83-57, 70)
P. sp. 2 (HI83-67, 68)
B-chromosome
polymorphism
Camponotus festinus*(HI83-24, 52)
C. sp. 2* (HI83-19)
C. sp. 7 (HI83-107)
C. sp. 8 (HI83-23)
Robertsonian polymorphism
C. sp. 9 (HI83-80, 81)
c. sp. 10 (HI83-41, 42)
Robertsonian polymorphism
C. sp. 11 (HI83-84)
C. sp. 12 (HI83-100)
C. sp. 13 (HI83-78)
Po/yrhachis il/audata(HI83-36)
P. hector (HI83-76, 112)
P. raste//ata (HI83-39, 40, 79)
Echinop/a sp. 1 (HI83-48, 64)
Paratrechina sp. 4* (HI83-34)
P. sp. 6 (HI83-92)
P. sp. 7 (HI83-33)
Preno/epis jerdoni*(HI83-26, 91)
B-chromosome
polymorphism

Chromonumber(ff) 2n18( 8)(15, 17,19) 30(19) 38
40
(20)
38,39
40
(13, 14)
52
18
18
(18)
(21) 4242(12) 2416
30
16(16,20,25,27)
30,31,
32, 34

N.B. The species with asterisks were observed also in the survey of 1982.

(HI83-97-108), and Templer Park (HI83-116-119). Among 119 colonies,
we were able to observe chromosome spreads in all samples except HI83-13,
46, 57, 63, 79, 89, and 119. The results are summarized in Table 1. In
addition, two species collected at Sungey Menyala Reserve, Negeri Sembilan,
in 1981 by Brown and Pong (Pachycondyla leeuwenhoeki and Eurhopalothrix

RESEARCHES CARRIED OUT IN 1983

69

n. sp.), were karyotyped separately by Brown; these have been added toTable 1.
Modes of Inheritance of X-Y Dissociation in Inter-Subspecies
Hybrids between BALB/c Mice andMus musculus molossinus
Yoichi MATSUDA!), Hirotami T. IMAI, Kazuo MORIWAKI
and Kyoji KONDO!)

Genetic analysis of the high frequency of X-V chromosome dissociation
found in primary spermatocytes of F I hybrids between Japanese wild mice(Mus musculus molossinus) and inbred laboratory mice (BALB/c) was attempted. The frequency of X-V dissociation (X//Y) in both BALB/c and
M. m. molossinus was lower than 30% (Low X/IV), while the value wasmore than 70% (High X/IV) in their F I hybrids. Two types of progeny(High X/IV and Low X/IV) appeared in the backcross between BALB/cand High X//Y males, although the frequency of Low X//Y progeny decreased with increasing numbers of backcross generations (26.5 % at N 2 ,13.2% at N a, 5.3% at N 4 , and 0% at N 5). Low X/IV sires produced only
Low X/IV mice. We hypothesize that at least one heritable factor whichis responsible for the end-to-end association of the sex chromosomes (temporarily symbolized as Sxa) is located on the common part of the X andY chromosomes. The Sxa allele of BALB/c is Sxa8. and that of M. m.
molossinus is Sxab. The genotype expected in High X//Y males is Sxa8./
Sxa b and in Low X//Y males and their parental stocks either Sxa8./Sxa8. or
Sxab/Saxb. The repeated segregation of Low X/IV progeny from High X/IV
sires is interpreted simply by assuming that crossing-over has occurred between the X and Y chromosomes. The gradual decrease in the recombinant
type mice (Low X/IV) during sequential backcross suggests the presence ofsome autosomal factors that suppress the crossing-over of the sex chromosomes and that do not seem to function in the inter-subspecies hybrids.
For details see Cytogenet. Cell Genet. 35: 209-215 (1983).

!)

Faculty of Agriculture, Nagoya Univeristy, Nagoya.

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Rates of Mammalian Karyotype Evolution by the
Karyograph Method
Hirotami T. IMAI, Takeo MARUYAMA and Ross H. CROZIER1)Using the karyograph method devised by Imai and Crozier (1980), we
attempted to estimate the rate of mammalian karyotype evolution. Denoting the chromosome number and arm number of the mean haploid karyotype (K i) in a given mammalian taxon by fii and ANi' respectively, thekaryotype can be represented at a point j(ANi, fii) on a two-dimensionallattice (karyograph), where the arm number is plotted against the chromosome number. If we assume that Ko(ANo, fi o) is the karyotype of primordial mammals, it is possible to define three types of karyological distance between the K o and K i • Namely, the distance defined by the difference in the arm numbers (DAN=ANi-ANo), the distance by the chromosome numbers (D,,=ni-nO)' and the karyonumber distance (DKN=D AN +D,,). Imai and Crozier (1980) found that mammalian karyotypes tend to
evolve, in a statistical sense, orthodirectionally toward increasing chromosome number and/or arm number mainly by centric fission and by pericentric inversion changing acrocentrics (A) to metacentrics (M) (pj. (AM».
The distances D" and DAN are, therefore, the total numbers of centric fissions and of pj. (AM) accumulated in the K i since the karyotype diverged
from the K o• We assumed that mammalian karyotype evolution beganfrom the karyotype K o=3M, which is the lowest-numbered karyotype
among extant mammals, about 100 million yr ago; i.e., the common ancestor has K o(A No =6, no=3) and the divergence time (t) is 100 million
yr. Now, the mean rate of arm number change (VAN), chromosome number change (V), and karyonumber change (VKN ) is given as VAN=DAN/t,
V,,=D,,/t, and V KN = V AN + V". We estimated the rate of Karyotype evolution using 1,070 mammalian species, and obtained V,,=0.19, VAN =0.27,
and VKN =0.46. In other words, the chromosome number (n), arm number(AN), and karyonumber (KN=n+AN) in mammals change, respectively,
at a rate of about one in every 5.3, 3.7, and 2.2 million yr. For details see
Am. Nat. 121: 477-488 (1983).

1)

School of Zoology, The University of New South Wales, Australia.

RESEARCHES CARRIED OUT IN 1983

71

Cytogenetic Analysis of Recombination in Males ofDrosophila ananassae
Muneo MATSUDA!), Hirotami T. IMAI and Yoshiko N. TOBARl1)

Cytogenetic studies of recombination in males of Drosophila ananassae
were carried out by examining F 1 males derived from the mating of marker
females, b se; bri ru of the BS stock, with males of two wild strains, TNG
and L8. The male recombination values in both sections b-se (chromosome2) and bri-ru (chromosome 3) are high in TNG F 1 but extremely low in
L8 Fl' We demonstrate the presence of chiasmata in TNG F 1 meles at afrequency capable of accounting for the observed recombination values.
A unique series of "iso-site aberrations" was also observed in TNG F 1
males. Because of a parallelism in the distribution pattern between thechiasmata and the iso-site aberrRtions, we propose that recombination inmales of D. ananassae is meiotic in origin and that the iso-site aberrations
are related to chiasma formation. For details see Chromosoma (Berl.)
88: 286-292 (1983).

Quantitative Analysis of Karyotype Alteration and Species
Differentiation in Mammals
Hirotami T. IMAI

A two-dimensional graph (species-karyotype graph or s-k a graph) was
devised for analyzing the role of karyotype alteration in speciation. Whenthe number of species and dissimilar karyotypes involved in a genus aredenoted as sand ka, the genus can be represented as a point (s, ka) on thegraph. If karyotype alteration always initiates speciation as suggestedby the stasipatric speciation model, the genus will fall in the area ka/s"?;.!.
On the other hand, if speciation occurs without karyotype alteration andkaryotype substitution proceeds subsequently in each of the resulting species,
the genus will fall in the area ka/s;;;'!. Among the 235 mammalian genera
examined, the genera distributed in the area ka/s> 1, ka/s< 1 and ka/s= 1
were 21 (8.9 %), 57 (24.3 %) and 157 (66.8 %). This result suggests thatspeciation without karyotype alteration predominates in mammals. Basedon the distribution patterns of mammalian genera on the s-k a graph, an
1)
Department of Biology, Tokyo Metroporitan University, 2-1-1, Fukasawa, Setagaya-ku, Tokyo 158, Japan.

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

overall tendency of the life cycle of mammalian genera to follow this se1)

2)

quence was suggested: k a/s=l, k a=s=l-'>k a/s<l, k a=l, s>l-'>k a/s<l,3)4)s>k a>l-'>k a/s=l, k a=s>l-'>k a/s=l, k a=s=l, that is, 1) speciation
without karyotype alteration, 2) substitution of karyotypes in each resulting
species, 3) climax of species differentiation, and 4) degeneration of genus
by extinction of species. The parapatric distribution of karyotypicallydistinct populations are interpretable under the model as a transitional stepin karyotype substitution. For details aee Evolution 37: 1154-1161 (1983).
Genetic Studies on Spontaneous Sister Chromatid Exchanges
in Spermatogenesis of Drosophila melanogaster
Masatoshi YAMAMOTO

Population cytogenetics has demonstrated that the DNA content of anorganism is not fixed. Especially the content of heterochromatin (C-band)or satellite DNAs seems to be most variable and it is known as C-bandpolymorphism. The variation of heterochromatin content causes alterations in the frequency of recombination over and above genic regulations.
Changing the amount of DNA can thus bring biologically important consequences. The mechanism how the DNA content is altered is however
yet to be solved.
The postulated mechanisms are (1) unequal meiotic recombination and(2) unequal sister chromatid exchanges (USCE). The former should beless likely the mechanism because the heterochromatin hardly undergoes
recombination in itself and further it does not explain the polymorphismof the Y chromosome and the B chromosome which often exists alone
without their homologs in a genome. The latter is an quite attractive
hypothesis and has recently been shown to occur spontaneously in a totally
heterochromatic ring Y chromosome in D. melanogaster. Neverthelessthe occurrence of spontaneous SCE's in the germ line is still a subject of
cytogenetical debates.
The question of the function of SCE has been set aside so far althoughextensive studies on the mechanism and the increase of frequency depending on the dose of chemical mutagens have been carried out.
Here I determined genetically the frequency ofspontaneous SCE in the spermatogenesis of D. melanogaster in order to test the hypothesis that the USCE

73

RESEARCHES CARRIED OUT IN 1983
Table 1. The progenies obtained from the cross zw+R61e190' X C(l)DX, ywf~
Phenotypez (lemon yellow) 0'
mosaic (lemon/wild) 0'
z+ (wild type) 0'
ywf 0'
ywfr:f.
z~

Super females
Total

No. of Flies
154663521
121328781
44

No. of Harem

5
2
8
1

40

287624is a mechanism in changing the content of chromosomal DNA. The fly
strain I used is zw+R61e19 which carries a mutant allele of zeste(z) and apartially duplicated white locus. zw+ males show wild type eye colour eventhough the genotype is z. On the other hand zw+ R61e19 which is duplicatedin a subsegment of the white locus shows lemon yellow eye colour in the male.
A single male of zw+R61e19 was crossed to 10 to 15 XX, ywjfemales andevery other two days they were transferred into a new bottle. The maleprogenies coming out of the cross are all carrying the paternal genotype.
The important point of this experiment is that the zw+R61e19 X chromosome
never go through the female genome. If the DNA content at the duplicatedregion of the white locus is altered it must be happened in the male genome,
namely in the single X chromosome. A change in the DNA content at
the specific locus can be easily monitored by the appearance of wild typemales showing brick red eye colour. The experimental crosses were repeated 480 times and 287,624 flies were checked the phenotype individually.
The results are shown in the table 1.
Among the total of 154,701 males 21 z+ males which should be considered
to be lost a part of the duplicated white locus are included. These males
could be derived from a gene mutation at the z locus or the spontaneouspartial deletion at the white locus, but the frequency 1.4 X 10- 4 is too highif it were the case. Furthermore the z+ males were confirmed not to havea mutation at the zests locus. These findings strongly suggest that spontaneous unequal sister chomatid exchanges (USCE) happened so that theregion was deleted out from the zw+R61e19. In addition, it is clear that the
USCE's occured in the spermatogonial mitosis not spermatocytes because
they came out as clusters from 2 harems of the cross.

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

The evidence showing that spontaneous USCE's occurs at mitotic divisions was also indicated from the mosaic (lemon yellow eye colour/wild
type eye colour) males.
It should be stressed that the frequency 1.4 X 10- 4 was measured in avery small euchromatic duplication. It can be thus concluded that in theheterochromatin where highly repeated DNA is abundant, the USCE's
must happen at the significantly higher rates. The molecular change
associated with this USCE is now under investigations.

RESEARCHES CARRIED OUT IN 1983

VI.

75

MUTATION AND MUTAGENESIS IN ANIMALS

Induction of Specific-Locus Mutations by High Temperature
Exposures in Newly Laid Eggs of the Silkworm
Akio MURAKAMI

Some sixty years ago, R. Goldschmidt (1927) reported that the frequencyof mutations in Drosophila increased by high temperature exposure. Since
then, there were numerous papers on the mutation induction by low orhigh temperature treatments in various organisms. In the silkworm, experimental induction of various abnormalities including androgenic andparthenogenic individuals as well as polyploids was attempted firstly by
Tanaka and his students. They treated eggs and pupae with low and hightemperatures. Their findings were confirmed and extended by Tazima(1939, 1947).
In the silkworm, it was established that spontaneous mutation frequencies
at some loci were higher than those at other loci, and were markedly dependent on seasons when experiment was carried out. This suggests thatthe temperature is one of the effective factors to increase the mutation
frequency. To examine this supposition, the present experiment was
carried out by treating newly laid eggs with various temperatures, 2.5°, 10°,20°, 25° (as the control), 30°, 35°, 37°, and 40°C. Newly emerged virgin
F I hybrid moths from the cross between CI08 female and Aojuku male werestored in a refrigerator at 5°C for 24 hrs. Those females were then matedto marker males homozygous for pe and re egg-colour genes for 4 to 5 hrs
at 25°C, separated each other and then allowed to lay eggs for 20 min.
The eggs collected were immediately incubated at various temperatures
for 90 min. During this period eggs proceeded their development from thevery late meiosis I of oocytes to completion of meiosis II or just before
syngamy.
The F I offsprings or eggs from all those crosses were expected to be black
wild-type eggs except for eggs after treatment with high temperatures at
35° to 40°C. In fact, no significant increase of abnormalities was observedin the experimental groups exposed at 2.5° to 25°C, but an increase ofabnormalities at the pe locus was recovered in ocoytes (and/or eggs) after

