NATIONAL INSTITUT£: OF GEN£:TICS
JAPAN

ANNUAL REPORT
No. 281977

Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka·ken, Japan
1978

Annual Report-of the-National Institute of Genetics
No. 28, 1977

Published by

The National Institute of Genetics, Japan
1978

CONTENTS
General statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff
Council"
Association for propagation of the knowledge of genetics
Projects of research for 1977
Researches carried out in 1977..............................

I.

12

Molecular Genetics
Inhibition of messenger RNA activity by methylated guanylicacid derivatives. MIURA, K., KODAMA, Y. and SHIMOTOHNO, K.
A novel 5'-exonuclease which degrades uncapped messenger
RNA. SHIMOTOHNO, K. and MIURA, K.
Nucleotide sequence around the replication origin of polyoma
virus DNA. SOEDA, E. and MIURA, K. .... .. . ..... .. ....
A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase j3'-subunit. SUGIURA, M.
Physiological studies on a temperature-sensitive Escherichia coli
mutant with an altered RNA polymerase j3'-subunit.
SUGIURA, M.
.. . . . . . . . . . . . . .
Joining of ribooligonucleotides with T4 RNA ligase and identification of the oligonucleotide-adenylate intermediate.
SUGIURA, M.
Joining of synthetic ribotrinucleotides with defined sequences
catalyzed by T4 RNA ligase. SUGIURA, M.
Molecular cloning of tobacco chloroplast ribosomal RNA genes.
SUGIURA, M. and KUSUDA, J.

II.

I
J
6
7
S

Microbial Genetics
Involvement of penicillin binding protein in cross-linking ofmurein. SUZUKI, H., NISHIMURA, Y. and HIROTA, Y.. . . . . .
Control of chromosome replication by an integrated ColEl
plasmid in Escherichia coli. YASUDA, S., NISHIMURA, Y.
and HIROTA, Y.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

III.

Biochemical Genetics and Immunogenetics
Estimation of genetic distance between the Japanese and theEuropean wild mice, M. m. molossinus and M. m. domesticus.
MORIWAKI, K. and MINEZAWA, M.
..............
Distribution of three H-2 antigenic specificities in various speciesof wild rodents. MORIWAKI, K. and SHIROISHI, T.. . . . . . . . .
Genetic regulation for the expression of erythrocytic H-2 antigens in a mouse intersubspecies hybrid. MORIWAKI, K.
and SHIROISHI, T.
Developmental changes of H-2 antigens on postnatal mouseerythrocytes. SHIROISHI, T. and MORIWAKI, K. ..... . . . . . .
Allelic constitution of Hbb locus in Asian house mouse subspecies. MINEZAWA, M. and MORIWAKI, K.
Geographical distribution of isocitrate dehydrogenase-l C in theJapanese wild mouse, Mus musculus molossinus. MINEZAWA,
M., MORIWAKI, K. and KONDO, K.
Electrophoretic survey for regional differences of blood proteinsin Ryukyu mice from the Southeast Asia. MINEZAWA, M.,
HIRAI, H., TSUCHIYA, K., SUDTO, P., Ku, T. Y., IKAwA, Y.
and MORIWAKI, K.
Comparative analyses of Japanese wood mice between Oki islands
and mainland from view points of biochemical and cytogenetics. HIRAI, H., UCHIDA, T. A. and MORIWAKI, K. ...
The genetic control of tryptophan pyrrolase in Drosophila melanogaster: Mechanism of suppresion. NAWA, S. and YAMADA,
M.A.
Structural and functional properties of Drosophila and Ephestia
tryptophan pyrrolase. NAWA, S. and YAMADA, M. A. . . . .
Genetic control of alcohol dehydrogenase in the Japanese speciesof Trillium. IHARA, M. and ENDo, T.
A new method for peroxidase isozyme stain. ENDo, T. . . . . . . . .

IV.

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39
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41

Developmental and Somatic Cell Genetics
Cell lineage and development of chimera hydra. SUGIYAMA, T.
and FUJISAWA, Too'"..
.. .. ..
Cellular composition of chimera hydra. SUGIYAMA, T. and
FUJISAWA, T.

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CONTENTS

Nematocyte differentiation in hydra. FUJISAWA, T. and SUGIYAMA, T.
Pyrimidine requirements of cultured rudimentary embryonic cells
of Drosophila melanogaster. KURODA, Y.
Enhancing effect of cellophane films on chondrogenesis of quaillimb-bud mesenchymal cells in culture. KURODA, Y. and
MATSUTANI, E. .
;..
Comparative study on mutagenic activity of various agents incultured human diploid cells. KURODA, Y.
Colony-forming activity of embryonic human diploid cells iIi
primary cultures. KURODA, Y.
Fundamental research on genetic monitoring of environmental
mutagens by using cultured human cells. KURODA, Y. ....
Isolation and characterization of X-linked maternal effect lethal
mutants in Drosophila melanogaster. YAMADA, M. A. andNAWA, S..............................................
On the number of mitotic cleavage division times in the silkworm(Bombyx mori). OHTSUKI, Y., KITAZAWA, T. and MURAKAMI, A.
Mosaicism of ganglion in the silkworm (Bombyx mori). KATSUKI,
M., WATANABE, I. and MURAKAMI, A.
Restriction of differentiation of pluripotent teratocarcinoma cells
in some allogeneic strains of mice. NOGUCHI, T.
Introduction of tWO and t w18 tailless mutations into LTjSv inbred
mice with a high incidence of ovarian teratomas. NOGUCHI,
T.
Abnormality in primordial germ cell proliferation in 129jter-Sv
inbred mice with a high incidence of testicular teratomas.
NOGUCHI, T.

v.

Cytogenetics
Does the potential of unscheduled DNA synthesis of mammals
correlate with their life-span? KATO, H.
Temperature dependent formation of sister chromatid exchange.
KATo, H.
A simple technique for observation of sister chromatid exchange
in vivo. KANDA, N. and KATo, H.

iii

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Some genetical aspects or supernumerary chromosomes in theblack rats. YOSIDA, T. H.
Some genetical aspects of C-band polymorphism in the black
rats. YOSIDA, T. H. and OeRIAI, Y.
Robertsonian fusion of acrocentric pairs no. 11 and 12 in black rats
obtained from Chichijima island, Japan. YOSIDA, T. Hoo . . .
Frequent occurrence of sex chromosome anomalies in F 2 hybrids
between geographical variants of the black rats. YOSIDA,
T. H.
Artificial insemination between Rattus norvegicus, R. annandalei
and R. losea. YOSIDA, T. H. and TAYA, C.
Candidates for a new experimental animal, II. The Indian spiny
mouse, Mus platythrix. YOSIDA, T. H.
Difference of nucleolar organizer regions in the black and Norway
rats. YOSIDA, T. H.
Cytogenetical identification of the X-chromosome in Bombyx
mori. MURAKAMI, A and IMAI, H. T.

VI.

Mutation and Mutagenesis in Animals
An exceptional pattern of mutation spectrum observed in theoffspring of mitomycin C treated females of a strain rb of thesilkworm. TAZIMA, Y.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...
Metabolic activities of silkworm microsome fraction on someindirect carcinogens with special regard to the differential
mutation response between silkworm and Drosophila. MURAKAMI, A., OOTO, M. and TAZIMA, Y . . . . . . . . . . . . . . . . . . . . .
Mutagenic effectiveness of an internal r-emitter, tritium. (4)RBE of tritium at low dose-rate range. TAZIMA, Y, ONIMARU, K. and FUKASE, Y
Experiments on the biological effects of ortho-phenylphenol
sodium salt for silkworm germ-cells. MURAKAMI, A,
FUKASE, Y, NISHIJIMA, H. and OOTO, M.
Changes of radiosensitivity to the induction of mutations inprimordial germ-cells during the gonad development offemale silkworm. MIKI, M. and MURAKAMI, A.
Toxicity and mutagenicity of cadmium and furylfuramide in
Drosophila melanogaster. INOUE, Y and WATANABE, T. K.. .

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CONTENTS

VII.

v

Radiation genetics and Chemical Mutagenesis in Microorganisms
and Plants
Repair enzymes for gamma-ray-damaged DNA in bacterial andhuman cells. INOUE, T., YOKOIYAMA, A. and KADA, T.....
Rec-assay with Bacillus subtilis spores. HIRANO, K., MATSUMOTO, H. and KADA, T. ..
Antimutagenic action of cobalt chloride in Escherichia coli Blr
WP2 try. KADA, To, HARA, M. and KANEMATSU, N
Antimutator effect of cobaltous chloride on a Bacillus subtiUs
mutator strain. OHTA, Y, INOUE, T. and KADA, To ..
Desmutagenic activities in vegetables and fruits. KADA, T.,
HARA, Mo and MORITA, K.
Purification and properties of a desmutagenic factor from plant(Brassica oleracea) for mutagenic principle of tryptophan
pyrolysateo INOUE, To, MORITA, Ko and KADA, T. ..
Dose response of maize to chronic gamma-ray irradiation. AMANO,
E. and UKAI, Y. o.
Measurement of waxyness in the induced wx mutants. AMANO,
Eo
Modification of radiation damage and variation of mutant sector
in maize with different kinds of radiations. FUJII, T.
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Population Genetics (Theoretical)

Theoretical study of genetic variability, assuming stepwise production of neutral and very slightly deleterious mutations.
MARUYAMA, To and KIMURA, Mo
Extension to the neutral mutation random drift hypothesis.
OHTA, T
On the gene conversion model as mechanism for maintenanceof homogeneity in systems with multiple genomeso OHTA,
T.
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Number of sterile loci in Drosophila melanogaster.
T. K. and LEE, W. Ho
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WATANABE,•••••••

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Evolutionary changes of inversion frequencies in a natural population of Drosophila melanogaster. INOUE, Y. and WATA-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

NABE, T. K. .

X.

,.................................

Evolutionary Genetics
Preponderance of synonymous changes as evidence for the neutral
theory of molecular evolution. KIMURA, M.
A new hypothesis for the direction of evolution among related
species of Drosophila. WATANABE, T. K. and KAWANISHI,
M.

XI.

XII.

88

Human Genetics
Reexamination of paternal age effect in Down's syndrome.
MATSUNAGA, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A genetic and epidemiologic study of polydactyly in humanembryos. SHIOTA, K. and MATSUNAGA, E. '" ..
Palindromic theory and the telomere of eukaryote chromosome.
NAKAGOME, Y.
Behavioral Genetics
Circadian locomotor activity of sensitive and unsensitive strainsof Drosophila melanogaster for noise environment. OSHIMA,
C. and LEE, W. H. . ..... ;.............................
Genetic variation of· flying ability of Drosophila melanogaster.
LEE, W. H. and WATANABE, T. K.
Effects of noise on the learning performance of mouse strains
with different learning abilities. FUJISHIMA, T.
Genetic analysis of a mutant showing abnormal behavior in thesilkworm. MURAKAMI, A. and OHNUMA, A.

XIII. Ecological Genetics
Observations of rice species and accompanying savanna plants
at the southern margin of Sahara desert. aKA, H. I.,
MORISHIMA, H., SANO, Y. and KOIZUMI, T.
The breeding behavior of Oryza perennis populations observed in
Thailand. aKA, H. I. and SANO, Y.
A competition experiment between Oryza perennis strains ingreenhouse. MORISHIMA, H. . . . . . . . . . . . . . . . . . . . . . . . . . . ..
The inheritance of copper tolerance in a hybrid population ofrice. MORISHIMA, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

90

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CONTENTS

Copper tolerance and competitive ability of Alopecurus aequalis
strains. MORISHIMA, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The self-persisting ability of soybean hybrid lines in semi-natural
conditions. OKA, H. 1.
Interactions among five summer annual weeds. MORISHIMA, H. . .
Variation in competitive ability among Oryza perennis strains.
SANO, y. and MORISHIMA, H.
The density response of Oryza perennis strains. SANO, Y.. . . ..
Ecological factors controlling the coexistence of Drosophila
simulans and D. melanogaster. KAWANISH, M. and W ATANABE, T. K.

XIV.

vii

107
107
108
109
110

110

Applied Genetics

Temperature responses of isogenic lines with different earlinessgenes of rice. YAMAGISHI, H., TSAI, K. H. and OKA, H. 1.. .
Noise sensitivity of wild and domestic Japanese quails as shownby the fertility of eggs. KAWAHARA, T.
Nitrogen fixation in rhizosphere of rice plant. HIROTA, Y.,
FUJII, T., SANO, Y. and lYAMA, S.. . . . . . . . . . . . . . . . . . . . . . ..
Analysis of genes controlling the F 1 sterility between Oryza
sativa and O. glaberrima. SANO, Y. ... . . . . . . .. . . . . . . . . ..
Breeding of isogenic lines of rice carrying gene markers in interchanged chromosome segments. SANO, Y.
Books and papers published in 1977 by research members
Abstracts of diary for 1977
Foreign visitors in 1977
Author index

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117
118
124
125
126

GENERAL STATEMENT
At a request of the Ministry of Education I delivered a lecture on
the present situation of recombinant DNA research in the presence of
H.1. H. the Crown Prince at the Palace on April 1st, 1977. Introducing
my own studies on planned chromosome translocation in the silkworm, I
talked on recent advances of genetic studies that had made possible to recombine DNA molecules of different species origin, its anticipated contribution to the advancement of both basic and applied sciences,its
conjectured potential hazards to plants and animals including man, andthe necessity of guidelines for the safe conduct of the research. My
talk was so often interrupted by the Prince's keen questions that thescheduled time was largely exceeded. H. 1. H. showed interests not only inbiological aspects, but also in the present situation of this research inadvanced countries, particularly with regard to obtaining public consent
for the promotion of the research. I was very much impressed with the
Prince's earnestness. I also talked about the Academy Forum of the U.S.
Academy of Sciences held in early March in Washington, D.C., which I
participated in. I also referred to the present status of the deliberation on
this issue at the Science Council of Japan, expecting that they would reachconclusion by autumn. Indeed, the Council adopted at its 74th general
assembly held in October, 1977, a statement that the recombinant DNA
researches should) be promoted in Japan, taking every possible care of thesafety of research'.V0rkers)as well as of the public.
It is my great regret~ to report here that Dr. Alice Flora Lilienfeld, anhonorary member of the Institute, passed away on July 14th, 1977. She
was 91 years old. She first came to Japan in 1929, and served as alecturer at the Faculty of Agriculture, Kyoto University, helping Professor
H. Kihara. During the World War II she returned to the United States toavoid the ravages of the war. After the peace was restored, she came again
to Japan in 1950 by the invitation of the Kihara Institute for BiologicalResearch. Since then she affiliated also with the National Institute of
Genetics as a foreign researcher. As she was proficient in several languages, she took, beside her own research on genetics of Medicago, charge ofreading the almost all papers written in English or German by the staff
members. In this regard we were greatly indebted to her.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Next, I should like to mention a few lines about the organization of theInstitute. The Institute was established in 1949 as a national center of thestudy of genetics under the jurisdiction of the Ministry of Education, when
any inter-university research institutes did not exist. The Institute has been
independent from any universities, and research staffs have been appointed
from qualified scientists in disregard of their academic affiliations. In themeantime several inter-university research institutes have been establishedwith the aim of common use of big research facilities and carrying out
cooperative researches. The administrative systems of those institutes areespeciaIIy suitable for cooperative researches. They can also take part in
the education of post graduate students. Our Institute is not granted suchprivileges. I think it is high time for us to consider seriously whether weshould reorganize the system as one of the inter-university research institutes
or keep the Institute as it is.
Completed with three laboratories, i.e., plant, animal and microbial,
the Genetic Stock Center has already started to carry out its task. The
budget for erecting the main building of the Center was approved this year,
and the construction work is now under way on the site recently purchasedfrom the City. No wonder, the Center will contribute to the further
development of the genetic researches as well as the biological sciences inbroad sense. Taking this opportunity I wish to express my deep gratitude
to the authorities concerned for their thoughtful and sympathetic assistance.

3

STAFF
Director

TAZIMA, Yataro, D. Ag.
Members1.

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

2.

Department of Cytogenetics
YOSIDA, Tosihide H., D. Sc., Head of the Department
The 1st Laboratory
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
KATO, Hatao, D. Sc.
The 2nd Laboratory
MORIWAKI, Kazuo, D. Sc., Head of the Laboratory
IMAI, Hirotami, D. Sc.

3.

Department of Physiological Genetics
OSHIMA, Chozo, D. Sc., Head of the Department
The 1st Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
WATANABE, Takao K., D. Sc.
The 2nd Laboratory
OSHIMA, Chozo, D. Sc., Head of the Laboratory
OISHI,* Kugao, Ph. D.

*

Visiting researcher

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

4.

Department of Biochemical Genetics
SUGIYAMA, Tsutomu, Ph. D., Head of the Department
The 1st Laboratory
NAWA, Saburo, D. Sc., Head of the Laboratory
YAMADA, Masa-Aki, M. Sc.
The 2nd Laboratory
OGAWA, Yoshito, D. Med., Head of the Laboratory
ENDO, Toru, D. Ag.
The 3rd Laboratory
SUGIYAMA, Tsutomu, Ph. D., Head of the Laboratory
FUJISAWA, Toshitaka, Ph. D.

5.

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

6.

Department of Induced Mutation
KADA, Tsuneo, D. Sc., Head of the Department
The 1st Laboratory
TUTIKAWA, Kiyosi, Acting Head of the Laboratory
INOUE, Tadashi, D. Sc.
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, M. Sc.

7.

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

STAFFThe 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
SHIOTA, Kohei, D. Med.
The 2nd Laboratory
NAKAGOME, Yasuo, D. Med., Head of the Laboratory
IINUMA, Kazuso, B. Med.

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.

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
FURUIeHI, Yasuhiro, D. Pha.
SOEDA, Eiichi, D. Ag.
The 2nd Laboratory
SUGIURA, Masahiro, D. Sc., Head of the Laboratory
SHIMOTOHNO, Kunitada, D. Pha.

11.

Genetic Stocks Center
Plant Section
FUJII, Taro, D. Ag., Head of the Laboratory

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

SANO, Yoshio, D. Ag.
Animal Section
YOSIDA, Tosihide H., D. Sc., Head of the Laboratory
NOGUCHI, Takehiko, D. Sc.

12.

Experimental FarmaKA, Hiko-Ichi, D. 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., Managing Director of the Institute of Physicaland Chemical Research

COUNCIL
FUJII, Takashi, Chairman, Emeritus Professor of University of TokyoIZEKI, Shoei, Vice Chairman, Director of National Research Institute of
Police Science
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
KONDO, Norio, Professor of Tokyo University of Agriculture
MISONOO, Keisuke, Director of National Institute of Radiological Sciences
MORIWAKI, Daigoro, Managing Director of the Institute of Physical andChemical Research
MOROHOSHI, Seijiro, Professor 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$ASA, Manabu, Director, National Institute for Environmental Studies$HINOZAKI, Nobuo, Director of Institute of Population Problems
TAKAHASHI, Ryuhei, Professor of Okayama University
UMEZAWA, Hamao, Head of the Department of Antibiotics, National
Institute of Health
ASSOCIATION FOR PROPAGATION OF THE KNOWLEDGE
OF GENETICS
MORIWAKI, Daigoro, President, Managing Director of the Institute of Physical and Chemical Research
MATSUNAGA, Ei, Managing Director, Head of the Human Genetics Department
YOSIDA, Tosihide H., Managing Director, Head of the Cytogenetics Department
KIHARA, 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, Head of the Physiological Genetics Department

PROJECTS OF RESEARCH FOR 1977
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 and IMAI)
Studies on recombination in the silkworm (MURAKAMI)
Genetic studies of the silkworm (TAZIMA and ONIMARU)
Radiation and chemical mutagenesis in the silkworm (TAZIMA andONIMARU)
Depatment of Cytogenetics

Studies on chromosomal evolution in rodents (YOSIDA)
Chromosome study on experimental tumors (YOSIDA)
Experimental breeding· and genetics of mice, rats and other wild rodents(YOSIDA and MORIWAKI)
Cytogenetical study on sister chromatid exchange in mammalian cells (KATO)
Cytogenetical and immunological studies on mouse plasma cell tumor(MORIWAKI)
Biochemical studies on serum transferrin variations in rodents (MORIWAKI)
Immunogenetical study on the histocompatibility antigens of wild rodents(MORIWAKI)
Cytogentical study of ants (IMAI)
Department of Physiological Genetics

Behavior genetics of Drosophila (OSHIMA and WATANABE)
Analysis of deleterious and inversion chromosomes in natural populationsof Drosophila melanogaster (WATANABE)
Studies on fitness of Drosophila under controlled environment (OSHIMA andWATANABE)
Genetic studies on the effects of adverse environments on Drosophila flies(OSHIMA and WATANABE)

PROJECTS OF RESEARCH FOR 1977

9

Department of Biochemical Genetics

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

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

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

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Mutation and differentiation studies of plant tissue culture (AMANa and
KADA)
Radiation and chemical interaction in the cells (KADA)
Genetic fine structure analysis in maize (AMANa)
Department of Human Genetics

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

Genetic regulatory mechanism of DNA replication in E. coli (HIROTA,
NIsmMuRA, and YASUDA)
Genetic regulatory mechanism of cellular division in E. coli (HIROTA)
Molecular genetics on DNA replication (YASUDA and HIROTA)
Molecular mechanisms of cell division in E. coli (SUZUKI, NISHIMURA andHIROTA)
Genetics of bacterial cell envelope (HIROTA, SUZUKI, and NISHIMURA)
Synthetic bacterial plasmid (YASUDA, and NIsmMuRA)
DNA replication origin of E. coli (YASUDA, 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 1977

11

Department of Molecular Genetics

Studies on the chemical structure of genome of viruses containing doublestranded RNA (MIURA, SUGIURA and SHIMOTOHNO)
Studies on the interaction between RNA polymerase and template nucleic
acid (MIURA, SUGIURA, SHIMOTOHNO, and SOEDA)
Studies on the primary structure of DNA (SOEDA and MIURA)
Studies on structure and function of messenger RNA (MIURA andSHIMOTOHNO)
Genetical and enzymological studies on E. coli RNA polymerase (SUGIURA)
Genetic Stocks Center

Studies and conservation of gennplasm resources in rice and wheat species(FuJII, SANO and OKA)
On the sex expression in monoecious plants (FuJII)
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)
Studies on teratocarcinogenesis in mice (NOGUCHI)RESEARCHES CARRIED OUT IN 1977I.

MOLECULAR GENETICS

Inhibition of Messenger RNA Activity by Methylated Guanylic
Acid Derivatives
Kin-ichiro MIURA, Yaeko KODAMA and Kunitada SHIMOTOHNO

Most eukaryotic mRNAs carry a "cap" at the 5'-terminal as
m 7 G5'ppp5'N(m)-. Since protein synthesis proceeds in the 5'.-73' direction of the template mRNA, the 5'-terminal modification may be related
to the initial step of protein synthesis. The cap (m 7 G 5'pp) can be deleted
by tobacco phosphodiesterase with no damage to the RNA chain. The
ability to synthesize protein and the ability to form the complex for the
initiation of protein synthesis in the cap-deleted mRNA are decreased
remarkably (Annual Rep. No. 27 (1976) p. 20; Shimotohno, K., Kodama,Y, J. Hashimoto and K. Miura, Proc. Natl. Acad. Sci. USA, 74: 2734
1977)). This experiment suggests that the role of the cap is in protein
synthesis, especially in the initiation step. Since the cap-eliminated mRNAloses its stability in cell extracts (Annual Rep. No. 27 (1976) p. 20;
Shimotohno, K., Kodama, Y, J. Hashimoto and K. Miura, Proc. Natl.
Acad. Sci. USA, 74: 2734 (1977)), the cap may function in protein
synthesis through stabilization of mRNA. However, there are some datasuggesting that the cap interacts with cellular components required forprotein synthesis. Several reports have described that the addition ofpm7 G
to the protein synthesizing system causes strong inhibition. To determine
if pm 7 G is specific inhibitor of protein synthesis, various methylated derivatives of guanylic acid were synthesized chemically and their inhibitory effects
were studied in wheat germ and E. coli cell-free systems.
The effect of adding 7-methylguanylic acid to the in vitro protein synthesizing system was studied by incorporation of labeled amino acids into
protein in the wheat germ cell-free extract with eukaryotic mRNAs, tobacco
mosaic virus RNA and cytoplasmic polYhedrosis virus mRNA. Protein
synthesis with both eukaryotic mRNAs was inhibited by 7-methylguanylicacid at concentrations higher than 0.5 mM. Prokaryotic mRNA, MS 2

RESEARCHES CARRIED OUT IN 1977

13

phage RNA, was not inhibited. The confronting nucleotide structure
m7GpppA, the capping structure at the 5'-terminal of eukaryotic mRNA,
was also inhibitory. However, GpppA and GppA, which do not containa methyl group at the 7-position of G, were not inhibitory.
Since the methylation of guanosine in the cap of mRNA seems to berequired for inhibition of protein synthesis, a variety of methylated derivatives of guanylic acid were prepared to test the inhibitory effects.
These were synthesized by Drs. M. Ikehara and J. Fukui in the Pharmaceutical Department of Osaka University. Strong inhibitors carry amethyl group at the 7-position, suggesting that the 7-methyl group is
specially important for inhibition of protein synthesis. However, all the
7-methyl guanylic acid derivatives did not show strong inhibition; 2,2,7trimethylguanylic acid and 7,8-dimethylguanylic acid showed weak inhibition, and 2,2,7,8-tetramethylguanylic acid did not give any inhibitory effect.
In these cases, the inhibitory effect of the 7-methyl group would be cancelled by steric hindrance or configurational change by other methylation
around the 7-methyl group.
7-methyl guanine carries positive charge differently from other purinebases. To determine the effect of the positive charge in inhibition ofprotein synthesis, other similar nucleotides were prepared. Neither 1methyladenylic acid nor 2-methylthio-7-methyl inosinic acid showed any
inhibition in a wheat germ protein synthesizing system. Thus, the methylation itself at the 7-position in guanine seems to be specifically required forinhibition rather than its positive charge. The confronting nucleotide
structure (cap) at the 5'-terminus of mRNA involves 2'-O-methylated
nucleotide or 6-methyladenylic acid as the first nucleotide of the RNA
chain. Addition of 2'-O-methylguanylic acid and 6-methyladenylic acid to
the wheat germ system did not cause inhibition for protein synthesis.
These results indicate that the inhibition of protein synthesis was causedby 7-methylguanylic acid residue in the confronting nucleotide structure.
It has been known that the 7-methylguanylic acid-deleted mRNA treatedwith tobacco pyrophosphatase loses its ability to form the initiation complex of protein synthesis. It was also shown that addition of 7-methylguanylic acid to the intact CP virus mRNA or the TMV RNA caused
inhibition of the formation of the initiation complex.
The 7-methylguanylic acid-blocking structure is commonly involved ineukaryotic mRNA, whereas it is not found in prokaryotic mRNA (Annual

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Rep. No. 27 (1976) p. 14). It was of interest to learn if inhibition ofprotein synthesis by 7-methylguanylic acid is specific for the eukaryotic
system. An in vitro protein synthesizing system was prepared from E.
coli. Using phage MS2 RNA and TMV RNA as mRNA, the effect of
7-methylguanylic acid was measured. There is no inhibition of protein
synthesis by 7-methylguanylic acid in the E. coli system, regardless ofwhether eukaryotic or prokaryotic mRNA is used. Thus, the inhibitionby 7-methylguanylic acid takes place in the step of the initiation complex
formation for protein synthesis with eukaryotic ribosomes. These suggestthat the m7 GMP residue in the cap of mRNA performs some specific rolein eukaryotic protein synthesis, especially in its initial step.
A Novel 5 /·Exonuclease which Degrades Uncapped Messenger RNA
Kunitada SHlMOTOHNO and Kin-ichiro MIURA

Our previous work using a wheat germ cell-free system showed that the
5' -terminal cap structure in eukaryotic mRNA is important in protein
synthesis, especially in the formation of the initiation complex. It wasalso shown that the cap structure protects mRNA from nuclease attackand stabilizes mRNA. In these experiments the intact mRNA was compared with uncapped mRNA, which was prepared by treatment withpyrophosphatase from cultured tobacco cells (Annual Rep. No. 27 (1976)
p. 20; Shimotohno, K., Kodama, Y., J. Hashiomoto and Miura, K., Proc.
Natl. Acad. Sci. USA, 74: 2734 (1977)). After incubation of uncappedmRNA with wheat germ extract (S-30 fraction), mRNA was degraded tonucleotides, while the intact mRNA was not. This result suggest thepresence of a 5'-exonuclease acting on the uncapped mRNA in wheat germ
extract. We therefore attempted to purify the enzyme and to characterize it.
The S-30 fraction (soluble fraction in centrifugation at 30,000 X g) ofwheat germ extract was centrifuged at 100,000 X g for 2 hrs to obtain apostribosomal fraction. The fraction containing the exonuclease activity
was obtained by the following procedures: column chromatographies with
DEAE-cellulose, Sepharacryl S-200 and hydroxyapatite. The enzyme
activity was surveyed employing two kinds of substrate, intact mRNA andthe decapped mRNA which had been treated with tobacco pyrophosphatase.
The exonuclease was purified about 20-fold at the final step of hydroxyapatite
chromatography.

