NATIONAL INSTITUTE OF GENETICSJAPANANNUAL REPORT
No. 261975•

Publi.hed bll

THE NATIONAL INSTITUTE OF GENETICS
Mi.ima, Sizuoka-ken, Japan1976

Annual Repor~of ~heNa~ional Ins~i~u~eof Gene~ics

No. 26, 1975

Published by

The National Institute of Genetics, Japan
1976

CONTENTS
General statement
Staff
Council.. ..
..
.. ..
.. .. .. . . .. ..
.. .. .. . . ..
Association for propagation of the knowledge of genetics
Projects of research for 1975 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
............................
Researches carried out in 1975

I.

II.

III.

I
3
7
7
8
12

Molecular Genetics
The modified structure at the 5'-terminus of mRNA in a bonemarrow cell which produces hemoglobin vigorously. MIURA,
K., FURVICHI, Y. and MIURA, Y. . . . . . . . . . . . . . . . . . . . . . . . .
Modified structure in messenger RNA of reovirus. SHIMOTOHNO,
K. and MIURA, K.
Modified structure in messenger RNA of rice dwarf virus. SHIMOTOHNO, K. and MIURA, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structure of the double-stranded RNA in a virus of Penicillium
chrysogenum. YAZAKI, K. and MIURA, K.
Mechanism of formation of the modified structure at the 5'terminus of mRNA of silkworm cytoplasmic polyhedrosis
virus. SHIMOTOHNO, K. and MIURA, K. . . . . . . . . . . . . . . . . . .
A new acid phosphatase from Escherichia coli cells. SUGIURA, M.
Induction of RNA ligase activity in bacteriophage T4-infected
Escherichia coli cells. SUGIURA, M. and SEGAWA, K.

16

Microbial Genetics
An Escherichia coli mutant defective in murein lipoprotein synthesis. NISHIMURA, Y., SUZUKI, H., YASUDA, S., IKETANI, H.
and HIROTA, Y.

17

Biochemical Genetics and Immunogenetics
A simplified method for reading hemagglutinations on a flatbottom microtitiration plate in the mouse H-2 assay. MORIWAKI, K., AOTSUKA, T. and SHIROISHI, T.
H-2 antigenic specificities in the Japanese wild mouse, Mus

12
12
13
13

14
15

20

ii

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

musculus molossinus. MORIWAKl, K., SHIROISHI, T., AOTSUKA,
T., MINEZAWA, M. and KONDO, K.
Studies on protein polymorphism of Japanese wild mouse, Mus
musculus molossinus. MINEZAWA, M., MORIWAKI, K. andKondo, K.

IV.

V.

Developmental and Somatic Cell Genetics
Estimation of genetic load and isolation of mutants of hydra fromsexual crosses. SUGIYAMA, T. and FUJISAWA, T.
Nematocyte deficient strains of hydra. FUJISAWA, T. and SUGIYAMA, T.
Regeneration deficient strains of hydra. SUGIYAMA, T. and
FUJISAWA, T.
A tissue-specific defect in developmentally lethal cells fromfused embryos in Drosophila melanogaster. KURODA, Y.. . . .
Reverse mutations of 8-azaguanine resistance to sensitivity inhuman Lesch Nyhan diploid cells in culture. KURODA, Y.. .
The effect of cell density on induction of 6-thioguanine resistantmutations in Chinese hamster cells in culture. KURODA, Y.
and SHIBUYA, T.
Changes in cell-to-cell cohesive activity during cell cycle of HeLa
53 cells in rotation culture. KURODA, Y.
Protective effect of vitamin E on bisulfite-caused inhibition of
aggregation of embryonic quail liver cells in culture.
KURODA, Y.
Effective lethal phase of the curly mutant in Drosophila melanogaster. KURODA, Y. and MINATO, K.
Cytogenetics
Population survey of B-chromosomes in black rats. YOSIDA,
T. H.
Further studies on the frequencies of chromosome polymorphismin pairs No.1, 9 and 13 of the black rats in Japan. YOSIDA,
T. H.
Frequencies of chromosome polymorphism in pairs No.1, 9 and
13 in the black rats distributed in the world. YOSIDA, T. H.
Karyotype and chromosome polymorphism in Indian gerbil
Tatera indica. YOSIDA, T. H. and OeHIAI, Y.

20

23

26
26
27
28
28

29
30

31
31

33

34
35
36

CONTENTS
Mechanisms for sister chromatid exchanges.

iii

KATO, H. . . . . . . . .

37

B I OBR mouse with a partially deleted Y-chromosome. MORIWAKI,
K. and SAKAKmARA, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VI.

VII.

Mutation and Mutagenesis in Animals
Mutagenic effectiveness of an internal f3 emitter, tritium. (2)Measurement of mutagenic effectiveness on germ cells ofBombyx. TAZIMA, Y., ONIMARU, K. and FUKASE, Y.
On an effective stage of chemical mutagens as detected by the silkworm oocyte system. TAZIMA, Y. and ONIMARU, K.
Nature of chemically induced mutations obtained by oocyte
system in the silkworm. TAZIMA, Y., ONIMARU, K. and
OHNUMA, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of incorporated amount of furylfuramide into eggplasm after injection into silkworm pupa. SHIMADA, Y.,
MURAKAMI, A. and TAZIMA, Y. ..
Differential stage sensitivity to the induction of mutations of
post-meiotic male germ-cells by mitomycin C in the silkworm.
MURAKAMI, A. and MUROTA, T.
Comparison of recombinogenic effects of radiomimetic alkylating
agents on oogenic cells in the silkworm. MURAKAMI, A. ..
Mutagenesis of furylfuramide in mature sperm of the silkworm.
MURAKAMI, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Spontaneous diabetes in Djungalian hamster, Phodopus sungorus.
YOSIDA, T. H.
Dominant black coat color in the black rat. YOSIDA, T. H.
Radiation Genetics and Chemical Mutagenesis in Microorganisms
and Plants
Comparative studies on DNA damages induced by gamma-ray
and 3H-decay. KADA, T., INOUE, T. and SADAIE, Y.
Enzymatic repair mechanisms of DNA damage induced by ionizing
radiation: Identification of a "cleaning enzyme". INOUE,
T. and KADA, T.
Enhanced mutagenesis, repair deficiency, mutator activity, andthermal prophage inducibility in DNA-8132 strains of Bacillus
subtilis. SADAIE, Y., NARUI, K. and KADA, T. . . . . . . . . . . . .
Detection of environmental mutagens and anti-mutagens by the

38

39
39

40

41

42
44
45
46
46

48

49

49

iv

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

rec-assay and reversion-assay procedures. KADA, T.
Waxy starch mutations in rice. AMANO, E.
Germination and early growth of some Podostemaceae seeds.
AMANO, E.............................................
Sex expression in monoecious-like variants in cucumber. FUJII, T.
Effects of gamma-rays and neutrons on the seedling and callus
growth in rice seeds. BARADJANEGARA, A. A., FUJII, T. and
AMANO, E.............................................
VIII. Population Genetics (Theoretical)
Mathematical contributions to population genetics. KIMURA, M.
Distribution of allelic frequencies in a finite population under
stepwise production of neutral alleles. KIMURA, M. and
OHTA, T.
Theoretical analysis of electrophoretically detectable polymorphisms: models of very slightly deleterious mutations.
OHTA, T. and KIMURA, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Moments for sum of an arbitrary function of gene frequency along
a stochastic path of gene frequency change. MARUYAMA, T.
and KIMURA, M.
The effect of selected linked locus on heterozygosity of neutral
alleles (the hitch-hiking effect). OHTA, T. and KIMURA, M.
Statistical analyses of Drosophila and human protein polymorphisms. OHTA, T.
The effects of linkage in multilocus system-overdominance.
YAMAZAKI, T.
Distribution of enzyme polymorphism and functional differencein Drosophila. YAMAZAKI, T. and MARUYAMA, T.
Distribution of gene frequency in a structured population. MARUYAMA, T.

IX.

Evolutionary Genetics
Spectra of heterozygosities and of evolutionary rates among
different loci. MARUYAMA, T. and YAMAZAKI, T.
Colonization of Drosophila simulans in Japan. WATANABE, T. K.
and KAWANISHI, M.

50
51
52
54

55
57

57

57

58
58
59
59
60
61

62
65

X.

XI.

CONTENTSv

Monitoring cancer families. MATSUNAGA, E.
Retinoblastoma in Japan: Follow-up survey of sporadic cases.
MATSUNAGA, E.
Classification with the size of C-bands in the human chromosome
variants and a case report of a de novo Yq-. IINUMA, K. and
MATSUNAGA, E.
Initiation of DNA replication in human chromosomes. NAKAGOME, Y.

66

Human Genetics

68
68

Behavioral Genetics
Noise effect on adult emergence and longevity of Drosophilamelanogaster. OSHIMA, C. and LEE, W. H.
Albinism and learning performance in mice. FUJISHIMA, T.

XII.

67

7I
71

Applied Genetics
Variations in copper tolerance among strains of wild and cultivated rice species. MORISHIMA, H. and aKA, H. 1.
Variations in copper tolerance among strains of barnyard grass.
MORISHIMA, H. and aKA, H. I. . . . . . . . . . . . . . . . . . . . . . . . . . .
The impact of copper pollution on the weed vegetation in Japaneserice fields. MORISHIMA, H. and aKA, H. 1.
Genetic loads in wild and domestic Japanese quails. KAWAHARA, T.
Esterase-D isozymes in Japanese quail. KAWAHARA, T., WATANABE, S. and SHIBATA, T.
Variation in susceptibility of Echinochloa crus-galli to a herbicide.
IYAMA, S.
On the efficiency of selection for elite trees. IYAMA, S. . . . . . . . . . .
Variations in anaerobic germinability of seeds found among wild
rice strains. SANO, Y.

79

Books and papers published in 1975 by research members. . . . . . . . . .
Abstracts of diary for 1975
Foreign visitors in 1975..........................
Author index

80
86
87
88

73
73
74
76
77
78
78

GENERAL STATEMENT
Dr. Daigoro Moriwaki retired from the Director's post at the end of
February, 1975. He was elected in April, 1969, as the successor of Dr.
H. Kihara and dovoted all his energy to the development of the institute
ever science. Especially he made strenuous efforts to characterize, inthe current of modern genetics, the divisional organization of the institutewith staff members who had taken root in the classical genetics. His idea
was to develop the whole fields of genetics in good harmony. He completed setting up of the 10th research department, Molecular Genetics, andthen planned to establish the Genetic Stock Center. He had also paid close
attention to the improvement and perfection of research facilities andequipments.
On March 1st I was appointed, assuming the heavy responsibility, the 4th
director of the institute of its 25 year's tradition established under the directorship of Dr. K. Oguma, Dr. H. Kihara and Dr. D. Moriwaki. I am firmly
determined to exert myself to the utmost for further development of theinstitute and sincerely solicit for the advice and cooperation of all persons
concerned.
"The institute aims at fundamental researches on the principle of genetics
as well as on its practical application." Needless to say, we should take up
essential research subjects among so many problems which may arise fromgenetic studies, so that we can contribute to the creation of new concepts. Furthermore, there are several problems which human being is confronted with-on food, population, environment, energy, etc. Researches
to meet such social needs of the times may be no less important than theformer. Innumerable research subjects are in front of us, but, our power totackle with them is only limited. Accordingly, we must carefully select toinvestigate essential problems of primary importance. We should not go
on in the same old rut.
The institute opens a door to the public one day every year during
Science and Technology Week in the middle of April. Visitors have been
increasing year after year. This year we had more than 7,000 visitors,
which recorded the highest figure since the foundation of the institute.
This indicates the deep interest of the people in genetic researches.
Another event open to the public annually is the public lecture, which was

2

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

held on November 1st at the lecture hall of the Science Museum in Tokyo.
Speakers were Dr. Y. Hirota, Head of the Department of Microbial Genetics
and Dr. Tomoko Ohta, Chief Researcher, Department of Population
Genetics. They talked on "Cell Division" and "Molecular Evolution,"
both of which gave a deep impression on the minds of the audience.
Until now several summer seminars had been held at irregular intervals.
This summer it was organized during July 7-9th by the staff members of the
Department of Population Genetics, with the cooperation of several scientists from outside the institute, entitled "Population Genetics and Molecular
Evolution." The participants came up to over 100 from all over Japanand the auditorium was full of enthusiastic atmosphere of learning.
Looking back the year from a budgetary aspect, i) the Genetic Stock Center,a new establishment, which started last year with the Plant section, wasgranted for adding the Animal section. Budgetary requirement for setting
up Microbial section has already been approved for the fiscal year 1976.
Accordingly, it will not take long before the Genetic Stock Center willfunction as we planned originally. ii) In addition to radiation laboratory
and radioisotope laboratory budget for setting up low level radioisotope
laboratories was also approved on the expenses for remodelling the oldannex building. It will offer a great convenience for molecular and microbial genetics groups. iii) The purchasing plan of the neighboring area,
where a junior high school once occupied, will be completed with the next
fiscal year budget. Now we are in a position to draw up our future plan ofbuilding construction with enough space. Herewith I wish to express my
deep gratitude to the authorities concerned for their assistance given by themfor the materialization of the necessary budget.

STAFF
Director

TAZIMA, Yataro, D. Ag.
Members

1. Department of Morphological Genetics
The 1st Laboratory
TAZIMA, Yataro, D. Ag., Head of the Laboratory
MURAKAMI, Akio, D. Ag., D. Sc.
ONIMARU, Kimiharu
The 2nd Laboratory
KURODA, Yukiaki, D. Sc., Head of the Laboratory
MINATO, Kiyoshi, 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
NAGAMI, * Shuzo, D. Ag.

* Research members under grant from other organization or visiting researchers.

4

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

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
IYAMA, 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
NOGUTI, Takehiko, 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.
SADAlE, Yoshito, M. Sc.

STAFF

7.

Department of Human Genetics
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Department
The 1st Laboratory
MATSUNAGA, Ei, D. Med., D. Sc., Head of the Laboratory
The 2nd Laboratory
NAKAGOME, Yasuo, D. Med., Head of the Laboratory
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
The 2nd Laboratory
SUZUKI, Hideho, 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
KIMURA, Motoo, Ph. D., D. Sc., Head of the Laboratory
OHTA, Tomoko, Ph. D., D. Sc.
The 2nd Laboratory
MARUYAMA, Takeo, Ph. D., D. Sc., Head of the Laboratory
YAMAZAKI, Tsuneyuki, Ph. D.

10.

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

11.

Genetic Stock Center
FUJII, Taro, D. Ag., Head of the Laboratory
SANO, Yoshio, D. Ag.

5

6

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

12. Experimental Farm
OKA, Hiko-Ichi, D. Ag., Head of the Farm
MIYAZAWA, Akira
13.

Department of AdministrationTEZUKA, Tomokazu, Head of the Department
OHTSUKA, Haruichi, Chief of the General Affairs Section
TAMATE, Shigeo, 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
LILIENFELD, Flora A., Ph. D.
SAKAI, Kan-ichi, D. Ag., Professor of Kagoshima University
TSUJITA, Mitsuo, D. Ag.
MORIWAKI, Daigoro, D. Sc., Managing Director of the Institute of Physicaland Chemical Research

COUNCILKIKKAWA, Hideo, Chairman, Professor of Hyogo College of Medicine
FUJII, Takashi, Vice Chairman, Emeritus Professor of University of Tokyo
EGAWA, Tomoji, Director of National Institute of Agricultural Sciences
IZEKI, Shoei, Director of National Research Institute of Police Science
KAYA, Seiji, Emeritus Professor of University of Tokyo
KIHARA, Hitoshi, Emeritus Professor of Kyoto University
KURODA, Toshio, Director of Institute of Population Problems
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
NAGAKURA, Saburo, Professor of University of Tokyo
SAKATA, Takeo, President of T. Sakata Company

COUNCIL

7

TAKAHASHI, Ryuhei, Professor of Okayama University
TANAKA, Nobunori, Professor of Teikyo University
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 1975
Department of Morphological Genetics

Genetic studies of the silkworm (TAZIMA and ONlMARU)
Radiation and chemical mutagenesis in the silkworm (TAZIMA and ONIMARU)
Cytogenetics in the silkworm (MURAKAMI and IMAI)
Studies on recombination in the silkworm (MURAKAMI)
Genetic studies on insect cells in tissue culture (KURODA and MINATO)
Developmental genetic studies on carcinogenesis in tissue culture (KURODA)
Genetics of somatic mammalian cells in culture (KURODA and MINATO)
Department of Cytogenetics

Studies on chromosomal evolution in rodents (YOSIDA and KATO)
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)
Cytogenetical study of ants (IMAI)
Department of Physiological Genetics

Behavior genetics of Drosophila (OSHIMA and WATANABE)
Analysis of delecterious and inversion chromosomes in natural populationsof Drosophila melanogaster (WATANABE)
Studies on fitness of Drosophila under controlled environment (OSHIMA andWATANABE)
Ecological genetic studies on the differentiation of Chrysanthemum species(NAGAMI)
Genetic studies on the effects of adverse environments on Drosophile flies(OSHIMA and WATANABE)

PROJECTS OF RESEARCG FOR 1975

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)
Genetic 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 (lYAMA)
Simulation studies on artificial selection (IYAMA)
Evolutionary studies in wild and cultivated rice species (OKA and MORISHIMA)
Ecological genetic studies in some grass species (MORISHIMA)
Genic analysis for isozyme variations in rice (ENDo and OKA)
Genetic effects of environmental pollution on plants (!YAMA, Morishima
and OKA)
Department of Induced Mutation

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

10

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

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

Genetic studies on common disorders with complex inheritance (MATSUNAGA)
Genetic studies on retinoblastoma (MATSUNAGA)
Clinical cytogenetic studies of congenital abnormalities in man (NAKAGOME
and IINUMA)
Molecular cytogenetic studies of human chromosomes (NAKAGOME)
Studies on human chromosome variants (IINUMA, MATSUNAGA and NAKAGOME)
Department of Microbial Genetics

Genetic regulatory mechanism of DNA replication in E. coli (HIROTA andNISHIMURA, and YASUDA)
Genetic regulatory mechanism of cellular division in E. coli (HIROTA)
Genetics of bacterial flagella (ENOMOTO and SUZUKI)
Transduction mechanism of phages (ENOMOTO)
Genetics of bacterial cell envelope (HIROTA, SUZUKI, and NISHIMURA)
Flagellar synthesis and its regulation in a cell-free system (SUZUKI)
Molecular genetics of flagellar synthesis and its regulatory mechanism(SUZUKI, ENOMOTO and HIROTA)
Department of Population Genetics

Theoretical studies of population genetics (KIMURA, MARUYAMA and OHTA)
Studies on molecular evolution from the standpoint of population genetics(KIMURA and OHTA)
Mathematical studies on the genetics of structured populations (MARUYAMA)
Linkage disequilibrium in finite populations (OHTA and KIMURA)
Experimental studies on protein polymorphism in Drosophila (YAMAZAKI)
Statistical studies on protein polymorphisms in natural populations (MARUYAMA and YAMAZAKI)PROJECTS OF RESEARCH FOR 1975

11

Department of Molecular Genetics

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

Studies and conservation of germplasm 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)

RESEARCHES CARRIED OUT IN 1975I.