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

treatment with relatively high temperatures at 30° __ 40°C.
Surprisingly a significant increase in mosaics of black (re+) and red(re) serosa cells and a few recessives at the re locus were detected, besides
the markedly high frequency of abnormalities at the pe+ locus of either mosaic
or recessive individual, when the eggs were treated with high temperatures
at 37° and 40°C. The occurrence of both mosaic and recessive abnormalities
at the re+ locus in consequence of the androgenic development could hardly
be interpreted, because the pe gene is epistatic to the re gene. The plausible
interpretation for this finding was that these abnormalities may be produced
by gene mutations and chromosomal aberrations induced with high temperature exposures, as reported in other organisms. Thus, it can be assumed from the present observation that, in silkworm females, the hightemperatures are one of important factors to increase egg-colour specificlocus mutations and that the deflection of frequency in spontaneous as well
as artificially-induced mutations at different seasons may be partly dependent on the temperature during experiments. This suggests that hightemperatures accelerated chemical reactions which produced the highfrequency of mutations.
At present, the mechanism responsible for the marked increase and/or
deflection of spontaneous mutations at the pe+ locus as compared with there+ locus is not clear, but it is likely to assume that, under the high temperature condition, the occurrence of the mosaics of black (pe+) and yellowish-white (pe) serosa cells may originate from the abnormal development of the androgenic nucleus in one part of the egg and the normal development of fertilized zygotes in the other part. Similarly, the recessive
abnormalities at this locus would have a tendency to arise from dispermic
androgenic development. It could not exclude a possibility, however, thata different location of genes on the chromosome may be also related to adifferential mutability: the pe gene on the proximal portion (5-0.0) and there gene on the middle portion (5-31.7).
A New Egg Colour Mutant, Light Brown Egg with Black Eyesin the Silkworm
Akio MURAKAMI and Yosoji FUKASE

In the course of studies on the chemically-induced mutagenesis withsilkworms, a number of recessive visible complete and mosaic mutants

RESEARCHES CARRIED OUT IN 1983

77

have been recovered by the specific-locus method using egg-colour genes,
pe and reo Among them a mosaic mutant composing of light brown andnormal black serosa cells was detected in oocytes after treatment of pupae
with a mycotoxin Aflatoxin B1 • To establish a complete mutant from thismosaic one, the mosaic mutant was first crossed to the double recessive
marker stock homozygous for pe and re loci and consequently about
twenty eggs for light brown were obtained in addition to a number of blackand yellowish-white eggs. In the next step, the female mutants showing
the light brown egg were mated to a tester male homozygous for re gene todiscard the marker chromosome having both pe and re genes and in consequence black and red eggs were obtained. To obtain the homozygous
individuals for the mutant factor, crosses between those black eggs were carried out and the offspring black, red and light brown eggs were obtained
instead of yellowish-white eggs.
It is of interest to note that the pigmentation of compound eyes in theinsect with the light brown eggs was hardly distinguished from that of thewild type black (+) eggs, but clearly different from the yellowish-white(pe) eggs. The intensity of the colour of serosa cells in the light brown eggs
showed yellowish-white until the first 72 hrs after oviposition, like as thatin the pe mutant eggs, but the pigmentation is gradually changed into lightbrown cloor peculiar to this new mutant phenotype by the early phase ofdiapause period. When a female of the light brown egg was crossed to amale of the wild-type black egg, the offspring all had light brown eggs withblack eyes, and the same result was obtained in the reciprocal cross. This
indicates that the mutant is recessive to the wild-type gene (pe+). Whenthe silkworm with light brown eggs was mated to a pe silkworm, all offspringin the F 1 generation, had a phenotype of the mutant, regardless of the crosssystem. This result shows that the factor for light brown was epistatic to
the pe. Such being the case, we named the mutant, light brown eggs withblack eyes, pe bw , as one member of multiple allelomorph at the pe+ gene
locus on the proximal region of the 5th chromosome (5-0.0). Although
the pigmentation of the pe mutant ganglia was pale pink, that of the lightbrown (pe bw ) egg was violet, compared with that of deep violet in the wildtype.
In addition to the pe bw mutant, we detected several varieties of the yellowish-white eggs with different intensities of the colour. Probably, thesevarieties at the pe locus could be explained on the basis of a single gene

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

variation. These facts indicate that these several varieties may be themultiple allelomorph of the pe+ gene and show various intensities of thecolour of the serosa and genglion cells. Such variant alleles of the pe+gene would arise as gene mutations rather than chromosomal aberrations.
If so, it could be said that Aflatoxin B1 was able to induce so called "genemutations" as well as chromosomal aberrations in germ cells of the silkwork.
For reference, a white egg lethal pel has long been regarded as one ofvariants of the pe+ gene. However, recently, viable yellowish-white eggs
was detected with a low frequency in this lethal suggesting that both thelethal and pink eye loci with yellowish-white eggs may be close together
with each other. Thus the allelomorph at the pe locus may have a polymorphic function controlling the pigmentation of serosa and ganglion cells
as well as of compound eyes.
Effects of Tritium on the Induction of Recessive Visible Mutationsin Primordial Germ-Cells of Silkworm Embryos:
The Nature of fi-Ray-Induced Mutants
Akio MURAKAMI

Environmental contaminations with internal .a-emitters arise in various
nuclear operations including nuclear fusion devices. Concequently, aninterest is increasing on the possible mutagenicity as well as other biologicaleffects of .a-radiation which were naturally or accidentally accumulated in
the body or the tissues with low doses and low dose-rates. In this connection, the primordial germ-cell systems using diapause silkworm embryos
have an advantage in detecting genetical effects by determining incorporated
radionucleides in the above-mentioned exposure condition. Evaluationof the relative mutagenic effectiveness or the RBE of .a-rays has been long
carried out by Tazima (Res. Rept. to the Ministry of Education, Science
and Culture of Japan, "The Fundamental Studies on the Biomedical Effectsof Tritium" [1978]). He reported that the effect of .a-rays was ca. twotimes more effective than the standard radiation. This RBE value inembryonic germ-cells indicates that .a-ray-induced mutants includes genetic
lesions other than gene mutations. In the present study, the progeny
tests were carried out to make clear the nature of .a-ray-induced mutantsin primordial germ-cells using [6- 3 H]thymidine (6T) as a model j1-emitter.

RESEARCHES CARRIED OUT IN 1983

79

The mid-stage female pupae of the wild-type ClOB strain were treated with
25 pCi/capita of 6T and mated to another wild-type Aojuku strain males toallow oviposition of radioactive eggs. The duration of incubation or diapause periods was about 7 months. Mter hatching, offsprings weregrown and then mated to the marker stock moths homozygous for eggcolour genes, pe and re, to detect the recessive visible mutations. We
obtained almost complete mutations, but rarely mosaic mutations.
The procedure for the progeny test was as follows. First yellowishwhite egg mutants in the pe+ locus, for example, were crossed with thetester strain homozygous for the re gene to obtain heterozygous chromosomes(black eggs) carrying the newly induced mutation and/or to discard thepe re tester chromosome. In the next step, the matings between the black
eggs were carried out to obtain the homozygous chromosomes for the
mutated locus and the two expected eggs, black and red, in the next generation. Lastly, the yellowish-white eggs as well as other phenotypic eggs
were tested for their viabilities and/or hatchabilities. Among 32 mutants
examined for both pe+ and re+ loci, 15 were found to be lethals, indicating
about half of them might have chromosomal aberrations. However, it
should be emphasized that the total number of mutants subjected to thetest was up to several thousands and that the majority of them (ca. 90%)
died before the progeny test at the stage of embryos. This clearly indicatesthat a large number of the mutants detected might be caused by chromosomal
damages.
The present observation is in agreement with the implications deduced
from the RBE value (ca. 2) of (3-rays obtained by the specific locus test inembryonic germ-cells. It has been long established that chromosomal
damages induced in pre-meiotic stage cells could hardly be transmitted to
the next generation. But the present finding may contrast with such a view.
Further studies are required to confirm these possibilities and also to elucidata the relative ability of (3-rays to induce gene mutations and chromosomal aberrations in primordial germ-line cells of the silkworm.
A New Type Maternal Inheritance in the Silkworm
Akio MURAKAMI

The first example of maternal inheritance in the silkworm was reported
by Toyama (1913). In this insect, there are a number of egg-color mutants,

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

WI> bI> b2 , pe, re, and others. The first three mutants showed the maternal
inheritance or determination: the phenotypic expression of the individual's
own genotype was delayed and segregation occurred in the Fa generation.
This unusual phenomenon was simply interpreted on the basis of the eggcytoplasmic contribution by mother genotype regardless of either dominantor recessive trait of her genotypes. Besides those egg-color mutants, thelast two pe (pink eyed white egg), re (red egg) and other egg-color mutants
showed the normal mendelian inheritance. Accordingly, these pe and regenes have been used for the specific-locus method to detect the mutations.
In the series of chemically- and physically-induced mutation experiments, numerous mutants at these gene loci have been detected and/or
preserved in our laboratory. However, the intensity of the color of theserosa cells in those mutants has a marked variation. For example,
mutants at the re locus have a broad color spectrum varying from yellowish-orange which is almost indistinguishable from the yellow of the pemutant to bright red. Since it is very difficult to designate accurately
each mutant with a pertinent term, for convenience each mutant was classified by a wave length unit adopted from the interference color chart for
NIKON polarizing microscope as follows: re (l100-1150m,u), re (10501100), re (1000-1050), re (975-1000), re (450-500), etc.
The bright red egg, mutant re (l100-1150,um), kept its phenotype
throughout subsequent generations when the mother is such a mutant.
Several mutants analysed so far are viable in the homozygous state, suggesting that they may be caused by gene mutations rather than chromosomal
deficiencies. When one of these mutants, re (1100-1150) was mated tothe black wildtype (re+) the F 1 offspring had all the black egg color regardless of the mutants used, confirming that the mutant behaved as aregular mendelian recessive mutant to its corresponding black wild-typeallele. Similar situations were observed in other re mutants. Consequently, each mutant may occupy the same locus of the re gene (5-31.7). In
the F 2 a segregation occurs within a single batch with a ratio of three black
eggs to one red eggs. When a re (1100-1150 m,u) female was matted to are (450-500 m,u) all offspring in the next generation showed a phenotypeof the re (1100-1150) mutant, for example. In the reciprocal cross, theoffspring showed a phenotype of the re (450-500) mutant. Similar situations were observed in the crosses between other re egg mutants. Thus,
this phenomenon may be limitted to be the allele to the gene locus for the

RESEARCHES CARRIED OUT IN 1983

81

phenotype of red eggs. The analysis of the behaviors of each mutant for
further generations was not yet completed however, it appears that the trait
has a tendency to behave as the maternal inheritance independent of its
wild-type allele. It is also suggested that the factor controlling these phenotypes is genetically equistatic each other. It is necessary to further analyse
what constitutes an essenstial factor for the phenomenon, to draw a final
picture for the present finding which may throw light on the mechansim(s)responsible for the unusual maternal inheritance.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

VII.

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICROORGANISMS AND PLANTS
Induction of Prophages in Spores of Bacillus subtilis by
Ultraviolet Irradiation from Synchrotoron
Orbital Radiation
Yoshito SADAIE and Tsuneo KAnA

Recently vacuum UV has become available from synchrotron orbitalradiation and can be used for studying the effect of vacuum UV on livingorganisms. Because vacuum UV is available only in a high vaccum, bacterial spores are appropriate materials for studying its effect on living cell.
We examined the induction of prophages (SP02) from lysogenic spores ofBacillus subtilis by SR UV (160 nm to 240 nm) and found that SR UV ataround 220 nm was most effective in the inactivation of spores and prophage
induction. SR UV at around 220 nm might produce spore specific photoproducts in DNA as the conformation of spore DNA is different from thatof vegetative cells and UV irradiation produces a unique photoproductfrom the thymine residues in spores (J. Radiat. Res. 25: 170-173, 1984).
Detection of Natural and Environmental Bioantimutagens
and their Mode of Actions
Tsuneo KADA and Tadashi INOUE

In the past few years, we found a number of new natural antimutagens
working against radiation- and chemically induced mutations in bacteria.
They include certain biometal compounds such as cobalt (proc. Japan
Acad. Ser. B, 5: 234, 1978; Mutation Res. 91: 41, 1981) and germanium(lnt. J. Radiat. BioI. 42: 653, 1982; Mutation Res. 125: 145, 1984), ingredients of plants such as cinnamaldehyde (Agric. BioI. Chern. 48: 1905,
1984; Mutation Res. 107: 219, 1983), coumarin and umbelliferone (Mutation Res. 117: 135, 1983), protoanemonin (Mutation Res. 116: 317, 1983)
and enmein (Agric. BioI. Chern. 48: 1647, 1984). Form animal origins,placenta tissues of mammals were shown to possess strong antimutagenicity
against gamma-ray-induced mutations (J. Rad. Res. 22: 297, 1981).

RESEARCHES CARRIED OUT IN 1983

83

The action spectra of certain antimutagens such as cobaltous chloride
are very wide and covers those of not only UV, gamma-ray and MNNG
but also a frame-shift mutagen (Trp-P-l) (Mutation Res. 95: 145, 1982)
indicated that the antimutagen might be working not at specific mutagenic
sites but as stimulator of error-free repair of non-specific DNA lesions.
Modification of the Activities of recA Protein by an
Antimutagenic Metal Compound, Cobaltous Chrolide
Tadashi INOUE and Tsuneo KAOA

In addition to a large number of mutagenic agents in our environment,
we also have various compounds which show antimutagenic activities incertain mutation assay-systems using bacteria and mammalian cells cultured in vitro. From the view points of both human health and fundamental
genetics, it is of interest to study the mechanisms by which the antimutagenic
compounds exhibit their activity. One of the potent antimutagenic compounds we have so far found is cobaltous chrolide. This antimutgen iseffective in both radiation- and chemically induced mutagenesis in Escherichia coli as well as in spontaneous mutagenesis in Bacillus subtiUs. In order
to elucidate the mode of action of this antimutagen, we examined its effects
on the purified recA rpotein in vitro. The recA protein of E. coli is the
product of the recA gene on which various cellular functions including
mutagenesis, recombination and repair profoundly depend. It promotesin vitro the homologous pairing of double-stranded DNA and linear singlestranded DNA, thereby forming a three-stranded joint molecule called
D-Ioop. The protein also catalyzes the hydrolysis of ATP which depends
upon single-stranded DNA or replicative form I DNA. Experimental
results showed that both D-Ioop formation and replicative form I DNAdependent ATPase activity was stimulated whereas the single-stranded
DNA-dependent ATPase activity was partially inhibited by the antimutagenic metal compound, suggesting that the antimutagenicity of this metalcompound is ascribed to the elevated activity of recombinational repair
which is thought to be error-free. This is also supported by the fact thatthe survival of mutagen-treated bacteria was enhanced by the posttreatment
with the antimutagen.
This work was done in collaboration with Drs. T. Shibata, O. Makino,
T. Ando (Ricken Institute, Wako) and T. Ohta (Institute of Environmental

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Toxicology, Tokyo).
Restorative Effects of the Human Placenta Extracts
on Gamma-ray-irradiated Mice
Tsuneo KADA and Hideo TEZUKA

Effects of the human placenta extracts were observed on mice which were
lethally exposed to gamma-ray. Placental tissues obtained from thirty
individuals were homogenized and mixed. The mixture was deproteinizedby treating with pronase and heating in boiling water, followed by centrifugation. The clear supernatant solution was stocked at -80°C. We
previously reported that such an extract showed an antimutagenic activity
on gamma-ray-induced reverse mutations in E. coli Bjr WP2 trp (J. Radiat.
Res. 22, 297, 1981).
Male BaCsF1 mice of 9 weeks old were purchased from Japan Charles
River Co. Ltd. and kept in this laboratory for a week before use. Wholebody irradiation was carried out with 10 Gy of gamma-ray from a 137Cs
source at a dose-rate of 0.5 Gyjmin. Within 40 minutes after irradiation,
0.3 ml or 0.6 ml of the placenta extract was injected i.p. into each mouse.
For the control mice the corresponding amount of physiological saline wasgiven. All the mice were housed in cages kept in an air-conditionned room