RESEARCHES CARRIED OUT IN 1977

15

Optimal conditions for the enzyme reaction were studied. The enzyme requires 0.2 mM Mg+ +. The optimal temperature and pH are 25°C
and pH 6.0. The enzyme hydrolyzes uncapped mRNA but cannot attack
intact mRNA. This result suggests that the enzyme recognizes the 5'terminal structure of the substrate RNA strictly. Using RNA substrates
which carry various structures at the 5'-termini, the degradation behavior
of RNA upon enzyme treatment Was tested in detail, and it was found thatthe enzyme cannot attack 5'-hydroxylated RNA or the 5'-capped RNA,.
though mono, di- or triphosphorylated RNA's were hydrolyzed.
Degradation products produced by this enzyme were analyzed by paper
electrophoresis. Only mononucleotides were detected. The mononucleotides obtained here were not converted to nucleosides by further incubation with 3' -phosphatase (Penicillium nuclease P), so it was concluded thatthe enzyme gives nucleoside 5'-monophosphates as final products from RNA..
Decapped mRNA was incubated with the enzyme for various times andthe degradation of RNA was followed by centrifugation in a glycerol
density gradient. It is suggested that the enzyme acts on RNA in an
exonucleolytic mode. Next the direction of hydrolysis by the enzyme wasstudied. Decapped CPV mRNA, which was labelled with 32p at the 5'terminus and 3H at the 3'-terminus, was incubated with this enzyme and the
kinetics of hydrolysis were observed. Under the optimal conditions forthis reaction, only radioactivity of 32 p was detected in the cold ethanolsoluble fraction. When the RNA was incubated with venom phosphodiesterase (Worthington Co.), the same percentages of radioactivity of 32p
and 3H were detected in the ethanol-soluble fraction at a given time ofincubation. Thus it was confirmed that the enzyme attacks RNA fromthe 5'-terminus in an exonucleolytic manner. This 5'-exonuclease activity
is different from that of any exonuclease reported previously. This exonuclease would degrade mRNA from the 5'-terminus only after the 5'-terminal
cap structure had been removed by an enzyme such as a specific pyro-·
phosphatase found in tobacco cells.
Nucleotide Sequence around the Replication Originof Polyoma Virus DNA
Eiichi SOEDA and Kin-ichiro MIURA

Structural studies on the DNA of small tumor viruses are proceeding

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

intensively as an initial step toward understanding the function of eukaryotic
DNA as well as elucidating the process of tumorigenesis. Mouse polYoma
virus has been well studied, as has simian virus 40 (SV 40). For SV 40
analysis of its nucleotide sequence has progressed repidly (W. Fiers, et a/
in Belgium and S. Weissman, et al. in U.S.A.). It is necessary to determine
the nucleotide sequence of polyoma virus DNA and compare it with thatof SV 40 to clarify the common structure for function. The starting pointof DNA replication has been located on both SV 40 and polyoma DNA.
Further, both the transcription starting points of "early" and "late" viralmRNAs are located at regions close to the origin of DNA replication.
Therefore, studies on the region around the replication origin will provide
much information on the initial step in DNA replication and RNA synthesis.
The DNA replication of polyoma virus is initiated at a unique site of theviral genome, which has been mapped at 71 ±3 map units. Some DNA
fragments obtained by restriction endonucleases (HapIl, HhaI and AluI)
cover the origin of DNA replication. Maxam and Gilbert have developed
a method for determining the nucleotide sequence in DNA by using DNA
fragments labeled at the 5' terminus with [32 P]phosphate by polynucleotide
kinase. Because it is necessary to label the terminal part of the DNA
fragment for this analysis, they labeled the 5' terminus with 32p by polynucleotide kinase. We have developed a technique for 3'-terminallabelingby the use of bacteriophage T4-induced DNA polymerase in place ofpolynucleotide kinase and have applied it to the polyoma DNA fragment
to assess the fidelity of the analysis method with the DNA polymerase;
the technique was found to be effective and convenient when compared withpolynucleotide kinase. The sequences deduced from the 3'-termini of thestrands of the fragment coincided almost with those derived from the 5'terminal labeling with polynucleotide kinase. The 32P-labeled fragment
was split and analyzed with gel electrophoresis according to Maxam andGilbert.
The nucleotide sequence thus derived is shown in a possible transient
secondary structure as follows.
A long cluster of pyrimidines is included between 24 and 41. In thisregion a characteristic true palindrome sequence of T-C-T-T-T-T-T-C-T(or A-G-A-A-A-A-A-G-A) from 26 to 34 is contained. This region issandwiched between two small symmetrical regions with a 2-fold rotational
axis (20-23 and 37-40). The sequenced fragment may be characterized

RESEARCHES CARRIED OUT IN 1977

17

Late strand
Early strand

also by the G+C)-rich region from 1 to 15 with a 2-fold rotational axisof symmetry, which possibly forms double hairpin loops, as well as theother (G+C)-rich region from 71 to 83.
Because mouse polyoma virus and SV 40 have similar characteristics,
there is a hypothesis that the two viruses are descended from the sameancestor. If this is the case, a common structure would be conserved in afunctionally important region of DNA. Comparing the polyoma nucleotide sequence analyzed here with the corresponding region in SV 40 DNA,
which was sequenced by Weissman et at., a characteristic symmetricalregion containing a five-A stretch from 28 to 32 in the polyoma late strand
fits the similar regions in SV 40 DNA. Other common long sequences
which were found both in polyoma and SV 40 DNA were C-A-G-A-GG-C, (from 46 to 52 in polyoma) and G-A-G-G-C-T-T (from 38 to 44 inpolyoma), the latter is also contained in phage ¢X174 DNA near the
replication origin. However, these are not symmetrical sequences.
There might be similarities among the nucleic acid structures having acommon function, even among very different organisms, as shown in thecloverleaf structure of tRNA. Recently Tomizawa et at. showed that the
replication origin of colicin El plasmid DNA contains a five-A stretch,where primer RNA switches to DNA, and both sides of this region areflanked by (G+C)-rich regions. This feature is quite similar to the structure of the polyoma DNA segment mentioned here. The A+T cluster
flanked by (G+C)-rich regions has been found also at the origin of
replication in phage ¢X174 DNA, although the region does not show
apparent symmetry. Such a characteristic structure may be common forthe starting of DNA synthesis in eukaryotes as well as in prokaryotes.
Moreover, it has been shown that the promoter region contains a common

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

sequence rich in the A· T pairs near the initiation point of transcription.
Because the cluster of the A· T base pairing must be loosened easier thanthe (G' C)-rich region, it is reasonable to say that the A· T cluster isinvolved in the starting point of DNA replication and RNA synthesis.
If the nucleotide sequence in the present segment of polyoma DNA isaligned with the corresponding region of SV 40 DNA by inserting somegaps, we can observe an extensive similarity. Fifty-eight nucleotides of the82 (70 %) of the polyoma segment are the same as SV 40. It is known thatrecombination occurs most frequently in this region; many variants withaddition and deletion around the replication origin have been isolated sofar, including viable and defective mutants. If such structural changes
occurred also in the course of evolution, it is likely that polyoma virus
and SV 40 have descended from a common ancestor.
This work was collaborated with Prof. Genki Kimura, Kyushu University School of Medicine. The contents were published in FEBS Lett. 79,
383-389 (1977) and Proc. Natl. Acad. Sci. USA 75, 162-166 (1978).
A Temperature-Sensitive Mutant of Escherichia coli withan Altered RNA Polymerase /3' Subunit
Masahiro SUGIURA

DNA-dependent RNA polymerase of E. coli (Ee 2.7.7.6) consists offour subunits and has the structure (X2(3(3'a. This enzyme plays a central
role in genetic transcription in bacteria. The isolation of mutants withaltered RNA polymerase is, therefore, essential to the study of transcription and its control mechanisms.
We have isolated several temperature-sensitive (ts) RNA polymerase
mutants by the procedure described previously (this Report, No. 27, pp.
22-24, 1976). One strain called JElO092 was studied in detail. The PI
transduction experiments showed that the ts cell growth was due to thets RNA polymerase and that the map order was argH-rif((3)-rpoC92(the ts mutation in JElO092). To identify the defective subunit in the ts
enzyme, the subunit reconstitution experiment was carried out by mixing
excess amount of a wild-type subunit with the ts enzyme. Only the ts
RNA polymerase mixed with wild-type (3' subunit resulted in temperatureresistant. This indicated that the ts mutation (rpoC92) was located in thegene for (3' subunit of the RNA polymerase.

RESEARCHES CARRIED OUT IN 1977

19

The RNA polymerase of JE10092 was easy to purify and was as stable as
the parent (PA3092) enzyme up to at least 6 months. The most prominent
characteristic of this enzyme was its reversible temperature sensitivity inin vitro reaction systems. When a reaction mixture containing the ts enzyme
was incubated for 10 min at 43° and then transferred to 30°C, the activity
was restored. No recovery was, however, observed when rifampicin wasadded simultaneously with the temperature sift-down. This indicated thathigh temperature affected the initiation of RNA synthesis. The ratios ofactivity at 43°C to 30°C differed significantly with templates used: T4DNA, 0.19, calf thymus-DNA, 0.31, T7-DNA, 0.34, A-DNA, 0.48, ¢X174
RF-DNA, 0.65. The results suggested that the 13' subunit played an
important role in promotor selection.
This work was done in collaboration with K. Segawa, K. Yoshinaga,
F. Yu, N. Ito, S. Yasuda and Y. Hirota, and was published in Biochem.
Biophys. Res. Commu. 76, 739-745 (1977) and was presented at the 11th
FEBS Meeting, Copenhagen, August 1977.
Physiological Studies on a Temperature-Sensitive Escherichia coli
Mutant with an Altered RNA Polymerase f3' -Subunit
Masahiro SUGIURA

Some in vivo properties of an E. coli temperature-sensitive (ts) mutant(JEI0092) with an altered RNA polymerase j3'-subunit were studied.
The mutant cells grew in L-broth at 30°C with a doubling time of 90 min.
When the log-phase culture was shifted to 43°C, the turbidity stopped toincrease within 1 hr and the colony-forming ability was reduced exponentially after an initial transient increase. The optimal growth temperature
was 32°C.
When the mutant culture was shifted from 30°C to higher temperature,the (3H)uridine incorporation decreased immediately to 85 % at 37°C, 55 %at 40°C and 6 % at 43°C as compared with that at 30°C. The observed
drop in (BH)uridine at high temperature was due to the reduction of therate of RNA synthesis which was examined by a pulse-labeling method.
RNA synthesis was recovered immediately upon transfer to 30°C. DNA
synthesis, protein synthesis and nucleoside triphosphate formation continued after the temperature shift-up.
The most prominent characteristic of this mutant was the immediate

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

cessation of RNA synthesis after the shift to the nonpermissive tempera_
ture. Many ts RNA polymerase mutants so far reported did not show suchan immediate block of RNA synthesis at high temperature. Therefore,
our mutant JEl0092 is more useful for studies on the subunit function andthe overall control of transcription within the cell.
This work was done in collaboration with K. Yoshinaga and was publishedin Biochem. Biophys. Acta 479, 172-179 (1977).
Joining of Ribooligonucleotides with T4 RNA Ligase and
Identification of the Oligonucleotide-Adenylate
Intermediate
Masahiro SUGIURA

RNA ligase has been found in T4-infected E. coli cells. Originally, theligase activity was found to promote the cyclization of ribopolynucleotidesof chain length around 40 particularly well and a minimum chain length
for cyclization was reported as being eight. Shorter oligonucleotides werejoined using an excess of the 3'-hydroxyl component.
T4 RNA ligase was purified from T4 amN82xE1l40-infected E. coli
A19 (RNase 1-), which was low in RNase activity. The RNA ligase wasfound to join A-A-A-A-A-A and 32 pU-U-U-U. In this reaction the pyrophosphate of *pU-U-U-U and pA was isolated by chromatography on a
RPC-5 column, besides the joined product and the starting materials.
This pyrophosphate was shown to be an intermediate in the joining
reaction because of the fact that coupling with A-A-A-A-A-A to give thedecanucleotide could be performed in the absence of ATP. The structureof the oligonucleotide-adenylate was determined by enzymatic digestion
with base-nonspecific nuclease and venom phosphodiesterase. Further
evidence for the proposed structure was obtained by isolation of the intermediate obtained by using *pU-U-U-U and [a_ 32P]ATP. This pyrophosphate gave *pA and *pU by treatment with venom phosphodiesterase.
The ligation reaction, therefore proceeds as follows,/pA
ATP+*pU-U-U-U -> 0
+ PPi"*pU-U-U-U/pA
A-A-A-A-A-A+O->A-A-A-A-A-A*pU-U-U-U + AMP"*pU-U-U-U

RESEARCHES CARRIED OUT IN 1977

21

This work was done in collaboration with E. Ohtsuka, S. Nishikawa andM. Ikehara, and was published in Nucleic Acids Res. 3, 1613-1623 (1966).
Joining of Synthetic Ribotrinucleotides with Defined
Sequences Catalyzed by T4 RNA Ligase
Masabiro SUGIURA

T4 RNA ligase has been shown to catalyze intra- and intermolecularjoining of the 5'-phosphate and the 3'-hydroxyl group of oligo and polynucleotides using ATP as cofactor.
We found that RNA ligase catalyzed the joining of pC-C-Ap with
C-A-A in the synthesis of C-A-A-C-C-Ap, which has the sequence of the
Escherichia coli tRNAfet 3'-end. pC-C-A to various synthetic ribotriplets, such as C-C-A, A-A-A, C-C-C, U-U-U, U-A-G, C-C-G and U-U-C,
was performed as well as joining to the partially substituted trimers witha photolabile o-nitrobenzyl group, C-Anbzl-A and C-C-AnbzI. The yields
were C-A-A-C-C-A (69 %), C-C-A-C-C-A (38 %), A-A-A-C-C-A (66 %),C-C-C-C-C-A (71 %), U-U-U-C-C-A (50 %), U-A-G-C-C-A (23 %), C-CG-C-C-A (43%) and U-U-C-C-C-A (46%). C-Anbzl-A was a slightly
poorer acceptor than C-A-A and C-C-Anbzl did not serve as an acceptor.
Recongnition of acceptor molecules by RNA ligase is discussed in terms
of affinity of ligonucleotides for the enzyme.
This work was done in collaboration with E. OHTSUKA, S. NISHIKAWA,
R. FUKUMOTO, S. TANAKA, A. F. MARKHAM and M. IKEHARA, and was
published in Eur. J. Biochem. 81, 285-291 (1977).
Molecular Cloning of Tobacco Chloroplast Ribosomal RNA Genes
Masahiro SUGIURA and Jun KUSUDA

Chloroplasts of higher plants contain ribosomes of similar sizes to thoseof prokaryotes. Chloroplast DNA exists as a circular molecule with amolecular weight of around 108 daltons and codes for its rRNA's. In
order to analyze the fine structure and the expression of chloroplast rRNA
genes, we have constructed recombinant molecules between E. coli plasmid
pMB9 and chloroplast rRNA genes from tobacco leaves.
Chloroplast DNA was purified from healthy mature tobacco leaves byusing differential and sucrose gradient centrifugations. The chloroplast

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

DNA was digested with restriction endonuclease EcoR!. The DNA
fragments were fractionated by 1 % agarose gel electrophoresis and trans_ferred to a Millipore filter according to Southern. Chloroplast 23S andl6S rRNA were paritially hydrolyzed by alkali and then labeled with 32p
at their 5'-ends by using T4 polynucleotide kinase and (r-32P)ATP. 32p_
rRNA probe was hybridized to the filter-bound DNA fragments. The
resulting autoradiograph indicated that the chloroplast rRNA hybridized
to the fragments of molecular weights of 1.9 X 106 and 2.8 x 106 daltons.
Total EcoRI fragments of the chloroplast DNA and pMB9 digested
with EcoRI followed by alkaline phosphatase treatment were joined using
T4 DNA ligase, and the ligated DNA was used to transform E. coli RBlOl.
373 tetracycline-resistant transformants were screened for the presence ofDNA complementary to (5'- 32 P)rRNA using the colony hybridization procedure developed by Grunstein and Rogness. The 14 clones hybridized tothe 32P-rRNA. The recombinant plasmids containing a 1.9 X 10 6 fragment(TC001) and a 2.8 X 106 fragment (TC309) were selected by agarose gelelectrophoresis followed by filter hybridization.

RESEARCHES CARRIED OUT IN 1977

II.

23

MICROBIAL GENETICS

Involvement of Penicillin Binding Protein in Cross-Linking of Murein
Hideho SUZUKI, Yukinobu NISHIMURA and Yukinori HIROTA

In order to elucidate the role of penicillin binding protein (PBP) inmurein biosynthesis, a series of multiple mutants having various combination of mutations in PBPs were constructed. Particulate fractions wereprepared from them and activities for murein synthesis were investigated byN-acetylmuramylpentapeptide-dependent incorporation of N-acetylglucosamine with UDP-linked precursors. The level of murein synthesis in vitro
was essentially unchanged in the mutants defective in PBP-Ia(ponA),
PBP-2(rodA) or PBP-3(ftsI). In contrast, practically no murein synthesis
in vitro was found in two independent mutants for PBP-Ib(ponB704 andponBI085), although lipid intermediates were synthesized at a level comparable to that in the wild type. In another PBP-I b mutant(ponB353) the
particulate fraction incorporated the precursor into murein at a lower level(about 60 % of the wild type). These different characteristics are specificfor each mutation while the general phenotype of these strains is the lackof PBP-I b. The in vitro products were analyzed for cross-linking bylysozyme digestion and subsequent separation of muropeptides by gelfiltration. The ratio of bis(disaccharide-peptides) to disaccharide-peptides
was about 0.15 in the ponB353 mutant, whereas it was 1.1±O.2 in thewild type and in the mutants defective in PBP-Ia, PBP-2 and PBP-3. The
sharp decrease in the degree of cross-linking in the ponB353 mutant indicatesthat the PBP-I b is involved in the reaction of murein cross-linking. The
level of the incorporation of substrate in the ponB353 was high enough toallow the interpretation that the extention of glycan-chain proceeded in theabsence of transpeptidation and to preclude the argument that the failurein cross-linking might result from the absence of extended glycan-chainsthat could serve as substrates for transpeptidation.
The degree of cross-linking in vivo was examined in dap- lys- auxotrophsof mutants defective in PBPs by following the incorporation of He_
diaminopimelic acid into lysozyme digests. In contrast to the in vitro
experiments, the degree of cross-linking in vivo in all three of the ponBmutants was almost the same as that in the wild type. A double mutant

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

carrying ponA ts ponB was shown to be temperature sensitive with respect
to growth, although the parental strains carrying a single mutation, either
ponAts or ponB, were not. This suggested that the loss of function of PBPIb(ponB) in vivo was bypassed by the product of the ponAts gene, PBP-la ts •Furthermore, the double mutation, ponAts ponB, did not decrease thedegree of cross-link in the murein synthesized in vivo at 42°, in spite of
causing cell lysis. In the presence of benzylpenicillin or cephaloridine at adose leading to cell lysis, synthesized murein still showed the normal
degree of cross-link, although gross murein synthesis was decreased to lessthan one third of the control in one hour. To explain these results, thefollowing working hypothesis was proposed.
There must be at least two reactions for cross-linking in vivo: one iscomparatively insensitive to p-lactam antibiotics and another is sensitive
to p-lactam antibiotics. Transpeptidation which is required for the lateralextention of the sacculus is supported by the former reaction which constitutes a major part of cross-linking reactions for cell elongation. PBP-lb
is involved in the latter reaction which is a primary target of p-lactam
action. The PBP-l b specific reaction is functional at a certain stage of thecell cycle and arrest of this reaction, either by action of p-lactams or as aresult of a genetic defect, would bring about a cleft on succulus leading tobulge formation and cell lysis. Extrusion of cytoplasm out of the cleft may
diminish or discontinue overall murein synthesis as a result of disorganization of structural contact of cytoplasmic membrane and murein sacculus.
Bulge formation appears to be localized at a specific site which has a capacity
to differentiate into a septum. Thus, the PBP-l b may function to cross-link
muropeptides at the potential division site developed at a certain stage ofthe cell cycle. The PBP-3 may act to direct the differentiation of this site
into a septum, alternatively these two PBPs may cooperate in the reaction
leading to septation. Evidence for such cooperation comes from studies
of a multiple mutatnt defective in both PBP-l band PBP-3. This mutant
was found to be osmotically sensitive, requiring a hypertonic medium
for proper progagation. Transfer of the mutant to ordinary nutrient brothcaused progressive cell lysis. This result suggests the possible requirementof cooperation of PBP-3 in murein synthesis at some specific stage of acell cycle.

RESEARCHES CARRIED OUT IN 1977

25

Control of Chromosome Replication by an Integrated ColEl
Plasmid in Escherichia coli
Seiichi YASUDA, Yukinobu NISHIMURA and Yukinori HIROTAThe ColEl plasmid is known as a unique replicon which has different
characteristics from those of other replicons, such as E. coli chromosome,
F and R plasmids. There are about 20 copies of ColE! plasmid DNA per
cell. Initiation of replication is not inhibited by chloramphenicol, andelongation is catalyzed by DNA polymerase I. Integration of ColEl
plasmid into the chromosome of E. coli Kl2 has not been reported, and no
integrative suppression by ColEl has been observed for a thermosensitive
DNA initiation mutation, danAt s .
We report here an integrative suppression of dnaAts mutation by a synthetic ColE l plasmid. When a plasmid, pLC26-3, carrying the ilv region of
the E. coli chromosome (Clarke, L. and J. Carbon 1976. Cell 9: 91) was
introduced into a dnaAts mutant, CRT46, thermoresistant revertants wereobtained at a frequency of about 4 X 10- 5 • The reversion frequency was
about 50-fold higher than that of CRT46 which did not carry the plasmid.
These revertants grew slowly at 42°C. This higher frequency of appearanceof thermoresistant reversion in the CRT46 which carries a ColE! plasmid
could be interpreted as an integrative suppression (Nishimura, Y, L. Caro,C. M. Berg and Y Hirota 1971. J. Mol. BioI. 55: 441) by the ColE! plasmid.
A homology in nucleotide sequences between the E. coli chromosomal
fragment in the plasmid and the corresponding region of host chromosome
could provide the recombination site. This would result in the formationof a fused replicon in which the ColEl plasmid is integrated into the hostchromosome. Such bacteria could replicate their DNA using the replication
machinery of the integrated CoEI replicon.
Immunity to colicin El in these revertants, a phenotype of E. coli carrying
ColEl genone, was co-transducible with the met gene, which is located near
the ilv gene, and the frequency of joint transduction of the two outside
markers was lowered. These results indicated that the synthetic ColEl
plasmid was integrated into ilv region and that the region was stretched to thecorresponding length of the plasmid. The profile of CsCl-ethidium bromide
density centrifugation of DNA extracted from the revertants showed theabsence of a satellite band which could be responsible for the plasmid DNA
in cytoplasmic state. This result confirmed that the plasmid DNA in therevertants was in an integrated state.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

III.

BIOCHEMICAL GENETICS AND IMMUNOGENETICS
Estimation of Genetic Distance between the Japanese and theEuropean Wild Mice, M.m.molossinus and M.m.domesticus
Kazuo MORIWAKI and Mitsuru MINEZAWA

Recently several investigators have reported circumstantial evidencessuggesting rather remote genetic distance between the Japanese wild mouse,
Mus musculus molossinus, and the European one, M.m.domesticus, fromwhich most of the laboratory mice likely originated. Those are thedifferences in gamma-globulin heavy chain genes (Liberman and Potter
1969, J. Exp. Med. 130: 519), Hbb alleles (Nishimura et al. 1973, Exp.
Animals 22: 187), satellite DNA contents (Rice & Straus 1973, P.N.A.S.
70: 3546), chromosome C band patterns (Dev et al. 1975, ChromoSoma
53: 335) and Isocitrate dehydrogenase-l alleles (Minezawa et al. 1976,
This report 26: 23) and also abnormal meiotic figures in F 1 hybrid between
the two subspecies (Roderick 1971, Mut. Res. 11: 59).
We attempted to estimate their genetic distance quantitatively using thedata on the allelic frequencies of various biochemical characters reported
previously by us (Minezawa et al. 1976, This report 26: 23) and otherworkers (Selander et al. 1969, Evolution 23: 379; Selander & Yang 1969,
Genetics 63: 653; Staats 1976, Cancer Res. 36: 4333). They are summarized in Table 1. Allelic frequencies of those 10 loci, 6 polymorphic and4 monomorphic (albumin, transferrin, erythrocytic esterase and prealbumin), were applied to Nei's equation (1975 Molecular population genetics
and evolution, North Holland) for calculating standard genetic distance (D).
D=- log J xyj,vJ~J;-

Calculated D is 0.041 between M.m.domesticus in Europe and that inNorth America, 0.327 between M.m.domesticus and M.m.musculus and0.514 between M.m.domesticus and M.m.molossinus. Nei (1975) estimatedD valuse between M.m.musculus and M.m.domesticus as 0.196 using 41loci reported by Selander et al. (1969). If we use more loci for the calculation of D between M.m.molossinus and M.m.domesticus, it should be
reduced to a considerable extent. Moreover, considering 'deme' structure
characteristic in wild mice, estimated D value could be around 0.3. Ac-

RESEARCHES CARRIED OUT IN 1977
Table 1.

-------Locus~ase-1

AlIeIic frequencies of six protein loci in the threesubspecies of mouse, Mus musculus
Name of subspecies and alIeIic frequencies
AlIele

M.m.
M.m.
M.m.
M.m.
molossinus musculus domesticus domesticus
in Japan in Europe in Europe in U.S.A.

Laboratory
mice

a
b
c

.74
.18
.07

.99
.01
.00

.00
1.00
.00

.01
.98
.01

.16
.84
.00

Esterase-2(Es-2)

a
b
c
d

.09
.03
.88
.00

.00
.07
.93
.00

.00
1.00
.00
.00

.09
.84
.01
.04

.08
.90
.02
.00

Esterase-3(Es-3)

a
b
c
d

1.00
.00
.00
.00

.00
.44
.56
.00

.00
.35
.65
.00

.00
.75
.23
.01

.33
.05
.62
.00

Esterase-5(Es-5)

a
b
c

.91
.01
.08

.95
.05
.00

.74
.26
.00

.85
.15
.00

.06
.94
.00

Isocitrate
dehydrogenase(Id-1)

a
b
c
d

.08
.78
.13
.01

.12
.88
.00
.00

.93
.07
.00
.00

.55
.46
.00
.00

.58
.42
.06
.00

Hemoglobinbeta-chain
(Hbb)

d
s
p

.40
.08
.60

.33
.67
.00

.16
.84
.00

.20
.80
.00

.53
.46

.01

194

69

30

1221

40

(Es-1)-

27

Number of samples
References-----------

Minezawa Selander
Selander Selander & Staats,
et al., 1977 et al., 1969 et al., 1969 Yang, 1969 1976~----------------.

cording to Nei (1975), divergence time is obtained by 5 x 106 D. D=O.3
gives approximately 1.5x 106 years. This value well reconcile with anotherestimation (Benveniste et al. 1977, J. Virol. 21: 849). Allelic frequency
data in Table 1 also strongly suggest that most of the laboratory mouseoriginated from the European wild mouse, M.m.domesticus.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Distribution of Three H-2 Antigenic Specificities in Various
Species of Wild Rodents
Kazuo MORIWAKI and Toshihiko SHIROISHIDistribution of three H-2 antigenic specificities, KD5, K8 and D13, invarious species of wild rodents was surveyed by a quantitative abbsorptionof monospecific alloantisera with either erythrocytes or spleen lymphocytesof the animals to be examined. Results are summarized in Table 1. The
alloantisera for detecting KD5, K8 and D13 were given by NIH, designa_
tions of which are D-5bAF, D-8 and D-13 respectively. Residual activityof each antiserum after the absorption was assayed by cytotoxicity test
using 51Cr labelled lymphocytes as targets obtained from either BlO or
BIO.D2 congenic mice. Percent residual activity is expressed as follows:
Released 51Cr cpm by absorbed antiserum - blank100
Released 51Cr cpm by unabsorbed antiserum - blank X+,

In Table 1,± and - correspond to three ranges of the average
percentage of 2 or 3 individuals, 0-20%, 20%-50% and 50%-100%.
Within Mus musculus species, those H-2 antigenic specificities were commonly observed. Previous studies have also demonstrated that H-l.5
specificity widely distributes both in M.m.domesticus (Europe) and M.m.
molossinus (Japan) (Mickova & Ivanyi 1976, Folia BioI. 22: 169; Moriwaki
et at. 1976, This report 26: 20). Out of M.musculus species, H-2.5 could
not be detected as far as the present survey is concerned, while H-2.8 has
widely been observed within subfamily murinae.
Though serologicalsurvey of common antigens in the wild rodents often involves complex
methodological problems such as crossreactivity of antibody, it should
be of great interest that the commonest specificity in Mus musculus species
does not exist in many other rodent species where the other H-2 specificity
not being very frequent in Mus species widely distributes.

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Genetic Regulation for the Expression of Erythrocytic H-2
Antigens in a Mouse Intersubspecies Hybrid
Kazuo MORIWAKI and Toshihiko SHIROISHI

So far mouse H-2 genes are to be expressed codominantly in most adulttissues. However, in F 1 hybrid between two mouse subspecies, Mus musculus
domesticus (European wild mouse and most laboratory mouse) and M.m.
molossinus (Japanese wild mouse), expression of erythrocytic H-2 antigens
introduced from domesticus is. frequently suppressed as previously described by us (Moriwaki & Shiroishi 1976, This report 27: 36). This suppression behaved like a dominant trait in F 1 hybrid. We examined further
the mode of H-2 expression in F 2 progenies.
Firstly, a BlO.D2 strain female was mated with a Mol.Aj (M.m. molossinus) male to obtain F 1 hybrids. Erythrocytic H-2 antigen specificities
detected are 4+,5- and 23- in the former, 4-,5- and 23- in the latter, and4-, 5- and 23- in the F 1 hybrids. Thus H-2.4 from BlO.D2 was clearlysuppressed on the erythrocytes of the F 1 hybrids as already stated, whereas
it was distinctly detected on their spleen lymphocytes.
Secondly, the F 1 male was mated with a BlO.BR female to produce F 2progenies. H-2 antigenic specificities of BlO.BR mouse are 4-,5+ and 23+.
In F 2 progenies, about half should have heterozygous H-2 chromosomes
such as BlO.BRjBlO.D2 and the other half BlO.BRjMol.Aj.
BIO.D2 (4+ ,5- ,23-) x Mol.Aj (4- ,5- ,23-)

I
BlO.BR (4-Y,23+)xF1 (4-,5-,23-/4-,5-,23-)

I
F2

I

"\
suppressed

I

I

BlO.BR/MoI.Aj
BIO.BR/BlO.D2(4- ,5+ ,23+ /4+ ,5- ,23-) (4- ,5+ ,23+ /4- ,5- ,23-)"\t
not suppressed

If H-2 chromosome of Mol.Aj has some dominant suppressing factor,
about 50 % of F 2 progenies can not express H-2.5 and H-2.23 specificities on
the erythrocytes. But our data on 16 F 2 progenies demonstrated that all ofthem definitely expressed H-2.5 and 23 specificities on the erythrocytes,
though H-2.4 positive individuals were 7 out of 16.

31

RESEARCHES CARRIED OUT IN 1977

This result excludes the possibility that a single dominant gene is governing suppression mechanism of H-2 antigens on erythrocytes in this case.
Developmental Changes of H-2 Antigens on Postnatal
Mouse Erythrocytes
Toshihiko SHIROISH and Kazuo MORIWAKI

Pizarro et al. already reported temporal patterns of development of H-2
antigens on erythrocytes (Pizarro et al. 1961). In their study, it wasdifficult to trace the quantitative developmental changes of H-2 antigens
exactly, because all their observation were conducted by a hemagglutination
method.
In the present work, we performed the quantitative absorption of antiH-2 sera, and subsequently assayed the residual activity by the cytotoxicity
method using 51Cr labelled lymphocytes. We examined KD3, D4, K5,
K23 specificities of H-2 antigens on postnatal BlO.A (H-2 a ) mouse erythrocytes. Results are summarized in Table 1. The quantity of antigens isexpressed in terms of absorbing capacity, which means % of absorbed
cytotoxic activity against the full cytotoxic activity of non-absorbed alloantisera.
Table 1.

Temporal patterns of development of H-2 antigens
on postnatal BlO.A erythrocytes

H-2 antigenic
specificities
examined

1 day

3 days8 days13 days

8 weeks

KD3
D4
K5
K23

11
18
23
12

45
33
11
53

37
44
53
42

66
65
85
50

76
90
100
72

*

absorbing capacity (%) in each stage*

Each stages are designated days after birth.