MOLECULAR GENETICS

The Modified Structure at the 5 ' -terminus of mRNA in a Bone
Marrow Cell which Produces Hemoglobin Vigorously
Kin-ichiro MIURA, Yasuhiro FURUIcm and Yasusada MIURA

As a novel modified structure was discovered by us at the 5' -terminusof messenger RNAs of cytoplasmic polyhedrosis virus and vaccinia virus
as written in the last issue of this annual report (No. 25,1974 p. 11), presenceof the similar structure was surveyed in a cellular mRNA. Since hemoglobin messenger RNA is well known as an instance of a eukaryotic mRNA
which contains an information for a single protein, the 5'-terminal structureof this mRNA was studied. In order to detect small amount of a modified
structure in a long RNA molecule, radioisotope labeling of RNAs with
(32P] and [methyl 3H] methionine was carried out. A good condition was
obtained with the primary suspension culture of bone marrow cell of ananemic rabbit. Major part of the mRNA molecules synthesized in thiscell is hemoglobin mRNA. Total RNA was extracted from cell and fractionated by an oligouridylic acid-Sepharose column, which can adsorb poly
A-containing mRNA molecule. The [methy13H] radioactivity was containedin the poly A-containing mRNA fraction. This RNA was analyzed as
mentioned in the last annual report (No. 25, 1974, p. 11). In the 5'-terminal
part isolated by the treatment of Penicillium nuclease Ph the methyl label
was involved as the structure m7G~'ppp~'Cm-N-. 7-methyl guanylic acidblocks the 5 ' -terminal nucleotide through two pyrophosphate linkages asshown in mRNAs of CP virus and vaccinia virus.
Modified Structure in Messenger RNA of Reovirus
Kunitada SmMoToHNO and Kin-ichiro MIURA

A modified structure of mRNA discovered in silkworm cytoplasmic
polyhedrosis virus was tested for human reovirus as another instance ofRESEARCHES CARRIED OUT IN 197513

the double-stranded RNA-containing virus. As reovirus contains RNA
polymerase in its coat, RNA is synthesized in a test tube, if the trypsintreated virus particles were added with four kinds of substrate NTP. If[methyl 3H] S-adenosylmethionine (SAM) was added to this system as amethyl donor, the synthesized mRNA contained [3H] methyl groups. In
order to identify the modified structure, [p 32P] GTP (which is incorporated
to the 5'-terminal nucleotide-see the last annual report No. 25, 1974, p. 14)
was also added to the reaction mixture. After all the terminal modified structure ofreovirusmRNA was determined as follows: m 7 G5'ppp5'Gm-C------.
Modified Structure in Messenger RNA of Rice Dwarf Virus
Kunitada SmMOTOHNO and Kin-ichiro MIURA

Rice dwarf virus (RDV) containing double-stranded RNA can synthesize
RNA in vitro as cytoplasmic polyhedrosis virus and reovirus. When[methyl 3H] S-adenosylmethionine was added to the reaction mixture, [3H]
methyl was incorporated to the 5'-terminal part of the RDV mRNA. The
terminal structure was identified as follows: m 7 G5'ppp5'Am_Y.
Structure of the Double-stranded RNA in a Virus of
Penicillium chrysogenum
Kazumori YAZAKI and Kin-ichiro MIURA

Recently virus particles which contain double-stranded RNA have beenfound in many kinds of fungi. The RNA extracted from these virus consistsof three or four different size segments. In order to get a view for organization of these genome segments and mechanism of gene expression, theterminal structure of these RNA segments in Penicillium chrysogenum Virus(Pc V) was investigated.
The 3'-terminus was labeled with [3H] by borohydride reduction. Twokinds of the 3'-terminal structure was identified as follows for every threesegment: ---G-U and ---Y-A.
The 5'-terminus was tried to label with [32P] phosphate by polynucleotide
kinase. Only one kind of the 5'-terminal nucleotide sequence A-G-Y- waslabeled. The efficiency of the labeling reached only about one mole for
one RNA duplex. This means that one strand of the duplex can be phosphorylated, but another strand cannot. The latter must carry the blocked

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

terminal structure as shown at the 5'-terminus of the mRNA strand ofcytoplasmic polyhedrosis virus and reovirus.
The nucleotide sequence of the unblocked 5'-terminus, A-G-Y-, is notcomplementary to both the 3'-terminal nucleotide sequences. To test thepossibility that the double-stranded RNA of Pc V may have a singlestranded part near the terminal region of a polynucleotide chain, the 3'[SH] labeled RNA and the 5'_[S2P] labeled RNA were mixed and treatedwith nuclease 8 1 from Aspergillus, which selectively cleves single-stranded
region in a polynucleotide chain under some conditions. Only [S2P] labeled
nucleotide was released from the double-stranded RNA. This means thatthe 3'-termini should be in a double-stranded state, but at least one 5'terminus, that is, the 5'-terminus of the unblocked chain carrying the sequence A-G-Y-, is in a single-stranded stretch.
Mechanism of Formation of the Modified Structure at the
5'-terminus of mRNA of Silkworm Cytoplasmic
Polyhedrosis Virus
Kunitada SHiMOTOHNO and Kin-ichiro MIURA

The modified structure including the 7-methylguanosine blocking through
5'-5'-pyrophosphate linkage and the ribose-methylation at 2'-position of
the first nucleotide was found at the 5'-terminus of mRNA of cytoplasmic
polyhedrosis (CP) virus (Annual Rep. No. 25 (1974) p. 12; Nature 253 (1975)
374-375). CP virus, which contains double-stranded RNA as a genome
and enzymes including RNA polymerase, phosphohydrolase and methylases,
is able to synthesize mRNA carrying the 5'-terminal modification in vitro
in the presence of a methyl-donor S-adenosylmethionine (SAM). The process of the formation of the modified structure at the 5'-terminus of thismRNA was studied.
The results show that methylation at the 5'-terminus of the mRNA occurs
at first on the blocking guanylate at 7-position of the base residue, and the
methylation at the 2'-position of the ribose moiety of the first adenylate
starts only after RNA chain has reached three nucleotide length.
In order to confirm the steps of methylation the confronting nucleotide
structure without methyl groups, G5'ppp5'A and G5'ppp5'G, were prepared bychemical synthesis and added to the reaction mixture containing CP virus.
The result shows that GpppA can be a substrate for the methylase associatedRESEARCHES CARRIED OUT IN 197515

to CP virus, but GpppG can not. This is not conflict with the 5'-terminal
structure of CP virus mRNA, m7GpppAm. The methylation enzyme in CP
virus seems to recognize the substrate structure strictly and to methylate
after the blocked structure is constructed. In fact, GTP in the reaction
mixture was not methylated. The methyl group incorporated into GpppA
was found only in 7-position of G, but not in 2'-position of A. Here again
it is clear that methylation at the 7-position of the blocking guanosine residue
is carried out at first, and the ribose moiety in the A residue is not methylated
before a few nucleotides linked to it.
Based on these results, the process of the 5'-terminal modification during
the synthesis of CP virus mRNA are written as follows:ATP~ GpppA~ m7 GpppA
SAM
GTP~ m 7 GpppA-G(with or without SAM)
m 7 GpppA-G+ UTP+CTP+SAM -~ m 7 GpppAm-G-U-C-----GTP +GpppA +
m 7 GpppA +This work was published in FEBS Letters 64: 204-208 (1976).
A New Acid Phosphatase from Escherichia coli Cells
Masahiro SUGIURA

There are many phosphatase activities in Escherichia coli cells. Some ofthem have been purified and characterized. During the course of polynucleotide kinase purification from bacteriophage T4-infected E. coli cells,
we found a new type of acid phosphatase activity. This enzyme was notbacteriophage T4-coded one because the activity was detected in uninfected
E. coli cell extract.
The acid phosphatase was purified from E. coli strain Al9 (RNase 1-)
by streptomycin sulphate fractionation followed by successive column chromatographies on DEAE-cellulose, phosphocellulose and Sephadex G-l00.
The purified enzyme had an optimal pH of 4.1 and was activated by magnesium and cobalt ions. The enzyme catalyzed the hydrolysis of nucleoside
5'-diphosphates and nucleoside 5'-triphosphates to nucleoside 5'-monophosphates, and nucleoside 3',5'-diphosphates to a mixture of nucleoside
3'-monophosphates and nucleoside 5'-monophosphates. Nucleoside 3'monophosphates and nucleoside 5'-monophosphates were not hydrolyzed

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

but nucleoside 2'-phosphates were hydrolyzed at slower rate. 5'- and 3'Terminal phosphates of RNA were removed by this enzyme. The phosphatase hydrolyzed p-nitrophenylphosphate but not bis-p-nitrophenylphosphate. This enzyme was differ from any of the E. coli phosphatases
previously reported and we tentatively called it acid polynucleotide phosphatase. This enzyme may be useful for the chemical analysis of nucleic
acids and their components because of its unique specificity.
Induction of RNA Ligase Activity in Bacteriophage
T4-Infected Escherichia coli Cells
Masahiro SUGIURA and Kazuko SEGAWA

RNA ligase has been isolated from bacteriophage T4-infected E. coli cells.
This enzyme catalyzes inter- and intra-molecular joining reactions of ribopolynucleotides. Deoxyribooligonucleotides were also joined by this enzyme action. Therefore, we investigated the relationship between DNA
ligase and RNA ligase. E. coli strain Al9 (RNase 1-, Su-) cells were
infected with phage T4 amber mutant of gene 30 (DNA ligase), am H39,
am en or am £605 and the RNA ligase activity was assayed. All mutant
phages induced the RNA joining activity comparable to wild-type phage.
This indicated that the induction of RNA ligase activity was nothing to dowith the structural gene of T4 DNA ligase. A T4 mutation called SP62,
which overproduced some of early enzymes in combination with DNAnegative mutation, did not cause any overproduction of RNA ligase. A
double amber mutant N82 X £1140 induced the highest activity of RNA
ligase among T4 DNA-negative mutants tested.

RESEARCHES CARRIED OUT IN 1975

II.

17

MICROBIAL GENETICS

An Escherichia coli Mutant Defective in Murein
Lipoprotein Synthesis
Yukinobu NISHIMURA, Hideho SUZUKI, Sei-ichi YASUDA,
Hiroko IKETANI and Yukinori HIROTA

The mutant lacking murein lipoprotein in the outer membrane has notbeen available so far. Such a mutant is expected to contribute very muchtoward the elucidation of function of the lipoprotein. During the genetic
manipulation of an F prime, we found a mutation defective in the production of the lipoprotein on the F prime. The gene necessary for the lipoprotein production was mapped at 36.5 min between uidA and aroD on the E.
coli linkage map.
It has been established that lpm t and its closely linked markers are mappedin the order of -man-uidA-lpm-aroD-pps-. An F prime carrying the genetic
loci of man and aroD was constructed by the cross between HfrB7 and
JE5507 (recAI, man, aroD), and introduced into the rec+ strain having lpm,
in order to obtain lpm-carrying F prime. Among transductional derivativesof this strain, we found two clones in which the lysozyme digests of theirmurein showed no electrophoretic band characteristic to the bound form of
the lipoprotein.
One of the clones designated as JE5508 isolated by transduction with PI
was examined further with respect to the lipoprotein. The electrophoretic
analysis of the outer membrane proteins solubilized with hot-SDS showedno band of the lipoprotein detectable by staining with Coomasie blue. Thus
the free form of the lipoprotein as well as the bound form was absent in thisstrain. This was further confirmed by labelling the cellular proteins withradioactive arginine. The cells of JE5508 were labelled with 3H_ or 14C_
arginine, mixed with those of the parental strain labelled with the counterspecies of the radioisotope, respectively, and disrupted to prepare themembrane fractions. The electrophoretic analyses showed that the radioactivity peaks found at the position of the bound or unbound lipoprotein in
the parental strain were missing in the mutant. The serological assay on
an Ouchterlony agar plate showed that materials cross-reacting with antilipoprotein serum was absent in the samples prepared from the mutant cells

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

by sonic-disintegration and subsequent solubilization with hot-SDS. The
messenber activity of RNA was examined if it could stimulate the synthesisof lipoprotein, by the cell-free system for protein synthesis followed by theserological purification of the product and the electrophoretic analysis.
The RNA extracted from the mutant did not stimulate the synthesis of thelipoprotein, whereas that from the parental strain directed the lipoprotein
synthesis in vitro at the level amounting to 1.9 % of the general protein
synthesis. These results indicate that the cells of JE5508 lacked the lipoprotein because of their inability to synthesize active mRNA for the lipoprotein.
The mutation leading to the loss of lipoprotein appeared to have occurredin the process of production of the F prime, since the merodiploid isolated
at first by the cross of HfrB7 and the rec- strain was shown to carry thismutation on the F prime by the transduction experiments. This mutation
will be referred to as lpo.
The colour of the colony of lipoprotein-less strains was found to becomedarker after prolonged incubation. This character was shown to be absolutely parallel with phenotypes identified by electrophoresis, and aided
the genetic analyses of the lpo mutation.
Our experimental results demonstrated that the map position of the lpo
was at 36.5 min on the Tayler's map (Bachmann B. J. et al., 1976, Bacteriol.
Rev., 40: 116), as determined by transduction with PI. The merodiploids
carrying Ipm+jF'lpo and Ipm-jF'lpo exhibited phenotypes of the wild andthe modification, respectively. The lpo mutation could be a delection
mutation which is extending over at least two loci, lpm and a hypotheticalstructural gene coding for polypeptide of the lipoprotein, lpp.
The physiological nature of this mutant was examined with isogenic
strains obtained by PI transduction. The /po mutant grew normally over
the wide range of growth conditions and remained susceptible to the following bacteriophages; A, ¢80, PI, P2, n, T2, T3, T4, T5, T6, T7, f 1, f2
and MS2. The mutant revealed hypersensitivity to EDTA, acriflavin andethidium bromide. The mutant leaked periplasmic enzymes; ribonuclease 1,
alkaline phosphatase, cyclic phosphodiesterase and penicillinase, more than
the wild type strain. The effect of EDTA was conspicuous. The additionof EDTA to cell suspension of the /po mutant brought about rapid cell lysis,
but this was rescued by the simultaneous addition of magnesium. The
passive transport of ,a-galactosides was not changed by the lpo mutation,
as observed by the measurement of the crypticity of ,a-galactosidase activity

RESEARCHES CARRIED OUT IN 1975

19of bacterial cells (Cohen, G. N. & Monod, J., 1957, Bacteriol. Rev., 21: 169).
From these physiological characteristics of the mutant, it was suggested
that the lipoprotein participates in maintaining the stability of the outermembrane structure by anchoring the murein layer of the cell, but does notin the vital processes of growth and division.
t/pm:

a gene for the altered lipoprotein (SUZUKI et aI., 1976 J. Bacterioi. 127: 1494).

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

III.

BIOCHEMICAL GENETICS AND IMMUNOGENETICSA Simplified Method for Reading Hemagglutinations on a Flat Bottom
Microtitiration Plate in the Mouse H-2 Assay
Kazuo MORIWAKI, Tadashi AOTSUKA and Toshihiko SmRorsm

The original polyvinylpyrrolidone (PVP) method of Stimpfling (Transpl.
Bull. 27: 109, 1961) has been successfully modified to the more dependable
form by Kaliss (Transpl. 15: 251, 1973). As his method is still laborious
in the larger scale assay, a simplified method for reading the hemagglutinations microscopically on a microtitration plate with flat bottom wells has
been developed in our laboratory. The recipe for working solutions followed
Kaliss. Fresh blood for target RBC was obtained from the orbital sinus
using a microhematocrit tube. We usually pour the full tube of blood (75 pI)
into 10 ml Alsever solution immediately after bleeding, mix gently and wash
three times with PBS by centrifugation at 2,000 rpm for 3 min. Final
volume was adjusted to make 0.75 % RBC suspension.
Titrations are performed in the Microtiter Plate for tissue culture (Cooke
Laboratory Products) with 96 flat bottom wells. One trop (25 pI) of 1.2 %(only for 1st row) or 0.6% PVP BSA was dispensed to each wells, then theantisera were serially diluted by 25 pI loops. Finally 25 pI RBC suspension
was dispensed to each wells and shaked for about 10 seconds by a Voltex
mixer. After incubation at 37°C for one hour and at 5°C overnight, eachplate was shaked again for 10 seconds just before reading. Microscopic
observation of the hemagglutinations was conducted by the inverted typemicroscope with X10 objective lens. Though the plate method seems to beslightly less sensitive than the original Kaliss' method, its operational
simplicity could enough compensate the small defect.
H-2 Antigenic Specificities in the Japanese Wild Mouse,
Mus musculus molossinus
Kazuo MORIWAKI, Toshihiko SmROlsHI, Tadashi AOTSUKA,
Mitsuru MINEZAWA1) and Kyoji KONDol )The Japanese wild mouse, Mus musculus molossinus has likely derived
1)

Nagoya University

RESEARCHES CARRIED OUT IN 1975

21

from an aboriginal mouse, Mus musculus manchu, which geographical distribution was quite distant from the other aboriginal mice, Mus musculus
wagneri and Mus musculus spicilegus (Schwarz and Schwarz, J. Mammal.
Table 1. Occurrence of twelve H-2 specificities in the Japanesewild mice, Mus musculus m%ssinus
H-2 antigenic specificities
SampleNo

Place ofcollection

Sex

1
2
3
4
5
1
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Teine
Teine
Teine
Sunagawa
Sunagawa
Sunagawa
Sunagawa
JohojiNatori
Niigata
Kanazawa
Kanazawa
Utsunomiya
Hattabata
Haranoya
Anjyo
Anjyo
Mizuho
Ohtsu
Ohtsu
Kochi
Kochi
Matsuyama
Matsuyama
Sasaguri
Sasaguri
Sasaguri
SasaguriHakozakiOkinoerabu Is.
Yonakuni Is.

M
F
F
M
F
F
F
M
M
M
F
F
M
M
F
M
M
M
M
M
M
F
M
F
F
M
F
F
F
M
M

Public1

Average freq.

3

5

8

11

Private
13

25

28

35

2

4

23

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

+ +
+ +
+ +

+

+ + +

+ +

+ +
+ +

+ +

+

+

+ +

+ +
+ +
+
+

+ + +
.11 .57 .53 .07 .03 .07 .03 .07 .03 .00 .03 .03

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

54: 59, 1945). These two subspecies are possible origins of the European
wild mice, Mus musculus musculus and Mus musculus domesticus, in which
many H-2 specificities, especially public ones being fairly common in thevarious inbred strains of mouse, have been detected with the higher frequency. The present investigation aims to survey serologically those H-2
antigenic specificities in the Japanese wild mice, Mus musculus molossinus,
from view point of H-2 gene evolution in the mouse species.
Assay of H-2 antigenic specificities was carried out using a modified
Kaliss method (Moriwaki et al. Experientia, in press) which allowed us todirectly observe PVP-hemagglutinations of the microtitration plate withflat' bottom by the inverted microscope. Twelve alloantisera employed inthis study were given by the Transplantation Immunology Branch, NIAID,
NIH. Twelve numericals listed in Table I represent antigenic specificities
against which each of the alloantisera mainly directed. In many cases thehemagglutination titers for each H-2 specificities in M. m. molossinus wererelatively lower than those in the standard mice.
Table 1 summarizes the present data on the H-2 specificities of M. m.
molossinus. For the comparison between M. m. molossinus and M. m.
musculus or M. m. domesticus, we adopted nine public specificities which
seem to be considerably common in the European and American wild mice,(Klein, Science 168: 1362, 1970. Mickova & Ivanyi, Immunogenetics ofthe H-2 system: 20, 1971) and three private specificities as well. Only two
public specificities, H-2· 3 and H-2' 5, were apparently common in theJapanese wild mice and the others were very rare.
Considering numerous differences in the biochemical characteristics between the Japanese and the European feral mice (Minezawa et al., This
Annual Report), the frequency differences in their H-2 antigenic specificities
may reflect a certain remote distance between them. In the other words,
H-2·3 and H-2' 5 public specificities likely are rather older evolutionarily
than the other. They might have their origin in a common ancester of thethree subspecies, M. m. molossinus, musculus and domesticus.

RESEARCHES CARRIED OUT IN 1975

23

Studies on Protein Polymorphism of Japanese Wild Mouse,
Mus musculus molossinus
Mitsuru MINEZAWA1), Kazuo MORIWAKI and Kyoji KOND0 1)

The protein polymorphism of Mus musculus molossinus in Japan was
surveyed on Hbb, Es-I, Es-5, Id-I, Es-2, Es-6, Amy-I, Amy-2, Amy-3,
Mod-I and Trf loci by gel electrophoresis, and Gus locus by spectrophotometry. Table I demonstrates the localities of collection, number of miceobserved in each locality and phenotypic distributions of Hbb, Es-I, Es-5
and Id-l loci.
Hbb locus (Hemoglobin): P, PC, D and PS types were observed, but
HbbS-gene was rare and Hbb d and Hbb P were common alleles in molossinus.
Es-Ilocus (Serum esterase): A, AB and B types were observed and A type
was considered to be popular among them. Es-5 locus (Serum esterase):
A, Band C types were found and the C type showed slower migrating band,
and was not yet observed in laboratory strains of mice. A type was common
one. Id-I (Kidney isocitrate dehydrogenase): 7 phenotypes, namely A,
AB, B, AC, BC, C and BD types, were observed. The C type or C band
was a slower migrating band than A band, and the D band was migrated
faster than B band. Both two types, C and D bands, were not seen in laboratory mice. The B type is considered to be common type of molossinus.
Results of the electrophoretic survey for other proteins, which are notshown in Table I, were as follows. Es-2 locus (Kidney esterase): A, B,
BC and C types were found. The C type was common in molossinus (49/60
observed). Es-6 locus (Kidney esterase): A, B, AB and C types wereobserved, and the C type was slower migrating band than B band. The
type A was common type. Amy-l locus (Salivary amylase): AB and B
types were observed and B type was common one. Amy-2 locus (Pancreaticamylase); A and B types were observed and B type was popular. Amy-3
locus (Pancreatic amylase): All the mice showed A type (49 mice). Mod-llocus (Liver supernatant malic enzyme): A and AB types were found, andA type was thought to be common type of molossinus. Trf locus (Serumtransferrin) and Gus locus (Liver j9-glucronidase): At these loci, all of themice observed are monomorphic, namely, B type at Trf and A type at Gus
respectively.
1)
Department of. Animal Breeding, Faculty of Agriculture, Nagoya University, Chikusaku, Nagoya~Phenotypic distributions of proteins, Hbb, Es-l, Es-5
and Id-l in Japanese Mus muscu/s m%ssinus

Table 1.