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Changes of survival in mice after irradiation with mcs r -rays.
5

RESEARCHES CARRIED OUT IN 198385

at 22°C with nutritional chow (Oriental Yeast Co. Ltd.) and tap water ad
libitum.
It was repeatedly found that more than 70 % of irradiated mice with theplacenta injection survived at least for 30 days. All the mice with saline
injection usually died between 10th and 20th days after irradiation. Since
promotion of DNA-repair by certain antimutagens was often found in
irradiated bacteria, the hypothesis that a similar effect might be involvedin the critical target tissue(s) is being examined to explain the present observation.
Effects of DBMA on the Gamma-ray Mutagenesis in Soybean
Taro FUJII

9,1O-dimethyl-I,2-benz(a)anthracene (DMBA) is a well-known tumor
initiator in mouse-skin carcinogenesis. Mutagenic activity of DMBA wasinvestigated with a soybean test system. Dry seeds were treated at theconcentrations of between 50 ,ug/ml to 2 mg/ml for 24 hrs in 4 experiments, and the mutation frequency was measured as the number of mutant
spots on two simple leaves of heterozygous plants. No difference in the
average number of spots per leaf was observed in the groups treated withwater or different concentrations of DMBA. DMBA has no mutagenic
activity in the present test system.
To see the effect of DMBA on the gamma-ray mutagenesis, combination
treatment of drug and gamma-ray exposures was conducted. This experiment was divided into 2 parts; in Exp. A, seeds were treated first withgamma-rays and then with DMBA, in Exp. B, seeds were treated first with
DMBA and then with gamma-rays. In Exp. A, the mutagenicity of gammarays was not affected by post-treatment of seeds with DMBA. The spotting
frequencies of 2.26 and 2.45 per leaf obtained when irradiated seeds were
post-treated with DMBA at 1 and 2 mg/ml, respectively, were similar to
those obtained when irradiated seeds were post-treated with water (2.66
spots per leaf). On the other hand, interesting results were observed inExp. B. Spotting frequency of 9.86 were obtained when water pre-soaked
seeds were irradiated with 150 R. However, frequency of spots per leaf
was much lower in DMBA pre-treatment lots than that of water pre-treatment lot. Although severe killing effect was observed at 2 mg/ml pretreatment plot, the spotting frequencies of 5.69 and 3.83, respectively, were

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

observed when 1 and 2 mg/ml DMBA pre-treated weeds were irradiatedwith 150 R of gamma-rays. Namely, decrease in mutation frequency was
noticed with DMBA pre-treatments, while most of chemical mutagens
enhance the damage or the mutation frequency induced by radiations.
Absence of Mutagenic Activity of BP in the Soybean
Mutation Test
Taro Fum and Tadashi INOUE

Mutagenic effect of benzo(a)pyrene (BP) in higher plants was studied using
the soybean test system. Concentrations of 5 to 400 ,ug/ml of BP wereapplied in the first experiment. There were no differences in the average
number of spots per leaf in treatment with water or 4 different concentrations of BP treatments. However, some abnormal gorwth or deformed
leaf development, without increase in germination ability, was observed at
highest concentration treatment. To examine the BP action on presoaking
seeds, combined treatments of water presoaking and BP were conducted(2nd experiment). Seeds presoaked for 2 to 6 hrs in water were immersed
in 50 to 200 ,ug/ml of BP for 24 hrs. No increase of spotting frequency was
noticed.
Activation by liver S9 mixture is necessary for BP to be mutagenic inbacterial mutation assay system. We therefore applied BP to dry seeds
together with rat liver S9-mixture. Severe toxic effects of the S9-mixture
were observed in this experiment. In spite of such a physiological damage
by the rat liver S9-mixture, the number of mutant spots did not differ
throughout all treatments. The results indicated that rat S9 is useless forthe soybean test system. It has been reported that microsomes from
Jerusalem artichoke tubes are capable of activating BP in vitro. We therefore examined whether the extract of Jerusalem artichoke is effective in our
soybean mutation test. In these experiments, dry or presoaked seeds weretreated with BP in the presence of plant S9-mixture, or seeds presoaked inplant S9-mixture were treated with BP in water. The average number ofspots in each experiment was similar to those of respective control plot.
Mutation induction by a mixture of BP with plant S9-mixture could not
be detected either in treatments for dry or water-presoaked seeds, or BP toseeds presoaked with the S9-mixture.
BP is known to have strong carcinogenic and mutagenic activity after

RESEARCHES CARRIED OUT IN 198387

metabolic activation with cytochrome P450 and NADPH. However, thepresent experiments indicated no mutagenic activity of BP irrespective of
the various combination treatments, Some hidden action of BP may not
be excluded because a concentration of 400 Itg/ml to dry seeds induced
some physiological damage, and when BP was mixed with rat liver S9mixture, a decrease in germination ability was paralled to an increase in BPconcentrations. We are currently investigating this observation in moredetail to see if mutagenic activity is present in the chemical because BP is
one of the environmentally significant substances having potential hazardous
effect on living organisms, and contamination in food stuffs has been noticed.
At present, our experiments demonstrated negative results.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

VIII.

POPULATION GENETICS (THEORETICAL)

Diffusion Model of Intergroup Selection, with Special Reference
to Evolution of an Altruistic Character
Motoo KIMURA

Let us assume a diploid species consisting of an infinite number of competing demes, each having N. reproducing members and in which mating is
at random. Then consider a locus at which a pair of alleles A and A' aresegregating, where A' is the "altruistic allele," which has selective disadvantage s' relative to A with respect to individual selection, but which isbeneficial for a deme in competition with other demes; namely, a deme having A' with frequency x has the advantage c(x-x) relative to the averagedeme, where c is a positive constant and x is the average of x over the
species.
Let ifJ=ifJ(x; t) be the distribution function of x among demes in thespecies at time t. Then, we have oifJjot=L(ifJ)+c(x-x)ifJ, where L is the
Kolmogorov forward differential operator commonly used in population
genetics [Le., L=(lj2)(02joX2)V~.-(ojox)M~.], and M~. and V~. stand forthe mean and variance of the change in x per generation within demes. As
to migration, assume Wright's island model and denote by m the migration rate per deme per generation. By investigating the steady state, inwhich mutation, migration, random drift, and intra- and interdeme selection balance each other, it is shown that the index D=cjm-4N.s' serves
as a good indicator for predicting which of the two forces (i.e., group selection or individual selection) prevails; if D>O, the altruistic allele predominates, but if D<O, it becomes rare and cannot be established in the species.
For details, see Proc. Natl. Acad. Sci. USA 80, 6317-6321.
Rare Variant Alleles in the Light of the Neutral Theory
Motoo KIMURA

Based on the neutral theory of molecular evolution and polymorphism,
and particularly assuming "the model of infinite alleles," a method is proposed which enables us to estimate the fraction of selectively neutral alleles

RESEARCHES CARRIED OUT IN 198389

(denoted by P neut) among newly arisen mutations. It makes use of dataon the distribution of rare variant alleles in large samples together withinformation on the average heterozygosity. The formula proposed isPneut=[He/(1-He)][loge (2fiq)/fi a(x<q)], where fiix<q) is the average number ofrare alleles per locus whose frequency, x, is less than q; fi is the average
sample size used to count rare alleles; He is the average heterozygosity per
locus; and q is a small preassigned number such as q=O.OI. The method
was applied to observations on enzyme and other protein loci in plaice,humans (European and Amerindian), Japanese monkeys, and fruit flies.
Estimates obtained for them range from 0.064 to 0.21 with the mean andstandard error P neut =0.14±0.06. It was pointed out that these estimates
are consistent with the corresponding estimate P neu lHb)=0.14 obtained
independently based on the neutral theory and using data on the evolutionary rate of nucleotide substitutions in globin pseudogenes together
with those in the normal globins. For details, see Molecular Biology &Evolution 1, 84-93.
Selective Constraint in Protein Polymorphism: Study of theEffectively Neutral Mutation Model by Usingan Improved Pseudosampling Method
Motoo KIMURA and Naoyuki TAKAHATA

To investigate the pattern of allelic distribution in enzyme polymorphism, with special reference to the relationship between the mean (H) andthe variance (VH) of heterozygosity, we used the model of effectively neutral
mutations involving multiple alleles in which selective disadvantage of mutant
alleles follows a r distribution. A simulation method was developed thatenables us to study efficiently the process of random drift in a multiallelicgenetic system and that saves a great deal of computer time. It is an improved version of the pseudosampling-variable (PSV) method [Kimura,
M. (1980) Proc. Natl. Acad. Sic. USA 77, 522-526] previously used tosimulate random drift in a diallelic system. This method will be useful for
simulating many models of population genetics that involve behavior of
multiple alleles in a finite population. By using this method, it was shown
that, as compared with the model of strictly neutral mutations, the present
model gives the reduction of both H and V H and an excess of rare variant
alleles. The results were discussed in the light of recent observations on

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

protein polymorphism with special reference to the functional constraintof proteins involved. For details, see Proc. Natl. Acad. Sci. USA 80,
1048-1052.
Theoretical Study on the Accumulation of Selfish DNA
Tomoko OHTA

The accumulation of selfish DNA in eukaryotic genomes was studied
from the standpoint of population genetics. Selfish DNA is assumed toreplicate itself within a haploid set. For the selectively neutral case, thefate of a single self-replicating DNA segment (unit) within a population
was investigated by the method of the probability generating function, andby Monte Carlo simulation, with special reference to the probability ofsurvival and average number of units per haploid set. For the selectively
deleterious case at the organismal level, the equilibrium between new
occurrence and selective elimination was studied, and the average and variance of the number of units per haploid set in the population was examinedby Monte Carlo simulation. It is shown that the process of self-replication (duplication-deletion) plays an essential role for the maintenance andelimination of selfish DNA. For details, see Genet. Res., Camb. 41, I-IS.
On the Evolution of Multigene Families
Tomoko OHTA

Multigene families are classified into three groups: small families as
exemplified by hemoglobin genes of mammals; middlesize multigene families,
by genes of mammalian histocompatibility antigens; and large multigene
families, by variable region genes of immunoglobulins. Facts and theories
on these evolving multigene families are reviewed, with special reference tothe population genetics of their concerted evolution. It is shown thatmultigene families are evolving under continued occurrence of unequal (buthomologous) crossing-over and gene conversion, and that mechanisms formaintaining genetic variability are totally different from the conventionalmodels of population genetics. Thus, in view of widespread occurrenceof multigene families in genomes of higher organisms, the evolutionary
theory based mainly on change of gene frequency at each locus would
appear to need considerable revision. For details, see Theor. Pop. BioI.

RESEARCHES CARRIED OUT IN 1983

91

23,216-240.
Time until Fixation of a Mutant Belonging to a
Multigene Family
Tomoko OHTATime until fixation of a mutant that occurs in one copy of a multigene
family was investigated from the standpoint of population genetics. Be<:ause of the complexity of the double process of random drift (on the chromosome and in the population), an approximate method based on the rateof steady decay of genetic variability is applied. The simple model ofgene conversion with constant gene family size is also used. The expectation based on the approximate method is shown to be valid by extensiveMonte Carlo simulations, and the results are useful for understanding themechanisms of turnover of multigene families, when comparison is available
between closely related species. For details, see Genet. Res., Camb. 41,
47-55.
Population Genetics of Multigene Families that are
Dispersed into Two or More Chromosomes
Tomoko OHTA and Gabriel A. DOVER

The evolution of multigene families whose members are dispersed into
two or more nonhomologous chromosomes is studied from the standpointof population genetics. By using a simple model of gene conversion, equilibrium and transient properties of the probability of identity of genes belonging to the family are investigated. Also, the time until fixation of amutant belonging to a subdivided multigene family is studied by an approximation method. It is shown that dispersion of the family into two or more<:hromosomes has a relatively minor effect on the extent of identity between
genes, unless the conversion rate between genes on nonhomologous chromosomes is extremely low, or unless the number of nonhomologous chromosomes on which gene members are dispersed is large. For details, see
Proc. Natl. Acad. Sci. USA 80, 4079-4083.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Gene Identity and Genetic Differentiation of Populationsin the Finite Island Model
Naoyuki TAKAHATA

A formula for the variance of gene identity (homozygosity) was derived
for the case of neutral mutations using diffusion approximations for thechanges of gene frequencies in a subdivided population. It is shown thatwhen gene flow is extremely small, the variance of gene identity for theentire population at equilibrium is smaller than that of the panmictic population with the same mean gene identity. On the other hand, althougha large amount of gene flow makes a subdivided population equivalent toa panmictic population, there is an intermediate range of gene flow in which
population subdivision can increase the variance. This increase resultsfrom the increased variance between colonies. In such a case, each colony
has a predominant allele, but the predominant type may differ from colony
to colony. The formula for obtaining the variance allows us to study suchstatistics as the coefficient of gene differentiation and the correlation ofheterozygosity. Computer simulations were conducted to study the distribution of gene identity as well as to check the validity of the analyticalformulas. Effects of selection were also studied by simulations. For
details, see Genetics 104, 497-512,

Evolutionary Dynamics of Extranuclear Genes
Naoyuki TAKAHATA and M. SLATKIN

We studied the evolutionary dynamics of extranuclear genes taking into
account simple kinds of selection, two modes of inheritance and the multiplicity of genomes within a cell. Particular attention was paid to theaccumulation of advantageous or deleterious mutations in an extranuclear
genome. Within-generation drift due to multiplicity of genome and nonMendelian segregation promotes the fixation of advantageous mutations
and prevents deleterious mutations from accumulating. We show alsothat the extent of paternal contribution makes little difference in the rate,but, in contrast, the configuration of the genome and the mode of transmission both make a large difference. These results are compatible withwhat is known about extranuclear genomes. For details, see Genet. Res.,
Camb. 42, 257-265.

RESEARCHES CARRIED OUT IN 1983

93

Linkage Disequilibrium of Extranuclear Genes under Neutral
Mutations and Random Genetic Drift
Naoyuki TAKAHATA

To evaluate the genetic constitution of extranuclear or non-Mendeliangenes, several formulas for the variance of linkage disequilibrium (nonrandom association of linked genes) were derived under the neutral mutation hypothesis. Two kinds of the variances are mainly considered. One
is the variance for the entire population and the other is the variance within
a single germ cell. At equilibrium, both variances differ from that of a
Mendelian population with the same parameters. In particular, when thecytoplasmic contribution from a male gamete {j is small, as is found in mostorganisms, the effect of recombination between extranuclear genes is weakbecause of low within-cell heterogeneity. Then, the total variance for theentire population is large. Other important quantities determining thevariances are the number of extranuclear genomes per cell, n, and the number of cell divisions in a germ cell line per generation, A. Unless (3=0,even a small contribution from a male gamete becomes significant depending on nand A. For details, see Theoretical Population Biology 24,
1-21.

