NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 301979

Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka.ken, Japan1980

Annual Reportof theNational Institute of Genetics
No. 30, 1979

Photo by Y. NakagomE

Published by

The National Institute of Genetics, Japan
1979

CONTENTS
General statement
Staff
Council....
.
.. ..
Association for propagation of the knowledge of genetics
Projects of research for 1979
Researches carried out in 1979 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

1
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12

Molecular Genetics

Relation of the structure of cytoplasmic polyhedrosis virus andthe synthesis of its messenger RNA. YAZAKI, K. and
MIURA, K. I.
Process of cap formation of meSsenger RNA by vaccinia virus
particles carrying an organized enzyme system. URUSHIBARA,
T. and MIURA, K. I.
Relation of stability and presence of the cap structure of mRNA.
YAMAGUCHI, K. and MIURA, K. I. . . . . . . . . . . . . . . . . . . . . . . .
Presence of single-stranded region adjacent to the protein-binding
5 ' -termini of adenovirus genome double-stranded DNA.
ARIGA, H., SHIMOJO, H., HIDAKA, S. and MIURA, K. I... . . . .
Purification of T4 RNA ligase and its application to 5S RNA
sequencing. SUGIURA, M., SUZUKI, M. and TAKAIWA, F. ..
Organization and expression of the ribosomal RNA gene cluster oftobacco chloroplasts. SHINOZAKI, K., KUSUDA, J., TAKAIWA,
F., TOHDOH, N. and SUGIURA, M.
Heterogeneity of the ribosomal RNA gene clusters in rice.
OONO, K. and SUGIURA, M.
Structure of Harvey murine sarcoma viral DNA: Nucleotide
sequence of the terminal repeats. SOEDA, E. and MARTIN,
M.A.................................................
Structural analysis of fibroin genes of silkworm and its relatives
by in situ hybridization. KUSUDA, J., ONIMARU, K., SAKATE,
S., SUZUKI, Y. and TAZIMA, Y... . . . . . . . . . . . . . . . . . . . . . . . .
A new method for construction of restriction map based on end

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

assignment. TAKEDA, Y., YAMADA, M. and HIROTA, Y.

II.

21

Microbial Genetics
A model for distribution of division sites in E. coli. YAMADA,
M., MARUYAMA, T. and HIROTA, Yo .
Transcriptional control of flagellar genes in Escherichia coli K-12.
KOMEDA, Yo
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Biochemical Genetics and Immunogenetics
Geographical distribution of two groups in Japanese wild population of medaka, Oryzias latipes. SAKAIZUMI, Mo, EGAMI,
N. and MORIWAKI, Ko .....
The purification and characterization of H-2 antigens of Japanesewild mouse. SHIROISHI, T., MORIWAKI, K. and TAKEUCHI,T.
H-2 antigenic specificities in BlO·MOL H-2 congenic mousestrains. MORIWAKI, K., SHIROISHI, To and SAGAI, T.....
Relationship between laboratory mice and the subspecies Mus
musculus domesticus based on restriction endonucleasecleavage patterns of mitochonrial DNA. YONEKAWA, H.,
MORIWAKI, Ko, GOTOH, 0., WATANABE, J., HAYASHI, J. 1.,
MIYASHITA, N., PETRAS, M. L.and TAGASHIRA Y.
Evolutionary relationships among five subspecies of Mus musculus based on restriction enzyme cleavage patterns.
YONEKAWA, H., MORIWAKI, K., GOTOH, 0., HAYASHI,
J.1., WATANABE, J., MIYASHITA, No, PETRAS,M. L. and
TAGASHIRA, Y....
In vitro hybridization of endosperm ADH in the Japanese andthe American species of Trillium. IHARA, M. and ENDO,
T .•.. o. '"
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31
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Developmental Genetics and Somatic Cell Genetics
In vitro cultivation of gonads from a female sterile mutant /s231
of Drosophila melanogaster. KURODA, Y. .
Effect of cell association on In vitro chondrogenesis of mesen-

45

chyme cells from quail limb-buds KURODA, Y. and MATSUTANI, E
Dose-rate effects of Trp-P-l on survival and mutation induction

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CONTENTSiii

in cultured human diploid cells. KURODA, Y.. . . . . . . . . . . . . .
Analysis of the decreased hatchability of eggs laid by flies rearedon excess yeast in Drosophila melanogaster. MINATO, K.. .
A normal diploid teratocarcinoma (OTTlOA-5) obtained froma 6-day embryo of BlO.A mouse. NOGUCHI, T., TAYA, C.
and MORIWAKI, K.
The establishment of some clonal cultures of a Tetratocarcinoma
derived from strain BlO.A mouse. NISHIMUNE, Y., MATSUSHIRO,A., OGISO, Y., TAYA, c., NOGUCHI, T.and MORIWAKI, K.. . .
Characterization of the lethal hybrid rescue (Lhr) gene of Drosophila simulans. TAKAMURA, T. and WATANABE, T. K.......
Gonad development and gametogenesis in the opposite sex of thesilkworm, Bombyx mori. YOSHIDA, H. and MURAKAMI, A. ..
Characterization of Is (1) MY-18 mutant rescued with thetransplantation of normal egg cytoplasm in D. melanogaster.
YAMADA, M. A. and NAWA, S........•........ ;.........

47
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52

Cytogenetics
F 1 hybrids between Mauritius and Oceanian type black rats
and their karyotypes. YOSIDA, T. H.
Nucleolus organizer regions (NORs) in 7 Rattus species andtheir differentiation and evolution. YOSIDA, T.H.. . . . . . . . . .
Chromosome constitution of Rattus villosissimus resulted from
Robertsonian fission. YOSIDA, T. H. . . . . . . . . . . . . . . . . . . . .
A consideration on relation between the karyotype evolution
and the mutagenic environment. YOSIDA, T. H. and PARIDA,
B. B. .....•.....................................
Finding of 1/12 translocation in the Lewis-strain rat. YOSIDA,
T.H. ..
Evolutionary aspects of mitochondrial DNAs in variant types of
black and Norway rats. HAYASHI, J. I., YONEKAWA., H.,
GOTOH, 0., TAGASHIRA, Y., MORIWAKI, K. and YOSIDA, T.

H.
Comparative studies on release of enzymes from platelets during
blood clotting in the black and Norway rats. EMORI, T.,
TAKAHASHI, M., NAGASE, S. and YOSIDA, T. H.
Establishment of cell lines from Millardia meltada and exami-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

nation of endogenous virus. SUGIYAMA, H., YUTSUDO,
M., TOYOSHIMA, K., YOSIDA, T. H. and MURATA, Y.......
Acquired resistance and immune responses of eight rat strains
to infection with Angiostrongylus cantonensis. YOSHIMURA,
K., AlBA, H., HIRAYAMA, N. and YOSIDA, T. H.
Karyotypes of four carangid fishes. MUROFUSHI, M. and YOSIDA,
T.H.
High incidence of urethan-induced pulmonary tumor in theintersubspecies hybrids between laboratory mouse andJapanese wild mouse; MORIWAKI, K., SHIROISHI, T. andMIYASHITA, N ..,.......................................
Urethan-induced chromosome aberrations in various mouse strains suggesting an inverse relationship between chromosomechanges and tumorigenesis. MORIWAKI, K., SHIROISHI, T.,
MIYASHITA, N. and SAGAI, T.
On the precocious XY separation" at the first meiotic.' metaphase of mouse spermatocytes. MATSUDA, Y., IMAI, H.
T., MORIWAKI, K. and KONDO, K. . . . . . . . . . . . . . . . . . . . . . .

VI.

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Mutation and Mutagenesis in Animals
In vitro complementation of repair enzyme activities in cellular
extracts from ataxia telangiectasia fibroblasts. INOUE, T.,
YOKOIYAMA, A. and KADA, T.
Human enzymes functioning in repair of gamma-ray-induced
DNA damage. INOUE, T. and KADA, T.
The mutagenic activity of pyrrolizidine alkaloids in the silkworm
germ-cells. MURAKAMI, A., FURUYA, T. and OZAWA, T. .. ..
Mutagenicity of two phosphoramides, TEPA and HMPA, in thesilkworm germ-cells. MURAKAMI, A. and OZAWA, T. .....
The mutagenicity test of some flavonoids in pupal germ-cells of
the silkworm, Bombyx mori. MURAKAMI, A. and OZAWA, T.
Strain differential activity for metabolic activation of 2-acety1aminofluorene in the silkworm. MURAKAMI, A. and GOTO,
M.
Mutagenic effects of X-rays at a dose down to 25 R in adultoocytes of the silkworm. MURAKAMI, A. and OZAWA,

T.

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CONTENTSv

X-ray-induced recombinational events between Z and W chromosomes in oogenic cells of the silkworm (Bombyx moriL.). MURAKAMI, A. and OHNUMA, A. .... . . . . . . . . . . . . . . . .
Chromosomal aberrations in Drosophila simulans. INOUE, Y. . . . .

7777VII. Radiation Genetics and Chemical Mutagenesis in Microorganisms
and Plants
Natural and environmental antimutagens. KADA, T., MOCHIZUKI, H., HARA, M., YOKOIYAMA, A., INOUE, T., KANEKO,
K., MINAKATA, H. and SADAIE, Y.
Antimutagenic effect of cobaltous chloride on various mutator
strains. INOUE, T., SHIMOI, K. and KADA, T.
Mutagenic effect of L-ethionine in soybean and maize. FUJII,

79
79

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Identification of newly induced waxy mutant gene in maize.
AMANO, E.
Phenotypic expression of Wx gene in cereals. AMANO, E. . . . . . . . .

81
82

VIII.

Population Genetics (Theoretical)

Efficiency of truncation selection.

CROW, J. F. and KIMURA,

M.

84

Fixation of a deleterious allele at one of two "duplicate" lociby mutation pressure and random drift. KIMURA, M. andKING, J. L.
Model of effectively neutral mutations in which selective constraint is incorporated. KIMURA, M.
Population genetics of multigene family with special reference
to decrease of genetic correlation with distance between
gene members on a chromosome. KIMURA, M. and OHTA,

T.
An extension of a model for the evolution of multigene families
by unequal crossing-over. OHTA, T.
Genetic variability maintained in a finite population undermutation and autocorrelated random fluctuation of selection intensity. TAKAHATA, N. and KIMURA, M. . . . . . . . . . . . .

84
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IX.

Population Genetics (Experimental)
New inversions of Drosophila melanogaster spreading over Japan.
INOUE, Y., TSUNO, K. and WATANABE, T. K.

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Inversion polymorphism in some African, New Guinean andPhilippine populations of D. melanogaster. INOUE, Y. and
WATANABE, T. K;.................

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X. Evolutionary Genetics
Courtship song and mating preference in Drosophila melanogaster, D. simulans and their hybrids. KAWANISHI, M. and
WATANABE, T. K.
Chromosome analysis of hybrid male sterility between D. simufans and D. mauritiana. TAKAMURA, T. and WATANABE, T.
K
Host-dependent evolution of three papova viruses. SOEDA,
Eo, MARUYAMA, T., ARRAND, Jo R. and GRIFFIN, B. E.
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Human Genetics
Hereditary retinoblastoma: host resistance and age at onset.
MATSUNAGA, E. .
Threatened abortion, hormone therapy and malformed embryos. MATSUNAGA, E. and SHIOTA, K.. . . . . . . . . . . . . . . . . . . .
Suppressed centromere and the loss of "centric dot" in dicentricchromosomes. NAKAGOME, Yo.
A new approach in the evaluation of chromosome variants inman (II and III). NAKAGOME, Y., AZUMI, J. I., OKA, S.
and MATSUNAGA, E. .
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Behavioral Genetics
Selection for the learning ability of mice. FUJISHIMA, T. ..
o' 100Genetic difference in oviposition cycfe in D. melanogaster.
TAKAMURA, T....
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Differentiation of oviposition thrusting power in D. melanogasterspecies sub-group. TAKAMURA, T. . ..
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Effect of UV treatment on the hatchability of buried and unburied eggs of D. melanogastero TAKAMURA, T.
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CONTENTS

XIII.

Ecological Genetics

Observations on rice species and accompanying weeds in the
hilly area of tropical Asia. OKA, H. I., MORISHIMA, H.
and SANO, Y.
An experiment on population growth of wild rice in greenhouse.
MORISHIMA, H.
Copper tolerance and reproductive competition in barnyard
grass. MORISHIMA, H. and OKA, H. I. . . . . . . . . . . . . . . . . . . ..
Copper tolerance and reproductive competition in four annual
weeds. MORISHIMA, H. and OKA H. I.
Variation in competitive ability among Oryza perennis strains.
SANO, Y. and MORISHIMA, H.
Neighbour effects between plants of Oryza sativa and O. glaberrima grown in mixture in Nigeria. SANO, Y. and MORISHIMA, H.

XIV.

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Applied Genetics

Effect of assortative mating on the multigenic character in a
finite population. IYAMA, S. . .. . . . . . . . . . . . . . . . . . . . . . . . ..
Changes in isozymes and some quantitative characters observedin hybrid populations of rice. MORISHIMA, H. and ENDO,

T.
Nitrogen fixation in the rhizosphere of cultivated and wild rice.
SANO, Y., FUJII, T., IYAMA, S., HIROTA, Y. and KOMAGATA,
K.
Analysis of genes controlling the F i pollen sterility between
Oryza sativa and O. glaberrima. SANO, Y.
Adaptation to high ambient temperature of Japanese quail.
KAWAHARA, T.
Domestication in Japanese quail, Coturnix coturnix japonica.
KAWAHARA, T,.
Books and papers published in 1979 by staff members
Abstracts of diary for 1979
. . . . . . . . . . . . . . . . . . . . . . . . . . ..
Foreign visitors in 1979
Author index
,...

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GENERAL STATEMENT
June 1st of this year was the 30th anniversary of the foundation of theInstitute. Wishing to make this occasion the turning point to further development of the Institute, and to thank those who exerted their endeavors
for the realization of the Institute as well as those who supported and encouraged us at all times, we planned the following colourful- program.
1. Commemoration ceremony' and reception
2. Memorial lecture
3. Publication of "Memoir in the Days of the Foundation"
4. Collection of important documents relating to the foundation
5. Memorial planting
The commemoration ceremony was a great success, attended by men of
distinction both in and out of the government agencies and institutions:among them attendance of Mr. Keijiro Inai, the Vice-Minister of Educac
tion, Science and Culture; Prof. Koji Fushimi, President of the Science
Council of Japan; Prof.- Seiji Kaya, former President of the Universityof Tokyo; Prof. Takashi Fujii, member of the Science and Technology Council, honoured us extremely.
Thanks to the contributions from Drs. Seiji Kaya, Kiyoshi Masui, Hitoshi
Kihara, Yosito Sinoto, Kiyoshi Okano, Heihachiro Miyayama and manyothers, the commemorative publication "Memoir in the Days of the Foundation" was full of valuable articles. It would never be possible for such articles to be contributed again from so many authoritative sources concerned.
The highlight among them was Prof. Kaya's article, in which Prof. Kaya, thethen Director of the Science Education Bureau of the Ministry of Education,
Science and Culture, described realistically the progress of the negotiation
between the General Headquarters (GHQ) and the Ministry of Education,
Science and Culture, in regard to the permission of the foundation of theInstitute. In answering the expectation of those farsighted leaders concerned with the establishment of the Institute we renewed our resolution
to endeavor all our efforts for further development of our science.
Commemorative lectures were given by Dr. Takashi Sugimura, Director of the National Cancer Center Research Institute, and Dr. James F.
Crow, Professor of the University of Wisconsin. Both of them talked to2

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

us enthusiastically and most impressively about their recent works entitled"Mutagenicity and carcinogenicity in our environment," and "Hybrid disgenesis and other strange genetic phenomena," respectively.
We are much indebted and grateful to those persons and corporations
who gave us financial support for achieving the above commemoration
program.
As to personal change in the staff, Dr. Chozo Oshima, Head of the Department of Physiological Genetics, retired on April 1st. Dr. Takeo Maruyama was appointed the new head. Dr. Oshima affiliated to the Institutein May, 1957, in succession to Dr. Taku KomaL Since then, Dr. Oshima
had made his efforts as a leader of Drosophila study group to develop population genetics and behavioral genetics of Drosophila. By the retirementof Dr. Oshima, all the heads of the three departments which existed at thetime of the founding have been brought to the third generation, and thestaff members have been quite rejuvenated.
The main building of the Genetic Stocks Center was completed in
March and the staff members have all moved to the new building and started
their activities there. The construction of the attached laboratories formice and silkworms were completed in this fiscal year.

STAFF
Director

TAZIMA, Yataro, D. Ag.
Members

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

2. Department of Cytogenetics
YOSIDA, Tosihide H., D. Sc., Head of the Department
The 1st Laboratory
YOSIDA, Toshihide, H., D. Sc., Head of the Laboratory
The 2nd Laboratory
MORIWAKI, KAZUO, D. Sc., Head of the Laboratory
IMAI, HIROTAMI, D. Sc.
3.

Department of Physiological Genetics
TAZIMA, Yataro, D. Ag., Head ·of the Department
The 1st Laboratory
WATANABE, Takao K., D. Sc., Head of the Laboratory
The 2nd Laboratory
TAZIMA, Yataro, D. Ag. Head of the Laboratory

4. Department of Biochemical Genetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
The 1st Laboratory
NAwA,Saburo, D. Sc., Head of the Laboratory
YAMADA, Masa-Aki, M. Sc.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
ENDO, Toru, D. Ag.
The 3rd Laboratory
SUGIYAMA, Tsutomu, Ph. D., Head of the Laboratory
FUJISAWA, Toshitaka, Ph. D.

5. Department of Applied Genetics
OKA, Hiko-IcHI, D. Ag., Head of the Department
The 1st Laboratory
OKA, Hiko-Ichi, D. Ag., Head of the Laboratory
KAWAHARA, Takatada, D. Ag.
FUJISHIMA, Tohru, D. Ag.
The 2nd Laboratory
IYAMA, Shin-ya, D. Ag., Head of the Laboratory
The 3rd Laboratory
MORISHIMA-OKINO, Hiroko, D. Ag., Head of the Laboratory

6. Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department
The 1st Labqratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
INOUE, Tadashi, D. Ag.
The 2nd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
The 3rd Laboratory
KADA, Tsuneo, D. Sc., Head of the Laboratory
AMANO, Etsuo, D. Ag.
SADAIE, Yoshito, D. Sc.

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
The 2nd Laboratory
NAKAGOME, Yasuo, D. Med., Head of the Laboratory
AZUMI, Jun-ichi, D. Sc.

STAFF

8. Department of Microbial Genetics
HIROTA, Yukinori, D. Sc., Head of the Department
The 1st Laboratory
HIROTA, Yukinori, D. Sc., Head of the Laboratory
YASUDA, Seiichi, D. Sc.
The 2nd Laboratory
HIROTA, Yukinori, D. Sc., Head of the Laboratory
NISHIMURA, Yukinobu, D. Sc.
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
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Laboratory
TAKAHATA, Naoyuki, D. Sc.

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

11. Genetic Stocks Center
OKA, Hiko-Ichi, D. Ag., Head of the Center
Animal Section
YOSIDA, TosihideH., D. Sc., Head of the Laboratory
NOGUCHI, Takehiko, D. Sc.
INOUE, Yutaka, D. Sc.
KUSUDA, Jun, D. Ag.
Plant Section
FUJII, Taro, D. Ag., Head ofthe Laboratory

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

SANO, Yoshio, D. Ag.
Microbial Section
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
KOMEDA, Yoshibumi, D, Sc.

12.

Experimental Farm
OKA, Hiko-IchiD. Ag., Head of the Farm
MIYAZAWA, Akira

13. Department of Administration
OISHI, Tatsunori, Head of the Department
OHTSUKA, Haruichi, Chief of the General Affairs Section
KIMURA, Susumu, 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.
TSUJITA, Mitsuo, D. Ag.
MORIWAKI, Daigoro, D. Sc., Emeritus Professor of Tokyo Metropolitan
University
OSHIMA, Chozo, D. Sc.

COUNCIL
FUJII, Takashi, Chairman, Emeritus Professor of University of TokyoINOUYE, Eiji, Professor of University of Tokyo
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
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
MUKAIBO, Takashi, President of University of TokyoNAGAKURA, Saburo, Professor of University of Tokyo

COUNCIL7OOSAWA, Fumio, Professor of Osaka University
SASA, Manabu, Emeritus Professor of University of TokyoSHINOZAKI, Nobuo, Director of Institute of Population Problems
TAKAHASHI, Man-emon, Professor of Hokkaido University
UMEZAWA, Hamao, Emeritus Professor of University of Tokyo
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Emeritus Professor of Tokyo Metropolitan
University
MATSUNAGA, Ei,Managing Director, Head of the Human Genetics Department.
YOSIDA, Tosihide H., Managing Director, Head of the Cytogenetics Department
K!HARA, Hitoshi, Manager, Emeritus Professor of Kyoto University
SINOTO, Yosito, Manager, Professor of International Christian University
WADA, Bungo, Manager, Emeritus Professor of University of TokyoTAZIMA, Yataro, Manager, Director of the National Institute of Genetics
OSHIMA, Chozo, Manager,

PROJECTS OF RESEARCH FOR 1979
Department of Morphological Genetics

Genetic studies on insect cells in tissue culture (KURODA and MINATO)
Developmental genetic studies on carcinogenesis in tissue culture (KURODA)
Genetics of somatic mammalian cells in culture (KURODA and MINATO)
Cytogenetics in the silkworm (MURAKAMI)
Studies on recombination in the silkworm (MURAKAMI)
Genetics of the silkworm (MURAKAMI, FUKASE and OHNUMA)
R~diation and chemical mutagenesis in the silkworm (TAZIMA and MURAKAMI)
Department of Cytogenetics

Studies on chromosomal evolution in rodents and other small mammals(YosmA)
Chromosome study on experimental tumors (YosmA)
Experimental breeding and genetics of mice, rats and other wild rodents(YosmA and MORIWAKI)
Cytogenetical and immunological studies on mouse plasma cell tumor(MORIWAKI)
Immunogenetical study on the histocompatibility antigens of wild rodents(MORIWAKI)
Cytogenetical study of ants (IMAI)
Cytogenetical study of fishes (YosmA)
Department of Physiological Genetics

Behavior genetics of Drosophila (WATANABE and TAKAMURA)
Evolutionary and ecological genetics of Drosophila (WATANABE and KAWANISHI)
Population genetics of Drosophila (WATANABE, INOUE, and TSUNO)
Department of Biochemical Genetics

Studies on transformation in higher organisms (NAWA and YAMADA)PROJECTS OF RESEARCH FOR 1980

9

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

Quantitative genetic studies in poultry (KAWAHARA)
Genetic studies in wild populations of Japanese quails (KAWAHARA)
Behavioral genetic studies in mice (FUJISHIMA)
Theoretical studies on breeding techniques (IYAMA)
Genetic studies of trees in natural forest (lYAMA)
Evolutionary studies in wild and cultivated rice species (OKA and MORISHIMA)
Ecological genetic studies in weed species (MORISHIMA)
Genic analysis for isozyme variations in rice (ENDO and OKA)
Genetic effects of environmental pollution on plants (IYAMA, MORISHIMA
and OKA)
Department of Induced Mutation

Molecular mechanisms of spontaneous, chemical- and radiation-induced
mutations (KADA, SADAIE and INOUE)
Environmental mutagens and carcinogens (KAnA, SADAIE, TUTIKAWA and
HARA)
Radiation genetics in mice (TUTIKAWA)
Biochemical factors involved in cellular repair of genetic damage (INOUE
and KADA)
Genetics of Bacillus subtilis (SADAIE and KADA)
Molecular mechanisms of unicellular differentiation in Bacillus subtilis

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30(SADAIE)
Radiation and chemical interaction in the cells (KADA)
Genetic fine structure analysis in maize (AMANO)
Department of Human Genetics

Genetic and epidemiologic studies on certain malformations in humanembryos (MATSUNAGA)
Genetic studies on retinoblastoma (MATSUNAGA)
Clinical cytogenetic studies of congenital abnormalities in man (NAKAGOME
and OKA)
Molecular cytogenetic studies of human chromosomes (NAKAGOME andAZUMI)
Studies on human chromosome variants (NAKAGOME, AZUMI and MATSUNAGA)

Department of Microbial Genetics

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

Theoretical studies of population genetics (KIMURA, MARUYAMA, OHTA,
and TAKAHATA)
Studies on molecular evolution from the standpoint of population genetics(KIMURA and OHTA)
Theoretical studies on the evolution of multigene family (OHTA)
Mathematical studies on the genetics of structured populations (MARUYAMA)PROJECTS OF RESEARCH FOR 1980

11

Department of Molecular Genetics

Studies on the structure of genome RNA of viruses (MIURA)
Studies on the primary structure of DNA (SOEDA and MIURA)
Studies on structure and function of messenger RNA (MIURA)
Genetical and enzymological studies on E. coli polymerase (SUGIURA)
Studies on T 4 RNA ligase (SUGIURA)
Cloning of eukaryotic genes and their structural analysis (SUGIURA andSHINOZAKI)
Genetic Stocks Center

Studies and conservation of germplasm resources in rice and wheat species(FuJII, SANO and aKA)
Specificity of mutagen tolerance in higher plants (FuJII)
Exploitation of genetic ability of nitrogen fixation in Graminae (FuJII, SANO,IYAMA and HIROTA)
Studies on genetic differentiation in rice (SANO)
Cytogenetic studies of Norway rats and establishment of the chromosomal
mutant stocks (YOSIDA)
Experimental breeding of wild rodents for establishment of the new experimental animals (YoSIDA)
Developmental genetic studies on mouse teratomas (NOGUCHI)
Studies on chromosomal polymorphism in Drosophila (INOUE)
Analysis of fibroin genes of silkworm and its relatives (KUSUDA)
Genetical study of flagellar formation in Escherichia coli K-12 (KOMEDA)
Basic studies on the gene purification and the construction of DNA banks(SUGIURA)RESEARCHES CARRIED OUT IN 1979I.

MOLECULAR GENETICS

Relation of the Structure of Cytoplasmic Polyhedrosis Virusand the Synthesis of its Messenger RNA
Kazumori Y AZAKI and Kin-ichiro MIURA

Cytoplasmic polyhedrosis virus (CPV) was observed in an electronmicroscope using a combination of staining and shadowing methods to see
genome nucleic acid and virus core protein simultaneously. With thismethod projections not only in positions horizontal to the grid, but also
vartical were clearly visualized. When the particle was mildly disrupted
with EDTA, genome double-stranded (ds)RNA was released from a projection. If the particle was previously fixed with glutaraldehyde and thendisrupted, dsRNA was released with a protein particle, which seemed tocorrespond to the base part of the projection. The protein particle wasin most cases at the end of the strand, which sometimes takes a supercoiled
structure. When CPV was incubated in medium adequate for mRNA
synthesis, swelling or deformation of projections was observed. After
CPV was incubated during mRNA synthesis, it was fixed by glutaraldehyde and disrupted by EDTA treatment succesively. Genome dsRNA wasreleased from a virion carrying a protein particle. In this case, protein
particles were attached not only to the edge of a strand, but also at various
positions along the strands. From these observations; we suggest thattranscription in this virus particle proceeds as follows: Genome dsRNA istranscribed by passing through the base part of the projection, where the
enzymes for mRNA synthesis are located. A completed mRNA is released
from the virion at the projection.
Process of Cap Formation of Messenger RNA by Vaccinia Virus
Particles Carrying an Organized Enzyme System
Toshiyuki URUSHIBARA and Kin-ichiro MIURA

Vaccinia virus mRNAs carry the cap structure m7 G 5 'pppAm- or m7 G5 ' .

RESEARCHES CARRIED OUT IN 197913

pppGm- at the5/-terminus, which is synthesized by a series of RNA polymerase and capping enzymes contained in the virus particle (Annual Rep.
No. 25 (1974) p. 14; Urushibara, T., Furuichi, Y., Shimotohno, K.and
Miura, K.: FEBS Lett. 49 (1975) 385). The process of the cap formation
at the 5/-terminus of mRNA was studied using an in vitro system undersimilar conditions to these of vaccinia virus multiplication in its host cell.
Mter adding a methyl-group donor, [methyl-3H] S-adenosylmethionine, theoligonucleotides, which were the de novo synthesized 5/-terminal part ofmRNA, were isolated from the RNA-synthesizing virion at appropriate timeintervals, and were analyzed. The 5/-5/ confronting nucleotides with 2/-0methylation, G 5'pppAm and G5'pppGm, were found with the completed
cap structure, m 7 G 5'pppAm and m 7 G5'pppGm. The confronting nucleotides with only 7-methyl guanine as a methylated component, m 7 G5'pppAand m7 G 5'pppG, were not detected at any incubation time, and it was concluded that methylation at the 2f-position of the 5/-terminal purine nucleoside of mRNA precedes methylation of the 7-position of the blocking guanosine. This result is different from that obtained using the enzymes isolated
from vaccinia virus (Moss et al., 1976) and also from the results obtaiJ?-ed
using other kinds of virus particles, which carry RNA polymerase and capping enzymes. These differences may be due to the specific organizationof a series of capping enzymes and RNA polymerase in each virus particle.
Relation of Stability and Presence of the Cap Structure of mRNA
Kazuko YAMAGUCHI and Kin-ichiro MIURA

On incubation of mRNA in a wheat germ extract, the mRNA lacking
5/-cap quickly degraded from the 5 f-terminus in an exonucleolytic way,whereas the intact mRNA remained stable. These results show that one of
the cap functions is to stabilize the mRNA to prevent its degradation by exonuclease attack (Annual Rep. No. 27 (1976) p. 20. Shimotohno, K., Kodama,Y., Hashimoto, J. and Miura, K. Proc. Nat. Acad. Sci. USA 74: 2734
(1977)). In addition to an enzyme removing the 5/-blocked structure in
RNA, an exonuclease, which degrades mRNA from the 5 f-terminus successively, was found in wheat germ extract (Annual Rep. No. 28 (1977) p.
p. 14). As the activity of protein synthesis in a cell depends on the state
ofmRNA, enzyme activity being concerned to stability of mRNA was compared in various kinds of cell.