No.
Mice
Okinawa
KyushuHakozaki
Sasaguri
MiyazakiTsushima
Shikoku
Kochi
Matsuyama
HonshuMine
Anjyo
Hattabata
HaranoyaKanazawa
Niigata
Johoji
Hokkaido
Nemuro
Teine
Total
n.t.:

Es-l

Hbb
P

PD D

PS n.t.

4

4

Es-5

~

Id-l

A AB B n.t. A

B C n.t. A AB B AC BC C BD n.t.

3

2

1

8 11
4 2
1
6

17
3
1

1

3

8
3

1
1

8
2

6
2
5
4
5
8
4

1
2

5

1

1

1

3

3
5

16

~

t'"'
:;l:l
t'T1
"d

0

26
11
1
11

6

8
3

8
2

6
2
5
5
5
8
4

1
2
5
2
3

14
4

1

117

35 21

10 7
1 10

3

7
5

1

11

5

1

5
2

7
3

1
5

1
2
1

11

6
1
1
1

2
5

~

1

0

4

>-l
....

"I1

~

0

not tested.

3

2
1
4

1

1

2
2
5
1

1
4

4

5
5
8
4

4
13
4

44

1

4

1

14
3

1

2

14
1 1

13 70 2125

1 70

2 12 33

5
4
5
8
4

1

13
1
1

3 28

5

>-l
t'T1

0

"I1

~
Z

t'T1
>-l

....

t/)

3
9

t/)

(j

1
7

~

Z
~
c:::

1 63

Z

P
N

'"

RESEARCHES CARRIED OUT IN 1975

25

Based on the results of survey of protein polymorphism, several clear
differences between molossinus and laboratory mice were able to be observed.
For examples, P type of Hbb locus is a rare variant in laboratory mice, butcommon in molossinus, and C type of Id-l and also C type of Es-5 are notobserved in laboratory mice. Besides the difference between molossinus
and laboratory mice noted above, some differences among localities wereseen in the ratio of protein types.

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

IV.

DEVELOPMENTAL AND SOMATIC CELL GENETICS

Estimation of Genetic Load and Isolation of Mutants of Hydra
from Sexual Crosses
Tsutomu SUGIYAMA and Toshitaka FUJISAWA

Sexual crosses were made between field strains of Hydra magnipapillata
collected from various localities in Japan, and viability and other charactersof the progenies were examined.
The results showed that the chance for the fertilized eggs to hatch andgrow to adults was generally very low due to genetic and non-genetic factors.
Statistical analysis showed that field strains of H. magnipapillata contained
an average of between 3.6 and 4.1 lethal equivalent units of recessive deleterious genes per gamate (between 7.2 and 8.2 per animal).
The results also showed that among the surviving F 1 progenies, there weremany types of developmentally defective strains such as mini strains, maxi
strains, multi-head strains, nematocyte deficient strains, regeneration deficient strains and male sterile strains. The defective traits of these strains
were stably transmitted to their progenies produced by asexual budding.
These strains, therefore, were all propagated asexually and maintained asclonal lines to be used later for further characterizations.
Nematocyte Deficient Strains of Hydra
Toshitaka FUJISAWA and Tsutomu SUGIYAMA

Nematocytes are hydra's stinging cells and they are located mainly on thetentacles where they are used primarily to capture and paralyse prey. Hydra
magnipapillata has four types of nematocytes (type A, B, C, and D) which
together account for one-third to one-fourth of the total body cells.
The nematocytes deficient strains of H. magnipapillata were isolated bycrossing male and female strains collected from the same ponds and examining their F 1 and F 2 progenies for the food capturing ability and thenematocyte compositions. Two strains selected from the six nematocyte
deficient strains were subjected to detailed analyses.
Nem-3, which virtually lacked type B nematocytes in its tentacles, wasfound to contain a greatly reduced level of the interstitial cells within its

RESEARCHES CARRIED OUT IN 197527

body. Since interstitial cells are known to be the precursors of the nematocytes, it is likely that the lack of the interstitial cells is responsible for thenematocyte deficiency of this strain.
Nem-4, which was deficient in type A nematocyte in its tentacles, contained a normal level of the interstitial cells. Several lines of evidence indicated that the primary defect of this strain is probably in the migration of
the matured type A nematocyte from the body column to the tentacles.
Regeneration Deficient Strains of Hydra
Tsutomu SUGIYAMA and Toshitaka FUJISAWA

Three strains of Hydra mgnipapillata selected from the seven regeneration
deficient strains isolated by sexual crosses were subjected to detailed examination.
The number of the tentacles regenerated per animal was used as a measureof the capacity for regeneration. Wild type strains generally had about 6
tentacles per animal and, after amputation of the head and the foot, theyregenerated practically the same number of tentacles within 5 to 6 days.
In contrast, one of the regeneration deficient strains (reg-I) regenerated only
about 40 % and the other two strains (reg-16 and reg-19) regenerated only
about 10 % of the original tentacles during the same period of time.
It was however found that the efficiency with which these strains regenerated depended greatly upon how the animals were disected. When reg-16 andreg-19 were further disected into smaller pieces after head and foot amputation, their ability to regenerate increased markedly, and they produced up
to 50 % of their original tentacles.
Tissues taken from the distal and proximal ends of regenerating animals
were transplanted to intact host animals to analyse their ability to regenerate
into head ("head determination"). It was found that head determination
took place within 24 hours after the head and foot amputation of the wild
type animals. This determination, however, was abnormal with reg-16
and reg-19. It was suggested that the regeneration deficiency of these strains
was due to this abnormal head determination, and that this in turn wasprobably caused by defective polarity gradient(s) in these strains.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

A Tissue-Specific Defect in Developmentally Lethal Cells
from fused Embryos in Drosophila melanogasterin Culture
Yukiaki KURODA

The fused (fu) strain is known to be an X-linked (1-59.5) recessive lethal
mutant. Homozygous or hemizygous eggs for the fu gene die during
embryogenesis when they have the cytoplasm given by homozygous mothers.
In the present experiment cells from these lethal embryos were cultured to
study when and what types of cells were affected by the factors produced ata specific locus (17D or l7E) of the fu gene on the X chromosome.
When the effective lethal phase of 605 embryos obtained from matings offulfu females with fulY males was examined, it was found that 0.5 % diedafter blastoderm formation, 5.0 % died after gastrulation, 8.6 % died afterhead and trunk segmentation, 4.0 % died after formation of a sac-like midgut,
0.7% died after chitinization, 76.0 % died after entrance of air in trachea,
5.3 % died after active movement and none became larvae.
When cells from fu embryos after gastrulation were cultured, spindleshaped muscle cells, flat polygonal epithelial cells, fibroblastic cells, andnerve cells were maintained in a functionally active state for longer thanthe effective lethal phase of the embryos. Some muscle cells contracted atregular intervals and others produced the syncytial complexes. Epithelial
cells grew slowly and a brown chitinous pigmentation was observed in thecytoplasm. After several days of cultivation baloon-like cellular spheres
which are suspected to be imaginal disc materials were formed.
In contrast to rather normal natures and functions of these cell types,
nerve cells from fu embryos were found to have some defects in extension
and branching of nerve fibers. The droplet formation on nerve fibers which
was found in cultures of wild-type embryonic cells and seemed to show somesecretions, was also not found in cultures of fu embryonic nerve cells.

Reverse Mutations of 8-Azaguanine Resistance to Sensitivity
in Human Lesch Nyhan Diploid Cells in Culture
Yukiaki KURODA

Human diploid cells derived from the elbow skin of an 11 year Lesch
Nyhan boy were treated with ethyl methanesulfonate (EMS) at various

RESEARCHES CARRIED OUT IN 1975

29

concentrations for 2 hours, incubated in normal medium for mutation expression time of 48 hours, and selected in HAT medium to induce reverse
mutations from 8-azaguanine (8AG) resistance to sensitivity.
To examine the effect of EMS on cell survival, the Do values of cells
treated EMS were calculated from the concentration-survival curves. The
Do values for 2 hour- and 14 day-treatments with EMS were 5.8 X 10- 2 M
and 4.8 X 10- 4 M, respectively. The Do value for 14 day-treatment with
8AG was 14 flgfml, which were about 20 times more resistant than that ofnormal human diploid cells.
When Lesch Nyhan cells were treated with 10- 3 M of EMS for 2 hours,
the induced mutation frequency of 8AG sensitive cells (surviving in HAT
medium) was 30.0 X 10- 5 • When the cells were treated with higher concentrations of EMS, the induced mutation frequencies increased gradually.
Cells treated with 10- 2 M EMS gave the induced mutation frequency of
54.9 X 10- 5 • This reverse mutation frequency of Lesch Nyhan cells by EMS
was about two times higher than the forward mutation frequency in normal
human diploid cells treated with the same concentration of EMS. This
indicates that the induction of reverse mutations may be more sensitive
than that of forward mutations in human diploid cells, as far as a sex-linked
recessive 8AG resistance (at the HGPRT locus) is concerned.
The Effect of Cell Density on Induction of 6-Thioguanine
Resistant Mutations in Chinese Hamster Cells
in Culture
Yukiaki KURODA and Tohru 5mBUYA

Chinese hamster lung cells, Don-6, were treated with ethyl methanesulfonate (EMS) for 16 hours, incubated for 48 hours in normal medium, andselected in medium containing 6-thioguanine (6TG). In this procedure fordetecting the induced 6TG resistant mutations, it was found that the replating of dissociated cells in new petri dishes after mutation expression
time in normal medium enhanced the induced mutation frequency of cells.
The number of replated cells in each petri dish also markedly affected theinduced mutation frequency: 4.4 replated cells per mm 2 of surface area of
petri dishes produced the highest induced mutation frequency among various
cell densities employed. The effects of replating and small cell density inenhancing the induced mutation frequency may be accounted by the reduc-30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

tion in cell-to-cell contact (metabolic cooperation) in monolayer culture.
The concentrations of 6TG used for selecting 6TG resistant cells did not
significantly affect the induced mutation frequency.
Two 6TG resistant cell clones, ET-I031 and ET-OOll, were isolated fromthe original 6TG sensitive cells after treatment with the mutagen. Their Do
values for 7 day-treatment with 6TG were 18.3 p,gjml and more, indicatingthat they were highly resistant to 6TG compared with the original cells.
The5k 6TG resistant cells showed a cross-resistance to 8-azaguanine (Do=40.2 p,gjml).
Changes in Cell-to-Cell Cohesive Activity during Cell
Cycle of HeLa 83 Cells in Rotation Culture
Yukiaki KURODA

The cell-to-cell contact is known to have a close relationship to the proliferation and differentiation of animal cells in development. In the present
experiment changes in cell-to-cell cohesive activity during cell cycle was
examined using synchronized cell population of HeLa S3 cells.
HeLa S3 cells were treated with 2.5 mM thymidine for 24 hours, cultured innormal medium for 10 hours, and treated with 0.5 p,gjml colchicine for 2
hours. Thus mitotic cells which showed a synchronization of 96.5 % werecollected, inoculated in 60-mm petri dishes in replicate, and incubated ina CO 2 incubator for various times. Cells in petri dishes were harvested
every 3 hours, and 3 X I O~ cells in 3 ml medium were transferred into 25 ml
Erlenmeyer flasks which were rotated at 70 rpm at 38"C for I hour. The
number of single cells remaining in cell suspensions was counted and the cellto-cell cohesive activity of cells at each phase of the cell cycle was compared.
The highest cell-to-cell cohesive activity was found at the M phase (0 hour
after cell collection) of cells. The cohesive activity of cells decreased gradually during the G 1 phase. At the early period of the S phase (12 hours aftercell collection) the cohesive activity increased again and reached the second
highest value. The cohesive activity decreased gradually at the late periodof the S phase (18 hours after cell collection). During the G z phase thecohesive activity showed a gradual increase and reached the highest valueof the M phase. The cyclic changes in the cohesive activity of cells may
have some correlations with the DNA and protein syntheses of cells during
cell cycle.

RESEARCHES CARRIED OUT IN 1975

31

Protective Effect of Vitamin E on Bisulfite-Caused
Inhibition of Aggregation of Embryonic Quail
Liver Cells in Culture
Yukiaki KURODA

Vitamin E is known to have a specific role in the redox reaction andantioxidant effects on various metabolic processes in higher animals. In
the previous experiment it was found that vitamin E counteracted the effectof bisulfite in reducing colony formation of cultured human cells. In thepresent work the protective effect of vitamin E on inhibition by bisulfite ofaggregation of dissociated embryonic quail liver cells was examined inrotation-mediated cell culture.
Liver cells from 7-day quail embryos were dissociated by the standard
procedure. Samples of 3 ml of cell suspension, containing 106 cells, wererotated at 70 rpm at 38°C for 24 or 48 hours. Then the aggregates were
harvested and their mean diameters were determined.
Bisulfite had a concentration-dependent inhibitory effect on the aggregation of cells, and in the presence of 10- 2 M bisulfite, the mean diameters of
aggregates formed after 24 and 48 hours were less than half those of aggregates in control cultures. Addition of 10- 7 M vitamin E significantly
reduced the inhibitory effect of bisulfite on cell aggregation. A concentration of 10- 5 M vitamin E had the most protective effect on aggregation ofembryonic liver cells. Further investigations on the mechanism of inhibition
by bisulfite and protection by vitamin E of aggregation of embryonic liver
cells are in progress.

Effective Lethal Phase of the Curly Mutant in DrosophilamelanogasterYukiaki KURODA and Kiyoshi MINATO

A number of studies on the phenotypic expression of genes during development of higher animals have been carried out by determining the specifictissue and time of the gene action. The lethal genes in D. melanogasterprovide a useful tool for analysing the tissue- and time-specificity of thegene action in development. In the present experiment the effective lethal
phase of the Curly (Cy) mutant (which involves inversions at 22Dl-2 and33F5-34Al) on the second chromosome was examined.

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

F 1 eggs obtained from matings of Cy/+ females with Cy/+ males wereallowed to develop on a moistened filter paper, and hatched larvae weregrown on the standard cornmeal-sugar-agar medium. The numbers of
eggs collected, hatched larvae, pupae and adult flies were determined.
Among 5063 eggs collected, 11.2 % died during embryonic development,
25.9% died during larval period, 4.1 % died during pupal period, and 58.7%
reached the adult flies. In adult flies the ratio of Cy/+: +/+ was about
2: 1 (1853: 962). These results indicate that Cy/Cy individuals may havebeen about one fourth of total eggs collected and most of them may havedied during larval period of development. Investigations on the tissuespecificity of the action of the Cy gene is also in progress.

RESEARCHES CARRIED OUT IN 1975

v.

33

CYTOGENETICS

Population Survey of B-chromosomes in Black Rats
Tosihide H. YOSIDA

Black rats (Rattus rattus) have been classified into 3 types according tochromosome numbers; Asian type with 2n=42, Oceanian type with 2n=38
and Ceylon type with 2n=40. B- or supernumerary chromosomes in Asiantype black rats have been reported by some investigators (Gropp, A. et al.
1970, Z. f. S6ugetier. 35: 363; Ray-Chaudhuri, S. P. and Pathak, S. 1970,
MCN, 11: 135; Yosida, T. H. and Sagai, T. 1975, Chromosoma 50: 283).
Distribution of the B-chromosomes and their types in the black rats were
investigated in the animals collected from several localities in the world.
Among 271 Japanese black rat (Rattus rattus tanezumi), 190 were collectedfrom five different localities of Japan (Misima, Nagasaki, Hokkaido, Niigata
and Tokyo), and the remaining 81 were obtained from our breeding colonyof the animals originated from Japanese black rats. Almost all black rats,
except one which came from Hokkaido, showed diploid chromosome numbers (2n=42). This' one rat showed 43 chromosomes including one small
submetacentric B-chromosome. Nine R. rattus flavipectus collected from
Hong Kong, 19 R. rattus mindanensis from Philippines, 14 R. rattus from
northern India and 20 R. rattus from northern Pakistan showed all exact
diploid number (2n=42). On the other hand, among 20 R. rattus diardii
from Malaysia, 11 rats showed 43 chromosomes with one B-chromosome and3 rats had 44 chromosomes with two B-chromosomes. These B-chromosomes are all small metacentrics and they were stained heavily by C-band
technique.
In the Oceanian type black rats (2n=38) two from Iran, 4 from Australia
and 10 from the United States showed normal diploid chromosome numbers(38). However, among 41 black rats from southern India (20 from direct
observation from collected rats, and 21 from our breeding colony originatedfrom the southern Indian rats), 8 rats had 39 chromosomes with one small
metacentric Band 2 rats showed 40 chromosomes by having 2 small metacentric B. In Sri Lanka, 13 Ceylon type black rats (R. rattus kandianus)
were collected. Among them one small metacentric B-chromosome wasfound in two rats.

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

Morphology of the B-chromosomes in these black rats was exactly thesame by the small metacentric type except only one found in Hokkaido,
Japan which showed the small submetacentric type. From these findings
it is likely that the small metacentric B-chromosome is common in theblack rats, and it may have developed somewhere in Asia and propagatedthere, from which they were distributed widely to the other localities irrespective of the karyotype evolution from 2n=42 to 40 and 38. One small submetacentric B-chromosome found in Hokkaido, northern Japan, must havebeen developed independently from the above B.
Further Studies on the Frequencies of Chromosome Polymorphism inPairs No.1, 9 and 13 of the Black Rats in Japan
Tosihide H. YOSIDA

Pair No.1 chromosome as well as Nos. 9 and 13 in the black rat (Rattus
rattus) were polymorphic consisting of A/A, A/S and S/S complexes withregard to acrocentric (A) and subtelocentric (S) centromeres. A population
survey of the No.1 chromosome polymorphism in 453 black rats collectedin various localities of Japan has been reported (Yosida et al. 1971, chromosoma 33: 30), but a numerical survey of No.9 and 13 chromosomes has not
yet been carried out, except for a small samples (16 rats) in Japan (Yosida
1971, this report 21: 55). A statistical survey of A/A, A/S and S/S types
in the three chromosome pairs (Nos. 1,9 and 13) has been carried out in 185
black rats in Japan. Among them 95 black rats came from four localities,
Sapporo (22 rats), Niigata (15), Misima (35) and Nagasaki (23), which arelocated in northern, northwestern, southeastern and southern districts ofJapan. Remaining 90 animals were derived from our breeding colony
originated from a few rats collected in Misima.
Frequencies (%) of three chromosome types (A/A, A/S and S/S) of thepair No.1 chromosome in total 95 black rats coming from the field were
75,24 and 1, respectively, those in the pair No.9 were 91, 5 and 4, respectively, but those in the pair No. 13 were 49,36 and 18, respectively. The pair
No.1 of all rats from Sapporo and Niigata carried the monomorphic A/Atype, while that in specimens from Misima showed A/A (54 %), A/S (43 %)and S/S (3 %) polymorphism, and that from Nagasaki by A/A (74 %), AjS(21 %). With regard to pair No.9, all 22 black rats from Sapporo were of
the A/A type, while in Niigata, Misima and Nagasaki populations the rats

RESEARCHES CARRIED OUT IN 197535

with AIS or SIS pair were observed together with a high frequency of the AlA
type. Frequencies of the pair No. 13 were markedly different from those ofpairs No.1 and 9, because in all four localities rats with AI A, A/S andSIS types were usually found. About one half of the rats were A/S andSIS types.
Frequencies of chromosome polymorphism in pairs No.1, 9 and 13 in
90 Japanese black rats bred in the laboratory were shown that rats withAIS and SIS types in all three chromosome pairs were observed at higher
frequencies than those in the natural population of Japan, although modeof the occurrence of three chromosome pairs was somewhat different ineach chromosome pair. Based on the above findings it is likely that thechromosome inversion of the pair No. 13 has occurred in earlier period thanthose of the other No.1 and 9 pairs, and thus the rats with the inverted pair
No. 13 chromosome have distributed widely in Japan.
Frequencies of Chromosome Polymorphism in Pairs No.1, 9 and 13
in 'the Black Rats Distributed in the World
Tosihide H. YOSIDA

Frequencies of three chromosome types (AlA, AIS and SIS) in the three
chromosome pairs (Nos. 1,9 and 13) in the black rats (Rattus rattus) collectedfrom 9 different localities in Southeast, Southwest Asia and Oceania were
investigated. Total 141 rats examined were as follows; 7 from Hong Kong,
19 from Philippines, 6 from Kualalumpur, Malaysia, 8 from northern India,
8 from southern India, and 22 from northern Pakistan showed the Asiantype idiogram (2n=42); 14 from Davis, United State, 13 from Brisbane,
Australia, 25 from southern India, 3 from southern Pakistan and 3 from
Iran showed Oceania type (2n=38) and the remaining 13 from Kandy, Sri
Lanka, showed Ceylon type idiogram (2n=40). The frequencies of chromosome polymorphism of the three chromosome pairs in the Hong Kong
population was similar to those of the Japanese one, but in the Philippine
rats most of the pairs No.1 and 9 were subte1ocentrics, and pair No. 13
was polymorphic, most of the animals showing an acrocentric pair. The
Malayan population was similar to the Philippines, but pair No.1 in the former was more polymorphic than the latter. In the northern Indian population, the chromosome polymorphism was rather few, pairs No.1 and 9
being subtelocentrics and the pair No. 13 acrocentrics. On the northern

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

Pakistan population pairs No.1 and 9 were also usually subtelocentrics,
but pair No. 13 was polymorphic showing high frequency of subtelocentricchromosomes. The Asian type rats of Mysor population in southern India
was slightly different from those of northern Indian and Pakistan ones, because pairs No.9 and 13 were acrocentrics.
Pair Nos. 1 and 9 in Oceanian and Ceylon type black rats were always
the subtelocentric homologous pair, but no polymorphism was found inthese chromosomes. Pair No. 13, however, was polymorphic in regard toacrocentric and submetacentrics. This was similar to the rats in northern
India and Pakistan. Frequencies of three chromosome types in pair No. 13
were similar to all rats distributed in southern India and Pakistan, Iran and
Sri Lanka. They were remarkable by showing high frequency of the acrocentric pairs. On the other hand, those of the pair No. 13 in the black rats
collected from Davis, U.S.A. and Brisbane, Australia, were quite different
from those of the Asian population by showing a high frequency of thesubte10centric pair. Based on the above findings it is likely that the pericentric inversion of pair No. 13 occurred in earlier period than that theother pairs No.1 and 9, and therefore it was involved widely in the black rats
distributed in the world.