Population Genetics of Extranuclear Genomes under the
Neutral Mutation Hypothesis
Naoyuki TAKAHATA

Population genetics of extranuclear genomes is further developed under
the neutral-mutation random-drift hypothesis, and the characteristic evolutionary aspects are summarized. Several formulae derived here are concerned with the variances of genetic variability (gene identity) at a single
extranuclear locus and the evolutionary distance between two isolated populations which is estimated from a comparison of homologous linked nucleotide sites. Two types of variance are considered; one is the variancein the entire population (VQ ) and the other is the variance within a single
germ cell (VH ). When compared with a Mendelian genetic system in apanmictic population, an extranuclear genetic system has the following
equilibrium properties: (1) the mean genetic variability is low despite thehigh multiplicity of the genome in a cell if the proportion of the cytoplasmic

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

contribution from the male's gamete is small, (2) the effect of recombination is small and a large amount of variance of linkage disequilibrium tends
to be maintained, (3) the overall relationship between the mean and varianceof genetic variability does not much differ but VQ(VH) is expected to besmall if the paternal contribution is small, and (4) the evolutionary distanceestimated depends on the extent of intrapopulational variation in a common ancestor population which in turn depends on within-cell variation.
I argue that there is an analogy between the model of extranuclear genomes
in a finite population and that of nuclear genes in a subdivided population.
The analogy helps our understanding of some properties in an extranuclear
genetic system. For details, see Genet. Res., Camb. 42, 235-255.

A Quantitative Genetic Model of Reciprocal Altruism:
A Condition for Kin or Group Selection to Prevail
Kenichi AOKIA condition is derived for reciprocal altruism to evolve by kin or group
selection. It is assumed that many additively acting genes of small effectand the environment determine the probability that an individual is areciprocal altruist, as opposed to being unconditionally selfish. The particular form of reciprocal altruism considered is TIT FOR TAT, a strategy
that involves being altruistic on the first encounter with another individualand doing whatever the other did on the previous encounter in subsequentencounters with the same individual. Encounters are restricted to individuals of the same generation belonging to the same kin or breeding group,
but first encounters occur at random within that group. The number ofindividuals with which an individual interacts is assumed to be the same
within any kin or breeding group. There are 1+1 expected encounters
between two interacting individuals. On any encounter, it is assumed
that an individual who behaves altruistically suffers a cost in personal fitness
proportional to c while improving his partner's fitness by the same proportion of b. Then, the condition for kin or group selection to prevail isr>{(cjb)-[l-(cjb)]IC}/{ I +[l-(cjb)]I[C+(TbjVb)]}if group size is sufficiently large and the group mean and the within-group
genotypic variance of the trait value (i.e., the probability of being a TITFOR-TAT strategist) are uncorrelated. Here, C, Vb, and T b are the popula-RESEARCHES CARRIED OUT IN 1983

95

tion mean, between-group variance, and between-group third central momentof the trait value and r is the correlation between the additive genotypicvalues of interacting kin or of individuals within the same breeding group.
The right-hand side of the above inequality is monotone decreasing in C
if we hold Tb/Vb constant, and kin and group selection become superfluous
beyond a certain threshold value of c. The effect of finite group size isalso considered in a kin-selection model. For details, see Proc. Natl.
Acad. Sci. USA 80, 4065-4068.
Stochastic Theory of Population Genetics
Takeo MARUYAMAStochastic models of population genetics are studied with special reference
to the biological interest. Mathematical methods are described for treatingsome simple models and their modifications aimed at the problems of themolecular evolution. Unified theory for treating different quantities is
extensively developed and applied to some typical problems of current
interest in genetics. Mathematical methods for treating geographicallystructured populations are biven. Approximation formulae and their accuracy are discussed. Some criteria are given for a structured population
to behave almost like a panmictic population of the same total size. Somequantities are shown to be independent of the geographical structure andtheir dynamics are described. Bulletin of Mathematical BIology 45: 512554.
Analyses of the Age of Genes and the First Arrival
Times in a Finite Population
Takeo MARUYAMA

The age of a mutant gene is studied using the infinite allele model in which
every mutant is new and selectively neutral. Based on a time reversal
theory of Markov processes, we develop a method of mathematical analysis
that is considerably simpler for calculating the various statistics of the age
than previous methods. Formulas for the mean and variance and for the
distribution of age are presented together with some examples of relevanceto cases in natural populations.-Theoretical studies of the first arrival timeof an allele to a specified frequency, given an initially monomorphic condi-

96

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

tion of the locus, are presented. It is shown that, beginning with an allele
that has frequency p= 1 or an allele with frequency p= 1/2N, there is an
initial lag phase in which there is virtually no chance of an allele with a
specified intermediate frequency appearing in the population. The distribution of the first arrival time is also presented. The distribution shows
several characteristics that are not immediately obvious from a consideration of only the mean and variance of first arrival time. Especially noteworthy is the existence of a very long tail to the distribution. We havealso studied the distribution of the age of an allele in the population. Again,
the distribution of this measure is shown to be more informative for several
questions than are the mean and variance alone. Genetics 105: 1041-1059.
Models of Evolution of Reproductive Isolation
Takeo MARUYAMA

Mathematical models are presented for the evolution of postmating andpremating reproductive isolation. In the case of postmating isolation it isassumed that hybrid sterility or inviability is caused by incompatibility ofalleles at one or two loci, and evolution of reproductive isolation occurs byrandom fixation of different incompatibility alleles in different populations.
Mutations are assumed to occur following either the stepwise mulation
model or the infinite-allele model. Computer simulations by using Ito's
stochastic differential equations have shown thRt in the model used the
reproductive isolation mechanism evolves faster in small populations thanin large populations when the mutation rate remains the same. In populations of a given size il evolves faster when the number of loci involved islarge than when this is small. In general, however, evolution of isolation
mechanisms is a very slow process, and it would take thousands to millions
of generations if the mutation rate is of the order of 10- 5 per generation.
Since gene substitution occurs as a stochastic process, the time required forthe establishment of reproductive isolation has a large variance. Although
the average time of evolution of isolation mechanisms is very long, substitution of incompatibility genes in a population occurs rather quickly
once it starts. The intrapopulational fertility or viability is always veryhigh. In the model of premating isolation it is assumed that mating preference or compatibility is determined by male- and femaic-limited characters, each of which is controlled by a single locus with multiple alleles, andRESEARCHES CARRIED OUT IN 1983

97

mating occurs only when the male and female characters are compatible
with each other. Computer simulations have shown that the dynamics ofpremating isolation mechanism is very similar to that of postmating isolation mechanism, and the mean and variance of the time required for establishment of premating isolation are very large. Theoretical predictions
obtained from the present study about the speed of evolution of reproductive isolation are consistent with empirical data available from vertebreteorganisms. Genetics 103: 557-579.
An Approach to Population and Evolutionary Genetic
Theory for Genes in Mitochondria andChloroplasts, and Some Results
Takeo MARUYAMA

We developed population genetic theory for organelle genes, using an
infinite alleles model appropriate for molecular genetic data, and considering
the effects of mutation and random drift on the frequencies of selectively
neutral alleles. The effects of maternal inheritance and vegetative segregation of organelle genes are dealt with by defining new effective gene numbers,
and substituting these for 2N. in classical theory of nuclear genes for diploidorganisms. We define three different effective gene numbers. The mostgeneral is N" defined as a function of population size, number of organelle
genomes per cell, and proportions of genes contributed by male and female
gametes to the zygote. In many organisms, vegetative segregation oforganelle genomes and intracellular random drift of organelle gene frequencies combine to produce a predominance of homoplasmic cells within
individuals in the population. Then, the effective number of organelle
genes is N. o , a simple function of the numbers of males and females and of
the maternal and paternal contributions to the zygote. Finally, when thepaternal contribution is very small, N eo is closely approximated by the
number of females, Nt. Then if the sex ratio is 1, the mean time to fixation or loss of new mutations is approximately two times longer for nuclear
genes than for organelle genes, and gene diversity is approximately fourtimes greater. The difference between nuclear and organelle genes disappears or is reversed in animals in which males have large harems. The
differences between nuclear and organelle gene behavior caused by maternal
inheritance and vegetative segregation are generally small and may be

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

overshadowed by differences in mutation rates to neutral alleles. For
monoecious organisms, the effective number of organelle genes is approximately equal to the total population size N. We also show that a
population can be effectively subdivided for organelle genes at migration
rates which result in panmixis for nuclear genes, especially if males migrate
more than females. Genetics 103: 513-527.
A Mathematical Model of Codon Substitution
Takashi GOJOBORI

A mathematical model for codon substitution is considered, taking into
account unequal mutation rates among different mucleotides and selective
constraint against amino acid changes. This model is constructed byusing a 61 x 61 transition probability martix for the 61 nonterminating
condons. Under this model, a computer simulation is conducted to study
the numbers of silent (synonymous) and amino acid-altering (nonsynonymous) mucleotide substitutions when the underlying mutation rates among
the four kinds of nucleotides are not equal. It is assumed that the substitution rates are constant over evolutionary time and the codon frequencies
are at equilibrium. Thus, the numbers of synonymous and nonsynonymous
substitutions both increase linearly with evolutionary time. It is shown
that, when the mutation rates are not equal, the estimate of synonymous
substitutions obtained by F. PERLER et aI's (Cell 1980) "Percent Corrected
Divergence" method increases nonlinearly, although the true number ofsynonymous substitutions increases linearly. It is, therefore, possible thatthe "saturation" of synonymous substitutions observed by PERLER et a/.
is due to the inefficiency of their method in detecting all synonymous substitutions. For details, see Genetics 105: 1011-1027.

RESEARCHES CARRIED OUT IN 1983

IX.

99

HUMAN GENETICS AND HUMAN CYTOGENETICS
Modern Medical Practice Versus Environmental Mutagens:
Their Possible Dysgenic Impact
Ei MATSUNAGA

We do not know how many mutations are being produced in humanpopulations by exposure to environmental mutagens. If these mutagens
caused a persistent rise in mutation rates, then ultimately there would be a
proportional increase in the frequency of a variety of genetic diseases, including those that are difficult to treat and that require life-long care ofaffected individuals. In contrast, modern medical practices are relaxing
selection pressure selectively with respect to disease, leading to a gradualincrease in the frequency of certain genetic and partly genetic diseases thatcan be effectively treated. The pattern of this increase would differ fromcondition to condition, depending upon the mode of inheritance and theextent to which selection is relaxed; except for some special cases, the anticipated increase would generally be slow. Additional economic burdens
on future society and families imposed by relaxed selection would mainly
involve expenditures for relatively inexpensive treatments, and not those for
expensive life-long care. Moreover, individuals treated successfully canbe expected to contribute productively to society. With education andcounseling for those who survive serious dominant and X-linked disorders,
and with the development of accurate, inexpensive prenatal diagnosis, thepresumed dysgenic effects of relaxed selection could be balanced. For
details, see Mutation Res. 114: 449-457, 1983.
Retinoblastoma and ABO Blood Groups
Ei MATSUNAGA and Kensei MINODA1)

Data for the ABO blood groups were obtained from records of 193
children with bilateral retinoblastoma and 346 with sporadic unilateral cases.
They were ascertained by a nationwide survey from 1975 to 1981, supported by the Children's Cancer Association of Japan. As controls, data
1)

Ophthalmologic Clinic, Tokyo M~tropolitanYoiku-In Hospital, Itabashi-ku, Tokyo,

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

by Fujita et al. (Jpn. J. Hum. Genet. 23: 63, 1978) were utilized. The
blood group distribution in either group of the patients was essentially thesame as the control. Furthermore, comparison was made of the patientswith early onset, who could be regarded as the most susceptible, with thegeneral population. The blood group distribution in those patients was
also close to the control. Thus, we conclude that the ABO blood groups
are not involved in the inherited host susceptibility to the development of
retinoblastoma. For details, see Hum. Genet. 63: 87, 1983.

Origin and Structure of a Minute Chromosome in Patientswith Congenital Malformations
Yasuo NAKAGOME and Yutaka NAKAHORI

It is not unusual to find a minute chromosome of unknown origin during
the course of a cytogenetic survey on patients with mental retardation
and/or congenital malformations. Because of its small size, the origin andstructure of it is usually not identifiable by the standard banding techniques
i.e., G, C or Q. Even the high resolution banding is often useless in theidentification of it.
In the present study, a total of 12 such patients were studied by some of
the new banding techniques in addition to the standard techniques. The
distamycin-DAPI (4'-6-diamidino-2-phenylindole) dual staining technique(Schweizer et ai. 1978) followed by the destaining and DAPI staining proved
itself to be the most useful of all, so far tested.
Table 1. Cases with a Minute Chromosome
47, XX, +i (15p)47, XY, +del (15) (q21.2q26.3)
47, XX, +psu die (15) (q12 or 13)
46, XX, -14, -15, +t(14qI5q), +psu die (15) (q12 or 13)
45, X/46, X, +psu die (13) (q13)/47, X, +psu die (13),+psu die (13)/47, X, del (Y) (qll), +psu die (13)46, X, i (Yp)46, X, del (Y) (q11)46, X, minute ring
47, XY, +psu die (14) or (21)45, X/46, X, +min
* mother of 2584.