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

CP virus mRNA that was labeled uniformly with [3H]UTP was preparedin vitro (Annual Rep. No. 22 (1971) p. 12, Shimotohno, K. and Miura, K.:
J. Biochem. 74 (1973) 117). A part of samples was then decapped withtobacco pyrophosphatase (Annual Rep. No. 27 (1976) p. 18, Shinshi, H.,
Miwa, M., Sugimura, T., Shimotohno, K. and Miura, K.: FEBS Lett.
65 (1976) 254). Either the native mRNA or the decapped mRNA wasincubated respectively with extract from various kinds of cell to assay degradation of mRNA. There are two types for kinetics of RNA degradation. In the extracts of wheat germ, baby hamster kidney cell and erythrocyte cell in bone marrow, the decapped mRNA was degraded faster than
the native one. However, in the extracts of muscle, liver, and reticulocyte,
the native mRNA was degraded at the same rate as the decapped mRNA.
Since reticulocyte was differentiated from erythrocyte in bone marrow cell,the state of enzymes relating to the stability of mRNA may change according
to differentiation.
Presence of Single-stranded Region Adjacent to the Protein-binding
5'-termini of Adenovirus Genome Double-stranded DNA
Hiroyoshi ARIGA, Hiroto SHIMOJO, So HIDAKA
and Kin-ichiro MIURA

The structural features of a linear duplex DNA of adenovirus are an
association of a protein at the 5' -termini of either strand. The replicationof adenovirus DNA proceeds through a displacement mechanism. The
origin and terminus of replication has been localized near the moleculartermini. However, the mechanism of the start of replication at the 5 /termini remains unclear. From these points, the structure of the region
near the 5' -termini is required to be clarified.
The [oH] thymidine-labelled Adenovirus 5 (Ad 5) DNA-protein complex
was prepared. On the other hand, Ad 5 DNA-(125I] protein complex was
prepared from cold Ad 5 DNA-protein complex by Bolton-Hunter reagentof 1251. Both the samples were digested with various kinds of enzyme. The
digests were analyzed by the following methods: gel electrophoresis, filter
binding assay, Benzoylated-Naphthoylated DEAE (BND)-cellulose column
chromatography, and gel filtration using Bio-gel A 1.5 m. With deoxyribonuclease or micrococcal nuclease digestion, DNA was degraded andprotein was released. With proteinase K or pronase digestion, protein was

RESEARCHES CARRIED OUT IN 197915

degraded and DNA was released. Since no change was observed in DNAprotein complex with ribonuclease treatment, ribonucleotides do not participate in linking of protein and DNA. The protein was separated fromDNA by nuclease Sl under the conditions for single-strand cleavage. Either DNA or protein remains in intact size with this treatment. This suggests that a protein-binding 5'-terminal part of DNA is in a short singlestranded state as written below.
protein 5'

3'0,--------------------------0
3'
5' protein

The presence of the similar single-stranded stretch at the 5'-terminus was
also observed in some Bacillus subtilis phages, which carries a protein at the
5'-terminus of DNA also (Hirokawa, et al). These specific structures may
playa common role for the initiation of replication of these DNAs. This
work. was published in FEBS Lett. 107 (1979) 355-358.
Purification of T4 RNA Ligase and its Application to 5S RNA
Sequencing
Masahiro SUGIURA, Mie SUZUKI' and Fumio TAKAIWA

A new method is developed for purifying the RNA ligase induced inphage T4-infected E. coli cells. It was found that T4 RNA ligase could bindto 2', 5'-ADP Sepharose in the presence of Mg2+ but not in its absence.
2', 5'-ADP Sepharose chromatography was introduced at the last step ofthe
purification procedure. The purified RNA ligase preparation gave one
major band on SDS-acrylamide gel electrophoresis and was estimated to
be over 80 % pure. The purified enzymt; had no detectable RNase activity
and was suitable for the joining of short. oligoribonucleotides as well as
RNA sequencing.
Tobacco chloroplast 5S RNA was purified by two cycles of polyacrylamide gel electrophoresis and labeled at its3'-end with 32p using the T4
RNA ligase and [5'32P]pCp. The [3' 32 P] 5S RNA was sequenced from the
3'-end by the chemical method. On the other hand, the 5S RNA was labeled at its 5'-end with 32p using T4 polynucleotide kinase and [r32P]ATP andthen sequenced from. the 5'-end by the enzymatic method. The complete
nucleotide sequence is shown as a possible secondary structure constructed

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

UAGG
AAC C
CCA
SI:PUAUUCUGGUGUCCCGUAGAGG
ACACCAAU
Uc
3':UAGGACCGCAG
cC
UCAUCUCAGGUGGUU
C
Cu G
AAUU
AAG
C C
G GG
A
U
U
G
A
CA
A G

A A
A

U

A A
G C
G U
C G
G U
U A
C G
C G
U G

G
G
G A

according to Hori. Tobacco chloroplast 58 RNA is 121 nucleotides longand its sequence resembles that of 58 RNA from blue-green algae. T4
RNA ligase purification was carried out with E. Ohtsuka, 8. Nishikawa,
H. Uemura and M. Ikehara in Osaka University and this work was published in FEB8 Letters 97: 73-76 (1979).
Organization and Expression of the Ribosomal RNA Gene Clusterof Tobacco Chloroplasts.Kazuo SHINOZAKI, Jun KUSUDA, Fumio TAKAIWA,

Naoki TOHDOH and Masahiro SUGIURA

We have cloned tobacco chloroplast rRNA genes using plasmid pMB9
as a vector. Recombinant plasmids pTCl, pTC309, pTC7, pTCP6, andpTCP243 contain most of 168 gene, part of 168 and 238 genes, 4.58 and
58 genes, 168 and 238 genes, and all four genes, respectively. Using theseplasmid DNAs, a fine physical map of the chloroplast rDNAwas constructedby cleavage with various restriction endonucleases and hybridization of therestriction fragments with 32P-labeled 168, 238, 4.58 and 58 RNAs. The
order of these genes is 168-238-4.58-58. The precise location of the 168
and 238 rRNA .genes was determined by annealing rRNA to linear pTCP6
DNA and observing the R-loops in an electron microscope. No intervening sequences were detected in these genes. The sizes of coding regions

4.55 RNAAATTCAAGA[G I\A<JGTC,KGC, Cr.AC,ACGAGC CGHTAT(AT TACGATAGGT GTCAAGTGGA AGTGCAGTGA TGTATGCAGC TGAGGCATCC TAACAGACCG

100:;.;:Ittl

I;I.l

GfA(;AC'lTGA5TTGTleC lACATG,KCT GATCAATTCG ATCAGGCACT CGCCATCTAT TTTCATTGTT CAAATCTTTG ACAACACGAA AAAACCATTG

200

~

::0

flCA·<CTCTT TCiACAACATG AAAAAACCAA AAGCTCTGGC CTCCcTcTCT ATCTATCCAA GGGATGGAAG GGCAGAGGCC TTTGGTGTCC CCTCCAGTCA

300

g
ttl

I;I.l

()

AGAA TTGGGG CC10CAATC ACTAGCCI.A rAT GCrTnCT CTeA TGCCTT TCTTCGTTCA T6GTTCGft[TA TTCTGGTGTC CTAGGCGTAG AGGAACCACA

400

>
::0
::0
.....

55 RNAm

500

0

TAACA(CTCT CATTeTfATT A(TTTTT(AA fATGAAAACG AAAAAAAAAA AAATGAAAAA TCAAAAGGTA cGTATTTATT CAAAACCCCA ATTGATGACA

600..,c::.....

'((CTTCTCT CCCACTTCAC ACCTCGGAAC <JCACCCfTCf TATAGAGATA AACGCGCCTT CACATCTTCT TAACCCGAAA TGGCTGGGGA GAGGAAAGGT

700

T(CTTTTTTT GAGGGTACTC CCGGGAACAG ATCCAGTGGA GACGGGGTGG CC

800

CCAATCcATC CCGAACTTGG TGGTTAAACT CTACTGCGGT GACGATACTG TAGGGGAGGT CCTGCGGAAA AATAGCTCGA CGCCAGGA]G ATAAAAAGCT0....Z\0....\0....

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

for the 23S and 168 genes are l,4x 106 and 0.84x 106 daltons, respectively.
The 16S and 23S genes are separated by a 1.3 X 106 dalton spacer. There is a0,45 X 106 dalton spacer between the 23S and 4.5S genes. Using plasmid
pTC7 DNA, the DNA sequences of the 4.5S and 5S RNA genes and theirsurrounding regions were determined according to the method of Maxamand Gilbert (see the figure). The coding regions of the mature 4.5S and
5S RNAs are composed of 103 and 121 base pairs, respectively, and thetwo genes are separated by the 256 base pair spacer.
The l6S, 23S, 4.5S and 5S RNAs are coded for by the same DNA strand
and the direction of transcription is from 16S to 5S. In vitro Transcription
experiments were carried out using the 1.9 X 106 dalton fragment isolated
from pTCl and E. coli RNA polymerase. Two RNA species with distinct
sizes were synthesized and both RNAs hybridized only to the coding strand.
This suggests that E. coli RNA polymerase can transcibeproperly the chloroplast rRNA genes. Parts of this work were presented at the Ozi International Seminar on Genetics and Evolutionary Aspects of Transcriptional
and Translational Apparatus held at Tomakomai on September 1979 andwere published in Molec. Gen. Genet. 178: 1-7 (1980), Gene 10: 95-103(1980) and Japan. J. Genet. 55: 121-125 (1980).
Heterogeneity of the Ribosomal RNA Gene Clusters in RiceKiyoharu OONO and Masahiro SUGIURA

Rice nuclear DNA was isolated from rice embryos (Oryza sativa L. var.
Mangetsumochi). The DNA was digested completely with EcoRI and thefragments were separated by 0.7% agarose gel electrophoresis. Two distinct bands could be identified in an ethidium bromide stained gel and thesize of these bands was estimated to be 5.2 X 106 and 5.0 X 106 daltons. The
DNA fragments in the gel were transferred to nitrocellulose filters and hybridized with rice 25S and 17S rRNAs labeled with 32P. Both intensities
of an ethidium bromide stained photograph and of an autoradiograph in
the 5.0 X 106 dalton band were higher than those in the 5.2 X 106 dalton band.
This indicates that the two fragments are not arranged alternatively and that
there is length heterogeneity in rice rDNA populations. From relative
intensities of the two bands and the known amounts of A EcoRI fragments
as internal standard, the population of the 5.2 X 106 and 5.0 X 106 dalton
bands was estimated to be approximately 0.08 % and 0.15 % of the total

RESEARCHES CARRIED OUT IN 1979

DNA, respectively.
76: 85-89 (1980).

19This work was published in Chromosoma (Ber!.)

Structure of Harvey Murine Sarcoma Viral DNA:
Sequence of the Terminal Repeats

Nucleotide

Eiichi SOEDA and Malcolm MARTIN*

The 5.4 kbp Harvey murine sarcoma virus (Ha-MuSV) RNA genome iscomposed of a 4.5 kbp insert of rat sequences flanked on the 5' and 3' termini by 0.1 kbp and 0.9 kbp of Moloney murine leukemia virus, respectively.
The rat sequences contain transforming genes.
The supercoiled DNA intermediates were extracted from NIH 3T3 mouse
cells infected with Ha-MuSV and cloned in Agt· WES AB vector system.
Three classes of Ha-MuSV DNA inserts were isolated with 6.6, 6.0 and
TCTAGAACCATCAGACGTTTC'iAGGGTGCCCCAAGG~CCTGAAATGACCCT~TGCCTTATTTAAACTAACCMTCAGTTCGCTTCtCTAGATCTTGGTAGTCTGCAAAGGTCCCACGGGGTTCCTGGACTTTACTGGGACACGGAATAAATTTGATTGGTTAGTCAAGCGAAG

eGA

cl'V"'"''"'''~~CCGAGCTCMTilAAAGAGCCCACAACCCCTCACTCG~Ct>=AGTCCTCCGATTGAGTCGCCCGGGT-.;;.;====_GGCTCGAGTTATTTTCTCGGGTGTTGGGGAGTGAGCCGfGCGGTCAGGAGGCTAACTCAGCGGGCCCACap
3'

ACCCGTATATCC~CCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTAAGTGATTGACT~..

TGGGCATATAGGTTATTTGGGAGAACGTCAACGTAGGCTGAACACCAGAGCGACAAGGAACCCTCCCAGAGGAGATTCACTAACTGATGG

PalyA
CGTTAGTGGGGGTCTTTCAT GCGTGGACCCCGCC'rGTGGGTTGGCGTGCTGCTTGAGTAGCGCCATTTACGGGCGTGGAAAAATACATAGCAATCACCCCCAGAAAGTA CGCACCTGGGGTGGACACCCAACCGCACGACGAACTCATCGCGGTAAATGCCCGCACCTTTTTATGTAT--t-RNApro

AC"""====-·GTTCGGATCAGGG"T-=="""-""""''''''''''T''''GAA=T'''A''''T'''GG'''G'''C''''CAAA''''''''''C"'AG"'G"'M=TA"'T"'C"'T""G"'TG"'G"'T"'AG"'C"'A"'G""T"'TC"'C"'T""GC'"CAAGCCTAGTCCCA"""""-'.~""'''-Ifo.!T"" .wTC",G",A""C...
TT",A",T.(\A",CC",C",G",GTJ;.T",T",G:£TC",C...T...
T""AT",A",G",A""CA",C",C~A:£TC",G",T..
C""AA",G""G",A..
CG",

TG

GC AAGAA~r.;.G;;;A;.:T;.:A.:j.:=;;':';:'::;:'::;+;;':';:'::;:'::;==:'::;';;';;';;';':

CCCG

cc

GG

GG CCCGGTTCTT

CTAT'1i.i~~"",",~';';:;':~::;';;':";':;~~~=';';:;':"","",~~~~~l;;.G~A=",C~CC",G",G:£TT.l.:.

-nq:;GATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTCTAGA,:wfCTACCAGGGGTCTACGCCAGGTCGGGAGTCGTCAGATCTo Direct Repeats+-+- Inverted Repeats

Fig. 1. Nucleotide sequence of the terminal repeats of Ha-MuSV DNA intermediates.
* DNA Recombinant Research Unit, NIAID, NIH, Bethesda, USA.

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

5.4 kbp long. Restriction enzyme digestion and electromicroscopic examination have indicated that the 5.4,6.0 and 6.6 kbp Ha-MuSV inserts containone, two and three copies, respectively, of nucleotide sequences derived
from the termini of the RNA genomes.
We have deduced the complete nucleotide sequence of the terminal repeats by the method of Maxam and Gilbert and compared with that of
Moloney sarcoma virus DNA integrated into the mink genome (R. Dhar
personal comm.).
The terminal repeat sequence is 583 nucleotide long and identified corresponding to minus stop DNA, putative RNA promoter, CAATAAAAG,
and signal polyadenylation, AATAAA (Fig. 1). An almost perfect 68 bp
duplication is present in the region where the viral DNA is integrated into
the host genome as a provirus.

Structural Analysis of Fibroin Genes of Silkworm and its
Relative by in situ Hybridization
Jun KUSUDA, Kimiji ONIMARU, Sakae SAKATEt, Yoshiaki SUZUKI*
and Yataro TAZIMAFibroins produced by Antheraea pernyi and Antheraea yamamai are mainlycomposed of glycine and alanine as is known for those of Bombyx mori.
These two amino acids comprise more than 70% of total amino acids in
Antheraea fibroin, but contents of amino acids other than glycine and alanine vary with species.
Suzuki et aZ. assumed that the fibroin gene of Bombyx mod comprises
predominantly guanine and cytosine since corresponding codons for glycine and alanine were known to have high G+C contents.
Futher, they were able to comfirm this assumption by sequencing a partof coding region of this gene after cloning to E. coli.
From this finding, fibroin genes of Antheraea species are considered to berich in guanine and cytosine and may be homologous, to some extent, tothat of Bombyx mori. If we could determined the nucleotide sequencesof the fibroin genes of related silkworm strains, it seems feasible to estimate,
therefore, the phylogenie relationship among the fibroin genes of those silkworm species.
* National Institute of Basic Biology.
tSericultural Experimental Station.

RESEARCHES CARRIED OUT IN 1979

21

At first, we attempted to examine the homology of the fibroin gene of
Antheraea pernyi to that of Bombyx mori by in situ hybridization using thecloned DNA, as a probe, containing the fibroin gene of Bombyx mori.
The DNA extracted from posterior and middle silk glands of the 5th
instar larvae of Antheraea pernyi was completely digested with EcoRI and
separated on 1.5% agarose gel. DNA fragments were transfered to nitrocellulose filters according to Southern's method and hybridized to the cloned
DNA (pFb29) which was labelled with 82p by nick translation. Radioactive probe hybridized to single fragment with molecular weight of 2.9 x
106 daltons, but the extent of hybridization was not so completely.
From these results, it was shown that the fibroin gene of Antheraea pernyi
has some homology to that of Bombyx mori.

A New Method for the Construction of a Restriction Map Basedon End Assignment
Yutaka TAKEDA, Masao YAMADA and Yukinori HIROTA

Construction of restriction map of DNA is one of the necessary steps inthe cloning of a desired gene and in the determination of nucleotide sequence.
Many works including restriction map of DNAs have been reported. The
authors digested the DNA with many restriction enzymes, measured molecular weight of the restriction fragments, puzzled about arrangement of thefragments and then constructed the restriction map. They sometimes usedpartial·digestion methods, fragment isolation and labeling of ends of larger
fragment.
The usual method for construction of a restriction map is mainly based on
the precise measurement of molecular weight of restriction fragments. However, there is uncertainty in the measurement of molecular weight by gel electrophoresis especially in the case of the fragments with larger or lower molecular weights, because the standard curve is not linear.
If we can know which restriction enzyme produces the end of eachfragment, it will give great benefit for construction of a restriction map. We
found that the ends of restriction fragments are easily assigned to restriction
enzyme by triple digestion; We report in this communication a systematic
procedure for construction of restriction map based on the assignment of end.

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

Procedure and rule

1) Choose 3 kinds of restriction enzymes, each of which cuts the DNA
at several sites. Let us assume A, Band C as restriction enzymes, and nA,n B and ne as numbers of cutting sites of the restriction enzyme in the DNA
respectively. The restriction fragments are numbered in the decending orderof molecular weight, i.e. A-I, A-2, ..... A-n A in the case of cutting withrestriction enzyme A. DNA is digested by all the possible combinationsof 3 restriction enzymes, and run in agarose or acylamide gel electrophoresis.
For easiness of comparison of restriction fragments, we recommened to use
the slab system for gel electrophoresis. This method can be extended so
as to use 4 or more restriction enzymes, but the number of digestion markedly
increases: i. e. 7 digestions are needed for 3 enzymes, 15 for 4 enzymes and
31 for 5 enzymes. Here we show the case of using 3 kinds of restriction
enzymes. The DNA is singly digested with each of A, Band C, doubly
digested with A+B, B+C and C+A, triply digested with A+B+C, andthen the restriction fragments obtained by the above 7 different digestion
mixtures are separated by gel electrophoresis.
2) Correspond the restriction fragment appeared in triple digesion to
the fragment in single or double digestion, and then determine which restrition enzyme produces the ends of each restriction fragment taking into consideration of rules described below.
rule 1. When a circular DNA such as a plasmid DNA is digested with arestriction enzyme A, fragments as many as nA are generated.
Fragments as many as n A+n B, and n A+nB+ne are generated
when a circular DNA is doubly digested with restriction enzymes
A and B (A+B), and triply digested with A, Band C (A+B+C),
respectively.
rule 2. Fragments obtained by doubly digesting with restriction enzyme A
and B are classified into 3 with respect to the nature of the end.
One has both ends cut by A (A-A), another has both ends cut by
B (B-B), and the other has one A end and one B end (A-B). We
designate the fragment with both ends cut by the same restriction
enzyme, such as A-A and B-B, as a homofragment, and the fragment with the ends cut by difierent enzymes, such as A-B, as a heterofragment. Homofragments appearing in the double digestion
can correspond to the fragment appearing in the single digestion.
On the other hand, heterofragments do not correspond to any

RESEARCHES CARRIED OUT IN 197923

fragments appearing in single digestion. It is worthy of saying thatthe reverse of this proposition is not always true. Any fragments
obtained double digestion whose molecular weight is different fromthat in single digestion must be heterofragments, but the fragment
in double digestion whose molecular weight is the same as that insingle digestion is not always homofragment except the fragmentwith lowest molecular weight among that in double digestion.
rule 3. Fragments obtained by triple digestion with A+B+C must appearin at least one of other 6 digestions. This fact is based on the fol·lowing fact. Fragments obtained by triple digestion with A+B+C
are classified into at most 6 with respect to the nature of the ends:
i.e. A-A, B-B, C-C, A-B, B-C and C-A. If a homofragment isobtained by triple digesion, the fragment must appear not only insingle digestion but also in double digestion. For example, if a A-A
fragment appears in triple digestion with A + B+C, the same fragfragment also appears in A, A+B and A+C. because there is neither B nor C site in the A-A fragment. If a heterofragment isobtained by triple digestion (i.e. A-B), it also appears in double
digestion with A + B, but not in any other digestions.
rule 4. In each digestion, the sum of the number of ends cutted by a restriction enzyme (i.e. by A) is twice number of site cut by the restriction enzyme i.e.n A ). This rule helps you to find mistakes happened
during assignment of end of fragment to enzyme. This rule isalso useful to correspond fragments between different digestion
when salts in reaction mixture make slight divergence in the mobility of restriction fragments in gel electrophoresis.
3) Determine whether or not there are sites cut by another restriction
enzyme on the restriction fragment by single or double digestion.
rule 5. If certain fragment obtained by double digestion (i.e. A + B) is
intact after triple digestion, the fragments do not have C site. On
the other hand, if the fragment dissappeared by triple digestion,
the fragments must have more than one C sites. When the fragment has more than two C sites, at least one C-C fragment appears
in triple digestion.
4) Determine the arrangement of each restriction fragment so as to forma circle.
rule 6. Cutting by restriction enzymes produces smaller fragments than

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

original fragment. In other words, each fragment obtained bytriple digestion is derived from larger fragment appeared in single
or double digestion than this, except that the fragment is corresponded to some fragment in single or double digestion.
rule 7. The end cut by a restriction enzyme must be connected with theend of other fragment cutted by the same enzyme.
How to arrange the fragment depends on each case. It is easy way to begin
to arrange the fragments around the largest fragment in triple digestion.
Another easier way is to begin to connect the fragment at the site by the
enzyme whose cutting site is few.
Precharacterization of ends of restriction fragments is convenient marker
for their arrangement. We applied this method to determine the restriction
map of pLC 39-47, and the result will be reported in the journal (Takeda
et al. Plasmid, in press).

25

RESEARCHES CARRIED OUT IN 1979II.

MICROBIAL GENETICSA Model for Distribution of Division Sites in E. coli
Masao YAMADA, Takeo MARUYAMA and Yukinori HIROTA

When rod shaped bacterial cells, such as Escherichia coli, grow, they elongate and then divide at around the longitudinal center of the bodies. The
mechanism reponsible for site determination is one of fundamental issues
to be solved.
Mathematical model

We have advanced a mathematical model accounting for the division site,which appears to be consistent with presently available experimental facts.
The model is essentially based on GiereI' and Meinhardt's pattern formation system. (GiereI', A. and H. Meinhardt. 1972 Kybernetik 12: 30) We
assume two substances, say A and H. Substance A stimulates production of
A .Itself, and H inhibits A. Thus we may call A activator and H inhibitor.
In reaction field the two substances differ greatly in their rates of difusion:
H is a fast diffusing material and A is slow. We assume the reaction ofthese two substances is governed by the following pair of differential equations:(1)oH =CA 2 -ot

H+D oZHv"

ox2

where t and x are time and space variables, D a and D It are diffusion
coefficients, f! and v are decaying constants, and p is a de novo produc~tion rate of A. The above equations are nonlinear and difficult foranalytic solutions. As in GiereI' and. Meinhardt, we studied the behaviorof the system by numerical method using a computor. It was confirmedthat the system leads to formation of high peaks of A in the field and thepeaks are separated from each other with a certain distance. Importantly
the pattern of the peaks of A is determined entirely by the values of para-

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

meter appearing in equation (1), but independent of the initial values. In
relation to the cell division, the peaks of A can be regarded as division sites.
Experimental result

Cells of temperature sensitive filament former, Its, grow and divide normally at nonpermissive temperature as wild type cells. However, at nonpermissive temperature they cannot divide and only form long filaments.
Some of the Its mutants, such as ItsA, ItsE and ItsI, recovered the ability
to divide when cultural temperature was shifted to permissive temperature
after 1 or 2 hr preculture at nonpermissive temperature. In a viscous medium, daughter cells could not move apart, so that the site of division could
be scored by measuring each cell length in one clone. A typical division site
found in long filaments was shown in Fig. I A and B. Distance between the
septation site and the adjacent cell end is not constant but rather proportional
to total length of the clone. We obtained the filaments with various length
by varing the cultural period at nonpermissive temperature. Filamentswith double length divided at central position only. Filaments with 4-folds
length divided into either 1: 3 or 1: 2: 1. Most filaments with 8-folds divided into 1: 7, I: 6: 1, 1: 2: 4: 1 or 1: 2: 2: 2: 1. Filaments with intermediate
length between 4 and 8, divided either 4-folds type or 8-folds type. No otherdividing pattern was observed. According the extension of cell length,
the division pattern changed discontinuously.
We compared the simulation results of high peaks of A appearing in afield of certain length to the actual division sites formed in bacteria of equivalent length. A typical case of the comparison is given in Fig. 1. Incidentally, when the model was simulated for a small field where at most one
peak of A is possible, it always formed at around the middle. On the other
hand, a slightly larger field permitted to form two peaks of A and they
were at both sites 25 % of the field length apart from each adjacent end.
According the increase of the length of simulated field, the pattern changed
discontinuously. The results obtained by simulation are in agreementwith the division pattern of filament cells.
The following two assumptions imposed upon our present model seem tobe very critical. One is that at the boundary we assumed A is absorbed, but
H is reflected. This assumption is necessary for the pattern of peaks being
independent of initial condition. Another critical assumption is Da 4:..D",which is needed to form sharp and stable peaks of A. To have such peaks,

RESEARCHES CARRIED OUT IN 1979

Al::::::i:::::::

Ct::::::::::::::~'---'--'
,
,
I

,

I

I

!

:

,,

,

27

~

.

~

..
,

'--'-----'
,
,
I

,

,

,

.

,

,
,

,

,

,

,

,

!

.

,

,
,

,

,,
,
,

t
t

,
,,
!

!

,
t

,

,
I

L-,
1
,

,
,

!

t
!

!

1

,

Fig. 1. Comparison of experimental results with simulated results. Left:
Division sites observed in filament cells. Cells of PAT84 (ftsA) were incubated at
42°C for 30 min (A) or 60 min (B), and then cultured at 30°C. Division occurred 20 or 30 min later and the sites were measured under microscope. Eachhorizontal line represents one clone and dots represent division sites observed.
Right: The result of simulation by computor. A set of parameter was substituted
into the equation (1) and calculated with computor. Each horizontal line represents the length of the field and dots represent the sites of stable peak of A.

it is necessary to assume the value of D" is one hundred times of D a or greater.
It is rather difficult to assume such two kinds of molecules to have a great

difference in diffusion coefficient in the identical medium. This can however be possible if H diffuses in cytoplasm, while A in membrane.
We clearly establish the mechanism in which even fluid material determines specific site in the cell and any rigid apparatus is not necessary essential.
Transcriptional Control of Flagellar Genes in Escherichia coli K-12.
Yoshibumi KOMEDA

Previous studies have defined 29 genes necessary for the synthesis of EsThis study analyzed the transcriptional
interaction of flagellar genes using the Mu-d (Apr, lac) phage (Casadaban,cherichia coli flagellar apparatus.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

M. J. and S. N. Cohen. 1979 PNAS 76: 4530) insertion mutants. The
flagellar mutants in which the phage were inserted were operon fusion mutants of flagellar genes to the lac genes. They allowed the measurement offlagellar operon expression by detection of beta-galactosidase activity. In
these mutants, the synthesis of the enzymes occurred constitutively. The
promoters whose operons coded for constituent proteins of a flagellum
showed higher activity. The lambda transducing phages carrying thesechromosomal fla-lac fusions were also isolated. They enabled us the extensive examination of Fla- effect onto expression of each flagellar operon.
This result shows that flagellar operons are divided into six classes: (l) flbB
operon, that controls all of the other flagellar genes. (2) flaU and flbe
operons, that are controlled by the flbB operon gene products and are notresponsible for other expression. (3)flbA operon, flaG operon,flaD operon,flaN operon, flaB operon, and flaA operon, that are under flbB operon control and are responsible for other expression. (4) flaZ operon, that is controlled by the gene products of the obove (l) (3) operons and is responsible
for hag transcription. (5) "mocha" operon, flaS operon, that are controlled by the gene products of the above (1) (3) operons. (6) hag operon.
The 'Fla- effects can be converted to a scheme for interaction of flagellar
genes using following assumptions. (i) If an "A" gene defect results to failure of "B" operon expression (examined by the expression of lacZ on j("B"-Interactionofflagellar genes
flaU.flbC---'"
flbB fial

flaD. flaN. flaG flaH.
flM flaW flaY flaK
flaX flaL flaY fl a

fla Z----)0 hag

8.

flaB flaC flaO flaE.
flaA flbD moW
flaR flaQ flaP'.

~ot

ch:l

lhaS flarJ

RESEARCHES CARRIED OUT IN 197929

lac phage), the "A" product is required for transcription of the "B" operon
and the "B" operon is situated after "A" in the sequence. (ii) If"C" operon
is not expressed on hosts carrying a "D" gene defect and an "E" gene defectand "E" is situated after "D" as shown in (i), the interaction sequence isdrawn as D~E~C. The scheme of interaction of flagellar genes was thus
drawn as shown below. The assumption of (i) does not specify the interaction mechanism with molecular terms and would be accessible. In second
assumption, we currently have no way to descriminate this sequential effectfrom another mUltiple effect. This assumption has practically no problem
until we can define molecular mechanism. The sequence in the figure has
two exceptional genes, flaE and motD. They were the members of those
operons which were controlled by theflbB operon genes. Unlike the othermembers, thejiaE defect had no effect to expression of group (5) operons andthe moW defect had no effect for transcription of any of the operons at all.
Taking into account of the observations of flagellar precursors by electron
microscopy (Suzuki, T. and Y. Komeda, unpublished), the sequence obtained
as above was consistent with an assembly pathway.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

III.