Karyotype and Chromosome Polymorphism in Indian gerbil
Tatera indica
Tosihide H. YOSIDA and Yuriko OCHIAI

A few Indian gerbils, Tatera indica, were collected from Mysor, India,
in 1972 by us, and then they were bred in this laboratory. Diploid chromosome number of this animals was 68 (33 autosome pairs and X, Y), among
which 50 acrocentrics (autosome pairs No. I to 25), 16 subtelo- or submetacentrics (autosome pairs No. 26 to 33) and a large metacentric X and a small
acrocentric Y being included. The smallest biarmed autosome pair No. 33
was remarkable by showing chromosome polymorphism with three chromosome types. Type A is a considerably large submetacentric, type B a smaller
submetacentric and type C the smallest subtelocentric. These types seems
to be developed by deficiency of the chromosome arm of the smallest pair
No. 33. Identification of the A and B types is really difficult because they
are the same submetacentrics. The type A, however, is almost the same
size as the pair No. 32, but the B was much smaller than that pair. Type CRESEARCHES CARRIED OUT IN 1975

37

is remarkable because of its smaller size than the above two and by having
a tiny short arm. This will be developed by breakage of the short arm of
the type B chromosome. Seven animals sampled at random from ourbreeding colony showed the following combinations; AB (1 rat), AC (2),BB (3) and BC (1). Six types were expected theoretically, but four types
were observed in the present materials. The other two types (AA and CC)
could be observed by sampling of more number of animals.
Mechanisms for Sister Chromatid Exchanges
Hatao KATO

By taking advantage of the fact that visible light (VL) induces strand
breaks only in bromodeoxyuridine (BrdU)-substituted DNA, and that those
breaks eventually lead to the formation of sister chromatid exchanges (SCEs),
the response of SCEs to VL was studied carefully in Chinese hamster chromosomes in which, out of four DNA strands, BrdU-substitution and occurredeither in one or three strands. The frequency of VL-induced SCEs did not
differ greatly between these two types of chromosomes. This apparently
contradicts to the expectation that the SCE frequency might be higher intrifilarly-substituted chromosomes than in the unifilarly-substituted ones
previded that each break has an equal opportunity to evoke an exchange
event, and seems to suggest that breaks induced in the post-replicational
regions of the trifilarly-substituted chromosomes do not contribute to the
induction of SCEs. On the other hand, when cells were submitted to caffeine
treatment after VL illumination, a drastic increase in the SCE frequency was
detected in the trifilarly-substituted chromosomes while a significant decreasein the unifilarly-substituted ones. It is very likely that breaks induced in
the post-replicational regions of DNA in the trifilarly-substituted chromosomes now participate in the exchange event, possibly due to interference ofrepair processes by caffeine treatment.
Based on these results, a working hypothesis is developed that the SCE
can arise by at least two different mechanisms, one operating at replicating
points probably utilizing the machinery of DNA replication, and the otheracting only in the post-replicational DNA portion, probably in a similar
fashion as assumed in a general model of crossing over in the eukaryote.
The majority of SCEs induced by VL would arise through the first mechanism
irrespective of whether chromosomes are unifilarly- or trifilarly-substituted

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

with BrdU. The second type of mechanism might operate only undersituation where two broken ends localized in two sister DNA duplexes caninteract. Such a situation would be provided if caffeine disturbs repairof breaks while permitting the displacement of single strands at the breakpoints.
The presence of these dual mechanisms may probably account for thediscrepancy encountered in the explanations of the induction of SCEs byvarious exogenous agents as well as spontaneous SCEs.
BIOBR Mouse with a Partially Deleted Y-chromosome
Kazuo MORIWAKI and Katsumi SAKAKIBARAA BlOBR mouse with a partially deleted Y-chromosome has been foundin the progenies of a non-pedigreed litter mate pair which was obtained
from the Jackson Laboratory, U.S.A. in 1968. Actually the karyotype wasobserved for the first time at the 3rd generation in our institute by using bonemarrow cell preparation, revealing about half size of Y-chromosome in
the 40 diploid chromosomes. G-banding pattern analysis of this mouse
failed to show any translocation of the Y-chromosome fragment to the otherchromosome. Observation of the spermatogonia has also shown no tetravalent meiotic chromosomes due to Y-autosome translocation.
This subline with a small Y-chromosome was designated BIOBR_ydel
and has been successfully propagated until now through sister-brother mating.
The litter size of them is almost the same as the normal BlOBR line, though
the sex ratio apparently deviated to the female. Recently the Y-chromosome
was able to be introduced to the three congenic strains of mouse for establishing BlO_Ydel, BlOA_Ydel and BlOD2-Ydel, respectively.
Furthermore, microscopic survey of the spermatozoa of BlOBR_ydel
mouse demonstrated significantly higher occurrence of mal-formation.
Whether the partial deletion ofY-chromosome could cause the malformationof the spermatozoa directly in the Y-carrying cells or indirectly throughthe male having the partially deleted Y remains unsolved.

RESEARCHES CARRIED OUT IN 1975

VI.

39

MUTATION AND MUTAGENESIS IN ANIMALS

Mutagenic Effectiveness of an Internal f3 Emitter, Tritium. (2) Measurementof Mutagenic Effectiveness on Germ Cells of Bombyx
Yataro TAZIMA, Kimiharu ONIMARU and Yosoji FUKASE

We reported in the last issue of this report (Ann. Rept. Natl. Inst. Genet.
No. 25) that mutagenic effect of tritium was able to be demonstrated by somatic mutation expressed in Fllarvae of Bombyx by means of intraperitoneal
administration of the isotope to mother moths. The method, however,
hardly permitted the estimation of induced mutation frequency. Hence, thisexperiment was undertaken with use of a germinal mutation system. 3H_
TdR in aqueous saline was injected into the body cavity of wild type femalesof a strain C108 at a mid-pupal stage (100 ,uCi/pupa). The average radioactivity transmitted to a deposited egg was 5.2 X 10- 9 Ci. Injected and noninjected (control) females were crossed to pe ok +,.. males. There were
164 days from injection to the hatching of F l eggs. Calculated exposure
dose was 3.17 rad per day and 519.5 rad in total.
F l larvae exhibited tiny ok spots at high frequency. Those females werecrossed to pe+ ok re males which made possible the assessment of germinal
mutation frequency at re locus. Cluster-like appearance of re mutants wasmarked. The mutation frequencies were 4.9 X 10- 5 and 4.1 X 10- 3 in thecontrol and treated group respectively, giving induced frequency of 7.96 X
1O-6/rad. By assuming Poisson distribution mutation frequency per cell
generation was calculated to be 1.21 X 1O- 6/rad.
On an Effective Stage of Chemical Mutagens as Detectedby the Silkworm Oocyte System
Yataro TAzlMA and Kimiharu ONIMARU

In the silkworm oocyte system (Tazima, 1973), chemicals' to be tested formutagenicity are injected at mid pupal stage, 5-6 days before emergence.
It was conjectured that injected chemical was incorporated maximum into
the oocytes after the injection at this stage and reacted with genetic materials
around meiotic metaphase which takes place just after oviposition. Ourfinding, that AF-2 was mutagenically effective even when it was injected five

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

days before supposedly sensitive stage, i.e. around meiotic metaphase, raised
a question, because the compound had been known to be easily metabolized
in bacterial cells or mammalian liver tissues. Immediate mutation responseof genetic materials to injected chemicals has been elucidated by an experiment using EMS, a biological half life of which was known about 48 hours
at 25°C. However, the obtained response patterns were not necessarily
consistent.
Experiment was, therefore, continued using DES, a biological half life ofwhich was known to be few hours. The chemical was injected into female
pupa at various developmental stages every 24 hours at the dose of 50 p,g/
pupa. The mutation response curve was clearly mono-modal representing
a peak at mid pupal stage, six days before emergence. Further experimentwith EMS, in which data were collected at daily basis regarding time interval
between injection and emergence instead of on average duration basis, also
exhibited clear cut mono-modal curves. It was concluded that genetic
materials in silkworm oocytes at mid pupal stage are in a state that can
respond to chemical mutagens. The system may, therefore, be applicable
also to the detection of chemical mutagens of short biological half life.

Nature of Chemically Induced Mutations Obtainedby Oocyte System in the Silkworm
Yataro TAZIMA, Kimiharu ONIMARU and Akio OHNUMAThe purpose of this experiment was to know whether there was qualitative
difference in the recovered mutation at specific loci by oocyte system between
those induced by chemical mutagens and by ionizing radiations. The experiments were performed by treating oocytes of the silkworm with chemical mutagens, EMS and DES. Mutations obtained at speficic loci were
examined of their spectrum with regard to lethality expressed in homozygote, so as to be compared with those induced by ionizing radiations.
Wild type females of strain CI08 were treated with those chemical mutagens
at mid pupal stage and mated to non-irradiated males of pe re, after emergence, to pick up mutations to pe (or re) in Fl. Of those mutants only
females were used to cross with + re (or pe +) males, so as to separate
the mutation-bearing chromosome from its homologue (pe re). All thosecrosses showed segregation of 1+: I re (or 1+: I pe) in the next generation.
After discarding the red (or pink) eggs, only mutation-bearing heterozygotes

RESEARCHES CARRIED OUT IN 197541

were isolated, and they were mated inter se.
The recovered phenotype (pe or re) appeared again in all mutant strains
with a frequency of approximately 25 %. Since the marker chromosome
used for the detection of the mutation were already been discarded andthere are no crossingover in the female heterozygotes in the silkworm,
the segregated phenotype could be regarded as a homozygote for a newly
arisen mutation.
Lethality was examined for all new mutations. For mutations which wereobtained first as mosaics, lethality test was performed only in female lines.
They were then crossed to pe re males and BF1 females of mutant phenotype
were subjected to the further analysis by the same procedure as mentioned
above. The results are given in the Table, together with those obtained forx- or r-ray irradiation in our previous experiments (Y. Tazima, Japan.
J. Genet. 36, Suppl. 50, 1961).
Table Results of lethality test for induced mutations in homozygotes
Homozygote
MutagenViableLethal

15
5

16
31

Semi-L

Trans.

Total_ _---...

EMS, DES
X-, r-rays

(2)

32
36

When compared to radiation-induced mutations, where lethal is overwhelming, it may be noted that the proportion of lethal is lower in chemicallyinduced mutants. This indicates that mutational lesions produced bychemical mutagens, as EMS and DES, are less drastic than those producedby X- or r-rays. Anyway, it should be noted that the proportion of lethal
is still fairly high after chemical treatment and even chromosome aberrationsof two hit events as translocation were induced after chemical mutagen
treatment.
Estimation of Incorporated Amount of Furylfuramide into Egg-plasm
after Injection into Silkworm Pupa
Yumi SmMADA, Akio MURAKAMI and Yataro TAZIMAIn the silkworm oocyte method, developed for mutagenicity testing, themost effective stage of injection of mutagenic compound was determined
to be 5-6 days before emergence. Screening of various mutagens has been

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

performed by utilizing this method. It seems to be essential for this method
to know exactly the amount of incorporated mutagenic compound into the
ovum for the evaluation of the mutagenic effectiveness. Using furylfuramide
which is labelled with radioactive HC this work has been carried out. Wildtype female pupae of a strain C108 were injected, 5 days before emergence,
into the body cavity with 0.025 ml each of 0.5 % CMC saline suspension of
14C-AF-2. Injected doses of the compound were 10 p.g and 20 p.g per pupa.
Treated females were mated to non-treated males. After oviposition,measurement of radioactivity was performed on urine, deposited eggs,
ovarioles with some remaining ova, body fluid, and the remnant body.
All those samples were well grinded and shaked with 2 ml distiled water and
0.1 ml of each sample was dissolved in a Bray scintillator for counting theradioactivity by scintillation counter. The results are given in the Table.
Table

FemaleMale

Proportion of radioactivity incorporated into deposited eggs,
remnant body, etc. after injection of 14C-AF-2,
five days before emergence
Remnant
body

Body
fluid

Genitalorgan*

Deposited
egg**

Urine

Total

6-7
8.2

2-3
2.9

2-3
0.0

6-7

82-83
88.9

100
100

* Including undeposited ova in ovariol for the female and testes and other genitalorgans before ejaculation for the male.
** On an average deposited eggs per batch were 467 in number and 280 mg in weight.

Radioactivity per egg grain (weighing 0.6 p.g) was calculated 0.02 % of
total amount of injected radioactivity of the chemical. With an assumptionthat chemical structure of the injected AF-2 is kept unchanged, it can be
estimated that the amount of AF-2 incorporated into deposited egg is 2 x 10- 3
pg (3.3 ppm) per egg grain, when 10 pg of AF-2 had been injected. The
amount is very close to that had been legally permitted for addition to meatsausages and soybean curd, maximum concentration being 5 ppm.
Differential Stage Sensitivity to the Induction of Mutations of
Post-Meiotic Male Germ-Cells by Mitomycin C in the Silkworm
Akio MURAKAMI and Tetsuro MUROTA

Mitomycin C (MC) is highly mutagenic for inducing egg-color specific

RESEARCHES CARRIED OUT IN 1975

43

locus mutations in the sperm of adult moths (Inagaki and Oster, 1969), but
not for dominant lethal mutations in the sperm at the mid-pupal stage orfive days before emergence (Murota and Murakami, 1975). In mice, accumulated evidences indicated that, whatever the basic explanation might be,some chemicals having the ability to produce dominant lethals also produce
so called point mutations and vice versa. If such the statement is applicableto the silkworm, the frequency of specific locus mutations in the sperm at
the mid-stage pupae by treatment with MC would not markedly increase asthat of dominant lethals, while the frequency of MC-induced dominantlethals in the sperm at the late stage of pupae (including adult moths) would
increase as that of specific locus mutations.
1-50 flg per individual of MC which had been dissolved in saline wasinjected into the abdomen of wild type (CI08 strain) silkworm males atdifferent developmental stages, mature larvae, mid- and late-stage pupae.
In the testes of mature larvae, there are a predominant number of latespermatids and sperm in addition to pre- and meiotic germ-cells. The mostadvanced germ-cells throughout the whole pupal stage (10-12 days at 25°C)
seems to be at the stage of sperm. The treated males were then mated with
non-treated marker females having double recessive egg-color genes, pe andreo The incidence of the dominant lethal mutations was detected by the
reduction in hatchability of fertilized eggs as compared with control eggs
produced from non-treated parents and that of the recessive visible mutations
was detected by the egg-color specific locus method.
The results of dominant lethal tests were indicated that LD 60 values ofsperm in mature larvae and late pupae were ca. 30 flg and 15 flg of MC per
individual, respectively. However, an LD 50 value of the sperm in mid-stage
pupae could not be estimated in doses lower than 50 flg since over this dose
female moths mated with male moths which are derived from the treatedpupae did not deposit a sufficient number of fertilized as well as unfertilized
eggs. Thus, the sperm in late-stage pupae was the most sensitive to MC
when judged by the incidence of dominant lethals, and followed by the germcells in mature larvae and mid-stage pupae. The results of the specificlocus mutation tests were indicated that the sensitivity to the induction of
mutations was quite similar to that to dominant lethals, suggesting thatthe frequency of MC-induced specific locus mutations in post-meiotic germcells is in parallel with that of dominant lethals. This may support theview that chemicals having the ability to induce dominant lethal mutations

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

also induce the specific locus mutations and vice versa, suggesting that thenature of the specific locus mutations is partly similar to that of the dominantlethal mutations which may be due to chromosome aberrations.
For the mechanism of the differential stage sensitivity to the induction of
those mutational incidences, three possible interpretations may be given:
the first is a differential susceptibility to the germinal selection of the damaged
DNA in the different post-meiotic germ-cell stages prior to fertilization (orpenetration of sperm into ova) and the second is a differential repairabilityof the damaged DNA molecules. Lastly, a combination of those abovementioned possibilities may be the cause of the mitomycin C stage sensitivity.
Comparison of Recombinogenic Effects of Radiomimetic Alkylating
Agents on Oogenic Cells in the Silkworm
Akio MURAKAMI

Recombinogenic effects of several radiomimetic alkylating agents having
a difference in functional arm numbers on silkworm larval prophase I
oocytes were compared in order to throw light on the mechanisms underlying
those of ionizing-radiations. This cell stage is showing the highest incidenceof X-ray induced recombinational events. One monofunctional alkylating
agent, ethyl methane sulphonate (EMS) and two polyfunctional ones, mitomycin C (MC) and trimethylmelamine (TEM), were used. For estimating
recombination frequencies the egg-color method (Murakami, this Report 23:
66-67 (1973» was employed. Mature female larvae (prophase I oocytes)
were treated with these alkylating agents.
The results indicated that no significant increase of recombinational eventsin prophase I oocytes treated with EMS was observed. On the other hand,when the oocytes were treated with MC and TEM, the frequencies of recombinations were clearly higher than those of controls as observed in
X-rayed oocytes. The present finding and others tend to suggest that
X-ray induced recombinational events may rise primarily through the double
strand breakage of DNA within the vicinity of the complementary strand
similar to the breakage event being derived from cross-links between the both
DNA strands in oocytes treated with polyfunctional alkylating agents.