2584,2587*
2667
2608,2493
2618
2646
2590
2611
2498
2630
2300

RESEARCHES CARRIED OUT IN 1983

101

In 6 cases, the minute chromosome was present as an extra chromosome(47, XX or XY plus a minute), in the 3 it replaced one of the normal complement, two cases showed mosaicism and a case had both Robertsoniantranslocation and, to our surprise, an extra psu die chromosome. In 9
cases, the origin and structure of the minute chromosome were determined(Table 1). In the two cases of i(l5p) and two cases involving Y, phenotypic abnormality of the propositus presumably had notheing to do with theminute chromosome. The former, in fact, included phenotypically normal
mother of the propositus. In other 5 cases, each minute chromosome involved euchromatic segment of an autosome and thus would have been
responsible for phenotypic abnormalities.
Cloning and Characterization of a Highly Y·specific
3.5 Kb Repeated Fragment
Yutaka NAKAHORI and Yasuo NAKAGOME

We attempted to obtain a highly V-specific DNA probe, presumablly
consisting of repeated sequences rather than a unique sequence. The former
is expected to be a very sensitive probe in the in situ hybridization-study inaddition to the standard Southern blot analysis.
The male specific repeated sequence was first reported by Cooke as a
3.4 Kb HaeIII fragment. Though there have been described multiple
families of repeated sequences on the human Y chromosome, this represents
the largest fraction of all and constitutes about 40 % of the total Y chromosome DNA.
It was presumed that the fragment has a 3.4 Kb interval for both HaeIII
sites and EcoRI sites. EcoRI-digested human male DNA was electrophoresed on polyacrylamide gel and a segment corresponding to the 3.4 Kbband (male-band) was cut out. DNA fragments were eluted from it andcloned in the EcoRI site of the plasmid pBR325. Clones were selected by
the colony hybridization technique using the nick translated male-band
DNA as a probe. Three independent clones proved to be highly malespecific in the Southern-blot analysis each being 3.5 Kb in length and gave
an identical restriction pattern. They were mainly constructed of the 5 bp
repeating unit (TTCCA) and its related sequences.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

Mitochondrial DNA Polymorphism in Japanese
Satoshi HORAI and Ei MATSUNAGA

The mitochondrial genome of human is a small (16.5 Kb), naked, circular,
supercoiled, maternally inherited DNA. Since Potter et al. (1975 Proc.
Natl. Acad Sci. USA 72: 4496) showed that a restriction enzyme can detectvariability in mammalian mitochondrial DNA (mtDNA) sequences, restriction enzyme analysis has become a powerful tool for detecting genetic
variation in humans (Brown and Goodman, 1979 Extrachromosonal DNA:
485; Brown, 1980 Proc. Natl. Acad. Sci. USA 77: 3605). In the present study
an attempt was made to obtain enough amount of purified mtDNA fromhuman placenta in order to examine mtDNA polymorphism with a large
number of restriction enzymes without using Southern blotting analys~s andend labeling method. A total of 120 placentas were obtained from Japaneseand closed circular mtDNA were prepared. Recoveries of closed circularform of mtDNA ranged from 200 to 200 f-lg per individual placenta. Preliminary studies with 8 restriction enzymes that recognize six base pairs
showed that 5 enzymes (£CaRl, HindIII, Pstl, HindI and XhoI) were polymorphic, while 3 enzymes (BamHI, KpnI and Xbal) were monomorphic.
Particularly, in the digestion with HindI, seven distinct cleavage patterns
were observed.
Differential Enzyme Activities in Human Esterase D Phenotypes
Satoshi HORAI and Ei MATSUNAGA

Recently, studies of families with hereditary retinoblastoma demonstrated
close linkage of the gene for this tumor with the ESD locus (Sparkes et al.
1983 Science 208: 971). Benedict et al. (1983 Science 219: 973) reported
a patient with retinoblastoma who had 50 % ESD activity in all normal cells
but no deletion of 13q14 at 550 band level, indicating that the smallest
constitutional chromosomal deletion within 13q14 is associated with susceptibility to retinoblastoma. This suggests that examination of ESD
activity is essential for the detection of a minute constitutional deletion or
inactivation of DNA segment involving the retinoblastoma locus. For thispurpose, however, it is necessary to know about the range of quantitative
variation in the enzyme activity among normal individuals. In the present
study, the ESD activity and phenotype were determined in blood from 200

RESEARCHES CARRIED OUT IN 1983

103

unrelated individuals. The distribution of different ESD phenotype is asfollows; ESD-l (39%), ESD2-1 (47%) and ESD-2 (14%). The gene frequencies of ESD*1 and ESD*2 are estimated at 0.625 and 0.375 respectively, which are not different from those of other Japanese populations inearlier reports. The mean enzyme activities of three phenotypes were
estimated at 276.7 (ESD-l), 216.6 (ESD2-1) and 171.5 (ESD-2) expressed as
10- 7 M methylumbelliferone producedjhjgHb, respectively. The activities
associated with a single ESD* 1 gene are estimated to be 60 % higher than
ESD*2.
Establishment of a Human Genomic Library with
MboI-Digested DNA
Yasuo NAKAGOME, Yutaka NAKAHORI and Jun KUSUDA

It is attempted to establish a human genomic library from which unique
DNA probes for RFLP (restriction fragment length polymorphism) studies,can be obtained. Lambda phage Charon 30 was chosen as it can take apassenger DNA fragment up to 19.10 kb long into its BamHI sites. DNA
was isolated from leukocytes of the senior author and partially digested
with a restriction enzyme Mbo!. Its recognition site is !GATe and a fragment generated with it can be inserted into a BamHI site with G!GATCC
sequence. After the insertion, BamHI sites may not be preserved, however,the passenger can be cut out with HpaI together with attached small piecesof lambda DNA on both ends (470 bp from the left arm and 770 bp fromthe right arm).
So far, about 300,000 recombinant phages have been obtained. Preparation of additional 700,000 clones are in the progress.

104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

X.

BEHA VIORAL GENETICS

The Behavioral Changes in Mice Continuously Reared under
Noise Conditions
Tohru FUJISHIMA

Mice continuously reared under noise conditions have been examined forthe behavioral changes in comparison with those reared under normal, or
non-noise conditions. The mice of the two groups were originally derived
from the same full sib families. It has been found previously that the micehaving been successively reared under noise conditions have come to showa chronic and non-recovering emotionality. It was also suggested in theprevious experiment that noise might have an influence on the mice at least
during the prenatal period. To examine the validity of this suggestion, a
portion of the pregnant mice of the non-noise group was moved to thenoise conditions and returned to the non-noise conditions at the first dayof birth. The result showed that the adult mice having experienced noise
conditions during their prenatal period expressed a high emotionality.
These experiments revealed that noise would have an influence upon themice during the prenatal period and arouse their non-recovering emotionality.

RESEARCHES CARRIED OUT IN 1983

XI.

105

ECOLOGICAL GENETICS

Observations on Wild and Cultivated Rices in Thailand
for Ecological-Genetic Study
Hiroko MORISHIMA, Yoshio SANO, Yo-Ichiro SATO
and Yoshiya SHIMAMOTO*

We stayed in Thailand from November 21 to December 24,1983, travellingin the North, North-East, South and Central Plain. The objective of thepresent tour was firstly to study population dynamics of wild rice, Oryza
perennis, and secondly to collect information and seed materials of Thai
native rice cultivars for understanding their phylogenetic status. For these
purposes, we made general survey of 131 wild and 92 cultivated rice sites,
and then permanent observation-sites were established for demographic
study. In each site, various items of habitat conditions (biotic and abiotic)
and characteristics of rice plants were recorded.
Wild rice: Both perennial and annual types were widely distributed allover the country. Their habitats, however, precisely segregated according
to water regimes conditioned by macro- and micro-topography. Introgression between wild and cultivated rices was prevailing particularly in the
direct-seeded fields. Hybrid derivatives were frequently observed in various
habitats such as in and at the fringe of deep-water rice fields, marshy waste
land, roadside ditch, forming hybrid swarms or uniform weedy populations
depending on their breeding system.
Demographic study: Seven permanent study-sites were chosen in thesuburb of Bangkok. Those sites were inhabited by perennial, annual andintermediate or weedy type of wild rice, and had contrasting environmental
conditions. In the permanent quadrats established in respective site,detailed records were taken in addition to general observation. Demography and population flux will be monitored in these sites.
Cultivated rice: Rice varieties grown in Thailand show a wide variationin adaptability to water condition (deepwater-lowland-upland) as well asin photoperiodic response. Further, it is known that Japonica type varieties
exist in the northern hilly areas. We found in the present trip that upland* Hokkaido University.

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

varieties grown in the South where upland fields are located less than 200300 m above sea level contained a high proportion of Japonica-like varieties.
This suggests that existence of Japonica type in tropics is not necessarily
due to its adaptability to highland as often argued, but due to adaptability
to upland condition.
Seed samples of 363 wild and 145 cultivated strains were collected for
future genetic study.
Invasion of Drosophila simulans into Inland Area and the
Effect of Winter Temperature on its Frequency
Takao K. WATANABE and Yutaka INOUE

A nine year survey of D. simulans population around the area of Mt.
Fuji revealed that the species gradually invaded inland from the Pacificcoast area since 1975. The frequencies of 12 autumn populations arepresented 'by the percent of D. simulans among D. melanogaster and D.
Table 1. Percents of D. simulans among D. melanogaster and D. simulans
in autumn populations around Mt. Fuji and average temperaturesof winter and summer in Mishima
Year
Bite
1975
Mishima
SusonoGotenba
Fujiyoshida
Otsuki
Katsunuma
Kofu
NirasakiKajikazawa
MinobuTomizawa
Fujinomiya
Mean
Winter*Summer*1976 1977

95
95
95
76
44
19
2
50
50
0
0
0
0.5 0.4
0
0
0
0
0
0
3
0
0
0
1
0
0
1
0
0
0
4
39
92
99
99
29.9 24.0 18.4
5.6 6.0 5.1
24.3 23.5 23.9

1978

1979

99
94
57
35
25
55
17
56
2
41
0.1
0.5
18
2
1
0
2
12
7
35
33
52
97
75
28.4 38.0
6.1
7.2
25.7 24.7

1980 1981
92
43
56
50
70
7
0
0
8
62
83
78
45.8
6.6
23.8

1982 1983

93
85
72
46
15
21
0
14
1
19
0
0.5
1
4
0.2 3
1
9
7
17
11
51
97
82
22.7 31.6
4.9 5.6
24.2 23.1

96
77
75
33
26
0
0.6
0.5
3
13
22
90
36.3
6.3
23.9

* Average temperatures CC) of winter (Dec.-Feb.) and summer (June.-Aug) in Mishima.

RESEARCHES CARRIED OUT IN 1983

107

simulans (Table 1).

D. simulans was not found in the inland area in 1975.
But it was found in every collection site in 1979. Thereafter the species
seemed to be colonized there with some temporal fluctuations of its size.
However, a remarkable reduction of the D. simulans frequency was observedin 1981 in every collection site. In order to correlate this phenomenon
with some environmental factors, we compared the summer and wintertemperatures preceding collection of flies with the frequency of D. simulans.
The summer temperature did not correlate with the mean frequency of D.
simulans but the winter temperature did significantly correlate with ti. Cold
winters decreased the size of D. simulans population relatively.

Biochemical Phylogeny of the Drosophila montium Subgroup
Seido OHNISHI and Takao K. WATANABE

Phylogenetic relationships among twenty nine species in the D. montium
subgroup were investigated by two electrophoretic techniques (2DE and
SGE). Based on data of genetic distances estimated by protein differences
detected by 2DE, a dendrogram was constructed (Fig. 1). In addition,
species specific allozymes (diagnostic alleles) were surveyed by SGE inorder to find a method to distinguish species easily. It was found thatsix enzymes (Aldox, G6pdh, Men, 6Pgdh, Idh and Est-6) are helpful forsystematics of this subgroup. For instance, if we run two enzymes of
Aldox and G6pdh in a starch gel, we can distinguish 26 of 29 species. And
if we add two enzymes (Men and Est-6) we can classify all 29 species by
the four enzyme systems.

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34
D.pennae
D. bockiD. kikkawai

2DE

D.leontia
D.liniD.lini-likeD.barbarae
D.jambulina
D. punjabiensis-Iike
D. punjabiensis
D. quadraria
D. triauraria

D. auraria
D. biauraria
D. new auraria

D. rufa
D. mayri
D. bicornuta
D. truncata
D. burlai
D. seguyi
D. bi rchii
D.serrata

D. baimaii
D.khaoyana
D. khaoyana-lik~
D. lacteicornis
D. parvula
D. rhopaloa-like

0.4

0.2

I

o

Genetic Distance (D)Fig. 1.

A dendrogram of 29 species of the D. montium subgroup.

RESEARCHES CARRIED OUT IN 1983

XII.

109

APPLIED GENETICS

Correlated Selection Responses in Characters andAJlozymes of Rice
Hiroko MORISHIMA

Three hybrid populations of rice, one between wild-annual and wildperennial type (Cross I) and the other two between cultivated and wild
type (Cross II, III), were propagated in bulk. In F s , the seeds were collected by two different methods; one from naturally shed seeds on the groundand the other from the seeds remaining on the panicles. The same procedure was repeated in each group until Fa. This resulted in selection forearly (shed-seed group) and late (non-shed seed group) flowering groups
in Cross I, while selection for shedding and non-shedding groups resulted
in Cross II and III. In F s and Fa, various metric characters, coloration ofplant organs and allelic frequencies at two isozyme loci, Pox-l and Acp-l,
were examined.
In F s , we failed to find out significant correlations between charactersand allozymes, except for a loose association of flowering time with Pox-l
in Cross I. In Fa, some correlated responses to selection and associations
between characters and allozymes were detected as follows. In Cross I(peren. X ann.), the early flowering group had shorter awns, greater valuesof seed productivity components, higher reproductive allocation, lower
regenerating ability, shorter anthers than the late flowering group. Thispattern of character association is consistent with that found between theperennial and annual types of wild rice except for few traits. In Crosses
II and III (cult. X wild), shedding group consistently showed longer awns
and more pronounced seed dormancy than the non-shedding group. Further, plants with white grain and those with straw-color hull increased during
F s to Fa in non-shedding group (characteristics of cultivated type), but
tended to decrease in shedding group.
The parents of each cross carried different alleles at Pox-l and Acp-l.
In Crosses I and III in which 2A and 4A alleles at Pox-l segregated, thefrequency of 2A generally increased during F s to Fa except in the lateselected group in Cross I. The plants homozygous for 2Aj2A and 4Aj4A

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

were compared in each cross regarding various character measurements.
Generally, 2AJ2A plants had greater values of seed productivity components,
higher reproductive allocation, shorter anthers, less pronounced seed dormancy, and in Cross I they flowered earlier than 4AJ4A plants. This isconsistent with the trend of character association found between the wild
perennial and annual types or between the wild and cultivated types. At
the Acp-ilocus, however, no such consistent trend was detected.
From these observations, the following points can be suggested: (1)There could be a key factor governing seed productivity which is locatedon a chromosome segment marked by Pox-i. (2) One of major genes controlling flowering time differeing between the wild perennial and annualtypes is linked also with Pox-i. (3) Seed shedding is partly correlated withawn length and seed dormancy, developmentally or by linkage, but seems
to be independent of seed productivity factor. A gene or genes controlling
seed shedding is linked with RcJrc locus for grain color as well as with oneof the three complementary genes for hull color.
Non-random association found among many adaptive traits and gene locican be attributed to various factors. The present experiment suggests
existence of two independent adaptive gene blocks, noe loading linked genes
for flowering time and seed productivity, and the other loading linked
genes for seed shedding, and grain and hull color. Selection and fixationof the combination of these two blocks could have brought about effective
and constant seed productivity which meets the basic requirement for thecultivated type.

The Genetic Basis of Hybrid Chlorosis Found in a Cross
between Two Japanese Rice CuItivars
Yo-Ichiro SATO and Seiji MATSUURA*

We incidentally found a case of hybrid chlorosis in the F 2 population ofa cross between two Japanese native rice cultivars, J-147 and J-321. Its
first symptom was a change in color of the third or fourth leaf-blade toyellowish. The chlorotic plants died within 30 days after germination,
hence no progeny seed was obtainable from them.
The F 2 population segregated into 849 normal and 63 chlorotic plants,*

Kochi University.