From these results, we found out that even new born mouse (l day)already expressed each H-2 antigens on erythrocytes at 10-20 % of adultmouse, generally they increased untill 8 weeks. It is, however, notablethat there are some difference of developmental patterns among each H-2
antigenic specificities examined in this experiment. For example, H-2DA
increased lineally from 1 day to 8 weeks, while H-2KD.3 and H-2K.23

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

decreased once from 3 days to 8 days and after 8 days increased again
untill 8 weeks. A similar pattern was observed in the development of H-2K.5
antigen, which decreased from 1 day to 3 days. The most striking findings
are that these quantitative dissmilarity with respect to their developmental
changes is observed within the H-2 antigens which have been believed toexist in the same H-2 molecule. Both H-2K.5 and H-2K.23 antigens aresupposed to be composed on the same molecule H-2K, neverthless thepatterns of development of these two antigens are different each other.
These findings are not quite agreement with Pizarro's observation.
They might reflect the possible existence of independent genes which
direct each H-2 antigens. But now we do not have any solid explanation
for this phenomenon.
Allelic Constitution of Hbb Locus in Asian House Mouse Subspecies
Mitsuru MINEZAWAl) and Kazuo MORIWAKIOne of the Asian mouse subspecies, Mus musculus molossinus (habitate
in Japan), has rather different genetic constitution from the Western hemisphare wild mouse (Minezawa et al. 1976, This Report 26: 23-25). Its
Hbb (hemoglobin beta-chain) locus consisted of mainly Hbb P and Hbb dalleles. But Hbb s allele was rare and considered to be introduced toJapanese wild population very recently, though it is common as well as
Hbb d in the Western hemisphare (Minezawa et al. 1977, This Report 27:
33-34). It is not yet clear whether the frequent occurrence of Hbb P allele
is unique to the Japanese population or general feature in the Asian mice.
This would be crucial ploblem to reveal the evolutionary process of Hbb
gene of Mus musculus and differentiation of Mus musculus subspecies.
Twenty three wild mice were collected from four Asian countries to
examine their Hbb types. Twelve were obtained from Philippines bycourtesy of Dr. J. S. Masangkay, 11 from Quezon and 1 from Batangas;
Six from Taichung in Taiwan by courtesy of Dr. T. Y. Ku; Three from
Lahore in Pakistan and two from Kabul in Afghanistan by courtesty of
Mr. H. Ikeda. Two fancy mice from Bangkok in Thailand were also
examined. The results are summarized in Table 1. Allelic constitution
similar to Japanese population was observed and the frequencies of Hbb P
and Hbb d fluctuated depending on the locality of collection. Putting to1)

Primate Research Institute, Kyoto University

33

RESEARCHES CARRIED OUT IN 1977
Table 1.

Phenotypes of hemoglobin beta-chain (Hbb) of
house mouse from five Asian countries

Country

No. ofMice

Taiwan (wild mouse)
Philippines (wild mouse)
Pakistan (wild mouse)
Afganistan (wild mouse)
Thailand (fancy mouse)

6
12
3
2

2

No. of Phenotype
P

PD

D

5
2

2
1
2

10
1

gether the other observations (Gilman 1974, Ann. N.Y. Acad. Sci. 241:
416-433; Morton and Tobin 1977, Biochem. Genet. 15: 101-108), Hbb
locus of wild mouse population in the Eastern Asia generally consisted ofHbb d and Hbb p alleles, and Hbb s alleles is rare or inexistent. This geographical distribution profile of Hbb alleles, especially inexsistence of Hbb sallele, is inconsistent with Gilman's hypothesis, which supposed coexistenceof two ancestral alleles, duplet which consist of two close linked genes, andHbbs-like singlet alleles. We could not find the cause of disappearence of
singlet allele from Eastern Asia. Another possible hypothesis is thatancestral Hbbs-like singlet allele was produced by deletion or suppressionof one component of the duplet genes. On the basis of the distributionof two alleles, Hbb P and Hbb s, Mus musculus suspecies would be classified
into two subspecies groups, such as Asian subspecies with Hbb p and Hbb dalleles and Western hemisphare subspecies with Hbb s and Hbb d alleles.
This classification is similar to that by Marshall who proposed two major
subspecies groups, castaneus group habitated in Eastern Asia andmusculus group habitated in Western hemisphare and Western Asia (1977Bull. Am. Mus. Nat. Rist. 158: 172-220). The disagreement between
Marshall and we is found in Afghanistan and Pakistan mice which wereclassified into musculus group and possessed Hbb P allele. This inconsistency is, however, well accounted by the fact that M. m. bactrianus
inhabitated in those area revealed many mixed characters of European
and East Asian subspecies, and intermingling process between two subspecies groups is now in progress in those area.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Geographical Distribution of Isocitrate Dehydrogenase-t e in
the Japanese Wild Mouse, Mus musculus molossinus
Mitsuru MINEZAWA1 ), Kazuo MORIWAKI and Kyoji KOND0 2)

We have preliminarily reported four alleles of isocitrate dehydrogenase(Id_r,b,c and d) locus in the Japanese wild mouse (Mus musclus molossinus)
population. Among them, Id-Ia and Id-Jb alleles were recorded fromseveral Western hemisphare pouplations and laboratory strains, whereas
Id-I c and Id-Id alleles were not except for rare case (probably Id-J<) in Hawaii(Minezawa et al. 1976, This Report No. 26: 23-25). As Id-I c allele wasobserved in another Asian subspecies Mus musculus castaneus fromThailand and Philippines, we considered this allele is specific to Asian mice.
Table l. Allelic frequency of isocitrate dehydrogenase (Id-l) of wildhouse mouse in 14 geographical regions of Japan
Allelic Freq. (%)

Regions

No. ofMice

Hokkaido
Mainland (Honshu)
TohokuHokuriku
Kanto
Tokai
Kinki
San-yoSan-inShikoku
Tsushima
KyushuOosumi Isis.
Amami Isis.
Okinawa

19

97.4

14
6
14
17
10
15

100.0
100.0
100.0
97.1
100.0
100.0
96.2
100.0
13.6
51.2

a

b

c

d

~--------

13

3.8

7
11

42
3
8
1

27.3
17.8
18.8

75.0
50.0

2.6

2.9

59.1
31.0
100.0
6.2
50.0

Geographical distribution of those Id-l alleles in the Japanese wild
mouse population was shown in Table 1, based on 181 mice collected from
7 islands (Hokkaido, Shikoku, Tsushima, Kyushu, Oosumi IsIs., Amami
IsIs. and Okinawa) and 7 regions of Mainland (Tohoku, Hokuriku, Kanto,
1) Primate Research Institute, Kyoto University
2) Faculty of Agriculture, Nagoya University

RESEARCHES CARRIED OUT IN 1977

35

Tokai, Kinki, San-yo and San-in). As a whole, Id-l b was the commonest
in Japan. The frequencies of four alleles, Id-Jb, Id-l c, Id-la and Id-l d , were
79.4 %, 13.1 %, 6.9 % and 0.6 %, respectively. In northern part of Japansuch as Hokkaido, Shikoku and Mainland except for San-in, Id-l locus
consists of Id-Jb allele except for two cases with Id-l BD phenotype.
Whereas, in southern part of Japan, this locus mostly consist of two or
three alleles, namely assortment of Id-JC, Id-Jb and Id-la. All three micecollected from Oosumi IsIs. showed Id-l c allele in homozygous state.
Those findings may suggest a possibility that an original allele of theJapanese wild mouse population was Id-Jb. Considering its geographicaldistribution and electrophoretical mobility, Id-l d allele seems to be produced from Id-l b by one or two amino acid substitution. On the otehr
hand, existence of Id-l c allele in either Asian continent and southern islandsof Japan, suggested that Id-JC allele was immigrated into Japan from
Asian continent through two routes, Korea to Tsushima route andTaiwan to Southeast archipelago route.

Electrophoretic Survey for Regional Differences of Blood Proteinsin Ryukyu Mice from the Southeast Asia
Mitsuru MINEZAWA1l, Hirohisa HIRAI, Kimiyuki TSUCHIYA2 l, Prajong SUDTO"l,T. Y. Ku 4J, Yoji IKAWA5) and Kazuo MORIWAKI

Recently Marshall (Bull. Am. Jus. Nat. Hist. 158: 172, 1977) has classified
Mus formosanus Kuroda, which habitats only in Taiwan and is characterizedby a sharply bicolored tail, into Mus caroli (Ryukyu mouse) based on
the morphological characters. As the measurements of external characters,
head and body length and tail length, of Mus caroli obtained from three
regions in the Southeast Asia, Okinawa, Taiwan and Thailand, revealed
rather large differences, we further examined several biochemical characteristics to clarify their intraspecies relationship. Total nineteen individuals
were obtained from the three regions, 5 from Naha city in Okinawa (bycourtesy of Dr. Kyoji Kondo), 6 from Taichung city in Taiwan and 8 from
1)

2)
3)
4)
5)

Primate Research Institute, Kyoto University
Hokkaido Public Health InstituteDepartment of Agriculture, Thailand
Plant Protection Center, Taiwan
Cancer Institute

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Lombri province in Thailand. Hemoglobin, serum esterases and serum
proteins were examined by starch- and acrylamide gel electrophoresis.
Hemoglobin: Mus caroli possess a unique hemoglobin beta chain among
Mus species (Gilman 1974, Ann. N.Y. Acad. Sci. 241: 416). All mice from3 regions exhibited this species specific hemoglobin on their electrophretic
patterns.
Serum esterases: Major serum esterase in the mice obtained fromTaichung showed a zymogram different from those from the other regions.
Serum proteins: Albumine and transferrin of the mice from the three
regions showed almost the same pattern. Whereas several minor serum
proteins observed in Taichung region were somewhat different electrophoretically from those in the other two regions.
Those results suggest a possibility that Mus caroli in Taiwan is genetically
remote to some extent from the other two Mus caroli in Okinawa andThailand, not in parallel with their geographical relationship.
Comparative Analyses of Japanese Wood Mice between
Oki Islands and Mainland from the View pointsof Biochemistry and Cytogenetics
Hirohisa HIRAI, Kazuo MORIWAKI and Teru Aki UeRIDA1 )At present the wood mouse of Hondo (Japanese mainland) is classified
into Apodemus speciosus speciosus and that of Dozen and Dogo in Oki
islands A.s.navigator. Hiraiwa et al. (1958, Sci. Bull. Fac. Agr. Kushu
Univ. 16: 547) have described that the tail ratio of navigator is lower thanthat of speciosus and the dead center represented by the distance between
the first upper molars of skull in specimens from Oki Is., especially from
Dozen, is longer than that of Hondo. Judging from this, they claimed
that the animals from Dozen can be distinguished from navigator of Dogoat a subspecies level. We attempted to dissolve this problem from thegenetical viewpoints.
As a preliminary survey, we compared A.s.navigator from Dozen andDogo with A.s.speciosus from Fukuoka and Mishima by using electrophoretic, immunological and cytogenetical techniques. Albumin, transferrin, hemoglobin, erythrocytic esterase, serum esterase-l and -2 were analysedby the thin layer acrylamide gel electrophoresis. Fifteen per cent acrylamide
1)

Faculty of Agriculture, Kyushu University

RESEARCHES CARRIED OUT IN 1977

37

separation gel dissolved in Tris-Hcl buffer (pH 8.9) was prepared in 1 mm
thick between two glass plates of 170 X 180 X 3 mm each. Spacer-sample
gel with 3.2 % acrylamide in Tris-Hcl buffer (pH 6.7) was put on that.
Thirteen samples were analysed at once usually by 4 hours run at 400 V
constant voltage under refrigeration. Coomasie brilliant blue was used forstaining protein on the gel. Esterase bands were detected by the incubationof gels at 37°C for 20 minutes in a mixture (pH 6.8) of 0.03 % naphthyl
acetate and 0.1 % fast blue RR salt.
Though there was no detectable difference in the six blood proteins
mentioned above among four groups of samples, one those of remarkableband (tentatively designated D-band) could be observed between albumin
and transferrin in two individuals obtained from Dogo. After treatment ofacrynol (Moriwaki et al. 1974, Experientia 30: 119), this protein remains
in the soluble fraction with transferrin. SDS-acrylamide gel electrophoresis
indicated molecular weight of this proteins approximately 50,000.
Antiserum against Apodemus serum proteins was prepared by immunizing rabbits with whole serum of A.s.speciosus collected from Fukuoka.
Immunoelectrophoresis was conducted on 0.85 % Agar gel using the antiserum. No visible difference was observed in the immunoelectrophoretic
patterns for the whole sera of speciosus and navigator suggesting that theunknown D-band protein has a unique antigenicity.
Chromosome G- and C-bands were observed by trypsin method (Seabright, 1971) and BSG method (Sumner, 1972), respectively. So far, nogeographical variation between Old and Hondo populations has been
detected.
The Genetic Control of Tryptophan Pyrrolase in Drosophilamelanogaster: Mechanism of Suppression
Saburo NAWA and Masa-Aki YAMADA

The enzyme tryptophan pyrrolase, which catalyzes the conversion of
tryptophan to formylkynurenine, is absent in vermilion (v) flies of Drosophila melanogaster. The Vermilion is known to be suppressed by arecessive mutation, SU(S)2 which is non-allelic to vermilion. Suppressedvermilion exhibits a partial restoration of tryptophan pyrrolase activity.
Various models of genetic interaction by the suppressor gene in the control
of Drosophila tryptophan pyrrolase have been proposed. Jacobson et al.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

presented evidence that the vermilion produces an active but altered tryptophan pyrrolase which is inactivated by a specific isoacceptor form oftyrosine tRNA. This inhibition was removed by digesting the tRNAwith
RNase T 1 • Similarily, the suppressor mutation, sues?, removes the tRNAand thus allows the v-tryptophan pyrrolase to function.
The recessive mutant a of Ephestia also lacks the activity of tryptophan
pyrrolase. Since we have used both a and v for the transformation experiment and obtained transformants in a but not in v, it is of interest to knowwhether the mutant a produces an altered but active enzyme which isinhibited by a RNA. In the homogenate of a, however, no appearance oftryptophan pyrrolase activity was observed by treatment with ribonucleaseT 1 or pancreatic RNase. Then the activation of enzyme from vermilion,
which has been used in the transformation experiment, was examined. No
tryptophan pyrrolase activity appeared in the v-homogenate when treatedwith RNase. It has been known that there are two kinds of vermilion
alleles, suppressible and unsuppressible. There may be a possibility thatthe v used is unsuppressible. Then, we obtained v; bw; SU(S)2, which isapparently brown eyed and has a detectable tryptophan pyrrolase activity.
By the elimination of SU(S)2 from this strain, a white eyed v; bw was produced which must be a suppressible vermilion allele. When the homogenate of this suppressible vermilion was treated with RNase, no tryptophan pyrrolase activity was observed. Furthermore, it was found that thehomogenate of Drosophila adults, either wild type or vermilion, shows astrong RNase activity. In the condition used for the assay of tryptophan
pyrrolase activity, almost all of the endogenous RNA were shown to be
degraded in acid soluble forms by the endogenous RNase within 30 minutes.
This means that if an altered enzyme is present in an inhibited form in thevermilion homogenate it should become active by elimination of the RNA
without any exogenous RNase, since incubation of 2 or 3 hours is usual
for tryptophan pyrrolase assay. Thus, the model which requires that thealtered polypeptides produced by suppressible vermilion mutants are inhibited by the RNA which is absent in sues?, finds no support in thepresent data.

RESEARCHES CARRIED OUT IN 1977

39

Structural and Functional Properties of Drosophila andEphestia Tryptophan Pyrrolase
Saburo NAWA and Masa-Aki YAMADA

The activity of tryptophan pyrrolase in the a+ /a heterozygote of Ephestia
was super-additive, being greater than half the tryptophan pyrrolase activity in the a+ /a+. Furthermore, when a/a extract was mixed with a+ /a+
extract, the normal enzyme level was enhanced. This a-material responsible for the super-additivity was found to be a protein which has the same
molecular weight as normal tryptophan pyrrolase. Exactly the same
phenomenon as in a was observed in vermilion of Drosophila. Moreover,
the a-protein of Ephestia was found to be able to enhance the activity of
Drosophila enzyme. In the same way, the v-protein of Drosophila could
enhance the Ephestia tryptophan pyrrolase. Drosophila tryptophan pyrrolase was fractionated in the same place as that for Ephestia tryptophan
pyrrolase on Sephadex gel filtration, indicating that both enzymes havethe same molecular weight. The a-protein as well as v-protein also showed
the same molecular weight as normal type tryptophan pyrrolase. When aconcentration of tryptophan pyrrolase, either of Drosophila or of Ephestia,
in the assay medium was reduced, for example, in one-half, the activity
decreased much less than half. When a volume of the reaction mixture
for the assay was reduced in half without changing amounts of the enzyme,
activity increased greatly.
These observations may be interpreted in terms of interaction between
aggregate enzyme and its subunits, on the assumption that the subunits
are inactive and the homomultimer composed of two or four identicalsubunits is the enzymatically active structure. An equilibrium of multimer~subunits may vary according to the concentration of enzyme protein.
The dilution of enzyme will bring the equilibrium to dissociate to subunits.
The a-protein or v-protein will prevent the normal type of tryptophan
pyrrolase, owing its molecular structure similar to the enzyme, from being
dissociated to subunits, indicating the super-additive effect. These relationship between Drosophila and Ephestia protein with regard to the superadditivity suggests a close resembrance in structure between Drosophila andEphestia tryptophan pyrrolase.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Genetic Control of Alcohol Dehydrogenase in
the Japanese Species of Trillium.
Masaaki IHARA and Tohru ENDO

Genome constitution of the Japanese species of Trillium (Liliaceae) has
been investigated with the aid of differential stainability in the Feulgen
reaction of the somatic chromosomes. For this reaction, the root tip orovular tissues are chilled for 2 to 4 days at 0° to 4°C. The karyotypes
documented to date are K1K1 in T. kamtschaticum (2n=1O), K 2 K 2TT in
T. tschonoskii (2n=20), and SSUU in T. smallii (2n=20) (cf. Haga,
Cytologia 16: 243, 1951). In investigating the mode of differentiation inbiochemical traits, the genetic control of alcohol dehydrogenase (ADH)
isozymes has been examined in the seeds of these three species and their
F 1 hybrids.
Zymograms for each group are depicted diagramatically in Fig. 1.

Fig. 1. ADH zymograms in Japanese species of Trillium (1-3) and their F 1
hybrids (4-9). The author's designation of the genomes in the endosperm andthat given by Haga (1951: in parenthesis) are shown for respective zymograms.
The numbers represent species and their cross; (1) kamtschaticum (K), (2)tschonoskii (T), (3) smallii (S), (4) K x T, (5) T x K, (6) T x S, (7) S x T, (8) S x K
and (9) KxS.

RESEARCHES CARRIED OUT IN 1977

41

The figure suggests that T. kamtschaticum and T. tschonoskii have different
zymograms which are monomorphic within each species. The ADH
zymogram of genome K~ and that of genome T are identical. In T. smallii,
we detected three discrete anodal bands, located at 3.5 cm (slow, darkly
staining), 4.0 cm (middle, intermediate staining), and 4.3 cm (fast, faintly
staining). Assuming that the middle band is a dimer derived fromhybridization between the slow and fast moving isozymes, and that theiractivity ratio is 1: m (l>m>O), we would expect a 1: 2m: m 2 ratio for thethree bands. The slow and fast moving isozyme may be specified byaIleles Adh s (genome S) and Adh u (genome U). If the m value were 0.2,
the ratio would be 1: 0.4: 0.04. This is comparable to the pattern and the
expected pattern even though the Adh u allele has low activity. The actionof Adh u might be extremely low in the F 1 of smallii (AdhSjAdhSjAdhUjAdh U)
X kamtschaticum (AdhKjAdh K). This may also be true for the reciprocal
cross. On the other hand it gives a reaction with appreciable intensity in
the F I of smalliix tschonoskii (AdhTjAdhTjAdhTjAdh T) and in this reciprocal cross. In all the F I hybrids, the zymographic pattern appears to be
subjected to binomial expansion. An example would be in the case of
TTTTK (tschonoskiixkamtschaticum), (4r+l/,:)2-+16rr: 8u:: llClC, where
rand IC are ADH isozymes which are specified by Adh T and Adh K,
respectively.
A New Method for Peroxidase Isozyme Stain
Tohru ENDO

A number of peroxidase staining methods have been reported in which
the staining reaction is due either to oxidation of phenols or amines withexogenous peroxide, or by Nadi reaction of amine-phenol combinations(Endo, Bot. Mag. Tokyo, 85: 147, 1972). The method presented here isbased on a new combination of amine and phenol compounds. The
reaction mixture consists of 2 mM eugenol (ca. 0.03 m!) and 0.5 mM
3-amino-9-ethylcarbazole (ca. 10 mg) which is completely dissolved in 10 ml
acetone. Then, 10 ml of 1 M Tris-acetic acid buffer, pH 4.0, 1 ml of 3 %hydrogen peroxide, and deionized water are added to 100 ml. Staining
is best accomplished in the dark, and the peroxidase bands are stable for atleast one week.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETiCS
Cell Lineage and Development of Chimera Hydra
Tsutomu SUGIYAMA and Toshitaka FUJISAWA

A mutant strain of Hydra magnipapillata (nf-I) was isolated which lacks
interstitial cells and their derivatives (nerve cells and nematocytes). This
strain arises by spontaneous loss of interstitial cells from its parental
strain (sf-I). Reintroduction of interstitial cells from other strains into
nf-I leads to the creation of chimeric strains that consisted of epithelial
cells derived from sf-I and interstitial cells and their derivatives from otherstrains.
In chimeras, interstitial or epithelial cells apparently maintain very
stable cell lineages; no indication was obtained that suggested interstitialcell differentiation into epithelial cells or dedifferentiation in the opposite
direction during the long courses of culture of the chimeras (up to one year).
Developmental characters of chimeras were examined and compared tothose of the epithelial cell (sf-I) and the interstitial cell donors. Almost
all of the chimera's characters examined (graowth rate, budding rate,tentacle numbers, polyp size, regenerative capacity, etc) closely resembled
those of the epithelial cell donor, but not of the interstitial cell donors.
This suggests that epithelial cells, rather than interstitial or nerve cells, arethe primary determinant of most, if not all, of hydra's developmental
characters.
Cellular Composition of Chimera Hydra
Tsutomu SUGIYAMA and Toshitaka FUJISAWA

The hydra (Hydra magnipapillata) tissue consists of 6 basic type of cells;
epitheliomuscular, digestive, gland, interstitial, nerve cells and nematocytes.
Mutants having abnormal cellular compositions are isolated through sexual
imbreeding of wild hydra. Chimeric hydra are produced by reintroducing
interstitial cells from other strains into a strain (nf-I) which lacks interstitial cells, nerve cells and nematocytes.
Analyses and comparisons of the cellular compositions of all thesestrains revealed that the numbers of nerve or interstitial cells in the

RESEARCHES CARRIED OUT IN 1977

43

chimeras were very similar to those in their interstitial cell donors. Sincechimeras and their interstitial cell donors share the same interstitial celllineages, this suggests that interstitial cells or their derivatives (nerve cells
and nematocytes) play major roles in determining the nerve and interstitial cell levels in the hydra tissues. It is suggested that some forms of
homeostatic feedback mechanisms are probably involved in regulating thelevels of these cell types.
Nematocyte Differentiation in Hydra
Toshitaka FUJISAWA and Tsutomu SUGIYAMA

In hydra, the general patterns of interstitial cell differentiation into fourtypes of nematocytes are well known. The interstitial cell divides synchronously and the resulting cells remain together in a nest as a syncytium. In
each nest the cells subsequently undergo synchronous differentiation toform nematocytes of one type. However, the question remains as to whatstage during cell differentiation the interstitial cells become determined to
produce specific types of nematocytes. This problem of cell determination
was studied statistically using mutant strains of Hydra magnipapillata,
which have abnormal nematocyte compositions. The four types of nematocyte nests of various sizes were identified and the numbers were scored ineach strain and the data were subjected to statistical analyses.
Based on the observed patterns of nematocyte differentiation, twomodels are put forward and compared statistically. In the first model, theinterstitial cells undergo cell divisions until they reach the stage at which
specific nematocyte types differentiate. In the second model, the interstitial cells are destined to differentiate into one of four types of nematocytes at an early stage, and then cell multiplication occurs. Variance test
favours the first model suggesting that the interstitial cells become determined to differentiate into the specific nematocyte types after they multiply.
Pyrimidine Requirements of Cultured rudimentary Embryonic
Cells of Drosophila melanogasterYukiaki KURODA

The rudimentary 39k (r g9k; 1-54.5) is a sex-linked recessive lethal gene.
Homozygous or hemizygous r S9k embryos produced from homozygous r 39k

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

mothers died during embryonic development. When cells obtained fromr 39k embryos prior to their effective lethal phase were cultured in K-17

medium supplemented with 0.1 mgjml fetuin and 15% fetal calf serum, acharacteristic defect was found in the maturation (chitinous pigmentation)of epithelial cells, although other types of cells from the same embryos
remained normal in their growth and differentiation in culture.
It has been reported that the r eggs could be rescured by injecting each
egg at the blastoderm stage with pyrimidine nuc1eosides (Okada, M., I. A.
Kleinman and H. A. Schneiderman. 1974 Develop. BioI. 37: 55). In thepresent experiment some pyrimidine nuc1eosides and intermediates in thepyrimidine biosynthetic pathway were added to the culture medium to test
their repair activity of the defect of r 39k embryonic epithelial cells.
Uridine at 2.5 X 10- 4 M and thymidine at 5 X 10- 3 M were effective inrepairing the r 39lc embryonic cell defect. Carbamyl phosphate at 10- 3 M,
carbamyl aspartic acid at 2.5 X 10- 4 M, dihydroorotic acid at 2.5 X 10- 4 M
and orotic acid at 2.5 X 10- 4 M, all had repair activities on the defact of
cultured r 39k embryonic cells. Dihydroorotic acid and orotic acid weremost effective, repairing r 39k defect almost completely. These results suggest that r 39k epithelial cells may be deficient in carbamyl phosphate synthetase which is the first enzyme of the pyrimidine biosynthetic pathway.
Enhancing Effect of Cellophane Films on Chondrogenesis of Quail
Limb-Bud Mesenchymal Cells in Culture
Yukiaki KURODA and Etsuya MATSUTANI

Mesenchymal cells from the limb-buds of avian embryos were known todifferentiate into cartilages or muscles in vitro. Thus they seemed suitablematerial for analyzing the mechanism of differentiation of common precursor cells into the two types of cells.
In the present study a very simple culture technique with a cellophane
film was found to have a marked effect in enhancing chondrogenesis ofmesenchymal cells from embryonic quail limb-buds in vitro. Mesenchymal
cells were obtained from the hind limb-buds of 3.5 day (stage 22-23) quail
embryos. Drops of 0.025 ml of cell suspensions were inoculated into
35-mm petri dishes with a micropipette (Gilson, P-200). Two strips ofcover glass were placed on either sides of the drop. After the petri dishes
were incubated for 4 hours, 2 ml of culture medium was added to the

RESEARCHES CARRIED OUT IN 1977

45

dishes. A cellophane film was placed on top of the glass spacers andcovered with a stainless steel ring. Thus the cells were cultured in a
microspace under a cellophane film.
It was found that the cellophane film markedly stimulated the process ofexpression of chondrogenesis of mesenchymal cells, which was estimatedquantitatively by the number of cartilage nodules and the amount of
toluidine blue absorbed into chondrocyte matrix. This stimulation ofchondrogenesis by cellophane films was found to be due to the accumulation of some factor(s) released from mesenchymal cells under cellophanefilms.
Comparative Study on Mutagenic Activity of Various Agentsin Cultured Human Diploid Cells
Yukiaki KURODA

In our previous works, the mechanism of mutation induction at the
8-azaguanine (8AG) resistant (HGPRT deficient) locus by ethyl methanesulfonate (EMS), furylfuramide (AF-2), phloxine and bisulfite in human
diploid cells was investigated.
In the present experiment, a-phenyl phenol (OPP), which is a fungicidal
substance for citrus fruits, and X-rays, which are physical agents for comparison, were examined for their mutagenic activities in human diploid cells.
OPP had a cytotoxic effect on human diploid cells at a concentration of
10- 6 M for 2 hour-treatment and at a concentration of 3 X 10- 7 M for 14
day-treatment. Cells were treated with OPP for 2 hours, cultured innormal medium for the mutation expression time of 48 hours, and thenumber of 8AG resistant cell colonies formed in the presence of 30 ,ug/ml
8AG was scored. No significant increase in the induced mutation frequency
was observed in cells treated with OPP at various concentrations from 10- 8
to 10- 6 M.
When cells were exposed to 0 to 1,000 rads of X-rays, the Do value
calculated from a dose-response curve for X-rays was 200 rads. The
frequency of 8AG resistant mutations increased rapidly as an increase in the
X-ray doses. X-rays at 300 rads were most effective in inducing 8AG
resistant mutations without extremely marked decreases in cell survival.
When the mutagenic activities of various chemical and physical agents on
induction of 8AG resistant mutations in human diploid cells were compared

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

at the doses giving about 50 % cell survival, the induced mutation frequencies were 24.4 X 10- 5 for EMS (10- 2 M), 58.4 X 10- 5 for AF-2 (10 ,ug!ml),24.3 X 10- 5 for phloxine (30,ug!ml), 11.7 X 10- 5 for bisulfite (10- 2 M), 0 for
opp (3 X 10- 7 M) and 17.1 X 10- 5 for X-rays (100 rads).
Colony-Forming Activity of Embryonic Human Diploid Cells
in Primary Cultures
Yukiaki KURODA

Cells dissociated from various organs of aborted human embryos atvarious gestation stages were cultured in 60-mm petri dishes in Ham's
medium F12 supplemented with 15 % fetal calf serum to examine theircolony-forming activity in primary cultures.
Among various organs from which cells were dissociated, lung cells
showed the highest colony-forming activity. Heart cells were difficult tobe dissociated completely into single cells and showed a lower colonyforming activity. Skin cells required a specific procedure for their pureisolation from embryos. Liver cells and kidney cells produced the extremely
low colony-forming activity.
The colony-forming activity of lung cells was largely dependent on theage of embryos from which cells were obtained. The colony-forming
activity was 0.022 in lung cells from a 6-week embryo, 0.126 in those froman 8-week embryo, and 0.771 in those from a lO-week embryo. Lung cells
from a 13-week embryo showed the highest colony-fanning activity of 1.688
among those from various ages of embryos tested. Lung. cells fromembryos older than 17-weeks showed a gradual decrease in their colony"forming activity under the same conditions employed. The colony-forming
activity of cells from a 24-week embryo was 0.007.
Lung cells dissociated from a lO-week embryo were cultured in TD-40
flasks in primary culture, and then inoculated in petri dishes in secondary
culture. The colony-forming activity of cells in secondary culture was
1.337, which were twice as high as that in primary culture (0.771). Whenlung cells cultured in TD-40 flasks in primary and secondary cultures were
inoculated in petri dishes in tertiary culture, their colony-forming activity
was 2.577, which were again twice as high as that in secondary culture.
Results indicated that embryonic human lung cells showed a rapid increasein their colony-forming activity in successive subcultivation for theirRESEARCHES CARRIED OUT IN 1977

47

initial culture period.
Fundamental Research on Genetic Monitoring of Environmental
Mutagens by Using Cultured Human Cells
Yukiaki KURODA

There are some advantages in the procedures using cultured humansomatic cells for monitoring genetic effects of environmental mutagens:direct effects of the mutagens on human cells could be quantitatively
detected within a relatively short period under strictly controlled conditions.
Thirty human embryos were obtained by artificial abortions in sevenmaternity hospitals in Misima. They were at various ages of gestationfrom 6 to 24 weeks. Lung cells dissociated from embryos at various
stages were cultured for one to three weeks in 60-mm petri dishes at 37°C
in the presence and absence of 30 ,ug/ml 8-azaguanine (8AG). The numberof colonies formed were scored and the frequency of 8AG resistant mutant
cells present in the original cell populations of embryonic lung was estimated.
The frequencies of mutant cells were 0 in a 6-week embryo, 1.6 X 10- 3 in
an 8-week embryo, 1.8 X 10- 3 in a lO-week embryo, 2.0 X 10- 3 in a 13-weekembryo, 7.9x 10- 3 in an 18-week embryo and 15.0x 10- 3 in a 24-weekembryo. If these frequencies (Yi) are supposed to increase linealy with anincrease in the age (t i ) of embryos, they are expressed as a simple formula:
Yi=at i . By using above data with estimated variances, the value of a wascalculated to be O.l4x 10- 3 • It was found that the calculated frequencies
of mutant cells in each age of embryos by using this a value coincided wellwith the observed frequencies in the clonal cultures. This indicates that
this procedure may be useful for monitoring somatic gene mutations inhuman environment.
Isolation and Characterization of X~linked Maternal Effect Lethal
Mutants in Drosophila melanogaster
Masa-Aki YAMADA and Saburo NAWA

In higher organisms, the cytoplasm of egg is produced by the female
genome. The cytoplasm of egg has an essential role for the early development of the zygote. The cytoplasmic defective mutants are considered to
be useful for the investigation on the interactions between the egg cytoplasm

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

and the zygote nucleus during the early development.
After the males of wild type fly (Oregon R) were fed on 2.5 X 10- 3 M
EMS in 1 % sucrose solution for 24 hours, female sterile mutants on Xchromosome were isolated by Muller-5 method. From the many fs(l)mutants, 26 strains of the maternal effect mutants were picked up anddivided into 16 complementation groups. All of the strains produced apparently normal eggs in shape which were unable to develop into larvaeeven when fertilized with wild-type sperms, and the development of theembryos stopped at various stages of embryogenesis. In some cases, theabnormality of embryogenesis also was observed at the stage of the cellular
blastula or inversination.
It was examined in 7 mutants (fs(l) MY-13, 18, 131, 152, 160, 170 and335) whether the embryos were rescued with the wild type cytoplasm injectedby the microinjection method. The cytoplasm of the wild type eggs was
injected into 300-600 eggs of each mutant and it was examined whether
larvae could hatch. In the most of mutants, the embryos were not rescued,
but in fs(1) MY-18, 13 larvae hatched from 598 eggs injected with thenormal cytoplasm. In the control experiment, when eggs were injected with
fs(l) MY-18 cytoplasm, no larvae were obtained. Thus, it is likely that acomponent of cytoplasm from normal eggs, but not from eggs of fs(l)MY-18, can rescue the developmental defect in the mutant. There is no
information about the identity of this component at present. Experiments
for the identification of the component are now in progress.
On the Number of Mitotic Cleavage Division Times in the Silkworm(Bombyx Mori)
Yoshiki OHTSUKI1), Toshio KlTAZAWA 1) and Akio MURAKAMI

Although a number of papers on the embryogenesis in the silkworm havebeen published, knowledge regarding the number of mitotic cleavage
divisions in eggs is meagre. Ten cyclic changes in radiosensitivity withreference to embryonic lethality in accordance with the development ofembryos were already observed and suggested that the number of synchronous cleavage division cycles occurred at least ten times. In addition tothese synchronous divisions, a few cleavage divisions are required to complete1).if Biology Division of the Sericultural Experiment Station, M.A.F.F., Suginami-ku,
Tokyo.