RESEARCHES CARRIED OUT IN 1975

45

Mutagenesis of Furylfuramide in Mature Sperm of the Silkworm
Akio MURAKAMI

Furylfuramide (FF) is proved to be mutagenic in pupal oocytes of thesilkworm (Tazima and Onimaru, 1973) as well as in many of microbes andmammalian cultured cells. However, it has been reported that FF is nonmutagenic in pupal spermatozoa of this insect (MURAKAMI and FUJIOKA.
MUTATION Res. 31, 266-267, 1975). These findings and others indicated thatin the silkworm genetic effect of FF is clearly dependent on the cell stage(or type) and mature sperm seems to be resistant to the chemical in contrast
to oocytes.
Current evidence indicates that although FF itself is a hydrophobic and/or
non-mutagenic compound, it becomes mutagenic (and hydrophilic) compound(s) after metabolic activation by rat liver homogenates. If this is thecase, FF may be not activated in the male pupae before reaches it the spermatozoa. To check this possibility mutagenic effects of FF on the spermatozoa
was investigated by means of the metabolic activation of the compound with
rat liver homogenates. Mutagenicity was determined by the egg-colour specific locus method. Livers from 10 week old male Wistar rats were used inthis experiment. The incubation mixture with containing required cofactors
and a supernatant from the rat liver homogenate were incubated in a shaking
incubator at 37°C for 15-30 min. For the experimental group FF was added
at concentration of 7.5 ftg per pupa. After incubation 0.025 mljpupa of
the reaction mixtures with or without FF was immediately injected into
wild-type (Aojuku) male silkworm mid-pupae, which were then mated tomarker females having pe and re gene loci.
The killing effect of FF on pupae was increased with doses of the compound and an estimated LD~o was around 5-15 ftg/pupa depending on bodyweight. While the treated FF did not show any significant killing effects
within the doses tested ranging from 0.5 to 50 ftg per pupa, indicating that
FF might be converted into non-toxic intermediates. The frequency ofthe egg-color specific locus mutation in males after treatment with the complete incubation mixture containing FF slightly but significantly increased
as compared with the control. Next, 5 or 10 ftg/pupa of FF labeled with
HC at carbon-3 of the acrylamide moiety was injected into the mid pupal
male. After emergence the radioactivity remained in testis, blood and theremnant part of the moth as well as in meconium was measured. The result

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

so far obtained indicating that a very slight radioactivity (0.2 %) was detected
in tests. A few percents of the activity was remained in blood as well as
in the remnant part of the moth. Above 90 % of the radioactivity was recovered in meconium, suggesting that almost treated FF was excreated.
Thus, it may conclude that the lack of mutagenic activity of FF in the
spermatozoa of the silkworm is absence of the incorporation of the compound probably due to lack of an activating enzyme(s) for FF in the male
pupal body or in the testis.

Spontaneous Diabetes in Djungalian Hamster, Phodopus sungorus
Tosihide H. YOSIDA

Djungalian or striped hairy footed hamsters, Phodopus sungorus, wereobtained by courtesy of Dr. H. E. Pogosiantz in 1967 and from one pair ofthem a colony of the animals was established in this laboratory. They weremaintained by inbreeding and/or outbreeding. Among 35 animals (18 femels
and 17 males) sampled at random from the colony, the spontaneous diabetes
was found at a high frequency; about one third (12 animals) among themshowed the heavy diabetes. The disease was found in both males and females
and also in adults any youngs. The diabete was found by reaction of theurine to the test paper (test-paper) prepared by Shionogi Pharmaceutical Co.
LTD., Japan. The urine reaction of the disease animals showed all serious
sympton asor
to the test paper. The behaviour of the diabetes
animals seems to be normal, and they are fertile. The genetical study ofthe disease is in progress now.

++ +++

Dominant Black Coat Color in the Black Rat
Tosihide H. YOSIDAOver five hundred black rats, Rattus rattus, were collected in several
localities of Japan. Their coat color was agouti except one black color.
The rat with the black coat was bred in the laboratory and then crosted to
the rats with the wild agouti rats. Then the rats were bred for several
generations. The black coat color in the Norway rat and the mouse isalways recessive character which is subjected by a non-agouti gene (a).
From the segregation ratio of the o,spring obtained by crossing between

47

RESEARCHES CARRIED OUT IN 1975

the wild and the black color, it was found that the black coat color in theblack rat was completely dominant as seen in Table 1.
Table 1. Segregation of coat color in the black rats
Offspring
Parents

No. of litters
examined

No. of rats
examined

Segregationwild: blackxwild(+1+)*

black(BLI+)

8

31

18
(+j+)

13
(BLj+)

blackx(BLj+)

black(BLI+)

7

36

15
(+1+)

21
(BLj+)(BLjBL)

blackx(BLjBL)(BLj+)(BLjBL)black(BLjBL)(BLjBL)(BLI+)

050(BLjBL)(BLj+)

x

wild
(+1+)

wild
(+1+)

* Probable conbination'of genes.

12

50

50

206

BL: dominant black gene.

206
(+1+)

0

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

VII.

RADIATION GENETICS AND CHEMICAL MUTAGENESISIN MICRO-ORGANISMS AND PLANTS
Comparative Studies on DNA Damage Induced by Gamma-ray
and 3H-decay
Tsuneo KADA, Tadashi INOUE and Yoshito SADAIE

In order to evaluate damage induced in DNA by ionizing radiations anddecay of radioisotopes incorporated in the cell, comparative studies havebeen carried out on inactivation of transforming DNA, induction and inactivation of spores of Bacillus subtilis, as well as on production of DNA
single-strand breaks of colicin EI plasmids by irradiation with gamma-ray
and treatment with 3H-glycerine.
In the transforming study, inactivation of an arginine marker was measured
using cells of the strain HI7 (arg, try) as recipient. For mutation induction,
spores were prepared on Schaeffer medium by cultivating the strain witha supprressible his mutation and histidine reversions were studied. Circular
DNA was extracted from E. coli harvoring colicin El plasmids and neutral
sucrose gradient sedimentation analyses were made on irradiated DNA
molecules. Irradiation or treatment with 3H-glycerine was done in appropriate buffer at O°e.
Based on the theoretical estimation that the absorbed energy of f3-rayfrom 3H (83 ,uCi/mlfhr) corresponds to 1 R of gamma-ray, we concluded that
relative effectiveness of 3H-decay is very similar to that of gamma-irradiation
with regard to inactivation of the arginine marker of transforming DNA andto production of DNA single-strand breaks, indicating that the 3H-decay
effects are solely explained by f3-irradiation.
On the other hand, at identical absorbed doses, 3H-decay was morethan 100 times more efficient in inactivating and in inducing mutation in
spores. These results were presented at the 1976 annual meeting of JapaneseSociety of Radiation Research.

RESEARCHES CARRIED OUT IN 1975

49

Enzymatic Repair Mechanisms of DNA Damage Induced by Ionizing
Radiation: Identification of a "Cleaning Enzyme"
Tadashi INOUE and Tsuuneo KAoA

Studies have been developed to elucidate enzymatic mechanisms of
repair of DNA damages induced by ionizing radiation. Previous studies(Biochi. Biophys. Acta 395 284, 1975) revealed the existence of endonucleolytic m exonucleolytic activities specific for gamma-irradiated DNA in
extracts of Bacillus subtilis cells, both promoting the priming activity ofirradiated DNA for type I DNA polymerase. The detailed properties ofthe endonuclease were described earlier (Biochim. Biophys. Acta 395 294,
1975).

Further studies were made on the enzyme which had exonucleolytic activity on gamma-irradiated DNA. The enzyme was purified more than
340 fold by means of salt fractionation and chromatographies on DEAEand phospho-cellulose. The purified enzyme preferentially degraded irradiated DNA exonucleolytically releasing mono-phosphorous substancesand produced effective priming sites for DNA polymerase. Hypothesis isunder examination in which this enzyme is a promissing candidate for the
exonucleolytic "cleaning" of damaged nucleotides which was produced withgamma-irradiation.

Enhanced Mutagenesis, Repair Deficiency, Mutator Activity,
and Thermal Prophage Inducibility in DNA-BI32 Strainsof Bacillus subti/is
Yoshita SADAIE, Kikuko NARUI and Tsuneo KAoA

The integrity of the bacterial chromosome is maintained by a numberof cellular components. Isolation of dna mutants is an attempt to clarify
the role of each component whose synthesis is controlled by respective
dna loci. We have found pleiotropic effects of a ts mutation dna-BI32
at permissive temperature which was isolated by Hara and Yoshikawa andmapped in the region of chromosome replication origin of Bacillus subti/is(Nature New BioI. 244, 200). This mutation arrested DNA synthesis (within
30 min) at a restricted temperature without any change in the purAjmetB
ratio and was classified to be neither of the initiation nor the continuation
type. We have found that strains carrying this mutation exhibited a lower

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

capacity for repairing radiation or chemical damage at a permissive temperature. Introduction of polA59 mutation (Gass et al. J. Bacteriol. 108, 364)
further enhanced the repair deficiency, indicating that the polA59 or the
dna-8132 blocks independent and complementary pathways. The dna-8132
mutation alone did not reduced cellular capacity of reactivation of UV
irradiated phages, whereas the polA59 mutation did. However, the effectof polA59 was enhanced in the presence of dna-8132. Therefore, the wildtype dna-8132 gene product appears to be involved in phage DNA repair
in the absence of polymerase I activity. Spores of dna-8132 strains wereresistant to UV while polA59 spores showed enhanced sensitivity to UV
irradiation. The mutations dna-8132 and polA59 did not affect the resistancyof spores to gamma-rays. Spontaneous reversion mutations of a suppressible nonsense his mutation were observed at a higher frequency in the
dna-8132 strains and the frequency was much enhanced in the simultaneouspresence of polA59 mutation. These results suggest that the dna-8132 geneproduct may be involved directly in the chromosome replication and repair.
SP02 lysogens carrying this mutation produced mature phages upon a temperature shift from 30 to 48°C. Phage production at a nonpermissive temperature suggests that there are few defects in the precursors of DNA synthesis in the mutant. Base change type mutations (from His- to His+) were
induced by chemicals and UV irradiation. Mutation was abnormallyenhanced in the dna-8132 polA59 double mutant strain. Thus it is suggested
that the dna-8132 gene product may be involved in a error prone repair andmutations observed may have originated from errors in the base selection.
Though the dna-8132 mutation was found to be linked to rec-43 (J. Bacteriol.
125, 489), no drastic reduction in recombination ability was observed in thestrains with dna-8132. Some of the results were reported earlier (J. Bacteriol.
126, 1037 and Ann. Rep. Natl. Inst. Genet. 25, 51).
Detection of Environmental Mutagens and by the Rec-assay
and Reversion-assay Procedures
Tsuneo KADA

We have been carrying out screening of chemical mutagens in our environment and detected upto now more than 30 new mutagens, by means of the
rec-assay and reversion-assay procedures, including phloxine, AF2, DAPA,
reaction products between NaN0 2 and sorbic acid, etc. (for review see T.

RESEARCHES CARRIED OUT IN 1975

51

KADA, Scientific Publication No. 12 of International Agency for Research
on Cancer, p. 105 1975 and Y. Shirasu et al. Proceeding of the 1976 Cold
Spring Harbor Symposium, in press). The rec-assay screening was further
carried out in this laboratory by Dr. N. Kanematsu of Gifu Dental College
on about 80 metal compounds and some 25 of them were found to be clearly
positive. They include compounds of chromium, arsenic, mercury, cadmium,etc. on which reversion-assays were made. Except those compounds whose
mutagenicities are already known, some compounds of rhodium (Rhcl 3) and
Vanadium (VOC1 2) were found to induce tryptophane reversions in Escherichia coli WP2 try. We have recently carried out the rec-assay screening on
about one hundred food samples, homogenates prepared from vegetables,
fruits, species and other natural food. Many species including garlic black
pepper, cinnamon, paprika, nutmeg, clove, oregano, mustard, and curry gave
positive results.
Waxy Starch Mutations in Rice
Etsuo AMANO

Induction of waxy starch mutations in rice plant, Oryza sativa var. Norin
No.8 has been continued and five new mutants were obtained in 1975.
Following mutagen treatment was effective. Unhulled seeds which had
been presoaked in water for 24 hours were soaked in 0.05 Methyl methanesulfonate (EMS) aequous solution for five hours. After the mutagen
treatment, seeds were rinsed and incubated in water for 20 hours. A 27°C
shaking incubator was used for these treatments. Seeds were sown in soil
in germinating flats and grown in them until harvest. A few panicles
were obtained from each treated seed. Harvested and dried panicles hulled
between two wooden plates with rough surfaces. Visually selected waxy
starch mutant grains were confirmed by iodine staining. Each panicle wasconsidered as a unit of mutation and all grains of the mutant segregating
panicles were kept for further examinations.
When Norin No.8 was treated, only a few endosperm mutants other thanwaxy starch could be detected visually. In the present experiments only
four opaque endosperm of different degrees and a case of short and roundgrains were found. Compared to those five possible endosperm mutations,
15 waxy starch mutants were detected, including a very leaky case (74wx3)
confirmation of which is under way. Of these 15 waxy mutants, seven were

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

leaky type of some degrees, or in other words mutations fixed somewehere
between non-waxy starch and fully waxy starch. Ten mutants were planted
in 1975, as M 2 or M a (one mutant) generations. In most of the mutants,
some unfavorable growing characters and/or low fertilities were accompanied.
However, segregation of these characters in homo- or heterozygotes of thewaxy mutants suggested that most of these unfavorable mutations accompained might locate in different chromosomes or very weakly linked.
These results were very alike to those of maize experiments and may be
helpful in practical mutation breeding, provided frequent and various
degrees of leaky mutations are taken into consideration.
It was noticed during examinations of hulled grains of heterozygotes forsegregation scoring that in many waxy mutants some of the non-waxy
grains could be classified as intermediate phenotype. In 1975, some of theseintermediate grains were planted separately and all of them turned out to beheterozygous for the waxy gene. In this case, about half of the remaining
non/waxy grains were also heterozygotes although the number of plants
examined was not so many. Further examination of grains of waxy heterozygotes revealed that most of the waxy starch mutants obtained in the present
experiments seemed to have gene dosage effect in endosperm. The expressions of heterozygotes differ from grain to grain and distributed rather
continuously between waxy and non/waxy. However, in some cases,
possible four classes of the genotype in triploid endosperm could be separated
though with difficulty. Quantitative measurement of the phenotype andconfirmation of these four classes are in progress. These intermediate
phenotypes differ in their genetics and expression from those of the leaky
waxy mutants. The latter ones showed uniform waxy characters within
the mutant and could be established as homozygous mutant lines. Intermediate phenotypes of heterozygotes were genetically unstable and always
segregated in the following generation into waxy, intermediate- and non-waxy
endosperm classes.
Germination and Early Growth of Some Podostemaceae Seeds
Etsuo AMANO

Seeds of Mourera fluviatilis and two other South American species ofPodostemaceae could successfully germinate in dilute nutrient solution.
The seeds were about 1/4 mm or smaller in length and had a layer of cells

RESEARCHES CARRIED OUT IN 1975

53

which imbibed water easily, swelled and seemed to secrete some kinds ofgluing materials. Thus they could be attached to a slide glass with a dropof distilled water. Once dried, the seeds sticked on the glass all throughthe surface disinfection procedure and incubation in the culture solution
which was basically 1/10 dilution of inorganic recipe of Murashige andSkoog (1962).
Newly obtained Podostemaceae seeds from Sri Lanka were tested withthe same culture methods. All five groups, or species (unidentified) had
capsules and seeds as small as the smaller species in Apinagia, a SouthAmerican genus. Sticking property of wet seeds was the same as those of
the South American species. Dry slide glasses on which seeds were attached were surface disinfected by immersing into mixture of one part ofethanol and one part of 3 % H 20 2 for five minutes. Glasses were thenrinsed twice in distilled water. Mter three days of incubation in culture
solution in Petri dish kept at 25°C and under continuous illumination,
germination started. Basic patterns of the germination were the same forall the species tested. Young plant emerged from seed coat with its rootend first. However, there was no conspicuous stem or root on the youngplant but only root hairs appeared on the outer end of the part where two
cotyledons met. The young plant sticked to the glass by these root hairs
after emergence from seed coat. Sri Lanka species were different in shapeof cotyledons and size of young plant from the South American species.
M. fulviatilis and two Apinagia species had needle or cylindrical cotyledons
which elongated after germination. Fully opened cotyledons reached to 2
to 5 mm from tip to tip. One species from Sri Lanka had cylindrical
cotyledons but four other species opened rather flat cotyledons. Seedlingsof all five Sri Lanka species were smaller than 1 mm. In all the species
tested, leaves were produced from basal part of the cotyledons, crosswise
and alternately, and grew in direction of either cotyledon. Disk shapedpart between cotyledons grew longer into elliptical form and then the growing
point rose in small angle. After this early flat and centipede like forms,
they start expressing their adult characteristics.
Fresh Podostemaceae seeds showed good germination under the present
culture condition, but for further growth they require ample volume of theculture solution and good aeration. Under these favorable condition
M. fulviatilis grew up to 3 cm in diameter and produced characteristic spinous
processes and papillae on their leaves. A species of Apinagia (A. longifolia)

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

reached to about 5 cm long with more than seven nodes. In still culture,
M. fulviatilis seedlings could survive in the aseptic culture solution in deep
Petri dishes for two years though growth was very slow. It had not beensuccessful yet to grow the plants in open culture.
This study has been made in cooperation with the Kihara Institute for
Biological Research, and seed materials were collected and kindly suppliedby the expedition parties of the Kihara Institute.
Sex Expression in Monoecious-Iike Variants in Cucumber
Taro FUJII

Sex expression in the F 1 plants obtained from the crosses between original
gynoecious strain (MSU) with male flowers of monoecious-like plant (MSU713-5-M) has already been reported (Ann. Rep. 25, 56-57). Make flower
development in the F 2 generation was examined successively using seeds
obtained by selfed monoecious-like F 1 segregants. The data are diagrammatically given as follows;
F 1 fruit No.

MSU-713-5 X MSU 713-5-M
5
1

I

I

No. of plants Gene>. Mono:(segregation)
17
6

I

Geno. Mono.
23
18

I

5-32
5-1
5-7
(49 male flo- (37 male flo- (60 male flowers overwers overwers over6 nodes)
10 nodes)13 nodes)F 2 fruit No.
(male flowers
in F 2 plant)

1-8(one male
flower ona node)No. of plants(segregation)

Geno. Mono. Geno. Mono. Geno. Mono. Geno. Mono.
7
3
8
18
5
10
5 10

I

I

I

__I~

Segregation of monoecious plants was observed in all the F 2 pupulations;even a plant with only one male flower (F 2 No. 1-8) produced a large numberof monoecious plants. Although more than half of plants in each strain
had shown genoecious habit in the F 1 generation, the number of monoecious
types exceeded genoecious types in the F 2 • This suggests that the tendency
to monoecism is promoted by selting.
The experiment was repeated again in the second experiment. The same

RESEARCHES CARRIED OUT IN 197555

original strain (MSU-713-5) was exposed to 30 and 50 kR of gammarays.
One of 34 M 1 plants in 30 kR lot developed one male flower; of 28 plants
in 50 kR lot, 3 plants, designated as MSU-50A, 50B and 50C, developed
male flowers similarly as in the first experiment. Selfed seeds were obtained
from these plants. Monoecious segregants in the F 1 generation were 19, 21
and 23 in 50A, 50B and 50C, respectively among each 34 plants.
As mentioned above, monoecious-like plants were frequently observedin the irradiated (M 1) generation. If they were mutants, either induced or
spontaneous, they would behaved as recessives. Moreover, their incidence
was much higher than the usual mutation frequency. Though the mechanisms involved are still not clear, the results of the present experiment suggestthat the monoecious-like variants might be due to a retain change in heredity.
The change from genoecism to monoecism might be attributed to a radiationinduced change in the gene(s) controlling growth hormone metabolism.
Effects of Gamma-rays and Neutrons on the Seedlingand Callus Growth in Rice Seeds
A. A. BARADJANEGARA, Taro FUJII and Etsuo AMANO

Seeds of rice c.v. Norin-8 and two radiation induced dwarf mutants
MGS-46 and -95 were used to investigate the effects of gamma- and 14 MeV
neutron-radiations in different culture systems. The following three different culture systems were adopted in the present study. (1) Irradiatedseeds were sown in soil. (2) The surface of irradiated seeds was sterilized
and the seeds were sown onto agar slant media in test tubes. Two types of
media, modified-White's (M-W) and modified-Eriksson's (M-E) were used.
(3) To examine effects of gamma-irradiation on the callus formation andgrowth, the third culture system was used consisting of the M-W and M-E
media with 20 mg/liter and 2 mg/liter 2,4-D supplimentation. Seedling
growth of irradiated seeds in soil, and in two types of synthetic media, as
well as the callus growth on 2,4-D supplemented media were measured as
an index of radiation damage. The seedling height in soil culture indicate
considerable decreases in growth with increasing of gamma-ray doses in theparent Norin-8, and similarly in two mutant strains. However, two mutant
strains were relatively more sensitive to the neutron treatment compared to
the Norin-8. The seedling growth on media was indicated that the seedlingheight in the control lots of MGS-46 and -95 on the M-E medium were

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

almost twice of those found on the M-W medium, while the control lots of
Norin-8 showed a slight difference between two types of media. In theirradiated lots, Norin-8 showed striking decrease in growth especially on
the M-W medium, while growth of two mutant strains had repressed slightly
on the same medium. The callus formation on the M-W varied with gammaray doses. In the unirradiated controls as well as in the lowest dose lots,
100 %callus induction was seen in all strains examined. The callus formation
ability was slightly reduced with 18 krad treatment and drastically depressed
at 27 krad exposure. On the other hand, 100 % callus growth formation
took place on the M-E medium in every lot. The callus growth was clearlyretarded after radiation treatment, and the reducing rates along with the dose
increment were almost the same as those observed in soil culture. Fromthese experiment, the M-E medium seem to be appropriate for both the seedling and callus growth of rice.