BIOCHEMICAL GENETICS AND IMMUNOGENETICS

Geographical Distribution of Two Groups in Japanese Wild
Population of Medaka, Oryzias /atipes
Mitsuru SAKAIZUMI, Nobuo EGAMI1) and Kazuo MORIWAKIThe medaka, Oryzias latipes, is a freshwater fish native to Japan andadjacent region. In the previous report it was suggested that the Japanesewild population of medaka is divided into two large groups by allozyme
study (Sakaizumi et al. 1980). After that samples from several localities
were appended, and more detailed distribution of these two groups wasfound.
Adult fish were collected from 31 different localities in Japan as follows(number of samples examined in parenthesis): Aomori (8), Hirosaki (5),Niigata (3), Ryotsu (3), Joetsu (3), Kaga (5), Ichinoseki (I), Fujisawa,Iwate-ken (1), Kesen-numa (I), Iwaki (I), Urizura, Ibaraki-ken (5), Mito(3), Tsunezumi, Iabaraki-ken (3), Hokota (3), Shimodate (3), Sakura (5),
Odawara (2), Mishima (5), Shizuoka (1), Iwata (1), Toyohashi (5), Ueno(5), Tanabe (5), Okayama (5), Tottori (I), Matsue (1), Hamada (I), Tsuma,
Oki Is. (1), Tokushima (1), Kazusa, Nagasaki-ken (5), Reihoku, Amakusa
Is. (5). Enzymes in each individuals were analyzed by acrylamide slab andagarose gel electrophoresis.
Geographical distribution and allele frequency of enzymes are shown in
Table 1. This result indicates that Japanese wild population of medaka is
divided into two groups, one is the fish collected from 6 localities (Aomori,
Hirosaki, Niigata, Ryotsu, Joetsu and Kaga) in northern coast of theJapan Sea (Northern population), and the other is the fish from the rest
part of Japan (Southern population). In addition, the heterozygosity ofnorthern population is extremely low compared with that of southern population, as shown in esterases, octanol dehydrogenase (Odh) and acid phosphatase (Acp). These findings suggest that these two populations wereisolated each other in fairly ancient times.

1)

Zoological Institute, Faculty of Science, University of Tokyo

RESEARCHES CARRIED OUT IN 1979
Table 1.

..---

Observed allele frequencies at the polymorphic loci of two large
populations of Japanese wild medaka

Locus

Aci<!phosphatase(Acp)

Alcohol dehydrogenase(Adh)

Esterase-l(£s-1)

Esterase-2(Es-2)

Esterase-3(Es-3)

Octanol dehydrogenase(Odh)

Phosphoglucomutase(Pgm)

Solbitol dehydrogenase(Sdh)

Superoxide
dismutase (Sod)
1)

2)
3)
4)
5)

31

Allele
a
b
a
b
a
b
c
a
b
c
a
b
c
a
b
c
d
a
b
a
b
a
b

Northern population1)
1.0

1.0
1.0

1.0
1.0

.759
.241 5)
1.0
.056
.944
1.0

Southern population2).703.293 3).957.043 4)
.200
.645
.155
.071
.179
.750
.278
.622
.100
.064
.786
.150
.007
.993
1.0

1.0

Twenty-seven individuals from 6 localities.
Seventy individuals from 25 localities.
All of the fish from Tanabe and Okayama were Acp-b.
Adh-b was found only in the fish from Toyohashi in southern population.
Odh-d was found only in the fish from Aomori.

The Purification and Characterization of H-2 Antigens of
J apaneseWild Mouse
Toshihiko SHIROIsm, Kazuo MORIWAKI and Takuji TAKEUCHI1)

Hitherto, many investigators have tried to caracterize the molecular structures of H-2K, D gene products. Furthermore, comparison of primary&tructures of H-2 antigens between two independent haplotypes have been
1)

Biological Institute, Tohoku University Aoba-yama Sendai.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

made by trypsin-digested peptide mapping and amino acid sequence analysis, considering extraordinary H-2 porymorphism. In those studies, however, H-2 antigens were isolated only from the established laboratory mouseand European wild mouse, Mus musculus domesticus, genetically related to
the laboratory mouse. In the present study, we attempted to analyze themolecular structures of H-2 antigens of Japanese wild mouse, Mus musculus
molossinus, genetically remote from the laboratory mouse, for elucidating
the difference of H-2 molecular structures from that of the laboratory mouse.
As the sources of H-2 antigens, we used spleen cells of the following strains;
BIO.BR, BIO. D2, BlO.MOL.Sg and BlO.MOL.YkB. The latter two strainsare congenic lines in which H-2 region on chromosome 17 of Japanese wild
mouse was introduced into BIO genetic background. Alloantisera utilized
for immunoprecipitation are listed in Table 1. The procedure used for the
Table 1. Alloantisera used for immunoprecipitation
Serum designation
D-23
D-4
MS-l
MS-2

Reciepient

Donor

Antigens detected(BI0 x LP.RIII)(BI0.AKM x 129)
(BI0.D2 x C3H.NB)(BlO.D2 x C3H.NB)

BI0.A(2R)
BI0.A
BI0.MOL.Sg
BI0.MOL.YkB

H-2K.23
H-2DA
undefined
undefinedisolation and purification of H-2 antigens pricipally followed Cillen and
Schwartz (1976) with a minor modification.
SDS-polyacrylamide gel electrophoresis of the immunoprecipitates from
BlO.BR, BlO.D2 and BlO.MOL. Sg exhibited H-2-1ike major peak (Fig. 1;
a, b, c). Though both BIO.D2 and BlO.MOL. Sg showed a single peak,
it was not demonstrable on BlO.MOL. YkB precipitate, instead broad peaks
corresponding to molecular weight from 25,000 to 45,000 were observed(Fig. Id). The major peak demonstrated on BlO.BR and BIO.D2 precitates were similarly 45,000 dalton, being consistent with many other reports.
The peak of BlO.MOL. Sg precipitate was approximately 50,000 dalton,
indicating the same position as marker protein (heavy chain of human IgG)
on gel pattern. This peak is apparently H-2 gene product of BlO.MOL.Sg
strain, because alloantiserum used for the immunoprecipitation was produced by immunization in which both donor and reciepient mouse havecommon BIO background with except of H-2 region. It is, therefore, notlikely that any surface molecules contaminated the precipitate through puri-

RESEARCHES CARRIED OUT IN 1979
H
200

L

~

a

H
50 -

100

33

b

1

1

25

0
:;:

~

H
100

L

C

!

""

.rl
0

...""
I

"'

"'~

50

25

0
2.5

5.0

7.5

2.5

5.0

7 .5

OM of Gel

Fig. 1. SDS-polyacrylamide gel electrophoresis patterns of iInmunopresipitatesfrom lysates of [SH-]-leucine labelled spleen cells. H and L represent the positionsof heavy and light chains of human IgG electrophoresed on the same gels. a BI0.D2,
b BIO.BR; c BlO.MOL.Sg, d BI0. MOL. YkB.

fication procedure. These results led us to conclusion that -the molecularsize of H-2 antigen of BIO.MOL. Sg is about 50.000 dalton larger thanthat of BIO.BR and BlO.D2 and, probably, many other laboratory mice.
Since the intact H-2 antigen solubilized from cell menbrane is glycoprotein,
there are two possibilities for the difference of molecular size between two
kinds of H-2 antigens mentioned above. First, peptide part is responsible
for the difference. And second, sugar part of the antigens contributes thedisparity of the molecular size. To examine which possibility is the caseremains unsolubled.
The rason that H-2 peak was not demonstrated in BIO.MOL.YkB precipitate could not be clarified in the present study. The electrophoresis of the
precipitate showed the peaks of 35,000 and 25,000 dalton, which seem to be
two subunits of Ia antigen encoded by H-2 complex region. Interestingly,
cytotoxicity test against the BIO.MOL. YkB lymphonode cells indicated that
this strain does not carry most of the Ia antigenic specificities observed in

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

many other strains (unpublished data). It seems to be concerned with thepresent result.

H-2 Antigenic Specificities in BIO· MOL H-2
congenic Mouse Strains
Kazuo MORIWAKI, Toshihiko SHIROISm and Tomoko SAGAI

Since 1975 we have developed several BlO·MOL H-2 congenic strains
by repeated backcrosses of the mice carrying No. 17 chromosome of Japanese wild mouse (Mus musculus molossinus) to BlO·BR and BlO·D2 strains.
As some of them has cometo congenic or semicongenic condition, we tried
to determine their H-2 specificities using alloantisera produced in the laboratory inbred strains. Table 1 summarizes the reactivity of BlO·MOL H-2
congenic strains in hemagglutination test against H-2 antisera produced
in inbred strains. As already reported by us (Moriwaki et a!., J. Immunogenet. 6: 97, 1979), H-2.5 showed relatively higher activity.H-2.8 wasquite strong in these congenic strains differing from the previous population
survey. BlO· MOL Okb exhibited no activities at all. In all of those strains,
we could rarely detected higher activity of private specificities.
100

9.
V>

'"

U

"0

'"
-"

....

,.
0

50

o 2~0--ti40;--'8;:;-0--ii'16no ----,,32;r,;0:---761n0:-".-~1;;;2';;";80'--;;;25ct6no-

Antiserum dilution

Fig. 1.

RESEARCHES CARRIED OUT IN 1979

35

For the cytotoxicity test, we employed a newly developed flat plate method(Shiroishi et ai, In Methods of Immunological Experiment, in press, in Japanese). The value of cytotoxicity activity of H-2 antisera is given by thefollowing cytotoxicity index (C.I.):
C.I. = Log2 N/1O

Where N is the highest antiserum dilution that killed more than 90 %of cells.
This index is 0 at N less than 20. Fig. 1 demonstrates an example.
Cytotoxic activity against BIO' MOL H-2 congenic mice is summarizedin Table 2. Again, several public specificities as H-2.l, 3, 5, 35 and 8 werequite strong, but not many private ones were detected. It is noteworthy
that each molossinus H-2 chromosome showed positive reaction in either
one of K or D end as to the known private specificities.
Moreover, BlO· MOL Okb exhibited definitely several H-2 specificies
in spite of their complete absence in hemagglutination test. This suggeststhat type I and type II on the erythrocytic H-2 expression could be controlled by MHC.

W

0-

I~

Table 1.
H-2 antigenic specificities
Public

K,D

H_2d

H-2s

H-2r

H-2f

K
31

D
12

KID
18

D
9

3

5

28

35

8

11

25

H-2b
K
33

0
0
1
0
0

0
0
5
0
0

3
2
4
0
4

Nt
Nt
0
0
Nt

Nt
1
0
6
0

3
8
0
0
8

8
0
8
0
0

0
Nt
0
Nt
Nt

1
1
0
0
1

0
0
0
0
0

1
1
0
0
0

1
0
0
0
0

0
0
0
0
6

41)

81)

62)

51)

31)

83 )

21)

31)

24 )

Nt

4 5)

46)

68)

BlO, MOL-H-2
congenic lines
BlO.MOL. TeA
BlO, MOL. TeB
BlO.MOL.Sg
BlO. MOL. Okb
BI0. MOL. YkB
BlO congenic lines
carrying H-2 haplotypesof inbred strains

PrivateK

The value of reactivity is given by following hemagglutination index (H,I.). H.I.=Logt NII0, where N is the highest antiserum dilution that hemagglutination is observed. Nt=not tested. 1)=BlO.A, 2)=BlO.BR, 3)=BlO.M, 4)=BlO, 5)=
A.SW, 6)=BI0.RIII.

~
~

z

»-

~Z

!
o
"lj

I
Q

z

9
w

o

Table 2.
H-2 antigenic specificities
PrivatePublic

iX'
t'rI

til

BlO. MOL-H-2
congenic lines
congenic lines
BlO.MOL.TeA
BlO.MOL.TeB
BlO.MOL.Sg
BI0.MOL.Okb
BlO.MOL.YkB
BlO congenic lines
carrying H-2 haplo~
types
of inbred strains

K

K,D

H_2d
K D

H-2q
K D

H-2 k
K D

D

H-2b
K D

-

--- --- --- --- ---

H-2S
K I)

H-2(
KID D

~

-- -

::r:

H~2r

1

3

5 28

35

8

11

25

13

33

2

31

4

19 12

17

30 23

32

18

9

1
2

0
0
5
3
0

2
2
5
0
1

5
6
0
4
0

6
6
5
3
6

1
1
2
0
0

0
0
3
0
0

0
0
0
0
0

3
3
0
1
1

0
0
0
0
0

0
0
0
5
0

0
0
0
0
0

0
0
0
0
0

0
0
0
0
0

0
0
0
0
0

0
0
0
1
0

0
1
2
1
0

0
0
0
0
5

4

0
0

0
0
1
2
0

0
0
1
0
0

0
0
0
0
0

iX'

()

t'rI

til

~

iX'
iX'

t'rI

0

0

c:::
>-oj

--'"'"
Z

4

6

5

3

7 4 0
BI0.A (H-2 a )35

6 4 7 5 2 5 4 4
3 6
55(BlO) (BlO.D2) (A.SW) (DBA/l)(BlO.BR) (BlO.RIII)(BlO.M)

-.l

...,

-.l

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

Relationship between Laboratory Mice and the Subspecies
Mus musculus domesticus Based on Restriction Endonuclease
Cleavage Patterns of Mitochondrial DNA
Hiromichi YONEKAWA*, Kazuo MORIWAKI, Osamu GOTOH*, Junko WATANABE*,
Jun-Ichi HAYASHI*, Nobumoto MIYASHITA, Michel L. PETRAS**
and Yusaku TAGASHIRA*The mitochondrial DNAs of house mouse, Mus musculus in twenty-fivedifferent laboratory strains showed identical cleavage patterns on digestion
with ten different restriction endonucleases (Table 1). These cleavage
patterns were identical to those of Canadian wild mice, M. m. domesticus
but clearly different from the patterns of three Asian subspecies, M. m.
bactrianus, M. m. castaneus and M. m. molossinus (Fig. 1). Based on comparison of the cleavage maps of the four subspecies, the time of devergence
between M. m. domesticus and three Asian subspecies was estimated to be

n IT

rI

.

T1 I'i'i'IT t

iIl 1

jiI

II Ij' I.I

c2stan2US I

TIhj

II I'j' I

7ino;ossinus I

n f

fI

Tj If I25

50

domesticus I
bactrianus I

TT

I

I

0

75

T

.Tl T
I

100

% Genome Length
Fig. 1. Clevage maps for mtDNAs from four subspecies of mice Cleavage sites
for individual restriction enzymes are identified by vertical lines topped with thefollowing symbols ... BamHI, 0 EcoRI, • HindU, 0 HindIII, • Pst! and 'V HpaI.
Positions of one of HindU sites are not certain in the maps of M. m. bactrianus.
Possible sites in this. case are represented by vertical dashed lines topped with theappropriate symbols. The linear map is arranged by assuming the 0 position tothe origin of DNA reprication; length is given in percent of total genome. The
direction of DNA replication is to the right.
* Saitama Cancer Center Research Institute, Saitama-ken** University of Windsor, Canada

Table· 1. The laboratory mouse strains used, their SOurce and their restriction enzyme cleavage patterns
Strains
SourceRestriction endonuclease used
EcoRI BamHI HindU HindUI HpaI
HpaU Haell HaeIII
Pst!
InbredAKR/MsBALB/cBALB/cC57BL/6J
C57BL/6N
C57BL/I0Sn
C3H/HeN
C3Hf/Bi
CBA/H
DBA/lJ
DBA/2
RIII/2J
129/Sv-Cl-C-P

FRG
DDD

DDO
DDY
NC
KK
MutantBeige (C57BL/6J)1)
TlALD(AKR)
Closed colonies
ddN
ICR
Swiss/Ms
SL

Stm
CRJ
Yok
Stm
CRJ
Ms
CRJ
Stm
Stm
Ms
Yok
Ms
Ms
Csk
Jms
Osb
Yok
Crk
Wak

*
*
*
*

*

*
*
*
*
*

*

*

*

*

*

*
*
*

*

*

*

*
*
*

*

*
*

*

*

*
*

*

*

ND

*

ND

*
*
*

*
*

*

*

*

ND

*
*
*
*
*

*

*

ND

*

ND

*

*

*

*

*
*

*
*
*

*
*

*
*

*

ND
ND

ND
ND

ND
ND
ND

*
*

*
*

*

*

*

*
*
*

*
*
*

*

*

*

*

*
*
*
*

*

*

*

*
*

*
*
*

*
*
*
*

*

*

*
*

*

*

*

*

*

*
*

*

*

*

*

*

*

*
*

*
*

*

*

*

*

*

*

*
*

*

*

ND

*

ND

*
ND

*

*

ND

*

ND

*

ND

ND

*
*
*

ND

*

*

*
*

*
*

ND

*

ND

Stm
Stm

*
*

*

*

*

*

JCL
JCL
Stm
Stm2)

*

*

*
*
*

*
*
*
*

*

ND

*

*

*

*
ND

*

*

*

*
*

*

*
*
*
*
*
*

*
*
*

*

*

*
*
*
*
*

*
ND

*
*
*

*

*

*
*
*

SmaI

*

*

ND
ND

*
*
*

ND

*

*

ND
ND

*
*
*

ND

*

*
*
*
*
*

ND
ND

~
:;0

~

VJ

Q
~
@
o

ND

~

*

......

*

*

Z

'"
-J

'"

*

*

w

'"

* The heterogeneities were not observed in the cleavage patterns.
Original strain is shown in parenthesis.
ND; Not done
CRJ; Charls River Japan
Crk; Dr. S. Sakai, Cancer Res. Inst., Kanazawa Univ.
Csk; DrsS. Suzuki & K. Tsuji, Central Res. Lab., Chugai Pharmaceutical Co., LTD
JCL; CLEA JAPANJms; Dr. K. Suzuki, Animal Breed. Unit, Inst. Med. Sci., Univ. Tokyo.
Ms; Nat!. Inst. Genet.
Osb; Dr. J. Kawamata, Res. Inst. Microbial Dis., Osaka Univ.
Stm; Dr. H. Shisa, Dept. Pathol., Saitama Cancer Center Res. Inst.
Stm2); Dr. Y. Houma, Dept. Chemotherapy, Stm
Yok; Dr. M. Nakagawa, Dept. Vet. Sci., NIH (Japan)
Wak; Dr. M. Nishimura, Wakayama Medical College

~

1)

;I>

Z

Z

c:

;I>

r
:;e

tg

o
:;e
>-l

o

"I1

~

o

~

t"'

Z

~
~

o"I1

i
~

w
o

RESEARCHES CARRIED OUT IN 1979

41

1-2.5 X 106 years.

These results suggest that the genetic background oflaboratory mice is mainly derived from M. m. domesticus.
Evolutionary Relationships among Five Subspecies of Mus musculus
Based on Restriction Enzyme Cleavage Patterns
Hiromichi YONEKAWA*, Kazuo MORIWAKI, Osamu GOTOH*, Jin-Ichi HAYASHI*,
Junko WATANABE*, Nobumoto MIYASHITA, Michel L. PETRAS**
and Yusaku TAGASHIRA*

The intra- and intersubspecific genetic distances between five subspecies
in the species Mus musculus were estimated from comparison of restriction
enzyme cleavage patterns or maps of mitochondrial DNA (mtDNA). The
subspecies used are an European subspecies,M. m. domesticus and Asiansubspecies, M. m. bactrianus, M. m. castaneus, M. m. molossinus and M.
rn. urbanus. Although nomenclature of M. m. urbanus has not been standardized, the nomenclature of the others are widely accepted. For eachsubspecies except M. m. urbanus, at least two local races trapped in distantly!separated localities were examined. Intrasubspecific heterogeneity was
found in the cleavage patterns of mtDNAs obtained from two races of M.
m. bactrianus and M. m. castaneus. In M. m. molossinus and M. m. domestious, however, no intrasubspecific heterogeneity was detected. From thecleavage patterns of the mtDNAs, the four subspecies except M. m. urbanus
could be clearly distinguished from each other but M. m. urbanus had identi;dalcleavage patterns to those of M. m. castaneus with several enzymes (Table'1).

Quantitative estimates of genetic distances between these local races orsUbspecies were obtained by comparing cleavage maps of the mtDNA with
,N;ll.rious restriction enzymes. Nucleotide divergence of mtDNA and time'of divergence between two local races were extimated to be less than 0.4 %;a.nd less than 0.15 X 106 years in M. m. bactrianus and less than 0.3 % andsO.lx10 6 years in M. m. castaneus, respectively. On the other hand, nucleoltide divergences between European subspecies M. m. domesticus and Asian;sijbspecies M. m. bactrianus and M. m. castaneus were 7.1 % and 6.4 %,Lr~spectively. The time of divergence were calculated from these values to.;be 2.5 and 2.3 X 106 years, respectively. It was also shown that the nucleo* Saitama Cancer Center Research Institute, Saitama-ken** University of Windsor, Canada

Table l. Mus musculus subspecies used, their collection localities
and their set of restriction enzyme cleavage patterns of mtDNAs
Subspecies

Condition

M. m. domesticus
M. m. bactrianus

M. m. castaneus

M. m. molossinus

M. m. urbanus
M.musculus
subsp.

(13)
(

(

( 1)

B
C

wild population
under inbreedinginMs
under inbreeding
inMs

Bm Ec H2 H3 He2 HpI Ps Bg Hp2 He3

F.

( 3) Windsor,CanadaA( 1) Skokholm Island d ) A. England( 7) Kabul, Afghanistan C

under inbreedingN

The set of restriction
enzyme cleavage patterns&)

M.

(97)

wild population

Collection
localities(25). wild populations
SK/Cam(Inbred strain)under inbreeding b )inMs
under inbreedinginMs
under inbreedinginMs
under inbreedinginMs
wild populations
wild populations
under inbreeding·)
inNgo(Mot)wild populations

A

D

Sex
(No. used)

.j:>.

A

~
~

>
t""

A
A

A
A

A
A

A
A

A
A

A
A

A
A

A
A

-

C

B

B

B

B

B

B

B

B

0

~

trJ

"t:I

~

) Lahole, Pakistan

C

C

B

B

B

B

B

B

C

C

0

( 2)

(71) Quezon City
Philippines( 1) Taichung, Taiwan

C
C

B
B

C
C

B
B

C
C

C
C

B
B

A
A

D
D

D
E

~
....

(12)
( 4)
( 3)

(11) Mishima,Japan(3) Hakozaki, Japan(14) Tsushima,Japan

D
D
D

B
B
B

A
A
A

B
B
B

C
C
C

A
A
A

B
B
B

A
A
A

A
A
A

F
F
F

Z

(

(

D

B

A

B

C

A

B

A

A

F

C

B

C

B

- -

B

A

-

C

C

B

B

B

B

B

-

( 5)

) Yonaguni Island
Japan
Banderuwela,
Sri Lanka( 1) Kota Kinabal e )Indonesea( 1) Kota Kinabal eJ( 4) Mauritius

C
A

D
A

C
A

-

A
A

-

-

( 7)

( 3) Seychelles

A

A

A

B

- - - A A
- A A

A

-

)

)

B

B

-

-

"I1

Z

0

>
t""
....
Z

[J).

>-oj
....

~trJ

0

"I1

0

trJ

Z

~
....

(j
[J).

bJ
oj

dJ
e)

Abbreviation used are Bm; BamHI, Ec; BeoRI, H2; HindIl, H3; HindIII, He2; Haell, HpI; HpaI, Ps; Pst!, Bg;
Bfll, Hp2; Hpall and He3; HaellI. Letters refer to the types of cleavage patterns in order of discovery in eachenzymes.
Ms; National Institute of Genetics.
Ngo; Dr. K. Kondo, Nagoya University.
Gift from Dr. M. Nishimura of Wakayama Medical College.
Gift from Mr. M. Harada of Osaka City Univeristy Medical School.

Z

9
0
""

RESEARCHES CARRIED OUT IN 197943

tide divergence and time of divergence between M. m. molossinus and other
two Asian subspecies were comparable with those between M. m. molossinus and M. m. domesticus (about 3.5 % and I X 106 years, respectively).
These results suggest that M. m. molossinus is situated at a unique evolutionary position in Asian subspecies. The values for intraspecific divergenceof Mus musculus mtDNA were comparable to those of Rattus mtDNAs
but smaller than Peromyscus mtDNA.

In vitro hybridization of endosperm ADH in the Japaneseand the American species of Trillium.
Masaaki IHARA and Toru ENDO

As shown in our earlier report (Ihara & Endo 1978), at least 5 loci areresponsible for the genetic control of the endosperm ADH of the Japanese
species. They are AdhF(K\ AdhS1(T); Adh S2 (Tl, Adhs(U) and AdhF(S).
Electrophoretic patterns are explained as follows: only one homodimer(KfKf) in T. kamtschaticum (2n=1O); two homodimers (PITSI, TS2TS2)
(+)
FF

FN1
/p1

-____

N2N~

N1N1\.
S2S2\'
SlSl=

--==

__

_

-

--

;;;;.;;;=;;::;;;;;;:.:;;:;;;;;;
K

K

K KT

K

K

K

KKK

+
T

/

+

T

E

/
E

+
C

/
C

+
Ch

/
Ch

E
+
Ch

g~lN2
:iYN1S1
L:S1S2

E/
Ch

Fig. 1. Zymograms showing endosperm ADH isozymes hybridized in vitro
with a fast moving isozyme from T. kamtschaticum and slow moving isozymes. Only
major bands are drawn in some zymograms for the sake of clarity. Enzyme extracts were made from 100 g of seeds in each sample, ground separately in a mortar
and pestle with 0.4 ml of a phosphate buffer (pH=6.8) containing 0.1 M 2-mercaptoethaNol, 0.1 M NaCI and 0.4 M sucrose; then the slurry was centrifuged at 12,000 rpm
for 60 min at O°C. The supernatant thus obtained was subjected to in vitro
hybridization at-30°C for more than 12 hr, followed by a 2-4 hr dialysis against
the extracting solution except NaCI at 4°C. A starch gel electrophoresis was conducted at 250 V for 4 hr at 8°C in a refrigerator. Abbreviation: K (kamtschaticum)
T (tschonoskii), E (erectum), C (cernuum), Ch (chloroperatlum), KT (kamt. x tsch.),K +T etc. (two enzyme extracts mixed after dissociation-reassociation had takenplace), KIT etc. (a mixture of two enzymes subjected to be the dissociation-reassociation process).

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

and a heterodimer (TSlTS2) in T. tschonoskii (2n=20); two homodimers(USUS, SfSf) and a heterodimer (USS f) in T. smallii (2n=20), respectively.
Meanwhile three American species were examined· by a starch gel electrophoresis. Trillium erectum (2n= 10) and T, cernuum (2n= 10) revealed only
an anodally moving slow band, similar to the Japanese slow one (e.g., TSlTsl
and USUS). In T. chloropetalum (2n= 10) three ban.ds were detected, eachat positions between UsUS and usUf isozyme bands, between USUf and
SfSf isozyme bands and near the position of usUf isozyme band of T.
smallii. Since no segregati9n of the pattern was seen in seeds of each fruit,
the ADH isozymes are probably controlled by a compound locus. Endosperm ADH isozymes may be dimers in the Japanese as well as the American
species of Trillium, which is demon.strable by means of in vitro hybridizationtechniques. Our results are shown in Figure I, proving thedimer
hypothesis.. The slow migrating isozymes. were common in their mobility with the Japanese and the American species and evert with the species ofParis(Ihara 1979), which may provide another dimension to be worked out
from molecular evolutionary point of views.

RESEARCHES CARRIED OUT IN 1979

IV.

45

DEVELOPMENTAL GENETICS· AND SOMATIC
CELL GENETICS
In Vitro Cultivation of Gonads from a Female Sterile
Mutant/s231 of Drosophila melanogasterYukiaki KURODA

Developmental studies on gonads including germ cells may provide some
important informations· for analyzing the maternal effects of sex-linked
recessive lethal genes and the cytoplasmic factors affecting the differentiaiion of cleavage cells in early embryogenesis. In the present work, anattem.pt has been made to .culture the pupal gonads from a female sterilemutant is231 to investigate the expression of abnormality in gonads which
may cause the female sterility in their development. The mutant is231(1-22.7) was kindly provided by Dr. R.C. King, Northwestern University and maintained in a stock balanced" by FM3.
.
Dechoriollated eggs were sterilized by 70 % ethyl alcohol and transferred
to autoclavedfoods. Pupae grown under sterile conditions were dissociated in physiological salt solution to isolate gonads. Ovaries and testes
thus obtained were cultured in T-5flasks with a coverslip in their bottom
in Medium K-17 supplemented with 15 % fetal calf serum and 0.1 ,ag!ml
fetuin.
Cells migrated from the peritoneal sheath of ovaries and testes proliferated
a.ctively and formed· cell sheets around· the original explailts. In single¢ystsof primary spermatocytes liberated from the intact testes, each process
of meiosis of spermatocytes, the development of spermatids and the differ~entiation of mature sperm were normally observed. In isolated ovaries,
however, no marked differentiation of oogonia and oocytes was observed.
Since it has been reported by King (1978) that is231 ovaries contained
numerous tum.or cells, the establishment of cell lines from these tumor cells
are under investigation.

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

Effect of Cell Association on In Vitro Chondrogenesisof Mesenchyme Cells from Quail Limb-buds
Yukiaki KURODA and Etsuya MATSuTANI

Mesenchyme cells obtained from limb-buds of chick embryos can differentiate into cartilage cells in vitro. These cells from chick embryos at stage
25 or later can form cartilage colonies, whereas those from younger embryos can not. This developmental stage coincides with the stage at
which limb-bud cells acquired the stabilization of chondrogenic ability
in situ.
We studied the cartilage-forming ability of mesenchyme cells obtained
from the hind limb-buds of quail embryos at stages 21-22. In the primaryclonal cultures, only fibroblastic cell colonies were formed, but no differentiated cartilage colonies were found. To obtain the cartilage colonies
from these cells, we examined the effect of cell association on stabilizationof the cartilage-forming ability of these cells.
The mesenchyme cells were pre-incubated as monolayers, as cultureswith gyratory shaking, as precipitated pellets or as cultures with reciprocate
shaking for 4-48 hours, then the cells were .dissociated and cultured for 14
days at low cell density (103 , 2.4 X 103 , 5 X 103 cells/35 mm dish) to test theirability to form cartilage cell colonies.
In mass monolayer cultures, the cells did not form multilayers during
this pre-incubation time. When the pre-incubated cells were cultured as
low cell density for 14 days, the colonies formed consisted entirely of fibroblastic cells. In contrast, when cells had been pre-incubated for 5 days,
they had formed multilayers, then formed cartilage cell colonies in cultures
at low cell density. These results suggest that the two-dimensional cell
association in monolayer cultures may not stabilize the chondrogenic abilityof mesenchyme cells.
When cells were pre-incubated with reciprocate shaking to prevent theircell association, the small number of cell colonies were formed in cultures
at low cell density, and afew cartilage cell colonies were found exceptionallyin cultures with the initial inoculum of 5 x 103 cells per dish. In gyratory
shaking. cultures, colonies of cartilage cells appeared after a minimum preincubation time of 12 hours, and increased in number with the increase in
the pre-incubation time to a maximum (12.8±2.2 cartilage colonies/5 X 103
initial cells) after pre-incubation for 24 hours.