RESEARCHES CARRIED OUT IN 197749

blastulation. The total number of cleavage divisions required would be atleast twelve to sixteen until the blastulation. After 10 or 11 cleavages, thenuclei reach the periphery of the egg and a blastula is formed. It is a wellknown fact that no cleavage division takes place in eggs from the completionof blastulation to gastrulation corresponding to the egg ages from 17 to48 hrs after oviposition. Accordingly, it may be easy to calculate the numberof cleavages that occur until the blastulation by counting the total numberof cleavage nuclei or cells in the blastula. This report aimes histologicallyto calculate the total number of cleavage divisions.
Eggs of the CI08 bivoltine race were used for the present experiment.
Eggs deposited within ten minutes were kept at 25°C after oviposition forobservations. The number of nuclei was counted in the eggs at 13 and72 hrs after oviposition to record the number of mitotic cleavage divisions
for blastoderm formation. Histological preparations of the 13-hr-old eggs
were made by a usual paraffin method and stained in Heidenhain iron
hematoxylin and the nuclei in the preparation were counted. The 72-hrold eggs (or just before the completion of pre-dormant stage) which had
already completed nuclear divisions were dissected in a mixture of 0.5 %
acetic acid, glycerin and distillated water (dw) (9: 1: 4). Embryos were
stained with acetocarmin (saturated solution of acetocarmin 1: dw 4) for
10 min on a glass printed the sectional pattern and covered with a thinglass and pressed softly to separate each cell. For both egg stages, thenumber of nuclei in all fields of micrographs of one preparate was counted.
The result of experiments indicated that the average number of nuclei inthe 13-hr-old egg calculated was 1,985, of which 697 were destined to
become the nuclei of embryonic and amnion cells and the rest of nuclei
become either serosa or yolk cells. In the embryo of 72-hr-old eggs, thenumber of nuclei counted was 12,668.
From these findings, it can be said that eleven divisions occur until
13 hrs after oviposition at 25°C and thereafter, at least, five more divisions
are required. Accordingly, the products of 16 mitotic cleavage divisions
ought to form the embryo (and/or blastoderm) with more than 10,400 but lessthan 20,800 nuclei.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Mosaicism of Ganglion in the Silkworm (Bombyx MOrl)
Motoya KATSUKl1), !tam WATANABE1) and Akio MURAKAMI

It is worthwhile to knwo how to control genetically the differentiationof the nervous systems in higher organisms. Since the nervous system of
the silkworm is composed of only 13 pairs of ganglia from brain toposterior abdomen and simple in their networks, it forms suitable material
for research this field. In addition to these advantages, it has been observed
independently by Murakami (unpublished observation) and by Tamazawa(unpublished observation) that the color of ganglia of wild-type stocks isblack or redish-black, while that of egg(or eye)-color mutants, such as pe,
W 1 and wz, is milky white.
In this report, we have tried to investigate the developmental relationship between these two traits, egg (or serosa membrane cell)- and ganglioncolor by using the pe gene. Egg-color mosaics were obtained by supercooling treatment. The wild type (CI08; pe+) were mated to the egg-color
mutant male homozygous for pe gene and three hours after ovipostion,
F 1 eggs (pe+ jpe) were cooled at -lODC for 24 hours and then incubated at
25°C. Six hundred and five egg-color mosaics having yellow (pe) andblack (pe+) serosa cells out of approximately 10,000 eggs tested weredetected. Among the mosaics eggs 41 hatched as larvae. For the convenience of experiments, the larvae were separated randomly into threegroups: 22 larvae were reared to the 5th instar 8 days and they were killed
in 80 % alcohol and dissected to observe the color of ganglia under an
ordinary binocular microscope. The others were furthter incubated toeither pupal or adult stage. Although the ganglion in larvae was clearlyvisible, in both pupal and adult stages the color was very faint so it could
not be clearly distinguished whether the color of ganglia is redish-black orwhite. The result of the observations on the ganglion of the 5th instar
larvae has been summarized in Table 1. As can be seen in the table,
eleven egg-color mosaics have had wild-type redish-black (pe+) ganglia.
The other 11 larvae have had mosaic colored ganglia, three of them showedmosaic ganglia as one half was redish-black (pe+) and the another half
was white (pe) in one side of a pair of the ganglion: two individuals had
mosaicism in the right side ganglion and one in the left side one. It
1)

Department of Molecular Biology, Keio University School of Medicine, Shinjukuku, Tokyo.

51

RESEARCHES CARRIED OUT IN 1977
Table 1.
Individual
1-11
12,13
14-16
17
18
19
20
21
22

B

Mosaicism of ganglia in the 5th instar larva

Sg t1

t2

t3

a1

a2 a3

a4 a5 a6 a7 a8 No.ofJarvae+ + + .,, + + + + + + + + +ill+ + + + + + + + + + + +

m

m

ill

+ + m

ill

ill

ill

m

+ +

m
m

m
m

+ ill

m

m

ill

ill

m

m

ill

m

ill

ill

m
m
m
m

m

m

ill

ill

ill

m

m
m

ill

ill

m

ill

ill

ill

N

N

+ + + + + + +ill+ + + ., + ++ ill + + + + + +ill
m ill m ill + + +

+
+
+
+

m

I

ill

m

m

m

m

ill

11
2
3
1
1
1
1
1
1

Abbreviations: B, brain; Sg, subesophagalganglion; t, thorax; a, abdomen; +, redishblack (pe+); -, white (pe); m, mosaics having redish-black and whiteparts in one of a pair of the ganglion.

was also observed that about 80 % of ganglionmosaics appeared on only
one side of a pair of the ganglia.
In summary, approximately 50 % of the egg-color mosaics detected were
The ganglion-color mosaics appearedalso ganglion-color mosaics.
randomly for each ganglion, while mosaics having for both side of a pairof ganglia were not found.
Restriction of Differentiation of Pluripotent Teratocarcinoma
Cells in Some Allogeneic Strains of Mice
Takehiko NOGUCHI

Although antigenic status of the early mammalian embryos is not atpresent well difined, some antigens have been proved to appear at specificstages of development. Searl et al. (1974 Transplantation 18: 136) reported
that development of blastocysts was inhibited if they were transplanted
into host mice differing from blastocyst donors either at combined H-2
and non H-2 loci, or at non H-2 loci alone. These facts imply that immunological barriers may be useful for analysis of early mammalian development. But collecting normal embryos in large numbers is not easy.
In order to overcome this difficulty embryoid bodies, the ascitic form ofmurine teratocarcinoma, were used as the substitutes for normal early
embryos because they are more easy to obtain in large numbers, andbecause early phase of their differentiation mimics that of the normal

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

embryos.
In order to see whether embryoid bodies are inhibited in their development in allogeneic hosts as in normal blastocysts, embryoid bodies of apluripotent teratocarcinoma cell line originated from a graft of 6 day
embryo of l29jSv-SICP strain were transplanted into the peritoneal cavity
of some allogeneic hosts. These hosts were BlOjSn, BlO.l29(6M)jSn and
BlOAjSgSn. All these mice share the same set of non H-2 histocompatibility antigen loci which are different from those of l29jSv-SICP.
When about 5 X 104 embryoid bodies were injected into BlOjSn males
whose H-2 haplotype is H-2 b , a little different from that of 129jSv-S1CP(H_2 bC), they did proliferate and formed solid tumors. About two months
were required for killing hosts. It was roughly twice as long as in thesyngeneic males. Embryoid bodies looked normal as far as light microscopic observation was concerned. Solid tumors were found in the similarsites as in syngeneic mice, mainly on the adipose tissues adherent to thepancreas, the mesentery and the testis. Histological examination of thesesolid tumors revealed that they were composed mainly of cells resemblingto embryonal carcinoma cells or to embryonic ectodermal cells. At a low
frequency rosette like structures of ectoderm-like cells could be observed.
Many dying cells were present in between the embryonic cells. Welldifferentiated tissues were rarely found. Embryoid bodies were alive andcapable of proliferating even after 3 successive transplantations into BlOjSn
males at intervals of about 50 days. If they were back-transplanted intosyngeneic 129 males at every end of the transplantations, they formed solid
tumors containing various kind of tissues.
When embryoid bodies were transplanted to BlO.129(6M)jSn males
which ha' e the same H-2 haplotype as 129 males (H_2 bC ), similar results
were obtained as in BlOjSn mice. But the rosette structures of ectodermlike cells 0 cured a little more frequent in this strain.
When embryoid bodies were injected into BlOAjSgSn males which havemore desparate H-2 haplotype(H-2 a ) from 129 than BlOjSn, abdominal
swelling due to increase in ascites fluid did not occured even 2 months after
the injection. Embryoid bodies were found as whitish materials in between
the viscera. Solid tumors remained very small. Many of them were under
rejection. Embryoid bodies which attached to the adipose tissues werealso being rejected.
Important points suggested by this experiment are (1) embryoid bodies

RESEARCHES CARRIED OUT IN 1977

53

may survive and multiply for a long period of time in certain allogeneic
mice, (2) differentiation of embryoid bodies may be restricted in allogeneic
mice, (3) the extent of the restriction may depend on differences of the hosts
from teratocarcinoma cells at H-2 as well as non H-2 histocompatibility
antigen loci.
Introduction of t w5 and t wl8 Tailless Mutations into LT/Sv Inbred
Mice with a High Incidence of Ovarian Teratomas
Takehiko NOGUCHI

Recessive lethal tailless mutations cause death of homozygous embryos atcharacteristic stages. If teratocarcinoma cell lines with these mutations
can be obtained, they may be useful materials for the analysis of early
development of mice.
Ovarian teratomas of LTISv mice derive from parthenogenetically activated oocytes. Most of the ovarian teratoma cells are known to be diploid.
Recent study of isozymes of teratomas obtained from a recombinant
inbred strain mice produced from LT/Sv and C57BL/6J revealed thatoocytes giving rise to teratomas had completed the first meiotic division(Eppig, J. et al. 1977, Nature 269: 517). If LT females are heterozygous
for a t allele, the genotype of their ovarian teratomas or teratocarcinomas
are expected to be +1+ or tit. Teratocarcinomas with tit may, therefore,
be obtained from +It LT females if the tit parthenonts survive upto thestage when the mother cells of the embryonal teratocarcinomas appear.
At present the mother cells are believed to be the totipotent embryonal
ectoderm cells of the early egg cylinder stage embryos.
In the present experiment tWO and t W18 were chosen. Embryos homozygous for tWO and for t W18 are known to show abnormalities at mid egg
cylinder stage and at primitive streak stage respectively. Developmental
potentiality of the hypothetical tWDlt WD teratocarcinomas would be null.
The hypothetical tW181tW18 teratocarcinomas would contain tissues derived
from ectoderm and endoderm because tW181tW18 embryos are known to die
due to the inability to form mesoderm.
LT/Sv females were mated with Tlt WD and Tlt W18 males. The offsprings
with normal tails (+10 were chosen, and backcrossed to LT females whichshow 50 % incidence of ovarian teratomas. In subsequent backcrossing thepresence of t was checked by intercrossing the offsprings to TI + mice.

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Until now +/t W5 offsprings at 5th backcross generation (B 5 ) and +/rV18
at B2 were obtained. Seventeen +/t W5 females at B4 and at B5 werecollected and autopsied at the age of 3 months. Three had benign teratomas. This low incidence may be due to the inability of the t W5 /t W5 parthenonts to develop. No teratocarcinomas was obtained yet.
Mice earring t W5 , t W18 and T were kindly gifted by Dr. K. Yanagisawa in
the Mitsubishi-Kasei Institute of Life Sciences.
Abnormality in Primordial Germ Cell Proliferation in 129/ter-Sv
Inbred Mice with a High Incidence of Testicular Teratomas
Takehiko NOGUCHIIn 129/terSv mice about 30 % of the males develop congenital testicular
teratomas (Stevens, L. C. 1973, J. Natl. Cancer lnst. 50: 235). Thesetumors derive from primordial germ cells. Proliferative pattern of PGC
in the fetal testes of this inbred strain was studied in comparison withthose of 129 substrains with much lower incidences of the tumor.
Total number of dividing POC estimated with hematoxiline-eosin stained
serial sections of the fetal testes were ploted along the developmental stages
from 12 days to 16 days of gestation. In substrains with tumor incidenceof less than 1 % (low strains) it made a curve with a peak at 13 days andwith a foot reaching to zero level at 15 to 16 days of gestation. However
the pattern of 129/terSv differentiated from that of the low strains. First
at the peak region many plots of total counts of dividing POCs scattered
below the peak of the low strains. Most of the tel' testes showed total
counts level of about 2/3 of that of the peak. This reduced numbers ofdividing POCs is attributable to the reduced numbers of total POCs
in tel' testes. About 1/5 of tel' testes showed count levels of only 1/3 to
1/5 of that of the peak. These testes had so much reduced number of
total POCs that they looked like testes of embryos earring homozygous
steel mutation which cause severe depletion of PGCs.
Second abnormal point was that the timing of entering in mitoticallyquiescent stage of PGC was shifted to older stage. Thus many plots
scattered above the foot of the low strains. This phenomenon seems to
be well corelated with teratocarcinogenesis in tel' testes. Testes withabnormal numbers of dividing PGCs at 16 days had about 8 times as muchtendency to have teratoma foci as testes with normal numbers. Further

RESEARCHES CARRIED OUT IN 197755

16 day testes with teratoma foci were found to have smaller total seminiferous tubule volume than testes without teratomas. As the seminiferous
tubules at this stage are filled with PGCs, smaller tubule volume indicatessmaller total PGC numbers. Thus the partial depletion of PGCs may
also be linked to teratocarcinogenesis.
When genital ridges from male 129 embryos are transplanted into adulttestes, they develop into testes and about 80 % of them have teratomas.
Histological examination of these grafts indicated that marked reduction of
pGC in the developing seminiferous tubules occured due to the death of
pGCs. Mitotically active stage of PGC were protracted remarkably.
Partial depletion and protraction of the mitotically active stage of PGC
in the developing seminiferous tubules may, therefore, have relevance toteratocarcinogenesis in the testes of 129 mice.
This work was started in the Jackson Laboratory (Maine, U.S.A.) in
1975 under the sponcership of Dr. L. C. Stevens, and was continued in thisInstitute.

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28v.

CYTOGENETICS

Does the Potential of Unscheduled DNA Synthesis of Mammals
Correlate with Their Life-Span?
Batao KATO

Hart and Setlow (PNAS 71: 2169, 1974) reported that abilities of diploidfibroblasts derived from seven species of mammalian species to performunscheduled DNA synthesis (a measure of excision-repair) after UV-irradia.
tion were correlated well with the life-span of the speices. Based on anassumption that UV is a good model insult which mimmics the normallyoccurring DNA damage, they have postulated that the potential for repair
of DNA damage would be one of crucial factors determining life-span of
mammals. However, several basic questions seem to remain unanswered.
Firstly, the number of species studied may be too small to generalize theirresults to Mammalia as a whole, which contains nearly 5,000 species.
Secondly, although they regarded that almost all mammalian diploid cells
have similar DNA contents, recent studies indicate clearly that this is notcorrect. The ability of unscheduled DNA synthesis is assessed by the
number of silver grains per non-S nucleus in autoradiographs but not perunit amount of DNA, hence neglection of the genome-size difference
among species may cause a serious bias concerning the true repair potential.
These questions prompted me to re-investigate the ability of unscheduled
DNA synthesis following UV-irradiation of 34 mammalian species inreference to their life-span and genome size.
Results are shown in Fig. 1. Cytophotometrical measurement of the
DNA content of cultured cells revealed that the genome sizes of mammals
here studied varied from 58 % to 141 % of that of human. Refering to theseresults, observed values for unscheduled DNA synthesis were corrected.
As shown in this figure, the mojority of species did not show any
correlation between the repair potential and their life-span. A typicalexample that contradicted to the reported correlation was the cases ofPrimates. The maximum life-spans of the tree shrew, the crab-eatingmonkey and man are 5 yr, 15.5 yr and 110 yr, respectively, but all of themshowed the same degree of repair potentials. Another good example wasfound in chiropteran species. The reported maximum life-spans of bats

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28v.

CYTOGENETICS

Does the Potential of Unscheduled DNA Synthesis of Mammals
Correlate with Their Life-Span?
Hatao KATO

Hart and Setlow (PNAS 71: 2169, 1974) reported that abilities of diploidfibroblasts derived from seven species of mammalian species to performunscheduled DNA synthesis (a measure of excision-repair) after UV-irradiation were correlated well with the life-span of the speices. Based on anassumption that UV is a good model insult which mimmics the normallyoccurring DNA damage, they have postulated that the potential for repair
of DNA damage would be one of crucial factors determining life-span of
mammals. However, several basic questions seem to remain unanswered.
Firstly, the number of species studied may be too small to generalize theirresults to lvlammalia as a whole, which contains nearly 5,000 species.
Secondly, although they regarded that almost all mammalian diploid cells
have similar DNA contents, recent studies indicate clearly that this is notcorrect. The ability of unscheduled DNA synthesis is assessed by the
number of silver grains per non-S nucleus in autoradiographs but not perunit amount of DNA, hence neglection of the genome-size difference
among species may cause a serious bias concerning the true repair potential.
These questions prompted me to re-investigate the ability of unscheduled
DNA synthesis following UV-irradiation of 34 mammalian species inreference to their life-span and genome size.
Results are shown in Fig. 1. Cytophotometrical measurement of the
DNA content of cultured cells revealed that the genome sizes of mammals
here studied varied from 58 ~~ to 141 %of that of human. Refering to theseresults, observed values for unscheduled DNA synthesis were corrected.
As shown in this figure, the mojority of species did not show any
correlation between the repair potential and their life-span. A typicalexample that contradicted to the reported correlation was the cases ofPrimates. The maximum life-spans of the tree shrew, the crab-eatingmonkey and man are 5 yr, 15.5 yr and 110 yr, respectively, but all of themshowed the same degree of repair potentials. Another good example wasfound in chiropteran species. The reported maximum life-spans of bats

57

RESEARCHES CARRIED OUT IN 1977

/

I

'"

/

/
/
I

/
/

-

/

,

•

/

o

/

•

/

o

•

/

/

x

/

.,

()

/
/

/

•

;/

..

()

/
/

x ()

/

/

()

()/

()

()

/

e

()

;
/

/

()

/

;

e

e

e

1 '----'-_--L-_.L---L_--L-_-'----L_-'--_-'----J._-'

60()[J[;;,,,/r, ti.c i,' (: Dr """,,,,)

Fig. 1. Relation of repair potential of life-span in 34 species of mammalian
species. The symbols denote groups of species as follows: x, Marsupialia; e, Insectivora; e, Chiroptera; ®, Primates; 0, Edentata; EB, Lagomorpha; (), Rodentia;
6, Cetacea; D, Carnivora; '7, Perissodactyla; T, Artiodactyla. A broken line
indicates a presumptive correlation between life-span and the ability of unscheduled
DNA synthesis.

range from 15 yr to 24 yr. The abilities of their fibroblasts to performunscheduled DNA synthesis varied from 5 to 40 ~{ of that of human and thiswas almost the same as that of insectivores that are known to be the mostshort-lived mammals (1.5 yr). The repair potential of the giant anteater(Edentata) was also found to be about 20% of that of human, whereas its
life-span is reported to be 24.5 yr. It is of interest to note that all of theseanimals are phylogenetically related. Among rodents, repair potentials
varied greatly independent upon their life-spans but tended to be similar
within a Family.
In the present study, five (shrew, mouse, rat, golden hamster and man)our of seven animals (the former plus cow and elephant) utilized by Hart

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

and Setlow were examined. Interestingly enough, uncorrected values fortheir repair potentials could be plotted on or near the presumptive correlation line. The values for two artiodactyls other than the cow were also
distributed near this line. These findings seem to suggest that the difference
in experimental systems between the present and the previous reports as
described in the legend to figure does not largely affect the results ofassessment of repair potentials, and may imply that the excellent correlation between the extent of unscheduled DNA synthesis and life-span of
the species observed by the previous workers could be a spurious one.
Repair potentials were determined by using diploid fibroblasts mostly
derived from lung tissues of adult animals whose exact ages were unknown.
Cultures were used for experiments at their earlier passages. For some ofanimals, cultures were established from both lung and epidermis. The
extents of unscheduled DNA synthesis in the skin and the lung cultures
were found to be comparable. To detect unscheduled DNA synthesis,
exponentially growing cells on coverslips were treated with 0.1 mM FUdR
for 1 h, washed with buffered saline and exposed to UV light (254 nm) at afluence of 10 Jm- 2 • Cells were then incubated for 4 h in medium containing 5,uCi/ml 3H-thymidine (spec. act. 12.6 Ci/mM) and processed for
autoradiographic preparation immediately thereafter.
Temperature Depllndent Formation of Sister Chromatid Exchange
Hatao KATO

There is evidence that sister chromatid exchange (SCE) in mitotic
chromosomes of higher organisms occurs spontaneously. In general, its
incidence in cultured mammalian cells ranges from 2 to 6 per metaphaseper cell generation at 3TC, and this frequency is at least 100 times higher
than that of spontaneous chromosomal aberrations. Its constant occurrence indicates that SCE is not a sporadic event but a phenomenon
occurring as a function of time under physiological conditions. Furthermore, it is assumed that any causes trigerring a process of SCE formation
would be intrinsic. Had the cause be intrinsic, either the occurrence of thecause per se or its biological consequence might be influenced largely bytemperature. Based on this consideration, effects of temperature on theincidence of SCE was studied by using cultured Chinese hamster cells.
Cells were subcultured into 25 cm 2 plastic culture flasks at a concentra-

RESEARCHES CARRIED OUT IN 1977

59

tion of J05 cells per flask. One day after subculturing, flasks were transferred to light-proof incubators adjusted at various temperatures. Afteran adaptation period of 24 h to each temperature 0.25 ,ug/ml bromodeoxyuridine was added to cultures, which were then grown for two roundsof cell cycle before fixation for chromosome preparation.
The incidence of SCE in these cultures varied greatly dependent on the
incubation temperature. The SCE frequency at 3\ 'c was 2.6 per cell,while it increased gradually with increasing temperature to reach 13.9 percell at 42°C. The frequency of cells having chromosomal aberrations suchas chromatid gaps and breaks was found to be 2 ~~ at temperature between
31°C and 39°C but it increased abruptly at 40'C and above. At 42°C all
metaphases were found to possess aberrant chromosomes.
The temperature dependence of the SCE formation is shown in the formof an Arrhenius plot in Fig. 1. The SCE frequency at each temperature
could be plotted nicely on a regression line which showed a break at 39°C.
The presence of this break seems to imply that the SCE formation cannot
be achieved by a single chemical process and that at least one of processes
might function to enhance SCE formation at temperature higher than 39°C.
Analyses of cell kinetics of cultures grown at various temperaturesindicated that the length of the DNA synthesic period (S) was shortened
gradually as the incubation temperature was raised from 31°C to 39"C.
However, above 39°C, it was elongated remarkably. It is very likely thatthe elongation of S is due to a disturbance in the process of DNA
replication, and that the disturbance is responsible for the promotion of
SCE formation.
In order to detect a cellular phas:: at which the temperature treatment
was most effective in enhancing SCE formation, cultures were synchronizedby the mitosis selection method at 37°C and exposed to 41 "C for various
time lengths during the first cell cycle. Results indicated that a signific<lI1t
increase in the SCE frequency was detectable only when the high temperature treatment was given during S phase. This finding also indicatesthat DNA replication is intimately related to the mechanism for spontaneous SeE.
The temperature dependent increase of the SCE frequency clearly showsthat the effect of temperature was not only to speed up an SCE formation
process but also to increase the number or amount of the intrinsic causesthat trigger the SCE process. Furthermore, the break in the regression line

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

15 ~
10 ~

3.15

3.20

3.25

3.30

Fig. 1. Arrhenius plot of frequencies of SCE in Chinese hamster cells.

in the Arrhenius plot at 39°C suggests strongly that a disturbance in the
DNA replication process at temperature higher than 39°C might exert an
additive effect on SCE formation.
A Simple Technique for Observation of Sister Chromatid Exchange
In Vivo
Naotoshi KANDA and Hatao KATO

In vivo observation of sister chromatid exchange by the BUdR-labeIing
method is hampered greatly by a rapid degradation of bromodeoxyuridine(BUdR) within the body of the host animal. To solve this problem,
BUdR is usually administered to the host either by repeated intraperitoneal
injections at an hourly interval for a period covering a whole cell cycle orby continuous infusion through the tail vein for one or two rounds of cell
cycle using a special pumping device.
In an attempt to avoid these laborious procedures, we have developed asimplified technique that consisted of a single intraperitoneal injection of
BUdR absorbed on activated charcoal and assured continuous supply of
BUd R to target cells throughout two rounds of replication cycle. In
brief, BUdR aqueous solutions were first prepared at various concentra-

RESEARCHES CARRIED OUT IN 1977

61

tions, and to these solutions, neutralized and sterilized activated charcoal
was added at a concentration of 100 mgjml. 1 ml or 0.5 ml of the BUdRactivated charcoal suspension was injected to each animal.
This technique was applied to observation of sister chromatid exchange
in tumor cells proleferating in the peritoneal cavity of the mouse and tomouse spermatogonial cells. In both types of cells, the minimum dose of
BUdR required for sister chromatid differentiation was found to be
5 mgjbody (6-8 week-old C3H mouse), and average exchange frequencies
were 4.5jcell in tumor cells and 1.8jcell in spermatogonia. The latter value
was identical to those obtained by using other techniques for the mouse
spermatogonia. Because of its simplicity, this technique seems to be usefulto study effects of chemical mutagens on germ-line cells as well as to study
meiotic processes in various animals.
Some Genetical Aspects on Supernumerary Chromosomesin the Black Rats
Tosihide H. YOSIDA

Presence of supernumerary of B-chromosomes has been described by
Gropp et af. (1971), Young and Dhaliwal (1972), Raman and Sharma(1974) and Yosida (1977) in the Asian type black rats with 2n=42.
According to these authors the number of supernumeraries varied from
one to four, and their shapes were usually metacentrics which weremorphologically indistinguishable from the regular metacentric autosomes.
Supernumeraries with the same morphology as found in the Asian typerat were also observed in the Ceylonese (2n=40) and the Oceanian typeblack rats (2n=38) (Yosida 1977). Genetic surveys of supernumeraries inmammalian species are rare, and no report on the genetics of supernumeraries
in the black rat.
The black rats used in the present study were originally obtained fromThailand and were bred for two years in this laboratory. Supernumeraries
were often observed in the offspring of these rats. To know the mode ofinheritance of the supernumerary chromosomes, parents with one supernumerary were mated and 37 offspring from 7 litters were obtained. Amongthem 31 rats had from 1 to 3 supernumeraries; 11 rats had 1 supernumerary, 14 had 2 and remaining 6 had 3 supernumeraries. From themating of a female with 2 supernumeraries and a male lacking a super-

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

numerary, 17 offspring from 5 litters were obtained. Among these rats,
13 had from 1 to 4 supernumeraries. In the third case 12 offspring (3litters) were obtained from a mating between a female with three supernumeraries and a male without the supernumerary. Among them 10 rats
had from I to 6 supernumeraries.
According to Raman and Sharma (1975) the supernumeraries in the black
rat do not show conjugation in the meiotic-I. From the above data it issuggested that the supernumerary would be moved to either one pole or theother without division in meiotic-I and that the non-disjunction of thesupernumeraries would occur at random in the meiotic-II phase. Thus thenumber of supernumeraries included in the offspring is higher as shown by
the above matings. Generally speaking, the number of rats with few
supernumeraries was considerably larger than the expected one, while thenumber of the rats without the supernumeraries was smaller. From theseresults it can be said that black rats with only a few supernumeraries havea selective advantage over those without supernumeraries.
Some Genetical Aspects of C-band Polymorphism in the Black Rats
Tosihide H. YOSIDA and Yuriko OCHIAI