RESEARCHES CARRIED OUT IN 1983

111

giving a good fitness to the 15: 1 ratio. Fa strains showing 1: 0, 3: 1 and15: 1 ratios were 72, 35 and 37, respectively. The Fa ratio fitted 7: 4: 4,
which was expected by assuming two independent recessive genes. The
data thus indicated that there was a set of duplicate genes independent ofeach other whose double-recessive combination causes chlorosis. They
were symboled eh-I-a and eh-I-b, tentatively.
Sato and Hayashi (1983) reported the presence of a set of complementarylethal genes, L-2-a and L-2-b causing F 1 weakness, and that the distributionof L-2-a was suggestive of the phylogenetic relationship among varietalgroups and the mode of dissemination of rice varieties. The examinationof distribution of the hybrid chlorosis genes is under way, and it may also
be useful for phylogenetic study.
Chromosomal Location of Pgi-2 Locus in Rice
Reiko SANO and Hiroko MORISHIMA

Forty isozyme loci of 14 enzyme species were so far proposed in wild
and cultivated rice (Second 1982), but formal genic analysis and linkage
studies were carried out for very limited loci. In the present study, chromosomal mapping of the isozyme loci differing in allelic frequencies between
the Indica and Japonica types of rice was attempted. Linkage relationshipsof six isozyme loci (Cat-I, Aep-I, Pox-2, Pgi-I, Pgi-2, Est-2) and eightmarker genes covering 5 chromosomes (Re, WX, dl> g, gl, 19, la ph) were
examined using the F 2 and B1F1 plants derived from several crosses by starchgel electrophoresis. Pgi-2 was found to be located on chromosome 6
as it was linked with wx and Est-2 with recombination values of 35 % and13%, respectively (wx-Est-2-Pgi-2). Linkage between Aep-I and Pox-2
which was already reported was reconfirmed in the present study. Fourloci other than Pgi-2 and Est-2 were found to be independent of the all
marker genes used. Our previous statement that Pgi-2 and wx was independent (Ann. Rep. 31: 117-118) was not correct.
Differential Regulation of waxy Gene Expressionin Rice Endosperm
Yoshio SANO

In order to examine the effects of different alleles on the gene expression

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

at the waxy locus, the Wx gene product wihch controls the synthesis ofamylose was isolated from endosperm starch of rice plants and analysedby electrophoretic techniques. The major protein bound to starch granules
was absent in most of waxy strains and increased with the number of Wx
alleles in triploid endosperms, suggesting that the major protein is theWx gene product. In addition to wx alleles which result in the absence ordrastic reduction of the Wx gene product and amylose, differentiation ofWx alleles seemed to have occurred among nonwaxy rice strains. At least
two Wx alleles with different efficiencies in the production of the major
protein as well as amylose were detected. This work was published in
Theor. Appl. Genet. 68, 467-374 (1984)
Temperature Effects on the Wx Protein Level and Amylose
Content in Rice Endosperm
Yoshio SANO, Masahiko MAEKAWA and Harumi KIKUCHI

It is well known that the waxy locus controls the synthesis of amylose
as endosperm starch of wx homozygotes almost lacks amylose. In maize,
the Wx gene specifies a starch-bound glycosyltransferase which is responsible for the production of amylose in the endosperm and pollen. On theother hand, amylose content in rice endosperm is considered to be one of
the major determinants of eating qualities. Since lower temperatureduring grain development increases the amylose content in Japanese cultivars, great efforts for decreasing amylose content have been made to improve the grain quality of local rice cultivars in the northern part of Japan.
Recently, it was reported in rice that one, two and three doses of Wx allelesin the endosperm showed a progressive increase in the level of the gene product called Wx protein as well as in amylose content and that the two allelesof wx a and Wx b control not only the quantitative level of Wx protein but
also amylose content. Thus, amylose content in rice endosperm seems tobe partly controlled by the Wx protein level. The objective of this study
was to investigate whether low amylose content observed in a low-amylose
mutant and in plants grown under low temperature is due to the reduced
level of Wx protein or not. Two rice strains, Shiokari and its low-amylose
mutant, were compared with regard to the levels of Wx protein and amylose
under different temperature regemes (Table 1). The mutant showed the
reduced level of Wx protein bound to starch granules compared to Shiokari,

RESEARCHES CARRIED OUT IN 1983

113

Table 1. Amylose content and the relative amount of Wx protein boundto starch granules in the two rice strains, Shiokari and SM-l
Location
Paddy field
Glasshouse

StrainShiokari
SM-l
Shiokari
SM-lHeadingdata

Amylosecontent

August 18
August 21
July 31
July 26

25.4
17.8
16.0
9.5

(%)

Relative
Mean temperature
amount offor 20 days
Wx protein after heading (0C)
100
52
43
18

21.1
21.8
26.8
25.5

showing that the gene carried by the mutant regulates the Wx protein level
as well as amylose content. In addition, lower temperature during grain
development increased the amount of Wx protein as well as amylose,
indicating that the genic expression at the wx locus is influenced by temperature. The present study suggests that the Wx protein level is controlled
by a trans-acting regulatory gene as well as temperature and that amylose
content decreases as the Wx protein level decreases, so far examined.
(J. Hered. in press)
Genetic Variation of Nitrogen Fixing Activity in the Progeny
Population of Rice Hybrid
Shin-ya IYAMA and Theo J. L. van HINTUM

The cross between two rice strains of different nitrogen fixing activity was
made in order to investigate the hereditary nature of the character in aprogeny population. From F 2 to F 5 the population was maintained by
self-pollination with single seed decent method. About 100 F 6 lines were
derived from the F 5 population in 1982. Ten plants per line were grown individually in a small plastic pot with 180 g of soil and their nitrogen fixing activity was measured by acetylene reduction method with gas-chromatography.
Acetylene reducing activity (ARA) was assayed one day after the heading ofeach plant. ARA of the lines was expressed by C 2 H 4 produced (nmol)/hour/g dry root. Wide variation among lines was found, ranging from 99
to 732 nmol, the average being 326 nmol. The variation among lines was
statistically highly significant, and some lines exceeded the parental range
in both directions.
In 1983, selfed F 7 progeny lines obtained from each of the F 6 lines weregrown and they were examined for estimating heritability of the nitrogen

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34
Table 1. Realized heritability of ARA of rice estimated from theselection for high and low ARA in the F o and the genetic
gain in the F 7 of T65 x C5444
Generation

+)

***

No. ofselectedlines

Means of
High
Low
(H)
(L)

II - L

10
20

551.6
483.3

185.2
210.6

368.4
272.7

10
20

1166.81076.1

611.8
627.6

555.0***(1.71)
448.0***(1.38)

Heritability(3 .43)+(2.54)
0.50
0.54Standardized values in the parentheses.
Significantly different from zero at the 0.1 % level.

fixing activity. As observed in the F o, wide variation among F 7 lines wasfound but the activity in 1983 was generally higher compared with that ofthe previous year. The variation ranged from 253 to 1584 nmol, the average
being 867 nmol. Ten or 20 lines each were selected from the F o population in both high and low directions, respectively, and means of the selected
F 7 progeny groups were compared. I-test showed that the difference
between the two groups selected to high and low directions was highly
significant, indicating that the selection was effective. Heritability value
was estimated by the ratio of the realized gain in the F 7 to the selection
differential in the F o• Since the seasonal variation of ARA was evident,
those measurements were standardized by the standard deviation of the
F o and F 7 populations, respectively, before being used for calculation.
Realized heritability or repeatability was estimated as around 0.5 (Table 1),
indicating that the selection based on the line means from the progeny
population is considerably effective. This estimation was in good accord
with the correlation coefficient between the F o in 1982 and the F 7 in 1983
calculated based on the whole lines used (r=0.47).
It was concluded from the above results that (1) ARA in the rhizosphereof rice plant is heritable and controlled by rice genotype. (2) Genes controlling ARA in the parents segregate among progenies through crossing of
two different genotypes and some progeny genotypes may exceed parental
performance through recombination. (3) One may improve ARA of riceby ordinary breeding procedure of hybridization and selection.

BOOKS AND PAPERS PUBLISHED IN 1983 BY STAFF MEMBERS

115

BOOKS AND PAPERS PUBLISHED IN 1983
BY STAFF MEMBERS
AOKI, K. 1983: A quantitative genetic model of reciprocal altruism: a condition for kin
or group selection to prevail. Proc. Natl. Acad. Sic. USA 80: 4065-4068.
BAGCHI, S.* and S. IYAMA 1983: Radiation induced developmental instability in
Arabidopsis thaliana. Theor. Appl. Genet. 65: 85-92.
BAVERSTOCK, P. R.*, M. ADAMS*, L. R. MAXSON* and T. H. YOSIDA 1983: Genetic
differentiation between cytotypes of the black rat, Rattus rattus. Genetics 105: 969983.
BIRKY, Jr., C. W.*, T. MARUYAMA, P. A. FUERST,* 1983: An approach to populationand evolutionary genetic theory of genes in mitochondria and chloroplasts. Genetics
103: 513-527.
ENDO, T. and H. MORISHIMA 1983: Rice. In "Isozymes in Plant Genetics and Breeding,Part B" (Ed. by S. D. Tanksley and T. J. Orton), p. 129-146, Elsevier (Amsterdam).
FUJII, T. 1983: Future prospects of mutation breeding with genetic engineering. Gamma
Field Symposia 20: 41-56.
FUJII, T., M. SHIZAKI*, H. FUJIKI* and T. SUGIMURA* 1983: Effect of TPA on the mutagenicity of caffeine in the soybean mutation test. Mutation Research 110: 263-269.
FUJII, T. and T. INOUE 1983: Mutagenic effects of a pesticide (Ekatin) in the soybean
test system. Env. Exp. Botany 23: 97-101.
GOJOBORI, T. 1983: Codon substitution in evolution and the "saturation" of synonymouschanges. Genetics 105: 1011-1027.
GOJOBORI, T. and M. NEI* 1983: Concerted evolution of immunoglobulin V H genes.
(Abstract) Genetics 104: S28.
GOJOBORI, T. see under LI, W.-H.
HASHIMOTO, Y., A. SUZUKI*, T. YAMAKAWA*, N. MIYASHITA* and K. MORIWAKI 1983:
Expression of GMI and GDla in mouse liver is linked to the H-2 complex on chromosome 17. J. Biochem. 94: 2043-2048.
HIROTA, Y. see under NAKAMURA, M.
HIROTA, Y. see under PARQUET, C.
HIROTA, Y. see under RICHET, E.
HIROTA, Y. see under TABATA, T.
IMAI, H. T. 1983: Quantitative analysis of karyotype alteration and species differentiation in mammals. Evolution 37: 1154-1161.
IMAI, H. T., T. MARUYAMA and R. H. CROZIER 1983: Rates of mammalian karyotype
evolution by the karyograph method. Amer. Nat. 121: 477-488.
IMAI, H. T. see under MATSUDA, M.
IMAI, H. T. see under MATSUDA, Y.
INOUE, T. see under FUJII, T.

116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34!NOUCHI, Y.*, T. OKUNO*, K. OISHI* and T. K. WATANABE 1983: Studies on the sexspecific lethals of Drosophila melanogaster. VI. An autosomal, recessive, nonmaternal effect female-specific lethal mutant. Jpn. J. Genet. 58: 525-530.
!YAMA, S., Y. SANO and T. FUJII 1983: Diallele analysis of nitrogen fixation in the rhizosphere of rice. Plant Science Letters, 30: 129-135.
!YAMA, S. 1983: Simulation experiment on the effect of mass selection in a small population. Animal Improvement Research, Proc. 4th Internat. SABRAO Congr. 177-181.
!YAMA, S. see under BAGCHI, S.
!YAMA, S. see under SAKAI, K. I.
!YAMA, S. see under TANIGUCHI, N.
KADA, T. 1983: Desmutagens: An overview. In "Carcinogens and Mutagens in theEnvironment" (Ed. by H. F. Stich). Vol. II: 63-83, CRC Press Inc.
KADA, T., K. AOKI* and T. SUGIMURA* 1983: Isolation of streptomycin-dependent
strains from Salmonella typhimurium TA98 and TAloo and their use in mutagenicity
tests. Environmental Mutagenesis 5: 9-15.
KADA, T., K. SHIMOI* and Y. SADAIE 1983: Metabolic cooperation between Bacillus
subtilis and Escherichia coli. Agric. BioI. Chern. 47: 2145-2148.
KADA, T. see under MITA, I.
KADA, T. see under OHTA, T.
KADA, T. see under SADAIE, Y.
KADA, T. see under SHIBUYA, H.
KANDA, N.* and K. MORIWAKI 1983: In vivo sister chromatid exchange analysis of amouse plasmacytoma cell line NP-38. Exp. Cell Res. 146: 417-421.
KIMURA, M. 1983: The Neutral Theory of Molecular Evolution. 367 p. Cambridge
University Press, (Cambridge).
KIMURA, M. 1983: The neutral theory of molecular evolution. !n "Evolution of Genes
and Proteins", (Ed. by M. Nei & R.K. Koehn), Sinauer. (Sunderland, Mass.), pp.
208-233.
KIMURA, M. 1983: Diffusion model of intergroup selection, with special reference toevolution of an altruistic character. Proc. Natl. Acad. Sci. USA 80: 6317-6321.
KIMURA, M. 1983: Rare variant alleles in the light of the neutral theory. Molecular
Biology and Evolution 1: 84-93.
KIMURA, M. and N. TAKAHATA 1983: Selective constraint in protein polymorphism:
Study of the effectively neutral mutation model by using an improved pseudosampling
method. Proc. Natl. Acad. Scie. USA 80: 1048-1052.
KOMINAMI, R.*, M. MURAMATSU* and K. MORIWAKI 1983: A mouse type 2 Alu sequence(M2) is mobile in the genome. Nature 301: 87-89.
KOMURA, H.*, H. MINAKATA*, H. MOCHIZUKI* and T. KADA 1983: Antimutagenicity
of human placenta extract: Chemical studies. In "Carcinogens and Mutagens in
the Environment" (Ed. by H. F. Stich), Vol. II: 85-99, CRC Press, Inc.
KURODA, Y. 1983. Mutations induced by environmental mutagens in human cells. (inJapanese). In "Environment and Human Body, II" (Eds., I. Chuma, S. Kondo
and H. Takebe), 87-104, Tokyo Univ. Press (Tokyo).
KURODA, Y. 1983: Comparison and evaluation of the strength of environmental mu-