RESEARCHES CARRIED OUT IN 197947

Cell pellets obtained by centrifugation were pre-incubated in centrifuge
tubes, then the cells were dissociated and incubated at low cell density.
Colonies of cartilage cells appeared after a minimum pre-incubation time of
16 hours and increased to a maximum (50.4±4.9/5 x 103) in cultures ofcells pre-incubated for 48 hours.
These results indicated that the formation of cartilage colonies depended
upon the type of cell association and duration in pre-incubation period.
Gyratory shaking or pellet cultures probably mimics the process of cellcondensation in the prospective chondrogenic region in the limb-buds.
This suggests that the three-dimensional cell association for a certain period
may stabilize the ability of mesenchyme cells to differentiate into cartilage
cells.
Dose-rate Effects of Trp-P-l on Survival and Mutation
Induction in Cultured Human Diploid Cells
Yukiaki KURODA

Trp-P-I is a tryptophan pyrolysis product contained in cooking and processing fish and meat and it has a strong mutagenic activity for Salmonella
typhimurium. In the present experiment, the dose-rate effects of Trp-P-l
On survival and mutation induciion in cultured human diploid cells were
examined.
When cells were treated with Trp-P~I at various concentrations for I, 2,
3" 8, 16 and 24 hours, the survival of cells decreased depending on concentrations and exposure times. When the cytotoxic effects of Trp-P-I were
~pmpared at the same product value of concentrations and exposure times,
4igher concentrations with shorter exposure times were more effective in reQ,ucing cell survival than lower concentrations with longer exposure times.
T:he concentrations of Trp-P-I giving 50% survival which were calculatedf.~()m concentration-survival curves for exposure times of 1, 2, 3, 8, 16 and~~hours were 13.6, 6.2, 3.5, 3.1, 1.2 and 1.1 ,ug/ml, respectively.
tCells treated with Trp-P-I at various concentrations for I, 2, 4 and 24
hPurs were cultured in normal medium for the mutation expression time
qf6. days, and replated cells were selected with 30 ,ug/ml of 8-azaguanine(M-O). The frequency of 8AG-resistant mutations increased as the conq~J1trations of Trp-P-l employed for each exposure time increased. TrpP-l at higher concentrations with shorter exposure times induced 8AG-

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

resistant mutations at higher frequency than that at low concentrations withlonger exposure times.
These results indicate that the effects of Trp-P-I on survival and mutation induction in cultured human diploid cells were dose-rate dependent.
This suggests that some degradation or inactivation may occur in TrpP-I during its incubation with human cells or that human diploid cells may
have some repair activity for their chemical-induced damages, as shownin the· case of radiation-induced damages.
Analysis of the Decreased Hatchability of Eggs Laid by Flies
Reared on Excess Yeast in Drosophila melanogaster
Kiyoshi MINATO

It is known that flies fed on excess yeast lay eggs at their maximum rate.
It was, however, found that the hatchability of these eggs decreased remark-ably. The similar phenomenon has also been observed by Robertson andSang (1946). Some analytical experiments were carried out on this phenomenon.
When twenty newly eclosedflies (Oregon R; 10 females and 10 males)
were reared at 25°C on agar-cornmeal-dead yeast-sugar food, the· surfaceof which was partially covered with the thick suspension of live Baker's
yeast in 3 X 10 cm glass tube, with the daily change of food, flies laid eggs
at a very high rate (50 eggs/female/day) after a few days. But the hatchability of these eggs decreased gradually, reaching about 50 % on the tenthday.
On the fourth day (the average hatchability was 70 %), the individual
female flies were separated and examined for the hatchability of their eggs
for one day. No flies showed zero hatchability of their eggs and all flies
showed evenly lowered hatchability, indicating that the decreased hatchability was not due to the presence Of unmated female flies in the population.
In addition to the egg hatchability, the larval and pupal mortalities were
examined for ten days. It was found that the decrease in the egg hatchability after several days was accompanied with an increased rate of larval
lethality, in which· many larvae showed little motility immediately after
hatching. The above findings· suggest that the decrease in the egg hatchability in this casel11ay be due to some physiological abnormality of eggs
during development.

RESEARCHES CARRIED OUT IN 1979

49

A Normal Diploid Teratocarcinoma (OTTlOA-5) obtained
from a 6-day Embryo of BI0.A Mouse
Takehiko NOGUCHI, Choji TAYA and Kazlio MORIWAKITransformation of multipotential cells of the mouse embryo can be experimentally induced by transplanting early embryos into the adult testis(Stevens, Develop, BioI. 21: 364, 1970) or under the kidney capsule (Solter,
et aI, Nature 227: 503, 1970). When 6-day embryos were transplanted
under the kidney capsule, inducibility of teratocarcinomas was several times
lower in strain BlO.AjSgSn mice than in strain 129 mice. Low inducibilityof the experimental teratomas in the mouse has been ascribed to low permissiveness of the host environment to the growth of embryonal carcinoma
cells.
A teratocarcinoma (OTTlOA-5) was induced from a strain BlO.A bytransplanting a 6-day embryo into the scrotal testis of a BlO.A X C3H Fl
llybrid male. After 3 succesive passages in the hybrid males, this teratocarcinoma has been serially transplanted in the syngeneic, BlO.A hosts.
Transplantabilities of OTTlOA-5 in BlO.A and the hybrid males were 86%
and 56% respectively. This result suggests that a mouse strain having low
inducibility of embryo-derived teratocarcinoma by the transplantation
method do not necessarily have low permissiveness to transplantable teratocarcinomas.
Chromosome G-band patterns of the stem cells in this teratocarcinoma
did not differ significantly from normal diploid cells during 5 serial transplantation generations (for 8 months). The solid tumors contained embryonal carcinoma cells and derivatives from all three germ layers. It has
been suggested that euploidy of teratocarcinoma stem cells may be associated with developmental totipotency in the blastocyst environment (Cronmiller and Mintz, Develop. BioI. 67: 465, 1978). Stem cells of this teratoca.rcinoma may, therefore, be promissing vehicles for deliberate intro41.lction of specific mutant genes into mice via blastocyst injection method(Mintz, and Illmensee, Proc. Nat. Acad. Sci., 72: 3585, 1975).

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

The Establishment of Some Clonal Cultures of a Tetratocarcinoma
Derived from Strain BIO.A Mouse
Yoshitake NISHIMUNE*, J Aizo MATSUSHIRO*, Yuko OGIso*, Choji TAYA,
Takehiko NOGUCHI and Kazuo MORIWAKITransplantable teratocarcinoma (OTTlOA-5) cells from strain BIO.A
mouse were adapted to growth in tissue culture, and two groups of clones
were established after 3 serial cloning. One group (18 clones) was cloned
by using feeder layer cells, and the other in the medium containing 10- 4 M
f3-mercaptoethanol without feeder layer. They could form solid tumors
containing various kinds of tissues, showing the multipotentiality.
The stability of the multipotentialities and the euploidy of the cultured
clones during their long-term propagation in vitro was examined. All of
the observed clones (A2ll, A425 and A222) which have been established on
feeder layer cells have normal diploid G-band patterns and also have themultipotentiality even after continuous culture in vitro for over three months(30 generations) or after freezing in liquid nitrogen. However, the clones(Bf372 1, Bf3838) established in the medium containing f3-mercaptoethanol
without feeder layer exhibited a Slight change in the G-band patterns, though
they have kept multipotentiality.
Thus, the cultivation of the clones of teratocarcinoma on feeder layercells seems to keep the stability of karyotype, but that in the medium con·taining p-mercaptoethanol may have the trend to emerge chromosomechanges. The multipotentiality seems to be more stable charactor than
the karyotype.
Characterization of the Lethal Hybrid Rescue (Lhr) Gene of
Drosophila simulans
Tsuguhiko TAKAMURA and Takao K. WATANABE

Lethal hybrid rescue (Lhr) gene (2-95) of D. simulans (Watanabe, Jap.
J. Genet. 54:325, 1979) was studied further by crossing it to several D.
melanogaster complex chromosome lines. Watanabe (1979) reported Lhr
rescued 100 % of lethal hybrid males from the cross melanogaster ~ X Lhrsimulans(; but that it could not rescue the female hybrid of the cross XX/Y
malanogaster~ X Lhr simulans(;. We repeated the latter cross again using*

Research Institute of Microbial Deseases, Osaka University

RESEARCHES CARRIED OUT IN 197951

two different XX/Y chromosomes in order to confirm that Lhr's rescue
lictivity was limited to males and that it cannot rescue XX/YS(ys: simulans Y)
hybrids. In the first cross with C(1)DX, which was used by Watanabe,
tid XX/ys females was rescued as expected. However, in the cross withe(l)RM, 33.3 % of the progenies were XX/ys females. Further, Lhr simu·tans was crossed to X/X/Y malanogaster. In this cross, usual lethal hybrids, xm(=melanogaster X)ys, xmxmys, xmymys, were all rescued. These
data shoW that Lhr does not work male specifically.
'>'C(I)DX is known of lacking bb+ locus on both arms, and also Y chromosome of D. simulans dose not carry bb+ locus (Ritossa and Atwood, P.
NtA. S. 56: 496, 1966), while melanogaster Y carry bb+ locus. Thus, thehybrid, C(1) DX/Ys, does not carry any bb+ locus which is known to coderibosomal RNA. Therefore it was natural that the hybrid could not be
rescued by Lhr because it did not carry r-DNA at all. This was convincedb)lthe cross between Df(l)bb-/FM4 melanogastery. and Lhr simulans(;.
(Dilly FM4/Ys male was rescued and no Df(1)bb-/ys were recovered. From
ttllthe crosses so far done, following two possibilities were extracted. 1)
Hybrids carrying simulans Yare all lethal and Lhr can rescue all such letFials. 2) Hybrids carrying simulans X are all viable except female hybrid of melanogaster(; X simulans y., and Lhr rescues all the hybrids without~artying simulans X. It is not yet answered which is the correct choice.
Gonad Development and Gametogenesis in the Opposite Sexof the Silkworm, Bombyx moriHARUO YOSHIDA 1) and Akio MURAKAMIIn the silkworm, Bombyx mori L., there have been often detected sexual
mosaics or gynandromorphs. These observations suggest that sex-hormonlike substances in this insect do not take part in the expression of the sexualcll~racters. Unfortunately, functions of gametes in the gynadromorph are~R~ble to investigate by mating experiments. It has been well known factsth.~t transplanted rudimentary gonads in the opposite sex silkworm are~bp~rent1y developed regradless of the sex of host and/or donor. How~~~r, biological information on the gametogenesis in the opposite sex host$~rIllS to be somewhat meagre. The aim of this experimental series is to\(;n '

4!i1~)"Chiba University of Commerce, Nishifunabashi, Chiba-ken

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

elucidate the effects of differential physiological condition on the gametogenesis and/or sexual differentiation of gonads in the insect by the exchangeable transplantation of larval gonads.
In the present study, the gametogenesis in the opposite sex was examinedby the mating experiments. On the second day of the fifth instar larvae,
one or two rudimentary gonads (ovaries or testes) were transplanted into
the body-cavity of larvae in the opposite sex. Operated larvae were normally metamorphosed as did non-operated control insects. After eclosion,
they were crossed with the non-operated another sex moths. They werealso dissected to observe the gonads in the opposite sex. No abnormal
fertility of spermatogenic cells in males which had been received an ovaryor two ovaries was observed on the basis of either the number of eggs or thefertility of eggs laid by the mated females. The function of oocytes developed in the testis-transplanted females was also not different from the control regardless of the number of testes transplanted. These observations
led to a conclusion that the gametes in recipients which had been transplanted opposite sex gonads can be developed normally. Immature oocytesin ovaries transplanted to the male larvae were apparently developed to almost normal mature oocytes and/or eggs in size. Growth of testes in thefemale was also very much of the control.
From these findings and others, it is likely that both non-transplanted
and transplanated gonads can be developed free from different environmental condition, suggesting that sexual differentiation of gonads is notinterfered under the sexualy different condition and/or by the sexual hormon-like substances.
Characterization of js(l) MY-IS Mutant Rescued with the Transplantation of Normal Egg Cytoplasm in D. melanogaster
Masa-Aki YAMADA and Saburo NAWA

For the investigation of the interactions between nucleus and cytoplasmof egg during early development, X-linked maternal effect mutant, fs(1)MY-18, was isolated with EMS treatment. The characters of this mutant
were as follows. I) The locus of this gene was 11.3 on X chromosome.
2) The viability was normal in both sexes. This shows that this gene has
no effect on the development from larvae to adults. 3) The eggs laid by
the homozygous females were normal in shape and size but unable to deve-RESEARCHES CARRIED OUT IN 1979

53

loP into larvae even when fertilized with wild type sperms. The development of eggs was stopped at various stages of embryogenesis and in the mostof dead embryos the morphorogical abnormalities were observed in theanterior region. 4) About 2 % of embryos from eggs laid by homozygous
females were rescured with the transplantation of normal egg cytoplasm(about 1 % of egg volume), which was effective even after stored for 3 days
at - 20°C. 5) This mutant was neither auxotrophic nor temperaturesensitive. 6) When the proteins of the ovary were analyzed by O'Farrell
two-dimensional electrophoresis, the mutant ovary was deficient in one
protein among about 230 protein spots detected. The isoelectric point of
the protein was similar to vitellogenin. But its molecular weight was smaller
than that of vitellogenin. It was conteined only in the insoluble fraction ofovary lysate in 0.8 % NaCl solution, being dissolved by adding urea. But
this protein was not detected in egg and other tissues of adult. It is considered that the protein is specific to ovary and acts during the oogenesis to
produce essential components in early development. Further biochemical
analysis is in progress.

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30v.

CYTOGENETICSF 1 Hybrids between Mauritius and Oceanian Type Black Ratsand their Karyotypes
Tosihide H. YOSIDA

Mauritius type black rats were remarkable by the presence of 8 extra
small acrocentric autosomes than the other black rats. A comparative
karyotype analysis between the Mauritius and Oceanian type rats haverevealed that those extra small acrocentrics of the Mauritius rat seemed to
be originated from Robertsonian fission of two small metacentric autosome
pairs, nos. 14 and 18, occurring in the Oceanian type rat. Thus, this mechanism induced the chromosome number of the Mauritius rat to be 2n=42(Yosidaet al. 1979, Chromosoma 75: 51-62). To obtain a evidence forthe relation between these two types, cross experiments have been carriedout between the Mauritius and Oceanian type rats. Six F 1 hybrids consisting 2 females and 4 males were thus obtained in two litters. Chromosome numbers in all F 1 individuals were found to be 40 as a result of a combination of each haploid set from both parents. The F 1 metaphase showed
the constant occurrence of large metacentric M 1 and M 2 pairs, indicatingthat they are of the Oceanian origin. The presence of 7 small acrocentrics
were remarkable in the hybrids. Among them three small acrocentrics,
or pair no. 13 and the Y chromosome, were common to the both type rats,
but the remaining 4 small acrocentrics were outstanding in the hybrid specimens, which were derived from the Mauritius rat.
The G-band analysis clearly showed that four small acrocentrics were
homologous to the long and short arms of the small metacentric pair nos.
14 and 18. Each one partner of the small metacentrics of pair nos. 14
and 18 in the hybrids could be originated from the Oceanian type rat, while
the small acrocentrics would be derivatives from the Mauritius type rat.
All other chromosome pairs in the F 1 hybrids were homologous. The present hybridization experiment seems to provide somewhat confirmative
evidence for the above presumption on that the Mauritius black rats were
derived from the Oceanian type rat by chromosome fission.

RESEARCHES CARRIED OUT IN 1979

55

Nucleolus Organizer Regions (NORs) in 7 Rattus Species andtheir Differentiation and Evolution
Tosihide H. YOSIDA

Nucleolus organizer regions (NORs) has been recognized as an importantorganization in the chromosome architecture, as well as are believed to bepossible sites ofrRNA genes. The NORs of the black rat (R. rattus) and the
Norway rat (R. norvegicus) have been investigated by Yosida (1978, Proc.
Jap. Acad., 54B: 522-527). Taxonomically the above species are closely
related with apparently similar karyotypes (Yosida et al. 1965, Chromosoma
16: 70-78; Yosida and Amano 1965, Chromosoma 16: 658-667): They had
42 chromosomes with 13 acrocentric or subtelocentric autosome pairs, 7
small metacentric autosome pairs and acrocentric X and Y chromosomes.
According to him (Yosida 1973, Chromosoma 40: 285-297), the karyotypeof the Norway rat was recognized as to be derived from one of the polymorphic karyotypes in the Asian type black rat. In spite of the similarityof karyotypes in the black and Norway rats, the NORs were different between these two species with regard to their sites in the chromosomes. In
the former species they were found in pair nos. 3, 8 and 13, while in thelatter they were in pair nos. 3, 12 and 13 (corresponded to pair nos. 3, 11
and 12 in the standard karyotype nomenclature of the Norway rat). The
present paper deals with the NORs in the other 5 Rattus species, R. exlans,
R. annandalei, R. losea, R. sabanus and R. neilli, in comparison with thoseof the black and Norway rats. Among the 7 Rattus species 5 species (R.
rattus, R. norvegicus, R. exulans, R. annuandalei and R. losea) had the same
chromosome number (2n=42) with similar karyotypes. In R. exulans
the NORs were found in the centromeric regions of pair nos. 3, 5 and 13.
Interestingly the NORs occurring in pair nos. 3 and 13 were common inthese three Rattus species, but the other one chromosome with the NORs
was different from each other. It was found in pair no. 8 in R. rattus,
pair no. 12 in R. norvegenicus and pair no. 5 in R. exulans. The NORs
in R. annandalei and R. losea were characterized by being located in two
chromosome pairs. In the former species they were in pair nos. 8 and 13,
whereas the latter species showed in pair nos. 3 and 13. The karyotype of
R. sabanus and R. neilli were considerably different from the above species.
In the former species the NORs were found in centromeric resions ofpair nos. 1, 3 and 5, while in the latter species they occurred in only two

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

chromosome pairs, nos. 1 and 3. Noticeable is the fact that the NORs
were found in the distal end of pair no. 9 in these two species. Thus,
these 7 Rattus species showed the NORs in pairs 1, 3, 5, 8, 9, 12 and 13.
Among them, the NORs occurring in no. 3 were most frequently (6/7),
those in no. 13 ranked second (5/7), and the NORs occurring in the otherpairs were observed in one or two species. It is noticeable that the NORs
are located in different chromosomes in each species. This feature is apparently species specific. Based on the above findings a conclusion would
be possible that the differentiation of the nucleolus organizer regions on
chromosomes is important to species differentiation.
Chromosome Constitution of Rattus villosissimus resulted from
Robertsonian Fission
Tosihide H. YOSIDA

Chromosomes of R. villosissimus has previously been investigated by
Barverstock et al. (1977) with a suggestion of the Robertsonian fission without banding analysis. In the present study the karyotype of this animal
was reexamined by the conventional and G-banding karyotype analysis
with a special attention toward a karyotypic comparison between this species
and Mauritius type black rat with the Robertosonian fission. Rattus villossimus (R. sordius villosissimus), were obtained through the courtesy of Dr.
C.H.S. Watts from Asutralia in 1972 and they had been bred in the author's laboratory.
The chromosome number of this species was determined as 2n=50 whichcontained two large metacentrics which were derived from the Robertsonian
fusion of acrocentric pair nos. 2 and 3 and also of acrocentric pair nos. 6
and 10. The karyotype peculiarity of this species was peculiar in the Rattus
species, because 26 small acrocentrics and two small metacentrics werealways included in the complement. Of the 26 small acrocentrics, two wereof pair no. 13, but the remaining 24 chromosomes (12 pairs) were suggested
to be derived from the centromeric fission of 6 small metacentric pairs (nos.
14 to 19). The other two small metacentrics were located at the pair no.20.
Under this situation, the karyotype of this specis is to correspond to thebasic karyotype of the Rattus species. Based on the arrangements of chrosomes due to the G-band characteristics it was revealed that twelve extra
small acrocentric pairs just corresponded in their banding patterns to eachRESEARCHES CARRIED OUT IN 197957

member of 6 small metacentric pairs (nos. 14 to 19) of the basic Rattus
karyotype.
In the course of cytogenetical studies of the black rats, we (Yosida et al.
1979a, b, Yosida 1979) have chanced to find the chromosome-fission mechanism in the Mauritius black rats. Basically they had the Oceanian karyotype but were exceptional by having extra 8 small acrocentrics (4 pairs),
which could have been resulted from the Robertsonian fission of two small
metacentric pair nos. 14 and 18 originally presented in the Oceanian typeblack rat. The karyotype comparison between the Mauritius type blackrat (R. rattus) and R. villosissimus revealed a similarity in their basic constitution. They had two large metacentric chromosomes, although the original elements forming two large metacentrics were dissimilar between two
species, and the number of extra small acrocentrics occurred by Robertsonian fission of small metacentrics. In the black rat the dissociation occurred in two metacentric pairs (nos. 14 and 18), whereas in the R. villosissimus it was taken place in six metacentric pairs (nos. 14 to 19). Based on thecomparative analysis of karyotypes among the Rattus species it is possible
to state that the Robertsonian fusion mechanism plays an important rolein karyotype evolution, but the Robertsonian fission takes also a part inevolutional changes of the karyotype and species.
A consideration on Relation between the Karyotype Evolution

and the Mutagenic Environment
Tosihide H. YOSIDA and B. B. PARIDA1)

It is widely accepted that structural rearrangements in chromosomes
have played a primary role in the development of variation of species. In
the process of speciation and evolution, two events, one being at the genic
level, and the other at the chromosomal level, are to be taken into consideration. The latter event deals primarily with changes of the phenotypicalkaryotype as a result of inversion translocation and others, and also those of
the inner-structural karyotypes revealed by heterochromatin, the nucleolusorganizer regions (NORs) and some others. Evidence favoring the species
differentiation at chromosomal level has been availably providing recent
karyological studies in three mammalian species; the black rat (Rattus
rattus), the Indian spiny mouse (Mus platythrix) and the Indian muntjac
1)

Visiting scientist from UtkaI University, India.

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30(Muntiacus munjak). All the species under consideration have widely been
distributing in Indian subcontinents. It seems very likely cytogeneticallythat their karyological changes in relation to evolution have occurres insouthern India. It has been shown that in the black rats, the Ceylonese typerats having 2n=40 and the Oceanian type rats having 2n=38 had occurredin the southern India in association with the sequential Robertsonian fusionof certain chromosomes in the Asian type black rats with 2n=42 (Yosida
et al. 1974, Chromosoma 45: 99-109). In the Indian spiny mouse (2n=26),
the mechanism of tandem fusion between two elements of certain acrocentrics occurring in the house mouse (Mus musculus) having 2n=40 seemed
to take part in their development (Yosida 1979, Proc. Jap. Acad. 55B:
270-274). The evolutional change of the Indian muntjac with 2n=6 (~)
from the Reeve's muntjac (Muntiacus reevesi) with 2n=46 has tentatively
understood as a sequence of tandem fusion of certain acrocentrics (Wurster and Benirschke 1970, Science 168: 1364-1366). To know the mechanism of such karyotype changes concentrately occurring in the Indian subcontinents is of utmost biological importance. Theoretically it seems probable that the changes have occurred in association with certain environmental mutagens. Such considerations are to be presented in the present
paper on karyotype evolution, species differentiation and environmental
mutagen with particular reference to the karyotypic rearrangements in thethree species of Indian mammals concerned here.
Three examples mentaioned above clearly indicate that the karyotype evolution, after a long duration, ultimately leads to subspecies and species differentiation. Evidence is interesting showing that the evolutionary events
concerned here are those occurring in the Indian subcontinent, especiallyin South India. Then a question arises as to factor or factors in India,
particularly environmental, which influenced the germ cells to cause thekaryotype evolution leading to species differentiation. There exist a few
areas in the world which embrace a high natural radioactivity. One of theseareas is said to occur in the Kerala State of South India (United Nations
Report, 1966). The carrier of the radioactivity is known to be black monazite sand in the coast, which is derived from igneous rock-minerals containing thorium phosphate. Thorium is known as a radioactive element
carrying a very long life (1.39 X 1010 years). This type of monazite sand is
known to occur in various concentrations in other parts of South India
including coastal areas of the Bay of Bengal like Gopalpur in Orissa State.

RESEARCHES CARRIED OUT IN 197959

Based on the above discussion on the karyotype evolution in the blackrats, Indian spiny mouse and Indian muntjac it makes possible that India,
particularly South India, might had been a platform where cytogeneticalevents such as chromosomal breakages, fusion, inversion and translocation
had taken place, probably under the natural mutagens. For the decided
statements the further studies should be demanded on mammalian faunain the above areas.
Finding of 1/12 Translocation in the Lewis-strain Rat
Tosihide H. YOSIDA

The present paper describes the translocation chromosomes observedin the Lewis-strain rat, along with data on its inheritance to their offspring.
Among six Lewis strain rats (3~ and 35' examined), 5 rats (2~ and 35')
had 42 chromosomes having the normal karyotype characteristic of the rat,
while the remaining one female rat was remarkable by having the heteromorphic pair no. 1. The short arm of one member of the pair no. 1 wasnormal in length, whereas that of the othe member was constantly longer
slightly than the normal partner. This rat had also a heteromorphic pairno. 12. The normal partner of the pair 12 was small subtelocentric, but
the other partner was autstanding in being very small metacentric. The
small metacentric pair 12 seemed to be derived from the deletion of thelonger arm of the original subtelocentrics. The brocken end of the pairno. 12 thus formed seemed to be translocated to the short arm of one partner
ofthe pair no. 1. The above relation between the pairs 1 and 12 was clearly
defined by G-banding staining of these chromosome pairs.
Tn a cross of the translocation female rat with the normal male, 17 rats
from 3 litters were obtained. Among them 9 rats showed the normal homologous pair nos. 1 and 12, while the remaining 8 rats had the 1/12 translocation. The rats with the 1/12 translocation and those with the normal
karyotype appeared in almost the same ratio in number as expected. The
partner of the chromosome with 1/12 translocation was normal as describedabove, but only a single rat was exceptional by showing mosaic cells; oneof them had the normal partner to the 1/12 translocation as described above,
whereas the other was characterized by the inversion partner. Details of
the mosaic rat will be reported later.

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

Evolutionary Aspects of Mitochondrial DNAs in variant Types ofBlack and Norway Rats
Jun-Ichi HAYASHI!" Hiromichi YONEKAWA1l, Osamu GOTOH1),
Yusaku TAGASHIRA1), Kazuo MORIWAKI and
Tosihide H. YOSIDA

Mitochondrial DNAs (mtDNAs) were prepared from varuous inbred
and wild Norway rats, Rattus norvegicus, and from three types of black rats,
Rattus rattus, (Asian type, Ceyloness type and Oceanian type). Intraand interspecies divergence of their mtDNA sequences were calculated
based on changes in restriction endonuclease cleavage sites. The extent
of intraspecies divergence of black rats (about 8 %) is much larger than thatof Norway rats (l %) and the mtDNA of Asian-type black rats resembles
the mtDNA of Norway rats more closely than it resembles the mtDNA ofother types of black rats. These results strongly suggest that during thecourse of intraspecies differentiation of black rats, probably long after
the separation of the three types of black rats, some Asian-type black rats
were isolated sexually and formed a new species, Norway rats. On thebasis of our observations we propose a hypothetical process to explain theevolution of animal mtDNA.
Comparative Studies on Release of Enzymes from Platelets during
Blood Clotting in the Black and Norway Rats
Toshihiro EMORI 2), Masakazu T AKAHASHI2 ), Sumi NAGASE2)
and Tosihide H. YOSIDA

Increase oflactate dehydrogenase (ECI. I. I. 28) and creatine kinase (EC2.7. 3. 2) in serum and plasma level during blood clotting was compared in
the black rat (R. rattusfiavipectus) and the Norway rat (R. norvegicus, JCLSD strain). Both the enzyme level increased by the release from platelets
in process of clotting in rat as previously described (Emori T. et ai. 1978,
Exp. Animals 27, 168), but the change in black rat was very slight.
Owing to search for the reason to prove the difference in this phenomenon,
physiological and chemical properties of blood from both the animals werecompared, and it was demonstrated that the. content of plasma fibrinogenin the black rat is higher than that in the Norway rat, while the platelet
1)

2)

Department of Biochemistry, Saitama Cancer Center Research Institute.
Sasaki Institute, Surugadai, Chiyoda-ku, Tokyo.

RESEARCHES CARRIED OUT IN 197961

number contained in each ml of blood and the enzyme activities in eachplatelet are not different between them. The analysis of the membraneproteins from platelets of both the animals by SDS-polyacrylamide gelelectrophoresis showed that the fractional value of glycoproteins was foundto be different between them.
Establishment of Cell Lines from Millardia meltada andExamination of Endogenous Virus
Haruo SUGIYAMA1!, Masuo YUTSUD0 1), Kumao TOYOSHIMA1J,
Tosihide H. YOSIDA and Yoshiaki MURATA2 )Millardia meltada, a member of Muridae, is a wild rodent found in fields
in India, and this rat was established as a useful laboratory animal by Yosida,
one of the present author (Yosida 1978, Lab. Anim. 12: 73-77). It is intermediate in size between a mouse and a rat, and its diploid cells have 50
chromosomes. It is still uncertain whether M. meltada is more closely re"lated to mice or to rats. There are, of course, no tumor virlogical observations on M. meltada at all. If M.meltada is to be used as a laboratory
animal for cancer research, it is especially interesting to know whether it
has endogenous type-C RNA virus, because various classes of oncovirus
have been isolated not only from laboratory rodents but also from wild
ones. The object of this work was (1) to establish cell lines from Millardia
embryos, (2) to induce endogenous virus from Millardia cells, and (3) toexamine whether Millardia cell DNA has any nucleotide sequence homologous with murine leukemia virus (MuLV) and rat endogenous virus.
For the above studies four cell lines were established from this animals;
an untransformed cell line (MM-D), a non"producer (NP) cell line trans~
formed with murine sarcoma virus (MM-CL4), and SV40-transformed
cell lines (MM-8, MM-663). The MM-D line is epithelioid and contactinhibited, whereas MM-CL4, MM-8, and MM-663 lines are fibroblastic
aNd grow piling up. No endogenous virus was induced in these cell lines
by a long-term culture with various inducers. Millardia cell DNA has
no nucleotide sequence homology with eDNA of either murine leukemia
virus or rat endogenous virus.
1)
Department of Tumor Viruses, Research Institute for Microbial Diseases, Osaka
University.
2)
Department of Parasitology, Nara Medical University.