A remarkable polymorphic variation of the heterochromatic C-bands in
the Japanese black rat (Rattlls ratfus tanezumi) has been reported by
Yosida and Sagai (Chromosoma 50, 283-300, 1975). The present study
deals with some genetic investigations in the C-band polymorphism of theseanimals bred in the laboratory. In 62 black rats examined, chromosome
pairs no. I, 3, 7, 8, lO, II and 13 were polymorphic with regard to thepresence or absence of the band. In pair no. I, 45.2 per cent showed
C-bands in both members of the pairs, 40.3 per cent were heteromorphic
clue to the presence or absence of the band, and in the remaining 14.5 percent C-bands were absent in both members of the pair. Pair no. I in theJapanese black rat was polymorphic for acrocentrics and subtelocentrics asreported previously. The C-band in pair no. 1 was always observed inacrocentric chromosomes.
/\. mode of inheritance of the C-band patterns from the parents to theoffsprings was examined in 24 rats (5 litters) derived from 5 different set ofparents. In a total of 288 chromosomes observed in the 24 offspring the
C-bancl positive and negative types were segregated 148: 140. This value

RESEARCHES CARRIED OUT IN 1977

63

closely corresponds to the expected one of 150.0: 137.5, and indicates that
the C-band polymorphism of the black rat is exactly transferred to the offspring by the Mendelian segregation ratio.
For the development of the large metacentric chromosome in the
Chichijima black rats, the following two explanations are possible: I)
Robertsonian fusion between pairs no. 11 and 12 independently occurredfrom the Ceylonese and the Oceanian type rats with large metacentrics byfusion of these chromosomes. 2) Ceylonese or Oceanian type black rats
migrated to this island resulting in hybridization with the Asian type rat.
A conclusive statement on the origin of the metacentrics however, will
require further studies using a larger number of specimens.
Robertsonian Fusion of Acrocentric Pairs no. 11 and 12 in Black
Rats Obtained from Chichijima Island, Japan
Tosihide H. YOSIDA

Three specimens of the black rat, Rattus rattus, were obtained from
Chichijima, one of the Ogasawara islands of Japan by courtesy of Mr.
Mitsuru Minezawa of the Institute of Primates, Kyoto University. Amongthem, one male (a) had 42 chromosomes, the typical Asian type idiogram.
Two others, one female (b) and one male (c) were found to have 41
chromosomes. Their karyotypes contained one large metacentric chromosome. All 10 cells from the band c showed the same metacentrics.
Karyotype analysis suggested that Robertsonian fusion between membersof pairs no. 11 and 12 resulted in the formation of the metacentrics.
Unpaired acrocentrics 11 and 12 seemed to be homologous to the long andshort arms of the metacentrics. G-band analysis clearly showed that thelarge metacentric chromosome consisted of the Robertsonian fusion ofchromosomes 11 and 12.
As previously reported by the present author, chromosome pairs no. I,
9 and 13 in the Asian type black rats are polymorphic with regard to theacrocentrics and subtelocentrics. The three Chichijima black rats also
showed a polymorphism of a similar nature for these chromosomes. Yosida
and Sagai (1975, Chromosoma, 50: 283-300) demonstrated that the Japanesebalck rat (R. rattus tanezumi) was distinguishable by having C-band polymorphism for the presence or absence of these bands. The present
materials were also polymorphic by showing C-bands of a type similar to

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

other fanezumi rats.
Frequent Occurrence of Sex Chromosome Anomalies in F 2 Hybrids
between Geographical Variants of the Black Rats
Tosihide H. YOSIDA

By their karyological characteristics, black rats (Rattus rattus) may beclassified into three geographical types, Asian (2n=42), Ceylonese (2n=40),
and Oceanian (2n=38). In any combination of these three types, F 1
hybrids are easily produced, but they seem to be semisterile, except for
those between Ceylonese and Oceanian types. Among the F 2 hybrids
between the Ceylonese and Oceanian types, rats with sex chromosome
anomalies have often observed. When the present author (Yosida 1977,
Cytogenet. Cell Genet. 19: 262-272) first observed the XO female in theF 2 hybrids, the occurrence of this abnormality was assumed to be sporadic.
In further observations another XO female and an XXY male were foundin the same hybrids. The sex chromosomal abnormalities as seen in the F 2
hybrids were never observed in non-hybrid offspring in Ceylonese andOceanian type black rats, so far as the observations were taken on our
breeding colonies.
All F 1 hybrids obtained by mating Oceanian and Ceylonese type blackrats invariably possessed 39 chromosomes. By mating the F 1 hybrids 22
F 2 offspring (11 females and 11 males) were produced. Among them 6 (2females and 4 males) were Oceanian type, 13 (8 females and 5 males) were
the F 1 type, and the remaining 3 (1 female and 2 males) were of the
Ceylonese type. The segregation ratio of these three types was very
similar to the theoretical one (5.5: 11.0: 5.5). Among the F 2 offspring
both females with the Oceanian type showed the same XO in the sex
chromosome complement. The one male with Ceylonese karyotype wasremarkable by having the XXY. The other females and males had thenormal sex chromosome pattern, XX and XY. Non-disjunction of sex
chromosomes in gametegenesis of the F hybrids may be the cause of thehigh occurrence of the sex chromosome anomalies in F 2 offspring.
j

RESEARCHES CARRIED OUT IN 1977

65

Artificial Insemination between Rattus norveg;clIs,
R. annandale; and R. losea
Tosihide H. YOSIDA and Choji TAYA

The results of artificial insemination between the female Norway rat(Rattus norvegicus) and the male black rat (Rattus rattus) have been previously reported. The results were that among 20 rats, in which theinsemination was successfully achieved, 9 rats (45 %) were successfully
pregnant (Yosida and Taya 1977, Jap. J. Genet. 52: 289-299). The
average number of uterine implanted eggs per rat was 2± 1.2. However,
all embryos always degenerated within two weeks after insemination.
The present study was undertaken to determine the success rate ofartificial insemination between the Norway rat and two other related
species, R. annandalei and R. losea. Some spercimens of Rattus annandalei were originally collected in Malaysia in 1972, and a colony has been
maintained in this institute for about six years. The chromosome numberof this species is 42 and the karyotype is similar to the black rat. Spermatozoa of R. annandalei were inseminated into the female Norway rats
by the same technique as that used for the baIck rat. Among 28 rats
which were successfully inseminated, only 5 rats (18 %) became pregnant.
An average of 1.7±1.2 eggs were implanted in the uterus of the pregnant
rats. The embryos usually perished within two weeks after insemination.
Based on the above investigations it is clearly shown that the pregrant ratio
by artificial insemination between the Norway rat and annandalei was lower
that between the Norway rat and the black rat. This means that theevolutionary distance between the Norway rat and annandalei is greaterthan that between the Norway rat and black rat.
Some of male and female Rattus losea were obtained from Thailand in
1976, and they were bred in the laboratory for two years. This species
also had 2n=42 and a similar karyotype to the Asian black rat. Twenty
four Norway rats were successfully inseminated with the spermatozoa of
R. losea. In this fertilization, no pregnant females were obtained. Inorder to determine whether or not the egg of the Norway rat was successfully fertilized by the spermatozoa of R. losea, 6 rats were dissected on the
5th day after insemination, and their eggs were examined. In all rats, 3 to
6 eggs were obtained and among them fertilized eggs were found in 5 rats.
However, they were only cell masses, whereas in the control a normal

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28
R. annandalei < - - - - > R. norvegicl/s < - - - - > R. losea

1

R. rattl/s

Fig. 1. An illustration of the evolutionary positions between the Norway rat(Rattl/s liorvegicl/s) and the other Rattl/s species (R. rattl/s. R. annandalei and R.
losea) as suggested by embryonic development after artificial insemination.

blastocyst was present by this time. These eggs, therefore, would not be
implanted in the uterus of the Norway rat. This experiment shows thatthe evolutionary relationship between norvegicus and losea is more distant
than that between nonegicus and annandalei. Based on the results of theartificial insemination between the Norway rat (R. norvegicus) and theother 3 species (R. rattus, R. annandalei and R. losea), the evolutionary
positions seem to be as illustrated in Figure 1.
Candidates for a New Experimental Animal, II.
The Indian Spiny Mouse, Mus platythrix.
Tosihide H. YOSIDA

Indian spiny mice, Mus platythrix, were originally collected in Mysor,
India in 1972, and have been successfully bred in this laboratory by brotherand sister matings for about 8 to 10 generations by our standard mouse
breeding system. The size of this animal is slightly larger than the house
mouse. The mean body weight of 19 females and 13 males, fully grown is36.1 g and 43.0 g, respectively. Laboratory breeding of this animal issimple and the litter size is 5.4. The gestation period averages 21 ± 1 days,
and the eyes open 13 to 15 days after birth.
The number of chromosomes in this species has been previously reported(Tsuchiya and Yosida, this Annual Report, 22: 51-52, 1971). The diploid
chromosome number is 26 and all autosome pairs and X and Y chromosomes are acrocentrics. Based on comparisons of chromosome G-bands
and chromosome lengths between the spiny mouse and the house mouse(Mus musculus), it was suggested that 8 autosome pairs (no. 1 to 8) in theformer species had probably occurred from the tandem fusion of several
combinations of two acrocentric pairs in the latter species. Pairs 9 to 12
and X and Y in the spiny mouse seemed to correspond to some autsomal
pairs and to the X and Y of the house mouse.

RESEARCHES CARRIED OUT IN 1977

67

By having fewer and larger chromosomes, the Indian spiny mouse may
prove to be more useful than the house mouse for some types of biomedical
research such as cytogenetic surveys.
Difference of Nucleolar Organizer Regions in theBlack and Norway Rats
Tosihide H. YOSIDA

Two species of genus Rattus, the black rat, R. rattus, and the Norway rat,
R. norvegicus, are characterized by the same chromosome number (2n=42)
and a similar karyotype. Based on comparative karyotype analysis ofthese two species, the present author (Yosida 1973, Chromosoma, 40: 285297) postulated that the Norway rat may have differentiated from one typeof polymorphic karyotype which occurs in the Asian type black rat. In
the black rat there are three geographical variants having 2n=42, 40 and
38 (Yosida et al. 1974, Chromosoma, 45: 99-109). Although the karyotypes are markedly different from each other, hybridization easily occurs
between these three types. Hybrids between the black and the Norway
rat, however, have never been obtained, even though artificial insemination
was employed (Yosida and Taya 1977, Jap. J. Genet. 52: 289-299).
Nucleolar organizers are known to be important structure of chromosomes in relation to the formation of the nucleolus as a site of ribosome
RNA synthesis. From a comparative study of the nucleolar organizer
regions of chromosomes of the black rat and the Norway rat, some evidence
indicating an evolutionary relation between these species may be suggested.
Observations show that both species have the silver stained nucleolarorganizer regions in three chromosome pairs. In the black rat the regions
are found in pairs no. 3, 8 and 13, although there are some polymorphic
changes in their occurrence. The nucleolar organizer regions are found inthese three chromosome pairs in all three geographic types of black rats.
In the Norway rats the nucleolar organizer regions were observed inchromosome pairs 3 and 13. This is similar to the pattern present in theblack rat. However, the organizer region is not present in pair no. 8 in
the Norway rat, but is observed in pair no. 12 (chromosome pairs no. 12
and 13 in the Norway rat described here correspond to pairs no. 11 and 12
in the Standard Karyotype System). This investigation indicates that thechange of the nucleolar organizer regions may by important in species

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

differentiation.
Cytogenetical Identification of tbe X-Cbromosome in Bomhyx Mor;
Akio MURAKAMI, Akio OHNUMA and H. T. IMAIThe karyotype in the silkworm Bombyx mori is n=28 with male homogamety (XX or ZZ) and female heterogamety (XY or ZW). The chromosomes are holocentric and small in size and do not show marked individual
variations. With recent developments in chromosome observation techniques which give better resolution, it is possible to observe greate cytological details of the chromosome during mitotic as well as meiotic division
stages in this insect (Murakami and Imai, 1974). However, we could not
distinguish morphological differences between sex-chromosomes and autosomes. But, we have been able to recognize either bivalent or trivalentchromosome due to a differential thickness of the chromosome pairs
and/or a differential chromosome pairing figure. Namely, two members(bivalent) of the trivalents are connected tightly along the entire length,
while one remained single (univalent) and is often loosely coiled.
Based on these observations, we have proposed a hypothesis that when ahypo-triploid male such as a 3A+XX is obtained, it would be possible toidentify morphologically the X-chromosome (as well as Y-chromosome)from autosomes. The following mating scheme has been devised to obtain
the hypotriploid male. Females having a Y-chromosome translocated withpe+ gene which is located on the 5th linkage group, T(Y: 5), and the 5thchromosome homozygous for pe (yellowish-white egg, 0.0) are crossed withtester males homozygous for sch (sex-linked chocolate, X-21.5) and pe.
The resultant F 1 hybrid females are treated with CO 2 gas at egg stages from(r p,+:

y_p.+~)

60 to 120 min after oviposition to induce tetraploid females,

'fk +

X~

~.

In this mating, it is expected that several other genotype of F 1 hybrids suchas diploid, triploid silkworms and so on would appear. The female moths
developed from the tetraploid eggs are backcrossed to the tester male
moths. In this cross, various classes of BF1 offspring can be detected:
females hatched from wild-type (black) eggs having a chromosome constitution either 3A+XXYY or 3A+XXY and hypo-triploid males derived

RESEARCHES CARRIED OUT IN 1977

69

from yellowish-white eggs having a chromosome constitution 3A+XX.
Males in the latter case would arise due to non-disjunction of sex-chromosomes. This 3A+ XX individual is the just expected hypo-triploid male.
Testes of this 3A+XX line male larvae of either 3rd or 5th instar wereused for chromosome observations. The improved air-drying techniquedeveloped by Imai et al. (1977) was adopted for chromosome observations.
Chromosome number of spermatogonial cells were analyzed for eightindividuals. The number of chromosomes in 71 metaphase stages collectedfrom 8 hypo-triploidal individuals was found to be 83 (3n-1). Theoretically assuming that all the autosomes in this hypo-triploid male shouldmake trivalents, while the X-chromosomes form bivalents and that chromosome condensation between trivalents and bivalents should not remarkably
differ. As a matter of course, the paired X-chromosome should be slender(2/3) in which as compared with the other trivalent autosomes (at pachytene
stage). In fact, one unusual chromosome pair was observed which was
slender while there were 27 thick chromosomes corresponding to trivalents.
This slender chromosome pair would be the bivalent of the X-chromosomes.
Chromosome size or relative percentage to the total length of the haploid
set was measured using 16 well spread pachytene stages. The result
obtained so far indicate that the X-chromosome is one of the member ofthe large chromosome group or the 3rd longer in a statistical sense.
This idea presented for the identification of the X-chromosome may bealso applied to other linkage groups in Bombyx. At present, chromosome
identification of the 5th linkage group of Bombyx is in progress.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

VI.

MUTATION AND MUTAGENESIS IN ANIMALSAn Exceptional Pattern of Mutation Spectrum Observed in the
Offspring of Mitomycin C Treated Females of a
Strain rb of the Silkworm
Yataro TAZIMAThe preponderance of mosaics among induced mutants has been known
to be a chracteristic feature of chemical mutagenesis. When mutations
were induced by ionizing radiation it was found that the number of induced
mosaics increased remarkably with the progress of spermiogenesis after
meiosis. Mutations induced by chemicals, however, manifest themselves
as mosaics even when treatment was performed before meiosis. For
instance, the proportion of mosaics is as high as 85-95 % when spermatids
had been treated with Mitomycin C.
The mutational response of female germ cells is quite different fromwhat is known for male germ cells. Although the incidence of mosaics,
after chemical treatment, exceeds whole-types, its proportion to total
mutants is usually far smaller than that for male germ cells, i.e. 60-70 %.
An exceptional case of mutation response, i.e. less mosaics than wholetypes, was discovered for rb, a strain most sensitive to ionizing radiation.
When female pupae of this strain had been injected with Mitomycin C theincidence of mosaics was less than that of whole types. Similar treatmentof other strains, Kansen, Aojuku, Sekko and ClO8 did not show such
distortion in the proportion of mosaics. Further, injection of EMS into
rb showed no distorted mutation spectrum.
In these experiments injection was performed from I to 6 days before
meiotic metaphase, when each chromosome were ready for meiotic division
after completing the formation of two chromatids. The high incidence ofwhole-type mutants could, therefore, be accounted for by either one of thefollowing alternative hypothesis (l) two helices of DNA were simultaneously
affected by Mitomycin Cor (2) one helix was affected and DNA replication
occurred once more before the completion of meiotic division. The
finding that the distortion was found in a radiation sensitive strain seems
to support the former hypothesis.

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71

Metabolic Activities of Silkworm Microsome Fraction on Some
Indirect Carcinogens with Special Regard to the Differential
Mutation Response Between Silkworm and Drosophila
Akio MU~AKAMI, Michiko GOTO and Yataro TAZIMA

Indirect carcinogens thus far most intensively investigated in the silkworm are N-dimethylnitrosamine (DMN) and N-acetylaminofluorene(AAF). To those compounds silkworm (Bombyx) and Drosophila were
found to respond differently as regard to mutation induction, i.e., 1) DMN
is clearly effective in producing recessive lethal mutations in Drosophila,
whereas it is not mutagenically active in Bombyx, 2) AAF has been reported
to be incapable of producing recessive lethal mutations in Drosophila,
although weak mutagenicity was detected by heterochromatic loci method
using bobbed. The silkworm system clearly detects a positive mutagenicityof this compound although only at a very high dose, 500 ,ugJinsect.
In order to reveal the mechanism(s) underlying the discordance, weinvestigated the metabolic activation potentiality of Bombyx microsome
fraction using the mutagenicity to Salmonella as a criterion.
Microsome fraction (9000 G supernatant) was prepared from the homogenates of Bombyx at various metamorphosic phases and the activity wasdetermined by mutagenicity of indirect carcinogens+microsome fraction to
Salmonella T98 and TlOO strains. The microsome fraction from the fifth
instar larvae was highly active on AAF, but those from pupae and eggs
were almost inactive. In contrast, the same fraction, even from fifth
instar larvae, did not activate DMN.
These results seemed to suggest that the lack of mutation response to
DMN in Bombyx could be attributed to the lack of metabolic activity on
DMN in silkworm microsome fraction. However, this assumption could
not apply to Drosophila, because Drosophila microsome was found toactivate AAF very effectively (Brewen and Nix,1978).
Accordingly, the above mentioned discrepancy in mutation response
between Bombyx and Drosophila appears to be due to mechanism(s) otherthan the metabolic activation capacity detected by Salmonella mutagenicity
testing systems.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Mutagenic Effectiveness of an Internal r-Emitter, Tritium(4) RBE of Tritium at Low Dose-Rate Range
Yataro TAZIMA, Kimiharu ONIMARU and Yosoji FUKASE

Experiments have been continued to estimate the mutagenic effectivenessof tritiated water (THO) at low dose-rate range paying special regards to
RBE. Materials and methods used for tritium experiment was almost
similar to those reported previously (this Report No. 27: 62). Tritiatedwater was injected to wild type female pupae of strain CI08 at doses of 60
and 30 ,uCi per pupa. Emerged females were mated to males of anotherwild type strain Aojuku and deposited eggs were subjected to acid treatment for artificial hatching. Average radioactivity transmitted to thedeposited egg we.e 27073.8 dpm and 14100.0 dpm, from which radiation
dose-rate absorved by germ cells were calculated to be ca. 7.28 and3.77 rad/day. The duration from injection to hatching, based on which
total irradiation dose was calculated, was 17.75 days. The calculated
absorbed doses were 129.3 and 67.3 rad respectively.
Gamma-ray irradiation was performed using special chronic irradiation
apparatus of I Ci 60Co source. Female pupae of strain CI08 were placed on
rotating trays, 165 cm and 228 cm apart from the 60CO source, at exactly
same time when THO injection was performed and they were continuously
irradiated there until emergence. Irradiation was continued even when
they were mating with males of strain Aojuku, depositing and throughout
egg stage until hatching except for a few hours' intermission for artificial
hatching. The net exposure time for r-rays was 16.6 days and radiation
dose-rates and total doses delivered (shown in parentheses) were 11.6 rad/day (193.0 rad) and 5.8 rad/day (97.3 rad).
Those treated individuals were raised and mated to partners with double
recessive markers, pe re, in order to assess the mutation frequency.
The results showed clearly linear relationship with increasing dose for
THO group but rather obscure for r-ray group. They were approximately
represented as follows:for irradiation of female
THO: Y=(1.58X+62)X 10-44
r: Y=(0.88X+62)X 10- 4
for irradiation of male
THO: Y=(2.04X+171) X 10- 4

RESEARCHES CARRIED OUT IN 1977

r: Y=(1.14X+171) x 10-\where Y denotes mutation frequency per 105 when absorbed energy X wasgiven in rad.
From these results RBE's of THO were calculated to be 1.8 for both sexes.
Experiments on the Biological Effects of Ortho-Phenylphenol
Sodium Salt for Silkworm Germ-Cells
Akio MURAKAMI, Yosoji FUKASE, Hiyorio NISHIJIMA and Michiko GOTO

Ortho-phenylphenol (OPP) has been permitted to use only as a fungicidal
agent for fruit packages and/or wrapping papers, but this agent was detected
in imported oranges in April, 1975. Consumer-organisations expressed
their concern about it use. One report gave positive DNA damaging effects
in micro-organisms (Nishioka and Ogasawara, 1977), whereas the otherreports showed no evidence for any genetical or cytogenetical effects in
microorganisms, mice and mammalian cultured cells (Ochi and Tonomura,
1977; Shirasu et al., 1976). In view of the gravity of the problem, as well
as even the remote doubt about the induction of genetic hazards by thisagent is likely to endanger the human population, a detailed investigation
was taken up to study the genetic and biological effects of this compoundin the silkworm (Bombyx mod L.).
An F I hybrid from a cross between wild-type silkworm strains CIOB andAojuku and a marker stock having double recessive egg-color genes, pe
and re, were used in all experiments. Ortho-phenylphenol sodium salt
(OPP.Na) used in this experiment was supplied by the National Institute ofHygienic Sciences as a test sample (lMS 74-20) for the Carcinogen
Screening Test Programme.
Toxicity or killing test for a single dose of OPP.Na was measured as the
reduction in survival (or adult emergence) ratio. LD 50 values at various
developmental stages of the silkworm were estimated from the doseresponse relationships. Genetic toxicity test was measured by the dominantlethal mutation and recessive visible mutation. Statistically significant
reduction in hatchability represents the occurrence of the dominant lethal
mutation. The latter type mutation was measured by the egg-color specificlocus test. Significant reduction in the average number of eggs deposited
per female moth which had been treated or untreated with the agent andmated to untreated or· treated male, respectively, indicates occurrence of74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

infecundity.
LD oo value on gram body weight basis was in the range of ca. 100 to
600 pg: the LD 50 is 230 pg in a day old larvae of the 5th instar, 100 pg in 4days old larvae of the 5th instar, 300 pg in mid-stage pupae and 600 pg inlate-stage pupae. The results obtained from the dominant lethal test in
spermatid and sperm following treatement of 5th instar day 3 male larvar
and mid-stage male pupae with OPP.Na indicated no significant increase ofthis class mutation above the spontaneous level at 2.2 to 414.3 pg/g bodyweight. Evidence for the induction of dominant lethal mutation in pupal
oocytes at the concentration tested ranging from 1.8 to 182.5 pg/g pupal
weight was not observed. This fungicide is shown to be non-mutagenic
with the specific locus test in both pupal sperm and oocytes between 2.2
and 182.5 pg per gram pupal weight. It should be noted, however, that
OPP.Na can cause high incidence of infecundity (or fecundity was reduced
to I /l 0 of the control level) for mid-stage female pupae after treatment withthis chemical at a concentration equivalent to ca. one-half of the LD 50
dose, but not for male pupae.
Changes of Radiosensitivity to the Induction of Mutationsin Primordial Germ -Cells During the Gonad Developmentof Female Silkworm
Mutsuo MIKI") and Akio MURAKAMI

The radiosensitivity to mutation induction in the female primordial germcells during the development of gonad in early stages of the silkworm
embryos has been investigated. Eggs were collected from actively laying
wild-type CI08 strain female moths crossed to the same strain of males atintervals of 30 min at 25°C. Developing embryos stages ranging from 44
to 212 hr after oviposition were irradiated at 12 or 24 hr intervals with a,constant single dose of 1000 R of 137CS gamma-rays (dose-rate at 300
R/min). Irradiated eggs were reared and crossed after emergence, to normal
males belonging to the marker stock homozygous for pe and reo The
mutational incidence was used as the criterion for checking radiosensitivity.
The incidence of mutations increased gradually with the development ofgonad in embryos from 68 to 116 hr after oviposition (or 24 hr before
blastokinesis), After blastokinesis the incidence of mutations decreased
1)

The Saitami-ken Sericultural Experiment Station, Kumagaya, Saitama-ken.

RESEARCHES CARRIED OUT IN 1977

75

markedly in the 188 hr-old embryo to the level of the 44 hr-old embryo.
The frequency of mutations in the 116 hr-old embryo showed to be thehighest and that was newly twice as high as the frequency of mutations in
the 68 hr-old embryo. The frequency of mutations again increased slightly
but significantly in the 212 hr-old embryo. According to histological observation of changes of the number of germ-cells during development ofgonad, it was observed that one division cycle in priomordial germ-cells
was required for an approximately 30 hr in embryos ranging from 68 to
128 hr after oviposition. The completion of gonad fromation was observedat the 116 hr-old embryo and blastokinesis was occurred 140 hr after
oviposition. However, after blastokinesis, the multiplication of primordial
germcells ceased temporarily until the onset of gonial divisions in older
embryos 2 days before hatching.
From these experimental results it can be concluded that changes in theradiosensitivity in priomordial germ-cells during gonad development isdependent on whether these stages of cell division or not as well as inother stage germ-cells of the silkworm.
Toxicity and Mutagenicity of Cadmium and Furylfuramide in
Drosophila melanogasterYutaka INOUE and Takao K. WATANABE

Cadmium (CdCI 2) and furylfuramide (AF-2) were fed to Drosophila
larvae throughly mixed with the standard medium of Drosophila. The
toxicity of CdCb has been detected by the concentrations of 50-75 ppm infecundity, 75-100 ppm in viability and 1-10 ppm in developmental time.
The toxicity of AF-2 has also been detected by the concentrations of
500-1,000 ppm both in fecundity and viability, and 0-200 ppm in deveopmental time. Thus, the developmental time is a most sensitive physiologicaltrait to test the toxicity of these chemicals.
The mutagenicity of these chemicals has been tested by the attached-X
method for the detection of mutations on the X chromosome. Statisticallysignificant mutations could not be found in both cadmium (50 ppm) andAF-2 (200 ppm) treatments by this method. Recessive lethal or sterile
mutations were accumulated in the second chromosome by the Cy-Pm
method for many generations. Cadmium and AF-2 again did not increase
the lethal and sterile mutation rates significantly. For details, see Japan.
J. Genetics 53, 183-189.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

VII.

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICRO-ORGANISMS AND PLANTS
Repair Enzymes for Gamma-Ray-Damaged DNA in
Bacterial and Human Cells
Tadashi INOUE, Akiko YOKOIYAMA and Tsuneo KADA

We have recently developed a new assay system for the detection of
enzymes functioning in the repair of gamma-ray-induced damages (Noguti,
T. and T. Kada 1975, Biochim. Biophys. Acta 395: 294; Inoue, T. and T.
Kada 1977, Biochim. Biophys. Acta 478: 234). In this system, the capacityof cellular extracts to enhance the priming activity of gamma-irradiatedcolicin E1 DNA for purified DNA polymerase I was measured. Using thesystem, we have successfully identified in extracts of Bacillus subtiUs cells
two kinds of enzymes (primer activating enzymes) which may function in
the repair of gamma-irradiated DNA. One of them was shown to be an
apurinic endonuclease and the other was a 'cleaning' exonuclease. Since
gamma-rays induce various types of lesion in base and sugar moieties aswell as scissions in the phosphodiester linkage, a wide variety of enzymes
which function in repair may be detectable by this assay system, provided
that the lesions are removed by excision-type repair passway in which
DNA polymerase plays a significant role.
More recently we have applied the system to detect enzymatic defect of
the human genetic disease ataxia telangiectasia which is characterized bytelangiectasia, immunodeficiency, an increased frequency of malignancy andhypersensitivity to ionizing radiation. Preliminary studies revealed thatan ataxia strain had substantially lower activity of the primer activating
enzyme than a normal strain (Inoue, T. et al. 1977, Biochim. Biophys. Acta
479: 497). Encouraged by these data, we examined nine ataxia strains
and six normal strains in collaboration with Prof. B. A. Bridges and Drs.
C. F. Arlett and A. R. Lehmann at the University of Sussex, as to theactivity of the primer activating enzyme. The preliminary experiments
yielded the results that all of the ataxia strains examined had lower
activity than normal strains confirming that some, if not all, of the causesof the disease are deficiency in the repair enzyme activity for gamma-ray-

RESEARCHES CARRIED OUT IN 1977

77

damaged DNA.
Rec-Assay with Bacillus subtilis Spores
Koichi HIRANO, Hisako MATSUMOTO and Tsuneo KADA

The rec-assay using Bacillus subtilis strains, M45 (Rec-) and H17 (Rec+)was tried with their spores on thin diffusion agar and remarkable improvements were made as to its sensitivity, quantitatively and reproducibility.
The typical procedures are as follows. 10 ml of the molten broth agar
(0.8% agar) containing about 2x 10 5/ml spores of the strain HI7 or M45
were poured at 42°C into an empty petri dish and solidified at room
temperature. A paper disk impregnated with chemical solution was placedon the surface of broth agar and incubated at 37°C for 20 hours. Thenthe diameter of inhibition zone appeared around the disk was measured andcompared for Rec+ and Rec- strains. For metabolic activation, the usual
59 fraction was prepared from PCB-treated rats and its 0.1-0.3 ml weremixed with broth agar. A cofactor solution containing 80 mg per ml ofNADP and 40 mg per ml of Glucose-6-phosphate was prepared and its
20 ,ul were impregnated in the paper disk and diffused simultaneously withthe drug (Details to be published as a full paper or in a chapter of "Chemical Mutagen" Vol. 6, Plenum).
Antimutagenic Action of Cobalt Chloride in
Escherichia coli B/r WP2 try
Tsuneo KADA, Masako HARA and Nobutake KANEMATSU

We found that MNNG (N-methyl-N'-nitro-N-nitrosoguanidin)-induced
mutations are remarkably reduced in Escherichia coli by the presence of
cobalt chloride. The strain E. coli B/r WP2 was used and reversions to
ptototrophy were studied. Broth-grown cells were washed with phosphatebuffer and treated with MNNG and plated after appropriate dilution on
broth-enriched (1 %) minimal agar containing cobalt chloride (CoCk 6H 20)
at different concentrations (0 to 25 ,ug/ml). In an experiment, the level ofsurvivals after the mutagen treatment was about 80 %. The presence of
cobalt chloride in the agar medium reduced drustically the frequence of
MNNG induced Try+ revertants; 686.8 per lOS survivals in the absence of
cobalt chloride, and 10.0 per 108 survivals in the presence of cobalt

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

chloride (20 ,ugjml). Similar observation was made with ultraviolet lightinduction of reversions in the same strain.
We are examing if the metal compound might block the mutagen-induced
error-prone repair function in the cell (Proc. Japan Acad. 54, Ser. B, 234,
1978).
Antimutator Effect of Cobaltous Chloride on a
Bacillus subtilis Mutator Strain
Yoshiko OHTA, Tadashi INOUE and TSlmeo KADA

We have recently reported that cobaltous chloride (CoC1 2) reduced
MNNG"induced mutation (Trp- -->Trp+) in Escherichi coli WP2 Bjr (Kada,
T. and N. Kanematsu 1978, Proc. Jpn. Acad. 54(B): 234). We therefore
examined whether CoC1 2 is effective on spontaneous mutation frequency ina Bacillus subtilis mutator strain. B. subtilis NIG 1125 (met his mut-l) is astrong mutator strain. The mutation mut-1 reduces DNA polymerase III
activity, and elevates spontaneous mutation frequency (Brazill, G. W. and
J. D. Gross 1973, Nature New BioI. 243: 241). Two mutation, met andhis possess the same suppressible nonesence codon (Okubo, S. and T.
Yanagida 1968, J. Bacteriol. 95: 1187).
Growing cells in Penassay broth were harvested and washed by centrifugation. Washed cells were plated on semienriched minimal agar plates
containing various concentration of CoC1 2 and 20 ,ugjml of L-methionine.
On the above plates, mutation frequency (His--->His+) was approximately
2.4 X 10- 5 , whereas on plates containing 0.1 mM of CoC1 2 , the frequency
was found to reduce to 1.2x 10- 6 •One explanation for the mode of action of CoC1 2 is that CoC1 2 inhibits
the cell division which is neccessary to induce the mutation. In order toexamine this possibility, the cells were plated on minimal agar whichcontained CoC1 2 and limited amount of nutrient broth. On these plates,
cells can divide only a few times due to the limitation of the broth. Afterthe incubation, cells were recovered from the plates and viable cells werescored to determine the number of cell division on the plates. Experimentswith the plates containing various amount of the broth and CoCl 2 yeilded
the results that CoCl 2 did reduce the mutation without the effect on thecell division.