RESEARCHES CARRIED OUT IN 1975

VIII.

57

POPULATION GENETICS (THEORETICAL)

Mathematical Contributions to Population Genetics
Motoo KIMURA

Mathematical theories have played a vital role in the development of
population genetics. In this paper, recent progress in the field of mathematical population genetics is reviewed, with special reference to the diffusion
models treating the stochastic behavior of mutant genes in a finite populations. Also, recent developments of theories treating linkage disequilibrium due to random drift are reviewed. For details see Genetics 79:
91-100 (1975).
Distribution of Allelic Frequencies in a Finite Population
under Stepwise Production of Neutral Alleles
Motoo KIMURA and Tomoko OHTA

A formula for the distribution of allelic frequencies in a finite population
is derived assuming stepwise production of multiple alleles. Monte Carloexperiments were performed to check the validity of the formula, andexcellent agreement was obtained between theoretical distribution and experimental results. The formula should be useful for analyzing genetic
variability in natural populations that can be detected by electrophoretic
methods. For details, see Proc. Nat. Acad. Sci. 72: 2761-2764.
Theoretical Analysis of Electrophoretically Detectable
Polymorphisms: Models of Very Slightly
Deleterious Mutations
Tomoko OHTA and Motoo KIMURAThe nature of mutation-selection balance was investigated using models
of mutation which assume stepwise production of new alleles in the allele
space occupied by one or two type genes and multiple slightly deleterious
mutations. The distribution of allelic frequencies and the effective number ofalleles at equilibrium were obtained based on deterministic treatments. It
was shown that they depend on the ratio between the selection coefficient

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

and the mutation rate (s/v). When this ratio is large, the frequencies ofthe deleterious alleles become too low to form polymorphism; however,when the ratio is small, such a mutation-selection balance becomes important
as a cause of polymorphism. In addition to analytical treatments, extensiveMonte Carlo experiments were performed to investigate the stochastic effectdue to small population size. When the effective population size (No) issmall so that both Nov and Nos are small, the allele distribution is indistinguishable from the neutral case, while it approaches the deterministic
equilibrium as No gets larger. The bearing of the present models on observations regarding protein polymorphisms was discussed. For details, see
Amer. Natur. 109: 137-145.
Moments for Sum of an Arbitrary Function of Gene Frequency
along a Stochastic Path of Gene Frequency Change
Takeo MARUYAMA and Motoo KIMURA

A diffusion model is developed to compute any moment of the sum ofan arbitrary function of the gene frequency along sample paths between
any two specified frequencies. This is used to calculate the mean age ofa mutant of frequency x, including or excluding the possibility of its having
been at value x= 1 during the process, and the total frequency of heterozygotes involving an allele since its origin. For details, see Proc. Nat.
Acad. Sci. 72: 1602-1604.
The Effect of Selected Linked Locus on Heterozygosity of
Neutral Alleles (the Hitch-hiking Effect)
Tomoko OHTA and Motoo KIMURA

A diffusion model was developed to investigate the effect of a mutant
substitution by natural selection on heterozygosity at a linked neutral locus.
Using this theory, we made extensive numerical analyses to compute theexpected total heterozygosity (i.e. the sum of the fraction of heterozygotes
over all generations until fixation or loss) at the neutral locus. It was shownthat the hitch-hiking effect is generally unimportant as a mechanism forreducing heterozygosity. The effect becomes significant only when therecombination fraction between the selected and the neutral marker loci issmaller than the selection coefficient. In order to check the validity of the

RESEARCHES CARRIED OUT IN 1975

59

mathematical theory, Monte Carlo experiments were performed, and theresults were in agreement. It has been suggested that linkage is important
only in transient small populations such as at the time of speciation. For
details, see Genet. Res. 25: 313-326.
Statistical Analyses of Drosophila andHuman Protein Polymorphisms
Tomoko OHTA

By using the distribution function of allelic frequencies which was recently
derived by Kimura and Ohta (P.N.A.S. 72: 2761) for the model of stepwise
production of neutral alleles, the observed protein polymorphisms of Drosophila and man are tested for fit to the theory of neutral protein variation.
The observed and theoretical distributions of alleles agree quite well except
for the excess of rare alleles in the actual distributions. In human polymorphisms, the alleles with frequencies less than 1 % are more numerous
than expected, whereas in Drosophila, those with frequencies less than 10%
are more numerous. It is pointed out that these results support the thesisthat mutational pressure rather than balancing selection is the main cause
for the maintenance of protein polymorphisms. For details, see Proc.
Nat. Acad. Sci. 72: 3194-3196.
The Effects of Linkage in Multilocus
System-Overdominance
Tsuneyuki YAMAZAKI

Computer simulations were performed with overdominant multiple alleles
under multiplicative fitness model among tightly linked loci. A new measureof non-random association between multi-allelic loci is introduced: x2 jN(n-l)
was introduced as a new measure of linkage disequilibrium which can also
be applied to multiple allele model, where N is the sample size, and n is
the number of alleles (n is the smaller one when the number of alleles aredifferent between two loci).
Findings of simulations are: (1) With multiple (three or four) alleles,
the approach to the equilibrium is slower and the amount of disequilibrium
established is weaker. (2) The number of complementary chromosomes isa function of number of alleles and population size. (3) As population size

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

increases the approach to the equilibrium is slower. (4) There is an optimum
selection coefficient to minimize the transient fixation probability of alleles
with linkage.
Distribution of Enzyme Polymorphism andFunctional Difference in Drosophila
Tsuneyuki YAMAZAKI and Takeo MARUYAMA

Published data on enzyme polymorphisms in Drosophila species were
examined with special reference to molecular evolution. These data wereused to test several rival hypotheses regarding the maintenance mechanismsof genetic variation in natural populations. Three different quantities wereused for these tests: (1) Number and frequency of alleles, (2) Distributionof heterozygosity, (3) Variance of heterozygosity.
The following conclusions were obtained from the above tests. (1) Analysis of the data collectively indicates that distributions of most of thesepolymorphisms are consistent with those expected under the selective neutrality of polymorphic alleles. (2) When the enzymes were classified into twogroups according to their substrate specificity, namely substrate-specific andsubstrate-nonspecific ones, it has become apparent that there are more than
one mechanism for the maintenance of these polymorphic alleles. It was
found that substrate-nonspecific enzymes are maintained by the balance
between the mutational production of selectively neutral alleles and random
genetic drift, but that polymorphisms of substrate-specific enzymes appear to
be maintained by the balance between mutational production of very slightly
deleterious alleles and selective elimination. (3) More detailed examination
by analyzing each enzyme separately has, generally speaking, confirmed theabove conclusion and has shown that the possibility of balancing selection
playing a major role for the maintenance of protein polymorphism seems
unlikely at any of the 22 enzyme loci examined. Among them the most
likely candidates whose rare alleles are found to be selectively disadvantageous are enzymes such as malate dehydrogenase, fumarase, octanol dehydrogenase, tetrazolium oxidase, and glutamate-oxaloacetate transaminase.
The distributions of the various quantities at alkaline phosphotase, isocitrate dehydrogenase, and adenylate kinase loci are relatively closer to those
expected under balancing selection than those of other enzymes. The consideration of the step-wise model of mutation rather than the infinite allele

RESEARCHES CARRIED OUT IN 197561

model further decreases the possibility of balancing selection.
Distribution of Gene Frequency in
a Structured Population
Takeo MARUYAMASteady state distribution of gene frequencies were obtained for populationsof general geographical structure. Briefly, it consists of a finite number ofcolonies of arbitrary, finite sizes, and mating takes place in each colony
independently and then migration occurs between colonies. We assume
that the population structure is fixed so that steady state exists.
Diallelie Case
Assume that there are two alleles Al and A 2 at a locus under consideration,and that mutation rate from Al to A 2 is u and the reverse rate is v. Let N ibe the size of colony i (2Ni genes) and Xi be the gene frequency of Al incolony i. Now let

where N= L; N i the total population size. Therefore X is the mean genefrequency of Al allele reference to the whole population. The X is a randomvariable and the change of this random variable forms a stochastic process.
Then the steady distribution of X is given by

(2)
I

I

H(X)=N ~ Xi(l-Xi)Ni •Infinite Allele Case of Kimura and Crow (1964)(3)(])(X)= ~Nu_(l_X)4Nu.X I (I-X I )/H(X I )H(X),where O<XI <X and H(X) is given by (2).

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

IX.

EVOLUTIONARY GENETICS

Spectra of Heterozygosities and of Evolutionary
Rates among Different Loci
Takeo MARUYAMA and Tsuneyuki YAMAZAKI

Kimura and Ohta (1971) suggest that protein polymorphism and protein
evolution should be regarded not as separate phenomena, but as two aspectsof a single process. They further argue that most of naturally occurring
polymorphisms are transient and due to random frequency drift of selectively
neutral mutants in finite populations. Correspondingly, Kimura (1968)
and King and Jukes (1969) have argued that most of molecular evolutionarychanges detected as differences in amino acid sequence in homologous proteins, are due to fixation of neutral mutants within each species. The purposeof this note is an attempt to examine the hypothesis. Crow and Maruyama(1971) showed that for neutral polymorphisms at a steady state the mutation
rate (u) times the population size (N) can be estimated from the local andglobal genetic variations:
1- 10 ,

4Nu=--_~-f(1)

where 10 is the probability that two random homologous genes in a local
region are identical by descent and/is the corresponding probability for two
genes randomly chosen from the entire population. Importantly, thisrelationship is independent of the population structure. On the other hand,under the neutrality hypothesis of molecular evolution, Kimura proved thatthe evolutionary rate (R) or the rate of amino acid substitution is exactly
equal to the mutation rate (v) and is independent of other factors. Both ofthese quantities are observable and are known to vary greatly among loci.
Considerable data from which homozygosity (local 10, and global/) can
be calculated are available in published literature. Dayhoff's Atlas ofProtein
Sequence and Structure (1972) gives the evolutionary rates for a number ofproteins. Since both of these quantities are proportional to mutation rate,
if they are really two different aspects of a single process, their distributions
spectra ought to be similar. The purpose of this note is to obtain the distributions from available data and to examine whether they appear to be

RESEARCHES CARRIED OUT IN 1975

63

identical or not. This possibility was discussed by Maruyama and Yamazaki (1973) and Harris et al. (1974).
It is convenient to compare the distributions on a logarithmic scale. Thenif u and v have the same distribution, the distribution patterns oflog (I -10)1/
and log R should be identical, but one is shifted by a constant (log 4N)from the other. Gene frequency data for some 200 loci were collectedfrom the literature and the value of (I -10)1/ was calculated for each locus.
We regard different species of the same enzyme as sample paths of theprocess and the average value as an estimate of 4Nu. There are 18 loci forwhich four or more sample paths are available in all Drosophila and only
these were used. They are presented Figure l(a). In Dayhoff's Atlas (p. 50),
the rate of amino acid substitution is given for each amino acid site. Therefore to make them comparable to polymorphism data which are generated
on a protein basis, the value (r) given in the Atlas is multiplied by the
number (n) of amino acid residues of that protein. On the neutrality hypothesis this value R( =rn) should be the mutation rate per locus per year.
Since the rates in Dayhoff's Atlas are calculated from mammalian data andmammals have approximately the same length of generation time, the valueof R should be proportional to mutation rate per gene per generation whichis therefore comparable to the value of u. The distribution of log R among
different proteins is presented in Fig. 1(b).
The variances of the two distributions were calculated. The variance ofthe estimated 4Nu values is 1.51 with 17 degrees of freedom, while that of
the evolutionary rates is 2.13 with 16 degrees of freedom. The varianceratio is 1.41. The data are undoubtedly insufficient to draw a definite
conclusion. Nevertheless if we take these statistics at face value and apply
the variance ratio test, the probability of finding this much difference is morethan 20 percent. Therefore the result can be regarded as a supporting
evidence for the hypothesis of Kimura and Ohta. Finally it is worth notingthat larger variance in the evolutionary rates is mainly due to one very
slowly evolving protein, histone. When histone is excluded, the two distributions have almost the same variance and a similar pattern. Furthermore,
if the displacement constant 15 is interpreted as log.4N, then N is about 106 ,not an unreasonable value for the average effective population number.
The proteins used in constructing these graphs are different, as are thespecies. The polymorphism graph being based entirely on Drosophila data
and the evolutionary rates from mammals. Therefore, our comparison of

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26(0) Protein polymorphism

109 (1-/0 ) (:: log u+ const.)(b) Evolutionary rate

-6 -5 -4 -3 -2 -I

0logR+15(= logv+15)
Fig. 1. (a) The distribution of the values of log. (average (1-[0)11) which is an
estimate of log. u+ log. 4N. The [0, }, u and N are the average local homozygosity,
the global homozygosity, the mutation rate and the population size respectively.
(b) The distribution of the values of log. R which is an estimate of log. v. The R isthe evolutionary rate and v is the mutation rate.

apples and oranges is meaningful as a test of the neutral hypothesis only if
the distribution of mutation rates is comparable for the two groups of proteinsin the two groups. It would be better to have the same proteins and thesame kinds of animals for comparison.
For this reason we realize that the evidence offered in this note is weak.
Furthermore, monomodal distributions skewed to the left are common for
logarithmically transformed data, and we have very little statistical power todistinguish differences in shapes. Doubtless there are other hypotheses that
could generate graphs of roughly the same shape.

RESEARCHES CARRIED OUT IN 1975

65

We offer this analysis as weak supporting evidence for a common explanation of both phenomena, but mainly as a new methodology. We hope thatin the future data on polymorphism and evolutionary rates for the same proteins will be obtained in sufficient abundance that this kind of comparison
can be useful.
As more data become available it should be possible to look for correlations between log R and log (1-10)1/ for individual proteins, and to see if
the displacement of one distribution from the other is larger for species withlarge population numbers than for those with small numbers.
Colonization of Drosophila simu/ans in Japan
Takao K. WATANABE and Masaoki KAWANISHI

In Japan Drosophila simulans had been known only in the Bonin IsIs.
(Ogasawara) since its discovery in 1936 (Komai 1937, DIS 8, 78). Ourcollection survey work of Drosophila from Katsunuma during the past
twelve years have failed to obtain simulans there. Unexpectedly, since 1972
D. simulans has been frequently collected in the mainland of Japan. Although the first landing place of simulans has remained unknown, thespecies has already been distributed in many places of Japan.
In November of 1975 Drosophila flies were collected at 29 sites in Yamanashi and eastern Shizuoka Prefectures. A total of 6129 flies was identified
as members of the family Drosophilidae. D. immigrans was most abundant
everywhere, and D. melanogaster and D. simulans followed. The latter two
species were nearly the same in the total number, though their relative
frequency varied among the sites. D. simulans tended to be frequent in thesites nearby the Pacific coast, e.g. Mishima (97 %simulans), whereas simulans
was not collected in the inland area, e.g. Katsunuma (100% melanogaster).
Yet, the declination of simulans frequency toward Katsunuma occurredalong several routes from the Pacific coast. For details see Proc. Japan Acad.
52: 191-194 (1976).

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

x.

HUMAN GENETICS

Monitoring Cancer Families
Ei MATSUNAGA

Individuals with certain genotypes or certain chromosomal aberrations
are known to be at increased risks of developing specific cancers. The
genotypes may be heterozygous for a dominant gene with high penetrance
in which multiple tumors (e.g., bilateral retinoblastoma and polyposis of
the colon) are developed, or they may be homozygous for a recessive genesuch as that for Fanconi's anemia and Bloom's syndrome in which leukemia
may develop. For common cancers such as those of the stomach, the breast
and the uterus, heredity plays in general a minor role, but studies with avariety of genetic markers, notably those related to enzyme variants and the
HLA system, may eventually identify specific genotypes with which cancer
formation is closely associated.
These 'cancer genotypes' are usually ascertained by routine diagnostic
procedures. If the genotype can be detected by a simple and cheap testwith high sensitivity and high specificity, and if some kind of effective treatment is available before they manifest the disease, screening populations orgroups at risk can be undertaken as a preventive measure. No such test is,however, at present available for genetic markers closely associated with
common cancers. For rare dominantly inherited cancers such as retinoblastoma and polyposis of the colon, population screening by clinical examination is obviously not feasible. However, since relatives of patients withthose cancers form a special group at risk, family screening as an extensionof genetic counseling would be rewarding; it may allow detection of thoseat the initial stage of tumor formation and may identify carriers of the geneamong apparently healthy individuals.
At present most cases of heritable cancers occur as a result of fresh mutations. Yet, it is clear that, with increasing success of medicine, more andmore cases inherited from the survivors will be emerging unless some preventive measures are taken. Therefore, monitoring of cancer families overa long term is of great importance, not only for the families themselves but
also for the whole community.

RESEARCHES CARRIED OUT IN 1975

67

Retinoblastoma in Japan: Follow-up Survey of Sporadic Cases
Ei MATSUNAGA•

Previously we presented some genetic and epidemiologic data obtainedby a population survey of cases of retinoblastoma which occurred in Hokkaido during the period from 1945 to 1957. Since then we carried out a followup survey of these patients, together with a new series of cases, that is, 206
inpatients with retinoblastoma who had been treated in seven university
hospitals from 1900 to 1940. While some of the results of preliminary
nature had been reported in the meantime, we have recently completed theanalysis of the data.
The five-year survival rates for the 206 patients were about 50 % forunilateral and 17 % for bilateral cases, whereas for 74 patients occurred in
Hokkaido from 1945 to 1958 the rates were 60 % for unilateral and 32 % forbilateral cases. The mean ages at diagnosis of unilateral cases were 34.8
months in the University Hospital series and 31.5 months in the Hokkaido
series, while the corresponding figures of bilateral cases were 15.0 months
and 17.1 months in the respective series. With respect to the prospect ofmarriage and fertility of the survivors of unilateral retinoblastoma, there wasno essential difference from the general population for the males, but for thefemales, the chance of marriage seemed to be slightly decreased and, whenmarried, the average number of children was significantly smaller than thatof the male survivors, probably because of more eager practice of familyplanning on the female side. However, among 28 sporadic unilateral cases
having children, only one was found to have transmitted the condition tochildren. Taking into account the distribution of number of children, thefraction of sporadic unilateral cases that can transmit the condition was
estimated at 5 %. As to sporadic bilateral cases, there were three survivors
who produced children, and each had transmitted the disease to theirchildren, suggesting that all sporadic bilateral cases are due to dominant
mutations.
The frequency of retinoblastoma in Japan was 4.5 X 10- 5 or once in every
22, 326 children who survived the first one year, and about 60 % of them wererepresented by the non-hereditary form. Based on these data, a revised
estimate of germinal mutation rate was given by the indirect method as
8 X 10- 6 per generation, and the rate of somatic mutation was estimated,according to the hypothesis by Knudson (1971), at 5 X 10- 6 per locus in the

68

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

course of an individual's development.
Details of the study will be published in the Jap. J. Ophthalmol. 20:
266-282.
Classification with the Size of C-bands in the Human Chromosome
Variants and a Case Report of a de novo YqKazuso IINUMA and Ei MATSUNAGA