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

Acquired Resistance and Immune Responses of Eight Rat Strains to
Infection with Angiostrongylus cantonensis
Kentaro YOSHIMURA1>, Hiroko AIBA1), Nakami HIRAYAMA2)
and Tosihide H. YOSIDA

Strain-dependent differences in susceptiability to a wide variety of parasitic infections have been found in various experimental animal models.
These differences are thought to be possibly determined by hostgenetic
factors among which immune responsiveness would be one of the most important components. However, only limited information is available ona possible association between the host susceptibility and immune responsiveness. Up to the present, however, no specific data are available on not
only differences in susceptibility to Angiostrongylus cantonensis of different
inbred rat strains but also the functional relationship between the host immune responses and susceptibiluty. As a prelude to such an exploration,
the present study was attempted to compare the susceptibility and immune
responses of eight inbred strains of rats, ACI (F102), BUF (F66), LEW9
(F17), NIG(F33), Tokyo (F50), WKA(F202), WKS(F14) and WjMs (F71),
to infection with A. cantonensis. Consequently, it was found that both
BUF and ACI strains acquired less protective immunity than other strains
and, conversely, Tokyo and NIF strains yielded higher resistance. The remaining strains showed various intermediate degrees of acquired resistance.
Striking strain differences in pattern of reagin production were found; Tokyo
was a high responder while ACI was a low responder. In contrast, thekinetics of indirect hemagglutination (IHA) antibody response was essentially the same in all the strains examined. The IHA antibody titers weregenerally high in both Tokyo and NIG. Histopathologically, ACI was
distinct from NIF in that fibrotic reactions around the nodules of developing
eggs in the lung parenchyma were almost undiscernible. These data suggestthat genetic factors would be involved in acquired resistance and reaginic
antibody responses to A. cantonensis infection in rats.

1)
Department of Parasitology, Iuntendo University School of Medicine, Bunkyo-ku,
Tokyo.
2)
Department of Parasitology, Azabu Veterinary College, Fuchinobe, Sagamiharashi, Kanagawa.

RESEARCHES CARRIED OUT IN 1979

63

Karyotypes of Four Carangid Fishes
Makoto MUROFUSHI1) and Tosihide H. YOSlDA

The present paper deals with the cytogenetical study of four carangid
fish species, Trachurus japonicus, Caranx equals, C. sexfasciatus, and Alectis
cilialis, obtained in the west coast of Izu Peninsula, Shizuoka Prefecture.
Diploid chromosome numbers from kidney cells in the four carangid fish~pecies were all 48. The karyotype of Alectis cilialis was the simplest form
consisting of all acrocentric pairs ranging from large to small in size. On
the other hand, the karyotypes of two Caranx species (C. equula and C.
sexfasciatus) were similar to the above species, except the largest pair (no. 1)
which was characterized by subtelocentrics. The other 23 pairs of these
species were acrocentrics. The remaining one species (Trachurus japonicus)
had the same chromosome number as the above three, but the karyotype
was markedly different from the other species. Fifteen pairs (nos. 1 to 15)
were biarmed chromosomes. Among them two pairs (nos. 2 and 11) weremetacentrics, and seven pairs (nos. 1, 6, 10, 12, 13, 14 and 15) were submetacentrics, and six pairs (nos. 3, 4, 5, 7, 8, and 9) were subtelocentrics. The
remaining nine pairs (nos. 16 to 24) were acrocentrics, although very shortarms were sometimes found in some chromosome pairs (nos. 16,20 and 22).
Sex chromosomes could not be identified in any of the four species examined.
Based on the karyotype characteristics it is clearly shown that two species(Caranx equula and C. sexfasciatas) with one subtelocentric (pair no. 1)
seems to be derived from Trachurus japonicus with the acrocentric pair bypericentic inversion. The third type of carangid species, Trachurus japonicus, which had fifteen biarmed pairs, might have been resulted from theinversion of the other acrocentric pairs included in the former species. Fromthe view point of the karyotype analysis, three species of Alectis and Carax
seemed to be closely related, but Trachurus is rather far from the above three.
High Incidence of Urethan-induced Pulmonary Tumor in theIntersubspecies Hybrids between Laboratory Mouse
and Japanese Wild Mouse
Kazuo MORIWAKI, Toshihiko SHlROlSHl and Nobumoto MIYASHlTA

Genetic regulatory systems governing developmental processes in mammal)

Faculty of Misima Junior College, Nihon University, Misima.

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

Iian forms seem to be considerably stable. In these systems, regulatory
genes and structural genes are possibly couple quite precisely which could
not allow the occurrence of viable mutations. In the intersubspecies hybrids (A X B), however, it is possibly assumed that a regulatory gene derived
from A subspecies can not completely control a structural gene from B subspecies and vise versa, if the divergence time between the two subspecies isfairly large. This is sometimes favorable for the development of tumors.
We have already estimated the divergence time between Japanese wild mouse,
Mus musculus molossinus, and the laboratory inbred mouse, most probably
Table 1.
Mousestrains

Number ofmouse observed

Pulmonary nodles
per mouse

ICR(ICR x MOL. TEN)F1
Mol. Ten1825

2.7±0.88
5.8±2.08
0.2±0.34

7

Urethan treatment: Subcutaneous injection, 1.0 mg!g body weight, 17-19th day ofgestation.
Scoring of nodules: 5 months age.

1.0

BALBJc

"'"'w
u
'"
W
P<

(I)

Z

o
H

E-<

."

'"

'"cow

0.5

."

W

~
o

U)

g
""

u

12

24

36

48

72

96

120

HOURS AFTER URETHAN TREATMENT

Fig. 1.

168

RESEARCHES CARRIED OUT IN 1979

65

Table 2.
Chromosome breaks per cell after
urethan treatment

Mousestrain-BALBjc
C. TenF1
Mol. Ten
B. Ten F l
C57BLjlO
Urethan dose:

0

12

24

36

48 (hr)

0.0
0.0
0.0

0.10
0.20
0.34
0.20
0.28

0.55
0.69
1.59
1.06
9.72

0.02
0.09
0.72
0.11
0.11

0.06
0.04

0.0

1.5 mgjgB.W. Age: 3 weeks.

originated from M. m. domesticus, roughly 1-2 X 106 years.
In the present study, molossinus mouse strain Mol. Ten, the laboratory
strain ICR and their hybrids were used for the transplacental administrationof 10 % urethan solution (1 mgjg B: W.) at around 17-19 days of gestation.
Five months after birth, the number of pulmonary tumor foci was scored.
Table 1 summarizes the results demonstrating a significantly higher susceptibility of F 1 hybrid to urethan-induced pulmonary tumor.
In order to examine whether such a high susceptibility to urethan in the
hybrids as shown above is related to the changes of genetic materials in theearlier stage of urethan action in vivo or not, we observed chromosome
aberrations in the bone marrow cells at the several points shortly after theurethan administration. In this experiment, we employed 3 weeks youngs
and urethan dose of 1.5 mgjg B.W. Chromosome breaks per cell werescored at 12, 24, 36 and 48 hours after subcutaneous urethan injection. As
shown in Fig. 1, a peak of chromosome bleaks sharply appears at 24 hours.
Comparing the effect of urethan on the chromosome aberrations at thisstage, we could not recognize any augmentation of them in the F 1 hybrids
between either BALBjc X Mol. Ten and C57BLjlO X Mol. Ten (Table 2).
This may suggest that the higher incidence of pulmonary tumors in the F 1
hybrids is probably caused by the later stage of tumor development.

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

Urethan-Induced Chromosome Aberrations in Various Mouse
Strains Suggesting an Inverse Relationship between
Chromosome Changes and Tumorigenesis
Kazuo MORIWAKI, Toshihiko SHIROIsm, Nobumoto MIYASHITA
and Tomoko SAGAI

It has been already known that A and BALB/c strains are highly susceptible to urethan-induced pulmonary tumor and C57BL is lowly susceptible.
Japanese wild mice seem to be quite resistance to the urethan action which
are demonstrated in this Ann. Rep. (Moriwaki et al.). Comparing the frequency of chromosome breaks in their bone marrow cells 24 hours after
the urethan administration, we obtained the results suggesting an inverse
relationship between chromosome aberration and tumorigenesis (Table 1).
As this result seems to be contradictiory to the present understanding on
the relation between chromosome changes and tumor development, we
examined the error-prone DNA repair which was said to probably concern
with tumorigenesis (Kondo, 1977). Treatment with caffeine, an inhibitor
Table 1.

0

12

24

36 (hr)Urethaninducedtumorigenesis

A/J
0.0
0.0

0.10
0.28

0.55
0.55
0.72
0.89

0.02
0.11

+++
++
+

0.0

0.34

1.56

0.72

±
±

Chromosome breaks per cell
Mouse strain
A/J
BALB/cC57BL/1O
Mol. Msm
Mol. Ten
Urethan dose:

1.5mg/gB.W.. Age: 3 weeks.
Table 2.

Treatment
ControlCaffeine
UrethanUrethan +caffeine

Aberrant cell percentage 24 hours
after urethan and caffeine treatment
0.00
0.01
19.7±7.923.9±8.3

Urethan dose: 1.5 mg/gB.W.
Caffeine dose: 0.1 mg/gB.W. at 0.8 and 16 hours after urethan injection, s.c.

RESEARCHES CARRIED OUT IN 197967

of error-prone DNA repair, could somewhat increase theurethan-induced
chromosome aberrations (Table 2).
This finding might imply that on the carcinogen treatment, error-prone
DNA repair introduce elevated tumorigenesis on one hand, but on the other
hand seemingly decreased chromosome aberrations by the DNA repair
even if it is error-prone.
On the Precocious XY Separation at the First Meiotic
Metaphase of Mouse Spermatocytes
Yoichi MATSUDA, Hirotami T. IMAI, Kazuo MORIWAKI,
and Kyoji KONDO")

Recently we found an unique phenomenon that the X and Y chromosomes
separate highly frequently (ca. 70 %) at meiotic metaphase I of the F 1 hybrid males between laboratory mice (BALBjc) and Japanese wild mice (Musmusculus molossinus). The same phenomenon was also observed in theF1 hybrids between BALBjc and various subspceis M. m. castaneus, M. m.
bactrianus, and M. m. urbanus (Table 1). On the other hand, the frequenciesof the XY separation were only 21.1 % in Japanese wild mice and 12.0 %inBALBjc. These observations suggest that the highly frequent separation of the XY is characteristic to the F 1 hybrids between subspecies in the
Table 1. Frequencies of the precociously separatedXY at the first meiotic
metaphase of spermatocytes in the wild and laboratory strain miceand their F I hybrids
Materials used
Japanese wild mice (M. m. molossinlls)
Laboratory mouse strain (BALB/c)
F I hybrids
BALB/c x M. m. molossinlls
BALB/c x M. m. castaneliSBALB/c x M. m. bactrianlls
BALB/c x M. m. llrbanlls
BALB/c x M. m. domesticlls

Number ofindividuals
examined

Number of% of thecells
separated XY
observed

30
18

3804
3175

21.1±21.6
12.0± 4.5

36
8
5
6
12

5324
800
628
722
1491

73.2± 9.6
81.9± 1.1
78.2± 6.4
67.3:1: 3.9
6.5± 2.3

1)Department of Animal Breeding, Faculty of Agriculture, Nagoya University,
Chikusaku, Nagoya

68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

.!!!

ID

.~100

..x
I1l

:a
1;j

••

>.
x

•
• ••
• ••••
•
• .:
•

....

.,."•••
• •
,

~ 00

50

••

•
100

"10 of the separated X. Y at metaphase I
Fig. 1. Correlation of the XY separation in spermatocytes compared between diakinesis and metaphase I.

genus Mus. In connection with this, one exceptional case was observedin the F i hybrids between BALBjc and M. m. domesticus, where the value
was extremely low (6.5 %). Such exceptional data may be consistently
interpretable by the generally accepted assumption that the laboratory inbred mouse strains probably originated from the European wild mice (M.
m. domesticus).
To know the initial stage of the XY separation, we compared the frequency of the separated X and Y chromosomes between diakinesis andmetaphase' 1. As demonstrated in figure I, there is a clear tendency thatthose individuals showing high separation values at metaphase I also showhigh values at diakinesis, though the latter is lower (about half) than theformer in most cases. These data suggest that the XY separation strats
at least at early diakinesis and accelerates during metaphase 1. Besides
it is well known fact that the X and Y chromosomes usually separate completely during the first meiotic anaphase stage. Therefore, we conclude
that the XY separation we found falls under the category of "precocious
separation", but does not mean non-pairing of the sex chromosomes at
zygotene stage.

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

VI.

69

MUTATION AND MUTAGENESIS IN ANIMALSIn Vitro Complementation of Repair Enzyme Activities in
Cellular Extracts from Ataxia Telangiectasia Fibroblasts
Tadashi INOUE, Akiko YOKOlYAMA and Tsuneo KADA

Ataxia telangiectasia is an autosomal recessive disease of man characterized
by telangiectasia, immunodeficiency, cancer-proneness, and hyper-sensititivity to ionizing radiation. We previously suggested that the hypersensitivity of ataxia cells to ionizing radiation may be ascribed to the deficiency
in some kind of repair enzymes, which are assayed in a newly developed
system using gamma-irradiated colicin £1 DNA and purified· DNA polymerase I.
In the present study, we applied this system to examine whether the extracts from various ataxia strains complement each other in vitro. The
strain used were AT3BI, AT5BI and AT4. AT4 was kindly donated by
Prof. Sasaki of Kyoto University. The experiments yielded the results that
AT3BI and AT4 complemented each other as well as AT3BI and AT5BI.
However, AT5BI and AT4 did not complement. These results suggestthat there are at least tow complementation groups in ataxia telangiectasia;
one contains AT3BI and AT4, and another contains AT5BI.
Human Enzymes Functioning in Repair of Gamma-Ray-Induced
DNA Damage
Tadashi INOUE and Tsuneo KADA

Many kinds of enzymes are though to be functioning in repair of DNA
damage especially of those which induced by ionizing radiation, since theradiation produces various types of lesion in base and sugar moieties aswell
as scissions in the phosphodiester linkage. In order to purifiy and characterize the repair enzymes, we developed an assay system for repair enzymes
in which the capacity of cellular extracts to enhance to priming activity of
gamma-irradiated DNA for purified DNA polymerase was measured. Application of this system to the cellular extracts of fibroblasts from patientswith genetic disorder ataxia telangiectasia revealed that the ataxia cells

70

RESEARCHES CARRIED OUT IN 1979

are deficient in some kind of repair enzyme activity (Biochim. Biophys,
Acta 479: 497). Ataxia telangiectasia is an autosomal recessive diseasecharacterized by hypersensitivity to ionizing radiation, telangiectasia, immunodeficiency and an increased frequency of malignancy. It is thereforeof interest to examine the relationship between these symptoms and enzymatic defect. Tissue culture cells, however, provide too little materials
for extensive enzymatic characterization, so we have thus turn to humanplacenta to study further the physical and catalytic properties of humanrepair enzymes.
The placental repair enzyme activity was separated into several distinct
fractions by means of column chromatographies. One of them was purified to approximately 400-fold by successive application of ammonium
sulfate fractionation, DEAE-cellulose column chromatography, gel-filtration and affinity chromatography on gamma-irradiated DNA-cellulose.
Though the preparation is not yet homogeneous, it significantly enhanced
the priming activity of gamma-irradiated DNA without any effect on unirradiated DNA. Neither phosphatase nor exonuclease activity was detectedin the preparation. The preparation exhibited endonucleolytic activity
to aCid/heat-treated DNA. This activity was detected only when Mg2+
was present in the reaction mixture.
The MutageDic Activity of Pyrrolizidine Alkaloids in
the Silkworm Germ-Cells
Akio MURAKAMI, Tsutomu FURUYA1) and Toshiko OZAWA

Pyrrolizidine alkaloids are widely distributed in plants. Many pyrrolizidine alkaloids are carcinogenic or carcinostatic in experimental animals
and mutagenic in various organisms including Drosophila. In silkworm
Bombyx mori, no information on the mutagenicity of the alkaloid has beenreported. The purpose of the present experiment was to investigate themutagenic activity of pyrrolizidine alkaloids in Bombyx: namely to test
whether or not the alkaloids induce recessive visible mutations in the pupal
germ-cells by the egg-color specific locus method.
The pyrrolizidine alkaloids tested in this experiment were clivorine, ligularidine and seneciphylline. The first two alkaloids were extracted from Li1)
School of Pharmaceutical Sciences, Kitasato University, Shirogane, Minato-ku,
Tokyo 108.

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

71

gularia dentate and the last one was from Sececio cannabifolius. Each com-pound was suspended in 0.5 %carboxyl methyl cellulose (CMC) with 0.85 %NaCl solution and were administered by injection into the abdomen of wildtype (CI08xAojuku)F 1 silkworms at the mid-stage pupa. They were thenll1ater individually to the marker stock homozygous for pe and re loci toll1easure the frequency of egg-color mutations. Seven concentrations ofclivorine were used: 0.001, 0.01, 0.1. 1.0, 10.0, 20.0 and 40.0 pg per capita.
Ligularidine was tested at two concentrations of 20 and 40 pg/capita andseneciphylline was tested only at a concentration of 20 pg/capita. A
0.025 ml of 0.5 % CMC solution per capita was applied to control.
The treatment of pupal oocytes and spermatozoa with clivorine at lower
concentrations ranged from 0.001 to 1.0 pg per pupa did not induce the eggcolor specific locus mutations. On the contrary, when applied high dosesof 10, 20 and 40 pg of clivorine per capita, a significant increase in the frequency of the egg-color mutations was observed. The non-induction of
mutations by the alkaloid at lower doses may be due to the fact that thiscompound is not sufficient to cause enough mutational events to result in
the recessive visible mutations, and suggesting that a threshold dose might
be existed in the dose-response relation. The frequency of egg-color mutations after ligularidine administration was slightly higher in the treatedthan in the control group. It is well known facts that both clivorine andligularidine are promutagens and the mutagenicities of these alkaloids are
dependent on the metabolic activation. Accordingly, silkworm pupae were
postulated to contain enzymes or an enzyme-like system capable of converting the parent alkaloid into a mutagenic substance. None of the tested
seneciphylline concentration at 20 pg/capita was increased the mutation
frequency.
The result of the present experiment suggests that the pyrrolizidine alkaloids with otonecine as the necine base were mutagenic to germ-cells ofsilkworms, while the alkaloid with retronecine as base was not mutagenic.
This mutagenic response of the pyrrolizidine alkaloid to the germ-cells of
the silkworm seems to have something in common with that of Salmonella
test system coupled with the mammalian metabolic activation system (Yamanaka, etal., 1979). In this context, the silkworm has proved to be a useful
insect species as well as Drosophila for biological monitoring of the pyrrolizidine alkaloid which requires metabolic activation for demonstration of
their mutagenic activity.

72

RESEARCHES CARRIED OUT IN 1979

Mutagenicity of Two Phosphoramides, TEPA and HMPA,
in the Silkworm Germ-Cells
Akio MURAKAMI and Toshiko OZAWA

Triethylene phosphoramide, TEPA, is widely used in the chemical industry.
TEPA andHMPA, hexamethyl phosphoramide, a nonalkylating analog
of TEPA, have been given attention on the biological properties as either
insect chemosterilants or cancer chemotherapeutic drugs. The mutagenicity of both phosphoramides has been demonstrated in various organisms
from bacteriophages to mammals. In Drosophila, these compounds induced
a high frequency of recessive lethal mutations, dominant lethals and otherchromosomal events. The genotoxicity of TEPA was about 100 times more
efficient than HMPA in the sperm of Bracon hebetor (R. M. Watrous, Brit.
Ind. Med. 4: 111 (1947». Domesticated silk moth (Bombyx mori), as oneof the mutagenicity testing insects, may differ from Drosophila and Braconin the metabolism and eating habits; thus, mutagenic response to chemicalcompounds is not.necessarilly expected to be identical in these insect species.
Therefore, we tested the mutagenic activity of these known promutagens,
TEPA and HMPA, on germ-cells of the silkworm using the egg-color
specific locus method.
.Both phosphoramides were dissolved in 0.85 % NaCI solution and wereadministered by injection method to the pupae of wild-type (CI08 X Aojuku)
F1silkworms at 0.5, l.0, 2.5, 5.0, 10.0 and. 25.0 ,ug/capita, they were matedwith marker stocks having a double recessive egg-color genes, pe and re,for evaluation of mutagenic activity of the compounds. The germ-cellstages in pupae are prophase I oocytes and spermatozoa for female andmale, respectively.
After TEPA treatment a significant increase of mutations at both pe andre loci was observed in pupal oocytes and spermatozoa. However, the increase was non-linear in relation to the doses studied. The genotoxicity of
TEPA for pupal germ-cells was more pronounced than that of Diethylsulfate (DES), often equalling or surpassing that of DES. On the contrary,with the positive result in other insect test systems, the frequency of eggcolor mutations after HMPA administration was not significantly increasedin the treated than in the control group, regardless of type of germ-cells.
This finding means that the silkworm pupa is lacking in enzymes or an enzyme system capable of converting or demethylating HMPA into muta-

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

73

genic intermediates. From the present experience and others, it can besaid that test organisms for the mutagenicity assay of environmental chemicals should include, at least one species having an ability to activate thegiven parent chemical for biologically active intermediate(s).
The Mutagenicity Test of Some Flavonoids in Pupal Germ-Cellsof the Silkworm, Bombyx Mori
Akio MURAKAMI and Toshiko OZAWA

The flavonoids are universally distributed among vascular plants including
mulberry tree, and the mutagenicities of several of them have been reported
in various mutation test systems. Most of the flavonoids are a so-called
promutagen, i.e., not mutagenic in itself, but it requires metabolic activation for conversion into a mutagenic substance. The silkworm feeds onleaves of the mulberry trees. The mulberry leaves contain a various kindof the flavonoids. It is of interest to test whether ot not the flavonoids are
mutagenic to the silkworm. This communication concerns the mutagenic
activity of four different flavonoids, quercetin, quercitrin a glycoside ofquercetin, rutin a rutinoside of quercetin and morin, in pupal germ-cells of
the silkworm by the egg-color specific locus method. All compounds tested
were purchased from Wako Pure Chemicals, Osaka.
Each chemical was dissolved in 0.025 ml of 0.5 % CMC solution, and applied by injection into the abdomen of mid-stage pupae of wild-type [CI08 X
Aojuku] F 1 silkworms in four different concentrations, 100, 200, 400 and
600 flg per capita for evaluation of the mutagenic effect. Control pupae
were given the same volume of 0.5 % CMC solution. After emergence,
they were allowed to mate with the marker stock homozygous for egg-color
genes, pe and re, and recessive visible mutations were scored. The numberof whoJe- and fractional-body mutants detected was recorded separetely
for each locus and the over-all frequency of mutations was calculated.
The frequency of egg-color recessive visible mutations at both loci in the
pupal oocytes and spermatozoa after the flavonols, quercetin and mori, administration was not significantly higher in the treated than in the control
group. These flavonols were mutagenic to Salmonella tests in the presence of mammalian metabolic activation systems (Brown, Muta. Res. 75:
243 (1980)). The mutagenicity of the two flavonol glycosides, quercitrin(quercetin-3-0-rham) and rutin (quercetin-3-0-glu-rham), were also not muta-

74

RESEARCHES CARRIED OUT IN 1979

genic in both sex germ-cells of the silkworm pupae.
Negative results with this test system could mean either that the flavonoids are not mutagenic in the silkworm germ-cells or that the flavonoids are
mutagenic but not detected as such by this procedure. The silkworm feeds
innoxiously on mulebrry leaves which contain a various kind of flavonoids.
In addition, isoquercitrin, a flavonoid, in mulberry leaves was identified as
one of biting factors in the silkworm (Hamamura, 1970). Morin showedalso the biting activity in the insect but not found in mulberry leaves(Hamamura, 1970). Such being the context, it would seem highly probable
that, in the long evolutional process, silkworms have been acquired· anability to take mulberry leaves without any detrimental effect to maintaintheir life. Accordingly, it can be said that the silkworm seems to be disqualified for the mutagenicity test of flavonoids and their related compounds regardless of the presence or absence of enzymes or an enzymelike
system capable of converting the parent compounds into a metabolic active
intermediate.
Strain Differential Activity for Metabolic Activationof 2-Acetylaminofluorene in the Silkworm
Akio MURAKAMI and Michiko GOTO

Many environmental chemicals defined as promutagens exert their mutagenic effect on the experimental organisms· only after their conversion into
active intermediates. The development of sensitive assay systems and/or
biological materials for such promutagens is urgently necessary. The present study aims to select the sensitive silkworm strains or stocks having ahighly metabolic activity for making promutagens to form mutagenic active
intermediates.
Microsomal S9 fractions from the 5th instar whole larva in seven stocks ofof the silkworm (Bombyx mori L.) were compared for their ability to metabolize parent chemicals to mutagenic substances. Drug metabolic activityof the S9 fraction was measured by the His+ Salmonella TA 98/silkworm
microsome system according to Sakamoto et al. (1977) with small modifications of the method developed by Ames et al. (1975). In this system,
the metabolic activity was assayed as the mutagenicity of the chemical to
Salmonella or the increase of His+ independent revertant colonies on minimum glucose media as compared with controls without the microsomal

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

75

59 fractions. 2-Acetylaminofluorene (AAF), one of typical promutagens,
was used as the test chemical. In Salmonella TA 98, AAF is not mutagenic
in the absence of the activation systems, but mutagenic in the presence of thesystems. The preparation of silkworm microsomal S9 fractions was carriedout as described by Ames et al. (1975). TheS9 fractions were obtained fromfive original stocks (C108, old C108, Aojuku, Nihonnishiki, and Kojiki),
an F 1 hybrid of Aojuku x C108 and a mutant stock, rb (red blood).
The results obtained in this study indicate that the AAF metabolic activity
among 7 stocks investigated was C108 as the highest activity and old C108,
F1 hybrid of Aojuku x C108, Aojuku, Nihonnishiki, rb, and Kojiki in thisorder regardless of sexes. S9 fractions taken from female larvae seemed tohave somewhat higher the activity for AAF metabolic activations than thoseof males except for the cases of Nihonnishiki and rb. The activity of S9
fractions taken from larvae of C108 and old C108 was three times higher
than that of liver S9 from rats which had been treated with an enzyme inducer polychlorinated biphenyl and that of those from F 1 hybrid of Aojuku
X ClOB, Aojuku, Nihinnishiki, and rb was almost the same as that of rat
livers. The S9 fraction from Kojiki alone was less in activity than that fromrat livers. In any case, it can be said from the present study that the AAF
metabolic activation system in the silkworm larval microsome S9 fractions
is considerably active.
Mutagenic Effects of X-Rays at a Dose Down to 25 R in Adult
Oocytes of the Silkworm
Akio MURAKAMI and Toshiko OZAWA

The greater use of X-radiation, radioisotopes in medicine, and of nuclear
power for electricity generation bring foward an important social problem
on the genetic effects of radiation at low dosage levels. However, little
information on this subject has been reported in higher organisms. Amongthem, available experimental results of Drosophila males provided evidence
that a linear relationship between doses and mutation frequencies can beapplied to low doses down to around 5 to 8R. Unfortunately, no sufficient study on the dose-response relations at low-level radiations in female
germ-cells has been reported. In the silkworm, the dose-response curvefor egg-color mutations induced by X- or gamma-rays for prophase I 00cytes was non-linear over the high dose ranges studied from 500 to 15000 R

76

RESEARCHES CARRIED OUT IN 1979

(Murakami, 1971) as reported for Drosophila oocytes (Parker, 1960). This
report communicates about the genetic effects of low-level X-radiation on
silkworm female germcells by the specific locus method. To make lowdose experiements, it requires a large number of individuals to be observedin order to find a reasonable number of mutants. For this silkworm prophase I oocytes in adults just before entering into meiotic division process
are suitable because collection of a large number of homogeneous materials
is easy and that this stage oocytes are fairly sensitive to radiation. In addition, the egg-color specific locus method as the mutation detection systemallows to easily detect the visible complete or mosaic mutants among a large
number of F 1 individuals or diapaused eggs.
The materials used in this experiment were newly emerged the wild-type(Cl08 x Aojuku) F 1 virgin female moths and were irradiated with various
doses of X-rays (180 kVp, 25 mA, 1.0 mm Al filter and a dose-rate at 300
Rjmin). Applied doses Were 25, 50, 100, 200, 300, 400 and 500 R. Nonirradiated female moths were used as control. Each locus mutation frequency was calculated as the total frequency of complete and mosaic
mutants.
It was clearly observed a locus differential sensitivity between the pe andre loci for both control and experiment groups. Spontaneous frequency
was almost 10 times higher at the pe locus than at the re locus as in otherexperiments reported elsewhere. At the re locus, a linear relationship between egg-color mutation frequencies and X-ray doses was observed with thedose over range studied and indicating that no threshold dose at this locus
in mutagenic effects of low doses of radiation down to 25 R. While, atthe pe locus, the frequencies of mutations at 25 and 50 R were no moredifferent from an extraordinarily high spontaneous mutation frequency which
estimated to be around a lOx 10-5 • This may be not due to mutational
incidence, but to abnormal fertilization such as androgenesis. Accordingly,
at thepe locus, the linear dose relationship was applied to a dose down to
no more than 100 R.
Large scale experiments at the re locus on the· mutagenic effects withfurther low-level radiations in the oocytes system of silkworms are in progress.

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

77

X-Ray-Induced RecombinationalEvents between Z and W
Chromosomes in Oogenic Cells of the Silkworm(Bombyx Mori L.)
Akio MURAKAMI and Akio OHNUMAThe physical induction of recominational events between Z and W chromosomes has been investigated in the female silkworm, in which it occurs
scarcely spontaneous crossing-overs. Females (sch+ jwpe+ : pejpe) having
a W-chromosome marked with pe+ gene and the 5th chromosome homozygous for pe (yellow-white egg: 5-0.0) received a single dose of X-rays(l,000 R) at various developmental stages from first instar larvae to latestage pupae. They were crossed to tester males (schjsch: pejpe) homozygous for both sch (sex-linked chocolate: 1-21.5) and pe genes.
The results indicated that X-rays clearly increase the occurrence of recombinational events in oocytes after irradiation of silkworms from thelate stage larvae to mid-stage pupae, but slightly increase in oogonia fromfirst to early third instal' larvae. The most sensitive germ-cell stage to Xray-induced recombinational events was observed in oocytes of early stage
pupae (153.4X 10-5 ) and that was delayed for a few days as compared withthe exchange type recombinations in the 5th chromosome. The frequencyof recombinants recovered in oocytes of late stage pupae was reduced to
the level of the first instal' larvae. The incidence of recombinational events
between sex-chromosomes was several times more frequent than that of
the 5th chromosome (Murakami, 1976).
Genetic analysis of the recombinants recovered is not yet completed, it
can be said, however, that the femaleness deciding factor(s) is localized in
the inside segment rather than the distal Portion of the W chromosome as
inferred by Tazima (1946). In addition, it can be deduced from the analysis
that the factor suppressing the maleness or development of male sex organs
and gametes may be located in the W-chromosome.
Chromosomal Aberrations in Drosophila simulans
Yutaka INOUE

Natural and radiation induced chromosomal aberrations were studied in
These sibling species are similar in morphology, but cytologically different by a single inversion in the right arm of

D. simulans and D. melanogaster.

78

RESEARCHES CARRIED OUT IN 1979

the third chromosome. In natural populations of D. simulans, no polymorphic inversions have been detected. Only four unique inversions werereported by Dobzhansky (BioI. Rev. 14: 339, 1939) and !toh (DIS 55: 64,1980). On the other hand, a total of eleven kinds of the polymorphic inversions were observed in D. melanogaster on the both arms of the second
and third chromosomes. In addition, the unique inversions were foundin frequency of 0.26% per autosome arm (Inoue and Watanabe, Jap. J.
Genet. 54: 83 1979). A total of 734 iso female lines of D. simulans fromJapan, Africa and Australia were examined and the four inversions wereobserved (In(3L)68D: 70C, In (3L)64B; 70C, In(3R)86A; 93D. In(3LR)
67F; 83£). Their frequency was 0.07 % per autosome arm and it was much
lower than that found in D. melanogaster population. Males from 16 geographical strains of Japan were irradiated by X ray (4000 rads) and mated to
non-irradiated females of the respective species. Chromosomal aberrations induced in the germ cells of the males were examined in F 1 larvae.
The frequencies of aberrations were 8 %(24/300) for D. melanogaster and
4 % (11/300) for D. simulans. The results were rather consistent with Lemke
et al. (DIS 53: 159 1978), but were different from those of Woodruff andAshburner (Am. Nat. 112: 456, 1978) in which they showed the frequency
was almost equal for the both species.