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79

Desmutagenic Activities in Vegetables and Fruits
Tsuneo KADA, Masako HARA and Kazllyoshi MORITA

Sorbic acid reacts with sodium nitrite and produces at least several recassay positive mutagenic nitroso- and nitro-compounds. Formation ofmany other mutagenic (and carcinogenic) nitroso-compounds is known infood. It may be fortunate if we have anti-mutagenic factors for the above
mutagens in our food. We found by means of the rec-assay procedures,that vegetable or fruits such as pumpkin or cabbage contain factors which
inactivate reaction products between sorbic acid and sodium nitrite.
Recently, it has been reported that smokes or pyrolysates of cigarettes,
fish or meat contain mutagens of high activities, and the pyrolysis productsof proteins and amino acids must share important parts of their total
mutagenic capacity probably related to their carcinogenicity. Our recent
observation revealed that juices prepared from cabbage, broccoli, green
pepper, egg plant, apple, burdock, shallot, ginger, pineapple and mint leaf
possess strong capacities of inactivating the mutagenicity of tryptophane
pyrolysis products. In addition, radish, sweet potato, grape, Japanese
ginger, cauliflower, beefsteak plant, enokidake mushroom and simeji
mushroom were moderatly effective. Among the above eleven samples thatinactivated the tryptophane pyrolysate efficiently, egg plant, burdock andbroccoli showed the widest spectra of inactivating other mutagenic aminoacids pyrolysates (Mutation Res. 53, 351, 1978; Agrc. BioI. Chern. 42,
1235, 1978).
Purification and Properties of a Desmutagenic Factor fromPlant (Brassica oleracea) for Mutagenic Principleof Tryptophan Pyrolysate
Tadashi INOUE, Kazuyoshi MORITA and TSllneo KADA

The salmonella/microsome mutagenicity test of Ames (Ames, B. N. et ai.
1975, Mutat. Res. 31: 347) has been widely employed for screening forchemical mutagens in our environment. By using the system, Sugimuraand his collegues (Sugimura, T. et ai. 1977, Proc. Jpn. Acad. 53: 58) haverecently found in pyrolysate of tryptophan an extremely potent mutagen,3-amino-I-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) whose mutagenicity iscomparable to that of aflatoxin B1 which is one of the most potent

:SO

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28,carcinogen so far found. Since the pyrolysis product is very likely to be
produced by cooking proteinous foods such as fish and meat, and since thecorrelation between mutagens and carcinogens have been generally ac,cepted, one can speculate that such a mutagenic pyrolysis product plays
some roles in carcinogenesis in human. It may therefore be very fortunate
jf we have desmutagenic factors for it in our environment especially infoods. With this view in mind we have examined some sixty species ofvegetables as to the capacity of their extracts to inactivate the mutagenic
principle and found that several kinds of vegetables including cabbage(Brassica oleracea) had considerable capacity to inactivate the principle(Kada, T. et al. 1978, Mutat. Res. 53: 351; Morita, K. et al. 1978, Agric.
BioI. Chem. 42: 1235).
The desmutagenic factor was purified from extract of cabbage leaves by
successive application to chromatographies on DEAE-cellulose, CMcellulose, Sephacryl S200, DEAE-Sephadex and CM-Sephadex. The final
preparation was approximately 360 times purified; and upon SDS-gel
electrophoresis, only one protein band was present and its position of
migration corresponded to a molecular weight of about 43,000. The
purified factor exhibits the absorption spectrum with maxima at 278, 404
and 497 nm and small shoulder at around 380 nm. The absorptions at thewave length maxima are present in the following ratio: 0.332, 1.000 and
0.089, respectively; The extinction coefficient E~o~~m was calculated to be
7.33. These spectral properties suggest that the desmutagenic factor contains a prosthetic group of a heme-like chromophore. We therefore
examined the effect of reducing agent on the spectrum. Treatment of thefactor with sodium hydrosulfite resulted in partial bleaching of the 404 nm
band and in shift the band to 436.5 nm. This result strongly indicates thatthe factor is a hemo-protein. Analysis of iron by the atomic absorption
revealed that the factor contained 0.624/kg of iron per mg of protein. If
we assume that the molecular weight of the factor is 43,000 as determinedby SDS-gel electrophoresis, it was calculated that 0.492 mole of iron per I
mole of the protein. In addition to Trp-P-2, the mutagenicity of Trp-P-l,which is 4-methyl derivative of Trp-P-2 and is also contained in pyrolysate of tryptophan, was effectively abolished by the factor. Ethydium
bromide and 2-aminoanthracene were also susceptible to the factor.
However, the factor had no effect on the mutagenicity of ICR-170
and AF-2. The mutagenicity of 1,2-diamino-4-nitrobenzene was greatly

RESEARCHES CARRIED OUT IN 1977

81

enhanced by the factor.
The mode of action of the factor on the mutagens is under investigation.

Dose Response of Maize to Chronic Gamma-Ray Irradiation*
Etsuo AMANO and Yasuo UKAI**

To study the mutagenic effects of low-dose rate chronic gamma-ray,maize (Zea mays) were planted in Gamma Field of Institute of Radiation
Breeding, NIAS, Ohmiya-machi, Ibaraki-ken. For examination of size ofmutant tissues in chronically irradiated plants, ygz/Yg 2 heterozygotes wereused, and for the response in lower dose rate range, wx/wx inbred line wasused to score possible mutants in pollen grains.
A mutant cell induced by irradiation would proliferate and develop toform a mutant tissue. The size of this mutant tissue might vary dependingon the growth after the induction of the mutation. If the size, or thenumber of cells involved, vary too much in chronically irradiated materials,
reliability of mutant pollen scoring would be reduced, for the pollen grains
were pooled in the scoring disregarding the position in the inflorescences.
To examine this, yg2/Yg 2 heterozygotes were planted in the Gamma Field
and compared with the semiacute irradiation. Semi-acute irradiation wasmade by irradiating 20 days old seedlings in the same Gamma Field forfive days, but with approximately 5 times higher dose rate upto 473 R/day.
For chronic irradiation, materials were planted in the Gamma Field at thesame age, and irradiated until harvest of leaves, dose rate ranging from
15.2 R/day to 83.0R/day. The final leaves were collected to examine themutant sectors. Lengths of the light colored sectors were measured andtheir relative locations on the leaf were recorded. The average size of themutant sectors of the semi-acutely irradiated materials were larger than thechronically irradiated ones, and many of the sectors extended from theirinitiation points to nearly tip of the leaf. Compared to this, most of thelight colored sectors on leaves of the plants which were chronically irradiated all through the growing stage were small. Only a few percent of thesectors exceeded five per cent of the leaf length. In scoring of the sectors,
only the sectors which exceeded 1 mm were adopted, but many smaller
sectors were also noticed. This might indicate that practically the size of***

Research sponsored by Ministry of Agriculture and Forestry.
Institute of Radiation Breeding.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

the mutant tissue were so small and homogeneous that mutant pollen
scoring might reflect the mutant frequency of the materials. Moreover the
mutatnt sector frequencies in the irradiated materials were 20 times morein chronic irradiation.
Mutant pollen frequencies were examined in chronically irradiated wx/wx
inbred line (No. 639). Pollen grains can be easily collected and examined
up to 106 • The waxy character can be visualized by iodine staining.
Normal non waxy (Wx) pollen grain stains blue black. Waxy mutant (wx)shows reddish brown color. But the color change by forward mutationfrom Wx to wx may not be separable from physiological damage of thepollen, i.e. the lightening of the stained color. The use of the wx/wx
enabled the detection of the second starch character gene, the amylose
extender (ae). The materials used were wx, Ae/wx, Ae. Mutation from
Ae to ae increases amylose like component in starch changing the color of
the iodine stain darker. Reverse mutation from wx to Wx would be rare,
but it would be detected likewise.
Male tassels were collected and fixed in 70 % alcohol before anthesis.
About 40 flolets per slide were taken out and homogenized in Nelson's
iodine solution (0.18 % I 2 in KI solution). The suspension was filteredthrough cheese cloth on the large slide glass. The number of the total
pollen grains were estimated by scoring the pollen at 15 places. Dark
stained mutant pollen grains were scored by scanning all the prefixed 50 X
75 mm 2 area. Distribution of the mutant frequency along the main axisof the tassel did not deviate very much, again supporting the results of theleaf sector scoring. The mutant frequency increased almost linearly up to
10 R/day and 1 X 10- 2 mutant frequency, in parallel with the pollen
sterility. The lowest dose rate that increased the mutant frequency were
3.8 R/day at 92 m from the source. It was twice in the distance and 1/4
in intensity for yg2 leaf sector scoring. The results indicated that pollen
grain scoring was a very sensitive mean to study the mutagenic effect oflow dose rate gamma-ray. Preliminary results showed linear increase ofmutant frequency with the dose rate.
Measurement of Waxyness in the Induced wx Mutants
Etsuo AMANO

Ethyl methanesulfonate (EMS) is a very effective chemical mutagen. It

RESEARCHES CARRIED OUT IN 1977

83

may induce base pair substitution in higher plants. This was shown bypresence of CRM in sh 1 mutants in maize by Chorey and Schwartz (1971,
Mut. Res. 12) in maize. Frequently leaky mutants which would be indications of missence mutation, were found in C locus, wx locus in maize (Zeamays) and wx mutants in rice plant (Oryza sativa). Measurements of thisleakyness were tried by modified blue value method for the iodine stained
starch solution. The measuring device consisted of stabilized incandescentlamp, thermal filter, sample cell, beam divider mirrors and two silicon
photo-cells with 430 nm or 660 nm interference filter. A 10 mm x 10 mmsample cell was deviced with a small mixing motor and inlet and outlet
tubes to enable continuous dilution of the sample solution. Ordinally blue
value method to measure the amylose, the Wx component, which stains
blue by iodine, use the absorption of 660 nm red light of the solution ofcertain concentration of total starch. The present measurement used the
430 nm blue light to determine the concentration of the starch solution.
DC amplifier and digital printer recorded the transmission value of 660 nm
at the instant when the iodine stained solution in the sample cell was diluted
to the concentration of 50 % transmission for 430 nm.
Each cereal grains were crashed and put into test tubes. Distilled water
was added to the tubes, 3 ml each for rice grains and 5 ml each for maizegrains. Tubes were autoclaved at 1 atm. for 15 min. Tubes were stirred
while warm. After cooling down to room temperature, 1 ml supernatantsolution was taken and mixed with 0.1 ml of 0.09% Iz-KI solition (1/2 of
Nelson's I 2-KI solution) in the sample cell. Then measured the trans~mission of 660 nm light at the 50% transmission concentration for 430 nm
light. The T-value in percent for 660 nm was used as index to show the
waxyness. The measuring system was as sensitive as to take a single ricegrain as a sample, thus distribution of the wx index among a population could
be analyzed. The index value of normal Wx starch was about 20 in rice and
17 in maize. The standard waxy mutants showed the indices, around 70
in rice and 65 in maize. In maize, five out of eight EMS induced mutants
examined showed nearly equal value to small deletion mutant wx R • The
rest of the mutants showed lower index values. In rice, four out of eight
EMS induced mutants showed the indices as high as the standard wx
mutant. Other four mutants showed lower index numbers.
Examinations of self pollinated F 2 populations in maize showed clear
3: 1 segregation indicating complete dominance of Wx over wx, regardless

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

of the wx mutant lines examined. Compared to this, in rice, segregations
were not so clear as in maize. Recessive wx group could be separated,
but Wx group distributed from standard Wx to almost half way to wx.
The frequency spectra indicated 2: I: I distribution suggesting gene dosage
effect in the triploid endosperm. The numbers would correspond to
WxWxWx and WxWxwx, Wxwxwx, and wxwxwx. This gene dosage
effect had been noticed visually in endosperm, and the progeny tests supported the explanation by gene dosage effect.
Modification of Radiation Damage and Variation of Mutant Sector
in Maize with Different Kinds of Radiations
Taro FUJII

Mutation from dominant Bz to recessive bz was detected as a phenotypicchange in the aleuron color of M 1 seeds in the cross of a recessive stockwith the irradiated pollen grains carrying the dominant gene. Large
variations in the mutated area of M 1 seed surface was noticed, viz. wholearea covered with mutated character-whole type, and mutated character
appeared in a limited part of seed surface-partial type. Furthermore,partial type shows wide variation in its sector size. To investigate thecorrelation between mutation type and radiation quality, partial type wasdivided into four categories; nearly-whole-almost all area of seed surface
covered with mutated character, 1/2-about 50 % is mutated area, 1/4about 25 % is mutated area, and dot-mutated character appeared as a
Table 1.

Mutation detected and radiation type
Frequency distribution in

Deline in
Whole: Partial mutation
frequency
Gamma-rays(Fractionation)

2
1.5" (Low dose rate)

2
2
2
1
1

"

14 MeV neutrons" (Fractionation)
Ultraviolet(Photoreactivation)
"

1
1
1
4-5
4-5

Yes (onlyin whole)
Yes
No
Yes

_._-_._-"-

the partial type (%)
Neary
whole

1/2

1/4

Dot

50
50

20
20

20
20

10
10

50
50
50
20
20

20
20
20
50
50

20
20
20
20
20

10
10

10
10
10

---_.---RESEARCHES CARRIED OUT IN 1977

85

dot, and frequency distribution of these categories was examined. Summary of the results are shown in Table 1. Dose range used in the present
study is 500-3000 R of gamma-rays, 250-680 rad of neutrons and 400012000 ergsjmm 2 of ultraviolet. No dosage effect was observed in eachtype of radiation on the ratio of whole and partial types, as well as in
the frequency distribution among the partial type.
Recovery of premutational damage was observed with fractionation
treatment merely in whole type in the gamma-ray induced mutations (40kRjh intensity). However, at low dose rate (l kRjh) mutation frequency
declined both in whole and partial types. On the other hand, mutation
frequency did not show any significant difference with single and frac·tionated treatments of neutrons. Nevertheless, whole and partial mutants
were always in the ratio of 2: I in ionizing radiations, except gamma-ray
fractionation treatment. The same frequency distribution in the partial
type was observed both in gamma-rays and neutrons.
In the ultraviolet induced mutations, ratio of the partial type is very
high, frequency distribution in the partial type is also quite different fromthose of ionizing radiations. Although the decline in mutation frequency
is very clear with photoreactivation treatment both in whole and partial
types, change in the frequency distribution in the partial type was notobserved either with or without photoreactivation treatment. Thus, thenature of damage caused by UV-irradiations which differ greatly from thatof ionizing radiations may reflect on the mutation type. Mechanism responsible for these mutation types are still obscure, and dual mechanism, lethal
hit, master strand or miss repair hypothesis have been proposed to explain
the mechanism of their origin. However, ratio of whole and partial,
and frequency distribution in different categories of partial mutations
shows a constant ratio according to the kind of radiation.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

VIII.

POPULATION GENETICS (THEORETICAL)

Theoretical Study of Genetic Variability, Assuming Stepwise Productionof Neutral and Very Slightly Deleterious Mutations
Takeo MARUYAMA and Motoo KIMURA

Mathematical treatments are presented that enable us to compute theamount of genetic variability maintained in a finite population, assumingthat mutations occur in stepwise fashion and that both selectively neutral
and slightly deleterious alleles are involved. Two numerical examples
show that, if very slightly deleterious mutations are prevalent, the amountof genetic variability increases much more slowly as the population number
increases than is the case when all the mutations are strictly neutral. (Fordetails see Proc. Nat. Acad. Sci. USA 75: 919-922, 1978).
Extension to the Neutral Mutation Random Drift Hypothesis
Tomoko OHTA

Effects of varying selective force (not fluctuating from time to time fora single mutant but varying over many newly arising mutants) on the
average heterozygosity and the evolutionary rate have been investigated under
the hypothesis assuming prevalence of very slightly deleterious mutations.
It has been shown that, as the population size increases, the rate of approach to the upper limit of heterozygosity under negative selection isslower for the case of varying selection coefficients as compared with thecase of constant selection. Also, an inverse relationship between theevolutionary rate and the population size is shown to hold if the selection
coefficients against mutant alleles follow an exponential distribution.
The theoretical distribution of genetic distance was studied using Monte
Carlo experiments assuming selective neutrality. It agrees reasonably wellwith the observed distribution in D. willistoni group. However, under the
more restricted condition of measuring the distance for polymorphic cases
only, there is a discrepancy in the distribution of distances between theactual observations and the theoretical expectations. It was pointed outthat the hypothesis of very slightly deleterious mutations (a modified

RESEARCHES CARRIED OUT IN 1977

87

neutral theory) offers a most natural interpretation for this kind of minor
discrepancy. Details were published in Proc. of The Second TaniguchiInternational Syrup. on Biophysics, "Molecular Evolution and Polymorphism" (ed. M. Kimura), pp. 148-167.
On the Gene Conversion Model as Mechanism for Maintenanceof Homogeneity in Systems with Multiple Genomes
Tomoko OHTA

The gene conversion model reported by Birky & Skavaril (Genet. Res.
27, 249, (1976)) has been analytically studied by using the theory of diffusion
models of Kimura (Jour. Applied Probability 1, 177, (1964)) in population
genetics. It has been shown that the fate of new mutations in systems withmultiple genomes may be satisfactorily treated by the diffusion model. For
details, see Genet. Res. 30, 89-91.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

IX.

POPULATION GENETICS (EXPERIMENTAL)

Number of Sterile Loci in Drosophila melanogaster
Takao K. WATANABE and Won Ho LEE

The number of loci which are potentially able to produce sterility genes
was estimated for D. melanogaster. The frequencies of induced andspontaneous sterility mutations and that of simultaneously occurred lethal
mutations were compared, together with some data from published literature. The mutagenic strength of the chemical treatment differed to someextent between experiments, and treated chromosomes. However, if
mutations to lethality and sterility are compared within each experimentand presented as relative mutation ratios, these values are useful for
estimating the standard mutation rate of sterility genes.
To standardize the mutagenic effects of various mutagens on various
chromosomes, the frequency of male-sterile (dm) or female-sterile chromosomes (df) was divided by the frequency of lethal chromosomes (1) in eachexperiment. Although the ratios dm/l or df/l were varialbe among experiments, they were apparently smaller than 1. A maximum likelihood
estimate was 0.153 for dm/l and 0.115 for df/l.
If we suppose that sterile and lethal mutations per locus occur approximately at the same rate, the number (Ns) of loci which are able to havesterile can be calculated, Ns=Nl x (d/l). Since the number of lethal loci(Nl) on the second chromosome is about 500 (Wallace 1950 PNAS 36: 654),
the number of potentially male-sterile loci is 500 x 0.153=76.5 and that offemale-sterile loci 500xO.115=57.5 on the second chromosome. For
details, see Genet. Res. 30, 107-113.
Evolutionary Changes of Inversion Frequencies in a Natural
Population of Drosophila melanogasterYutaka INOUE and TAKAO K. WATANABE

Inversion polymorphism in natural population of Katsunuma has beenstudied for more than ten years. The frequency of polymorphic inversions
was relatively high at sixties where the average frequency was about 25 %,RESEARCHES CARRIED OUT IN 197789'

whereas it decreased at seventies to be about 15 %. Simultaneous reductions of several kinds of inversions suggest that natural selection was
acting against such inversions (2Lt, 2RNS, 3LP, 3RMo) in similar way andit became stronger in these years.
On the other hand, an inversion (3RP) has survived for these ten years
without any frequency change. Two new inversions, 2LW (28e; 32C)
and 3LY (68F; 75C), have recently appeared and settled by the frequency
of about 3 % in the population. These three inversions must have different adaptive strategies from the above inversions, since they have survived rather constantly or more adaptively in these years.
The reduction of some inversion frequencies in Katsunuma has changed
the order of relative abundance of four polymorphic inversions in four majorautosome-arms. The order was 2Lt>2RNS>3RP>3LP in the sixties, but
it became 3RP>2RNS;;;;;2Lt>3LP in the seventies. The 2Lt decreased so
rapid that its frequency became close to or somewhat less than thefrequency of 2RNS whose decreasing rate was relatively slow.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

x.

EVOLUTIONARY GENETICS

Preponderance of Synonymous Changes as Evidence for the
Neutral Theory of Molecular Evolution
Motoo KIMURA

According to the neutral mutation-random drift hypothesis of molecular
evolution and polymorphism proposed by the author in 1968 (Kimura,
Nature 217, 624), most mutant substitutions detected through comparative
studies of homologous proteins (and the nucleotide sequences) are theresults of random fixation of selectively neutral or nearly neutral mutations.
This is in sharp contrast to the orthodox neo-Darwinian view that practically all mutant substitutions occurring within species in the course ofevolution are caused by positive Darwinian selection.
Following the neutral theory, we consider two types of mutational changes,
neutral and deleterious, and assume that the probability of a mutation being
neutral (not harmful) depends on the functional constraint of the molecule.
Namely, the stronger the constraint the smaller the probability of selective
neutrality.
It is known that a large fraction (roughly 2/3) of nucleotide substitutions
at the third positions of the codon are synonymous, that is, they do notcause amino acid changes in proteins. Since natural selection acts throughphenotypes, and particularly, through the function of the protein, it isexpected that synonymous changes are subject to much weaker natural
selection, if any, than nucleotide changes causing amino acid changes.
This means that, if the neutral theory is valid, the rate of mutant substitutions at the third positions of the codon must be very high.
Recently, it has become possible to estimate the evolutionary rate ofsynonymous mutant substitutions by using data obtained from comparative studies of messenger RNA sequences.
Salser et at. (Fedn. Proc. 35(1), 23 (1976)) have presented a comparisonof homologous parts from the fragments of the human and rabbit hemoglobin mRNA sequences. Using their data, it is estimated the rate ofnucleotide substitutions per site per year at the third position of the codon isk unc =(2.3±1.l)X 10- 9 •RESEARCHES CARRIED OUT IN 1977

91

This is a very high evolutionary rate comparable with that of the fibrinopeptides.
A similar but more interesting report comes from Grunstein et at. (J.M.B.
104, 351 (1976» who compared histone IV mRNA sequences of two sea
urchin species. It is well known that histone IV is by far the most highly
conserved protein with the rate of amino acid substitution k aa =0.006 X 10- 9
per year. Their report now shows, however, that many synonymouschanges have occurred in the gene coding for this protein in the course ofevolution. Using Grunstein et at.'s data, the following estimate has beenobtained for the rate of nucleotide substitutions at the third nucleotide
position of the codon in the histone IV genes.

k nuc =(3.7±1.4) X 10- 9 •These estimates conform remarkably well with the framework of theneutral theory. Let k be the rate by which mutant genes are substituted
in the species in the course of evolution, and let 10 be the fraction of molecular mutants that are selectively neutral. Since, for neutral mutants, therate of mutant substitution is equal to the mutation rate, we havek=vrlowhere Vr is the total mutation rate. According to the neutral theory theprobability (/0) of a random change being selectively neutral is larger the
weaker the functional constraint with the result that k in the above equation
gets larger. It is clear that the maximum evolutionary rate is attained
when 10=1, that is, when all the mutations are neutral. Now, the highevolutionary rates observed at the third position of the codon can be
explained from the neutral theory by assuming that the majority ofsynonymous changes are selectively neutral (Note that roughly 2/3 ofrandom nucleotide substitutions at the third position of the codon aresynonymous). On the other hand, if we adhere to the selectionist position
that practically all the mutant substitutions in evolution are caused bypositive natural selection, there can be no upper limit to the evolutionaryrate at the molecular level (as directly set by the mutation rate vr).
In my opinion, various observations suggest that as the functional constraint diminishes the rate of evolution converges to that of the synonymous
substitutions. If this turns out to be true, such a convergence or plateauingof molecular evolutionary rates will turn out to be strong supporting

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

evidence for the neutral theory.

For more details, see Nature 267, 275-

276 (1977).

A New Hypothesis for the Direction of Evolution among
Related Species of Drosophila
Takao K. WATANABE and Masaoki KAWANISHI

Drosophila females usually choose their mating partners if males ofdifferent species are present, while males generally show no mate preference. The degree of successful matings between pairs of reciprocal
hybrid matings often differs and this indicates the direction of evolution
among the sbling species.
For example, D. melanogaster females were always more successful inmating with D. simulans and D. mauritiana males than the reciprocals.
On the other hand, simulans females were more successful in mating withmauritiana males than the reciprocal cross, but were less successful withmelanogaster males than the reciprocal. Finally, mauritiana females werealways less successful in mating with both melanogaster and simulans
males than were the reciprocal crosses. If we hypothesize that females ofa newly evolved species tend to reject males of the original (ancestral)species, the direction of evolution would be melanogaster~simulans-+
maUl"itiana.

The direction of evolution in the virilis group of Drosophila has been
discussed on the basis of cytological (Stone et at. 1960, PNAS 46: 350),
morphological (Throcknorton 1962, UTP 6205: 207), and electrophoretical(Hubby and Throckmorton 1965, Genetics 52: 203) studies. Investigationsof these different features have resulted in the conclusion that virilis is theoriginal species and th other species have been derived as follows: virilis-+
novamexicana~americana~texana, and virilis~lacicola and montana. This
relation can be perfectly substantiated by the present hypothesis by using
data of Patterson et al. (1947 UTP 4720: 7, 1949 UTP 4920: 7). That is,females of a derived species discriminate and reject males of the ancestral
species whereas females of the ancestral species readily accept males of thederived species.
Mating preference and cytological data indicate the same direction ofevolution in the mulled group of Drosophila (Wasserman 1963, Am. Nat.
97: 333; Patterson 1947, UTP 4720: 32). The phylogeny of D. willistoni

RESEARCHES CARRIED OUT IN 1977

93

group established from the electrophoretical differences (Ayala et at. 1974,
Evolution 28: 576) was almost exactly substantiated by our method when it
was applied to the data (Buria et al. 1949, Evolution 3: 300; KorefSantibanez 1972, Evolution 26: 326).
Our hypothesis is based on the assumption that a newly derived species
would have gained some cortship pattern in addition to the ancestral one.
Because of this the derived females show strong discrimination against
males of ancestral species which lack the full courtship pattern, but derived
males are still accepted by the ancestral females which require a partial(ancestral) courtship pattern.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

XI.

HUMAN GENETICS

Reexamination of Paternal Age Effect in Down's Syndrome
Ei MATSUNAGA

Paternal age distribution for 1279 cases of Down's syndrome born in
1952-1968 was compared with the corresponding distribution for thegeneral population, taken from the vital statistics data and corrected forthe maternal age as well as for the year of birth of the patients. Although
there was no difference in the mean paternal age, the two distributions
differed significantly, largely due to the excess of fathers aged 55 years andover and to the deficit of those aged 40-44 years in the patients born tomothers aged 30 years and over. The overall pattern of the relative incidenceof Down's syndrome with advancing paternal age, with maternal age controlled, seems to be consistent with the hypothesis proposed by Stene et al.
(1977 Ann. Hum. Genet. 40: 299); it increased from 0.8 for fathers aged
20-24 years slowly up to 1.2 for those aged 45-49 years, though with anintermediate drop to 0.8 at the age of 40-44 years, and then sharply to 2.4
for those aged 55 years and over. This rising pattern of the relative incidencewith paternal age was essentially the same for the patients born in 19521960 and for those born in 1961-1968, although the slope was less steep
in the latter than in the former group. In addition, for those patients born
to younger mothers (under 30), the relative incidence increased lineally up
to the paternal age of 39. For details, see Hum. Genet. 40: 259-268 (1978).
A Genetic and Epidemiologic Study of Polydactyly in Human Embryos
Kohei SHIOTA and Ei MATSUNAGA

Among the large collection of human conceptuses in the Human EmbryoCenter for Teratological Studies, Kyoto University, 129 embryos with
polydactyly were found in the period from 1962 to 1974. The overall
incidence of this anomaly in human embryos was 0.35 %, which must be
an underestimate because the denominator includes a large number ofdamaged embryos. A significant increase was noted in 1971 and 1972,and some exogenous factors, most probably ascertainment bias, were sus-

RESEARCHES CARRIED OUT IN 197795

pected to be responsible. About three quarters of the present cases had
preaxial polydactyly in contrast to the predominance of postaxial type in
Negro and Caucasian populations. The ratio unilateral to bilateral affection was approximately 2; 1. The mean maternal age did not differ
significantly from that of the general embryonic population. Maternal
pregnancy order had no effect upon the causation of this malformation.
The case mothers were shown to have more frequent miscarriages thancontrols, suggesting the possible effect of some kind of maternal predisposition. No association was verified with paternal age, parental consanguinity nor with maternal medical history, including irregular menstrual
cycles, and smoking and drinking habit. None of the cases was found tobe familiaL Dominant inheritance does not seem a major cause andcomplex genetic mechanisms or unknown exogenous factors may be
responsible for a majority of the Japanese cases of polydactyly. Details
have been published in Jap. J. Human Genet. 23: 173-192 (1978).
Palindromic Theory and the Telomere of Eukaryote Chromosomes
YaSilO NAKAGOME

The known DNA polymerases act in the 5' to 3' direction and require afree 3'-OH as a primer. Thus the mechanism of replication of the 5' end ofa linear DNA molecule remains unclear. A few models, including palindrome models of Cavalier-Smith and Bateman, have been proposed(Nature, 250: 467, 1974 and 253: 379, 1975).
In eukaryote chromosomes, a telomere behaves differently from a broken
end in that it has no tendency to join with the latter. Cavalier-Smithpointed out that the existence of "terminal palindrome could explain
several intriguing properties of the telomeres." If telomeres have a structure
proposed by Bateman, 50 % of them consist of DNA with two newly
syntheiszed strands and the rest with two old strands (see Fig. 1 ofBateman). If the Cavalier-Smith's model is the case, each telomere consists of each one of old and new strand. In only the Bateman model, atelomere with two old strands can arise.
In the present study, human diploid cells (WI-38) were incubated in thepresence of BUdR for the length of S+G2 phase. Metaphases were
stained with acridine orange. The experiment was designed to detect
intense fluorescence of regions with two old strands. The technique was.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

essentially the same as that used in our previous work (Nakagome, Exp.
Cell Res., 106: 457, 1977). In most metaphases, chromosomes showed dull
and homogeneous appearance and none of more than 1000 telomeres
fluoresced intensely. In some of metaphases, chromosomes showed R.
band pattern, presumably representing cells with long generation time.
It was reported that single T2 phage was detectable by the use of acridine
orange (Mayor and Hill, 1961). The virus has a linear DNA with 18.2X
10 4 nucleotide pairs which exceeds the largest palindrome (2 X 104) isolated
so far from eukaryotes by biochemical means. However, it does notpreclude the possibility that telomeres contain longer palindromic sequence.
There are only a total of 92 telomeres in an interphase nucleus and a
specific sequence, if any, is not likely to be detected by biochemical means.
It may be that telomeres of human chromosomes either have no DNA
with two old strands (Bateman structure) or have one which is too small
to be detected.