Heteromorphism of the secondary constrictions in the human chromosomes
has been well known, and a new staining method, C-banding, has allowed
quantitative evaluation of the size of heterochromatin blocks observed inthese regions. After the method described by Sumner, A.T. (Exp. Cell
Res. 75, 304, 1972), a total of 52 persons with normal karyotypes havebeen examined. The common size of the C-band of No.1 was defined asslightly larger than that of No.9. The latter had commonly a C-band withthe similar size of its short arm. The C-band of the No. 16, in most cases,
extended to the half of the long arm. Increases or decreases in the length ofthese secondary constrictions were expressed by symbols, h + or h -, respectively. Without these definitions, such various combinations with apparent
similarity as h/h-, h+/h or h+/h- could not have been distinguished.
The frequencies of Ih+, Ih-, 9h-, 16h+ and 16h- were 0.02,0.13,0.14,
0.01 and 0.06, respectively.
A cytogenetic study on a patient with congenital anomalies revealed adeletion on the distal long arm of the Y chromosome. The propositus had
severe psychomotor retardation, muscular hypotonicity, high-arched palate,
hypertelorism, low-set ears, short upper limbs and hypogenitalism. He hada small Y chromosome, and Q-banding showed a deletion of the fluorescing
long arm of the Y. The parents had normal karyotypes and the father's
Y had a commonly observed fluorescing portion of the long arm. Resultsfrom family studies on the red cell antigens and other genetic markers
including haptoglobin, transferrin and isoenzymes showed no inconsistency
for paternity.
A part of this study was reported in the 20th annual meeting of the Japan
Society of Human Genetics (1975).
Initiation of DNA Replication in Human Chromosomes
Yasuo NAKAGOME

While autoradiography has contributed much to the study of DNA

RESEARCHES CARRIED OUT IN 197569

replication of mammalian chromosomes, the resolving capacity of the technique is rather limited. An alternative method with a higher resolution is
now available. The fluorescence of the chromosome stain Hoechst 33258
is quenched by the incorporation of 5-bromodeoxyuridine (BrdUrd) in theDNA. Thus when BrdUrd is given during a part of the S-phase of a cell
cycle and the chromosomes at the next mitosis are examined, the segments ofchromosome containing BrdUrd appear as less fluorescent bands (Latt, 1973).
A modification of this technique involving Giemsa staining has also beenused. DNA replication of both random and partially synchronized cultures
has been studied using these techniques. The G-positive bands (Paris Conference) of chromosomes appear to continue DNA replication until lastpart of the S-phase (Grzeschik et al. 1974; Epplen et al. 1975), while Gnegative bands seem to start and finish replication earlier than the former(Kim et al. 1976; Epplen et al. 1976). On the other hand, Stubblefield (1975)concluded that there was no positive correlation between time of replication
and G-band pattern in Chinese hamster chromosomes.
In the present study, DNA replication within the first 10 minutes of the
S-phase was studied using synchronized human diploid cells (WI-38). A
combination of two techniques, mitotic cell detachment (Terasima & Tolmach,1963) and 5-fluoro-2'-deoxyuridine (0.1 ,ugjml) treatment (Priest et al. 1967),
was used to obtain good synchronization at the border of the G 1- and the Sphase. Cells entered the S-phase by the addition of small amount of thymidine (1.45 ,ugjml). After 10 minutes BrdUrd (50-150,ugjml) was administered. The "initiation sites" could be distinguished from the rest by more
intense fluorescence when stained either with Hoechst 33258 or acridine
orange. Alternatively, modified Hoechst-Giemsa technique was employed(Perry & Wolff, 1974).
The former two methods revealed exactly the same results. It appearedthat every chromosome in the human genome, including late-replicating X,
had segment(s) which initiated DNA replication within the first 10 minutesof the S-phase. The position, the shape and the size of these segments
corresponded to those of Q (G)-negative bands suggesting that each ofthem constitutes a basic unit of initiation of DNA replication. It does notnecessarily mean, however, that all of them initiate DNA replication at thistime. Q-negative secondary constriction of Nos. 1, 9 and 16 start replication late in the S-phase. They have been shown to contain satellite DNAs
(Jones, 1973).

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

When treated by the Hoechst-Giemsa method, several chromosome arms
(1p, 5q, 6p, llq and 12q) showed inconsistent pattern, i.e., the G insteadof the expected R-type bands were observed in most cells. In control
cultures that were treated similarly as the initiation-pattern experiment
except that BrdUrd was not administered, some cells showed G-bands.
Unusual pattern was also observed by Epplen et al. (1975) in a few arms including lp. It may be that some chromosome arms have a tendency toshow G-bands when stained with this technique irrespective of incorporationof BrdUrd (for details, see Nakagome: Exp. Cell Res., in press).

RESEARCHES CARRIED OUT IN 1975

XI.

71

BEHAVIORAL GENETICS

Noise Effect on Adult Emergence and Longevity of
Drosophila melanogaster
Chozo OSIDMA and Won Ho LEE

Four strains, which have homozygous second chromosomes extracted
from a natural population of Ishigaki-jima in 1973, were used in this experiment. Adult flies of these two strains emerged with circadian rhythms andof other two strains emerged with arhythmicity under light and dark 12: 12
environment. Many eggs, laid by many female flies for 12 hours, were
performed to develope under uncircadian rhythmic white and pink or 2000
cycle pure noise (NQ, NQ 4: 4, 8: 8) 100 phon, constant dark (DD) andtemperature (25°C) environments.
A total number of flies, emerged under noise environments was found tobe .fairly more than that under noiseless environment. Such a stimulus ofwhite and pink noise was assumed to be stronger than pure noise. The
noise accelerated remarkably the developmental rate of larvae and pupae,
and also the aging of adult flies. Longevity of adult flies was clearly reducedby the noise in addition to constant dark and temperature environments.
No significant difference was observed between the responses of four
strains for the stimulus of noise environments, but two strains showing
circadian rhythms in adult emergence, developed a little more quickly thanother two strains showing arhythmic emergence.
Albinism and Learning Performance in Mice
Tohru FUJISHIMA

The relationship between coat color and learning performance, (discriminated avoidance, avoidance and discrimination) was examined inmouse hybrids. The four-way hybrids obtained from crosses among four
inbread strains, C3H (agouti), SWM (albino), C57L (leaden) and DI03
(albino), were classified according to their coat colors into agouti, cinnamon
and albino groups. The results indicated that albino mice, as comparedThis work was supported by a grant from the Ministry of Environment Agency

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

with pigmented ones were generally inferior in avoidance learning performance (p<O.05). However, when compared with their pigmented sibs
from the same litter, the albinos were not inferior.
The inferiority in learning performance of albino mice to the pigmented
ones has been reported by several workers. But the data of the present experiment indicated that the inferiority of albinos could differ according to
the performances of inbred strains used and the analytical procedures
employed.

RESEARCHES CARRIED OUT IN 1975

XII.

73

APPLIED GENETICS

Variations in Copper Tolerance among Strains of Wild and Cultivated
Rice Species
Hiroko MORISIDMA and H. I. OKA

Forty strains belonging to Oryza perennis, O. sativa, O. breviligulata
and O. glaberrima were tested for copper tolerance in two plots of gravel
culture, in the same manner as were the barnyard grass strains. O. perennis
showed a wide range of variation in copper tolerance. Some Asian annualtypes appeared to be highly tolerant, showing better performance in 6 ppm
copper plot than in the control. The tolerance of sativa strains was comparable to that of susceptible perennis strains. O. breviligulata and O.
glaberrima gave a medium degree of tolerance. Measuring copper uptakeof the plants showed that O. perennis generally had a higher content (40140 ppm) than O. sativa (30-60 ppm), and those with lower content tended
to have a higher tolerance. But, copper uptake and tolerance were uncorrelated among the whole strains tested.
Further, 50 Fa lines from non-tolerant sativa (108) x tolerant perennis
strain (WI06, annual) were tested for copper tolerance. The results suggested that a few genes controlling tolerance could be involved and one ofthem, a recessive gene, was linked with the gene for pericarp coloration, Rd.
The Fa lines also showed a wide range in copper uptake, but it was uncorrelated with copper tolerance. Also the copper tolerance and performancein the control plot appeared uncorrelated. Perhaps, there are several genes
controlling different physiological responses which take part in copper
tolerance.
Variations in Copper Tolerance among Strains of Barnyard Grass
Hiroko MORISHIMA and H. I. OKA

The seeds of Echinochloa crusgalli (6X) and E. oryzicola (4X) were collected from copper polluted and control rice fields at Ohta (Gumma prefecture),
Tsuru (Y amanashi prefecture) and some other places, and also from a slag
deposit of Hanaoka copper mine (Akita prefecture). The progeny of asingle plant was considered a strain. This year, 69 strains were tested for

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

copper tolerance using two plots of gravel culture, one (control) with normal
culture solution (Matsushima's prescription) while the other containing
6 ppm of copper (CuS0 4 ) in addition. The copper tolerance of the plants
was shown by the copper-added: normal ratio in performance (in plantheight, tiller number, dry matter weight, root weight, etc., measured atmaturity). The data showed that: 1) A population could be highly
heterogeneous not only for copper tolerance but also for other characters.
2) The frequency of tolerant plants was higher in polluted than in control
fields. 3) When both 6X and 4X plants exist in the same population, 4X
plants generally had higher tolerance than 6X plants. 4) Tolerant strains,when tested without copper, generally had a lower performance than nontolerant ones. This resulted in a negative correlation between copper
tolerance and the performance in normal conditions. 5) The plants in thecopper-added plot showed at maturity a copper content ranging from 20 to
200 ppm (above-ground part, dried). The copper content was negatively
correlated to tolerance, suggesting that the tolerance was at least partly
due to a reduced copper uptake.
The Impact of Copper Pollution on the Weed Vegetation in
Japanese Rice Fields
Hiroko MORISffiMA and Hiko-Ichi OKA

In several districts in Japan, river water was polluted by the drain ofcopper mines located on the upper stream resulting in the pollution of rice
fields irrigated with the river water. Though the copper content of the soil
(to the depth of 20 cm) is not very high (maximum about 450 ppm), a large
area of rice farms, for instance, over 6,000 ha in the basin of the Watarase
River (north of Tokyo), has become polluted. The grain yield of rice begins
to significantly decline at about 150 ppm copper while those of wheat andbarley at about 80 ppm. Under the auspice of the Environment Agency,
we are working on the genetic effect of soil pollution on the weeds of rice
fields. Our work includes observations of Echinochloa crusgalli and Alopecurus aequalis (with plants from collected seeds) and other weed species(with plants from soil-buried seed stock). The latter part of the work isoutlined in this paper.
Soil samples were taken from rice fields in two districts, Ohta, Gumma
prefecture, polluted by the Watarase River (Asio mine) and Tsuru, Yama-

RESEARCHES CARRIED OUT IN 197575

nashi prefecture, polluted by the Ohata River (Takara mine). So far, 16
polluted (50 to 310 ppm) and 13 control (less than 30 ppm, from nearest
unpolluted rice fields) were examined. The number of plants arising fromthe soil samples in two months, sorted into species, were recorded, and theplants of several major species were tested for growth rate on normal soil
and copper tolerance (as shown by the ratio of growth rates on coppercontaining and normal soils). The results are summarized as follows:
1) General-The number of plants of different species were examinedassuming that their distributions were lognormal. The rice fields in different localities had a unique composition of weed vegetation (as shownby significant correlations between localities) which markedly differed fromthat of upland fields. Trends to positive or negative association of species
were significant in 9 out of 66 species combinations, when the correlations
among 12 major species were examined.
2) Diversity and dominance-The polluted soil samples produced agreater number of weed plants but a smaller number of species than thecontrols. Evaluating species diversity by the quantity of information(H = - ~PI log. PI) indicated that diversity was significantly reduced bycopper pollution. On the other hand, dominance (in terms of I-H/Hma ,,;Hmax=-log.ft) was found to be promoted by pollution. In a polluted rice
fields, the site nearby water inlet has a higher content of copper than wateroutlet. The above differences between polluted and control fields weresimilarly recognized when water inlet and outlet of the same fields werecompared.
3) Change of vegetation-Polluted rice fields generally gave larger
numbers of plants of Vandellia angustifolia, Lindernia procumbens, Rotalaindica and Fimbristylis miliacea and a smaller number of plants of Mazus
japonicus than unpolluted controls. The same pattern of changes was also
found from a comparison of water inlet and outlet of the same fields.
Analysis of variance of the data proved the significance of these changes.
Significant changes in species association due to pollution were found in
4 species combinations.
4) Copper tolerance-Cyperus difformis was highly tolerant while
Fimbristylis miliacea was most susceptible, and other species (among thosegiven above) appeared to be intermediate. The evolution of tolerant
plants in the polluted fields was recognized in three species, i.e., Vandellia
angustifolia, Mazusjaponicus and Cyperus difformis. Thus, neither the mean

76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

tolerance for species nor within-species differentiation of tolerant plants
did account for the above-mentioned increase and decrease in plant numberof different species. Perhaps, a relatively low level of copper pollution
as presently observed has its impact on sociological interactions among
species.
5) Growth of tolerant plants-When tested on normal soil, the tolerant
plants of Cyperus difJormis and Vandellia angustifolia had a lower growthrate than non-tolerant ones. This resulted in a negative correlation between
tolerance and performance on normal soil. It seems that the change in
metabolism increasing copper tolerance is disadvantageous in normal
conditions.

Genetic Loads in Wild and Domestic Japanese Quails
Takatada KAWAHARA

Birds of a wild strain reared for 2 to 4 generations in cages, which showedchanges in characters toward the domesticated type as reported elsewhere(Ann. Rep. 1974), were compared with a domestic strain, and their reciprocal F 1 hybrids were investigated. The initial numbers of matings were
191 wild x wild (W), 85 domestic X domestic (D), 83 domestic ~ X wildd' (DW) and 108 wild ~ X domestic d' (WD), and the F 1 birds were
subjected to full-sib mating. The amount of genetic load in terms oflethal equivalent was estimated by the regression method (Morton et aT.
1956 Proc. Natl. Acad. Sci. U.S. 42: 855).
The estimated lower (2B) and upper [2(A + B)] limits of lethal equivalent
per zygote, and BfA ratios as well, were calculated for various traits fromthe populations with F=O and F=0.25 (Table 1). Lethal equivalents inwild strain were higher than those in domestic strain in all the traits examined. The domestic strain may have established themselves by eliminating a part of detrimental genes during their history of domestication. However, the F 1 hybrids had approximately the same lethal equivalent as of thedomestic strain. All estimates of the BfA ratio were lower than 14.

RESEARCHES CARRIED OUT IN 1975

77

Table l. Estimates of lower and upper limits of lethal
equivalent per zygote, and of BIA ratio
Lethal equivalent per zygote
Trait

StrainBIA

Lower limit

Upper limit(2B)[2(A+B)l

Fertility

W
D
DW
WD

0.80
0.24
0.44
1.12

1.46
0.48
0.86
1.48

1.23
1.03
1.06
3.07

Hatchability

W
D
DW
WD

5.24
3.48
3.60
2.56

6.32
3.82
4.04
3.22

4.85
10.27
8.24
3.91

Viability

W
D
DW
WD

7.22
3.78
4.10
3.32

7.76
4.20
4.54
3.74

13.49
9.16
9.50
7.81

Egg production
rate

W
D
DW
WD

2.18
1.24
0.88
1.24

3.20
1.46
1.28
1.58

2.16
2.05
2.19
3.77

Fitness index(Total)

W
D
DW
WD

15.44
8.74
9.02
8.24

18.74
9.96
10.72
10.02

4.68
7.16
5.31
4.63

Viability: up to 20 weeks after hatching; egg production rate: hen-day basis up
to 60 days after first egg; fitness index: fertility x hatchability x viability x egg
production rate.

Esterase-D Isozymes in Japanese Quail
Takatada KAWAHARA, Seiki WATANABE and Takeshi SHIBATA•

The erythrocytes and eleven internal organs from four strains, one wild
and three domestic (DN, DS and DM), of Japanese qualis were examinedfor esterase-D isozymes by the use of starch gel electrophresis.
Three bands were detected in a region. No difference was found among
the zymograms from erythrocytes and various organs.
The results of mating experiment indicated that these three bands were
controlled by codominant autosomal alleles symbolized Es-DF and Es-Ds.

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

Three domestic strains differed in the frequency of these alleles e.g., the frequency of Es-DF was 0.264 in DN, 0.275 in DS and, 0.568 in DM, thedifferences being statistically significant. The frequency in the wild strain
was 0.521.

Variation in Susceptibility of Echinochloa crus-galIi
to a Herbicide
Shinya IYAMA

Echinochloa crus-galli collected from various parts of Japan were investigated regarding its susceptibility to herbicide containing propanil which
can kill it but does not damage rice plants. Lines were established on thebasis of individual plants collected in the rice fields around Sendai (Northern
Japan), Konosu, Ohta and Mishima (Central) and Kochi (Western Japan);twenty to thirty lines from each locality (or population) were tested. Fifty
to 100 seedlings per line were grown in a plastic tray, and at the age of two
weeks they were sprayed with a given amount of propanil solved in water.
The susceptibility was evaluated by percent survival in two weeks after thetreatment.
The results showed that I) all the populations had a wide variation inpercent survival (0 to 100 %) and 2) they differed in average suceptibility,
being high in Konosu, Ohta and Mishima and low in Sendai and Kochi.
No line as resistant as the rice plant was found from the present material.

On the Efficiency of Selection for Elite Trees
Shinya IYAMAThe efficiency of a selection procedure for elite trees was investigated bycomputer simulation. The procedure is a generally used one for selecting
elite trees from a standing forest: a candidate tree is compared with themean of best three (m') taken from 20 to 30 adjacent trees regarding thevolume of single tree, and if it exceeds a criterion, say 1.5 times as much as
the mean, the candidate is selected. The intensity of selection resulting
from this procedure is determined by the relative value of population mean(m) and its standard deviation (s) when a fixed number of adjacent trees aremeasured. In the case of twenty trees including a candidate, the intensity

RESEARCHES CARRIED OUT IN 1975

79

of selection is theoretically expected to be 0.32 %, 0.06 % and 0.01 % whenm=2s, 3s and 4s, respectively. It is suspected that by this procedure, selection is often based on a lower criterion than expected m' resulting in a reduction in effectiveness of selection. Computer simulation revealed that the
reduction in the m' was 13.2%, 17.3% and 25.6% when m=2s, 3s and 4s,
respectively.

Variations in Anaerobic Germinability of Seeds found among
Wild Rice Strains
Yoshio SANO

It is well known that the seed of rice can germinate in water by anaerobic
respiration. But, how this capacity does differ according to genotypes is
not known. The seeds of 40 strains of Oryza perennis (wild) stored for 10
to 15 years at 0°_5°C were tested in petri-dishes with moistened filter paper
and in flasks filled with water at 25°C, with the seeds of a few strains of cultivated species (0. sativa and O. glaberrima) taken as the controls. The
results showed that: 1) Generally, the seeds of O. perennis did not germinate
well in water suggesting that they require more oxygen than those of cultivated species. 2) The American form of O. perennis showed this trend
most strongly, while the African form showed better germination of submerged seeds. The Asian strains, which may be the wild progenitor ofcultivated rice, appeared to be intermediate. 3) The capacity of anaerobic
germination seemed to be a function of seed dormancy as the germinability
differed according to the period of cold storage. Relatively new seeds (lessthan 10 years in cold storage) showed a low rate of germination in water.

80

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

BOOKS AND PAPERS PUBLISHED IN 1975
BY STAFF MEMBERS( 1) ARIGA, I. and A. MURAKAMI 1975: Induction of recombinants by mitomycin C inoogenesis of the silkworm (Bombyx mori L.). (In Japanese) J. Sericult. Sci. Japan
44: 154-160.
(2) CmYo, H., Y. NAKAGOME, I. MATSUI, Y. KUROKI, H. KOBAYASHI and K. ONO 1975:
Two cases of 8p trisomy in one sibship. Clinical Genet. 7: 328-333.
( 3) CHIYO, H., Y. KUROKI, I. MATSUI, K. YANAGIDA and Y. NAKAGOME 1975: A 6p
trisomy detected in a family with a "giant satellite." Human Genet. 30: 63-67.
(4) CHOO. J. 1975: Genetic studies on the phototactic behavior in Drosophila melanogaster 1. Selection and genetic analysis. Jap. J. Genet. 50: 205-215.
(5) CHOO, J. 1975: Genetic studies on the phototactic behavior in Drosophila melanogaster 2. Correlated response: lethal frequency and eclosion rhythm. Jap. J. Genet.
50: 361-372.
(6) CHOO, J. 1975: Genetic studies on walking behavior in Drosophila melanogaster1. Selection and hybridization analysis. Can. J. Genet. Cytol17: 535-542.
( 7 ) ENOMOTO, M. and B. A. D. STOCKER 1975: Integration, at hag or elsewhere, of
H2 (phase-2 flagellin) genes transduced from Salmonella to Escherichia coli. Genetics 81: 595-614.
(8) FREIRE-MAlA, A. W-H. LI and T. MARUYAMA 1975: Genetics of acheiropodia(the handless and footless families of Brazil). VII. Population dynamics. Amer.
J. Human Genet. 27: 665-675.
(9) FURUICHI, Y. and K. MIURA 1975: A blocked structure at the 5'-terminus ofmRNA of cytoplasmic polyhedrosis virus. Nature 253: 374-375.
(10) HAYATSU, H., Kyu CHARN CHUNG, T. KAoA and T. NAKAJIMA 1975: Generation of mutagenic compounds by a reaction between sorbic acid and nitrite.
Mutation Res. 30: 417-419.
(11) IINUMA, K., T. OHZEKI, K. OHTAGURO, E. HIGASHIHARA, A. TANAE and Y. NAKAGOME 1975: Y-chromatin positive cells in the smear preparations of the gonad
from an XX male. Human Genet. 30: 193-196.
(12) IINUMA, K., Y. NAKAGOME and M. HIGURASHI 1975: A de novo translocationt(6q+ : 15q-) in a boy with trisomy 21. Jap. J. Human Genet. 20: 147-151.
(13) IMAI, H. T. 1975: Evidence for non-random localization of the centromere on
mammalian chromosomes. J. theor. BioI. 49: 111-123.
(14) IMAI, H. T. and M. KUBOTA 1975: Chromosome polymorphism in the ant, Pheidole
nodus. Chromosoma (Berl.) 51: 391-399.
(15) KAoA, T. 1975: Mutagenicity and carcinogenicity screening of food additives by
the rec-assay and reversion procedures; in "Screening tests in chemibal carcinogenesis" (ed. Montesano, R., Bartsch, H. & Tomatis, L., Lyon). IARC (International Agency for Research on Cancer), Scientific Publication No. 12: 105-115.