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

VII.

79

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICROORGANISMS AND PLANTS

Natural and Environmental Antimutagens
Tsuneo KADA, Hajime MOCHIZUKI, Masako HARA, Akiko YOKOIYAMA,
Tadashi INOUE, Kenji KANEKO, Hiroyuki MINAKATA
and Y oshita SADAIE

A number of environmental antimutagens were detected by assaying, infour bacterial systems, capacities for reducing; (a) UV- or gamma-ray-induced reverse mutations in E. coli WP2 Bjr try, (b) high spontaneous histidine
reversions in B. subtilis strain NIG1125 possessing an altered DNA polymerase III, (c) spontaneous reversions in Salmonella typhimurium TAlOO
possessing a plasmid pKMIOI, and (d) streptomycin-dependent~inde­
pendent mutations in strains derived from Salmonella TA98 and TAlOO.
The followings are examples that were concluded as antimutagens; somebio-essential metal compounds such as cobaltous chloride, extracts fromplacenta tissues (human, monkey, dog, rabbit, rat, mouse); extracts fromJapanese green tea plants; extracts from leaves of some lily species; wateror methanol extracts of some crude drugs; etc. 5-Fluorouracil was used
as a positive control.
Antimutagenic Effect of Cobaltous Chloride on Various
rvIutator Strains
Tadashi INOUE, Kayoko SHIMOI and Tsuneo KADA

We previously reported that the presence of cobaltous chloride in posttreatment media markedly diminished the MNNG- as well as gamma-rayinduced reverse mutations in Escherichia coli WP2 Bjr trp. In order toelucidate the detailed mechanisms of the antimutagenesis by cobaltous chloride, we examined the effect of the metal compound on the following four
mutator strains whose molecular bases for mutator activity are well characterized. (1) Bacillus subtilis NIG 1125 (mut-l). The mut-l cells produce
an error-prone DNA polymerase III which is a chromosome replicating
enzyme of the bacterium. (2) E. coli SG 902 (dna E). This strain is an

80

RESEARCHES CARRIED OUT IN 1979

analogue of B. subtilis NIG 1125, i.e., the mutator activity is ascribed to temperature sensitive error-prone DNA polymerase III. (3) E. coli DM 1187
(lexA spr tif sfi). In this strain, the error-prone DNA repair pathways(SOS-functions) appear to be expressed constitutively without any inducing
signals. (4) T4 L56 (ts). This phage also produces an error-prone DNApolymerase. The error-proneness is attributed to decrease activity of proofreading exonuclease (3' to 5' exonuclease) of the enzyme.
Among these mutator strains, B. subtilis NIG 1125 and E. coli DM 1187
were found to be very susceptible to the metal compound with respect to theirspontaneous mutability. The mutation frequency of histidine reverse mutation was reduced by the metal compound to one-twentyth and one-seventhin B. subtilis NIG 1125 and E. coli DM 1187, respectively. However, in
E. coli SG 902 and T4 L56, which possess mechanisms for mutator activity
very similar to those of B. subtilis NIG 1125, no antimutagenic activity ofthe metal compound was detected.
From these data, we suggest that the metal antiinutagen may correct
error-proneness of DNA replicating enzymes by improving their fidelity
in the DNA synthesis, but the susceptibility of the enzyme to the metal compound is species specific.
Mutagenic Effect of L-ethionine in Soybean and Maize
Taro FUJII

Ethionine, a methionone analogue, is a well known carcinogen inducing
predominantly liver-cancer. It is known that about 80 % of the carcinogens
have mutagenic activities whereas a number of carcinogenic substances remain non-mutagens. A lack of mutagenic activity of ethionine has been
documented in mammalian and microbial test systems, except in Bacillus
subtilis assay (Kada et al., 1980, Chemical Mutagens 6: 149). Strain T-219
of soybean with a marker Yl1 and maize heterozygouns for the Yg 2 -gene
were· used, and· mutagenicity of L-ethionine was examined. In the experiment with soybean, seeds were soaked in 0.5-2.5 mg/ml concentrations andincrease in mutation frequency was measured by the increase in numbers of
spots per leaf. Increase of spot frequency was observed with higher concentrations, and repeated experiments with concentrations ranging 0.252.0 mg/mI, also showed consistent results. Spot frequencies from these
two experiments showed clear dose-related frequency relationships. To

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

81

obtain the threshold dose of the substance for mutation induction, much
lower concentrations, viz., 0.05-0.20 mg/ml, were applied. No increase inspot frequency was observed with these low concentration treatments.
Maize seeds were treated with concentrations ranging 0.025-5.0 mg/m!.
The frequency of somatic mutation of the Yg 2-gene, characterized by yellowgreen stripes, did not differ between the water. steeped sample and thosetreated at rather lower concentrations. At 1.0 mg/ml treatment, however,an appreciable increase of striping frequency per leaf was observed, and thefrequency increased linearly at higher concentrations. These results indicate that L-ethionine induces somatic mutations in maize at concentrations
higher than 1.0 mg/ml. In spite of many negative results, the present resultswith soybean and maize demonstrate that L-ethionine has mutagenic activity in higher plants. Frequencies of three spot types due to forward andback mutations, and somatic crossing over in soybean, and forward mutation frequency in maize, induced commonly by physical or chemical mutagens, were increase with L-ethionine treatments.
Identification of Newly Induced Waxy Mutant Gene in Maize
Etsuo AMANa

Among many marker genes in maize, waxy (wx) is a very clear and dependable endosperm marker. It is one of the few loci which have beenstudied their intralocus fine structure. The enzyme produced from thislocus has been reported. Mutations can be induced in this locus by seedtreatment with a chemical mutagen, EMS, or less frequently with ionizing
radiations. The newly induced wx mutant gene could be detected in endosperm after pollination with tester wx pollen. But this pollination not only
upset purity of the stock used but also complicated isolation of the new wx
gene. The mutant kernels detected contain both tester wx and newly induced wx genes. They can be separated by back cross to original Wx stock
followed by self pollination in the next generation. By this way, new wx
or tester wx used can be brought into homozygotes respectively. The
problem is how to distinguish new wx mutant line from the tester wxline
in the siblings. There are some flanking markers available on the chromosome 9 where wx locates. However, since they are some distances apartfrom wx locus, in either side of wx, they can only help as a primary proof
for the new wx gene. The best guarantee will be the characteristics. of the

82

RESEARCHES CARRIED OUT IN 1979

new wx gene itself. Mutant site or position of the alteration in wx cistron
may be one of the factors for identification, but it requires detailed examination of fine structure analysis. Polypeptide analysis or DNA base se"quence analysis will be the ultimate proof of the new wx mutant gene, butthey are much more laborious tasks. For practical purposes, leakyness of
wx phenotype seemed to be a good help. Phenotype of wx mutant is expressed as decreased level of amylose fraction in starch. This changes thecolor of staining by iodine from dark blue to reddish-purple. Some of the
EMS induced wx mutants showed intermediate color suggesting that theymight be miSsense mutation. These intermediate or leaky phenotype could
serve as a proof for the new wx mutant gene. When such intermediate
phenotype was so clear that it could be noticed by visual examination, it iseasy to separate the new wx mutant from tester wx. For nearly complete
wx mutant, dual wave length photometry was applied. In some of thetested mutant, aleuron layer was colored to purple or brown by other marker
genes. However, the photo-metrical measurements of iodine stained endosperm starch solution were not disturbed very much, and some possible"slightly intermediate" wx mutant could be separated.
Phenotypic Expression of Wx Gene in Cereals
Etsuo AMANO

Waxy (wx) starch mutants which can synthesize no or only small amountof amylose component of starch, are found in several species of cereal crops.
They can be induced artificially with radiations or more efficiently withchemical mutagens. During examinations of these waxy mutant, genedosage effect in endosperm was noticed in rice. In the mutant segregating
populations, M 3 , M" or F 2 , endosperms could be classified visually into 1
full waxy, 1 intermediate waxy and 2 nonwaxy groups. The last group still
had some deviation in turbidity in endosperm. Dual wavelength analysis
which was a modified blue value method and was developed to measure thewaxy phenotype quantitatively, supported the results of visual classification.
Such gene dosage effect was shown neither in maize nor barley, in whichclear and theoretical 3: I segregations were observed both in visual andinstrumental examinations.
In F 2 population of Oryza perennis (Wx) x Oryza sativa (wx) segregation
ratio of Wx and wx was distorted probably due to low seed fertility or small-ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

83

ness in numbers of endosperms examined. However, as for the gene dosage
effect, there was no intermediate phenotype. In a F 2 population of Wx/wx
heterozygote of Oryza glaberrima, another cultivated rice species, segregation pattern was the same as other maize or barley. Only Wx genes in
Japonica type Oryza sativa, NorinNo. 8 and Taichung 65, showed the genedosage effect producing wx heterozygotes with intermediate phenotypes.
Heterozygosities of these intermediate kernels, selected visually, were confirmed in the following generation where all of them segregated in 2: I: 1
ratios.
These results may suggest that at least Wx genes of some of the Japonica
rice have different degree of dominance over wx, from other starchy cereal
or even from other Oryza relatives. In general, fully matured storage starchin plants contains about 25 % amylose. It would be interesting to supposethat a single normal Wx gene could synthesize amylose up to 25 ~~, but bysone kind of mutation occurred during the evolution, Wx gene in Japonica
rice lost some of its function so that full three Wx genes were required inendosperm to synthesize the normal amount of amylose. To test this hypothesis, a wide survey of the gene dosage effect of Wx gene in other varieties of rice is in progress.

84

RESEARCHES CARRIED OUT IN 1979

VIII.

POPULATION GENETICS (THEORETICAL)
Efficiency of Truncation Selection
James F. CROW and MOTOO KIMURA

Truncation selection is known to be the most efficient form of directional
selection. When this is modified so that the fitness increases linearly overa range of one or two standard deviations of the value of the selected character, the efficiency is reduced, but not greatly. When truncation selection iscompared toa system in which fitness is strictly proportional to the character
value, the relative efficiency of truncation selection is given by f( c)/(J, in whichfCc) is the ordinate of the frequency distribution at the truncation point and (]is the standard deviation of the character. It is shown, for mutations affect,
ing viability in Drosophila, that truncation selection or reasonable departures therefrom can reduce the mutation load greatly. This may be one
way to reconcile the very high mutation rate of such genes with a small mutation load. The truncation model with directional selection is appropriate
for this situation because of the approximate additivity of these mutations.
On the other hand, it is doubtful that this simple model can be applied to
all genes affecting fitness, for which there are intermediate optima and antagonistic selection among components with negative correlations. Whether
nature ranks and truncates, or approximates this behavior, is an empiricalquestion, yet to be answered. For details, see Froc. Nat!' Acad. Sci. USA
76, 396-399.

Fixation of a Deleterious Allele at One of Two"Duplicate" loci by Mutation Pressure
and Random Drift
Motoo KIMURA and Jack L. KING

We consider a diploid population and assume two gene loci with twoalleles each, A and a at one locus and Band b at the second locus. Mutation from wild-type alleles A and B to deleterious alleles a and b occurs with
mutation rates Va and Vb' respectively. We assume that alleles are com·pletely recessive and that only the double recessive genotype aabb showsANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

85

a deleterious effect with relative fitness I-e. Then, it can be shown that
if V",>Vb mutant a becomes fixed in the population by mutation pressure anda mutation"selection balance is ultimately attained with respect to the Bib
locuS alone. The main aim of this paper is to investigate the situation inwhich V",=Vb exactly. In this case a neutral equilibrium is attained and either
locuS can drift to fixation for the mutant allele. Diffusion models are developed to treat the stochastic process involved whereby the deleterious mutant
eventually becomes fixed in one of the two duplicated loci by random sampling drift in finite populations. In particular, the equation for the averagetime until fixation of mutant a or b is derived, and this is solved numericallyfor some combinations of parameters 4Nev and 4Nee, where v is the mutation rate (V",=Vb=V) and N e is the effective size of the population. Monte
Carlo experiments have been performed (using a device termed "pseudo
sampling variable") to supplement the numerical analysis. For details,
see Proc. Nat!' A cad. Sci. USA 76, 2858-2861.
Model of Effectively Neutral Mutations in which
Selective Constraint is Incorporated
Motoo KIMURA

Based on the idea that selective neutrality is the limit when the selective
disadvantage becomes indefinitely small, a model of neutral (and nearly
neutral) mutations is proposed that assumes that the selection coefficient(s') against the mutant at various sites within a cistron (gene) follows a r
distribution; f(s')=aPe-as's,p-I/T(~), in which a=Ns' and 8' is the mean
selection coefficient against the mutants (8'>0 ;1:;:;;:~>0). The mutation
rate for alleles whose selection coefficients s' lie in the range between 0 and
1/(2Ne), in which N e is the effective population size, is termed the effectively
neutral mutation rate (denoted by v e ). Using the model of "infinite sites"in population genetics, formulas are derived giving the average heterozygosity (he) and evolutionary rate per generation (kg) in terms of mutant substitutions. It is shown that, with parameter values such as ~=0.5 and"3"=0.001, the average heterozygosity increases much more slowly as N e
increases, compared with the case in which a constant fraction of mutations are neutral. Furthermore, the rate of evolution per year (k1) becomes
constant among various organisms, if the generation span (g) in years isinversely proportional to .;N e among them and if the mutation rate per

86

RESEARCHES CARRIED OUT IN 1979

generation is constant. Also, it is shown that we have roughly kg=v.. The
situation becomes quite different if slightly advantageous mutations occur
at a constant rate independent of environmental conditions. In this case,the evolutionary rate can become enormously higher in a species with a verylarge population size than in a species with a small population size, contrary
to the observed pattern of evolution at the molecular level. For details,
see Proc. Natl. Acad. Sci. USA 76, 3440-3444.
Population Genetics of Multigene Family with Special
Reference to Decrease of Genetic Correlation withDistance between Gene Members on a Chromosome
Motoo KIMURA and Tomoko OHTA

A mathematical method is developed which enables us to treat exactly
the process of coincidental evolution under mutation, unequal intrachromosomal crossing-over as well as ordinary crossing-over between homologous
chromosomes.in a finite population of the effective size N. It makes useof finite difference equations involving two quantities denoted by .ft, andrpi' in whichfi is the identity coefficient of two gene members that are i steps
apart on the same chromosome and rpi is that of two members i steps aparton two homologous chromosomes. When the number of genes (n) per
family is large, the finite difference equations can be approximated by ordinary second-order differential equations which can then be solved analytically. Results obtained by the present method are compared with the corresponding results previously obtained by one of us (Ohta, Genet. Res. 31:
13-28) using conventional diffusion models of gene frequency changes in
population genetics. For details, see Proc. Natl. Acad. Sci. USA 76,40014005.
An Extension of a Model for the Evolution of Multigene
Families by Unequal Crossing-overTomoko OHTA

Evolution of a multigene family is studied by using approximate diffusion
model. It is assumed that the multigene family is undergoing continuousinterchromosomal unequal crossing-over, mutation and random frequency
drift. The equilibrium properties of the probability of gene identity (identity

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

87

coefficient) are investigated, using two measures: identity probability within
and between chromosomes. The measures represent average identity coefficient of genes within a family in one chromosome and that of gene families
between two homologous chromosomes. The means, the variances and thecovariance of these two measures of identity coefficient are obtained. It isshown that the means and the variances are generally smaller than those prepredicted in the previous model assuming intrachromosomal (sister chromatid) unequal crossing-over (Ohta, Genet. Res. 31: 13-28). For details,
see Genetics 91: 591-607.
Genetic Variability Maintained in a Finite Population under
Mutation and Autocorrelated Random Fluctuationof Selection Intensity
Naoyuki TAKAHATA and Motoo KIMURA

By using the diffusion equation method, the level of genetic variability
maintained under mutation pressure in a finite population is investigated,assuming autocorrelated random fluctuation of selection intensity. An
appropriate mathematical model was formulated to treat the change of genefrequencies, incorporating mutation pressure and fluctuating selection. Extensive Monte Carlo simulation experiments were also performed to supplement the theoretical treatments. Excellent agreement between the two
results suggests the validity of the present diffusion model for the autocorrelated selection. One of the most important findings from the simulation
studies is that mutations and random sampling drift largely determine thelevel of genetic variability, and that the presence of autocorrelated selection
can significantly lower genetic variability only when its strength, as measured by the cumulative variance of selection intensity, is larger than about
103 times the mutation rate. It is pointed out that the effects of both mutations and random sampling drift have to be incorporated in order to assess
the role of various factors for the maintenance of genetic variability in natural
populations. The present finding is favorable to the neutral theory of molecular evolution. For details, see Proc. Natl. Acad. Sci. USA 76, 5813-5817,
1979.

88

RESEARCHES CARRIED OUT IN 1979

IX.

POPULATION GENETICS (EXPERIMENTAL)

New Inversions of Drosophila melanogaster spreading over Japan
Yutaka INOUE, Kendo TSUNO and Takao K. WATANABE

A new inversion on the left arm of the second chromosome, In(2L)W28C;
32C, was firstly found in Katsunuma in 1972. Thereafter, it has been detected there in frequency of about 3 or 4 percent which is the next most frequent inversion to In(2L)t on the arm. The W inversion was also foundin other eastern Japan than the Katsunuma; Sapporo (1977), Tochigi (1978,1979), Niigata (1978), Shiojiri (1977, 1978, 1979), Mishima (1978), Ito (1975),
Shimoda (1975). Association between some electromorphs and the W ininversion was examined among 48 W inversions ever found in the KatsuKatsunuma; 42 was a-GpcfF-Adh F, 5 was a-GpdF-Adh s and 1 was a-Gpd sAdh F.
Another new inversion on the left arm of the third chromosome, In(3L)
Y68F; 75C, was firstly found in the Katsunuma in 1975. Thereafter, it has
been detected there in frequency of about 2 or 3 percent which is surpassing
the old inversions, 3LP and 3LM, on the same arm. The distribution of theY inversion is relatively wider than the W inversion; from Sapporo (1977)
to Tottori (1979) and Osaka (1979). Association between l::St-6S and the Y
inversions seemed to be exclusive in the Katsunuma (1979) population.
These two inversions, Wand Y, might have occurred in some middle
part of Japan in comparatively lately and is spreading over Japan. Papulations carrying these inversions have also carried the other polymorphic
old inversions simultaneously. This situation suggests that a new inversion can survive to become a polymorphic inversion where the old inversions already exist.
Inversion Polymorphism in some African, New Guinean
and Philippine Populations of D. melanogasterYutaka INOUE and Takao K. WATANABE

A total of 70 Drosophila melanogaster females were collected at six localities of Africa, New Guinea and Phillippines in 1979. They were indivi-

;l>-

I
:;>;:lTable 1.

~...,:;>;:lFrequencies of inversions detected in some African, New Guinean and Philippine populations of
D. melanogaster% of polymorphic inversion/chromosome arm

No. oftested lines

No. of inversion
carrying lines

2Lt

2RNS

3LP

3LM 3RP3RC 3RK

3RMo

Mombasa, Kenya

4

1*

0

0

0

0

0

0

0

0

Nairobi, Kenya

27

14

18.5

0

1.9

0

7.4

3.7

5.6

0

Tananarive, Madagascar

13

7

26.9

0

0

0

0

3.9

0

0

41.7

25.1

8.3

58.3

0

0

0

Locality

Wau, Papua New Guinea

6

6

33.3

Port Moresby, Papua New Guinea

18

18

66.7

47.2

44.4

2.8

83.3

0

0

0

Manila, Philippines

2

1

0

0

25.0

0

25.0

0

0

0

* Unique endemic inversion:

In (3L)62D-68A.

~

~

::l

o

~

r

~
~

~

o"I:I
Cl

~

dg
~

w
o

00

\0

90

RESEARCHES CARRIED OUT IN 1979

dually sent to Japan as isofemale strains. One larva was sampled from eachstrain and examined the salivary gland chromosomes. Table I shows theresult. Every population carried inversions although the number of testedlines was small. Most populations except Mombasa were characterized
by the maintenance of polymorphic inversions. Among them, two New
Guinean populations showed the highest level of the inversion contents whencompared with the Ishigaki, Japan (Inoue and Watanabe, Jap. J. Genet.
54,69. 1979) and the Florida, U.S.A. (Mettler et al., Genetics, 87: 169, 1979)
populations. In(3R)K is restricted its distribution in Africa and U.S.A.,
and it was confirmed here. Other polymorphic inversions are spread over
the world, but the lacks of In(3R)Mo in every locality and In(3R)C in New
Guinea may be ascribed to the small sample of the present study (Supported
by MEJ 404149 & 504344).

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

X.

91

EVOLUTIONARY GENETICS

Courtship Song and Mating Preference in Drosophilamelanogaster, D. simulans and their Hybrids
Masaoki KAWANISHI and Takao K. WATANABE

Intra-specific variations of the courtship song (inter-pulse interval: ipi)
of Drosophila melanogaster and D. simulans males were investigated. The
variation among strains was small in D. melanogaster (31.3-34.8 msec, n=14), whereas it was much larger in D. simulans (42.8-56.8 msec, n=55). The
average ipi was 32.8 msec for D. melanogaster and49.l msec for D. simulans,
and the distributions between the two sibling species were non-overlapping.
Courtship song (ipi) and mating preference of hybrids between D; melanogaster and D. simulans were investigated. The ipi of hybrid males carrying
the X chromosome either of D. simulans or of D. melanogaster is intermediate
between that of the two parents, suggesting that the autosome determination
with incomplete dominance. On the other hand, the mating preferences ofhybrid males measured by the number of copulated pairs within 30 minutes
differed by the X chromosomes carried by the males; the hybrid males carrying the X chromosome of D. simulans copulated with D. simulans females
exclusively while the hybrid males carrying the X chromosome of D. melanogaster preferred to copulate with D. melanogaster females. These mating
successes were primarily due to male mating discriminative abilities, since thehybrid males with the D. simulans X did not court D. melanogaster females
while the hybrid males with the D. melanogaster X rather couted D. melanogaster females though the discrimination was weak.
Chromosome Analysis of Hybrid Male Sterility between
D. simulans and D. mauritiana
Tsuguhiko TAKAMURa and Takao K. WATANABEInterspecific cross between two sibling species, D. simulans and D. mauritiana, produces fertile females and sterile males (David et £11., J. Genet. 62:
93, 1976). We set up a line of D. simulans marked with y, bw, st on the first,
second, and third chromosome respectively. This simulans was crossed to92

RESEARCHES CARRIED OUT IN 1979

wild mauritiana and F 1 females were backcrossed to y: bw: st simulans.
Segregated 8 types of males were examined whether seminal vesicles contained motile sperm or not as a criterion of fertility
In the cross between F 1 females of simulansc.f X mauritianaC!; and marked
simulans, fertility of male progenies were: (y: bw: st) 16.5 %, (y: bw)6.67 %,(y: st)23.76 %, (y) 7.55 %, (bw: st) 3.88 %, (bw)l.92 %, (st) 3.91 %, (+) 0.97 %.
In the cross, reciprocal F 1 females X marked simulans, the fertilities were10.00%, 4.00%, 11.00%, 3.00%, 2.00%, 0.00%, 1.00%, 2.00%, for eachphenotype respectively. If we hypothesize additivity in the contribution ofeach chromosome homozygous for marked chromosome, the contribution of
the first chromosome, for example, to the fertility is calculated by lj4[(y: bw:st)-(bw: st)+(y: bw)-(bw)+(y: st) - (st)+(y) - (+)]. The chromosomal
contributions calculated in such away are 11.00, -1.78,7.72 for 1st, 2nd,
and 3rd chromosome respectively in the first cross and 5.75,-0.25, 3.75 in
the reciprocal one. These figures means that the first and third chromosome contribute largely to male fertility but the second chromosome does notor even worse when it becomes homozygous with simulans chromosome.
Even (y: bw: st) males from the first cross recovered its fertility only 16.5 %though the three chromosomes and cytoplasm are all simulans. This wasprobably because the breakage of coadapted systems by the crossing over in
the F 1 females. To conferm this, recombination ratio in the F 1 hybrid was
obtained between y and w, and y and v on the first chromosome. Crossing
over value between yand w was 5.34% in the hybrid while 9.74% in thecontrol simulans. Between y and v was 29.36 % in F 1 and 40.53 in simulans.
Thus the crossing over takes place in high frequency in F 1 females though theratio was smaller than that in simulans. The reason why there is a difference between reciprocals are not answered yet.
Host-dependent Evolution of Three Papova Viruses
Eiichi SOEDA, Takeo MARUYAMA, John R. ARRAND*
and Beverly E. GRIffin*

A marked similarity has been observed in the organization of genomic
information between two of the papova viruses, SV40 and polyoma virus(Py), suggesting that these viruses may have diverged from a common ancestor during evolution. However, distinct differences between them and also

* Imperial Cancer Research Fund Laboratories, London, EnglandANNUAL REPORT OF NATIONAL INSTITUTE OFGENETICS NO. 30

93

between them and the human papova virus, BKV, are observed, for example,
in their interactions with several animal cells and the immunological crossreaction between viral proteins. Results from DNA-DNA hybridization
experiments suggest that homologies between the viral DNAs are small,
although the search for homology has depended largely on the stringencyof the experimental conditions. It has also recently been pointed out thatin stringent hybridization conditions, greater than 85 % homology is required for the observation of double-strandedness, and that the use of lessstringent conditions reveals more extensive homology between Py and SV40
than had earlier been reported. Nonetheless, the above data did not seemto allow detailed discussion of the relationships between papova viruses.
We therefore turned to a comparison of the nucleotide sequences of the viralgenomes and found striking homologies around the DNA replication origins
(a)

TCyr)

3x107

Fossil• __ , __ ,_,MonkeyGlobinTkaa

0.09._ ••••oMouse Man

Monkey

Cd)

(e)

(b)

T"n

DNA

0.38 __ •• __ ._ .. --.-...

Mouse

SV40
{monkey}

ProteinTka.

0.42 - •• ------ -------

Py

o

BK

SV40

Py

(mouse) (man)(monkey)(mouse)Fig. 1. Phylogenetic trees for hosts and for viruses. a. For the hosts,
based on palaeontological evidence; each branch of the tree represents evolutionary
divergence time. b. For the homologous globin amino acids; each branch represents the evolutionary distance measured by /CT. c. For the viruses basedon nucleotide sequence; each branch represents the evolutionary distance measured
by AT. d. For the viruses based on the amino acid sequence comparisons, eachbranch represents the evolutionary distance measured by /CT. The dotted horizonzontal lines indicate the relative points where the branch should occur if the molecular evolutionary distances among the viruses agreed completely with the branchingsof the hosts. As the dotted lines show only relative locations, one of the two lines
can be placed at an arbitrary location. We have therefore adjusted the branchings
between Py and SV40 (BKV) to the same height of the primates-rodent branching in a.

\0

-I>-

Table 1. Comparison of nucleotide and amino acid sequences among
the three virus species BKV, SV40 and polyoma virus (Py)
Virus lineage
Py/SV40

Py/BKV

BKV/SV40

Gene
Small tLarge T
VP2/3
Small tLarge T
VP2/3
Small tLarge T
VP2/3

Probability of identity
pn
pa
DNA
Protein
0.453
0.501
0.501
0.470
0.425
0.469
0.709
0.721
0.764

0.306
0.423
0.434
0.322
0.407
0.341
0.709
0.753
0.758

Linearized evolutionary
distance*
2J.T

2kT

DNA

Protein

0.98
0.82
0.82
0.92
1.09
0.92
0.37
0.35
0.28

1.25
0.89
0.86
1.19
0.93
1.12
0.35
0.29
0.28

No. ofnucleotides
examined
513
1,881
885
513 t
1,380t
5 tot
516
1,533
534

* This is divergence time (T) multiplied by rate (J. or k).
t
The line-up of homologous peptides of BKV/Py is very similar to that oberved in Py/SV40. Approximation to maximum homology of BKV/Py was mainly obtained by substitution of homologous peptides of BKV/SV40 into those found for
Py/SV40.

~

:;e

~

Vl

~

:;e

~

o

~

Z
....
\0
-l
\0

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

95

of these viral species. Recently, the complete sequences of SV40, Py and
BKV DNAs have been elucidated, providing insight into the evolutionary
relationships of these viruses.
BKV, SV40 and Py are easily isolated from healthy human, African greenmonkey and mouse tissues, respectively. They grow in vitro to high titreson cells from these species; therefore, we consider these to be their natural
hosts. From the DNA sequence homology, we have calculated relative
evolutionary distances by the method of Zuckerkandel and Pauling (Table1), and constructed a phylogenetic tree for the viral species. For the hosts,
the evolutionary time and the relationships between species have been well
documented from palaeontological studies, based on the fossil record (Fig.
1).

From coincidence of the phylogeny, we conclude that each of these viral
species has evolved from a common ancestor and diverged with· its hostorganism.
This work has been reported in Nature, 285, 165-167 (1980) and waspresented at the Imperial· Cancer Research Fund Tumor Virus Meeting· on
SV40, polyoma and adenoviruses, Cambridge, England 1979.

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RESEARCHES CARRIED OUT IN 1979XI.