BOOKS AND PAPERS PUBLISHED IN 1983 BY STAFF MEMBERS

117

tagens. (in Japanese). Environmental Information Science 12: 39-49.
KURODA, Y. 1983: Characteristics of mutagen tests in cultured human cells. (in Japanese).
Environmental Mutagen Research Communications 5: 6-11.
KURODA, Y. see under MIKAMI, K.
KORO, T. *, M. YOKOYAMA·, T. TAGUCHI·, Y. KITAMURA· and K. TUTIKAWA 1983: Effectof the SI' mutant allele on the production of tissue mast cells in mice. J. Hered.
74: 375-377.
LI, W.-H. * and T. GOJOBORI 1983: Rapid evolution of goat and sheep globin genes
following gene duplication. Mol. BioI. Evol. 1: 94-108.
MARUYAMA, 1.*, A. YAMAMOTO*, T. MARUYAMA and Y. HIROTA 1983: The fine architecture and function of the gene coding for PBP-3 of Escherichia coli. In "The Target
of Penicillin" (Ed. by Hakenbeck, Holtje, and Labischinski), 393-402, Walter deGruyter & Co. (Berlin, New York).
MARUYAMA, T. 1983: Stochastic theory of population genetics. Bull. Math. BioI. 45:
521-524.
MARUYAMA, T. and P. A. FUERST 1983: Analysis of the age of genes and the first arrival
times in a finite population. Genetics 105: 1041-1059.
MARUYAMA, T. see under BIRKY, Jr., C. W.
.MARUYAMA, T. see under NEI, M.
MATSUBARA, T.* and Y. NAKAGOME 1983: High-resolution banding by treating cellswith acridine orange before fixation. Cctogenet. Cell Genet. 35: 148-151.
MATSUDA, M.*, H. T. IMAI and Y. TOBARI· 1983: Cytogenetic analysis of recombination in males of Drosophila ananassae. Chromosoma 88: 286-292.
MATSUDA, Y.*, H. T. IMAI, K. MORIWAKI and K. KONDO* 1983: Modes of inheritanceof X-V dissociation in inter-subspecies hybrids between BALB/c mice and Mus
musclus molossinus. Cytogenet. Cell Genet. 35: 209-215.
MATSUNAGA, E. 1983: Perspectives in mutation epidemiology 5. Modern medical
practice versus environmental mutagens: their possible dysgenic impact. Mutation
Res. 114: 449-457.
MATSUNAGA, E. and T. MINODA* 1983: Retinoblastoma and ABO blood groups. Hum.
Genet. 65: 87.
MATSUNAGA, E. 1983: Retinoblastoma: A model for the study of carcinogenesis in
humans. Jpn. J. Hum. Genet. 28: 57-71 (in Japanese with English summary).
MATSUNAGA, E. 1983: Genetics of retinoblastoma. In: Ganka MOOK 19, "Tumorsof the Eye" (Eds. S. Mishima, I. Tsukahara, Y. Uemura and K. Minoda), 1-12 (inJapanese), Kanehara, Tokyo.
MATSUNAGA, E. 1983: Genetic background of multiple primary cancers, with specialreference to retinoblastoma. In "Basic and Clinical Aspects of Multiple Primary
Cancers" (Eds. K. Suemasu and S. Sato), 59-79 (in Japanese), Life Science Centre,
Tokyo.
MIKAMI, K.* and Y. KURODA 1983: Enhancement of aggregation of Chinese hamster
V79 cells in rotation culture by 12-O-tetradecanoyphorbol-13-acetate. Cell Structure
Function, 8: 193-198.
MIURA, K. I. see under YAZAKI, K.

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34
MIURA, K. I. see under MORINAGA, T.
MITA, I.., Y. SADAIE and T. KADA 1983: Deoxyribonucleic acid repair in competentcells of Bacillus subtilis. J. Bacteriol. 155: 933-936.
MIYAMURA, T.·, H. JIKUYA· and E. SOEDA 1983: The genomic structure of human papovavirus JCV; Nucleotide sequence containing replication origin and entire coding
region of small T antigen. J. Virol. 45: 73-79.
MORINAGA, T.·, M. IKEGAMI· and K. I. MIURA 1983: Infectivity of the cloned DNAs
from multiple genome components of bean golden mosaic virus. Proc. Japan Acad.
59B: 363-366.
MORISIDMA, H. and Y. SANO 1983: Neighbor effects observed in inter- and intra-specificmixtures of rice: A review and presentation of new data. In "Frontiers of Researchin Agriculture" (Ed. by S. K. Roy), p. 110-127, Ind. Stat. Inst., Culcutta.
MORIWAKI, K. see under HASIDMOTO, Y.
MORIWAKI, K. see under KANDA, N.
MORIWAKI, K. see under KOMINAMI, R.
MORIWAKI, K. see under MATSUDA, Y.
MORIWAKI, K. see under SAKAI, S.
MORIWAKI, K. see under SAKAIZUMI, M.
MUROFUSID, M.·, S. NISHIKAWA· and T. H. YOSIDA 1983: Cytogenetical studies on fishes.
V. Multiple sex chromosome mechanism (XX-Y) found in two dragonet fishes. Proc.
Japan Acad. 59(B): 58-61.
MUROFUSHI, M.·, S. NISHIKAWA· and T. H. YOSIDA 1983: Cytogenetical tudsies on
fishes. IV. Karyotypes of six species in the sparoid fishes. Japan. Jour. Genetics
58: 361-367.
MUROFUSHI, M.· and T. H. YOSIDA 1983: Cytogenetical studies on fishes, VII. XX-Y sex
chromosome mechanism newly found in the snake eel, Muraenichthys gymnotus(Anguilliform, Pieces). Proc. Japan Acad. 60(B): 21-23.
MUROFUSHI, M.·, S. NISHIKAWA· and T. H. YOSIDA 1983: Cytological studies on fishes.
VII. Karyological comparison of two species in the sandperch fishes. La Chromosome (11)-35: 1122-1125.
MUROFUSHI, M.·, S. NISHIKAWA and T. H. YOSIDA 1983: Cytogenetical studies on
fishes, VI. Karyological comparison of four species in the dragonet fish. La Chromosome (11)-35 (in press).
MUROFUSHI, M.·, S. NISHIKAWA and T. H. YOSIDA 1983: Cytogenetical studies on fishes,
IX. Karyological comparison of two species of snipefishes. Jap. Jour. Genet. 59:
155-158.
NAKAGOME, Y., T. MATSUBARA· and H. FUJITA· 1983: Distribution of break points instructural rearrangements. Amer. J. Hun. Genet. 35: 288-300.
NAKAMURA, M.·, I. MARUYAMA·, M. SOMA·, J. KATO·, H. SUZUKI· and Y. HIROTA 1983:
On the process of cellular division in Escherichia coli: nucleotide sequence of thegene for penicillin-binding protein 3. Mol. Gen. Genet. 191: 1-9.
NEI, M.· and T. MARUYAMA 1983: Models of evolution of reproductive isolation.
Genetics 103: 557-579.
NISHIMURA, Y. see under RICHET, E.

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OHNISHI, S.*, M. KAWANISHI* and T. K. WATANABE 1983: Biochemical phylogenies ofDrosophila: Protein differences detected by two-dimensional electrophoresis. Genetica
61: 55-63.
OHNISHI, S.*, K. KIM* and T. K. WATANABE 1983: Biochemical phylogeny of the Drosophila montium species subgroup. Jpn. J. Genet. 58: 141-151.
OHTA, T. 1983: Multigene families and their implications for evolutionary theory. In"Synergetics, from Microscopic to Macroscopic", (Ed. by E. Frehland), pp. 133-139.
OHTA, T. 1983: Theoretical study on the accumulation of selfish DNA. Genet. Res.
Camb. 41: 1-15.
OHTA, T. 1983: Time until fixation of a mutant belonging to a multigene family. Genet.
Res. Camb. 41: 47-55.
OHTA, T. 1983: On the evolution of multigene families. Theor. Pop. BioI. 23: 216-240.
OHTA, T. and G. A. DOVER 1983: Population genetics of multigene families that are dispersed into two or more chromosomes. Proc. Natl. Acad. Sci. USA 80: 4079--4083.
OHTA, T.,* K. WATANABE*, M. MORIYA*, Y. SHIRASU* and T. KADA 1983: Analysis of
the antimutagenic effect of cinnamaldehyde on chemically induced mutagenesis in
Escherichia coli. Mol. Gen. Genet 192: 309-315.
OHTA, T.,* K. WATANABE*, M. MORIYA*, Y. SHIRASU* and T. KADA 1983: Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli. Mutation Research 107: 219-227.
OHTA, T.,* K. WATANABE,* M. MORIYA*, Y. SHIRASU* and T. KADA 1983: Anti-mutagenic effects of coumarin and umbelliferone on mutagenesis induced by 4-nitroquinoline N-oxide or UV irradiation in E. coli. Mutation Research 117: 135-138.
PARQUET, c.,* B. FLoURET*, M. LEDUC*, Y. HIROTA and J. VAN HEIJENOORT* 1983:
N-acetylmuramoyl-L-alanine amidase of Escherichia coli K12: possible physiological
functions. Eur. J. Biochem. 133: 371-377.
RICHET, E.,* Y. NISHIMURA, Y. HIROTA and M. KOHIYAMA* 1983: Escherichia coli
uvrD mutants with thermosensitive DNA-dependent adenosine triphosphatase I(helicase 11). Mol. Gen. Genet. 192: 378-385.
SADAIE, Y. and T. KADA 1983: Formation of competent Bacillus subtilis. J. Bacteriol.
153: 813-821.
SADAIE, Y. and T. KADA 1983: Effect of septum-initiation mutations on sporulationand competent cell formation in Bacillus subtilis. Mol. Gen. Genet. 190: 176-178.
SADAIE Y. see under KADA. T.
SADAIE, Y. see under MITA, I.
SAKAI, K. I., S. IYAMA, S. HAYASHI,* S. IWAGAMI*, A. MIYAZAKI* and K. TOMITA* 1983:
Genetic differentiation in natural stands of Cryptomeria japonica. Crop Improvement Research, Proc. 4th Internat. SABRAO Congr. 489-501.
SAKAI, S.,* K. MORIWAKI*, S. MIGITA*, K. SUDO*, K. SUZUKI*, D. Y. Lu*, C. WANG*
and M. TAKAHASHI* 1983: Structural polymorphism of murine factor B controlled
by a locus closely linked to the H-2 complex and demonstration of multiple alleles.
Immunogenetics 18: 117-124.
SAKAIZUMI, M.,* K. MORIWAKI and N. EGAMI* 1983: Allozymic variation and regional
differentiation in wild populations of the fish Oryzias latipes. Copeia 1983: 311-318.

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34
SANO, Y. 1983: A new gene controlling sterility in F 1 hybrids of two cultivated rice species.
Its association with photoperiod sensitivity. J. Hered. 74: 435-439.
SATO, Y. and K. HAYASHI* 1983: Distribution of the complementary genes causing
F 1 weakness in the common rice and its wild relatives. I. L-2-a gene in Asian native
cultivars. Jpn. J. Genet. 58: 411-418.
SHIBUYA, H.,* Y. NISHI, T. KADA and N. INUI* 1983: Mutagenicity of cyclophosphamide in mammalian cells treated in vitro and in utero. Jpn. J. Genet. 58: 23-31.
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 (Ber!.) 87: 1-8.
SHIRAISHI, Y.* and T. H. YOSIDA 1983: Analysis by the three-way differentiation of
Bloom syndrome sister chromatid exchanges in bromodeoxyuridine-substituted
chromosomes. Proc. Japan. Acad. 58(B): 227-230.
SHIRAISHI, Y.,* T. H. YOSIDA and A. A. SANDBERG 1983: Analyses of bromodeoxyuridine-associated sister chromatid exchanges (SCE) in bloom syndrome based on cellfusion: Single and twin SCEs in endoreduplication. Proc. Nat!. Acad. Sci. USA
80: 4369-4373.
SOEDA, E. see under MIYAMURA, T.
SOEDA, E. see under WADA, A.
SUGIYAMA, K.,* S. NAGASE and T. H. YOSIDA 1983: Chromosomal localization of the
rat albumin gene in normal and analbuminemic rats determined by in situ hydridization. Jpn. J. Genet. (in press).
SUGIYAMA, T. 1983: Isolating hydra mutants by sexual inbreeding. In "Hydra: Research methods". (Ed. by H. M. Lenhoff). Plenum, (New York).
SUGIYAMA, T. see under TAKANO, J.
SUZUKI, H.* and T. H. YOSIDA 1983: Frequency of sister-chromatid exchanges depending on the amount of 5-bromodeoxyuridine incorporated into parental DNA. Mut.
Res. 111: 277-282.
SUZUKI, H.,* T. KANEHISA* and T. H. YOSIDA 1983: Chromosome location of 18S and
28S ribosomal RNA genes in the Amami black rats by in situ hybridization. Proc.
Japan Acad. 59(B): 215-218.
SUZUKI, H.* and T. H. YOSIDA 1983: Relation between the sister chromatid exchanges
and metabolic perturbation of deoxyribonucleotide synthesis. Jpn. J. Genet. 58:
369-372.
SUZUKI, H. * and T. H. YOSIDA 1983: Association of sister chromatid exchanges and chromatid breaks in Chinese hamster D-6 and lung cultured cells treated with hydroxyurea
and some other chemicals. Cytologia (in press).
TABATA, T.,* A. OKA*, K. SUGIMOTO*, M. TAKANAMI*, S. YASUDA and Y. HIROTA 1983:
The 245 base-pair oriC sequence of the E. coil chromosome directs bidirectional
replication at an adjacent region. Nucleic Acids Research, 11: 2617-2626.
TAKAHATA, N. 1983: Linkage disequilibrium of extranuclear genes under neutral mutations and random genetic drift. Theor. Pop. Bio!. 24: 1-21.
TAKAHATA, N. 1983: Population genetics of extranuclear genomes under the neutral
mutation hypothesis. Genet. Res. 42: 235-255.