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XII.

97

BEHAVIORAL GENETICS

Circadian Locomotor Activity of Sensitive and Unsensitive Strainsof Drosophila melanogaster for Noise Environment
Chozo OSHIMA and Won Ho LEE

The circadian locomotor activity of an individual male fly in a small
glass chamber (4 X 10 X 50 mm) was recorded continuously by the electronic
actograph. The record was usually taken for a week; the former half
period was under light and dark daily cycle (L D) with dusk and dawn,
and the latter half period was under constant dark (D D) and temperature
was kept constant 25°C. The locomotor activity of flies of about ten
homozygous strains for the second chromosome was observed to be daily
rhythmic and showed two high peaks at dusk and dawn under L D
environment. On the other hand, the free running locomotor activity offlies continued at low lebel during the subjective light period under D D
environment. Under both L D and D D environments, the locomotor
activity of flies stoped usually for about 6 or 7 hours of common dark
period. Then, the pure sound (2000 cycle, 100 phon) was projected for 5
hours of the rest time.
Male flies of 3 homozygous strains 168, 145 and 146 felt the noise andmoved not only for 5 hours, but also their excitement continued after
several hours. On the contrary, male flies of 3 homozygous strains 16,
49 and 83 felt it a bit or not at all. The former strains were assumed to besensitive and the latter ones were unsensitive (resistant) for the noise
environment.
The most sensitive strain 168 and unsensitive strain 83 were crossed
reciprocally. The locomotor activity of hybrid male flies was recorded.
Their sensitivities for noise were middle between both parental strains, butclose to the unsensitive one and their characteristics of locomotor activity
rhythm at dusk and dawn under L D environment were similar to thesensitive one. From the result, the sensitivity for noise environment ispresumed to be a recessive character.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

Genetic Variation of Flying Ability of Drosophila melanogaster
Won Ho LEE and Takao K. WATANABE

The flying ability of Drosophila flies was measured by a glass cylinder
apparatus, similar to Benzer's flight-tester (Scientific Am. 229: 24, 1973).
The inside wall of the cylinder was coated with. liquid paraffin. Thirty
flies of one sex, 20r 3 days old, were gently aspirated into the apparatus.
If flies can fly they strike on the wall from upper region (5) to lower region(2) according to their flying ability and fix there, and if they can not theyfall to the bottom (1).
A total of 52 second chromosomes was isolated from Katsunuma of
1975. Homozygotes for each chromosome line were examined in theapparatus. Variations of flying ability in the homozygotes ranged from
2.6 to 4.4 with an average value of 3.88. Two lines showed very weak
flying ability but no flightless lines were detected. Analyses of variance
was significant among 52 lines, suggesting the flying ability was geneticallycontrolled.
Correlations between the flying ability and other genetic traits such as
walking ability, viability, body weight and wing length were not at all
statistically significant. Thus, genes controlling flying ability were in largepart different from those controlling other genetic traits examined above.
Effects of Noise on the Learning Performance of Mouse
Strains with Different Learning Abilities
Tohru FUJISHIMA

The effects of noise on the learning performance of mice was examined
under the following experimental conditions. Two strains, C3HjHeMs
and SWMjMs, were exposured to one-hour pink noises of 100 phon six
times at one hour intervals every night for a period of one or three weeks,
and they were tested with an automated Y-maze apparatus, using buzzer
and lamp as the conditioned stimuli and electric shock as the unconditioned stimulus. In a previous experiment, the C3H showed the best
discriminatory ability, while the SWM showed the best avoidance ability
among several inbred strains tested (cf. Ann. Rep. 25: 82).
Both of the two strains showed no remarkable depression in learning
performance in the first training session after one-week or three-week

RESEARCHES CARRIED OUT IN 1977

99

exposures to the noise environment. However, the two strains differed intheir responses to noise. In the second training session, the SWM showed aslight depression in avoidance performance, while the C3H showed alittle improvement in this performance. The learning test following an
additional one-week exposure to noise, however, indicated significant
depressions of performance in both strains, especially in avoidance performance. It was also found that noise made the mice emotional or
nervous. They showed no acclimatization to the noise environment withregard to activity and learning performance.
Genetic Analysis of a Mutant Showing Abnormal
Behavior in the Silkworm
Akio MURAKAMI and Aldo OHNUMAFor a better understanding of animal behaviors, it is favourable toanalyze mutants which show abnormal behavior in organisms with a simple
nervous system. With this in view, observations were made to detectmutants in the silkworm, Bombyx mori. We report here the occurrence ofa sex-linked recessive mutation showing abnormal behaviors regarding tomuscle response in the larvae and sexual response in adults of Bombyx.
In this mutant line intersegmental membranes in all segments of larvae,
specially in the aged larvae, are abnormally soft and pliant when compared with the normal stocks. Accordingly, the inability of the larva tokeep a normal carrieage may be due to the disorder of neuromyal junction.
It is also interesting to note that the mutant male moths can not respond toa sex-attractant of pheromone which is secreted from an alluring gland offemale moths, although they are able to flutter when these males are hungwith fingres. This suggests that this line male moth has a normal wingmuscular system. It is further observed in this line infecundity andsterility caused by the abnormal sex-behavior pattern-its inability to mate.
In spite of those abnormalities, this mutant silkworm develops normallywithout any significant reduction of viability. These evidences clearlysuggest that the mutational trait may primarily affects the central nervous
system or brain controlling behaviors in general rather than a special
ganglion affecting partial behavior.
The abnormal syndrome was detected at first in only female offsprings
from a cross between a normal female and an F 1 male in which the female

100 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

had been X-irradiated with a 1,000 R, the males did not show abnormal
syndrome. However, subsequent sib-matings of those PI offspring produced larvae in the ratio 1 normal: 1 abnormal regardless of sex, suggesting that this syndrome is controlled by a single sex-linked recessive
factor (or gene) spli (soft and pliable). To verify this point, females
hemizygous for spli were crossed with heterozygous males (splij +), theoffsprings were wild-type and abnormally soft and pliable larvae in theratio of 1: 1 regardless of sex. When wild-type female (+ j +) werecrossed with homozygous males (splijspli), abnormally soft and pliable
female larvae and wild-type male larvae were obtained. In addition,matings of abnormal females hemizygous for spli and wild-type males(+ j +) produced only wild-type larvae for either male or female. Homozygous male larvae for spli have shown a typical syndrome the muscular
dystrophy. It can concluded from these experimental results and others
that this syndrome is under the control of a single recessive gene, spli,
which is located on the X-chromosome. Preliminary investigations indicatethat the spli is located between known gene loci sch (sex-linked chocolate:21.5) and e (elongate: 36.4) on the X-chromosome. However, this has
to be confirmed further.
Histological and electrophysiological analysis for this mutant line are in
progress.

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XIII.

101

ECOLOGICAL GENETICS

Observations of Rice Species and Accompanying Savanna Plants
at the Southern Margin of Sahara Desert
H. T. aKA, H. MORISHIMA, Y. SANO, and T. KOIZUMI*In the early dry season (October-November) of 1977, we have travelledin the interior of Nigeria and other West African countries for ecologicalgenetic studies of rice species and accompanying savanna plants. Our
trip was planned and realized under a grant for overseas field research of
the Ministry of Education, and with the generous cooperation of theInternational Institute of Tropical Agriculture (IITA, Nigeria), Office de laRecherches Scientifique et Technique Outermer (ORSTOM, Chad andIvory Coast), Institut de Recherches Agronomiques Tropicales (IRAT,
Ivory Coast), and other agricultural institutions. Our objectives were to
make observations on the diversity of plants as influenced by natural
envrionment and human activity, and to collect the seeds of wild andcultivated rice species.
The seed samples collected were: Oryza sativa, 36; O. glaberrima, 31;
O. perennis subsp. barthii (= O. longistaminata), 27; O. breviligulata, 20,
and O. punctata, 8. Each accession consisted of seed samples taken at
random from 20 to 30 plants of a pouplation, either on a single plant basis
or in bulk. The seeds were shared with the units in charge of germplasm
conservation of the IITA and ORSTOM.
Observations were recorded at 77 selected sites, of which 28 were swampy
depressions (where wild rice species grew), 23 were upland savannas, and26 were rice fields. Records were taken at each site with regard tophysiography, water condition, degree of disturbance by man, grazing by
animal, height and cover of major plant species, habitat segregation, andwithin-population variations in visually distinguishable characters. For
rice fields, the method of cultivation was recorded in addition. On the basisof herbarium specimens collected at these sites, a total of 117 species wereidentified by Dr. 1. O. Akobundu of the IITA. Further, for a survey ofburied seed populations, soil samples were taken from 27 sites. Although* Research assistant, Tokyo Gakugei University, Department of Geography.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

experiments with the collected materials are under way, the following facts
were pointed out from our field notes.
a) Savanna vegetation: Latitude is an important factor in determining
natural vegetation as well as cropping system, as the higher the latitude,
the shorter the rainy season and the more uncertain the rainfall. The
proportion of annual species at a site tended to be high at higher latitude.
Total biomass estimate (plant height X cover) tended to be small at higher
latitude. Generally, species diversity was correlated with total biomass,
though it differed according to topographic conditions and habitat disturbance.
b) Wild rice species: Oryza perennis subsp. barthii had rhizomes in all
27 populations observed. The populations were homogeneous in outward
appearance except for a difference in awn color (red vs. white), even though
they are known to have a large amount of enzymatic variations. In many
places, the plants formed clumps as the result of their rhizomatous propagation. Some of the plants showed pollen and/or seed sterility.
The annual species, O. breviligulata, was found in swampy depressions in
the Sudan and Sahel zones. In the area where the plants form big
populations as in Chad and northern Cameroun, the people collect thegrain as their food. Scattered plants of this species exhibiting somecultivated characters (resembling O. glaberrima) were found in disturbed
habitats like abandoned rice fields. Despite their quite different breeding
strategies, O. breviligulata and O. perennis subsp. barthii were often sympatric. But they showed a tendency to habitat segregation as the former
species grew at a lower zone (some 10 cm or less) than the latter species.
O. punctata seemed to be divisible into two ecotypes, one adapted tomarshes shaded by trees, and the other adapted to open habitats andgrowing often sympatrically with O. breviligulata.
c) Cultivated rice: There were different types of rice fields which weredivisible into upland, rainfed lowland, and river flood plain types, inaddition to modernized irrigated fields. The rice fields of peasant farmers
were generally undulated but not sectioned by dykes. In a majority of
those fields, the two species, O. sativa and O. glaberrima, were mix-planted
in varying proportions. Although governmental agencies recommend
planting sativa cultivars, many farmers recognize that the traditional
glaberrima rice is more tolerant to drought, submergence, and other stresses.
The farmers rather willingly accept mixing of the two species and theirRESEARCHES CARRIED OUT IN 1977

103

different varieties.
Irrigable rice fields have been open in some places in the semi-arid inland.
There, we learned that the semi-dwarf improved Indicas of O. sativa introduced from Asia were generally inadaptive. This is possibly because thevarieties selected for high yielding potential under a good cultural management have not been selected for tolerance to water stresses.
d) Character variations within rice populations: The seed samples
collected from the fields showed that the populations were highly polymorphic in visually distinguishable characters. Evaluating the diversity inseed type by the amount of information (H = - I; Pi log. Pi), we found thatin the fields where glaberrima and sativa were mix-planted, the diversity
within glaberrima populations was highest when the relative frequency ofglaberrima plants was 50-60 percent. Its paraboric regression line on therelative frequency was highly significant. A similar trend was also foundfor sativa populations. We found further that the within-population diversity was in both species correlated with species diversity in the field
which was estimated (also by H) from percent biomass for each of
coexisting species (rice and weed). These relationships suggest that thecomplexity of the biotic emvironment as shown by species diversity plays
a role in maintaining polymorphisms in rice populations. It was alsosuggested that the temporarily changing water conditions and microtopographic undulations in the field impose a pressure of disruptive selection
on the rice populations and plant community as a whole and result ingreater species and genotypic diversity. The interrelationship foundbetween genetic variation within rice populations and community structure,
though the dynamics are difficult to elucidate, may be of some use inconsidering work for the conservation of genetic resources.
e) Other crops than rice: In addition to rice, sorghum, millet, pearl
millet, maize, cowpea, groundnut, cotton, etc. are grown in the rainyseason. In many places, two or more crop species were found to bemix-planted. According to a report from the Institute of Agricultural
Research, Ahmadu Bello University, intercropping in certain conditions
was advantageous in increasing total yield. In cowpea, the presence of
cereals in the field reduced pest damages when no pesticide was sprayed.
The cowpea seeds obtained from local markets were various in size andcolor pattern in the same manner as were rice seeds. Presumably, both
genetic heterogeneity in a species and species mixing play similar roles in104 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

increasing the biotic complexity of the community and serve as stabilizing
mechanisms. Attempts of agricultural modernization by monoculturing
a single crop may encounter with various hazards unless the agroecosystems are carefully investigated and managed.
The Breeding Behavior of Oryza perennis Populations
Observed in Thailand
H. I. OKA and Y. SANO

We have been working on variations in the breeding systems (Oka andMorishima 1967, Evolution 21: 249), adaptive mechanisms (Oka 1976,
Evolution 30: 380), and the pattern of resource allocation (Sano andMorishima 1977, Ann. Rep. 27: 92) of a wild rice species, Oryza perennis
Moench. These investigations concern the variation in perenniality among
strains of this species. But it has remained unwarranted if the populations
actually exhibit perennial or annual habit in their natural habitats and how
the proportion of sexual and asexual propagations is. To confirm thispoint, Oka has observed eight populations of O. perennis in Thailand in theearly part of the rainy season (19-28 June 1976) when the wild rice plant
had a height of 20-50 em.
The sites selected for observation were waste lowlands between roadembankment and rice field. Such places are regularly disturbed by man inaccordance with the management of rice field, and are inundated forseveral months as the water level rises in the river. At the time of visit inJune, most of the sites were still dry or shallowly flooded. At each site,l-m2 quadrats were set down, and each O. perennis plant was dug up to
examine the underground part as to whether it had sprouted from a stem
node of an old plant (ratooning) or from a seed. The result showed thatone population consisted wholly of seedlings, 5 consisted of ratooned
plants, while two others were mixtures of both. The seedling population
had a larger number of buried seeds than the others. It was in a temporary swamp parched in the dry season, while ratooned populations were
found in more deeply inundated sites. This suggested the role of water
stress in conditioning the propagation behavior.
The plants raised from the seeds of these populations were observed for"reproductive effort" (percent seed weight to total dry-matter weight) andother various attributes of adaptive mechanisms (by Sano). The result

RESEARCHES CARRIED OUT IN 1977

105

proved that the seedling population was of the annual type and the
ratooned populations were of the perennial type, while those performing
mixed sexual and asexual propagations were of intermediate perennialannual type. An observation of isoenzymic variations controlled byalleles at two loci (PXl and ACP1) indicated that the populations showing
mixed breeding behavior were highly polymorphic and heterozygous. Oneof them consisted of 9 separate clumps which differed in characters. Such
populations should have a high evolutionary potentiality. It was suggested
that the wild progenitor of O. sativa could be such intermediate populationsof O. perennis.
A Competition Experiment between Oryza perennis Strains in Greenhouse
Hiroko MORISHIMA

To observe how wild rice strains compete each other through theirvegetative growth and reproduction, three strains, i.e., Wl20 (a perennial
type from India), W593 (a perennial type from Malaya), and W630 (anannual type from Burma) were tested for their interactions. They wereplanted in pure stands and in 1: 1 mixtures of all binary combinations at
3 densities (20- X 20 cm, 10- X 10 cm, and 5- X 5 cm), in 1976 summer in a
concrete bed filled with newly prepared garden soil in a greenhouse. After
seed shedding, the above-ground part of the plants were harvested forrecording several character values, and a half of each plot was cultivatedwith a hoe. Then, the concrete bed was not watered for 6 months until
irrigated in 1977 May. The numbers of buried seeds, seedlings, andratooned plants were recorded in the second (1977) season.
The effect of competition as shown by the changes in performance ofthe first generation plants exhibited a circular order of relationships among
the three strains, that was ,-W630 <WI20<W593 <:-1' Contrary to expectation, the competition effect was stronger in widely spaced planting
than in dense planting. The survivorship (percent ratooned plants in the·second season) was 40 % for W120, 84 % for W593, and 2 % for W630.
The mean number of buried seeds per soil sample (10- X 10 cm, to a 5 em
depth) was 4.5 for W120, 7.9 for W593, and 23.3 for W630. In the second
season, the annual strain (W630) produced a much larger number ofplants than the perennial strains (W120 and W593). The cultivated halfof the plots had larger number of seedlings than the other half.

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

The weeds in the first season, which arose from their buried seeds in thesoil, significantly differed in the number of species as well as in the total
number of plants according to the strains of O. perennis planted. One ofthe purposes of this experiment was to learn to what extent the conditionsin natural habitats can be simulated in a greenhouse. Many difficulties were,
however, experienced for achieving this purpose.
The Inheritance of Copper Tolerance in a Hybrid Population of Rice
Hiroko MORISHlMA

An Oryza sativa strain sensitive to copper (108, an Indica from Taiwan)
and an O. perennis strain highly tolerant to copper (W106, an annual typefrom India) were crossed, and their F 4 lines, 30 in total number, were tested
for copper tolerance in gravel culture (with cultural solution containing 0
and 5 ppm Cu). Copper tolerance was shown by toxic-normal ratios in
performance which was obtained by snythesizing measurements for leafstem dry weight and three other characters. The susceptible parent (l08)gave a ratio of 80 %, while the tolerant parent (WI06) gave 190 %, and thevalues for F 4 lines were distributed between the parental values. The
parent-offspring regression (heritability) was estimated to be 0.36 (significant at 5 % level) by comparing the data with those from a similar test of
F g lines (recorded in 1975).
In general, the plants collected from the field show a tendency to "tradeoff" or negative association of tolerance with the performance in normal
condition. But the hybrid-derived lines presently tested showed no such
trend. The absence of trade-off was also recognized in a hybrid populationof barnyard grass.
Measurements of copper content in the leaf-stem and root of the plants
in copper-treated plot revealed significant differences due to lines. But thecopper content and copper tolerance were not correlated among the lines.
In contrast, in barnyard grass, copper content in the above-ground part ofthe plants was negatively correlated with copper tolerance. It seems thatgenes controlling various physiological processes regulate the expression of<copper tolerance in plants.

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107

Copper Tolerance and Competitive Ability of Alopecurus aequalis Strains
Hiroko MORISffiMA

Strains obtained from copper polluted and unpolluted control fields in
Gumna and Yamanashi Prefectures, 23 in total number, were tested forcopper tolerance. The seed of each strain was sown in pots (l/50 m 2) filledwith soils containing 0 and 250 ppm copper, with two replications. As
reported previously (Ann. Rep. 27: 90), their copper tolerance was evaluatedon the basis of viability in copper-containing soil as compared with that incontrol soil, after the plants were partly uprooted by frost needles in winter.
Strains from polluted fields tended to have higher viability than thosefrom control fields when they were subjected to frost needles in coppercontaining soils, and the heritability of viability shown by parent-offspring
regression was 0.34 (significant at 5 % level). Unlike the cases in barnyard
grass and other summer annuals, the comparison of performance between
the plants in toxic and normal soils did not seem to be useful for evaluating
copper tolerance in this species. Tolerant strains with a high winterviability showed a high copper co ~.tent in the root, but showed no such
trend as to the copper content in the leaves and stems.
Further, the 32 strains were tested for their competitive response to mixplanted milk vetch (Astragalus sinicus) in pots filled with normal soil.
The competitive ability against milk vetch showed a negative correlation
with copper tolerance shown by winter viability (r= -0.50, significant at
5 % level). This serves as an example of "trade-off".

The Self-persisting Ability of Soybean Hybrid Lines
in Semi-natural Conditions
Hiko-Ichi OKA

Glysine soja, the putative wild progenitor of cultivated G. max, naturally
occurs in abandoned fields on the campus of this institute. It was foundthat if the seed was scattered on a grassland, a new population was establishedthere and persited several years. In contrast, the cultivated soybean can
not exist if not seeded by man. To look into the difference in selfpersisting ability, two soybean cultivars, Chung-Hsing 2 and 3 (selected fromhybrids for wide adaptability at Taichung) were crossed with the pollen
grains of a wild strain obtained from the campus. The F 2 plants raised in

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

the 1975 season were observed for various characters and 30 plants showing
different combinations of traits distinguishing the wild from cultivated plants
were selected. The characters recorded were vine-climbing on bamboo
sticks, pod twistedness, pod dehiscence, single seed weight, etc.
The F 3 lines were sown at three sites (A: experimental field, B: abandoned
field, and C: grassland along the edge of a field) in April 1976. At eachsite, a line was represented by 16 plants raised in a l-m 2 plot with 1.5 ill
spacing from the neighboring plots. At sites A and B, a 1.8 m bamboo
stick was stood beside each plant to let it climb up. After recording
characters, the plants were left in the field and were allowed to shed seed.
Then, the field was left untouched and the number of plants naturally
growing in the 1977 season was counted for each line.
The result showed that the number of self-seeded plants per m Z was 0 to
720 (702 for G. soja) at site A, and 0 to 293 (552 for G. soja) at site B. At
site C, a 0 to 100 % range of coverage by the soybean plants was observed
among the 30 lines though the number of plants was not recorded. The
self-persisting abilities thus recorded at three sites were intercorrelated
indicating that they were genotypically controlled. Further, the lines
were planted with a 7 m spacing in 1977 to measure the distance of seeddispersal. How the abilities of self-persistence and seed dispersal areassociated with the characters recorded will be examined by analysing the1978 data.
Interactions among Five Summer Annual Weeds
Hiroko MORISHIMA

To observe the pattern of interactions among different weed species, thisexperiment was conducted with 9 strains belonging to five species of summer
annual weed, i.e., Erechtites hieracifolia (Compositae, symboled D), Arthraxon hispidus (Gramineae, K), Setaria viridis (Gramineae, E), Digitaria
adsendens (Gramineae, M), and Echinochloa crus-galli (Gramineae, H).
The 9 strains were tested in pure and mix (1: 1 with a tester) stands; a K
and a M strain were used as the testers. The seeds of these species collectedin the fall months of 1976 were sown on cultivated field plots (each 1 m2)
with four replications. Starting one month after seeding, two of the fourplots were walked over 5 times a week for three months, while the othertwo plots were left untouched as the controls.

RESEARCHES CARRIED OUT IN 1977

109

The number of plants and dry matter weight per unit area, as well as thedry weight of greatest and smallest 5 single plants were recorded at seedmaturity by harvesting the above-ground part of the plants. The data
indicated first that the effect of walking over markedly differed according to
species. The number of D plants sharply decreased due to walking over, but
M showed no change in number. Second, the effect of mix-planting on survivorship also differed according to species combination and walking over.
In terms of dry matter weight per unit area, M was the strongest competitor while D was the weakest in the control plots. In the walked-overplots, E was as strong as M in competition. Thirdly, the difference in dry
weight between the greatest and smallest plants would be an expression ofphenotypic plasticity. This difference was greatest in K and smallest in D,
and tended to be larger in pure than in mix-planted plots.
Variation in Competitive Ability among Oryza perennis Strains
Yoshio SANO and Hiroko MORISHIMAWith the view to investigate the role of competition in adaptive strategy
of wild rice, a mix-planting experiment of Oryza perennis strains was
carried out. Thirty four perennis and two sativa strains were tested in
mix-planting with a test-strain which was an Asian annual type of O.
perennis (W 107). Dry plant weight at maturity and several other characters
were recorded for each strain. The competitive ability of a strain wasshown by (XI2-XU)-(X21-X22), where Xu and X l2 stand for dry plantweight of strain 1 in pure and in mixed stand with 2, etc.
Among Asian perennis strains, the annual and intermediate annual-perennial types tended to have higher competitive ability than the perennial type.
The American strains had a low competitive ability. A positive correlation
existed between the competitive ability and "reproductive effort" (proportionof seed weight to total plant weight).
Based on covariations between competitive ability and other growthmeasurements, path coefficients were computed to estimate their contributions to the competitive ability. About 68 percent of the variance ofcompetitive ability could be accounted for by three measurements taken frompure stands, density response (coefficient a, in the foregoing report), dry
plant weight at 93 days after germination, and percent plant cover at 70 days.
A simillar experiment with an O. sativa strain (Taichung 65) was reported

110 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

last year (Ann. Rep. 27: 92). The positive association between competitive
ability and reproductive effort found from the present experiment was notin the experiment conducted last year. The competitive ability with a sativa
tester showed a negative correlation with reproductive effort. This indicatesthat the competitive response of perennis strains greatly differ according to
the competitor. Presumably, this difference comes from different growth
patterns and plant types as were also observed among the wild rice strains.
The Density Response of Oryza perennis Strains
Yoshio SANG

It is an established principle that the effect of standing density on plants
is represented by WNa=K, where W is mean single plant weight, N is density
or the number of plants per unit area, K is a constant, and a is a coefficient ofdensity response. To investigate the variations in density response, 23
strains of Oryza perennis (showing a perennial-annual continuum) and 2
O. sativa strains were tested at three density levels. The seedlings of eachstrains were planted at the rate of 2,4, and 16 plants per pot (1/50 m 2), with
2 replications. At the maximum tillering stage (93 days after planting),
the plants were harvested and were measured for the dry weight of leaves
and roots. From the data, the a values were computed in respective
strains. The a value for the above-ground part ranged from 0.51 to 1.10,
and that for the root from 0.39 to 1.04 among the strains tested. In thevalue for root weight, the difference due to strains was statisticallysignificant.
The density response as shown by a was positively correlated with thecompetitive ability of the strains with an annual strain of O. perennis (W
107 from India). Density response is an expression of phenotypic plasticity.
The data indicate that the wild rice strains showing different breeding habits
and adaptive mechanisms differ in density response.

Ecological Factors Controlling the Coexistence of Drosophila
simulans and D. melanogaster
Masaoki KAWANISHI and Takao K. WATANABE

The two sibling species, D. simulans and D. melanogaster, are believed tocoexist in nature sharing food and space. Using banana bait traps, Dro-RESEARCHES CARRIED OUT IN 1977

111

sophiIla flies were monthly collected from October 1975 to November 1976
at three ecologically different sites in Mishima. A total of 11,276 specimens
involving 29 species of Drosophilidae were collected. The relative abundance of D. simulans and D. melanogaster was apparently different at different
sites; the former prefers a semi-domestic niche and the latter a domestic one.
The seasonal abundance of these species was compared. D. simulans
peaked in population size twice a year, in July and September, while D.
melanogaster showed only one population peak, in August. However,this seasonal separation of the sibling species seems to be a temporary
and transient phenomenon, since the optimum temperature for D. simulans
is the same for D. melanogaster. In fact, in some populations at Sapporo
and Hiroshima, where D. simulans was very scare, D. melanogaster showeda clear bimodal seasonal activity, avoiding mid-summer. Thus, D. melanogaster inevitably restricts its breeding season to the mid-summer in
Mishima to lessen the competition with D. simulans. For details, see Jap.
J. Ecol. 27, 279-283.

112 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

XIV.

APPLIED GENETICS

Temperature Responses of Isogenic Lines with Different
Earliness Genes of Rice
H. YAMAGISHl*, K. H. TSAI** and H. 1. OKA

The number of days to heading of rice plants is controlled by various
genes. It was concluded from repeated experiments that the major
earliness genes in photoperiod insensitive Japonica cultivars were E (8thlinkage group) which moved up the time of floral initiation about one
week and m (independent of E) which intensified the effect of E (Tsai
1976, Japan. J. Genet. 51: 115). A number of isogenic lines with different
alleles at these two loci were established from the experiments. In Taiwan,
the m gene produced stronger effect in the winter (first crop) season than in
the summer (second crop) season, and it promoted heading in winter evenwhen combined with e.
Seven isogenic lines carrying gene combinations Em, Em+, em, and em+
were tested in two growth chambers (one adjusted at 30 e in day and25°e in night, and the other adjusted at noe in day and l7°e in night;
both illuminated with about 4,000 lux light for 14 hours a day), an ordinary
greenhouse (temperatures varying between 30° and l8°e, under natural
0

Table l.

Comparison of heading date among isogenic lines
with different earliness genes
Growth chamber

Genotype
e m+ (T65)
Eam+
Eb m +Eam
Eb mem

25°_30°17°_22°o (120)*0(160)
-20
-23
-32
-16
+11

-14
-15
-27
-18
+8

Greenhouse
18°_30°Outdoors,summer
18°_30°Outdoors,
11.5 h/day
18°_30°o (108)0(112)
-6
-8
-27
-20

0(104)
-7
-7
-14

-9

-1

-41
-41
-50
-33
-3

-10

* Number of days to heading in Taichung 65* Vegetables Experiment Station, Tsu City.
** Food and Crop Research Institute, National Chung-Hsing University, Taichung.

RESEARCHES CARRIED OUT IN 1977

113

days in winter), and in two concrete beds (outdoor, one with 11.5 hour-days,
and the other under natural daylength, in summer). The result of thisexperiment demonstrated that the heading-promoting effects of E and mmarkedly differed according to the environment. The mean number ofdays to heading of the four genotypes under respective conditions are given
in Table 1, in which the number of days obtained for Taichung 65 (em+)
was taken as zero. In the growth chambers, particularly in the lowtemperature chamber, the m gene appreciably retarded heading whencombined with e, though it promoted heading in the greenhouse and field.
This indicates that the effect of m is sensitive to temperatures. The
epistatic effect of man E was much stronger when m was combined with Ea(from northern China) than with E b (from Hokkaido). The heading dateof lines with Ea m was retarded by short days.
Noise Sensitivity of Wild and Domestic Japanese
Quails as Shown by the Fertility of Eggs':'
Takatada KAWAHARA

When expose to noise, wild strains of Japanese quail showed a more
remarkable depression in egg laying performance than the domestic control(Ann. Rep. 27: 95). The effect of noise on the fertilities of four mating
combinations between the wild and domestic birds was investigated thisyear. A total of 92 matings between the wild (W) and domestic (D) strains,
WxW, WxD, DxW, and DxD, were examined. The birds at an ageof 130 days were subjected to a noise treatment: a buzzer of 95 phonsworked 12 times a day, once for one hour for 20 days. Egg fertilites wereTable 1. Effects of buzzer treatment on fertilityof wild and domestic strains
MatingFertility (%)

Female x MaleNo. ofmatings
testedPretreatment

During noise
treatment

Posttreatment

WxW
WxD
DxW
DxD

47
10
25
10

70.3
74.6
79.0
96.2

57.4
69.0
63.7
98.8

63.5
78.5
81.4
93.4

.. _--------._------"------~*

This research was supported by a grant from the Environment Agency.