HUMAN GENETICS

Hereditary Retinoblastoma: Host Resistanceand Age at Onset
Ei MATSUNAGA

Age-specific incidence data from 244 bilateral and 31 familial unilateralcases of retinoblastoma were analyzed. In the bilateral cases, a high intraclass correlation between ages of the patients at diagnosis in the right andleft eyes was noted, even after removal of the bias due to the fact that thediagnosis for each eye is never independent. This finding indicated strongly
that the age at onset was largely determined by host factors common to both
eyes. The mean ages of the patients at diagnosis of the inherited cases
among the ,bilateral or unilateral group varied consistently with parental
phenotype. This finding implied that inherited host resistance played an
important role in the latency period for the gene carriers. The fraction ofcases not yet diagnosed fitted well to a negative exponential distribution,and in the less susceptible group with hereditary unilateral cases, tumor formation may have been suppressed about twice that in the most susceptiblegroup with bilateral cases. It was argued that the presumed second hit
initiating tumor formation in the gene carriers was not a mutational process,but probably an error in the process of differentiation that could be suppressed completely in the most resistant group who could remain unaffected,
but that could not be suppressed completely in the less resistant group.
For details, see J. Natl. Cancer Inst. 63: 933-939, 1979.

Threatened Abortion, Hormone Therapy and
Malformed Embryos
Ei MATSUNAGA and Kohei SmoTA

Causal relations between maternal genital bleeding, supportive hormone
therapy and external malformations of the embryos were investigated withspecial reference to the critical period of oraganogenesis. This was doneusing morphological and obstetrical data obtained by Nishimura and his
associates from 667 undamaged embryos derived from induced abortions

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

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whose mothers had genital bleeding in early pregnancy. In addition, datafrom 90 embryos with polydactyly and 38 with limb reductions in the Nishimura collection were used for case history studies. Evidence was presented
to demonstrate that, for major malformations such as eNS anomalies,
cleft lip, polydactyly and limb reductions, maternal genital bleeding was nota cause but a consequence of the conception of an abnormal embryo. No
indication was revealed that exogenous female hormones currently used inJapan for preventing miscarriages could produce major malformations recognizable at the embryonic stage, including limb reductions, nor salvage theseverely malformed embryos. This does not however mean to exclude thepossible relationship of progestogens/estrogens intake during early pregnancy with an increased incidence at birth of certain internal and/or externalmalformations. It was suggested that most, if not all, of the minor anomalies observed at certain embryonic stages are kinds of normal variants
without any functional impairment of embryonic development. For details,
see Teratology 20: 469-486, 1979.
Suppressed Centromere and the Loss of "Centric Dot"in Dicentric Chromosomes
Yasuo NAKAGOME

In 1979, Eiberg (Nature 248: 55, 1974) described a technique called Cdband which stained two small dark spots on both sides of a centromere(kinetochore). The Cd denoted a centric dot and presumably represented
either kinetochore itself (Evans, H. J. & Ross, A.: Nature 249: 861, 1974)
or condensed chromatin related to a kinetochore (Roos, U. P.: Nature
254: 463, 1975).
In 1976, Nakagome et al. described a child with multiple congenital anomalies caused by an unusual translocation resulting a dicentric chromosome:tdic (7: 15)(p21; pll)(Nakagome, Y. et al.: Clin. Genet. 9: 621, 1976). Bothcentromeres of a no. 7 and a no. 15 were included in the tdic chromosome
but it assumed monocentric appearance. Only the centromere derived of
the 7 showed characteristics of a centromere. The other was presumably
not functioning [pseudodicentric chromosome; psu dic (7)t (7; 15) (p21;pll)]. In a C-banded (CBG) metaphase, both active and inactivated (suppressed) centromeres were positively stained. When stained with the Cdband technique, however, only the active (derived of a no. 7) centromere was

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RESEARCHES CARRIED OUT IN 1979

positive. No Cd positive material was detected at the site of inactivatedcentromere. Later, Mattei et at. (Hum. Genet. 50: 33, 1979) and Maraschio
et at. (Hum. Genet. 54: 265, 1980) reported similar results.
In the present study, three cases of pseudo-dicentric chromosome, 3 casesof i(Xq) and a case of r(6) (P25q27) were examined using a variety of banding
techniques including GTG, QFQ, AT-specific(DAPI), distamycin-DAPI,
CBG, Ag-I and Cd. In a case of Xq+, karyotype was found to be 46, X,
psu dic(X) (pter~cen~q22:: q22~pter), i.e., one of the two centromeres
were inactivated. Both active and inactive centromeres were stained positively by the C-band but the Cd staining revealed dark spots only on the siteof an active centromere. A case of psu dic(21) was found to have a karyotype 46, XX, psu dic(21)(pter~cen~q22::q22~pter). Both ends of the psu
dic were C-positive. The Ag-I staining also revealed nucleolus-organizing
regions on both ends, however, the Cd staining revealed dark spots at only
the site of an active centromere. In a 3rd case, the karyotype was identified
as 47, XY, +psu dic(9) (q2101) based on several different banding techniques (Abe et ai., Ann. Genet. 20: 111, 1977). Both centromeres were Cpositive and brightly fluoresced in a distamycin-DAPI stained metaphase.
In only one of them, were Cd spots detected.
In 2 of 3 cases of i(Xq), a C- and Cd-positive site was detected on the i(Xq).
In one case, two C-positive but only one Cd-positive site was detected on
an i(Xq). The two C-positive sites were very close each other and sometimes they appeared fused. According to Hsu, L.Y.F. et ai. (J. Med. Genet.
15: 222, 1978), of 30 cases with i(Xq), 21 showed two C-positive spots. In
all of them, only one constriction (centromere) was visible. No Cd-bandstudies were carried out. In a r(6), some of the metaphases contained adobule-sized ring. They always had two C or Cd spots in accordance withthe presence of two visible centromeres. No single sized rings had two C
or Cd spots.
We do not still know the exact nature of Cd-positive material, however, it appears that the detection of Cd-positive material corresponds to thepresence of an active centromere and the loss of the former to the supression of the latter. In a case of dicentric i(Xq), whether the presence of two
active centromeres is compatible with the survival of the dicentric due tothe close proximity of both centromeres, is to be determined.

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A New Approach in the Evaluation of Chromosome
Variants in Man (II and III)
Yasuo NAKAGOME, Jun-ichi AZUMI, Shigehiro OKA
and Ei MATSUNAGA

A new approach is proposed in the evaluation of chromosome variants
in man. A scanning microdensitometer was used in the determination of
the size (area) of a variant and results were assigned into five classes based on
the difierence from an average in terms of standard deviation. In the firstpart of the present series, results abtained in C variants of nos. 1, 9 and 16
were described (Azumi et al., Jap. J. Hum. Genet. 24: 99, 1979).
In the present study, the procedure was applied to LBA-treated metaphases. LBA stood for late-replicating band detected by BUdR-acridine
and intended to detect variable segments on the basis of their DNA-replicating pattern. It was shown to be very effective for chromosome pairslacking established Q or C variable segments, e.g., nos. 2, 5, 6, 7, 8, 10, 11,
12,17,18,19 and 20 (Nakagome et al., Hum. Genet. 38: 307, 1977). Metaphases were obtained from twelve unrelated individuals. Chromosomes
were identified based on their Q-band pattern. The size (area) of an LBAbright centromeric heterochromatin (variants) was measured using a microdensitometer, expressed relative to that of euchromatic segment of the given
chromosome, and were assigned into five classes as described in the firstpart (for example those within ± 1SD of an average was assigned into class3).
A total of 87 LBA-variants were detected in twelve individuals, i.e., each of
twelve pairs with no established variable segment revealed 4 to 10 variantsby the present method. Acrocentrics with Q-variable sites, i.e., pairs 13, 14,
15, 21 and 22, were also studied. A total of 47 LBA-variants were detected (for details, see Azumi et al. (part II) Hum. Genet. 55: 75, 1980 and
Oka et al. (part III) Hum. Genet. 55: 327,1980).

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RESEARCHES CARRIED OUT IN 1979XII.

BEHAVIORAL GENETICS

Selection for the Learning Ability of Mice*
Tohru FUJISHIMA

A selection has been made bidirectionally for the discriminated avoidance
performance (DAR, %) of mice (60-70 days of age) in the second of two
daily training sessions, each comprising fifty learning trials. The fundamental stock was constructed from crossing of four inbred mouse strains (C3H/
HeMs, SWMjMs, C57L/JMs and D103jMs), each having different characteristics of DAR measured with an automated Y-type maze apparatus (cf.
Ann. Rep. 25: 85).
The data for the fifth selected generation were obtained in this year. They
indicated that the cumulative genetic gain for the average of males and females was 10.8, and the difference in avoidance between the DAR High line
and the DAR Low line (High line minus Low line) was 20.9, while the difference in discrimination was -3.5. They also showed that the superiorityof the Low line over the High line in discrimination was more considerable
in males than in females. These results suggested that the selection forhigher DAR resulted in the higher avoidance ability of mice rather than thehigher discriminatory ability.

Genetic Difference in Oviposition Cycle in D. melanogaster
Tsuguhiko T AKAMURA

By artificial selection, two lines which are different in oviposition site
preference was set up (Takamura and Fuyama, Behav. Genet. 10, 105, 1980).
One prefers paper placed on the medium (P line) and the other prefers medium (M line). The oviposition behavior of these two lines were observedand compared in a small chamber which contained a cube medium halfof which was covered with paper. Two clear difference was detected between these two lines.
Before oviposition, D. melanogaster females always probe the surface of* This work was supported by a grant from the Ministry of Education (No. 339026).

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substrate with its ovipositor and if the site is judged suitable, they deposit
eggs. The first difference was seen in the frequency of the first probing
which took place on the paper. Ninety-five percent of the probing of thep line took place on the paper half but only 60 % for the M line.
There are three sequels after first probing took place on the paper. 1)
Oviposition on the paper, 2) Repetition of probing until the fly comes to themedium half, then oviposition, 3) Giving up oviposition after several probings. P line went into these three sequels almost in the same frequency.
But M line never deposit on the paper even if the first probing was releasedon the paper, that is, the frequency of 1) is zero. 2) was 64.2 % and 3)
35.8 %. If the first probing was started on the medium (5 % for P line and
40% for M line), both line deposit eggs 100% on the medium. The eggs
which are not laid in the first cycle after probing on the paper, about 30 %95 X 0.53) for P and about 20 % (60 X 0.36) for M, are recycled into the oviposition cycle from the beginning again. If we denote this recycling ratio r,
and the proportion of eggs deposited on the paper after the first cycle ap
and that on the medium am, the over all egg production on the paper is expected with ap/l-r and on the medium am/l-r.
It was known that tarsus amputation deprived discriminating ability ofof P line and ovipositor caute1ization reduced that of M line. If the firstprobing is released by the sensory imput from the tarsi, and tarsus amputation makes the frequency of the first probing on the paper and on the medium
equal, this strongly influence P line. If ovipositor cauterization renders
the frequency of three sequels equal after probing on the paper, it influences
M line largly. And these hypothesis meet well to the experimental results
cited above (Takamura and Fuyama, Behav. Genet. 10 1980, Takamura,this report, 29).
Consequently, there are at least two components in the oviposition. behavior of D. melanogaster which was genetically controlled and changeable
by artificial selection. One is where the first probing takes place, whichmust be mediated by tarsus sensory organs and the other is actual oviposition
released by the sensory imput from ovipositor. These two factors mightplay important roles in the choice of oviposition site of D. melanogaster.

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RESEARCHES CARRIED OUT IN 1979

Differentiation of Oviposition Thrusting Power in D. melanogaster
Species Sub-group
Tsuguhiko T AKAMURA

In D. melanogaster it was found that there were large amount of genetic
variation in the tendency of inserting eggs during oviposition (Takamura,lap. l. Genet. 55: 91, 1980). The character was tested easily by letting flies
oviposit on the medium half of which was covered with paper. If the fly
has strong tendency to bury eggs, they does not deposit on the paper because it is impossible to insert eggs into it. They only lay eggs on the medium
half. However if the fly does not gave such a strong tendency, it deposit
eggs also on the paper in a certain proportion. Using this method, twosiblings of D. melanogaster, D. simulans and D. mauritiana, were examined
whether there are intraspecfic genetic variations or not. Also 6 members ofmelanogaster species sub-group were compared in the tendency of burying
eggs.
Little genetic variation was found among 40 and 17 isofemale lines ofD. simulans and D. mauritiana respectively. Almost all the lines laid eggs
on the medium half. This is a striking contrast to D. melanogaster. Then,
more than 60 flies of each 6 species of melanogaster species sub-group wereindividually examined for their oviposition site preference either paper or
medium. Except melanogaster all the species laid eggs on the medium.
This means D. melanogaster is a exceptional species in the group that onlyshows genetic variation of the character.
The strength of egg,inserting tendency is measured more purely by thefollowing method. After oviposition on the medium the surface is treatedby NaOH for 20 minutes. After that the surface is washed by tapping
water. This treatment washed away the unburied eggs. So the proportion
eggs left after the NaOH treatment shows the degree of strength of the burying tendency. In order to compare the strength of the thrusting power of
the 6 species, NaOH treatment experiments were done using mediums ofdifferent agar concentrations ranging from 0.8 % to 4 %. The larger theconcentration, the harder the medium became. The sequence in the thrusting power of the 6 species was, D. teissieri-:;;;'D. malanogaster (line prefering
paper) <D. melanogaster (line prefering medium);;;D. yakuba<D. simulans<D. mauritiana<D. erecta. Except two cosmopolitan species, melanogaster and simulans, 4 others are African endemic species. Some of them

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are known to have specific host plants. It was postulated that in the processof speciation and habitat differentiation of the 6 species, the increase anddecrease of oxiposition thrusting power played an important role.
Effect of UV Treatment on the Hatchability of Buried andUnburied Eggs of D. melanogaster
Tsuguhiko T AKAMURA

Takamura (Japan J. Genet 55: 91) reported that there was a large amountof genetic variation in natural population of D. melanogaster in the tendencyof egg-insertion when they oviposit. Some isofemale lines from Katsunuma
showed strong tendency to bury eggs but some did not. However nothing
was known as to the adaptive meaning of the behavioral difference and any
difference in any fitness components of these two egg condition has not been
examined either. One possible environmental facter causing differencein fitness is ultra violet lay. Here reported is a effect of UV treatment on
hatchability of eggs laid by flies of different tendency of inserting eggs. Usedlines, M8, M9 (having strond tendency to bury eggs), P8 and P9 (having
not so strong tendency to bury eggs) were derived from the artificially selectedlines for their oviposition site preference (Takamura and Fuyama Behav.
Genet. 10, 1980)
UV, 25 erg!mm 2!sec., was irradiated for 110 second on the surface of
medium where eggs were already laid by above 4 lines. Number of eggs
which did not hatch after 36 hours of storation of the medium in a humidified container at 25°C was counted. The hatchabilities were 5.34± 1.68,
7.25±2.31, 47.94±2.37, and 58.19±2.35 for P8, P9, M8, M9 lines respectively. While control viabilities with no UV treatment were 87.35±4.10,
94.15±3.54, 96.36±0.78, 82.67 ± 1.96 for each line in the same order. Thereis a possibility that M line eggs are physiologically resistant to UV. Thus,
the UV treatment were done on eggs deposited on the paper, where it is impossible for flies to bury eggs. In this case the hatchability was around 10 %in both P and M lines. Therefore, the large difference in hatchability after
UV treatment was due to the effect of insertion of eggs into the medium.
The medium protected the eggs from the UV. Of course, there is no possibility for eggs to be exposed to such a strong UV used in the experimentin natural condition but if the dosage of UV and its effect has strong correlation, week natural UV may cause difference in fitness such as hatchability,

104

RESEARCHES CARRIED OUT IN 1979preadult viability, and fertility between buried and unburied eggs or larvae
and adults from these eggs. However, it dose not explain the polymorphismof the character in natural population.

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105

ECOLOGICAL GENETICS

Observations on Rice Species and Accompanying Weedsin the Hilly Area of Tropical Asia
Hiko-Ichi OKA, Hiroko MORISHIMA and Yoshio SANO

In the eary dry season (October-November) of 1979, we travelled in the
hilly areas of Nepal, India and Thailand for ecological genetics studies ofrice species and accompanying weeds. Our trip was realized under a grantfor overseas field research of the Ministry of Education. Our objectives
were: a) observations on the diversity of rices and weeds influenced by environmental conditions, b) reexamination of the place and conditions of theinitiation of rice domestication, and c) collection.of seed samples of wild andcultivated rice species for future studies. Observations were recorded at
36 sites for wild rice and at 52 sites for cultivated rice. At each site, recordes
were taken regarding physiography, degree of disturbance and water condition of the habitat, and plant height and percent cover of rice species andmajor weed species. In rice fields, conditions of cultivation were also recorded. As to weeds, a total of 147 species were identified by using herbarium specimens taken at each site. Extracts from our field notes arebriefly summarized below.
I) Oryza perennis (Wild): a) This species was frequently found inmarshy lowlands but never in the hilly area except in the Jeypore Tract,
Orissa,. In the foothill of the Himalayas (Terai), wild rice was not found.
b) Niche differentiation between the perennial and annual types of O. perennis
was observed. The perennial type was found in deep swamps which appeared to be relatively stable, while the annual type was found in temporary
shallow swamps which were parched in the dry season and frequently disturbed by man. The perennial type tended to be associated with perennialcompanion species, while the annual type with annual species. In a small
marsh near Bhbaneswar, Orissa, we observed an annual type growing on thefringe and a perennial type in the center. c) Intermediate perennial-annualtypes were observed in disturbed habitats, and their habitats showed a highdiversity of coexisting plant species. d) In the rice fields directly seeded,
plants of O. perennis were often found occurring as weeds. They appeared

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RESEARCHES CARRIED OUT IN 1979

to have absorbed genes from O. sativa and have become weedy types adapted
to the conditions of rice fields. e) We observed in Thailand that a roadsidepopulation of perennial type became extinct as replaced by sedge species
possibly because of environmental pollution and disturbance, and thatan annual population declined along with an increase of perennial weed
species, probably because of relaxation of habitat disturbance.
2) Oryza sativa (Cultivated): a) Different types of rice culture were observed; lowland fields with varying water depths, terraced fields (upto 2000 m
in Nepal), and upland fields. In accordance with diverse environments,
different varieties were grown. b) Several seed characteristics were examined
to estimate the Indica-Japonica differentiation. Varieties collected from thehilly area in Nepal were mostly of Japonica type, and some from nothern
Thailand appeared to be intermediate Indica-Japonica types. Among native
varieties of Nepal and Manipur, negative phenol reaction, long apiculus
hair and round spikelet shape were frequently found, but these characters
were not inter-correlated so as to make the distinction ofIndica and Japonica
types possible. c) Populations of O. sativa grown in the hilly area werepolymorphic in seed characters, but no trend to Indica-Japonica differentiation could be recognized within populations. In the highland of Nepal,
farmers sometimes grew mixture of two varieties. Populations collected innothern Thailand were glutinous or mixtures of glutinous and non-glutinous
plants. d) Within-populational variability as shown by the generalized
variance of length and width of spikelets tended to be positively correlatedwith species diversity of coexisting weed species.
We came to the view that the Terai region might be not the place of originof cultivated rice, since there was found no wild rice which could be theprogenitor of cultivated rice, although some authors (e.g. Chang 1976) considered the foothill of the Himalayas to be the homeland of cultivated rice.
During this trip, 37 populations of O. perennis, and 51 populations and 190
strains of O. sativa were collected. They are preserved in our Genetic
Stocks Center.
An Experiment on Population Growth of Wild Rice in Greenhouse
Hiroko MORISHIMA

In order to examine how a perennial and an annual strain of Oryza perennis interact in their vegetative and reproductive phases, and how they respondANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

107

to habitat disturbance, two strains, W120 (perennial) and W106 (annual),
were planted in pure stand and in mixture in concrete beds prepared in agreenhouse. The planting was made in 1978 summer with two replications.
At maturity, the seeds were left to natural shedding, and biomass was recorded for the wild rice and also for naturally emerged weeds. Then, thefield was divided into disturbed (the plants were cut and the soil was dugwith a hoe) and non-disturbed plots. The fields were not watered during thewinter-spring season to simulate the water condition in their natural habitats. In the second season, the following characters were recorded; numberof surviving plants, number of recruited plants from soil-buried seeds,
number of buried seeds per unit area, and dry weights of vegetative and reproductive parts of the plants. The results obtained are summarized asfollows:
1) The perennial strain was a strong competitor against the annual strain
in their vegetative growth. 2) With weeds, however, the annual strain
showed a strong competitive ability than the perennial strain. 3) About
40 % of annual plants survived after seed shedding because of a low seedfertility caused by low temperatures in winter. In the non-disturbed annual
plot, the population consisted of both original and recruited plants, whilein the disturbed plot the plants were exclusively recruited ones. 4) The survival of perennial plants was about 75 %, but they produced no seedling.
Their dry matter production was generally greater in non-disturbed plot
than in disturbed plot, but their reproductive effort (proportion of panicle
weight to total plant dry weight) was markedly higher in the disturbed plot.
5) In the non-disturbed plot where the two strains were mixed, the population in the second year consisted of perennial plants only, while in the disturbed plot both the perennial and annual strains coexisted.
The data thus indicated that perennial habit (vegetative propagation)
was advantageous in stable environment, and annual habit (seed reproduction) was advantageous in disturbed environment.
Copper· Tolerance and Reproductive Competition in Barnyard Grass
Hiroko MORISffiMA and Hiko-Ichi OKA

The strains of barnyard grass (Echinochloa crus-galli), C and D, were grownin 1978 in copper-polluted (ca 300 ppm) and control plots, and their seeds
were left to natural shedding. Strain C was tolerant of copper as compared108

RESEARCHES CARRIED OUT IN 1979

with D, but was a weak competitor with D in vegetative growth. This year,
the plants occurring from naturally sown seeds were observed for plant
number and biomass at maturity. Records were also taken for other weed
species naturally emerged. In general, strain D was found to dominate
over C. In the polluted plot, however, C was not inferior to D so much.
The dry matter weights of C and D coexisting on the same sampling sitewere negatively correlated among sites in the control plot, but they werepositively correlated in the polluted plot. This indicated that the mode ofcompetitive interaction between the two strains was affected by copper
treatment. The total biomass of other weed species showed no significant
difference between the polluted and control plots. Comparing the two plots,
however, we found marked difference in the relative dominance of component species suggesting a change in community structure caused by copper
treatment.
Copper Tolerance and Reproductive Competition in
Four Annual Weeds
Hiroko MORISHIMA and Hiko-Ichi OKA

Last year (1978), the seeds of Cyperus difformis (Cy), Fimbristylis miliacea(Fm), Centipeda minima (Ce) and Vandellia angustifolia (Va) were sown in
mixture (1: 1) in all possible binary combinations on normal and toxic(200 ppm copper) soils prepared in trays. Their seeds were left to natural
seeding from which the plants occurred in the second (this) year. In boththe first and second years, observations were recorded for their number andtotal dry weight at seed maturity. Their relative dominance was evaluatedby the ratio of total dry weight of coexisting two species. On the normal
soil, their dominance was higher in the order Fm=,=;:Cy>Ce>Va in the firstyear, and Cy;?::Fm;?::Ce>Va in the second year. Phenotypic plasticity,which was greater in the order Cy>Fm>Ce>Va, could have played somerole in their adaptation under the competitive condition. On the toxic soil,
dominance relation was Cy>Ce>Fm>Va in both the first and second
years. This was in agreement with the order of their copper tolerance,
Cy>Ce>Fm=,=;:Va, which was evaluated on the basis of "toxic-normal
ratio" in plant weight in pure stand.

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Variation in Competitive Ability among Oryza perennis Strains
Yoshio SANO and Hiroko MORISHIMATo examine how competitive ability as shown by neighbour effect is associated with the variation in reproductive strategy, a mix-planting experiment
was carried out with 26 strains of Oryza perennis (wild) ranging between
perennial and annual types and two strains of O. sativa. They were tested
in pure stand and mixture with a test-strain in a concrete bed with automatic shortday control. As the test-strain, a strain of Asian perennial typeof O. perennis, W120, was used; many plants having the same genotype wereobtained from regeneration of stem cuttings taken from one plant of thestrain, and they were planted in each row alternately with the seedlings ofthe strain to be tested with an interval of 10 cm. The dry weight at seedmaturity was recorded in each strain. Competitive ability was estimatedin terms of (Xij-Xii)-(Xji-Xjj), where Xij is the measurement of strain
i in mixture with j (test-strain) and Xii represents that in pure stand.
The strains tested showed a wide range of variation in competitive ability.
In general, perennial wild strains were stronger competitor with the teststrain than annual wild strains. A negative correlation (r= -0.41,significant at 5 % level) was found between competitive ability and reproductive
effort (proportion of seed weight to total plant dry weight in pure stand).
As reported previously (Ann. Rep. 27: 92 & 28, 109), the same set of strains
had been tested for competitive ability with two different test-strains, asativa strain (Taichung 65) and an annual wild strain (W107). The results
obtained with different test-strains were not consistent; when tested witha perennial atrains, perennial strains were stronger in competition than annual
strains, while annual strains were stronger than perennial strains when tested
with an annual strain. The sativa test-strain can be considered as a perennial type. This complex of variations in competitive ability seems to haveresulted from differentiation of O. perennis strains in adaptive strategy, andits analysis is left for studies in the future.
Neighbour Effects between Plants of Oryza sativa andO. glaberrima Grown in Mixture in Nigeria
Yoshio SANO and Hiroko MORISHIMA

Two rice species, Oryza glaberrima and O. sativa, are often planted in mix-

110

RESEARCHES CARRIED OUT IN 1979

ture in West Africa. Seeds of coexisting plants of the two species, sampledfrom a upland and a lowland field in Nigeria and multiplied in Misima, wereused for this experiment; the material consisted of four strains, G 1 (glaberrima, upland), G 2 (glaberrima, lowland), Sl (sativa, upland), and S2 (sativa,
lowland). The four strains were tested in a mixture-diallel experiment on
a controlled soil moisture gradient to examine neighbour effect, which wasevaluated in terms of (log Xij-Iog Xii) where Xij stood for the dry weightof plant i neighboured by j. The data showed that, although lowland strains
were generally strong competitos with upland strains, the neighbour effects
markedly differed according to strain combinations and water conditions.
In the mixture of two species collected from the same field, O. sativa tended
to be a strong competitor against O. glaberrima under wet conditions, but
was not so under dry conditions. This trend was particularly obvious whenupland strains of the two species were in mixture. On the other hand, lowland strains of the two species appeared to be cooperative in mixture, showing a RY.T. higher than unity (R.Y.T.= 1/2(Xij/Xii+XjdXjj); relative
yield total). These changes in neighbour effect suggest th.at the responses
to neighbouring plants differ according to water conditions.

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111

APPLIED GENETICS

Effect of Assortative Mating on the Multigenic Character ina Finite Population
Shinya IYAMA

Computer simulation was carried out to inquire into the effect of assortative
mating on the genetic structure in a finite population when assortment was
based on the character controlled by many genes. Assortative mating was
assumed to take place on the quantitative character governed by 31 equallyeffective independent genes. No other selection was not assumed in the simulation. In a randomly mating population of finite size, it has been known
that the genetic variability decreases with generations and finally the population reaches a fixation into a single genotype. The results of simulation
showed that the assortative mating accelerated the speed of loss of variability in the population (Table 1). Though the temporary increase in
genetic variability was observed in the early generations of assortative mating, but differentiation was not attained and the population became fixed
into any single genotype between both extremes. When the more similarity
Table 1. Percentage of fixed loci in successive generations of assortative matingbased on the character governed by 31 equally effective independent genes.
Initial gene frequency=0.5. Average of 10 runs.
Population size=40
Generation

10
20
30
40
50
100
150

Assortative
mating*
II
I
4.0
21. 8
43.5
59.2
77.4
99.2
100.0

0.7
9.7
15.5
31.0
37.4
72.3
91.0

Randommating**(0)0
1.4 (1.1)
6.5 (5.9)
14.3 (13.3)
18.4 (21.8)
57.1 (57.1)
77.0 (77.0)

Population size =200
Generation

50
100
150
200
300
400
500

Assortative
mating*
I
II
0
31.6
78.6
90.3
96.3
97.2
100.0

0
3.7
29.0
47.5
73.3
86.6
94.5

Randommating**0(0)
0.7 (1.1)
7.1 (5.9)
12.3 (13.3)
32.3 (30.1)
43.9 (45.0)
58.1 (57.1)* I; Perfect assortative mating; II; Assortment included three adjacent classesof phenotype to both sides. ** Theoretical values are shown in parentheses.