BOOKS AND PAPERS PUBLISHED IN 1975

81(16) KADA, T., T. Nooun and Y. SADAlE 1975: DNA repair in Bacillus subtilis; Comparative studies with gamma-rays and ultraviolet light. Anais da Academia
Brasileira de Ciencias 45: SuppI. 179-184.
(17) KATO, H. and H. SIllMADA 1975: Sister chromatid exchanges induced by mitomycin
C: a new method of detecting DNA damage at chromosomal level. Mutation
Res. 28 : 459-464.
(18) KIMURA, M. and T. OHTA 1975: Distribution of allelic frequencies in a finite population under stepwise productoin of neutral alleles. Proc. Nat. Acad. Sci. U.S.A.
72: 2761-2764.
(19) KIMURA, M. 1975: Mathematical contributions to population genetics. Proc.
XIII Inter. Congo of Genetics. Part II. Genetics 79 (Supplement): 91-100.
(20) KIMURA, M. 1975: An introduction to theoretical population genetics. "Foundations of Human Genetics" (In Japanese) Iwanami Lecture Series in Modern Biology
6 (ed. by Kimura, M.), Iwanami Shoten.
(21) KING, J. L. and T. OHTA 1975: Polyallelic mutational equilibria. Genetics 79:
681-691.
(22) KITAMURA, Y., T. KAWATA, K. TSUCHlYA and K. MORIWAKI1975: Range ofxenogeneic donors whose hematopoietic cells can form colonies in macrophage layer ofmice. Exp. Hemat. 3: 383-388.
(23) KOToKu, K. and A. MURAKAMI 1975: Strain difference in larval phototactic behavior of the silkworm (Bombyx mori L.). (In Japanese) Environ. Control in BioI. 13:
95-103.
(24) KURODA, Y. 1975: Mutagenesis in cultured human diploid cells. III. Inductionof 8-azaguanine-resistant mutations by furylfuramide. Mutation Res. 30: 229-238.
(25) KURODA, Y. 1975: Movement of cells in tissue reconstruction of animal cells in
culture. (In Japanese) Heredity (Tokyo) 29 (5): 25-31.
(26) KURODA, Y. 1975: The present status and prospective view of tissue culture
studies in the field of genetics. I. (In Japanese) Tissue Culture (Tokyo) 1(2): 55-61.
(27) KURODA, Y. 1975: On the tissue and organ formation of animal cells in culture.
(In Japanese) Tissue Culture 1 (5): 248-251.
(28) KURODA, Y. 1975: Mutagenesis in cultured human diploid cells. IV. Inductionof 8-azaguanine resistant mutations by phloxine, a mutagenic red dye. Mutation
Res. 30: 239-248.
(29) KURODA, Y. 1975: Mutagenesis by phloxine to 8-azaguanine resistance in cultured
human diploid cells. Mutation Res. 31: 264-265.
(30) KURODA, Y. 1975: Participation of intercellular materials in enhancing aggregation of embryonic quail liver cells. Cell Structure and Function 1: 111-118.
(31) KURODA, Y. 1975: Protective effect of vitamin E on reduction in colony formation of cultured human cells by bisulfite. Exp. Cell. Res. 94: 442-445.
(32) KURODA, Y. 1975: Gene expression in development (abstract). Teratology 12:191.
(33) KURODA, Y., A. YOKOIYAMA and T. KADA 1975: Radiosensitization of cultured
mammalian cells by 5-iodouridine. Int. J. Rad. BioI. 27: 247-257.
(34) KURODA, Y., A. YOKOlYAMA and T. KADA 1975: Isolation and characterizationof variant clones of Chinese hamster cells after treatment with irradiated 5-iodouri-

82

(35)
(36)

(37)

(38)

(39)
(40)

(41)
(42)
(43)
(44)
(45)

(46)

(47)
(48)

(49)
(50)
(51)

(52)

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26
dine. Mutation Res. 33: 285-298.
MARUYAMA, T., and J. F. CROW 1975: Heterozygous effects of X-ray induced mutations on viability of Drosophila melanogaster. Mutation Res. 27: 241-248.
MARUYAMA, T., J. F. CRow and J. PANDEY 1975: Heterozygous effects of X-ray
induced mutations on viability of Drosophila melanogaster: Addendum. Mutation
Res. 27: 255.
MARUYAMA, T. and M. KIMURA 1975: Moments for sum of an arbitrary function ofgene frequency along a stochastic path of gene frequency change. Proc. Nat. Acad.
Sci. U.S.A. 72: 1602-1604.
MATSUNAGA, E. and E. IKEDA 1975: Genetic composition of the Ainu: Fingerprints. Anthropological and genetic studies on the Japanese. JIBP Synthesis
2: 285-288.
MATSUNAGA, E., S. SAWAGUCHI and T. HONNA 1975: Estimation of heritability ofliability to indirect inguinal hernia. Jap. J. Human Genet. 20: 197-200.
MATSUNAGA, E. 1975: Genetic consequences of abortion. In "Genetics and the
Quality of Life" (ed. by C. Birch & P. Abrecht), 130-146, Pergamon Press(Australia).
MATSUNAGA, E. 1975: Demographic trends, family planning and their genetic
implications. (In Japanese) Tokyo Jour. Med. Sciences 83: 330-343.
MATSUNAGA, E. 1975: Are congenital malformations increasing in Japan? (InJapanese) Kosei-no-Shihyo 22(7): 11-17.
MATSUNAGA, E. 1975: Population problems and the quality of life. (In Japanese)
Jour. Home Econ. 26: 89-96.
MATSUNAGA, E. and K. KOBAYASHI 1975: Monitoring for fertility trend towardstabilization of population. (In Japanese) Bull. Populatoin Assoc. Japan 9: 42-43.
MIURA, K., Y. FURUICHl, K. SHlMOTOHNO, T. URUSHlBARA, K. WATANABE and
M. SUGIURA 1975: Modification at the 5 ' -terminus of messenger RNA strand.
Les Colloq. de INSERM (EMBO Symp.) 47: 153-160.
MIURA, K., Y. FURUICHl, K. SHIMOTOHNO, T. URUSHlBARA and M. SUGIURA 1975:
Synthesis of viral messenger RNA carrying a unique modified structure. Proc. 10th
FEBS Meeting, Paris, 1975,95-108.
MORISHIMA, H. 1975: Variation in the pattern of growth and its significance in
breeding. Recent Advances in Breeding Science 16: 65-70. (In Japanese)
MORISHIMA, H. 1975: Mortality variation and its adaptive significance in perennial
ryegrass and orchardgrass varieties. J. Japan. Grassl. Sci. 21: 26-23. (In Japanesewith Eng. Summary)
MORISHIMA, H. 1975: The mechanisms of weediness found in dallisgrass population.
Japan. J. Breed. 25: 265-274. (In Japanese with Eng. Summary)
MORISHIMA, H. and H. I. OKA 1975: Comparison of growth pattern and phenotypic plasticity between wild and cultivated rice strains. Jap. J. Genet. 50: 53-65.
MORIWAKI, K., H. KATO, H. T. IMAI, K. TSUCHlYA and T. H. YOSIDA 1975: Geographical distribution of twelve transferrin alleles in black rats of Asia and Oceania.
Genetics 79: 295-304.
MUKAI, T., R. A. CARDELLINO, T. K. WATANABE and J. F. CROW 1975: The genetic

BOOKS AND PAPERS PUBLISHED IN 1975

83

variance for viability and its components in a local population of Drosophila melanogaster. Genetics 78: 1195-1208.
(53) MURAKAMI, A. 1975: Analysis of a chromosome specific genetic instability in thesilkworm, Bombyx mori. Mutation Res. 27: 411-414.
(54) MURAKAMI, A. 1975: Mutagenic action of ethyl methanesulfhonate in oogenesisof the silkworm, Bombyx mori L. Jap. J. Genet. 50: 179-187.
(55) MURAKAMI, A. and H. FUJIOKA 1975: Mutagenesis of furylframide in mature
sperm of the silkworm. Mutation Res. 31: 266-267.
(56) NAKAGOME, Y. and H. KOBAYASm 1975: Trisomy of the short arm of chromosome
10. J. Medical Genet. 12: 412-414.
(57) NAMIKI, M. and T. KADA 1975: Formation of ethylnitrolic acid by the reaction ofsorbic acid with sodium nitrite. Agr. BioI. Chem. 39: 1335-1336.
(58) NAWA, S., M. YAMADA and Y. OHTA 1975: Hereditary changes in Capsicum annuum L. III. Induced by DNA treatment. Jap. J. Genet. 50: 341-344.
(59) NEI, M. and T. MARUYAMA 1975: Lewontin-Krakauer test for neutral genes.
Genetics 80: 395.
(60) NEI, M. ,T. MARUYAMA and R. CHAKRABORTY 1975: The bottleneck effect andgenetic variability in populations. Evolutoin 29: 1-10.
(61) NOGUTI, T. and T. KADA 1975: Studies on DNA repair in Bacillus subtilis I. A
cellular factor acting on r-irradiated DNA and promoting its priming activity for
DNA polymerase I. Biochim. Biophys. Acta. 395: 284-293.
(62) NOGUTI, T. and T. KADA 1975: Studies on DNA repair in Bacillus subtiUs II.
Partial purification and mode of action of an enzyme enhancing the priming activityof r-irradiated DNA. Biochim. Biophys. Acta. 395: 294-305.
(63) OHTA, T. 1975: Statistical analyses of Drosophila and human protein polymorphisms. Proc. Nat. Acad. Sci. U.S.A. 72: 3194-3196.
(64) OHTA, T. and M. KIMURA 1975: Theoretical analysis of electrophoreticallydetectable polymorphisms: models of very slightly deleterious mutations. Amer.
Nat. 109: 137-145.
(65) OHTA, T. and M. KIMURA 1975: The effect of selected linked locus on heterozygosityof neutral alleles (the hitch-hiking effect). Genet. Res. Camb. 25:313-326.
(66) OKA, H. I. 1975: Experimental studies on the origin of cultivated rice. Genetics
78: 475-486.
(67) OKA, H. I. 1975: Yield stability, its mechanisms and selection. Recent Advancesin Breeding Science 16: 41-45. (In Japanese)(68) OSHIMA, C., W. H. LEE, T. KAWAHARA and T. FUJISmMA 1975: Genetic studies on
the effects of adverse environments on plants and animals. I. Effects of noise
environments in insects, birds and animals. Environmental research in Japan, 1975
Environment Agency, 1-2.
(69) PAl, C. T., T. ENDO and H. I. OKA 1975: Genic analysis for acid phosphataseisozymes in Oryza perennis and O. sativa. Can. J. Genet. CytoI. 17: 637-650.
(70) SAWAGUCHI, S., E. MATSUNAGA and T. HONNA 1975: A genetic study on indirect
inguinal hernia. Jap. J. Human Genet. 20: 187-195.
(71) SmNoDA, T. 1975: Comparative structural studies on the light chains of human

84

(72)
(73)
(74)
(75)
(76)
(77)
(78)
(79)
(80)

(81)
(82)

(83)
(84)

(85)
(86)
(87)

(88)
(89)
(90)

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26
immunoglobulins. J. Protein Ka with the Inv (3) allotypic marker. J. Biochem.
77: 1277-1296.
SHINODA, T. 1975: Antibody structure and antigen-antibody reaction. Gunma
Symp. Endocrinol. 12: (in press).
SLATKIN, M. and T. MARUYAMA 1975: The influence of gene flow on genetic distance. Amer. Nat. 109: 597-601.
SLATKIN, M. and T. MARUYAMA 1975: Genetic drift in a cline. Genetics 81:
209-222.
SUZUKI, H. and T. lINo 1975: Absence of messenger ribonucleic acid specific forflagellin in non-flagellate mutants of Salmonella. J. Mol. BioI. 95: 549-556.
TAZIMA, Y., H. DOIRA and H. AKA! 1975: The domesticated silkmoth, Bombyx
mori. In "Handbook of Genetics" (ed. R. C. King), Plenum Press, New York.
TAZIMA, Y. and K. ONIMARU 1975: Silkworm oocyte system for testing mutagenicity
and nature of chemical. Mutation Res. 31: 270-271.
TAZIMA, Y., T. KADA and A. MURAKAMI 1975: Mutagenicity of nitrofuran derivatives, including furylfuramide, a food preservative. Mutation Res. 32: 55-80.
TAZIMA, Y. 1975: Introductory remark on environmental mutagens and teratogens
in man. Teratology 12 (2): 189.
TUTIKAWA, K. and T. KADA 1975: Studies on the mutagenicity of furylfuramide:
results of the host-mediated rec-assay and dominant lethal test in mice. Mutation
Res. 31: 271.
URUSHIBARA, T., Y. FURUICHI, C. NISHIMURA and K. MIURA 1975: A modified
structure at the 5'-terminus ofmRNA of vaccinia virus. FEBS Letters 49: 385-389.
SCHWARZ, U., A. RYTER, A. RAMBAcH, R. HELLIO and Y. HIROTA 1975: Processof cellular division in Escherichia coil: differentiation of growth zones in the sacculus.
J. Mol. BioI. 98: 749-759.
WATANABE, S., H. YOSHIDA and T. KAWAHARA 1975: Serum amylase isozymes in
the Japanese quail. (In Japanese) Jap. Poult. Sci. 12: 67-70.
WATANABE, S., S. KONDO and E. MATSUNAGA (ed.) 1975: Anthropological andgenetic studies on the Japanese. JIBP Synthesis Vol. 2, 1-337. Tokyo University
Press.
WATANABE, T. K. 1975: A new sex-transforming gene on the second chromosomeof Drosophila melanogaster. Jap. J. Genet. 50: 269-271.
WATANABE, T. K. and S. OHNISHI 1975: Genes affecting productivity in natural
populations of Drosophila melanogaster. Genetics 80: 807-819.
YAMAZAKI, T. and T. MARUYAMA 1975: Isozyme polymorphism maintenance mechanisms viewed from the standpoint of population genetics. Isozyme IV Genetics
and Evolution. (ed. by C. L. Markert), 103-114, Academic Press.
YOSHIDA, Y. and K. MORIWAKI 1975: Specific marker chromosomes involving atranslocation (12-15) in a mouse myeloma. Proc. Jap. Acad. 51: 588-592.
YOSIDA, T. H. and T. SAGAr 1975: Variation of C-bands in the Chromosomes ofseveral subspecies of Rattus rattus. Chromosoma (Berl.) 50 283-300.
YOSIDA, T. H. 1975: Spontomeous diabetes in Djungalian hamster, Phodopus
sugOUS. Medicine and Biology 91: 267-269.

BOOKS AND PAPERS PUBLISHED IN 1975

1975: Chromosomal alteration and development of experimental
tumors. Handbuch der allgemeinen Pathologie. Tumors II. Springer-Verlag(Berlin): 677-753.
(92) YOSIDA, T. H. 1975: Diminution of heterochromatic C-bands in relation to thedifferentiation of Rattus species. Proc. Jap. Acad. 51(8): 659-663.
(91)

YOSIDA, T. H.

85

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 26

ABSTRACTS OF DIARY FOR 1975
February 14 226th Meeting of Misima Geneticists' ClubApril
12 119th Biological Symposium
20 120th Biological SymposiumMay'
16 121st Biological Symposium
19 227th Meeting of Misima Geneticists' ClubJune
16 228th Meeting of Misima Geneticists' Club20 229th Meeting of Misima Geneticists' ClubJuly
14 230th Meeting of Misima Geneticists' Club
November 15 122nd Biological Symposium

FOREIGN VISITORS IN 1975

87

FOREIGN VISITORS IN 1975
February 23-28
April

20-22
24-25

•

May

19

June

23

September

12

CROW, J. F., Laboratory of Genetics, University of
Wisconsin, U.S.A.
LI, W.-H., Center for Demographic & Population
Genetics, University of Texas, U.S.A.
KIRK, R. L., Dept. of Human Biology, Australian
National University, Australia.
RANGASWAMI, G., Vice-Chancellor, Tamil Nadu Agricultural University, India.
STORMONT, C. J., Dept. of Veterinary Microbiology,
University of California, U.S.A.
VILLIERS, J. N., University of Stellenbosch, South
Africa.

88

ANNUAL REPORT NATIONAL INSTITUTE OF GENETICS NO. 26

AUTHOR INDEXAMANO, E
51,52,55
AOTSUKA, T
20
BARADJANEGARA, A. A. .
55
Fum, T. .
54, 55
FunsAwA, T
26, 27
FunsHIMA, T............•............ 71
FUKASE, Y
39
FURUICHI, Y. .
12
HrroTA, Y
17
IKETANI, H
17
IINUMA,K. .
68
INOUE, T.48, 49
IYAMA, S
78
KAoA, T. .
.48, 49, 50
KATO, H
37
KAWAHARA, T. .
76,77
KAWANISHI, M
65
KIMURA, M
57,58
KONDo, K. .
20, 25
KURODA, Y
28, 29, 30, 31
LI, W.-H
71
MARUYAMA, T. .
58, 60, 61, 62
MATSUNAGA, E
66, 67, 68
MINATO, K
31
MINEZAWA, M
20, 23
MWRA, K. .
12, 13, 14
MWRA, Y12
MORISHIMA, H. .
73, 74MORIWAKI, K,20, 23, 38
MURAKAMI, A.41, 42, 44, 45

MUROTA, T.42
NAKAGOME, Y•...................... 68
NARUI, K.49
N~HIMURA, Y
17
OCHIAI, Y. .
36
OHNUMA, A. .
40
OHSHIMA, C. .
71
OHTA, T
57,59
OKA, H. I.
73, 74
ONIMARU, K. .
39, 40SADAIE, Y.48
SAKAKIBARA, K. .
38
SANO, Y79
SEGAWA, K
16
SHIBATA, T
77
SHIBUYA, T. .
29
SHIMADA, Y
41
SHIMOTOHNO, K.
12, 13, 14
SHIROISHI, T. .
20
SUGWRA, M. .
15, 16SUGIYAMA, T
26,27
SUZUKI, H•.......................... 17
TAZIMA, Y. .
39, 40, 41
WATANABE, S
77
WATANABE, T. K. .
65
YAMAZAKI, T
59, 60, 62
YASUDA, S
17
YAZAKI, K. .
13
YOSIDA, T. H
33,34,35,36,46

...

I

..
lE~iI~~1i1f~fflijt3t~m

m 26-J}IlBfU 51 ~ 12 Jj 20 B fflill
IlBfU 51 ~ 12 Jj 25 B J!fi

J!fi~

EEl Hb ~

* iB

~~~

~

~

1iIjz:)I~~iJf~ml13

lifi

iJ&

1iIjz:3I~~iJf~J'f113

11.

ffliUJ'f:~ OOI~tit)(Mk$lllutt

JI( Jj( ~ Ii,~
1

:tr JJ: iJ&
m18: jffj mm~ 3-8-8

ffl.U~

Jt!:Jj(~liml8:jffjmm~

3-8-8

J! fi m ~3Li:I~~iJf~m

T411 ftJllIiIJ/i\=llIsm~m 1111~
(0559) (75) 0771

'll

ft*

to HakoneNt
1:25,000•"•