BOOKS AND PAPERS PUBLISHED IN 1983 BY STAFF MEMBERS

121

TAKAHATA, N. and M. SLATKIN* 1983: Evolutionary dynamics of extranuclear genes.
Genet. Res. 42: 257-265.
TAKAHATA, N. 1983: Gene identity and genetic differentiation of populations in thefinite island mode!. Genetics 104: 497-512.
TAKAHATA, N. see under KIMURA, M.
TAKANAMI, M.,* T. TABATA,* A. OKA,* K. SUGIMOTO,· H. SASAKI" S. YASUDA and Y.
HIROTA 1983: The Escherichia coli origin of replication: essential structure forbidirectional replication. In "Mechanisms of Replication and Recombination", 257273, Alan R. Liss Inc. (New York).
TAKANO, J.* and T. SUGIYAMA 1983: Genetic analysis of developmental mechanisms in
hydra. VIII. Head-activation and head-inhibition potentials of a slow-buddingstrain (L4). J. Embryo!. expo Morph. 78, 141-168.
TANIGUCHI, N.,* K. SUMANTADINATA* and S. IYAMA 1983: Genetic change in the firstand second generations of hatchery stock of black seabream. Aquaculture, 35:
309-320.
TUTIKAWA, K. and K. TUTIKAWA* 1983: Developmental disorders of chromosomallyunbalanced zygotes in mice. Teratology 23: 344.
TUTIKAWA, K. see under KORO, T.
WADA, A.,. M. YAMAMOTO* and E. SOEDA 1983: Automatic DNA sequencer: Computer-programmed microchemical manipulator for Maxam-Gilbert sequencing
method. Rev. Sci. Instrum. 54: 1569-1572.
WADA, M. and T. H. YOSIDA 1983: A simple and applicable chromosome technique forsex identification of the bird. Proc. Japan Acad. 59(B): 219-222.
WADA, M. and T. H. YOSIDA· 1983: Karyotype of the Honshu sika deer, Cervus nippon
centralis. La Chromosomo (II)-35: 1117-1121.
WATANABE, T. K. and M. KAWANISHI 1983: Stasipatric speciation in Drosophila. Jpn.
J. Genet. 58: 269-274.
WATANABE, T. K. see under INOUCHI, Y.
WATANABE, T. K. see under OHNISHl, S.
YASUDA, S. and T. TAKAGI* 1983: Overproduction of Escherichia coli replication proteins by the use of runaway-replication plasmids. J. Bacteriol. 154: 1153-1161.
YASUDA, S. see under TABATA, T.
YAZAKI, K.,* T. KURITA* and K. I. MIURA 1983: Demonstration of stepwise coiling of
nucleoprotein in adenovirus core by application of critical point drying. J. Virol.
Methods 6: 119-125.
YOSIDA, T. H. 1983: Chromosome differentiation and species evolution in rodents.
"Chromosomes in Evolution of Eukaryotic Group, I." (Ed. by A. K. Sharma andA. Sharma), 147-176. CRC Press (Boca Raton, Florida).
YOSIDA, T. H. 1982: Chromosomal alteration and the development of tumors, XXV.
Quantitative changes of nucleolar organizer regions (NORs) in the Indian spiny
mouse tumors developed by 3-methylcholanthrene. Proc. Japan Acad. 58(B): 169172.
YOSIDA, T. H. 1982: Cytogenetical studies on insectivora, II. Geographical variationof chromosomes in the house shrew, Suncus murinus (Soricidae), in East, Southeast

122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34
and Southwest Asia, with a note on the karyotype evolution and distribution. Jpn.
J. Genet. 57: 101-111.
YOSIDA, T. H. 1983: Karyotype envolution and tumor development. Cancer Genet.
and Cytogenet. 8: 153-179.
YOSIDA, T. H. 1983: Sequentiality of chromosome evolution in mammals. Proc. Jap.
Acad. 59(8): 5-8.
YOSIDA, T. H. 1983: Parallelism of karyotype evolution in mammals. Proc. Jap. Acad.
59(8): 78-82.
YOSIDA, T. H., Y. HAYASHI and H. SUZUKI 1983: Remarkable decrease of C bands andNORs in the black rats collected from Amami and Tokunoshima Islands in southern
Japan. Proc. Japan Acad. 59(8):211-214.
YOSIDA, T. H. 1983: Rate of offspring with chromosome aberrations in the black and
Norway rats after r-irradiation. Proc. Japan Acad. 59(8): 263-266.
YOSIDA, T. H. 1983: Chromosome changes and transformation in cell and animal
tumor models. 13 Intern. Cancer Congo Part. C, Biology of Cancer (2): 269-278.
YOSIDA, T. H. 1983: Karyotype of a Ceylonese type black rat with several chromosome
alterations born after r -irradiation. Proc. Japan Acad. 59(8): 308-311.
YOSIDA, T. H. 1983: Studies on the karyotype differentiation of the Norway rat, VI.
Linkage analysis of the bared (ba) gene to the inversion pair no. 1 chromosome.
Proc. Japan Acad. 59(8): 351-354.
YOSIDA, T. H. 1983: Studies on the karyotype differentiation of the Norway rat, VII.
Chromosomes involving translocation in offspring born after r -irradiation. Proc.
Japan Acad. 60(8): (in press).
YOSIDA, T. H. 1984: Studies on the karyotype differentiation of the Norway rat, VIII.
Translocation between autosomes 9 and 18 occurred in a F-344 strain rat after rirradiation and its transmission to offspring. Proc. Japan Acad. 60(8): 50-53.
YOSIDA, T. H. and K. KAWAHARA 1983: Chromosomal alteration and the developmentof tumors, XXVI. A mammary cancer of the domestic cat charcterized by hypodiploid and hypotetraploid modes, with a special note on increase of diploid cells
after deep freezing. Jpn. J. Genet. 58: 1-9.
YOSIDA, T. H., T. SADAIE and Y. SADAIE 1984: Somatic and meiotic chromosomes ofthe small free-living nematode, Canorhabditis elegans. Proc. Japan Acad. 60(8):54-57.
YOSIDA, T. H., H. SUZUKI* and H. KUSANO* 1983: Sister chromatid exchanges in theuridine requiring Chinese hamster cell line, UR-216, cultured in the uridine rich or
starved medium. Proc. Japan Acad. 59(8): 223-226.
YOSIDA, T. H., M. WADA* and O. G. WARD 1983: Karyotype of a Japanese raccoon
dog with 40 chromosomes including two supernumeraries. Proc. Japan Acad.
59(8): 267-270.
YOSIDA, T. H., M. WADA*, O. G. WARD*, and D. H. WURSTER-HILL 1984:* Further
studies on the Japanese raccoon dog karyotypes, with a special regard to somatic
variation of B-chromosomes. Proc. Japan Acad. 60(8): 17-20.
YOSIDA, T. H., M. WADA, Y. SAKAI and N. KONDO 1983: Determination of the sexchromosome in a sex-reversed peacock and its karyotype. Proc. Japan Acad. 59(8):BOOKS AND PAPERS PUBLISHED IN 1983 BY STAFF MEMBERS
304-307.
YOSIDA, T. H. see under BAVERSTOCK, P. R.
YOSIDA, T. H. see under MUROFUSHI, M.
YOSIDA, T. H. see under SHIRAISHI, Y.
YOSIDA, T. H. see under SUGIYAMA, K.
YOSIDA, T. H. see under SUZUKI, H.
YOSIDA, T. H. see under WADA, M.

123124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

ABSTRACTS OF DIARY FOR 1983
January
March
April
June
July
AugustSeptember
OctoberNovember

December

24
27
9
17
1
28
8
11
19
24
30
9
1
13
20
25
17
28
29
5

283rd Meeting of Misima Geneticists' Club
284th Meeting of Misima Geneticists' Club
285th Meeting of Misima Geneticists' Club
201st Biological Symposium
202nd Biological Symposium
286th Meeting of Misima Geneticists' Club
203rd Biological Symposium
204th Biological Symposium
287th Meeting of Misima Geneticists' Club
205th Biological Symposium
206th Biological Symposium
207th Biological Symposium
208th Biological Symposium
209th Biological Symposium
210th Biological Symposium
211 th Biological Symposium
212th Biological Symposium
213th Biological Symposium
288th Meeting of Misima Geneticists' Club
214th Biological Symposium

FOREIGN VISITORS IN 1983

125

FOREIGN VISITORS IN 1983
Jui. 29, '82-Jui. 28, '83
January

26

February

7-Mar.31

March

12-Mar. 13
20-21

April
May

1
24-26
22-May 21, '84

June

8

8
8
8
8

August15
6-Dec.3
11-Mar. 11, '84
24-25

September

7-10

Huang, J.-T., Institute of Sericultural Research, China.
Jefferson, Roland M., U. S. National Arboretum, U.S.A.
Cai, Y.-M., Institute of Sericultural Research, China
Sueoka, N., University of Colorado, U.S.A.
Weil, T. H., Institut de Biologie Moleculaireet Cellulaire, France.
Manna, G. K., Kalyani University, India
Ullmann, A., Institut Pasteur, France
Yoo, I.-D., Institute of Agricultural Sciences,
Korea
Oakberg, Eugene F., Oak Ridge National
Laboratory, U.S.A.
Shaw, William P., Kettering Research Laboratory, U.S.A.
Nazerali, Aly-Raza, The United Nations
University, Tokyo.
Kokke, Robert, The United Nations University, Tokyo.
Soyasa, Chandra H., The United Nations
University, Tokyo.
Bengt, O. B., University of Lund, Sweden.
Baradjanegara, Abudul A., National Atomic
Energy Agency, Indonesia.
van Hintum. T. J. L., Agricultural University in Wagenigen, Netherlands.
Novotny, Jiri, The Massachusetts General
Hospital, U.S.A.
Green, Maurice, University of St. Louis,
U.S.A.

126 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

October

1-31
4-5
4-5
10-16
14
18-Nov.25
21-22

Crow, James F., University of Wisconsin,
U.S.A.
Ward, Oscar G., University of Arizona,
U.S.A.
Wurster-Hill, Doris H., Dartmouth Medical
College, U.S.A.
David, Charles N., Universitat Miinchen,
Germany.
Ohno, S., City of Hope Research Center,
U.S.A.
Watterson, Geoff, University of Monash,
Australia.
Hawkes, J. G., University of Birmingham,
U.K.

24-31
29
November

9
17-18
21
21-Sept. 24, '84
24
24
24
26

26-29

Fuerst, Paul A., Ohio State University,
U.S.A.
Cherry, Flore F., University of Tulane,
U.S.A.
Lewis, Herman W., National Science Foundation, U.S.A.
Walker, Charles W., University of New
Hampshire, U.S.A.
Kasturi-Bai, A. R., Karnataka State Sericultural Development Institute, India
Zhu, N.-K., Institute of Environmental
Chemistry, China.
Jalaludn, B. S. S., Univeriti Pertanian Malaysia.
Mohammod, M. O. R, University of Malaya, Malaysia.
Rahim, M. S. A., University of Malaya,
Malaysia.
Tahir, M.
) International Center for
Srivastava, J. P. A . I
I Rh'Somaroo, R H.
gncu turaesearc 10
Nachit, M. M.
the Dry Areas, Syria.
Nei, M., University of Texas, U.S.A.

FOREIGN VISITORS IN 1983

27-29
December

5
15

127

Goldberg, Alfred L., University of Harvard,
U.S.A.
Nevo, Eviatar, Haifa University, Israel
Arnould, Daniel, Belgium Embassy, Belgium.

128

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 34

AUTHOR INDEXADAMS, M •.•..•.•.••.••...•.•••••. .46

KUSUDA, J •••••••••••.•••••••••• 44, 103

AOKI, K .•..••.•••..•.••••••......... 94

MAEKAWA, M •••••.••.•.•.•••••••.• 112

AsWATHANARAYANA, N. V

MARUYAMA, T .••.••.••..... 70, 95, 96, 97
MATSUDA, M •.•••••••.•••••••••••••• 71

58
BAVERSTOCK, P. B.
.46
BONHOMME, F
32
BROWN, Jr., W. L.
66
CROZIER, R. H. • ••.•.•...•.•.....•.. 70
DEGUCHI, Y
64
DOVER, G. A •..........•.........••• 91
Fum, T35, 85, 86,
FUJISAWA, T •..••••.•.........•..... 41
FUJISHIMA, T. • .•.......•.........•. 104
FUKASE, Y
76
FURillCHI, M .....••••.•....•........ 14,
GOJOBORI, T ••.....••.•........•.•••• 98
HARA, H •..•••......••........... 21, 22
HIRASHIMA, T. . •.....•.......••••.•• 34
HIROTA, Y
19, 20, 23
HORAI, S •.•••...••..•..•.•......... 102
IMAI, H.T66, 69, 70,71
INOUE, T •..•.••••.••••.••...•• 82,83,86
INOUE, Y
106
IYAMA, S •..••...••••.•.••..•.••.... 113
JIKUYA, H. • .•..•••••..•..•••.•..... 13
KADA, T
22,38,82,83,84
KAGIMOTO, M •..•......••.•.......•. 16,
KANEHISA, T. • •••••.•••••.......... .45
KATO, J.-1. .•••••••..••.••..•..•..•. 20
KIKUCHI, H. • •.....•••............. 112
KIMURA, M •••••••..••••.•..••.•• 88,89
KOHIYAMA, M ..•••••...••.••••...... 20
KOMINAMI, R
31
KONDO, K •.••••.••••.••...••••....• 69
KONDO, N •.•••••..•••••••.••..••••• 61
KUBOTA, M
66
KUMARI, P •..••...•••••..•.••.•..... 58
KURIHARA, Y
32
KURODA, Y
37, 38, 39

MATSUDA, Y

69
99,102
MATSUURA, S •••••••.•.•..•••.•.•••• 110
MAXSON, L. R. • ••.....•.•..•....••. .46
MIKWS, G. L. G ••.......•......•••.. 41
MATSUNAGA, E

MINATO, K •.••••...•••••.•......... 40
MINODA, K ••.••...•.•••.•....•.•.... 99
MIYASHITA, N ••••••..••••••••• 25, 26, 29
MIYOSHI, J ••..••••.....•.•.•.••.•••. 16
MORISHIMA, H ••••••.•••••. 105,109,111
MORIWAKI, K •••••••••• 25, 26, 28, 29,30,

31, 32, 34, 69
75, 76, 78, 79
MURAMATSU, M •••••••••••••••••••.• 31
MUROFUSHI, M •••••••••..••••• 62,63,64
NAKAGOME, Y
100, 101, 103
NAKAHORI, Y
100,101,103
NAKAYAMA, N •.••.•.••••••••••...... 13
NATORI, T. • •••••••••.....•••.••••.. 34
NAwA, S
35
NEvo, E31
NISHIKAWA, S •••••••••••••••••••• 62,63
NISHIMURA, A
23
NISHIMURA, Y
20
NITo, S
29
NOGUCHI, T ••....•.•••••••••••.••.•. 44
OHNISHI, S
107
OHTA, T .•.••.••...•..........••• 90,91
OKA, A ••••••.•..•.•.••........•.•.. 19
ONIMARU, K. • •.••.•••••••••••..•••• 44
RrCHET, E .•••••••...•.••••..•••••.. 20
SADAIE, T •.•••••••...••••••••••••••• 65
SADAIE, Y
22, 65, 82
SAGAI, T •••••••••••..••.•.••.. 25,26,28
MURAKAMI, A.

AUTHOR INDEX129

SAKAI, Y •....••.•.•••.........•.... 61

TEZUKA, H.42
SAKAKI, Y. . .•....•.....•.••...••••. 16
SANDBERG, A. A
65
SANO, R
111
SANO, Y35, 105 111, 112
SASAKI, H •....•......•.••...•••••• , .19
SATO, Y105, 110
SlllMADA, Y ••...•••••...•••..•••••.. 37
SmMAMoTo, Y ••.••••.•••••..•.•.•.. 105
SIDRAIsm, Y. • •••.•..••••••.•••••••• 65
SmROIsHI, T •.....••••.••...•..... 25,28
SLATKIN, M
92
SOEDA, E
13,14,16,17
SUGAWARA, E
38
SUGIMOTO, K
19
SUZUKI, H •.••••.•.. 20, 21, 22, 25, 31, 45
TABATA, S ••••..••.••••••••..•..•••• 19
TAKAHATA, N
89, 92, 93
TAKANAMI, M
19
TAYA, C30

THO, Y.P•••••.•....••••.•.•.••.•... 66

SAKAIZUMI, M

84

71
17
UEDA, T21
VAN HiNTUM, T. J. L.
113
WADA, M. Y
53,54,55,56,57,59,60,61
WARD, O. G
53
WATANABE, S ••.......•.•••••••...•.• 17
WATANABE, T. K
106,107
WURSTER-HILL, D. H •.•.•.•.•.•••••.. 53
YAMADA, M. A
35
YAMAMOTO, M
41, 42,72
YAMASHITA, O
44
YASUDA, S
13,14,19
YOKOIYAMA, A
38
YONG, H.-S...•...••••••..••••.•.••• 66
YosmIKE, K
17
YOSIDA, T. H .•. .45, 46,47,48,49,50,51,
52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65

TOBARI, Y. N
UCHIDA, S.

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