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

required for assortment, the faster the fixation. It was unlikely that theassortative mating resulted differentiation of genotypes even for multigenic
character when the population was of finite size.
Changes in Isozymes and Some Quantitative Characters
Observed in Hybrid Populations of Rice
Hiroko MORISHIMA and Tom ENDO

The wild rice, Oryza perennis, has many alleles at various loci controlling
isozymes, but only a part of the variation is found in cultivated rice, O.
sativa, derived from the former species. To look for the reason why domestication results in reduction of allelic variations, hybrid populations from wild
and cultivated strains were propagated in bulk from F 2 to F 6 generation,and the frequencies of alleles at two loci (PXl and ACP1) controlling peroxidase and acid phosphatase isozymes in the F 6 population, respectively, werecompared with those observed in Fa.
The data showed that two alleles, Pxi A and ACPi, tended to increase, bothbeing possessed by the sativa parents. In Fa, the seeds for the next generation were harvested by two methods, one from the seeds remaining on
panicles at maturity and the other from the seeds shed on the soil, and thesame procedure was taken in each group for successive generations. In
general, the genotype of populations shifted towards the cultivated typein response to the pressure of cultivation. The populations raised from theseeds remaining on panicles more rapidly shifted to cultivated type than thosefrom shed seeds, in certain quantitative characters such as degree of shedding and those concerning yielding potential. Further, the former populations differed in the frequencies of isozyme alleles from the latter. It
appeared that a gene for earliness was linked with ACP1'
Nitrogen Fixation in the Rhizosphere of Cultivatedand Wild Rice
Yoshia SANO, Taro FUJII, Shinya IYAMA, Yukinori HIROTA,
and Kazua KOMAGATA

To look into variations among genotypes of N z fixing ability in the rhizosphere of paddy rice, a modified assay system using the acetylene reduction technique was devised in order to measure large numbers of plants
easily. Acetylene reducing activity was detected immediately after incu-

RESEARCHES CARRIED OUT IN 1979113

bation and increased almost linearly.
The activity measured by the present method was compared with that obtained by the whole plant assay system previously reported (Ann. Rep. 28:
114). Correlation between the readings obtained by the two methods from
21 cultivars was highly significant (r=0.74, d.f. = 19), indicating that the
detachment of the shoot did not seem to affect the acetylene reducing ability
in the rhizosphere when the activity was measured within 2 hr of incubation.
The present assay system was successfully used to examine the N z fixing
ability of a number of strains. Among 47 O. sativa strains, acetylene reducing ability per plant was significantly correlated with the activity per g
dry root but not with dry root weight. This suggested that the activity perplant is independent of the quantity of root. Then acetylene reducing
activity per g dry root was compared among strains. The activity showeda wide range of variation from 81 to 2148 nmol per hr per dry root. In
addition, the activities of O. sativa and O. glaberrima strains (cultivated)were significantly higher than those of O. perennis and O. punctata strains.
To assess seasonal and diurnal changes in acetylene reducing ability, a ricestrain C5444 which showed a relatively high activity, was used to measure
the activity at different stages. At each stage, four plants were examined
six times every 4 hrafter noon. The activity was low in the early stages ofgrowth and gradually increased as development advanced. Maximum
activity was observed during flowering though the diurnal fluctuation also
increased after flowering. The activity tended to be higher at night thanin the daytime.
Nitrogen fixing bacteria were isolated from the rhizosphere of C5444 on
a nitrogen-free agar medium. Isolated bacteria was subcultured several
times with the same media, and N z fixers were purified. They were spiral,filamentous, or rod shapes. The results indicated that various species ofN2 fixers existed in the rhizosphere of rice.
Analysis of Genes Controlling the F 1 Pollen Sterility
between Oryza sativa and O. glaherrima
Yoshio SANOThe Pi plants between the two cultivated rice species, Oryza sativa andO. glaberrima, are highly pollen-sterile although the chromosomes normallypair in meiosis. They can be backcrossed as some embryosacs remain

114

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

functional. As reported previously (Ann. Rep. 25: 85 & 27: 80), isogenic
F1-sterile lines having the genetic background of sativa (Peiku, Acc. 108,
an Indica type from Taiwan) and of glaberrima (W025 from Guinea) parent
were isolated from BsF 2 plants, respectively. They were self-fertile andshowed semi-sterility in the F 1 plants when crossed with the respective parental strains. But the F 2 plants were fully fertile. This F 1 sterility was favorably explained by a "one locus sporo-gametophytic interaction" modelof sterility genes, which assumes that Acc. 108 and W025 have Sf Sf S2
S2 and S1 S1 S: S:, respectively, and that if a S1 or S2 gene is prepresent
in the maternal tissue, gametes with Sf or S: deteriorate.
A following-up experiment was initiated in 1975 by using a Japonica
strain (Taichung 65 from Taiwan) as the sativa parent and W025 as theglaberrima parent, following the same procedure as in the preceding experiment. The backcrossing experiment showed a different pattern of segregation from that observed before. Out of 51 B9 F 1 plants, 27 were fertileand 24 were semi-sterile, giving a 1: 1 ratio. When the semi-sterile BsF1
plants were pollinated as the pollen parent, all the B9 F 1 plants were semisterile. It was also observed that the partially pollensterile plants had a seedfertility exceeding 90 percent and their selfing produced fertile and semisterile plants in a 1: I ratio. This indicates that megaspores were not affected by the sterility genes.
This F 1 sterility can be explained by assuming that Taichung 65 and W025
have S: S: and S3 S3' respectively, and that if a S3 gene is present in the maternal tissue, pollen with S: deteriorates (one locus sporo-gametophytic interinteraction). In addition, the heading date of plants having S3 S: or S: S:
was about 10 days earlier than those having S3 S3' This difference was notobserved in shortday conditions, suggesting that a recessive gene (se) controlling photosensitivity (carried by W025) was closely linked with the S3
gene.

Adaptation to High Ambient Temperature of Japanese quail
Takatada KAWAHARA

F 1 hybrid birds of domestic Japanese quail were used for preliminarily
examining their adaptability to the high ambient temperature. The numberof female .quails examined was 36 birds for temperature treatment and 51
birds for non-treatment control, respectively. The experiment was carried

RESEARCHES CARRIED OUT IN 1979115

out for one hundred and forty days from 231 to 370 days after hatching withthree consecutively changed ambient temperatures. For the pre-temperature
treatment, the temperature was kept at 25°C±2°C (relative humidity: 60±
10%) for 17 days. Then it was gradually changed to 35°C within 6 days,
for the first high temperature treatment, where it was kept at 35°C±2°C(50± 10 %) for 23 days, then again changed to 39°C within 3 days for thesecond high temperature treatment, where it was kept at 39°C±2°C (45±10%) for 20 days and returned to 25°C within 2 days. After then, the temperature was kept at 25°C±2°C (60± 10 %) for 69 days. The control group
was reared under fixed temperature conditions: 25°C±2°C and relative
humidity 60±1O%. Photoperiod applied was 18-hour light and 6-hour
darkness throughout the experiment. Food (20 % crude protein) and water
were supplied ad libitum.
Almost all birds showed a panting under either of the two high temperature treatments. Body weight also decreased under high ambient temperature treatment, to 89.2 % at 35°C and to 86.7 % at 39°C of those of thecontrol. Egg production rate of both groups in the pre-treated period(25°C) was ranging from 65.3 % to 84.7%. During the first high temperature treatment (35°C) it decreased to minimum 36.1 % (control: 75.5%
to 89.2 %), and during the second high temperature treatment (39°C), it
decreased further to minimum 20.8 % (control: 79.4 % to 88.2 %). But theegg production rate was recovered in the later period of high temperature
treatment, to 60 % at 35°C and to 52 % at 39°C, respectively. When temperature conditions returned from 39°C to the moderate temperature 25°C,
the egg production rate recovered to the level of control in 20 days. Egg
weight was reduced under high temperature conditions, the average being
8.50 g for 35°C treatment (9.24 g in a control) and 8.17 g for 39°C treatment(9.29 g in a control). Differences between the treated and the control
groups were statistically significant at the 1% level. When temperature
conditions returned from 39°C to the moderate temperature 25°C, the egg
weight rapidly recovered to the level of the control.
Under the ambient temperature conditions as high as the body temperature, egg production rate and egg size of domestic Japanese quails decreased
but egg production rate recovered gradually in the later period of the treatment.

116

RESEARCHES CARRIED OUT IN 1979

Domestication in Japanese quail, Coturnix coturnix japonica
Takatada KAWAHARA

According to tradition, Japanese quail has been domesticated for the last
several hundred years in Japan. The birds were loved for its countrified crowby the Samurai caste in the feudal days before the close of 19th century, but
after the opening of the present century, they have become economically important for producing eggs and meat. Recently, they achieved rapid progressas an experimental animal. However, the domestic Japanese quails arevery similar in appearance to the wild ones, and it is often impossible todistinguish clearly one from the other. Both groups are very different insome physiological as well as growth properties.
This experiment was carried out to find the genetic changes in wild Japanese quails reared for ten generations under domestic conditions. Examination was made on several characters of the birds which had been reared
without conscious selection since they were captured in the field. Eggs
laid during the ages of 20 to 35 weeks were used to obtain the birds for the
next generation.
The results indicated that the most striking changes of the quails toward
the domestic type occurred several characters: to early sexual maturity andincreased fertility, hatchability, viability, egg production rate, fitness index(fertility x hatchability x viability x egg production rate) and body size.
Decrease in the variability of various characters was also observed in progressive generations. The degree of change in performance with progress ofgeneration was measured by the linear regression of a given trait on generation. Number of generations required to reach the level of domestic
birds was estimated for various traits based on the regression. It ranged
from 12 to 64 generations. In fitness index, for instance, it was estimatedas 24 generations.

BOOKS AND PAPERS PUBLISHED BY STAFF MEMBERS

117

BOOKS AND PAPERS PUBLISHED IN 1979
BY STAFF MEMBERSAKATSUKA, A., O. NISHITANI, T. KITAGAWA, A. KAGEYAMA, I. INANAand Y. NAKAGOME
1979: Trisomy 9 mosaicism with punctate mineralization in developing cartilages.
Eur. J.. Pediat. 131: 271-275.
ARIGA, H., H. SHIMOJO, S. HIDAKA and K. I. MIURA 1979: Specific cleavage of theterminal protein from the adenovirus 5 DNA under the condition of single-strand
scission by nuclease SI. FEBS Lett. 107: 355-358.
AZUMI, J., Y. NAKAGOME and E. MATSUNAGA 1979: A new approach in the evaluationof C-positive variants in man. Jap. J. Hum. Genet. 24: 99-104.
AZUMI, J., I. FURUTA, M. IWAI and G. KOHAMA 1979: Two cases of 4p-syndrome
with cleft palate. Sapporo Med. J. 48 (2):218-221.
CROW, J. F. and M. KlMURA 1979: Efficiency of truncation selection. Proc. Natl.
Acad. Sci. USA 76: 396-399.
GUTMAN, G. A. and K. MORIWAKI 1979: Rat kappa-chain allotypes. II. Multiple specificities detected by RI-1b antisera and their distribution among Rattus rattus andother Asian rodents. Immunogenet. 8: 139-151.
HAYASHI, J. I., H. YONEKAWA, O. GOTOH, Y. TAGASHIRA, K. MORIWAKI and T. H.
YOSIDA 1979: Evolutionary aspects of variant types of rat mitochondrial DNAs.
Bioch. Biophys. Acta 564: 202-211.
HIDAKA, S., K. SHIMOTOHNO, K. MIURA, Y. TAKANAMI and S. KUBO 1979: Nucleotide
sequence near the 5'-terminal of cucumber mosaic virus RNA No. 5 segment.
FEBS Lett. 98: 115-118.
HIROTA, Y., S. YASUDA, M. YAMADA, A. NISHIMURA, K. SUGUMOTO, H. SUGISAKI, A. OKA
and M. TAKANAMI 1979: Structural and functional properties of the Escherichia coli
origin of DNA replication. Cold Spring Harbor Symposium on Quantitative Biology.
43: 129-138.
INOUE, T., H. MATSUDA, K. SHIMURA, Y. HAMAZAKI, I. KIKUTA, K. IINUMA and Y.
NAKAGOME 1979: A ring chromosome 9 in .an infant with malformations. Hum.
Genet. 50: 231-235.
INOUE, Y. 1979: The fate of polymorphic inversions of Drosophila melanogaster transferred to laboratory ,conditions. Jap. J. Genet. 54: 83-96.
INOUE, Y. and T. K. WATANABE 1979: Inversion polymorphisms in Japanese natural
populations of Drosophila melanogaster. Jap. J. Genet. 54: 69-82.
KADA, T., T. INOUE, A. YOKOIYAMA and LB. RUSSELL 1979: Combined genetic effects
of chemicals and radiation. Proc. 6th Intern. Congo Radiation Res. 711-720.
KANDA, N. and T. H. YOSIDA 1979: Identification of the facultative heterochromatic
X chromosomes of 25 rodent species. Cytogenet. Cell Genet. 24: 12-22.
KIMURA, M. and J. L KING 1979: Fixation of a deleterious allele at one of two "duplicate" loci by mutation pressure and random drift. Proc. Natl. Acad. Sci. USA 76:

118

ANNUAL REPORT OF NATONAL INSTITUTE OF GENETICS NO. 30

2858-2861.
KIMURA, M. 1979: Model of effectively neutral mutations in which selective constraint
is incorporated. Proc. Natl. Acad. Sci. USA 76: 3440-3444.
KIMURA, M. 1979: The neutral theory of molecular evolution. Scientific American
241 (5): 94-104.
KIMURA, M. and T. OHTA 1979: Population genetics of multigene family with specialreference to decrease of genetic correlation with distance between gene members on
a chromosome. Proc. Natl. Acad. Sci. USA 76: 4001-4005.
KOMEDA, Y. and T. IINO 1979: Regulation of expression of the flagellin gene (hag) in
Escherichia coli K-12: Analysis of hag-lac gene fusions. J. Bacteriol. 139: 721-729.
KURODA, Y. 1979: Procedure for detecting mutagens in cultured cells. (In Japanese)
Tissue Culture (Tokyo) 5: 137-147.
KURODA, Y. 1979: Insect tissue culture in genetic research. (In Japanese) Tissue Culture(Tokyo) 5: 297-309.
KURODA, Y. 1979: III vitro cultivation of insect cells. (In Japanese) Protein, Nucleic
Acid and Enzyme (Tokyo) 24: 1305-1315.
MATSUNAGA, E. and K. SHIOTA 1979: A reply to Doctor Shepard's letter to the editor.
Teratology 19: 127_128.
MATSUNAGA, E. 1979: Hereditary retinoblastoma: Host resistance and age at onset.
J. Natl. Cancer Inst. 63: 933-936.
MATSUNAGA, E. and K. SHIOTA 1979: Threatened abortion, hormone therapy and malformed embryos. Teratology 20: 469-480.
MATSUNAGA, E. and K. SHIOTA 1979: A response to Doctor D. T. Janerich's letter tothe editor. Teratology 20: 485-486.
MINEZAWA, M., K. MORIWAKI and K. KONDO 1979: Geographical distribution of Hbb P
allele in the Japanese wild mouse, Mus musculus molossinus. Jap. J. Genet. 54:
165-173.
MIURA, K., Y. KODAMA, K. SHIMOTOHNO, T. FUKUI, M.IKEHARA, I. NAKAGAWA andT. HATA 1979: Inhibitory effect of methylated derivatives of guanylic acid for protein synthesis with reference to the funtionalsttucture of the 5'-cap in viral messenger
RNA. Biochim. Biophys. Acta 564: 264-274.
MONoz, L. E., Y. SADAIE and R. H. DOl 1978: Spore coat protein of Bacillus subtilis:
Structure and precursor synthesis. J. BioI. Chem. 253: 6694-6701.
MORISHlMA, H. and H. I. OKA 1979: Genetic diversity in rice populations of Nigeria:
Influence of community structure. Agro-Ecosystems 5: 263-269.
MOTOJIMA, K., I. YAMATO, Y.ANRAKU, A. NISHIMURA aNd Y. HIROTA 1979: Amplification and characterization of the proline transport carrier of Escherichia coli K-12
by using proP hybrid plasmids. Proc. Natl. Acad. Sci., USA 76: 6255-6259.
MORIWAKI, K., T. SHIROISHI, M. MINEZAWA, T. AOTSUKA and K. KONDO 1979: Frequency distribution of histocompatibility-2 antigenic specificities in the Japanese wild
mouse genetically remote from the European subspecies. J. Immunogenetics 6: 99113.
MUROFUSHI, M. and T. H. YOSIDA 1979: Cytogenetical studies on fishes, I. Karyotypes of four filefishes. Jap. J. Genet. 54: 191-195.

BOOKS AND PAPERS PUBLISHED BY STAFF MEMBERS

119

MUROFUSHI, M. and T. H. YOSIDA 1979: Cytogenetical studies on fishes, II. Karyotypes of four carangid fishes. Jap. J. Genet. 54: 367-376.
NAGASAWA, M. and T. MARUYAMA 1979: An application of time reversal of Markov
process to a problem of population genetics. Adv. Appl. Prob. 11: 457-478.
NAKAYAMA, T., L. E. MUNOZ, Y. SADAIE and R. H. DOl 1978: Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis. J. Bacterio!.
135: 952-960.
NOGUCHI, T. 1979: Mouse teratomas; as a model for the study of cell differentiationand neoplasia. (In Japanese) Exp. Anim. 28: 121-125.
NOGUCHI, T. 1979: Teratoma (129, LT mice). (In Japanese) Handbook of animal desease models, p670-674. ISHIYAKU-SHUPPAN (Tokyo)
OOAKA, T., H. IKEDA, K. MORIWAKI, A. MATSUZAWA and M. MIZUNO 1979: Genetic
resistance to murine leukemia and sarcoma viruses in strain G and Japanese wild
mice. Oncogenic Viruses and Host Cell Genes, 39-44, Academic Press.
OKA, H. I. 1977: The ancestors of cultivated rice and their evolution. Meeting on
African rice species, IRAT-ORSTOM, pp. 57-62, Paris.
OKA, H. I. 1977: Genetic variations of Oryza glaberrima, their survey and evaluation.
ibid. pp. 77-86.
OKA, H. I. and K. H. TSAI 1978: The use of isogenic lines in breeding and genetic research: A review with special reference to experiments in rice. SABRAO J. 10(2):130-142.
OHTA, T. 1979: An extension of a model for the evolution of multigene families by unequal crossing-over. Genetics 91: 591-607.
SAKAI, S., K. MORIWAKI, S. AMANO, J. I. HAYAKAWA, T. KAIDOH and M. TAKAHASHI
1979: Allotypes of C3 in laboratory and wild mouse distinguished by alloantisera.
J. Immunology 123: 216-221.
SANO, Y., Y. E. CHU and H. I. OKA 1979: Genetic studies of speciation in cultivatedrice. I. Genic analysis for the F 1 sterility between O. satiFa L. and O. glaberrima
Steud. Jap. J. Genet. 54: 121-132.
SOEDA, E. and K.MIURA 1979: Common structural features in DNA around thereplication origin of papovaviruses, mouse polyoma virus, simian virus 40 and human BKV. FEBS Lett. 101: 359-363.
SOEDA, E., J. R. ARRAND, N. SMOLAR and B. E. GRIFFIN 1979: Polyoma virus DNA.
I. Sequence from the early region that contains the viral origin of replication andcodes for small, middle and (part of)large T antigens. Cell 17: 357..,.370.
SOEDA, E. 1979: The primary sequence of the late region of polyoma virus DNA. II.
The expression of the late genes and comparison with DNA sequences of SV40 and
BKV. Nucleic Acids Res. Symp.Series 6: sI57-160.
SOEOA, E., J. R. ARRAND and B. E. GRIFFIN 1979: Polyoma virus, The early region
and its T-antigens. Nucleic Acids Res. 7: 839-857.
SUGIMOTO, K., A. OKA, H. SUGISAKI, M. TAKANAMI, A. NISHIMURA, Y. YASUDA and Y.
HIROTA 1979: Nucleotide sequence of Escherichia coli K-12 replication origin.
Proc. Nat!. Acad. Sci., USA, 76: 575-579.
SUGIURA, M., M. SUZUKI, E. OHTSUKA, S. NISHIKAWA, H. UEMURA and M. IKEHARA 1979:

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30
Purification of T4 RNA ligase by 2', 5'-ADP sepharose chromatography. FEBS
Letters 97: 73-76.
SUGIURA, M. and J. KusuDA 1979: Molecular cloning of tobacco chloroplast ribosomal RNA genes. Molec. Gen. Genet. 172: 137-141.
SUGIURA, M., J. KusuDA and K. SHINOZAKI 1979: Cloning of tobacco chloroplast ribosomal RNA genes. J. Supramolecular Structure, 9, Supplement 3: 148.
SUGIYAMA, H., M. YUTSUDO, K. TOYOSHIMA, T. H. YOSIDA and Y. MURATA 1979: Establishment of cell lines from the wild rodent Millardia meltada and test for endogenous virus. Gann 70: 297-303.
SUGIYAMA, T. and T. FUJISAWA 1979: Genetic analysis of developmental mechanisms in
hydra. VI. Cellular composition of chimera hydra. J. Cell Sci., 35: 1-15.
SUGIYAMA, T. and T. FUJISAWA 1979: Genetic analysis of developmental mechanisms in
hydra. VII. Statistical analyses of developmental-morphological characters andcellular compositions. Develop., Growth and Differ., 21: 361-375.
TAKAHATA, N. and T. MARUYAMA 1979: Polymorphism and loss of duplicate gene l:)Xpression: A theoretical study with application to tetraploid fish. Proc. Nat!. Acad.
Sci. USA 76: 4521-4525.
TAKAHATA, N. and M. KIMURA 1979: Genetic variability maintained in a finite population under mutation and autocorrelated random fluctuation of selection intensity.
Proc. Nat!. Acad. Sci., USA 76: 5813-5817.
TAZIMA, Y. 1979: Consequences of the AF-2 incident in Japan. Environmental Health
Perspectives, 29, 183-187.
UEDA, S., H. ITO, H. MASAI and T. KAWAHARA 1979: Abnormal organization of thecerebellar cortex in the mutant Japanese quail, Coturnix coturnix japonica. Brain
Research 177: 183-188.
WATANABE, T. K. and M. KAWANISHI 1979: Mating preference and the direction ofevolution in Drosophila. Science 205: 906-907.
WATANABE, T. K.1979: A gene that rescues the lethal hybrids between Drosophila melanogaster and D. simulans. Jap. J. Genet. 54: 325-331.
YOSIDA, T. H. 1979: Genetic aspects of polymorphic C-bands in chromosomes of theblack rat (Rattus rattus tanezumi) in Japan. Cytologia 44: 265-274.
YOSIDA, T. H. 1979: Sex chromosome anomalies in F 2 hybrids between Oceanian and
Ceylonese type black rats. Jap. J. Genet. 54: 27-34.
YOSIDA, T. H. 1979: Population cytogenetics of the black rat. Proc, XIV Intern.
Congress of Genetics, Moscow, 1978 (Symposium). (in press).
YOSIDA, T. H. 1979: Karyotype of F 1 hybrids between Mauritius and Oceanian typeblack rats. Proc. Japan Acad. 55, Ser. B: 275-279.
YOSIDA, T. H. 1979: Possible evidence for the karyotype evolution of the Indian spiny
mouse due to tandem fusion of the house mouse chromosomes. Proc. Japan Acad.
55, Ser. B: 270-274.
YOSIDA, T. H. 1979: Karyotype of Indian spiny mouse resulted from the tandem fusionof some of the house mouse chromosomes. Cytologia (in press).
YOSIDA, T. H. 1979: A comparative study on nucleolus organizer regions (NORs) in
7 Rattus species with special emphasis on the organizer differentiation and species

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121

evolution. Proc. Japan Acad. 55, Ser. B.: 481-486.
YOSIDA, T. H. 1979: The karyotype of Rattus villosissimus resulted from Robertsonian
fission of small metacentrics. Proc. Japan Acad. 55, Ser. B.: 497-501.
YOSIDA, T. H., H. KATO, K. TSUCHIYA, K. MORlwAKI, Y. OCHIAI and J. MONTY 1979:
Black rats from Mauritius with a new karyotype involving the Robertsoan fission(a preliminary note). Proc. Japan Acad. 55, Ser B.: 120-125.
YOSIDA, T. H., H. KATO, K. TSUCHIYA, K. MORlwAKI, Y. OCHIAI and J. MONTY 1979:
Mauritius type black rats with peculiar karyotypes derived from Robertsonian fissionof small metacentrics. Chromosoma (Berl.) 75: 51-62.
YOSIDA, T. H., K. MORlwAKI, H. KATO, K. TSUCHIYA, M. SABARATONAM, K. D. ARULPRAGASAM and H. E. FERNANDO 1979: Note on distribution of Ceylonese type blackrats in Sri Lanka. Proc. Japan Acad. 55, Ser. B.: 351-356.
YOSIDA, T. H. and C. TAYA 1979: Studies on hybridization in rodents, II. Histological observations on the hybrid embryos developed by artificial insemination between
Norway rats and black rats. Jap. J. Genet. 54: 371-378.
YOSHIKURA, H., Y. NAITO and K. MORlwAKI 1979: Unstable resistance of G mouse
fibroblasts to ecotropic murine leukemia virus infection. J. Virology 29: 1078-1086.
YOSHIMURA, K., H. AlBA, N. HIRAYAMA and T. H. YOSIDA 1979: Acquired resistanceand immune responces of eight strains of inbred rats to infection with Angiostrongylus cantonensis. Jap. J. Vet. Sci. 41: 245-259.

ABSTRACTS OF DIARY

122

ABSTRACTS OF DIARY FOR 1979
February

March

April
May

July
AugustSeptember
October
December

9
13
23
28
1
8
12
15
23
30
19
2
21
25
13
28
3
14
6
27
25
25
3

254th Meeting of Misima Gene ticists'Club
255th Meeting of Misima Geneticists' Club
256th Meeting of Misima Geneticists' Club
144th Biological Symposium
145th Biological Symposium
257th Meeting of Misima Geneticists' Club
258th Meeting of Misima Geneticists' Club
259th Meeting of Misima Geneticists' Clubl46th Biological Symposium
260th Meeting of Misima Geneticists' Clubl47th Biological Symposium
l48th Biolobical Symposium
l49th Biological Symposium
l50th Biological Symposium
26lst Meeting of Misima Geneticists' Club
262nd Meeting of Misima Geneticists' Club
l5lst Biological Symposium
152nd Biological Symposium
153rd Biological Symposium
154th Biological Symposium
155th Biological Symposium
156th Biological Symposium
263rd Meeting of Misima Geneticists' Club

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30 123

FOREIGN VISITORS IN 1979
January

February
March
April
May

June
AugustSeptember

October

LAMSEEJAN, S., Kasetsart University, Thailand
CAMPIRANAN, A, Kasetsart University, Thailand
10
SUEOKA, N., University of Colorado, U.S.A
28-March 1
RAMEL, c., University of Stockholm, Sweden
1
PITTENDRIGH, C. S., Stanford University,
U.S.A.
PRICE, C. A, University of Rutgers, U.S.A.
21
SOKAL, R. R, University of New York, U.S.A
2
SORIANO, J. D., University of the Philippines,
17
Philippines
SEARLE, T., MRC Radiobiology Unit, England
19
SCHAECHTER, M., University of Tufts, U.S.A
19-21
24-June 5
CROW, J. F., University of Wisconsin, U.S.A
HAYNES, R. H., University of Yoke, Canada
1
14
NAKADA, D., University of Pittsburgh, U.S.A.
MILLER, J. R., University of Columbia,
14-15
Canada3-March 30, '80 PARIDA, B. B., University of Utkal, India
5-6
BARLOW, B. A, University of Flinders, Australia
ROJANAPO, W., Nat'l Cancer Institute, Thailand
HARLAN, J., University of Illinois, U.S.A.
1-3
RUFFlE, J., Centre d'Hemotypologie du CNRS,
15FranceSCHMID, D.O., Miinchen University, Germany
22
PAVAN, C., University of Sao Paulo, Brazil
PATERNIANI, University of Sao Paulo, Brazil
IKUTA, H., University of Sao Paulo, Brazil
24-27
POULSON, D. F., University of Yale, U.S.A.

10

124

FOREIGN VISITERS

25-26
November

8de GROUCHY, J., Lab. de Cytogenetique,
Hopital des Enfants-Malades, France
WArSON, J. D., Cold Spring Harbor Laboratory, U.S.A.

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 30

125

AUTHOR INDEXAlBA, H

62
81,82
ARIGA, H
14
ARRAND, J. R. .
92
AZUMI, J. I.
99
CROW, J. F
84
EGAMI, N
30
EMORI, T
60
ENDO, T
.43, 112
Fum, T. .
80, 112FUJISHIMA, T
100
FURUYA, T
70
GOTO, M
74
GOTOH, 038, 41, 60
GRIFFIN, B. E. .
92
HARA, M
79
HAYASHI, J. I.
38, 41, 60
HIDAKA, S. .
14
HIRAYAMA, N
62
HIROTA, Y. .
21, 25, 112
IHARA, M.43
IMAI, H. T. .
67
INOUE, T • . . . . . . . . . . . . . . . . . . . . . . . 69, 79
INOUE, Y.
.
77,78
IYAMA, S
111,112
KADA, T69, 79KANEKO, K. .
79
KAWAHARA, T. .
114, 116
KAWANISHI, M
91
KIMURA, M. • •••••••...... 84, 85, 86, 87
KING, J. L.
84
KOMAGATA, K.
112
KOMEDA, Y. .
27
KONDO, K.
67
KURODA, Y. .
45, 46, 47
KUSUDA, J. .
16, 20MARTIN, M. A. .
19
MARUYAMA, T
25, 92
MATSUDA, Y. .
67
AMANO, E.

.

96, 9950
MATSUTANI, E. . ..............•...... 46
MINAKATA, H. .
79
MINATO, K.
.48
MIURA, K. I.
12, 13, 14
MIYASHITA, N • . . . . . . . . . . . . 38, 41, 63, 66
MOCHIZUKI, H. .
79
MORISHIMA, H. .
105, 106, 107, 108,
109,112MORIWAKI, K. .
30, 31, 34, 38,41,
49, 56, 60, 63, 66, 67
MURAKAMI, A
51,70,72,73,74,75,77
MURATA, Y. .
61
MUROFUSHI, M. .
63
NAGASE, S. .
60
NAKAGOME, Y. .
97, 99
NAwA, S • . . . . . . . . . . . . . . . . . . . . . . . . . . 52
NISHIMUNE, Y. .
50
NOGUCHI, T. .
.49, 50
OGISO, Y
50
OHNUMA, A. .
77
OHTA, T
86
OKA, H. I.
105, 107, 108
OKA, S
99
ONIMARU, K
20
OONO, K • . . . . . . . . . . . . . . . . . . . . . . . . . . 18
OZAWA, T • . . . . . . . . . . . . . . . 70,72,73,75
P ARIDA, B. B. .
57
PETRAS, M. L.
38, 41SADAIE, Y
79
SAGAI, T.
. . . . . . . . . . . . . . . . . . . • . . 34, 66
SAKAIZUMI, M. .
30
SAKATE, S
20
SANO, Y. .
105, 109, 112, 113
SHIMOI, K. . . . . . . . . . . . . . . . . . . . • . . . . . . 79
SHIMOJO, H. .
14
SHlNOZAKI, K. .
16
SHIOTA, K.
96
MATSUNAGA, E

MATSUSHIRO, A.

AUTHOR INDEX

126

31, 34, 63, 66
19, 92SUGIURA, M.
.
15, 16, 18
SUGIYAMA, H .. ,, .61
SUZUKI, M
15
SUZUKI, Y. .
20
TAGASHIRA, Y. .
83, 41, 60
TAKAHASHI, M. .
60
TAKAHATA, N
87
TAKAIWA, F
15,16
TAKAMURA, T
50,91,100,102,103
TAKEDA, Y
21
TAKEUCHI, T • . . . . . . . . . . . . . . . . . . . . . . . 31
TAYA, C.
.49, 50TAZIMA, Y
20
TOHDOH, N. .
16

SHIROISHI, T
SOEDA, E.

.

TOYOSHIMA, K.

61
88
URUSHIBARA, T • . . . . . . . . . . . . . . . . . . . . . 12
WATANABE, J
38, 41WATANABE, T. K.
50,88,91
YAMADA, M
21, 25
YAMADA, M. A.
52
YAMAGUCHI, K
13
YAZAKI, K. .
12
YOKOIYAMA, A
69, 79YONEKAWA, H.
.
38, 41, 60
YOSHIDA, H
51
YOSHIMURA, K. .
62
YOSIDA, T. H
54,55,56,57,59,
60, 61, 62, 63
YUTSUDO, M. .
61

TSUNO, K.

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