114 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

recorded in three periods of 9 days each, which were chosen on the basis of
the time from fertilization to egg laying. Those were noiseless pre-treatment
period, during noise treatment, and noiseless post-treatment period.
As shown in Table 1, the matings ofWxW, WxD, and DxW had reduced fertilities in response to noise treatment. But the D X D mating
showed no significant change. In other words, when the wild strain wasused as either male or female parent, fertility was reduced. The wild strain
used for this experiment had been reared in the cage for 10 generations after
the capture of original birds in the field. The birds showed remarkablechanges in productive and behavioral characters toward the domestic typeas the result of "unconscious selection" in the cage (Ann. Rep. 24: 72;
Kawahara, T. 1976, Experimental Animals 25: 351). Yet, they retained
the higher noise sensitivity of the wild type as was demonstrated in thisexperiment.
Nitrogen Fixation in Rhizosphere of Rice Plant
Yukinori HIROTA, Taro FUJII, Yoshio SANa and Sinya IYAMAIn order to investigate the nitrogen fixing ability in paddy rice, the following experiment were carried out.
(A) Improvement of an assay system.
So far various assay systems have been reported to investigate nitrogen
fixing activity of rice rhizosphere. The result of them showe dconsiderable
time lag before the nitrogen fixing activity appeared, arousing a question
whether the activity measured was due to a real nitrogen fixation of ricerhizosphere or not. In our system, that of an intact plant was assayed underanaerobic conditions as described below. Whole sample of an intact plant
which included the soil and water or rice rhizosphere and the bulk of
relatively undisturbed root system was enclosed in a vessel so that the
exposure of rhizosphere to the air was minimized. Rigorous displacementof gas phase in the vessel with a gas-mixture of helium and acetylene (9: 1
in volume) was achieved two times repeatedly by a evacuation pump andflushing the gas-mixture up to 1 atm. Then the vessel was kept for 30 min.
to equilibrate the system with the gas, then it was evacuated and the gasmixture was flushed again. The vessel was incubated under the laboratory
room light condition at 28°C and fractions of the gas phase were withdrawn as a function of time using syringe for assay. Amount of ethylene

RESEARCHES CARRIED OUT IN 1977

115

produced from acetylene were measured with a gas-chromatography.
Considerable activity of acetylene reduction was found in certain strainsof rice. The production of ethylene from acetylene proceeded linearly andno time lag of the reaction was observed. When acetylene was not added
to the system, no ethylene was produced. Soil without plant showed no
detectable acetylene reducing activity during 6 hours of incubation andlater small but significant amount of ethylene production was observed.
Aerobic condition for the acetylene reduction assay showed lower activity
than anaerobic condition. Thus, our assay system should be considered
to measure the nitrogen fixing activity in rice rhizosphere properly.
(B) Genetic variation of nitrogen fixation.
The nitrogen fixation activity of rhizosphere of 50 strains of rice wasmeasured. The strains used were those collected from various places in thetropical Asia and preserved at the Genetic Stocks Center in this institute.
The plants were grown in pots until they reached to the flowering stage andthe activity of these intact plants, two each a strain, was measured under
the assay condtions described above. A wide range of variation wasobserved among strains (Table 1). Moreover, strong correlation (r=0.895, significant at the 1 % level) was observed between root weight andnitrogen fixing activity within a strain. Namely, the larger the amount orroot per unit area, the higher the activity was observed. This means thatexistence of rice root is apparently essential in nitrogen fixation by bacteria
in paddy field.
Table 1.

Variation in acetylene reducing activity of 50 rice strains
ethylene produced (n mole/plant/hour)

No. of strains

0-200-400-600-800-1000-1200
13 23 8 2 3
1

Total number ofstrains observed50---'-----------------------(C) Rice-bacterial association.
Seeds of a rice strain which exhibited a high activity of nitrogen fixation
were sterilized and seeded on sterilized soil, and grown under the germfree growth condition. The activity of nitrogen fixation was measured.
No activity of acetylene reduction was detectable on the sterile plant,suggesting the presence of microbial involvement in the activity. An
anaerobic bacterium, rod shaped, spore formers at the cell terminus, gram
positive and biotin require as a growth factor was isolated. It is likely to116 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

be a Clostridium. It is known however that from a single soil sample one
can isolate many species of soil bacteria capable of fixing nitrogen, and it
is difficult to judge whether or not this bacterium is responsible for thenitrogen fixation of rice rhizosphere. A systematic examination of nitrogen
fixing bacteria in the rhizosphere of rice in our experimental field are in
progress.
Analysis of Genes Controlling the F 1 Sterility between
Oryza sativa and O. glaberrima
Yoshio SANO

Experiments on the genetic basis of the F 1 sterility between two cultivatedrice species, O. sativa and O. glaberrima, have been continued since 1966
(Ann. Rep. 24: 67, 25: 85, & 27: 80). In the previous work, an Indica
strain (108 from Taiwan) was used as the sativa parent, and from its crosswith a glaberrima strain (W 025 from Sierra Leone), isogenic F)-sterile lines
with the genetic background of either parent were established and wereused for crossing experiments. The result proved that the F 1 sterility wasat least partly controlled by alleles at a certain locus which produced aparticular sporo-gametophytic interaction in heterozygote (Ann. Rep. 27: 80).
A following-up experiment was initiated in 1975 by using a Japonica
strain, T65, as the sativa parent, and W025 as the glaberrima parent in thesame manner as in the preceding experiment. The backcross generations
so far observed (up to B6) showed a different pattern of segregation from thatobserved before. Namely, out of 52 B5 F 1 and 23 B6 F 1 (75 in total), 33 weresemi-sterile and 41 were fertile, giving a 1: 1 ratio. Since two of the lines
showing the same pattern of segregation had the cytoplasm of glaberrima
parent, the possibility that the sterility is controlled by gene-cytoplasm
interaction may be rouled out.
This type of segregation has been observed throughout backcrossing
experiments between distantly related sativa varieties (aka 1974, Genetics
77: 521). On a series of evidence, the partial F 1 sterility between sativa
cultivars was attributed to duplicate gametic lethals. Possibly, a similar
genic system is involved in the F 1 sterility between the two rice species.
Yet, it seems plausible to assume that various genic systems contol the F jsterility between two sibling species, and most dominant one is selected
when the backcross method is used for isolating isogenic F)-sterile lines.

RESEARCHES CARRIED OUT IN 1977

117

Breeding of Isogenic Lines of Rice Carrying Gene Markers
in Interchanged Chromosome Segments
Yoshio SANOThis work was initiated by Oka (Ann. Rep. 24: 66), and has been continued for 11 years. Isogenic lines of Taichung 65 having different genemarkers, respectively, were early established. The genes chosen as markers
were nl (neck leaf), be (brittle culm), g (long empty glume), 19 (ligulelessness), gl (glabrousness), Rc (brown pericarp), wx (glutinous endosperm),
etc. The pleiotropic effects of these genes on metric characters weresometimes significant but trivial in general (Ann. Rep. 25: 84). If thesegene markers are involved in interchanged chromosome segments, theisogenic lines carrying them may be more useful testers for genic analysis.
For this purpose, crosses were made between isogenic marker lines andisogenic translocation lines (Ann. Rep. 27: 102).
This work is still under way, partly with the correlation of Dr. S. Satoof the Ryukyu University. So far, 7 lines carrying a marker in a translocatedsegment (T2 U, T39 u, T44 u, T55 u, T52 nl , T54 bc , and T55 1u) were isolated. In
many of them, the recombination value between marker and the point ofinterchange was about 10 % or less. These will be used for observing thegenetic control of attributes of adaptive mechanisms.

118 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

BOOKS AND PAPERS PUBLISHED IN 1977
BY STAFF MEMBERS
ABE, T., M. MORITA, K. KAWAI, S. MISAWA, T. TAKINO, H. HASHIMOTO and Y. NAKAGOME
1977: Partial tetrasomy 9 (9pter->9q2101) due to an extra iso-dicentric chromosome. Ann. Genet. 20: 111-114.
CHANG, T. T. and H. I. OKA 1976: Genetic variousness in the climatic adaptation of ricecultivars. Proc. Symp. Climate and Rice, IRRI, 87-111.
HIGURASHI, M., M. SEGAWA, I. MATSUI, K. IrNUMA and Y. NAKAGOME 1977: Screening
for autosomal aberrations. Acta Paediat. Scandinav. 66: 501-504.
HIROTA, Y., H. SUZUKI, Y. NISHIMURA and S. YASUDA 1977: On the process of cellulardivision in Escherichia coli: a mutant of E. coli lacking a murein-lipoprotein. Proc.
Nat. Acad. Sci. USA 74: 1417-1420.
IMAI, H. T., R. H. CROZIER and R. W. TAYLOR 1977: Karyotype evolution in Australianants. Chromosoma (Bed.) 59: 341-393.
INOUYE, S., N. LEE, M. INOUYE, H. C. Wu, H. SUZUKI, Y. NISHIMURA, H. IKETANI and
Y. HIROTA 1977: Amino acid replacement in a mutant lipoprotein of the Escherichia
coli outer membrane. J. Bacteriol. 132: 308-313.
INOUE, T. and T. KADA 1977: Studies on DNA repair. Ill. Identification of an exonuclease which enhances the priming activity of r -irradiated DNA by "cleaning
damaged ends". Biochim. Biophys. Acta 478: 234---243.
INOUE, T., K. HIRANO, A. YOKOIYAMA, T. KADA and H. KATO 1977: DNA repair
enzymes in ataxia telangiectasia and Bloom's syndrome fibroblasts. Biochim.
Biophys. Acta 479: 497-500.
ISONO, K., A. G. CUMBERLIDGE, S. ISONO and Y. HIROTA 1977: Further temperaturesensitive mutants of Escherichia coli with altered ribosomal proteins. Molec. gen.
Genet. 152: 239-243.
KADA, T., Y. SADAIE and M. HARA 1977: Analysis of mutagen-antimutagen reactions
in food additives by the rec-assay and reversion-assay procedures. Proc. 2nd Int.
Conf. on Environm. Mutagens, Edinburgh.
KANEMATSU, N., M. HACHINOHE, D. HATANAKA, K. SHIBATA, Y. KURODA and H.
KAWAHARA 1977: Effect of metal ions on tissue culture cells. I. Effect of metal
ions on colony formation of Chinese hamster Don cells. Japan. J. Oral Sur. 23: 1-7.
KATO, H. 1977: Mechanisms for sister chromatid exchanges and their relation to the
production of chromosomal aberrations. Chromosoma (Bed.) 59: 179-191.
KATO, H. 1977: Spontaneous and induced sister chromatid exchanges as revealed by
the BUdR-labeling method. Intern. Rev. Cytol. 49: 55-97.
KATO, H. and A. A. SANDBERG 1977: Effects of herpes simplex virus on sister chromatid
exchange and chromosome abnormalities in human diploid fibroblasts. Exp. Cell
Res. 109: 423-427.
KATO, H. and A. A. SANDBERG 1977: The effect of sera on sister chromatid exchanges inBOOKS AND PAPERS PUBLISHED IN 1977

119

vitro. Exp. Cell Res. 109: 445-448.
KAWANISHI, M. and T. K. WATANABE 1977: Ecological factors controlling the coexistenceof the sibling species Drosophila simulans and D. melanogaster. Jap. J. Ecol. 27:
279-283.
KIMURA, M. (ed.) 1977: Molecular Evolution and Polymorphism. Proceedings of theSecond Taniguchi International Symposium on Biophysics. Nat. lnst. Genet.
(Misima).
KIMURA, M. 1977: Preponderance of synonymous changes as evidence for the neutral
theory of molecular evolution. Nature 267: 275-276.
KIMURA, M. 1977: Genetic codes and the laws of evolution as the bases for our understanding of the biological nature of man. Proc. of the 5th International Conf. on
the Unity of the Sciences, Washington, D.C. (1976), 621-630.
KIMURA, M. 1977: Causes of evolution and polymorphism at the molecular level. Proc.
of the 2nd Taniguchi International Symp. on Biophysics (ed. by M. Kimura), 1-28.
KIMURA, M. 1977: Essai sur notre passe, notre present et notre avenir: Ie point de vue
d'un geneticien des populations. Mem. de l'Acad. des Sci., Inscriptions et BellesLettres de Toulouse 139: 103-107.
KIMURA, M. 1977: The neutral theory of molecular evolution and polymorphism.
Scientia 112: 687-707.
KIMURA, M. and T. OHTA 1977: Further comments on "counter-examples to a neutral
hypothesis". J. Mol. Evol. 9: 367-368.
KURODA, Y. 1977: Vitamin E counteracting the bisulfite inhibition of aggregations ofembryonic quail liver cells in culture. Cell Structure and Function 2: 211-218.
KURODA, Y. 1977: Studies on Drosophila embryonic cells in vitro. II. Tissue- andtime-specificity of a lethal gene, deep orange. Develop. Growth and Differ. 19: 57-66.
KURODA, Y. 1977: Embryonic development of insects. (In Japanese) Zoku-SaibogakuTaikei 2. Animal Cell Science (ed. by K. Ogawa et al.), 341-366, Asakura-Shoten(Tokyo).
KURODA, Y. 1977: CeIl-to-ceIl interactions. (In Japanese) "Cell Biology" (ed. by K.
Oda) Bunkodo Medical Monograph Series, 275-291, Bunkodo (Tokyo).
KURODA, Y., A. YOKOIYAMA and T. KADA 1977: Selection and characterization of a 5iodouridine-resistant variant in cultured Syrian hamster cells. Jap. J. Genet. 52:
133-147.
LEE, W. H. and T. K. WATANABE 1977: Accumulation of deleterious genes in a cage
population of Drosophila melanogaster. Genetics 86: 657-664.
MARUYAMA, T. 1977: Stochastic Problems in Population Genetics (Lecture Notes in
Biomathematics, 17), Springer-Verlag (Berlin).
MARUYAMA, T. 1977: Human genetic load. "Gene-Environment Interaction in Common Diseases" (eds. by E. Inouye & H. Nishimura), 41-64.
MARUYAMA, T. 1977: Comments on genetic load. Ibid. 223-224.
MATSUDA, H., N. TAKAHATA and T. GOJOBORI 1977: A diagnosis of the mechanism ofprotein polymorphism. Proc. of the 2nd Taniguchi Internat'l Symp. on Biophysics(ed. by M. Kimura), 168-188.
MATSUHASHI, M., Y. TAKAGAKI, I. N. MARUYAMA, S. TAMAKI, Y. NISHIMURA, H. SUZUKI,

120 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28
U. OGINO and Y. HIROTA 1977: Mutants of Escherichia coli lacking in highly
penicillin-sensitive D-alanine carboxypeptidase activity. Proc. Nat. Acad. Sci. USA
74: 2976-2979.
MATSUNAGA, E. 1977: Following retinoblastoma patients: Carcinogenesis and mutation.
(In Japanese) Hokkaido Med. J. 52: 83-89.
MATSUNAGA, E. and K. SHIOTA 1977: Use ofprogestagens for prevention of miscarriages
and malformed embryos. Teratology 16: 113-114.
MATSUNAGA, E. and K. SHIOTA 1977: Holoprosencephaly in human embryos:
Epidemiologic studies of 150 cases. Teratology 16: 261-272.
MATSUNAGA, E. and K. SHIOTA 1977: Epidemiologic study of holoprosencephaly inhuman embryos. Proc. 5th Int. Conf. Birth Defects (Excerpta Medica Internat'l
Congress Series, No. 426), 101.
MATSUNAGA, E. and H. FUJITA 1977: A survey on maternal age and karyotype in
Down's syndrome in Japan, 1947-1975. Hum. Genet. 37: 221-230.
MORISHlMA, H. 1977: Numerical approaches to the species problem: Example in wild
and cultivated rice species. Proc. Jap. Soc. Plant Tax. 3: 5-9.
MORISHlMA, H. and H.1. OKA 1977: The impact of copper pollution on weed communities in Japanese rice fields. Agro-Ecosystems 3: 131-145.
MORISHlMA, H. and H. 1. OKA 1977: The impact of copper pollution on barnyard grass
populations. Jap. J. Genet. 52: 357-372.
MORIWAKI, K., T. AOTSUKA and T. SHIROISHI 1977: A simplified method for reading
hemagglutinations on a flat-bottom microtitration plate in the mouse H-2 assay.
Experientia 33: 673-674.
NAKAGOME, Y. 1977: Initiation of DNA replication in human chromosomes. Exp.
Cel1 Res. 106: 457-461.
NAKAGOME, Y., S. OKA and E. MATSUNAGA 1977: LBA technique in the detection ofchromosome variants. II. Chromosomes except for those with Q variants. Hum.
Genet. 38: 307-314.
NAKAGOME, Y., S. OKA and M. HIGURASHI 1977: Quinacrine banding without afluorescence microscope. Lancet 2: 139-140.
NAKAGOME, Y, T. KITAGAWA, K. IINUMA, E. MATSUNAGA, T. SHINODA and T. ANDO 1977:
Pitfalls in the use of chromosome variants for paternity dispute cases. Hum. Genet.
37: 255-260.
NIKAlDO, H., P. BAVOIL and Y. HIROTA 1977: Outer membranes of gram-negative
bacteria. XV. Transmembrane diffusion rates in lipoprotein-deficients mutants ofEscherchia coli. J. Bacteriol. 132: 1045-1047.
NISHIMURA, H. and K. SHIOTA 1977: Summary of comparative embryology and teratology. "Handbook of Teratology" (ed. by J. G. Wilson and F. C. Fraser), 3:
119-154, Plenum Press (New York).
NISHIMURA, H., K. SHIOTA, T. TANIMURA, T. MIZUTANI, M. MATSUMOTO and M. UEDA
1977: Levels of polychlorinated biphenyls and organochlorine insecticides inhuman embryos and fetuses. Paediatrician 6: 45-57.
NISHIMURA, Y., Y. TAKEDA, A. NISHIMURA, H. SUZUKI, M. INOUYE and Y. HIROTA 1977:
Synthetic ColEl plasmids carrying genes for cell division in Escherichia coli. Plasmid

BOOKS AND PAPERS PUBLISHED IN 1977

121

1:67-77.
OGATA, K., K. IINUMA, K. KAMIMURA, R. MORINAGA and J. KATO 1977: A case reportof a presumptive +i(l8p) associated with serum IgA deficiency. Clin. Genet. 11:
184-188.
OKA, S., Y. NAKAGOME, E. MATSUNAGA and M. ARIMA 1977: LBA technique in the'detection of chromosome variants. I. Chromosomes with known sites of Q variants.
Hum. Genet. 39: 31-37.
OKA, S., Y. NAKAGOME, F. TERAMURA, F. HosoNo and K. KATAOKA 1977: Trisomy!
partial monosomy mosaicism of no. 13 pair [46, XX, -13, +rob(13q13q)/46,XX,
r(13)(pllq34)]. Jap. J. Hum. Genet. 22: 73-78.
OHTA, T. 1977: Genetic variation in multigene families. Nature 267: 515-517.
OHTA, T. 1977: On the gene conversion model as a mechanism for maintenance of
homogeneity in systems with multiple genomes. Genet. Res. 30: 89-91.
OHTA, T. 1977: Extension to the neutral mutation random drift hypothesis. Proc. of the
2nd Taniguchi International Symp. on Biophysics (ed. by M. Kimura), 148-167.
OHTSUKA, E., S. NISHIKAWA, R. FUKUMOTO, S. TANAKA, A. F. MARKHAM, M. IKEHARA
and M. SUGIURA 1977: Joining of synthetic ribotrinucleotides with defined sequences
catalyzed by T4 RNA ligase. Eur. J. Biochem. 81: 285-291.
SADAIE, Y., K. NARUI and T. KADA 1977: Differential effects on a DNA-synthesis
mutation on UV-induced mutation yields in vegetative cells and spores of Bacillus
subtiUs. Photochem. PhotobioI. 26: 161-162.
SANO, Y. 1977: The pollination systems of Melitotus species. OEcol. Plant. 12: 383-394.
SHIMOTOHNO, K. and K. MIURA 1977: Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus. J. Biochem. 81: 371-379.
SHIMOTOHNO, K" Y. KODAMA, J. HASHIMOTO and K. MIURA 1977: Importance of 5'terminal blocking structure to stabilize mRNA in eukaryotic protein synthesis.
Proc. Nat. Acad. Sci. USA 74: 2734-2738.
SHIOTA, K. and E. MATSUNAGA 1977: An epidemiological study of polydactylism inhuman embryos. Teratology 16: 122.
SHIRASU, Y., M. MORIYA, K. KATO, F. LIENARD, H. TEZUKA, S. TERAMOTO and T. KADA
1977: Mutagenicity screening on pesticides and their modification products: a basisof carcinogenicity evaluation. "Origins of Human Cancer" (ed. by H. H. Hiatt &others) Book A: 267-285, Cold Spring Harbor Lab.
SOEDA, E., K. MIURA, NAKASO, A. and G. KIMuRA 1977: Nucleotide sequence aroundthe replication origin of polyoma virus. FEBS Lett. 79: 383-389.
SUGIURA, M. and K. YOSHINAGA 1977: A thermosensitive mutant of E. coli with analtered RNA polymerase subunit. 11th FEBS Meeting, Copenhagen, Aug., 1977,
Abstracts A2-4-206.
SUGIURA, M. and K. MIURA 1977; Transcription of double-stranded RNA by Escherichia
coli DNA-dependent RNA polymerase. Eur. J. Biochem. 73: 179-184.
SUGIURA, M., K. SEGAWA, K. YOSHINAGA, F. Yu, N. ITO, S. YASUDA and Y. HIROTA 1977:
A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase{3' subunit. Biochem. Biophys. Res. Comm. 76: 739-745.
SUGIYAMA, T. and T. FUJISAWA 1977: Genetic analysis of developmental mechanisms in122 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NU. 28
hydra. I. Sexual reproduction of Hydra magnipapillata and isolation of mutants.
Develop. Growth and Differ. 19: 187-200.
SUGIYAMA, T. and T. FUJISAWA 1977: Ibid. III. Characterization of a regeneration
deficient strain. J. Embryo!. Exp. Morpho!. 42: 65-77.
SUZUKI, Y., K. ONO, S. OKA, T. MATSUBARA, M. ARIMA and Y. NAKAGOME 1977: A caseof (13q; 18q) translocation with proximal13q monosomy. Hum. Genet. 38: 337-341.
TAZIMA, Y. 1977: Stabilinosti i nestabilinosti genetycheski system na primere chelko_
pryada. Ploblemi Eksperimentalinoi Biologii, Akademia Nauk SSSR, 122-131.
TSUJI, H., H. KATO and K. MORIWAKI 1977: Unsually high incidence of spontaneouschromosomal aberrations in mouse primary cell culture. Jap. J. Genet. 52: 65-71.
WATANABE, S., T. SHIBATA and T. KAWAHARA 1977: Esterase-D isozymes in Japanese
quai!. (In Japanese) Jap. Poultry Sci. 14: 66-70.
WATANABE, T. K. 1977: Genetic variation in reproductive ability observed in natural
populations of Drosophila melanogaster. Adv. in Invertebrate Reprod. (ed. by
K. G. Adiyodi & R. G. Adiyodi) 1: 25-35, Peralam-Kenoth (Kerala).
WATANABE, T. K. and T. WATANABE 1977: Enzyme and chromosome polymorphisms inJapanese natural populations of Drosophila melanogaster. Genetics 85: 319-329.
WATANABE, T. K. and W. H. LEE 1977: Sterile mutation in Drosophila melanogaster.
Genet. Res. 30: 107-113.
WATANABE, T. K. and M. A. YAMADA 1977: Effects of SR factor and da gene on theviability of the hybrid between Drosophila melanogaster and D. simulans. Jap. J.
Genet. 52: 9-14.
WATANABE, T. K., W. H. LEE, Y. INOUE and M. KAWANISHI 1977: Genetic variation of
the hybrid crossability between Drosophila melanogaster and D. simulans. Jap. J.
Gent. 52: 1-8.
YAGI, Y., NAKAMURA, I. TOMITA and K. TUTIKAWA 1977: Teratogenicity and distribution of di(2-ethylhexyl) phthalate in mice. J. Toxic. Sci. 2: 317.
YAGI, Y., Y. NAKAMURA, I. TOMITA, K. TUTIKAWA and N. SHIMOI 1977: Embryotoxicityof phthalate esters in mouse. Proc. of 1st Inter. Congr. on Toxicology (Toronto), 33.
YASUDA, S. and Y. HIROTA 1977: Cloning and mapping of the replication origin ofEscherichia coli. Proc. Nat. Acad. Sci. USA 74: 5458-5462.
Y AZAKI, K. and K. MIURA 1977: Terminal structure involving a single-stranded stretch
in the double-stranded RNA from Penicillium chrysogenum virus. Virology 82:
14--24.
YOSIDA, T. H. 1977: Frequencies of chromosome polymorphism in pairs no. 1, 9 and
13 in three geographical variants of black rats, Rattus rattus. Chromosoma (Ber!.)
60: 391-398.
YOSIDA, T. H. 1977: Supernumerary chromosomes in the black rat (Rattus rattus) andtheir distribution in three geographical variants. Cytogenet. Cell Genet. 18: 149-159.
YOSIDA, T. H. 1977: Karyological studies on hybrids between Asian, Ceylonese andOceanian type black rats, with a note on an XO female occurring in the F 1 generation.
Cytogenet. Cell Genet. 19: 262-272.
YOSIDA, T. H. 1977: New inversion of the pair no. 3 chromosome in a black rat.
Experientia 33: 1022-1023.

BOOKS AND PAPERS PUBLISHED IN 1977
YOSIDA, T. H. and C. TAYA 1977:

Studies on interspecific hybridization in the rodents.
I. Artificial insemination between the Norway rat (~) and black rat «5') and the"resulting karyotypes in the hybrid blastocysts. Jap. J. Genet. 52: 289-299.
YOSillNAGA, K. and M. SUGIURA 1977: Physiological studies on a temperature-sensitive
Escherichia coli mutant with an altered RNA polymerase p'-subunit. Biochim.
Biophys. Acta 479: 172-179.

124 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

ABSTRACTS OF DIARY FOR 1977
January
February

24
14
23
March
9
16
25
April
18
June
1
22
August29
September 7
9
October
5
18
November 2
17
22
26
December 5
16

241st Meeting of Misima Geneticists' Club
242nd Meeting of Misima Geneticists' Club
125th Biological Symposium
243rd Meeting of Misima Geneticists' Club
244th Meeting of Misima Geneticists' Club
245th Meeting of Misima Geneticists' Club
126th Biological Symposium
246th Meeting of Misima Geneticists' Club
127th Biological Symposium
247th Meeting of Misima Geneticists' Club
128th Biological Symposium
129th Biological Symposium
130th Biological Symposium
131st Biological Symposium
132nd Biological Symposium
248th Meeting of Misima Geneticists' Club
133rd Biological Symposium
134th Biological Symposium
135th Biological Symposium
136th Biological Symposium

FOREIGN VISITORS IN 1977

125

FOREIGN VISITORS IN 1977
February
April

23
7-14
18-19
22-23
June 29-July 12
September 6-7
October
9
13

GROS, F., L'Institut Pasteur, France.
CROW, J. F., University of Wisconsin, USA.
FITCH, W. M., University of Wisconsin, USA.
AMBLER, R. P., University of Edinburgh, UK.
JONEJA, M. G., Queen's University, Canada.
SCHNEIDERMAN, H. A., University of California, USA.
STERN, H., University of California, San Diego, USA.
CONSTANS, J., Centre National de la Recherche Scientifique, France.
November 8
SIDDIQUI, K. A., Atomic Energy Agricultural ResearchCenter, Pakistan.
23
NARAYANSWAMI, N., Commissioner for Sericulture,
Government of India.
23-24 FRAENKEL-CONRAT, H., University of Calfornia, USA.
26
SPIRIN, A. S., Institute of Protein Research, USSR.
December 5
MAHER, V. M., Michigan State University, USA.
MCCORMICK, J. J., Michigan State University, USA.
5
16
ARLETT, C. F., University of Sussex, England.

126

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 28

AUTHOR INDEX. ......•............•......... 81, 82

AMANO, E.

MATSUNAGA, E.40, 4184, 11442, 43
98
72, 7371, 7377, 7935, 3677
23, 25, 114
40
35
68
76, 78, 79
75, 88114
76, 78, 79
60
77
56, 58, 60
50
113
92, 11086, 90
.48
12
101
34
35
.43, 44, 45, 46, 47
21
88, 97, 98
86
77
94

MATSUTANI, E

44

ENDO, T.

..

FUJII, T.

..

FUJISAWA, T.

..

FUJISHIMA, T
FUKASE, Y
GOTO, M.

..

HARA, M.

..

HIRAI, H.

.

HIRANO, K.

..

HIROTA, Y
IHARA, M
IKAwA, Y
IMAI, H. T.

.

INOUE, T.

..

INOUE, Y.

..

IYAMA, S

KAnA, T. ..
KANDA, N
KANEMATSU, N.

.

KATO, H. ..
KATSUKI, M.

..

KAWAHARA, T
KAWANISHI, M.
KIMURA, M.

..

..

KITAZAWA, T. •KODAMA, Y
KOlZUMI, T.

..

KONDO, K

Ku, T. Y
KURODA, Y.

.

KUSUDA, J
LEE, W. H.

.

MARUYAMA, T.

..

MATSUMOTO, H.

.

MIlU, M
MINEZAWA, M
MIURA, K.

.

74
26, 32, 34, 35
12, 14, 15...101,105,106,107,108,109
79
MORIWAKI, K.
26, 28, 30, 31, 32, 34, 35, 36
MURAKAMI, A. ...48, 50, 68, 71, 73, 74,99
NAKAGOME, Y
95
NAwA, S37, 39,47
NISHIJIMA, H
73
NISHIMURA, Y. ..
23, 25NOGUCHI, T.
..
.51, 53, 54
OCRIAl, Y
62
OHNUMA, A
68, 99
OHTA, T.
..
86, 87
OHTA, Y78
OHTSUKI, Y.48
OKA, H.1.
101, 104, 107, 112
ONlMARU, K.
..
72
OSHIMA, C. ..
97
SANO, Y. . .. 101,104,109,110,114,116,117
SHIMOTOHNO, K.
..
12, 14SHIOTA, K.
..
94
SHIROISHI, T.
..
28, 30, 31
SOEDA, E
15
SUDTO, P
35
SUGIURA, M
18, 19,20,21
SUGIYAMA, T.42, 43SUZUKI, H
23
TAYA, C65
TAZIMA, Y. .
70, 71, 72
TSAl, K. H.
.
112
TSUCHIYA, K
35
UCHIDA, T. A. ..
36
UKAI, Y81
WATANABE, 1. .......•.•......•......••........ 50
WATANABE, T. K
75, 88, 92, 98,110
YAMADA, M. A.
37, 39,47
YAMAGISHI, H. . ...•...•.......•............. 112
YASUDA, S
25
YOKOIYAMA, A.
..
76
YOSIDA, T. H ....... 61, 62, 63, 64, 65, 66, 67
MORISHIMA, H.

MORITA, K

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